Immunity and Inflammation in Health and Disease: Emerging Roles of Nutraceuticals and Functional Foods in Immune Support 9780128054178, 0128054174

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Immunity and Inflammation in Health and Disease: Emerging Roles of Nutraceuticals and Functional Foods in Immune Support
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Table of contents :
Front Cover
Immunity and Inflammation in Health and Disease
Copyright Page
Dedication
Contents
List of Contributors
Preface
I. Innate and Adaptive Immune Systems: Components and Regulation
1 Innate and Adaptive Immunity: Barriers and Receptor-Based Recognition
1.1 Introduction
1.2 The Components of the Immune Response (External and Internal Elements)
1.3 The Innate Immune System (Cell Types, Signaling)
1.4 Inflammation and Innate Immunity
1.5 Induction of Adaptive Immunity (Antigen Presentation as a Key Event)
1.6 Cell Types in the Adaptive Immune Response 1.7 Crosstalk Between the Innate and Adaptive Immune Responses1.8 The Vascular Endothelium as a Converging Site for Both Innate and Adaptive Immunity
1.9 Chronic Diseases of the Innate and Adaptive Immune Systems
1.10 Improving Immunity for Prevention and Care
1.11 Summary
References
2 Innate Immunity at Birth: Implications for Inflammation and Infection in Newborns
2.1 Introduction
2.2 Microbiome in Shaping the Newborn Immune System
2.3 Mucosal Immunity at Birth
2.4 The Innate Immune Cells of the Newborn
2.4.1 Neonatal Neutrophils
2.4.2 Neonatal Monocytes 2.4.3 Neonatal Macrophages2.4.4 Neonatal Dendritic Cells
2.4.5 Neonatal Natural Killer (NK) Cells
2.5 Soluble Plasma Components
2.6 Conclusion
References
3 Redox Signaling and the Onset of the Inflammatory Cascade
3.1 Introduction
3.2 ROS Generation During the Onset of the Inflammatory Cascade
3.3 Redox Control of Inflammatory Mediators
3.4 Redox Control of Antioxidant and Anti-inflammatory Transcription Factors
3.5 Emerging Role of Peroxiredoxins in the Onset of the Inflammatory Cascade
3.6 Conclusions and Perspectives
References II. Reactive Oxygen Species, Oxidative Stress and Immune Cell Activation4 Reactive Oxygen Species, Oxidative Damage and Cell Death
4.1 Introduction
4.2 Endogenous ROS Production
4.3 Exogenous ROS Production
4.4 Physiological Role of Reactive Oxygen Species
4.5 Oxidative Stress
4.6 Oxidative Damage is Outcome of Oxidative Stress
4.7 Cell Death
4.8 Conclusions
References
5 Mitochondrial ROS and T Cell Activation
5.1 Introduction
5.2 Reactive Oxygen Species
5.3 Sources of Mitochondrial ROS
5.4 Regulation of mROS
5.5 Targets of mROS
5.6 Mitochondrial ROS in T Cell Activation 5.7 SummaryReferences
6 Overcoming Oxidants and Inflammation: Endothelial Targeting of Antioxidants to Combat Chronic Inflammatory Disease
6.1 Introduction
6.2 Inflammation and Disease
6.2.1 Vascular Endothelium, Reactive Species, and Inflammatory Agents
6.2.2 Markers of Oxidative Stress and Inflammation
6.3 Antioxidants as therapeutics
6.4 Antioxidant Targeting Strategies
6.5 Nanocarrier-Mediated Delivery of Antioxidants
6.5.1 Liposomes
6.5.2 Polymer Nanocarriers
6.5.3 Magnetic Nanoparticles
6.5.4 Lipid Nanoparticles and Complexes
6.6 Conclusions
References

Citation preview

IMMUNITY AND INFLAMMATION IN HEALTH AND DISEASE

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IMMUNITY AND INFLAMMATION IN HEALTH AND DISEASE Emerging Roles of Nutraceuticals and Functional Foods in Immune Support Edited by

SHAMPA CHATTERJEE University of Pennsylvania School of Medicine, Philadelphia, PA, United States

WOLFGANG JUNGRAITHMAYR University Hospital Zurich, Zu¨rich, Switzerland; Medical University Brandenburg, Brandenburg, Germany

DEBASIS BAGCHI University of Houston College of Pharmacy, Houston, TX, United States

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1800, San Diego, CA 92101-4495, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright r 2018 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress ISBN: 978-0-12-805417-8 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisition Editor: Nancy Maragioglio Editorial Project Manager: Billie Jean Fernandez Production Project Manager: Kiruthika Govindaraju Cover Designer: Limber Matthew Typeset by MPS Limited, Chennai, India

Dedication

Dedicated to the memory of my grandmothers Pratibha (Ganga) and Kamala (Dulu) both of whose humility and goodness has been a guiding light. —Shampa Chatterjee Dedicated to my family, in particular to my father, who always has an open ear, a profound understanding, and comprehensive advice on issues of humanity and far beyond. —Wolfgang Jungraithmayr Dedicated to My Respected and Beloved Brother, Late Brigadier Dr. T.K. Roy, Kolkata, India, A Man Who Always Believed in Hard Work and Discipline. —Debasis Bagchi

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Contents

List of Contributors Preface

xiii xv

3. Redox Signaling and the Onset of the Inflammatory Cascade JOSE P. VAZQUEZ-MEDINA

I

3.1 Introduction 3.2 ROS Generation During the Onset of the Inflammatory Cascade 3.3 Redox Control of Inflammatory Mediators 3.4 Redox Control of Antioxidant and Anti-inflammatory Transcription Factors 3.5 Emerging Role of Peroxiredoxins in the Onset of the Inflammatory Cascade 3.6 Conclusions and Perspectives References

INNATE AND ADAPTIVE IMMUNE SYSTEMS: COMPONENTS AND REGULATION 1. Innate and Adaptive Immunity: Barriers and Receptor-Based Recognition

37 37 38 39 40 41 41

PRIYAL PATEL AND SHAMPA CHATTERJEE

1.1 Introduction 1.2 The Components of the Immune Response (External and Internal Elements) 1.3 The Innate Immune System (Cell Types, Signaling) 1.4 Inflammation and Innate Immunity 1.5 Induction of Adaptive Immunity (Antigen Presentation as a Key Event) 1.6 Cell Types in the Adaptive Immune Response 1.7 Crosstalk Between the Innate and Adaptive Immune Responses 1.8 The Vascular Endothelium as a Converging Site for Both Innate and Adaptive Immunity 1.9 Chronic Diseases of the Innate and Adaptive Immune Systems 1.10 Improving Immunity for Prevention and Care 1.11 Summary References

3

II

5 5 6

REACTIVE OXYGEN SPECIES, OXIDATIVE STRESS AND IMMUNE CELL ACTIVATION

8 8 8

4. Reactive Oxygen Species, Oxidative Damage and Cell Death

9

NANDINI GHOSH, AMITAVA DAS, SCOTT CHAFFEE, SASHWATI ROY AND CHANDAN K. SEN

4.1 Introduction 4.2 Endogenous ROS Production 4.3 Exogenous ROS Production 4.4 Physiological Role of Reactive Oxygen Species 4.5 Oxidative Stress 4.6 Oxidative Damage is Outcome of Oxidative Stress 4.7 Cell Death 4.8 Conclusions References

10 10 11 11

2. Innate Immunity at Birth: Implications for Inflammation and Infection in Newborns BALLAMBATTU VISHNU BHAT AND SELVARAJ MANOJ KUMAR KINGSLEY

2.1 Introduction 2.2 Microbiome in Shaping the Newborn Immune System 2.3 Mucosal Immunity at Birth 2.4 The Innate Immune Cells of the Newborn 2.5 Soluble Plasma Components 2.6 Conclusion References

15 15 16 17 27 28 29

45 45 47 48 48 49 50 52 52

5. Mitochondrial ROS and T Cell Activation KARTHIK B. MALLILANKARAMAN

5.1 Introduction 5.2 Reactive Oxygen Species 5.3 Sources of Mitochondrial ROS

vii

57 58 58

viii

CONTENTS

5.4 Regulation of mROS 5.5 Targets of mROS 5.6 Mitochondrial ROS in T Cell Activation 5.7 Summary References

59 59 60 61 62

6. Overcoming Oxidants and Inflammation: Endothelial Targeting of Antioxidants to Combat Chronic Inflammatory Disease ELIZABETH D. HOOD

6.1 Introduction 6.2 Inflammation and Disease 6.3 Antioxidants as Therapeutics 6.4 Antioxidant Targeting Strategies 6.5 Nanocarrier-Mediated Delivery of Antioxidants 6.6 Conclusions References

65 66 67 68 69 74 75

7. Oxidative Signaling in Chronic Obstructive Airway Diseases

9. The Biological Role of NADPH Oxidases in Ischemia-Reperfusion Injury Mediated Pulmonary Inflammation ASHISH K. SHARMA

9.1 Introduction 9.2 Free Radicals and Oxidative Stress in Lung IschemiaReperfusion Injury 9.3 NADPH Oxidase in Lung Inflammation and IschemiaReperfusion Injury 9.4 Cell-Specific Role of NADPH Oxidases in Pulmonary Inflammation 9.5 Role of NOX Isoforms and Pharmacological Inhibitors in Lung IR Injury 9.6 Conclusions References

119 120 120 121 122 123 123

10. Receptor Blockade of CD26/DPP4 as a Therapeutic Strategy Against I/R Injury and Lymphocytic Inflammation and its Clinical Implications WOLFGANG JUNGRAITHMAYR

TANIA A. THIMRAJ, LEEMA GEORGE, SYED ASRAFUZZAMAN, SWAPNA UPADHYAY AND KOUSTAV GANGULY

7.1 7.2 7.3 7.4

Introduction Sources of Reactive Oxygen Species (ROS) in the Lung Antioxidant Defense System in Lung Mechanism of Oxidative Stress Mediated Chronic Obstructive Airway Disease (COAD) Pathogenesis 7.5 ROS-Regulated Downstream Signaling Pathways in COADs 7.6 ROS-Mediated Effects in COADs 7.7 Antioxidant-Based Therapeutic Approaches for COADs References Further Readings

79 82 83 86 87 88 88 90 97

10.1 Introduction 10.2 CD26/DPP4 as a Therapeutic Target in Experimental Models Against I/R-Injury 10.3 Modulation of CD26/DPP4 Against Lymphocytic Inflammation 10.4 Clinical Implications for CD26/DPP4 as a Therapeutic Target in I/R Injury and Lymphocytic Inflammation References

127 127 130 130 131

11. DPPIV/CD26 as a Target in Anti-inflammatory Therapy GWENDOLYN VLIEGEN AND INGRID DE MEESTER

III INFLAMMATORY SYSTEMS: MECHANISTIC PATHWAYS OF STIMULATION AND SUPPRESSION

11.1 Introduction to Dipeptidyl Peptidase IV 11.2 DPPIV in the Immune System 11.3 Effects of DPPIV/CD26-Inhibitors in Autoimmune and Inflammatory Disease 11.4 DPPIV/CD26-Inhibitors 11.5 Conclusion References

8. Immune Responses in the Upper Respiratory Tract in Health and Disease

12. Redox Sensitive Transcription via Nrf2-Keap1 in Suppression of Inflammation

DEREK B. MCMAHON AND ROBERT J. LEE

ELANGO BHAKKIYALAKSHMI, DORNADULA SIREESH AND KUNKA M. RAMKUMAR

8.1 Overview of Upper Respiratory Physiology and Innate Immunity 8.2 Regulation of Immune Responses by Sensory Receptors in the Sinonasal Cavity 8.3 Impairment of Upper Respiratory Immunity and Disease 8.4 Conclusions and Remaining Questions Acknowledgments References

101 105 110 111 112 112

12.1 12.2 12.3 12.4

Introduction Islet Inflammation in Diabetes Components of the Nrf2-Keap1-ARE Machinery Mechanism and Regulation of the Nrf2-Keap1-ARE Pathway 12.5 Role of Nrf2 in Diabetes and Its Complications 12.6 Role of NF-κB in Diabetes and Its Complications

133 134 136 138 141 141

149 150 151 153 154 155

CONTENTS

12.7 Crosstalk Between Nrf2 and NF-κB Pathways in Diabetes 12.8 Role of Nrf2 Activators: Rescue From Islet Inflammation 12.9 Perspectives References

155 156 157 158

IV IMMUNITY AND INFLAMMATION: COMMON THREADS OF MULTIFACTORIAL DISEASES 13. Monogenic Defects of Toll-Like Receptor Signaling and Primary Immunodeficiency TRAVIS M. SIFERS AND VENKATESH SAMPATH

13.1 Introduction: The Genetic Theory of Infectious Disease 13.2 A Renaissance in Innate Immune System Research 13.3 Drosophila Toll, Interleukin-1 Receptor and the Discovery of the TLR Superfamily 13.4 TLR Signaling Networks 13.5 TLR3 Signaling via the Alternative (TRIF-dependent) Pathway 13.6 Monogenetic Primary Immunodeficiencies of the Canonical Pathway and Susceptibility to Pyogenic Bacterial Infections 13.7 Monogenetic Primary Immunodeficiencies of the Alternative Pathway and Susceptibility to Herpes Simplex Encephalitis 13.8 Monogenic Defects in NF-κB Signaling Leads to a Broad Spectrum of Infectious Susceptibility 13.9 Conclusion References

165 165 166 166 167

169 170 172 172

AMY K. SCHAEFER, JAMES E. MELNYK, ZHAOPING HE, FERNANDO DEL ROSARIO AND CATHERINE L. GRIMES

175 175 178 182 183

15. Inflammation and Calcification in the Vascular Tree; Insights Into Atherosclerosis CARLA S.B. VIEGAS AND DINA C. SIMES

15.1 Introduction 15.2 Atherosclerotic Lesion. Composition and Clinical Outcome 15.3 Inflammation in Atherosclerosis

191 192 195 196 198 198

16. From Inflammation to Cancer: Opportunities for Chemoprevention via Dietary Intervention JEONG-SANG LEE, EUN-JI LEE, HYE-KYUNG NA AND YOUNG-JOON SURH

16.1 Introduction 16.2 Inflammation and Cancer 16.3 COX-2 as a Key Player in the Pathogenesis of Inflammation-Associated Cancer 16.4 Transcriptional and Posttranscriptional Regulation of COX-2 Expression 16.5 Intracellular Signaling Cascades in Aberrant COX-2 Induction 16.6 The Oncogenic Potential of COX-2-Drived PGE2 and its Metabolic Inactivation 16.7 Nutritional/Dietary Manipulation of Aberrant Inflammatory Signaling in the Management of Cancer Acknowledgments References

203 203 204 204 205 206 208 208 208

167

14. Pathogen- and Microbial- Associated Molecular Patterns (PAMPs/MAMPs) and the Innate Immune Response in Crohn’s Disease

14.1 Introduction 14.2 Innate Immune Receptor Recognition of Bacteria 14.3 Bacteria and the Human Microbiome 14.4 Diagnosis and Treatment References

15.4 Calcification Associated With Atherosclerosis 15.5 Vascular Calcification 15.6 Vitamin K as a Potential Therapeutic Target for Atherosclerosis 15.7 The Vicious Cycle of Inflammation and Vascular Calcification 15.8 Final Remarks References

ix

189 189 190

17. Inflammation in Bullous Pemphigoid, a Skin Autoimmune Disease FRANK ANTONICELLI, SE´BASTIEN LE JAN, JULIE PLE´E AND PHILIPPE BERNARD

17.1 Clinical and Biological Aspects of Bullous Pemphigoid 17.2 From Autoimmunity to Inflammation and Blister Formation 17.3 From Inflammation to Autoimmunity and Therapeutic Response 17.4 Conclusion References

213 215 217 219 219

18. Inflammation in Systemic Immune Diseases: Role of TLR9 Signaling and the Resultant Oxidative Stress in Pathology of Lupus CHHANDA BISWAS

18.1 Introduction 18.2 TLR Family 18.3 TLRs Immunomodulate Both Innate and Adaptive Responses 18.4 TLR Adaptor Proteins and Signal Transduction 18.5 MyD88-Dependent Signaling 18.6 TRIF-Dependent TLR Signaling 18.7 TLRs and Sub-Cellular Localization-Specific Function 18.8 TLR9, Self-Recognition and Role in Autoimmune Diseases

223 224 225 226 227 228 228 228

x

CONTENTS

18.9 Oxidative Stress, Mitochondrial Inefficiency, Tissue Damage, and TLR9 Activation 229 18.10 A Collapse in Disposal of Cellular Debris in SLE Pathogenesis 229 18.11 Systemic Lupus Erythematosus (SLE) 230 18.12 Altered CD3-T-cell Receptor (CD3-TCR) Signaling in SLE T-cells 231 18.13 Enhanced Aggregation of Lipid Rafts and TCR Activation in SLE T-cells 231 18.14 Enhanced CD44-ERM/ROCK (Rho Associated Protein Kinase) Pathway in SLE Manifestation 231 18.15 Lack of Interleukin 2 (IL-2) and Peripheral T-cell Tolerance in SLE 232 18.16 Overall ROS Level is Heightened in T-cells Isolated From Lupus Patients and Lupus Mouse Models 232 18.17 Global Hypomethylation in SLE CD41 T-cells 232 18.18 Diet, Commensal Microbiota and Autoimmunity 233 18.19 Summary 233 References 234

References Further Reading

254 255

21. Micronutrients in Skin Immunity and Associated Diseases SE K. JEONG, SUNG J. CHOE, CHAE J. LIM, KEEDON PARK AND KYUNGHO PARK

21.1 Introduction 21.2 Skin Structure 21.3 Immunity in the Skin 21.4 Role of Certain Micronutrients in Skin Immunity 21.5 Conclusions Acknowledgments References

257 257 258 259 266 267 267

22. Mycobiome and Gut Inflammation: Implications in Gut Disease ELIZABETH A. WITHERDEN AND DAVID L. MOYES

V NUTRACEUTICALS IN BOOSTING IMMUNE SUPPORT AND AS THERAPEUTICS FOR INFLAMMATORY DISEASES

22.1 Host Microbiota and the “Superorganism” Hypothesis 22.2 Fungal Interactions 22.3 Studying the Mycobiome 22.4 The Mycobiome 22.5 Concluding Remarks References

271 272 273 274 277 278

23. Flavonoids in Treating Psoriasis

19. Anti-inflammatory and Anti-microbial Properties of Achillea millefolium in Acne Treatment

MARCO BONESI, MONICA R. LOIZZO, FRANCESCO MENICHINI AND ROSA TUNDIS

RAHUL SHAH AND BELA PEETHAMBARAN

19.1 19.2 19.3 19.4 19.5 19.6 19.7

Introduction Inflammation Etiology and Pathology of Acne vulgaris Management of Acne vulgaris The Molecular Basis for the Anti-acne Activity Isolation of Anti-acne Compounds From A. “Moonshine” Identification and Characterization of the Novel Alkamide 19.8 Comparison of other anti-acne sources to the novel alkamide from A. “Moonshine” 19.9 Conclusions References

241 241 242 242 246 246 246 247 247 247

JUN NISHIHIRA, MIE NISHIMURA, TOMOHIRO MORIYA, FUMIHIKO SAKAI, TOSHIHIDE KABUKI AND YOSHIHIRO KAWASAKI

249 249

281 282 283 283 290 290

24. Probiotics and Anti-inflammatory Processes in HIV Infection: From Benchside Research to Bedside MARCELLA REALE AND KATIA FALASCA

24.1 Introduction 24.2 Overview of Probiotics and Their Mode of Action 24.3 Probiotics and HIV References

20. Lactobacillus Gasseri Potentiates Immune Response Against Influenza Virus Infection

20.1 Introduction 20.2 Immunogenic Role of Probiotics Against Influenza Virus Infection 20.3 Clinical Intervention With Probiotics to Boost Immune Response 20.4 Perspectives for Research on Probiotics in the Immune System Acknowledgments

23.1 Introduction 23.2 Psoriasis: A Chronic Immunemodulated Inflammatory Disease 23.3 Conventional Therapies to Treat Psoriasis 23.4 Flavonoids in Treating Psoriasis 23.5 Conclusion References

295 295 301 303

25. Chronic Inflammation in Asthma: Antimalarial Drug Artesunate as a Therapeutic Agent THAI TRAN, YONGKANG QIAO, HUIHUI YOU AND DOROTHY H.J. CHEONG

250 253 254

25.1 Artesunate: A Potent Drug Beyond Its Antimalarial Property References

309 316

xi

CONTENTS

26. Nutrition as a Tool to Reverse Immunosenescence?

319

ANIS LARBI, OLIVIER CEXUS AND NABIL BOSCO

26.1 Introduction 26.2 Epidemiological, Physiological and Clinical Features of Aging 26.3 Nutrition and Weight Loss in Older Adults 26.4 Immunobiology of Aging 26.5 Prevention by Nutrition: The Growing Importance of Functional Food in Older Adults 26.6 Functional Food to Improve Immunity: A Closer Look at Claims and Clinical Reality 26.7 Functional Food With Proven Clinical Efficacy to Ameliorate Elderly Immunity 26.8 Conclusions and Future Directions References

RAMESH POTHURAJU, VENGALA RAO YENUGANTI, SHAIK ABDUL HUSSAIN AND MINAXI SHARMA

319 319 320 321 324 327 328 332 332

NEW PERSPECTIVES AND FUTURE DIRECTIONS

Introduction Importance of Functional Foods Role of Gut Microbiota in Health Effect of Fermented Milk by Probiotics on Obesity and Inflammation 29.5 Fermented Milk Products 29.6 Effect of Fermented Milk (FM) Products in Obesity 29.7 Conclusion Acknowledgments References Further Reading

389 390 390 391 394 396 398 398 398 401

RAVI KIRAN PURAMA, MAYA RAMAN, PADMA AMBALAM, SHEETAL PITHVA, CHARMY KOTHARI AND MUKESH DOBLE

30.1 Introduction 30.2 Colorectal Cancer and Gut Microbiota: Implications of Metabolites Acknowledgments References Further Reading

27. Therapeutic Interventions to Block Oxidative Stress-Associated Pathologies NUPOOR PRASAD, PRERNA RAMTEKE, NEERAJ DHOLIA AND UMESH C.S. YADAV

Introduction Oxidative Stress-Induced Diseases Traditional and Novel Therapeutic Targets Modern Approaches to Understand Oxidative Stress-Induced Pathologies 27.5 Regulatory Role of Nutraceuticals and the Paradoxes 27.6 Drugs in Clinical Trials for Oxidative Stress-Induced Pathologies 27.7 Problems and Limitations Associated With Nutraceuticals 27.8 Conclusion and Future Directions Acknowledgments References Further Reading

29.1 29.2 29.3 29.4

30. Prebiotics and Probiotics in Altering Microbiota: Implications in Colorectal Cancer

VI

27.1 27.2 27.3 27.4

29. Fermented Milk in Protection Against Inflammatory Mechanisms in Obesity

341 342 350

403 403 410 410 413

31. Naturopathy Lifestyle Interventions in Boosting Immune Responses in HIV-Positive Population PRADEEP M.K. NAIR AND HYNDAVI SALWA

352 355 356 356 357 358 358 362

31.1 Introduction 31.2 Naturopathic Approach Towards HIV-Positive Individuals 31.3 Lifestyle Modification Before ART 31.4 Lifestyle Modification During ART 31.5 Discussion 31.6 Conclusion References

415 416 418 418 419 420 420

28. Phytochemicals as Anti-inflammatory Nutraceuticals and Phytopharmaceuticals

32. Eating Habits in Combating Disease: Nutraceuticals and Functional Foods at the Crossroads of Immune Health and Inflammatory Responses

MELANIE-JAYNE R. HOWES

SHAMPA CHATTERJEE AND DEBASIS BAGCHI

28.1 Introduction 28.2 Flavonoids 28.3 Terpenoids 28.4 Steroidal Aglycones and Saponins 28.5 Curcumin 28.6 Stilbenes 28.7 Phenolic Acids 28.8 Conclusion Acknowledgments References

363 364 368 377 378 379 380 381 382 382

32.1 Introduction 32.2 Inflammatory and Immune Responses in the Pathology of Communicable and Noncommunicable Diseases 32.3 Diet Microbiota and Immune Responses 32.4 Functional Foods in Reversing Metabolic Syndrome as Well as in Improving Malnutrition-Induced Immune Impairment 32.5 Between Deprivation and Overconsumption: Maintaining a Balanced Diet to Combat Disease

423

424 425

426 426

xii 32.6 Diet, Immunity and Advancing Age 32.7 Good Eating Habits 32.8 Nutrigenomics: Personalized Nutrition to Combat Disease 32.9 The Take-Home Message 32.10 Summary and Conclusions

CONTENTS

427 428 429 429 430

Acknowledgments References

Appendix Index

430 430

433 441

List of Contributors

Padma Ambalam Christ College, Rajkot, Gujarat, India Frank Antonicelli University Reims, France Syed Asrafuzzaman Odisha, India

Utkal

of

Champagne-Ardenne,

University,

Debasis Bagchi University of Houston Pharmacy, Houston, TX, United States Philippe Bernard Reims, France

University

of

Bhuvaneswar, College

of

Elizabeth D. Hood University of Pennsylvania School of Medicine, Philadelphia, PA, United States Melanie-Jayne R. Howes Royal Botanic Gardens, Kew, Surrey, United Kingdom Shaik Abdul Hussain National Dairy Research Institute, Karnal, Haryana, India Se

Champagne-Ardenne,

K. Jeong Seowon University, NeoPharm Co., Ltd., Daejeon, Korea

Cheongju,

Korea;

Elango Bhakkiyalakshmi SRM University, Kattankulathur, Tamil Nadu, India

Wolfgang Jungraithmayr University Hospital Zurich, Zurich, Switzerland; Medical University Brandenburg, Neuruppin, Germany

Chhanda Biswas Children’s Hospital of Philadelphia, Philadelphia, PA, United States

Toshihide Kabuki Japan

Marco Bonesi University of Calabria, Rende, Italy

Yoshihiro Kawasaki Saitama, Japan

Nabil Bosco Nestle Research Centre, Singapore, Singapore Olivier Cexus Agency for Science Technology Research (ASTAR), Biopolis, Singapore

and

Charmy Kothari

Megmilk Snow Brand Co. Ltd., Saitama, Megmilk Snow Brand Co. Ltd.,

Christ College, Rajkot, Gujarat, India

Shampa Chatterjee University of Pennsylvania School of Medicine, Philadelphia, PA, United States

Anis Larbi Agency for Science Technology and Research (ASTAR), Biopolis, Singapore; University of Sherbrooke, Sherbrooke, Canada; National University of Singapore (NUS), Singapore, Singapore; Nanyang Technological University (NTU), Singapore, Singapore; El Manar University Tunis, Tunis, Tunisia

Dorothy H.J. Cheong National University of Singapore, Singapore, Singapore

Se´bastien Le Jan University Reims, France

Sung J. Choe Yonsei University, Wonju, Korea; University of California, San Francisco, CA, United States

Eun-Ji Lee

Scott Chaffee The Ohio State University Wexner Medical Center, Columbus, OH, United States

Amitava Das The Ohio State University Wexner Medical Center, Columbus, OH, United States Ingrid De Meester

University of Antwerp, Wilrijk, Belgium

Fernando Del Rosario AI DuPont Nemours Children’s Hospital, Wilmington, DE, United States Neeraj Dholia

of

Champagne-Ardenne,

Seoul National University, Seoul, South Korea

Jeong-Sang Lee Jeonju University, Jeonju, South Korea Robert J. Lee University of Pennsylvania School of Medicine, Philadelphia, PA, United States Chae J. Lim

Incospharm Corporation, Daejeon, Korea

Monica R. Loizzo

University of Calabria, Rende, Italy

Francesco Menichini University of Calabria, Rende, Italy

Central University of Gujarat, Gujarat, India

Mukesh Doble Indian Institute of Technology Madras, Chennai, Tamil Nadu, India

Karthik B. Mallilankaraman National Singapore, Singapore, Singapore

Katia Falasca University “G.D’Annunzio” Chieti-Pescara, Chieti, Italy

Selvaraj Manoj Kumar Kingsley Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India

Koustav Ganguly

Karolinska Institutet, Stockholm, Sweden

University

of

Leema George SRM University, Kattankulathur, Tamil Nadu, India

Derek B. McMahon University of Pennsylvania School of Medicine, Philadelphia, PA, United States

Nandini Ghosh The Ohio State University Wexner Medical Center, Columbus, OH, United States

James E. Melnyk United States

University of Delaware, Newark, DE,

Catherine L. Grimes University of Delaware, Newark, DE, United States

Tomohiro Moriya Japan

Megmilk Snow Brand Co. Ltd., Saitama,

Zhaoping He AI DuPont Nemours Children’s Hospital, Wilmington, DE, United States

David L. Moyes Kingdom

King’s College London, London, United

xiii

xiv

LIST OF CONTRIBUTORS

Sungshin Women’s University, Seoul,

Amy K. Schaefer University of Delaware, Newark, DE, United States

Pradeep M.K. Nair Ministry of AYUSH, Government of India, New Delhi, India

Chandan K. Sen The Ohio State University Wexner Medical Center, Columbus, OH, United States

Jun Nishihira Japan

Hokkaido Information University, Ebetsu,

Rahul Shah University of Sciences, Philadelphia, PA, United States

Mie Nishimura Japan

Hokkaido Information University, Ebetsu,

Ashish K. Sharma University of Virginia, Charlottesville, VA, United States

Hye-Kyung Na South Korea

Kyungho Park University of California, San Francisco, CA, United States; Hallym University, Chuncheon, Korea

Minaxi Sharma ICAR-Central Institute of Post-Harvest Engineering and Technology (CIPHET), Ludhiana, Punjab, India

Priyal Patel University of Pennsylvania School of Medicine, Philadelphia, PA, United States

Travis M. Sifers Children’s Mercy Hospital, Kansas City, MO, United States

Keedon Park Incospharm Corporation, Daejeon, Korea

Bela Peethambaran PA, United States

University of Sciences, Philadelphia,

Dina C. Simes

University of Algarve, Faro, Portugal

Dornadula Sireesh Nadu, India

SRM University, Kattankulathur, Tamil

Sheetal Pithva Christ College, Rajkot, Gujarat, India Julie Ple´e France

University of Champagne-Ardenne, Reims,

Young-Joon Surh Korea

Seoul National University, Seoul, South

Ramesh Pothuraju National Dairy Research Institute, Karnal, Haryana, India; University of Nebraska Medical Center, Omaha, Nebraska, United States

Tania A. Thimraj Nadu, India

SRM University, Kattankulathur, Tamil

Nupoor Prasad Central University of Gujarat, Gujarat, India Ravi Kiran Purama NIT Calicut, Calicut, Kerala, India Yongkang Qiao National Singapore, Singapore

University

of

Singapore,

Maya Raman Indian Institute of Technology Madras, Chennai, Tamil Nadu, India Kunka M. Ramkumar Tamil Nadu, India Prerna Ramteke India

SRM University, Kattankulathur,

Central University of Gujarat, Gujarat,

Marcella Reale University “G.D’Annunzio” Chieti-Pescara, Chieti, Italy Sashwati Roy The Ohio State University Wexner Medical Center, Columbus, OH, United States Fumihiko Sakai Megmilk Snow Brand Co. Ltd., Saitama, Japan Hyndavi Salwa Ministry of AYUSH, Government of India, New Delhi, India Venkatesh Sampath Children’s Mercy Hospital, Kansas City, MO, United States

Thai Tran National University of Singapore, Singapore, Singapore Rosa Tundis

University of Calabria, Rende, Italy

Swapna Upadhyay Sweden

Karolinska

Institutet,

Stockholm,

Jose P. Vazquez-Medina University of Pennsylvania School of Medicine, Philadelphia, PA, United States Carla S.B. Viegas

University of Algarve, Faro, Portugal

Ballambattu Vishnu Bhat Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India Gwendolyn Belgium

Vliegen

University

Elizabeth A. Witherden United Kingdom

of

Antwerp,

Wilrijk,

King’s College London, London,

Umesh C.S. Yadav Central University of Gujarat, Gujarat, India Vengala Rao Yenuganti National Dairy Research Institute, Karnal, Haryana, India Huihui You National University of Singapore, Singapore, Singapore

Preface

Euge`ne-Ernest Hillemacher’s painting on the cover of this book shows the English physician Edward Jenner injecting a small boy with cowpox to protect him against smallpox. This concept of inoculating a person with a benign or weak form of a pathogen to induce immunity from virulent forms of a similar virus is widely regarded as the foundation of immunology, a modern scientific discipline devoted toward understanding the immune system. The word immune originates from the Latin “immunis,” a combination of in (not) and munis (ready for service) and thus translates in modern medical parlance into “exempt from a disease.” This exemption or protection arises in part due to endogenous cells, collectively called immune cells that are capable of mounting an attack on foreign agents or pathogens (bacteria, fungus, and virus) either via mechanisms of “immediate recognition” or “priming for recognition” i.e. processes that “prime” immune cells for recognition of pathogens. The body’s immune system is made up of innate (i.e., inborn or immediate) and adaptive (acquired or primed) immune systems. The innate immune system is the first line of defense against pathogens as it acts immediately upon pathogen attack or injury, while the adaptive immune system is triggered on later. The “immediate recognition” pathway involves cells of the innate immune system that can “sense” microbial danger by recognizing molecular patterns in agents associated with pathogens. Recognition is in the form of binding of these molecular patterns to receptors that exist a priori on immune cells; such binding leads to the activation of complex signaling cascades that in turn mobilize a vast repertoire of immune cells. These cells either engulf pathogenic particles or initiate the generation of mediators that drive the destruction of pathogens. “Priming for recognition” on the other hand is an indirect process involving cells of the adaptive immune system. These cells need to be “primed to detect” to distinguish foreign from self. Priming occurs when fragments from pathogenic material are processed by a subset of cells to trigger the generation of appropriate receptors for these pathogens on adaptive immune cells. Later when these adaptive immune cells differentiate and proliferate, they possess a specific receptor for that particular fragment of the pathogenic material. Upon subsequent attack by the same pathogen, this “priming for recognition” pathway enables the expanded population of adaptive immune cells to “recognize” the pathogen and initiate an attack on it or stimulate the production of antibodies against it. This pathway forms the basis of the protection conferred by immunization or vaccination. In other words, Jenner’s cowpox inoculation induced a stable memory population of adaptive immune cells that conferred protection against subsequent smallpox infection. Both the innate and adaptive immune systems rely on recognition of the “non-self” from the “self” antigens and on the ability to activate mediators that can recruit a larger repertoire of immune cells. These mediators are inflammatory molecules that can attract immune cells to the region of injury and infection as well as to damaged, injured or infected tissue. This causes inflammation, a typical physiological response to infections and tissue injury that drives pathogen killing as well as tissue repair processes to restore homeostasis at infected or damaged sites. However, an exaggerated or an aberrant response by the innate and adaptive immune system can lead to chronic inflammation and autoimmune disease, respectively. Indeed, chronic inflammation gives rise to a significant number of noncommunicable diseases such as cardiovascular disease, obesity and diabetes; while autoimmune malfunction (that arise from nonrecognition of self-antigenic peptides) drives rheumatoid arthritis, asthma, systemic lupus and a host of other diseases. It is becoming increasingly clear that the inflammation-immune response, necessary as it is for protection from infection and injury, cannot be allowed to go on an “overdrive.” Regulation of this response is crucial to homeostasis. However, regulation or intervention at both cellular and molecular levels in the immune-inflammation processes necessitates an understanding of the signaling pathways in the form of multiple transcription factors, negative and positive feedback loops as well as synergistic mechanisms that participate in “immune overdrive” and in its inhibition.

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PREFACE

Our purpose in this book has been to highlight signaling pathways associated with specific diseases that share the inflammation-immune pathway, as well as to showcase mediators that can amplify or dampen the immune and inflammation response. In doing so, we aim to reinforce the message that regulation or modulation of “immune-mediated inflammation,” either by molecular switches or by nutrition-lifestyle changes, forms the basis of health and well-being. Food consumption has undergone a massive transformation in the course of the last century. Further, modern urban, technology-driven lifestyles have altered patterns of physical activity. This “high calorie intake -sedentary activity” paradigm impacts metabolism and has the potential to drive a low-grade chronic inflammation. At the same time, undernutrition in the form of malnourishment is well established to drive immunosuppression and impair infection resistance. While micronutrients in our diet are important determinants of immune status, dietary components such as long- chain omega-3 fatty acids, antioxidant vitamins, flavonoids, prebiotics and probiotics can modulate chronic inflammatory conditions. These nutrients have been reported to decrease inflammatory mediator production through effects on cell signaling and gene expression (omega-3 fatty acids, vitamin E, plant flavonoids), by quenching the production of damaging oxidants (vitamin E and other antioxidants), and by promoting gut barrier function (prebiotics and probiotics). Indeed most human studies have correlated analyses of habitual dietary intake with systemic markers of inflammation. Integrated human epidemiological studies with large cohorts will likely provide further evidence of inflammationhealth/disease associations, and the ability of diet to positively modulate inflammation. This would be a crucial step in developing diets with an intention to block chronic inflammatory and immune pathologies. The book has been thematically divided into six sections. The first three sections cover the various cellular and molecular players of the immune-initiated inflammation paradigm and include the events that lead to the onset of metabolic, aging, and autoimmune related diseases. The chapters of the fourth section deal with the ramifications of a robust and excessive inflammatory response. Section five showcases the role of dietary nutrients in playing a balancing role between host defense and immune support. The sixth section comprises chapters that envision paradigm shifts in the field, whereby new futuristic directions are discussed. Shampa Chatterjee, Wolfgang Jungraithmayr and Debasis Bagchi

S E C T I O N

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C H A P T E R

1 Innate and Adaptive Immunity: Barriers and Receptor-Based Recognition Priyal Patel and Shampa Chatterjee University of Pennsylvania School of Medicine, Philadelphia, PA, United States

1.1. INTRODUCTION Vertebrates have evolved an immune system to stave off pathogens in the form of bacteria, fungi, viruses, parasites, or other unwanted vicious biological invasions. The vertebrate immune system comprises a complex array of receptors and signals that can sense or recognize microbial danger and respond via signaling cascades that drive inflammation and associated processes for direct killing of pathogens (Abrahimi et al., 2016). Traditionally, classified into innate (in-born or non-specific) and adaptive (acquired or specific) immunity (Figs. 1.11.3), it is now becoming increasingly clear that these are not discrete responses that operate in isolation (Akira and Takeda, 2004; Akira et al., 2006; Amadi et al., 2005); rather these two immune signaling pathways are synergistic with extensive crosstalk whereby mobilization of various immune cells follows a temporal sequence of immune activity (Fig. 1.2) from non-specific attack on pathogens in general to the development of “immunological memory” for specific pathogens. Therefore, in recent times these immune compartments are being considered as one functional unit (Bales and Kraus, 2013). While the innate immune system responds rapidly, taking minutes to hours to “recognize” patterns in the invading pathogen, the adaptive system takes days to weeks to respond as it uses immunological memory for recognition of antigen fragments specific to pathogens. But once the recognition of the pathogen is in place, the adaptive immune system enables the host to recognize a subsequent attack by the same pathogen (Barnes and Karin, 1997). In this first chapter, we provide a snapshot of the contents of the book. We introduce the vertebrate immune system, the components of this system, the signals that activate various immune cells to trigger the killing of bacterial, fungal and viral pathogens and the networks of communication that facilitate a bridging of innate and adaptive immunity. Next, we focus on the importance of the vascular wall, specifically the endothelium that lines the vessel wall in driving immune and inflammation responses via recruitment of immune cells as well as via antigen presentation. We also discuss the pathology of inflammatory diseases that are associated with unrestrained immune response and highlight the immune-inflammation-health-disease paradigm, which requires a better understanding in order to improve immunotherapy for various diseases. Finally, we highlight the necessity to integrate the inflammation and immune response so as to reconcile the need for defense against pathogens with protection against inflammation-driven chronic diseases. The roles of regulators and modulators (ligands that block inflammation signals and/or nutrients that activate immune responses) that can optimize the inflammation-immune processes are also reviewed.

Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00001-9

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Copyright © 2018 Elsevier Inc. All rights reserved.

Immune system Innate Physical Phagocytes • Skin • Monocytes • Cough reflex • Macrophages • Tears (enzymes in • Neutrophils tears) • NK cells • Mucosal layer • Dendritic Cells • Stomach acids

Adaptive • Helper T-cells

B-lymphocytes • Clonal B-cells

• Suppressor T cell

• Memory B cells

T-lymphocytes

• Cytotoxic T cells

FIGURE 1.1 The immune system has numerous components both physical and immunological. The innate immune system is a nonspecific defense mechanism that comes into play almost immediately or within minutes to hours of a pathogen’s (antigen) appearance in the body. Phagocytic cells are activated by the antigens released by the pathogens. The adaptive immune system processes the antigens first. Adaptive signaling facilitates recognition and once the antigens are recognized, the adaptive immune system creates an army of immune cells specifically designed to attack that antigen.

FIGURE 1.2

Cells of the innate and adaptive immune system. Monocytes, macrophages, neutrophils and dendritic cells (DC) are key effector cells of the innate immune system. The response of these cells is rapid but short-lived. Natural Killer (NK) T cells (NKT) traditionally considered part of the innate immune response are now being accepted to possess the characteristics of cells of the adaptive immune system. T and B lymphocytes and plasma cells constitute the main cellular components of adaptive immunity. T lymphocytes upon contact with antigen presenting cells (APC) differentiate into cytotoxic T cells (CTL) which then express cytokines and cytolytic enzymes that drive cell death.

FIGURE 1.3 The interaction between an APC and T lymphocyte is crucial to activation of T-cells. The first step is the antigen presentation on APCs. Certain unstimulated cells such as the naive endothelium does not express antigens; upon activation antigens are produced. Next, co-stimulatory molecules are produced on the APCs. These bind to their corresponding counterparts on the T cell. The antigens presented to the T cell bind to the T cell receptor. Source: Adapted From Gujar SA et al. Oncolytic virus-mediated reversal of impaired tumor antigen presentation. Front Oncol. 4 , 2014, 77.

1.3. THE INNATE IMMUNE SYSTEM (CELL TYPES, SIGNALING)

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1.2. THE COMPONENTS OF THE IMMUNE RESPONSE (EXTERNAL AND INTERNAL ELEMENTS) The immune system in vertebrates is a collection of host defenses ranging from the external barrier provided by mucosal and epithelial cells to recognition of pathogens by highly specific receptors that are genetically encoded on immune cells. External barriers are characterized by defenses such as the cough reflex, enzymes in tears and skin oils, the mucosal layer that traps bacteria and small particles, the skin, and stomach acids. Pathogen recognition, on the other hand, is more complex and driven by cellular machinery that responds to pathogens by recognizing conserved motifs in pathogens, as well as a number of other indicators of cell stress or death. The cells of the innate immune system (Fig. 1.1) are dendritic cells, monocytes, macrophages, polymorphonuclear neutrophils (PMN), granulocytes, and natural killer T cells (NKT). In addition to these sentinels, the skin, the pulmonary epithelial and gut epithelial cells also act as an interface between the tissues and the environment. During a pathogenic attack, the innate immune system is our first line of defense. It responds rapidly via receptors that recognize patterns either on pathogens or on molecules released by them. Recognition is in the form of binding of these molecules released by pathogens (also called alarmins) to receptors on immune cells leading to the activation of signaling pathways that attack the invading microbes (Bianchi, 2007; Bishayee, 2009). The receptors of the innate immune system are germ line encoded by specific genes within the DNA while the receptors of the adaptive immune system generated in developing lymphocytes (that occurs in the bone marrow and thymus) involve different variants of the genes encoding the receptor molecules. Each lymphocyte expresses receptors for only one specific antigen; the millions of lymphocytes in the body carry receptors for millions of antigens or antigenic peptides specific for pathogens. Thus lymphocytes that encounter an antigen to which their receptor binds will be activated to proliferate and differentiate (Fig. 1.3). This population of lymphocytes will be available to recognize the pathogen upon subsequent attack (Bonifaz et al., 2002). The components of the immune response can be divided into four major categories: inducers (bacterial products such as LPS, oxidative cell damage), receptors (TLRs, NLRs), mediators (cytokines, chemokines, eicosinoids) and effectors (immune cells that respond to inflammatory mediators). These are discussed in the next section.

1.3. THE INNATE IMMUNE SYSTEM (CELL TYPES, SIGNALING) The innate immune cells recognize inducers or exogenous pathogenic agents; this recognition is facilitated by receptors that can detect molecular patterns that are expressed by pathogens but are foreign to mammalian cells. Several pattern-recognition receptors (PRR) have been identified and studied over the past few years (Bishayee, 2009). These are (1) the Toll-like receptors (TLRs), (2) NOD-like receptor proteins (NLRPs), (3) C-type lectin receptors (CLRPs), and (4) RIG-1- like receptors (RLRs). These receptors can detect conserved ligand motifs called alarmins on pathogens or on moieties released by pathogens. Alarmins are of two types: those that are associated with pathogens are termed as pathogenassociated molecular patterns (PAMPs), and those that are released from non-pathogenic damage (sterile injury) are called damage associated molecular patterns (DAMPs). Both PAMPs and DAMPs bind to PRRs (i.e., receptors) on immune cells that lead to the capturing (endocytosis) of the recognized viral or bacterial particles into the cell followed by their lysosomal degradation. The PAMP/DAMP-PRR binding results in the recruitment of adaptor proteins and the subsequent activation of a signal transduction cascade that activates mediators or proinflammatory transcription factors including NFκB (Bradley et al., 1993). The subsequent inflammatory cascade facilitates recruitment of effectors or immune cells and pathogen removal or killing (Abrahimi et al., 2016; Bishayee, 2009). The innate immune system consists of multiple cell types (Fig. 1.2) that participate in engulfing pathogens as well as in triggering inflammatory signals that drive pathogen removal. These cells are: 1. Polymorphonuclear Neutrophils (PMN) are a category of leukocytes (white blood cells) that have granules (hence PMN are considered granulocytes) within them containing proteolytic enzymes to break down bacterial protein. PMN participate within a few minutes of microbial attack or injury and later in the debridement of the injured tissue. These cells are termed as initial responders and are followed by monocyte and macrophage recruitment into the area of infection or injury (Calder et al., 2009).

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2. Monocytes are leukocytes and phagocytes (a large cell that can engulf bacteria) that are in the circulation and that upon chemokine/cytokine cues reach the area of pathogen attack or injury. Monocytes have plasticity and are environmentally regulated to differentiate into macrophages or dendritic cells (Calder, 2006; Carmeliet, 2003). 3. Macrophages are large phagocytes that are considered as prolific “eaters” as they engulf pathogenic particles, apoptotic (dead) cells and fragments of injured tissue. Macrophages produce large amounts of some cytokines (e.g., prostaglandin E2, interleukins or IL-6, IL-10), creating a cytokine milieu consistent with chronic low-grade inflammation (Carmeliet, 2003, 2005). 4. Natural killer cells (NK) are lymphocytes (a subclass of leukocytes) in the circulation that recognize virallyinfected and neoplastic cells as non-self (via their detection of major histocompatibility complex or MHC class I self antigens) (Chassaing and Gewirtz, 2014). 5. Dendritic cells: These cells lie at the interface of innate and adaptive immunity (Fig. 1.2) and sense antigens either from self or from external pathogenic materials and ingest these. Once ingested, these antigens are degraded and processed as peptide fragments which are then displayed on the dendritic cell surface within MHC class I and II molecules. The dendritic cells are considered as professional antigen presenting cells (APC). These cells migrate to lymph nodes (and spleen) where they can be in contact with cells of the adaptive immune system specifically T-lymphocytes. When T-lymphocytes come in contact with APCs such as DCs, they bind to these cells via T-cell receptors (TCRs). The antigens presented by DCs drive an APC-T cell binding via the TCRs. Overall, DCs participate in defense by their production of immune-enhancing cytokines and the mobilization of innate lymphocytes (NK, NKT, γδT) (Chen et al., 2008; Chinen and Buckley, 2010; Cines et al., 1998; Cooke et al., 2016). Collectively the innate immune cells play a major role in anti-pathogen defenses either by direct engulfment of bacterial or viral pathogens or by upregulation of an inflammation cascade that leads to production of oxidants that cause pathogen destruction.

1.4. INFLAMMATION AND INNATE IMMUNITY The first phase of the immune response comprises recognition of bacterial or viral pathogens by immune cells followed by either ingesting (phagocytosis or endocytosis) them or by activating a signaling cascade that causes production of oxidants to obliterate pathogens. In the second phase, the damaged tissue in the vicinity of the infection as well as damaged extracellular matrix material, and cellular detritus are ingested and removed by the immune cells. The third phase consists of repair of the damaged cells and tissue around the infection (Davidson, 2010). The first phase or when innate immune cells recognize infection occurs via a complex cascade where PRRs on immune cells ligate alarmins, i.e., pathogen-associated molecular patterns (PAMPs), such as microbial nucleic acids, lipoproteins, and carbohydrates, or damage-associated molecular patterns (DAMPs) released from cells injured by sterile insults (heat, oxidant-induced damage, etc.). Activated PRRs then oligomerize and assemble large multi-subunit complexes that initiate signaling cascades that trigger the release of factors, promoting further recruitment and adherence of PMN and other immune cells. The various players in the innate immune response are: 1. Alarmins (inducers) a. PAMPs: include microbial surface components like endotoxins (LPS), proteins, lipoproteins, surface polysaccharides, glycoproteins, bacterial cell wall components such as β-glucan and α-mannan, components of the peptidoglycan bacterial cell wall and the bacterial protein flagellin, bacterial and viral nucleic acids, motifs within bacterial DNA, dsRNA, etc. (Abrahimi et al., 2016; Bishayee 2009). b. DAMPs are endogenous molecules induced in cells that “face” a sterile (by oxidants such as reactive oxygen species or ROS) attack. DAMPs may also be released during cell death. DAMPs include high mobility group box-1 (HMGB-1), a DNA binding nuclear protein that binds to a cell surface receptor called receptor for advanced glycation end products (RAGE). RAGE ligation by HMGB1 leads to activation of a downstream signaling cascade that appears to be involved in activation of the cells of the adaptive immune system (Dempsey et al., 2003). RAGE is often considered a potential link between adaptive and innate responses, as RAGE ligation may affect adaptive immune responses (DiPietro, 1995).

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2. Pattern recognition receptors a. Toll Like Receptors (TLR): the major PRRs in cells are transmembrane proteins containing leucine-rich repeats that recognize bacterial and viral PAMPs. These receptors are members of the TLR family that recognize PAMPs/DAMPs in the extracellular such as TLR1, TLR2, TLR4, TLR5, TLR6, and TLR11 or in the intracellular milieu such as in endo-lysosomes (TLR3, TLR7, TLR8, TLR9, and TLR 10). TLR induced signaling occurs via adapter proteins that possess the Toll/interleukin-1 receptor (TIR) domain. These adaptor proteins, such as myeloid differentiation primary response protein (Myd88), TIR receptor domain-containing adapter protein (TIRAP), TIR domain containing adapter-inducing interferon beta (TIRF) and TIR containing adapter-inducing interferon beta-related adapter molecule (TRAM) activate a signaling pathway that drives the production of cytokines and chemokines that have anti-viral and antibacterial action (Dunzendorfer et al., 2004; Edele et al., 2007) (Fig. 1.4) b. NLRP (NOD-like Receptor Protein): Certain members (NOD1,2 and 3) of the NLPR family of cytosolic PRRs can bind to PAMPs/DAMPs and activate NF-κB activation or secretion of the pro-inflammatory cytokines IL-1β and IL-18 (NLRP1, NLRP3 and NLRC4) via a process termed as inflammasome activation. This occurs when the PAMP-NLRP binding leads to the assembly of a multimeric complex comprising NLRP, adaptor protein ASC and inactive zymogen pro-caspase-1. The inflammasome acts as a scaffold where pro-caspase-1 is cleaved to active caspase-1, protease that cleaves (activates) the precursor interleukins to cytokines (pro-IL-1β and pro-IL-18). This leads to expression of IL-1β and IL-18 which are able to induce an inflammatory form of cell death known as pyroptosis. c. The RIG-like receptor (RLR) family of PRRs are intracellular receptors for RNA viruses and comprise three members namely RIG-I, melanoma differentiation factor-5 (MDA5), and laboratory of genetics and physiology-2 (LGP-2). Activation of the RLR pathway triggers innate antiviral responses, mainly through expression of inflammatory cytokines such as interferons (Type I IFN-α, β) limit viral replication (Edfeldt et al., 2002; Fan et al., 2003; Fearon and Locksley, 1996).

FIGURE 1.4 (A) Innate immune signaling via Toll-like Receptor 4. Toll-like receptors (TLRs) link the pathogen with the host cell. LPS, one of the main pathogen-associated molecular patterns (PAMPs) of pathogenic bacteria, is recognized by the host through TLRs, resulting in activation of multiple downstream cell signaling cascades. The TLR-PAMP interaction recruits specific adaptor molecules which then bind the interleukin (IL)-1 receptor associated kinase (IRAK), initiating a signaling cascade. The four adaptor proteins, including myleloid differentiation primary-response protein 88 (MyD88), TIR domain-containing adaptor-inducing interferon β (TRIF), MyD88 adapter-like/TIR domaincontaining adaptor protein (Mal/TIRAP), and TRIF-related adaptor molecule (TRAM), contain TIR domains that can be recruited. (B) Adaptive immune signaling. The surface of the APC expresses antigens. A T-cell binds to MHC-antigen complexes. This causes activation of the T-cell and it releases cytokines. The CD8 1 cells are activated to cytotoxic lymphocytes (CTL) that cause dissolution (cytolysis) of the bacterial/viral particles. The CD4 1 cells activate T-helper (TH) cells, some of which differentiate into memory cells that respond upon subsequent attack by the same pathogen. TH cells also cause differentiation of B cells into cells that produce antibodies against the antigen of the pathogen.

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1.5. INDUCTION OF ADAPTIVE IMMUNITY (ANTIGEN PRESENTATION AS A KEY EVENT) A key event of the adaptive immune response is antigen presentation (Fig. 1.3). This occurs when a subset of immune cells called dendritic cells (DC) capture and process antigens emanating from foreign agents and particles. The processed antigens are then presented within major and minor histocompatibility complexes (MHCclass I and II) on the cell surface. DCs migrate to the lymphoid organs where they arrive in the regions populated by T-lymphocytes (often referred to as T-cells). Besides DCs, other antigen presenting cells (APC) are macrophages, thymic epithelial cells, etc. However ROS and/or chemokines such as interferon gamma produced during inflammation are reported to confer several cell types such as endothelial, epithelial, etc., the ability to express MHC-II and process and present antigens (Frantz et al., 2005). These antigenic peptides on MHC-I and/or II are recognized by T cell receptors (TCR). The peptide-MHCIITCR binding forms the basis of adaptive immune response. Thus, antigen presentation is a pivotal event. T cells activated by DCs differentiate and/or proliferate in the thymus and possess a specific receptor for a fragment of the antigen. Fig 1.3 summarizes the APC-T cell interaction that occurs via the antigen-receptor binding. Antigen-bearing DCs need to be in contact with T cells for periods ranging from one to more days. Sustained triggering by the antigen causes a proliferative phenotype in T cells (Girodon et al., 1997; Greenfeder et al., 2001; Grimm et al., 2002).

1.6. CELL TYPES IN THE ADAPTIVE IMMUNE RESPONSE All cells of the immune system originate in the bone marrow. These are myeloid (neutrophils, basophils, eosinpophils, macrophages, and dendritic cells) and lymphoid (B lymphocyte, T lymphocyte and Natural Killer) cells as shown in Fig. 1.2. The bone marrow myeloid progenitor (stem) cells give rise to erythrocytes, platelets, neutrophils, monocytes/macrophages and dendritic cells whereas the lymphoid progenitor (stem) cell gives rise to the NK, T and B lymphocytes. For the development of T cells, the precursor T cells must migrate to the thymus where they undergo differentiation into two distinct types of T cells, the CD41 T helper cell and the CD81 precytotoxic T cell. Within the T-helper cells, there are two types, i.e., the TH1 cells, which help the CD81 precytotoxic cells to differentiate into cytotoxic T lymphocytes (CTL), and TH2 cells, which help B cells differentiate into plasma cells, which secrete antibodies. B and T-lymphocytes form the basis of the adaptive response. In their inactive and immature form, these cells circulate in the peripheral blood. However, once they recognize pathogens presented by an APC, the selection, growth, and differentiation of the lymphocyte begins. TH1 cells produce IFN-γ while TH2 cells produce IL-4, IL-5, and IL-13. B and T cells are small but can be distinguished by their cell surface receptors which are known as immunoglobulins (B-cell receptors) and T-cell receptors (TCR). Lymphocyte differentiation and proliferation results in the development millions of different kinds of receptors for both B-cells and T-cells(Barnes and Karin, 1997; Gupta et al., 1998). When T cells recognize antigens or fragments of antigens from pathogens that are presented to them, they initiate direct attack of antigen-bearing cells by a cytotoxic T lymphocyte population or by stimulation of B cells to produce antibodies against the antigens. Additionally, TH cells also produce interferon gamma and other cytokines leading to inflammation and bacterial cell kill.

1.7. CROSSTALK BETWEEN THE INNATE AND ADAPTIVE IMMUNE RESPONSES As we have mentioned earlier, the innate immune cells possess receptors that recognize patterns associated with pathogens (Fig. 1.4A). These receptors are germ line encoded and therefore exist prior to pathogen attack. This enables an immediate response to bacteria, virus or other infection and injury. However this also implies that these cells have a non-specific repertoire of sensing which allows the detection of a broad range of pathogens but does not facilitate the detection of a particular bacteria or virus. Specific pathogen detection is a function carried out by the cells of adaptive immune system namely the T- and B- lymphocytes that possess receptors that can bind to specific peptide fragments associated with a particular pathogen. These receptors are expressed

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because of earlier priming and immunological memory whereby APC-T cell interaction and subsequent differentiation and proliferation of a subset of T cells drives pathogen killing (Fig. 1.4B). Straddling these two branches of the immune response are the natural killer T (NKT) cells (Fig 1.2). These cells have characteristics of both innate and adaptive systems in that they possess pattern recognition receptors as well as immunoglobulins or T-cell receptors. The crosstalk between these two systems is bidirectional, i.e., innate immune system activates the adaptive system and vice versa. While innate immune cells such as dendritic cells activate T cells via antigen presentation, adaptive immune cells activate cells of the innate immune system via IFN-γ produced by T-helper cells that in turn activate macrophages, dendritic cell subsets. While innate components are the first line of defense, components of adaptive immunity mobilize more slowly. This is because adaptive immunity is the form of antigen presentation, APC-T cell binding, clonal expansion; and proliferation as well as formation of cytotoxic T cells is a part of the sequence of a prolonged pathway (Fig. 1.4B). This pathway also utilizes the complement cascade and adhesion molecules, which evolved as part of innate immunity. Consequently, shared mediators unite innate and adaptive immunity function as parts of an integrated immune system.

1.8. THE VASCULAR ENDOTHELIUM AS A CONVERGING SITE FOR BOTH INNATE AND ADAPTIVE IMMUNITY As integral components of the immune system, blood vessels are important in the surveillance of self. They play key roles in lymphocyte circulation and act as portals between tissue and blood compartments. Endothelial cells (ECs) form a single cell layer called the endothelium, which lines the vasculature and lymphatic systems forming a semi-permeable barrier between blood or lymph within vessels and the surrounding tissues. The endothelium is a highly specialized, dynamic, disseminated organ with many essential functions in physiological processes. Besides serving as a physical barrier, ECs have a wide array of functions which are characterized into three major categories: trophic, tonic, and trafficking. ECs also have important immunological functions (Hahn and Schwartz, 2009). Cells of the immune system function to defend against invasive foreign pathogens and detrimental endogenous materials. Inflammation can be seen as a vascular response (Hansson et al., 2002), where ECs become activated, display increased leakiness, enhanced leukocyte adhesiveness, and procoagulant activity, and form new vessels (Harvey et al., 2008). Thus, an immune response resulting in inflammation depends upon the ability of the microvasculature to either recruit or prevent the indiscriminate influx of immune cells into a tissue. Compared with large blood vessels, the microvascular bed constitutes the bulk of the overall endothelial surface, covering an area B50 times greater than that of all large vessels combined (Hawiger et al., 2001). Due to their location, ECs are one of the first cells to interact with microbial components in the circulation. EC recognition and response may be integral to early innate immune system activation. In fact, like DCs, ECs are reported to express both TLRs and NLRs as well as express chemokine receptors (Huffman et al., 2007; Ingulli, 2010). Specifically, ECs have been shown to secrete the proinflammatory cytokine interleukin-8 (IL-8) in an NLPR1-dependent manner in response to microbial stimulation. Innate immune signaling occurs via PAMPs and DAMPs binding to receptors (TLRs) on the host cells. In this context, it needs to be mentioned that TLR1 immunoreactivity was observed in atherosclerotic endothelium (Italiani and Boraschi, 2017). TLR2 has also been detected on atherosclerotic endothelium, expressed by ECs and markedly up-regulated in vascular inflammation (Italiani and Boraschi, 2017). ECs upon stimulation with LPS, TNF-α and IFN-γ express TLR2 mRNA and protein in a NF-κB- and MyD88-dependent manner (Jakubzick et al., 2017; Kawai and Akira, 2009). The upregulation of TLR2 in EC is via neutrophil NADPH oxidase and neutropenic mice (mice without neutrophils) have been found to show decreased endothelial TLR2 expression (Jakubzick et al., 2017). This indicates a crosstalk between polymorphonuclear neutrophils and ECs that would enhance vascular defenses by up-regulating TLR2. The impact of ECs on adaptive immunity may be exerted through their interaction with T cells. Although ECs cannot replace T and B cells, it is becoming clear that ECs can express MHC I and II class molecules and process antigens or fragments of antigens from pathogens, and can thus under some conditions act as APC. This has been reported and ECs from different species express accessory molecules required for antigen presentation, including CD80, CD86, ICOS-L, programmed death ligand 1, programmed death ligand 2, LFA-3, CD40, and CD134L (Lloyd and Hessel, 2010). As shown in Fig. 1.3, the accepted paradigm is that professional APCs such as

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DC present antigens to activate the recipient’s naı¨ve T lymphocytes. The T lymphocytes thus activated cause production of inflammatory cytokines and other enzymes that cause cytolysis, injury and onset of graft rejection (Loo and Gale, 2011; Mahabeleshwar et al., 2006). The APC-T lymphocyte interaction is thus pivotal to the onset of adaptive immune response (Majno, 1998). Apart from professional APCs, other cell types can also be stimulated to be transformed into APCs. For instance, endothelial cells activated by IFN-γ or by hydrogen peroxide show expression of antigens MHC I and II and co-stimulatory molecules (Manach et al., 2004; Marelli-Berg and Jarmin, 2004; Matzinger, 2007). These moieties are the hallmarks of an APC as they facilitate APC-T cell interaction (Fig. 1.2). It is now acknowledged that the endothelium is not just a passive target of immune response but an active player in T-lymphocyte activation (Medzhitov and Janeway, 1997). However, the mechanism by which the endothelium is transformed into an APC during organ storage has not been elucidated. EC-immune signaling also drives the process of angiogenesis (the formation of new blood vessels from preexisting ones) which is integral to most immune-mediated conditions. As inflammation evolves, vessels expand to supply nutrients to sustain the accumulation of activated immune cells in the affected tissues (Medzhitov, 2008; Morgan et al., 1999), thus both innate and adaptive immune responses promote angiogenesis. Additionally, immune cells such as macrophages with their tissue macrophages foster vessel growth, remodeling, or regression (Hansson et al., 2002; Murdoch et al., 1999). Their proangiogenic activity is mediated by various TLRs acting in synergy with adenosine A2A receptors that up-regulate VEGF production (O’Neill et al., 2013).

1.9. CHRONIC DISEASES OF THE INNATE AND ADAPTIVE IMMUNE SYSTEMS The major function of the immune system is to recognize the non-self antigens or antigenic peptides. However an exaggerated or an aberrant response can lead to inflammation as well as to autoimmune disease (that arises from non-recognition of self antigenic peptides). Chronic inflammatory pathologies such as atherogenesis and autoimmune disorders such as rheumatic fever, rheumatoid arthritis, ulcerative colitis, myasthenia gravis, etc., are all manifestations of an exaggerated inflammation-immune response whereby cytokines and chemokines released by stimuli such as injury, denudation of vascular layer, or hormonal imbalance, accumulation of fat cells, i.e., adipocytes and/or pathogenic attack lead to recruitment of activated immune cells to the site of lesions, thus amplifying and perpetuating the inflammatory state. Chronic or long-term inflammation drives repair and remodeling process that alter cellular and tissue function. The remodeling of the vessel that occurs with atherosclerosis involves large numbers of macrophages and T cells at the site of the atherosclerotic plaque. Thus recruitment is driven by local topology (Parisi et al., 2017) as well as by modified lipoproteins and cholesterol, while in diseases such as dermatitis or psoriasis where a bacterium like streptococcus is involved, TLR 2 activation in response to this infection leads to a high expression of TNF-α, IL-12, IL-23, and IL 17, which in turn produces psoriatic lesions (Gupta et al., 1998). In the case of asthma, an allergen challenge provokes the influx of T helper cells (specifically activated TH2) into the airways causing an increase in the levels of TH2-type cytokines and recruitment of immune cells such as eosinophils (Piotti et al., 2014; Pober et al., 1983). Airway inflammation leads to airway hyperresponsiveness and epithelial cell proliferation linked tissue remodeling. There is also clinical evidence to suggest that respiratory viral infection is also linked to the initial development of asthma as well as exacerbations that might perpetuate the disease (Pober et al., 1983). Low-grade inflammation also characterizes metabolic syndromes and associated disorders (obesity, diabetes, etc.). Normally PMN and macrophages that show uncontrolled activity during chronic inflammation are strongly implicated as having a causal role in cancer development (Polonsky et al., 2016; Pulendran et al., 2001). NLRP inflammasomes also participate in driving several neurologic and metabolic diseases. Misfolded protein aggregates and aberrant accumulation of certain metabolites associated with other diseased states function as DAMPs and activate the NLRP3 inflammasome, which plays a critical role in the initiation and progress of inflammation (Pulendran, 2005; Reith et al., 2005).

1.10. IMPROVING IMMUNITY FOR PREVENTION AND CARE Studies have shown that lack of adequate nutrition is a major cause of immune dysfunction. A study from Spain (Riquelme et al., 1997) suggests that up to 85% of elderly patients with community-acquired pneumonia are malnourished. Similarly studies have shown that complete recovery form pathogen-induced infections (even

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though the bacteria or protozoa were present) is possible via nutritional intervention in the form of micronutrients, aminoacids, simple lipids, etc. (Sager et al., 2017). On the other hand, excessive calorie intake stimulates adipose tissue growth and promotes abdominal obesity, which along with other factors (smoking, exposure to environmental pollutants etc.) can activate the immune pathway leading to onset of inflammation. A hallmark of most chronic immune diseases is the elevated levels of inflammatory cytokines, chemokines and inflammatory growth factors. Studies carried out in individuals following calorie restrictions (enrolled in the Calorie Restriction Society, whose members voluntarily observe calorie-controlled diets) showed that consuming lower than baseline diet calories (B1800 calories/day) over several years decreased serum levels of TNF-α, proinflammatory cytokines and lipoprotein profiles (Santangelo et al., 2007). Studies conducted in Australia among postmenopausal women demonstrated that the risk of death from inflammatory disease (digestive, respiratory, nervous system, and endocrine disorders) was nearly three times greater among women consuming a high-GI diet (a high glycemic index diet consists of high carbohydrate and sugar intake) compared with women eating a low-GI diet (Saresella et al., 2016). Intake of trans fatty acids was also found to drive inflammation. Trans 18:2 fatty acids were observed to be integrated into human aortic endothelial at twice the rate as cis 18:2 fatty acids, causing the cells to clump together and bind to arterial walls, stimulating the release of proinflammatory cytokines (Schmidt et al., 2001). Interventions in the form of vitamin and mineral supplements cause abrogation of certain agents that are high in individuals suffering from inflammatory diseases. A supplement of retinol, β-carotene, thiamine, riboflavin, niacin, pyridoxine, folate, iron, zinc, copper, selenium, iodine, calcium, magnesium, and vitamins B12, C, D, and E when provided to healthy adults over a 12-month-long, double-blind, randomized, placebo-controlled trial, showed an elevated immune function in the form of an increase in CD4 1 T numbers and NK cell activity. Importantly, infectious illness days were reduced from a mean of 48 in the placebo group to 23 in the group that received the supplement (P 5 .002), and antibiotic use was lowered from an average of 32 to 18 days (P 5 .004) (Shanker et al., 2015). Fish oil supplements (4 g/day for a minimum of 6 weeks) in the diet resulted in significantly decreased plasma levels of TNF-α with subjects with type 2 diabetes and in reduced cellular content of proinflammatory cytokines IL-1 beta, IL-6, and IL-8 in healthy subjects (Steinman and Hemmi, 2006). In subjects with rheumatoid arthritis, fish oil intake caused symptom relief (Steinman and Hemmi, 2006). Vitamin C intake in plasma levels correlated with reduced proinflammatory prostaglandins and C-reactive protein (CRP) in a small clinical study of healthy subjects. Plasma vitamin C levels inversely correlated with symptoms of rheumatoid arthritis. Supplementation with 1 g/day of vitamin C decreased measures of oxidative stress and improved endothelial function in a clinical study of individuals with hypertension (Tam and Jacques, 2014). Polyphenols (found in fruits, vegetables, grains, chocolate, coffee, olive oil, and tea) in the diet show powerful anti-inflammatory effects. Laboratory investigations, clinical trials, and prospective studies suggest that polyphenols inhibit enzymes decrease proinflammatory cytokine production, and block the activity of proinflammatory signaling systems (Wolinsky, 1980; Yamazaki et al., 2006; Zhou et al., 2016).

1.11. SUMMARY The highly specialized mammalian immune system consists of two distinct arms, innate and adaptive immunity. The cells of the innate immune system that function via recognition of molecular patterns that are expressed by pathogens also prime the cells of the adaptive immune system. The innate immune cells respond within minutes to hours of a pathogen attack. Adaptive immune responses are slow to develop upon first exposure to a new pathogen, as specific clones of B and T cells have to become activated and have to expand. However the cells of the adaptive immune system remember previous encounters with specific pathogens so that these pathogens are easily combated in subsequent infection episodes. In subsequent chapters, innate and adaptive immune signals and their role on regulating inflammation and the how these form the common basis of multifactorial diseases will be discussed. Our understanding of these immune mechanisms and preventative measures will allow for options beyond vaccinations and drug development.

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2 Innate Immunity at Birth: Implications for Inflammation and Infection in Newborns Ballambattu Vishnu Bhat and Selvaraj Manoj Kumar Kingsley Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry, India

2.1. INTRODUCTION The neonatal period is the most crucial phase of life and survival during this period is of paramount importance. Infections account for 36% of the four million neonatal deaths every year (Lawn et al., 2005). Immunologic immaturity is one of the major reasons for the increased susceptibility of neonates to early onset sepsis (Shane and Stoll, 2014). The diminished fetal immune system may be an adaptive mechanism to prevent premature rejection by the mother (Clapp, 2006). The key players in adaptive immunity are T-lymphocytes that express markers CD4 or CD8. T-lymphocytes expressing CD4 are also known as helper T cells, and these are the most prolific cytokine producers. This subset can be further subdivided into Th1 and Th2. The Th1-type cytokines produce proinflammatory responses while Th2-type cytokines are primarily anti-inflammatory in their effects. The newborn immune response is skewed towards anti-inflammatory Th2 response, which increases their susceptibility to a diverse range of intracellular pathogens. Toll-like receptors (TLRs) are a major family of pattern recognition receptors (PRRs) that has a vital role in the innate host defense and initiation of the adaptive immune responses. The reduced infection control in newborns is associated with the impaired TLR signaling and diminished response to bacterial cell wall components (Sadeghi et al., 2006). The newborn innate immune cells have shown several unique characteristics that sometimes exceed but most often fall short of the required optimal response. The microbiome and epigenetic influences regulate the development of immunity in early life. The ontogeny of immunity in early life involves several complex mechanisms and the naı¨ve nature of the newborn immunity increases its susceptibility to inflammation and infections.

2.2. MICROBIOME IN SHAPING THE NEWBORN IMMUNE SYSTEM Microbial colonization of infant microbiota begins at birth and it depends largely on the transfer of maternal microbiota during delivery. Due to several factors like maternal antibiotic usage, invasive procedures at birth, hospital environment stress, neonates, especially very low birth weight infants, are characterized by dysbiosis (Groer et al., 2015). The maternal microbiomes including the placental microbiome influence the increased incidence of preterm births (Vinturache et al., 2016). There were significant variations in the composition of placental microbiota in low birth weight and normal weight neonates (Zheng et al., 2015). Moreover when compared to babies born though vaginal route to normal weight mothers, those born to obese mothers had their microbiota enhanced in bacteroides and reduced Acinetobacter, Enterococcus etc (Mueller et al., 2016). This implies that maternal body mass index affects the development of infant microbiome during vaginal delivery. Epigenetic changes induced by in-utero variations modulate the early microbial colonization and the gastrointestinal (GI) tract development which increases the susceptibility of newborns to infection (Cortese et al., 2016). CD711 erythroid cells are immunosuppressive, but they also prevent exaggerated activation of immune cells in intestines where Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00002-0

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frequent colonization with commensal microbes occurs after birth (Elahi et al., 2013). The disruption of the host intestinal microbiota may increase the susceptibility of neonates to necrotizing enterocolitis (Elgin et al., 2016). The breast milk given to newborns comprises of carbohydrates, immunoglobulins, cytokines and other immunomodulatory factors (Bertino et al., 2012). It is also a source of several microbes such as Lactobacillus sp. and Bifidobacterium sp., which promote the development of newborn intestinal microbiota (Jeurink et al., 2013). Human recombinant lactoferrrin has shown to alter the fecal microbiome in very low birth weight infants (Sherman et al., 2016). Newborn mice that are undernourished had depleted microbiota with minimal diversity and were lacking in numerous microbial genetic pathways, which resulted in repressed energy exudation from dietary components (Preidis et al., 2015). Neonatal malnutrition inhibits body weight gain and reduces NO production which elevates the viability of macrophages infected with methicillin resistant Staphylococcus aureus, and also promotes reactive oxygen species (ROS) production which may enhance systemic infection with S. aureus (de Morais et al., 2016). Early life exposure of newborn mice to a mixture of microbial extracts improved survival significantly after infection with Streptococcus pneumoniae. Preexposure to microbial extracts improved their ability to suppress bacterial growth, inhibited pneumococcal blood invasion, and enhanced the colonization of bacteria in the airway, improving airway resistance (Yasuda et al., 2010). It was later found that intratracheal exposure to microbes improved the opsonic activity of bronchoalveolar lavage fluid, which improved phagocytosis of alveolar macrophages (AMs). The maturation of dendritic cells (DCs) (CD11c 1 expressing cells) in the airway was also enhanced on microbial exposure (Kasahara et al., 2012). Probiotic bacteria could modify the intestinal microbiome in newborns to tackle allergic disease. Probiotic strains such as Bifidobacterium bifidum induced partial maturation of cord blood DCs and those matured DCs stimulated T cells to secrete IL-10 and IL-4 (Niers et al., 2007). Pregnant mouse dams exposed to antibiotics had reduced level of microbes in the intestines. The newborn pups had reduced levels of neutrophils and granulocyte macrophage progenitor cells in bone marrow and in the circulation (Deshmukh et al., 2014). The antibiotic exposure also led to a deficit in IL-17 producing intestinal cells and secretion of granulocyte colony stimulating factor (G-CSF), which led to granulocytopenia impairing the neonatal defense to Escherichia coli and Klebsiella infections. The recruitment of normal microbiota to antibiotic treated neonatal mice restored the IL-17 generation from innate lymphoid cells (ILCs) in the intestine and elevated the number of neutrophils and granulocyte macrophage colony stimulating factor (GM-CSF) levels, through the TLR4 and MyD88 pathway (Deshmukh et al., 2014). Newborn mice born to antibiotic treated mice during pregnancy showed reduced IFN-γ producing CD8 1 T cells. This showed that maternal antibiotic treatment alters the development of infant GI microbiota which weakens the antiviral immune response (Gonzalez-Perez et al., 2016).

2.3. MUCOSAL IMMUNITY AT BIRTH There is increasing evidence that the diverse and complex microbiota in early life influences the development of mucosal immunity in newborns (Malmuthuge et al., 2015). From a relatively sterile in-utero environment, the newborns are exposed to the challenges of pathogens and microbes at birth. They are mainly protected by maternal antibodies at the mucosal surfaces (Harris et al., 2006). The recognition of pathogens is facilitated via evolutionarily conserved structures on pathogens called pathogen associated molecular patterns (PAMPs) that bind to their respective PRRs. This binding of PAMPs to their respective receptors forms a major mechanism of innate mucosal immunity. Newborns depend on the transplacental transfer of IgG in-utero and the immunoglobulins IgA, IgG, IgM in mother’s milk. Secretory IgA at the mucosal surfaces mediates the transport of commensal bacteria across the intestinal epithelia, limits the intestinal penetration of commensal bacteria, and helps maintain the hostmicrobiota symbiosis (Harris et al., 2006; Rol et al., 2012). The mucosal secretions from intestinal, genital or respiratory tracts contain significant amounts of immunoglobulin G (IgG). Recently a contrasting role for IgG in the neonatal period has been found which may limit the mucosal adaptive immunity. The maternally acquired IgG and IgA act against the gut microbiota and repress the mucosal T helper cell responses in early life (Koch et al., 2016). The neonatal Fc receptor (FcRn) within the mucosal tissues has significant role in triggering local immune responses needed for antitumor immunosurveillance (Baker et al., 2013). Transgenic mice that overexpress FcRn receptor show improved production of anti-thymocyte globulin, which enhanced the humoral response in rabbits (Baranyi et al., 2013). The FcRn receptor transports the IgG from the intestinal barrier to the lumen and then redirects the IgG-antigen complex to the lamina propria across the intestinal barrier. This is followed by the antigen presentation by DCs to CD41 T cells in lymphoid structures (Yoshida et al., 2004).

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Interleukin 23 (IL-23) responsive group III ILCs are well known to play a role in adult immune homeostasis; in neonates ILCs upon activation by IL-23 in neonatal intestines reportedly produced more proinflammatory mediators like IFN-γ and GM-CSF (Chen et al., 2015). The ILCs accumulate under the intestinal epithelium and aggregate along with neutrophils and cause intestinal bleeding and death in newborns. Depletion of ILCs using antibodies reduced mortality in neonatal mice (Chen et al., 2015). Gut mucosal injury in neonatal mice is characterized by macrophage rich leukocyte infiltration and is recruited to the inflamed neonatal gut mucosa by the chemokine CXCL5 (MohanKumar et al., 2012). A combination of multiple cell types regulates immunity and inflammation. Other prototypical members of the ILC family such as the natural killer (NK) cells, DCs as well as CD41 and CD81 T cells participate in neonatal immunity. The profound NK cell percentage in intestinal epithelial compartment (IEC) during early life reduced other intestinal epithelial cell subpopulations such as CD31 cells. After weaning, the NK cell percentage declined and the CD41 CD81 regulatory T cells were the predominant cells in IEC (Pe´rez-Cano et al., 2005). CD1031 DCs influence the oral tolerance in neonates and the superantigen Staphylococcal enterotoxin given to neonatal mice increased the FoxP3 expression in stimulated T cells of small intestines, improving oral tolerance by CD1031 DCs. This is mediated by the increased expression of retinal aldehyde dehydrogenase enzyme (RALDH) in DCs, which metabolizes Vitamin A (Stern et al., 2013). Newborn piglets respond to oral antigens with immunity, when administered along with adjuvants like CpG-oligodeoxynucleotides (CpG-ODN) (Pasternak et al., 2014). Dietary bovine lactoferrrin alters the mucosal and systemic immune cell response, improving the levels of immunoglobulins and cytokines produced in neonatal piglets indicating a role for lactoferrrin in strengthening the neonatal immune response (Comstock et al., 2014).

2.4. THE INNATE IMMUNE CELLS OF THE NEWBORN The innate immune system is the first line of host defense during infection and is responsible for the initial recognition of pathogen and generation of potent proinflammatory response. The innate immune response is mainly mediated by phagocytic cells such as granulocytes and macrophages, cytotoxic NK cells, and antigenpresenting cells such as the DCs. The phenotypic and functional characteristics of each of these cell types during the neonatal period and their innate immune response to various pathogens, stimulants and inflammatory mediators are discussed below.

2.4.1. Neonatal Neutrophils Polymorphonuclear neutrophils (PMN) are some of the first cells to reach the sites of inflammation and execute pathogen clearance. Thus high neutrophil count is an index of inflammation. In the umbilical cord blood, a high proportion of immature neutrophils was found with reduced expression of complement receptor 3 and L-selectin (Rebuck et al., 1995; Reddy et al., 1998). Neonates that were exposed to labor showed higher neutrophil counts of approximately 3500 neutrophils per microliter (Christensen et al., 2012). Late onset low neutrophil count or neutropenia was observed in very low birth weight infants, with counts decreasing from ,1500 μL21 at ,3 weeks of age to 500 μL21 at 34 months of age (Vetter-Laracy et al., 2014). During infection in neonates the release of immature or band forms of neutrophils is common (Linderkamp et al., 1998; Schelonka et al., 1995). The negative regulators of hematopoiesis, such as transforming growth factor beta 1 (TGF-β1), macrophage inflammatory protein-1α (MIP-1α) and IL-8, were found to be reduced in cord blood mononuclear cells at the mRNA and proteins levels, which may contribute to the impaired defense in neonates (Satwani et al., 2005). Administration of recombinant GM-CSF and G-CSF significantly improves the bone marrow myeloid progenitor pool, neutrophil storage pool and circulating neutrophil counts in neonatal rats (Satwani et al., 2005). The neutrophil cytoplasmic granules are filled by the antimicrobial proteins and peptides (Levy, 2000). Bactericidal permeability increasing protein (BPI) is found in the primary granules of neutrophils and has great attraction to the lipid A moiety of LPS, and neutralizes the inflammatory effects of LPS. It has been found that human cord blood-derived neutrophils are deficient/lack BPI, and this contributes to the diminished antibacterial activity of neonatal neutrophils (Levy, 2002; Levy et al., 1999; Qing et al., 1996). The diminished adhesion, chemotaxis and migration of neonatal neutrophils may contribute to the increased susceptibility of neonates to infections. The expression of adhesion molecules like L-selectin (CD62L) and beta-2 integrin (CD18) was low in neutrophils during the early life period after birth (Harris et al., 1985; Lee and Kehrli, 1998). There was also correlation of

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adhesion molecule CR3 (CD11b/CD18) with gestational age, and the deficiency was more profound in premature neonates (McEvoy et al., 1996). Moreover, the PMN adherence and chemotaxis are more severely affected in stressed neonates than healthy neonates and increase their susceptibility to bacterial infections (Krause et al., 1986). The levels of Rac-2, a guanosine triphosphate binding protein was lower in cord blood neutrophils, which may contribute to the impaired migration and chemotaxis of neonatal neutrophils (Meade et al., 2006). Lipoxin A4, which is associated with inhibiting the chemotaxis, respiratory burst and promoting apoptosis of adult neutrophils, had no such effect in neonatal neutrophils. The anti-inflammatory activity of lipoxin A4 is mediated by peroxisome proliferator activator receptor gamma (PPAR-γ), whose expression was found to be reduced in neonatal neutrophils (Weinberger et al., 2008). Neonatal PMNs had abundant arginase 1 protein, which reduced arginine I levels in neonatal plasma. Exogenous arginine I enhanced the neonatal lymphocyte proliferation through an IL-2 independent pathway (Yu et al., 2014). The induction of IL-8 during normal labor promotes the chemotaxis of neonatal neutrophils and their transmigration was higher than the newborns from cesarean sections (Yektaei-Karin et al., 2007). In slight contrast, it has been recently found that term neonates do not show reduced chemotaxis and only preterm neonates show reduced chemotaxis compared to adults (Birle et al., 2015). The expression of complement regulatory proteins CR1 (CD35), CD55 in resting neonatal neutrophils was significantly higher than adults, but upon stimulation there was marked increase in expression in only adult neutrophils (Lothian et al., 1997). There was differential expression of several proteins in neonatal neutrophils and these were associated with the impaired function of neutrophils in proteasome, lysosome, phagosome, leukocyte transendothelial migration and NETosis (Zhu et al., 2014). The phagocytosis ability of neutrophils from full-term and preterm newborns was impaired at birth but improved significantly three days after birth (Filias et al., 2011). Preterm infants have lower numbers of monocytes and neutrophils with phagocytic potential than term infants, which may increase their susceptibility to bacterial infections. However, the phagocytosis ability of those cells from preterm infants was found to be stronger (Prosser et al., 2013). The levels of phagocytic monocytes and neutrophils and degree of bacterial uptake between cord blood and peripheral blood of ,24 h newborns correlated with each other. The addition of exogenous rabbit complement (RbC) enhanced the phagocytosis ability of neutrophils and monocytes of preterm and term infants (Prosser et al., 2013). The hematopoietic growth factors such as GM-CSF, G-CSF, M-CSF, which regulate the phagocytosis of leukocytes, were found to be reduced at the mRNA and protein levels in cord blood mononuclear cells (Satwani et al., 2005). Stimulation through the Fc gamma receptor is a method used to improve the phagocytosis and other neutrophil functions. Neutrophils from newborn infants and preterm infants expressed the Fc gamma receptor (CD64) during bacterial infections, which may represent a crucial mechanism to enhance neutrophil phagocytosis (Fjaertoft et al., 1999). The enhanced neutrophil CD64 expression is a sensitive marker of culture positive neonatal sepsis (Soni et al., 2013). The endogenous expression of caspase 1/11 promotes excessive inflammation and suppresses the protective immunity in newborns. Removal of Caspase 1/11 in neonatal mice expanded the bone marrow and splenic hematopoietic stem cell pool and increased the levels of G-CSF in the peritoneum after sepsis (Gentile et al., 2015). Increased recruitment of neutrophils to the peritoneum and enhanced phagocytosis by neutrophils was also observed. The mechanism by which neutrophils kill bacteria extracellularly is through the formation of neutrophil extracellular traps (NET) called NETosis (Brinkmann et al., 2004). Previously it was stated that neonates show impaired NET formation, which decreases their antimicrobial defenses (Yost et al., 2009). Later on it was found that neonates do indeed form functional NETs, but after an extended time of stimulation (Marcos et al., 2009). Usually, once they are done with their job of phagocytosis, neutrophils rapidly undergo apoptosis. This pathway in neonates is not well regulated and may be the reason for the increased incidence of inflammatory diseases in newborns. This was evident from the reduced proapoptotic proteins Bax, Bad, Bak, caspase 3 expression and activity in neonatal neutrophils (Hanna et al., 2005; Song et al., 2011). The sustained expression of FLICE inhibitory protein (FLIP) in neonatal PMNs was associated with their resistance to apoptosis (Rashmi et al., 2011). The surviving neonatal neutrophils secrete inflammatory mediators associated with chronic inflammation such as IL-8 involved in PMN activation and recruitment and also higher levels of MIP-1α (Nguyen et al., 2010a,b). Neonatal PMNs exhibited decreased sialic acid binding immunoglobulin-like lectin (Siglec-9) and Src homology domain 2 containing tyrosine phosphatase-1 (SHP-1), which interact with one another and promote apoptosis (Rashmi et al., 2009). The decreased responsiveness of neonatal neutrophils to Fas ligand and decreased expression of Fas receptor contribute to their prolonged survival and reduced apoptosis (Hanna et al., 2005; Song et al., 2011). HSV-1 infection significantly enhances the apoptosis of neonatal neutrophils by activating the Fas and amplified release of Fas ligand. The neutrophil apoptosis was less in neonates exposed to labor because of the expression of TNF-α in those neutrophils (Weinberger et al., 2007). The cytotoxic abilities of newborn PMNs are

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equivalent to adult PMNs, and they could be improved by cytokines or antibodies (Stiehm et al., 1994). Stimulated neonatal neutrophils with prolonged survival produced more reactive oxygen intermediates and also showed enhanced cytotoxic functions, which may contribute to the pathogenesis of inflammatory disorders in neonates (Koenig et al., 2005). During pregnancy, in addition to myeloid-derived suppressor cells (MDSCs), polymorphonuclear cells (PMNs) also induce T cell immunosuppression by arginine depletion induced by arginase (Yu et al., 2014). Increased phosphorylation of protein tyrosine kinase p53/56 lyn, confined in membrane granule fractions of neonatal neutrophils, may impair the ability of neonates to respond to gram negative infections (Yan et al., 2004). Human newborn PMNs in response to LPS expressed diminished levels of the downstream adapter protein MyD88 and reduced p38MAPk phosphorylation (Al-Hertani et al., 2007). The TLR2 mediated production of IL-10 in neonates inhibits the neutrophil recruitment to sites of infection, and blocking IL-10 signaling restores their migration (Andrade et al., 2013). The neutrophil STAT-1 and STAT-3 phosphorylation in response to IFN-γ stimulation and STAT-5 phosphorylation following GM-CSF stimulation were reduced in neonates compared to adults (Nupponen et al., 2013). Neonatal neutrophils are insensitive to the anti-inflammatory effect of Vitamin D which may be due to the reduced expression of Vit D receptor and 1α hydroxylase (Hirsch et al., 2011). The administration of CpG-ODN has shown to significantly improve the innate and adaptive immune response and could prevent neonates from infection and improve immune response to vaccines. Intramuscular administration of CpG-ODN to neonatal foals increased the IFN-γ mRNA expression and reduced the degranulation when compared to untreated neutrophils (Cohen et al., 2014). Drugs such as milrinone, piclamilast, urinastatin, ketamine deactivated the activated neutrophils, reduced the inflammatory response and improved microcirculation in neonates during inflammation (Craciun et al., 2013). The in-utero exposure of neonatal rats to maternal betamethazone and intra-amniotic LPS increased and sustained the neutrophils in lungs and disrupts alveolarization (Lee et al., 2013). Treatment with combination of drugs, betamethasone and indomethacin reduced the proinflammatory cytokines such as TNF-α and IL-6, while increasing IL-10 in neonatal cord blood mononuclear cells in response to gram positive pathogens (Ernst et al., 2015). Maternal diet could influence the functions of immune cells and reprogram them to be more effective in early life. Fish oil supplementation during pregnancy reduced the leukotriene-4 production in neonatal neutrophils and reduced proinflammatory responses (Prescott et al., 2007). This shows the influence of maternal intake of polyunsaturated fatty acid included foods in immune reprogramming of fetus in-utero or early life (Fig. 2.1).

FIGURE 2.1 Diminished neutrophil functions in the neonatal period. Neutrophils show impaired adhesion and migration in response to pathogenic stimuli. Altered innate functions such as phagocytosis, degranulation and NET formation increases the susceptibility of newborns to infection.

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2.4.2. Neonatal Monocytes Monocytes are the largest leukocytes in the circulation. They can differentiate into DCs and macrophages. A high monocyte count is indicative of chronic infection or inflammation. At birth the monocyte counts are significantly elevated in neonates when compared to adults. Depending on the gestation age (22 weeks to 42 weeks) the mean value of monocyte counts increased linearly and the reference range at 40 weeks was 3003300 μL21 (Christensen et al., 2010). The monocyte counts gradually increased during the first 2 weeks of life. In human blood there are several monocyte populations classified according to their expression of antigens, i.e., the CD1411CD162 classical monocytes, the CD141CD161 proinflammatory monocytes and the human leukocyte antigen (HLA-DR) expressing monocytes. In preterm and full-term neonates, the CD1411CD16 2 and CD14 1 CD16 1 monocytes were significantly elevated, but in preterms the HLA-DR expression was reduced (Schefold et al., 2015). The level of expression of CD16 (Fc gamma R III) in cord blood monocytes was reduced and the percentage of CD14--dim monocytes expressing HLA-DR was also less among cord blood monocytes (Murphy and Reen, 1996). The expression of Toll-like receptor 4 (TLR4) (a pivotal receptor for innate immune signaling) and HLA-DR expression in neonatal monocytes showed a gestational age-dependent increase (Wisgrill et al., 2016). Infants with infection had a lower percentage of HLA-DR 1 monocytes than non-infected infants (Juskewitch et al., 2015). The neonatal monocyte HLA-DR expression varies with allergen-specific immune responses (Upham et al., 2004). In LPS and IFN-γ stimulated neonatal monocytes from term and preterm infants, the expression of co-stimulatory molecules CD80 increased, whereas CD86 decreased (Pe´rez et al., 2010). Neonatal monocytes expressed defective MHC II antigen presentation, reduced MHC II surface expression and T cell responses, but the decreased MHC II expression on neonatal monocytes does not correlate with defective antigen presentation (Canaday et al., 2006). The beta chemokine receptor CCR5 expression is diminished in neonatal monocytes and it gradually increases during monocyte differentiation to macrophages. The phagocytosis ability of monocytes in newborn infants did not show any difference from adults at birth, though preterms had fewer phagocytes (Filias et al., 2011). Interestingly, the CD64 expression and phagocytic capacity of very low birth weight infants were higher than preterm or term infants (Hallwirth et al., 2004). Pretreatment of neonatal monocytes with GM-CSF significantly increased the superoxide anion O2-production in response to PMA, but M-CSF could not enhance the anti-fungal activity of neonatal monocytes against Candida albicans (Gioulekas et al., 2001). Treatment of neonatal monocytes with polyunsaturated fatty acids (PUFAs) induced the activation of caspases 3, 8, and 9 and amplified monocyte apoptosis (Sweeney et al., 2007). PUFAs exerted a dose-dependent effect on cord blood monocyte survival and at higher concentrations ( .100 μM) they induced profound cell death (Sweeney et al., 2001). This suggests that maternal diet including PUFAs could influence the immune response in newborns. Removal of immune effector cells after the elimination of pathogens is crucial in preventing sustained inflammation and associated disorders. Phagocyte-induced cell death (PICD) accounts for this effective removal of immune effectors. Upon infection with E. coli, cord blood monocytes showed diminished PICD that was characterized by the diminished caspase 8, caspase 9 and CD95L expression after phagocytosis (Gille et al., 2008). In neonatal cord blood monocytes, the anti-apoptotic Bcl-XL proteins were found to be upregulated, which reduced PICD and sustained inflammation in neonates (Leiber et al., 2014). The TLR mediated inflammatory response is regulated differently by different pathogens. Differential TLR2 and TLR4 expressions were observed in stimulated cord blood and peripheral blood with various pathogens (Sugitharini et al., 2014). Following stimulation with TLR ligands like LPS and bacterial lipoproteins, neonatal monocytes produced reduced TNF-α compared to adults, but in response to TLR7/8 ligand R-848 (resiquimod) the TNF-α release was similar from newborn and adult monocytes (Levy et al., 2004). The production of IL-10 by human neonatal blood mononuclear leukocytes was reduced following LPS/TNF-α stimulation and this may be associated with the reduced TNF-α receptors and TNF-α production by neonatal monocytes (Chheda et al., 1996). In contrast, LPS/IFN-γ stimulated cells of the monocyte lineage from neonatal foals produced more IL-10 than adults and there was no significant difference in IL12p35 and IL12p40 levels (Sponseller et al., 2009). Exogenous IL-10 added to LPS stimulated monocytes, inhibited the proinflammatory cytokine release and activator protein-1 DNA binding activity, while increasing the STAT-3 nuclear phosphorylation (Chusid et al., 2010). Another study has shown that neonatal cord blood mononuclear cells produced comparable levels of IL-6, IL-1β, IL-10, IL-13, lesser IL-23, MCP-1 and higher IL-8 than adult cells (Sugitharini et al., 2014). The TLR8 agonists, imidazoquinolines induced the TLR pathway transcriptome activation and production of Th1-type cytokines TNF-α and IL-1β from neonatal monocytes and MoDCs through adenosine refractory and caspase 1 dependent pathways (Philbin et al., 2012). The activation of ERK1/2 and NF-κβ was impaired in neonatal monocytes subpopulations following stimulation with TLR agonists. The intracellular TNF in non-classic monocytes of preterm infants

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was repressed and this functional suppression of non-classic monocytes contributes to the increased susceptibility to bacterial infections (Wisgrill et al., 2016). The STAT-6 phosphorylation by IL-4 was comparable in neonatal monocytes and adults (Nupponen et al., 2013). In response to R-848, the p38 MAP kinase phosphorylation was also similar between newborn and adult monocytes (Levy et al., 2004). In response to TNF-α or bacterial stimulation the NF-κβ phosphorylation was higher in preterm neonates than in term neonates. The p38 phosphorylation was higher in all neonates whereas the STAT-1 phosphorylation by IFN-γ or IL-6, STAT-3 by IL-6 and STAT-5 by GM-CSF was reduced in all neonates compared to adults (Nupponen et al., 2013). This shows the profound activation of the inflammatory pathways in preterm and term neonates, which may contribute to the unwarranted inflammation and tissue injury. The expression of genes regulated by Interferon regulatory factor-3 (IRF-3) or Type I IFNs in cord blood monocytes stimulated with LPS or Listeria monocytogenes was suppressed, when compared to adult monocytes (Lissner et al., 2015). The deficit in IRF-3 activity in neonates leads to the diminished expression of IFN-dependent genes and weakens the innate immune response against infections. Respiratory Synctial Virus (RSV) infection of neonatal monocytes stimulates the synthesis of IRF-1 and enhanced the transcription and translation of IL-1β, secreting more of the soluble protein (Takeuchi et al., 1998). In response to different types of dengue virus, neonatal monocytes produced lesser levels of cytokines TNF-α, IL-6 and IL-1β when compared to adults (Valero et al., 2014). The oxidative stress parameters such as NO, MDA, and SOD were also reduced in neonatal monocytes, suggesting reduced anti-oxidant response of neonatal monocytes (Valero et al., 2013). The CCR5 receptor expression corresponded with the increased susceptibility of neonatal monocytes/macrophages to HIV infection (Zylla et al., 2003). BCG vaccination improved the viability of monocytes and promoted the uptake of Mycobacterium tuberculosis but was unable to enhance the killing of ingested pathogen (Sepulveda et al., 1997). Gene expression analysis in the first 45 min of LPS stimulated cord blood and adult monocytes showed differential expression of 168 genes, of which 95% were overexpressed in adults (Lawrence et al., 2007). This pattern changed after two hours, as several differentially expressed genes were more upregulated in cord blood monocytes than adults. This suggests that the expression of several genes in neonatal monocytes is delayed but it improves rapidly within a short time to reach adult levels (Lawrence et al., 2007).

2.4.3. Neonatal Macrophages Macrophages play a key role in controlling initial infection by recruiting neutrophilic granulocytes and secretion of chemokines (Beck-Schimmer et al., 2005; Cailhier et al., 2006). Neonatal macrophages from mice showed a profound granular morphology which was not seen in adults (Winterberg et al., 2015). Neonatal murine peritoneal macrophages showed a marked deficit in the expression of MHC II, F4/80 and co-stimulatory molecules CD86 and CD80 (Winterberg et al., 2015). The expression of macrophage lineage marker CD11b was diminished in neonates while the expression of CD68 in neonatal macrophages was almost equivalent to adults. Neonatal AMs exhibit diminished phagocytosis, which impairs bacterial clearance and promotes sustained infections. They have been shown to secrete higher prostaglandin E2 and reduced leukotriene B4, which promotes host defense, but this does not account for the impaired phagocytosis in neonatal AMs (Ballinger et al., 2011). In contrast, neonatal monocyte-derived macrophages showed enhanced phagocytosis compared to adults; this is due to the impaired tyrosine phosphatase SHP-1 signaling (Lawrence and Koenig 2012). The expressions of Toll-like receptors (TLR2, TLR4, TLR9) in neonatal macrophages were significantly reduced compared to adult macrophages (Winterberg et al., 2015). Upon stimulation of neonatal murine peritoneal macrophages with four different TLR ligands, they expressed higher chemotaxis promoting genes (Winterberg et al., 2014). This leads to the abundant recruitment of neutrophils which may cause over-inflammation and tissue injury in neonates. The differentiation of macrophage DC progenitors into regulatory macrophages is favored by the lymph node stromal cells in neonates. These regulatory macrophages possess reduced T cell co-stimulatory molecules and MHC II molecules and suppress T cell proliferation via secretion of nitric oxide (Zhang et al., 2012). In response to increasing doses of LPS, neonatal macrophages produced increasing levels of TNF, IL-6, Rantes, MCP-1, MIP-1α and the levels of MIP-1α, MCP-1 and IL-6 were enhanced in neonatal macrophages compared to adults (Winterberg et al., 2015). The pro-inflammatory immunoregulatory chemokine, macrophage migration inhibitory factor (MIF) was 10 times higher in newborns than adults. Upon stimulation with E. coli or GBS in newborns, MIF was highly expressed, which promotes the p38 phosphorylation, ERK1/2 activation and secretion of cytokines like TNF-α (Roger et al., 2016). Lipoteichoic acid (LTA) induces production of TNF-α, IL-6, IL-12 and ERK1/2 phosphorylation in cord blood macrophages (Cheng et al., 2013). Neonatal macrophages in

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early life show elevated expression of IL-27 which increases IDO activity. Following IL-27 stimulation of neonatal macrophages, they phosphorylate STAT-1 and STAT-3, which control IDO activity and the subsequent immunosuppression (Jung et al., 2016). Neutralization of IL-27 in neonatal macrophages improved their ability to limit bacterial replication (Kraft et al., 2013). The exposure of neonatal mice to mild hyperoxia increased the number of immune cells (macrophages) in lungs without altering the lung structure (Bouch et al., 2015). The depletion of lung macrophages in neonatal mice caused pneumonia, characterized by neutrophilic inflammation and increased Th1 cytokines; when accompanied by defective mucus clearance it increased their susceptibility to pneumonia (Saini et al., 2016). The impaired Th1 function weakens the neonatal host defense mediated by macrophages and contributes to increased susceptibility to infections. AM activation was age dependent and IFN-γ exposure stimulated neonatal AM to advance RSV clearance while AM depletion represses weight gain in neonatal mice infected with RSV (Eichinger et al., 2015). Following influenza A virus infection, TLR3, TLR7, TLR8, IFN-β and TNF-α mRNA expressions were significantly enhanced in neonatal porcine AMs (Delgado-Ortega et al., 2014). The ability of neonatal macrophages to stimulate T cell proliferation was reduced compared to adults (Winterberg et al., 2015). Neonatal macrophages are crucial for heart regeneration and neoangiogenesis following myocardial infarction. Depletion of macrophages in neonates resulted in defunct ability to regenerate myocardia, forming fibrotic scars and suppressing angiogenesis (Aurora et al., 2014). Neonatal mice expand the resident cardiac macrophages of embryonic origin and reduce inflammation and promote cardiac myocyte proliferation and angiogenesis. The adult heart contains similar embryonic derived macrophages, but they are replaced by monocyte-derived macrophages after injury which triggers a proinflammatory type deleterious response (Lavine et al., 2014). Recently it has been found that arterial macrophages originate embryonically from CX3CR1 1 precursors and after birth from the circulating monocytes (Ensan et al., 2016). In the adult stage, arterial macrophages are self renewed and are replenished during inflammation or on exposure to bacteria. The CX3CR1-CX3CL1 interactions control the arterial macrophage survival in adulthood (Ensan et al., 2016). The expression of CXCL13 that influences B cell homing was absent in neonatal macrophages from peritoneal cavity (Winterberg et al., 2015). In mice the deletion of maternal very low-density receptor (VLDL) produced defective milk with reduced levels of platelet activating factor acetyl hydrolase (PAFAH) (Du et al., 2012). This elevates the proinflammatory platelet activating factors causing neonatal inflammatory disorders like alopecia. The VLDL deletion weakens the phospholipase A2 group 7 expression in macrophages, which represses PAFAH secretion (Du et al., 2012). Prenatal supplementation of Vitamin D3 reduced the intracellular LL-37 expression of cord blood monocytederived macrophages but the ex-vivo bactericidal capacity was not affected (Raqib et al., 2014). Neonatal rats delivered from mother rats fed on low protein diet, were of low body weight from the fifth day of birth until adulthood when compared to those from high protein diet mothers (Costa et al., 2016). The dietary limitation during lactation modifies the oxidant function of AMs in newborns and increases susceptibility to C. albicans infection (Costa et al., 2016). Prenatal alcohol exposure causes alveolar macrophage dysfunction and impairs the innate immune defenses in newborns. The in-utero ethanol exposure leads to increased fatty acid ethyl ester (FAEE) in lungs of the neonatal guinea pig and the increased FAEE causes alveolar macrophage dysfunction (Mohan et al., 2015). Nicotine exposed AMs in neonatal mice elevated the expression of TGF-β1 and IL-13 and modulated the resting state of AMs to alternate activation with diminished phagocytosis (Wongtrakool et al., 2012) (Table 2.1).

2.4.4. Neonatal Dendritic Cells The activation and development of DCs play a crucial part in developing the immune system in early life. In neonates there is a delay in the maturation of follicular DCs that mediate the development of antibody secreting lymphocytes. The delayed maturation of certain DCs in neonates results in limited production of IL-12, which is crucial for the differentiation of Th1 cells (Zaghouani et al., 2009). The neonatal CD8α 1 DC compartment expands and reaches adult size by day 7, but the CD41 DC increase gradually by three weeks (Sun et al., 2003). DCs constitutively express the cell surface hematopoietic markers CD45, MHCII and CD11c. Conventional DCs (cDCs) consist of two major subsets: CD1031 CD11b 2 and CD11b1 cDCs and the expression of CD103 assists in the identification of the two CD11b1 subsets in the lamina propria. The number of conventional DCs and the ratio of CD1031 DCs to CD11b1 DCs in neonatal lungs were low and plasmacytoid DCs (pDCs) were more lowly compared to adults. In contrast, it has been shown that pDCs were equivalent to adult levels in newborns and only cDCs were reduced (Kollmann et al., 2009). CD1031 DCs are involved in the development of oral tolerance by converting naı¨ve T cells into T reg cells. This is by their ability to translate vitamin A to retinoic acid by RALDH (Stern et al., 2013). The DC population in mesenteric lymph nodes of neonatal mice following RSV I. INNATE AND ADAPTIVE IMMUNE SYSTEMS: COMPONENTS AND REGULATION

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TABLE 2.1 Quantitative, Phenotypic and Functional Characteristics of the Various Innate Immune Cells of the Newborn Cell type

Quantitative characteristics

Phenotypic characteristics

Functional significance

Neutrophils

• At birth the neutrophil count rises before settling down to normal ranges in the first week of life. • Rapid depletion of mature PMN reserves during infection leads to release of immature band forms

• Low expression of adhesion molecules Lselectin and CD11b/CD18 (Mac-1) • Reduced expression of proapoptotic proteins caspase 3, Bax, Bak

• Decreased adhesion, reduced chemotaxis and migration • Phagocytosis ability is impaired at birth, but improved within the first 3 days after birth • Reduced MPO and respiratory burst activity • Impaired/Delayed NETosis

Monocytes

• At birth the monocyte concentration • TLR4 and HLA-DR exp. showed a is higher and stabilizes over the first gestational age-dependent increase 2 weeks • CD80, CD86, and MHC II expression was reduced in neonatal monocytes • Reference range at 40w was 3003300 μL21

• Phagocytosis ability is comparable to adults in full-term infants, not preterms • Diminished PICD after phagocytosis (reduced CD95L, caspase 8, 9) • Reduced anti-oxidant response

Macrophages • Presence of resident cardiac macrophages of embryonic origin • Depends on the number of monocytes which differentiate into macrophages

• Profound granular morphology • Reduced MHCII, F4/80, CD80, CD86 • TLR2, TLR4, TLR9 expression reduced

• Phagocytic functions were normal in neonatal macrophages except in alveolar macrophages • Retarded ability to induce T cell proliferation

Dendritic cells

• pDCs equivalent to adults but cDCs, • mDCs and pDCs from cord blood show mDCs were reduced reduced MHC II, ICAM-1, CD80, CD86 • Ratio of mDCs:pDCs is 1:3

• Diminished Th1 response • Impaired IFN-γ production and antiviral response • Impaired antigen presentation to T cells

Natural killer cells

• Average abs cell count 500 mm23 in • Cord blood NK cells express higher term neonates inhibitory receptor CD94/NKG2A, lower activating receptor NKG2C, LIR-1, KIR • Elevated at birth, decline then after and gradually increase to adult • Low expression of CD62L and ICAM-1 levels by 5 years • CD56 bright subset slightly higher in cord blood NK cells

• Cord blood NK cells secrete diminished TNF-α, IFN-γ • Cytotoxic efficiency of cord blood NK cells threefold lesser than adults • Ability to perform ADCC and FCγRIII expression was comparable to adults

Soluble plasma components

• Serum levels of MBLs, ficolins and MBL serine proteases were reduced • Decreased levels of fibronectin • Higher adenosine in neonatal plasma • Low levels of APPs and AMPs

• Reduced MBL increase risk of sepsis • Fibronectin deficiency weakens opsonic functions and antibacterial activity • Elevated adenosine favor antiinflammatory response

2

infection was predominantly composed of CD1031 DCs and lesser CD11b1 DCs. These neonatal lung migratory DC populations showed defective transport and processing of soluble antigen during RSV infection. They also showed limited expression of co-stimulatory molecules. CD86 expression increased with age after infection, while CD80 were consistently at the low range which contributes to the impaired CD81 T cell response in neonates (Ruckwardt et al., 2014). MoDCs from umbilical cord blood in response to influenza A infection, showed reduced expression of CD40, CD80, CD86 and HLA-DR compared to adult peripheral blood. The cord blood MoDCs produced lesser TNF-α, but IL-6 or IFN-α levels were not affected. Their ability to stimulate CD31 T cell proliferation was also repressed by influenza A infection (Lin and Lee, 2014). Neonatal CD1231 pDCs are intrinsically deficient and needed stimulation with the TLR9 ligand CpG A, to produce significant amounts of IFN-α, and CpG B DNA for upregulation of co-stimulatory molecules CD80/86 to adult levels (Gold et al., 2006). The neonatal FcRn receptor is required for effective cross presentation of IgG containing immune complexes (IgCs) by CD8 (2) CD11b (1) MoDCs. FcRn assists in the intracellular sorting of IgCs to the proper sites for effective cross presentation and also traps the antigen, preventing its degradation in acidic loading compartment (Baker et al., 2011). The FcRn receptor within DCs is also crucial for the activation of mucosal CD81 T cells to provide antitumor immunity (Baker et al., 2013). Endosomal FcRn enhanced the cross presentation of Ova ICs to I. INNATE AND ADAPTIVE IMMUNE SYSTEMS: COMPONENTS AND REGULATION

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CD41 T cells effectively, but the FcRn in phagosomes showed variation in Ag presentation between macrophages and DCs. The presentation of phagocytosed OvA ICs to CD41 T cells in macrophages was enhanced (which is attributed to the acidic phagosomal pH in macrophages) and it remained unaffected in DCs where phagosomal pH remained unaltered (Liu et al., 2011). Following in-vitro exposure to anti-CD3, neonatal lung explants showed increased production of Th2 cytokines and GATA3 by a subset of cells which were CD31 , CD4, and CD8 double negative T-lymphocytes (Roux et al., 2011). Neonatal basophils in mice produce IL-4, which is captured by the IL4Rα/IL13Rα1 heteroreceptor expressed by DCs, which subsequently downregulates IL-12. This leads to the overexpression of the heteroreceptor in differentiating Th1 cells which cripples Th1 immunity on subsequent exposure to antigen in neonates (Dhakal et al., 2015). The transcriptional activation of IL-12p35 gene at the mRNA level was severely impaired in LPS stimulated neonatal moDCs and a defect in nucleosomal remodeling repressed the IL-12p35 transcription at the chromatin level (Goriely et al., 2004). The defect in production of IL-12 by neonatal DCs results in impaired ability to trigger IFN-γ secretion by T cells (Goriely et al., 2001). Impaired expression of IFN-β and IFN inducible genes was found in neonatal blood and moDCs stimulated with LPS (Aksoy et al., 2007). This is associated with the impaired IRF-3 activity in neonates. The IRF-3 DNA binding activity and its association with the co-activator CREB-binding proteins was reduced in neonatal moDCs. IRF-3 is also related to the production of IL-12p70 and may contribute to the reduced Th1 responses in newborns (Aksoy et al., 2007). The Th2 bias in newborns reduces the Th1 protective immunity, and modulation of DCs response could provide novel solutions for restoration of Th1 protective immunity in newborns. TLR agonists are used to stimulate the Th1 responses in newborns. moDCs activated by TLRs in combination with Dectin-1 induce strong secretion of IL-12p70 and favor the differentiation of naı¨ve T cells towards IFN-γ producing Th1 cells (Lemoine et al., 2015). The synergistic use of TLR agonists (poly (I:C) and R-848 or LPS and R-848) increased the IL-12p70 production by neonatal DCs and their supernatants induced IFN-γ production from autologous naı¨ve T cells (Krumbiegel et al., 2007). R-848 stimulated cord blood antigen-presenting cells to produce more TNF-α, IL-6, IL-10, IL-12, IFN-γ, while CpG-B induced higher production of TNF-α and IL-6 (Schu¨ller et al., 2016). Neonatal BCG primed lung DCs, triggered production of Th1 cytokines from adult naı¨ve CD4 1 T-lymphocytes, but was only able to evoke a Th2-type response from neonatal naı¨ve lymph node T cells (Roux et al., 2011). Stimulation of neonatal porcine blood DCs with TLR ligands viz., LPS (TLR4), poly (I:C) (TLR3), imiquimod (TLR7), CpG-ODN (TLR9) significantly increased the expression of co-stimulatory molecules CD80/86 expression, and also induced higher production of immune cell recruiting chemokines such as CCL-2, CCL-4, CXCL-10. Imiquimod and CpG class A stimulation produced more TNF-α, IL12p40 and IFN-α by neonatal DCs (Auray et al., 2013). Neonatal porcine DCs stimulated greater proliferation of adult lymphocytes, while their potential to trigger lymphoproliferation was lost when co-cultured with neonatal lymphocytes (Auray et al., 2013). The adjuvant LTK63 promoted the maturation of follicular DCs, improved the germinal center reaction and sustained antibody secreting cells to vaccine, in the bone marrow (Bjarnarson et al., 2012). Infection of DCs with outer membrane protein A upregulates the expression of the protein CD47 and its ligand thrombospondin-1 (TSP-1). The ligation of CD47 with TSP-1 prevents the maturation and cytokine production of DCs. The ablation of CD47 in neonatal mice by siRNA technique prevented them from neonatal meningitis (Mittal et al., 2010). Enhancing the number of intestinal CD103 1 DCs using FLT-3L significantly reduced the susceptibility of neonates to infection. The recruitment of CD103 1 DCs produced IFN-γ and IL-12 in the lamina propria and lymph nodes, limiting intracellular parasite multiplication (Lantier et al., 2013). Bryostatin-1 and calcium ionophore promote the maturation of cord blood MoDCs and enhance activation of alloreactive neonatal T cells and IFN-γ production (Do et al., 2004). Neonatal pDCs from cord blood efficiently responded to influenza A virus, HIV or HSV by significant production of TNF-α, IFN-γ and chemokines (Zhang et al., 2013). pDCs in neonates formed an extended CD2 1 compartment that did not affect the antiviral IFN-α response. Within this CD2 1 pDCs compartment a subpopulation of CD5 was found that produced IL-12p40, but this subpopulation was profoundly decreased in neonatal pDCs than in adults (Zhang et al., 2013). RSV infection in newborns induced restricted type I IFN responses and pDC responses, and either IFN-α pretreatment or adoptive transfer of adult pDCs reduced the viral load and IL4-Rα in Th2 cells during reinfection (Cormier et al., 2014). When neonatal moDCs were stimulated by recombinant protein subunit vaccine candidate BBG2Na against RSV, they induced antigen specific CD4 1 T cell proliferation and differentiated into non-polarized CD4 T cell effectors (Matthews et al., 2007). Trivalent influenza vaccine cosignals T cells to produce IFN-γ in neonates, which involves the engagement of pDCs by TLR7 agonists triggering Th1 responses in an IL-12 independent manner and IFN-dependent manner (Zhang et al., 2014). Improved cross presentation of antigen to CD81 T cells by these cord blood pDCs was found (Zhang et al., 2014) (Table 2.2).

I. INNATE AND ADAPTIVE IMMUNE SYSTEMS: COMPONENTS AND REGULATION

TABLE 2.2 Effect of Various Stimulants in Modulating the Innate Immune Response of Immune Cells in the Newborn Stimulant

Effect/significance

LPS (TLR4) and R-848 • LPS/R-848 responses were attenuated in very preterm neonates and this improved with age (TLR7/8) • LPS and R-848 improved the TNF-α, IL-6 and IL-12-23p40 levels and IL-6 levels were higher than adults in term newborns

Reference Marchant et al. (2015)a

R-848 and CpG-B

• Stimulation with R-848 elevated TNF-α, IL-6, IL-10, IL-12, and IFN-γ in cord blood APCs • CpG-B induced higher secretion of TNF-α and IL-6 from cord blood APCs

Schu¨ller et al. (2016)

LPS

• LPS stimulated release of IL-1β was reduced in cord blood cells of low birth weight newborns which correlated with reduced expression of TLR4 and MyD88

Singh et al. (2014)b

LPS and CpG-ODN (TLR9)

• Expression of CD80/HLA-DR in mDCs reaches adult levels within 3 m, 69 m for monocytes and pDCs • Following LPS stimulation, TNF-α, IL-10, and IL-12p70 reached adult levels within 69 m • Cord blood stimulated with CpG-ODN produced increased levels of IL-6, IL-8, IL-1β, IL-10 • Following CpG stimulation, type I IFN dependent chemokines IP-10 and CXCL-9 increased gradually

Nguyen et al. (2010a)

Zymosan (TLR2)

• Zymosan induced comparable levels of inflammatory cytokines TNF-α, IL-6,8,10, and IL-1β in cord blood monocytes, DCs and moDCs compared to adults

Nohmi et al. (2015)c

R-848(TLR7/8), 3M-002 (TLR7/8), 3M-003 (TLR8)

• All the TLR8 agonists increased TNF-α in neonatal blood, IL-12 in neonatal monocytes • Upregulation of CD40 on neonatal mDCs • Induce p38 MAPk phosphorylation in neonatal monocytes

Levy et al. (2006)d

Pam3CSK (TLR1/2), • All three TLR(14) ligands induced comparable levels of TNF-α and enhanced IL-6 in Poly (I:C) (TLR3), LPS mononuclear cells from cord blood • Expression of TLR4 mRNA in neonatal cord blood cells was higher than adults

Liao et al. (2013)e

Poly (I:C)/R-848 LPS/R-848

• Combined TLR agonists use induces maturation of neonatal moDCs • Induces slender increase in neonatal IFN-γ, IL12p70 production • TLR4/8 combination induced adult like IL-12p40, higher IL-1β in neonatal DCs

Krumbiegel et al. (2007)

Interleukin-15

• IL-15 enhances natural killer activity and antibody-dependent cellular cytotoxicity in neonatal cells

Nguyen et al. (1998)f

Imidazoquinoline (3M-002)

• IMQ induced more TNF-α and IL-1β from neonatal monocytes • IMQ TLR8 agonists activate newborn monocyte and DCs through adenosine refractory and caspase 1 dependent pathways

Philbin et al. (2012)

Imidazoquinolines analog hybrid 2 (TLR8)

• Hybrid 2 better than R-848 in stimulating higher TNF-α, IL-1β in cord blood monocytes

Ganapathi et al. (2015)g

Pam3CSK4

• Following TLR1/2 stimulation, IL-8 secretion by neonatal PMNs was elevated than adults

Thornton et al. (2012)h

Poly (I:C) (TLR3), • Following TLR(3,4,7) stimulation, cord blood production of IFN-γ and IL12p70 was reduced and Escherichia coli (TLR4), this increased to adult levels by 1 month of age loxoribine (TLR7), • The potential of neonatal blood to produce Th1-type cytokines in response to LPS remains CpG-ODNA (TLR9) impaired up to 1 month of age

Belderbos et al. (2009)

VTX-294 (TLR8)

• VTX-294 induced higher TNF-α and IL-1β in newborn cord blood than R-848 • Effective induction of co-stimulatory molecules HLA-DR and CD86 on moDCs

Dowling et al. (2013)i

CpG-ODN (TLR9)

• CpG pretreatment was protective in newborn mice against RSV infection, with reduced viral load and improved Th1 responses at secondary adult RSV infection • It induced transient rise in MHC II and CD80 expression on CD11c 1 cells • Increased IFN-γ production by NK cells following RSV infection

Yamaguchi et al. (2012)j

a

Marchant, E., Kan, B., Sharma, A., van Zanten, A., Kollmann, T., Brant, R., et al., 2015. Attenuated innate immune defenses in very premature neonates during the neonatal period. Pediatr. Res. 78, 492497. b Singh, V.V., Chauhan, S.K., Rai, R., Kumar, A., Rai, G., 2014. Decreased toll-like receptor-4/myeloid differentiation factor 88 response leads to defective interleukin-1β production in term low birth weight newborns. Pediatr. Infect. Dis. J. 33, 12701276. c Nohmi, K., Tokuhara, D., Tachibana, D., Saito, M., Sakashita, Y., Nakano, A., et al., 2015. Zymosan induces immune responses comparable with those of adults in monocytes, dendritic cells, and monocyte-derived dendritic cells from cord blood. J. Pediatr. 167, 155162.e1e2. d Levy, O., Suter, E.E., Miller, R.L., Wessels, M.R., 2006. Unique efficacy of Toll-like receptor 8 agonists in activating human neonatal antigen-presenting cells. Blood 108, 12841290. e Liao, S.L., Yeh, K.W., Lai, S.H., Lee, W.I., Huang, J.L., 2013. Maturation of Toll-like receptor 1-4 responsiveness during early life. Early Hum. Dev. 89, 473478. f Nguyen, Q.H., Roberts, R.L., Ank, B.J., Lin, S.J., Thomas, E.K., Stiehm, E.R., 1998. Interleukin (IL)-15 enhances antibody-dependent cellular cytotoxicity and natural killer activity in neonatal cells. Cell. Immunol. 185, 8392. g Ganapathi, L., Van Haren, S., Dowling, D.J., Bergelson, I., Shukla, N.M., Malladi, S.S., et al., 2015. The imidazoquinoline toll-like receptor-7/8 agonist hybrid-2 potently induces cytokine production by human newborn and adult leukocytes. PLoS One 10, e0134640. h Thornton, N.L., Cody, M.J., Yost, C.C., 2012. Toll-like receptor 1/2 stimulation induces elevated interleukin-8 secretion in polymorphonuclear leukocytes isolated from preterm and term newborn infants. Neonatology 101, 140146. i Dowling, D.J., Tan, Z., Prokopowicz, Z.M., Palmer, C.D., Matthews, M.A., Dietsch, G.N., et al., 2013. The ultra-potent and selective TLR8 agonist VTX-294 activates human newborn and adult leukocytes. PLoS One 8, e58164. j Yamaguchi, Y., Harker, J.A., Wang, B., Openshaw, P.J., Tregoning, J.S., Culley, F.J., 2012. Preexposure to CpG protects against the delayed effects of neonatal respiratory syncytial virus infection. J. Virol. 86, 1045610461.

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2. INNATE IMMUNITY AT BIRTH: IMPLICATIONS FOR INFLAMMATION AND INFECTION IN NEWBORNS

2.4.5. Neonatal Natural Killer (NK) Cells NK cells can be categorized into several populations on the basis of the expression of surface markers CD16 and CD56. The two major subsets of NK cells are the CD56brightCD16dim- and CD56dimCD161. The NK cell percentage at birth in very preterm neonates (,30 weeks) was reduced compared with term neonates; this is associated with increased late onset infection (Ma et al., 2014). The absolute count and percentage of NK cells increase with gestational age (Pe´rez et al., 2007). Throughout the suckling period in neonatal rats, the level of NK cells in the intraepithelial intestinal compartment was higher. Apart from the day of birth, during the first 2 weeks of life most intestinal NK cells expressed CD8 (Pe´rez-Cano et al., 2005). It has been earlier shown that cord blood NK cells are functionally and phenotypically immature and CD161 CD56 2 NK cells in cord blood are precursors of the more mature NK cell phenotypes CD161 CD561 (Gaddy and Broxmeyer, 1997). Recently, it has been found that the percentage of neonatal NK cells expressing the phenotype CD56 1 CD16 1 CD3- in CD45 high or CD45 dim populations were comparable or higher than those from adult peripheral blood (Lo´pez et al., 2009). The CD56 bright and CD56 dim NK cell populations were found to be similar in cord blood and peripheral blood (Tanaka et al., 2003). Only few CB NK cells expressed L-selectin (adhesion molecule), which is an indicator for immature NK cells (Dalle et al., 2005). The terminal differentiation marker CD57, was found to be significantly lower in neonatal NK cells than adult peripheral blood (Tanaka et al., 2003). The NK cell activity was significantly enhanced in the vaginally delivered newborns rather than those delivered by cesarean sections and under influence of maternal anesthesia (De Amici et al., 1999). The activation and expansion of invariant NK T cells (iNKT) in the third trimester led to late preterm birth and neonatal mortality (Louis et al., 2016). The inhibitory killer Ig like receptor (KIR) repertoire in neonatal NK cells is diverse but is not biased towards identification of cognate HLA class I. Functional education of neonatal NK cells by autologous HLA class I ligands precedes structural modulation of KIR repertoires (Scho¨nberg et al., 2011). In response to TLR9 ligation, neonatal lymphoid progenitors unlike adult cells were unable to produce functional NK cells (Vadillo et al., 2014). NK cells act on strained cells through the production of cytokines. NK cells express CD107a during degranulation and release lytic granules composed of perforins and granzymes (Tomescu et al., 2009). The expression of perforin and granzyme in CB NK cells was higher than adult NK cells (Dalle et al., 2005). Neonatal NK cells expressed adult levels of adhesion glycoproteins CD18 and CD44 and Fc receptor for IgG. Neonatal NK cells exhibit diminished antibody mediated conjugation, which may account for the reduced antibody-dependent cellular cytotoxicity (Kohl et al., 1999). The NK cell cytotoxicity in murine models is regulated by MHC class I receptors, Ly49 and CD94/NKG2 (Kubota et al., 1999). Only very few neonatal NK cells express Ly49, though most of them showed NKG2 expression, which implies that expression of the inhibitory receptor NKG2A precedes Ly49 in NK cell development in early life (Kubota et al., 1999). The expression of CD69 and CD107a has also been associated with elevated NK cell cytotoxicity (Clausen et al., 2003; Tomescu et al., 2009). Neonatal NK cells in response to LTA and costimulation with IL-15 showed elevated expression of CD69 and CD107a (Cheng et al., 2013). Neonatal NK cells that express cognate killer cell immunoglobulin-like receptor show enhanced cytotoxicity and degranulation (Scho¨nberg et al., 2011). The cytotoxic potential of CB NK cells against NK sensitive targets was enhanced when stimulated by IL-15 and IL-2 (Bradstock et al., 1993; Dalle et al., 2005). The cytotoxic effector molecule expressed by NK cells in fetal mice is the tumor necrosis factor related apoptosis inducing ligand (TRAIL). The immature cytotoxic NK cells were characterized by expression of TRAIL. The antitumor surveillance in neonatal mice was solely dependent on TRAIL dependent apoptosis rather than perforin mediated killing (Takeda et al., 2005). CD56-ve NK cells from cord blood showed decreased inhibitory and activating receptors when compared to CD56 1 ve cells (Jacobson et al., 2013). Neonatal NK cells promote pathogenic T cell response at various levels of the pathogenesis of autoimmune disease (Setiady et al., 2004). Neonatal NK cells lack the Ly49C/I receptors and are hypersensitive to ZP3 antibody, which triggers neonatal autoimmune ovarian disease in neonatal mice (Rival et al., 2014). Neonatal spleen contained predominantly NK 1.1 TCRV beta (2), which produced IFN-γ, while depletion of NK cells prevented from neonatal autoimmune ovarian disease (Setiady et al., 2004). Maternal preeclampsia caused significant rise in the cord blood NK cells (CD3 2 / CD561 161 ) compared to those without preeclampsia (Bujold et al., 2003). NK cells in an infant’s liver and biliary ducts promote the increased expression of cytotoxicity genes, and in neonatal mice they act via NK group 2D receptor causing tissue-specific injury in experimental biliary atresia (Shivakumar et al., 2009). In normal infants, the CD56 dim population was found to be predominant with lower expression of granzyme B, reduced production of IFN-γ and chemokines RANTES, MIP-1α and MIP-1β (Jacobson et al., 2013). The neonatal antibody responses against RSV infection are diminished by the production of IFN-γ by NK and T cells. This implies that the induction of a potent cellular immune response in neonatal life may also negatively influence the

I. INNATE AND ADAPTIVE IMMUNE SYSTEMS: COMPONENTS AND REGULATION

2.5. SOLUBLE PLASMA COMPONENTS

27

FIGURE 2.2 Innate immunity at birth. The various innate immune components of a newborn and their diminished functional response to pathogens have been shown.

antibody response (Tregoning et al., 2013). Following influenza virus stimulation of NK cells of small for gestational age neonates, they showed significantly diminished antiviral cytokines like IFN-γ, TNF-α and perforins. The antiviral activity of NK cells was positively correlated with birth weight (Li et al., 2013). Neonatal NK cells prevent HIV replication through a chemokine arbitrated mechanism. Their ability to suppress HIV replication was equivalent to those of neonatal CD8 1 T cells and better than adult NK cells (Bernstein et al., 2004). The potential of neonatal NK cells to establish intercellular extensive contacts and form immunological synapse was diminished although they were more strongly activated following contact with K562 cells (Ribeiro-do-Couto et al., 2003). Neonatal calves in response to stimulation with BCG infected DCs induced a population of NK like CD8 1 cells to produce IFN-γ (Hope et al., 2002) (Fig. 2.2).

2.5. SOLUBLE PLASMA COMPONENTS Newborn plasma has lower levels of various soluble plasma proteins that impact innate immunity. The range of neonatal complement components was reduced and ranged from 10% to 70% of the levels in adult plasma (McGreal et al., 2012). The complement activity increased with gestational age with lower activity in cord blood and increased titers in the first week after birth (Grumach et al., 2014). The complement regulators including the C1 inhibitor were below adult levels in premature babies and cord blood, which slightly increased in 5-day old, whereas others such as factor H, factor I, properdin were below adult levels in newborns irrespective of the gestational age (Grumach et al., 2014). The complement components C3 and C9 are at low levels in human and murine newborns, which weakens the bactericidal activity. The neonatal deficiency of complement components restricts the ability of newborns to inhibit the replication of various bacterial strains in blood (Levy et al., 2000). At the onset of infection in neonates the anaphylatoxin C3a increases considerably which may lead to vasodilation and increased microvascular permeability (Zilow et al., 1993). When newborn mice are immunized with C3b conjugated antigen it enhances the antibody response (Pihlgren et al., 2004). Lactic acidosis can be used to

I. INNATE AND ADAPTIVE IMMUNE SYSTEMS: COMPONENTS AND REGULATION

28

2. INNATE IMMUNITY AT BIRTH: IMPLICATIONS FOR INFLAMMATION AND INFECTION IN NEWBORNS

activate the complement factors C3a and C5a in neonates, although not as profoundly as in adults (Hecke et al., 2001). There was profound depletion of complement component C9 in neonatal rats in the first few days of birth, which increased significantly in the first three weeks after birth. Likewise, supplementing with C9 improved the bactericidal and hemolytic ability of neonatal rat serum and improved survival of neonatal rats with E. coli infection (Lassiter et al., 1997). Reduced expression of complement receptor 2 in B cells of neonates affects the antibody response to thymus independent antigens (Griffioen et al., 1993). The elevated levels of complement activation fragment Bb in early pregnancy correlates with the occurrence of spontaneous preterm birth less than 34 weeks gestation (Lynch et al., 2008). As they develop, T-lymphocytes (which are an important adaptive immune cell type) are polarized into producing different patterns of cytokines. The newborn immune response is biased against Th1 polarizing cytokines (i.e., IL-12 and IFN-γ) which are pro-inflammatory. Possibly some components of the neonatal plasma may influence this behavior. When exposed to cord blood plasma, mononuclear cells produced less IL12-p70 and higher IL-10; it was identified that two distinct factors in the neonatal plasma were responsible for these diverse effects (Belderbos et al., 2012). In contrast, adult PBMCs cultured along with neonatal plasma diminished the release of IL-10 (Belderbos et al., 2009). Transfer of anti-platelet antibodies from immune thrombocytopenia mothers through breastfeeding is associated with sustained and prolonged neonatal thrombocytopenia (Hauschner et al., 2015). Mannan binding lectins (MBLs), ficolins recognize the microorganisms and activate the lectin pathway of complement through the MBL associated serine proteases. The levels of cord blood MBL and lectin pathway proteins were found to be reduced, increasing the susceptibility of newborns to sepsis (Luo et al., 2014; Schlapbach et al., 2010). The MBL deficiency and reduced serum MBL levels in neonates increases their susceptibility to respiratory infections (Speletas et al., 2015). Likewise, I-ficolin deficit is strongly associated with prematurity, low birth weight and infections (Swierzko et al., 2009). The reduced fibronectin in neonatal plasma leads to the decreased function of the reticuloendothelial system and makes them more prone to sepsis (Polin, 1990). There was significant deficiency of antimicrobial proteins and peptides in cord blood plasma, which increases their susceptibility to invasive bacterial infections (Strunk et al., 2009). The increased expression of adenosine and prostaglandins in newborns impairs the antimicrobial responses of innate immune cells. Especially in very low birth weight newborns the adenosine levels are elevated, which correlates with prematurity (Panfoli et al., 2016). Adenosine suppresses production of IL-12 and TNF-α, through adenosine A2a receptor dependent and independent mechanisms and weakens the Th1-type response (Hasko´ et al., 2000). Adenosine suppresses the CD11b expression in cord blood polymorphonuclear leukocytes (Hou et al., 2012). Recombinant MIF used at neonatal concentrations opposes the adenosine, TNF production and PGE2 mediated suppression of ERK1/2 activation in newborn monocytes infected with E. coli (Roger et al., 2016). CD711 erythroid cells in neonatal mice have immunosuppressive nature due to expression of arginase-2 which depletes L-arginine. Treatment of murine neonates with anti-CD71 antibodies reduced the bacterial load in spleen after bacterial challenge but it was not due to the decrease in immunosuppressive CD71 1 erythroid splenocytes, which suggests neonatal CD711 erythroid cells have little effect in reducing neonatal infection and inflammation (Wynn et al., 2015).

2.6. CONCLUSION The development of immunity in early life involves diverse factors and mechanisms that shape the immune system of an individual. The newborns depend on the innate immunity at birth to ward off the threat of microbes and pathogens. The bias against Th1 proinflammatory response makes them more prone to infection from intracellular pathogens. The placental microbiome has great influence in the development of newborn immunity and also has shown to influence the preterm births. The behavior of immune cells in the neonatal period shows that, though they are inexperienced they can mount mature responses at various times and with additional costimulatory agents. The retarded vaccine responsiveness in neonates could be overcome by agents like R-848 that trigger TLRs. Newborns provided with proper nutrition and breast milk have shown to develop stronger immune characteristics than undernourished neonates. The type of delivery and environmental conditions at birth could also potentially affect the immune development of newborns. Better elucidation of the complex mechanisms underlying the newborn immunity could help us script more efficient strategies to improve the immunity of newborns.

I. INNATE AND ADAPTIVE IMMUNE SYSTEMS: COMPONENTS AND REGULATION

REFERENCES

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C H A P T E R

3 Redox Signaling and the Onset of the Inflammatory Cascade Jose P. Vazquez-Medina University of Pennsylvania School of Medicine, Philadelphia, PA, United States

3.1. INTRODUCTION Reactive oxygen species (ROS) produced in vivo by endothelial, inflammatory and immune cells by various cellular pathways, have two faces; first is its role in oxidative stress and tissue injury, and second is its participation in redox signaling. Inflammation encompasses both these aspects of ROS function. The onset of the inflammatory cascade is a coordinated event that occurs in part via redox signaling events that cause the recruitment and adherence of immune cells to the site of infection or injury. Once immune cells arrive at this site, they produce ROS. Indeed immune cells, dendritic cells, epithelial cells and endothelial cells all produce ROS upon activation during either infectious or sterile tissue damage. ROS are critical effectors that participate in a plethora of redox-regulated cellular events eventually leading to the resolution of inflammation. Excessive ROS generation, however, results in chronic inflammation, inflammatory tissue damage, organ failure and the development of a variety of chronic inflammatory diseases. This manuscript reviews the current knowledge on the role of redox signaling in the onset of inflammation.

3.2. ROS GENERATION DURING THE ONSET OF THE INFLAMMATORY CASCADE The primary enzymatic system that generates ROS during the onset of inflammation is the NADPH oxidase, a family of seven enzymes expressed in numerous cell types that differ in their organelle-specific localization, type of ROS generated, and control of their activity (Brandes et al., 2014). NOX2 is the main NADPH oxidase isoform expressed in phagocytes, dendritic cells and endothelial cells (Segal et al., 1981; Forman et al., 1980; Elsen et al., 2004; Jones et al., 1996). NOX2 is quiescent under resting conditions with the intrinsic membrane (gp91phox and p22phox) and cytosolic subunits (p47phox, p67phox, p40phox, and rac) confined to their respective compartments. During the onset of inflammation, NOX2 is activated in response to a variety of stimuli such as ligation of specific pathogen recognition receptors by microbial products, opsonized particles, and integrin-dependent adhesion (Gantner et al., 2003; El-Benna et al., 2016; Panday et al., 2015). Activation of NOX2 leads to translocation of the cytosolic components to the plasma membrane, the assembly of the oxidase complex, and the generation of superoxide radicals. In phagocytes, superoxide radicals generated by NOX2 are essential for antimicrobial host defense, as they are dismutated into hydrogen peroxide that is then used by myeloperoxidase to produce potent microbicidal metabolites in the presence of chloride and bromide (Markert et al., 1985). Furthermore, NOX2 is also crucial for the formation of neutrophil extracellular traps that help target pathogens (Remijsen et al., 2011). Patients with chronic granulomatous disease (CGD), an inherited disorder characterized by defective phagocytic NOX2-dependent superoxide generation due to mutations in different NOX2 subunits, suffer from life-threatening infections requiring antibiotic therapy for survival (Hohn and Lehrer, 1975). Besides phagocytes, NOX2 is also well established as a source of ROS in vascular endothelial cells. Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00003-2

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Mitochondria, xanthine oxidase, and uncoupled nitric oxide synthase are additional sources of ROS that may play a role in the onset of inflammation (Mittal et al., 2014). Although not directly related to host defense, these mechanisms could be involved in the propagation of vascular inflammation. Under inflammatory conditions, vascular oxidative stress promotes the opening of endothelial cell junctions and the migration of inflammatory cells across the endothelial barrier. Of note, however, is evidence demonstrating that NOX2 knockout mice (gp91phox null) show decreased vascular inflammation in sterile models of sepsis (Menden et al., 2016; Gandhirajan et al., 2013).

3.3. REDOX CONTROL OF INFLAMMATORY MEDIATORS A key event in the onset of the inflammatory cascade is the generation of proinflammatory cytokines and adhesion molecules. There are several pathways stimulated by either exogenous pathogen-associated molecular patterns (PAMPs) or endogenous damage-associated molecular patterns (DAMPs) responsible for the onset of inflammation. Specific receptors (collectively termed pattern-recognition receptors or PRRs) that recognize both PAMPs and DAMPs initiate cellular cascades that ultimately result in either the synthesis or the maturation of different cytokines and adhesion molecules (Takeuchi and Akira, 2010). Among those receptors, the cell surface Toll-like receptors (TLRs), which mediate NF-κB-dependent transcription of inflammatory cytokines/chemokines, cellular adhesion molecules (CAM) such as VCAM-1, ICAM-1 and E-selectin among others, are well characterized (Kawai and Akira, 2010). In contrast, NOD-like receptors (NLRs) or inflammasomes, which detect intracellular signals, are emerging as sensors with a crucial role in the onset of inflammation (Franchi et al., 2009). The regulation of all these molecules is multifactorial. Aside from TLR-dependent pathways, interleukin-1 (IL-1), tumor necrosis factor (TNF-α), and NOX2-derived hydrogen peroxide can activate NF-κB (Kaul and Forman, 1996; Mercurio and Manning, 1999). Redox-dependent activation of Nf-κB is rather complex and can happen through several distinct pathways including activation of MAP kinases, serine or tyrosine phosphorylation of NF-κB’s repressor I-κB or direct oxidation of cysteine residues in the p50 subunit of NF-κB. Moreover, redox-regulation of NF-κB is distinct depending on the intracellular location of NF-κB during initial activation (cytoplasm) or DNA binding activity (nucleus) (Kabe et al., 2005; Morgan and Liu, 2011; Na and Surh, 2006). Besides this, inflammatory cytokines that are known activators of NF-κB such as TNF-α and IL-1 can also increase NOX2-dependent ROS generation (Kim et al., 2010; Yang et al., 2007). Therefore, it is likely that there are several cellular events, increased ROS generation among them, that initiate and sustain NF-κB activation during inflammatory conditions. Blaser and colleagues recently reviewed the interconnected mechanisms of redox- and TNF-α-mediated activation of NF-κB during the onset of inflammation (Blaser et al., 2016). Another critical player in the inflammation pathway is the inflammasome. The NLRP3 (NOD-like receptor containing pyrin domain 3) is the most well-characterized inflammasome. NLPR3 controls the activation of caspase-1 leading to the processing and secretion of interleukin-1β (IL-1β) and IL-18, and ultimately to inflammatory cell death or pyroptosis (Martinon et al., 2009). Upon activation by DAMPs or PAMPs, NLRP3 oligomers form a multi-complex protein with the adaptor protein ASC and activated caspase-1. The formation of this complex results in the maturation of pro-IL-1β and pro-IL-18 (Sutterwala et al., 2007). NLPR3 inflammasomes can be activated by several mechanisms including ATP and K1 efflux, Ca21 signaling, endoplasmic reticulum stress, lysosome destabilization, deubiquitination, and increased ROS generation (Abais et al., 2015). Many studies point at ROS as important mediators of NLRP3 inflammasome activation. The crystal structure of NLRP3 contains a highly conserved disulfide bond in the protein-interacting domain, between Cys8 and Cys108, which is sensitive to oxidation and therefore to redox control (Bae and Park, 2011). The source of ROS responsible for NLRP3 activation, however, remains controversial. As discussed above, NOX2 is most likely the primary source of ROS during the onset of inflammation. The role of NOX2-derived ROS in NLRP3 inflammasome activation is nevertheless unclear. While peripheral blood mononuclear cells isolated from CGD patients lacking expression of p22phox secrete normal amounts of IL-1β (van Bruggen et al., 2010), neutrophils isolated from CGD patients with mutations in gp91phox release little amounts (Gabelloni et al., 2013). Similarly, NLRP3 inflammasome activation occurs in bone marrow-derived macrophages isolated from gp91phox knockout mice (Hornung et al., 2008) but not in lung endothelial cells (Xiang et al., 2011). Thus, the requirement of NOX2derived ROS for NLRP3 inflammasome activation is likely cell-type specific.

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As discussed at the beginning of this chapter, there are alternative sources that generate ROS during the onset of inflammation and could certainly compensate for the lack of NOX2. Aside from mitochondria and xanthine oxidase, lipoxygenases and cyclooxygenases, ROS-generating enzymatic systems that release inflammatory mediators such as prostaglandins and leukotrienes, have been linked to IL-1β secretion (Amaral et al., 2012; Barbera-Cremades et al., 2012). The participation of these systems in NLRP3 inflammasome activation is yet to be understood and undoubtedly warrants further investigation. Similarly, several conflicting reports exist regarding the precise role of ROS in NLRP3 activation. ROS may participate in both, “priming” of the inflammasome by contributing to pro- IL-1β synthesis through activation of NF-κB (Bauernfeind et al., 2009), and in post-translational regulation via oligomerization of the inflammasome complex, caspase-1 activation and IL-1β secretion (Abais et al., 2013). The role of this two-step process in particular cell types expressing different levels of pro- IL-1β (e.g., neutrophils vs endothelial cells) remains unknown but sheds some light on the distinct roles of endothelial and phagocytic NLRP3 inflammasomes in acute and chronic inflammation.

3.4. REDOX CONTROL OF ANTIOXIDANT AND ANTI-INFLAMMATORY TRANSCRIPTION FACTORS As discussed throughout this chapter, increased ROS generation is a primary event in the onset of the inflammatory cascade. Aside from activating proinflammatory pathways such as NF-κB and NLRP3 inflammasomes, increases in local ROS production, chiefly hydrogen peroxide, result in the activation of redox-sensitive transcription factors that control the expression of antioxidant and anti-inflammatory genes. Among those, Nrf2 (nuclear factor erythroid-derived related factor 2) and AP-1 (activator protein-1) are the most well studied. Under basal conditions, cytosolic Nrf2 is bound to its repressor protein Keap1. This interaction targets Nrf2 for ubiquitin conjugation and further proteasomal degradation. Increases in cytosolic ROS result in the oxidation of Cys273 and Cys288 residues in Keap1, promoting its dissociation from Nrf2 and resulting in the nuclear translocation and binding of Nrf2 to the electrophilic response element (EpRE, also called antioxidant response element). Binding of Nrf2 to EpRE initiates the transcription of several antioxidant and anti-inflammatory genes (Itoh et al., 1997). Nrf2 can also be activated by a non-canonical pathway consisting of p62-dependent sequestration and autophagic degradation of Keap1 (Komatsu et al., 2010), and by phosphorylation in a Keap1-independent manner (Rada et al., 2011). Besides stimulating the transcription of antioxidant defenses that are crucial to counteract increased ROS generation, Nrf2 controls the expression of a series of genes that contribute to limiting inflammation and suppresses NF-κB activation by modulating the redox environment (Ahmed et al., 2017). For example, Nrf2 activation increases heme oxygenase-1 (HO-1) expression. HO-1 catalyzes the degradation of heme into free iron, carbon monoxide (CO) and biliverdin, contributing to the clearance of proinflammatory free heme as well as to the production of anti-inflammatory bilirubin and CO (Alam et al., 1999). HO-1 also blocks NF-κB-dependent transcription of vascular adhesion molecules in endothelial cells (Soares et al., 2004). Similarly, Nrf2 limits IL-6 and IL-1β transcription by binding to the proximity of these genes and thus inhibiting the recruitment of RNA polymerase II, a process that is independent of the suppression of NF-κB (Kobayashi et al., 2016). Furthermore, recent evidence suggests that Nrf2 is involved in the regulation of NLRP3 inflammasomes (Liu et al., 2017). It is yet not clear, however, whether Nrf2 activates or suppresses NLRP3. One the one hand, Nrf2 can inactivate NLRP3 by a mechanism that involves Nrf2-regulated NQO1 expression (Liu et al., 2017). On the other hand, bone marrow-derived macrophages isolated from Nrf2 knockout mice show a dramatic decrease in NLRP3 inflammasome activation and IL-1β secretion in a model of peritonitis (Zhao et al., 2014). Thus Nrf2 may play both protective and pathogenic roles during the onset of inflammation depending, perhaps, on specific cell types or disease conditions. Activator protein-1 (AP-1) is a transcription factor formed by heterodimers of the Jun, Fos, ATF and MAF family of proteins that bind to TPA or cAMP response elements. Cytokines, chemokines, growth factors, hormones and multiple types of stress including oxidative stress can activate AP-1. Activation of AP-1 induces the transcription of several genes involved in a variety of contrasting cellular responses such as proliferation, apoptosis, survival (including antioxidant defense), and differentiation. Activation of the AP-1 complex requires phosphorylation of specific components by stress-responsive MAP kinases such as JNK, p38, and ERK (Karin et al., 2001). Several studies show that endogenous hydrogen peroxide increases the activity of these kinases (Whitmarsh and Davis, 1996) and thus activates AP-1 in intact cells (Iles et al., 2002). Recent evidence suggests that other ROS

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FIGURE 3.1 Redox-regulated events that take place during the onset of the inflammatory cascade. Binding of DAMPs and PAMPs to their respective PPRs induces NOX2-driven ROS generation. ROS are important mediators that control the expression and release of adhesion molecules, cytokines and chemokines through the NF-κB and NLRP3 inflammasome pathways. Dysregulated ROS production leads to oxidative stress and ultimately to cell death. ROS also activate redox-regulated transcription factor Nrf2 initiating the transcription of antioxidant enzymes that help balance increased ROS generation during the onset of inflammation. Aside from contributing to ROS scavenging and the modulation of redox signaling, peroxiredoxins can act as DAMPs and PAMPs contributing to sustaining the inflammatory response. DAMPs: damage-associated molecular patterns. PAMPs: pathogen-associated molecular patterns. PPRs: pattern-recognition receptors. NOX2: NADPH oxidase type 2. NF-κB: nuclear factor kappalight-chain-enhancer of activated B cells. NLRP3: NOD-like receptor containing pyrin domain 3. Nrf2: nuclear factor erythroid-derived related factor 2.

such as nitric oxide can also activate AP-1 (Srivastava et al., 2016). As is the case of Nf-κB and Nrf2, the onset of inflammation, which results in increases in ROS generation, likely induces the activation of AP-1.

3.5. EMERGING ROLE OF PEROXIREDOXINS IN THE ONSET OF THE INFLAMMATORY CASCADE Peroxiredoxins are a highly conserved family of cysteine-dependent peroxidases that reduce hydrogen peroxide, lipid hydroperoxides, and peroxynitrite. They are ubiquitously expressed and thus have emerged as one of the most important peroxide scavenging enzymes along with catalase and glutathione peroxidases. One particular feature of peroxiredoxins is that they are susceptible to hyperoxidation. Hyperoxidation of peroxiredoxins causes its catalytic inactivation which results in increased localized hydrogen peroxide levels that promote redox signaling events such as the activation of MAP kinases and redox-regulated transcription factors. Peroxiredoxins are thus primary players in two processes of crucial importance in the onset of the inflammatory cascade: ROS scavenging and peroxide signaling (Rhee, 2016; Perkins et al., 2015). Peroxiredoxin 6 (Prdx6) is an enzyme that, in contrast to peroxiredoxins 15, expresses phospholipase A2 (PLA2) activity in addition to its peroxidase activity. The PLA2 of Prdx6 is needed for NOX2 activation in endothelial cells and phagocytes (Chatterjee et al., 2011; Vazquez-Medina et al., 2016). Pharmacological inhibition of the PLA2 activity of Prdx6 ameliorates inflammation during acute lung injury (Lee et al., 2014). Similarly, Prdx6 knockout reduces inflammation but increases hepatic damage and hydrogen peroxide levels in a model of ischemia/reperfusion. These results reflect the significant role of Prdx6 in both peroxide scavenging and signaling during inflammatory conditions. Peroxiredoxins may also play other roles in the onset of inflammation. Recent evidence shows that TLR and TNF-α signaling stimulate the oxidation of peroxiredoxins targeting them for release via the exosomal pathway (Mullen et al., 2015; Salzano et al., 2014). Extracellular peroxiredoxins can act as DAMPs and PAMPs binding to TLRs and thus contribute to sustaining a proinflammatory response that involves the Nf-κB-mediated production

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of cytokines (Shichita et al., 2012; Choi et al., 2013; Kuang et al., 2014; Riddell et al., 2010). This emerging role of peroxiredoxins as amplifiers of inflammatory signaling highlights the potential dual role of these enzymes as pro- and anti-inflammatory mediators and undoubtedly warrants further investigation.

3.6. CONCLUSIONS AND PERSPECTIVES The onset of the inflammatory cascade results in increased ROS production by immune cells, dendritic cells, epithelial cells and endothelial cells. ROS generated during the onset of inflammation are crucial for host defense and contribute to the activation of redox-regulated pro- and anti-inflammatory mediators. ROS can activate NF-κB and NLRP3 inflammasomes through several mechanisms. Therefore, ROS are important for the synthesis, maturation or release of inflammatory cytokines, chemokines, and adhesion molecules. Similarly, ROS can activate pathways mediated by redox-sensitive transcription factors that control the expression of genes crucial to limit inflammation and to prevent oxidative stress. Among those genes, peroxiredoxins play a primary role in ROS scavenging and are thus key modulators of redox signaling. Paradoxically, recent evidence suggests that peroxiredoxins can also act as DAMPs and PAMPs contributing to sustaining the inflammatory response (Fig. 3.1). Redox signaling is, therefore, a common denominator of many crucial cellular events that occur during the onset of the inflammatory cascade. Elucidating the intriguing contrasting roles of redox signaling during the onset of inflammation could provide valuable knowledge for the understanding and treatment of inflammatory diseases.

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Nuclear factor E2-related factor-2 negatively regulates NLRP3 inflammasome activity by inhibiting reactive oxygen species-induced NLRP3 priming. Antioxid. Redox Signal. 26 (1), 2843. Markert, M., Glass, G.A., Babior, B.M., 1985. Respiratory burst oxidase from human neutrophils: purification and some properties. Proc. Natl. Acad. Sci. U.S.A. 82 (10), 31443148. Martinon, F., Mayor, A., Tschopp, J., 2009. The inflammasomes: guardians of the body. Annu. Rev. Immunol. 27, 229265. Menden, H.L., et al., 2016. Nicotinamide adenine dinucleotide phosphate oxidase 2 regulates LPS-induced inflammation and alveolar remodeling in the developing lung. Am. J. Respir. Cell Mol. Biol. 55 (6), 767778. Mercurio, F., Manning, A.M., 1999. NF-kappaB as a primary regulator of the stress response. Oncogene. 18 (45), 61636171. Mittal, M., et al., 2014. Reactive oxygen species in inflammation and tissue injury. Antioxid. Redox Signal. 20 (7), 11261167. Morgan, M.J., Liu, Z.-G., 2011. 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4 Reactive Oxygen Species, Oxidative Damage and Cell Death Nandini Ghosh, Amitava Das, Scott Chaffee, Sashwati Roy and Chandan K. Sen The Ohio State University Wexner Medical Center, Columbus, OH, United States

4.1. INTRODUCTION Oxygen radicals such as singlet oxygen, superoxide radicals, peroxyl radicals, hydroxyl radicals, etc., and certain non-radical oxidizing agents such as hydrogen peroxide, ozone, hypochlorous acid that can easily be converted into oxygen radicals are collectively known as Reactive Oxygen Species (ROS) (Chekulayeva et al., 2006; Phaniendra et al., 2015). ROS are small and highly reactive molecules that function as “redox messengers” in intracellular signaling and regulation when maintained at proper cellular concentrations (Circu and Aw, 2010). At excess physiological levels, ROS can induce oxidative modification in other oxygen species, proteins, or lipids resulting in cellular damage and cell death (Wu and Cederbaum, 2003). This excess ROS in a physiological system is referred to as oxidative stress (Wu and Cederbaum, 2003). Hence, alteration of ROS levels in the intracellular space has a significant impact on health and disease. ROS could be produced endogenously by normal cellular metabolism and inflammatory process or due to exogenous exposure or ingestion of environmental toxicants. ROS have long been implicated in oxidative damage and in eventual cell death by myriad pathways. The following sections summarize current literature on these topics to provide an overall glimpse of the impact of ROS in biological systems.

4.2. ENDOGENOUS ROS PRODUCTION Production of reactive oxygen species (ROS) is an unavoidable consequence of aerobic metabolism (Sharma et al., 2012). ROS are produced through stepwise reduction of molecular oxygen (O2) by electron-transfer reactions or high-energy exposure (Sharma et al., 2012; del Rio et al., 2006). ROS are also produced as a byproduct of various metabolic pathways or as a consequence of attack by pathogens that may enhance ROS generation due to disruption of cellular homeostasis (Sharma et al., 2012). At high concentrations, ROS are extremely harmful and may lead to cellular damage and death. ROS are mainly produced in cytosol, mitochondria, peroxisomes, microsomes, neutrophils and macrophages. However, other cellular compartments such as endoplasmic reticulum, lysosomes and plasma membrane can also generate ROS (Sharma et al., 2012). ROS production in the cytosol: Intracellular ROS can be produced by several soluble cellular components, such as thiols, hydroquinones, catecholamines, and flavins, as they are able to undergo redox reactions (Freeman and Crapo, 1982). Moreover, catalytic activity of several cytosolic enzymes, such as xanthine oxidase (XO) also contributes to the production of ROS. Xanthine dehydrogenase (XDH) catalyzes the oxidation of hypoxanthine to xanthine; the xanthine is then converted to uric acid in healthy tissues via NADP1 as an electron acceptor. Xanthine dehydrogenase can be converted to xanthine oxidase by reversible sulfhydryl oxidation or by irreversible Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00004-4

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proteolytic modification. Xanthine oxidase plays a crucial role in catabolism of purines in certain species such as humans. Xanthine oxidase then catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid producing H2O2 and transferring electrons to molecular oxygen producing the superoxide radical (McCord et al., 1985). ROS production in mitochondria: Mitochondria, in aerobic cells, play an essential role in maintaining energy homeostasis by controlling oxidative phosphorylation. However, during this process, the leakage of electrons from the electron transport chain in mitochondria leads to the formation of ROS. The univalent auto-oxidation of the electron carriers such as NAD1, FMN, and FAD molecules of the electron transport chain results in the formation of H2O2 by superoxide dismutase (SOD) which subsequently forms hydroxyl radical in the presence of free iron or copper (Fenton reaction, Fig. 4.1). The involvement of the inner mitochondrial membrane carriers in the generation of ROS has thereby linked mitochondria with several diseases and aging (Sharma et al., 2012). Complexes I and III are the main sites involved in mitochondrial ROS production; however, recently it has been reported that Complex II might be involved in ROS generation through succinate dependent and glycerol-3phosphate dependent ROS production as shown in rat tissues (Quinlan et al., 2013). Several oxidoreductases located in mitochondrial membranes, such as monoamine oxidases, dihydroorotate dehydrogenase, α-glycerophosphate dehydrogenase, succinate dehydrogenase, and α-ketoglutarate dehydrogenase complex, have shown to generate ROS (Sharma et al., 2012; Quinlan et al., 2013; Andreyev et al., 2005; Hauptmann et al., 1996). ROS production by peroxisomes: Peroxisomes are small, membrane-enclosed organelles that contain enzymes that are involved in several metabolic pathways, such as α-oxidation, β-oxidation, and ω-oxidation, synthesis of lipid compounds such as etherphospholipid biosynthesis, amino acid and glyoxylate metabolism (Singh, 1997). During these processes, the peroxisomes generate H2O2 that diffuses through the peroxisome membrane to the surrounding medium. The peroxisomes contain high concentrations of catalase to convert H2O2 to H2O. However, high catalase content cannot prevent the release of H2O2 into the cytosol. Peroxisomes also contain xanthine oxidase (Angermuller et al., 1987) that further contribute in the generation of H2O2 which may give rise to hydroxyl radicals in presence of excess free copper and iron ions and low antioxidant levels via the Fenton reaction. Hence, peroxisomes are a potential source of such highly reactive species. ROS production by microsomes: The microsomes are a small (around 20200 nm) heterogeneous set of vesicles formed from the endoplasmic reticulum when the cells are disrupted. The production of reactive oxygen intermediates by human microsomes is a growing field of research. Microsomes are enriched in a variety of cytochrome P450 (CYPs) proteins such as CYP1A1, 1A2, 2B6, and 3A4. Among all the cytochromes, CYP3A4 was the most active P450 in production of superoxide anion radical and catalyzing NADPH oxidation. CYP1A1 was the

FIGURE 4.1 The Fenton reaction is the production of ROS.

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next most reactive CYP, whereas CYP1A2 and 2B6 displayed a comparable, lower activity. CYP3A4 in human liver microsomes contributes significantly to the generation of ROS (Puntarulo and Cederbaum, 1998). ROS production by endoplasmic reticulum: The endoplasmic reticulum (ER) is involved in multiple functions, such as xenobiotic metabolism, synthesis, folding, and transport of fatty acids, calcium storage (Koch, 1990), and lipid metabolism. Xenobiotic metabolism and introduction of double bonds in fatty acids during its synthesis may lead to ROS production. The process of detoxifying xenobiotics and converting them into more water soluble compounds to facilitate their urinary or biliary excretion is known as xenobiotic metabolism. The microsomal cytochrome P450-dependent monooxygenase system (NADPH-cytochrome c reductase) of the endoplasmic reticulum membrane is a major source of H2O2 (Gillette et al., 1957). The ER also facilitates fatty acid desaturation with the help of a multi-enzyme system composed of desaturase, NADH cytochrome b5 reductase, and cytochrome b5 that transfers electrons from NADH cytochrome b5 reductase to the desaturase. Desaturase then introduces carbon-carbon double bonds into fatty acids using one molecule of O2 and forming two molecules of H2O (Gupta et al., 2012). However, during this process NADH cytochrome b5 reductase could leak electrons to molecular oxygen (O2) to make H2O2 (Samhan-Arias and Gutierrez-Merino, 2014). Most of the proteins synthesized in the ER undergo the folding that is facilitated via oxidation of free sulfydryl groups resulting in the incorporation of disulfide bonds. The process of oxidative protein folding is driven by protein disulfide isomerase (PDI), which is a member of the thioredoxin family, endoplasmic reticulum-resident protein (Ero1p), which is tightly associated with FAD moiety and molecular oxygen as the source of oxidizing equivalents. Electrons are transferred from PDI to molecular oxygen by Ero1p in a FAD-dependent reaction, resulting in ER protein folding-induced oxidative stress (Tu and Weissman, 2002). By theoretical calculation, it was estimated that 25% of cellular ROS produced during ER protein synthesis results from thiol oxidation via PDI and Ero1 (Tu and Weissman, 2004). ROS production by plasma membrane: The plasma membrane is composed of phospholipids and can therefore be oxidatively modified by ROS. The plasma membrane plays a major role in cellular processes including cell adhesion, ion conductivity, and cell signaling (Meo et al., 2016). The outer oxidizing environment outside of the plasma membrane makes it a key site for generation of free radicals. The ROS produced by the dysfunctional cells (Skulachev, 1996), cause oxidative damage of membrane components unless efficient antioxidant systems are operative. Oxidation of lipids and structurally important proteins can increase plasma membrane permeability resulting in a decrease in transmembrane ion gradients, loss of secretory functions, and inhibition of cellular metabolic processes (Meo et al., 2016). In addition to lipid oxidation, the metabolism of arachidonic acid (AA) also induces ROS production. AA released from membrane phospholipids is metabolized by membrane associated enzymes lipoxygenase and cyclooxygenase to form products such as prostaglandins, thromboxanes, and leukotrienes that cause production of free radicals and ROS (Cho et al., 2011). However, the main source of ROS are the members of the membrane-bound enzyme NADPH oxidase family, specifically NADPH oxidase 2 in the phagocytic cells that kill bacterial intruders (Babior, 1999). ROS production by lysosomes: Lysosomes are organelles that interact with and degrade macromolecules from the secretory and phagocytic pathways (Sun-Wada et al., 2003). The optimal acidic pH of the lysosome in rat liver membranes is maintained by flavins, ubiquinone, and a b-type cytochrome (Arai et al., 1991) that form a functional electron transport system that support proton accumulation within lysosomes (Gille and Nohl, 2000) to maintain an optimal pH that is required for acidic hydrolases (Sun-Wada et al., 2003). Hans Nohl and Lars Gille, in 2013, discussed that ubiquinone that is associated with this redox chain contributes to unilateral proton distribution via redox-cycling (Nohl and Gille, 2005). This study showed that almost 70% of total lysosomal ubiquinone was in a divalent (reduced) state and that a FAD containing NADH dehydrogenase was involved in the transfer of reducing equivalents from cytosolic NADH to ubiquinone through a b-type cytochrome (Nohl and Gille, 2005). Oxygen was found to be the terminal electron acceptor of this novel lysosome redox chain regulating the acidification of the lysosomal matrix. In this novel proton-pumping redox chain, oxygen was in a trivalent reduced state giving rise to the hydroxyl radicals (  OH) (Nohl and Gille, 2005).

4.3. EXOGENOUS ROS PRODUCTION Apart from the ROS production through the endogenous system, ROS is also produced by external agents including environmental factors such as nutrient deficiency, aerobic exercise, as well as by pollutants (smoke, tobacco) and by UV light and radiation, etc.

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Nutrient deficiency: Numerous dietary nutrients such vitamin C, vitamin E, glutathione, carotenoids, coenzyme Q, flavonoids and certain minerals such as copper, manganese, zinc serve as enzymatic and non-enzymatic antioxidant systems in cells and regulate ROS balance. These nutrients might directly scavenge the singlet oxygen (e.g., β-carotene, lycopene, superoxide dismutase, vitamin C) or remove the hydroxides (catalase, glutathione peroxidases). Some nutrients act as chain-breaking antioxidants by reacting with peroxyl radicals (e.g., vitamin E, coQ10) to terminate the cyclic generation of peroxyl radicals. The deficiency of these nutrients may cause accumulation of free radicals (Krumova and Cosa, 2016). Aerobic exercise: Sjodin et al. (1990), hypothesized that high intensity exercise could lead to generation of potentially harmful free radicals that may potentially lead to a loss of cell viability and to cell necrosis and could initiate the skeletal muscle damage and inflammation (Sjodin et al., 1990). Exercise increases the oxygen consumption 10- to 15-fold above resting levels and this may result in a temporary increase in the rate of production of mitochondrial free radicals that can induce onset of an inflammatory signaling cascade similar to that which occurs with injury or infection (Camus et al., 1994). The exercise-induced tissue injury and phagocytic activation can further contribute to the generation of ROS. In the case of strenuous exercise such as running a marathon, these processes may be more prominent. Lipid and protein oxidation induced by high-intensity exercise have been clearly demonstrated to induce lipid and protein peroxidation (Alessio, 1993; Ji, 1995). However, evidence supporting increased free radical attack on DNA during exercise is still limited. Pollution and UV light: Several in vitro studies have confirmed that benzo[a]pyrene (BaP), a common environmental pollutant and UVA synergistically induce oxidative DNA damage as measured 8-OHdG and H2O2 production (Shyong et al., 2003). Other studies support the hypothesis that interaction of BaP and UVA generates ROS such as H2O2 which further induce oxidative DNA damage, leading to carcinogenesis. K.E. Burke and H. Wei in 2009 provided experimental evidence that BaP can generate reactive oxygen species (ROS) when exposed to UVA radiation by acting as a photosensitizer leading to genetic damage, photo damage and carcinogenesis (Burke and Wei, 2009). Radiation: Ionizing radiation (X-rays, γ-rays) or UV light can cause radiolysis of water to produce ROS. ROS in turn can react with redox active metal ions, thereby inducing oxidative stress (Biaglow et al., 1992; Chiu et al., 1993). Signal transduction molecules are activated, resulting in the expression of radiation responserelated genes (Birben et al., 2012). Tobacco smoking: Cigarette smoke consists of oxidants, free radicals and organic compounds. Upon inhalation of cigarette smoke, some endogenous inflammatory pathways are activated which eventually leads to accumulation of neutrophils and macrophages, which further augments the oxidant-induced injury (Church and Pryor, 1985).

4.4. PHYSIOLOGICAL ROLE OF REACTIVE OXYGEN SPECIES Despite its role in injury and in driving the pathology of several oxidative stress related disease, ROS participate in numerous physiological processes like cell growth, necrosis, apoptosis, protease activities and gene expression (Zuo et al., 2015; Dillard et al., 1978). ROS plays an important role in the maintenance and alteration of cellular processes governing hormone concentration, chemical equilibrium and enzyme activation (Powers and Jackson, 2008). The effects of ROS are evident in the skeletal muscle, the immune system and the nervous system. However, the most beneficial effects of ROS are encountered in the immune system and in wound healing. ROS is a critical player in cellular warfare and helps to combat invading pathogens, thereby governing the immune responses (Bedard and Krause, 2007). Produced by both neutrophils and macrophages, ROS plays a key role in fighting infections associated with wounds (Babior, 2000; Babior et al., 2002). In fact, deficiency of nitric oxide at the wound site during diabetes mellitus is one of the factors which leads to increased wound complications in hyperglycemia (Witte et al., 2002a,b). This dysfunction can be overcome by nutrient supplementation as in the case of use of preparations of fermented papaya (Collard and Roy, 2010; Dickerson et al., 2012). In fact, low levels of oxidants in biological systems are not necessarily deleterious since several oxidants could actually serve as signaling messengers, which in turn regulate cellular signaling (Sen and Roy, 2008).

4.5. OXIDATIVE STRESS At the point when the level of ROS exceeds the endogenous antioxidant defense mechanisms, the cell is said to be in a state of oxidative stress (Sharma et al., 2012). An increased generation of free radicals and/or reduced

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physiological activity of antioxidant defenses against free radicals results in disturbances in a normal redox state within the cell environment. This could lead to damage to all components of the cell. Severe oxidative stress can cause cell death. Classical parameters to characterize oxidative stress in biological samples involve increased levels of radicals and oxidants, decreased levels of antioxidants both lipid soluble and small molecular water soluble ones, imbalance in the cellular redox system, and oxidative damage to cellular components such as lipids, proteins and DNA.

4.6. OXIDATIVE DAMAGE IS OUTCOME OF OXIDATIVE STRESS Enhanced ROS production during oxidative stress leads to oxidation of proteins, lipid peroxidation, enzyme inhibition and DNA damage, all of which can collectively activate pathways that are programmed to lead to cell death. Protein oxidation: High levels of ROS may lead to the direct or indirect modification of the proteins compromising its activity. Modification of proteins can lead to protein unfolding and altered conformation, altered interactions with biological partners and modified turnover (Davies, 2016). Proteins can be modified directly through nitrosylation, carbonylation, disulfide bond formation, and glutathionylation or indirectly by conjugation with breakdown products of fatty acid peroxidation (Yamauchi et al., 2008). High levels of ROS can also lead to protein fragmentation by oxidation of amino acid residue side chains, oxidation of the protein backbone (Berlett and Stadtman, 1997), and formation of protein-protein cross-linkages. In tissues with high oxidative stress, high levels of carbonylated proteins are used as markers for protein oxidation (Moller and Kristensen, 2004). High levels of carbonylated proteins have been observed in case of various diseases such as rheumatoid arthritis, Alzheimer’s disease, diabetes, sepsis and chronic renal failure (Dalle-Donne et al., 2003). Different amino acids have varied susceptibility to ROS attack; for example, thiol groups, sulfur containing amino acids and methionine residues are highly susceptible to oxidation (Kelly and Mudway, 2003). Conformational changes, protein unfolding and degradation can results from sulfhydryl groups or methionine residues of proteins oxidation (Dean et al., 1985). Enzymes containing metals may undergo metal catalyzed oxidation that could lead to enzyme inactivation (Stadtman, 1990). Lipids: When ROS levels are high, i.e., beyond the cellular (homeostatic) threshold, it can induce lipid peroxidation in both cellular and organellar membranes that may inactivate membrane-bound receptors and enzymes, affecting normal cellular functioning (Girotti, 1985). The metabolism of malondialdehyde (MDA), a lipid peroxidation product of unsaturated fatty acids, leads to cell membrane damage and inactivation of many cellular proteins by forming protein cross-linkages (Siu and Draper, 1982). Oxidative stress is further aggravated by the production of lipid-derived radicals that themselves can react with and damage proteins and DNA. Under stressful conditions, lipid peroxidation levels correlate with ROS-mediated cellular damage (Halliwell and Gutteridge, 2007). The unsaturated (double) bond on the phospholipid molecules and the ester linkage between glycerol and the fatty acid serve as the common sites for ROS-induced oxidation. Hence, the polyunsaturated fatty acids (PUFAs) of the phospholipids membranes are particularly sensitive to oxidation by ROS. An imbalance in the antioxidant and oxidant ratio could lead to peroxidation of many polyunsaturated fatty acids, leading a cyclical chain reaction. DNA: Excess ROS can cause oxidative damages to both the sugar and base moieties of DNA resulting in deoxyribose oxidation, strand breakage, a variety of modifications in the organic bases of the nucleotides yielding subsequent mutations, and DNA protein crosslinks. DNA damage may cause alterations in the encoded proteins resulting in malfunctions or complete inactivation of the proteins encoded in these genes. Sugar damage mainly results from hydrogen abstraction from deoxyribose while oxidative attack to DNA bases generally involves  OH addition to double bonds (Sharma et al., 2012).  OH generates various products from the DNA bases which mainly include C-8 hydroxylation of guanine. Hydroxyguanine is the most commonly observed product. DNA protein crosslinks are formed when  OH attacks on either DNA or proteins associated with it (Sharma et al., 2012). If replication or transcription precedes repair, DNA protein crosslinks may be lethal. Lack of protective protein, histones, and close locations to the ROS producing systems is the reason for mitochondrial and chloroplast DNA to be more susceptible to oxidative damage (Sharma et al., 2012). Excess ROS may lead to permanent damage to the DNA despite DNA repair system.

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4.7. CELL DEATH The most common forms of cell death are apoptosis, necrosis and autophagy. However several novel mechanisms for cell death in vivo have been identified and characterized in the last few years (Fig. 4.2). Apoptosis, necrosis and autophagy: There are two distinct apoptotic pathways that cells progress through when exposed to excess levels of ROS, and those pathways are known as the intrinsic and extrinsic pathways (Ozben, 2007). The extrinsic pathway is a Fas activation pathway. The Fas receptor is a “death” receptor expressed on the surface of cells. Fas activation has been associated with ROS generation. Fas activation leads to the recruitment of Fas-associated protein along with caspase 8 and death domain, which initiates the apoptosis process (Gupta et al., 2012). The intrinsic pathway, also known as the caspase cascade, is due to the release of cytochrome c via ROS, often as a result of mitochondrial damage. Cytochrome c is released by activation of pore-stabilizing proteins (Bcl-2 and Bcl-xL) and by deactivation of pore-destabilizing proteins, (Bcl-2-associated X protein, Bcl-2 homologous antagonist/killer) (Martindale and Holbrook, 2002). Pro-apoptotic proteins are released as a result of any DNA damage or oxidative stress (Maiuri et al., 2007). Mitochondrial damage also leads cells to progress towards apoptosis, making mitochondria a suitable target for therapies such as cancer treatment (Fulda et al., 2010). While excessive ROS drives cell towards apoptosis, it may also facilitate necrosis, which is a pathway of uncontrolled cell death (Hampton and Orrenius, 1997). In the presence of an inflammatory environment, in the form of chemokines such as TNF-α, ROS accumulates and ultimately cause the cells to swell and eventually leak their contents. Besides apoptosis and necrosis, recent research has also shown a relationship between ROS levels and autophagy, which is another source of cell destruction (Gibson, 2010). Excessive ROS is a critical factor of both autophagy and apoptosis, as the accumulation ultimately results in irreversible DNA damage. Autophagy is a self-catabolic physiological process that leads to the degradation of any damaged cell organelles or proteins via lysosomes (Shrivastava et al., 2011). When cells are faced with any sort of oxidative stress and excessive ROS, cells activate autophagy as a means for degradation and disposal of harmful cell organelles without necessarily leading the cells to apoptotic pathways (Scherz-Shouval and Elazar, 2007). When cells digest too much of their own organelles, this leads to an autophagic cell that may lose the ability to regulate autophagic genes in the future (Xie and Klionsky, 2007). Cell death via autophagy is incompletely understood, but as more information emerges, signaling molecules of the autophagy pathway can serve as potential therapeutic targets.

Autophagy: ROS acts as a signal transducer and irreversibly oxidizes DNA and other critical cellular biomarkers

Apoptosis: Induced in inflammatory cells, i.e. neutrophils, macrophages, and eosinophils, leading to damaged DNA and protein

Cornification: Small proline-rich proteins (SPRR) in cell envelope are responsible for ROS quenching, preventing damage to the skin

Entosis: Accumulated ROS in vacuoles can trigger cells to recruit autophagy proteins necessary for cell death

Anoikis: ROS serves as a cell adhesion mediator that allows cells to avoid programmed death

Cell death

Paraptosis: As a result of mitochondrial Ca2+ influx, ROS production leads to mitochondrial membrane loss of function

Pyronecrosis: ROS stimulates inflammasome production, which downstream disrupts the cell ion gradient leading to cell lysis

Mitotic catastrophe: Triggered by oxidative stress, ROS enhances p53 function and programmed cell death

Wallerian degeneration: Neuronal degradation as a result of an accumulation of ROS in absence of Sarm1

Necrosis: Initiated via TNF, causes swelling of cells and eventual leakage of contents

Excitotoxicity: leads to lethal influx of Ca2+, resulting in excessive ROS production and cell death

Pyroptosis: ROS leads to inflammsome complex production, which causes the production of inflammatory cytokines and further cell damage

FIGURE 4.2 A summary of the myriad cell death pathways that are activated by ROS and oxidative stress.

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As excessive ROS regulates these pathways of cell death, it is clear that the apoptosis, necrosis and autophagy are not mutually exclusive (Scherz-Shouval and Elazar, 2007). Indeed autophagy is activated with depolarization of the mitochondrial membranes that also facilitates apoptosis. Crosstalk between these processes could provide potential targets for therapies, especially cancer. Cornification: Cornification is a term used to describe a type of programmed cell death, occurring in the epidermis in keratinocytes, termed corneocytes, which contain a combination of proteins, i.e., keratine, loricrin, and lipids that are necessary for the barrier function of the epidermal layer, including elasticity and resistance. Small proline-rich proteins (SPRR) are present in the cell envelope, and they are critical for ROS quenching, which prevents further damage to the skin (Vermeij and Backendorf, 2010). Cornification or keratinization is thus a model of cell death in the skin whereby a cornified envelope replaces the plasma membrane of differentiating keratinocytes (Candi et al., 2005). Cornification involves the activation of specific caspases, which is a hallmark of programmed cell death. New insights into the molecular mechanisms of cornification are increasing our understanding of this unique form of programmed cell death, which is associated with barrier malfunctions (Kroemer et al., 2005). Mitotic catastrophe: Mitotic catastrophe is a mechanism of cell death that is employed as a result of an error in the mitosis process, i.e., uneven distribution of chromosomes (Vitale et al., 2011). This process is known to precede the other cell death pathways (Castedo et al., 2004). As a result of the dysregulation of the cell cycle, cells may experience morphological changes, i.e., micronucleation and multinucleation; however, to this point, there is no definitive lexicon to describe these changes (Castedo et al., 2004; Roninson et al., 2001; Okada and Mak, 2004). Mitotic catastrophe ultimately leads to two cell death pathways, apoptosis and necrosis (Vakifahmetoglu et al., 2008). Due to the ambiguity when describing the errors in the mitotic catastrophe process, the NCCD (Nomenclature Committee on Cell Death) endorses the use of specific phrases to indicate the step of mitosis where the error occurs, i.e., “cell death occurring during metaphase” (Kroemer et al., 2005). ROS is produced in the presence of oxidative stress, which aids p53 function and directs the cells towards programmed cell death (Hung et al., 2013). Anoikis: Anoikis is a process of cell death that results from attachment-dependent cells losing contact with the surrounding extracellular matrix (Gilmore, 2005). Anoikis relates to a very specific mechanism of cell death; however, much of the pathways are common with classical apoptosis (Grossmann, 2002). It is becoming difficult to find the clear definition of anoikis as it continues to be discussed in literature; nonetheless, NCCD suggests the name based on its historical significance. Anoikis essentially refers to a variety of processes that occur in cells when detachment is lost, allowing for progress into apoptotic pathways. ROS acts as a critical cell adhesion mediator, regulating which cells are destined for a programmed cell death (Giannoni et al., 2008). It is critical to understand whether different processes of cell death are also involved when attachment-dependent cells lose contact, such as pathways that are involved with caspase inhibitors and other uncontrolled cell death scenarios (Kroemer et al., 2005). Excitotoxicity: Excitotoxicity is a type of cell death occurring with damage and death of neurons. Specifically, overactivation of glutamate leads to an accumulation of intracellular calcium, which causes the opening of the N-methyl-D-aspartate Ca21 permeable channel. This drives an increase in cytosolic calcium. The accumulation of excess calcium in the cytosol leads to an accumulation of excess ROS, ultimately leading to the activation of lethal signaling pathways (Orrenius et al., 2003; Siegel et al., 1999). Excitotoxicity shares common mechanisms with traditional pathways of cell death such as apoptosis and necrosis. Glutamate activation leads to release of the matrix metalloproteinase-9 (MMP-9), a protease that promotes neuronal cell death. However due to the involvement of other cellular regulators, i.e., nitric oxide, the process of excitotoxicity is not to be considered a distinct cell death modality (Melino et al., 1997). Wallerian degeneration: Wallerian degeneration is an example of the various types of cellular degradation processes that occur in the nervous system. This process involves the degeneration of axon distal to the cell, without any damage to the cell body (Luo and O’Leary, 2005; Raff et al., 2002). Wallerian degeneration is not traditionally considered a pathway of cell death as the cells are able to remain alive (Raff et al., 2002). The axons are able to regenerate, which may affect some of the events surrounding Wallerian degeneration (Rotshenker, 2011). Paraptosis: Paraptosis differs from classical apoptosis and necrosis (Sperandio et al., 2000) in that it involves discrete pathways such as that initiated by insulin-like growth factor receptor I. Phases of paraptosis involve both cytoplasmic vacuolization and the swelling of mitochondria (Sperandio et al., 2000). Mitochondrial dysfunction and Ca21 overload has been reported to contribute to hesperidin-induced paraptosis (Yumnam et al., 2016). Paraptosis does not share any additional characteristics of apoptosis, as caspase inhibitors or the presence of antiapoptotic proteins, i.e., Bcl-2-like, were found to be ineffective in the prevention of this pathway (Sperandio et al., 2000, 2004).

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Pyroptosis: Pyroptosis is an inflammatory-driven, caspase-1 dependent programmed cell death that is often caused by the invasion of a pathogen or various other pathological stimuli. The first literature involving pyroptosis involved macrophages that were invaded with Salmonella typhimurium (Brennan and Cookson, 2000). Activation of caspase-1 is a result of pathogen invasion, i.e., Pseuodomonas aeruginosa, Shigella flexneri, and its activation of the NOD-like receptor protein (NLRP) inflammasome pathway (Fink et al., 2008). Pyroptosis may also be activated (as in the case of Bacillus anthracis) via other NLR proteins, such as Nalp1 (Fink et al., 2008). Pathogens are capable of developing mechanisms to prevent the onset of pyroptosis. In another scenario, macrophages stimulated with lipopolysaccharide (LPS), despite the presence or absence of available ATP, are driven towards pyroptosis with ASC protein (an adaptor protein for the inflammasome activation) as a mediator (Fernandes-Alnemri et al., 2007). ASC forms a complex known as pyroptosome, a supramolecular cytoplasmic complex, by pairing with caspase-1 (Fernandes-Alnemri et al., 2007). These examples serve to show there are multiple pathways that lead to pyroptosis. The inflammasome complex is reportedly generated by the presence of ROS (Harijith et al., 2014). This complex leads to the eventual release of IL-1β, a major proinflammatory cytokine, as well as IL-18 (Martinon and Tschopp, 2007). The morphological characteristics observed of macrophages undergoing pyroptosis show similarities with both classical apoptosis, as well as necrosis cell death (Labbe and Saleh, 2008). Pyronecrosis: A relatively newly discovered form of cell death, pyronecrosis shares morphological similarities to necrotic cell death. Pyronecrosis requires NLRP3 and ASC (the inflammasome complex), as well as lysosomal protease cathepsin B (Willingham et al., 2007). Pyronecrosis is independent of caspase-1, yet is associated with a proinflammatory environment, particularly IL-1β. Pyronecrosis is involved with monocytic cells infected by S. flexneri along with the release of the cytokine and damage associated molecular pattern protein HMGB1 (high mobility group box-1) (Willingham et al., 2007). These pathways are still under investigation (Martinon et al., 2007) but ROS is known to facilitate the formation of inflammasome complex (Amaral et al., 2016). Entosis: Entosis is a non-apoptotic process of cell death that involves the invasion of one cell by a neighboring cell. This term originated from a study involving Huntington’s disease whereby lymphoblasts were undergoing cellular cannibalism (Kroemer et al., 2005). The process of entosis entails a cell integrating itself into a neighboring host cell, and then programming itself to die within the phagosome (Overholtzer et al., 2007). An accumulation of ROS in the cell’s vacuoles allows for the recruitment of autophagic proteins necessary for cell death (Liu and Levine, 2015). The cells involved in entosis do not necessarily resemble those undergoing apoptosis and autophagy, as represented by MCF-7 cells, breast cancer cells, both of which lack caspase-3 and beclin-1, which are important in apoptosis and autophagy (Overholtzer et al., 2007). The process of entosis seems to be provoked by the detachment of cells from their extracellular matrix surroundings. Entosis does not appear to be inhibited by antiapoptotic proteins, i.e., Bcl-2 or Z-VAD-fmk as well. The cell that invades the neighboring host appears normal after ingestion, nevertheless studies indicate their eventual disappearance as a result of lysosomal degradation. Studies have shown the ingested cells retain the capability of cell division within their host (Overholtzer et al., 2007). However, there is a paucity of data on entosis and its characterization, so that it is not clear whether it is completely independent from other modes of cell death (Kroemer et al., 2005; Doukoumetzidis and Hengartner, 2008).

4.8. CONCLUSIONS The complex biology of ROS-induced signaling arises due to its multiple cellular and molecular targets, each of which triggers signals that have both physiological and patho-physiological ramifications. Several of these pathways, such as those of apoptosis, necrosis and autophagy, as well as the inflammasome-related signaling intersect and may provide potential targets to accelerate or block cell death.

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5 Mitochondrial ROS and T Cell Activation Karthik B. Mallilankaraman National University of Singapore, Singapore, Singapore

5.1. INTRODUCTION Stimulation of the T cell receptor (TCR) by foreign antigens through antigen-presenting cells (APCs) drives the T cells into rapid proliferation and differentiation, wherein naive T cells expand clonally and differentiate to become effector T cells. Traditionally, TCR stimulation results in PLC-γ1 induction that causes T cells to proliferate and differentiate via Inositol 3,4,5-triphosphate (IP3) mediated rise in cytosolic Ca21 and downstream activation of Ca21-dependent transcription factors. These transcription factors are nuclear factors of activated T cells (NFAT), NF-κB and AP-1 and are well established to control the T cell activation-induced gene expression. While NFAT is predominantly Ca21 dependent, NF-κB and AP-1 are promiscuous and can be activated by low physiological levels of reactive oxygen species (ROS) that is generated transiently during the T cell activation process (Droge, 2002; Kaminski et al., 2010, 2012) Thus, oxidative signal can be considered to be indispensable for T cell activation (Droge, 2002; Kaminski et al., 2010; Devadas et al., 2002). Together with the Ca21 influx, low levels of ROS constitutes the nominal requirement for activation-induced expression of genes such as interleukin 2 (IL-2), a major autocrine factor for T cell proliferation (Meuer et al., 1984), IL-4 and CD95L (Kaminski et al., 2007, 2010; Devadas et al., 2002; Gulow et al., 2005). During T cell activation, ROS is produced from different sources such as NADPH oxidases (NOX2, DUOX2), lipoxygenases (Jackson et al., 2004; Kwon et al., 2010; Los et al., 1995), and the mitochondrial respiratory chain (Kaminski et al., 2007, 2010, 2012; Yi et al., 2006). T cell activation is a highly energy demanding process, where mitochondrial ATP production plays a pivotal role. To meet the high energy demand during the activation process, T cells increase their mitochondrial biogenesis and respiratory chain activity (D’Souza et al., 2007). Increased mitochondrial respiration results in the rapid increase in mitochondrial ROS (Grayson et al., 2003). Activation of T cells is immediately followed by clonal expansion through rapid proliferation and differentiation. While physiological levels of ROS play an important role in T cell activation, uncontrolled high-levels of ROS affect the clonal expansion and result in removal of activated cells (Hildeman et al., 1999). A fine balance between these two events of activation and expansion requires optimal levels of ROS. ROS also drives apoptosis in activated T cells either via activation-induced cell death (AICD) or activated cell autonomous death (ACAD) (Meuer et al., 1984; D’Souza et al., 2007; Hildeman et al., 1999; Brenner et al., 2008; Stranges et al., 2007). Apoptosis is facilitated by the AICD mechanism triggered predominantly by Fas-L which itself is induced by ROS (Stranges et al., 2007). Activation caused autonomous death (ACAD) also mediates apoptosis (Hildeman et al., 1999) but this is via pro- and anti-apoptotic members of Bcl-2 family proteins (Hildeman et al., 2003b). The ROS driven apoptosis serves to regulate the number of activated T cells. Interestingly, ROS have been implicated in down regulation of Bcl-2 in activated T cells (Hildeman et al., 2003a). Additionally, the increase in mitochondrial content during early activation could contribute to apoptosis through cytochrome c release via Ca21 and ROS-dependent mitochondrial permeability transition pore opening (Crompton, 1999). Thus, alteration in the status of mitochondria and ROS could transform the activation, kinetics and population of activated T cell and thereby modulate T cell homeostasis to regulate the adaptive immune response. This chapter will focus on the sources and targets of ROS, specifically on the oxidants of mitochondrial origin and the mechanisms of oxidant-induced effects on T cell activation.

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5.2. REACTIVE OXYGEN SPECIES ROS are small short-lived molecules derived from oxygen (O2) that are chemically highly reactive and readily oxidize other molecules in vicinity. The extreme reactivity is contributed by the unpaired electrons (radicals). Hydrogen peroxide (H2O2), superoxide (O22  ), hydroxyl radical (OH  ), hypochlorous acid (HOCl), lipid peroxides (ROOH), singlet oxygen (1O2), and ozone (O3) are the common forms of ROS (Winterbourn, 2008). Superoxide and hydrogen peroxide are the most important forms of ROS involved in the regulation of many biological processes. Most of the forms of intracellular ROS are derived primarily from O22  . O22  once produced, rapidly react with surrounding molecules or get converted to H2O2 by superoxide dismutases (SODs) (Miller, 2012). Compared to O22  , H2O2 is more stable, less reactive, can diffuse rapidly in the cellular microenvironment, and has the ability to cross cell membranes. ROS are of exogenous or endogenous origin; exogenous sources of ROS, includes ultraviolet and gamma radiation, air pollutants such as smoke, chemicals such as hydrogen peroxide as well as several drugs. Endogenous sources of ROS are generated from enzymes often compartmentalized in mitochondria, plasma membrane, phagosomes, endoplasmic reticulum, peroxisomes, and cytosol. Among these regulatory ROS generators -NADPH oxidases or NOX enzymes and the mitochondrial ROS are the major contributors of cellular superoxide and hydrogen peroxide. NOX-derived ROS is mostly involved in ROS-dependent pathogen killing and is expressed highly in phagocytic cells such as macrophages, neutrophils and dendritic cells, and in low levels in B cells, NK cells, mast cells and eosinophils (Babior, 1984). In these cell types, ROS are produced by the phagocytic NADPH oxidase (PHOX), an enzyme consisting of several subunits (Quinn and Gauss, 2004). NOX-2 (gp91phox) the extensively studied isoform, and is expressed at either the plasma or phagosomes’ membrane. Interestingly, six homologs of gp91phox (NOX-2) have been identified in different tissues: NOX-1, NOX-3, NOX-4, NOX-5, dual oxidase 1 (DUOX-1), and DUOX-2 (Lambeth, 2004; Bedard and Krause, 2007). Activation of cellular receptors by several ligands such as tumor necrosis factor (TNF-α), platelet derived growth factor (PDGF), granulocyte macrophage colony stimulating factor (GM-CSF), angiotensin, insulin, chemokines that bind G protein-coupled receptors, complement component 5a (C5a), lysophospholipids, and leukotriene B4, as well as by cell adhesion triggers the NOX production (Nathan and Cunningham-Bussel, 2013). Because of the widespread expression of NOX and DUOX isoforms across organelles, different cell types, and organisms, NOX enzymes have traditionally been considered to be the major sources of ROS that drive myriad cellular signaling (Brown and Griendling, 2009). However, work over the past decade or more shows that ROS produced by the mitochondrial electron transport chain (ETC) or mitochondrial metabolic enzymes also mediates a wide range of signal transduction (Hamanaka and Chandel, 2010; Sandalio et al., 2013). It has to be noted that the physiological low levels of mitochondrial ROS are important for signal transduction and are much different from that of the signaling cascades activated during oxidative stress. Since the specificity and functional role of mROS is dependent on its levels, stringent regulation of mROS is crucial for its proper function in cellular signaling. The physiological low levels of mitochondrial ROS are kept in balance by antioxidant systems in the cell.

5.3. SOURCES OF MITOCHONDRIAL ROS Mitochondria form the major source of endogenous ROS and a significant amount of mitochondria ROS (mROS) is generated by its aerobic respiration (Kowaltowski et al., 2009; Finkel, 2011, 2012). Mitochondrial inner membrane harbors the respiratory chain complexes, which transfer electrons (received from reducing equivalents coming of the Krebs cycle such as NADH and succinate), across the complexes to the final electron acceptor, the molecular oxygen (O2). O2 is reduced to H2O upon receiving four electrons from the electron transport chain (ETC). Nonetheless, the respiratory chain is not perfectly ideal, and sometimes electron slippage occurs from the ETC, reduces the molecular O2 that is readily available in the vicinity and reduces to superoxide (O2 2  ) or Hydrogen peroxide (H2O2). Traditionally, Complex I (NADH dehydrogenase) and complex III (ubiquinone-cytochrome c reductase) are the major sites for mitochondrial O2 2  production that carries out various cellular signaling functions (Drose and Brandt, 2012; Lambert and Brand, 2009). In recent years many other sources of mitochondrial ROS have been identified making a total of 11 different sources of mROS (Mailloux, 2015). Each source of mROS has a different redox potential at which ROS is produced. Based on this, Brand and colleagues recently suggested a classification of mROS producing enzymes broadly into two different isopotential subgroups namely (1) NADH/NAD isopotential group and (2) QH2/Q isopotential group (Quinlan et al., 2013, 2014).

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The NADH/NAD isopotential group consists of four enzymes: Complex I (NADH dehydrogenase), 2-oxoglutarate dehydrogenase (Odh), Pyruvate dehydrogenase (PDH), and Branched-chain oxo-acid dehydrogenase phosphatase (Bckdh). The QH2/Q isopotential group is made up of seven enzymes; Complex III (ubiquinonecytochrome c reductase), Succinate dehydrogenase (Complex II), Electron-transfer flavoprotein:ubiquinone oxidoreductase (ETFQO), proline dehydrogenase, dihydroorotate dehydrogenase, Succinate-coenzyme Q reductase (SQR), and sn-glycerol-3-phosphate dehydrogenase (sn-G3PDH). The ROS production in the NADH/NAD isopotential group is dependent on the concentration of NADH, whereas in the QH2/Q isopotential group it is subject to reduction of Q to QH2. Notably, in the QH2/Q group the major sources of ROS are attributed to reverse electron transport (RET) as they arise from Complex II, and III (Quinlan et al., 2013, 2014; Goncalves et al., 2014).

5.4. REGULATION OF mROS Prolonged or excessive ROS production can lead to the impairment of cellular functions, cell death, senescence, or malignant transformation (Mailloux, 2015; Finkel and Holbrook, 2000; Reuter et al., 2010). The rates of mROS production from the different enzymatic sources are highly dependent on nutrient availability, mitochondrial redox status, and availability of ADP. Since the magnitude of mROS dictates its specificity and functional consequences, strict regulatory mechanisms are essential to harness and employ in specific cellular signaling. Thus the mROS signaling is controlled by several regulatory mechanisms. Antioxidants form the first and foremost regulatory component in mROS regulation. While low levels of ROS are of physiologic significance, excessive mROS could potentially cause damaging effects. In order to avoid the damages caused by excess ROS, mitochondria harbor a number of antioxidants. Dismutases such as SOD2, which rapidly and spontaneously dismutate superoxide to hydrogen peroxide, form the major pool of antioxidants. Other scavenging enzymes such as peroxiredoxins (PRX) undergo oxidation in the presence of H2O2 at an active cysteine site and then subsequently reduced by thioredoxin, thioredoxin reductase and NADPH. Of the six mammalian peroxiredoxin isoforms, PRX 3 and 5 are expressed in mitochondria. Although the activities of intracellular antioxidants and ROS scavengers determine the ROS levels, the magnitude of mROS generated also rely on mitochondrial bioenergetics. Specifically, mitochondrial membrane potential could modulate the ROS production wherein the increased membrane potential (Δψm) is believed to favor the production of ROS, whereas mitochondrial uncoupling agents that dissipate Δψm lower the mROS production. Furthermore, defects in components of the ETC that lead to disruption of electron flow could also modulate the ROS production. In addition, most of these enzymes that participate in ROS production have thiol residues that are close to or adjacent to ROS producing centers which indicates that the redox signaling may mechanistically control the ROS production (Mailloux et al., 2014). For instance, 2-oxoglutarate dehydrogenase produces both superoxide and hydrogen peroxide at the same time and these ROS deactivate the enzyme and thus ROS production by a negative feedback mechanism (Tretter and Adam-Vizi, 1999; Starkov et al., 2004). Other factors such as mitochondrial fission and fusion and assembly of Krebs cycle enzymes and respiratory complexes also could influence ROS production (Yu et al., 2006; Winge, 2012).

5.5. TARGETS OF mROS ROS can interact with a wide range of macromolecules and modulate its function. Increasing evidence suggests that ROS can cause reversible post-translational modifications in mitochondrial proteins and thereby regulates many signaling pathways. H2O2 is a highly stable ROS and acts as the major signaling messenger that has the ability to traverse across cellular or organelle membranes (Quinlan et al., 2012). H2O2 reacts preferentially with thiol groups (SH) on cysteine residues to form sulphenic acid by a process called sulfenylation (Ilbert and Bonnefoy, 2013). Sulfenylation may lead to further post-translational modifications, such as glutathionylation when sulphenic acid reacts with GSH, disulfide bond formation when sulphenic acid reacts with adjacent thiols, and sulfinilation when sulphenic acid reacts with amides to form sulphenyl amides (Lagoutte et al., 2010; Watt et al., 2010). Sulfenylation and the subsequent modifications can bring changes in the protein conformation, either leading to the activation or inactivation of the catalytic center or confer other functional alterations of the sulfenylated protein, thus altering its role in the signaling pathway. Interestingly, sulfenylation could be reversed by glutothioredoxins and thioredoxins (Shabalina et al., 2014; Harel et al., 2014). Multiple classes of proteins have been

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shown to be regulated by sulfenylation, including phosphatases and kinases (such as PTP1b, PTEN, and MAPK), transcription factors and histone deacetylases, antioxidant enzymes and heat-shock proteins, proteases and hydrolases, ion channels and pumps, adaptor molecules and cytoskeleton components (Goncalves et al., 2014; Lagoutte et al., 2010; Watt et al., 2010; Lane, 2014).

5.6. MITOCHONDRIAL ROS IN T CELL ACTIVATION Mitochondrial ROS are gradually gaining recognition as important signaling mediators in wide range of cellular processes, such as metabolic adaptation, adaptive responses to hypoxia, cellular differentiation, autophagy and regulation of innate or adaptive immunity (Finkel, 2012; Sena and Chandel, 2012). More recently, the importance of mitochondria and mitochondrial-derived free radicals in T cell physiology is being recognized. Mitochondrial metabolism plays a crucial role in determining the fate of T cell differentiation into memory and regulatory phenotypes (Gerriets and Rathmell, 2012; Wang and Green, 2012). Several studies in the last decade have implicated the importance of mitochondria in the immunological synapse, where mitochondria centrally act as calcium buffers, thus determining the amplitude and duration of Ca21 signals and also provide a strong ATP gradient for TCR-mediated phosphorylation events (Hoth et al., 1997; Baixauli et al., 2011; Quintana et al., 2006, 2007). This buffering of Ca21 occurs through the uniporter channel and is dependent on intact mitochondrial membrane potential (Mallilankaraman et al., 2012a,b). Hence a defect in mitochondrial membrane potential either due to mtDNA depletion or mitochondrial uncoupling will result in dampened TCR-induced Ca21 signal due to a reduction in the ability to buffer and prolong Ca21 influx (Hoth et al., 2000; Koziel et al., 2006). T cell activation-induced mitochondrial Ca21 uptake is also known to stimulate mitochondrial function by activating the Ca21 dependent enzymes of the TCA (tri-carboxylic acid) cycle (Carafoli, 2012). The initial phase of T cell activation triggers the proliferation of naive T cells. This process is associated with an increase in mitochondrial DNA (mtDNA) content, mitochondrial mass and oxidative phosphorylation (OXPHOS) activity (D’Souza et al., 2007; Darzynkiewicz et al., 1981; Frauwirth and Thompson, 2004). The rapid increase in OXPHOS activity leads to increased mROS production, which in turn drives T cell activation-induced ROS production (Kaminski et al., 2007, 2010; Yi et al., 2006; Nagy et al., 2003). The receptor on T cells, the TCR and the costimulatory molecule CD28, when activated, lead to mitochondrial hyperpolarization as well as ROS overproduction. T cell activation-induced mROS production induces chemokines, CD95L and antioxidant genes via the activation of transcription factors NF-κB- and AP-1 (Nagy et al., 2003). Furthermore, TCR -induced mROS is known to participate in AICD and ACAD. The requirement of intact mitochondria for T cell activation-induced ROS production was revealed using mtDNA-depleted Jurkat T cells (ps-ρ0 phenotype), and in mtDNA-depleted human T cells (by prolonged ciprofloxacin exposure) (Kaminski et al., 2007, 2010). Cells devoid of mtDNA showed diminished levels of activation-induced ROS, which resulted in decreased expression of IL-2, IL-4 and CD95L. In addition the mtDNA-less cells were protected from CD95L-dependent AICD. Several in vitro studies have implicated the requirement of mROS for T cell activation and subsequent proliferation and clonal expansion; however these findings have not been confirmed in in vivo model systems. Although there are 11 known sources of mROS, Complex I and III seem to be the main sources that contribute in T cell activation and associated signaling. Complex I releases ROS towards the matrix while the Complex III releases ROS towards the intermembranous spaces. Several studies have shown that inhibition of Complex I (with rotenone, a potent inhibitor of complex I) blocks T cell activation-induced mROS production (Kaminski et al., 2007, 2010, 2012; Yi et al., 2006) and subsequent IL-2, IL-4 and CD95L gene expression (Kaminski et al., 2007, 2010; Bauer et al., 1998). Because rotenone also interferes with centrosomal function and tubulin assembly (Brinkley et al., 1974; Diaz-Corrales et al., 2005), other inhibitors of Complex I (piericidin A and metformin (an anti-diabetic and mild complex I blocker)) have also been used and these too conformed the role of ROS released via Complex I in T cell activation (El-Mir et al., 2000; Horgan et al., 1968). Studies were also carried out using knock-down of an essential complex I assembly factor NDUFAF1 (Vogel et al., 2005). These revealed that NDUFAF1 abrogated T-cell activation-induced ROS generation. Superoxide generated at Complex I is converted into H2O2 by matrix localized SODs (MnSOD, SOD2) (Kaminski et al., 2007, 2010). H2O2 can cross mitochondrial membranes and diffuse into the cytoplasm to participate in oxidative signal transduction. T Cell activation leads to an increased superoxide levels in the mitochondrial matrix (Kaminski et al., 2012; Sena and Chandel, 2012). The increased superoxide could potentially trigger MnSOD, a major matrix antioxidant (Kaminski et al., 2012;

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Kiessling et al., 2010). Interestingly, MnSOD content and activity was shown to be upregulated with T cell activation. MnSOD, upregulated during the late phase of a TCR-induced response, is considered a critical regulator of oxidative signal generation (Kaminski et al., 2012). Thus, intact, functional respiratory complex I is indispensable for the mitochondrial ROS production during T cell activation. Intact respiratory complex III is shown to be required for antigen-specific (TCR- and CD28-mediated) T cell activation-induced mitochondrial ROS generation. T cell specific conditional knockout of complex III subunit RISP, showed impairment in CD41 and CD81 T-cell responses. In this study it was suggested that complex IIIderived mitochondrial ROS release to be dependent on Tcell activation-induced mitochondrial Ca21 uptake and up-regulation of the Krebs cycle. Further, the complex III-derived ROS was suggested to enhance the activation of NFAT and Ca21-dependent transcription. Thus complex III mediated mROS production is crucial for T-cell activation (Kaminski et al., 2012; Sena and Chandel, 2012). Inhibition of both Complex III (inhibitor: antimycin A) and Complex IV (inhibitor: NaN3) potentiates TCR-induced mROS production. T cell activation is followed by a metabolic switch from mitochondrial ATP dependent to aerobic glycolysis similar to that of the Warburg effect observed in cancer cells (Wang et al., 1976; Warburg, 1956; Vander Heiden et al., 2009). Glucose influx is critical for the glycolytic phase of the activation process to potentiate TCR-triggered transcription (Jacobs et al., 2008; Stentz and Kitabchi, 2005). Interestingly, in in vitro expanded human T cells, TCRinduced mROS production is complemented by a metabolic switch closely resembling the Warburg effect. Glucose uptake and cellular ATP concentration rise while mitochondrial respiration coupled ATP production declines. Mitochondria display cristae rearrangements closely reminiscent of ROS release and low respiratory activity (Arismendi-Morillo, 2011). This immediate metabolic change and redirection of glycolytic flow due to the induction of ATP-independent phospho-enol pyruvate phosphatase activity leads to activation of the mitochondrial glycerol3-phosphate shuttle via the inner membrane localized GPD2 (glycerol-3-phosphate dehydrogenase 2) (Kaminski et al., 2012; Vander Heiden et al., 2009). GPD2 knock-down abrogates the mROS production, NF-κB activation and the downstream NF-κB-dependent transcription (Kaminski et al., 2012; Vander Heiden et al., 2009). This suggests that the metabolic switch from mitochondrial respiration to aerobic glycolysis mediates mitochondrial ROS production. Activated GPD2 is shown to directly reduce ubiquinone and release mROS via complex I. Thus, GPD2 acts as a major player in T cell activation-induced ROS release during the metabolic switch. In addition to GPD2, TCR-triggered metabolic switch also activates another glycolytic enzyme, ADPGK (ADPdependent glucokinase). The PKC-dependent ADPGK activity is triggered shortly after TCR-mediated stimulation. Modulating the ADPGK levels by either knock-down or over-expression reveal the crucial role of ADPGK in activation-induced ROS production. Although ADPGK is an ER resident protein with its active site exposed on the cytosolic side, its activation could enhance the glycolytic flux and thus possibly contribute to mROS production (Kaminski et al., 2012; Vander Heiden et al., 2009).

5.7. SUMMARY ROS are well established to play a crucial role in cellular signal transduction and mitochondria as a source of endogenous ROS, are increasingly being evaluated. There are 11 different sites within mitochondria that act as centers of ROS production but till date our knowledge on ROS, T cell activation and T cell induced mROS production is limited to Complex I and III and to some extent complex IV. ROS is regulated tightly by antioxidant mechanisms within mitochondria that comprise antioxidant enzymes and ROS scavengers. There is growing interest in antioxidant-mediated mROS regulation in T cell activation process. Most of the studies are focused on SODs that reside within mitochondrial matrix. While the involvement of respiratory complexes in mROS production is convincing, one should remember all these studies are limited to in vitro conditions. Among the varied reasons for this limitation is the lack of highly sensitive experimental techniques that could measure compartmentalized ROS in a spatiotemporal manner. Further, the lack of knockout mouse models where mitochondrial ROS is compromised is another major limitation. This is because the respiratory complexes are indispensable in mice. Despite these limitations, new evidence of the crucial role of mitochondrial oxidants in T cell activation process is gradually emerging. Functional respiratory complex I (and complex III), and MnSOD activity as well as the metabolic switch from mitochondrial ATP to aerobic glycolysis triggering the GDP2 and ADPGK constitute the crucial players in T cell activation process. Nevertheless, more research is needed to further understand the in vivo molecular mechanisms of T cell activation-induced mitochondrial ROS production.

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6 Overcoming Oxidants and Inflammation: Endothelial Targeting of Antioxidants to Combat Chronic Inflammatory Disease Elizabeth D. Hood University of Pennsylvania School of Medicine, Philadelphia, PA, United States

6.1. INTRODUCTION In vascular disease research, the interactions between chronic inflammation, endothelial dysfunction, and oxidative stress have been studied extensively, although in light of numerous unfavorable antioxidant therapy trials, much remains to be understood. While systemically administered antioxidants may provide some advantage in the treatment of chronic oxidative stress, they have not yet shown any tangible benefits in the treatment of acute and dangerous conditions including ischemia-reperfusion, adult respiratory distress syndrome (ARDS), and sepsis (Christofidou-Solomidou and Muzykantov, 2006). Additionally, the mechanisms and overall functionality of oxidants are not as straightforward as good versus bad. In moderate concentrations and balanced within the confines of cellular functions, oxidative agents participate in the regulation and perpetuation of healing and homeostatic regulation. Oxidative stress is caused by a surplus of reactive oxygen species (ROS) acting in concert with the resulting inflammation; this creates mutually reinforcing pathways that underlie the mechanisms of numerous human pathological conditions. Small molecule antioxidants provide some utility for the alleviation of chronic oxidative stress, but to date, have largely failed to provide tangible benefits in the treatment of acute, dangerous conditions that lack pharmacotherapy. These conditions include acute vascular and pulmonary pathologies characterized with high morbidity and mortality such as sepsis, ischemia/reperfusion, and ARDS (Janssen-Heininger et al., 2008). Megadoses of non-enzymatic antioxidants may alleviate modest, chronic oxidative stress, but are only marginally effective in acute severe conditions including ischemia-reperfusion, inflammation, and radiation injury (Christofidou-Solomidou and Muzykantov, 2006). Additionally, large doses of antioxidants, particularly fatsoluble vitamins, provoke toxicity concerns (Fuhrman, 2000). Antioxidant enzymes (AOE) such as superoxide dismutase (SOD) and catalase, (decomposing superoxide and hydrogen peroxide (H2O2) respectively) are by their nature vastly more potent antioxidants than small molecule vitamins, but, to the detriment of their efficacy, are eliminated from the blood within minutes of administration (Muzykantov, 2001b). Modifications have been sought to improve the pharmacokinetics (PK) of these compounds. Some enhancement of bioavailability has been achieved by conjugation of enzymes with polyethylene glycol (PEG), PEG-based pluronics, and encapsulation in liposomes. These modifications prolong circulation of AOE, thus enhancing their efficacy in treatment of some forms of inflammation and conditions associated with elevated ROS level in plasma and tissue parenchyma (Yi et al., 2010). This chapter briefly explores the potential benefit of therapeutic antioxidants as treatment for acute inflammatory pathologies of the vasculature, those antioxidants and their functions, and their means of targeting delivery by means of nanomedicine.

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6.2. INFLAMMATION AND DISEASE As mentioned, the imbalance of ROS and RNS creates oxidative stress as the innate antioxidant defense mechanisms are overwhelmed. Although acute inflammation is the body’s necessary and vital response to injury and infection, unchecked chronic inflammation is a link between cardiovascular and a myriad of other diseases as listed above (Awan and Genest, 2015; Vaziri and Rodriguez-Iturbe, 2006). The many and varied pathologies affected and/or driven by oxidative stress include (1) cardiovascular diseases, including atherosclerosis, hypertension and heart failure (Siekmeier et al., 2007; Thomson et al., 2007; Vaziri and Rodriguez-Iturbe, 2006); (2) neurological diseases, such as Alzheimer’s, Parkinson’s, Huntington’s and multiple sclerosis (Patel, 2016); (3) pulmonary disease, such as chronic obstructive pulmonary disease (COPD) and asthma (Kirkham and Barnes, 2013; Papi et al., 2006); (4) acute conditions associated with ischemia-reperfusion including ischemic stroke (Kirkham and Barnes, 2013) and organ transplantation (Thuret et al., 2014); (5) cancer (Sosa et al., 2013); (6) rheumatic diseases (e.g., arthritis) (Firuzi et al., 2006); and (7) others including but not limited to metabolic disorders, kidney disease, psychiatric disorders, and obesity (Saso and Firuzi, 2014). According to the National Institute for Health (NIH), cardiovascular, lung, and blood diseases, when taken together, are by far the leading causes of deaths in the United States. The successful pharmacotherapy of inflammation is a globally important and challenging goal.

6.2.1. Vascular Endothelium, Reactive Species, and Inflammatory Agents The vascular endothelium is a monolayer of endothelial cells that provides a barrier and regulatory interface between circulating blood and the tissues beneath. Healthy vascular endothelial cells maintain the fluidity and flow of circulating blood, control permeability across the vessel wall, and regulate leukocytes (Pober and Sessa, 2007), thus regulating blood vessel function and subsequently vascular health and disease. Endothelial cells represent both a source and a target of ROS in conditions involving vascular oxidative stress and inflammation. Endothelial abnormalities include ischemia, infectious agents, cytokines, ROS and other mediators are implicated in cardiac, pulmonary, cerebral, and peripheral vascular pathology (Thomas et al., 2008). Dysfunction of endothelium in disease states results in phenotypes that include spontaneous hemorrhage, vasospasms, hypertension, and precipitate atherosclerotic events (Craige et al., 2015). Dysfunction of the endothelium has a key role in myriad pathologies, and thereby, presents a prime target for therapeutic intervention (Shuvaev et al., 2015). Reactive oxygen species (ROS) (including superoxide O2  2, H2O2, nitric oxide NO, peroxynitrite ONOO2, and hydroxyl radical  OH) play an important role in vascular pathophysiology (Muzykantov, 2001b). Superoxide is produced in the mitochondrial respiratory chain and several cell enzyme systems including NADPH oxidase (NOX) by the reduction of molecular oxygen (Fig. 6.1). The NOX family is a group of multicomponent transmembrane proteins that facilitate electron transport across membranes to molecular oxygen to form ROS. NOX also reacts with NO producing peroxynitrite, decreasing the NO pool, which interferes with vascular tone and disrupts homeostasis (Craige et al., 2015). Furthermore, ROS produced by NOX in the lumen of endothelial endosomes in response to cytokines are implicated in signaling for proinflammatory endothelial activation (Oakley et al., 2009). The pathology of oxidative stress and inflammation must be considered at the cellular level. Both surplus of ROS or deficiency of antioxidant machinery can lead to oxidative damage to lipids, proteins, and DNA in vascular cells provoking harmful consequences (Christofidou-Solomidou and Muzykantov, 2006). Superoxide,

FIGURE 6.1 Oxygen reduction. Xanthine oxidase (XO), NADPH oxidase (NOX), and mitochondria produce single electron transfer converting molecular oxygen to superoxide anion (O2  2) that rapidly dismutates into hydrogen peroxide (H2O2) through the action of SOD, spontaneously, or as the end product of Nox4  H2O2 and is then converted to water and oxygen by catalase (Cat), peroxiredoxin (Prx), and glutathione peroxidase (Gpx). Adapted from Craige, S.M., Kant, S., Keaney, J.F., Jr., 2015. Reactive oxygen species in endothelial function  from disease to adaptation. Circ. J. (79), 11451155.

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spontaneously or by action of SOD, may dismutate into hydrogen peroxide. Hydrogen peroxide produces the extremely reactive hydroxyl radical in the presence of transition metals or hypochlorous acid by action of myeloperoxidase (MPO). Cellular delivery of antioxidant enzymes catalase and glutathione peroxidases protect cells against hydrogen peroxide. Further exacerbation of harmful effects occur when pathological mediators (e.g., cytokines, described further below) cause excessive endothelial ROS production inciting inflammatory abnormalities (JanssenHeininger et al., 2008) and further aggravating the vicious cycle of inflammation (Li et al., 2009; Oakley et al., 2009; Ushio-Fukai, 2009).

6.2.2. Markers of Oxidative Stress and Inflammation During endothelial pathological activation, cell adhesion molecules (CAMs such as vascular cell adhesion molecule, VCAM-1, intercellular adhesion molecule, ICAM-1, selectins, etc.) are upregulated, further propagating inflammation via recruitment, adhesion, and migration of activated leukocytes, thus enhancing vascular permeability and thrombosis (Pober and Sessa, 2007). Interrupting this damaging pathway may alleviate vascular damage. Markers of oxidative stress and inflammation have been employed to monitor pathological changes and therapeutic effect with varied degrees of specificity, sensitivity, and ultimate utility in clinical and preclinical studies. A suitable marker of oxidative stress or inflammation should be detectable using practically feasible means, should reflect underlying pathogenic processes, and respond to therapeutic intervention. Inflammation biomarkers, useful as tools to measure efficacy of antioxidant drugs (Germolec et al., 2010), include leukocytes and other defense cells, such as cellular adhesion molecules (CAMs) and their receptors, cytokines (TNF-α, chemokines, etc.), and acute phase proteins (APPs such as C-reactive proteins, complement factors etc.). Neutrophils are a primary cellular inflammation mediator of acute response, having granules loaded with enzymes, peptides, and proteins that kill and digest bacteria and foreign matter. One prominent product of neutrophils, MPO, is thus considered a biomarker of both oxidative stress and severity of inflammation (Alegre et al., 2002). Endothelial CAMs support rolling, firm adhesion, and transmigration of leukocytes to sites of inflammation, including P- and E-selectins, and cell adhesion molecules ICAM-1 and VCAM-1 (Golias et al., 2007). P-selectin, along with von Willebrand factor, is found in Weibel-Palade bodies, endothelial storage granules important in hemostasis and inflammation. Upon cell activation P-selectin localizes to the vesicle membrane and, triggers release of vesicle contents into the milieu and protein expression on cell surface. Endothelial P-selectin plays a role in the cell’s interaction with leukocytes, in thrombosis, and in clot degradation. E-selectin is synthesized in endothelial cells during the acute phase of inflammation and serves as a low-affinity anchor decelerating neutrophil rolling. Cell adhesion molecules ICAM and VCAM participate in firm adhesion of leukocytes to cells, binding to integrins expressed on the leukocyte surface. Both ICAM and VCAM expression is regulated by mediators of inflammation, such as cytokines. Detection of E-selectin, ICAM, and VCAM using imaging, microscopy and tissue analysis methods have diagnostic potential for clinical management of vascular inflammatory diseases (Blankenberg et al., 2001) and in animal studies (Germolec et al., 2010). Cytokines regulate cells involved in innate and adaptive immune responses. The principle cytokines regulating inflammation include TNF, interleukins 1α, 1β, 2, and 6, IFN-γ, TGF-β, among others. Analysis of cytokines and other inflammatory mediators (i.e., proinflammatory transcription factor NF-κB) produced with a particular pathology provides mechanistic and clinical relevant information (Corsini and House, 2010).

6.3. ANTIOXIDANTS AS THERAPEUTICS Antioxidants, by definition, are entities that inhibit oxidation, and are numerous and varied. These molecules and compounds counteract ROS produced through metabolism and other biochemical processes in all cells and tissues. As previously described, large portions of ROS are produced via activated phagocytes and to a lesser extent, within the endothelial and smooth muscle cells of the vasculature. Among their many functions, antioxidants decompose ROS, block lipid peroxidation, and scavenge oxidants. These compounds can be categorized into either nonenzymatic or antioxidant enzymes. AOE are highly specific and detoxify ROS with remarkable efficacy. Nonenzymatic antioxidants are generally small molecules that nonspecifically provide equimolar quenching of oxidants or reduction of oxidized biomolecules. The main source of these small molecules in the body is dietary. Generally considered nontoxic, these small molecules may alleviate

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mild oxidative stress in sufficiently large doses. However, these agents generally fail in tests of severe oxidative stress management, presumably due to the inability to localize to sites of injury (Christofidou-Solomidou and Muzykantov, 2006; Muzykantov, 2001a). Principal among nonenzymatic antioxidants includes ascorbic acid (vitamin C), a major water-soluble antioxidant obtained through dietary sources. Among the oil-soluble compounds are tocopherol (vitamin E), retinoids (vitamin A) and carotenoids (e.g., lycopene and β-carotene), as well as coenzyme Q10 (CoQ10), carotenoids, and polyphenols that function as antioxidants in myriad processes in the cells and tissues to reduce oxidative stress throughout the body. Glutathione, an intracellular thiol, is a multifunctional antioxidant, particularly in reduced form (GSH). GSH is found in all tissues, having an important role protecting the lower airspaces of the lung and the epithelial layer (Papi et al., 2006). GSH also aids in detoxification of pulmonary oxidative damage from endogenous and exogenous toxins (Christofidou-Solomidou and Muzykantov, 2006). The sulfhydryl groups in N-acetylcysteine (NAC) interact with free radicals resulting in the nonoxidizing end product NAC disulfide. NAC scavenges hydroxyl radical (OH  ), H2O2, and hypochlorous acid (HOCl). Upon deacetylation, NAC reverts to cysteine, a precursor of GHS synthesis, thereby replenishing the glutathione system. Polyphenol antioxidants are numerous, with over 4000 species (Quideau et al., 2011). Generally water-soluble, polyphenols include tannins, flavonoids, and phenolic acids, are characterized by the presence of multiple aromatic ring groups, and, as a group, have many and varied antioxidant functions. The antioxidant and pro-apoptotic properties (Sandur et al., 2007) of the polyphenol curcumin has been examined for treatment of hepatocellular carcinoma (Darvesh et al., 2011). Resveratrol, a natural polyphenol with strong antioxidant and free-radical scavenging properties, found in the skins of grapes and other fruits, has been studied extensively for antioxidant protective and anticancer properties (Pezzuto, 2011). As mentioned above, AOE are highly potent and specific, with high affinities and rates of reaction that detoxify ROS with a high efficacy. As importantly for therapy, AOE are not consumed in reaction with ROS unlike nonenzymatic antioxidants. As examples, SOD accelerates superoxide anion conversion into H2O2, while catalase reduces the latter and into oxygen and water (Muzykantov, 2001b). Catalase degrades over 40 million molecules of H2O2 per second (Stenesh, 1998) demonstrating how AOE interventions offer efficacy for ROS quenching not attainable by small molecule antioxidants.

6.4. ANTIOXIDANT TARGETING STRATEGIES Cell and antigen specific targeting is the key to overcoming challenges of treating the damaging effects of unchecked ROS and inflammation. This is a broad and active area of research. Indeed, a literature search of the topic reveals myriad applications, diseases of interest, and modalities of directing therapies. Routes of administration include, but are not limited to, transdermal, oral, ocular, and intravenous. Vehicles of delivery are many, as are the vectors by which localization is directed. Targets investigated may be generalized to organs, as highly specified as the individual organelles (e.g., mitochondria, endosomes, or nuclei) within specific cell types, or individual molecular sites of interest (Silva et al., 2016). A significant obstacle facing the translation of antioxidant therapies into the clinical domain is the inadequate delivery of these agents to their intended site of action. As mentioned, the vascular endothelium in particular represents a key therapeutic target in oxidative damage associated with ischemia and inflammation (Muzykantov, 2001a). The uneven results of the last several decades of antioxidant research include large scale clinical trials studies (Siekmeier et al., 2007; Suarna et al., 2006; Thomson et al., 2007), which have demonstrated that for antioxidant therapy to work, it must: (1) localize to the cells suffering oxidative stress, (2) effectively detoxify the culprit ROS, and (3) do so within an appropriate time-frame for therapy. Due to their transient and focal nature, ROS act on a nanometer scale. Therefore, optimal delivery of antioxidants would localize within targeted endothelial cells. In order to direct and focus therapeutics to sites of oxidative stress and inflammation, diverse carriers and drug delivery systems have been engineered to the delivery of antioxidants in the vasculature. Also, timing is key to successful antioxidant therapy; antioxidant interventions in acute pathological conditions (e.g., radiation injury, ischemia-reperfusion and acute respiratory distress syndrome) should act over a few hours to a few days, whereas treatment of chronic conditions such as atherosclerosis should last many weeks and months (Delles et al., 2008; Dziubla et al., 2006; Ratnam et al., 2006). While the exact requirements vary in specific disease conditions, nanoscale drug carriers provide great promise to achieve these goals.

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Drug conjugates embody the simplest form of direct antioxidant targeting, comprising the attachment of a targeting moiety such as an antibody to an antioxidant for targeted delivery. An example of this is the conjugation of AOE with antibodies to cell surface determinants including Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM) and ICAM which enables their specific delivery to endothelial cells lining vascular lumen, the key target of oxidants (Muzykantov et al., 1996; Muzykantov et al., 1999; Nowak et al., 2007). Rapid endothelial internalization of PECAM-targeted conjugates enables detoxification of intracellular ROS (Muro, Wiewrodt, et al., 2003; Wiewrodt et al., 2002). Anti-PECAM/catalase and anti-PECAM/SOD conjugates bind to endothelial surface determinants, detoxify endothelial ROS, significantly boosts AOE binding, uptake, and protective effects in vitro, and in animal models (endotoxin-induced inflammation, ischemia-reperfusion and angiotensin-II induced vasoconstriction) (Shuvaev et al., 2009; Shuvaev et al., 2011). Endocytosis of ICAM- and PECAM- targeted AOE allows quenching of signaling proinflammatory ROS in the endosomes, otherwise inaccessible to antioxidants in the milieu (Shuvaev et al., 2011). Catalase immunoconjugates were used to address the problem of organ donor shortage for lung transplant. Warm ischemia causes graft dysfunction in lungs harvested after cardiac arrest, with ROS damaging the pulmonary endothelium. A 6-h reperfusion with anti-PECAM catalase conjugates ameliorated graft function and reduced lipid peroxidation, alveolar leakage and edema in an animal transplant model (Preissler et al., 2011). Complexation with PEG-containing pluronics has been reported to increase cellular permeability of diverse drugs in cultured cells (Batrakova et al., 1999) and in vivo (Batrakova et al., 1999; Batrakova et al., 2001). Although the mechanism of intracellular transport remains to be defined, no deleterious effects have been noted (Batrakova et al., 2004). For example, SOD-pluronic conjugates were reported to deliver enzymatically active SOD to neuronal cells more effectively than naked SOD or PEG-SOD without neuronal toxicity (Yi et al., 2010). While conjugation of drugs to antibodies (or other proteins) facilitate targeted delivery and controlled cellular uptake (Casi and Neri, 2012), conjugation to hydrophilic polymers, such as PEG, can limit renal filtration, cellular uptake, proteolytic degradation, and immunogenicity (“stealth” technology) (White et al., 1989). However, the uniformity of such conjugates may be suboptimal for safe widespread clinical use. Inactivation of labile biotherapeutics during conjugation, in circulation, and in target cells represents an additional challenge.

6.5. NANOCARRIER-MEDIATED DELIVERY OF ANTIOXIDANTS As mentioned earlier, the functionality of a therapeutic, however powerful, is limited by its ability to access focal sites of disease or injury. In the case of enzymes, once injected in their native state, they may be cleared from circulation rapidly due to proteolytic degradation, renal filtration, and/or clearance by the reticuloendothelial system (RES). Nor do they have any inherent facility to reach their therapeutic target’s cellular compartments. Use of nanocarriers (NCs) may help to overcome some of these challenges. A basic prototype NC is a vesicle or solid core delivery unit that improves the PK of loaded drug cargo and limits its nonspecific interactions en route to the therapeutic target. Advanced multifunctional NCs contain additional elements designed to provide targeting, tracing, local drug release, and/or enhanced intracellular delivery (Fig. 6.1). The NCs serve to enhance the benefit/risk ratio and enable novel therapeutic mechanisms by optimizing the localization and timing of the drug action at the desired site in the body (Cheng et al., 2012). Arguably, the most feasible application of NC-mediated therapeutics lies in the transient treatment of serious acute conditions such as ischemia-reperfusion, ARDS, and sepsis. Due to NC size (up to hundreds of nanometers), and the need for expediency in these conditions, vascular injection is the most suitable route for NC administration. The vascular route also offers direct access to endothelial cells, which as already noted, regulate key functions including leukocyte recruitment, and represent an important target for anti-inflammatory interventions. Herein is a brief review of vehicular delivery of antioxidants, AOE, and some equivalent mimetics to the vasculature. The focus is on those types of therapeutics whose effects either require or can benefit significantly by use of a delivery vehicle. For example, curcumin, a component of turmeric and a naturally occurring powerful antioxidant that is highly insoluble and poorly bioavailable has been studied extensively as a nanocarrier candidate therapeutic (Hani and Shivakumar, 2014). Diverse drug delivery systems have been designed or adopted for antioxidants, with rather mixed results in preclinical animal studies and clinical trials, including large scale clinical trials studies (Siekmeier et al., 2007; Suarna et al., 2006; Thomson et al., 2007).

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FIGURE 6.2 Antioxidant nanoparticle types. Particles are shown to have nanoparticle core structures, entrapped or integrated therapeutic cargo, and external targeting ligands. Adapted from Hood E.D., Shuvaev, V.V., Muzykantov, V.R., 2016. Targeted antioxidant interventions for vascular pathologies. In: Dziubla, T., Butterfield, D.A. (Eds.), Oxidative Stress and Biomaterials, pp. xiii, 389 pages.

Many studies are in the early development phase, with promising prototypes transitioning from in vitro proof of concept to animal studies looking at PK and efficacy of the interventions. Numerous formulations of nanoparticles and nanocarriers used in vascular delivery have been developed to date. The principle forms include drug conjugates targeting species (e.g., antibody, minibody, and peptide) or polymers; liposomes; solid lipid, magnetic and polymeric nanoparticles; and dendrimers (Fig. 6.2). Each platform exhibits specific, engineered features including diverse geometry, functional moieties, size, surface charge, stability, and kinetics of drug release details of which have been reviewed (Hood et al., 2011; Hood et al., 2016; Simone et al., 2008). Overall, however, to avoid retention in the microvasculature, the diameter of spherical carriers for vascular delivery is generally ,300 nm, and the surface charge is close to neutral (Hood et al., 2011).

6.5.1. Liposomes Liposomes, the most highly devoloped nanocarriers, are phospholipid bilayer vesicles surrounding an aqueous core that were originally described in the 1960s (Johnson and Bangham, 1969; Johnson et al., 1971; Johnson et al., 1973). The nature of the particle allows for drugs to be entrapped in the inner volume, conjugated to the surface, or intercalated in the bilayer. In the 1980s, liposomes were proposed as a means to protect antioxidants from clearance and deactivation in vivo, and to facilitate access to desired cells and tissues (Tanswell and Freeman, 1987). The amphiphilic nature of the liposome’s bilayer structure allows the delivery of both hydrophilic and hydrophobic agents including lipid soluble antioxidants incorporated within the hydrophobic bilayer including vitamin E (TOHs and tocotrienols), ubiquinones, retinoids, carotenoids, flavonoids, soy isoflavones, and synthetic butylated hydroxytoluene (BHT) among many others (Stone and Smith, 2004). Likewise, water-soluble antioxidants such as ascorbate, urate, and glutathione have been studied as liposomal cargoes, enclosed in the hydrophilic core of the liposome. Inclusion of tocopherol and other oil-soluble antioxidant vitamins into the lipophilic inner membrane of liposomes has been shown to lend greater antioxidant efficacy, while also serving to protect the light and oxidation-sensitive molecules from damage by environmental exposure (Gonnet et al., 2010). As within the other drug classes for targeted delivery, antioxidant liposomes are further developed than other nanoparticle platforms in this area of research. Hydrophobic antioxidants including vitamin E, ubiquinones, retinoids, carotenoids, flavonoids and BHT have been incorporated within the liposomal bilayer to improve their solubility and provide protection for the drug (Hood et al., 2011; Stone and Smith, 2004). The hydrophilic antioxidant, GSH, was encapsulated in the aqueous volume of liposomes and liposome-like spherical selfassembly nanoparticles and shown to protect cells from oxidative stress in vitro (Williams et al., 2009). N-acetylcysteine, a hydrophilic derivative of cysteine, has antioxidant activities, and replenishes GSH in tissues. NAC has a low bioavailability, likely due to acetylation, which necessitates the use of vehicular drug delivery systems. Suntres et al. found that liposomal NAC protected against liver injury in rats in a sepsis model

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better than free NAC (Mitsopoulos et al., 2008). A liposomal formulation of NAC, a precursor of the antioxidant gluthathione, showed a higher protective potency than the free drug when delivered intratracheally in a rat model of acute respiratory distress syndrome (Fan et al., 2000). A combination of tocopherols and NAC in liposomes were administered to guinea pigs after exposure to a mustard gas and provided a protective effect in the lungs (Mukhopadhyay et al., 2009). Delivery of curcumin has been studied (Bansal et al., 2011). Among the techniques employed to deliver the lipophilic antioxidant, inclusion within dimyristoyl phophatidylcholine (DMPC) liposomes that were targeted to prostate membrane antigen using antibodies showed enhanced anticancer properties as compared to free curcumin (Thangapazham et al., 2008). Liposomal delivery of resveratrol reduced vascular intimal thickening after endothelial injury in rats (Hung et al., 2006). A combination of resveratrol and curcumin was shown to reduce prostate cancer in a mouse model by liposomal delivery (Narayanan et al., 2009). GSH and polyethylene diacrylate (PEGDA) oligomers created liposome-like spherical self-assembly nanoparticles which have been shown to protect cells from oxidative stress in vitro (Williams et al., 2009). In early work by Freeman et al., liposomal delivery of SOD to cultured endothelial cells increased SOD activity compared to the controls cell treated with free SOD. Cells that received liposomal SOD were more resistant to oxidative damage by hyperoxia (Freeman et al., 1983). The same group reported protective effect against exposure to high respiratory oxygen in newborn rats treated with daily injections of liposomes containing SOD and catalase (Tanswell and Freeman, 1987). Further, they have reported enhanced delivery of SOD encapsulated in pH sensitive liposomes containing surfactant protein A (SP-A) to primary fetal pulmonary epithelial cells via SP-A receptors (Briscoe et al., 1995). Hypertension caused through Ang II activation of membrane-bound NADPH oxidase generating superoxide was reduced in rats treated with liposomal SOD (Laursen et al., 1997). Studies in a rat arthritis model showed that SOD liposomes inhibited edema more effectively than naked SOD (Corvo et al., 2002). The incorporation of an acetylated hydrophobic derivative of SOD, Ac-SOD, improved loading efficiency of the enzyme compared to unmodified SOD. Since the Ac-SOD localized to the bilayer and 50% of the enzyme was exposed to the exterior, the site of activity was focused at the surface of the liposome or “enzymosome,” instead of within the aqueous interior. The change in conformation reduced the effect of release rate on the activity of the liposome and increased bioavailability of the enzyme (Gaspar et al., 2003). This effect of SOD localization within the liposome structure was tested in a rat adjuvant arthritis model comparing PEGcoated liposomes with either Ac-SOD or plain SOD. The circulation time of the PEGylated liposomes increased regardless of the SOD type included, and a faster anti-inflammatory effect was observed with the As-SOD PEG liposomes versus the plain SOD PEG liposomes (Gaspar et al., 2007). Varying methodology and content of liposomal SOD formulations allows modulating the loading efficacy, localization within the liposomal compartments and enzymatic activity of resultant SOD/liposomes (Xu et al., 2012). NADPH-oxidase inhibitor MJ33 loaded into liposomes targeted to the endothelium by PECAM antibody accumulated in the endothelial cells, inhibited ROS production, and provided more potent protective effects than nontargeted counterparts against oxidative stress and inflammation in vitro, ex vivo in perfused mouse lungs and in mice in vivo (Fig. 6.3) (Hood et al., 2012). Similarly targeted liposomes loaded with EUK-134, a SOD/ catalase mimetic, bound to endothelial cells in culture and pulmonary vasculature in mice and provided

FIGURE 6.3 Targeted delivery of antioxidant nanocarriers to the endothelium. Adapted from Hood, E.D., Shuvaev, V.V., Muzykantov, V.R., 2016. Targeted antioxidant interventions for vascular pathologies. In: Dziubla, T., Butterfield, D.A. (Eds.), Oxidative Stress and Biomaterials, pp. xiii, 389 pages.

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anti-inflammatory effects in mouse model of endotoxin lung injury, whereas untargeted IgG/EUK liposomes neither deliver cargo to endothelium nor provided protection (Howard, Greineder, et al., 2014). ICAM-1 and VCAM-1 targeted echogenic liposomes filled with NO have been used to detect inflammatory changes in atherosclerotic lesions using contrast-enhanced ultrasound imaging. Acoustic enhancement was enabled by the pretreatment of the endothelium with targeted NO-loaded echogenic liposomes combined with localized ultrasound activation, providing improved detection of site-specific inflammatory changes (Kee et al., 2014; Kim et al., 2015).

6.5.2. Polymer Nanocarriers Polymer-based NCs include dendrimers, polymeric micelles and nanoparticles, and filomicelles. Their structural materials include both synthetic (e.g., poly(lactic-co-glycolic acid), PEG) and biological polymers (e.g., chitosan, poly(nucleic acids)) as well as blends of different polymers conjugated into diblocks, triblocks and higher order copolymers. Such versatility helps to individualize the NC properties and may even allow for design of NCs with several compartments, multiple layers, or controlled surface heterogeneity (Kohler et al., 2012; Labouta and Schneider, 2010; Misra et al., 2012; Pochan et al., 2011). Dendrimers (Fig. 6.2) are repetitively branched multimolecular spherical complexes in the 10100 nm range, characterized by a high level of precision in synthesis and structure, which have been adopted and tested for antioxidant and anti-inflammatory agents (Wijagkanalan et al., 2011). Drugs can be coupled to the active end groups or encapsulated into the meshwork, yet the loading capacity is generally lower than for other NCs. Polymeric micelles and nanoparticles are typically based on amphiphilic block copolymers, which form a distinct structure while in solution: the hydrophobic and hydrophilic portions of the polymer form the core and the corona, respectively (Kataoka et al., 2001). Technically, micelles have “dynamic” cores, meaning that there is an exchange of polymers among the various aggregates, whereas nanoparticles have solid or “frozen” cores (Nicolai et al., 2010). Yet, because the core state may change depending on the conditions—temperature, pH, solution— this distinction is more conceptual than practical. Due to higher material stability and stealth features (polymeric particles can be virtually 100% PEGylated), these carriers may exhibit longer PK and residence time in tissues versus lipid-based carriers. The polymeric materials degrade via hydrolysis, with the rate and mechanism (e.g., surface erosion vs bulk dissolution) dependent on the carrier size, content, structure, and environment. Encapsulation of the bioactive proteins in a polymer shell can provide protection from proteolytic inactivation, thereby extending therapeutic duration. For instance, using a standard water/oil/water emulsification method B7 wt% of SOD or catalase was loaded into poly(lactide-co-glycolide) (PLGA) polymer microspheres. By using PEG400 as a stabilizer, these particles (sizedB1015 μm) released active enzyme for over 50 days (Giovagnoli et al., 2005). However, their size exceeded the necessary circulation limit of ,500 nm. Although AOE are highly potent and specific, they are also, unfortunately, labile and easily inactivated during nanocarrier formulation conditions. Furthermore, they are prone to proteolytic inactivation in vivo, which limits their therapeutic duration in the lysosomes (Dziubla et al., 2005; Muro, Cui, et al., 2003). In order to overcome these challenges, biodegradable polymeric nanocarriers (PNC) were developed for the delivery of AOE. Polymeric carriers have been used for AOE delivery, in particular, to protect the cargo enzymes against inactivation. Nanoparticle design considerations must include consideration of the effects, if any, of the carrier and its byproducts, on the function of the drug cargo. More specifically, an acidic environment is known to inhibit enzymes, and this may happen in the lysosomes or other areas of acidosis, including carrier-induced acidosis (i.e., the release of lactic acid during the degradation of PLGA). To avoid this, nanocarriers can be synthesized from polymers that do not produce acidic groups during degradation. For example, SOD-loaded polyketal microspheres injected in the cardiac muscle have been reported to alleviate myocardial ischemia-reperfusion (I/R) injury (Seshadri et al., 2010). SOD-loaded polyketal particles introduced via intratracheal route alleviated bleomycin induced pulmonary injury (Fiore et al., 2010). However, these carriers are too big for intravascular delivery. In order to decrease the size of nanocarriers made through these standard methods, increased shear rates are needed during the last emulsification step, potentially inactivating the enzyme. However, a significant burst release (B50% to 60%) within the first few hours suggests that the majority of protein loading was limited to surface absorption, not incorporated within the particle interior (Giovagnoli et al., 2005). Introducing a freezing step during the primary emulsification ameliorates enzyme inactivation and enhances internal loading in PNC (Dziubla et al., 2005; Dziubla et al., 2008). When using an amphiphilic encapsulating

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polymer (e.g., poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PEG-PLGA)), PNC produced through this method were 200300 nm in size and contained 10 times the mass of catalase compared to synthesis without the cryogenic step. Also, these carriers maintained B25% of the original catalase activity after 24 hours incubation with a proteolytic enzyme, while free enzyme lost .90% activity within the first hour (Dziubla et al., 2005). This loading mechanism and protection was independent of the enzyme used, yet substrate diffusivity through the polymer shell greatly determined the ability of internally loaded enzyme to express its function (Dziubla et al., 2008). In another approach, several proteins, including SOD, were covalently functionalized with vinyl groups followed by free-radical polymerization in presence of other diacrylate monomers, which resulted in encapsulation of protein molecule in a nanometer thick (B 5 nm) polymer shell. Surface properties and the degradation rate of the polymer shell were controlled by monomer selection. SOD nanocapsules prevented cell death in a paraquat injury model, in vitro, suggesting that the polymer shell is permeable to the substrate molecules (e.g., O2  2) (Yan et al., 2010). Complexation of therapeutic compounds with PEG-containing pluronics has been reported to increase cellular permeability of diverse drugs in cultured cells (Batrakova et al., 1999) and in vivo (Batrakova et al., 1999; Batrakova et al., 2001). Although the mechanism of intracellular transport remains to be defined, no deleterious effects have been noted (Batrakova et al., 2004). For example, SOD-pluronic conjugates were reported to deliver enzymatically active SOD to neuronal cells more effectively than naked SOD or PEG-SOD without neuronal toxicity (Yi et al., 2010). Batrakova et al developed a macrophage-driven system for the delivery of AOE to the brain. Catalase was electrostatically complexed with a cationic block copolymer, polyethyleneimine-poly(ethylene glycol) (PEI-PEG). These “nanozymes” in which the PEI-PEG shielding protected the enzyme within the structure were phagocytized by macrophages, targeted by the subsequent migration of the cell to the inflamed brain in a mouse model of Parkinson’s disease (Batrakova et al., 2007). Testing morphological and biochemical parameters of the brain injury showed promising therapeutic efficacy of this delivery system (Brynskikh et al., 2010). Similarly formed SOD nanozymes alleviated neuronal oxidative stress after local administration in the CNS in rodents (Rosenbaugh et al., 2010). The mechanisms and utility of these fortuitous delivery means deserves rigorous studies. Nanoparticles composed of amphiphilic redox polymers were investigated for the treatment of oxidative stress-driven liver disease in a mouse model. Orally administered, the radiolabeled redox nanoparticles disintegrated in the stomach. The resultant redox polymers were traced to the liver, and therein showed alleviation of oxidative stress, inflammation, and fibrosis (Eguchi et al., 2015). Some nanoparticles have innately antioxidant properties without a therapeutic cargo. Cerium oxide nanoparticles, or nanoceria, mimic SOD and catalase activities, with redox cycle reacting between Ce(31) and Ce(41) oxidation states (Celardo et al., 2011). Furthermore, nanoceria delay photoreceptor cell degeneration in rodent models and prevent pathological retinal neovascularization in mutant mice. After a single intravitreal injection, it was shown that nanoceria were rapidly taken up by the retina and were retained for four months, and no acute or long-term negative effects were observed on retinal function or architecture (Wong et al., 2013).

6.5.3. Magnetic Nanoparticles Magnetic nanoparticles (MNPs) have been proposed for delivery of biotherapeutics. Chorny et al. reported delivery of plasmid DNA loaded in magnetically driven biocompatible polymeric MNPs composed of oleatecoated magnetite and surface modified with PEI oleate ion-pair complexes enabling DNA complexation. They also reported that paclitaxel loaded MNPs could be delivered to arterial stents in rats using a magnetic field: target accumulation was an order of magnitude higher in magnetically treated animals versus nonmagnetic controls (Chorny et al., 2009). Capitalizing on this, similarly formulated MNPs have been used to load either active SOD or catalase through a controlled precipitation method incorporating two aqueous phases. The loading of AOE into MNP bypasses the use of the strong sheer for emulsion or organic solvents, which helps to reduce the inactivation of the AOE. The formulation loaded active enzyme cargo efficiently and protected it from proteolytic degradation in vitro. MNPs demonstrated a protracted release of their cargo protein in plasma. Additionally, magnetically-delivered, catalase-containing MNPs protected endothelial cells in vitro, with 62 6 12% cells rescued from H2O2 induced cell death versus 10 6 4% under nonmagnetic conditions (Chorny et al., 2010). Additionally, PECAM-directed Ab/MNPs loaded with SOD or catalase accumulated in the pulmonary endothelium (33% injected dose at 30 min) in a mouse model. Catalase-loaded endothelial-targeted particles alleviated

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the pulmonary edema and leukocyte infiltration in a mouse model of endotoxin-induced lung injury, whereas targeted, SOD-loaded carriers mitigated LPS-induced lung inflammation (Hood et al., 2014).

6.5.4. Lipid Nanoparticles and Complexes Increased interest in the use of solid lipid nanoparticles (SLNs) relies on the advantages of lipid materials with regards to biocompatibility, biodegradation, controlled release, easy scale-up, and low cost. Additionally, these nanoparticles show excellent physical and chemical stability providing greater protection against degradation of drugs and the ability to load hydrophobic, hydrophilic, and amphiphilic macromolecules (Souto and Muller, 2010). SLNs contain a lipid core stabilized by emulsifiers and, if needed, decorated by PEG. When prepared with physiological lipids and FDA-approved emulsifiers, SLNs are biocompatible. Their properties (size, drug loading and release, and stability) are tightly controlled by ingredient composition (Kheradmandnia et al., 2010). For instance, SLNs based on highly crystalline lipids may show reduced drug loading but also a more sustained drug release. Depending on the cargo properties and medical goal, using a combination of lipids or even introducing a liquid lipid (to make nanostructured lipid carriers or NLCs) may be desirable (Wissing et al., 2004). An example of antioxidant SLNs, the novel dual-bioactive nanoparticle with antioxidant/anti-inflammatory properties, was developed based on the interactions of tocopherol phosphate and the manganese porphyrin SOD-mimetic, MnTMPyP. This entirely bioactive nanoparticle was shown to retain its SOD-like activity as intact particles, and to release in a slow and controlled manner. Conjugation of anti-PECAM antibody to the nanoparticles provided endothelial targeting and potentiated nanoparticle-mediated suppression of inflammatory activation of these cells manifested by expression of VCAM, E-selectin, and IL-8 (Howard et al., 2014). Batrakova et al. developed a macrophage-driven system for the delivery of AOE to the brain (Batrakova et al., 2007). Catalase was electrostatically complexed with the cationic block copolymer, PEI-PEG. These nanozymes in which the PEI-PEG shielding protected the enzyme within the structure were phagocytized by macrophages, targeted by the subsequent migration of the cell to the inflamed brain in a mouse model of Parkinson’s disease (Batrakova et al., 2007). Testing morphological and biochemical parameters of the brain injury showed promising therapeutic efficacy of this delivery system (Brynskikh et al., 2010). Similarly formed SOD nanozymes alleviated neuronal oxidative stress after local administration in the CNS in rodents (Rosenbaugh et al., 2010).

6.6. CONCLUSIONS This is brief review of targeted antioxidant therapy highlights some valuable developments in nanoformulations used to optimize the localization and timing of antioxidant action. Solving these delivery issues will enable novel anti-inflammatory interventions—nanoformulations of biotherapeutics and antioxidants—potentially transforming the management of patients with acute inflammatory conditions, especially those with poor outcomes. Combining the rational design of the carriers to fit both the pathophysiology of the condition and the requirements of the antioxidant cargo, and an ideal target selection are necessary to succeed in this goal. An important aspect of the rational design is a careful balance of the targeting platform’s advantages and disadvantages. On the plus side, a nanotherapeutic formulation may enable targeting, intracellular delivery, and localized activation; however, there may be unintended consequences such as harmful tissue accumulation in organs (e.g., liver, and kidneys), unintended activation of host immunity, or damage to the targeted cells themselves. Adaptations to formulations such as the development of PEG stealth technology in liposomes, and the reengineering of immunogenic targeting species have been made in order to address these and other issues. Still, many of the challenges of nanotoxicology must be addressed before nanotherapeutics for treatment of nononcological diseases can be meaningful clinically. For example, although nanocarriers may only degrade into biodegradability into nontoxic components, this does not equate to NC biocompatibility. Also, since nanoparticles circulate in the bloodstream, their accessibility to diverse tissues and cells, and cellular uptake—the very features enabling its delivery functions—also potentiates their ability to harm, which is more critical in the treatment of inflammation than in oncology. The complexity of nanotherapeutics aggravates these and other safety concerns while the translational challenges include difficulties of industrial production and control of size, structure, activity and homogeneity as well as regulatory complications and costs. Given these obstacles, in order to move forward towards translatable antioxidant targeted therapeutics, the focus should be on scenarios that would produce the greatest

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probability of success. In this context, nanotherapeutics that enable only incremental improvements over existing treatments, or create novel platforms for the sake of themselves should be set aside for those that will quantitatively improve the outcomes with the least potential for toxicity. Chronic repetitive IV administered treatments are more likely to cause adverse effects; therefore the effective translation of anti-inflammatory nanocarriers would lie in the treatment of acute conditions, at least initially. Successful vascular delivery of antioxidants enhanced using nanoparticles targeting to locations critically important for their desired effects may also ultimately shift the current models of pharmacological management of the myriad inflammation-driven diseases.

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C H A P T E R

7 Oxidative Signaling in Chronic Obstructive Airway Diseases Tania A. Thimraj1, Leema George1, Syed Asrafuzzaman2, Swapna Upadhyay3 and Koustav Ganguly3 1

SRM University, Kattankulathur, Tamil Nadu, India 2Utkal University, Bhuvaneswar, Odisha, India 3 Karolinska Institutet, Stockholm, Sweden

7.1. INTRODUCTION Asthma and chronic obstructive pulmonary disease (COPD) are the most prominent chronic obstructive airway diseases (COADs) worldwide that pose a huge socioeconomic and disease burden to both developed and developing nations. COPD alone is the fourth leading cause of death globally and accounts for three million deaths every year (WHO, 2017). Nearly 65 million people around the world suffer from COPD (WHO, 2015) and 334 million people suffer from asthma (The Global asthma report, 2014). COPD and asthma together account for almost 82 billion euros lost annually due to the cost of healthcare and loss of productivity in Europe (ERS white book 2013). The burden of asthma in terms of disability and premature death is greatest among children approaching adolescence (ages 1014) and the elderly (ages 7579) (The Global asthma report 2014). In 2008, asthma accounted for more than 10.5 million missed school days, and adults missed more than 14 million days of work in the United States (CDC, 2013). Oxidative stress has been implicated as a significant contributor for the pathogenesis of COADs (Elmasry et al., 2015, Kirkham and Barnes, 2013, MacNee 2005a,b, Gerritsen et al., 2005., Dutt et al., 2011, Cho and Moon, 2010). Increased lung reactive oxygen species (ROS) levels and an imbalance between the generation and elimination processes due to both exogenous and endogenous factors are critical for COAD pathogenesis. Cigarette (tobacco) smoke is the single most predominant cause of COPD worldwide. Indoor air pollution, particularly in the form of biomass combustion is another important contributor to COPD development (Salvi and Barnes, 2010, Kodgule and Salvi, 2012, Balcan et al., 2016, Olloquequi and Silva, 2016). Sources that trigger the onset of asthma include both indoor and outdoor pollutants. Indoor pollutants comprise particulate matter (PM) such as pollen, spores, bacteria, plant and animal debris, nitrogen oxides, second-hand smoke, and allergens from pests, pets, and molds (Breysse et al., 2010, Diette et al., 2008), benzene, carbon monoxide (CO), formaldehyde, naphthalene, polycyclic aromatic hydrocarbons (PAH), radon, trichloroethylene and tetrachloroethylene (WHO, 2010). Benzene, 1-3 butadiene, CO, lead, nitrogen dioxide from combustion, industrial waste, power plant exhaust and ozone (Sahiner et al., 2011) are outdoor sources. These pollutants are classified as exogenous sources of ROS in the lung. Mitochondrial respiration (subcellular level), neutrophils, eosinophils and macrophages (cellular level) are the important endogenous sources of ROS generation in the lung. Lipid peroxidation (Rahman et al., 2002, Waseem et al., 2012) and DNA damage (Neofytou et al., 2012, Ceylan et al., 2006, Caramori et al., 2011) mediated disruption of cell signaling and enzymatic activity are the major effects of oxidative stress observed in COADs. The lung, being the primary port of entry for inhaled exogenous ROS sources has a well-defined antioxidant defense mechanism. Disruption of redox homeostasis in the lung is a key pathogenic event for COAD

Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00007-X

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FIGURE 7.1

Schematic representation of the role of redox imbalance in chronic obstructive airway disease (COAD) development. CD, cluster of differentiation, COPD, chronic obstructive pulmonary disease, HNE, 4-hydroxy-2-nonenal, H2O2, hydrogen peroxide, MDA, malondialdehyde, OH2, hydroxide, ONOO2, Peroxynitrite, O22, superoxide, ROS, reactive oxygen species, 3NT, 3 Nitrotyrosine, 8-IP, 8 isoprostane, 8-OHdG, 8-Oxo-2’-deoxyguanosine, 8-OHG, 8-Hydroxyguanosine.

development. Fig. 7.1 illustrates the various sources and mechanisms of oxidative stress mediated COAD development. It is evident that oxidative stress plays a central role in the pathophysiology of COADs and therefore antioxidant-based therapeutic modalities are being investigated to combat redox imbalance in COADs. Spin traps, thiols, nonenzymatic antioxidants and enzymatic antioxidant mimetics are the potential therapeutic strategies. In this chapter we will discuss the role of oxidants and their downstream signaling in COPD and asthma, along with the current therapeutic approaches using antioxidant treatment modalities.

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7.1.1. Chronic Obstructive Pulmonary Disease The global initiative for chronic obstructive lung disease (GOLD) defines COPD as a common preventable and treatable disease characterized by persistent airflow limitation that is usually progressive and associated with an enhanced chronic inflammatory response to noxious particles or gases in the airways and lung (GOLD, 2012). COPD encompasses chronic bronchitis as well as emphysema. In chronic bronchitis, obstruction of small airways, inflammation, mucus gland enlargement, excess mucus production, and goblet cell hyperplasia (Saetta et al., 2000, Willemse et al., 2004, Hogg et al., 2004, Vestbo et al., 2013) is accompanied by a continuous cough of more than three months duration (Fabbri et al., 2003, Vestbo et al., 2013). Emphysema on the other hand involves enlargement of airspaces, destruction of lung parenchyma, loss of lung elasticity and closure of small airways (MacNee 2005a,b, Timmins et al., 2012). Overall, cellular changes such as replacement of clara cells with mucus producing cells, infiltration of mononuclear cells and accumulation of macrophages and neutrophils (Retamales et al., 2001, Keatings et al., 1996, Pesci et al., 1998, St˘anescu et al., 1996, Hoenderdos and Condliffe, 2013) along with increase of T lymphocytes are some notable features of COPD (Majo et al., 2001). Oxidative stress and inflammation play a pivotal role in the pathogenesis of COPD (Drost et al., 2005). Several studies have shown that oxidative stress is increased in the lungs of patients with COPD compared to healthy subjects (MacNee 2005a,b, Barreiro et al., 2010, Paredi et al., 2002, Kirkham and Barnes, 2013, Rahman and Adcock, 2006). Despite cigarette smoke exposure being the single major cause of COPD, cessation of smoking does not immediately prevent the progression of the disease primarily due to the continued presence of oxidative stress and the associated downstream signals (Louhelainen et al., 2009). Endogenous factors, such as autoimmunity or persistent infection have been also implicated in the production of ROS and pathogenesis of COPD even after exposure to cigarette smoke ceases (Cosio et al., 2009, Taraseviciene-Stewart et al., 2006). COPD is associated with chronic inflammation that occurs in the form of accumulation of innate immune cells such as neutrophils and macrophages in the peripheral airways and lung parenchyma. This potentially leads to lung injury besides causing progressive narrowing of the airways and shortness of breath (Kanner et al., 2001, Saetta et al., 1997). Enhanced or abnormal inflammatory response to inhaled particles or gases, beyond the normal protective inflammatory response in the lungs is a characteristic feature of COPD and can cause lung injury (Saetta et al., 2001). Inflammation has been observed in lung and bronchial biopsies of all cigarette smokers (MacNee 2005a,b). Alveolar macrophage and neutrophils are activated in the alveolar space in response to cigarette smoke (Sharafkhaneh et al., 2008). Biomass combustion, cigarette smoke, and other exogenous sources of ROS invoke inflammatory responses that result in ROS production. Thus, oxidative stress induced alterations in the lung over a prolonged period of time result in chronic inflammation that in turn leads to COPD.

7.1.2. Asthma Asthma is one of the most common chronic diseases affecting children and young adults. Its impact on working-age adults and activity limitation in the elderly has been widely recognized (Reddel et al., 2015). It is a heterogeneous disease, usually characterized by chronic airway inflammation (GINA 2016) initiated by exposure to environmental factors (Wenzel 2012a,b). The respiratory symptoms include recurrent episodes of wheeze, shortness of breath, chest tightness, cough and variable expiratory airflow limitation which is reversible either spontaneously or with treatment. Cough varies over time and intensity (Reddel et al., 2015). Asthma is phenotypically classified as mild, moderate and severe (Bousquet et al., 2000). Mild to moderate asthma or allergic asthma, is defined by airway hyper-responsiveness of the bronchioles (Zuo and Clanton, 2005) increased mucus production (Nabe et al., 2013), eosinophilic inflammation, epithelial cell desquamation, and airway remodeling (Comhair et al., 2005). Besides cells of the innate immune system, adaptive immune cells such as T helper (Th) 2 cells initiate allergic immune responses in mild to moderate asthma (Lloyd and Hessel, 2010, Murdoch et al., 2010). Severe asthma is nonallergic but display asthmatic symptoms in response to other stimuli such as diesel exhaust particles (DEP), viruses, and cigarette smoke (Thomson et al., 2004; Ghio et al., 2012). Th17 cells initiate neutrophilic inflammation in severe disease (Linde´n et al., 2001, 2014). Increased incidences of bronchial asthma have been reported in areas with high air pollution (Ercan et al., 2006, Comhair et al., 2005). Both indoor pollution and traffic exposure have been associated with an increased risk of asthma in children (McConnell et al., 2006). Gilliland and colleagues (2001) associated in utero exposure to maternal smoking and asthma, independent of environmental tobacco smoke exposure after birth and estimated that elimination of in utero exposure to maternal smoking would prevent 5%15% of asthma cases in children. Living close to a major road increases the risk of asthma in children irrespective of socio-demographic characteristics (McConnell et al., 2010).

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7.2. SOURCES OF REACTIVE OXYGEN SPECIES (ROS) IN THE LUNG ROS are unstable molecules with unpaired electrons, like superoxide anion (O2  2) and hydroxyl radicals (  OH), as well as derivatives of oxygen that do not contain unpaired electrons, e.g., hydrogen peroxide (H2O2), hypochlorous acid (HOCl), peroxynitrite (ONOO-) and ozone (O3) and are capable of initiating oxidation. Both exogenous and endogenous sources of ROS have been implicated in COADs. The respiratory tract is in direct contact with the external environment, which contains exogenous ROS sources such as air borne PM, cigarette smoke, DEP, fumes and dust (Upadhyay et al., 2014). The endogenous sources of ROS include normal essential metabolic processes such as cellular respiration.

7.2.1. Exogenous Sources Airborne PM is an important pollutant of urban atmosphere that has been linked to adverse health effects of the respiratory system. Hydrocarbons, sulphate and nitrate particles, elemental and organic carbon, biological endotoxins, metals and ions constitute PM (deFranco et al., 2016). Fuel combustion from vehicles, power plants, industry, household and biomass are the primary sources of PM (WHO). The size and BET (Brunauer, Emmett and Teller) surface area of PM plays an important role in its ability to enter the lungs (Kreyling et al., 2009), trigger oxidative stress and downstream inflammatory effects (Stoeger et al., 2006, Schmid and Stoeger, 2016). PM10 with a diameter of less than 10 μm evade mucocilliary clearance and enters the lower airways. PM2.5 having smaller particle size (,2.5 μm) effectively enters into the distal lung (Brunekreef and Holgate, 2002). Nanoparticles are PM with diameter less than 0.1 μm. Owing to their size nanoparticles effectively enter the alveolar region, evade macrophage clearance (Upadhyay et al., 2014) and enhance neutrophilic lung inflammation (Inoue et al., 2006). DEPs are the major components of airborne PM in the atmosphere of major cities (Mazzarella et al., 2007). They induce inflammation in the airways and proinflammatory response in macrophages and airway epithelial cells (Diaz-Sanchez et al., 1997). Cigarette (tobacco) smoke is a rich source of oxidants. It contains particles and vaporized agents including highly reactive carbon and nitrogen species suspended in a gaseous form. These oxidants cause the activation of inflammatory cells, which upon activation produce free radicals and add to the existing oxidant burden (Valavanidis et al., 2009). Electronic cigarettes (ecigarettes) are considered to be less harmful than the conventional tobacco cigarettes. However, studies indicate that e-cigarettes also cause pulmonary function impairment (Lappas et al., 2016, Valkali et al., 2014). Ecigarettes are devices that allow users to inhale an aerosol containing nicotine. Nicotine is a known carcinogen and apart from nicotine, e-cigarettes also contain toxic chemicals including cadmium, lead, acrolein, propylene glycol and glycerol (Grana et al., 2014). E-cigarette smoking has been associated with increased oxidative stress and inflammation (Schweitzer et al., 2015). In developing countries biomass exposure (wood burning stoves), second-hand smoke and indoor air pollution are major causes of COPD (Kodgule and Salvi, 2012). In these countries, the number of female cigarette smokers is much lower than the number of male cigarette smokers (Hitchman and Fong, 2011). However, the COPD prevalence is almost equal among both men and women (WHO 2015). For example, in India, the male to female ratio of COPD is nearly 1.5:1.0 despite the smoking ratio being 10:1 (Jindal et al., 2002). This has been attributed to the exposure of women to indoor air pollution in the form of combustion of solid fuel like crop residues, animal dung, wood and coal for cooking and heat which is common in the household of low and middle income countries (Salvi and Barnes, 2010). The pollutants from biomass combustion include a gaseous phase and a particle phase. Noxious gases such as CO, sulphur dioxide and nitrogen dioxide constitute the gaseous phase, and particles of PM10 and PM2.5 and nanoparticles make up the particulate phase (Torres-Duque et al., 2008). PAH, chlorinated dioxins, arsenic, lead, fluorine and vanadium are also found in biomass smoke (Salvi and Barnes, 2010). Burning solid fuels results in the production of very high levels of PM10 ranging between 3003000 μg/m3 and cooking may reach levels as high as 10,000 μg/m3 (Kogule, 2010). Air pollution is a reservoir of oxidizing agents such as ozone, nitrogen dioxide and PM. Ozone has been demonstrated to cause increased oxidative stress, epithelial injury and immunological diseases (Groves et al., 2012, Hollingsworth et al., 2007, Wiegman et al., 2015). Reaction of ozone with unsaturated fatty acids and cell membranes produce lipid ozonation products that are small, diffusible and relatively stable (Sheffield et al., 2015). Exposure to ozone has been associated with the progression of asthma and COPD (Uysal and Schapira, 2003). Prolonged exposure to exogenous sources of ROS results in the damage to cellular structure and function, a key feature of COAD pathogenesis.

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7.2.2. Endogenous Sources Cellular and subcellular metabolic activities are the major sources of endogenous ROS generation. At the subcellular level, mitochondrial respiration is the primary source of ROS production. The inner mitochondrial membrane contains an electron transfer system for ATP production, thereby continuously generating O2  2; the outer membrane produces large amount of H2O2 in the mitochondrial matrix as well as in the cytosol. Another source of ROS is the NADPH oxidase, a 5-subunit molecule found in the neutrophil. Immune cells such as neutrophils, eosinophils and macrophages are important cellular sources of ROS in the lung. Besides these, cells of the adaptive immune system such as CD8 1 T lymphocytes and Th cells have been implicated in COPD pathogenesis (Saha and Brightling, 2006). Neutrophil activation leads to the respiratory burst, which in turn results in the production of ROS that are responsible for the bactericidal activity. Myeloperoxidase and eosinophil peroxidases are produced by neutrophils and eosinophils respectively. These peroxidases, required for bacterial clearance, in turn produce hypohalous acids, HOBr and HOCl. Hypohalous acids are required for host defense against infections and they produce OH2 radicals. Eosinophilic airway inflammation has been reported in 10%40% of COPD cases during stable disease as well as during exacerbations (George and Brightling, 2016, Eltboli et al., 2014). Bronchial biopsies taken from patients during acute exacerbations and compared with stable COPD shows a 30-fold increase in the total number of eosinophils and a 3-fold increase in neutrophils (Saetta et al., 1994).

7.3. ANTIOXIDANT DEFENSE SYSTEM IN LUNG The lungs depend on strong antioxidant defense systems to combat the elevated levels of ROS produced by the various exogenous and endogenous sources. An imbalance between the ROS produced and the antioxidants to protect the lung against this surge results in oxidative stress. Innate deficiencies in the antioxidant defense system have been implicated in COPD and asthma pathogenesis.

7.3.1. Enzymatic Antioxidants Catalase (CAT), glutathione redox system, hemeoxygenase (HMOX1) and superoxide dismutases (SODs) are the primary enzymatic antioxidants in lung. Polymorphisms in glutathione S transferase mu1 (GSTM1), GSTP1 and SOD3 have been associated with a decline in lung function in COPD (Fisher et al., 2015). Table 7.1 summarizes the major enzymatic lung antioxidants, their cellular localization, phenotype in mouse models and association to COPD and asthma. Superoxide Dismutase (SOD) SODs act as the first line of defense against ROS (Sen et al., 2010). SOD1 (CuZnSOD), SOD2 (MnSOD), and SOD3 (extracellular, Cu-Zn) are the three forms of SODs. They catalyze the dismutation of two superoxide molecules into hydrogen and oxygen. SODs are the primary enzymatic antioxidants against superoxide molecules, and not only play a role in the primary defense against free radicals but also in protection against the progression of oxidant mediated lung injury. They are involved in cell homeostasis against superoxide molecules produced as a byproduct of normal cellular activities such as mitochondrial respiration. SOD1 is predominantly expressed in the cytosol but is also highly expressed in the nucleus and other organelles such as the lysosome (Chang et al., 1995, Crapo et al., 1992). SOD2 (mitochondrial) is highly expressed in the alveolar type II epithelial cells and alveolar macrophages. These cell types play a major role in protecting the lung against free radicals (Chang et al., 1995). SOD1 and SOD2 act as the bulk scavengers of superoxide in the cell cytosol and in the mitochondria. Impairment of SOD2 and SOD3 activity have been associated with asthma pathophysiology (Comhair et al., 2005) and increased oxidant mediated injury (Poonyagariyagorn et al., 2014, Carlson 1995, Folz et al., 1999). Gene polymorphisms for SOD2 are associated with COPD (Pietras et al., 2010). SOD3 is located in the extracellular matrix but it is also found in the cell junctions of airway epithelial cells and around the surface of vascular and airway smooth muscle cells (Oury et al., 1996,1994). SOD3 is involved in redox mediated signal transduction and in regulation of nitric oxide-mediated signaling across extracellular spaces (Folz and Crapo., 1994). In humans, SOD3 polymorphisms have been also associated with lung function decline

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TABLE 7.1 Major Lung Enzymatic Antioxidants and Their Association to Chronic Obstructive Airway Diseases (COADs) Like Asthma and Chronic Obstructive Pulmonary Disease (COPD) Antioxidant

Cellular localization

Targets

Knockout phenotype (2/2)

Cat

Association to COADs

Catalase (CAT)

Peroxisome, cytosol and mitochondria

Hydrogen peroxide (H2O2)

Glutathione peroxidase 1 (GPx1)

Cytosol and mitochondria

GPx(2/2) mice exhibit increased cellular H2O2, peroxynitrite sensitivity to H2O2 (de Haan et al., 2004)

Heme oxygenase 1 (HMOX1)

Smooth endoplasmic reticulum

Heme

Superoxide dismutase 1 (SOD1) [Cu-ZnSOD]

Mitochondria

Anion Sod1(2/2)mice exhibit: superoxide, 1. Increased sensitivity to hyperoxia peroxynitrite 2. Increased levels of ROS and malondialdehyde (MDA) post injury (Yoshida et al., 2000) 3. Decreased respiratory quotient (Dupuis et al., 2004)

SOD1 activity is reduced in the airway epithelium of asthmatics (DeRaeve et al., 1997)

Superoxide Dismutase 2 (SOD2) [MnSOD]

Cytosolic mitochondrial membrane

Sod2(2/2)mice exhibit: Anion superoxide, 1. Complete postnatal lethality peroxynitrite (Strassburger et al., 2005) 2. Hyperoxia (Huang et al., 2001) 3. Abnormal aerobic respiration (Melov et al., 1999)

Gene polymorphisms for SOD2 are associated with COPD (Pietras et al., 2010). Inactivation of SOD2 in the airways of asthmatics contributes to airway remodeling and hyper reactivity (Comhair et al., 2005).

Superoxide dismutase 3, extracellular (SOD3) [Cu-ZnECSOD]

Extracellular

Anion superoxide

CAT is decreased in bronchiolar epithelium of smokers with COPD (Betsuyaku et al., 1. Abnormal redox activity (Feinstein et al., 2013) 1967) 2. Increased sensitivity to oxidant mediated tissue injury (Ho et al., 2004) mice exhibit:

Hmox1(2/2) mice exhibit: 1. Increase in intracellular H2O2 and cytosolic ROS (Jais et al., 2014) 2. Increased lipid peroxidation (Yet et al., 1999) 3. Increased susceptibility to lung inflammation (Poss and Tonegawa, 1997)

SOD3(2/2)mice exhibit: 1. Increased conducting airway volume (Ganguly et al., 2007) 2. Increased sensitivity to hyperoxia (Carlsson et al., 1995) 3. Increased susceptibility to asbestos mediated lung injury and inflammation (Fattman et al., 2006) as well as bleomycin induced pulmonary fibrosis (Kliment et al., 2008)

GPx activity is increased in COPD patients (Biljak et al., 2010) Increased in the sputum of asthmatic patients (Ryter et al., 2006) and in the alveolar macrophages from smokers’ lungs (Maestrelli et al., 2001). Decreased levels associated with increased susceptibility to emphysema (Markus Exner et al., 2004).

The level of circulatory SOD3 is significantly decreased in patients with COPD (Young et al., 2006). SOD3 polymorphisms are associated with declined lung function in children (Ganguly et al., 2009) and COPD patients (Dahl et al 2008, Sørheim et al., 2010, Juul et al., 2006).

and increased risk for COPD (Young et al., 2006, Wilk et al., 2007). Two SOD3 variants (rs2536512-exon1-Thr/Ala and rs699473-promoter-C/T; allelic frequency B45%) have been associated with decreased forced expiratory volume in 1 s (FEV1) and maximum expiratory flow at 25% volume (MEF25) in children (Ganguly et al., 2009). SOD3 polymorphisms are also associated with declined lung function in COPD (Dahl et al., 2008, Sørheim et al., 2010). Mice lacking SOD3 have increased dead space volume (Ganguly et al., 2007). Transgenic mice overexpressing SOD3 are resistant against emphysema development induced by cigarette smoke and elastase (Yao et al., 2010). R213G missense mutation in SOD3 has a protective role and smokers with R213G amino acid exchange did not develop COPD (Juul et al., 2006,Young et al., 2006). The level of SOD3 is significantly decreased in the sputum of patients with COPD (Stark et al., 2008). Further, protection against the development of COPD in smokers has been observed when SOD3 activity is enhanced (Young et al., 2006).

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Catalase (CAT) Catalase is a tetrameric heme protein and detoxifies H2O2 into oxygen and water. It is a metalloprotein oxidoreductase enzyme and efficiently scavenges H2O2 when it is present in high concentrations (Day 2009, Cao et al., 2003). It is present in peroxisomes. Catalase can metabolize small molecules such as H2O2, methyl hydroxyperoxide and ethyl hydroxyperoxide (Deisseroth and Dounce, 1970, Reid et al., 1981) but not larger molecules such as lipid hydroxyperoxide products of lipid peroxidation (Comhair and Erzurum, 2010). Catalase protein and activity is decreased in asthma (Ghosh et al., 2006). Both transcript and protein levels of catalase are decreased in bronchiolar epithelium of smokers with COPD (Betsuyaku et al., 2013).

Glutathione Peroxidase (GPx) The glutathione system is a major thiol antioxidant system of the lung. The glutathione system consists of reduced glutathione (GSH), oxidized (GSSG) and glutathione peroxidase (GPx). It is a major thiol-disulphide redox buffer of the cell and scavenges H2O2 in addition to catalase; it can also eliminate large toxic peroxide molecules such as lipid peroxides (Masella et al., 2005). GPx is the key enzyme responsible for the reduction of H2O2 in the glutathione system. The reaction involves reduction of H2O2 into H2O by oxidizing glutathione to oxidized/disulphide form (GSSG). The GSSG formed is subsequently reduced back to GSH by glutathione reductase. GSSG breaks down into amino acid components for cellular uptake and recycling (Halliwell and Gutteridge.,1990). The GSH recycling mechanism prevents the depletion of cellular thiol antioxidants (Comhair and Erzurum, 2010). Very high amounts of extracellular and intracellular GPx and micromolar levels of GSH are found in the alveolar epithelial lining fluid (Cantin et al., 1987). Glutathione reductase activity is decreased while GPx activity is increased in patients with COPD when compared to healthy individuals (Biljak et al., 2010). The GSH/GSSG ratio is markedly lowered in epithelial lining fluid of asthmatic patients (Comhair and Erzurum, 2010).

Hemeoxygenase (HMOX) Hemeoxygenase is an enzyme that catalyzes the first and rate-limiting step in heme degradation reaction and produces CO, iron and biliverdin. HMOX1 and HMOX2 are the two known genetically distinct isozymes (Paine et al., 2010) and have both antioxidant anti-inflammatory functions. HMOX1 is inducible whereas HMOX2 is constitutive and noninducible (Trakshel et al., 1986). HMOX1 is present in very low levels in most cells and tissues and the levels increase during an oxidative stress condition (Keyse et al., 1990). The levels of HMOX1 are increased in the sputum of asthmatic patients (Ryter and Choi 2006) and in the alveolar macrophages from smokers’ lungs (Maestrelli et al., 2001). Upregulation of HMOX1 is associated with decreased airway inflammation, mucus production and airway hyperresponsiveness in ovalbumin-sensitized guinea pigs (Almolki et al., 2004). Polymorphism in HMOX1 promoter have been associated with reduced HMOX1 expression and increased susceptibility to emphysema (Markus Exner et al., 2004).

7.3.2. Nonenzymatic Antioxidant Vitamin E and C are the major nonenzymatic antioxidants. Vitamin E inhibits oxidation of cell membrane lipids by reacting with lipid radicals produced during lipid peroxidation reaction. α-tocopherol and gamma tocopherol are two forms of vitamin E. α-tocopherol is the predominant vitamin E in tissues, however gamma tocopherol is more effective as an antioxidant against lipophilic electrophiles (Jiang et al., 2001). Serum levels of Vitamin E are decreased during COPD exacerbations (Tug et al., 2004). The Women’s Health Study found a 10% reduction in the risk of developing chronic lung disease over a 10-year supplementation period in women using 600IU of vitamin E on alternate days (Agler et al., 2011). Vitamin C can directly scavenge superoxide molecules to hydroxyl molecule (Comhair and Erzurum, 2010). Upon supplementation of vitamin C, protection against oxidative stress and emphysema was observed in vitamin C-deficient mice (Koike et al., 2014). Lin et al., following a case control study, reported that COPD patients had lower dietary intake and lower serum levels of vitamin C than healthy controls (Lin et al., 2010).

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7.4. MECHANISM OF OXIDATIVE STRESS MEDIATED CHRONIC OBSTRUCTIVE AIRWAY DISEASE (COAD) PATHOGENESIS It is now well established that ROS-induced cellular injury, damage and downstream signaling pathways affect cellular structure and function. Structural changes need relatively high ROS concentration while lower levels cause changes in cellular processes including proliferation, apoptosis and necrosis (Day and Suzuki, 2005). Lipid peroxidation and DNA damage are structural effects of oxidative stress while disruption of cell signaling and enzymatic activity affect cellular function (Tkaczyk and Vizek, 2007). ROS-mediated effects such as protein nitration, lipid peroxidation and DNA damage contribute to the pathogenesis of COADs (Henricks and Nijkamp, 2001).

7.4.1. Lipid Peroxidation Oxidation of membrane phospholipids by ROS results in production of lipid hydroperoxide molecules within the cell membrane. This in turn impairs membrane function, inactivates membrane bound receptors and enzymes and increases tissue permeability (Barnes and Celli, 2009). Reactive aldehydes and isoprostanes are two products of lipid peroxidation. Reactive aldehydes are cytotoxic and they form adducts with histone deacetylase 2 (HDAC2) and extracellular proteins collagen and fibronectin. Formation of adducts leads to induction of specific cellular responses such as cell signaling. Acrolein, malondialdehyde (MDA), 4 hydroxy 2 nonenal (4-HNE) are examples of reactive aldehydes (Esterbauer et al., 1991). Isoprostanes are prostaglandin like compounds that are formed by free radical mediated peroxidation of essential fatty acids. Isoprostanes are stable compounds, excreted in urine and are used as accurate markers of oxidative stress (Morrow et al., 1995, Halder and Bhattacharyya, 2014). Increased levels of 8-isoprostane have been detected in bronchoalveolar lavage (BAL) of asthmatic patients. Children with severe asthma have increased lipid peroxidation (Fitzpatrick et al., 2014).

7.4.2. Protein Nitration Nitric oxide degradation by rapid reaction of nitric oxide with superoxide anion results in the formation of peroxynitrite (ONOO2) (Beckman and Koppenol, 1996). Protein nitration results due to the formation of ONOO2, which in turn adds a nitro group to the third position adjacent to the hydroxyl group of tyrosine to produce nitrotyrosine in proteins. Immune cells such as macrophages and neutrophils release NO and O2.2 into phagocytic vacuoles as a means of generating peroxynitrite to destroy endocytosed bacteria (Pacher et al., 2007). Tyrosine nitration damages epithelial cells in the lung and increased levels of nitrotyrosine have been detected in breath condensate of asthmatics and in patients suffering from COPD (Baraldi et al., 2003). Smokers with COPD have a higher average nitrotyrosine modification in many proteins relative to smokers without COPD (Jin et al., 2011).

7.4.3. DNA Damage ROS affects chromatin topology, promotes histone acetylation and inactivates HDAC2 through nitration of bases leading to promotion of proinflammatory gene expression. The most common byproducts of DNA damage are 8 hydroxyguanine (8-OH-Gua) and 8 hydroxy 2’ deoxyguanosine (8-OH-dG) (Valavanidis et al., 2009). ROS dependent, cigarette smoke induced nucleic acid oxidation in alveolar fibroblasts is involved in the pathogenesis of emphysema (Deslee et al., 2010). Expression of 8-OHdG is significantly increased in the peripheral lung of smokers compared to nonsmokers (Caramori et al., 2011, Igishi et al., 2003). Immunohistochemical analysis shows increased 8-OHdG in bronchiolar epithelial cells and alveolar epithelial cells following cigarette smoke exposure in animal models. 8-OHdG levels are increased in COPD patients compared to nonsymptomatic smokers and nonsmokers (Tzortzaki et al., 2012). Inefficiency of DNA repair is a common finding in COPD. Studies show that cigarette smoke causes DNA damage and impaired double strand break repair which is aggravated in patients with COPD and emphysema (Pastukh et al., 2011, Aoshiba et al., 2012). Immunostaining for DNA double strand markers gamma H2AX shows increased expression in lung tissue of asthmatics (Chan et al., 2016) along with an increase in DNA repair proteins Rad57, KU 70 and PAR.

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7.5. ROS-REGULATED DOWNSTREAM SIGNALING PATHWAYS IN COADs Transcription factors on activation regulate the expression of proinflammatory genes by binding to the DNA (Barnes and Adcock, 1998). ROS activates several redox sensitive transcription factors and consequently their downstream signaling cascades which in turn enhance airway inflammation in COAD (Øvrevik et al., 2015). Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), mitogen activated protein kinases (MAPK) and activator protein 1 (AP1) are the most studied redox sensitive transcription factors and their increased activity has been reported in asthma as well as COPD.

7.5.1. Nuclear Factor Kappa-Light-Chain-Enhancer of Activated B Cells (NFκB) NFκB is a transcription factor family composed of two subfamilies, NFκB proteins and Rel proteins (cRel, RelA, RelB, drosophila Dorsal and Dif). NFκB on dimerization with Rel subfamily activate transcription of target genes (Gilmore et al., 2006). The activity of NFκB is regulated by interaction with inhibitor of κB (IκB) proteins. Binding of IκB to the NFκB dimers block their nuclear localization sequence function and the dimer is thereby retained in the cytoplasm. NFκB plays a central role in proinflammatory signaling and stress response by regulating gene networks including cytokines, chemokines and adhesion molecules (Hayden and Ghosh, 2008). NFκB activation pathways involve the canonical or the alternative pathways. The classical pathway is activated by dimerization with RelA or cRel containing complexes; and the alternative pathway is activated by TNF family of cytokines (excluding TNFα) such as CD40 ligand, B cell activating factor, receptor of NFκB. ROS stimulates NFκB in the cytoplasm of the cell and inhibits its activity in the nucleus (Kabe et al., 2005). Increased markers of NFκB have been shown in the airways of asthma and COPD patients. Adults with severe to moderate asthma have higher levels of the NFκB p65 protein activation compared to the healthy subjects. Exposure to DEP results in the increased activation and nuclear translocation of NFκB (Li et al., 2011). NFκB activity is increased in sputum macrophages and bronchial biopsies of COPD patients during stable COPD as well as during exacerbations.

7.5.2. Mitogen-Activated Protein Kinases (MAPK) MAPK is a family of proteins involved in signal transduction from the cell membrane to the nucleus (Boutros et al., 2008). ERK (Extracellular signal regulated kinases), JNKs (Jun amino terminal kinases) and p38/SAPKs (Stress activated protein kinases) comprise the MAPK family and are involved in cell growth, cell proliferation and cell death (Cuadrado and Nebreda, 2010, McKay and Morrison, 2007). MAPK pathways are activated by ROS. Exposure of cells to exogenous H2O2 leads to the activation of MAPK pathways and intracellular H2O2 accumulation resulting in the prolonged activation of JNK pathway (Kamata et al., 2005). Glutamate-induced oxidative stress leads to the prolonged activation of ERK pathway by degradation of MKP1, an inhibitor of MAPKs (Choi et al., 2006). The JNK and P38 regulate the expression of proinflammatory cytokines and chemokines (Clark et al., 2009). Airway epithelial cells and macrophages exposed to cigarette smoke extract activate p38 MAP kinase pathways (Cheng et al., 2009). Activation of the p38 MAPK in alveolar macrophage has been reported in COPD, and increased activation of p38 MAPK by lipopolysaccharide is reported in macrophages of patients with severe asthma (Bhavsar et al., 2008).

7.5.3. Activator Protein 1 (AP1) AP1 is a heterodimeric complex of Fos (cFOS, Fos-B), Fos related antigen-1 [FRA1] and FRA2, Jun (JUN, JUNB and JUND) and activating transcription factor (ATF). AP1 proteins are members of the basic leucine zipper (bZIP) transcription family, characterized by a basic leucine rich area that is involved with dimerization with other transcription factors. Jun and Fos proteins are targets for JNK and ERK MAP kinases. On activation, ERK and JNK phosphorylate and activate AP1 proteins (Whitmarsh and Davis, 1996). AP1 regulates gene expression in response to various stimuli such as cytokines, growth factors, and stress signals. AP1 and NFκB cooperatively regulate inflammatory genes that are over-expressed in asthma (Barnes and Adcock, 1998). Cigarette smoke exposure modulates the activation of AP1 family members and is associated with chronic inflammation (Walters et al., 2005) and increased mucin production (Thorley and Tetley, 2007). FRA1/AP1 transcription factor plays a significant role in regulation of cigarette smoke induced lung macrophagic inflammation in mice (Vaz et al., 2015). Cigarette smoke induces FRA1 in bronchial epithelial cells at the transcription level (Zhang

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et al., 2005). Elevated levels of Fra1 expression have been reported in the lung of mice that have developed emphysema on exposure to cigarette smoke (Hirama et al., 2007).

7.6. ROS-MEDIATED EFFECTS IN COADs ROS results in direct oxidative damage to lung cells and triggers the release of various inflammatory mediators and cytokines. Inflammatory and immune cells in the airways, such as macrophages, neutrophils, and eosinophils release increased amounts of ROS in asthmatic and COPD patients (Barnes, 2008, Holgate and Polosa, 2008, Holgate et al., 2009, Comhair and Erzurum, 2010, Hogg et al., 2004, Raphine et al.,1997). The activated inflammatory cells in the airways release ROS such as O2  2, H2O2, HOCl, and  OH (Chanez et al., 1999). The H2O2 molecules amplify the initial injury by serving as a substrate for eosinophil peroxidase and myeloperoxidase which are proteolytic enzymes contributing to apoptosis of structural cells (Demedts et al., 2006). ROS also evokes bronchial hyperreactivity, mucous secretion, stimulate tracheal smooth muscle contraction, increase vascular permeability and increased synthesis of chemo attractants which are consistent with asthma and COPD phenotype (Rahman and Adcock, 2006, Rahman, 2012a,b). Enzymatic and nonenzymatic antioxidant deficiencies have been described in COADs (Duthie et al., 1991, Rahman and MacNee, 2000, Drost et al., 2005). BAL fluid of smokers contains reduced levels of vitamin E compared to nonsmokers (Pacht et al., 1986). An increase in the activity of GPx and a decrease in SOD is observed in patients with COPD (Bukowska et al., 2015). Genetic polymorphisms in several antioxidant genes such as CAT, GSTM1, GSTP1 and SOD2 have been associated with asthma and COPD. The presence of the minor allele (CT or TT) of the CAT-262C . T polymorphism increases the risk of new-onset asthma and decreases the antioxidant activity of catalase (Islam et al., 2008). Functional variants of both GSTP1 and GSTM1 are associated with adolescent onset of asthma (Islam et al., 2009). GSTP1 homozygous Val/Val genotype is associated with increased asthma risk (Tamer et al., 2004). Deletion mutations in GSTM1 have been associated with the development of emphysema in smokers and increased susceptibility to COPD (Cheng et al., 2004). Protein modifications of SOD2 in the asthmatic airways lead to the loss of SOD activity, which in turn triggers apoptosis and loss of airway epithelial cells contributing significantly to airway remodeling and hyperreactivity of asthma (Comhair et al., 2005). SOD3 mimics have proven to diminish airway inflammation in asthma animal models (Chang and Crapo, 2002). Lower lung function in COPD has been linked to polymorphisms in SOD3, and enhanced SOD3 activity has shown to increase protection against COPD development among smokers (Young et al., 2006). Markers of oxidative stress and carbonyl stress in COADs include elevated concentrations of nitrotyrosine (Comhair and Erzurum, 2010) and lipid peroxidation products, such as 8-isoprostane (Borill et al., 2008), 4-HNE (Rahman et al., 2002), and MDA (Antus, Kardos, 2015). 3- bromotyrosine, a product of eosinophilic peroxidase and eosinophils, was found to be three times higher in BAL fluid of individuals with asthma compared with the control subjects (Wu et al., 2000). In the intensive care unit, the level of 3-bromotyrosine in airways of severe asthma patients was found to be 100-fold higher than that in individuals hospitalized for causes other than asthma (MacPherson et al., 2001). Children with asthma have increased levels of MDA and lower than normal levels of glutathione (Ercan et al., 2006). CO and myeloperoxidase, markers of oxidative stress found in the exhaled breath condensate (EBC) (Kazmierczak et al., 2015) are increased during exacerbations of COPD (Kirkham and Barnes, 2013, Dekhuijzen et al., 1996), and in patients with very severe form of COPD (Kostikas et al., 2003). The levels of lipid peroxidation products such as 8- isoprostane (Pratico et al., 1998) and exhaled ethane (Paredi et al., 2000) are increased in sputum, exhaled air condensate in smokers and in patients with COPD as well as in asthmatic children (Baraldi et al., 2003) and adults (Montuschi et al., 2000). Carbonyl adducts, such as 4-HNE, have been observed in both the lung and systemically in muscle fibers of subjects with COPD (Rahman et al., 2002., Barreiro et al., 2005). Their levels correlated with disease severity as indicated by a decline in FEV1. Studies show that patients with severe COPD have the lowest GPx activity and the highest plasma MDA levels.

7.7. ANTIOXIDANT-BASED THERAPEUTIC APPROACHES FOR COADs Glucocorticoids are the main anti-inflammatory-based treatment option for asthma currently in use, and it is a relatively ineffective treatment for COPD (Barnes, 2011). Glucocorticoids are a class of corticosteroids that suppress inflammation (Barnes, 2011, Rhen and Cidlowski, 2005). Reduced number of eosinophils, T-lymphocytes,

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mast cells and dendritic cells have been observed in the EBC of asthmatics along with reduced levels of H2O2 following inhaled glucocorticoids treatment (Kharitonov et al., 2001a,b). However, owing to the differences in the nature of inflammation between asthma and COPD, even high doses do not reduce inflammation in COPD (Ferriera et al., 2001, Verhoeven et al., 2000) and some studies show glucocorticoid resistance in asthmatics (Corrigan and Loke, 2007., Loke et al., 2005). This has led to investigations aimed at discovering therapeutic strategies to overcome glucocorticoid resistance in COPD and asthma. Based on the involvement of oxidative stress in the pathogenesis of COADs, antioxidant therapies are considered to re-establish the redox imbalance. Two therapeutic approaches based on antioxidants have been reported for COADs; these involve limiting the release of free radicals and increasing antioxidant activity to detoxify ROS. The first involves the use of non enzymatic antioxidants such as spin traps or thiols, while the second uses enzymatic antioxidant mimetics as potential therapeutic strategies for pulmonary disease (Rahman, 2008, 2012a,b, Cazzola et al., 2010).

7.7.1. Nonenzymatic Antioxidants Antioxidant scavenging can be augmented by dietary antioxidant supplementation particularly of vitamins, trace elements and specific amino acids that have antioxidant effects and are precursors or cofactors for antioxidant enzymes (Villegas et al., 2014). Deficiencies in several antioxidant vitamins and amino acids have been observed in critically ill patients: ascorbic acid (vitamin C), a monosaccharide redox catalyst; and tocopherols/ tocotrienols (vitamin E), fat-soluble vitamin protect membranes from lipid peroxidation radicals. Antioxidant vitamin E has been shown to reduce oxidative stress in patients with COPD (Steinberg and Chait, 1998). The enhanced respiratory burst of smoker’s leucocytes obtained from both blood and air spaces can be reduced by administration of a mixture of vitamins E, C, 13-carotene, and selenium. However, vitamin E supplementation alone failed to reduce superoxide release from smoker’s macrophages (Brennan et al., 2000). Spin Traps Spin traps are chemical agents that can quench free radicals to form measurable stable end products. They are used for studying reactions involving free radicals (Biswas et al., 2013), and was developed as a means to study chemical and biological reactions of free radical production (Kirkham and Rahman, 2006). Spin traps trap a free singlet electron and form a stable product which can be measured, and have a nitrone or nitroxide-containing molecule that has been demonstrated to directly react with ROS/RNS at the site of inflammation when administered therapeutically (Chabrier et al., 1999). The initial shortcoming of having a very short half-life and production of hydroxyl radical has been overcome by use of organic molecules with electron withdrawing properties around the core pyrroline ring (Shi et al., 2005). Although spin traps have been used as therapeutic antioxidants for diseases such as stroke, Alzheimer’s and Parkinson’s, they are yet to be studied in COPD and asthma. Thiols Thiols are a group of organic compounds that contain a sulphydryl group and are important antioxidant components. N-acetyl-L-cysteine (NAC) is an acetyl derivative of the amino acid, cysteine and is a strong reducing agent. NAC may play a therapeutic role in COPD management due to its ability to increase intracellular GSH. NAC can also interact directly with oxidants and is used as a mucolytic agent to reduce mucus viscosity and improve mucociliary clearance. Long-term use of NAC (600 mg twice daily) in patients with moderate to severe COPD prevents exacerbations (Zheng et al., 2014). A clinical trial with oral administration of NAC (600 b.i.d for 2 months) reduced the oxidant burden in airways of stable COPD patients (DeBenedetto et al., 2005). Though there is some evidence that the administration of NAC provides benefit to COPD patients, its use as a maintenance therapy is debatable (Decramer et al., 2001). N-acystelyn is a lysine salt of NAC, has antioxidant properties and can reduce ROS levels and ROS mediated inflammatory events. It enhances GSH levels twice as effectively as NAC and has a neutral pH unlike NAC which is acidic (Gillisen et al., 1997), and does not cause any side effects when administered directly into the lung as aerosols in healthy volunteers. Erdosteine, fudosteine and carbocysteine are three thiols that possess both antioxidants and mucolytic properties and reduce bacterial adhesiveness. Patients who received erdosteine had significantly fewer COPD exacerbations and show less hospitalization time compared to the placebo group (Moretti and Fagnani, 2015). Fudosteine is a mucoactive agent (Komatsu et al., 2005), cysteine-donating compound that increases the cysteine levels of the cells and has greater bioavailability than NAC. It also inhibits mucin 5AC (MUC5AC) triggered mucin

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production and may be useful in controlling stress-related mucus secretion in COPD patients (Rhee et al., 2008). Prevention of airway hyperresponsiveness, accumulation of lymphocytes in airways and reduction in goblet cell hyperplasia, subepithelial collagenization and basement membrane thickening were observed in mice treated with fudosteine following an ovalbumin challenge (Ueno-lio et al., 2013). Carbocysteine is another mucoactive drug with in vitro free radical scavenging and anti-inflammatory properties. It improves airway mucus clearance and has anti-inflammatory effects including antioxidant properties. Carbocysteine has been clinically used to treat COPD patients and has shown that long-term use reduced the rate of exacerbations in COPD patients but not during short-term use (Zheng et al., 2008).

7.7.2. Enzymatic Antioxidant Mimetics Restoration of altered antioxidant enzyme activity can be achieved by small molecules possessing catalytic properties otherwise known as enzyme mimetics that mimic the activity of a larger enzyme-based molecule. These new antioxidants provide an alternative approach to attenuate oxidant-related lung injury in vivo. They get distributed more easily to tissues, reach higher concentrations in intracellular domains and avoid the problem of antigenicity. SOD Mimetics There are three types of manganese-based SOD mimetics namely macrocyclic ligands, metaloporphyrins and salens. Salens are reported to have catalase like activity and hence can neutralize the H2O2 in the cells along with their ability to decompose toxic ONOO2 (Sharpe et al., 2002). Significant decrement in the levels of markers of oxidative stress in the lungs, and suppression of emphysema development has been observed in response to M40419 (macrocyclic ligand) in animal models of lung diseases (Tuder et al., 2003). AEOL10113 (metalloporphyrin) was found to inhibit both airway inflammation and airway hyper-reactivity in an ovalbumin challenged model of airway inflammation (Chang and Crapo, 2002). In addition, AEOL10150 (metalloporphyrins) was demonstrated to inhibit cigarette smoke-induced lung inflammation and decrease lipid peroxidation and generation of ONOO2 following cigarette smoke exposure in rat models (Smith et al., 2002). Salens protect against cigarette smoke-mediated oxidative stress in the rat (Luchese et al., 2007). GPx Mimetics Ebselen is a selenium-based organic complex, which can mimic the activity of GPx (Rahman, 2012a,b). It is a strong antioxidant, which increases the efficiency of glutathione and has a strong neutralizing effect against peroxynitrite radicals. Ebselen prevents lipopolysaccharide-induced airway inflammation (Haddad et al., 2002, Zhang et al., 2002) and cigarette smoke-induced inflammation (Duong et al., 2010) in vivo. BXT-51072 and BXT51077 are low molecular weight, orally active, organoselenium GPx mimetics. BXT-51072 and BXT-51077 increase the rate of peroxide metabolism, inhibits inflammation and oxidative damage by preventing the activation of inflammatory mediators (Moutet et al., 1998). To summarize, it is well documented that oxidant imbalance is a key feature in the pathogenesis and progression of COADs like asthma and COPD. Oxidative stress and proinflammatory response complement each other in COAD pathogenesis. Genetic predisposition like variations and altered regulation of the antioxidant genes and redox sensitive transcription factors may also render susceptibility to COADs. Dietary supplementation particularly of nonenzymatic antioxidants such as vitamin E and C are suggested as preventive measures for COAD development. Thiols, and antioxidant mimetics like spin traps, are also being explored as potential therapeutic options for COADs. Further studies with particular focus on the effect of oxidative stress during lung development may provide novel insights into COAD pathogenesis.

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Further Readings Antus, B., Kardos, Z., 2015. Oxidative stress in COPD: molecular background and clinical monitoring. Curr. Med. Chem. 22, 627650. Aoshiba, K., Koinuma, M., Yokohori, N., Nagai, A., 2003. Immunohistochemical evaluation of oxidative stress in murine lungs after cigarette smoke exposure. Inhal. Toxicol. 15, 10291038. Baraldi, E., Giordano, G., Pasquale, M.F., Carraro, S., Mardegan, A., Bonetto, G., et al., 2006. 3-Nitrotyrosine, a marker of nitrosative stress, is increased in breath condensate of allergic asthmatic children. Allergy 61, 9096. Barnes, P.J., 2001. New treatments for chronic obstructive pulmonary disease. Curr. Opin. Pharmacol. 1, 217222. Barreiro, E., Rabinovich, R., Marin-Corral, J., Barbera`, J.A., Gea, J., Roca, J., 2009. Chronic endurance exercise induces quadriceps nitrosative stress in patients with severe COPD. Thorax. 64, 1319. Borrill, Z.L., Roy, K., Singh, D., 2008. Exhaled breath condensate biomarkers in COPD. Eur. Respir. J. 32, 472486. Caramori, G., Romagnoli, M., Casolari, P., Bellettato, C., Casoni, G., Boschetto, P., et al., 2003. Nuclear localisation of p65 in sputum macrophages but not in sputum neutrophils during COPD exacerbations. Thorax. 58, 348351. Cazzola, M., Floriani, I., Page, C.P., 2010. The therapeutic efficacy of erdosteine in the treatment of chronic obstructive bronchitis: a metaanalysis of individual patient data. Pulm. Pharmacol. Ther. 23, 135144. Cazzola, M., Page, C.P., Calzetta, L., Matera, M.G., 2012. Pharmacology and therapeutics of bronchodilators. Pharmacol. Rev. 64, 450504. Cosio, B.G., Mann, B., Ito, K., Jazrawi, E., Barnes, P.J., Chung, K.F., et al., 2004. Histone acetylase and deacetylase activity in alveolar macrophages and blood mononocytes in asthma. Am. J. Respir. Crit. Care. Med. 170, 141147. Day, B.J., 2004. Catalytic antioxidants: a radical approach to new therapeutics. Drug Discov. Today 9, 557566. Day, B.J., 2008. Antioxidants as Potential Therapeutics for Lung Fibrosis. Antioxid. Redox Signal. 10, 355370. Decramer, M., Dekhuijzen, P.N., Troosters, T., van Herwaarden, C., Rutten-van Molken, M., van Schayck, C.P., et al., 2001. The Bronchitis Randomized On NAC Cost-Utility Study (BRONCUS): hypothesis and design. BRONCUS-trial Committee. Eur. Respir. J. 17, 329336. Farsalinos, K.E., Voudris, V., Poulas, K., 2015. Are metals emitted from electronic cigarettes a reason for health concern? A risk-assessment analysis of currently available literature. Int. J. Environ. Res. Public Health. 12, 52155232. Giembycz, M.A., Lindsay, M.A., 1999. Pharmacology of the eosinophil. Pharmacol. Rev. 51, 213340. Gilliland, F.D., 2009. Outdoor air pollution, genetic susceptibility, and asthma management: opportunities for intervention to reduce the burden of asthma. Pediatrics. 123 (Suppl 3), 73. II. REACTIVE OXYGEN SPECIES, OXIDATIVE STRESS AND IMMUNE CELL ACTIVATION

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Gilliland, F.D., Berhane, K., Li, Y.-F., Rappaport, E.B., Peters, J.M., 2003. Effects of early onset asthma and in utero exposure to maternal smoking on childhood lung function. Am. J. Respir. Crit. Care. Med. 167, 917 924. Gilliland, F.D., Islam, T., Berhane, K., Gauderman, J.W., McConnell, R., Avol, E., et al., 2006. Regular smoking and asthma incidence in adolescents. Am. J. Respir. Crit. Care. Med. 174, 1094 1100. Hayden, M.S., Ghosh, S., 2012. NF-kappaB, the first quarter-century: remarkable progress and outstanding questions. Genes Dev. 26, 203 234. Hoenderdos, K., Condliffe, A., 2013. The neutrophil in chronic obstructive pulmonary disease. Am. J. Respir. Cell Mol. Biol. 48, 531 539. Holgate, S.T., Davies, D.E., Powell, R.M., Howarth, P.H., Haitchi, H.M., Holloway, J.W., 2007. Local genetic and environmental factors in asthma disease pathogenesis: chronicity and persistence mechanisms. Eur. Respir. J. 29, 793 803. Islam, T., Breton, C., Salam, M.T., McConnell, R., Wenten, M., Gauderman, J.W., et al., 2010. Role of inducible nitric oxide synthase in asthma risk and lung function growth during adolescence. Thorax. 65, 139 145. Jatakanon, A., Uasuf, C., Maziak, W., 1999. Neutrophilic inflammation in severe persistent asthma. Am. J. Respir. Crit. Care Med. 160, 1532 1539. Jiang, L., Diaz, P.T., Best, T.M., Stimpfl, J.N., He, F., Zuo, L., 2014. Molecular characterization of redox mechanisms in allergic asthma. Ann. Allergy Asthma Immunol. 113, 137 142. John-Schuster, G., Hager, K., Conlon, T.M., Irmler, M., Beckers, J., Eickelberg, O., et al., 2014. Cigarette smoke-induced iBALT mediates macrophage activation in a B cell-dependent manner in COPD. Am. J. Physiol. Lung Cell Mol. Physiol. 307, L692 706. Karin, M., Ben-Neriah, Y., 2000. Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity. Annu. Rev. Immunol. 18, 621 663. Keyse, S.M., 2000. Protein phosphatases and the regulation of mitogen-activated protein kinase signalling. Curr. Opin. Cell. Biol. 12, 186 192. Kreyling, W.G., Semmler-Behnke, M., Moller, W., 2006. Ultrafine particle-lung interactions: does size matter?. J. Aerosol Med. 19, 74 83. Loch, T., Vakhrusheva, O., Piotrowska, I., Ziolkowski, W., Ebelt, H., Braun, T., et al., 2009. Different extent of cardiac malfunction and resistance to oxidative stress in heterozygous and homozygous manganese-dependent superoxide dismutase-mutant mice. Cardiovasc. Res. 82, 448 457. MacNee, W., 2001. Oxidative stress and lung inflammation in airways disease. Eur. J. Pharmacol. 429, 195 207. MacNee, W., Rahman, I., 2001. Is oxidative stress central to the pathogenesis of chronic obstructive pulmonary disease? Trends Mol. Med. 7, 55 62. Martindale, J.L., Holbrook, N.J., 2002. Cellular response to oxidative stress: signaling for suicide and survival. J Cell Physiol. 192, 1 15. Matera, M., Calzetta, L., Peli, A., Scagliarini, A., Matera, C., Cazzola, M., 2005. Immune sensitization of equine bronchus: glutathione, IL-1β expression and tissue responsiveness. Respir. Res. 6, 1 7. Mazzarella, G., Ferraraccio, F., Prati, M.V., Annunziata, S., Bianco, A., Mezzogiorno, A., et al., 2007. Effects of diesel exhaust particles on human lung epithelial cells: an in vitro study. Respir. Med. 101, 1155 1162. McConnell, R., Islam, T., Shankardass, K., Jerrett, M., Lurmann, F., Gilliland, F., et al., 2010. Childhood incident asthma and traffic-related air pollution at home and school. Environ. Health. Perspect. 118, 1021 1026. Montuschi, P., Kharitonov, S.A., Barnes, P.J., 2001. Exhaled carbon monoxide and nitric oxide in COPD. Chest. 120, 496 501. Noguera, A., Batle, S., Miralles, C., Iglesias, J., Busquets, X., MacNee, W., et al., 2001. Enhanced neutrophil response in chronic obstructive pulmonary disease. Thorax. 56, 432 437. Odajima, N., Betsuyaku, T., Nagai, K., Moriyama, C., Wang, D.-H., Takigawa, T., et al., 2010. The role of catalase in pulmonary fibrosis. Respir. Res. 11, 183. Osama, E., Mona, B., Ian, P., Peter, C., Chris, B., 2014. Eosinophilic chronic obstructive pulmonary disease is not associated with helminth infection or exposure. J. Pulm. Respir. Med. 04, 179. Piade´, J.J., Jaccard, G., Dolka, C., Belushkin, M., Wajrock, S., 2015. Differences in cadmium transfer from tobacco to cigarette smoke, compared to arsenic or lead. Toxicol. Rep. 2, 12 26. Poli, G., Leonarduzzi, G., Biasi, F., Chiarpotto, E., 2004. Oxidative stress and cell signalling. Curr. Med. Chem. 11, 1163 1182. Rahman, I., 2002. Oxidative stress and gene transcription in asthma and chronic obstructive pulmonary disease: antioxidant therapeutic targets. Curr Drug Targets Inflamm Allergy 1 (3), 291 315. Rahman, I., 2005a. Oxidative stress in pathogenesis of chronic obstructive pulmonary disease: cellular and molecular mechanisms. Cell Biochem. Biophys. 43, 167 188. Rahman, I., 2005b. The role of oxidative stress in the pathogenesis of COPD: implications for therapy. Treat Respir. Med. 4, 175 200. Ryter, S.W., Choi, A.M., 2009. Heme oxygenase-1/carbon monoxide: from metabolism to molecular therapy. Am. J. Respir. Cell Mol. Biol. 41, 251 260. Saetta, M., Di Stefano, A., Turato, G., Facchini, F.M., Corbino, L., Mapp, C.E., et al., 1998. CD8 1 T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease. Am. J. Respir. Crit. Care. Med. 157, 822 826. Sano, H., Tohda, Y., Adachi, M., Fukuda, T., Ohta, K., Shoji, S., et al., 2012. Preventive effect of carbocysteine on exacerbation of asthma, GAIA randomised, placebo-controlled multi-centre study. Eur. Respir. J. 40. Sircar, G., Saha, B., Bhattacharya, S.G., Saha, S., 2014. Allergic asthma biomarkers using systems approaches. Front. Genet. 4, 308. Stanescu, D., Sanna, A., Veriter, C., Robert, A., 1998. Identification of smokers susceptible to development of chronic airflow limitation: a 13year follow-up. Chest. 114, 416 425. Turner, S.W., Palmer, L.J., Rye, P.J., Gibson, N.A., Judge, P.K., Cox, M., et al., 2004. The relationship between infant airway function, childhood airway responsiveness, and asthma. Am. J. Respir. Crit. Care. Med. 169, 921 927. Vakali, S., Tsikrika, S., 2014. E-Cigarette acute effect on symptoms and airway inflammation: comparison of nicotine with a non-nicotine cigarette. Tobacco Induced Dis. 12 (Suppl. 1), A35. Valavanidis, A., Vlachogianni, T., Fiotakis, K., Loridas, S., 2013. Pulmonary oxidative stress, inflammation and cancer: respirable particulate matter, fibrous dusts and ozone as major causes of lung carcinogenesis through reactive oxygen species mechanisms. Int. J. Environ. Res. Public Health 10, 3886 3907. Vroman, H., van den Blink, B., Kool, M., 2015. Mode of dendritic cell activation: The decisive hand in Th2/Th17 cell differentiation. Implications in asthma severity? Immunobiology 220, 254 261.

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8 Immune Responses in the Upper Respiratory Tract in Health and Disease Derek B. McMahon and Robert J. Lee University of Pennsylvania School of Medicine, Philadelphia, PA, United States

8.1. OVERVIEW OF UPPER RESPIRATORY PHYSIOLOGY AND INNATE IMMUNITY The upper respiratory tract, termed the sinonasal cavity, comprises the nose and paranasal sinuses, which form a labyrinthine structure that has been hypothesized to exist to lighten the skull, enhance vocal resonance, and/or generate thermal insulation (Dalgorf and Harvey, 2013). While we do not yet fully understand all of the functions of the sinonasal cavity, it is nonetheless the initial contact point for many environmental insults, including pollutants and infectious agents. In addition to sampling air for olfactory sensation, the sinonasal cavity filters and humidifies inspired air to protect the lower airways. The sinonasal cavity is thus the front line of respiratory defense. When it fails, upper airway infections can seed lower airway infections. Additionally, upper airway inflammation in disease such as chronic rhinosinusitis (CRS) and allergy can be intimately linked to lower airway inflammatory diseases such as asthma (Meena et al., 2013). Notably, an emerging novel concept that will be highlighted in this chapter is the discovery that bitter and sweet taste receptors are used by both taste cells of the tongue as well as cells involved in immune surveillance. Taste receptors were first discovered in the airways within the cilia of ciliated epithelial cells, which, along with mucin-secreting goblet cells, are one of the two major respiratory epithelial cell types from the nose down to cartilaginous bronchi. Taste receptors are also expressed in dedicated epithelial solitary chemosensory cells (SCCs), long known to exist in the gills of fish but only recently discovered in mammals. The taste receptors in both ciliated epithelial cells and SCCs both serve important immune roles (Lee et al., 2012, 2014b; Lee and Cohen, 2014a; Lee and Cohen, 2015b), highlighted below. This first section will describe the general innate defense mechanisms of the sinonasal cavity. The regulation of these defenses by various receptors will be discussed in Section 2, focusing on both toll-like and taste receptors. Finally, in Section 3, we will focus on specific diseases that affect upper respiratory immunity.

8.1.1. Mucociliary Clearance as the Foundation of Sinonasal Innate Immunity The nose and sinuses (termed the sinonasal cavity) trap and filter inspired particulates and pathogens. This is routinely accomplished by mucociliary clearance (MCC) (Knowles and Boucher, 2002), a specialized physical defense unique to the airway, complementing the barrier formed by epithelial cells surrounded by tight junctions (Fig. 8.1). MCC has two major components: mucus production and transport. The airway surface liquid (ASL) lining the respiratory tract consists of a top layer of antimicrobial-rich mucus gel, formed by glycosylated soluble mucins, including Muc5AC and Muc5B, produced by goblet cells (Jackson, 2001; Groneberg et al., 2003; Rogers, 2003) and submucosal glands (Kirkham et al., 2002; Martinez-Anton et al., 2006). This mucus layer traps inhaled pathogens and irritants via a wide range of carbohydrate side chains coating the web of mucins, creating a “combinatorial library of binding sites” (Knowles and Boucher, 2002). The mucus layer rests on top of a less-viscous periciliary layer (PCL) surrounding the cilia of airway epithelial cells (Fig. 8.2) that beat rapidly. Constant and Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00008-1

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Trapped pathogens Airway mucus Clearance

Mucus secretion

Ciliated epithelial cells

AMP secretion ROS/RNS generation

Coordinated ciliary beating Fluid secretion

Goblet cell

Chemokines (IL-8, MIP-1, MCP-1, RANTES, etc.)

Cytokines (IL-1β, IL-6, TNFα, etc.)

FIGURE 8.1 Overview of sinonasal innate immune mechanisms. In healthy tissue, respiratory epithelial cells are linked by tight junctions to form a protective physical barrier. Mucus overlays the epithelium and traps inhaled pathogens and debris, which are then removed by mucociliary clearance (MCC). Constant beating of motile cilia drives the pathogen-laden mucus toward the oropharynx, where it is then cleared by expectoration or swallowing. MCC is further regulated by secretion of mucus as well as ion and fluid transport, which controls the mucus viscosity. MCC is complemented by the generation of reactive oxygen and nitrogen species (ROS and RNS, respectively) and the production of antimicrobial peptides (AMPs). During more chronic exposure to pathogens, epithelial cells secrete cytokines (e.g., interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF) α, and others) and chemokines (e.g., IL-8, macrophage inflammatory protein (MIP)-1, monocyte chemotactic protein (MCP)-1, regulated on activation normal T expressed and secreted protein (RANTES)) to activate inflammatory pathways and recruit dedicated immune cells.

FIGURE 8.2 Airway epithelial motile cilia. (A) Differential interference contrast image showing cultured airway epithelial ciliated and nonciliated cells. Scale bar is 20 μm. (B) The beat cycle of each motile creates unidirectional force against the airway epithelial mucus layer. Cilia beat on average 810 cycles per second (810 Hz), but beating can range from 5 to 15 Hz depending on anatomical location and whether cells are in a stimulated or resting state. The coordination of hundreds of cilia per cell and higher level organization of groups of hundreds to thousands of ciliated cells creates a metachronal wave that drives mucociliary transport. (C) Immunofluorescence image showing airway tissue stained for the cilia-enriched tubulin isoform β-tubuin IV.

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coordinated ciliary beating (B815 Hz) (Salathe, 2007) facilitates transport of particle- and pathogen-laden mucus to the oropharynx, where it is swallowed or expectorated, and cleared from the airway (Cohen, 2006; Antunes et al., 2009; Stevens et al., 2015). Mucin proteins are large thread-like glycoproteins with peptide backbones and oligosaccharide side chains (Stevens et al., 2015) that can directly bind surface adhesions on microorganisms (Lamblin et al., 1991), including Streptococcus pneumoniae, Haemophilus influenzae (Davies et al., 1995; Hakansson et al., 1996), Moraxella catarrhalis (Reddy et al., 1997; Bernstein and Reddy, 2000), Pseudomonas aeruginosa (Vishwanath and Ramphal, 1985; Prince, 1992) and Pseudomonas cepacia (Sajjan and Forstner, 1992). Ciliated cells also express membrane-tethered mucins, including Muc1, 4, and 15 (Hattrup and Gendler, 2008). Their functions are less well understood than soluble mucins, but tethered mucins may form a lubricating periciliary brush-like structure keeping the mucus and PCL layers separated (Button et al., 2012). MCC is regulated by a diverse array of neurotransmitters (e.g., adenosine trisphosphate, acetylcholine, etc.) and neuropeptides (vasoactive intestinal peptide, substance P, etc.) that regulate fluid and mucus secretion (Lee and Foskett, 2014) and ciliary function (Workman and Cohen, 2014), as well as receptors for bacterial products (Lee et al., 2012) and mechanical stresses (Winters et al., 2007; Zhao et al., 2012). Inefficient MCC can cause or exacerbate respiratory disease. Several pathogens produce compounds that impair ciliary motion or coordination, including H. influenzae, S. pneumoniae, Staphylococcus aureus, Aspergillus fumagatus, and P. aeruginosa (Stevens et al., 2015). Hypoxia created by mucostasis or anatomical obstruction may also affect MCC by inhibiting ion transport and/or promoting polypogenesis to create anatomical obstructions (Stevens et al., 2015). Genetic and acquired defects of MCC in disease are discussed in Section 3.

8.1.2. Epithelial Cells as Immune Effectors: Generation of Antimicrobial Peptides and Radicals In addition to transporting mucus, sinonasal epithelial cells also produce substances with direct antipathogen effects such as lysozyme, lactoferrin, antitrypsin, defensins, and surfactants (Ooi et al., 2008; Ramanathan and Lane, 2007; Fig. 8.1). Most of these substances are tonically secreted, but their expression can be upregulated during infection (Parker and Prince, 2011) or after epithelial damage due to plasma extravasation (Persson, 1991). Of these substances, lysozyme is a small (B14 kDa) cationic protein that is secreted by most exocrine glands, including airway submucosal glands (Klockars and Reitamo, 1975). Lysozyme disrupts bacterial cell walls by catalyzing the hydrolytic degradation of the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetyl-D-glucosamine. Additionally, binding of lysozyme to the bacterial cell wall facilitates phagocytosis of the bacteria by host macrophages (Parker and Prince, 2011). Lysozyme is potent against gram-positive bacteria and also displays mild reactivity with gram-negative bacteria and fungi (Stevens et al., 2015). Currently, it is unclear whether lysozyme levels are increased (Woods et al., 2012) or decreased (Kalfa et al., 2004; Tewfik et al., 2007) in CRS. Lactoferrin was first identified as a component in milk, then later discovered in the secretions of various other exocrine glands (Parker and Prince, 2011). Lactoferrin functions as a chelator of iron, which is an important micronutrient essential for bacterial and fungal metabolism and growth (Parker and Prince, 2011). Bacteria also use iron to catalyze mucin degradation, causing a disruption in the protective mucosal barrier; thus, lactoferrin acts to inhibit this invasive activity (Clamp and Creeth, 1984)]. In gram-negative bacteria, lactoferrin causes the release of lipopolysaccharide (LPS) from the bacterial cell wall, directly damaging its structural integrity (Prokhorenko et al., 2009; Legrand and Mazurier, 2010; Legrand, 2012). In addition to these effects, lactoferrin also binds conserved microbial structures known as pathogen-associated molecular patterns (PAMPs), including LPS and bacterial unmethylated CpG DNA, thus preventing them from interacting with mammalian cells, a process that would normally have deleterious consequences (Puddu et al., 2009; Legrand and Mazurier, 2010; Legrand, 2012). Lactoferrin can also stimulate an immune response by interacting with surface receptors on both innate (natural killer cells, macrophages, basophils, neutrophils, and mast cells) and adaptive (antigen-presenting cells and lymphocytes) immune cells (Legrand and Mazurier, 2010; Legrand, 2012). Finally, lactoferrin can also bind to viral receptors or the viruses themselves, preventing entry of RNA and DNA viruses into host cells (van der Strate et al., 2001; Sano et al., 2003). The collectin (collagen-lectin) family of proteins, which include surfactant proteins and mannose-binding protein, exhibit antimicrobial properties against various airway bacteria (Sano and Kuroki, 2005; Woodworth et al., 2006, 2007a,b; Skinner et al., 2007). Collectins contain calcium-dependent carbohydrate-binding domains that recognize and bind to PAMPs, including LPS, and trigger bacterial clearance (Sano and Kuroki, 2005; Woodworth et al., 2006, 2007a,b; Skinner et al., 2007). LL-37, also known as cathelicidin (Bals et al., 1998b), is a 37-amino acid

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peptide produced by human nasal mucosa (Kim et al., 2003; Chen and Fang, 2004a) that has broad antibacterial activity, including potency against Pseudomonas biofilms when evaluated using animal models of CRS (Chennupati et al., 2009). LL-37 is thought to function by neutralizing gram-negative LPS, thus having potential anti-inflammatory properties (Golec, 2007). Interestingly, transcription of LL-37 is upregulated through the interaction of the bioactive form of vitamin D, 1,25-dihydroxycholecalciferol, with the vitamin D receptor (Yim et al., 2007). Sinonasal epithelial cells express 1-α-hydroxylase, the enzyme critical for the synthesis of bioactive vitamin D; sinonasal epithelial cells can convert inactive vitamin D precursors to bioactive vitamin D and thus enhance LL-37 production (Sultan et al., 2013). Therefore, vitamin D production is thought to play an important role in airway innate immunity (Hansdottir and Monick, 2011) in CRS and allergic rhinitis patients (Akbar and Zacharek, 2011; Bacchetta et al., 2013). The sinonasal epithelium also expresses members of the α-defensin and β-defensin families of proteins (Lee et al., 2002; Chen and Fang, 2004b). The expression of these proteins is upregulated in response to bacterial or viral exposure (Harder et al., 2000; Dauletbaev et al., 2002; Proud et al., 2004). Defensins are small, cationic proteins thought to induce the formation of pores in the membranes of gram-positive and gram-negative bacteria, and fungi. Defensins also bind to viruses, preventing their entry into host cells (Vareille et al., 2011). For example, β-defensins 2 and 3 have been shown to directly bind to viruses, which activates cytokine production and recruits immune cells to the location of infection (Schutte and McCray, 2002). Notably, the function of cationic defensins is inhibited under high ionic strength conditions (Bals et al., 1998a; Singh et al., 1998). This observation has led some to hypothesize that abnormalities in epithelial ion transport, as described in cystic fibrosis (CF) patients, may reduce defensin function through variations in ASL electrolyte concentration (Tarran et al., 2001). Antibodies, including immunoglobulins (Ig) A and G, are produced by mucosa-associated lymphoid tissue (MALT) of the inferior and middle turbinates, providing another layer of host defense (Wu and Russell, 1997; Fokkens and Scheeren, 2000). Dendritic cells aid antibody production by presenting mucosal antigens to T cells, which then engage B cells in the MALT, resulting in the synthesis of antibodies (Rampey et al., 2007). Secretory serous cells of submucosal glands secrete IgA into the airway through an intracellular transport system. IgA receptors expressed on the basolateral membranes of serous cells (Goodman et al., 1981; Kaliner, 1992) bind to IgA in the serosal interstitial fluid. This IgA/receptor complex (termed secretory IgA) is then transcytosed through the cell and then released into the airway. Interestingly, selective IgA deficiency is associated with recurrent sinonasal infections (Sethi et al., 1995). IgA and IgG function to opsonize pathogens. Additionally, aggregation of IgA molecules activates the classical complement pathway (Lamm, 1997; Pilette et al., 2001). IgE, which plays a central role in allergy, and IgM, a low affinity yet high avidity antibody, are also present in nasal secretions (Butcher et al., 1975; Miller et al., 1979; Swart et al., 1991; Biewenga et al., 1995) and likely also contribute to mucosal defense. Sinonasal mucus also contains nonprotein antimicrobial agents such as antimicrobial lipids and reactive oxygen species (ROS). Antimicrobial lipids, including cholesteryl linoleate and cholesteryl arachidonate, which are increased in CRS (Lee et al., 2010), may contribute to the antimicrobial properties of nasal secretions, though their mechanism of action is not fully understood (Do et al., 2008). The sinonasal mucosa also generates ROS, such as hydrogen peroxide (H2O2), which directly damages bacterial proteins and DNA. Lactoperoxidase (LPO; Wijkstrom-Frei et al., 2003) is an enzyme that functions as an antibacterial agent by catalyzing the oxidation of substrates by H2O2. Airway epithelial ciliated cells produce H2O2 through the action of NADPH oxidase isoforms, including DUOX1 and DUOX2 (Forteza et al., 2005; Fischer, 2009). Thiocyanate is an important substrate for the LPO/hydrogen peroxide defense system, i.e., secreted across the epithelium and oxidized via LPO to hypothiocyanite, an antibacterial, antifungal, and antiviral compound (Fragoso et al., 2004). Thiocyanate is of particular interest in CF, as the CF transmembrane conductance regulator (CFTR) ion channel may regulate airway epithelial thiocyanate transport, directly linking ion transport with host defense (Conner et al., 2007). CFTR defects may reduce thiocyanate secretions, leading to an impaired airway innate defense (Moskwa et al., 2007; Thomson et al., 2010; Lorentzen et al., 2011). Reactive nitrogen species, mainly nitric oxide (NO), are also produced by the sinonasal epithelium and thought to be critical for upper airway immunity (Gustafsson et al., 1991; Haight et al., 1999; Maniscalco et al., 2007). NO was first discovered in 1991 and identified in human breath (Gustafsson et al., 1991), with the primary source originating from the paranasal sinuses (Maniscalco et al., 2007). NO is generated by the enzyme NO synthase (NOS), which is expressed in the cilia and microvilli of the sinonasal epithelium (Deja et al., 2003; Degano et al., 2011), and most abundantly in the maxillary sinus (Deja et al., 2003). NO rapidly diffuses inside bacteria, where its reactive derivatives, S-nitrosothiols and peroxynitrites, function to damage bacterial DNA, membrane lipids, and any enzymes containing thiol groups or metal cofactors (Fang, 1997; Marcinkiewicz, 1997).

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NO is also effective against viral pathogens through inhibiting viral replication (Vareille et al., 2011). In the airway tissue, NO activates gualylyl cyclase to produce cyclic-GMP, activating protein kinase G, which increases ciliary beating, leading to increased MCC. Though some studies have observed increased NO elevates host defense in vivo (MacMicking et al., 1995; Fang, 1997; Grasemann and Ratjen, 2012), other studies have suggested overly elevated NO levels can actually be detrimental (Nathan, 1997). Several clinical studies have investigated sinonasal NO as a diagnostic, prognostic, or efficacy indicator in sinonasal disease, although confounding factors, such as the wide range of NO measurement methods or heterogeneous population studies, limit the conclusions that can currently be drawn (Phillips et al., 2011).

8.2. REGULATION OF IMMUNE RESPONSES BY SENSORY RECEPTORS IN THE SINONASAL CAVITY It has been proposed that the immune system is the mammalian “sixth sense” (Blalock, 2005; Blalock and Smith, 2007; Bedford, 2011). The upper respiratory tract utilizes several types of receptors to sense invading pathogens and react to them. We will focus on Toll-like receptors (TLRs), long known as important regulators of immune responses, and taste receptors, which have been recently identified as immune players.

8.2.1. Toll-Like Receptors and Other Pattern Recognition Receptors In addition to being the engines driving MCC, airway epithelial cells function in immune detection via the well-studied TLRs (Muir et al., 2004; Greene and McElvaney, 2005; Ramanathan and Lane, 2007; Ooi et al., 2008), which recognize conserved structures called PAMPs, (Parker and Prince, 2011), including lipoteichoic acid from gram-positive bacteria and LPS from gram-negative bacteria (Ramanathan and Lane, 2007; Ooi et al., 2008). TLRs have long been known to activate antimicrobial compound production. Sinonasal epithelial cells express at least 10 TLR isoforms (Lane et al., 2006a), and changes in expression have been observed in CRS (Lane et al., 2006b). TLRs are located both on the cell surface and in intracellular endosomes, which allows them to play an important role in detecting viruses, including rhinoviruses, RSV, and influenza. TLR signaling cascades involve intracellular proteins MyD88, TIRAP, TRAM, and TRIF, which activate transcription factors and interferon response factors that activate transcription of mucins (Parker and Prince, 2011), antimicrobial peptides (AMPs) (Lee et al., 2014b), cytokines, chemokines, and type I interferon (Vareille et al., 2011) (Fig. 8.3A). The secretion of these proinflammatory mediators is an important link between innate and adaptive immunity. TLRs also respond to molecules released after cell damage (Miyake, 2007), termed damage-associated molecular patterns (DAMPs), which can exacerbate inflammation. TLR polymorphisms have been linked to differential severity of inflammatory lower respiratory diseases (Kanagaratham et al., 2011; Nuolivirta et al., 2013; Koponen et al., 2014; Zuo et al., 2015; Lauhkonen et al., 2016), but it remains to be determined if TLR polymorphisms affect CRS susceptibility or severity by altering responses to PAMPs (Hamilos, 2014) or DAMPs (Van Crombruggen et al., 2013). Additionally, cytoplasmic helicases RIG-1 and MDA5 recognize RNA viruses by detecting intracellular viral double-stranded RNA (vRNA) to activate interferon responses (Parker and Prince, 2011; Stevens et al., 2015). Because TLR and helicase responses activate transcription, they occur over the course of several hours. Enhanced TLR-mediated AMP mRNA upregulation can take up to 12 h (Ramanathan and Lane, 2007; Ooi et al., 2008; Lee et al., 2014b). As we will describe below, many innate defense pathways in the sinonasal cavity are also regulated by taste receptors, which activate more rapid responses within minutes. Taste receptors may thus constitute the early-warning arm of sinonasal innate immunity, while TLRs are critical for sustained responses during prolonged infection.

8.2.2. Taste Receptors Looking at the immune system as our sixth sense, it is fitting that it utilizes components of sensory signal transduction. Chemosensory G-protein-coupled receptors (GPCRs) that were originally identified as taste receptors have now been found in many tissues outside the tongue, including the brain, heart, lung, and sinonasal cavity (Lee and Cohen, 2015b). On the tongue, taste GPCRs signal information to the brain regarding the nutritive value and/or potential toxicity of ingested foods (Margolskee, 1993, 2005). Our sensory perception of foods and

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FIGURE 8.3 Receptors involved in regulation of sinonasal immunity. (A) Toll-like receptors (TLRs) and other classical pattern recognition receptors (PRRs) recognize pathogen-associated molecular patterns (PAMPs) and stimulate innate immune responses. Bacterial PAMPs (e.g., lipopolysaccharide (LPS) and lipotechoic acid (LTA)) and viral PAMPs (single-strand (ss) RNA and double strand (ds) DNA and RNA) recognized by specific TLRs are indicated in the figure. Intracellular PRRs such as retinoic acid-inducible gene 1 (RIG-1) and melanoma differentiation-associated protein 5 (MDA5) also recognize dsRNA from RNA viruses. Stimulation of PRRs results in signaling cascades that activate transcription factors such as nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and interferon regulatory factor (IRF) that upregulate production of antimicrobial peptides (AMPs), proinflammatory cytokines (IL-8, chemokine CaC motif ligand 20 (CCL20), granulocyte-macrophage colony-stimulating factor (GM-CSF)), and components of the interferon (IFN) response, including Chemokine (CaXaC Motif) Receptor 3 (CXCR3) to stimulate T helper cell 1 (Th1 responses). (B) G-protein-coupled taste family 2 receptors (T2Rs) expressed in ciliated epithelial cells stimulate phospholipase C (PLC) isoform β2 to produce inositol trisphosphate (IP3) and activate IP3 receptor (IP3R)-mediated endoplasmic reticulum (ER) calcium release. Calcium-dependent NOS activation results in NO production. This NO diffuses into the ASL and has direct antibacterial effects, as well as acts as an intracellular signaling molecule to stimulate ciliary beat frequency through guanylyl cyclase activation, production of cyclic guanidine monophosphate (cGMP), and activation of protein kinase G (PKG), which directly phosphorylates ciliary proteins (Salathe, 2007; Stout et al., 2007). (C) The specific T2R isoform T2R38 is expressed in sinonasal cilia. Immunofluorescence (performed as published (Lee et al., 2012)) is shown for the cilia-enriched tubulin isoform β-tubulin IV in green (left) and T2R38 in magenta (right). (D) T2R38 detects acyl-homoserine lactones (AHLs) secreted by gram-negative bacteria such as P. aeruginosa. T2R38 activates NO production to enhance ciliary beating and directly kill bacteria as described above (Lee et al., 2012, 2014a). Because the NOS activation occurs within seconds and is Ca21-dependent, it is likely that the NOS isoforms involved are members of endothelial NOS (eNOS) family (Forstermann and Sessa, 2012), known to be expressed in the airway (Shaul, 2002).

beverages is called flavor (Beauchamp and Mennella, 2011), a complex sensation of taste, smell, texture, and sometimes pain (Viana, 2011; Wise et al., 2013). There are actually only five well-defined types of tastes that are detected by the taste buds of the tongue (Breslin and Huang, 2006): sweet, salty, sour, bitter, and umami, the taste of savory amino acids.

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Salty and sour are detected by the epithelial sodium channel (ENaC) and acid-sensing ion channels (ASICs), respectively (Lee and Cohen, 2015b). Sweet, savory, and bitter are detected by GPCRs. The taste type 1 receptor (T1R) family, evolutionarily related to metabotropic glutamate receptors (Bjarnadottir et al., 2005), contains three isoforms (T1R1, T1R2, and T1R3) that hetero-oligomerize to form receptors for umami (T1R1 1 T1R3) and sweet (T1R2 1 T1R3) (Lee and Cohen, 2015b), although T1R3 homo-oligomeric sweet receptors have been proposed to exist in pancreatic beta cells (Nakagawa et al., 2013; Kojima et al., 2014; Medina et al., 2014; Nakagawa et al., 2014) and adipocytes (Masubuchi et al., 2013). Umami receptors are activated by L-amino acids, while sweet receptors are activated by sugars (e.g., glucose, fructose, sucrose), artificial sweeteners (e.g., aspartame, sucralose, cyclamate), and some D- amino acids (e.g., D-tryptophan). Humans have at least 25 isoforms of taste type 2 receptors (T2Rs) that detect diverse chemical structures as bitter (Meyerhof et al., 2010), including alkaloids (e.g., strychnine, quinine, nicotine), isothiocyanates from green vegetables, and lactones (e.g., chlorogenic acid-derived lactones in coffee) (Lee and Cohen, 2015b). T2R bitter and T1R sweet GPCRs are expressed far beyond the tongue (Lee and Cohen, 2015b), including the brain, pancreas, bladder, and testes. These have been termed extraoral taste receptors. Further studies are needed to identify all of the physiological ligands for these receptors, but because many medications are bitter (Mennella et al., 2013), extraoral T2Rs may be a mechanism underlying some off-target drug effects (Clark et al., 2012), reinforcing the need to better understand the role of these receptors in human biology. A role for T2Rs in immunity is intriguing, as T2Rs have a high density of genetic variation (Kim et al., 2005) that contributes to complex individual taste preferences for bitter foods and beverages like green leafy vegetables (Li and Zhang, 2014), coffee (Hayes et al., 2011), scotch (Lanier et al., 2005), and beer (Lanier et al., 2005). It has long been thought that genetic components underlie susceptibility to certain infections (Lionakis et al., 2014), including respiratory infections (Greisner and Settipane, 1996; Cohen et al., 2006; Hamilos, 2007). As described below, data now show that T2Rs do serve immune roles, and genetic variation in at least one T2R contributes to susceptibility to infection. Human bronchial epithelial cells were first shown to express T2Rs within their motile cilia that activate calcium responses to increase ciliary beating (Shah et al., 2009). It was proposed that this process exists to clear noxious chemicals from the airway. This was novel, as traditionally there are two distinct classifications of animal cell cilia. Primary or sensory cilia are expressed on nearly every cell type in the body with one single primary cilia per cell, containing a 9 1 0 microtubule structure and functioning in sensory roles (Singla and Reiter, 2006; Satir and Christensen, 2008; Takeda and Narita, 2012). Motile cilia exhibit a 9 1 2 microtubule structure, occurring at 100300 per cell (Salathe, 2007), and has long been thought to be solely responsible for mechanical transport of fluid, as in the airway and during development of embryonic left-right asymmetry in vertebrates (Babu and Roy, 2013). Identification of chemosensory receptors within motile cilia suggests that they also serve a sensory role. Human sinonasal epithelial cells also express T2Rs localized to motile cilia (Lee et al., 2012). Cilia-localized T2R stimulate low-level calcium responses that activate NOS to drive robust intracellular NO production (Lee et al., 2012) via a signaling pathway utilizing important components of the canonical taste signaling, including TRPM5 and phospholipase C isoform β2 (PLCβ2) (Fig. 8.3B). This NO acts as a second messenger to increase MCC and also diffuses into the ASL with direct bactericidal effects (Lee et al., 2012, 2014a). One specific T2R isoform, T2R38, is expressed in cilia throughout the sinonasal cavity (Fig. 8.3C), where it acts as a receptor for physiological concentrations of two P. aeruginosa acyl-homoserine lactone quorum-sensing molecules, N-butyryl-L-homoserine lactone (C4HSL) and N-3-oxo-dodecanoyl-L-homoserine lactone (C12HSL) (Lee et al., 2012, 2014a). Because many gram-negative bacteria secrete AHLs (Li and Nair, 2012), this is likely a general immune mechanism against many pathogenic gram-negative bacteria. TAS2R38 is one of the most well-studied TAS2R genes (Lee and Cohen, 2013, 2014a, 2015b,a), with two common polymorphisms in Caucasians, one encoding a functional receptor and one encoding a nonfunctional receptor, resulting from differences in amino acid positions 49, 262, and 296. Functional T2R38 contains proline (P), alanine (A), and valine (V) residues while nonfunctional T2R38 contains alanine (A), valine (V), and isoleucine (I) at these positions, respectively (Bufe et al., 2005). Homozygous AVI/AVI individuals (B30% frequency in Caucasians) are nontasters for the T2R38-specific agonists PTC (also known as phenylthiourea or PTU) and 6-propyl-2-thiouracil (PROP) (Bufe et al., 2005). PAV/PAV individuals (B20% frequency in Caucasians (Bufe et al., 2005)) are termed super tasters for these agonists, while AVI/PAV heterozygotes have varying intermediate levels of taste (Bufe et al., 2005; Lipchock et al., 2013). AHL-induced antibacterial responses of human sinonasal epithelial cells correlate with these polymorphisms. Epithelial cells derived from PAV/PAV supertaster individuals exhibited markedly enhanced NO production, MCC, and bacterial killing compared with AVI/ PAV heterozygote or AVI/AVI nontaster cells (Lee et al., 2012). Furthermore, PAV/PAV T2R38 supertasters are less susceptible to gram-negative sinonasal infection (Lee et al., 2012), are less susceptible to CRS (Adappa et al., 2013,

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2014; Mfuna Endam et al., 2014), and may have better outcomes after sinus surgery than PAV/AVI or AVI/AVI patients (Adappa et al., 2015). Beyond the T2Rs in cilia, the upper airway also contains dedicated SCCs (Lee and Cohen, 2014b, 2015b), illustrated in Fig. 8.4. SCCs were first found in fish, then later in alligators, mice and rats, and humans (Lee and Cohen, 2014b, 2015b; Lee et al., 2014b). Human sinonasal SCCs express both T2R bitter and T1R2/3 sweet taste receptors (Lee and Cohen, 2014b, 2015b; Lee et al., 2014b). Activation of T2Rs (likely T2Rs 10, 46, and/or 47) in SCCs stimulates a calcium wave that spreads to the surrounding cells through gap junctions (Lee et al., 2014b). In humans, this response activates robust and immediate (within 5 min) secretion of already-synthesized AMPs, including β-defensins (Lee and Cohen, 2014a, 2015b; Lee et al., 2014b). It is yet unknown which, if any, pathogen-produced products activate human SCC T2Rs, though the strong antimicrobial response evoked by their stimulation suggests that they are indeed activated in response to infection. Microbes secrete numerous products in addition to AHLs, including exotoxins, metabolic products, and other quorum-sensing molecules, such as autoinducer 2 (AI-2, Pereira et al., 2013) and autoinducer peptides (Abraham, 2006; Frederix and Downie, 2011). Further identification of T2Rs expressed in SCCs and screening of these T2Rs with bacterial and fungal compounds will help eludicate what bitter products are secreted by pathogens. Additionally, while many components of T2R-initiated SCC signaling are similar in humans and mice, stimulation of mouse nasal SCCs does not cause release of AMPs. In mice, SCC signaling activates trigeminal neurons causing apnea (Tizzano and Finger, 2013) and inflammation (Saunders et al., 2014). Human sinonasal SCCs appear to be linked to more local innate immune responses. Interestingly, SCC calcium responses are blocked in a dose-dependent fashion by sweet receptor stimulation via apical sugars, such as glucose and sucrose, or nonmetabolizable artificial sweeteners, such sucralose (Lee et al., 2014b). Bitter and sweet taste receptors in human and mouse SCCs appear to function in antagonistic physiological roles. The concentration of glucose normally found in ASL (B 0.5 mM), is sufficient to partially activate

FIGURE 8.4 Nasal solitary chemosensory cell (SCC)-dependent regulation of sinonasal innate immunity. Bitter chemicals are secreted by microbes during infection, yet unidentified but distinct from AHLs, activate the T2R bitter receptors expressed in SCCs, which stimulates a calcium (Ca21) response that propagates to surrounding epithelial cells via gap junctions (Lee et al., 2014b). In human, but not mouse, sinonasal epithelial cells, this calcium signal causes the surrounding cells to secrete antimicrobial peptides (AMPs), including β-defensins, which directly kill both gram-positive and gram-negative bacteria (Lee et al., 2014b). ASL glucose (B0.5 mM in healthy individuals (Lee et al., 2014b)) normally attenuates T2R-mediated signaling through activation of T1R2/3 sweet receptors, except during times of infection, when bacteria likely decrease ASL glucose by consuming and metabolizing it. Reduction of ASL glucose relieves the T1R2/3-mediated inhibition of T2R signaling and AMP secretion (Lee et al., 2014b). In mice, SCC activation results in acetylcholine (ACh) release and activation of trigeminal neurons (Saunders et al., 2014); it remains to be determined if this mechanism also exists in human nasal epithelium. For purposes of simplicity and clarity, T2R receptors present in ciliated cells are not shown.

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T1R2/3 expressed in human nasal SCCs and attenuate antimicrobial responses. While this concentration is 10100-fold lower than the concentrations required to activate T1R2/3 in in vitro heterologous expression systems (Li et al., 2002) or to activate T1R2/3-dependent sweet taste (Schiffman et al., 1999), T1R sweet receptors expressed in pancreatic β-cells (Kyriazis et al., 2012) and gut endocrine (Jang et al., 2007) also respond to similar lower sugar concentrations. Extraoral T1R2/3 appears to be tuned to lower sugar concentrations that are physiologically relevant to the tissues in which they are found. Sinonasal T1R2/3 sweet receptors are likely activated by glucose that is always present in the ASL, albeit at low levels in healthy individuals. Glucose is tonically leaked through the epithelium via paracellular pathways, with reuptake via apical glucose transporters such as facilitative-diffusion GLUT transporters as well as sodium-linked glucose transporters (SLGTs) keep healthy ASL glucose around 0.5 mM or less, or approximately 10-fold below fasting serum levels (Lee et al., 2014b; Lee and Cohen, 2015b). T1R2/3 may act as a rheostat to control the magnitude of the T2R response depending on the glucose concentration in the ASL. Depletion of ASL glucose concentration via bacterial glucose consumption may signal the onset of a bona fide infection and play a role in the activation of T2R-mediated AMP secretion. The T1R2/3 sweet receptors in SCCs may function to desensitize SCC T2Rs to bitter compounds secreted by some bacteria during low-level colonization, but this desensitization is relieved when bacterial numbers increase enough to cause depletion of ASL glucose. A model of this proposed mechanism is shown in Fig. 8.4. The clinical implications of this hypothesis are described below. While this chapter focuses solely on taste receptors in epithelial cells, taste receptors may also serve immune roles in other airway cell types (Table 8.1). T2R38 has also now been found in human neutrophils (Maurer et al., 2015), where it activates chemotaxis in response to AHLs. It was also recently demonstrated that chemosensory brush cells of the rodent urethra, which express T2R bitter receptors, respond to bitter compounds and heatinactivated uropathogenic Escherichia coli, resulting in ACh release to activate the bladder detrusor muscle and trigger expulsion (Deckmann et al., 2014). It is likely that this is just the tip of the iceberg in our knowledge of taste receptors in mammalian immunity.

TABLE 8.1 Airway Expression of Taste Receptors Airway region

Cell type

Nose and Sinuses

Solitary Chemosensory Cell

Receptor type

Function

Endogenous ligands

Refs.

T2R Antimicrobial peptide Bitter secretion (human); breathReceptors holding and inflammation (mouse)

Bacterial acyl-homoserine lactone quorum-sensing molecules (mouse); unknown (human)

Finger et al. (2003), Sbarbati and Osculati (2003), Osculati et al. (2007), Gulbransen et al. (2008), Tizzano et al. (2010, 2011), Braun et al. (2011), Barham et al. (2013), Lee et al. (2014b), Saunders et al. (2014)

T1R Attenuate antimicrobial Sweet secretion (human); unknown Receptors (mouse)

Airway surface liquid glucose

Lee et al. (2014b)

Ciliated Epithelial Cell

T2R Nitric oxide production; cilia Bitter beating; direct bactericidal Receptors effects

Bacterial acyl-homoserine lactone quorum-sensing molecules (T2R38)

Lee et al. (2012, 2014a)

Trachea Chemosensory Brush Cell

T2R ACh release to stimulate trigeminal neuron activation Bitter Receptors and breath-holding (mouse); unknown (human)

Bacterial AHL quorumsensing molecules (mouse); unknown (human)

Sbarbati and Osculati (2005), Krasteva et al. (2011, 2012), Saunders et al. (2013)

Bronchi Ciliated Epithelial Cell

T2R Cilia beating Bitter Receptors

Unknown

Shah et al. (2009)

Unknown

Deshpande et al. (2010), An et al. (2012), Grassin-Delyle et al. (2013); Robinett et al. (2014)

Smooth Muscle T2R Bronchodilation Bitter Receptors

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8.3. IMPAIRMENT OF UPPER RESPIRATORY IMMUNITY AND DISEASE When upper respiratory innate defenses fail, chronic and acute infection can result. Several genetic and acquired defects in upper respiratory immune mechanisms are linked to respiratory disease, described below.

8.3.1. Acute and Chronic Rhinosinusitis Acute and CRS account for B1 in 5 antibiotic prescriptions in adults and are in the top 10 diagnoses for outpatient office visits (Orlandi et al., 2016). CRS is a disease of chronic infection and inflammation of the nose and sinuses (Cohen, 2006; Antunes et al., 2009), frequently requiring prolonged medical therapy and severely decreasing quality of life (Cohen, 2006; Antunes et al., 2009; Stevens et al., 2015). CRS affects 8%10% of the population in the United States (Orlandi et al., 2016), accounting for annual direct healthcare costs .$8 billion and an annual direct economic burden .$20 billion when factors such as absenteeism (missed work days) and presenteeism (reduced productivity at work) are taken into account (Bhattacharyya, 2011; Bhattacharyya et al., 2011; Settipane et al., 2013; Smith et al., 2015). CRS patients report worse quality-of-life scores for physical pain and social functioning than those suffering from chronic obstructive pulmonary disease, congestive heart failure, or angina (Gliklich and Metson, 1995; Khalid et al., 2004). In addition to reducing quality of life, sinonasal infections can seed lower airway infections and/or exacerbate existing lower airway diseases (Hens and Hellings, 2006). Identifying novel therapeutic targets to stimulate endogenous immune responses is particularly important in light of the rising prevalence of antibiotic-resistant bacteria in patients with CRS (Ramanathan and Lane, 2007; Bhattacharyya and Kepnes, 2008; Manes and Batra, 2012; Kennedy and Borish, 2013; Marcinkiewicz et al., 2013; Rujanavej et al., 2013). While twin studies and genome-wide association study linkage analyses have suggested genetic components to CRS (Cohen et al., 2006; Hamilos, 2007), validated markers of the disease are largely unknown (Stevens et al., 2015). T2R38, described above, is one of the most validated genetic modifiers (Lee et al., 2012; Adappa et al., 2013, 2014, 2015; Lee and Cohen, 2015b,a). However, several acquired innate immune defects have been associated with CRS. Acquired ciliary dysfunction is thought to occur through exposure to environmental or microbial toxins and/or inflammatory stimuli (Gudis et al., 2012). Epithelial barrier defects (Soyka et al., 2012) and NOS polymorphisms (Zhang et al., 2011) have also been linked to CRS. While the exactly relationship between the onset of inflammation and infection are debated in CRS (Stevens et al., 2015), using pharmacology to increase ciliary beating or enhance fluid secretion to thin mucus remains an attractive therapeutic strategy for enhancing MCC in multiple types of CRS, including CF-related CRS, described below.

8.3.2. Cystic Fibrosis In CF, defects in the apical membrane CFTR anion channel result in impaired salt and water secretion into the epithelium (Lee and Foskett, 2014) as well as possibly enhanced absorption (Boucher, 2002), creating dehydrated ASL, thick airway mucus, and impaired MCC. CF patients frequently develop severe recurrent sinonasal infections (Illing and Woodworth, 2014; Kang et al., 2015; Stevens et al., 2015) that may play a critical role in disease pathology by seeding or exacerbating lung infections. The upper airways of CF patients with sinusitis are frequently colonized with P. aeruginosa, a common pathogen implicated in CF lung disease. Moreover, nasal polyps are common in CF-related CRS, which may further impair quality of life. It remains to be determined whether current state-of-the art drugs designed to correct mutant CFTR folding defects (lumacaftor) or defective ion channel function (ivacaftor) (Quon and Rowe, 2016) will ameliorate CF-related CRS as well as lung disease.

8.3.3. Primary Ciliary Diskinesia The rare genetic disease primary ciliary dyskinesia (PCD), also known as immotile cilia syndrome, is a genetic multiorgan disorder of cilia dysfunction that severely impairs MCC (Czaja and McCaffrey, 1998; Carlen et al., 2003; Noone et al., 2004; Plesec et al., 2008). PCD patients typically present with chronic and recurrent airway and middle ear infections. Because embryonic nodal cilia are essential for the normal left-right asymmetry of organ development, approximately 50% of PCD patients have situs inversus, and most PCD patients are infertile from loss of Fallopian tube ciliary beating in females and sperm motility in males (Noone et al., 2004; Plesec et al., 2008). Several genetic mutations that result in abnormal cilia function and/or structure underlie PCD and

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directly impaired MCC and increased incidences of upper respiratory infections (Gudis and Cohen, 2010). Kartagener’s syndrome is a subgroup of PCD marked by the triad of chronic sinusitis, situs inversus and bronchiectasis, caused by genetic defects in the dynein arms of axonemal microtubules (Daniels and Noone, 2015; Lobo et al., 2015). Other subsets of PCD patients may demonstrate normal ciliary structure but random ciliary orientation, and thus, despite normal motility of individual cilia, MCC is impaired by ineffective coordination (Cohen, 2006; Gudis and Cohen, 2010).

8.3.4. Diabetes Mellitus Diabetics are more prone to some airway infections than nondiabetics (Koziel and Koziel, 1995; Garnett et al., 2012), including upper respiratory P. aeruginosa infections (Zhang et al., 2014). Patients with poorly controlled diabetes mellitus who have elevated blood glucose (hyperglycemia) have increased flux of glucose into their ASL and elevations in ASL glucose concentrations (Baker et al., 2007; Pezzulo et al., 2011). High ASL glucose can increase bacteria growth by providing more sugar for bacterial metabolism (Baker et al., 2007; Kalsi et al., 2009; Garnett et al., 2012, 2014; Patkee et al., 2016). Data described above suggest that high ASL glucose also facilitates infections by repressing T2R-mediated responses in SCCs through over-activation of T1R2/3 sweet receptors (Lee and Cohen, 2014a; Lee et al., 2014b). This could predispose patients with elevated ASL glucose to infection by limiting SCC responses to bitter bacterial molecules. Supporting a role for high ASL glucose in airway pathology, the mean glucose concentrations in nasal secretions from CRS patients are approximately three- to fourfold higher than healthy individuals (P ,.01) (Lee et al., 2014b), likely due to increased glucose leak from epithelial damage caused by chronic infection and inflammation (Garnett et al., 2012). It has been demonstrated in vitro that proinflammatory mediators increase paracellular glucose flux in bronchial cells and disrupt tight junctions in sinonasal cells (Rogers et al., 2011; Soyka et al., 2012). Diabetes mellitus and its frequent comorbidity, obesity, are associated with increased systemic inflammation and increased cytokines levels (Navarro-Gonzalez and Mora-Fernandez, 2011; Tilich and Arora, 2011; Jin and Flavell, 2013; Prajapati et al., 2014; Khodabandehloo et al., 2016; Ku and Bae, 2016), overstimulation of which can result in cell stress, tissue damage, and impairment of immunity. Inflammation in both CRS and diabetes elevates ASL glucose, simultaneously increasing bacterial growth and repressing innate defenses, creating a vicious cycle of infection. Topical application of T1R2/3 antagonists like lactisole (found in coffee beans (Jiang et al., 2005)) may restore the ability of sinonasal epithelial cells to mount appropriate antimicrobial responses to bitter bacterial molecules, and may be beneficial for some patients while avoiding conventional antibiotics. It also remains to be determined if there is any correlation between susceptibility to airway infections in diabetes with polymorphisms in the TAS1R2 and TAS1R3 genes, which encode T1R2 and T1R3. TAS1R polymorphisms can alter the response of T1R2/3 to sugars (Mennella et al., 2005; Fushan et al., 2009; Bachmanov et al., 2014). Increased sugar sensitivity of T1R2/3 in the airway might lead to increased repression of T2R signaling in SCCs, limiting antimicrobial responses. A role for T1R2/3 sweet receptors in airway disease is supported data from CRS patients and healthy individuals showing allele frequency differences of .10% for 16 different TAS1R single nucleotide polymorphisms (Mfuna Endam et al., 2014). Further studies of TAS1R genetics in airway disease are needed.

8.4. CONCLUSIONS AND REMAINING QUESTIONS The upper airway epithelium has multiple lines of defense to protect the respiratory tract from infection. The foundation is the physical process of MCC, which is complemented by the innate chemical defenses of AMPs and radicals (H2O2, NO). The respiratory epithelium has an important role in releasing chemokines and cytokines in response to infection signals like PAMPs that activate TLRs. This serves as an important link between innate and adaptive immune responses by recruiting dedicated innate immune cells such as macrophages, as well as stimulating adaptive immune cells such as antigen-presenting dendritic cells that go on to activate T cells and B cells. Efficient eradiation of infection by both innate and adaptive mechanisms begins with the sensing of pathogens by the epithelial cells. Thus the study and identification of novel pathogen receptors, such as taste receptors, is useful for identifying new ways to stimulate endogenous immune responses to eradicate infections and potentially reduce the use of conventional antibiotics.

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When sinonasal defenses fail, infection and inflammation of resulting respiratory disease can significantly impact quality of life, particularly in CRS. Several serious systemic diseases such as CF, PCD, and diabetes mellitus are associated with CRS, but research is still needed to identify other genetic markers and modifiers that may help to predict susceptibility and/or best treatment regiments for various patient subsets. Finding novel therapeutics for upper respiratory infections is needed to reduce our reliance on antibiotics and to slow the rise of antibiotic-resistant microorganisms. As described above, emerging data suggest that T2R bitter and T1R sweet taste receptors constitute a novel sentinel detection system in the upper airway epithelium. Multiple taste receptors are expressed in different airway cell types that regulate antibacterial defense mechanisms (Lee et al., 2012, 2014b) that may be promising therapeutic targets. The widespread tissue distribution of extraoral bitter and sweet receptors may also directly link bitter and sweet compounds in foods with activation or repression of various immune defense responses. More research is needed to examine the role of bitter and sweet taste receptors in both the upper and lower airways as well as other tissues and organ systems.

Acknowledgments Research described here was supported by the National Institutes of Health (1R03DC013862) and the University of Pennsylvania Department of Otorhinolaryngology.

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Vareille, M., Kieninger, E., Edwards, M.R., Regamey, N., 2011. The airway epithelium: soldier in the fight against respiratory viruses. Clin. Microbiol. Rev. 24, 210229. Viana, F., 2011. Chemosensory properties of the trigeminal system. ACS Chem. Neurosci. 2, 3850. Vishwanath, S., Ramphal, R., 1985. Tracheobronchial mucin receptor for Pseudomonas aeruginosa: predominance of amino sugars in binding sites. Infect. Immun. 48, 331335. Wijkstrom-Frei, C., El-Chemaly, S., Ali-Rachedi, R., Gerson, C., Cobas, M.A., Forteza, R., et al., 2003. Lactoperoxidase and human airway host defense. Am. J. Respir. Cell. Mol. Biol. 29, 206212. Winters, S.L., Davis, C.W., Boucher, R.C., 2007. Mechanosensitivity of mouse tracheal ciliary beat frequency: roles for Ca2 1 , purinergic signaling, tonicity, and viscosity. Am. J. Physiol. Lung. Cell. Mol. Physiol. 292, L614624. Wise, P.M., Wolf, M., Thom, S.R., Bryant, B., 2013. The influence of bubbles on the perception carbonation bite. PLoS One. 8, e71488. Woods, C.M., Lee, V.S., Hussey, D.J., Irandoust, S., Ooi, E.H., Tan, L.W., et al., 2012. Lysozyme expression is increased in the sinus mucosa of patients with chronic rhinosinusitis. Rhinology. 50, 147156. Woodworth, B.A., Lathers, D., Neal, J.G., Skinner, M., Richardson, M., Young, M.R., et al., 2006. Immunolocalization of surfactant protein A and D in sinonasal mucosa. Am. J. Rhinol. 20, 461465. Woodworth, B.A., Neal, J.G., Newton, D., Joseph, K., Kaplan, A.P., Baatz, J.E., et al., 2007a. Surfactant protein A and D in human sinus mucosa: a preliminary report. ORL. J. Otorhinolaryngol. Relat. Spec. 69, 5760. Woodworth, B.A., Wood, R., Baatz, J.E., Schlosser, R.J., 2007b. Sinonasal surfactant protein A1, A2, and D gene expression in cystic fibrosis: a preliminary report. Otolaryngol. Head Neck Surg. 137, 3438. Workman, A.D., Cohen, N.A., 2014. The effect of drugs and other compounds on the ciliary beat frequency of human respiratory epithelium. Am. J. Rhinol. Allergy. 28, 454464. Wu, H.Y., Russell, M.W., 1997. Nasal lymphoid tissue, intranasal immunization, and compartmentalization of the common mucosal immune system. Immunol. Res. 16, 187201. Yim, S., Dhawan, P., Ragunath, C., Christakos, S., Diamond, G., 2007. Induction of cathelicidin in normal and CF bronchial epithelial cells by 1,25-dihydroxyvitamin D(3). J. Cyst. Fibros. 6, 403410. Zhang, Y., Endam, L.M., Filali-Mouhim, A., Bosse, Y., Castano, R., Desrosiers, M., 2011. Polymorphisms in the nitric oxide synthase 1 gene are associated with severe chronic rhinosinusitis. Am. J. Rhinol. Allergy. 25, e4954. Zhang, Z., Adappa, N.D., Lautenbach, E., Chiu, A.G., Doghramji, L., Howland, T.J., et al., 2014. The effect of diabetes mellitus on chronic rhinosinusitis and sinus surgery outcome. Int. Forum Allergy Rhinol. Zhao, K.Q., Cowan, A.T., Lee, R.J., Goldstein, N., Droguett, K., Chen, B., et al., 2012. Molecular modulation of airway epithelial ciliary response to sneezing. FASEB J. 26, 31783187. Zuo, L., Lucas, K., Fortuna, C.A., Chuang, C.C., Best, T.M., 2015. Molecular regulation of toll-like receptors in asthma and COPD. Front. Physiol. 6, 312.

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9 The Biological Role of NADPH Oxidases in Ischemia-Reperfusion Injury Mediated Pulmonary Inflammation Ashish K. Sharma University of Virginia, Charlottesville, VA, United States

9.1. INTRODUCTION Lung transplantation provides a curative hope for many with end-stage pulmonary disease, but the long-term survival and outcome remain the poorest of any solid organ transplant with survival estimates demonstrating approximately 50% mortality after 5-year posttransplant (Yao et al., 2007). One of the major complications is lung ischemia-reperfusion (IR) injury following transplantation, which imposes a significant threat to graft and recipient survival thereby causing primary graft dysfunction (PGD). IR injury is one of the main causes of primary graft failure and significantly increases the recipient risk for acute rejection and graft-versus-host disease. Multivariate analysis of long-term graft function has implicated IR injury as an independent predictor for bronchiolitis obliterans syndrome, which is the most common cause of long-term morbidity and mortality after lung transplantation (Heumuller et al., 2008). IR-induced lung injury is characterized by nonspecific alveolar damage, lung edema, and hypoxemia occurring within 72 h after lung transplantation (de Perrot et al., 2003). Hypothermic organ storage is associated with oxidative stress, sodium pump inactivation, intracellular calcium overload, iron release, and cell death that induces cell surface expression patterns and proinflammatory mediators for leukocyte activation during the reperfusion period (de Perrot and Keshavjee, 2003). This inherent response mechanism implicates IR injury as a primary determinant of both immediate and long-term graft survival. The pathogenesis of lung IR injury is multifactorial and involves the generation of reactive oxygen species (ROS) which can be detected during ischemia (Minamiya et al., 1998) and reperfusion (Kennedy et al., 1989). One of the major candidates of intracellular ROS production has been shown to be NADPHdependent oxidases that are present in polymorphonuclear leukocytes as well as epithelial and endothelial cells in the lung. Although ROS derived from vascular NADPH oxidase (NOX) has been implicated in the pathophysiology of many cardiovascular diseases, their role in lung IR injury, specifically in the context of immune cells and leukocytes as well as resident lung cells, is still being defined. In a recent clinical study, oxidative stress regulatory genetic variation in lung transplant recipients and donors has been shown to contribute to the risk of PGD after lung transplantation (Cantu et al., 2014). In particular, this study and other experimental studies have demonstrated a critical role of NOXs in lung IR injury and PGD. This review focuses on providing a broad overview of the experimental studies conducted to analyze the role of various NOX isoforms in lung IR injury. This information will be further substantiated by summarizing proposed molecular signaling pathways and modulation of gene and protein expression of cytokines and transcription factors in relation to NOX-dependent oxidases during acute lung injury after IR.

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9.2. FREE RADICALS AND OXIDATIVE STRESS IN LUNG ISCHEMIA-REPERFUSION INJURY Lung IR injury is a complex pathological phenomenon encompassing various cellular, biochemical, and molecular mechanisms. One of the key signaling pathways involving multiple cell types includes oxidative stress due to the generation of ROS. Several groups have previously demonstrated that inhibiting the enzymes involved in ROS generation can dramatically reduce the tissue injury and the proinflammatory profile after lung IR (Fu et al., 2008; Lee et al., 2013; Reyes et al., 2006; Wolf et al., 2008). A burst of ROS production occurs immediately upon reperfusion of hypoxic cells including antigen-presenting cells like macrophages, epithelial and endothelial cells as well as neutrophils (Chen et al., 2014; De Pascali et al., 2014; Geudens et al., 2008; Nanavaty et al., 2002; Omasa et al., 2003; Sharma et al., 2014; Sugimoto et al., 2012; Tan et al., 2013). The antioxidant defense capabilities of the lung are unable to cope with the increase in ROS leading to altered cellular metabolic function and redox signaling. Oxidative stress due to ROS causes proinflammatory cytokine release and enhanced transcription of numerous genes resulting in inflammation, cell injury and neutrophil recruitment and activation in the lung after IR (Fisher et al., 1991; Gedik et al., 2012; McCourtie et al., 2008; Sharma et al., 2007; Simon et al., 2012; Wang et al., 2013). Reperfusion of ischemic tissue results in ROS generation such as superoxide (O2•2), hydrogen peroxide (H2O2) and the hydroxyl radical (  OH) that leads to oxidative damage in the lung tissue (Al-Mehdi et al., 1994, 1997; Ayene et al., 1992; Eckenhoff et al., 1992; Fisher et al., 1991; Zhao et al., 1997). This oxidative burst can lead to increased adherence of neutrophils to the endothelium (McIntyre et al., 1995). The release of ROS not only induces cellular lipid membrane peroxidation and the production of inflammatory cytokines, but also plays a role in regulating the activity of several antioxidant enzymes (e.g., glutathione peroxidase, catalase and superoxide dismutase) as well as key transcription factors such as NF-κB and activator protein-1 (AP-1) (Cho et al., 2006; Morimoto, 1993; Schreck et al., 1992). One of the key ROS-producing enzymatic elements has been described to be the NOX-family of NOXs, which are ubiquitously present in most cells and can be rapidly activated during IR injury.

9.3. NADPH OXIDASE IN LUNG INFLAMMATION AND ISCHEMIA-REPERFUSION INJURY The NOX family members are transmembrane proteins that transport electrons across biological membranes to reduce oxygen to superoxide (Bedard and Krause, 2007; Sumimoto, 2008). Since the advent of neutrophilic NOX2 (an NOX isoform), several other isoforms (NOX 1-5) and Duox-1 and -2 have since been characterized that differ in their tissue distribution and mode of activation (Lambeth, 2004). This oxidase consists of at least six components as follows: Nox2, p22phox, p67phox, p47phox, p40phox, and the GTPase Rac. In resting cells, p67phox, p47phox, and p40phox exist in the cytosol as a complex, whereas p22phox and Nox2 form the heterodimer cytochrome b558, which is located in the plasma membrane (Pacquelet et al., 2008). Activated NOX is a multimeric protein complex consisting of at least three cytosolic subunits of p47phox, p67phox, and p40phox, a regulatory small molecular weight G protein Rac1 or Rac2, and a membrane associated cytochrome b558 reductase made up of gp91phox and p22phox. The specific cytosolic regulatory proteins of the NOX complex, i.e., p40phox, p47phox, and p67phox have been implicated in organ-specific inflammatory pathways using knockout mice. Specifically, studies using p47phox knockout mice demonstrated that chimeras created by bone marrow transplantation implicate NOX-generated ROS from bone marrow-derived cells leading to lung inflammation and IR injury (Yang et al., 2009). Another important cytosolic subunit required for NOX activation is Rac1 which is associated with vascular cell adhesion molecule-1 on endothelial cells to induce ROS generation culminating in loss of cell-cell adhesion and transendothelial leukocyte migration (van Wetering et al., 2003). The patriarch of the NOX protein family is gp91phox (NOX2). Along with p22phox in plasma membrane, gp91phox operates as an electron transferase shuttling electrons from NADPH in the phagocyte cytoplasm across two nonequivalent hemes buried in the membrane to O2, the electron acceptor, thereby generating superoxide anion (Babior, 2004; Cross et al., 1995). The assembly, translocation, and binding of the cytosolic subunits to the gp91phox/p22phox catalytic complex are facilitated and organized by p47phox. The sequential activation of p47phox involves serine phosphorylation, conformational changes in the SH3 domain, membrane translocation and subsequent binding with the proline-rich region of p22phox (El-Benna et al., 2009; Sheppard et al., 2005). A multitude of protein kinases including various isoforms of PKC, PKA, and MAP kinases have been shown to regulate p47phox phosphorylation and NOX activation in cells like human neutrophils, lung endothelial cells and alveolar type II epithelial cells (Chowdhury et al., 2005; Dang et al., 2006, 2011; Dewas et al., 2000; Leverence et al., 2011; Raad et al., 2009). III. INFLAMMATORY SYSTEMS: MECHANISTIC PATHWAYS OF STIMULATION AND SUPPRESSION

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9.4. CELL-SPECIFIC ROLE OF NADPH OXIDASES IN PULMONARY INFLAMMATION The biological and pathological aspects of various NOX/DUOX enzymes within various lung cell types have been previously elucidated (van der Vliet, 2011). NOX, which is present in immune cells (macrophages, T cells and neutrophils) as well as the lung resident cells (epithelial and endothelial cells), utilizes NADPH as a substrate to generate superoxide from molecular oxygen. Recent studies have demonstrated a cell-specific role of the NADPH-oxidase enzyme complex in ROS generation after IR (Goyal et al., 2004; Jackson et al., 2004; van der Vliet, 2008; Yang et al., 2009; Yao et al., 2007). Among the bone-marrow derived cell population, a pivotal role of NOX2 isoform in invariant natural killer T cells (iNKT) cells has been implicated to modulate cytokine production, neutrophil infiltration and inflammation (Sharma et al., 2016). Similarly, NOX-derived ROS from polymorphonuclear leukocytes, i.e., neutrophils, can contribute to inflammation and acute lung injury (Grommes and Soehnlein, 2011). Moreover, NOX isoforms associated with lung endothelial cells have been also shown to regulate ROS generation during reoxygenation after lung IR injury (Al-Mehdi et al., 1998; Zhao et al., 1997). Superoxide is usually rapidly converted to hydrogen peroxide (H2O2) or can react with nitric oxide (King et al., 2000) to generate peroxynitrite (ONOO2). The upregulation of NOX can lead to regulation of transcription factors such as NF-κβ and AP-1, increased expression of proinflammatory cytokines (CXCL1, IL-17, TNF-α) and neutrophil enzymes like myeloperoxidase (MPO) culminating in inflammation and lung IR injury (Aratani et al., 2012; Fisher et al., 2002; Naidu et al., 2003; Wei et al., 1999; Yang et al., 2009) (Fig. 9.1). Although the role of NOX isoforms in T cell, alveolar macrophage and neutrophil activation during lung IR injury is poorly understood, the modulation of epithelial cell activation and endothelial cell barrier permeability by NOX has been well defined by numerous studies. Among the various resident cell subtypes that can mediate pulmonary inflammation, a pivotal role of endothelial cells has been described in detail. A recent study also demonstrated a marked reduction in lung edema in NOX2-deficient mice postulating a protective role of NOX-deletion in lung IR injury (Weissmann et al., 2012). In this study, a mechanistic model was shown in lung endothelial cells wherein NOX-2-derived production of

FIGURE 9.1 Molecular signaling pathways in the pathogenesis of lung IR injury. NOX activation occurs secondary to IR and results in reactive oxygen (ROS) production. The signal transduction pathways induced by ROS-production involve transcription factor regulation culminating in inflammatory cytokine production as well as loss of endothelial permeability. Subsequently, this results in lung edema, inflammation and tissue injury.

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superoxide, activation of phospholipase C-γ, inhibition of DAG kinase and DAG-mediated activation of TRPC6 result in increasing endothelial permeability and cause lung IR injury. Another mechanism of NOX-dependent endothelial cell activation during lung IR injury has been shown to be via ATP-dependent K 1 channels (Zhang et al., 2005). Furthermore, previous studies have also indicated that activation of an endothelial cell-associated NOX may be the primary mechanism for ROS generation during reoxygenation after lung ischemia (Al-Mehdi et al., 1998; Zhao et al., 1997). Lung ischemia has been shown to induce endothelial cell depolarization because of shear stress alterations that activate NOX activity via Rac1 and phosphoinositide-3-kinase (PI3K)-dependent mechanism (Zhang et al., 2008). Moreover, cellular interactions between lung endothelial cells and neutrophilderived oxidants have been shown to induce lung injury after hemorrhagic shock in mice, which is mediated by NOX (Xiang et al., 2011). Taken together, an important role of NOXs in epithelial and endothelial cells to regulate lung IR injury symbolizes the critical role of this ROS-forming protein complex in resident lung cells. In addition to pulmonary endothelial cells, alveolar type II epithelial cell activation can also be mediated by NOX-dependent mechanisms. A significant role of p47phox subunit of NOX was demonstrated in alveolar type II epithelial cell-produced CXCL1 in a murine hilar clamp model of lung IR injury (Sharma et al., 2014). Furthermore, this study showed that the synergistic effect of proinflammatory cytokines, i.e., iNKT cell-produced IL-17 and alveolar macrophage-produced-TNF-α on alveolar type II epithelial cells-produced CXCL1 expression in the bronchoalveolar fluid was abolished by apocynin treatment and in p47phox knockout mice thereby signifying a crucial role of NOX-dependent pathway in resident lung cells to attenuate inflammation after IR injury. Also, another NOX isoform, DUOX2 has been shown to be mainly expressed in type II epithelial cells and can regulate hyperoxia-induced ROS production thereby causing caspase-mediated apoptosis, including ERK and JNK phosphorylation (Kim et al., 2014).

9.5. ROLE OF NOX ISOFORMS AND PHARMACOLOGICAL INHIBITORS IN LUNG IR INJURY Among the various isoforms of NOXs (NOX1-5 and Duox-1 and -2), most of the studies using experimental animal models of lung IR injury indicate that majority of the ROS production and oxidative injury with lung IR is due to NOX2 activation (Fig. 9.2). A significant protection has been shown in NOX2 knockout mice as well as in wild-type mice pretreated with the NOX inhibitor, apocynin or MJ33 (inhibitor of peroxiredoxin6 PLA2 activity) (Lee et al., 2013; Yang et al., 2009). These studies demonstrate that the majority of ROS production after lung IR is mediated via NOX2, and that phosphorylation of the p47phox is a critical event for the NOX complex

FIGURE 9.2 Schematic summary of NOX2 activation-mediated lung inflammation. Superoxide/ROS is produced in cells after stimulation by ischemia-reperfusion. NOX2 (gp91phox) is simulated by phosphorylation-activated p47phox or p67phox, in conjunction with activated Rac. NOX2 activation leads to superoxide and reactive oxygen species (ROS) production causing proinflammatory cytokine secretion and neutrophil infiltration resulting in lung inflammation and injury. Increase in cyclic AMP (cAMP) and protein kinase A (PKA) or inhibition of protein kinase C (PKC) activation by pharmacological interventions can lead to blocking of p47phox or gp91phox phosphorylation, thereby inhibiting NOX2 activation and downstream signaling events.

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activation (Shimoyama et al., 2005; Zhang et al., 2008). The ROS production that was not derived from NOX2 subunit of NOX could be from other potential sources of superoxide anion production such as NOX1 activation, the mitochondrial respiratory chain, or xanthine oxidase. A recent study involving lung transplant patient blood samples demonstrated a significant role of NOX3 in donor-recipient interaction that increases the risk of PGD (Cantu et al., 2014). As NOX3 and NOX5 are not present in rodent lung and NOX4 is constitutively active, the contributory role of other NOX isoforms still needs to be clearly defined after lung IR injury. Pharmacological inhibition of NOX can be achieved by various agents known to inhibit NOX-dependent ROS production. Among these, diphenyleneiodonium is a commonly used inhibitor of NOX2 in vitro, but may not be specific, and its toxicity related to general inhibition of flavoproteins would preclude its use as a therapeutic agent (Jaquet et al., 2009). Previous studies have documented that the pharmacological antagonism of the NOX complex by apocynin is shown to be protective against lung IR injury (Dodd and Pearse, 2000; Pearse and Dodd, 1999; Zhu et al., 2008). Apocynin has been shown to be the most specific inhibitor, and it can potently ameliorate lung injury associated with IR (Dodd and Pearse, 2000; Yang et al., 2009; Zhu et al., 2008). A key mechanism of apocynin-mediated protection is via the prevention of the translocation of cytosolic p47phox to NOX2 in the membrane of leukocytes, monocytes, and endothelial cells (Barbieri et al., 2004; Heumuller et al., 2008; Lapperre et al., 1999). However, the protective effects of apocynin may not be limited to NOX inhibition alone since it has been shown to inhibit cytochrome P-450 (Pietersma et al., 1998) and thromboxane synthase (Engels et al., 1992). Interestingly, both cytochrome P-450 and thromboxane have been shown to mediate lung IR injury (Bysani et al., 1990; Lu et al., 1997). Previous studies with lung endothelial cells, lung alveolar macrophages, and PMNs have shown that the PLA2 activity of Prdx6 is required for activation of NOX2 and the subsequent production of ROS, and that inhibition of PLA2 activity could be achieved by MJ33, a known inhibitor of the PLA2 activity of Prdx6 (Chatterjee et al., 2011; Ellison et al., 2012; Lee et al., 2013). The therapeutic effectiveness of these pharmacological agents of NOX2 inhibition is yet to be deciphered in acute lung IR injury, and the importance of this pathway is substantiated by experimental studies that demonstrate a beneficial effect on lung inflammation.

9.6. CONCLUSIONS Recent studies have revealed a crucial role of various isoforms of NOX in the initiation and progression of lung inflammation and injury. The physiological role of specific NOX isoforms in the realm of pulmonary IR injury provides pivotal insights into their pathological role in diseases and offers potential opportunities for therapeutic applications. Further studies of the biological role of NOXs will be more informative when studies encompassing the specific genes can be conditionally deleted in specific cell types (e.g., by inducible Cre recombinase under the control of a cell-specific promoter to delete a targeted/floxed gene) to pinpoint the exact role of specific NOX isoforms in particular cell subtypes. However, current studies represent a remarkable ability of the NOX pathway to regulate lung IR injury, thereby representing an important signaling pathway to modulate inflammation and tissue injury.

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Role of reactive oxygen species in reperfusion injury of the rabbit lung. J. Clin. Invest. 83, 13261335. Kim, M.J., Ryu, J.C., Kwon, Y., Lee, S., Bae, Y.S., Yoon, J.H., et al., 2014. Dual oxidase 2 in lung epithelia is essential for hyperoxia-induced acute lung injury in mice. Antioxid Redox Signal. 21, 18031818. King, R.C., Binns, O.A., Rodriguez, F., Kanithanon, R.C., Daniel, T.M., Spotnitz, W.D., et al., 2000. Reperfusion injury significantly impacts clinical outcome after pulmonary transplantation. Ann. Thorac. Surg. 69, 16811685. Lambeth, J.D., 2004. NOX enzymes and the biology of reactive oxygen. Nat. Rev. Immunol. 4, 181189. Lapperre, T.S., Jimenez, L.A., Antonicelli, F., Drost, E.M., Hiemstra, P.S., Stolk, J., et al., 1999. Apocynin increases glutathione synthesis and activates AP-1 in alveolar epithelial cells. FEBS Lett. 443, 235239. Lee, I., Dodia, C., Chatterjee, S., Zagorski, J., Mesaros, C., Blair, I.A., et al., 2013. 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C H A P T E R

10 Receptor Blockade of CD26/DPP4 as a Therapeutic Strategy Against I/R Injury and Lymphocytic Inflammation and its Clinical Implications Wolfgang Jungraithmayr1,2 1

University Hospital Zurich, Zurich, Switzerland 2Medical University Brandenburg, Neuruppin, Germany

10.1. INTRODUCTION Dipeptidyl peptidases (DPPs, CD26) were first assigned a role in inflammatory disorders more than three decades ago. One of the first inflammatory diseases in which DPPs have been found was in the synovial membrane from patients with rheumatoid arthritis (Kamori et al., 1991), suggesting an immunological role for this group of peptidases in inflammation disturbances. Specifically, DPP4 was found on the surface of inflamed human synovium, proposing potential approaches to the pharmacological manipulation of inflammation by specific enzyme inhibitors (Walsh et al., 1993). Ischemia-reperfusion (I/R) injury is a form of inflammation that reflects various key features of inflammation such as tissue injury and the involvement of various immune cells. During the development of I/R injury, a plethora of proteins are involved and many of them are substrates to CD26/DPP4 cleavage. Thus, inhibiting these substrates by CD26/DPP4-inhibitors (gliptins) can potentially ameliorate inflammatory diseases. In the following chapter, we will focus on the emerging role of CD26/DPP4 as a target for attenuating I/R injury, and also on reducing lymphocytic inflammation. We first elaborate on experimental evidence in inflammation in different organ systems, followed by addressing potential clinical applications.

10.2. CD26/DPP4 AS A THERAPEUTIC TARGET IN EXPERIMENTAL MODELS AGAINST I/R-INJURY The concept of CD26/DPP4 modulation gained increased attention when the protein glucagon-like peptide 1 (GLP-1) was found to be a key substrate of CD26/DPP4 enzymatic cleavage for a successful treatment of type 2 diabetes. GLP-1 stimulates insulin secretion through activation of GLP1 receptors in islet β-cells. Inhibitors of DPP4, the enzyme responsible for the inactivation of GLP1 which indirectly stimulates GLP1 receptors by increasing the concentration of endogenous GLP1, target GLP-1 (Ahren, 1998). These positive effects of GLP-1 have been shown later to exert protective effects on the cardiovascular system, as type 2 diabetes is strongly associated with an increased risk of cardiovascular disease. Hence there is considerable interest in strategies that reduce cardiovascular morbidity and mortality in diabetic subjects. A study by Ban and colleagues showed that mice developed increased left ventricular pressure and coronary flow post I/R injury. GLP-1 also restored functional recovery and cardiomyocyte viability of isolated hearts and dilated preconstricted arteries. GLP-1 administration during reperfusion was reported to reduce ischemic damage after ischemia-reperfusion and increased cGMP release, vasodilatation, and coronary flow (Ban et al., 2008). Further evidence for a cardiovascularImmunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00010-X

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protective role of CD26/DPP4 inhibition was provided by Sauve´ and colleagues, demonstrating that sitagliptin, a competitive inhibitor of DPP4 and the GLP-1R agonist liraglutide upregulated levels of cardioprotective proteins in nonischemic myocardium. These data suggested a role for GLP-1 in the context of enhanced survival via DPP4 inhibition or genetic ablation of DPP4 (Sauve et al., 2010). Others demonstrated a cardioprotective action against I/R injury also in DPP4-deficient rats, mediated through both GLP-1 receptor-dependent and receptorindependent mechanisms (Ku et al., 2011). Some of these mechanisms seem to be mediated via the GLP-1 receptor-Protein kinase A pathway (Hausenloy et al., 2013). In general, however, cardioprotective effects on I/R injury were not through modulation and enhancing of GLP-1 alone. DPP4 enzyme activity inhibition also modulates the activity of cardioactive peptides such as brain natriuretic peptide, neuropeptide Y, and stromal cell-derived factor-1α (SDF-1α) (Drucker, 2007; Jungraithmayr et al., 2011). In this context, there is a series of research done showing that SDF-1α functions as a robust substrate of dipeptidylpeptidase-4 cleavage. Thus, reduced cleavage leads to an enhanced recruitment of CXCR-4 1 circulating progenitor cells, and thereby attenuated myocardial I/R injury (Hocher et al., 2013). Alternatively, SDF-1α was directly given into the ischemic myocardium to prove the effectiveness of SDF-1α to show similar effects (Kanki et al., 2011). SDF-1α has potential pleiotropic effects, protecting from I/R injury while simultaneously stimulating repair by recruiting stem cells to the site of injury. Among numerous mechanisms occurring in the intracellular and extracellular environments during I/R injury, reactive oxygen species (ROS) play a major part in the pathogenesis of I/R injury. The imbalance between the cellular formation of free radicals and cellular capacity to defend against them can cause tissue injuries. In this direction, ROS play an essential role in both the organ injury and repair processes. In a more mechanistic approach using a clinically relevant I/R swine model, Chinda and colleagues showed the pivotal role for ROS in I/R injury. They found that preventing cardiac mitochondrial dysfunction that occurs due to severe oxidative stress with I/R by DPP-4 inhibition decreased ROS production and prevented ROS-induced cardiac mitochondrial depolarization (Chinda et al., 2013; Chinda et al., 2014). The DPP4-inhibitor Linagliptin and the GLP-1 activator Liraglutide reduced ROS production and also inhibited proinflammatory cytokines underscoring the importance of GLP-1 in the attenuation of cardiac I/R injury (Wang et al., 2016). Using another dipeptidyl peptidase-4 inhibitor (Alogliptin), others investigated the correlation between the development of an aneurysm and the presence of ROS, showing convincing antioxidant action by CD26/DPP4-inhibition. Aneurysms develop as a result from chronic inflammatory processes. Experiments in rats revealed that aneurysms became less dilated, showed reduced ROS production and also decreased mRNA expression of matrix metalloproteinases (MMPs) without affecting the glucose metabolism (Bao et al., 2014). In a very interesting experimental study performed by the highly experienced group in CD26/DPP4 and its inhibitors (Ingrid de Meester from the University of Antwerp), I/R injury in the kidney upon CD26/DPP4-inhibition was investigated. The kidney harbors relevant amounts of DPP4 including its substrates and is thus a suitable target for CD26/DPP4-inhibition. Authors studied the DPP4 inhibitor vildagliptin on the outcome of I/R- injury-induced acute kidney injury in rats in a model of 30-min unilateral renal ischemia followed by contralateral nephrectomy. Authors could not prove a direct regeneration of renal I/R injury in this experimental setup; instead, they showed a reduced apoptosis and a decrease in mRNA expression of the proinflammatory marker CXCL10, along with improved functionality and significantly reduced tubular necrosis in the affected kidney (Glorie et al., 2012). Interestingly, similar antiapoptotic effects were observed with the DPP4inhibitor alogliptin acting via GLP-1-mediated effects in a cisplatin-induced mouse acute kidney injury model (Katagiri et al., 2013). A recent publication that focuses on the interaction between DPP4 and adenosine deaminase, a natural ligand of DPP4, showed that alogliptin suppresses I/R injury via the adenosine receptor- and cAMP response element-binding protein-dependent signaling pathway (Ihara et al., 2015). The experimental evidence on the protective effect of DPP4 inhibition is not only limited to cardiac and renal I/R injury studies; there is also relevant work being done in the field of lung I/R injury associated with transplant (Tx) in rat and mice lung transplant models. Indeed, our own group provided the first evidence that the inhibition of intragraft DPP4 enzymatic activity significantly reduced I/R-associated pulmonary injury, as reflected by a functionally improved lung transplant organ (Zhai et al., 2007). Soon thereafter, we were able to identify another important peptide responsible for a protective effect in lung I/R-injury, namely vasoactive intestinal peptide (VIP). This peptide accumulated in predominantly alveolar macrophages and exerted effects towards improved functional and morphological parameters (Zhai et al., 2009; Jungraithmayr et al., 2009). The concept that VIP displays anti-inflammatory properties in I/R injury could be shown by our group in both experimental models, the rat and the newly introduced mouse model of lung transplantation by our group (Jungraithmayr et al., 2011; Jungraithmayr et al., 2009). In line with the aforementioned data showing SDF-1α to be a relevant substrate for CD26/DPP4 inhibition against I/R-injury in experimental cardiac studies, we were also able to identify SDF-1α to be responsible for improving lung I/R injury in a preclinically relevant mouse lung transplant model: the systemic DPP-4 inhibition

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of lung transplant recipients resulted in an increase in the protein concentration of SDF-1α in all compartments, plasma, spleen and lung. Furthermore, the frequency of cells bearing the SDF-1α receptor CXCR4 rose significantly in the circulation and also in the lungs of DPP-4-inhibited recipients. As a result, enhanced coexpression of CXCR4/CD34 in the grafts of animals treated with vildagliptin, the stem-cell markers Flt-3 and c-kit increased significantly. These mechanisms resulted in an improved morphology of lung grafts revealing less alveolar edema when compared with untreated recipients (Jungraithmayr et al., 2011). In our most recent work, we extended our studies to prove a prolonging anti-inflammatory effect of CD26/DPP4-inhibition in mouse lung transplants up to 14 days: most likely via SDF-1α—mediated effects, pro-inflammatory cytokines such as TNF-α decreased and inflammation-mediating immune cells such as macrophages, T and B cells decreased in functionally improved lung transplants. As an underlying mechanism, endothelial adhesion molecule or ICAM (intercellular adhesion molecule) considered as a key factor in recruitment and adherence of neutrophils, was absent in CD26/DPP4-inhibited transplant lungs, implying diminished immune cells infiltration into the transplanted organ thus resulting in reduced inflammation (Jang et al., 2016). The latter work was broadly discussed within the scientific community, supporting the relevant role of CD26/DPP4-inhibition in its anti-inflammatory activity (Williams and Denlinger, 2016; Date, 2016). Other plausible mechanisms of protection via DPP4-inhibition include antiatherogenic effects on vascular macrophages that occurs via decreased senescence and apoptosis in the vascular endothelium (Matsubara et al., 2012). In a most recent publication from this month (April, 2017), researchers also found neuroprotective effects upon DPP4-inhibition in a model of global cerebral ischemia-reperfusion-induced neuronal injury. The compound Genistein, an isoflavonoid phytoestrogen with DPP4-inhibiting properties, was able to suppress the increase in cerebral oxidative stress, reduced neuronal apoptosis, and increased cellular viability, most likely via a GLP-1mediated pathway (Rajput et al., 2017). Taken together, experimental studies in CD26/DPP4-inhibition targeted therapies provide broad evidence for anti-inflammatory therapy by CD26/DPP4-inhibition, not only in the heart but also in the lung, kidney, and most recently in brain experimental models (Fig. 10.1). These effects are largely mediated via substrates of CD26/ DPP4, first of all GLP-1, but also other relevant proteins such as SDF-1α and VIP. Main effector mechanisms include the reduction of ROS, reduced apoptosis, and decreasing proinflammatory cytokines. Those effects observed may also have been related to off-target effects rather than to the hypothesized mechanism of

FIGURE 10.1 Gliptins in experimental models with their respective mechanisms and effects acting via CD26/DPP4-inhibition.

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CD26/DPP4 inhibition, preservation of stromal-derived factor 1, and recruitment of progenitor cells; however, as CD26DPP4-inhibitors are in safe and routine clinical use against type 2 diabetes, results from these studies may have immediate clinical benefits.

10.3. MODULATION OF CD26/DPP4 AGAINST LYMPHOCYTIC INFLAMMATION Beside its enzymatic function, CD26 has potent costimulatory properties displaying robust effects on signaling lymphocytic activation involving migratory and memory responses of T cells (De Meester et al., 1999; Fleischer et al., 1997; Morimoto and Schlossman, 1998). Crosslinking of CD26 and CD3 can induce T cell costimulation and IL-2 production by CD261 T cells. Early work on CD26 as a costimulatory molecule relevant in inflammatory diseases stems from Hegen and colleagues who showed that the function of CD26 on T cells is dependent on the expression of the T cell receptor complex (Hegen et al., 1993). While some studies show that the costimulatory activity hinges on the enzymatic activity of CD26/DPP4, others can provide support for the hypothesis that signaling events in T cells involving costimulation with CD26 and ecto-ADA and the synergism observed upon ADA binding to CD26 occur independently of the catalytic activities of these cell surface molecules (Jeanfavre et al., 1996). Recognizing the costimulatory nature of this molecule, experimental studies in transplantation were subsequently initiated to explore the therapeutic potency of the inhibition of this costimulatory molecule in tissue inflammation. One of the first experiments in rat heart and lung transplantation taking advantage of inhibiting CD26 was performed by Korom and colleagues showing that the inhibition of CD26-costimulation can reduce T cell infiltration and thus reduce transplant rejection-related inflammation injury (Korom et al., 1997a; Korom et al., 1997b). Following the exploration of lymphocytic inflammation in lung transplant rejection, our group extended these findings in showing that CD26 costimulatory blockade promotes lung allograft acceptance via reduced T cell infiltration and an enhancement of alternatively activated macrophages, a lesser expression of the proinflammatory cytokine IL-17, and an increased expression of the anti-inflammatory cytokine IL-10 (Yamada et al., 2016). In studies exploring other organs, the role of CD26 costimulation was expanded to investigate transplant tolerance in the liver for the attenuation of liver chronic inflammation, cellular damage, regeneration and fibrosis (McCaughan et al., 2000). Other studies performed in inflamed synovia of patients with rheumatoid arthritis were found to contain activated T cells suggesting that T cells with high levels of CD26 antigen preferentially migrate into rheumatoid synovium to induce inflammation and tissue destruction (Mizokami et al., 1996). The role of CD26 is not limited to classical inflammatory diseases such as rheumatoid synovitis and autoimmune thyroiditis alone; CD26 has been assigned a major role as a targetable costimulatory molecule in the inflammatory process during graft versus host disease (GVHD). Indeed, administration of humanized antihuman CD26 monoclonal antibody decreased the severity of GVHD and prolonged survival without loss of engraftment of human T cells (Hatano et al., 2013). These data indicate a role for CD26 in the regulation of GVHD and point to CD26 as a novel target for therapeutic intervention in this disease.

10.4. CLINICAL IMPLICATIONS FOR CD26/DPP4 AS A THERAPEUTIC TARGET IN I/R INJURY AND LYMPHOCYTIC INFLAMMATION There is serious potential to translate experimental data into pre- and clinical applications. However, the paucity of studies limits the applicability of CD26/DPP4-inhibitors that can range in use from treatment of type 2 diabetes towards target areas of I/R-injury and lymphocytic inflammation. CD26 has the potential to function as a valuable marker of rejection post-transplant as CD26 is expressed on activated cells. Korom and colleagues studied the cellular and enzymatic expression posttransplantation and provided thereby a putative indicator for immunomodulation and rejection in patients following kidney transplantation (Korom et al., 2003). However, this has not been investigated clinically, but trials are underway, including several preliminary trials that are currently being carried out at our department. Other translational aspects are largely limited to evidence from cardiac I/R injury (Chattipakorn et al., 2016), with the potential for transfer to areas with inflammational implication. However, the base is set for initiating clinical trials, as there is a large body of data for the CD26/DPP4-inhibitors Vildagliptin and Sitagliptin for the attenuation of I/R-injury via reducing oxidative stress, mitochondrial dysfunction, and apoptosis. In the clinical transplant set-up, the concept

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of donor organ preconditioning by a CD26/DPP4-inhibitor could be easily performed and its effect in ameliorating I/R-injury and thus primary graft dysfunction in organ recipients estimated. As SDF-1α plays a central role as a substrate of CD26/DPP4 for acute and chronic cardioprotective effects, there is a high potential for therapeutic manipulations to protect against lethal tissue injury in the clinical setting (Bromage et al., 2014); however, this is yet to be shown. Besides their glucose-lowering activity, gliptins display a manifold cardiovascular impact. This impact is due to the presence of GLP-1 receptors in human cardiac myocytes, boosting the interest of researchers to properly establish the relevance of the gliptin-induced changes in clinical settings. These changes are explained by reasonable molecular mechanisms including the improvement of endothelial function, decrease of inflammatory markers, reduction of I/R injury in experimental models, prevention of left ventricular remodeling, and a modest decrease in blood pressure. Though the gliptins are considered safe in clinical use, there is some concern about serious side effects, particularly in combination with other drugs, such as the development of severe angioedema when gliptins are coadministered with ACE-inhibitors or angiotensin receptor blockers (Brown et al., 2009). Caution is therefore recommended when designing clinical trials with established CD26/DPP-inhibitors. Overall, results from basic science on CD26/DPP4-inhibitors are encouraging. As yet these have not translated into clinical evidence, probably due the pleiotropic enzymatic effects of DPP4 (Fadini et al., 2015). The results of placebo-controlled phase IV trials have been rather disappointing in terms of their cardioprotective actions. With regard to the application to other organs, such as lung or liver, we can only extrapolate from clinical evidence gained in the cardiovascular field. But the time has come to transfer this knowledge to these organs and related diseases.

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Jang, J.H., Yamada, Y., Janker, F., De Meester, I., Baerts, L., et al., 2016. Anti-inflammatory effects on ischemia/reperfusion-injured lung transplants by the cluster of differentiation 26/dipeptidylpeptidase 4 (CD26/DPP4) inhibitor vildagliptin. J. Thorac. Cardiovasc. Surg. Jeanfavre, D.D., Woska Jr., J.R., Pargellis, C.A., Kennedy, C.A., Prendergast, J., et al., 1996. Effect of deoxycoformycin and Val-boroPro on the associated catalytic activities of lymphocyte CD26 and ecto-adenosine deaminase. Biochem. Pharmacol. 52, 17571765. Jungraithmayr, W., De Meester, I., Matheeussen, V., Baerts, L., Arni, S., et al., 2011. CD26/DPP-4 inhibition recruits regenerative stem cells via stromal cell-derived factor-1 and beneficially influences ischaemia-reperfusion injury in mouse lung transplantation. Eur. J. Cardiothorac. Surg. Jungraithmayr, W., Oberreiter, B., De Meester, I., Wiedl, T., Inci, I., et al., 2009. The effect of organ-specific CD26/DPP IV enzymatic activity inhibitor-preconditioning on acute pulmonary allograft rejection. Transplantation. 88, 478485. Jungraithmayr, W.M., Korom, S., Hillinger, S., Weder, W., 2009. A mouse model of orthotopic, single-lung transplantation. J. Thorac. Cardiovasc. Surg. 137, 486491. Kamori, M., Hagihara, M., Nagatsu, T., Iwata, H., Miura, T., 1991. Activities of dipeptidyl peptidase II, dipeptidyl peptidase IV, prolyl endopeptidase, and collagenase-like peptidase in synovial membrane from patients with rheumatoid arthritis and osteoarthritis. Biochem. Med. Metab. Biol. 45, 154160. Kanki, S., Segers, V.F., Wu, W., Kakkar, R., Gannon, J., et al., 2011. Stromal cell-derived factor-1 retention and cardioprotection for ischemic myocardium. Circ. Heart Fail. 4, 509518. Katagiri, D., Hamasaki, Y., Doi, K., Okamoto, K., Negishi, K., et al., 2013. Protection of glucagon-like peptide-1 in cisplatin-induced renal injury elucidates gut-kidney connection. J. Am. Soc. Nephrol. 24, 20342043. Korom, S., de Meester, I., Belyaev, A., Schmidbauer, G., Schwemmle, K., 2003. CD26/DPP IV in experimental and clinical organ transplantation. Adv. Exp. Med. Biol. 524, 133143. Korom, S., De Meester, I., Onodera, K., Stadlbauer, T.H., Borloo, M., et al., 1997a. The effects of CD26/DPP IV-targeted therapy on acute allograft rejection. Transplant. Proc. 29, 12741275. Korom, S., De Meester, I., Stadlbauer, T.H., Chandraker, A., Schaub, M., et al., 1997b. Inhibition of CD26/dipeptidyl peptidase IV activity in vivo prolongs cardiac allograft survival in rat recipients. Transplantation. 63, 14951500. Ku, H.C., Chen, W.P., Su, M.J., 2011. DPP4 deficiency preserves cardiac function via GLP-1 signaling in rats subjected to myocardial ischemia/reperfusion. Naunyn Schmiedebergs Arch. Pharmacol. 384, 197207. Matsubara, J., Sugiyama, S., Sugamura, K., Nakamura, T., Fujiwara, Y., et al., 2012. A dipeptidyl peptidase-4 inhibitor, des-fluoro-sitagliptin, improves endothelial function and reduces atherosclerotic lesion formation in apolipoprotein E-deficient mice. J. Am. Coll. Cardiol. 59, 265276. McCaughan, G.W., Gorrell, M.D., Bishop, G.A., Abbott, C.A., Shackel, N.A., et al., 2000. Molecular pathogenesis of liver disease: an approach to hepatic inflammation, cirrhosis and liver transplant tolerance. Immunol. Rev. 174, 172191. Mizokami, A., Eguchi, K., Kawakami, A., Ida, H., Kawabe, Y., et al., 1996. Increased population of high fluorescence 1F7 (CD26) antigen on T cells in synovial fluid of patients with rheumatoid arthritis. J. Rheumatol. 23, 20222026. Morimoto, C., Schlossman, S.F., 1998. The structure and function of CD26 in the T-cell immune response. Immunol. Rev. 161, 5570. Rajput, M.S., Sarkar, P.D., Nirmal, N.P., 2017. Inhibition of DPP-4 activity and neuronal atrophy with genistein attenuates neurological deficits induced by transient global cerebral ischemia and reperfusion in streptozotocin-induced diabetic mice. Inflammation. Sauve, M., Ban, K., Momen, M.A., Zhou, Y.Q., Henkelman, R.M., et al., 2010. Genetic deletion or pharmacological inhibition of dipeptidyl peptidase-4 improves cardiovascular outcomes after myocardial infarction in mice. Diabetes. 59, 10631073. Walsh, D.A., Mapp, P.I., Wharton, J., Polak, J.M., Blake, D.R., 1993. Neuropeptide degrading enzymes in normal and inflamed human synovium. Am. J. Pathol. 142, 16101621. Wang, X., Ding, Z., Yang, F., Dai, Y., Chen, P., et al., 2016. Modulation of myocardial injury and collagen deposition following ischaemiareperfusion by linagliptin and liraglutide, and both together. Clin. Sci. (Lond.). 130, 13531362. Williams, R.D., Denlinger, C.E., 2016. Single-agent management of diabetes and ischemia reperfusion injury. J. Thorac. Cardiovasc. Surg. Yamada, Y., Jang, J.H., De Meester, I., Baerts, L., Vliegen, G., et al., 2016. CD26 costimulatory blockade improves lung allograft rejection and is associated with enhanced interleukin-10 expression. J. Heart Lung Transplant. 35, 508517. Zhai, W., Cardell, M., De Meester, I., Augustyns, K., Hillinger, S., et al., 2007. Intragraft DPP IV inhibition attenuates post-transplant pulmonary ischemia/reperfusion injury after extended ischemia. J. Heart Lung Transplant. 26, 174180. Zhai, W., Jungraithmayr, W., De Meester, I., Inci, I., Augustyns, K., et al., 2009. Primary graft dysfunction in lung transplantation: the role of CD26/dipeptidylpeptidase IV and vasoactive intestinal peptide. Transplantation. 87, 11401146.

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11 DPPIV/CD26 as a Target in Anti-inflammatory Therapy Gwendolyn Vliegen and Ingrid De Meester University of Antwerp, Wilrijk, Belgium

The first part of this chapter will elaborate on dipeptidyl peptidase IV (DPPIV/CD26, EC 3.4.14.5) and the roles this enzyme plays in the immune system. We focus on relevant data and refer for more details to the reviews by (Klemann et al., 2016; Lambeir et al., 2003; Waumans et al., 2015). This forms the mechanistic base for the second part in which we look into the effects of therapeutically used DPPIV/CD26-inhibitors, also called gliptins, on the outcome of several inflammatory diseases, including type 1 diabetes, psoriasis and rheumatoid arthritis. In the last part, naturally occurring DPPIV/CD26-inhibitors will be discussed. For now, these are mostly studied in the setting of type 2 diabetes, but it can be expected that the interest in these compounds will be expanded to other fields, including inflammatory diseases.

11.1. INTRODUCTION TO DIPEPTIDYL PEPTIDASE IV DPPIV was originally identified in 1966 by Hopsu-Havu and Glenner as glycyl-prolyl-β-naphthylamidase (Hopsu-Havu and Glenner, 1966). Throughout the years, it was also discovered that DPPIV is the same as adenosine deaminase binding protein and cluster of differentiation (CD) 26 (De Meester et al., 1994; Morrison et al., 1993; Ulmer et al., 1990). DPPIV/CD26 is a serine exopeptidase that is capable of releasing dipeptides from the N-terminus of peptides, with preferentially Pro or Ala at the P1 position and a free amino acid on the P2 position ¯ et al., 1974). DPPIV/ (Yoshimoto et al., 1978). Pro and hydroxyproline at the P1ʹ position are not tolerated (Oya CD26 is closely related to the enzymes DPP8 and DPP9 (Abbott et al., 2000; Olsen and Wagtmann, 2002). DPPIV/CD26 is a type II membrane protein with 766 amino acids and a molecular weight of 110 kDa (Tanaka et al., 1992). The gene is located on the long arm of chromosome 2 (2q24.3) (Abbott et al., 1994). It consists of a cytoplasmic region (residues 16), a hydrophobic membrane-spanning domain (residues 728), and an extracellular domain comprising the remaining residues (Tanaka et al., 1992). DPPIV/CD26 has nine potential N-glycosylation sites (Aertgeerts et al., 2004a, 2004b; Lambeir et al., 2003). Dimerization of the enzyme is necessary for full activity (Chien et al., 2004). DPPIV/CD26 consists of two domains, an 8-bladed β-propeller domain (residues 61495) located at the N-terminus, and C-terminally there is the α/β hydrolase domain (residues 3955 and 497766) (Aertgeerts et al., 2004b), which is a typical fold for peptidases belonging to clan SC (Rawlings et al., 2012). The catalytic triad consists of Ser630, Asp708, and His740 (Hiramatsu et al., 2003) and lies at the interface of the two domains. There are two possible ways for substrates to access the active site (Aertgeerts et al., 2004b; Hiramatsu et al., 2003). First, a substrate can enter the active site via the top of the tunnel created by the β-propeller domain. Second, a large side opening is formed by blade-1 of the β-propeller domain and the C-terminal domain. Physiological substrates most likely use this second option to access the active site (Aertgeerts et al., 2004b; Hiramatsu et al., 2003). In 1970 a soluble form of human DPPIV/CD26 was found in the serum (Hopsu-Havu et al., 1970), but cellular sources of plasma DPPIV/CD26 and mechanisms of release have not been fully elucidated to date. Soluble DPPIV (sDPPIV/CD26) lacks the transmembrane region and starts at residue 39 (Ser) (Iwaki-Egawa et al., 1998). Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00011-1

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DPPIV/CD26 does not contain a cleavable signal sequence and secretion cannot be influenced by brefeldin A treatment, which implicates that DPPIV/CD26 follows a nonclassical secretion mechanism and is probably shed from the cell membrane (Raschke et al., 2013; Ro¨hrborn et al., 2014). Bone marrow derived cells such as leukocytes or macrophages (Wang et al., 2014) are reported as a source, but also adipocytes and skeletal muscle cells can contribute (Lamers et al., 2011; Raschke et al., 2013). The use of general broad spectrum protease inhibitors for matrix metalloprotease (MMP)s, cysteine proteases and serine proteases has led to a decrease in the release of sDPPIV from human vascular smooth muscle cells (SMC) (Ro¨hrborn et al., 2014). In adipocytes this was only true for the general MMP inhibitor. Combinations of these inhibitors did not result in an additive effect, suggesting that a proteolytic cascade leads to DPPIV/CD26 release. MMP1, MMP2 and MMP14 are implicated in the shedding of DPPIV/CD26 from SMC and MMP9 is involved in the release from adipocytes (Ro¨hrborn et al., 2014). DPPIV/CD26 has a wide tissue distribution with protein expression being highest in the kidney and the small intestine (Darmoul et al., 1994; Kettmann et al., 1992; The Human Protein Atlas, n.d.). Expression of DPPIV/ CD26 on immune cells will be discussed in a next section. Many substrates of DPPIV/CD26 have been identified, examples are neuropeptide Y (NPY), peptide YY (PYY), several chemokines and the incretin hormones glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide (GIP) among others (Lambeir et al., 2003; Mentlein et al., 1993a, 1993b). GLP-1 and GIP are probably best known, since preventing their rapid inactivation by inhibition of DPPIV/CD26 or augmenting the incretin effect by DPPIV/CD26-resistant analogues ameliorates type 2 diabetes (T2D) (Deacon et al., 1995; Kieffer et al., 1995; Mentlein et al., 1993b; Pauly et al., 1996; Pospisilik et al., 2002).

11.2. DPPIV IN THE IMMUNE SYSTEM DPPIV/CD26 can exert many effects in the immune system not only through modifications of substrates, but also through interactions with other molecules. Moreover, DPPIV/CD26 is also relatively widely expressed on cells of the immune system as discussed further. Closely related enzymes of DPPIV/CD26 have also been implicated in the immune system and are extensively reviewed in (Waumans et al., 2015).

11.2.1. Cells Originally, DPPIV/CD26 was characterized as a T-cell (activation) marker (Fleischer, 1994; Scho¨n et al., 1984; Scho¨n and Ansorge, 1990; Tanaka et al., 1992; Willheim et al., 1997), now it is known that DPPIV/CD26 is expressed on immune cells of both the innate (e.g., monocytes, macrophages, dendritic cells, natural killer [NK] cells) and the adaptive immune system (T- and B-cells). Low expression levels of DPPIV/CD26 were found on monocytes, macrophages and dendritic cells (Zhong et al., 2013); and also basophils were shown to express DPPIV/CD26, while mast cells do not (Valent et al., 1990). Unactivated B-cells showed a low expression of CD26, but this increased greatly after stimulation with pokeweed mitogen and Staphylococcus aereus (Bu¨hling et al., 1995). Similarly, upon activation with interleukin (IL)-2, NK cells showed an increased expression of DPPIV/ CD26 (Bu¨hling et al., 1994). The knowledge on the expression of DPPIV/CD26 on the surface of different subsets of T-cells expanded in parallel with the discovery of new T-cell subsets. Originally, it was described on a subset of CD41 memory cells, namely CD41 CD291 CD45RO1 cells which respond maximally to recall antigens such as tetanus toxoid (TT) and stimulate IgG synthesis from B-cells (Morimoto et al., 1989). DPPIV/CD26 can be found on both CD41 and CD81 T-cells (Bengsch et al., 2012; Hatano et al., 2013; Morimoto et al., 1989). Later on, analysis of CD26 expression on different subsets of CD41 T-cells (Th1, Th2, Th17, Treg) revealed that the expression, expressed as mean fluorescence intensity, was highest on CD41 cells producing cytokines typically for Th17 cells, e.g. IL-17, IL-22, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor (TNF) (Bengsch et al., 2012). Cells of the Th1 and Th2 subsets showed an intermediate expression, while regulatory T-cells (identified by the CD25hiCD127-FOXP31 phenotype or IL-10 production) had the lowest levels of CD26. More than 90% of Th17 cells could be found in the fraction containing the CD2611CD41 T-cells (Bengsch et al., 2012). DPPIV/CD26 is also expressed on a subset of mucosal-associated invariant T-cells (Sharma et al., 2015). Taken together, DPPIV/ CD26 is expressed on various immune cells, is upregulated after activation and is observed to be highly expressed on T-cells, especially Th17 cells.

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11.2.2. Substrates DPPIV/CD26 has many substrates; for some of them their involvement in the immune system is obvious, such as inflammatory chemokines, for others the link is less clear at first sight. Many chemokines contain the N-terminal X-Pro or X-Ala motif which is preferred by DPPIV/CD26. The N-terminal region in chemokines is essential in triggering the receptor. Modifications herein can result in analogues that lose their chemotactic activity, but can still bind their receptor and act as antagonists (Baggiolini, 1998). DPPIV-mediated truncation caused the loss of chemotactic activity of several chemokines, namely, stromal cell-derived factor 1/CaXaC motif chemokine 12 (SDF1/CXCL12) (Crump et al., 1997); regulated on activation, normal T-cell expressed and secreted/CaC motif chemokine ligand 5 (RANTES/CCL5) (Proost, 1998), interferon gamma-induced protein 10/CaXaC motif chemokine 10 (IP-10/CXCL10) (Proost, 2001), interferon-inducible T-cell alpha chemoattractant/CaXaC motif chemokine 11 (I-TAC/CXCL11) (Proost, 2001), monokine induced by gamma interferon/CaXaC motif ligand 9 (Mig/CXCL9) (Proost, 2001), eotaxin/CaC motif chemokine 11 (CCL11) (Forssmann et al., 2008) and CaC motif chemokine ligand 14 a (974) (CCL14a(974)) (Richter et al., 2009). However in certain instances, chemotactic activity was preserved or modified; such is the case for granulocyte chemotactic protein 2/CaXaC motif ligand 6 (GCP-2/CXCL6) (Proost, 1998) and macrophage-derived chemokine/ CaC motif chemokine 22 (MDC/CCL22), which is truncated twice by DPPIV/CD26 resulting in the removal of 4 N-terminal residues (Proost, 1999). Macrophage inflammatory protein (MIP)-1α/CaC motif ligand 3-like 1 (CCL3L1) can be cleaved by DPPIV/CD26; both forms are equally potent in attracting eosinophils and neutrophils, but have an altered receptor specificity (Struyf et al., 2001). Also cleavage of MIP-1β/CaC motif ligand 4 (CCL4) leads to an altered receptor specificity (Guan et al., 2004, 2002). Overall, this N-terminal processing of chemokines by DPPIV/CD26 can result in an altered inflammatory response and it is difficult to estimate the in vivo contribution of DPPIV/CD26 to chemokine biology. For a review on DPPIV/CD26 in chemokine biology, we refer to (Mortier et al., 2016). Processing of NPY and PYY results in altered receptor affinity; after truncation by DPPIV/CD26 these peptides lose their activity at the Y1-receptor, but retain agonists for Y2 and Y5 (Mentlein, 1999). This Y1-receptor plays a role in immune system, signaling through the receptor results in inhibition of T-cell activation and activates antigen-presenting cells (Wheway et al., 2005). Also GLP-1 has been identified as being implicated in various inflammatory diseases, having antiinflammatory effects through regulation of several molecular pathways in multiple organs as reviewed in (Campbell and Drucker, 2013; Lee and Jun, 2016).

11.2.3. Interactions With Other Molecules Next to its enzymatic functions, DPPIV/CD26 can also bind other molecules. One of these molecules is adenosine deaminase (ADA) which can bind with DPPIV/CD26 on T-cells (De Meester et al., 1994; Kameoka et al., 1993). Neither the enzymatic activity of ADA, nor the DPPIV activity is necessary for this association (Dong et al., 1996). Binding occurs in the cysteine-rich domain where five residues have been identified to be necessary, namely L294, L340, V341, A342, R343 (Abbott et al., 1999; Dong et al., 1997). Glycosylation of DPPIV/CD26 is not required for ADA binding (Aertgeerts et al., 2004a). The complex of DPPIV/CD26 and ADA reduces the inhibitory effect of adenosine on the activation of T-cells (Dong et al., 1996). DPPIV/CD26 can also bind to proteins of the extracellular matrix such as collagen and fibronectin (Cheng et al., 2003; Hanski et al., 1985; Lo¨ster et al., 1995). Unfortunately not much progress has been made on the possible involvement of DPPIV/CD26 in the biology of the extracellular matrix (Baerts et al., 2015).

11.2.4. DPPIV/CD26 as a Costimulatory Molecule of T-cells Besides the recognition of antigen presented by MHC-molecules on antigen presenting cells by the T-cell receptor (TCR), cosignaling molecules (costimulatory or coinhibitory) define the activation status and function of the T-cell (Chen and Flies, 2013). A plethora of these cosignaling molecules are already discovered, amongst which DPPIV/ CD26. Crosslinking of CD26 and CD3 with their respective immobilized antibodies resulted in an increase of proliferation of T-cells and IL-2 production (Dang et al., 1990a, 1990b; Tanaka et al., 1992). Mutant DPPIV/CD26 in which the active site Ser was replaced by Ala, had a reduced production of IL-2, although it was still higher than in control transfectants. This suggests that the enzymatic activity contributes to, but is not essential for, DPPIV/CD26 mediated costimulation (Tanaka et al., 1993). Work in the 1990s used antibodies targeted to DPPIV/CD26 to elucidate its role in

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T-cell activation, but the identification of natural ligands was primarily done a decade later. In 1995 it was suggested that ADA binding to DPPIV/CD26 could serve as a costimulatory signal (Martı´n et al., 1995). Indeed, in 2005 it was reported that the interaction between ADA bound to adenosine receptors on dendritic cells with DPPIV/CD26 expressed on lymphocytes resulted in an increased production of interferon-γ, TNF-α and IL-6 and was not dependent on the enzymatic activity of ADA (Pacheco et al., 2005). Discovery of caveolin-1 as a ligand followed the observation that sDPPIV/CD26 upregulated the expression of CD86 on TT-pulsed monocytes (Ohnuma et al., 2001). A mutant DPPIV/CD26 in which Ser630 was replaced by Ala did not show an upregulation of CD86, which implicates that other factors, most likely substrates, are involved. The interaction between DPPIV/CD26 on T-cells and caveolin-1 on TT-loaded monocytes leads to phosphorylation of caveolin-1, which in turn leads to dissociation of Tollip and IRAK-1; IRAK-1 is phosphorylated in the cytosol and consequently activates NF-κB, followed by upregulation of CD86 that can interact with CD28 (Ohnuma et al., 2005, 2004). The interaction between DPPIV/CD26 and caveolin-1 not only initiates signaling pathways in the antigenpresenting cells, but also in the T-cell itself (Ohnuma et al., 2011, 2008). Crosslinking of DPPIV/CD26 on DPPIV/ CD26-transfected Jurkat cells resulted in increased tyrosine phosphorylation of several signaling molecules which are involved in the downstream events following stimulation of the TCR (Hegen et al., 1997). It was also demonstrated that the membrane-associated guanylate kinase-like (MAGUK) molecule CARMA1 is bound to the cytoplasmic tail of dimeric DPPIV/CD26, and ligation of DPPIV/CD26 with caveolin-1 recruits it to lipid rafts where a complex of CARMA1-Bcl10-MALT1 is formed. The latter then becomes a hub for NF-κB activation, involving a ubiquitin-dependent IKK activation (Ohnuma et al., 2007). In addition, it is possible that CD26/DPPIV interacts with other molecules when it is recruited into lipid rafts (Ishii et al., 2001; Ohnuma et al., 2008). On the one hand, blockade of DPPIV/CD26 co-stimulation by soluble caveolin-1-Ig fusion protein induces anergy in CD41 T-cells (Ohnuma et al., 2009), while on the other hand DPPIV/CD26 costimulation induces the expression of early growth response 2 and IL-10, possibly as a regulating mechanism (Hatano et al., 2015).

11.2.5. Effects of DPPIV/CD26 on the Migration of Immune Cells Migration of immune cells from the blood into the tissue via the endothelial layer is critical for the immune response to a pathogen or foreign body lodged in the tissue. sDPPIV/CD26 has been shown to enhance transendothelial T-cell migration, with the mannose 6-phosphate/insulin-like growth factor-II receptor being the receptor of sDPPIV/CD26 on the endothelial cell surface. Addition of a DPPIV-negative mutant of sDPPIV/CD26 did not result in an enhancement of the transendothelial migration of T-cells, suggesting that one or more substrates are most likely responsible for the effects seen (Ikushima et al., 2002). In contrast, the migration of CD41 T-cells was reduced when CD2611 T-cells were added in transwell experiments containing the chemokines CXCL9, CXCL10, CXCL11 and CXCL12. This effect could be inhibited with the DPPIV/CD26-inhibitor P32/98. This might represent a negative feedback mechanism for the otherwise pro-inflammatory Th17 cells (Bengsch et al., 2012). Eotaxin-1 or CCL11 is an eosinophil-specific chemokine, which is truncated by DPPIV/CD26. Recruitment of eosinophils to the site of injection of CCL11 was higher in DPPIV-deficient or DPPIV-inhibited (Ile-thia) rats than in wild-type F344 rats (Forssmann et al., 2008). In murine and human neutrophils DPPIV/CD26 has been described as a chemorepellent, although some cells tend to move towards DPPIV/CD26. Also in this case the effect could be blocked with the inhibitor diprotin A (Herlihy et al., 2013). Barreira da Silva et al., showed in vivo that DPPIV/CD26-inhibition leads to the preservation of the active form of chemokine CXCL10, resulting in the recruitment of lymphocytes into the parenchyma of tumors (Barreira da Silva et al., 2015). In conclusion, the overall in vivo effect of DPPIV/CD26-inhibition might be quite complex as it affects several signaling pathways in the immune system, making it difficult to predict an outcome. Inhibition may or may not result in additive effects and the end result will be highly dependent on the setting tested. The clinical experience so far does not point to severe alterations in the overall immune response (Aso et al., 2015; Price et al., 2013; Sromova et al., 2016; White et al., 2010).

11.3. EFFECTS OF DPPIV/CD26-INHIBITORS IN AUTOIMMUNE AND INFLAMMATORY DISEASE Although DPPIV/CD26 can have multiple effects in the immune system and it has been suggested on numerous occasions that inhibition could be beneficial in several autoimmune and inflammatory diseases, clinical

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studies on this subject are scarce. In recent years, the focus has been mainly on gliptins for the treatment of vascular disease (Remm et al., 2015). For the following section, PubMed and ClinicalTrials.fgov were searched using the terms gliptins and rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, psoriasis, lupus, type 1 diabetes, asthma, ischemia reperfusion injury or autoimmune disease. We focused on the in vivo effect of the therapeutically approved DPPIV/CD26-inhibitors in different animal models and patients.

11.3.1. Preclinical Studies Gliptins have been widely studied in ischemia reperfusion injury (IRI) of the heart, lung and kidney in mice, rats, pigs, and dogs. These studies all come to the same conclusion: that the gliptin used provides protective effects against IRI and improves the function of the studied organ (Chang et al., 2013, 2015; Chen et al., 2013; Chinda et al., 2014, 2013; Chua et al., 2014; Glorie et al., 2012; Ihara et al., 2015; Jungraithmayr et al., 2012). The involvement of DPPIV/CD26 in IRI is reviewed in (Matheeussen et al., 2012). There are a limited number of preclinical studies with gliptins in other animal models. Administration of the DPPIV/CD26-inhibitor sitagliptin in ovalbumin sensitized and challenged mice resulted in a attenuation of the airway inflammation and remodeling, suggesting that sitagliptin might be beneficial in the treatment of asthma (Nader, 2015; Nader et al., 2012). Anagliptin was used in dextran sulfate sodium-induced colitis in mice as a model for inflammatory bowel disease. The DPPIV/CD26-inhibitor improved body weight loss and disease activity in the recovery phase (Mimura et al., 2013). In murine experimental autoimmune myocarditis linagliptin demonstrated reduced levels of pro-inflammatory cytokines and suppressed cardiac fibrosis (Hirakawa et al., 2015).

11.3.2. Clinical Studies A large population-based cohort study in T2D patients showed that patients who were already on metformin and started with gliptins had a reduced risk of autoimmune disease compared to patients starting with other antidiabetics (Kim et al., 2015). Reports on more specific disease areas are mentioned in the paragraphs below. 11.3.2.1. Type 1 Diabetes The rationale for the use of DPPIV/CD26-inhibitors in type 1 diabetes (T1D) is based on its effect on incretin hormones. Since T1D is an autoimmune disease, it would not be surprising that if beneficial effects are observed in this setting, this is also partially mediated through its immune effects. Addition of a gliptin to insulin therapy results in improved glycemic control, reduced insulin requirements, while preserving the glucagon counterregulation during hypoglycemia (Ellis et al., 2011; Farngren et al., 2012; Hari Kumar et al., 2013; Lima-Martı´nez et al., 2014). Since several clinical trials with gliptins in T1D are still ongoing, our knowledge on this matter will probably be expanded in the following years. 11.3.2.2. Psoriasis A case study on the effect of DPPIV/CD26-inhibitors in psoriasis show that after initiation of the DPPIV/ CD26-inhibitor sitagliptin in a patient with T2D, the psoriasis improved without lowering HbA1c (Nishioka et al., 2012). In another case, sitagliptin was given to a psoriatic patient without diabetes, which also improved the psoriasis (Lynch et al., 2014). However, in one case report a patient with T2D prescribed with sitagliptin developed a psoriasiform eruption after the sixth dose of sitagliptin (Mas-Vidal et al., 2012). Thus, no clear pattern or effect of DPPIV/CD26-blockade in psoriasis patients has emerged yet. 11.3.2.3. Rheumatoid Arthritis There are also several case reports on gliptins and rheumatoid arthritis (RA). Patients taking sitagliptin to manage their T2D, presented within a time frame of several months with RA (Sasaki et al., 2010; Yokota and Igaki, 2012). One of these patients showed to be genetically predisposed and another patient was in a state of RA remission before starting the therapy with a DPPIV/CD26-inhibitor (Sasaki et al., 2010; Yokota and Igaki, 2012).

11.3.3. Immunological Side Effects Although immunological effects are desirable in patients with autoimmune and inflammatory disease, they may be a concern in patients treated with gliptins for T2D. As reviewed in (Karagiannis et al., 2014), earlier

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studies reported an increased risk of all-cause infections (Amori et al., 2007; Richter et al., 2008), but later metaanalyses could not find significant differences in infection risk with placebo or active comparators (Karagiannis et al., 2012; Liu et al., 2014; Yang et al., 2016). The effects on the very long run are not clear for now and should be kept in mind (Klemann et al., 2016; Stulc and Sedo, 2010). A limited number of studies address the effect of DPPIV/CD26-inhibition on immune function (Aso et al., 2015; Price et al., 2013; Sromova et al., 2016; White et al., 2010). Although the results do not fully align, probably due to different dosing, study time and disease setting, differences in the T-cell subsets were seen, but seem to be of a temporary nature (Aso et al., 2015; Price et al., 2013; Sromova et al., 2016). Taken together, these data are reassuring that the use of DPPIV/CD26-inhibitors does not result in overt alterations in the functioning of the immune system as far as can be judged after 10 years of therapeutic use.

11.4. DPPIV/CD26-INHIBITORS 11.4.1. The Gliptins, Therapeutically Used DPPIV/CD26-Inhibitors Diprotin A (Ile-Pro-Ile) and diprotin B (Val-Pro-Leu) were the first inhibitors of DPPIV to be identified from bacteria in 1984 (Umezawa et al., 1984), although later it was shown that they are actually substrates with a low turnover rate (Rahfeld et al., 1991). Linking DPPIV to T2D stimulated research for the development of DPPIVinhibitors, with sitagliptin being the first gliptin to reach therapeutic approval in 2006 (FDA, 2006; Kim et al., 2005). Several others gliptins are currently approved or under investigation. See Tables 11.1 and 11.2 for an overview of the currently approved gliptins and those which are in clinical trials (Adis Insight, n.d., European Medicines Agency, n.d., Galvus Approval Status, n.d., U.S. Food and Drug Administration, n.d.; GlaxoSmithKline, n.d.; Jiangsu HengRui Medicine Co., n.d.; Kenilworth, 2016; McCormack, 2015; McKeage, 2015; Pfizer, n.d.; Phenomix, n.d.; Roche, n.d.; Rosenstock et al., 2011; Sheu et al., 2015). DPPIV/CD26-inhibitory peptides can be derived from food proteins, but other substances originating from plants can also have inhibitory properties. Experiments in this context usually focus on glucose homeostasis, since DPPIV/CD26 inhibition is already therapeutically used in T2D. However, these compounds might find an application as a nutritional intervention therapy, not only as an addition to conventional diabetes treatment but also in other settings (Power et al., 2014).

11.4.2. Naturally Occurring DPPIV/CD26-Inhibitors 11.4.2.1. Food Proteins Many food proteins have already been evaluated in vitro for their DPPIV-inhibitory characteristics. Hydrolysates from, for example, quinoa (Chenopodium quinia Willd.) (Nongonierma et al., 2015), hemp, pea, rice and soy (Nongonierma and FitzGerald, 2015), amaranth (Amaranthus hypochondriacus L.) (Velarde-Salcedo et al., 2013) and common bean (Phaseolus vulgaris) (Mojica et al., 2015) have been identified as being capable to inhibit DPPIV/CD26. Besides these, peptides derived from milk proteins (Tulipano et al., 2011; Uchida et al., 2011; Uenishi et al., 2012), egg (Van Amerongen et al., 2009) and gelatin (Kuo-Chiang Hsu, Yu-Shan Tung, 2013; Li-Chan et al., 2012) have been described to inhibit DPPIV/CD26. When IC50 values are reported for individual compounds, these are in general around 1000-fold higher than the clinically approved. DPPIV/ CD26-inhibitors (μM vs nM range) (Augeri et al., 2005; Eckhardt et al., 2007; Feng et al., 2007; Kim et al., 2005; Villhauer et al., 2003). In vivo studies evaluating the effects of orally administrated hydrolysates or specific peptide inhibitors are rare and mainly focus on the antidiabetic effect, mostly tested in an oral glucose tolerance test, and do not always report whether plasma DPPIV/CD26 is inhibited (Huang et al., 2014; Uchida et al., 2011; Uenishi et al., 2012; Van Amerongen et al., 2009). It is not clear whether in vivo DPPIV-inhibition contributes to the observed effects.

11.4.2.2. Components Derived From Plants Grape seed-derived procyanidins (GSPE) were tested for their ability to inhibit DPPIV/CD26. In vitro and in vivo an inhibitory effect could be seen, but only on the intestinal DPPIV/CD26-activity, not on plasma DPPIV/CD26. These findings possibly suggest that the GSPE could not reach the systemic circulation (Gonza´lezAbuı´n et al., 2012). It would be interesting to study if the intestinal inhibition of DPPIV/CD26 is sufficient to

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TABLE 11.1 Overview of Approved DPPIV/CD26-Inhibitors Name

Trade name

Standard dosage

Year of approval

Alogliptin

Vipidia/Nesina

25 mg/day

EMA: 2013 FDA: 2016 Japan: 2010

Anagliptin

Suiny

2 3 100 mg/day

EMA: / FDA: / Japan: 2012

Evogliptin

Suganon

5 mg/day

EMA: / FDA: / Korea: 2015

Gemigliptin

Zemiglo

50 mg/day

EMA: / FDA: / Korea: 2012

Gosogliptin

SatRx

20 mg/day

EMA: / FDA: / Russia: 2016

Linagliptin

Trajenta/Tradjenta/Trazenta

5 mg/day

EMA: 2011 FDA: 2011 Japan: 2013

Omarigliptin

Marizev

25 mg once a week

EMA: /a FDA: /a Japan: 2015

Saxagliptin

Onglyza

5 mg/day

EMA: 2009 FDA: 2009 Japan: 2013

Sitagliptin

Januvia

100 mg/day

EMA: 2007 FDA: 2006 Japan: 2009

Teneligliptin

Tenelia

20 mg/day

EMA: / FDA: / Japan: 2012

Trelagliptin

Zafatek

100 mg once a week

EMA: /b FDA: /b Japan: 2015

Vildagliptin

Galvus

2 3 50 mg/day

EMA: 2007 FDA: /c Japan: 2013

a

No marketing applications ongoing in US and EU. Discontinued development in US and EU due to costs associated with gaining approval. Application discontinued when FDA requested additional data. EMA, European Medicines Agency; FDA, U.S. Food and Drug Administration (European Medicines Agency, n.d., Galvus Approval Status, n.d., U.S. Food and Drug Administration, n.d.; Kenilworth, 2016; McCormack, 2015; McKeage, 2015; Pfizer, n.d.; Rosenstock et al., 2011; Sheu et al., 2015).

b c

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TABLE 11.2 Overview of DPPIV/CD26-Inhibitors in Clinical Trials (Adis Insight, 2016.; GlaxoSmithKline, n.d.; Jiangsu HengRui Medicine Co., n.d.; Phenomix, n.d.; Roche, n.d.) Gliptin

Latest stage of development

Carmegliptin

Phase II (2008)

Denagliptin

Phase II/III (2006)

Dutogliptin

Phase III (2010)

Melogliptin

Phase II (2009)

Retagliptin

Phase I (2015)

Comment

Withdrawn, unfavorable preliminary data

Further development discontinued. No indication of reason

have an effect in diabetes. Several compounds from culinary herbs have been identified as inhibitors of DPPIV/ CD26. Flavonoids such as cirsimaritin, hispidulin and naringenin, present in Mexican oregano and marjoram, have been reported with IC50 values of 0.43, 0.49, and 2.5 μM, respectively (Bower et al., 2014). Compounds with DPPIV/CD26-inhibitory features were isolated from Fagonia cretica L. and Hedera nepalensis K. Koch with the most potent compounds having IC50-values ranging from 23.5 to 57.9 μM (Saleem et al., 2014). Berberine has also been shown to inhibit DPPIV/CD26 in vitro with an IC50 of 13.3 μM (Al-masri et al., 2009). Anthocyanins fractions isolated from blueberry-blackberry wine blends showed IC50 values ranging from 0.07 μM to more than 300 μM. Several phenolic compounds commonly found in citrus, berry, grape and soybean were also evaluated for their DPPIV/CD26-inhibitory characteristics. The most potent compounds found were resveratrol (IC50 5 0.6 nM), luteolin (IC50 5 0.12 μM), apigenin (IC50 5 0.14 μM) and flavone (IC50 5 0.17 μM) (Fan et al., 2013). The low IC50 value of resveratrol is remarkable, since sitagliptin has an IC50 value of 18 nM (Kim et al., 2005). The dietary intake of resveratrol is in the milligram range, but supplements containing doses up to 100 mg are available. After the oral ingestion of 25 mg of resveratrol it is rapidly metabolized and only trace amounts (levels lower than 5 ng/mL corresponding to 22 nM) could be found (Walle et al., 2004). Even though plasma concentration of resveratrol is low, it would still be high enough to obtain efficient DPPIV/CD26-inhibition. 11.4.2.3. Probiotics Several probiotics have been identified as being able to inhibit DPPIV/CD26 in vitro. However, the ability of a probiotic to survive and colonize the intestine of the host is also a very important factor to consider, since this will determine if it can be an effective DPPIV/CD26-inhibitor in vivo. Zeng et al. studied cell-free intracellular extracts of 13 Bifidobacteria strains, from these strains, B. adolescentis IF1-11 and B. bifidum IF3-211 showed the greatest DPPIV/CD26 inhibitory effect. These strains were also quite stable in simulated gastric and bile juices and showed good adhesion to Caco-2 cells (Zeng et al., 2016b). The same group also studied 21 Lactobacillus strains for their possible DPPIV-inhibitory effects (Zeng et al., 2016a). Cell-free excretory supernatans and cell-free intracellular extracts were tested and the strains with the highest DPPIV-inhibitory effect were submitted to simulated gastrointestinal conditions and their adhesion to HT-29 cells. Several strains were found to show concentration-dependent DPPIV-inhibition and should be able to survive in the gastrointestinal tract of the host. Trypsin treatment of the cell-free excretory supernatants resulted in increased inhibition of DPPIV/CD26, suggesting that peptide compounds are secreted and that their processing is necessary for an optimal effect (Zeng et al., 2016a).

11.4.3. Oral Bioavailability When considering the use of naturally occurring DPPIV/CD26-inhibitors as nutraceuticals, the oral bioavailability of these compounds is important. Peptides and proteins are known to be rapidly hydrolyzed when they pass through the gastrointestinal tract. Although, di- and tripeptides are capable of crossing the intestinal epithelium by the PepT1 H1/Peptide cotransporter, most of these di- and tripeptides are hydrolyzed by peptidases into amino acids once they are in the enterocyte (Miner-Williams et al., 2014). Only a minority can cross the basolateral membrane in their intact form. There is also little evidence that peptides having four amino acids or more can be absorbed from the gastrointestinal tract (Miner-Williams et al., 2014). Also procyanidins have a low oral bioavailability. A study in humans using 2.0 g of a procyanidin-rich grape seed extract showed serum levels of

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procyanidin B1 of 10.6 nM 2 h after intake (Sano et al., 2003). Since the daily intake of procyanidins has been estimated to be 95 mg per day, it is highly unlikely that this can reach therapeutic levels (Wang et al., 2011).

11.5. CONCLUSION There is no doubt about the involvement of DPPIV/CD26 in immune regulation, and DPPIV/CD26-inhibitors deserve further investigation as potential therapeutics for the treatment of certain inflammatory conditions. Limited clinical data already suggest beneficial effects of gliptins in T1D and psoriasis. Clinical trials in these and other inflammatory diseases will enlighten us in the future on their applicability as an anti-inflammatory therapy. Many naturally occurring DPPIV/CD26-inhibitors are already described, but their effects after oral application in diabetes and other conditions should be further explored and special attention should be given to the bioavailability. Also important to note, none of the aforementioned studies on natural DPPIV/CD26-inhibitors has tested the selectivity of the compounds towards other closely related enzymes, such as DPP8 and DPP9. This has to be kept in mind when attributing effects to DPPIV/CD26, since these may be mediated by the other enzymes. More in vivo studies are necessary to evaluate whether these natural compounds can be used as an add-on to conventional therapy as a functional food or as a nutraceutical. These compounds can also form leads for the development of more potent and selective DPPIV/CD26-inhibitors as has been exemplified in (Li et al., 2016; Zhang et al., 2011).

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C H A P T E R

12 Redox Sensitive Transcription via Nrf2-Keap1 in Suppression of Inflammation Elango Bhakkiyalakshmi, Dornadula Sireesh and Kunka M. Ramkumar SRM University, Kattankulathur, Tamil Nadu, India

12.1. INTRODUCTION Metabolic syndrome is becoming more common for the past few decades with its risk factors closely linked to overweight/obesity and lack of physical activity. The improved life expectancy has led to an increased health burden related to metabolic disorders, resulting in an increased pressure on healthcare services. Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The pathogenic processes involved in the progression of diabetes range from autoimmune assault of pancreatic β-cells with failure in insulin secretion, to abnormalities that result in resistance to insulin action at target tissues (American Diabetes, 2009). Major contributing factors for β-cell damage include mitochondrial dysfunction, glucolipotoxicity, inflammation, oxidative and endoplasmic reticulum stress (Prentki and Nolan, 2006). Pancreatic β-cell death is a crucial event in the pathogenesis of both type 1 diabetes (T1D) and type 2 diabetes (T2D). In T1D, autoimmune attack of β-cells causes progressive cell death, whereas the pathogenesis of T2D mainly involves insulin resistance leading to β-cell failure (Alberti and Zimmet, 1998). Intraislet expression of inflammatory mediators such as Interleukin (IL)-1β and tumor necrosis factor (TNF)-α that are characteristic of both T1D and T2D, trigger cascade of events leading to β-cell apoptosis and progressive β-cell loss (Donath et al., 2003). Accumulated evidences point to a key role of inflammation in the development of β-cell dysfunction (Collier et al., 2011; Eizirik et al., 2009; Quan et al., 2013; Wang et al., 2010). Inflammation in β cells is double edged; on the one hand it is an essential event in β-cell repair and regeneration, while on the other hand chronic inflammation can be deleterious as it causes the activation of autoinflammatory reactions that damage pancreatic islets (Donath et al., 2009). Moreover, proinflammatory signaling pathways inhibit insulin signaling, thereby providing a link between inflammation and insulin resistance (Schenk et al., 2008). Islet inflammation mainly exhibits architectural abnormalities, macrophage infiltration, and increased expression of cytokines such as IL-1β, CC motif chemokine ligand 2 (CCL)-2, and TNF-α that collectively impair the functions of β-cells as well as alter the β-cell mass eventually promoting an inflammatory response that leads to cell death (Donath et al., 2009; Ehses et al., 2009). Inflammatory process triggers the cascade of events via transcription factor nuclear factor (NF)-κB, which is the hallmark of β-cell death. As NF-κB induced proinflammatory cytokines are the major cause of inflammatory tissue injury that becomes deleterious to the pancreatic islets (Salem et al., 2014), the inhibition of this pathway has become a promising strategy to fight against islet inflammation. Conversely, β-cells have innate mechanisms to tolerate a variety of stress conditions including oxidative stress and inflammation. It includes “Nrf2-Keap1ARE” pathway, a key cellular defense mechanism, to fight against a variety of stress conditions by regulating an array of cytoprotective genes (Yagishita et al., 2014). Moreover diabetic conditions experience defective Nrf2dependent signaling that sensitizes β-cells to environmental insults (Masuda et al., 2015). Nuclear factor erythroid 2related factor 2 (Nrf2) is a crucial transcription factor, and a master regulator of chemical and oxidative stress, whereas Kelch-like ECH-associated protein 1 (Keap1) is the negative regulator of Nrf2 (Zhang, 2006). The Nrf2-Keap1 system mainly regulates the antioxidant enzymes, superoxide dismutase Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00012-3

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(SOD), glutathione S-transferase (GST), glutathione peroxidase (GPx), glutamate-cysteine ligase (GCL), catalase (CAT), thioredoxin (TRX) and detoxifying enzymes, NAD(P)H quinine oxidoreductase 1 (NQO1), and hemeoxygenase-1 (HO-1) (Taguchi et al., 2011). For the past decade, research has been geared towards the understanding of the anti-inflammatory role of Nrf2 that mainly speculates that an array of Nrf2 downstream target genes suppresses the expression of proinflammatory cytokines, such as interleukins and TNF-α (Itoh et al., 2004). Few reports also suggested that HO-1 and PrxI (Peroxiredoxin I) influence the inflammatory process (Ishii et al., 2000; Itoh et al., 2004). Moreover, the activation of redox sensitive transcription factor, NF-κB is limited by the reducing environment. Conversely, oxidative/nitrosative stress promotes the phosphorylation and degradation of IκB, the inhibitor of NF-κB. Nrf2 initiates the reducing environment by upregulating intracellular GSH levels and GSHdependent enzymes favoring the inhibition of NF-κB (Banning and Brigelius-Flohe, 2005). Hence, Nrf2 activation represents a promising strategy to safeguard the system against various stress conditions including inflammation. With this background information, this review delineates the mechanism and regulation of Nrf2-Keap1-ARE pathway, the importance of Nrf2 as a therapeutic target in islet inflammation with a focus towards the role of Nrf2 in islet biology.

12.2. ISLET INFLAMMATION IN DIABETES A major contributing factor for autoimmune-mediated T1D is the proinflammatory cytokine, Interleukin (IL-), IL-1β (Mandrup-Poulsen, 1996). It initiates Fas-mediated apoptosis in β-cells by triggering NF-κB signaling (Maedler et al., 2002). Moreover, elevated glucose concentrations also induce β-cell apoptosis via IL-1β production (Donath et al., 2008). Interestingly, pancreatic sections of T2DM patients showed elevated levels of IL-1β when compared to nondiabetic individuals (Welsh et al., 2005). Hence, these findings identify IL-1β as a major causative factor for β-cell dysfunction and its associated islet inflammation (Fig. 12.1).

FIGURE 12.1 Islet inflammation. Long-term exposure of β-cells to increased glucose and free fatty acids promotes the activation of inflammatory response. IL-1β, the major proinflammatory cytokine produced by the β-cells attract the immune cells. Macrophages are also activated by elevated levels of glucose, chemokines (CXCL1 and monocyte chemoattractant protein-1 or MCP-1) and cytokines (IL-1β, TNF-α, IL-6) triggering proinflammatory NF-κB pathway leading to β-cell apoptosis.

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Moreover, elevated glucose levels in short term confer β-cell proliferation, whereas in long-term the levels promote Fas-mediated β-cell apoptosis triggered by IL-1β. Conversely, Fas signaling also mediates β-cell proliferation when FLICE-inhibitory protein, a caspase 8 inhibitor, is active (Maedler et al., 2006). An elevated level of IL-1β initiates the cascade of events by self-induction which mainly promotes the release of chemokines and triggers the activation of NF-κB and pancreatic and duodenal homeobox 1 that finally recruits the macrophages (BoniSchnetzler et al., 2008; Schumann et al., 2007). Hence, the macrophage cells cause the release of higher levels of IL-1β, decrease β-cell mass, impair β-cell function and initiate islet inflammation (Donath et al., 2009). Dyslipidemia, leptin and circulating cytokines are major causative factors that link obesity to β-cell failure. The influence of dyslipidemia on the β-cells will depend on the lipid profile where free fatty acids and lipoproteins play a key role in β-cell apoptosis (Bardini et al., 2012). Long-term exposure of β-cells to saturated fatty acids such as palmitate mainly affects the β-cell turnover and function (Maedler et al., 2001). Lipoproteins play an important role in β-cell survival and function, where high density lipoprotein is cytoprotective in nature, while very low density lipoprotein and low density lipoprotein remain to be proapoptotic factors (Cnop et al., 2002; Fryirs et al., 2010). Leptin shares its structural similarity with other proinflammatory cytokines and henceforth turns on the receptor-mediated signaling cascade (Lord, 2006). In nonobese diabetic mice, leptin was found to accelerate autoimmune responses, thereby providing a link between T1D and T2D (Matarese et al., 2002). In addition, adipocytes release TNF-α and IL-6 and modulate β-cell survival whose mechanism of action remains unclear (Fain, 2006; Wang et al., 2010). Few reports identified that islet inflammation is chiefly characterized by macrophage infiltration (Chawla et al., 2011; Ehses et al., 2007; Kugelberg, 2013). Ehses et al., studied islet inflammatory process in nonobese GotoKakizaki rats and identified increased islet expression of IL-1β and other proinflammatory cytokines (IL-6, TNFα), a number of chemokines including (chemokine (C-X-C motif) ligand (CXCL)1, macrophage inflammatory protein (MIP-1α), MCP-1, together with immune cell infiltration (Ehses et al., 2009). Interestingly, few clinical studies reported increased islet-associated CD68 1 cells in T2D patients (Ehses et al., 2007; Richardson et al., 2009). Therefore, insulin-sensitive tissues undergo autoinflammatory process, which also targets other organs including eye, kidney and the vasculature. Hence, inflammation in the tissues not only initiates β-cell loss but also is involved in also the pathogenesis of diabetes complications.

12.3. COMPONENTS OF THE NRF2-KEAP1-ARE MACHINERY 12.3.1. Antioxidant Response Element Antioxidant response element (ARE), also termed as the electrophile response element, is a cis-regulatory element or enhancer sequence, which is found in the promoter region of several genes encoding detoxification enzymes and cytoprotective proteins. The core sequence of ARE includes 50 -TGACNNNGC-30 and responds mainly to oxidative stress inducers (Rushmore et al., 1991). Under conditions of oxidative stress, Nrf2 dissociates from Keap1, translocates into the nucleus, forms heterodimer with small Maf family of transcription factors, and binds to the ARE to transcriptionally activate antioxidant genes (Itoh et al., 1997). Bach1 (BTB and CNC homology1) is a transcriptional repressor of ARE. Under normal physiological conditions, Bach1 forms a dimer with Maf protein and prevents Nrf2 binding to ARE. Also Bach1 undergoes rapid nuclear export and proteasomal degradation in response to Nrf2/ARE inducers (Dhakshinamoorthy et al., 2005).

12.3.2. Cullin3-Rbx1 E3 Ubiquitin Ligase (Cul3-Rbx1-E3-Ligase) Generally, proteins are targeted to proteasomal degradation via 26S proteasome by covalent attachment of polyubiquitin moieties (Bonifacino and Weissman, 1998). Conjugation of ubiquitin is mediated by serial activation of E1, E2 and E3 ubiquitin ligase. Here E2 and E3 are found to coordinate the transfer of ubiquitin to the substrate protein, and E3 generally promotes substrate specificity (Hershko and Ciechanover, 1998). Interestingly, Cullin-3 is targeted to ubiquitination substrates via adapter proteins containing the BTB domain including the kelch repeat domains of Keap1 (Furukawa et al., 2003; Xu et al., 2003; Cullinan et al., 2004). Under basal condition, Nrf2 levels are regulated by Keap1 protein that binds to Cul3-Rbx1-E3-ligase and directs Nrf2 for ubiquitination-proteosomal degradation (Villeneuve et al., 2010).

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12.3.3. Small Maf (sMaf) Proteins sMaf proteins, composed of MafF, MafG, and MafK, possess a basic leucine zipper (bZIP) domain that is essential for homo-/hetero-dimerization with other bZIP transcription factors (Kannan et al., 1823; Motohashi et al., 2004). Since sMaf proteins lack the transactivation domain, its homodimer complex serves as transcription repressors (Toki et al., 1997), whereas Nrf2 forms heterodimer with sMaf proteins because of its specificity, thereby binding to high affinity ARE promoters of antioxidant and detoxifying genes (Itoh et al., 1997; Li et al., 2008).

12.3.4. Nuclear Factor Erythroid 2Related Factor 2 (Nrf2) Nrf2, a key transcription factor is essential for maintaining cellular homeostasis. It is a 66-kDa (605 amino acids) cap “n” collar (CNC) protein with a bZip DNA binding motif that is characteristic of NF-E2. Nrf2 contains six highly conserved domains named Nrf2-ECH homology domains (Neh1-6) (Bryan et al., 2013; Li et al., 2012) (Fig. 12.2). Neh1: The Neh1 domain lies in the C-terminal region and corresponds to bZip motif necessary for dimerization with sMaf and binding to DNA. This domain also comprises the nuclear localization sequence (NLS, residues: 494511), which is necessary for the nuclear localization of Nrf2 (Itoh et al., 1997).

FIGURE 12.2

(A) Components of Nrf2-Keap1-ARE machinery. The association of Nrf2 with Keap1 requires the association of CUL3RBX1-E3 ligase complex. Cul3 then recruits BTB (broad complex/tramtrack/bric-a`-brac) domain of Keap1 protein for homodimerization. The BTB domain is a proteinprotein interaction domain of Keap1. The dimeric assembly of the KEAP1-CUL3 complex is essential for the regulation of Nrf2, which contains two main degrons, a high affinity (ETGE) motif and a low affinity (DTG) motif separated by seven lysine residues for ubiquitin-mediated proteasomal degradation. (B) Domain structure of Nrf2 and Keap1. Nrf2 consists of 6 domains, Neh 16. Neh1 contains the basic leucine zipper (L-zip) region for dimerization with small Maf proteins. Neh2 contains DLG (weak affinity) and ETGE (strong affinity) motifs, responsible for interaction with Keap1. The seven lysine residues between these motifs are indispensable for Keap1dependent polyubiquitination and proteasomal degradation of Nrf2. Neh35 contains residues important for transcriptional activity of Nrf2 where both CBP and CHD6 are transcriptional coactivators. (CBP: cAMP response element binding protein-Binding Protein; CHD6: chromoATPase/helicase DNA binding protein family 6). Neh6 functions as degron, recognized by Trcp, to mediate degradation of Nrf2 in the nucleus (Trcp: Transducin repeats-containing proteins, an E3 ligase). Keap1 consists of five domains. The two proteinprotein interaction motifs, the BTB (Broad complex, Tramtrack and Bric-a`-Brac) domain and the Kelch containing DGR (double glycine repeats) domain, are separated by the intervening region (IVR). The BTB domain together with N-terminal region of IVR mediates homodimerization of Keap1 and Cullin-3 (CUL3) binding. The Kelch domain and the C-terminal region mediate Keap1 interaction with the Neh2 domain of Nrf2.

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Neh2: The highly conserved Neh2 domain lies at the N-terminal region of the protein. It serves as a negative regulatory domain in Nrf2 transcriptional activity. Neh2 contains DLG and ETGE motifs which correspond to the two binding sites for the Keap1 Kelch domain that facilitates the formation of a complex composed of one molecule of Nrf2 and two molecules of Keap1 (Katoh et al., 2005). The presence of seven lysine residues within Neh2 allows for negative regulation of Nrf2 transcriptional activity via proteasome-mediated Nrf2 degradation (Zhang et al., 2004). The presence of a serine residue (Ser40) in the Neh2 domain is essential for the release of Nrf2 from Keap1. Phosphorylation at Ser40 is required for Nrf2 to dissociate from Keap1 and thus inhibit Keap1-mediated ubiquitination. However, Ser40 is reported to be nonessential for Nrf2 stabilization and accumulation in the nucleus (Bloom and Jaiswal, 2003; Huang et al., 2002). Neh3: The Neh3 domain of Nrf2 is among members of the CNC bZIP transcription factors. It is located at the C-terminus of the protein and is essential for the transactivation of ARE genes by Nrf2 (Nioi et al., 2005). Neh4 and 5: The Neh4 and Neh5 domains are considered transactivation domains that cooperatively bind to cAMP response element binding (CREB) protein (CBP), which has been shown to be an essential coactivator for many transcription factors (Katoh et al., 2001). Neh6: The Neh6 domain, which is located in the middle of Nrf2, has been reported to be associated with redox-insensitive degradation of Nrf2 (McMahon et al., 2004).

12.3.5. Kelch-Like ECH-Associated Protein 1 (Keap1) Keap1 is a 69-kDa actin-binding protein and composed of 624 amino acid residues, 27 of which are cysteine residues (McMahon et al., 2004). Keap1 consists of five distinct domains: (1) the N-terminal region (NTR), (2) the broad complex, tram track and bric-a-brac (BTB) domain, (3) the intervening region (IVR), (4) the double glycine repeats (DGR) or Kelch domain, and (5) the C-terminal region (CTR) (Canning et al., 2015; Itoh et al., 2004). BTB: The BTB domain is an evolutionary conserved domain also found in actin-binding proteins and zinc finger transcription factors. Keap1 forms homodimer through the BTB domain that is required for binding to Nrf2 (Cleasby et al., 2014). In addition, the BTB domain is also responsible for the interaction between Keap1 and Cullin3-Rbx1 E3 ubiquitin ligase (Cul3-E3-ligase) (Canning et al., 2013). IVR: The cysteine rich IVR is sensitive to oxidation and the nuclear export signal motif, and is necessary for Keap1 activity (Canning et al., 2015). In the IVR domain of Keap1, four especially reactive cysteine residues have been identified: Cys257, Cys273, Cys288, and Cys297. Cys273 and Cys288 are essential for Keap1-dependent ubiquitination of Nrf2 and Keap1-mediated repression of Nrf2 activity (Dinkova-Kostova et al., 2002; Zhang and Hannink, 2003). Both the BTB and (IVR) domains were shown to be essential for Nrf2 degradation (Chauhan et al., 2013). DGR: The Kelch domain consists of six repeating Kelch motifs (KR1KR6) that form a six-bladed β-propeller structure. The Kelch domain is where Keap1 binds to the Neh2 domain of Nrf2 (Li et al., 2004).

12.4. MECHANISM AND REGULATION OF THE NRF2-KEAP1-ARE PATHWAY Keap1 functions as a master regulator of the Nrf2-Keap1-ARE pathway by controlling the steady state level of Nrf2 based on cellular redox conditions (Zhang, 2006). Under basal conditions, Nrf2 is bound to Keap1 and targeted for ubiquitination and proteasomal degradation by Cul3-E3-ligase, with a t1/2 of less than 20 min (Kobayashi et al., 2004). The rapid turnover of Nrf2 prevents the unnecessary expression of Nrf2 target genes. At the initiation of the regulatory process, Keap1 forms homodimer via its BTB domain as discussed earlier. The Neh2 domain of Nrf2 comprises two binding motifs: the high affinity ETGE and the low affinity DLG motifs. The ETGE and DLG motifs each binds to a separate Kelch domain in the Keap1 dimer (Tong et al., 2006). Under basal condition, the Nrf2-Keap1 system follows the two-site substrate recognition model, where the binding of each motif to a kelch domain is necessary for the ubiquitination of Nrf2 that leads to its rapid degradation by 26S proteasome. The α-helix region with seven lysine residues located between the binding motifs drives Nrf2 for ubiquitination-proteasomal degradation (Tong et al., 2006). Under induced conditions, the Keap1Nrf2Cul3 assembly is disturbed, and as a consequence, Nrf2 gets stabilized in the cytoplasm (t1/2 of up to 200 min) and translocates to the nucleus and triggers the cytoprotective genes (Tong et al., 2006) (Fig. 12.3).

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FIGURE 12.3 Nrf2-Keap1 pathway. In a basal state, Nrf2 is polyubiquitylated by Kelch-like ECH-associated protein 1 (Keap1)cullin 3 (CUL3) complex. Keap1 is a substrate adapter protein and CUL3 is an E3 ubiquitin ligase. This polyubiquitylation results in proteasomal degration of Nrf2. In induced state, the stimulus from variety of insults promotes the dissociation of Nrf2 from Keap1-Cul3 complex. Further Nrf2 translocate into the nucleus, where it becomes transcriptionally active after binding with antioxidant responsive element (ARE) along with one of its partner, Maf proteins thereby triggering an array of antioxidant and detoxifying genes.

12.5. ROLE OF NRF2 IN DIABETES AND ITS COMPLICATIONS Accumulated evidence suggests the pivotal role of oxidative stress and inflammation in the pathogenesis of diabetes resulting in the development of micro- and macrovascular complications (van den Berg et al., 2001; Vikram et al., 2014). Hyperglycemia has been shown to induce islet inflammation mainly due to the perturbed glucose metabolism, mitochondrial production of ROS, initiating the apoptotic and inflammatory signaling cascades (Elmarakby and Sullivan, 2012). The Nrf2-Keap1-ARE pathway has been shown to play an important role in cytoprotection and the regulation of energy metabolism, which has led to interest in the pathway as a potential target for the prevention and treatment of metabolic diseases such as diabetes. Notably, Nrf2 levels have also been shown to be lower in prediabetic and diabetic patients as compared to patients without diabetes. The nuclear fractions from peripheral blood mononuclear cells of these patients showed diminished Nrf2 expression with elevated levels of oxidative stress markers (Jimenez-Osorio et al., 2014). The role of Nrf2 in the regulation of metabolism and blood glucose levels has generated interest in targeting the pathway for the prevention and treatment of diabetes. In addition to metabolic regulation and the pathogenesis of diabetes, Nrf2 appears to have an important role in reducing oxidative damage associated with diabetic complications. For instance, hyperglycemia causes elevated ROS production and, thus, oxidative damage to the vasculature that directly contributes to the progression of diabetic cardiomyopathy. Multiple studies have shown experimental evidence that demonstrates the involvement of Nrf2 in diabetic cardiomyopathy and nephropathy (Bai et al., 2013; Cui et al., 2012; Miao et al., 2012). Streptozotocin treated-Nrf2-null mice were determined to be more susceptible to oxidative damage and renal impairment than wild-type mice (Choi et al., 2014; Jiang et al., 2010). The protective role of Nrf2 in diabetic nephropathy suggests that activation of Nrf2 could be used to prevent or impede the advancement of the disease. Bardoxolone methyl (CDDO-Me) is an activator of Nrf2 and its potential has been evaluated in T2DM patients with chronic kidney disease (Pergola et al., 2011). Interestingly, curcumin ameliorates macrophage infiltration by inhibiting NF-κB activation and proinflammatory cytokines in diabetic nephro- and retinopathies (Kowluru and Kanwar, 2007; Soetikno et al., 2011). Inline, using a reporter protein complementation assay

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developed in our laboratory for in vitro and in vivo screening of Nrf2 activators, we also identified pterostilbene (PTS) as a potential activator of Nrf2 (Ramkumar et al., 2013). Our recent reports identified the potential of PTS to activate Nrf2 and its downstream target genes to safeguard the pancreatic β-cells from apoptosis (Bhakkiyalakshmi et al., 2014). Further, the antidiabetic property of PTS highlighted its crucial role in protecting pancreatic islets against oxidative insult, thereby reinstating islet architecture, β-cell mass and function (Elango et al., 2016). Hence, several Nrf2 activators including sulforaphane, resveratrol, pterostilbene, curcumin, etc., are reported to play a pivotal role during diabetic conditions (Bhakkiyalakshmi et al., 2015). This chapter henceforth emphasizes the role of a few Nrf2 activators in the context of islet inflammation.

12.6. ROLE OF NF-κB IN DIABETES AND ITS COMPLICATIONS In T1D mellitus, pancreatic β-cells are the target of autoimmune attack mediated in part by cytokines, such as IL-1 and IFNs that activate the NF-κB signaling (Cardozo et al., 2001). These proinflammatory cytokines trigger apoptotic cascade of events leading to β-cell dysfunction (Eizirik and Mandrup-Poulsen, 2001). Previous studies indicated that IKK-β plays the major role in development of insulin resistance (Arkan et al., 2005), and highlighted the role of NF-κB in the pathogenesis of T2D (Cai et al., 2005). Arkan et al., further identified that inhibition of IL-1 signaling ameliorated inflammation-induced hyperglycemia (Arkan et al., 2005). Enhanced NF-κB activity was found to impair vascular function by Poly (ADP-Ribose) Polymerase 1 (PARP-1), specificity protein 1 (Sp-1), and COX-2dependent mechanisms in type 2 diabetic mice (Kassan et al., 2013). Clinically, T2D patients exhibited a more pronounced increase in NF-κB activity and JNK phosphorylation that develop dysregulated glucose disposal in the context of systemic inflammation (Andreasen et al., 2011). Several studies have also highlighted the effect of inflammation and NF-κB signaling in diabetic complications including nephro-, neuro-, retino-, and cardiomyopathies (Ganesh Yerra et al., 2013; Lorenzo et al., 2011; Romeo et al., 2002; Sanz et al., 2010).

12.7. CROSSTALK BETWEEN NRF2 AND NF-κB PATHWAYS IN DIABETES Enhanced NF-κB activity during the hyperglycemic state is associated with excess production of proinflammatory cytokines such as IL-1β, IL-6, TNF-α, COX-2 and iNOS that are prerequisite mediators for the initiation and amplification of inflammatory processes (Li et al., 2008). Reduced Nrf2 activity results in impaired antioxidant defense and is characterized by decline in SOD, CAT and GSH levels. In addition, it decreases the production of detoxifying enzymes such as, HO-I and NQO1, leading to nitrosative and oxidative stress (Kansanen et al., 2013), which contributes to increased advanced glycation end products formation and protein kinase C activation and increased peroxynitrite-mediated Poly ADP Ribosyl PARP activation resulting in apoptosis (Brownlee, 2005). The manifestations of inflammation and oxidative stress can cumulatively cause the structural damage that can lead to functional as well as biochemical deficits, which are major characteristics of islet inflammation (Imai et al., 2013). Oxidative stress involves cascade of events viz. the activation of IκB kinase (IKK), phosphorylation of IκB (inhibitor of NF-κB) and hence targets IĸB for polyubiquitination-mediated proteasomal degradation, resulting in the release of NF-κB, which then migrates into the nucleus and binds with the κ region (van den Berg et al., 2001). With the help of other coactivators and histone acetyltransferases (HAT), NF-κB causes the transcription of proinflammatory mediators such as IL-6, TNF-α, cyclooxygenase-2 (COX-2), interleukin-1 (IL-1), intracellular adhesion molecule (ICAM), and inducible nitricoxide synthase (iNOS) (Lawrence, 2009). Nrf2 pathway inhibits the activation of NF-κB pathway by increasing antioxidant defense and HO-1 expression, which efficiently neutralizes ROS and detoxify toxic chemicals and hence, reduces ROS-mediated NF-κB activation (Rushworth and O’Connell, 2004). Nrf2 pathway also inhibits NF-κB-mediated transcription by preventing the degradation of IκB-α. Similarly, NF-κB-mediated transcription reduces Nrf2 activation by reducing the ARE-dependent gene transcription, decreases free CREB binding protein (CBP) by competing with Nrf2 for CH1-KIX domain of CBP (Liu et al., 2008). NF-κB also enhances the recruitment of histonedeacetylase (HDAC) to the ARE region by binding to Mafk and hence interferes with the transcriptional facilitation of Nrf2 (Bellezza et al., 2010; Wakabayashi et al., 2010) (Fig. 12.4).

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FIGURE 12.4 Crosstalk between the Nrf2 and NF-κB pathways. Inflammation-mediated Nrf2 activation can lead to the production of antioxidant enzymes and detoxifying enzymes such as Superoxide Dismutase (SOD), catalase (CAT), Glutathione peroxidase (GPx), Hemeoxygenase-1 (HO-1) and NADPH quinone oxidoreductase (NQO1). NF-κB is involved in inflammation, immune function, cellular growth and apoptosis. p65 is a Rel protein with transactivation efficiency whereas its partner p50 lacks transcriptional activity. Inflammatory response activates IκB kinase (IKK) and phosphorylates NF-κB, and drives polyubiquitination-proteasomal degradation of IKK resulting in the release of NF-κB. It then translocates into the nucleus and binds with the DNA thereby transcriptionally activating proinflammatory cytokines including such as IL-6, tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), IL-1, intracellular adhesion molecule (ICAM) and inducible nitricoxide synthase (iNOS). Nrf2 can inhibit NF-κB signaling and vice versa at their transcription level mainly via protein-protein interaction and opposite functions.

12.8. ROLE OF NRF2 ACTIVATORS: RESCUE FROM ISLET INFLAMMATION 12.8.1. Resveratrol (RES) Resveratrol (trans-3,40 ,5-trihydroxystilbene) is a polyphenol abundant in grapes and has recently emerged as a therapeutic target for many disorders, including diabetes (Movahed et al., 2013). RES is well-known as an activator of Nrf2 and sirtuin-1 (Hasko´ and Pacher, 2010; Kim et al., 2011) and prevents inflammation by inhibiting NF-κB-mediated production of inflammatory cytokines (Guo et al., 2014). It inhibits CCR6, which encodes chemokine (CC motif) receptor (CCR) 6 in the splenocytes, leading to minimal pancreatic islet infiltration in diabetes. RES downregulates CCR6 expression on multiple inflammatory cell types, and blocks the migration of immune cells to the pancreas (Lee et al., 2011). Recently Cheng et al. (2015) reported that RES has the potential to improve insulin levels and promote Nrf2 expression in methylglyoxal-treated mice and remains to be a therapeutic target against β-cell dysfunction.

12.8.2. Dh404 Bardoxolone Methyl Analog (dh404), a novel synthetic triterpenoid derivative, remains a novel Nrf2 activator with a therapeutic potential for various diseases including diabetes (Tan et al., 2011). Recently, Li et al. (2015) identified that dh404 promotes translocation of Nrf2 from the cytoplasm into the nucleus in isolated islet cells. Pharmacological activation of the Nrf2 pathway significantly increased HO-1 expression, improved islet yield, viability, and function after transplantation. A study in HO-1-deficient mice has shown HO-1 to play a critical role in the anti-inflammatory process (Kapturczak et al., 2004). Furthermore, it has been reported that HO-1 upregulation led to protective effects on β-cells against various apoptotic stimuli including cytokines and Fas (Ribeiro et al., 2003; Tobiasch et al., 2001).

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Recently, Masuda et al., investigated the effect of dh404 in human islet cells. dh404 significantly increased expression of the key antioxidants enzymes that are downstream targets of Nrf2 (NQO1, HO-1, and GCLC), decreased inflammatory mediators in islets (IL-1β, IL-6, IFN-γ and MCP-1) and conferred protection against oxidative stress and inflammation in β-cells (Masuda et al., 2015).

12.8.3. Curcumin Curcumin (CUR), NF-κB inhibitor and an antioxidant, is the principal curcuminoid found in turmeric (Curcuma longa Linn.) and a well-known Nrf2 activator (Yang et al., 2009). In diabetes, CUR promotes islet survival and reduces ROS production by inhibiting poly ADP-ribose polymerase-1 activation, NF-κB translocation and IkB phosphorylation, without affecting normal islet function (Meghana et al., 2007). A few studies also highlighted that CUR treatment decreased lymphocyte infiltration in pancreatic islets, increased islet regeneration and insulin secretion (Chanpoo et al., 2010). It is noteworthy that CUR was reported to elevate the expression of HO-1, NQO1 through Nrf2-mediated mechanism, thus improving the outcome of islet transplantation (Zhang et al., 2013).

12.8.4. Vitamin D Vitamin D is a fat soluble secosteroid that interacts with a specific nuclear receptor similar to other steroid hormones, and plays a central role in calcium and phosphate homeostasis (Holick, 2007). The risk factors of vitamin D deficiency include severely impaired renal function, hypoalbuminemia, urine protein/ urine albumin concentrations, and diabetes. Moreover, vitamin D levels in CKD patients are affected by various factors, including physical activity, nutritional and inflammatory status, diabetes, and urinary protein excretion (Obi et al., 2015). Recently Nakai et al., identified that Vitamin D activates the Nrf2-Keap1 antioxidant pathway and ameliorates nephropathy in diabetic rats (Nakai et al., 2014). Moreover, Vitamin D facilitates the secretion of insulin from pancreatic β-cells, and its deficiency may be related to impaired insulin secretion and insulin resistance leading to β-cell dysfunction (Kostoglou-Athanassiou et al., 2013; Talaei et al., 2013). Though the potential of Nrf2 activators in diabetes have been extensively studied, their role in inflammation still remains unclear. Yet, in recent years Nrf2 activation has been proved as an effective strategy to safeguard pancreatic β-cells to reduce the burden of diabetes incidence. Interestingly, recent reports from our laboratory have elaborately discussed the role of Nrf2 activation against β-cell apoptosis. Our extensive research on Nrf2 has identified Pterostilbene (PTS) as a potent activator of Nrf2 through high throughput cell-based assays. We also identified the crucial role of PTS in diabetic mice, which maintains glucose homeostasis by regulating the key enzymes of carbohydrate metabolism. Notably, our studies also identified the potential of PTS in recovering β-cell mass and its functions via Nrf2-mediated mechanisms. Based on the reports from our laboratory and other researchers, the promising activities of Nrf2 warrant exploration in its importance in the context of inflammation in diabetes. Further research contributions are warranted to ascertain the role of Nrf2 in islet inflammation and to highlight its potential as a novel therapeutic target in diabetes.

12.9. PERSPECTIVES Small molecule Nrf2 activators prevail as an effective pharmacological target in the treatment of diabetes and its complications and remain as a potential therapeutic strategy for diabetes management. Though the available antidiabetic drugs provide broad spectrum molecular mechanisms to safeguard multiorgans, their effectivity in the protection of pancreatic islets remains unclear. Moreover, while the preclinical outcomes of antidiabetic drugs are positive, these do not translate into efficacious agents in long-term clinical trials, which is disappointing. In recent years, Nrf2 activation has become a promising strategy to avert diabetic complications, and a few Nrf2 activators have also been considered for translational research. Ample evidence has also identified the association of Nrf2 with inflammatory response. However, the mechanistic studies on the role of Nrf2 in islet inflammation need rigorous attention in the field of diabetes.

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13 Monogenic Defects of Toll-Like Receptor Signaling and Primary Immunodeficiency Travis M. Sifers and Venkatesh Sampath Children’s Mercy Hospital, Kansas City, MO, United States

13.1. INTRODUCTION: THE GENETIC THEORY OF INFECTIOUS DISEASE Infectious diseases have been a scourge for mankind throughout their existence and were an important cause of a limited lifespan as recently as the last century (Casanova and Abel, 2005; Quintana-Murci et al., 2007). Adoption of hygienic practices, antibiotics, and universal immunization has contributed significantly to the doubling of the human lifespan by the late 20th century (Quintana-Murci et al., 2007). Variability of infectious disease susceptibility between human hosts has been suspected almost a century ago (Casanova et al., 2011). Pioneering work in the last century by multiple investigators identified various classical immunodeficiency syndromes such as chronic granulomatous disease and severe combined immunodeficiency (Nunoi and Matsuda, 1992; Ochs et al., 1973). More recently, the esteemed immunologist Jean-Laurent Casanova postulated that in the future, human immunogenetics would create a new paradigm in understanding our susceptibility to infectious disease. Casanova recalls Pasteur’s microbial theory of infectious disease and how it externalized disease, but argues that there is a growing body of experimental and epidemiological evidence that genetic variations within a population may have much more to do with why some people succumb to illness while others do not (Quintana-Murci et al., 2007; Casanova et al., 2011). While many of the immunodeficiencies characterized in the 20th century focused on genes that regulated development of adaptive immunity (recombination-activating gene or RAG1, adenosine deaminase or ADA) or neutrophil function (NADPH oxidase 2 or NOX2), immunodeficiency arising from inborn errors of innate immune signaling have come to the forefront in this century.

13.2. A RENAISSANCE IN INNATE IMMUNE SYSTEM RESEARCH Temporally coinciding with the work demonstrating the importance of genetic variation on infectious disease, the early part of the 21st century has seen an explosion in knowledge regarding the human immune system. This is demonstrated by the growing excitement over what was once thought to be a genetically ancient and clinically obsolete aspect of human immunity, the innate immune system. The innate immune system consists of cells and proteins that are ready and armed to fight microbes at the portal of entry, and as such differs from the adaptive immune system. In general, innate immune responses are non-specific, and devoid of memory (Janeway and Janeway, 2001). The adaptive immune system is dependent on processing of foreign antigens by innate immune cells, is typically more specific, and exhibits memory in directing future responses. Almost all cell types have the potential to participate in innate immunity along with phagocytes, dendritic cells and natural killer cells, whereas adaptive immunity is primarily mediated by T- and B-cell lymphocytes (Janeway and Janeway, 2001; Dempsey et al., 2003). No aspect of the innate immune system better represents its entirety than pattern recognition receptors (PRRs). Human Toll-like receptor 4, the archetypal PRR discovered in 1997, spurred intense research into these conserved proteins that initiate specific innate immune responses to respond to structural motifs in pathogens (Medzhitov et al., 1997). To date, four families of PRRs have been discovered, but their complex role in both Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00013-5

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the innate and adaptive immune system remains largely unknown (Akira et al., 2006). In this review, we will focus on one such family of PRRs, the Toll-like receptors (TLRs), their role in pathogen recognition, how monogenetic defects lead to specific infectious disease susceptibility in infancy and childhood, and management of these diseases. In doing so, our goal is not only to inform the reader of these rare but significant conditions, but to demonstrate the importance of these discoveries in pursuit of a clearer understanding of human immunity and diseases resulting from TLR genetic defects.

13.3. DROSOPHILA TOLL, INTERLEUKIN-1 RECEPTOR AND THE DISCOVERY OF THE TLR SUPERFAMILY Toll-like receptors (TLRs) derive their name from their counterpart protein toll in drosophila melanogaster (fruit fly). Subsequent to the initial characterization of its role in dorso-ventral patterning in the fruit fly in 1985, it was discovered that the Toll gene product was a transmembrane receptor whose cytoplasmic domain shared 135 base pairs with the human interleukin-1 receptor (IL-1R) (Gay and Keith, 1991). This led to the discovery of TLRs in humans (Medzhitov et al., 1997); shortly thereafter, the role of TLR4 in mediating specific innate immune responses to lipopolysaccharide (LPS), a cell-wall component of Gram-negative bacteria was established (Poltorak et al., 1998). Currently, .11 mammalian TLRs have been identified and they function as receptors for conserved microbial motifs in bacteria, fungi and viruses (Casanova et al., 2011; O’Neill, 2008). The TLRs are a subfamily of the Toll receptor/Interleukin-1 receptor (TIR) superfamily, which is made up of 24 proteins that all share the cytosolic TIR domain (O’Neill, 2008). Extracellular binding to TIRs by pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), i.e., danger signal proteins that are produced with infectious and sterile injury result in dimerization and activation of the TLR signaling cascade with subsequent activation of transcription factors that regulate immunity and inflammation. Genetic defects in many members of this cascade have been described and are outlined in the sections below.

13.4. TLR SIGNALING NETWORKS TLR signaling is mediated through two different pathways, which confer pathogen specific immunity; these pathways are commonly referred to as canonical and alternative (Parker et al., 2007; Akira, 2006). Canonical signaling is the primary pathway for all TLRs with the exception of the viral-sensing TLR3, and it also mediates

FIGURE 13.1 Cartoon (simplified) of TLR signaling pathway highlighting genes implicated in primary immunodeficiency: Bacterial motifs recognized by TLRs1, 2, and 4 induce MyD88-dependent signaling. Viral dsRNA recognized by endosomal TLR3 activate TRIFdependent signaling. Genes in red have been implicated in immunodeficiency. TLR, Toll-Like Receptor; GPB, Gram-positive bacteria; GNB, Gram-negative bacteria; IRAK4, Interleukin receptor associated kinase 4; MYD88, Myeloid differentiation factor 88; NFKB, nuclear factor kappa-B; IκB-α, Inhibitor of kappa B-alpha; TRIF, TIR-domain containing adapter-inducing interferon β; IKK, IκB kinase; IKK-γ (NEMO), TRAF6/3, TNF receptor associated factor 6 and 3; UNC93B1, Unc-93 Homolog B1.

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IL-1 receptor-dependent IL-1, IL-18 and IL-33 signaling (Casanova et al., 2011). Stimulation of this pathway results in the activation of both nuclear factor κB (NF-κB) and mitogen-activated protein kinases (MAPK), leading to the synthesis of pro-inflammatory cytokines IL-1β, -6, -8, -12 and tumor necrosis factor alpha (TNFα) (Picard et al., 2011). With receptor binding, the cytosolic TIR domain recruits the cytosolic adapter proteins myeloid differentiation factor 88 (MyD88) and TIRAP/Mal (TLR2 and TLR4 only), resulting in the formation of a signaling complex called the Myddosome, comprising MyD88 and two IL-1R associated kinases, IRAK4, and IRAK2 (Gay et al., 2011). This results in phosphorylation, activation, and dissociation of the IRAKs from the receptor-adapter complex. The IRAKs in turn activate tumor necrosis factor receptor-associated factor-6 (TRAF-6) that propagates signaling through the Mitogen-activated protein kinases (MAPK) and the Inhibitor of Kappa B kinase (IKK) hetero-trimeric complex constituted of IKK-α, IKK-β, and IKK-γ (NEMO) (Akira, 2006). IKK phosphorylates Inhibitor of Kappa B-alpha (IKBA), resulting in its degradation and dissociation from NF-κB, facilitating transcriptional induction of NF-κB-dependent program. Four inherited monogenetic primary immunodeficiencies resulting from genetic defects in the canonical TLR signaling pathway have been described. Mutations in IRAK4 and MyD88 result in signaling defects restricted to the above pathway and confer bacterial disease susceptibility, while genetic defects in NEMO (IKK-γ) and IKBA have a complex phenotype and impair signaling through the classical and alternative TRIF-dependent pathway as described below (Picard et al., 2003; von Bernuth et al., 2008; Courtois et al., 2003; Doffinger et al., 2001).

13.5. TLR3 SIGNALING VIA THE ALTERNATIVE (TRIF-DEPENDENT) PATHWAY Toll-like receptor 3 (TLR3) is expressed in multiple cells including immature dendritic cells, epithelial cells and fibroblasts. Its localization is cell specific but is limited to either the cell surface, or more frequently, the intracellular endosome compartment (Yu and Levine, 2011). The ectodomain of TLR3 forms a horseshoe structure that binds viral double-stranded ribonucleic acid (dsRNA) molecules of certain size. TLR3 is one of four intracellular TLRs that binds nucleic-acid-like structures, along with TLR7, TLR8 and TLR9 (Ahmad-Nejad et al., 2002; Heil et al., 2003; Matsumoto et al., 2003). TLR3 signaling is unique in that it is independent of the MyD88-mediated canonical pathway, and it is involved in anti-viral immunity. Binding of dsRNA (or polyinosinic-polycytidylic acid [poly(I:C)] a synthetic analog to dsRNA) results in activation of the TIR-domain-containing adapter inducing interferon-β (TRIF) adapter complex (also known as Toll-IL-1 receptor (TIR) domain-containing adaptor molecule-1 [TICAM-1]). TRIF/TICAM-1 activation results in its association with the regulatory protein TNF receptor-associated factor 3 (TRAF3), which recruits the protein kinase TANK-binding kinase-1 (TBK1) to the TIR complex (Hacker et al., 2006; Oganesyan et al., 2006). TRAF3 also activates the IKK-complex and subsequent NFκB nuclear translocation (Hacker et al., 2006). Activated TBK1, along with the non-canonical I-kappa-B kinase epsilon (IKKE), results in phosphorylation, and activation of IRF3 leading to entry into the nucleus and initiation of type-1 interferon gene transcription which is essential for antiviral responses (Sharma et al., 2003; Wathelet et al., 1998; Taniguchi et al., 2001). In addition to the TRIF-IRF3 axis necessary for TLR3-mediated viral responses, UNC93B1, an endoplasmic reticulum membrane-spanning protein involved in intracellular trafficking and processing of TLR3, TLR7, TLR8 and TLR9, is necessary for dsRNA sensing and signaling of TLR3 (Brinkmann et al., 2007; Kim et al., 2008; Lee et al., 2013; Saitoh and Miyake, 2009).

13.6. MONOGENETIC PRIMARY IMMUNODEFICIENCIES OF THE CANONICAL PATHWAY AND SUSCEPTIBILITY TO PYOGENIC BACTERIAL INFECTIONS Interleukin-1 receptor associated kinase-4 (IRAK-4) plays an essential role in the canonical TLR signaling cascade and NF-κB activation. IRAK-4 is a serine-threonine kinase acting downstream from TLR dimerization. IRAK-4 deficiency is caused by homozygous or compound heterozygous mutations in the IRAK-4 encoding gene and its inheritance pattern is considered autosomal recessive. The disease was first discovered in 2003; currently more than 50 cases have been confirmed (Picard et al., 2011; Picard et al., 2003). Capucine Picard and colleagues first described three unrelated male patients with recurrent pyogenic bacterial infections. These children were otherwise healthy. Recurrent infections started soon after birth but became less frequent with age. The patients had normal serum antibody titers against protein and polysaccharide antigens. In a multitude of cell lines tested, all were found to be poor responders to lipopolysaccharide (LPS), IL-1β and IL-18, but not to tumor necrosis factor-α (TNFα) consistent with impaired TLR pathway signaling (Picard et al., 2003). MyD88 is a cytosolic adapter

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molecule that aids in the connection of the IKK complex with TLRs after receptor binding and is necessary for signal transduction within the canonical pathway following TLR ligand binding (Picard et al., 2011; Muzio et al., 1997; Medzhitov et al., 1998). Genetic mutations resulting in MyD88 deficiency as a cause of primary immunodeficiency disease were first discovered in 2008 (von Bernuth et al., 2008). In their seminal study, von Bernuth and colleagues identified MyD88 mutations in nine children from five kindreds who all had presented with recurrent, invasive pyogenic bacterial infections and who were negative for IRAK4 deficiency by genetic testing. Genetic studies reviewed both homozygous and compound heterozygous mutations in the MyD88 gene, some of which had normal MyD88 protein suggesting functional MyD88 deficiency (von Bernuth et al., 2008). Presently, more than 20 cases had been confirmed from .7 kindreds (Picard et al., 2011; Picard et al., 2010). Kindreds of MyD88 deficiency patients are consistent with an autosomal recessive inheritance pattern.

13.6.1. Immune Phenotype and Laboratory Diagnosis Patients with MYD88 and IRAK4 genetic defects are phenocopies with respect to their immune phenotype. Routine immunodeficiency work-up can be normal in these children. There are no overt deficits in leukocyte maturation or production, and antigen-specific T- and B-cells responses are essentially normal (von Bernuth et al., 2008; Picard et al., 2010; Ku et al., 2007b). The classic defect is the failure of peripheral white blood cells to produce IL-6 in response to IL-1β, but not TNF-α stimulation. Further, peripheral blood cells fail to shed CD62 ligand from granulocytes in response to TLR bacterial ligands like lipopolysaccharide (TLR4), flagellin (TLR5) or lipopeptides (TLR2) (Picard et al., 2011; Picard et al., 2010). Many patients have elevated IgG4 and IgE levels without showing any evidence of chronic allergy (Picard et al., 2010). Impaired responses to pneumococcal vaccination evident by low titers of IgG and IgM are found in some patients. In suspected cases, diagnosis is confirmed by detection of homozygous or compound heterozygous mutations in IRAK-4 or MyD88 along with a lack of IL-6 production in whole blood or lack of CD62L shedding in granulocytes when activated by TLR/IL-1R agonists (Picard et al., 2003; von Bernuth et al., 2008; von Bernuth et al., 2006).

13.6.2. Clinical Phenotype, Natural History and Management Despite the broad defect in the immune phenotype, patients with IRAK-4- and MyD88-deficiency are susceptible to a narrow spectrum of bacteria, while exhibiting normal resistance to parasites, fungi, viruses, and many bacteria. Meningitis, sepsis, arthritis, osteomyelitis, and abscesses are typically caused by Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa (Picard et al., 2010). Invasive bacterial disease presents early in the life of children with IRAK4-/MyD88-deficiency with approximately 90% of patients experiencing bacterial disease before the age of two (Picard et al., 2010; von Bernuth et al., 2012). Inflammatory markers and clinical signs of infection are also often delayed or absent. Picard and colleagues examined common signs of inflammation in the neonatal, infantile and childhood periods in patients who presented with confirmed invasive bacterial disease. Among their findings, infants and children often presented with normal temperature (43% and 50% respectively), normal c-reactive protein levels (52% and 44% respectively), and normal leukocyte counts (60% and 88% respectively) (Picard et al., 2010). Meningitis is the most common invasive infection documented (43.1% of all documented infections), followed by sepsis (20.4%), arthritis (15.6%), deep tissue abscesses (14.3%) and osteomyelitis (6.1%) (Picard et al., 2010). Interestingly, despite pneumococcal infections being most frequent, recurrent pneumonia is not a common finding (Picard et al., 2010). Invasive disease is typically recurrent up to the time of puberty, but no further invasive infections have been recorded after this period, leading to speculation that the maturation of the adaptive immune system becomes protective in these patients (Bouma et al., 2009; Cardenes et al., 2006; Enders et al., 2004; Ku et al., 2007a). Noninvasive bacterial infections are also common in these patients. The most common sites of infection are the skin (recurrent cellulitis, folliculitis, furuncles) as well as sinus and upper respiratory tract infections (recurrent otitis media, pharyngitis, gingivitis) (Picard et al., 2010). These infections continue into adulthood despite prophylactic antibiotics (Picard et al., 2010). Streptococcus pneumoniae, staphylococcus aureus, and pseudomonas aeruginosa are by far the most isolated pathogens in all bacterial disease of these patients. The three combined to account for 84.9% of all infectious and 87% of all invasive bacterial infections in IRAK4-deficient patients studied. Similarly, in MyD88-deficiency patients, the three pathogens accounted for 81% of all bacterial infections and 78.8% of all invasive bacterial disease (Picard et al., 2010). It is important to note that no invasive infections

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occurred from viral, fungal, mycobacterial or parasitic organisms, although some patients have had typical courses of common viral and fungal infections (Picard et al., 2010). Infants should be immunized with conjugated and unconjugated S. pneumoniae vaccine, H. influenzae conjugated vaccine, and N. meningitidis conjugated and unconjugated vaccines (Picard et al., 2011; Bousfiha et al., 2010). Prophylactic antibiotic therapy in childhood is recommended, while intravenous or subcutaneous IgG therapy may also decrease the incidence of invasive bacterial infections. Parents should be educated to have a low threshold to seek medical care with suspected infections and empirical parenteral antibiotics should be used as a first line of treatment with suspected bacterial infections. Unrecognized, mortality from these conditions can be between 30-50% but with intensive prophylaxis and empirical therapy mortality can be reduced significantly (Picard et al., 2010). As adults, patients generally are more resistant to severe infections and prophylaxis may be relaxed. It is important that genetic defects are not fully penetrant, as siblings of patients carrying same mutations can remain asymptomatic.

13.7. MONOGENETIC PRIMARY IMMUNODEFICIENCIES OF THE ALTERNATIVE PATHWAY AND SUSCEPTIBILITY TO HERPES SIMPLEX ENCEPHALITIS Perhaps the most relatable example in support of both the genetic theory of infectious disease and the specificity of TLR immunity has been elucidated by uncovering inborn errors relating to the pathogenesis of herpes simplex-1 viral encephalitis (HSE). HSE is the most common cause of sporadic viral encephalitis in the Western world with an annual incidence of two to four cases per one million individuals per year (Whitley and Kimberlin, 2005; Skoldenberg et al., 1984; Najioullah et al., 2000). Primary infection by HSV-1 makes up about one-third of all HSE cases and is the most common pediatric form; adults typically develop HSE as a result of reactivation of latent HSV from the trigeminal nerve (Whitley and Kimberlin, 2005; Skoldenberg et al., 1984; Nahmias et al., 1982; Whitley and Gnann, 2002). While HSV-1 infection is nearly ubiquitous, it rarely results in HSE and, until recently, the mechanisms of its pathogenesis had confounded investigators. Multiple studies found no known associations between variations in HSV-1 virulence or environmental risk factors predisposing patients to the disease (Whitley and Kimberlin, 2005; Baringer, 2008; Bergstrom et al., 1990; Izumi and Stevens, 1990; Norberg et al., 2004; Whitley et al., 1982). Supporting a potential contribution of human genetics to the pathogenesis of HSE, a study of French kindreds with HSE found a high incidence of parental consanguinity (Abel et al., 2010; Casrouge et al., 2006). This hypothesis was further supported by the fact that HSE had been found in patients lacking signal transducer and activator of transcription (STAT)-1 and nuclear factor kappa B (NF-κB) essential modulator (NEMO) (Dupuis et al., 2003; Rudd et al., 2006; Chapgier et al., 2009; Puel et al., 2006). STAT-1 gene product acts downstream from IFNα/β receptors. It is not selective to TLR-signaling and thus is out of the purview of this review. NEMOdeficiency is described in detail below. Both patients suffered from severe mycobacterial disease because of impaired IFN-γ-mediated immunity and from HSE because of impaired IFN-α/β- and λ-mediated immunity (Casanova and Abel, 2002; Haller et al., 2006). Expanding on the above information, in 2006, Casrouge and colleagues assessed IFN-α/β and IFN-λ production of peripheral blood cells (PBMCs) stimulated with HSV-1 in a cohort of otherwise healthy French children with sporadic HSE. Two unrelated patients were found to have markedly diminished IFN production patterns similar to mice with UNC93B-deficiency. Both cases had confirmed mutations in the UNC93B1 gene that was not found in 100 healthy European controls. After the identification of UNC93B-deficiency, Zhang and colleagues published results on two French children with HSE and no UNC93B-deficiency that were found to have heterozygous mutations of the TLR3 gene (Zhang et al., 2007). Since then, firm evidence has been established for the pivotal and disease-specific role of the TLR3-TRIF dependent pathway in conferring immunity to HSE with mutations in TRAF3, TRIF/TICAM1, IRF3 and TBK1, all being reported in patients with HSE and blunted production of type-1 interferons (Andersen et al., 2015; Perez de Diego et al., 2010; Sancho-Shimizu et al., 2011; Herman et al., 2012).

13.7.1. Pathogenesis and Immune Phenotype As stated earlier, TLR3 signals through the adapter TRIF to activate IRF-3 and NF-κB transcriptional programs resulting in the production of type-1 interferons that are critical to anti-viral immunity. In vivo dsRNA activates TLR3 signaling. HSV-1 is a dsDNA virus that utilizes dsRNA intermediates for viral replication. More

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specifically, TLR3 is highly expressed in resident cells in the CNS and peripheral nerves that are utilized by neurotropic viruses like HSV-1 to infect the central nervous system (Bsibsi et al., 2002). Indeed, CNS-resident cells, particularly neurons and oligodendrocytes are suspected of conferring impaired HSV-1 immunity in TLR3pathway deficiencies. Both neurons and oligodendrocytes in UNC93B-deficiency as well as neurons in TLR3deficiency were shown to display impaired IFN-β and IFN-λ production in response to HSV-1 (Lafaille et al., 2012). These cells showed an increased rate of infection and replication of HSV-1 and were rescued when transfected with the corresponding wild-type allele. As a general rule, fibroblasts—which, like the CNS are ectodermally derived—of patients with TLR3 pathway mutations show diminished production of IFN-β, IFN-λ and IL-6 when stimulated with neurotropic viruses like HSV-1 and vesicular stomatitis virus (VSV), and specific TLR3 agonist poly (I:C) when compared to wild-type controls, whereas response to both TNF-α and IL-1β are normal. These cells also show increased rates of viral replication and more rapid cell death when inoculated with HSV-1 and VSV. UNC93B- and TLR3-deficient patients are identical in their immunologic response to TLR3 agonists (Casrouge et al., 2006; Zhang et al., 2007). These mutations also result in impaired IRF-3 and NF-κB dimerization and nuclear translocation. Autosomal dominant TRAF3-deficieny was identified in one patient with a history of HSE. Fibroblasts stimulated with poly (I:C) show abolition of IFN-β, IFN-λ and IL-6 production; NF-κB nuclear translocation was also impaired. In contrast to UNC93B- and TLR3-deficient fibroblasts, IRF-3 dimerization was less strongly affected, suggesting TRAF3-deficiency affects the TLR3 pathway in terms of NF-κB activation and, to a lesser extent, IRF-3 (Perez de Diego et al., 2010). TRIF-deficiency patients also have shown the pattern of impaired cellular responses to TLR3 agonists described above. The TRIF protein also serves as an adaptor for the MyD88-independent pathway downstream from TLR4, yet no other infections, opportunistic or otherwise, were found in TRIF-deficiency with impaired TLR4 signaling, suggesting this pathway is largely redundant in host defense (Sancho-Shimizu et al., 2011). TBK1-deficiency and IRF3-deficiency also have been shown to display impaired INF-β production, although NF-κB activation is theoretically preserved since these proteins are downstream of the alternative pathway’s activation of NF-κB (Andersen et al., 2015; Herman et al., 2012). Like IRAK4 and MyD88-deficiency patients, those with monogenic inborn errors in TLR-pathway signaling have seemingly normal development of the principal myeloid and lymphoid leukocyte subsets and apparently normal T-cell and B-cell responses to antigens, at least in vitro. In all identified patients to date, there is no evidence of other infectious susceptibility and these patients are otherwise healthy and well appearing. HSV-1 primary infections in these patients are not particularly severe, and there is no association between TLR3-pathway signaling defects and systemic HSV-1 infectious disease. This is thought to be due to the fact that most mutations result in normal or only moderate deficits in interferon production by circulating PBMCs and keratinocytes (Zhang et al., 2007). Most of the TLR3-pathway signaling defects have also been identified in the kindreds of patients studied who did not have HSE, displaying a pattern of incomplete penetrance which is consistent with the typical sporadic, rather than familial, pattern of HSE infections (Zhang et al., 2007; Perez de Diego et al., 2010; Sancho-Shimizu et al., 2011). Insights into the role of type-1 interferons in HSE has also led to the investigation of recombinant IFN-α treatment along with acyclovir in pediatric patients with HSE. Indeed, rates of viral replication were normalized in UNC93-deficient fibroblasts pretreated with recombinant IFN-α2b (Casrouge et al., 2006). Further supporting the potential role of treating these patients with exogenous IFN-α, TRIF-deficient fibroblasts infected with both VSV and HSV-1 showed markedly reduced rates of cell death after being pretreated with IFN-α (Sancho-Shimizu et al., 2011). Together, these studies have solidified the critical role of TLR3-dependent IFN immunity in the pathogenesis of HSE and provide proof-of-principle that HSE may result from a new group of monogenic immunodeficiencies. They speak to a paradigm shift in our understanding of the innate immune system, showing exquisite disease specificity both in terms of the pathogen and the location of clinical manifestation.

13.8. MONOGENIC DEFECTS IN NF-κB SIGNALING LEADS TO A BROAD SPECTRUM OF INFECTIOUS SUSCEPTIBILITY Up until this point we have explored relatively recently discovered primary immunodeficiencies where defects in a single gene confer susceptibility to specific infectious disease. These discoveries have provided insight into exquisite specificity of clinical phenotypes resulting from defects in innate immune signaling due to redundancies inherent to TLR signaling pathways. While NF-κB activation is impaired in both IRAK4/MyD88-deficiencies and TLR3-pathway signaling defects, the impairment is pathway specific. Globally, NF-κB is a pluripotent

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transcription factor that regulates inflammation and cell-survival responses in response to a plethora of immune receptor families, including those triggered by the tumor necrosis factor receptors (TNF-R), T- and B-cell lymphocyte receptors, IL-1Rs and TLRs. Because of this, mutations that inhibit NF-κB nuclear translocation irrespective of the pathway being activated have broad and variable phenotypic features. Impaired NF-κB signaling caused by hypomorphic genetic defects in the NF-κB essential modulator (NEMO) gene (also known as IKBKG or IKKγ) as the cause of previously well described anhidrotic ectodermal dysplasia with immune deficiency (EDA-ID) was first reported in 2000 and determined to have X-linked inheritance (Zonana et al., 2000). Three years later, an autosomal dominant form of EDA-ID with hypermorphic mutation of IκBα gene causing gain-of-function enhanced inhibition of cytosolic NF-κB. Both mutations have broad immunologic and clinical phenotypes that are dependent on the degree of NF-κB inhibition.

13.8.1. Pathogenesis and Immune Phenotype IKBKG/NEMO is a regulatory subunit of the IKK complex, which is necessary for NF-κB activation and was first isolated in 1998 (Rothwarf et al., 1998). NF-κB remains inactive in the cytosol due to interaction with inhibitor of kappa B (IκB). IκB phosphorylation is mediated by the IKK complex in response to various cellular stimuli including inflammatory cytokines such as IL-1β, bacterial lipopolysaccharide, viruses and stress; and results in its ubiquitination and proteolytic degradation, leading to NF-κB dissociation and translocation to the nucleus (Karin and Ben-Neriah, 2000). Complete loss-of-function mutations of NEMO/IKKγ result in early embryonic death in males and a severe genodermatosis in females known as incontinentia pigmenti (IP) (Smahi et al., 2000). Incomplete, hypomorphic mutations of the NEMO/IKKγ gene result in X-linked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID, also known as hypomorphic ectodermal dysplasia with immunodeficiency [HED-ID]). Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD-EDA-ID) results from mutations in the gene that codes IκBα subunit result in a gain-of-function inhibitory process by preventing the IKK complex to free NF-κB from IκB in the cytosol thereby retarding its nuclear translocation (Courtois et al., 2003). Although both of these PIDs are inborn errors of NF-κB signaling, IκBα-deficient patients have a more complete abolishment of NF-κB activation as T-cell receptor-dependent activation is also attenuated, resulting in lymphocyte subset population aberrances in addition to TLR signaling deficiency (Courtois et al., 2003; Bustamante et al., 2008). While dysregulation of adaptive immune responses in IκBα deficiency is not discussed here, this is an example of the interdependence of our innate and adaptive immune systems and the difficulty in categorizing certain immunodeficiencies as either innate or adaptive.

13.8.2. Clinical Phenotype, Natural History and Management XL-EDA-ID patients suffer from multiple serious infections, which begin early in childhood, and the diverse immunological phenotype drives the broad clinical presentation of these patients. In a 2008 analysis of 72 patients with NEMO mutations, 32 different mutations were identified. 81% of patients had associated ectodermal dysplasia and 76% suffered from serious pyogenic bacterial infections, 39% with mycobacterial disease, 19% with serious viral infections and 21% with noninfectious inflammatory disease (Hanson et al., 2008). In this cohort, there was a 36% mortality rate in childhood with average age of death of 6.4 years. Rough estimates suggest that approximately one-third of NEMO-deficient patients suffer from sepsis, one-third from deep tissue abscesses, one-third from recurrent pneumonia which progresses to bronchiectasis, 18% with meningitis or encephalitis and one-fourth with serious gastrointestinal infections (Picard et al., 2011). Infections typically begin within the first year of life. Noninfectious inflammatory enterocolitis has also been reported in a few patients with NEMOdeficiency presenting as intense abdominal pain that responded to high dose steroids after failing to improve with broad-spectrum antibiotics (Cheng et al., 2009). As previously stated, developmental features are a prominent clinical aspect of NEMO-deficient inborn errors of immunity. While a significant minority of patients display no dysmorphic features, upwards of three-fourths of those with mutations in the NEMO gene and immune deficiency display some degree of ectodermal dysplasia with sparse hair, abnormal development of dentition (tooth agenesis or conical teeth) and hypohidrosis due to absence of eccrine sweat glands (Picard et al., 2011; Doffinger et al., 2001; Zonana et al., 2000). Immune manifestations often predate the detection of ectodermal dysplasia though and as stated, some patients may not have these developmental findings (Orange et al., 2004). Interestingly, and consistent with the above narrow-spectrum monogenetic innate immunodeficiencies, individual hypomorphic mutations in the NEMO gene seem to confer

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specific infectious susceptibility—further suggesting that these innate immune pathways have specific defense functions and that additional pathways are likely redundant (Hanson et al., 2008).

13.8.3. Management of NEMO and IκBα Deficiencies In patients with impaired B-cell immunity, intravenous or subcutaneous IgG (IVIG/SCIG) treatment should be utilized. Those who display functional B-cell immunity should be immunized with S. pneumoniae conjugated and unconjugated vaccines, H. influenzae conjugated vaccine and N. meningitidis conjugated and unconjugated vaccines. All patients should receive prophylactic antibiotics. Patients and physicians of patients with both NEMO and IκBα-deficiencies should be instructed to initiate empirical parenteral antibiotic treatment against the most common organisms (S. pneumoniae, S. aureus, P. aeruginosa, and H. influenzae) without delay nor regard to inflammatory parameters. Despite prophylaxis, these infections can become rapidly fatal. Patients with more severe, combined immunodeficiency may be considered for hematopoietic stem-cell transplantation (HSCT) but safety is far from certain and there is only a paucity of published data (Abbott et al., 2014; Dupuis-Girod et al., 2006; Fish et al., 2009; Klemann et al., 2016; Permaul et al., 2009). In fact, some suggest that frequently engraftment is difficult and post-transplant complications may be due to intrinsic barriers to successful engraftment in these patients (Fish et al., 2009).

13.9. CONCLUSION Monogenic defects of the TLR signaling pathway are an important cause of primary immunodeficiency in childhood. Characteristic features include presentation with recurrent invasive and non-invasive infections in infancy, increased vulnerability to a narrow spectrum of bacterial and viral pathogens, improvement of the clinical phenotype with maturation of the adaptive immune system, and significantly improved outcomes with a combination of early diagnosis combined with antimicrobial prophylaxis (Picard et al., 2011; Picard et al., 2010; Sancho-Shimizu et al., 2007). The subtle and restricted immunological phenotype with incomplete penetrance represents a diagnostic challenge, as routine immunodeficiency screening may not identify these diseases. Awareness of these conditions with specific testing of TLR signaling pathway in conjunction with genetic tests are required to diagnose these conditions. Discovery of immunodeficiency phenotypes related to yet uncharacterized genes in this pathway, as well the determination of spectrum and burden of infectious disease arising from TLR pathway genetic defects, remain significant topics for future research.

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14 Pathogen- and Microbial- Associated Molecular Patterns (PAMPs/MAMPs) and the Innate Immune Response in Crohn’s Disease Amy K. Schaefer1, James E. Melnyk1, Zhaoping He2, Fernando Del Rosario2 and Catherine L. Grimes1 1

2

University of Delaware, Newark, DE, United States AI DuPont Nemours Children’s Hospital, Wilmington, DE, United States

14.1. INTRODUCTION Crohn’s disease is a debilitating inflammatory bowel disorder caused by a misregulated immune response. Crohn’s disease cases are on the rise, particularly among pediatric patients (Gasparetto and Guariso, 2013; Kappelman et al., 2013). Those afflicted suffer from chronic inflammation along their gastrointestinal tract that can lead to a number of symptoms including abdominal pain, bloody diarrhea, fatigue, and malnutrition. The underlying cause of Crohn’s disease is not well understood, and current treatments rely on suppression of inflammatory symptoms. Genetic factors were first suspected to play a role in 1996, when scientists discovered a susceptibility locus on chromosome 16 (Hugot et al., 1996). In 2001 a genome-wide association study identified the gene CARD15, encoding the protein nucleotide-binding oligomerization domain-containing protein 2 (NOD2), to be implicated in disease pathogenesis (Hugot et al., 2001; Ogura et al., 2001a). Subsequently, NOD2 was identified to be of the same family as apoptotic protease-activating factor 1 (APAF1) and nucleotide-binding oligomerization domain-containing protein-1, proteins involved in regulating apoptosis and activating the NF-κB pathway, respectively (Inohara et al., 2001; Ogura et al., 2001b). In addition to genetic factors, Crohn’s disease is suspected to include environmental risk factors such as microbial dysbiosis (Seksik et al., 2003; Sokol et al., 2006; Swidsinski et al., 2002) which brings focus to the interface between human innate immune receptors, like NOD2, and the bacterial components they recognize. A more complete understanding of the relevant receptors and their recognition of, and response(s) to, bacterial cell components will aid in unraveling the underlying mechanisms and pathogenesis of Crohn’s disease, leading to better strategies for treatment and diagnosis. In this chapter we discuss the role of the innate immune receptor NOD2 in Crohn’s disease pathogenesis, key observations that contribute to a better understanding of the disease, and how these observations can be used to improve diagnosis and potential therapies for patients.

14.2. INNATE IMMUNE RECEPTOR RECOGNITION OF BACTERIA The innate immune system is our body’s first line of defense against pathogens. We rely on innate immune receptors to recognize general structural patterns of -non-self- and respond rapidly, distinguishing these receptors from the adaptive, clonal recognition system (Janeway, 1989). The innate immune receptors, also known as pattern recognition receptors (PRRs), recognize and respond to patterns found on pathogens, commonly referred Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00014-7

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to as pathogen-associated molecular patterns (PAMPs), and more recently microbial-associated molecular patterns (MAMPs) since commensal bacteria share many of the same molecular signatures (Didierlaurent et al., 2006; Medzhitov and Janeway, 1997). Upon recognition of a MAMP, a PRR will trigger inflammation via a signaling cascade that culminates with the production of proinflammatory cytokines and chemokines to mediate a response to invading pathogens and aid in tissue repair. Families of PRRs are based on protein domain homology and include toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-like receptors (RLRs), C-type lectin receptors (CLRs), and AIM2-like receptors (ALRs) (Brubaker et al., 2015). PRRs vary in their location, the patterns they recognize, and the pathways they initiate, providing a wide gamut of general protection to the host. Misrecognition of MAMPs or failed PRR responses can lead to inflammatory diseases such as allergies, asthma, atherosclerosis, and inflammatory bowel diseases (Inohara et al., 2005; Minnicozzi et al., 2011; Xavier and Podolsky, 2007). This section focuses on the NLR protein NOD2, as mutations to this immune receptor correlate to an increased susceptibility for Crohn’s disease, an inflammatory bowel disease.

14.2.1. Bacterial Cell Wall The bacterial cell wall consists of peptidoglycan, an essential protective barrier for bacterial cells that encapsulates the cytoplasmic membrane of both Gram-positive and Gram-negative bacterial cells. Peptidoglycan is a rigid, highly conserved, complex structure of polymeric carbohydrates and amino acids. The carbohydrate polymer consists of alternating β-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid residues (Fig. 14.1). The N-acetylmuramic acid residues are typically attached to three to five amino acids which are often cross-linked through their side chains, giving the peptidoglycan a web-like appearance. The necessity and highly conserved nature of peptidoglycan makes for an ideal MAMP for NOD2 and other innate immune receptors to recognize bacterial cells.

14.2.2. NOD2 as an Innate Immune Receptor As a class of PRRs, NLR proteins are tasked with identifying MAMPs and regulating an appropriate immune response (Benko et al., 2008). NLR proteins share a tripartate domain structure that consists of N-terminal protein-protein interaction domain(s) (i.e., caspase activation and recruitment domains (CARDs) or pyrin domains), a central nucleotide-binding oligomerization domain (NOD or NBD), and a C-terminal leucine rich repeat (LRR) domain. NLRs are predominantly found in the cytosol, making them important intracellular detectors of MAMPs (Girardin et al., 2002; Strober et al., 2006). NOD2 is a 1040 amino acid cytosolic innate immune receptor found primarily in intestinal epithelial cells, Paneth cells, monocytes and dendritic cells (Berrebi et al., 2003; Gutierrez et al., 2002; Hisamatsu et al., 2003; Ogura et al., 2001b, 2003; Rosenthiel et al., 2003). It consists of two N-terminal CARD, a central NOD domain and a C-terminal LRR domain. The receptor recognizes bacterial cell wall fragments and initiates signaling cascades through the NF-κB and MAPK pathways that mature as the inflammatory response.

14.2.3. NOD2 Mutations Correlate With Crohn’s Disease NOD2 was discovered when a susceptibility locus for Crohn’s disease on chromosome 16 (Hugot et al., 1996; Ohmen et al., 1996) was mapped to the CARD15/NOD2 gene, which encodes for the receptor (Hugot et al., 2001; Ogura et al., 2001a). Mutations to the C-terminal LRR domain were demonstrated to increase the risk for Crohn’s disease, and two missense variants and a frameshift variant (R702W, G908R, and 1007fsinsC) were highlighted as

FIGURE 14.1 Structure of peptidoglycans.

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FIGURE 14.2 Muramyl dipeptide (MDP), a fragment of bacterial cell wall.

the most common NOD2 mutations found in Crohn’s disease patients (Hugot et al., 2001; Lesage et al., 2002; Ogura et al., 2001a). Additionally, NOD2 was demonstrated to signal through the NF-κB pathway in the presence of bacterial components, whereas the frameshift variant exhibited reduced signaling, establishing a link between bacterial recognition and the development of Crohn’s disease (Ogura et al., 2001a). This focuses the discussion of Crohn’s disease pathology on NOD2 mutations and the human microbiome.

14.2.4. Recognition of Muramyl Dipeptide by the Innate Immune System and NOD2 Muramyl dipeptide (MDP) is a fragment within the highly conserved structure of the bacterial cell wall (Fig. 14.2). MDP can appear in several forms due to the carbohydrate modifications bacteria introduce to their peptidoglycan; however, the core molecule has long been recognized as the smallest known peptidoglycan fragment to activate an immune response (Ellouz et al., 1974). Since its discovery, various synthetic MDP analogs have been produced in an attempt to better understand the immunoactive nature of the fragment (Chedid et al., 1976; Hasegawa et al., 1980, 1981; Hiebert et al., 1983; Kiso et al., 1980; Kobayashi et al., 1980; Okumura et al., 1983). In the early 2000s, and shortly after its discovery, NOD2 was hypothesized to be the innate immune receptor for MDP, as it is able to generate a NOD2-dependent immune response. MDP is ubiquitously present in both Gram-positive and Gram-negative bacteria, which led to the conclusion that the NOD2 protein is a general sensor for bacteria and not specific to a particular type (Girardin et al., 2003). Studies were done to explore the mechanism of signaling established NOD2 as a cytosolic protein capable of initiating an inflammatory response by signaling through the NF-κB and MAPK pathways; however, biochemical evidence was unavailable to demonstrate that a true interaction was occurring between NOD2 and MDP (Abbott et al., 2004; Magalhaes et al., 2011; Yang et al., 2007) until 2012 when two labs independently demonstrated that NOD2 binds directly to MDP (Grimes et al., 2012; Mo et al., 2012).

14.2.5. Reduced Signaling of the Crohn’s Disease-Associated NOD2 Mutants The Crohn’s disease-associated NOD2 mutants demonstrate a reduced ability to signal an inflammatory response in the presence of MDP (Coulombe et al., 2009; Girardin et al., 2003; Inohara et al., 2003; Mohanan and Grimes, 2014). This is not the result of a reduction in gene expression, since the cellular mRNA levels of the mutants are comparable to that of wild-type NOD2. The source of this inability is therefore pinpointed to the reduced stability of the mutants, as they are shown to have a significantly lower half-life in cells compared to wild-type NOD2 (Mohanan and Grimes, 2014).

14.2.6. The NOD2 Receptor Is Stabilized by Proteins The NOD2 receptor is naturally an unstable protein, with a cellular half-life below that of an average protein (Melnyk et al., 2015). NOD2 is known to interact with the molecular chaperones HSP70 and HSP90, and demonstrates an increased stability in their presence. Additionally, MDP-stimulated NOD2-dependent NF-κB signaling

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is improved in the presence of these molecular chaperones indicating a potential correlation between receptor stability and signaling (Lee et al., 2012; Mohanan and Grimes, 2014). Signaling is also increased in the presence of nucleoporin 62 (p62), an ubiquitin-binding protein that selectively targets proteins for autophagy. This appears to be the result of p62’s ability to stabilize NOD2 oligomerization by forming NOD2/p62 oligomers (Park et al., 2013). As noted above, the mutants have a diminished response to MDP when compared to WT NOD2. Mutant loss of function is rescued however, in the presence of HSP70 suggesting that the decrease in signaling is a consequence of the instability of the Crohn’s-associated mutants and not the result of diminished ligand binding (Mohanan and Grimes, 2014).

14.2.7. The NOD2 Response in Mice The NOD2 response is altered in NOD2 knockout mice and mice containing NOD2 variants associated with Crohn’s disease. The studies revealed that NOD2 is required for recognition of MDP in an animal model (Kobayashi et al., 2005; Pauleau and Murray, 2003). Although NOD2 knockout mice did not respond to MDP, these mice are commonly reported to have an increased susceptibility for infections by microbial pathogens (Shaw et al., 2009, 2011), coinciding with evidence that NOD2 plays a key role in antimicrobial adaptive immunity (Kobayashi et al., 2005; Magalhaes et al., 2008). It is also worth noting that the bacterial composition in NOD2 knockout mice is different from WT NOD2 mice (Mandot et al., 2012; Nabhani et al., 2016). Mice produced with the equivalent variant to the 1007fsinsC NOD2 but without the TAG termination codon, and therefore an additional 42 amino acids not found in the proper 1007fsinsC NOD2 mutant, demonstrated an increased NF-κB response to MDP (Maeda et al., 2005). Interestingly, however, mice with the 1007fsinsC NOD2 mutant with the proper TAG termination codon indicated no response to MDP and a lower production of proinflammatory cytokines compared to WT NOD2 mice (Kim et al., 2011). These works highlight the effects of NOD2 and the mutants in an animal model; however, much work is still necessary to fully understand the complexity of WT and mutant NOD2 receptor activation and signaling.

14.3. BACTERIA AND THE HUMAN MICROBIOME 14.3.1. The Human Microbiome At birth humans are colonized by an initial microbial community and by the age of three our bodies are settled by a stable community of bacteria that make up our microbiome. In fact, estimates suggest that there are as many bacterial cells residing in our human body as we have human cells, and a large majority of these are found in our gut (Sender et al., 2016). The normal microbiome of a healthy adult consists of a variety of bacteria that benefit the human host in a number of ways (Human Microbiome Project, 2012). A healthy microbiome promotes bacterial antagonism and a healthy immune system, a healthy gastrointestinal tract, metabolism and synthesis of vitamins and nutrients, detoxification of carcinogens and regulation of drug metabolism as well as many other benefits (Berg, 1996; Turnbaugh et al., 2007). A variety of factors (disease, environment, diet, antibiotics, age, health, etc.) can cause a dramatic shift in the microbiome which can lead to chronic microbial dysbiosis, a condition that often correlates with gastrointestinal diseases or disorders (Carding et al., 2015; Marsland and Gollwitzer, 2014). Crohn’s disease is correlated with gastrointestinal microbial dysbiosis, indicating an interplay of genetic, microbial and environmental factors in disease pathogenesis (Kostic et al., 2014).

14.3.2. The Importance of the Microbiome to the Host Early research revealed numerous morphological differences between germ-free and conventional, or colonized, rodents establishing the importance of the microbiome (Berg, 1996). Most notably, germ-free rodents were observed to have a significantly enlarged cecum, large variability in the arrangement of intestinal crypts, longer microvilli, (Berg, 1996; Gustafsson and Maunsbach, 1971), a thinner intestinal wall and variations in mucin composition (Berg, 1996; Szentkuti et al., 1990). Germ-free rodents present multiple immunological deficiencies suggesting that bacterial colonization is vital in proper structural and functional development of the innate immune system. Specifically, germ-free rodents display lymphoid tissue with impaired immunologic function due to a reduced presence of immunologically competent cells (Bauer et al., 1963), as well as fewer and smaller gutassociated lymphoid tissues, and reduced expression of antimicrobial proteins (Round and Mazmanian, 2009). Additionally, germ-free rodents exhibit a decreased inflammatory response, a delayed antigen-specific immune

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response, (Berg, 1996) and a reduced capacity for bacterial clearance (Round and Mazmanian, 2009; Zachar and Savage, 1979). Studies of immunodeficient or genetically susceptible rodents showed that those raised in germfree environments did not develop gastrointestinal inflammation, whereas rodents introduced to nonpathogenic, or a normal bacteria developed intestinal inflammation (Bhan et al., 1999; Rath et al., 1996; Sellon et al., 1998; Taurog et al., 1994). It is important to note that inflammation was not caused by pathogenic bacteria, but normal enteric bacteria induced differential gastrointestinal inflammation (Rath et al., 1996). These animal models confirmed the importance of bacteria in inflammatory bowel diseases and led to a clinical focus on the bacterial composition of the human gastrointestinal tract.

14.3.3. Crohn’s Disease and Chronic Microbiome Dysbiosis Crohn’s disease is commonly associated with gastrointestinal microbiome dysbiosis (Table 14.1). A healthy microbiome is responsible for priming the host immune system and maintaining a healthy immune response. Gastrointestinal colonization by foreign microbes is known to introduce mucosal irritation, thus potentially contributing to disease pathogenesis in genetically susceptible individuals. The development of Crohn’s disease is not well understood; however it is likely the result of a combination of factors involving the host microbiome and a genetic predisposition (Gevers et al., 2014; Khor et al., 2011; Manichanh et al., 2012). It is therefore unsurprising that many studies have observed microbiome differences between Crohn’s patients and healthy individuals (Gevers et al., 2014; Keighley et al., 1978; Seksik et al., 2003; Sokol et al., 2006; Swidsinski et al., 2002). Crohn’s patients typically exhibit an altered and less diverse composition of bacteria in their microbiome compared to a healthy individual (Ott et al., 2004; Sokol et al., 2009; Walker et al., 2011; Willing et al., 2009). The differences in the microbiome are summarized in the table below (Table 14.1). Different bacterial compositions cause up- or downregulation of the involved pathways, creating a noticeable shift in protein and metabolite compositions in Crohn’s disease patients compared to healthy individuals. For example, shifts in oxidative stress pathways were identified in Crohn’s patients, and bacterial species that were increased in Crohn’s disease patients were unique to glycerophospholipid and lipopolysaccharide metabolism, and those species decreased in Crohn’s disease patients were unique to amino acid, carbohydrate, and nucleotide metabolism (Gevers et al., 2014; Morgan et al., 2012). TABLE 14.1

Bacteria Observed to be Decreased (k) or Increased (m) in Crohn’s Patients Compared to Healthy Individuals

Firmicutes

References

Firmicutes, Erysipelotrichales, Bifidobacteriaceae, Clostridia, k Aomatsu et al. (2012), Blaser et al. (1984), Gevers et al. (2014), Clostridiales, Lachnospiraceae, Ruminococcus/Blautia, Gophna et al. (2006), Joossens et al. (2011), Manichanh et al. (2006), Faecalibacterium, Roseburia, Coprococcus, Eubacterium, R. hansenii, Marchesi et al. (2007), Sokol et al. (2006, 2008, 2009), Swidsinski et al. R. gnavusa, C. leptum, C. nexile, C. bolteae, C. coccoides, E. rectale, (2005), Walker et al. (2011) C. comes, R. intesinalis, D. invisus, F. prausnitzii, B. bifidum, B. longum, B. adolescentis, C. bolteae Gemellaceae, Veillonellaceae, Streptococcus, Lactobacillus, M. avium, M. paratuberculosis, V. parvula, G. morbillorum, R. gnavusa

m Autschbach et al. (2005), Gevers et al. (2014), Joossens et al. (2011), Keighley et al. (1978), Kirkwood et al. (2009), Tanaka et al. (1991)

BACTEROIDETES Bacteroidetesa, Bacteroidales, B. vulgatus, B. caccae

k Dicksved et al. (2008), Gevers et al. (2014), Gophna et al. (2006), Ott et al. (2004), Seksik et al. (2003), Swidsinski et al. (2002), Walker et al. (2011)

B. fragilis, Prevotella

m Keighley et al. (1978), Swidsinski et al. (2005)

PROTEOBACTERIA Proteobacteria, Neisseriaceae, Pasteurellaceae, Enterobacteriaceaea, Haemophilus, Eikenella, E. coli, H. parainfluenzae, E. corrodens

m Darfeuille-Michaud et al. (1998, 2004), Gevers et al. (2014), Gophna et al. (2006), Keighley et al. (1978), Liu et al. (1995), Martin et al. (2004), Ott et al. (2004), Seksik et al. (2003), Swidsinski et al. (2002), Walker et al. (2011)

FUSOBACTERIA Fusobacteriaceae, F. nucleatum

m Gevers et al. (2014)

a

Reports of an increase and decrease.

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14.3.4. The Effects of Bacterial Cell Wall Fragments and Metabolites on the Host Bacteria possess an arsenal of glycosidases, endopeptidases and carboxypeptidases known collectively as peptidoglycan hydrolases. These enzymes are responsible for trimming, modifying and cleaving the bacteria’s peptidoglycan, and are suspected to play significant roles in bacteria morphology and disease pathogenesis. For every bond in peptidoglycan there exists a hydrolase capable of cleaving it; however, not all hydrolases are expressed in every species of bacteria, and differences in hydrolase expression often vary between species (Frirdich and Gaynor, 2013; Silhavy et al., 2010; Wyckoff et al., 2012). These variations cause different bacteria to produce different types of cell wall metabolites, and the types of metabolites produced directly affect which receptors and pathways of the human immune system are activated. Additionally bacteria associated with disease pathogenesis, like those found in Crohn’s disease patients, express different proteins and produce different metabolites that support their invasive nature, thus causing detectable chemical and proteomic shifts in the microbiome of Crohn’s disease patients (Table 14.2). This is also reflected in the immune activation and protein expression profile of some of Crohn’s disease patients, as their immune activation is observably different compared to that of healthy individuals (Marchesi et al., 2007; Meelu et al., 2014; Shkoda et al., 2007). A noticeable shift in cytokine production is also observed in the gastrointestinal tract. In general, cytokine expression is increased in the presence of bacterial fragments (Maeda et al., 2005) and is mainly responsible for the intestinal inflammation found in inflammatory bowel diseases (Muzes et al., 2012; Strober and Fuss, 2011; Strober et al., 2010). Cytokines have variable effects depending on the T-helper cell subsets present, which vary depending on the specific bacterial antigens the gastrointestinal tract encountered previously (Cua et al., 1996). The dysregulated immune response in Crohn’s disease causes a loss of tolerance to enteric commensal bacteria resulting in a greater shift in the immune response (Duchmann et al., 1995; Mow et al., 2004; Sartor, 2006).

TABLE 14.2

Altered Effector Molecules Decreased (k) or Increased (m) in Crohn’s Patients

Metabolites

References

acetate, butyrate, methylamine, trimethylamine, total cholesterol, LDL cholesterol, retinol, prostaglandin F1α, hippurate, campesterol, lathosterol

k Hrabovsky et al. (2009), Jansson et al. (2009), Levy et al. (2000), Marchesi et al. (2007), Williams et al. (2010)

alanine, leucine, lysine, valine, triacylglycerol, glutathione, malondialdehyde, triglyceride, C peptide, dopaquinone, 4-hydrozyphenyl-acetylglycine, (Z)/4/hydroxyphenylacetyaldehyde-oxime, tyrosine, tryptophan, glycocholate, taurocholate, trihydroxy-6β-cholanate, Clycochenodeoxycholate, palmitic acid

m Al-Jaouni et al. (2000), Jansson et al. (2009), Levy et al. (2000), Marchesi et al. (2007)

CHEMOKINES RANTES (regulated on activation normal T cell expressed and secreted), SLC (secondary lymphoid tissue chemokine / CCL21), ELC (EBI1 ligand chemokine / CCL19), MCP-1 (monocytes chemoattractant protein-1), CXCL16

m Banks et al. (2003), Diegelmann et al. (2010), Kawashima et al. (2005), Mazzucchelli et al. (1996)

CYTOKINES GM-CSF, IL-3, IL-4, IL-5

k Atreya et al. (2000), McClane and Rombeau (1999), Pullman et al. (1992)

TNF-α (cachectin), G-CSF (granulocyte colony-stimulating factors), IL-1, IL-1β, IFN-γ, IL-8, IL-12, IL-23, IL-18, IL-27, IL-17, IL-21, IFN-γ, IL-6

m Atreya et al. (2000), Banks et al. (2003), Cappello et al. (1992), Gross et al. (1992), MacDonald et al. (1990), Mahida et al. (1989), McClane and Rombeau (1999), Monteleone et al. (2005), Murch et al. (1993), Sartor (2006), Stevens et al. (1992)

PROTEINS carbonyl reductase, keratin 19, apolipoprotein A-1, apolipoprotein B

k Levy et al. (2000), Shkoda et al. (2007)

HMGB1, α1-antitrypsin, α1-acid glycoprotein, C-reactive protein, Rho GDI α, L-lactate dehydrogenase, CCR7 (chemokine receptor 7)

m Al-Jaouni et al. (2000), Gross et al. (1992), Kawashima et al. (2005), Shkoda et al. (2007), Vitali et al. (2011)

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14.3.5. Invasive Bacteria Can Modify Their Core Peptidoglycan Structure to Manipulate the Host Immune Response In many species, bacteria will modify the carbohydrate residues of their peptidoglycan after insertion into the cell wall (Fig. 14.3). Some of the more common modifications are present in pathogenic bacteria with many offering the bacteria resistance to the host immune system. Methods employed to resist digestion by host lysozyme involve N-deacetylation of the N-acetylglucosamine and N-acetylmuramic acid residues and attachment of acetyl groups and wall teichoic acids to the alcohol at the 6-position of the N-acetylmuramic acid (Bera et al., 2005, 2007; Bishop et al., 2007; Boneca et al., 2007; Brown et al., 2013; Vollmer, 2008). Conversely, actinomycetes like mycobacterium convert some of their UDP-N-acetylmuramic acid to N-glycolylmuramic acid during cell wall biosynthesis, which are then incorporated into their peptidoglycan. The function of the N-glycolyl modification is unknown; however, it is believed to promote lysozyme and antibiotic resistance (Coulombe et al., 2009; Raymond et al., 2005). It is noteworthy that many strains of bacteria express the enzymes necessary to decorate their peptidoglycan with multiple types of these modifications to manipulate the host immune response. Finally, the muramic acid δ-lactam modification is commonly found in bacterial endospores. In order for this modification to occur, the muramic acid residue must be both N-deacetylated and without a peptide on its acid functionality, thus allowing amide bond formation to introduce the cyclic δ-lactam. The muramic acid δ-lactam modification is recognized by germination-specific hydrolases and is necessary for the endospore outgrowth process (Popham et al., 1996).

14.3.6. N-Substitution on MDP Effects NOD2 Signaling and Stability The MDP commonly found in Mycobacterium, N-glycolyl MDP, induces a larger NOD2-dependent inflammatory response than the more common N-acetyl MDP, thus suggesting that the receptor may be most finely tuned for sensing this fragment (Coulombe et al., 2009). N-deacetylated MDP which is commonly found in immune resistant pathogenic bacteria such as Listeria and Staphalococcus is not recognized by and does not stabilize the NOD2 receptor, and therefore does not trigger a NOD2 specific immune response. The stabilization effects of MDP vary depending on N-substitution of the ligand and appear to be correlated with a ligand’s ability to activate the receptor (Bishop et al., 2007; Boneca et al., 2007; Coulombe et al., 2009; Melnyk et al., 2015). This has implications in disease pathogenesis and the innate immune system’s ability to respond to bacteria, as bacteria that are able to elude detection and clearance by the innate immune system possess an improved ability to persist in the host.

FIGURE 14.3 Structure of peptidoglycan with some common modifications.

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14.4. DIAGNOSIS AND TREATMENT Crohn’s disease is very difficult to diagnose and treat, and is currently incurable. The presenting symptoms are often nonspecific, and may consist of abdominal pain, chronic diarrhea, anemia, growth failure or malnutrition. Diagnosis commonly requires a combination of serologic tests such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and complete blood count (CBC), imaging (magnetic resonance enterography or barium studies) and endoscopy with biopsies (Kilcoyne et al., 2016). Serologic tests for anti-Saccharomyces cerevisea antibodies IgA and/or IgG (ASCA), have been found to be predictive for a more aggressive, stricturing/penetrating type of Crohn’s disease (Ryan et al., 2013); however, a positive result is not always indicative of Crohn’s disease.

14.4.1. Biomarkers The differences observed between bacterial composition of the microbiome, protein expression, and metabolites of Crohn’s disease patients and healthy individuals suggest the promise of employing a Crohn’s-specific biomarker to diagnose the disease. If reliably detectable, such a biomarker would offer a more robust and less invasive approach for diagnosing Crohn’s disease. Currently, the inflammatory protein calprotectin has been proposed as a biomarker for detecting Crohn’s disease. Calprotectin is easily measured in stool samples and is indicative of intestinal inflammation (Roseth et al., 1992; Vieira et al., 2009). Unfortunately, fecal calprotectin is correlated with many pathological diseases dealing with inflammation and therefore is not specific to Crohn’s disease (Dhas et al., 2012; Gaya and Mackenzie, 2002). While not a promising stand-alone biomarker for Crohn’s disease, calprotectin has been suggested for use after diagnosis as a way to monitor intestinal inflammation to determine Crohn’s disease activity and response to treatment (D’Haens et al., 2012; Turvill, 2014); however, on its own it is not a promising biomarker for Crohn’s disease (Lehmann et al., 2015). Studies thus far have highlighted promising options for monitoring disease activity after diagnosis and treatment (Schaffler et al., 2016); however, more research is necessary to identify a Crohn’s-specific biomarker.

14.4.2. Current Treatments A majority of treatments for Crohn’s disease, including steroids, anti-inflammatory agents like mesalamine, immunosuppressants and antibiotics, focus on mediating the intestinal inflammation, modulating the immune system, and shifting the fecal microbiome rather than eradicating the disease. These treatments therefore offer short-term solutions, since patients can suffer exacerbations during therapy requiring additional intervention and treatments. In severe cases of refractory to available pharmacotherapy, surgery may be required. Recent scientific advancements have expanded our knowledge of the disease, bringing to light new areas for diagnostic and therapeutic innovations (Tontini et al., 2015). The advent of biologic agents ushered a new era of treatment for Crohn’s disease by targeting and inhibiting proteins that are essential in the propagation of intestinal inflammation, such as tumor necrosis factor (Forrest et al., 2014), inhibiting interleukins, such as IL-12 and IL-23 (Khanna et al., 2015), or preventing the adhesion of leukocytes to endothelial cells which mitigates inflammation (Singh et al., 2016). Advancements in these treatments are ongoing (Berns and Hommes, 2016; Colman and Rubin, 2014; Deepak and Bruining, 2015; Ungar and Kopylov, 2016) and as we learn more about the underlying mechanisms of the disease, more promising therapeutic targets are becoming apparent.

14.4.3. Proposed Pharmacoperones Recent therapies have highlighted the use of pharmacoperones (pharmacological chaperones) for rescuing unstable or misfolded disease mutants (Amaral, 2006; Bernier et al., 2004; Janovick et al., 2011; Muchowski and Wacker, 2005). The ideal pharmacoperone for Crohn’s disease would be a small molecular scaffold specific for NOD2 that can facilitate protein folding and stability. Thus far pharmacoperones have been applied towards diseases such as Alzheimer’s disease, Parkinson’s disease, familial amyotrophic lateral sclerosis, Huntington’s disease, and cystic fibrosis, all of which result from misfolded, unstable, or aggregated proteins (Amaral, 2006; Bernier et al., 2004; Muchowski and Wacker, 2005). Biochemical studies have suggested that the Crohn’s diseaseassociated NOD2 mutants are in fact unstable, and are able to be stabilized and have their activity rescued in the presence of a chaperone protein (Mohanan and Grimes, 2014). Therefore, treating Crohn’s disease by targeting the Crohn’s disease-associated NOD2 mutants with a specific pharmacoperone serves as a potential strategy for

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treating the genetic aspect of the disease rather than simply mediating the symptoms. Additionally, combining this type of therapy with a probiotic treatment to remedy the unhealthy shift in microbiome composition observed in Crohn’s disease patients would offer a two-pronged approach to remedying the disease at its roots.

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Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation: evidence in Crohn disease and experimental colitis in vivo. Nat. Med. 6, 583588. Autschbach, F., Eisold, S., Hinz, U., Zinser, S., Linnebacher, M., Giese, T., et al., 2005. High prevalence of Mycobacterium avium subspecies paratuberculosis IS900 DNA in guy tissues from individulas with Crohn’s disease. Inflamm. Bowel. Dis. 54, 944949. Banks, C., Bateman, A., Payne, R., Johnson, P., Seron, N., 2003. Chemokine expression in IBD. Mucosal chemokine expression is unselectively increased in both ulcerative colitis and Crohn’s disease. J. Pathol. 199, 2835. Bauer, H., Horowitz, R.E., Levenson, S.M., Popper, H., 1963. The response of the lymphatic tissue to the microbial flora. Studies on germfree mice. Am. J. Pathol. 42, 471483. Benko, S., Philpott, D.J., Girardin, S.E., 2008. The microbial and danger signals that activate Nod-like receptors. Cytokine. 43, 368373. Bera, A., Herbert, S., Jakob, A., Vollmer, W., Gotz, F., 2005. Why are pathogenic staphylococci so lysozyme resistant? The peptidoglycan O-acetyltransferase OatA is the major determinant for lysozyme resistance of Staphylococcus aureus. Mol. Microbiol. 55, 778787. Bera, A., Biswas, A., Herbert, S., Kulauzovic, E., Weidenmaier, C., Peschel, A., et al., 2007. Influence of wall teichoic acid on lysozyme resistance in staphylococcus aureus. J. Bacteriol. 189, 280283. Berg, R.D., 1996. The indigenous gastrointestinal microflora. Trends Microbiol. 4, 430435. Bernier, V., Lagace, M., Bichet, D.G., Bouvier, M., 2004. Pharmacological chaperones: potential treatment for conformational diseases. Trends Endocrinol. Metab. 15, 222228. Berns, M., Hommes, D.W., 2016. Anti-TNF-α therapies for the treatment of Crohn’s disease: the past, present and future. Expert. Opin. Investig. Drugs. 25, 129143. Berrebi, D., Maudinas, R., Hugot, J.-P., Chamaillard, M., Chareyre, F., De Lagausie, P., et al., 2003. 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C H A P T E R

15 Inflammation and Calcification in the Vascular Tree; Insights Into Atherosclerosis Carla S.B. Viegas and Dina C. Simes University of Algarve, Faro, Portugal

15.1. INTRODUCTION Cardiovascular diseases (CVDs) remain the leading cause of death globally with an estimated 17.3 million deaths in 2012, representing 31% of all global deaths. According to recent reports from the World Health Organization, an estimated 7.4 million deaths were due to coronary heart disease and 6.7 million were due to stroke (Townsend et al., 2016). The number of deaths caused by CVD not only is higher than any other condition, but in many countries also represents double the number of deaths caused by cancer. Atherosclerosis is the dominant cause of CVD including myocardial infarction, heart failure, stroke and claudication. In Westernized societies it is the underlying cause of about 50% of all deaths. Atherosclerosis is a progressive and multifactorial process with numerous etiologies that work synergistically to promote lesion development, and characterized by the accumulation of lipids and fibrous elements in the large arteries. The progression of an atherosclerotic lesion is believed to be a dynamic process that involves several cellular and acellular processes that interact intimately with each other. These include endothelial dysfunction, inflammatory conditions, calcification, oxidative stress, cell growth and proliferation, lipoprotein modifications, macrophages modification, extracellular matrix (ECM) synthesis and degradation, and plaque rupture. Both inflammatory and vascular smooth muscle cells (VSMCs) have a preponderant role, contributing to plaque composition and stability. The plaque composition rather than its size, is believed to be crucial for the clinical outcome of atherosclerosis (Bennett, 2007; Finn et al., 2010). Atherosclerotic plaques consist of an accumulation of VSMCs, macrophages, T lymphocytes, dendritic cells, and mast cells, together with extracellular lipid, collagen and matrix. The outcome of the disease is directly related to the possibility of plaque rupture with the exposure of highly thrombogenic, red cell-rich necrotic core material that may suddenly cause life-threatening coronary thrombosis. This chapter briefly overviews current concepts and pathophysiological mechanisms involved in atherosclerosis, with a particular emphasis on inflammation and calcification processes, evidencing their interplay in a pathological vicious cycle driving disease progression.

15.2. ATHEROSCLEROTIC LESION. COMPOSITION AND CLINICAL OUTCOME It is generally accepted that atheroma is initiated by lipoprotein cholesterol complexes trapped beneath the endothelium, which cause an inflammatory response, resulting in the recruitment of phagocytic cells to the lesion, with possible deposition of fatty streaks. These features are characteristic of type I and II early Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00015-9

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atherosclerotic lesions, which although not clinically significant, may progressively develop to type III intermediate lesions characterized by the accumulation of lipid-rich necrotic debris and VSMCs. A fatal clinical event may occur in the presence of type IV or V lesions when these reach a size sufficient to obstruct the lumen of an artery (Stary, 2000). Both these advanced atherosclerosis lesions are histologically characterized by a dense accumulation of extracellular lipids, although differing from each other in the nature of the fibromuscular layer that faces the lumen above the lipid core, also named the cap of the lesion. In type IV lesions, the cap shows an increased thickening of the intima although maintaining a similar composition as the normal intima. On the other hand, in type V lesions, new fibromuscular layers are produced in places where tissue was disrupted by accumulated lipid, hematoma and organized thrombotic deposits. Cap portions or layers generated by disease and added to the preexisting part, ultimately lead to an increase in the thickness and stiffness and contribute to the narrowing of the lumen. Changes in the composition and structure of the atherosclerotic plaque are in fact reported to be the major triggers of plaque stability and vulnerability. Although atherosclerosis can result in decreased blood flow due to stenosis, the major threat appears to be atherothrombosis involving the damage and rupture of the plaques, leading to local platelet activation and aggregation, with local occlusion or distal embolisms that are the major triggers to CVD, particularly myocardial infarction (Leys, 2001). Approximately two-thirds of the acute coronary events occur due to the rupture of lesions with noncritical stenosis and are characterized by specific histological features conferring vulnerability (Batty et al., 2016). Vulnerable plaques liable to undergo rupture generally have a thin fibrous cap due to loss of VSMCs, a larger necrotic core and lipid component, and increased number of inflammatory cells. The maintenance of the fibrous cap reflects matrix production and degradation, and is influenced by a complex combination of factors and crosstalk interactions between, among others, inflammatory cell products, neovascularization and calcification.

15.3. INFLAMMATION IN ATHEROSCLEROSIS Atherosclerosis is no longer regarded as a lipid storage disease; it is increasingly being considered as a chronic inflammatory condition, where both cells of the innate and adaptive immune system play a crucial role in its pathogenesis. Inflammatory mechanisms are involved in all phases of atherosclerosis, from the initial recruitment of circulating leukocytes to the arterial wall, development and progression of plaques, until its eventual rupture that might result in thrombotic complications (Ghattas et al., 2013). Activation, dysfunction and structural alterations of the endothelium and their constituting endothelial cells (ECs) are at the basis of atherosclerosis initiation. Factors triggering ECs activation include smoking, hypertension, hyperglycemia, obesity, insulin resistance, and a high saturated-fat diet. In the case of an atherogenic diet, the initiating event is mediated by the subendothelial retention of lipid components such as low-density lipoprotein, that will be oxidized by oxygen radicals (reactive oxygen species such as superoxide hydrogen peroxide, superoxide anion, etc.) and enzymes (such as myeloperoxidase and lipoxygenases), inducing the expression of chemokines such as (CC motif) ligand 2 (CCL2), and adhesion molecules such as E-selectin and vascular cell adhesion molecule-1 (VCAM-1). The expression of adhesion molecules, particularly VCAM-1, promotes the adhesion of immune cells of both the innate immune system (monocytes, macrophages and neutrophils) as well as those of the adaptive immune system (T lymphocytes), and activated platelets to the endothelium. While activated platelets secrete additional chemokines and interact with leukocytes to further increment immune cell infiltration, monocytes penetrate the intima of the vessel wall through diapedesis, where they will maturate into macrophages or dendritic cells. Macrophages will engulf modified lipoproteins accumulating cholesterol esters, thus becoming foam cells. Simultaneously, macrophages multiply and release several growth factors, cytokines and chemokines that regulate monocyte/T cell infiltration and contribute for the proinflammatory signals amplification. Many proinflammatory cytokines, such as interferon-γ (IFN-γ), IL-1α and IL-1β, IL-4, IL-6, IL-12, IL-15, IL-18, TNF family members (such as TNFα), and MIF, and anti-inflammatory cytokines, such as IL-10 and TGF-β family members (TGF-β1, BMPs, GDFs), have been shown to be released by macrophages in atherosclerotic plaques (reviewed in Legein et al., 2013). Sustained inflammation, together with other altered physiological processes such as oxidative stress will result in macrophage apoptosis and necrosis. Inefficient clearance of macrophage cell debris results in the formation of a necrotic lipid core within the plaque, which can induce plaque vulnerability. In addition, inflammation has been shown to directly contribute to the formation of a thinner fibrous cap by interfering with the integrity of interstitial collagen matrix, through inhibition of new collagen fibers formation by VSMCs or by stimulating the destruction of existing collagens (Amento et al., 1991; Sukhova et al., 1999; Horton et al., 2001) (Fig. 15.1).

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FIGURE 15.1 Schematic illustration representing the development and progression of an atherosclerotic lesion, from the initial stage of endothelial activation through plaque rupture and thrombosis.

15.4. CALCIFICATION ASSOCIATED WITH ATHEROSCLEROSIS Calcification is considered a hallmark of atherosclerosis and it has been estimated that over 70% of atherosclerotic plaques observed in the aging population are calcified. Calcification of atheroma in the coronary arteries is ubiquitous, and the measurement and quantification of calcium deposits is being used as a surrogate marker of coronary atherosclerosis and as predictor of cardiovascular events. According to the American College of Cardiology Foundation/American Heart Association, measurement of coronary artery calcium (CAC) represent a valuable prognostic tool for cardiovascular risk assessment in asymptomatic adults at intermediate and low-to-intermediate risk (Greenland et al., 2010; Goff et al., 2013). CAC screening for the early detection of atherosclerosis leads to more accurate risk stratification of intermediate-risk groups, allowing for appropriate initiation of lipid-lowering therapies and lifestyle preventative measures. Calcium mineral deposits that make up calcified arteries are histomorphologically indistinguishable from bone. Thus, the process marking its generation is akin to other remodeling processes in the body. Although calcification has been historically associated with advanced stages of atherosclerosis, mainly due to the limitations on in vivo and in vitro imaging techniques, recent studies have shown that calcium deposition is an early event in the atherosclerotic process (Roijers et al., 2008; Roijers et al., 2011). Small punctate calcifications (microcalcifications) have been detected in the early stage of atherosclerosis, namely in preatheroma type I lesions of the human coronary arterial wall and commonly found in more advanced lesions as in type II to IV. Also, deposition of microcalcifications in the apolipoprotein E-deficient mice was shown to be an early event in the atherosclerotic aortic wall (Aikawa et al., 2007). The extent of calcification has been shown to correlate with plaque burden (Johnson et al., 2006), and atherosclerotic lesions at risk of rupture have been associated with positive remodeling, presence of a large necrotic core, macrophage infiltration, and microcalcifications (Finn et al., 2010; Batty et al., 2016; Ehara et al., 2004). Nevertheless, the role and contribution of calcification to plaque stability and rupture is still under debate (Aluganti Narasimhulu et al., 2016; Thilo et al., 2010; Otsuka et al., 2014). It has been proposed that atherosclerotic plaque proceeds through progressive stages where instability and rupture can be followed by calcification (Vengrenyuk et al., 2006; Maldonado et al., 2012). Deposits of large calcified

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material, usually associated with thick fibrous cap, may function to effectively separate the residual inflammatory site from the vascular lumen, providing mechanical stability and thereby protecting against rupture. On the other hand, microcalcifications are described to predispose the thin fibrous cap plaque rupture by increasing biomechanical stress. In fact, strong correlations between microcalcifications and risk of coronary artery plaque instability and rupture have been established, and indicate that microdeposits of Ca-rich material are likely to play a pathogenic role in the chain of events leading to overt atherosclerotic lesions and plaque instability. These microcalcifications have been suggested to be one of the earliest events in the development of atherosclerotic lesions, and to be actively involved in inflammatory responses (Chatrou et al., 2015), causing VSMCs apoptosis, one of the major contributors to the formation of a thin fibrous cap. In addition, to these processes, apoptotic cell fragments and cholesterol crystals can act as a nidus for crystallization, further promoting calcification.

15.5. VASCULAR CALCIFICATION Vascular calcification (VC) is a disease of disordered mineral metabolism in which calcium mineral, mostly in the form of hydroxyapatite, is deposited in the ECM of the vascular tree. VC can be induced in response to the loss of mineralization inhibitors and to dysregulated or inappropriate environmental stimuli. VC is ubiquitously present in several vascular disease processes, and can be divided into three distinct entities according to the vascular component and the specific site of occurrence within the vascular wall (Kalra and Shanahan, 2015; Demer and Tintut, 2014; Chen and Moe, 2015; Towler, 2013): (1) intimal calcification, characterized by mineral deposition associated with inflammatory cells in the vicinity of lipid or cholesterol deposits within atherosclerotic plaques, and exclusively associated with atherosclerosis; (2) medial calcification, also known as Mo¨nckeberg’s sclerosis, appearing as organized mineral deposition along the elastic lamellae in the absence of lipid or cholesterol deposits and without the involvement of inflammatory cells, and frequently associated with aging, diabetes mellitus, and chronic kidney disease; and (3) cardiac valve calcification (CVC), affecting the cardiac valve leaflets and contributing to stenosis, commonly associated with hyperlipidemia, mechanical stress, proinflammatory factors and aging. CVC is a major feature of calcific aortic valve disease sharing similarities, in terms of disease process, with atherosclerosis (Fig. 15.2). Medial and intimal calcifications have been associated with different clinical outcomes, different types of treatment strategies and prognosis, in addition to a distinguishable set of morphological features. Nevertheless, both types of calcification can be found simultaneously in patients, and studies using animal models have also provided evidence that both VC and atherosclerosis share some common pathomechanisms (reviewed in Amann, 2008). However, although increasing knowledge on VC has been achieved in the last century, mostly based on experimental models, the understanding of the mechanism of atherosclerotic calcification in humans remains limited and controversial, requiring additional research efforts to open the possibility of specific prevention of lesion formation and treatment.

15.5.1. Pathophysiological Mechanisms of Vascular Calcification For many years considered a simple passive process of mineral precipitation leading to apoptosis and calcification, it is now known that VC is an active, naturally occurring, and highly controlled cell-mediated process involving VSMCs osteochondrogenic differentiation and resembling developmental skeletal formation.

FIGURE 15.2 Schematic representation of the different types of vascular calcification occurring in the vascular tree, i.e., medial, intimal and valve calcification.

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A considerable amount of research data collected over the last 20 years has shown us that (1) hydroxyapatite, the main mineral form present in bone, is also the principal mineral found in VC (Duer et al., 2008; Schmid et al., 1980); (2) bone-like tissue, including bone trabeculae, osteoblast-like cells and marrow stromal cells have been identified in calcified arteries and heart valves (Bunting, 1906; Hunt et al., 2002); (3) matrix and mineralizationregulatory proteins involved in bone formation are expressed by calcifying VSMCs and associated with human calcified plaques (Shanahan et al., 1994, 1999, 2000); (4) extracellular vesicles (EVs) known to be the initial sites of primary nucleation during bone mineralization, are also found in calcified atherosclerotic plaques and shown to be crucial to in vitro calcification of VSMCs (Reynolds et al., 2004; Kapustin and Shanahan, 2012; Cui et al., 2016); and (5) tartrate-resistant acid phosphatase-positive multinucleated osteoclast-like cells are present at sites of VC (Jeziorska et al., 1998; Oksala et al., 2010). Altogether these findings support the currently accepted parallelism between pathological VC and physiological developmental osteogenesis. Although many aspects concerning the pathogenesis of VC are still unclear, pathological features, such as release of calcifying EVs, proliferation and differentiation of resident VSMCs, loss of anticalcific mechanisms, VSMCs apoptosis, endothelial dysfunction, oxidative stress, increased ECM remodeling, and chronic inflammation, are well described to contribute for the development of calcific lesions. Despite the agreement that VC is a multifactorial process, consensus on factors and conditions leading to the initiation of calcification, as well as to the sequence of events and interdependency of these processes, are still debatable. Still, many questions remain open especially when it involves the understanding of how intimal calcification is initiated. Current hypothesis postulates that (1) osteochondrogenic differentiation of VSMCs is required before calcification initiates (Speer et al., 2009); (2) apoptotic bodies (ABs) and cellular debris act as a nidus for calcification (Proudfoot et al., 2000; Clarke et al., 2008); (3) loss of calcification inhibitors produced by VSMCs allows the nucleation of calciumphosphate with formation of microcalcifications (Chatrou et al., 2015; Kapustin et al., 2011); (4) inflammation triggers and precedes calcification (Aikawa et al., 2007; Abdelbaky et al., 2015); (5) calcification is responsible for recruitment of inflammatory cells and amplification of inflammatory reactions (Chatrou et al., 2015; Nadra et al., 2005). In fact, it is most likely that the answer to this question includes a complex interplay among these processes occurring simultaneously or in a stepwise manner, contributing for the development and progression of VC. Furthermore, it is now clear that differences exist between intimal atherosclerotic and medial calcification processes, and that atherosclerotic calcification cannot be dissociated from inflammatory processes at atherosclerotic lesions occurring either in inflammatory cells or VSMCs, as will be further discussed in this chapter. It is, however, well accepted that VSMCs play a key role in the initiation and regulation of pathological VC. The plasticity of VSMCs allows their osteogenic conversion in the presence of osteogenic signals, with consequent changes from contractile to synthetic physiological roles. This transformation might be attributed to the common mesenchymal origin between VSMCs and osteoblasts and chondrocytes (Iyemere et al., 2006). In a normal physiological environment and despite the fact that serum is supersaturated in relation to calcium and phosphate concentrations, mineralization only occurs in bone, cartilage and teeth. Undesirable mineral formation is controlled due to the existence of inhibitory mechanisms to prevent ectopic calcification. Also, essential steps in the pathological calcification pathway involve VSMCs osteochondrogenic differentiation with upregulation of bone mineralization-regulatory genes, and spontaneous release of EVs (reviewed in Chen and Moe, 2015). Under physiological conditions, local and systemic inhibitors of mineral formation act to prevent widespread tissue calcification by influencing osteochondrogenic differentiation, formation of calcifying competent EVs, and ECM crystal growth. 15.5.1.1. Role of Calcifying Extracellular Vesicles The earliest phase of VC is believed to occur via the secretion of EVs that nucleate calcium phosphate crystals, in a process sharing many similarities to that observed during skeletal mineralization (reviewed in Cui et al., 2016). These EVs initially thought to be resulting from the budding of plasma membrane and called matrix vesicles, were recently shown to be exosomes with an endosomal origin arising from intracellular multivesicular bodies of living VSMCs (Kapustin et al., 2015). Calcification can also be initiated by apoptotic bodies (ABs) from dying VSMCs that act as nucleating structures for calcium crystal formation (Proudfoot et al., 2000; Clarke et al., 2008). In healthy arteries, EVs released by VSMCs are devoid of mineral, while in calcified arteries mineralcontaining EVs localize in proximity to elastin and collagen fibrils (Kapustin et al., 2011; Tanimura et al., 1983; Schlieper et al., 2010). These EVs are heterogeneous in size and mineral content, and in atherosclerotic plaques may originate from VSMCs, ECs, and leukocytes, including macrophages, lymphocytes and granulocytes (Leroyer et al., 2007). Mineralizing EVs contain a nucleation core consisting of calcium-phosphate-phospholipid complexes and proteins, such as Anx6, that promote mineralization, and the first crystalline calcium crystals are

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formed inside the lumen of EVs. The following deposition of mineralizing EVs in the ECM, with propagation of mineral crystals from EVs, was shown to form the first nidus for mineral nucleation leading to ECM calcification (Kapustin et al., 2011). These studies clearly showed that the release of calcifying EVs was a crucial event for VSMCs mineralization. Inhibition of EVs release was able to decrease EVs production and to prevent VSMCs calcification, while the addition of EVs to calcifying VSMCs enhanced calcification. The competence of EVs for directing the mineralization process seems to depend on many factors acting either alone or in combination (Kapustin et al., 2015). Elevated extracellular levels of calcium and/or phosphate, cytokines such as TNF-α, and growth factors such as PDGF-BB were shown to increase EVs release. Contrarily, IL-6, IL-10, and TGF-β1 decreased EVs production (Kapustin et al., 2011). Importantly, EVs released by healthy VSMCs are loaded with mineralization inhibitors that can be locally produced, as is the case of matrix gla protein (MGP) and gla rich protein (GRP), or uptake from circulation, like fetuin-A. Depletion of these mineralization inhibitors has been associated with increased mineralization capacity of EVs (Kapustin et al., 2011; Viegas et al., 2015). 15.5.1.2. VSMCs Osteochondrogenic Differentiation VSMCs have the capacity to undergo osteochondrogenic transformation in a process sharing many similarities with bone development. This is characterized by loss of smooth muscle lineage markers such as SM22α and SMα-actin, and increased expression of genes known to be master players in osteogenesis, including BMPs, Runx2 (Cbfa-1), Msx2, osterix, Sox9, osteocalcin, and alkaline phosphatase, among others (reviewed in Kalra and Shanahan, 2015; Demer and Tintut, 2014; Chen and Moe, 2015). This VSMCs differentiation is characterized by a conversion from a contractile to a synthetic phenotype promoting ECM mineralization, and can be triggered by pathological signals such as inflammatory cytokines, mineral imbalance, oxidized lipids and oxidative stress. Increased expression of these bone and cartilage-related proteins has been widely described in atherosclerotic plaques and in calcifying VSMCs (Bostro¨m et al., 1993; Tyson et al., 2003). Master transcription factors such as Msx2, Runx2, osterix and Sox9 are involved in a cascade of genetic programming inducing downstream matrix components such as collagen I, alkaline phosphatase, osteopontin and osteocalcin, designating cells for osteoblast/chondrocyte lineages. BMP2 is probably one of the earliest genes involved in the cascade of events leading to osteochondrogenic differentiation, acting upstream and via Msx2 and Runx2, but also through binding of MGP and interfering with levels of available MGP, hampering its role as a mineralization inhibitor (Bostro¨m et al., 2011). A combination of increased expression of osteogenic-related proteins and deficiencies in calcification inhibitors is in fact the perfect situation for the development and progression of VC. 15.5.1.3. Mineralization-Regulating Proteins. Role of MGP, GRP and Fetuin-A The notion that VC is a naturally occurring process that must be actively inhibited arises from in vivo and in vitro studies showing that the lack of certain inhibitors causes VC (Schinke and Karsenty, 2000), and implies that a tight control must exist between calcification promoters and inhibitors to maintain equilibrium avoiding undesirable pathological calcification. In a healthy organism VC is controlled because VSMCs synthesize or uptake from circulation natural mineralization inhibitors, which can counterbalance mineralization promoters. From the numerous identified molecules with mineralization inhibitory function, we will highlight the role of MGP, fetuin-A (or alpha 2-Heremans-Schmid glycoprotein, AHSG), and GRP, due to their potential involvement at multiple levels in the complex etiology of VC. MGP and fetuin-A are well recognized VC inhibitors (Schurgers et al., 2013; Jahnen-Dechent et al., 2011) with a preponderant role at tissue and systemic levels. While MGP is a vitamin K-dependent protein (VKDP) synthetized by VSMCs and ECs in vascular tissues, fetuin-A is a liver-derived blood cysteine protease inhibitor uptake from circulation by VSMCs. Functional in vivo and in vitro models have established their vital importance in VC. Knockout mice for MGP die within 8 weeks of birth due to massive vascular mineralization affecting the main arteries (Luo et al., 1997), and restoration of MGP expression in VSMCs of MGP (2/2) mice rescued the arterial mineralization phenotype (Murshed et al., 2004). Fetuin-A deficient mice combined with a calcification-sensitive mouse strain results in progressive and lethal calcification of soft tissues, including kidneys, skin, heart and vasculature (Schafer et al., 2003). Although the mechanisms underlying MGP and fetuin-A anticalcific function might have distinct molecular pathways, both are involved in the inhibition of mineral growth and VSMCs osteochondrogenic differentiation. Through its calcium-binding Gla residues, MGP directly interacts with calcium crystal inhibiting its growth. Fetuin-A binds small clusters of calcium and phosphate in blood forming a soluble protein mineral particle (called fetuin-mineral complex (FMC) or calciprotein particle (CPP)), preventing mineral growth, aggregation and precipitation. The mineralization capacity of VSMCs-derived EVs depleted of MGP and fetuin-A, has been associated with a deficient inhibition capacity of calcium phosphate crystals nucleation.

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FIGURE 15.3 Illustration representing the molecular mechanism of vascular calcification proposed to be involved in the shift from a physiological to a pathological state.

The binding of MGP to BMP-2 and fetuin-A to TGFs and BMPs contributes to a decreased effect on mineralization by impairing osteochondrogenic differentiation. GRP is the newest member of the VKDP family, characterized by the presence of an unprecedented 15 putative Gla residues in human, and recently shown as an inhibitor of calcification in the cardiovascular (Viegas et al., 2015) and articular systems (Cavaco et al., 2015). GRP calcium-binding properties and association with calcification processes indicate that its function might be associated with prevention of calcium-induced signaling pathways and to a direct mineral-binding capacity leading to an inhibition of the crystal formation/maturation. As in the case of MGP and fetuin-A, GRP is also involved in the mineralization-competence of VSMCs-derived EVs and in VSMCs osteochondrogenic differentiation (Viegas et al., 2015). Moreover, GRP was also shown to participate in the crosstalk between inflammation and calcification of articular tissues in osteoarthritis, acting as an anti-inflammatory agent (Cavaco et al., 2015). Treatments with GRP, either alone or as coating basic calcium phosphate (BCP) crystals, decreased the proinflammatory response of articular cells (Cavaco et al., 2015), while fetuin-A bound to mineral crystals has also been shown to decrease the proinflammatory response of neutrophils and macrophages, when compared to naked crystals (Smith et al., 2013; Terkeltaub et al., 1988). It is interesting to note that additional lines of evidence seem to point to a close relationship between fetuin-A, MGP and GRP, which might act as a powerful anti-mineralization system: (1) fetuin-A is bound to MGP in rat FMC (Price et al., 2002), although a similar interaction in human-derived FMCs has not yet been demonstrated; (2) fetuin-A, MGP and GRP are all involved in VSMCs mediated EVs mineralization process (Kapustin et al., 2011; Viegas et al., 2015); (3) a MGP-fetuin-A complex was described in chondrocyte-derived EVs (Wallin et al., 2010); and (4) a protein complex found associated with the mineral phase of calcified aortic valves was described to contain these three proteins and EVs components, such as annexin A2 and S100A9 (Viegas et al., 2015). This putative protein complex including two VKDPs might represent a novel potential therapeutic target for the inhibition of VC by functional modulation through vitamin K (Fig. 15.3).

15.6. VITAMIN K AS A POTENTIAL THERAPEUTIC TARGET FOR ATHEROSCLEROSIS The role of vitamin K in arterial calcification has been widely demonstrated, and consistently associated to its function as cofactor for γ-carboxylation reaction of VKDPs. The calcification inhibitory function of MGP is strictly dependent on its γ-carboxylation status (Schurgers et al., 2013), and only γ-carboxylated GRP was shown to have calcification inhibitory properties (Viegas et al., 2015). Increased calcification in warfarin (a vitamin K antagonist) treated rats was accompanied by decreased levels of MGP expression while increased accumulation of the nonfunctional undercarboxylated protein form (ucMGP) (Price et al., 1998). Increased MGP accumulation in

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atherosclerotic plaques was recently shown to reflect increased ucMGP accumulation, which correlated with location and quantity of calcification in different stage atherosclerotic lesions (Chatrou et al., 2015). Although the relation between γ-carboxylation and GRP in atherosclerosis is yet unknown, increased levels of undercarboxylated GRP (ucGRP) was found associated with pathological calcification-related diseases, such as calcified aortic valve stenosis, osteoarthritis and certain cancers (reviewed in (Viegas and Simes, 2016)). Studies in vivo have demonstrated that treatments with warfarin result in accelerated VC, which could be inhibited by simultaneous vitamin K treatments (Price et al., 1998; Price et al., 1982; Kru¨ger et al., 2013), and vitamin K2 was able to inhibit warfarin-induced arterial calcification (Spronk et al., 2003). In the population-based Rotterdam study, vitamin K2 intake was found inversely related to all-cause mortality and severe aortic calcification (Geleijnse et al., 2004). In addition to its essential role as cofactor for γ-carboxylation, vitamin K is also known to exert additional functions, such as anti-inflammatory, transcriptional regulator of osteoblastic genes, and inhibition of tumor progression (Ohsaki et al., 2010). In vivo studies have shown that high vitamin K status was associated with lower concentrations of inflammatory markers (Ohsaki et al., 2010; Shea et al., 2007) and dietary supplementation suppressed LPS-induced inflammation (Ohsaki et al., 2006). Furthermore, vitamin K has been associated with a decreased production of proinflammatory cytokines in vitro, such as IL-1β, IL-6, TNFα, and osteoprotegerin, and this effect was proposed to be mediated via the inactivation of NFkB signaling pathway (Ohsaki et al., 2010; Fujii et al., 2015). These in vitro studies suggest a direct effect of vitamin K, exerting its anti-inflammatory activity through blocking of reactive oxygen species generation, thereby protecting against oxidative stress. The involvement of vitamin K in anticalcification, antioxidant and anti-inflammatory processes, highly relevant in the context of atherosclerosis, makes it a promising preventive and therapeutic agent.

15.7. THE VICIOUS CYCLE OF INFLAMMATION AND VASCULAR CALCIFICATION In the context of ectopic calcification-dependent diseases such as atherosclerosis, inflammation and calcification are no longer regarded as discrete events, rather it is accepted that calcification and inflammation are intimately correlated, and involve complex interplay and crosstalk in a positive feedback loop that drives disease progression. In a cohort study that included subjects from the Framingham Heart Study who were free of clinically apparent CVD, C-reactive protein (CRP) levels were associated with coronary artery calcification in both men and women, even after adjustment for age, individual risk factors and Framingham risk score (Wang et al., 2002). While macrophages are key players signaling ECM degradation and resident tissue cells differentiation and calcification (Tintut et al., 2002; Ikeda et al., 2012), microcalcifications have been proposed to be involved in macrophage recruitment in the early stages of atherosclerosis (Chatrou et al., 2015). Also, BCP crystals are reported to be able to stimulate macrophages to produce proinflammatory cytokines affecting VSMCs differentiation, in a calcification-inflammation pathological feeding cycle (Nadra et al., 2005, 2008). The accumulation of macrophages within the vascular wall has been consistently colocalized with calcium deposits and associated with various phases of calcification in atherosclerotic lesions, with an important role in the initiation of atherosclerosis development and VC (Chatrou et al., 2015). Several lines of evidence indicate that inflammation triggers and precedes osteoblastic activity and the release of calcifying EVs from VSMCs, promoting the calcification process. In vivo, a real-time association of inflammation and early calcification was demonstrated in the ApoE(2/2) knockout mice, by mapping osteogenesis to sites of inflammation in the aorta in very early stages of atherosclerosis (Aikawa et al., 2007). These activities were found to evolve in close proximity, overlapping at border regions and increasing with plaque progression. Increased osteogenic activity was found associated with increased alkaline phosphatase activity and other mineralization-regulating proteins such as Runx2 and osteocalcin. Microcalcifications were present in EVs and associated with cholesterol crystals. Also, studies performed on a large cohort of individuals without aortic valve stenosis showed that early valvular inflammation plays an important role in the adverse pathophysiological process of valve calcification (Abdelbaky et al., 2015). The effect of inflammation on the VC process is described to be mostly mediated by macrophages, and likely to occur at multiple interconnected levels: (i) production of high levels of matrix metalloproteinases (MMPs), cysteine endoproteases and cytokines, which will enhance elastin and collagen degradation, leading to remodeling and structural changes of the ECM that can be a nidus for hydroxyapatite crystal formation (New and Aikawa, 2011; Li et al., 2012; Aikawa et al., 2009); (ii) the release of osteogenic-like factors capable of inducing VSMCs

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phenotypic changes (Tintut et al., 2002; Ikeda et al., 2012); (iii) release of calcifying EVs loaded with mineralization related factors, capable of accelerating ECM calcification (New et al., 2013); (iv) and through induction of VSMCs apoptosis (Boyle et al., 2003). The release of soluble factors by activated macrophages, such as IL-1β, IL-4, IL-6, IL-8, TNFα, TGFβ, among others, has long being reported to affect atherosclerosis, VSMCs osteogenic activity and calcification. Inhibition of TNFα in ApoE(2/2) mice was shown to reduce by 50% the size of aorta lesions (Bra˚ne´n et al., 2004), and the results of overexpression of the IL-1 natural inhibitor IL-1Ra or of subcutaneous administration of recombinant human IL-1Ra in ApoE (2/2) mice, clearly showed a reduced size of atherosclerotic lesions (Merhi-Soussi et al., 2005). TNFα exerts multiple effects during atherogenesis, including increasing permeability of ECs, promoting monocyte adhesion, inducing macrophage differentiation, and promoting foam cell formation. TNFα has a pivotal role in enhancing VSMCs calcification through the activation of NFκB, leading to a decreased expression of ankyloses protein homolog (ANKH) that controls pyrophosphate (PPi) efflux, accompanied by a reduction in PPi export (Zhao et al., 2012). Decreased levels of extracellular PPi might be also attributed to the effect of TNFα, and also IL-1β, on increased expression of TNAP, likely mediated through the effect of peroxisome proliferatoractivated receptor γ (PPARγ) (Lencel et al., 2011). In addition, TNFα and also IL-1β are reported to enhance VSMCs osteogenicity by increasing BMP-2 and reducing MGP expression (Ikeda et al., 2012). Increased BMP-2 expression potentiates VSMCs osteochondrogenic differentiation that can be aggravated due to the absence of MGP to inhibit BMP-2 osteogenic signaling, and lack of MGP promotes calcification through the release of calcifying competent EVs. Interestingly, a novel and direct role of macrophages on calcification has been recently proposed (New et al., 2013), and might be related to the capacity of macrophages to release calcifying EVs loaded with mineralization related factors and capable of accelerating ECM calcification. Based on this evidence we could speculate that some microcrystals found at sites of mineral deposition may be independent of VSMCs action, but originate directly from inflammatory cells, likely to play a role in the initiation of microcalcification. Not only do macrophages have an effect on VSMCs but VSMCs can also induce a proinflammatory mechanism in macrophages, acting synergistically in the formation of calcification. Although not explored in this chapter, it should be noted that VSMCs also have an active role in propagating inflammation during atherosclerosis development (reviewed in Lim and Park, 2014). It is known that inflammatory reactions have a profound effect on calcification; conversely, increased matrix degradation and calcification are also widely described to promote proinflammatory responses. Thus it has been suggested that pathological calcification is not solely a passive consequence of chronic inflammatory diseases but is actively involved in a positive feedback loop of calcification and inflammation that enhances disease progression. Most evidences supporting this notion have been collected from studies in degenerative arthritis, where BCP crystals are currently considered a damage-associated molecular pattern. The underlying mechanism is thought to be the signaling to the immune system of a state of stress (Sokolove and Lepus, 2013), and potentially contributing to inflammation through stimulation of articular cells (Liu et al., 2009; Rosenthal, 2011). Besides their effect on synoviocyte proliferation, along with production of inflammatory cytokines, MMPs and prostaglandins, BCP crystals also induce articular chondrocytes to produce prodegradative soluble factors, such as nitric oxide and promote apoptosis. Production of MMPs and chondrocyte apoptosis contribute to cartilage destruction, while cartilage degradation products drive inflammatory events in a pathological mineralization-inflammation tissue degradation cycle. Additionally, BCP crystals were shown to directly interact with macrophages, inducing a proinflammatory response with increased TNFα, IL-1β and IL8 production, which are capable of stimulating the activation of ECs and recruitment of mononuclear cells (Nadra et al., 2005). The effect of HA crystals on increased TNFα secretion by macrophages was shown to be dependent of NFκB activation (Nadra et al., 2008). Furthermore, BCP crystals induce VSMCs apoptosis, after phagocytosis and lysosome dissolution, probably due to the increase in intracellular calcium levels (Ewence et al., 2008), while secondary CPP particles induce an upregulation of TNFα accompanied by increased calcification (Aghagolzadeh et al., 2016). In fact, smaller calcium crystals have been shown as stronger inducers of proinflammatory responses (Nadra et al., 2008), and might explain why microcalcifications are more associated with predisposition of plaque rupture than large calcified material that is usually observed associated with more advanced lesions. Additionally, a coating of BCP particles with fetuin-A and GRP, resembling CPPs, was shown to decrease the production of cytokines by macrophage and articular cells when compared to naked crystals (Cavaco et al., 2015; Smith et al., 2013). This additional evidence reinforces the proposed interplay between mineralization inhibitors and inflammatory processes, ultimately leading to aggravate the pathogenesis of VC that contributes to the acute thrombotic complications of atherosclerosis (Fig. 15.4).

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FIGURE 15.4

Schematic representation of the molecular processes leading to a pathological calcification-inflammation cycle. Interactions between macrophages, vascular smooth muscle cells (VSMCs) and microcalcifications.

15.8. FINAL REMARKS Over the past years, numerous studies have highlighted the multiple pathophysiological mechanisms involved in atherosclerosis, shifting the concept from a lipid storage disease through a calcification-related chronic inflammatory disease. Nowadays there is no doubt that inflammation and calcification play a crucial role in the pathophysiology of atherosclerosis and its acute clinical complications. Increasing our current knowledge on the complex molecular mechanisms of both processes and how they modulate the disease process, will certainly benefit the course of atherosclerosis. Accepting that inflammatory and calcification pathways are interconnected and interdependent implies the existence of signals acting as crosstalk factors halting disease progression. These findings and efforts to further determine the nature and role of these signals should provide a conceptual framework for the development of future effective early therapeutic and preventive intervention measures.

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Evidence that serum alpha 2-HS glycoprotein is a potent and specific crystal-bound inhibitor. Arth. Rheumatol. 31, 10811089. Thilo, C., Gebregziabher, M., Mayer, F.B., Zwerner, P.L., Costello, P., Schoepf, U.J., 2010. Correlation of regional distribution and morphological pattern of calcification at CT coronary artery calcium scoring with non-calcified plaque formation and stenosis. Eur. Radiol. 20, 855861. Tintut, Y., Patel, J., Territo, M., Saini, T., Parhami, F., Demer, L.L., 2002. Monocyte/macrophage regulation of vascular calcification in vitro. Circulation. 105, 650655. Towler, D.A., 2013. Molecular and cellular aspects of calcific aortic valve disease. Circ. Res. 113, 198208. Townsend, N., Wilson, L., Bhatnagar, P., Wickramasinghe, K., Rayner, M., Nichols, M., 2016. Cardiovascular disease in Europe: epidemiological update 2016. Eur. Heart. J. pii: ehw334. Tyson, K.L., Reynolds, J.L., McNair, R., Zhang, Q., Weissberg, P.L., Shanahan, C.M., 2003. 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C H A P T E R

16 From Inflammation to Cancer: Opportunities for Chemoprevention via Dietary Intervention Jeong-Sang Lee1, Eun-Ji Lee2, Hye-Kyung Na3 and Young-Joon Surh2 1

Jeonju University, Jeonju, South Korea 2Seoul National University, Seoul, South Korea 3 Sungshin Women’s University, Seoul, South Korea

16.1. INTRODUCTION Cancer arises as a consequence of accumulation of mutations, especially oncogenes and tumor suppressor genes, and epigenetic alterations. In spite of substantial progress in chemotherapy and radiotherapy, cancer is still the leading cause of death worldwide. One of the rational and effective approaches to control cancer is chemoprevention, which is defined as the use of relatively nontoxic chemical substances of either natural or synthetic origin to impede, arrest or reverse carcinogenesis (Sporn, 1976; Sporn et al., 1976). The successful implementation of chemoprevention depends on a mechanistic understanding of carcinogenesis at the molecular, cellular and tissue levels. An important component of chemoprevention is to design and test new agents that act on specific molecular and cellular targets (Sporn and Suh, 2002). Notably, inflammation has been implicated in each stage of carcinogenesis, and is considered as an important target for cancer chemoprevention. A broad spectrum of anti-inflammatory substances, particularly those regulating aberrant inflammatory signaling, has been identified as potential candidates for chemopreventive agents (Chun and Surh, 2004).

16.2. INFLAMMATION AND CANCER Inflammation may result from microbial infection or noninfectious physical/chemical insults. It is a part of the response to tissue injury and comprises multifactorial networks of chemical signals initiated and maintained in order to heal the afflicted tissue (Coussens and Werb, 2002). A part of the complex inflammation cascade is the activation and directed recruitment of leukocytes (neutrophils, monocytes, and eosinophils) to the sites of tissue damage. In this context, the inflammatory response is similar to a wound-healing process in many aspects. Tumor growth is often considered as a wound that does not heal; thus tumor tissue can be thought of as a case of inflammation (Dvorak, 1986). In general, physiological inflammation is self-limiting as seen in wound healing. In contrast, chronic inflammation results from a persistent production of the proinflammatory factors or failure of mechanisms required for resolving the inflammatory response (Coussens and Werb, 2002). The functional or causal relationship between inflammation and cancer has been suspected for a long time. In 1863, Virchow found the presence of leukocytes in malignant tissues and hypothesized that tumors might arise from sites of chronic inflammation (Balkwill and Mantovani, 2001). Tumor cells produce various cytokines and chemokines that attract leukocytes, such as neutrophils, macrophages, dendritic cells, mast cells, and lymphocytes. Hence, an inflammatory component of a developing neoplasm may

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include a diverse leukocyte population (Coussens and Werb, 2002). The inflammatory tumor microenvironment enhances cancer cell proliferation, survival, migration, and angiogenesis (Greten et al., 2004). Persistent inflammation enhances cellular turnover, especially in the epithelium, and provides a selection pressure that results in the emergence of cells prone to undergo malignant transformation. Cytokines, chemokines, reactive oxygen species (ROS), and growth factors modulate microbial populations that colonize the host. Thus, therapeutic opportunities exist to target the causative microbes, the production of inflammatory mediators, and consequent epithelial cell responses. Such approaches could be of value in order to reduce the risk of inflammation-associated malignancies. Evidence for the implication of inflammation in neoplastic progression comes mainly from studies with nonsteroidal anti-inflammatory drugs (NSAIDs). NSAIDs, such as indomethacin and sulindac, have been shown to inhibit malignant transformation in many animal models and cultured cells. A number of population-based studies that prolonged use of these drugs reduces a colon cancer risk by 40%50%, and can also be preventive for other malignancies (Baron and Sandler, 2000; DuBois and Smalley, 1996; Garcia-Rodriguez and Huerta-Alvarez, 2001; Winde et al., 1997). Although a wide array of proinflammatory mediators have been identified as potential factors modulating carcinogenesis, the molecular and cellular mechanisms linking inflammation and cancer remain poorly understood. The following section will address inappropriate activation of some proinflammatory signal transduction pathways involved in promoting malignant transformation, with particular focus on cyclooxygenase-2 (COX-2) and its regulators.

16.3. COX-2 AS A KEY PLAYER IN THE PATHOGENESIS OF INFLAMMATION-ASSOCIATED CANCER COX or prostaglandin H2 synthase, is a key enzyme that catalyzes the rate-limiting step in prostaglandins (PGs) biosynthesis from the substrate arachidonic acid (Wotherspoon et al., 1991). COX exists in at least two isoforms, designated as COX-1 and COX-2. COX-1 is a housekeeping enzyme, being constitutively expressed in almost all mammalian tissues, and mediates physiological responses, such as cytoprotection of the stomach, regulation of renal blood flow and platelet aggregation. In contrast, COX-2 is barely detectable under normal physiological conditions. Like other early-response gene products, COX-2 can be induced rapidly and transiently by proinflammatory mediators and mitogenic stimuli including cytokines, endotoxins, growth factors, and oncogenes. The COX-2-derived PGs, such as PGE2, PGF2α, PGD2, and PGI2, are synthesized in a broad spectrum of tissue types and act as key mediators of inflammatory responses (Williams et al., 1999). PGs also play critical roles in carcinogenesis, and elevated levels of distinct PGs have been observed in various types of human cancers (Vanderveen et al., 1986; Verma et al., 1980). PGs induce ornithine decarboxylase activity, which is known as a hallmark of tumor promotion (Verma et al., 1980). Of the PGs produced by induced COX-2 activity, PGE2 has been shown to play a most prominent role in carcinogenesis. Exposure of various cancer cells to exogenous PGE2 enhanced tumor cell proliferation (Bortuzzo et al., 1996; Castellone et al., 2005; Qiao et al., 1995). Extensive clinical and experimental studies conducted over the last few decades have provided convincing data that link COX-2 with tumorigenesis. Inappropriate upregulation of COX-2 has been implicated in the pathogenesis of various types of malignancies, such as head and neck cancer, urinary bladder cancer, skin cancer, colorectal carcinoma, gastrointestinal cancer, and pancreatic cancer (Chan et al., 1999; Grubbs et al., 2000; Pentland et al., 1999; van Rees et al., 2002; Yip-Schneider et al., 2000). Moreover, COX-2 overexpressing transgenic mice are highly prone to develop tumors (Liu et al., 2001; Neufang et al., 2001), while COX-2 knock-out animals are less susceptible to experimentally-induced tumorigenesis (Chulada et al., 2000; Oshima et al., 1996; Tiano et al., 2002). Transgenic mice overexpressing COX-2 in mammary glands, skin or stomach developed tumors in these organs to a greater extent than did the wild-type mice (Muller-Decker et al., 2002; Neufang et al., 2001; Oshima et al., 2004). In contrast, genetic ablation of COX-2 suppressed the intestinal carcinogenesis (Oshima et al., 1996). Either administration of selective COX-2 inhibitors or the functional inactivation of the COX-2 in ApcΔ716 knockout mice, a murine model of human adenomatous polyposis, reduced both the number and the size of intestinal polyps (Oshima et al., 1996, 2001). Based on these findings, it is conceivable that targeted inhibition of abnormal upregulation of COX-2 is an effective and promising strategy for cancer chemoprevention (Subbaramaiah et al., 1997).

16.4. TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION OF COX-2 EXPRESSION Although the precise molecular mechanism underlying COX-2 expression is not fully elucidated, the roles of aberrant activation of intracellular signaling mediated via serine-threonine kinases, such as mitogen-activated IV. IMMUNITY AND INFLAMMATION: COMMON THREADS OF MULTIFACTORIAL DISEASES

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protein (MAP) kinases, protein kinase C (PKC), phosphatidyl-inositol-3-kinases (PI3K), and Akt/protein kinase B (PKB) and the down-stream transcription factors in the induction of COX-2 have been documented (Surh, 2003). Overexpression of COX-2 appears to be a consequence of both increased transcription and enhanced mRNA stability (Dannenberg et al., 2001; Shao et al., 2000). Oncogenes, tumor suppressor genes, growth factors, proinflammatory cytokines, chemotherapy, and tumor promoters stimulate COX-2 transcription via PKC and Rasmediated signaling (Smith et al., 2000). The expression of COX-2 is regulated by a distinct set of transcription factors, depending on the stimulus and the cell type. In general, the promoter region of the COX-2 gene contains a TATA box and various putative transcriptional regulatory elements, such as nuclear factor-κB (NF-κB), activator protein-1, CCAAT/enhancer binding protein, cyclic AMP response element binding protein, etc. Therefore, activation of aforementioned transcription factors, either alone or in combination, results in an elevated COX-2 expression (Smith et al., 2000; Subbaramaiah et al., 2002a). Although many factors enhance COX-2 transcription, much less is known about negative modulators. One such modulator is the tumor suppressor protein p53. Overexpression of wild-type p53 markedly suppressed transcription of COX-2 (Subbaramaiah et al., 1999). According to this study, p53 appears to inhibit COX-2 transcription by competing with TATA-binding protein for binding to the TATA box (Subbaramaiah et al., 1999). Several studies with human tumors highlight the importance of p53 status as a determinant of COX-2 levels (Ristimaki et al., 2002). For instance, COX-2 levels are higher in cancers of the esophagus, stomach, lung and breast that express mutant rather than wild-type p53 (Ristimaki et al., 2002). These findings suggest that the balance between activation of oncogenes and inactivation of tumor suppressor genes affects expression of COX-2. In addition, other modifications to the COX2 gene, such as hypermethylation of the COX-2 gene promoter, can alter transcription, and thereby affect levels of COX-2 in human gastric carcinoma cell lines (Song et al., 2001). There is also a growing body of evidence that posttranscriptional mechanisms determine COX-2 levels in neoplastic tissues. The 30 -untranslated region (UTR) of COX-2 mRNA contains a series of Shaw-Kamen sequences (AUUUA, also known as AU-enriched elements) that confer mRNA instability (Zhang et al., 2000). Oncogenes, cytokines, growth factors and tumor promoters induce COX-2 expression by enhancing mRNA stability in addition to stimulating transcription (Zhang et al., 2000). In human colon cancer, overexpression of COX-2 is a consequence of both increased transcription and decreased mRNA turnover (Dixon et al., 2001). Augmented binding of HuR, an RNA binding protein, to the Shaw-Kamen sequences of COX-2 is responsible, at least in part, for an observed increase in mRNA stability in colon cancer (Dixon et al., 2001). MicroRNAs have emerged as important posttranscriptional regulators in many cellular processes. mi-RNAs are a class of small noncoding single RNAs that regulate the translation of their target genes. Recently, mi-RNAs have been revisited as they have an important role in linking inflammation and cancer (Schetter et al., 2010). A small change of specific mi-RNA expression can provoke a dramatic change in inflammation (Baek et al., 2008; Bhaumik et al., 2009; Selbach et al., 2008). Some mi-RNAs have been found to be involved in the posttranscriptional regulation of COX-2 expression. For instance, miR-101a and miR-199a suppress COX-2 expression by binding to the COX-2 30 UTR in a murine model of endometrial cancer (Chakrabarty et al., 2007). Such mi-RNAmediated posttranscriptional regulation of COX-2 expression has also been identified in human colon cancer cells, tissue, liver metastases (Strillacci et al., 2009), and serous carcinoma (Hiroki et al., 2010).

16.5. INTRACELLULAR SIGNALING CASCADES IN ABERRANT COX-2 INDUCTION Protein kinases are enzymes that covalently attach phosphate to the side chain of either serine/threonine or tyrosine residue of specific proteins. Such phosphorylation of proteins can control the enzymatic activity, the interaction with other proteins and molecules, the localization in the cell, and the propensity for degradation by proteases. The MAP kinase signaling pathway consists of three distinct groups of well-characterized serine-threonine protein kinases that include extracellular signal-regulated protein kinases 1/2 (ERK1/2), c-Jun NH2-terminal kinase/stress-activated protein kinase and p38 MAP kinase. Intracellular proinflammatory responses are mediated via the activation of one or more members of MAP kinase family proteins. The activated form of each of the above MAP kinases amplifies the signal cascades through phosphorylation of down-stream molecules that include IκB, activating transcription factor-2, Elk, mitogen and stress-activated kinase (Kang et al., 2005) and transcription factors, thereby altering the expression of target genes including COX-2 (Chun and Surh, 2004; Deak et al., 1998). MAP kinases phosphorylate specific serines and threonines of target protein substrates and regulate the cellular activities ranging from gene expression, mitosis, movement, metabolism, and programmed death (Cobb, 1999). The phosphorylation of substrate proteins by MAP kinases functions as a switch to turn on or off the activity of the substrate protein. Protein phosphatases remove the phosphates that were transferred to the IV. IMMUNITY AND INFLAMMATION: COMMON THREADS OF MULTIFACTORIAL DISEASES

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protein substrate by the MAP kinases (Johnson and Lapadat, 2002). The action of MAP kinases and protein phosphatases reciprocally and rapidly alters the behavior of cells as they respond to changes in their environment. It has been shown that MAP kinases are important regulators in the signaling pathways leading to proto-oncogene expression (Cobb, 1999; Pages et al., 2000; Thomson et al., 1999). Some activated MAP kinases may translocate to the nucleus, where they phosphorylate the target proteins (Coso et al., 1995). Several MAP kinase family members play a role in chemical carcinogen-induced cox-2 gene expression (Arbabi et al., 2000; Nomura et al., 2000; Yang et al., 2000). Interestingly, activation of ERK1/2 and p38 MAP kinase stabilizes COX-2 mRNA in addition to stimulating transcription (Zhang et al., 2000). In a variety of cells treated with different inducing agents, specific inhibitors of p38 MAP kinase block the accumulation of COX-2 mRNA (Pouliot et al., 1997; Reiser et al., 1998). It has been demonstrated that 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression in mouse skin is mediated via activation of either ERK or p38 MAP kinase (Chun and Surh, 2004; Chun et al., 2003, 2004). Besides MAP kinase signaling cascades, other upstream kinases also contribute to an aberrant expression of COX-2. Pharmacological inhibition of either ERK or PI3K/Akt or expression of a dominant negative Akt partially blocked the K-Ras-induced expression of COX-2 (Zhang et al., 2000). A regulatory role of PI3K/Akt signaling in aberrant expression of COX-2 and PGE2 synthesis in endometrial cancer cells was also reported (St-Germain et al., 2004). In this study, two endometrial cancer cells (RL 952 and Ishikawa) expressing mutated PTEN exhibited elevated levels of COX-2 protein and its mRNA transcript as compared to cells expressing wild-type PTEN (HEC 1-A). Compared to the nontumorigenic keratinocytes (HaCaT), human skin cancer (HSC-5) cells were found to exhibit not only elevated levels PI3K activity but also COX-2 protein expression (Takeda et al., 2004). In contrast, interleukin (IL)-1β-induced expression of COX-2 protein and its mRNA transcript in human colon cancer (HT-29) cells was mediated via the MAP kinase-NF-κB axis, but not by the PI3K-mediated pathway (Liu et al., 2003). The induction of COX-2 in rat intestinal epithelial (IEC-6) cells transfected with an inducible K-RasVal12 cDNA was mediated via activation of both ERK and Akt/PKB (Sheng et al., 2001). In another study, treatment of PTEN-mutated RL 952 and Ishikawa cells with wortmanin and LY294002, specific inhibitors of PI3K-Akt signaling, downregulated IκBα phosphorylation, reduced NF-κB activity and decreased COX-2 expression (St-Germain et al., 2004). The persistent activation of PKCs also mediates inappropriate induction of NF-κB activation and subsequently COX-2 expression. For instance, induction of NF-κB activity in tumor necrosis factor (TNF-α)-treated human pulmonary epithelial (A549) cells was mediated by PKCε (Catley et al., 2004). PKC-mediated NF-κB activation was further supported by the observation that in PKCζ-deficient cells, NF-κB was transcriptionally inactive, and the phosphorylation of the RelA/p65 subunit of NF-κB in response to TNF-α was severely impaired (Duran et al., 2003). Huang and colleagues also demonstrated that the TNF-α-induced COX-2 promoter activity in human lung epithelial (NCI-H292) cells was attenuated by dominant-negative mutation of upstream kinases including PKCα, NF-κB-inducing kinase (NIK), and IκB knase (IKK)α/β (Huang et al., 2003). Furthermore, the induction of epidermal COX-2 expression and PGE2 synthesis was increased significantly in K5-PKCα transgenic mice treated with TPA as compared to wild-type mice (Wang et al., 2001). In addition to the aforementioned serine-threonine kinases, aberrant activation of cytosolic tyrosine kinases contributes to COX-2 overexpression. The TNF-α-induced COX-2 expression and PGE2 production in NCI-H292 cells were associated with activation of NF-κB via upstream protein tyrosine kinase, PKCα, NIK and IKK1/2 (Chen et al., 2000). Simultaneous treatment of human gingival fibroblast cells with extracellular growth factor (EGF) and IL-1β resulted in an enhanced COX-2 mRNA levels and synergistic stimulation of PGE2 biosynthesis, which was diminished by tyrosine kinase inhibitors, herbimycin A and PD 153035 hydrochloride (Yucel-Lindberg et al., 1999). IL-1β-induced COX-2 expression and PGE2 production in A549 cells were also inhibited by tyrosine kinase inhibitors, tyrphostin or erbstatin (Akarasereenont and Thiemermann, 1996). Genistein, a soy isoflavone with tyrosine kinase inhibitory activity, diminished-induced COX-2 expression and PGF2α production in J774.2 macrophages (Akarasereenont et al., 1994). Activated PKC and Ras signaling pathways were shown to regulate COX-2 expression at the transcriptional level (Marks et al., 2000). Moreover, Ras signaling was found to be involved in elevated COX-2 expression in HER-2/neu-positive cultured human mammary epithelial cells (184B5/HER) as compared to HER-2/neu-negative (184B5) cells (Subbaramaiah et al., 2002b).

16.6. THE ONCOGENIC POTENTIAL OF COX-2-DRIVED PGE2 AND ITS METABOLIC INACTIVATION As mentioned earlier, PGE2 is an inflammatory lipid mediator produced as one of the major products of the arachidonic acid metabolism catalyzed by COX-2 during inflammation (Nakanishi and Rosenberg, 2013).

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Production of PGE2 increases upon inflammatory insults, and its levels are abnormally elevated in various human malignancies (Rigas et al., 1993; Wang and Dubois, 2010) including breast cancer (Schrey and Patel, 1995). There are multiple lines of compelling evidence to support that COX-2-derived PGE2 plays a key role in inflammation-associated cancer progression (Marnett and DuBois, 2002). PGE2 exerts its function by binding to specific cell surface receptors, called EP receptors (Hull et al., 2004; Reader et al., 2011). As schematically illustrated in Fig. 16.1, PGE2 induces activation of EP receptors, leading to acceleration of cancer progression through modulation of several intracellular signaling pathways (Greenhough et al., 2009; Legler et al., 2010; Wang et al., 2007). Thus, PGE2 interaction with one of its receptors, EP4 activated EGF receptor signaling during gastric tumorigenesis (Oshima et al., 2011). While overproduction of PGE2 stimulates proliferation of various cancer cells, confers resistance to apoptosis of cancerous or transformed cells, and accelerates metastasis and angiogenesis, excess PGE2 undergoes metabolic inactivation which is catalyzed by NAD1-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) (Fig. 16.1). In this context, 15-PGDH has been speculated as a physiological antagonist of COX-2 and a tumor suppressor (Backlund et al., 2005). Thus, overexpression of 15-PGDH protects against experimentally-induced carcinogenesis and renders the cancerous or transformed cells susceptible to apoptosis by counteracting oncogenic action of PGE2 (Ding et al., 2005; Tai, 2011). Silencing of 15-PGDH is observed in some cancer cells, which is associated with epigenetic modification, such as DNA methylation and histone deacetylation, in the promoter region of 15-PGDH (Backlund et al., 2008; Wolf et al., 2006). A variety of compounds capable of inducing the expression of 15-PGDH have been reported, which include the histone deacetylase inhibitors, NSAIDs, and peroxisome proliferator-activated receptor-gamma agonists (Na et al., 2011). Therefore, 15-PGDH may hence be considered as a novel molecular target for cancer chemoprevention and therapy.

FIGURE 16.1 Formation, metabolism and oncogenic function of PGE2. Arachidonic acid (AA) released from the membrane bound phospholipid by phospholipase A2 (PLA2) activity is converted to PGE2 by COX-2. PGE2 is transported out of and into cells by multiple drug resistance-associated protein 4 (MRP4). Extracellular PGE2 interacts with a particular EP receptor and triggers various signal transduction pathways leading to cancer promotion and progression. PGE2 is further metabolized by 15-PGDH to 15-keto PGE2 that counteracts oncogenic functions of PGE2.

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16.7. NUTRITIONAL/DIETARY MANIPULATION OF ABERRANT INFLAMMATORY SIGNALING IN THE MANAGEMENT OF CANCER Significant clinical and experimental research conducted over the last few decades suggests that dietary constituents can prevent multiple forms of cancer. The rapid progress in our understanding of the cellular signal transduction pathways has paved the way to elucidating the molecular mechanisms of cancer prevention by dietary constituents. Since the cellular signaling network often goes awry in various disease processes, including cancer, it is fairly rational to target intracellular signaling cascades for achieving chemoprevention. Research directed toward elucidating the underlying molecular mechanisms of chemoprevention by dietary phytochemicals has recognized components of signal transduction pathways as potential targets (Chun and Surh, 2004; Kundu and Surh, 2005; Surh and Kundu, 2005; Surh et al., 2004, 2005). Among the ever-increasing list of dietary chemopreventive agents, anti-inflammatory phytochemicals are of particular interest as inflammation is closely associated with carcinogenesis. The chemopreventive potential of the majority of anti-inflammatory dietary phytochemicals has been attributable to their inhibition of abnormally elevated COX-2 expression/activity (Surh and Kundu, 2007). However, COX-2 is involved in production of not only oncogenic PGE2, but also some anti-inflammatory and inflammation resolving substances, particularly those derived from omega-3 polyunsaturated fatty acids (Groeger et al., 2010). Considering dual functions of COX-2, induced expression/activity of 15-PGDH, an enzyme that partly counteracts COX-2 action by converting PGE2 into nononcogenic metabolite, 15-keto PGE2 may be a more rational strategy for the chemoprevention of inflammation-associated carcinogenesis. The identification of dietary components that can potentiate the 15-PGDH expression/activity merits further investigation.

Acknowledgments This study was partially supported by the National Research Foundation of Korea (KRF) grant funded by the Korean government (Ministry of Science, ICT and Future Planning) (No. 2011-0030001 awarded to Y.-J. Surh and No. 2015R1C1A1A01053520) to J.-S. Lee).

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17 Inflammation in Bullous Pemphigoid, a Skin Autoimmune Disease Frank Antonicelli, Se´bastien Le Jan, Julie Ple´e and Philippe Bernard University of Champagne-Ardenne, Reims, France

17.1. CLINICAL AND BIOLOGICAL ASPECTS OF BULLOUS PEMPHIGOID Bullous pemphigoid (BP) is a rare autoimmune dermatosis that belongs to the pemphigoid group of diseases. This group of diseases also includes pemphigoid gestationis, mucous membrane pemphigoid, linear IgA disease as well as a number of rare disorders, which are all characterized by skin subepidermal blistering due to the disruption of the dermal-epidermal junction induced by the binding of autoantibodies directed against the components of the basal membrane zone. BP is not only the most common autoimmune subepidermal blistering disease within this group, but also represents the most frequent autoimmune blistering disease in general, including in European countries (Di Zenzo et al., 2007) and in France (Joly et al., 2012). BP is an acquired autoimmune disease that typically affects the elderly, with an onset after 60 years of age, although it can occur at any age, even during childhood. The relative risk for patients over 90 years of age appears to be about 300-fold higher than for those 60 years of age or younger, with roughly an equal occurrence in men and in women (Marazza et al., 2009). Clinically, BP is characterized by the occurrence of tense blisters of variable sizes that erupt most of the time on an erythematous urticarial skin, with a predilection for the flexural areas of the skin. The most commonly concerned areas are the inner thighs and upper arms, but any part of the skin surface can be involved such as the trunk and extremities, which also are frequently affected. However, in some patients, urticarial lesions are the sole manifestation of the disease although it is still not understood why the disease progresses in some patients and not in others (Illustrated in Fig. 18.1). The cause leading to immune skin dysregulation in BP is not well understood, apart from some medicinal induction of the disease. However, the first step of BP is characterized by the generation of autoantibodies on which the disease progresses, whether it is a mild or a severe disease. Following the autoimmune trigger(s), the mechanisms downstream to the breakdown of immune tolerance have been at least in part elucidated. BP is an immune mediated disease associated with both a humoral and a cellular response directed against the BP antigen 180 (BP180, BPAG2, or type XVII collagen) and the BP antigen 230 (BP230 or BPAG1), which are both components of a junctional adhesion complex called hemidesmosome. In physiological conditions, these two proteins assure the adhesion of the basal layer of keratinocytes to the dermis, creating the dermal-epidermal junction. However, in BP pathophysiological conditions, the generation of circulating and tissue-bound autoantibodies against these hemidesmosomal proteins is thought to be the critical event on which the dermal-epidermal junction disrupts (Illustrated in Fig. 18.2) (Giudice et al., 1992; Stanley et al., 1988). Both BP180 and BP230 have been cloned and characterized at the molecular level. The cloning of these proteins revealed that BP180 and BP230 are the products of distinct and unrelated genes (Giudice et al., 1992; Stanley et al., 1988; Diaz et al., 1990). Also, the cellular localizations of these proteins are distinct. While BP180 is a transmembrane protein with a large collagenous extracellular domain, BP230 is a cytoplasmic protein belonging to the plakin family. Structural analysis showed that BP230 is predicted to contain a central coil-coiled domain region flanked by two globular end domains (Tanaka et al., 1991; Sawamura et al., 1991; Green et al., 1992). In contrast, BP180 is a type II orientation transmembrane protein. The amino-terminal region of BP180 localizes to the intracellular hemidesmosomal Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00017-2

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FIGURE 18.1 Bullous pemphigoid. (A) Erythematous urticarial lesions. (B) Blister arising on inflammatory skin.

FIGURE 18.2 Proposed inflammatory mechanism of subepidermal blister formation induced by BP180 autoantibody binding.

plaque, and its carboxy-terminal portion projects into the extracellular milieu constituting the dermal-epidermal junction. The extracellular region of BP180 contains 15 peptide segments with an amino acid sequence characteristic of the collagenous protein family (Giudice et al., 1991; Li et al., 1993). Therefore, the ectodomain is structurally composed of 15 collagen domains separated one from another by noncollagenous sequences, the largest and last noncollagenous domain (NC16A) of BP180 being located within the upper lamina lucida immediately subjacent to the keratinocyte cellular membrane, while the first noncollagenous is positioned at the C-terminal region of BP180 and colocalizes within the anchoring filaments of the basal membrane. As a transmembrane protein, the extracellular part of BP180 makes it a preferred antigenic target in BP, and most of BP patients display in their serum autoantibodies that bind to the NC16A domain of BP180 (Giudice et al., 1993). Nevertheless, additional antigenic sites exist all along BP180 sequence, both on the extracellular and the intracellular domains. Autoantibodies directed against these additional regions of BP180 are detected in up to 40% of the BP sera, and autoantibodies against the intracellular BP230 antigen are found in approximately 60% of BP patient sera (Giudice et al., 1993, 1994; Zillikens et al., 1997; Perriard et al., 1999). Of note, the spectrum of clinical BP presentations varies according to the autoantibody titers. Mild disease may display only excoriated, eczematous or urticarial lesions (either localized or generalized), while more intense pruritic eruptions with widespread blister formation are representative of disease progression. Nevertheless, in severe as in mild diseases, skin inflammation seems to play a critical role, although the pathophysiological mechanisms involved must at least partly differ in pure erythematous/pruritic skin forms and in skin with tense blisters.

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In both mild and severe diseases, systemic corticosteroids represent the most endorsed treatment. For patients with generalized disease, a regimen of oral prednisone at a dose of 0.51 mg/kg/day usually controls the disease within 1 or 2 weeks. This dose is then progressively tapered down over a period of 69 months, or occasionally longer. However, prolonged administration of corticosteroids in the elderly is associated with significant side effects. Recent large controlled studies have emphasized the role of potent topical corticosteroids, which appeared to control even generalized BP with the same efficacy as oral corticosteroids, and, most importantly, with fewer systemic side effects (Joly et al., 2002, 2009). The use of immunosuppressive drugs is a matter of debate. Some clinicians prefer to use them only as second-line therapy, when corticosteroids alone fail to control the disease, or if the latter are contraindicated. Although the majority of patients finally go into clinical remission with treatment, the disease is often accompanied by significant morbidity with a profound impact on the quality of life, and poor general health related to old age is often associated with a poorer prognosis. Recently, the definition of a BP disease area index has been established to measure and assess disease extent, activity, severity, and therapeutic response (Murrell et al., 2012). Disease relapse when the treatment is tapered down highlights the need to better understand the pathophysiological process as the treatment options are limited in this patient, and in those with counter-indications/side-effects for the classical treatments. The following paragraphs are aimed at drawing up an inventory of the inflammatory molecules, and at characterizing their potential role in the pathophysiological processes associated to the BP disease.

17.2. FROM AUTOIMMUNITY TO INFLAMMATION AND BLISTER FORMATION Although blisters develop mainly on inflammatory skin, BP is first of all an autoimmune disease of which autoantibodies production is the critical step. Various animal models have provided strong evidence that autoantibodies against BP180 are pathogenic and trigger a blistering skin disease that closely mimics human BP. Pathogeny of BP180 autoantibodies were evidenced through several clinical and experimental observations (Kasperkiewicz et al., 2012). For instance, the autoantibodies BP180 NC16A serum titer is correlated with disease severity (Do¨pp et al., 2000; Haase et al., 1998); histological examination of skin biopsy specimen shows the presence of antibody-antigen complexes localized at the dermal-epidermal junction in the vicinity of tissue injury; in gestational pemphigoid, a disease closely related to BP, the transplacental transfer of autoantibodies against BP180 from the mother to the neonate can result in a transient bullous eruption; the use of in vivo neonatal and adult mouse models have provided strong evidence for the pathogenic role of autoantibodies in BP (Liu et al., 1993; Schulze et al., 2014), especially human autoantibodies against the NC16A domain are able to induce a blistering disorder that reproduced all the key features of BP in a humanized mouse model (Nishie et al., 2007); and finally the use of in vitro human skin to highlight key steps in the detachment of the epidermis from the dermis upon autoantibodies binding (Sitaru et al., 2002). All these studies and techniques helped in demonstrating that antibodies against the ectodomain of BP180 are pathophysiologically critical and contribute to tissue damage. Nevertheless, as mentioned above, some patients present neither blister nor erosive lesions. It was previously thought that absence of blisters corresponded to the early stage of the disease. However, it appears now that in some BP patients the disease will never progress to the bullous stage. In all cases, the primary event leading to the breakdown of tolerance and autoantibodies production is required and obviously involves lymphocytes recruitment and activation. BP disease has been associated with both a humoral and a cellular response. However, it was shown that blister formation relies more on cells from the innate immunity than from the adaptive immunity (Chen et al., 2002). Then, one can hypothesize that the autoantibodies-associated humoral response is required to establish the cornerstone on which both mild and severe diseases progress, while a supplementary step is required to involve the inflammatory-associated T-cell response in large blister formation that characterizes the severe forms of BP. The binding of anti-BP180 autoantibodies to their target antigen on keratinocytes triggers a signal transduction event that results in the secretion of IL-6 and IL-8 (Schmidt et al., 2001). Of note, IL-8 but not IL-6 expression is under the control of HSP90, a protein implicated in inflammatory responses suggesting that different signaling cascades are triggered upon autoantibodies binding (Tukaj et al., 2014). The release of these proinflammatory cytokines may keep the local inflammatory process on. Furthermore, it was shown that the binding of autoantibodies conducted to the depletion of BP180 from the keratinocytes surface (Iwata et al., 2009). Decrease in the expression of this hemidesmosomal antigen upon the humoral immune response is associated with a weakening of cellmatrix adhesion properties in the skin, and then could be responsible for the first clinical signs of BP observed in patients with pruritic/erythematous skin lesions.

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The bullous stage of BP is characterized by the development of vesicles and bullae mostly on erythematous skin together with urticarial and infiltrated papules and plaques. The blisters are tense, up to 14 cm in diameter, contain a clear fluid with an inflammatory infiltrate, and may persist for several days, leaving eroded and crusted areas. As noted above, BP severity varies according to the autoantibody titers. Then, one can imagine, that when reaching a certain limit, reduced skin barrier immunity resulting from autoantibody-induced skin fragility may give rise to further inflammatory triggers that may account for subepidermal blister formation. Indeed, an enhanced level of IL-6/IL-8 release upon high immune complexes formation associated or not with such supplemental triggers may result in a cascade of events that activate the inflammatory T-cell response. Such cascade involves complement activation and the recruitment of inflammatory cells that release even more inflammatory mediators responsible for the separation of the dermis from the epidermis. The use of mice deficient in different inflammatory cell types highlighted that mast cells, macrophages, and neutrophils play a direct role in subepidermal blistering in experimental BP (Chen et al., 2001). The classical pathway of the complement activation plays a major role in the development of the disease (Gammon et al., 1981), namely through the generation of C5a that in turn activates mast cells (Chen et al., 2001). Following autoantibody binding to keratinocytes, both complement activation and mast cells degranulation are critical to disease progression in their roles of neutrophil recruitment (Chen et al., 2001; Borrego et al., 1996; Iwata and Kitajima, 2013; Zebrowska et al., 2014). Interestingly, autoantibodies from BP patients predominantly belong to the noncomplement fixing IgG4 subclass, while autoantibodies of the IgG1 subclass are only present at lower concentrations (Bird et al., 1986; Bernard et al., 1990; Skaria et al., 2000). Then, it would be of interest in the future to analyze variations of the IgG1/IgG4 ratio in patients with mild and severe diseases. Upon activation, mast cells can produce a variety of mediators that induce an inflammatory cascade directly associated with the recruitment and activation of innate immune cells at the skin lesional site. The subsequent local cytokine storm may further activate both infiltrated and resident cells by an autocrine/paracrine signaling, leading to an amplified inflammatory spreading response that could participate to disease severity. Then, the exploration of the cytokines content in both serum and blister fluids from BP patients during the evolution of pathological conditions could represent a valuable approach in understanding BP progression. Mast cells produce a variety of cytokines in BP blister fluids such as interleukins (ILs), IL-1, IL-2, IL-5, IL-6, and TNF-α that have been linked directly or indirectly to neutrophil influx (Galli et al., 1991; Baba et al., 1976; Takiguchi et al., 1989; Endo et al., 1992; Schmidt et al., 1996a,b). Of note, no significant differences in the sera of BP patients versus those of normal subjects were reported for IL-1β, while a very elevated concentration of this cytokine was observed at the skin lesional sites, suggesting a local synthesis for this cytokine (Ameglio et al., 1998; Schmidt et al., 1996c). The possible pathogenic role for IL-1β in BP was then emphasized by the demonstration of a correlation between IL-1β and the number of skin lesions (Ameglio et al., 1998). Of note, a large increase in IL-1receptor antagonist, a competitor of IL-1 for the IL-1 receptor in the serum and in the blister fluid of BP patient was reported, suggesting that feedback mechanisms could occur to control the effects of this cytokine in BP disease progression (Schmidt et al., 1996c). Such inflammatory control could be reinforced by the secretion of other anti-inflammatory cytokine such as IL-10. Indeed, high IL-10 concentration has been described in the blister fluid of BP patients as compared to healthy sera (Schmidt et al., 1996b; Ameglio et al., 1998; D’Auria et al., 1999). Other anti-inflammatory cytokines could also participate to this regulation. Previous studies showed that concentration of TGFβ-1 was similar in the serum of BP patients and control, while low levels were detected in the blister fluid (Ameglio et al., 1998; Giacalone et al., 1998). Interestingly, TGFβ-1 was shown to increase the expression of both BP antigens in keratinocytes suggesting that in situ expression of this cytokine could limit the autoantibodies-induced BP180 depletion at the surface of keratinocyte (Sollberg et al., 1992). It would also be of interest to know whether such controls could constitute another level, discriminating patients with mild disease from those with disease on progress. Besides these potential feedback controls, cytokine expression may amplify the inflammatory response by recruiting polymorphonuclear cells at the site of lesion. Actually, the above-mentioned increased IL-8 release by keratinocytes upon binding of BP180 autoantibodies (Schmidt et al., 2001) is also a well-known chemoattractant for neutrophils. Furthermore, it has been largely demonstrated that along with other proinflammatory cytokines such as TNF-alpha and IL-6, increased IL-8 concentration may arise from both tissue resident cells and infiltrated cells upon IL-1β stimulation, therefore leading to an amplification of inflammatory cell recruitment. This fits with the fact that in situ IL-8 concentration correlated with the number of lesions (Ameglio et al., 1998). In this setting, a higher concentration of IL-8 was shown in the blister fluid of BP patients as compared to BP serum, which was even higher than in control serum (Ameglio et al., 1998; Inaoki and Takehara, 1998; Schmidt et al., 1996a). Conversely to IL-8, RANTES, a chemokine of the CC subgroup that chemoattracts and degranulates

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eosinophils, displays blister fluid concentration inferior to those observed in serum (D’Auria et al., 1998a). However, despite its low concentration in the blister fluid, RANTES expression has been significantly correlated with IL-5 concentration and with the number of lesions (D’Auria et al., 1998a). This is of interest as IL-5 is a potent eosinophil differentiation and activation factor, whose participation in the blistering processes has been well documented (Borrego et al., 1996). Like most of the inflammatory mediators, elevated levels of IL-5 have been described in the blister fluid of patients with BP, relatively to BP sera, which is even higher that control sera (Ameglio et al., 1998; D’Auria et al., 1998b; Endo et al., 1992). Therefore, the IL-8—RANTES/IL-5 balance could be representative of the concentration of neutrophils and eosinophils in the blister fluids, which have been shown to vary according to disease progress. The direct correlation between disease severity and the number of infiltrating neutrophils (Liu et al., 1997) suggests that neutrophils are the main innate cells that directly cause tissue injury at the dermal-epidermal junction, leading to BP skin blisters. Activated neutrophils secrete proteolytic enzymes known to degrade the extracellular matrix. The two main enzymes involved in blister formation are the human leukocyte elastase (HLE) and the metalloproteinase 9 (MMP-9), as a deficit of either of these two enzymes blocks blister formation in mice (Liu et al., 1998, 2000a). HLE is the main protease involved in the separation of the dermal-epidermal junction (Liu et al., 2000a; Sta˚hle-Ba¨ckdahl et al., 1994). Besides, MMP-9 inactivates the α1-proteinase inhibitor, a physiological inhibitor of HLE, therefore leading to unmitigated HLE degradation activity of the extracellular matrix proteins (including BP180), and subsequently to the separation of dermis from the epidermis (Liu et al., 2000b) (Fig. 18.2). Noteworthy, the disruption of the basal membrane occurring precisely at the hemidesmosomal structure is quite amazing considering that the very localized dermal-epidermal junction degradation occurs irrespective of the large spectrum of substrate of these proteases, which degrade various extracellular matrix proteins besides BP180 (Verraes et al., 2001; Shimanovich et al., 2004; Sta˚hle-Ba¨ckdahl et al., 1994), and that several other proteolytic enzymes, including cathepsin G, plasminogen activators, plasmin, MMP-2 and MMP-13 have also been evidenced in the biopsy specimens of BP patients (Briggaman et al., 1984; Gissler et al., 1992; Kramer and Reinartz, 1993; Liu et al., 2005; Schmidt et al., 2004). Following polymorphonuclear activation, skin inflammation and tissue injury are amplified by the recruitment of additional neutrophils that release even more proinflammatory cytokines and proteases, resulting in local tissue damage of the dermal-epidermal junction. It was recently shown in a large release of IL-17 by neutrophils in the blister fluids of BP patients as compared to BP sera and controls (Le Jan et al., 2014). Along IL-17 secretion, the serum and blister fluid of these patients also displayed high levels of IL-22 and IL-23 (Le Jan et al., 2014; Ple´e et al., 2015). Both IL-17 and IL-23 enhanced MMP-9 secretion from several leukocyte cell types. Noteworthy, proteolytic degradation of molecules within this area also participates to the inflammatory mechanisms bound to blister formation. Indeed, it was shown that protease-induced matrix degradation generated the release of the Proline-Glycine-Proline (PGP) peptide, which further supply the inflammatory amplification loop through the recruitment and activation of neutrophils (Le Jan et al., 2014). On top of participating in the innate inflammatory response at site of lesion, the production of peptides from collagenous molecules, especially from BP180, has also been shown to interfere on the adaptive response associated to BP (see below).

17.3. FROM INFLAMMATION TO AUTOIMMUNITY AND THERAPEUTIC RESPONSE BP is a dermatosis of autoimmune origin, which clinically initiates the formation of tense blisters subsequently to the recruitment of inflammatory innate cells. In turn, the inflammatory mediators release by these infiltrated cells and by tissue resident cells may shape the quality of the adaptive autoimmune response. Both autoreactive T-cells and B-cells responses have been found in patients with BP. The humoral response is generally polyclonal directed against several epitopes of BP180 and BP230, although most of BP patients generate autoantibodies against the NC16A domain of BP180. These BP180 NC16A autoantibodies belong to the IgG1 and IgG4 as well as to the IgE subclasses (Do¨pp et al., 2000; Bernard et al., 1990; Laffitte et al., 2001; Nieboer and van Leeuwen, 1980; Messingham et al., 2014a; van Beek et al., 2015; Ujiie, 2015). Th1 type cytokines preferentially induce IgG1 and/or IgG3, while Th2 type cytokines preferentially induce IgG4 and IgE isotypes. In BP patients, both Th1 and Th2 cytokines types have been detected. For instance, concentration of IFN-γ, a Th1 cytokine, was found elevated in blister fluid but not in the serum, therefore arguing for a local regulation/orientation of the adaptive immune response towards a Th1 profile (Ameglio et al., 1998). Also, CXCL10, another Th1 cytokine, was shown to be increased in the serum of BP patients along with disease severity (Nakashima et al., 2007), and CXCL10 concentration is even higher at the site of lesion (unpublished data). Meanwhile, other studies found in the blister high

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concentration of eotaxin-1 and eotaxin-3 as well as IL-5 fluids, which favor the Th2 T-cell polarization (Wakugawa et al., 2000; Gounni Abdelilah et al., 2006; Gu¨nther et al., 2011), supporting that both Th1 and Th2 autoreactive lymphocytes are involved in the pathophysiological in situ processes associated to BP progression. Besides the Th1 and Th2 lineages, it has also been reported the expression of Th17 and Treg at the skin lesional site of BP (Arakawa et al., 2011). In this setting, IL-17 expression was demonstrated in the epidermis and in influxed cells in dermis (Zebrowska et al., 2013). However, it has to be noted that, although high concentrations of IL-6 and IL-23 were also demonstrated in the blister fluids, the presence of the Th-17 lineage could not be confirmed (Le Jan et al., 2014). Besides, as the number of Treg is reduced in BP (Arakawa et al., 2011; Antiga et al., 2014), IL-10 secretion is more likely to be expressed by the Th2 orientated lymphocytes than from these Treg cells. In setting with the importance of the Th2 lineage in BP, it has been recently showed an overexpression of IL-21 in the serum of BP patients, a cytokine produced by follicular T helper cells, which are a key modulator of B cell activation and autoantibody production (Li et al., 2013). The requirement of T-cell interaction to produce anti-BP180 NC16A IgG in vivo was confirmed by use of an inhibitor of CD40-CD40L interaction (Ujiie et al., 2012). Altogether this suggests that both Th1 and Th2 are the main T-cell lineages associated to BP, and that the locale inflammatory response participates to the shaping of the adaptive autoimmune response. Such reciprocal interplay could further be affected by different factors such as age of the patients, acute-onset of the disease, disease severity and tissue microenvironment. Blister formation is associated with matrix components proteolysis, and peptides issues from tissue degradation can directly affect T-cell orientation. Notably, it has been recently shown peptides sequences spanning the extracellular NC16a immunogenic region of BP180 shaped the specificity of the T-cell responses (Pickford et al., 2015). In setting with the pathophysiology observed in BP, a complex pattern of responsiveness was associated with these peptides, with heterogeneous T-cell responses to NC16a and its constituent peptides with only a tendency towards a Th2 orientation profile. Another study using overlapping peptides of the NC16A sequence showed IgG production with T-cells from BP patients but not from controls, suggesting that T-cells must have been educated, and therefore that the cytokine profile of BP serum could be of importance (Thoma-Uszynski et al., 2006). Although these studies were restrained to the NC16A domain to BP 180, many other fragments of both BP180 and BP230, as well as other proteolyzed matrix molecules during the active phase of BP might play a role both in the orientation of the autoimmune and of the inflammatory responses. The interest of analysis cytokines concentration in BP goes further in the scientifically deciphering of BP pathophysiological mechanisms. Indeed, measuring cytokines serum content could help in deciphering BP patients’ response to treatment. For instances, remission of the BP patients was associated with increased frequency of IL-10producing cells (Teraki et al., 2001). Also, biotherapies have shown their clinical relevance in many other inflammatory diseases either when patients are intolerant or nonsensitive to the reference treatment. In general, BP treatment is still based on systemic immunosuppression, and the increased mortality in these fragile people might be at least in part attributed to these immunosuppressive treatments (Ludwig et al., 2013). Actually, about 30% of BP patients have an insufficient therapeutic response to corticosteroid, which corresponds to the first line of treatment that has been grounded on clinical practice (Joly et al., 2002, 2009; Di Zenzo et al., 2004; Roujeau et al., 1984; Guillaume et al., 1993; Beissert et al., 2007). Furthermore, when used at high dose and for a long period of time, the use of corticosteroids in the elderly is associated with significant side effects. As BP is an autoimmune disease associated with both a humoral and a cellular response, the use of classical immunosuppressive drugs is a matter of debate. Some clinicians prefer to use them solely as second-line therapy when corticosteroids alone fail to control the disease, or if the latter are contraindicated, while others tend to use them as additional immunomodulatory therapeutic agents to the corticotherapy in BP patients with a difficult disease to control as in chronic inflammatory diseases such as psoriasis, Crohn’s disease or rheumatoid arthritis (Toosi and Bystryn, 2010). However, these immunosuppressants also have several side effects or contraindications in elderly patients (Du-Thanh et al., 2011). Nevertheless, corticosteroids are very effective therapy most of the time in controlling BP disease at the time of diagnosis. Thus, biotherapies may be potential candidates for maintenance therapy in BP patients by allowing the reduction of the cumulative doses of corticosteroids, limiting the deleterious associated side effects thereof. Therefore, based on all these pathophysiological studies, the identification of biologics that target cytokines involved in the cascade of events bound to disease progress may represent new therapeutic horizons for BP with a good satisfactory benefit/risk ratio (Ludwig et al., 2013; Ple´e et al., 2015). For instance, sustained or even enhanced level of IL-17 and IL-23 in the serum of BP patients during the first months of follow-up contributed to the prediction of a poor disease outcome (Ple´e et al., 2015). However, it still needs to be demonstrated whether targeting these cytokines could significantly improve the outcome of those BP patients. Besides cytokines, biologic targeting autoantibodies could also demonstrate benefits for BP patients. Indeed, a therapeutic trial in BP with omalizumab, an anti-IgE monoclonal antibody, resulted in substantial improvement in the disease (Messingham et al., 2014b).

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17.4. CONCLUSION Work performed over the last decades shows that BP progression is not driven by only one key molecule, and that a panel of inflammatory molecules participate in an intricate network. Actually, no cytokine is specific for one single disease and increased levels of different molecules may be found in practically all inflammatory diseases. This evidence presumes that differences in the characteristics of the lesions are more likely to be linked to the profile of cytokines, determined by specific causes inherent to the disease, and to their quantitative variations in association with disease progression. Furthermore, most of the cytokines are increased at the skin lesional level suggesting that tissue environment plays a critical role in the local synthesis. The inflammatory response is also present and highlighted by circulating cytokines in the serum of BP patients, which, besides reflecting tissue injury, could also participate in the priming of immune cells before their extravasation to the site of the lesion. In this way, the serum represents a practical means to follow disease progression and therefore to adapt therapy for patients nonsensitive to the classical corticosteroid treatment. Although many other investigations are still required to fully understand the pathophysiological inflammatory mechanisms associated to BP and propose new effective biotherapies, BP disease can be summed up as follows: an initial unknown trigger leading to hemidesomosomal breakdown tolerance and to autoantibodies generation against BP 180 and BP230. This autoimmune process generates a local inflammatory response associated with skin fragility, followed most of the time by the activation of an amplification inflammatory feedback loop through the activation of the complement cascade, the recruitment and the activation of inflammatory cells belonging to the innate immune response, that releases large amounts of cytokines and proteases. The proteases are responsible for direct tissue degradation and blister formation, while the panel of cytokine and of released peptides could be responsible for disease severity by recruiting and shaping both Th1 and Th2 lymphocytes. The Th1 associated cytokines could then maintain the adaptive cellular T-cell inflammatory phenotype favoring the innate associated inflammatory response, while the Th2-type cytokines could sustain the humoral response from which the inflammatory response is ignited.

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C H A P T E R

18 Inflammation in Systemic Immune Diseases: Role of TLR9 Signaling and the Resultant Oxidative Stress in Pathology of Lupus Chhanda Biswas Children’s Hospital of Philadelphia, Philadelphia, PA, United States

18.1. INTRODUCTION Innate immune system is the first-line host defense specified to confine infection in the early hours after exposure to the microbial infection (Hoffmann et al., 1999). The innate immune system evolved to discriminate infectious nonself from noninfectious self utilizing a number of germ-line encoded receptors (Janeway, 1992; Medzhitov and Janeway, 2000), among which Toll-like receptors (TLRs) are the most extensively studied. The surfaces of microorganisms including bacteria, viruses, fungi, protozoa, and parasites typically bear pathogenic molecular structure in the form of conserved and repeating patterns commonly known as pathogen associated molecular patterns (PAMPs) (Janeway and Medzhitov, 2002; Meylan et al., 2006). In 1989 Charles Janeway proposed the concept of pattern recognition receptors (PRRs) (Janeway, 1989; Medzhitov and Janeway, 1997b), because TLRs could recognize these patterns (PAMPS), and were specified to the family of PRRs (Armant and Fenton, 2002). Signal transmission by TLRs is central to the innate host defense against many pathogenic microorganisms (Janeway, 1992; Medzhitov and Janeway, 1997a). Later, Charles Janeway’s attribution to the concept of linking innate with adaptive immune responses (Medzhitov and Janeway, 1999) remained a pivotal conceptual discovery. In 1991, the name Toll was coined due to the serendipitous discovery of sequence homology between the cytosolic domains of TLRs with the cytosolic domain of Drosophila melanogaster protein Toll. Initially the only known function of Toll was to promote dorsoventral polarity in the developing D. melanogaster embryo (Gay and Keith, 1991). With the advent of more studies, the importance of TLR-PAMP interaction in mounting and modulating immune responses was increasingly established. Medzhitov et al. (1997). Stimulation of TLRs triggers activation of specific transcription factors culminating in the expression of specific cytokines along with costimulatory molecules. These events ensure pathogen clearance by recruiting various cells of the innate immune system and further by shaping pathogenspecific adaptive immunity (Akira et al., 2001). Mice lacking TLRs or adapter molecules associated with TLR signaling have severe defects in their ability to control certain pathogens, often resulting in death. Genome-wide association studies identified receptor polymorphisms in humans that are associated with increased susceptibility to infectious and autoimmune diseases (Medvedev, 2013; Takeda et al., 2003). Although TLR activation is important for host defense, an exaggerated innate immune response with very high circulating levels of pro-inflammatory cytokines characterized as cytokine storm can lead to systemic inflammation, multiorgan failure, and if not treated to the death of the host. In addition, chronic activation of TLRs can also lead to development of autoimmune diseases, such as systemic lupus erythematosus (SLE) and inflammatory bowel disease with a greater propensity in genetically predisposed individuals. There are thirteen TLRs identified so far, and therefore the complexity of TLR function lies in that it acts as a double-edged sword. On one hand, TLR activation is a prerequisite to mobilize protective immunity. On the other hand, if misregulated it results in a perturbed immune response. This culminates in an imbalance in anti-inflammatory and

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proinflammatory responses either inhibiting or promoting disease progression. Cytokine storm also termed as macrophage activation syndrome (MAS) exhibits a complex network of proinflammatory cytokines including interleukin-12 (IL-12) and interferon gamma (IFNγ) leading to systemic immunopathology. MAS is quite complex with a poorly understood etiology, but is thought to be due to an excessive interaction of CD81 T-cells with antigen presenting cells (APC) mounting a feed-forward loop of amplified inflammation. Although rare, MAS is a fatal complication in a number of rheumatic, oncologic, infectious, and genetic disorders, and activation of TLR9 has been incriminated in the manifestation MAS (Behrens et al., 2011; Weaver and Behrens, 2014). Self-tolerance, a property distinguished by the failure of innate immunity to recognize its own protein and antigens and therefore failure to mount an autoimmune response, is quite extraordinary in terms of self-preservation. However, more studies changed the understanding of the nature of pathogen recognition and signaling mechanism in innate defenses [see review (Mogensen, 2009)]. In 1963, earlier than the discovery of PRRs, at least three reports indicated that exposure to nucleic acids derived from pathogens or host was able to produce interferon (IFN) in fibroblasts (Jensen et al., 1963; Isaacs et al., 1963; Rotem et al., 1963). However, the receptors for nucleic acids were not obvious until these studies came into light (Alexopoulou et al., 2001; Diebold et al., 2004; Kumagai et al., 2008; Lee et al., 2008). It became clear that host immune receptors can sense both nonself -DNA and endogenous circulatory or cytoplasmic DNA as evidenced by the formation of auto-DNA-antibodies (Lamphier et al., 2006; Hornung and Latz, 2010). Therefore, TLRs, though conceptually are meant for exogenous pathogen interaction, also recognize self-antigens associated with danger (danger associated molecular patterns or DAMPs) (Matzinger, 1994). DAMPs, also known as alarmins, are released from cells undergoing necrosis or apoptosis. They are broadly specified to self-molecules, which could be of various types, but have high specificity for particular immune sensors for recognition (Jounai et al., 2012; Jounai et al., 2012). DAMP release from damaged cells occurs presumably in an inhospitable condition arising from excessive oxidative stress-driven production of reactive oxygen species (ROS). Increased production of ROS is well described to have originated from metabolic adaptation coupled to increased energy metabolism by mitochondrial oxidative phosphorylation. This process is associated with mitochondrial hyperpolarization accompanied by disruption of mitochondrial membrane potential, and if not restrained accounts for cellular damage. ROS-generated intermediates with their potent oxidation power perturb the constitutive property of cellular components including proteins, lipids and DNA. This perturbation in structure alters the functional output by these molecules resulting in disruption of cellular homeostasis, and leads to cellular damage by various cellular mechanisms including autophagy, apoptosis or necrosis (Tang et al., 2012). Thereby, the released cellular components/DAMPS in the circulation or within the cytoplasm, upon recognition by specific immune receptors—mostly by antigen presenting cells —are able to evoke systemic autoimmune responses. The autoimmune responses arising from reaction with DAMPs are often referred to as sterile infection because apparently there is no direct evidence of presence or interaction with exogenous microbial products. More recently, activation of TLR9 was highlighted in noninfectious insults such as ischemiareperfusion, and further validated by the use of COV08-0064, a TLR9 antagonist, that prevented ischemia reperfusion injury in nonsteatotic and steatotic mice livers (Shaker et al., 2016; Arumugam et al., 2009). Therefore, it is imperative that a tight regulation of TLR9 activation is central for optimal and protective immune responses. The determining factors for the optimal signal are various, starting from the type and level of pathogens, type of TLR stimulated (whether surface or intracellular TLR, single or more than one); nature of the adapter protein complexes; the specific way signals are transmitted, regulated and integrated at the level of allosteric assembly of TLRs; posttranslational modifications; and subcellular trafficking of the molecules of the signaling complexes. Since the discovery of TLRs, significant progresses have been made to understand the mechanisms underpinning the regulation of TLR signaling. These studies lead us to appreciate that in addition to genetic predisposition, epigenetic and environmental factors also influence TLR signaling. Systematic understanding of the effect of these factors on TLR-driven immune responses is critical for prognosis and therapeutic prevention of immune diseases. The aim of this chapter is to discuss the molecular mechanisms of regulation of TLRs and their role, TLR9 in particular, in immunomodulatory responses. The discussion is also extended to understand oxidative stressmediated signaling and their influences on TLR9 signaling in SLE pathogenesis.

18.2. TLR FAMILY TLRs because of their property to recognize pathogen-associated molecular motifs/patterns are also called pattern recognition receptors (PRRs). They are highly conserved from Drosophila to humans. These receptors are expressed in innate immune cells such as dendritic cells, macrophages and natural killer (NK) cells, in lymphocytes including

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TABLE 18.1 This Table Comprehensively Provides Information on Subcellular Localization of Each TLR, Its Ligand(s) and Microbial Sources, Whether Forms Homodimer or Heterodimer, and Specific Adaptor Molecules That it Recruits TLR

Cellular localization

Hetero- or homo-dimer Ligands

Microbial source

Adapter protein(s)

TLR1

Cell surface

TLR1/TLR2

Lipopeptides

Bacteria, mycobacteria

TIRAP/MyD88

TLR2

Cell surface

TLR2/TLR2 TLR2/TLR1 TLR2/TLR6

Zymosan Peptidoglycans Lipoteichoic acids Lipoarabinomannan Porins Envelope-glycoproteins

Fungi Gram-positive bacteria Gram-positive bacteria Mycobacteria Neisseria Viruses

TIRAP/MyD88

TLR3

Endolysosomal compartment

TLR3/TLR3

dsRNA

Viruses

TRIF

TLR4

Cell surface Endolysosomal compartment

TLR4/TLR4

LPS LPS

Gram-negative bacteria Gram-negative bacteria

TIRAP/MyD88 TRAM

TLR5

Cell surface

TLR5/ TLR5

Flagellin

Bacteria

MyD88

TLR6

Cell surface

TLR6/TLR2

Diacyl lipopeptides Lipoteichoic acid

Mycoplasma Gram-positive bacteria

TIRAP/MyD88

TLR7

Endolysosomal compartment

TLR7/TLR8

ssRNA, dsRNA

Viruses, bacteria

MyD88

TLR8

Endolysosomal compartment

TLR8/TLR7

ssRNA, dsRNA

Viruses, bacteria

MyD88

TLR9

Endolysosomal compartment

TLR9/TLR9

Unmethylated CpG DNA Bacteria, protozoa, viruses MyD88

TLR10 Cell surface

TLR10/TLR10 TLR10/TLR2

Unknown



MyD88

TLR11 Endolysosomal compartment

TLR11/TLR11 TLR11/TLR12

Frofilin, Flagellin

Apicomplaxan parasite

MyD88

TLR12 Endolysosomal compartment

TLR12/TLR12 TLR12/TLR11

Profilin

Apicomplaxan parasite

MyD88

TLR13 Endolysosomal compartment

TLR13/TLR13

Bacterial 23S rRNA

Gram-negative bacteria

MyD88

VSV8

Virus

T- and B-cells, and also in some nonimmune cells such as fibroblasts and epithelial cells. The human genome encodes ten TLRs, TLR1 to TLR10, and the murine genome encodes twelve TLRs, TLR1 to TLR9, TLR11, TLR12 and TLR13 (Table 18.1). These receptors are type I transmembrane glycoproteins and share a conserved structural arrangement including an N-terminus ectodomain composed of tandem of leucine-rich repeats (LRR), a helical transmembrane domain and a C-terminus cytoplasmic Toll/interleukin 1 (IL-1) receptor (TIR) domain (Fig. 18.1). The ectodomain consists of varying numbers of LRRs and resembles a solenoid bent into a horseshoe shape (Brodsky and Medzhitov, 2007). These ectodomains are highly variable between TLRs and are directly responsible for the recognition of a wide variety of pathogen-associated motifs including lipopolysaccharides (LPS), lipopeptide, cytosine-phosphate-guanine (CpG) DNA, flagellin, imidazoquinoline, and ds/ssRNA (Table 18.1).

18.3. TLRs IMMUNOMODULATE BOTH INNATE AND ADAPTIVE RESPONSES TLR ligands such as bacterial CpG and synthetic Cytidine-phosphate-guanosine (CpG) ligonucleotides (CpG-ODN) and therefore their receptor, TLR9, drew attention because they activate cells of both the innate immune system [antigen presenting cells (APCs)] (Bendigs et al., 1999) and of the adaptive immune system (lymphocytes) independent of APCs (Hartmann et al., 1999). CpG-ODN is a known mitogen for B-cells (Jabara et al., 2012; Krieg et al., 1995). Several studies provide evidence for the view that the direct recognition of microbial ligands by TLRs on dendritic cells (DCs) plays a prominent role in the initiation of the adaptive immune response and for directing polarization to a Th1 response. DCs especially are mediators of pro-inflammatory interleukin-12 (IL-12) production in response to TLR9 activation (Behrens et al., 2011; Behrens, 2008). Depending on the gene transcription the signaling pathways and responses are very different between the cells types and

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FIGURE 18.1 Diagram of TLRs. TLRs, type I transmembrane glycoproteins showing a conserved structural arrangement including an Nterminus ectodomain composed of tandem of leucine-rich repeats (LRR), a helical transmembrane domain and a C-terminus cytoplasmic Toll/interleukin 1 (IL-1) receptor (TIR) domain. The ectodomain consists of varying numbers of LRRs and resembles a solenoid bent into a horseshoe shape.

also between early versus late peak of gene activation. The initial peak within three hours of CpG ligation to TLR9 transcribes genes that are associated with activation of the immune system, typically by upregulation of NF-kB; at the second distinct peak, after almost five days of activation, transcription of genes that are associated with cell division are enhanced (Klaschik et al., 2010). The CpG-driven immune responses at the early phase essentially require activation of stress kinases including Jun N-terminl kinases JNK1/2 and p38 pathway for the cytokine release of tumor necrosis factor alpha (TNFα), IL-12 and IL-6, because inhibition of p38 severely impairs these biological responses (Takeshita et al., 2001). Furthermore, endolysosomal maturation was essential for this signaling indicating activation of endolysosomal TLRs. Along with the secretion of cytokines, CpG stimulation also upregulates costimulatory cell surface molecules, which paves the signaling pathways for adaptive immune responses. Recent studies discuss the novel regulation of TLRs on the homeostasis and immunity of different Tcell subtypes including CD41CD251T regulatory cells (Treg) and interleukin (IL)-17-producing CD41 T-cells (T helper type 17) [see in review (Liu et al., 2010)]. A direct involvement of TLRs in T-cell-mediated immunity is considered an important role of TLRs in the occurrence of autoimmune diseases, infectious diseases and graft rejection. The important effects of TLRs in T-cell-intrinsic components also prompted many scientists to explore novel vaccine adjuvants including CpG towards modulation of desired immune responses in an efficient way in various disease scenarios (Lipford et al., 1997; Houot and Levy, 2009; Gershan et al., 2015). We will discuss in more detail the T-cell-mediated signaling and consequences in SLE pathogenesis.

18.4. TLR ADAPTOR PROTEINS AND SIGNAL TRANSDUCTION The involvement of TLR adapters downstream of TLR ligation is very important in skewing immune responses. The TLRs have an N-terminal ligand binding domains and the C-terminal cytoplasmic TIR domains (Fig. 18.1). The TIR domains are highly conserved consisting of approximately 200 amino acids. Sequence analysis revealed the presence of three highly conserved TIR regions among the different members of the family: box 1 (FDAFISY), box 2 (GYKLC-RD-PG), and box 3 (a conserved W tryptophan, surrounded by basic residues). Sitedirected mutagenesis and deletion analysis have shown that the TIR domain is essential for TLR signaling. Upon receptor ligation, structural reorganization facilitates dimerization of TLRs. This step promotes TIR-TIR dimer assembly in the cytoplasm to create a unique interface, which allows recruitment of the TIR domain of the adapter protein downstream. The TLR dimers usually are homodimers, but some can form heterodimers (Table 18.1). The TIR domain is highly conserved not only in TLRs but also in TLR adapter proteins responsible for the downstream TLR signaling (Ohnishi et al., 2009; Li et al., 2005; Jiang et al., 2006). Recruitment of the proper adapter protein(s) determines the specificity of the responses by initiating a number of signaling pathways leading to the transcription of specific genes, particularly nuclear factor-κB (NF-κB), a crucial and a central step to the innate immune-stimulatory responses (Fig. 18.2). TLR signaling constitutes a family of five adapter proteins: myeloid differentiation primary response gene 88 (MyD88), MyD88-adapter-like (MAL; also know as TIRAP), TIR-domain-containing adapter protein including IFNβ (TRIF; also known as TICAM1), TRIF-related IV. IMMUNITY AND INFLAMMATION: COMMON THREADS OF MULTIFACTORIAL DISEASES

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FIGURE 18.2 Overview of TLR signaling pathways. The signaling cascade downstream of various TLRs illustrating that all TLRs for signal transmission recruits MyD88 excepting for TLR3, which partners with TRIF. Further downstream recruitment of various kinases such as IRAK1, 2, and 3; and E3 ligases either TRAF3 or 6, differentially induce different transcription factors. For instance MyD88/IRAK1, IRAK2, IRAK4 recruit TRAF6 and induces NFkB, CREB or AP-1. Alternatively, TRIF/TRAF3 pathway induces IFN-specific transcription factors for the release of type I IFNs.

adapter molecule (TRAM; also known as TICAM2) and sterile α-and armadillo-motif-containing protein (SARM) (O’Neill and Bowie, 2007). As illustrated in Fig. 18.2, most TLRs are dependent on MyD88 except for TLR3, which requires TRIF for signaling (Akira et al., 2003; Takeuchi et al., 1999). Cells lacking MyD88 are unresponsive to all TLRs but TLR3. Briefly, these adapters can trigger two main pathways, either via MyD88 or TRIF, which lead predominantly but not exclusively, to the production of inflammatory cytokines, or type I IFNs (IFNα/β) (De Nardo, 2015). A brief description of MyD88 and TRIF-mediated signaling and signaling consequences follows.

18.5. MyD88-DEPENDENT SIGNALING MyD88 directly couples to the intracellular TIR domains of TLR dimer or via MAL in the case of TLR2 and TLR4, whereas for the activation TLR9 and TLR7 MAL is not essential. A study in 2006 by Kagan et al (Kagan and Medzhitov, 2006) demonstrated MyD88 localization to the lipid- enriched (PI(3)P) and (PI(3,5)P2 on endosomal membrane and that MyD88 essentially was required for the activation of TLR7 and TLR9 in response to natural stimuli such as DNA from herpes simplex virus. MyD88 via its death domain (DD) at the N-terminus forms a protein-protein interaction with a family of four DD-containing serine/thronine kinases, IL-1 receptor associated kinases (IRAKs). IRAK1, IRAK2, and IRAK4 display intrinsic kinase activity. While IRAKM, also known as IRAK3, is kinase inactive and is thought to act as a negative regulator (Kobayashi et al., 2002). The MyD88/IRAK4 interaction promotes IRAK4 autophosphorylation and subsequent DD-DD mediated recruitment of IRAK1 or IRAK2. When in close proximity to IRAK4, IRAK1 and IRAK2 are phosphorylated, which mediates their activation. IRAK1 is important in early activation of NK-κB, whereas IRAK2 and its kinase activity appears more critical for sustained TLR responses. Activation of the IRAKs allow transient recruitment of the E3ubiquitin (Ub) ligase, TNFR-associated factor 6 (TRAF6) to the receptor complex, followed by its subsequent activation and release into the cytosol where it forms a complex with TAK1, TAB1, and TAB2/3. This step activates an IKK complex consisting of NEMO, IKKα, and IKKβ, leading to phosphorylation and degradation of the inhibitor IκB. Thereby the release and nuclear translocation of NF-κB initiates transcription and production of stimulus specific cytokines. Simultaneouly, TAK1 triggers the MAP kinase pathway thereby mediating AP-1 and CREB activation, which are also critical for cytokine gene expression. Although MyD88-dependent pathway predominately culminates in cytokine production in plasmacytoid dendritic cells (pDCs), MyD88 signaling also leads to the expression of type I IFNs via IRAK1-mediated activation of the transcription factor IRF7 (Takeda and Akira, 2004). IV. IMMUNITY AND INFLAMMATION: COMMON THREADS OF MULTIFACTORIAL DISEASES

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18.6. TRIF-DEPENDENT TLR SIGNALING TRIF triggers a specific signaling cascade downstream of TLR3 and TLR4. The latter is unique as it induces both MyD88 and TRIF-dependent pathways. On the cell surface TLR4/MyD88 requires copartnership with MAL, following its translocation to lysosome TLR4 prompts signaling with copartnership from TRAM and TRIFmediated signaling (Fig. 18.2).

18.7. TLRs AND SUB-CELLULAR LOCALIZATION-SPECIFIC FUNCTION Based on sub-cellular localization, TLRs are broadly subdivided into those that are localized at the cell surface (TLR1/2/4/5/6), activated particularly by lipid and protein ligands; and those that are located in the endosomal compartment (TLR3/4/7/8/9), where they are poised to recognize nucleic acids. In a simplistic view, the extracellular microbial antigens are recognized by the cell surface TLRs (TLR1/2/4/5/6); in contrast, the intracellular microbial antigens such as DNA/RNA, for recognition need to interact with the host intracellular TLRs. The endolysosomal TLRs are membrane-trafficked in a highly regulated and specific manner by the conventional secretory pathway. After synthesis, most TLRs require proper assembly by a direct association with the endoplasmic reticulum (ER) folding machinery including a complex of glucose-regulated protein (GRP94) and CNPY3 (canopy 3). TLR3 is exceptional, as genetic disruption of either GRP94 or CNYP3 did not affect TLR3 localization in the endolysosome and also its function. After assembly in the ER, Unc93 homolog B1 (UNC93B1), a multipass transmembrane protein, renders membrane trafficking of most lysosomal TLRs from ER egress to golgi transit by remaining associated with TLRs until their transport to the endolysosome (Lee et al., 2013). Sorting and trafficking of TLRs to endosomes and lysosomes are specified by the tyrosine-based signals present within the cytoplasmic TIR domain of the TLRs (Chockalingam et al., 2012) and also by the tyrosine-based motifs in UNC93B1 (Pelka et al., 2014). These signals allow sorting by further employing the complex machinery of endolysosomal adapter proteins, AP1 and AP2, requiring their association with UNC93B1 (Lee et al., 2013). In the endolysosomal acidic pH, TLR9 processing by cathepsin-mediated cleavage of N-terminal ectodomain is compulsory for delivering a fully matured, stable and functional TLR (Park et al., 2008). In response to nucleic acids, functional TLR9 either as homodimers or heterodimers align a structural interface competent for the recruitment of the adapter proteins for downstream signaling. The endolysosome-specific function of TLR9 was supported by pharmacologic inhibition of golgi using brefeldin A; this blocked membrane traffic via golgi and resulted in the inactivation of TLR9. Additionally inhibition of endolysosomal acidification by either Bafilomycin A or chloroquine abrogated pH-sensitive endosomal cleavage and simultaneously TLR9 signal transmission (Park et al., 2008). The fact that endolysosomal TLRs including TLR9 recognize nucleic acids, their highly regulated intracellular localization is considered as the key protective measure to avoid easy access to self-recognition, and therefore to prevent autoimmunity in response to DAMPS. Nonetheless, in the past decades, a considerable amount of evidence suggests that TLRs actually can recognize self-nucleic acids leading to autoimmune diseases. TLR9, being the key receptor for double-stranded DNA (dsDN) (Kumagai et al., 2008), could drive autoimmune diseases particularly systemic lupus erythematosus (SLE), and drew attention of many immunologists (Christensen et al., 2005; Ghaly et al., 2013; Lartigue et al., 2006). Reports also suggest that TLR9 signaling, depending on the expression level and availability of UNC93B1, can alter energy metabolism pathways leading to differential outcome. For instance, downstream of TLR9 ligation, a very low level of UNC93B1 as shown in cardiomyocytes renders alternative activation of 5’ adenosine monophosphate-activated protein kinase (AMPK) instead of MyD88, which presumably is protective to the ischemia-reperfused cardiomyocytes. A metabolic switch from mitochondrial oxidative phosphorylation to glycolysis is indicated for the survival signal-induced beneficial outcome (Lee et al., 2013; Shintani et al., 2013). These studies clearly indicate that TLR9 signaling is an important arm of innate immunity and repair mechanism; therefore, therapeutic modulation of TLR9 bears significant importance.

18.8. TLR9, SELF-RECOGNITION AND ROLE IN AUTOIMMUNE DISEASES For self-preservation it is necessary for the immune system to avoid self-recognition. TLR9 initially was identified as the receptor for single-stranded CpG-rich motifs. CpGs are cytosine and guanine (CG) sites in DNA with a phosphate moiety in between. Cytosines in CpG dinucleotides can be methylated to form 5-methylcytosine by

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the enzyme DNA methyl transferases—a regulatory mechanism that is largely studied by the field epigenentics. An epigenetic trait is described as “stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence” (Berger et al., 2009). The extent of methylation can alter the transcriptional fate and thus expressional fate of the ultimate DNA-product. The hypomethylated form of CpG is recognized by TLR9 and is frequent in bacteria and rare in vertebrates with the exception of mitochondrial DNA, which evolutionarily derived from bacterial DNA. Therefore with cell damage, release of mitochondrial DNA can trigger TLR9 signaling. However, how the intracellular TLRs gain access to their ligands is not well understood. The evidence that TLR9 can be activated by the addition of CpG in the extracellular medium indicates that cells can transport nucleic acids from extracellular compartment to TLR containing intracellular compartment. Behrens et al. (2011) demonstrated in mice that repeated CpG injection developed macrophage activation syndrome (MAS), exhibiting cytokine storm (particularly IL-12 and IFNγ) without having any genetic defect. The mice with repeated CpG injection also produced large amounts of IL-10, apparently thought to induce partial resolution of the TLR9-driven MAS and inflammation, thus requiring repeat CpG injection for a sustained inflammation. Furthermore, development of CpG-induced MAS was TLR9-dependent because TLR9 knockout mice were protected from MAS pathophysiology. How CpG DNA is internalized and delivered to TLR9 is not well understood; the consensus understanding is that this is achieved by cell membrane-regulated phagocytosis or pinocytosis (Chaturvedi and Pierce, 2009). Later, the receptor for advanced glycation end-products (RAGE) was identified as a direct binder to CpG, and as a promoter of CpG internalization in plasmocytoid dendritic cells (pDCs) (Krieg, 2007). RAGE is the known receptor for high mobility group protein 1 (HMGB1) that can directly bind to CpG DNA (Tian et al., 2007; Sirois et al., 2013); it is to be noted that HMGB1 is one among many other DAMPS. Internalization of HMGB1-CpG DNA by the receptor RAGE results in activation of plasmacytoid dendritic cells (pDCs). Another study where co-crystal structure of RAGE with DNA supports the concept that RAGE can bind directly to nucleic acid via charged sugar phosphate backbone in a sequence independent manner (Koch et al., 2010). Based on more studies (Jozefowski, 2012; Jozefowski et al., 2012; Zhu et al., 2009; Kissick et al., 2014) there seemingly are additional scavenger receptors for CpG internalization to the intracellular TLR9.

18.9. OXIDATIVE STRESS, MITOCHONDRIAL INEFFICIENCY, TISSUE DAMAGE, AND TLR9 ACTIVATION Overall cellular oxidative stress is increased in SLE. Oxidative stress refers to a state accompanied with an elevated level of reactive free radicals of oxygen molecules commonly known as reactive oxygen species (ROS). A disproportionate level of ROS is generated by excessive energy metabolism by mitochondrial electron transport chain (ETC)/respiratory chain at the expense of reducing equivalents produced in tricarboxylic acid cycle (Fig. 18.3). During excessive mitochondrial respiration, the steady state mitochondrial membrane potential is disrupted due to excessive proton leaks through the inner membrane without the ATP production reflecting mitochondrial inefficiency and low level of energy. ROS in excess, if not scavenged by a set of antioxidant enzymes such as superoxide dismutases or catalase and so on (Fig. 18.3), rapidly oxidizes cellular building blocks such as lipids, proteins, and DNA. Such oxidative modification of DNA is very immunoreactive and can potentially lead to tissue damage either by autophagy, necrosis, or apoptosis (Wang and Law, 2015; Perl et al., 2004). The paradox of redox biology is that elevation of ROS can cause direct damage, while at moderate level it also modulates signaling pathways crucial for the maintenance of physiological functions. ROS activates transcription factors such as the nuclear factor erythroid 2-related factor 2 (Nrf2), one of the master transcriptional regulators of antioxidant enzymes. The specific signal transpired by these antioxidants enzymes enable a feed forward adaptive reprogramming to recovery, and promote a survival advantage in oxidative and environmental stress (Biswas et al., 2014).

18.10. A COLLAPSE IN DISPOSAL OF CELLULAR DEBRIS IN SLE PATHOGENESIS Disposal of cellular debris by a well-orchestrated enzyme-based safeguard system during tissue destruction is an integral part of the host immune and repair mechanism. With autoimmune disorders a severe breakdown of this system is observed allowing for accumulation of a disproportionate level of cellular debris and pathogenic DNA. For instance, constitutive hyper-methylation of CpG by DNA methyl transferases ensures escape of DNA by TLR9 recognition. Increasing evidence relates altered DNA methyl transferases particularly a lower level of DNA methyl transferase with SLE that was observed in a group of ethnic African American women with SLE (Wiley et al.,

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FIGURE 18.3 Energy metabolism pathways, production of ROS and ATP. This illustration demonstrates utilization of energy substrate (glucose) by glycolytic pathway followed by TCA cycle and the reducing equivalents produced entering the electron transport chain yielding protons (H1), reactive oxygen intermediates, hydrogen peroxide (H2O2) and oxide radical (O2  2). This scheme also demonstrates the set of antioxidant enzymes, superoxide reductase (SOD), catalase, glutathione peroxidase (Gpx), thioreductase (TR), peroxireductase (Prx), that neutralize the reactive oxygen species.

2013). Also an epigenetic chromatin remodeling leading to global hypomethylation was observed in CD41 T-cells in a group of SLE patients (Balada et al., 2008). Additionally, the availability of nucleases assures degradation of extracellular and intracellular self-DNA and thereby aids their escape from detection by the nucleic acid sensors. SLE pathogenesis is notably associated with the inability of the nucleases to degrade chromatin from SLE patients (Leffler et al., 2015; Walport, 2000). This facet was first recognized when a correlation was found between excessive levels of deoxyribonuclease inhibitor in human sera and the occurrence of antinuclear antibodies (Frost and Lachmann, 1968). During the past few years, among several mechanisms, the role of neutrophil extracellular traps (NETs) has also been proposed in the etiopathology of SLE. In extreme situations neutrophils, as a last resort, release NETs and ensure control of microbial infection by sequestering them, and also by antimicrobial activities via granular components in the trap. NETs are loaded with modified nuclear and mitochondrial DNA, which were found highly immunomodulatory in the pathogenesis of SLE (Pieterse et al., 2015).

18.11. SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) SLE is a chronic inflammatory disease characteristically exhibiting loss of tolerance to self-antigens, and production of high titer of autoantibodies directed particularly against naı¨ve DNA. It is a highly heterogeneous autoimmune disorder of multifactorial etiology including genetic, epigenetic, gender and immunoregulatory and environmental. Approximately 90% of individuals affected with SLE are female, predominantly of childbearing age. Prevalence of SLE in United States of America is estimated to be in the range of 0.05% and 0.1% of the population. The disease overwhelmingly affects African Americans (0.282% African Americans in comparison to 0.038% white women and 0.066% white men). Almost 50% of SLE patients exhibit severe complications of the disease, including nephritis, central nervous system vasculitis, pulmonary hypertension, interstitial lung disease, and stroke. Lupus nephritis (LN) is among the most common clinical complications of SLE, occurring in up to 74% of patients and accounting for significant morbidity and mortality particularly among ethnic minorities (Danchenko et al., 2006). An excessive deposition of autoimmune complex in the glomeruli is known to lead complement activation, chronic inflammation and renal insufficiency in the manifestation of LN as supported by histopathology, and the presence of proteinuria and cellular casts. A key feature of SLE is collapse in innate and adaptive responses that

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triggers loss of self-tolerance. Since excessive production of autoantibodies is attributed to SLE pathogenesis, initially B-cells were classically thought to be the primary mediators of this disease. Recent studies provide compelling evidence that it is majorly the amplified activation of T-cells that perpetuates enhanced activation of B-cells through stimulation to differentiate, proliferate and mature. In comparison with healthy subjects, T-cells isolated from SLE patients are abnormal in phenotypes and function featuring aberrant signaling, cytokine secretion and tissue homing (Kammer et al., 2002; Moulton and Tsokos, 2011), and therefore, T-cells are a therapeutic target in SLE. The biochemical and molecular studies over the last three decades broadened our knowledge about the anomalies associated with SLE T-cells’ aberrant signaling, and are described below.

18.12. ALTERED CD3-T-CELL RECEPTOR (CD3-TCR) SIGNALING IN SLE T-CELLS In SLE T-cells, CD3-T-cell receptor (TCR) signaling is dysregulated. TCRs remain as covalently bound heterodimers of TCR α and β chains. These chains are further associated with CD3 complex comprising four isoforms δ, ε, γ, and ζ. The former three isoforms belong to the immuglobulin (Ig) superfamily and the C-terminal intracellular region of each contains an ITAM (immunoreceptor tyrosine-based activation motif) motif. The ζ chain consists of a long intracytoplasmic domain that contains three ITAM domains. TCR signaling is initiated upon recognition and ligation of the TCR with the MHC-peptide presented by the antigen presenting cells (APCs). This results in auto phosphorylation of ITAM residues, which facilitates recruitment of tyrosine kinase Zap-70 (ζ-chain associated protein 70). In SLE T-cells, the level of CD3 ζ-chain is decreased significantly and instead there is an excessive expression of an analogous protein, FCγR (common γ chain of Fcε receptor). The FCγR does not participate with Zap-70 in TCR signaling, instead recruitment of spleen tyrosine kinase (Syk). The FCγR-Syk signaling that operates through Phospholipase C gamma 2 (PLCγ2) is 100 times more intense than the usual CD3ζ-Zap70 interaction, and this alteration in SLE T-cells amplifies the downstream signaling. In animal models inhibition of Syk or restoration of CD3ζ corrected the aberrant signaling by SLE T-cells. Following TCR activation the role of phosphoinositide 3 kinases (PI3K) has also been extensively implicated in SLE pathogenesis. CD41 T-cells isolated from the mouse lupus model showed increased activation of PI3K, and pharamacologic intervention of PI3K could resolve the intensity of lupus symptoms in these mice. Therefore, PI3K is also considered to be a therapeutic target in SLE (Takeuchi et al., 2004; Liossis et al., 1998).

18.13. ENHANCED AGGREGATION OF LIPID RAFTS AND TCR ACTIVATION IN SLE T-CELLS Lipid rafts are cholesterol-rich domains found on the cell surface, and normally aggregation of lipid rafts appears especially at the site of TCR-antigen ligation. This ensures inception of optimal immunological synapse necessary for maximum T-cell activation and antigen-specific downstream signaling. However, freshly isolated T-cells from SLE-prone mice harbor preclustered lipid rafts. Amplification of T-cell activation in lupus-prone mice can be attained by treatment with cholera toxin, a facilitator of lipid raft aggregation, exacerbating lupus pathology and the disease onset can be delayed by disrupting lipid rafts by statin-based drugs that block cholesterol synthesis (Jury and Kabouridis, 2004; Deng and Tsokos, 2008).

18.14. ENHANCED CD44-ERM/ROCK (Rho ASSOCIATED PROTEIN KINASE) PATHWAY IN SLE MANIFESTATION CD44 is a cell surface glycoprotein and participates in cell-cell interaction, cell adhesion and migration. CD44 gene is highly conserved and can produce various isoforms owing to alternate splicing. CD44 splice variants containing variable exons are designated CD44v. CD44 is referred to as HCAM (homing cell adhesion molecule). Activation of CD44 is initiated by binding to the common ligand hyaluronic acid (HA). The adhesion and migration property by CD44 involves the participation of ERM protein complex (Ezrin/radixin/moesin). The phosphorylation of ERM by ROCK promotes recruitment of ERM to the intracellular domain of CD44, and the downstream signaling by CD44/ERM/ROCK pathway facilitates adhesion and migration of T-cells. Exhibition of higher expression level of CD44, particularly CD44v3 and CD44v6 and pERM is observed in both CD41 and CD81 T-cells isolated from SLE patients. Also increased levels of HA and CD44 are observed in damaged kidney of SLE patients and

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lupus-prone mouse models. Further in a lupus-prone mouse model pharmacologic inhibition of ROCK proves to reduce pERM, and thus the intensity of lupus. These studies do suggest that altered output by CD44/ERM/ROCK pathway do have an association in the SLE pathogenesis (Isgro et al., 2013; Comte et al., 2015).

18.15. LACK OF INTERLEUKIN 2 (IL-2) AND PERIPHERAL T-CELL TOLERANCE IN SLE Several studies, including in vitro antigen activation of T-cells that are isolated from both lupus mouse model and from human SLE patients, support that they are defective in IL-2 production. IL-2 production is the signature characteristic of antigen-activated T-cells: necessary to promote growth, survival and differentiation of the activated T-cells. The importance of IL-2 is also implicated in the differentiation of helper T-cells—type 1 (Th1) and type 2 (Th2)—also differentiation and expansion of CD81 Cytotoxic effector T-cells and regulatory T-cells (Tregs). Manifestation of autoimmunity and peripheral T-cell tolerance observed in the specific knockout mice such as IL-22/2 and IL-2 receptor (IL-2r2/2) is attributed to the lack of Tregs in the periphery particularly of insufficient differentiation of CD41 T-cells into IL-17 secretory T (Th17) cells. Exhibition of the above factors owing to the lack of IL-2 in SLE patients is considered to exacerbate the level of infection, increase autoimmunity and mortality rate; and particularly due to a breakdown in the activation induced cell death—an important step in the elimination of activated and potentially autoreactive T-cells. Now the question is whether oxidative stress has any role in the altered signaling of T-cells that is associated with SLE pathogenesis. Recent studies indicate an association between oxidative stress-driven T-cell dysfunction and SLE pathogenesis (Lieberman and Tsokos, 2010).

18.16. OVERALL ROS LEVEL IS HEIGHTENED IN T-CELLS ISOLATED FROM LUPUS PATIENTS AND LUPUS MOUSE MODELS The noxious effects of ROS cause the following consequences: (1) shift of intracellular redox (i.e., decrease GSH/ GSSH ratio) condition, (2) oxidative modification of lipid, protein and DNA, (3) gene activation of oxidative stress and gene mutation related to antioxidant enzymes. Hallmark changes in peripheral T-cells isolated from SLE patients and also from lupus prone mouse model exhibit high levels of ROS characterized by mitochondrial hyperpolarization, elevated mitochondrial membrane potential, decreased oxidative phosphorylation, depletion of ATP and cytoplasmic alkalinization (Gergely et al., 2002). Simultaneously reduced levels of antioxidant enzymes such as catalases (CAT), superoxide dismutases (SOD) and glutathione peroxidase (Gpx) necessary for enzymatic elimination of reactive oxygen intermediates (ROI) were associated with the occurrence of SLE. This state also correlated with a decrease GSH/GSSH ratio (Perl et al., 2004). These peripheral T-cells clearly are consuming higher levels of oxygen through mitochondrial respiration, which is the major source of ROS release. The addition of metabolic modulator could switch in energy metabolism pathways from mitochondrial respiration to glycolysis (Warburg theory), was able to normalize dysfunction in CD41 T-cell isolated from SLE mouse models (Lopez-Lopez et al., 2014). Mitochondrial dysfunction in T-cells also permits the release of highly diffusible inflammatory hydroperoxides, which, by spreading oxidative stress to other intracellular organelles and through blood streams, makes the condition more dysfunctional and inhospitable. Oxidative modification of self-antigens triggers autoimmunity, and the degree of such modification of serum proteins shows striking correlation with disease activity and organ damage in SLE. In T-cells from patients with SLE and lupus-prone animal models, glutathione, the main intracellular antioxidant, is depleted. Based on these studies, antioxidant therapy with the supplementation of N-acetylcysteine has shown improvement by suppressing SLE in lupus-prone mice. Furthermore, pilot studies in patients with SLE have yielded positive results, which warrant further validation (Perl et al., 2004).

18.17. GLOBAL HYPOMETHYLATION IN SLE CD41 T-CELLS A number of genetic polymorphisms is associated with lupus (Delgado-Vega et al., 2010). Discordance in the development of lupus in twins suggests that the disease is not entirely genetic in origin. Interestingly, the finding of DNA methylation differences between the twins with the discordant for lupus indicates association of epigenetic factors in SLE pathogenesis. More recent studies strongly support an epigenetic contribution to the pathogenesis of SLE, particularly a global DNA hypomethylation in T-cells is considered the main incriminating factor (Lei et al., 2009). Transcription of genes is regulated mostly by repression when hypermethylation occurs on the IV. IMMUNITY AND INFLAMMATION: COMMON THREADS OF MULTIFACTORIAL DISEASES

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cytosine of CG repeats located within promoter region, a site between 1000 and 800 bp upstream of transcription initiation site (Hughes et al., 2010). In fact methylation is a very active regulatory mechanism and is key to many cellular responses from environmental stimuli including hypoxia, hormonal signaling, and viral latency and reactivation (Hattori et al., 2015; Chen et al., 2009; Blazkova et al., 2009). A study by Jeffries et al. (2011) compared a genome-wide methylation study on CD41 T-cell in lupus patients with those of normal healthy controls. This study reported 236 hypomethylated and 105 hypermethylated CG sites in CD41 lupus T-cells compared to control cells, consistent with widespread DNA methylation changes in lupus T-cells. Interestingly hypomethylation of CD9 was evident; a potent costimulatory signaling molecule in T-cells. Genes involved in folate biosynthesis, which plays a role in DNA methylation, were overrepresented among hypermethylated genes. Among many genes this study further reported alteration of methylation status of RAB22A, STX1B2, LGALS3BP, DNASE1L1 and PREX1, that correlates with disease activity in lupus patients. These studies suggest a role of epigenetic modification-driven DNA hypo-methylation and abnormal expression of methylation-related genes to the dysfunctionality of CD41 T-cells with SLE pathogenesis.

18.18. DIET, COMMENSAL MICROBIOTA AND AUTOIMMUNITY Growing evidence suggests that commensal microbiota in the gastrointestinal tract influences development of autoimmunity. However the cellular and molecular mechanism linking changes in gut microbiome with the development of self-reactive adaptive immunity is unclear. In preclinical models of autoimmunity the severity of disease was minimized by antibiotics treatment or in mice growing in germ-free facility. The Gram-positive spore-forming segmented filamentous bacteria (SFB) influenced by a single micronutrient, vitamin A (Kau et al., 2011), could impact the development of autoimmune arthritis (Wu et al., 2010), and experimental autoimmune encephalomyelitis (Lee et al., 2011), while was protective from type 1 diabetes (Kriegel et al., 2011). These pioneer studies linked these effects to excessive T helper 17 (Th17) cell responses consistent with the Th17-inducing function of SFB. Since chronic activation of TLRs is evidenced in many autoimmune diseases, it is likely that TLRs are the missing link between the gut microbiome and development of autoimmunity. More studies suggest that the mechanisms that limit immune activation belong to potential synergistic actions from both host and bacterial effector molecules. A recent review (see review (Valentini et al., 2014)) highlighted the role of gut microbiota in synergy with TLR activation to influence immune system by modulating number and function of effector and regulatory T-cells. Since humans have coevolved with commensals it is likely that those who are genetically predisposed to autoimmunity also harbor such gut microbiota that may help to sever or reduce the disease onset. Therefore, it will be important to determine the factors that modulate gut microbiome. In light of this, diet is important because dietary changes can affect both composition and function of the gut microbial community (Vieira et al., 2014). Food significantly changes the composition of the gut microbiota and its genetic makeup (Goodman et al., 2011; Muegge et al., 2011). For instance, caloric restriction is well documented being beneficial in preventing a multitude of autoimmune diseases (Jolly, 2004). However, it is unclear if these are the direct consequences of caloric restriction or are indirect consequenses of modulation of gut commensals. Meta analysis and comparison of fecal microbiomes between obese and lean humans and between monozygotic and dizygotic twin pairs concordant for leanness or obesity provide deep understanding into this inter-relationship (Turnbaugh et al., 2006; Ley et al., 2006; Turnbaugh et al., 2009). The observation that the human microbiota of an obese host that was significantly different from that of a lean host could induce weight gain when transplanted into germfree mice was outstanding. The recent thought is that RNA and DNA viruses within a mammalian host can reside both in eukaryotic and prokaryotic cells, for instance gut commensal bacteria. Therefore, the common interferon-α (IFN-α) signature in the peripheral blood of SLE patients suggests that it may have developed from viral-triggered flares and hence represents a novel target for therapies (Lichtman et al., 2012). These studies together provide us with various possibilities and to contemplate how diet, microbiome and virome interactions within a host could interplay in the pathogenesis of SLE (Fig. 18.4). The advances made in this field still do not completely clarify the molecular mechanisms that account for the interaction of the microbiota-immune system and need further attention.

18.19. SUMMARY Advances in the understanding of the innate immune system have revolutionized our understanding of cellular and molecular mechanism in the regulation and function of TLRs. The unique endo-lysosome-specific TLR9 IV. IMMUNITY AND INFLAMMATION: COMMON THREADS OF MULTIFACTORIAL DISEASES

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FIGURE 18.4 Oxidative stress mediated modulation of immune responses and crosstalk with gut microbiome. An illustration depicts how oxidative stress prompts cellular damage and release of DMAPs that evokes chronic activation of TLR, and autoimmune diseases. This scheme also shows a connection between gut microbiome and oxidative stress-mediated cell damage and DAMP release , and also PAMPs release from microbial degradation in amplifying autoimmune responses.

activation to avoid self-recognition and the role of TLR in linking innate with adaptive responses ensure clearance of infection. Auto-immune responses are triggered when the safeguard mechanisms are broken down under oxidative stress-driven over-activation of mitochondria and vice versa. Furthermore, oxidative and epigenetic modification of cellular components leading to tissue damamge and release of DAMPs complicate SLE. SLE pathogenesis is accompanied by a characteristic T-cell pathology displaying global hypomethylation of genes particularly in CD41 T-cell population and a proinflammatory SLE signature in over-production of IL-17 and IFNα. Extensive studies were carried out in characterizing T-cell pathology in SLE settings with few studies on antigen presenting cells (APCs). The fact that TLR9 activates T-cells directly and by APC-mediated antigen presentation and costimulation, show that more studies are needed on APCs for better understanding of SLE pathology. Owing to the heterogeneous nature of the clinical manifestations, SLE still remains to be a challenge to clinicians and researchers. In the past decade multiple studies found a link between commensal microbiota and activation of innate and adaptive immune responses, which is implicated as a major compounding factor in various autoimmune-related diseases. This is supported by a complete resolution of inflammatory signals in mice treated with antibiotics or grown in germ-free condition. Further studies on cellular and molecular mechanisms could help in strategic and therapeutic design to impede SLE progress. A stratification of lupus patients based on clinical symptoms and immunological dysfunctions, targeting disease development is very crucial. This is particularly important for the design of clinical trials, where the correct recruitment and sub-grouping of patients may directly impact the outcome measures. Pharmacogenomic and pharmacogenetic studies will ultimately be essential to identify patients that will most likely benefit from a specific treatment.

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19 Anti-inflammatory and Anti-microbial Properties of Achillea millefolium in Acne Treatment Rahul Shah and Bela Peethambaran University of Sciences, Philadelphia, PA, United States

19.1. INTRODUCTION Acne vulgaris affects 80% of adolescents and at times persists well into adulthood, resulting in scarring and hyperpigmentation. Acne develops in the sebaceous follicles on the cheeks, forehead, chin, and on the back. It is caused due to a combination of increased sebum production and abnormal hyperproliferation of keratinocytes. Inflammation is an important part of acne development as it is seen to develop during and even before the comedogenesis, which is the process of accumulation of sebum, enlargement of follicles, and build-up of keratinous material.

19.2. INFLAMMATION Inflammation is described as “the succession of changes which occurs in a living tissue when it is injured provided that the injury is not of such a degree as to at once destroy its structure and vitality” (Sanderson et al., 1871), or “the reaction to injury of the living microcirculation and related tissues” (Spector et al., 1963). However, in ancient times inflammation was recognized as a part of the healing process. Inflammation is characterized by signs such as: rubor (redness), calor (increased heat), tumor (swelling), dolor (pain), and functio laesa (loss of function). Redness and heat are due to increased blood flow to the inflamed area; swelling is a result of accumulation of fluid; pain is due to release of chemicals that stimulate nerve endings; while loss of function is a result of the combination of all the factors. Inflammation exists through the lifecyle of acne lesion and hence is an important part of the treatment of acne. Histological and immunological evidence, derived from analysis of skin samples from patients, supports that inflammation is a fundamental process in the development of acne lesions. The analyzed skin samples had elevated levels of CD31 and CD41 T-cells in the perifollicular and papilliary dermis and there were increased macrophages in these papules (Jeremy et al., 2003). Studies in early lesions have shown upregulation of inflammatory mediators such as E-selection, vascular adhesion molecule-1, interleukin-1, and integrin (Jeremy et al., 2003; Layton et al., 1998). Current drugs used for treatment of acne such as retinoids are effective in treating noninflammatory lesions (Eichenfield et al., 2010) by downregulating Toll-like receptor-2 and interleukin-10 expression (Tenaud et al., 2007; Liu et al., 2005). Gene array expression studies from skin biopsies of six acne patients showed that out of 211 upregulated genes the majority were involved in matrix remodeling and inflammation (Trivedi et al., 2006).

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19.3. ETIOLOGY AND PATHOLOGY OF ACNE VULGARIS Acne vulgaris (cystic acne or simply acne) is a common human skin disease, occurring mostly during the onset of puberty. It is mainly a disorder of the pilosebaceous unit. It affects almost 4050 million Americans and is estimated to occur in 85% of people at least some point in their lives. Analysis has also shown prevalence of acne in individuals in the beginning or during puberty (Bhambri et al., 2009). Acne occurs in areas of skin with large number of sebaceous follicles, which are found on the cheek, forehead, and back (Tanghetti, 2013). Acne is generally characterized by areas of skin with seborrhea, comedones, papules (pinheads), pustules (pimples), nodules and possibly scarring. The precise mechanism of acne is not known but is thought to involve four main pathogenic factors: follicular keratinization, increased sebum production, presence of Propionibacterium acnes, and inflammation (Bhambri et al., 2009). Apart from the above mentioned factors, which are central to acne, genetic and hormonal changes, birth control pills, certain medication, stress, use of oily cosmetics are also some of the major factors that can cause acne by creating a favorable environment for the growth of P. acnes in the follicles. Acne occurs due to blockage in the follicles resulting in the formation of comedones. The earliest microscopic lesion observed is the microcomedones. Microcomedones are the precursors that can develop into both noninflammatory and/or inflammatory lesions. They are characterized by follicular plugging which is caused due to follicular keratinization and reduced desquamation of keratinocytes in the pilosebaceous unit. Recently, the role of biofilm formation by P. acnes leading to formation of microcomedones has also been hypothesized. Over time, the microcomedones develop into comedones that further develop into blackheads and whiteheads. Lack of oxygen in these plugged follicles creates an anaerobic environment suitable for the growth of P. acnes. The cell wall and the biological by-products of these flourishing bacteria act as chemo-attractants and proinflammatory mediators leading to inflammation. The inflammatory cells diffuse from the follicle to the surroundings, secreting enzymes that rupture the follicular walls. These inflammatory mediators stimulate a localized immune response resulting in the formation of pustules, while a more intense inflammation is accompanied by formation of comedonal acne(Harper, 2004; Bhambri et al., 2009). Several factors influence the development of acne. These include infection of P. acnes and Staphylococcus epidermidis, natural environmental insults that produce free radical scavenging activity, skin type, and hormonal imbalance. There are also resulting effects post-acne formation that include inflammation and scarring of tissues. P. acnes are gram-positive, anaerobic bacteria that are present in the normal skin and are found in the sebaceous follicle. The obstructed lipid rich lumen with decreased oxygen tension in the comedones makes it an ideal environment for the bacteria to flourish. These bacteria hydrolyze the sebum triglycerides producing free fatty acids. P. acnes also encourages the proliferation of other aerobic, gram-positive bacteria S. epidermidis, which reside normally on the skin. Both these bacteria are found to colonize the acne prone areas.

19.4. MANAGEMENT OF ACNE VULGARIS There has been no significant change in the treatment and management of acne vulgaris for the last twenty-five years. Topical retenoids have been the drug of choice for acne treatment, but there have been problems of skin irritation leading to decreased patient compliance. Isotretinion, approved in 1982, is to date the most effective acne medication, clearing the acne lesions in 85% of the users. However its use causes teratogenic effects and psychiatric disturbances, and so has been a topic of discussion for long time ultimately leading to low patient compliance. Oral contraceptives have been accepted by women as an alternative medication for acne treatment. The contraceptives increase the production of sex hormone-binding globulin that binds to free androgens and reduces the amount of free testosterone, improving the acne lesions. However, these contraceptives also have their own set of side effects. Antibiotics have long since played an integral role in acne treatment and management. Once widely used tetracyclin has fallen off due to increasing cases of antibiotic resistance by P. acnes. A low dose of doxycycline is able to reduce the inflammation in acne but it does not decrease the microbial colony counts. However, longterm use of antibiotics has raised concerns about colony development of other pathogens and microbial resistance. A recent report has found a threefold presence of Streptococccus pyogenes in the orthopharynx of patients treated with systemic and topical antibiotics compared to acne patients not receiving it, raising concerns of growth of other pathogens.

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Topical benzoyl peroxides are also widely preferred in acne treatment since they do not cause issues of bacterial resistance. A combination of antibiotics with benzoyl peroxides may decrease the antibiotic resistance to the coadministered antibiotic. Even prolonged use of benzoyl peroxides have some serious side effects including dryness of skin, flaking, painful itchiness, making the skin more sensitive to sunlight and many others. Some other treatments include light, laser and photodynamic treatment for acne. However, long- term clinical studies have not been reported (Harper, 2004)

19.4.1. Achillea millefolium Achillea millefolium, commonly known as yarrow, belongs to the family Asteraceae. It is the most widespread and one of the most widely used medicinal plants in the world. The word “Achillea” refers to the Greek hero Achilles, who is believed to have carried the plant to treat the wounds of the soldiers, while the word millefolium meaning thousand-leaf, refers to the multipinnate leaves. The genus Achillea includes 110114 species, largely native to Eurasia, with some native to North America and Africa. However, hybridization and infra- specific variability have complicated the taxonomy and so until now there was no single broadly accepted classification. A. millefolium and related species form a complicated and a frequently hybridizing polyploidy complex (including diploid through octaploids). This data is confirmed via using biomarkers such as internal transcribed spacer region (ITS), trnL-trnf, which is a region of the chloroplast genome and AFLP data (Figs. 19.119.4).

FIGURE 19.1 Achillea “Moonshine” plants. namethatplant.files.wordpress.com.

FIGURE 19.2 Reduction of TNF -α cytokine production in cells exposed to P. acnes and then treated with 50 μg/ml of petroleum ether extract from A. “Moonshine”. Published in Shah R., Patel A., et al., 2015. Anti-acne activity of Achillea ‘Moonshine’ petroleum ether extract. J. Med. Plant Res. 9(27), 755763.

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FIGURE 19.3 Reduced IL-8 content in cells exposed to P. acnes and then treated with 50 and 100 μg/mL of petroleum ether extract from A. “Moonshine”. Published in Shah R., Patel A., et al., 2015. Anti-acne activity of Achillea ‘Moonshine’ petroleum ether extract. J. Med. Plant Res. 9 (27), 755763.

FIGURE 19.4 Proposed structure-N-(21-hydroxy-21-(piperidin-1-yl) henicosa-17, 19-diyl-1-yl) acetamide derived from NMR and MS data.

A. millefolium is native to Europe, according to the narrowest circumscription and includes three subspecies: subsp. millefolium, subsp. Alpestris, and subsp. ceretanum. Several other microspecies belong to the A. millefolium complex. A. lanulosa is the native North American yarrow, recognized at the species level and itself is sometimes divided into multiple species. The North American polyploids are different from the A. millefolium, in that the North American species have a closer or a direct relationship to diploids while the latter are generally tetraploid. However, the two have been used interchangeably and various American literatures refer to A. millefolium without making clear as to which species it is being referred to. The plants of the A. millefolium group are perennial plants with height up to 100 cm. The leaves are multipinnate with small flower heads arranged in corymbs. The flowers are white, creamish-yellow to pink ray florets. The volatile oil glands are present on the stem, leaves and the ray and disc florets (Rauchensteiner et al., 2004; Applequist and Moerman, 2011).

19.4.2. Phytochemistry of A. millefolium Many recent chemical studies have fully identified the phytochemistry of A. millefolium samples, although some fail to distinguish between species and subspecies. Proazulenes that belong to the class of sesquiterpenes, are the blue-colored compounds present in the essential oils and common to Achillea sect. millefolium. The subspecies alpestris is the only European species that contains chamazulene, the active compound also present in Matricaria chamomilla L. Studies have shown the presence of chamazulene in A. millefolium; however, it is not clear whether the population has been identified as subsp. millefolium. A large number of guaianolides are also present in proazulene containing species of the A. millefolium group.

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A total of 95 compounds have been identified from the essential oils of yarrow by gas chromatography. There were differences in the compounds depending on the type of the polyploidy and so the volatile oils have proven to be an important characterization tool. The major flavonoids in the plant include 7-O-glycosides and 7-O-malonylglycosides of epigenin and luteolin, as well as rutin, schaftoside, and isoschaftoside. Studies on A. lanulosa and A. asiatica have shown the presence of alkamides, which are different from those isolated from European A. millefolium complex indicating an independent line of evidence for a relationship. Proline, stachydrine, betaine, betaconine are also isolated from the aerial parts of yarrow. The herb is also identified for the presence of polyacetylenes, amides, sterols, tannins and coumarins (Chandler et al., 1982; Rauchensteiner et al., 2004; Falconieri et al., 2011; Saeidnia et al., 2011).

19.4.3. Bioactivity of A. millefolium—Anti-inflammatory and Anti-acne effects Anti-inflammatory effects: The use of A. millefolium has been a long tradition especially in the treatment of gastrointestinal and hepato-biliary disorders. It has also been used for skin inflammation and for healing wounds (Willuhn, 2002). It is one of the oldest known botanicals used by humans. Due to the complex chemical composition, yarrow possesses a variety of biological activities. Some of them include wound healing, respiratory infections, anti-hemorrhagic, anti-microbial, anti-inflammatory, and gastrointestinal infections (Akram, 2013). The major compounds in yarrow that contribute to the anti-inflammatory properties include phenolic acids, tannins, flavonoids, essential oil, and sesquiterpenoids lactones. Sesquiterpenes are known to cause anti-inflammation by inhibition of arachidonic acid metabolism (Kastner et al., 1993). Antiphlogistic activities were seen in fractions from Achillea enriched with flavonoids and dicaffeoylquinic acids. The anti-inflammatory mechanism of this extract included inhibition of human neutrophil elastase known to be associated with inflammatory process at IC50 values of 20 μg/mL and dicaffeoylquinic acids at IC50 of 72 μg/mL. The in vitro anti-inflammatory activity was also established through the inhibition of matrix metalloproteinases (MMP-2 and -9). These proteases are involved in psoriasis and atopic dermatitis and in inflammatory bowel diseases such ulcerative colitis and Crohn’s disease (Wiedow et al., 1992; Baugh et al., 1999). Azulenes, which comprises almost half of yarrow’s chemical composition, is a powerful anti-inflammatory phytochemical. It is recommended that three cups a day of Achillea tea prepared with 1.5 g crude drug, equal to 900 mg extract, would cause the anti-inflammatory effect. In our laboratory, we studied A. “Moonshine”, a hybrid, ornamental plant between A. clypeolata 3 A. “Taygetea”. It was introduced by Alan Bloom of the Bressingham Gardens in England in the 1950s. It is one of the most popular yarrows in American gardens. The plant is 12 ft in height with gray-green foliage and flattopped, dense inflorescence which is lemon yellow and 23ʹ in diameter. The flowers appear in early June and continue until September. A. Clypeolata is one of the parent species endemic to Romania and Bulgaria and native to the Balkan Peninsula. While the other parent species, A. aegyptiaca (A. taygetea Boiss & Heldr or Egyptian yarrow) is native to Europe; however, it came from Egypt. Anti-acne effect: There have been no scientific reports on use of A. “Moonshine” for cosmetics. Our research study screened this plant for its anti-acne activity and characterized the compounds present in Achillea “Moonshine” that contribute to the anti-acne activity (Shah et al., 2015). Anti-acne activity of any plant extract should consider treating most of the causative effects of acne. In our study, we determined the anti-bacterial activity of A. “Moonshine” extracts against the two acne-causing organisms (P. acnes and S. epidermidis), free radical scavenging activity, anti-tyrosinase, anti-inflammatory potential as well as cytotoxicity against human skin cells to identify the most potent extract possessing antiacne activity (Shah et al., 2015). A. “Moonshine” was extracted in four solvents: petroleum ether, ethyl acetate, ethanol and water. The most promising activity was determined in the petroleum ether extract. The minimum inhibitory concentration (MIC) value for the petroleum ether extract was 0.83 mg/mL against P. acnes and 0.37 mg/mL against S. epidermidis. The IC50 values for the petroleum ether extract for free radical scavenging activity and tyrosinase inhibition was 64.81 ug/mL and 0.033 mg/mL respectively (Shah et al., 2015). The extract was also able to decrease the inflammatory cytokines like TNF-α and IL-8 in a dose-dependent manner and showed no cytotoxicity against dermal fibroblasts. These results suggested the presence of active anti-acne phytochemicals, making it a novel plant candidate for the treatment of acne. The petroleum ether extract was further fractionated by column chromatography and one active compound was isolated.

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19.5. THE MOLECULAR BASIS FOR THE ANTI-ACNE ACTIVITY P. acnes are gram-positive anerobic bacteria that thrive in pilosabeaceous units. The bacterium drives inflammatory responses which initiates acne pathogenesis. P. acnes secreted lipases digest sebum triglycerides into fatty acid that leads to production of anti-microbial peptides such as Human defensin 1 and 2, cathelicidin and granulysin (Dessinioti et al., 2010). Toll-like receptors (TLR2 and TLR4) are pattern recognition receptors that activate immune response, are also seen to be activated by P. acnes, which leads to production of chemokines and cytokines such as IL-1, IL-6, IL-8, and TNFα (Jugeau et al., 2005). These cytokines induce metalloproteinases (MMP) which play a role in inflammation, dermal matrix destruction and hyperpigmentation post-acne treatment. A. “Moonshine” extracts were able to decrease the inflammatory cytokines like TNF-α and IL-8 in a dosedependent manner. The petroleum ether extract of A. “Moonshine” showed an increase in TNF-α and IL-8 production. Cocultures of cells with bacteria and extracts (50 μg/mL) suppressed the production of these cytokines (Shah et al., 2015). There was an elevated level of TNF-α cytokine production in 100 μg/mL of extracts which was also observed in other studies (Kim et al., 2008). Hyper-keratinization is promoted by P. acnes by activating production of integrin (cell adhesion protein and flaggrin which is mostly observed in the sebaceous duct and infundibulum). P. acnes acquires resistance to antibiotics by producing biofilm lining that improves its adherence, thus promoting hyper-keratinization. Hence, it is important to have an extract that has high potency for P. acnes, which was demonstrated, by the petroleum ether extracts from A. “Moonshine” (Shah et al., 2015).

19.6. ISOLATION OF ANTI-ACNE COMPOUNDS FROM A. “MOONSHINE” To isolate and identify the active phytochemicals, various analytical techniques were incorporated such as column chromatography, thin layer chromatography (TLC), TLC bioautography, HPLC, mass spectrometry and NMR (Shah et al., 2016). Using the column chromatography technique, the contents of the petroleum ether extract were first separated. Subsequently, the fractions showing same TLC profiles were pooled together. To know which of these fractions had potent antiacne activity, TLC bioautography against P. acnes was conducted. The fractions that showed zones of inhibition for growth of P. acnes in TLC bioautography assay were fractionated using HPLC. HPLC allowed separation of the fractions into individual peaks. The peaks were collected and tested again for their anti-microbial activity by TLC bioautography. The active compounds collected were then subjected to mass spectrometry and NMR for structure elucidation. Of the two compounds isolated, one compound was identified as N-(21-hydroxy-21-(piperidin-1-yl) henicosa-17, 19-diyl-1-yl) acetamide. This compound belongs to the class—Alkamides. Using NMR and mass spectrometry, the compound isolated from bioactivity-guided fractionation contributing to the anti-P. acnes was proposed as an alkamide named N-(21-hydroxy-21-(piperidin-1-yl) henicosa-17, 19-diyl-1-yl) acetamide.

19.7. IDENTIFICATION AND CHARACTERIZATION OF THE NOVEL ALKAMIDE The novel antiacne alkamide has a UV λ max 253nm, FT-IR (hexane) Vmax 3428.53, 2088.85, 1643.92 cm21. The High Resolution ESI Mass Spectrometry measured a mass of 445.12 m/z (calculated for C28H47NO3 445.356) (Shah et al., 2016). Structurally, natural alkamides commonly have an aliphatic, cyclic or aromatic amine residue, and a C-8 to C18 saturated or unsaturated chain (including double or triple bonds, or both) acid, which can also be aromatic. The nature of the acid (carbon chain lengths, unsaturation level, stereochemistry, etc.) and the amine residues are characteristic of each family and genus of plants such that these characteristics serve as chemotaxonomic criteria. The typical structure of the alkamides is known to contribute to the anti-microbial, anti-viral, larvicidal, insecticidal, diuretic, pungent, analgesic, cannabimimetic and antioxidant activities. (Rios, 2012). The genus Achillea is rich in olefinic and acetylenic pyrrolidides, piperidides, and the corresponding dehydro derivatives (piperideides, pyrrolideides) which are basically alkamides with five- or six-membered cyclic or aromatic rings with nitrogen. The distribution of cyclic amides is restricted to tribe Anthemidae. Most polyacetylenic cyclic amides

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found in this tribe consist of pyrrolidides and piperidides whereas polyacetylenic piperideides are rare. (Bohlman, 1988; Bohlmann et al., 1973; Christensen, 1992; Greger, 1988, 1984).

19.8. COMPARISON OF OTHER ANTI-ACNE SOURCES TO THE NOVEL ALKAMIDE FROM A. “MOONSHINE” Plant-derived remedies are gaining increasing popularity for acne treatment. Both topical and oral formulations consisting of plants and herbs are investigated for acne treatment. Many patients turn to these alternative therapies for acne management when the conventional treatments fail to cure acne. These are also sometimes used as adjuvant therapies along with the traditional treatments. Some of the common plants used for acne treatment and management include Aloe vera, Azadirachta indica, Curcuma longa, Hemidesmus incidus, Gossypium barbadense, essential oils of Eucalyptus radiate, and Melaleuca alternifolia and many others (Kanlayavattanakul and Lourith, 2011). There are many options available to incorporate these botanicals into routine acne care. These include monotherapy in the form of creams and facial washes, incorporation into cosmetics to allow continuous application and adjuvant therapy to increase the efficacy of standard treatments. Such options continue to appeal to patients because of their safety and high efficacy. Though there are several herbal remedies, there is a constant search for the specific phytochemicals that are responsible for the beneficial effects of plant extracts (Fisk et al., 2014). None of the plants mentioned have a specific compound or group of compounds identified that could be chemically synthesized. The novel alkamide isolated from petroleum ether extract of yarrow is easy to synthesize in a laboratory setting, increasing the possibility of using it as a drug. Modifications on this compound and its increased efficacy to treat acne could be tested. The knowledge of the structure of a novel anti-acne compound also aids in the synthesis on an industrial scale. Also, hydroalcohol extracts of A. millefolium (500 mg/kg) significantly reduced pain in the abdomen of rats, hence, the extracts also have pain-relieving attributes that supplement its use for wounds and acne.

19.9. CONCLUSIONS Antibacterial activity against P. acnes and S. epidermidis was observed in methanol, diethyl ether and petroleum ether extract of A. “Moonshine” (Shah et al., 2015). However, the petroleum ether extract showed more potency to P. acnes combined with present anti-inflammatory compounds that are vital for the treatment of acne. In other published studies extracts from Achillea has been successfully demonstrated to act as analgesic, anti-ulcer, choleretic, hepatoprotective, and in healing wounds. The diversity and complexity of the phytochemicals found in yarrow are responsible for the broad spectrum of the medicinal activity. The pharmacological effects are mainly due to its oil, proazulenes, sesquiterpene lactones, flavonoids, and dicaffeoylquinic acids. A study in my laboratory has also isolated a novel alkamide that shows anti-microbial activity against organisms that cause acne. Within the Achillea species the challenge has been to categorize medicinal traits to specific variety due to lack of an accepted taxonomical nomenclature. Yarrow, though known to be safe for topical use, has shown to cause allergic reactions in some individuals. The German Commission has approved A. millefolium extract as a biological additive in cosmetic products. Yarrow has been reportedly used in 65 cosmetic formulations at a concentration from 0.5%2%. Extracts from Achillea are not irritating to skin, which was shown in a clinical test that use of 0.1% extract was not an irritant. Traditionally yarrow has been used to stimulate uterine contractions. To confirm its traditional use as an abortifacient, rats were administered 56 times the human dose and it was shown to reduce fetal weight and increase placental weight (Boswell-Ruys et al., 2003). However, extracts from Achillea have been used externally in the form of a sitz bath, to compress skin inflammation, to heal wounds and treat bacterial infections. The review here provides details on the advances in the phytochemistry and pharmacological aspects of Achillea as anti-inflammatory and anti-acne medicine.

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Christensen, L.P., 1992. Acetylenes and related compounds in Anthemideae. Phytochemistry. 31 (7). Dessinioti, C., Katsambas, A.D., 2010. The role of Propionibacteriumacnes in acne pathogenesis: facts and controversies. Clin. Dermatol. 28 (1), 27. Eichenfiled, L.F., Jarrat, M., Schlessinger, J., et al., 2010. Adapalene 0.1% lotion in the treatment of acne vulgaris: results from two placebocontrolled, multicenter, randomized double-blind, clinical studies. J. Drugs. Dermatol. 9, 639646. Falconieri, D., Piras, A., et al., 2011. Chemical composition and biological activity of the volatile extracts of Achillea millefolium. Nat. Prod. Commun. 6 (10), 15271530. Fisk, W.A., Lev-Tov, H.A., et al., 2014. Botanical and phytochemical therapy of acne: a systematic review. Phytotherapy Res. 28 (8), 11371152. Greger, H., 1984. Alkamides: structural relationships, distribution and biological activity. Planta. Med. 50, 366. Greger, H., 1988. Comparative phytochemistry of the alkamides. In: Lam, J. et al., Eds., Chemistry and Biology of Naturally-Occurring Acetylenes and Related Compounds (NOARC) Bioactive Molecules 7, 159178. Harper, J.C., 2004. An update on the pathogenesis and management of acne vulgaris. J. Am. Acad. Dermatol. 51 (1, Supplement), 3638. Jeremy, A.H., Holland, D.B., Roberts, S.G., et al., 2003. Inflammatory events are involved in acne lesion initation. J. Invest. Dermatol. 121, 2027. Jugeau, S., Tenaud, I., Knol, A.C., Jarrousse, V., Quereux, G., Khammari, A., et al., 2005. Induction of toll-like receptors by Propionibacteriumacnes. Br. J. Dermatol. 153 (6), 11051113. Kanlayavattanakul, M., Lourith, N., 2011. Therapeutic agents and herbs in topical application for acne treatment. Int. J. Cosmet. Sci. 33 (4), 289297. Kastner, U., Sosa, S., Tubaro, A., Breuer, J., Ru¨cker, G., Della Loggia, R., et al., 1993. Anti-edematous activity of sesquiterpene lactones from different taxa of the Achillea millefolium group. Planta Med. 59, A699. Kim, S., Kim, J.Y., Lee, N., Hyun, C.G., 2008. Antibacterial and anti-inflammatory effects of Jeju medicinal plants against acne-inducing bacteria. J. Gen. Appl. Microbiol. 54 (2), 101106. Layton, A.M., Morris, C., Cunliffe, W.J., Ingham, E., 1998. Immunohistochemcial investigation of evolving inflammation in lesions of acne vulgaries. Exp. Dermatol. 7, 191197. Liu, P.T., Krutzik, S.R., Kim, J., Modlin, R.L., 2005. Cutting edge:all-trans retinoic acid down-regulates TLR2 expression and function. J. Immunol. 174, 24672470. Rauchensteiner, F., Nejati, S., et al., 2004. The Achillea millefolium group (Asteraceae) in Middle Europe and the Balkans: a diverse source for the crude drug Herba Millefolii. J. Tradit. Med. 21 (3), 113119. Rios, M., 2012. Natural alkamides: pharmacology, chemistry and distribution. Drug Discovery Research in Pharmacognosy, Prof. Omboon Vallisuta (Ed.). Saeidnia, S., Gohari, A.R., et al., 2011. A review on phytochemistry and medicinal properties of the genus Achillea DARU. J. Facul. Pharmacy Tehran Univer. Med. Sci. 19 (3), 173186. Sanderson J.B. (1871). A system of Surgery. London Longmans: Green and Co, 2. Shah, R., Patel, A., et al., 2015. Anti-acne activity of Achillea ‘Moonshine’ petroleum ether extract. J. Med. Plant Res. 9 (27), 755763. Shah, R., Patel, T., Tettamanzi, M.C., Rajan, J., Shah, M., Peethambaran, B., 2016. Isolation of a novel piperidide from Achillea ‘Moonshine’ using bioactivity guided fractionation for the treatment of acne. J. Med. Plant Res. 10 (30), 495504. Spector, W.G., Willoughby, D.A., 1963. The inflammatory response. Bacteriol. Rev. 27, 117149. Tanghetti, E.A., 2013. The role of inflammation in the pathology of acne. J. Clin. Aesth. Dermatol. 6 (9), 2735. Tenaud, I., Khammari, A., Dreno, B., 2007. In vitro modulcation of TLR-2, Did and IL-10 by adapalene on normal human skin and acne inflammatory lesions. Exp. Dermatol. 16, 500506. Trivedi, N.R., Gilliland, K.L., Zhao, W., et al., 2006. Gene array expression profiling in acne lesions reveals marked upregulation of genes involved in inflammation and matrix remodeling. J. Invest. Dermatol. 126, 10711079. Wiedow, O., Wiese, F., Streit, V., Kalm, C., Christophers, C., 1992. Lesional elastase activity in psoriasis, contact dermatitis, and atopic dermatitis. J. Invest. Dermatol. 99, 306309. Willuhn, G., 2002. Millefolii herba. In: Wichtl (Ed.), Teedrogen und Phytopharmaka. pp. 399400.

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C H A P T E R

20 Lactobacillus Gasseri Potentiates Immune Response Against Influenza Virus Infection Jun Nishihira1, Mie Nishimura1, Tomohiro Moriya2, Fumihiko Sakai2, Toshihide Kabuki2 and Yoshihiro Kawasaki2 1

Hokkaido Information University, Ebetsu, Japan 2Megmilk Snow Brand Co. Ltd., Saitama, Japan

20.1. INTRODUCTION 20.1.1. General Aspects of Probiotics Focusing on Immunity 20.1.1.1. Interaction of Microbiota and Probiotics in the Intestinal Immune System The intestinal microbiota consist of more than 1000 species with a collective weight of about 1 kg in the human intestine; their colonization begins immediately after birth (O’Hara and Shanahan, 2006). Among them, symbiotic bacteria benefit the host by providing a nutrient supply, defending against pathogens, and aiding in the development and function of the intestinal immune system. During the past few years, it has been well documented that the intestinal microbiota can be positively modulated by the administration of bacteria or bacterial substrates, which might lead to a significant modulation of the immune system (Sanchez et al., 2015; Scott et al., 2015). To date, a substantial number of studies have been performed using probiotics as potential modulators of the intestinal microbial community. Probiotics are defined as live microorganisms that confer a health benefit on the host when administered in adequate amounts (FAO/WHO, 2002). Probiotic bacteria, which mainly belong to the class of lactic acid bacteria (LAB), are beneficial for both human and animal health. In particular, lactobacilli, which produce lactic acid, are commonly applied when fermenting many vegetables, meats, and dairy products. These bacteria can affect the composition and activity of the intestinal microbiota. Currently, there is a general consensus that orally administrated probiotic bacteria contribute to immune homeostasis by adjusting the microbial balance or by interacting with the host immune system (Corthe´sy et al., 2007; Matsuzaki et al., 2007; Ouwehand, 2007). However, not all probiotic strains have the ability to modulate the immune system in humans, even when the immunomodulatory functions of the strains have been confirmed by extensive in vitro or in vivo mouse model studies (Rask et al., 2013; Van Puyenbroeck et al., 2012). In this chapter, the authors introduce a general view of probiotics and discuss the effect of the oral administration of yogurt containing LAB, particularly Lactobacillus gasseri SBT2055, on boosting vaccine-specific antibody responses in humans. The future direction of probiotic studies involving the intestinal immune system is then discussed.

20.2. IMMUNOGENIC ROLE OF PROBIOTICS AGAINST INFLUENZA VIRUS INFECTION The human immune response is composed of innate immunity that functions as the body’s first line of defense, and adaptive immunity, which refers to the acquired immune response or specific immunity. Innate Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00020-2

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Copyright © 2018 Elsevier Inc. All rights reserved.

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immunity consists of the recruitment of neutrophils, and natural killer (NK) cells that then release cytokines and chemokines. Indeed type I interferon (IFN) and interleukins (IL) -1, -6, -12, and 16 are representative of innate cytokine and innate lymphoid cells that play roles in innate immunity against virus-infected cells, respectively. The influenza virus is a major cause of severe respiratory tract infection. Influenza epidemics occur almost every winter, and the social and economic damage caused by influenza pandemics is substantial. The search for safe and cost-effective alternatives to reduce the risk of influenza virus infection is ongoing. Among various potential candidates, the use of probiotics is one possible way to prevent influenza virus infection. In this context, our group recently conducted a clinical trial in humans to confirm the effectiveness of a probiotic strain in improving immune function (Nishihira et al., 2016). One reliable method for assessing immune function in healthy subjects is a vaccine challenge, in which the guidelines on scientific requirements in the form of European Food Safety Authority Panel are included for health claims related to gut and immune function (European Food Safety Authority Panel on Dietetic Products, Nutrition and Allergies, 2011).

20.3. CLINICAL INTERVENTION WITH PROBIOTICS TO BOOST IMMUNE RESPONSE 20.3.1. Clinical Trial to Prove Enhanced Immunity With the Intake of LAB A series of mouse studies demonstrated that oral administration of LG2055 conferred a protective effect against influenza A virus infection through the induction of antiviral genes by type I IFN signaling (Nakayama et al., 2014) and IgA production (Sakai et al., 2014). We thus performed human trials to evaluate the effects of the probiotic Lactobacillus gasseri SBT2055 (LG2055) in boosting immune responses in healthy vaccinated subjects that received a trivalent influenza vaccine (Nishihira et al., 2016). LG2055 was originally isolated by the Milk Science Research Institute, Megmilk Snow Brand Co., Ltd. (Tokyo, Japan). Subjects were randomly divided into two groups, an active yogurt (LG2055) group and a control yogurt (placebo) group. All of the subjects consumed 100 g/day of either the active yogurt or control yogurt for 16 weeks. A blood sample was collected at week 0 (base line) before the intake of the first study yogurt. After a pre-vaccination period of four weeks, all of the subjects were administered the trivalent influenza vaccine against influenza virus A/H1N1, A/H3N2, and B/BX-51B. Virus quantitation was carried out by monitoring hemagglutination inhibition (HI) titers and immune response readout was via detection of natural killer cell activity and myxovirus resistance A (Mxa) gene expression, which is one of the antiviral genes stimulated by type I or type III Interferons in peripheral blood mononuclear cells. We found that HI antibody titers against A/H1N1 and B were significantly higher in the LG2055 group compared with the placebo group (Table 20.1). The seroprotection rate (HI titer $ 1:40) against B at week 11 was also significantly higher in the LG2055 group than in the placebo group (data not shown). On the other hand, there were no differences in the seroconversion rate (HI titer $ 4-fold increase) between the placebo group and the LG2055 group. From these data, we concluded that LG2055 was effective in potentiating the vaccine against at least A/H1N1 and B.

20.3.2. Immunoglobulin Profiles in Response to LAB Total immunoglobulin G (IgG) in plasma is an important biomarker for assessing immune function in healthy subjects. We found that the IgG level in plasma was higher in the LG2055 group than in the placebo group (Fig. 20.1A). Notably, the effect of LG2055 intake on plasma IgG was greater in subjects whose IgG levels were relatively low than in subjects whose IgG levels were relatively high (Fig. 20.1B, 20.1C). These data indicate that LG2055 intake is more effective in increasing plasma IgG levels in subjects with low plasma IgG. As for IgA, it is well recognized that it plays important roles in host defense against mucosally transmitted pathogens by preventing commensal bacteria from binding to epithelial cells and neutralizing toxins to maintain homeostasis at mucosal surfaces (Fagarasan, 2008). In our clinical trial, LG2055 intake not only increased plasma IgA (Fig. 20.2A) but also stimulated sIgA in saliva (Fig. 20.2B). The authors hypothesized that LG2055 stimulates humoral immunity, as indicated by the production of IgG in plasma and IgA in mucosal tissues. As for the mechanism of the increased antibody production in response to LG2055, we reported previously that LG2055 stimulated the production of cytokines from dendritic cells and IgA production from B cells, which

V. NUTRACEUTICALS IN BOOSTING IMMUNE SUPPORT AND AS THERAPEUTICS FOR INFLAMMATORY DISEASES

TABLE 20.1

Change in the HI Antibody Titers to Influenza Vaccine During the Study Period HI antibody titers

Antigen

Group

W0

W4 vaccination

W7

W11

W16

A/H1N1

Placebo

15.1

15.3

84.2

63.7

53.1

(95% CI)

(12.218.7)

(12.418.9)

(67.6104.9)

(51.479.4)

(42.167.0)

LG2055

20.6

21.1

115.7

87.5

66.5

(95% CI)

(16.425.8)

(16.626.7)

(91.1146.8)

(70.2109.1)

(53.083.5)

Placebo

41.5

34.3

132.1

145.4

142.0

(95% CI)

(31.854.1)

(26.244.8)

(102.0171.1)

(112.1188.7)

(109.7183.9)

LG2055

43.4

34.0

138.1

151.9

133.1

(95% CI)

(33.755.9)

(26.244.2)

(108.2176.2)

(117.7196.0)

(103.9170.5)

Placebo

13.1

12.9

28.1

27.3

26.0

A/H3N2

B





(95% CI)

(11.315.3)

(10.915.1)

(24.432.4)

(23.831.3)

(22.629.82)

LG2055

15.0

16.9

33.3

34.0

29.1

(95% CI)

(12.717.7)

(14.320.0)

(28.239.2)

(29.239.5)

(24.834.2)

The number is the geometric mean of the HI antibody titers, with the 95% confidence interval in parentheses. comparisons.

(B)

(C)

60

80

# 60 40

20

## #

140

Δ Plasma IgG (mg/dL)

Δ Plasma IgG (mg/dL)

Δ Plasma IgG (mg/dL)

#

120 100 80 60 40

0

0

–20 W7

20 0 –20 –40

–80

W0

W16

40

–60

20

W0

P , 0.05, MannWhitney U test between-group

160

(A)

100



W7

W16

W0

W7

W16

LG2055

Placebo

FIGURE 20.1 Change in total IgG levels in plasma from the baseline (W0). (A) Data represent total subjects (placebo: n 5 94, LG2055: n 5 94), (B) low-IgG-layer subjects with less than the median value (1209 mg/dL) (placebo: n 5 49, LG2055: n 5 45), and (C) high-layer subjects with more than the median value (placebo: n 5 45, LG2055: n 5 49). Data are presented as means 6 standard error for the LG2055 group (closed circle) and placebo group (open circle), #: P , 0.05, ##: P , 0.01, t test, between-group comparisons. (B) 14

#

Δ Plasma IgA (mg/dL)

12

P=0.0536 Δ salivary IgA rate (μg/min)

(A)

10 8 6 4

2 0 –2 –4

0 –10 P=0.070

–20 –30 –40 –50

W0

W7

W16

Placebo

W0

W7

W16

LG2055

FIGURE 20.2 Total IgA in plasma and secretory IgA in saliva. (A) Changes in the plasma total IgA concentration from the baseline (W0). (B) Changes in the sIgA secretion rate in saliva from the baseline (W0). Data are presented as means 6 standard error for the LG2055 group (closed circle) and placebo group (open circle). #: P , 0.05, between-group comparisons by t test.

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20. LACTOBACILLUS GASSERI POTENTIATES IMMUNE RESPONSE AGAINST INFLUENZA VIRUS INFECTION

strongly indicates that the Toll-like receptors (TLR)-2 signal is critical for this stimulation (Sakai et al., 2014). Consistent with our results, another research institute reported that LAB stimulates cytokine production via TLRs on macrophages and dendritic cells (Weiss et al., 2010). Taken together, these results suggest that LG2055 helps augment nonspecific and vaccine-specific antibody production by functioning as an adjuvant.

20.3.3. Enhancement of NK Cell Activity and Antiviral Activity by LAB Several reports have shown that the administration of LAB stimulates NK cell activity (Namba et al., 2010; Makino et al., 2010; Nishimura et al., 2015). A previous study showed that daily intake of Lactobacillus casei Shirota (LcS) increased NK cell activity, and the effects of LcS were remarkable in subjects with low baseline NK cell activity (Takeda and Okumura, 2007). Moreover, LcS intake prevented smoking-induced NK activity reduction (Reale et al., 2012), and the dietary intake of yogurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 increased NK cell activity in subjects with low baseline NK cell activity (Makino et al., 2010). In our clinical trial, we reported that the change in NK cell activity from the baseline (W0) to week 7 in the LG2055 group was significantly greater than that the placebo group (Fig. 20.3A). Of note, in the low NK cell activity layer at the baseline, the LG2055 group was significantly higher at week 7 and showed a strong tendency toward higher levels at week 16 than the placebo group (Fig. 20.3B). As for the higher NK cell activity layer, the group-by-time interaction and group-effect were not statistically significant (data not shown). In within-group (A)

(B) 12

#

6.0

#

10 Δ NK activity (%)

NK activity (%)

4.0 2.0 0.0 –2.0

8 6 4 2

P=0.084

0 –4.0 –6.0

-4 W0

W7

2

Δ NK activity (%)

W7

W16

(D)

4

0 –2 –4 –6 –8 –10

W0

W16

Relative MxA gene expression (fold)

(C)

–2

W0

W7

W16

Placebo

2.5

∗∗

2.0

##



1.5 1.0 0.5 0.0

W0

W7

W16

LG2055

FIGURE 20.3 Changes in NK cell activity and Myxovirus resistance protein A (MxA) gene expression. Changes in NK cell activities are shown in figures (A), (B), and (C). Data are presented as (A) “total subjects” (placebo: n 5 94, LG2055: n 5 94), (B) “low-activity-layer” subjects with less than the median value of NK cell activity at W0 (34%) (placebo: n 5 39, LG2055: n 5 53), (C) “high-activity-layer” subjects with the median value or greater than the median value (placebo: n 5 55, LG2055: n 5 41). In (D), Myxovirus resistance protein A (MxA) gene expression in PBMC is shown. qRT-PCR was performed using total RNA from PBMC (placebo: n 5 38, LG2055: n 5 38). Relative MxA gene expression was normalized by β-actin gene expression. All data are presented as means 6 standard error for the LG2055 group and placebo group. #: P , 0.05, ##: P , 0.01, between-group comparisons by t test.  : P , 0.05,  : P , 0.01, within-group comparisons by Bonferroni multiple comparisons test.

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20.4. PERSPECTIVES FOR RESEARCH ON PROBIOTICS IN THE IMMUNE SYSTEM

253

comparisons, the LG2055 group and the placebo group at week 16 both showed significant decreases from the baseline (Fig. 20.3C). Taken together, these findings suggest that increases in NK cell activity following the administration of LG2055 are likely to be more effective in subjects with low NK cell activity. IFN-induced human myxovirus resistance protein A (MxA) exhibits broad-spectrum antiviral activity, including against influenza A virus (Haller and Kochs, 2011). The expression of MxA is widely used as a surrogate marker for IFN activity in various experimental and clinical settings (Haller and Kochs, 2011). Moreover, MxA is known as an important component of the early innate immune defense in humans. To date, there have been many reports suggesting that the administration of LAB stimulates innate immunity, e.g., type I IFN production (Arimori et al., 2012; Sugimura et al., 2013). Consistent with these reports, we demonstrated that LG2055 raised the level of MxA gene expression at week 16 compared with the placebo (Fig. 20.3D). In within-group comparisons, the LG2055 group at weeks 7 and 16 showed significant increases in MxA gene expression from the baseline. Based on these data, LG2055 can be said to increase- MxA gene expression through the induction of type I or III IFN production.

20.4. PERSPECTIVES FOR RESEARCH ON PROBIOTICS IN THE IMMUNE SYSTEM 20.4.1. Molecular Mechanism by Which LAB Potentiates Immune Response It is generally accepted that the intestinal microbiota shapes the gut immune response in times of health and disease in various ways, such as through host genetics, lifestyle, and early colonization at birth. Dysbiosis, i.e., having a disordered microbiota population, is caused by cytokine imbalance among immune cells exemplified by effector T helper (Th) cells consisting of Th1, Th2, and Th17 cells. The increased incidence of immune-mediated disorders, e.g., atopic dermatitis and asthma, in developed countries could be due to alterations in the microbiota (O’Hara and Shanahan, 2006). Probiotics act on several cell types, including epithelial cells, dendritic cells, and T-cells, and have the ability to limit inflammation through the induction of regulatory T-cells (Treg). The molecular mechanism for the amelioration of bowel disorders involves the induction of interleukin-10 and transforming growth factor-β-expressing T cells, the induction of Treg, and the inhibition of nuclear factor-kβ activation (Round and Mazmanian, 2009) The chemical composition of the molecular effectors in probiotics is very diverse and consists of proteins that are secreted into the extracellular milieu or localized on the surface of the bacteria, as exemplified by lowmolecular-weight peptides, amino acids, cell-wall polysaccharides or components, bacterial DNA, or short-chain fatty acids (Macpherson and Harrisk, 2004; Turroni et al., 2013). Probiotics, such as LG2055, may exert their Hypothetical mechanisms for enhancement of innate and adaptive immunity

Target cells Dendritic cell or Macrophage

LG2055

TLR2 or TLR9

Mediators

Phenotypes

TGF-β

Antibody production

IL-6 IL-10

Plasma cell

Type I Interferon TLR2

Antiviral gene expression

? Naïve B cell

NK cell activity

FIGURE 20.4 Proposed mechanisms for enhancement of innate and adaptive immunity.

V. NUTRACEUTICALS IN BOOSTING IMMUNE SUPPORT AND AS THERAPEUTICS FOR INFLAMMATORY DISEASES

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20. LACTOBACILLUS GASSERI POTENTIATES IMMUNE RESPONSE AGAINST INFLUENZA VIRUS INFECTION

beneficial properties in a broad range of ways via direct cell-to-cell contact in the human gut, the secretion of diverse molecules that act as the final mediators of probiotic crosstalk, or through cross-feeding mechanisms. The precise mechanism of action, however, has been scarcely elucidated, thus further investigation is required.

20.4.2. Clinical Application of Probiotics for Disease Prevention It is well recognized that influenza virus infection is highly dangerous to specific populations such as pregnant women, diabetes patients, infants, and the elderly. In addition, immune responses are weakened by specific lifestyle attributes, such as obesity (Smith et al., 2009) and stress (Locke et al., 1984). Overall, maintaining robust innate and adaptive immunity is thought to be important for the prevention of a variety of diseases, particularly acute infectious illnesses, such as influenza virus infection. Thus, it is highly significant to show the potential of probiotic LG2055 against influenza virus infection through both innate and adaptive immunity. Our current hypothesis is shown in Fig. 20.4. It would be worthwhile to assess the occurrence of any similar effect even without vaccination in a large-scale randomized control trial investigation.

Acknowledgments The authors would like to thank Ms. Aiko Tanaka for the preparation of this manuscript, and to the staff of the Health Information Science Center, Hokkaido Information University for making the clinical trial of LG2055 successful.

References Arimori, Y., Nakamura, R., Hirose, Y., Murosaki, S., Yamamoto, Y., Shidarak, O., et al., 2012. Daily intake of heat-killed Lactobacillus plantarum L-137 enhances type I interferon production in healthy humans and pigs. Immunopharmacol. Immunotoxicol. 34, 937943. Corthe´sy, B., Gaskins, H.R., Mercenier, A., 2007. Cross-talk between probiotic bacteria and the host immune system. J. Nutr. 137, 781S790S. European Food Safety Authority Panel on Dietetic Products, Nutrition and Allergies, 2011. Guidance on the scientific requirements for health claims related to gut and immune function. EFSA J. 9, 19841995. Fagarasan, S., 2008. Evolution, development, mechanism and function of IgA in the gut. Curr. Opin. Immunol. 20, 170177. FAO/WHO, 2002. Guidelines for evaluation of probiotics in food: report of a joint FAO/WHO working group on drafting guidleines for the evaluation of probiotics in food. London, Ontario, Canada. Haller, O., Kochs, G., 2011. Human MxA Protein: an interferon-induced dynamin-like GTPase with broad antiviral activity. J. Int. Cytokine Res. 31, 7987. Locke, S.E., Kraus, L., Leserman, J., Hurst, M.W., Heisel, J.S., Williams, R.M., 1984. Life change stress, psychiatric symptoms, and natural killer cell activity. Psychosom. Med. 46, 441453. Macpherson, A.J., Harrisk, N.L., 2004. Interactions bewtween commensal intestinal bacteria and the immune system. Nat. Rev. Immununol. 4, 478485. Makino, S., Ikegami, S., Kume, A., Horiuchi, H., Sasaki, H., Orii, N., 2010. Reducing the risk of infection in the elderly by dietary intake of yoghurt fermented with Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1. Br. J. Nutr. 104, 9981006. Matsuzaki, T., Takagi, A., Ikemura, H., Matsuguchi, T., Yokokura, T., 2007. Intestinal microflora: probiotics. J. Nutr. 137, 798S802S. Nakayama, Y., Moriya, T., Sakai, F., Ikeda, N., Shiozaki, T., Hosoya, T., et al., 2014. Oral administration of Lactobacillus gasseri SBT2055 is effective for preventing influenza in mice. Sci. Rep. 4, 46384642. Namba, K., Hatano, M., Yaeshima, T., Takase, M., Suzuki, K., 2010. Effects of Bifidobacterium longum BB536 administration on influenza infection, influenza vaccine antibody titer, and cell-mediated immunity in the elderly. Biosci. Biotechnol. Biochem. 74, 939945. Nishihira, J., Moriya, T., Sakai, F., Kabuki, T., Kawasaki, Y., Nishimura, M., 2016. Lactobacillus gasseri SBT2055 stimulates immunoglobulin production and innate immunity after influenza vaccination in healthy adult volunteers. J. Funct. Foods Health Dis. 6, 544568. Nishimura, M., Ohkawara, T., Tetsuka, K., Kawasaki, Y., Nakagawa, R., Satoh, H., et al., 2015. Effects of yogurt containing Lactobacillus plantarum HOKKAIDO on immune function and stress markers. J. Trad. Complement. Med. 6, 275280. O’Hara, A.M., Shanahan, F., 2006. The gut flora as a forgotten organ. EMBO. Rep. 7, 688693. Ouwehand, A.C., 2007. Antiallergic effects of probiotics. J. Nutr. 137, 794S797S. Rask, C., Adlerberth, I., Berggren, A., Ahre´n, I.L., Wold, A.E., 2013. Differential effect on cell-mediated immunity in human volunteers after intake of different lactobacilli. Clin. Exp. Immunol. 172, 321332. Reale, M., Boscolo, P., Bellante, V., Tarantelli, C., Di Nicola, M., Forcella, L., et al., 2012. Daily intake of Lactobacillus casei Shirota increases natural killer cell activity in smokers. Br. J. Nutr. 108, 308314. Round, J.L., Mazmanian, S., 2009. The gut microbiome shapes intestinal immune responses during health and disease. Nat. Rev. Immununol. 9, 313323. Sakai, F., Hosoya, T., Ono-Ohmachi, A., Ukibe, K., Ogawa, A., Moriya, T., et al., 2014. Lactobacillus gasseri SBT2055 induces TGF-β expression in dendritic cells and activates TLR2 signal to produce IgA in the small intestine. PLoS. ONE. 9, e105370e105380. Sanchez, B., Gueimonde, M., Pena, A.S., Bernardo, D., 2015. Intestinal micorobiota as modulators of the immune system. J. Immunol. Res. 2015, 159094159097. Scott, K.P., Antoine, J.M., Midtvedt, T., van Hemert, S., 2015. Manipulating the gut microbiota to maintain health and treat disease. Microbial Ecol. Health Dis. 26, 2587725886.

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Smith, A.G., Sheridan, P.A., Tseng, R.J., Sheridan, J.F., Beck, M.A., 2009. Selective impairment in dendritic cell function and altered antigenspecific CD8 1 T-cell responses in diet-induced obese mice infected with influenza virus. Immunology. 126, 268279. Sugimura, T., Jounai, K., Ohshio, K., Tanaka, T., Suwa, M., Fujiwara, D., 2013. Immunomodulatory effect of Lactococcus lactis JCM5805 on human plasmacytoid dendritic cells. Clin. Immunol. 149, 509518. Takeda, K., Okumura, K., 2007. Effects of a fermented milk drink containing Lactobacillus casei strain Shirota on the human NK-cell activity. J. Nutr. 137, 791S793S. Turroni, F., Serafini, F., Foroni, E., Durani, S., O’Connell, M.M., Taverniti, V., et al., 2013. Role of sortase-dependent pili Bifidobacterium bifidum PRL 2010 in modulating bacterium-host interactions. Proc. Natl. Acad. Sci. U. S. A. 110, 1115111156. Van Puyenbroeck, K., Hens, N., Coenen, S., Michiels, B., Beunckens, C., Molenberghs, G., et al., 2012. Efficacy of daily intake of Lactobacillus casei Shirota on respiratory symptoms and influenza vaccination immune response: a randomized, double-blind, placebo-controlled trial in healthy elderly nursing. Am. J. Clin. Nutr. 95, 11651171. Weiss, G., Rasmussen, S., Zeuthen, L.H., Nielsen, B.N., Jarmer, H., Jespersen, L., et al., 2010. Lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a Toll-like receptor-2-dependent mechanism. Immunology. 131, 268281.

Further Reading Pachner, A.R., Bertolotto, A., Deisenhammer, F., 2003. Measurement of MxA mRNA or protein as a biomarker of IFNbeta bioactivity: detection of antibody-mediated decreased bioactivity (ADB). Neurology. 61, S24S26.

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C H A P T E R

21 Micronutrients in Skin Immunity and Associated Diseases Se K. Jeong1,2, , Sung J. Choe3,4,, Chae J. Lim5, Keedon Park5 and Kyungho Park4,6 1

Seowon University, Cheongju, Korea 2NeoPharm Co., Ltd., Daejeon, Korea 3Yonsei University, Wonju, Korea 4 University of California, San Francisco, CA, United States 5Incospharm Corporation, Daejeon, Korea 6 Hallym University, Chuncheon, Korea

21.1. INTRODUCTION The cutaneous immune system protects our bodies against infection and diseases, and the major components of cutaneous immune responses are innate immunity and adaptive immunity (Bangert et al., 2011). While innate immunity is an immediate, nonspecific defense system, adaptive immunity is an antigen-specific immune response which involves the action of B and T lymphocytes (Bangert et al., 2011; Parkin and Cohen, 2001). Emerging evidence demonstrates that nutrition status, especially from micronutrients, could modulate the immune system in skin, contributing to decreased susceptibility to infection and diseases in the skin (Park, 2015; Pappas et al., 2016). This chapter highlights recent insights into skin immunity and the interplay between selected micronutrients, immune responses, and associated skin diseases.

21.2. SKIN STRUCTURE Human skin is composed of three layers: epidermis, dermis, and subcutis (Fig. 21.1) (Richmond and Harris, 2014). The outer layer of skin is the epidermis that is further separated into four sublayers, from the innermost layer, stratum basale, upwards to the surface stratum corneum, caused by levels of keratinocyte differentiation with vitamin C and calcium (Richmond and Harris, 2014; Savini et al., 2002; Bikle et al., 2012). The stratum corneum provides a major role in protecting against external challenges. Besides keratinocytes, epidermis contains melanocytes, which produce melanin to determine skin color and to protect the skin from UV irradiationmediated damages, and Langerhans cells, a type of dendritic cell (DC) that regulates immune responses in the skin (Richmond and Harris, 2014; Brenner and Hearing, 2008; Seneschal et al., 2012). The dermis is located underneath the epidermis and is made up of blood vessels, lymph vessels, nerves, hair follicles, sweat glands, sebaceous glands, and dermal fibroblasts (Richmond and Harris, 2014). Soft tissues, e.g., collagens, elastin and structural proteoglycans, which are produced by fibroblasts, are responsible for making our skin elastic, flexible and structurally firm (Richmond and Harris, 2014; Driskell and Watt, 2015). In addition, certain immune cells, such as DCs, mast cells, macrophages, B-cells, and T-cell subtypes, are critical components of the skin’s immune system (Bangert et al., 2011; Parkin and Cohen, 2001). The innermost layer of the skin, the subcutis is made up of connective tissue and fat, which help to modulate several biological functions (Richmond and Harris, 2014); i.e., (1) controlling body temperature; (2) conserving energy for the body; and (3) protecting the internal organs. *

Se Kyoo Jeong and Sung Jay Choe contributed equally to this work.

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FIGURE 21.1 Structure of human skin. Intact human skin was subjected to hematoxylin and eosin staining. Scale bar, 200 μm.

21.3. IMMUNITY IN THE SKIN Overall skin integrity is maintained by numerous biological responses, including the immune system (Bangert et al., 2011; Elias, 2007). Innate immunity is the first line of defense against infection (Parkin and Cohen, 2001; Elias, 2007). Epidermal keratinocytes produce innate immune elements, called antimicrobial peptides (AMPs), such as cathelicidin AMP and β-defensins, which directly inactivate invading pathogens by disrupting cell membranes (Elias, 2007; Park et al., 2016; Kim et al., 2014). In addition to antimicrobial activities, AMPs regulate the production of inflammatory cytokines/chemokines (Elias, 2007; Kolls et al., 2008), and these function as immunomodulators to regulate the development of certain skin inflammatory diseases, e.g., psoriasis (Morizane and Gallo, 2012), atopic dermatitis (Harder et al., 2010), and rosacea (Yamasaki and Gallo, 2011). Major mediators of adaptive immune responses in the skin are B and T lymphocytes (Bangert et al., 2011; Parkin and Cohen, 2001; Elias, 2007). B lymphocytes produce antibodies that recognize and bind to foreign pathogens/substances (antigen) to neutralize them. In contrast, cell-mediated immunity is carried out by different subtypes of T lymphocytes (Bangert et al., 2011; Parkin and Cohen, 2001): (1) CD81 T cells (cytotoxic T lymphocytes) directly kill cancerous and virus-infected cells (Parkin and Cohen, 2001); (2) CD41 T cells, which are known as helper T cells, help to enhance the activity of other immune cells by releasing cytokines (Parkin and Cohen, 2001); and (3) Regulatory T cells (Treg) suppress immune responses, resulting in the prevention of skin autoimmune diseases (Parkin and Cohen, 2001; Dejaco et al., 2006). Together, appropriate interaction of both innate and adaptive immunity helps to protect the host against infection, cancer, and inflammatory disorders.

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21.4. ROLE OF CERTAIN MICRONUTRIENTS IN SKIN IMMUNITY Nutrition is an important factor in the maintenance of several skin functions, including immune responses (Park, 2015; Pappas et al., 2016; Richmond and Harris, 2014). In particular, emerging evidence reveals that specific micronutrients play critical roles in the development and activation of the immune system, protecting the host against infection and certain skin diseases (Bangert et al., 2011; Park, 2015; Richmond and Harris, 2014). The implication of selected micronutrients, especially vitamin A, C, D, and E, in skin immunity and associated diseases is discussed below.

21.4.1. Vitamin A Vitamin A is a class of fat-soluble micronutrient that includes retinol, retinyl ester, and provitamin A carotenoids, such as α-/β-carotene and β-cryptoxanthin (Wolf, 2008; Elias et al., 1981). It is known to regulate a number of biological functions (Fig. 21.2); e.g., normal growth, apoptosis (Wolf, 2008), and immunity (Mora et al., 2008) in several tissues, including skin (Elias et al., 1981; Lee et al., 2009). While vitamin A and provitamin A carotenoids are ingested through food from animal and plant sources, respectively, both forms are required for metabolic conversion into retinaldehyde, followed by the formation of retinoic acid by aldehyde dehydrogenase (Richelle et al., 2006). Retinoic acid then binds to its nuclear hormone receptors, the retinoic acid receptor (RAR) or the retinoid X receptor (RXR) to aid physiological functions (Park, 2015; Elder et al., 1991). In the presence of retinoic acid, the RAR and RXR form homo- or heterodimers and react with certain consensus DNA regions, known as retinoid acid response elements (RARE) or retinoid X response elements (RXRE), leading to mediation of transcriptional regulation (Park, 2015; Njar et al., 2006). While both RAR and RXR are expressed in major skin

FIGURE 21.2 Metabolism and molecular mechanism of vitamin A in skin function. RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response element; RXRE, retinoid X response element; Th, helper T cells; Treg, regulatory T cells.

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cell types (Elder et al., 1991), e.g., keratinocytes, fibroblasts, melanocytes, and immune cells such as Langerhans cells, retinoic acid modulates various biological actions: (1) proliferation (Gibbs et al., 1996); (2) increased keratinocyte differentiation (Gibbs et al., 1996); (3) suppression of transepidermal water loss (Elias et al., 1981); (4) suppression of ROS-generated DNA damage and oxidative stress (Kitamura et al., 2002); and (5) modulation of proinflammatory cytokines (Wolf, 2002), through binding to RARs and/or RXRs (Figs. 21.2 and 21.3). Prior studies revealed that retinoic acid plays an important role in modulating both innate and adaptive immunity (Mora et al., 2008; Raverdeau and Mills, 2014). Retinoic acid stimulates the development and differentiation of DCs via its effect on transcriptional factors, e.g., interferon regulatory factors, basic leucine zipper ATFlike transcription factors, and NF-κB subunits, which closely guide DC development/differentiation (Raverdeau and Mills, 2014; Saurer et al., 2007). While a Notch-dependent signaling pathway can regulate activity/expression of DC developmental-transcription factors, retinoic acid potentially influences the Notch signaling (Raverdeau and Mills, 2014). But further studies are required to evaluate the detailed mechanism involved in retinoic acid-driven DC development and differentiation. In addition, vitamin A and its metabolites regulate biological responses of T- and B-lymphocytes, major components of the adaptive immune system (Mora et al., 2008; Raverdeau and Mills, 2014; Ross, 2012). Retinoic acid promotes differentiation of CD41 T cells toward helper T (Th) 2 cells, resulting in increased production of Th2 type cytokines or an elevated ratio of Th2 cytokines relative to Th1 cytokines by attenuating Th1-mediated responses (Ross, 2012; Stephensen et al., 2002). Furthermore, retinoic acid suppresses Th17 cell development in the presence of increased levels of TGF-β, an essential factor that is required for the development of Th17 (Ross, 2012; Mucida et al., 2007). Retinoic acid is also a positive factor in the differentiation of Treg, which is known to suppress T-cells (Ross, 2012; Mucida et al., 2007). Together, the retinoic acid-mediated increase in the ratio of Treg relative to Th17 cell population contributes to the enhancement of immune homeostasis. Moreover, retinoic acid stimulates the maturation and differentiation of B-cells and increases antibody production by controlling transcriptional factors, such as paired box 5, B-cell lymphoma 6, B lymphocyte-induced maturation protein-1, and x box-binding protein 1 (Ross et al., 2011).

FIGURE 21.3 Roles of antioxidant vitamins in photoprotection. AP-1, activator protein 1; MMPs, matrix metalloproteinases; ROS, reactive oxygen species; TGF-β, transforming growth factor beta.

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Several lines of evidence suggest that retinoic acid contributes to the prevention/treatment of several skin inflammatory diseases, including psoriasis (Table 21.1) (van de Kerkhof, 2006). Psoriasis is a common, chronic inflammatory, systemic skin disease characterized by erythematous papules and plaques with a silver scale (Fig. 21.4) (Hsu et al., 2012). Although the pathogenesis of psoriasis is not fully understood, multiple factors have been suggested to play a major pathogenic role. In particular, T lymphocyte-mediated responses are currently considered as a key inducer of the phenotype and the pathophysiology of psoriasis, caused by multiple cellular mechanisms: (1) hyperproliferation of keratinocytes induced by T-cells (Cai et al., 2012); (2) induction of Th17 cell development and IFN-γ-induced infiltration into skin (Cai et al., 2012; Kagami et al., 2010); and (3) Th1/Th17 TABLE 21.1

Roles of Vitamin A in Skin Diseases

Disease

Key molecular mechanisms of action

Psoriasis

• • • • •

Acne

• Enhances skin immune system • Activates T-cell functions by increasing CD1d expression (Tenaud et al., 2007) • Activates dendritic cells to stimulate antimicrobial activity (Tenaud et al., 2007) • Suppresses development of Propionibacterium acnesinduced Th17 differentiation (Agak et al., 2014) • Attenuates expression of MMPs and other inflammatory factors (Jalian et al., 2008)

Chronic hand eczema

• Inhibits production of nitric oxide and inflammatory cytokines (Xu and Drew, 2006) (e.g., TNF-α, IL-1β and IL-12)

Aging & wound healing

• Promotes proliferation of keratinocytes and fibroblasts (Gibbs et al., 1996; Varani et al., 2000) • Inhibits expression of MMPs and matrix-degrading enzymes (Varani et al., 2000)

Nonmelanoma cancer

• Reduces regulation of proto-oncogenes (Schule et al., 1991) • Increases expression of p53 and proapoptotic caspases (Mrass et al., 2004) • Stimulates apoptotic pathways through the b-Raf/MEK/ERK signaling (Cheepala et al., 2009)

Suppresses production of inflammatory cytokines & growth factors (Wolf, 2008; Raverdeau and Mills, 2014) Inhibits keratinocyte proliferation (Jean et al., 2011) Enhances keratinocyte differentiation (Jean et al., 2011) Increases the ratio of Th2 cells relative to Th1 cells (Ross, 2012; Stephensen et al., 2002) Increases the ratio of Treg relative to Th17 cell population (Ross, 2012; Mucida et al., 2007)

FIGURE 21.4 Clinical and histologic aspects of psoriasis. Panel A shows the typical lesions of plaque type psoriasis. The lesion shows thick silvery-white scales with dryness, pruritus and redness. Panel B (hematoxylin and eosin staining) indicates typical histologic aspects of psoriatic lesion, characterized by thickening of the epidermis, parakeratosis, elongated rete ridges, and increased infiltration of neutrophils in the epidermis.

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cell-mediated increase in production of inflammatory cytokines/chemokines, such as tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin (IL)-17, and IL-23 (Cai et al., 2012; Kagami et al., 2010; Krueger and Bowcock, 2005). There are a variety of treatments available to patients with psoriasis depending on the severity of the disease, including topical treatments, phototherapy, systemic therapy, or biological/immunomodulatory medications (Hsu et al., 2012). Of the variety of topical medications, synthetic retinoic acids, such as isotretinoin, acitretin and tazarotene, are often used in combination with topical corticosteroids, since these acids attenuate skin irritation and augments steroid effects (Gollnick and Menter, 1999). A few possible mechanisms of isotretinoin, acitretin and tazarotene have been proposed: (1) binding to RAR and/or RXR, followed by transcriptional regulation of psoriasis-associated cytokine and growth factor production (LeMotte et al., 1996); and (2) regulating genes targeting keratinocyte proliferation and differentiation (Dogra and Yadav, 2014). In addition, as described above, since retinoic acid has been shown to elevate the ratio of Th2 cytokines relative to Th1 cytokines, and preferentially to contribute to the induction of Treg over Th17 cells, retinoic acid treatment for patients with psoriasis likely modulates overall immune responses by utilizing the shift from Th1 to Th2 response and from Th17 to Treg population with a decrease in Th1/Th17 cytokine production. While topical application of retinoic acid (adapalene) is also effective in the treatment of mild-to-moderate acne, oral retinoic acid (isotretinoin) has been used to treat severe acne (Zaenglein et al., 2016). CD1d is a cell surface receptor that plays a critical role as an antigen-presenting molecule and is responsible for the development of cutaneous inflammation (Tenaud et al., 2007; Chen and Ross, 2007). Previous studies reveal that retinoic acid enhances the epidermal immune system by increasing CD1d expression and by decreasing production of certain inflammatory cytokines, resulting in activating the communication of T-cells and DCs to stimulate antimicrobial activity against Propionibacterium acnes, a Gram-positive bacteria linked to the pathogenesis of acne (Tenaud et al., 2007; Chen and Ross, 2007). Moreover, although retinoic acid is also being used in the prevention and treatment of other skin diseases, such as nonmelanoma skin cancer (Lens and Medenica, 2008), atopic dermatitis (Mihaly et al., 2011), ichthyosis (Digiovanna et al., 2013), and wound healing (Hunt, 1986), a detailed regulatory mechanism of retinoic acid associated with the skin’s immune system remains under investigation.

21.4.2. Vitamin C Vitamin C is an essential micronutrient for humans, although they are unable to synthesize vitamin C (or acid) endogenously due to a lack of L-gulono-gamma-lactone oxidase, an enzyme that catalyzes the last step of vitamin C biosynthesis (Nishikimi et al., 1994). Since distribution of vitamin C is quite different in various human tissues/organs, it moves across cell membranes through sodium-vitamin C cotransporters to maintain optimal concentration (115 μg/mL in serum) (Tsukaguchi et al., 1999). Interestingly, vitamin C is detected in the stratum corneum, the outermost layer of the epidermis, forming a gradient with decreasing concentration from inner layers to the surface, contributing to the enhancement of epidermal barrier integrity (Weber et al., 1999). Vitamin C is well known as an effective antioxidant that cooperates with other antioxidants, including vitamin E, to protect the skin against a UV irradiation-induced increase in oxidative stress (Fig. 21.3) (Chen et al., 2012). Dermal collagen is composed of specific amino acids, e.g., glycine, proline, arginine, and hydroxyproline, and is a key factor for maintaining the structural integrity of skin (Murakami et al., 2012). UV irradiation stimulates production of proinflammatory cytokines, e.g., TNF-α, IL-1, IL-6 and IL-8, and matrix metallopeptidases, the major enzymes responsible for degradation of dermal collagen by activating certain transcriptional factors, such as protein-1 and NF-κB (Chen et al., 2012). In addition, a UV irradiation-induced increase in reactive oxygen species (ROS) significantly suppresses expression of transforming growth factor-β, a signaling mediator that promotes collagen formation (Quan et al., 2001). Vitamin C has been proven to stimulate collagen biosynthesis through different mechanisms: (1) increased expression of intercellular enzymes of collagen synthesis (Pinnell, 1985; Tajima and Pinnell, 1996); (2) increased stabilization of procollagen mRNA (Geesin et al., 1988); (3) induction of hydroxylation of lysine and proline amino acids, a critical step in the formation of type I collagen in the lumen (Peterkofsky, 1991); (4) acting as a cofactor of lysyl hydroxylase (procollagen-lysine 5-dioxygenases) responsible for collagen stabilization and cross-linking (Myllyla et al., 1984); and (5) stimulated lipid peroxidation (Darr et al., 1993). Vitamin C appears to inhibit the growth of nonmelanoma skin cancers, e.g., basal cell carcinoma (Fig. 21.5) and squamous cell carcinoma (Fig. 21.6), by suppressing overall RNA, DNA, and protein synthesis (Table 21.2) (Bronsnick et al., 2014; Lupulescu, 1991). Previous studies have largely focused on examining the protective role of vitamin C on UV irradiation-mediated skin damage and diseases (Table 21.2), e.g., oxidative stress,

L-ascorbic

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FIGURE 21.5 Clinical and histologic features of cutaneous basal cell carcinoma. Panel A shows typical lesions of nodular type basal cell carcinoma on the nasolabial fold. The lesion shows round nodules with ulceration and bleeding. Panel B (hematoxylin and eosin staining) shows the typical histologic aspects of basal cell carcinoma, characterized by development of multiple islands/nests of basaloid cells.

FIGURE 21.6

Clinical and histologic aspects of cutaneous squamous cell carcinoma. The histopathological picture of large, sun-induced squamous cell carcinoma shows pink-red nodules with erosion, ulceration, and crusting (Panel A). Hematoxylin and eosin staining reveals the typical histologic features of squamous cell carcinoma, which is characterized by increased atypical epithelial cells, and clusters of keratinized cells (squamous eddies) (Panel B).

TABLE 21.2

Roles of Vitamin C in Skin Diseases

Disease

Key molecular mechanisms of action

Aging & wound healing

• Suppresses photo-aging through removing free radicals (Chen et al., 2012) • Promotes collagen synthesis via following mechanisms: • Increased stabilization of procollagen mRNA (Geesin et al., 1988; Peterkofsky, 1991) • Induction of hydroxylation of lysine and proline (Peterkofsky, 1991) • Acting as a cofactor of lysyl hydroxylase (Myllyla et al., 1984) • Stimulated lipid peroxidation (Darr et al., 1993) • Inhibits overall DNA, RNA and other protein synthesis (Lupulescu, 1991)

Nonmelanoma cancer

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wrinkling, aging, wound healing, and dry skin. Further studies are needed to understand how vitamin C contributes to skin immunity.

21.4.3. Vitamin D UVB irradiation stimulates production of previtamin D3 from 7-dehydrocholesterol (provitamin D3), followed by the formation of 1,25-dihydroxyvitamin D3 (1,25D3) by the action of hydroxylase enzymes, 25-hydroxylase (CYP27A1) and 25-hydroxyvitamin D3 1-α-hydroxylase (CYP27B1), in the epidermis (Fig. 21.3) (see reference (Christakos et al., 2016) for details). Similar to vitamin A, the active form of vitamin D, 1,25D3, modulates multiple biological functions through binding to its nuclear hormone receptor, the vitamin D receptor (VDR), followed by complex formation with the RXR (Mora et al., 2008; Christakos et al., 2016). In the presence of 1,25D3, the VDR/RXR complex interacts with a consensus DNA region known as vitamin D response element (VDRE) and modulates the transcription of specific genes (Mora et al., 2008; Christakos et al., 2016). While both VDR and RXR are expressed in several types of cells, e.g., keratinocytes, fibroblasts, monocytes, macrophage, DCs, and T lymphocytes (Bouillon et al., 2008), 1,25D3 is currently recognized as an important modulator of innate and adaptive immune responses. As a first line of defense for protecting the host against infections, the epidermis produces innate immune element called AMPs, including cathelicidin antimicrobial peptide (CAMP) (Gombart et al., 2005). Two possible cellular mechanisms have been demonstrated to explain CAMP induction in epithelial cells (including skin): (1) Toll-like receptor (TLR) activation following bacterial infection increases expression of hydroxylase enzyme, CYP27, which generates 1,25D3 endogenously, resulting in stimulation of CAMP production via VDR activation (VDR-dependent mechanism) (Gombart et al., 2005; Park et al., 2011); and (2) endoplasmic reticulum stress-mediated increase in cellular sphingosine-1-phosphate stimulates CAMP production through activation of NF-κB and C/EBPα (VDR-independent mechanism) (Park et al., 2011, 2016). Vitamin D has also been shown to modulate other components of innate immunity, such as immune cell proliferation/development and inflammatory cytokine production (Lagishetty et al., 2011; White, 2012). Together, vitamin D stimulates cutaneous innate immunity (Fig. 21.7). In addition to innate immunity, vitamin D plays an integral role in adaptive immunity in skin (Prietl et al., 2013). As described earlier, T lymphocytes are a major component of the adaptive immune system (Fig. 21.3) (Mora et al., 2008). Vitamin D has been shown to inhibit the development and function of Th1 cells, which are mainly involved in activating macrophages and inflammatory responses, and Th17 cells, which play a critical role in the pathogenesis of psoriasis (Prietl et al., 2013). Instead, vitamin D enhances the generation and function of Treg, a major component of the immune suppressive factor (Jeffery et al., 2009). As such, it has been strongly suggested that vitamin D helps in preventing and treating autoimmune skin diseases, including psoriasis (Table 21.3) (Mostafa and Hegazy, 2015).

FIGURE 21.7 Vitamin D regulates cutaneous immunity. DC, dendritic cell; RXR, retinoid X receptor; Th, helper T cells; Treg, regulatory T cells; VDR, vitamin D receptor; VDRE, vitamin D response element.

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TABLE 21.3

265

Roles of Vitamin D in Skin Diseases

Disease

Key molecular mechanisms of action

Psoriasis

• Reduces expression of β-defensins (Peric et al., 2009) • Suppresses development of Th1 and Th17 cells (Prietl et al., 2013) • Increases the ratio of Treg relative to Th17 cell population (Jeffery et al., 2009)

Acne

• Stimulates antimicrobial activity against Propionibacterium acnes (Youssef et al., 2011) • Suppresses development of Propionibacterium acnesinduced Th17 differentiation (Agak et al., 2014)

Nonmelanoma cancer

• Inhibits the hedgehog signaling pathway (Bijlsma et al., 2006)

Melanoma

• Promotes growth inhibition of melanoma cells (Evans et al., 1996)

Infectious diseases

• Increases production of cathelicidin and β-defensins (Gombart et al., 2005; Youssef et al., 2011)

FIGURE 21.8 Vitamin E modulates both innate and adaptive immunity. Cox, cyclooxygenase; NADPH oxidase, nicotinamide adenine dinucleotide phosphate-oxidase; NK cells, Natural killer cells.

Besides immune responses, vitamin D enhances skin barrier functions by modulating keratinocyte proliferation/differentiation and hair follicle cycles (Bikle et al., 2004; Hawker et al., 2007; Demay et al., 2007).

21.4.4. Vitamin E Vitamin E is a lipophilic, powerful antioxidant that includes both tocopherols and tocotrienols. Originating from different isoforms of vitamin E, α-tocopherol is the most abundant form in the skin (Richelle et al., 2006). Vitamin E is primarily accumulated in the sebaceous glands, and it is delivered to the dermis and epidermis through sebum, which is an oily substance secreted by the sebaceous glands (Vaule et al., 2004). In the skin, vitamin E levels in the epidermis are higher than in the dermis, contributing to the enhancement of epidermal barrier functions, including antimicrobial defense (Shindo et al., 1994; Provinciali et al., 2011; Thiele et al., 2001). In addition to vitamin C, vitamin E primarily protects the skin against damage induced by UV irradiation-mediated free radicals and ROSs via previously demonstrated mechanisms (Fig. 21.3): (1) suppression of lipid peroxidation, which potentially causes cellular damage and subsequently leads to impaired immune responses (Moriguchi and Muraga, 2000); (2) absorption of UV light to reduce the production of free radicals (Kagan et al., 1992); (3) stimulation of the synthesis of glutathione, a highly important antioxidant and detoxifier (Masaki et al., 2002); and (4) attenuation of DNA damage and tumor formation (Fantappie et al., 2004). Furthermore, the combination of vitamin C and vitamin E has a synergistic effect in preventing UV damage to the skin, due to vitamin C-mediated increases in the stability of vitamin E in the skin (Chen et al., 2012). Vitamin E has been shown to enhance innate and adaptive immunity, and accordingly, leads to reduced susceptibility to certain infections, especially in the elderly via the following cellular mechanisms (Fig. 21.8): (1) decreased expression of prostaglandin E2 produced by activated macrophages (Jiang, 2000; Meydani et al., 1988); (2) improved cytotoxic activity of natural killer cells (Meydani et al., 1988); (3) increased function of other innate V. NUTRACEUTICALS IN BOOSTING IMMUNE SUPPORT AND AS THERAPEUTICS FOR INFLAMMATORY DISEASES

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TABLE 21.4

Roles of Vitamin E in Skin Diseases

Disease

Key molecular mechanisms of action

Aging & Wound healing

• Suppresses expression of MMPs and growth factors (Azzi et al., 2004) • Inhibits collagenase production by inhibiting PKC activation (Ricciarelli et al., 1999) • Restores impaired growth factor expression by Inhibiting lipid peroxidation (Altavilla et al., 2001)

Atopic dermatitis

• Reduces prostaglandin production and serum IgE concentration (Fogarty et al., 2000)

Nonmelanoma cancer

• Removes UV irradiation-mediated free radicals (Burke et al., 2000) • Prevents the development of UV-induced immunosuppression (Gensler and Magdaleno, 1991)

Melanoma

• Promote endoplasmic reticulum stress-mediated apoptosis (Montagnani Marelli et al., 2016)

Other cancers

• • • •

Removes free radicals (Lopez-Torres et al., 1998) Inhibits cell proliferation and angiogenesis (Gysin et al., 2002; Wells et al., 2010) Enhances apoptotic pathways (Sylvester, 2007) Suppresses activity of HMG-CoA reductase (Khor and Ng, 2000)

FIGURE 21.9 Clinical and histologic features of atopic dermatitis. Panel A shows initial lesions of early-onset atopic dermatitis involving the cheek, perioral, and periorbital region in an infant. Hematoxylin and eosin staining (panel B) reveals the typical histologic aspects of subacute to chronic stages, which are characterized by spongiotic area with focal parakeratosis in the epidermis. Prominent perivascular infiltration of lymphocytes and eosinophils are detected in the dermis.

lymphocytes, including neutrophils (de la Fuente et al., 1998); (4) protecting T-cells against a CD95 ligandmediated increase in cell death (Li-Weber et al., 2002); and (5) an increase in thymic lymphocyte proliferation through stimulated T-cell differentiation (Moriguchi, 1998). This evidence suggests that vitamin E improves both innate and adaptive immune responses and reduces risk of infection in aged subjects. As such, vitamin E has been employed in the treatment of patients with certain skin impairments or diseases (Table 21.4), e.g., wound healing (Altavilla et al., 2001), atopic dermatitis (Fig. 21.9) (Fogarty et al., 2000), and skin cancers (Montagnani Marelli et al., 2016; Burke et al., 2000).

21.5. CONCLUSIONS Micronutrients, especially the selected vitamins discussed above, modulate multiple biological functions, including cutaneous immune responses that protect the host from infection and associated skin diseases, such as

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psoriasis, atopic dermatitis, acne, and skin cancers. In particular, these vitamins currently are used in topical medications for the treatment of certain skin diseases: (1) topical retinoic acid (adapalene, tretinoin, isotretinoin, and tazarotene) improves the clinical appearance of acne, photodamage, actinic keratosis, and premalignant conditions; (2) topical vitamin D (calcipotriol) is effective in the treatment of psoriasis, vitiligo, ichthyosis, alopecia areata, and porokeratosis; and (3) antioxidant vitamins (vitamin C and vitamin E) serve as protection against UV light-induced damages and diseases, e.g., aging, dryness, and nonmelanoma skin cancers. But these vitamins are still mostly used in cosmetic products due to their low stability in topical formulations and/or insufficient evidence of treatment efficacy (Telang, 2013; Thiele and Ekanayake-Mudiyanselage, 2007). In addition to topical applications, the dietary influence of these vitamins in the prevention and treatment of several skin diseases has been investigated in clinical trials. However, previous studies have shown conflicting results, and no correlation between dietary vitamins and skin diseases has been adequately demonstrated (Bronsnick et al., 2014; Murzaku et al., 2014). Further studies are required to fully understand the contribution of selected vitamins on skin immunity. Moreover, we need to consider intensive interventional/clinical trials with these vitamins in order to generate sufficient evidence for the effectiveness of dietary supplementation and to develop potential therapeutic agents for the treatment of various skin diseases.

Acknowledgments The authors thank Ms. Joan Wakefield (NCIRE-VA Medical Center, University of California, San Francisco) for superb editorial assistance. This research was supported by Hallym University Research Fund, 2017 (HRF-201703-004). This research was financially supported by the Ministry of Trade, Industry and Energy (MOTIE) and Korea Institute for Advancement of Technology (KIAT) through the Promoting Regional specialized Industry (Grant No. R0004413).

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C H A P T E R

22 Mycobiome and Gut Inflammation: Implications in Gut Disease Elizabeth A. Witherden and David L. Moyes King’s College London, London, United Kingdom

22.1. HOST MICROBIOTA AND THE “SUPERORGANISM” HYPOTHESIS With the successful sequencing of the Human Genome Project (HGP) in 2003, we discovered that there were only about 25,000 genes in the human genome. Given that there are 23,000 genes in the chicken genome, 14,000 in the Drosophila melanogaster genome and 59,000 genes in the corn genome, this was somewhat unexpected. The years since publication of the human genome have seen a reassessment of the genetic makeup of complex organisms. Most significant has been the rise of interest in epigenetics and the host microbiome. With the advent of Next Generation Sequencing (NGS) technologies that arose from the HGP, techniques to investigate the human microbiota (the microbes resident in or on the human host) have taken a huge leap forward. No longer dependent on needing to culture the organisms in the laboratory to identify them, we can now extract their DNA and identify them based on genome sequences. Research into the microbiota (the complete microbial community in a location) has highlighted that there are 10 times more microbial cells in or on the human body, than human cells. Even more startling are the estimates of the number of distinct microbial genes (many of which are related to metabolism and nutrient processing) within the microbiome (total of all the microbial genes/genomes within the microbiota), with current estimates suggesting this number is at least 3.3 million (Qin et al., 2010). This, then, has led to the hypothesis that humans are “superorganisms.” That is, we are an organism made up of numerous mutually interdependent smaller organisms and their genomes. These organisms include the billions of bacterial, protozoan, viral and fungal cells that collectively make up the human microbiota, and whose genomes comprise the human microbiome. These cells, and more importantly their genes, play pivotal roles in the continuum between health and disease, through the modulation of fundamental human physiological processes including; metabolism, energy acquisition, immune modulation and even neurological development (Ghannoum and Mukherjee, 2013; Seed, 2015). The early era of microbiome research concentrated on identifying the constituent microbes that makes up these communities. Given the vast array of different genes contained within the microbiome, over the past 510 years, researchers have begun to focus on trying to understand the complex role the human microbiome plays in health and disease, by profiling the microbial communities and genes resident at different body sites in individuals with and without disease. Most of these studies have focused on the most abundant proportion of the microbiome, the bacteria, identifying a general trend of reduced diversity and richness in a number of inflammatory and chronic disease states. In contrast, both the mycobiome and virome, the fungal and viral portions of the microbiome, and the roles they play in the development of health and disease in humans have been poorly characterized to date (Ghannoum and Mukherjee, 2013; Mukherjee et al., 2015; Seed, 2015). Elucidating the role that the mycobiome plays in the etiology of health and disease is important for a number of reasons. First, the incidence of fungal infections has increased over recent decades, with fungi being increasingly associated with previously well-characterized chronic diseases, such as cystic fibrosis and asthma (Nguyen et al., 2015), as well as inflammatory bowel disease (IBD) (Iliev et al., 2012; Ott et al., 2008), and obesity (Mar Rodriguez et al., 2015). Second, there is increasing evidence to suggest that fungi play an important role in Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00022-6

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both the modulation of the host immune response (Iliev et al., 2012; Moyes and Naglik, 2012) and in disease progression through important fungalfungal, fungalbacterial, and fungalhost interactions (Ghannoum and Mukherjee, 2013; Mukherjee et al., 2015).

22.2. FUNGAL INTERACTIONS 22.2.1. BacterialFungal Interactions Both commensal fungi and bacteria have been found colocalized within specific patches of the gut (Iliev et al., 2012). Given their colocalization, it is not surprising, therefore, that there is extensive interaction between fungi and bacteria, and these interactions are, predictably, diverse incorporating both positive and negative interactions. Many of these interactions are about competition for resources and niches, with each microbe trying to influence others in the community to its own advantage. Bacteria and fungi can influence each other both directly and indirectly through physical interactions (such as biofilm formation on fungal surfaces and mixed-species biofilm formation (Hogan and Kolter, 2002), coaggregation (Peleg et al., 2008)), exchange/secretion of small chemicals, (such as bacterial and fungal quorum sensing molecules), use of microbial metabolites for growth/nutrition, changes in the environment (e.g., pH), and alteration of the host immune response (e.g., segmented filamentous bacteria driving innate and adaptive immune responses (Ivanov et al., 2009)). These interactions can lead to inhibition of growth, reduced fungal and/or bacterial cell viability (e.g., through the secretion of antimicrobial compounds or uptake of toxins or through nutrient depletion). The ability of some fungi to grow in different morphological states also complicates matters. Pseudomonas aeruginosa forms a dense biofilm on Candida albicans filaments and kills them (Hogan and Kolter, 2002). However, it neither binds nor kills C. albicans when it grows in its yeast form. Nonetheless, these interactions are not necessarily antagonistic to fungi and bacteria. Mixed species/kingdom biofilms, in particular, can prove to be a boon to both fungi and bacteria, providing protection for all species against external attack from antimicrobial drugs or the host immune response. One potential effect of these mixed interactions is to enhance or attenuate virulence of either the fungi or the bacteria. One of the earliest recorded studies investigating the impact of a multi-kingdom infection reported that preinfecting mice with a sublethal dose of Escherichia coli resulted in attenuation of host mortality in subsequent C. albicans infections (Gale and Sandoval, 1957). However, notably, if these two members of the healthy gut microbiome are given together, then virulence is increased, probably in an endotoxin-mediated fashion (Akagawa et al., 1995). As well as E. coli, P. aeruginosa infection has also been shown to precede life-threatening cases of candidaemia in human burn victims (Neely et al., 1986). Given this, it is self-evident that the fungal and bacterial communities within the microbiota influence each other in a delicate balance, even within healthy individuals. Whatever these interactions are, it is clear that they are of great importance in maintaining a healthy and balanced microbiota. If, e.g., the bacterial component is reduced or even removed completely, then issues to do with fungal growth are seen. For example, germ-free mice are extremely susceptible to infection by Candida species (Naglik et al., 2008), while antibiotic treatment that disrupts normal bacterial communities drives colonization and overgrowth by fungal species, particularly Candida (Dollive et al., 2013; Mason et al., 2012; Noverr et al., 2005). The mycobiome is not limited to bacterialfungal interactions alone and often involves interactions between various fungal strains; indeed a recent study investigating the mycobiome in HIV patients indicated that the commensal Pichia species was antagonistic to the pathogenic Candida, Aspergillus, and Cryptococcus species (Mukherjee et al., 2014).

22.2.2. HostFungal Interactions As well as other members of the microbial communities, fungi will also interact with the host through a variety of different mechanisms. Interactions with the host innate immune system occur through recognition of fungal pathogen associated molecular patterns (PAMPs) by pattern recognition receptors on immune cells such as neutrophils, macrophages and dendritic cells. These PAMPs are most commonly carbohydrate moieties, such as β-glucans, chitin and mannan structures, and it has become evident in recent years that the dominant receptors for these fungal PAMPs are the C-type lectin receptors, such as dectin-1 and dectin-2. Even more recently, the role for epithelial cells in discriminating between commensal and pathogenic states of fungi has begun to be elucidated, with host and fungal mechanisms being identified (Moyes et al., 2010, 2014, 2016; Naglik et al., 2014). The effect of these different interactions is to either suppress or activate specific phenotypes of the immune

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response—in particular type-17 immunity, leading to activation of TH17 T cells and other interleukin-17 (IL-17) expressing cells known to be an important immune defense mechanism against fungal infections. Host immune responses to pathogens fall broadly in to two categories—innate and adaptive, with innate immunity being nonspecific, little/no memory and immediate, while adaptive immunity is specific, has memory, and because of this is delayed in its appearance. Cells of the innate immune response include phagocytic cells, such as neutrophils and macrophages, whilst adaptive immune responses are generated by T-cells (coordinating cells) and B-cells (antibody producing cells). T cells encompass a spread of different phenotypes, each involved in combatting different forms of infection. For example, intracellular pathogens are combatted by TH1 T cells, helminth/ protist infections by TH2, and extracellular/mucosal infections by TH17. T cells also play a significant role in the resolution and dampening of immune responses (such as at mucosal surfaces) through another phenotype, Treg. Despite the two branches of an immune response (innate and adaptive) being comprised of different components, there is extensive communication between the two, and they often act to amplify or inform each other. This gives rise to descriptions of an immune response being TH1 or type-1 immunity etc. These different immune responses are generally categorized by the cytokines associated with them; hence, TH1 immunity is particularly associated with Interleukin-12 (IL-12) and interferon-γ (IFN-γ), TH2 with IL-4, IL-5 and IL-13 and TH17 with IL-23, IL-17A, IL-17F and IL-22, whilst Treg have been associated with IL-10 and Transforming Growth Factor-β (TGF-β). Defects in production, signaling or autoimmunity resulting in autoantibodies to IL-17 result in a condition known as chronic mucocutaneous candidiasis, where patients suffer from chronic Candida infections throughout their body (Boisson et al., 2013; Ling et al., 2015; Puel et al., 2011). Other type-17 immune cytokines, such as IL-22 have also been shown to be important, with mice lacking IL-22 being more susceptible to gastrointestinal candidiasis (De Luca et al., 2010). It is noteworthy, however, that the TH1 cytokines IL-12 and IFN-γ have also been suggested as playing a role in aspects of anti-Candida immunity, suggesting an important contribution of this form of immune response (Underhill and Iliev, 2014). What is notable is that both the TH1 and TH17 responses have been closely associated with IBD and in particular Crohn’s disease (Muzes et al., 2012; Strober and Fuss, 2011).

22.3. STUDYING THE MYCOBIOME Traditional approaches for analyzing host-associated fungi relied on culture-dependent techniques. These processes were restricted to detecting easily culturable, and highly abundant fungal species at the body site of interest, such as Candida spp. isolated from the oral mucosa, or gastrointestinal tract (Ott et al., 2008). Advances in NGS technology have allowed for culture-independent identification processes, where fungal organisms of low abundance and those uncultivable by routine culture techniques can be identified in human samples (Cui et al., 2013). As a consequence of this advancement it was shown that nonculturable fungi actually account for a large proportion of the mycobiome, similar to what was seen in early bacterial studies. Methods for profiling the mycobiome using NGS technology include; targeted rRNA sequencing and metagenomic whole genome sequencing (Dollive et al., 2012). In brief, both targeted and metagenomic sequencing approaches rely on the isolation of intact genomic DNA from an environmental sample of interest, prior to sequencing, followed by assignment of the sequencing reads to a microbial taxa by comparison to curated databases (Dollive et al., 2012; Seed, 2015). Current eukaryotic rRNA gene targets include the 18S rRNA gene and the internal transcribed spacer (ITS) region, which is internal to the 18S, 5.8S and 28S rRNA operon of eukaryotes. The 18s rRNA gene is present and conserved in all eukaryotes, and is therefore not 100% fungal-specific. As a result, many different 18S rRNA PCR primer sets have been developed to maximize fungal specificity, and reduce human 18S rRNA gene amplification. Furthermore, in studies using 18S rRNA amplicons for fungal profiling it has been shown that the 18S rRNA gene has insufficient hyper-variable regions for the discrimination of fungal isolates at lower taxonomic levels (Dollive et al., 2012). As a result numerous authors now use the ITS gene target, as it is more fungal-specific, and is thus a more powerful tool, allowing greater discrimination between more closely related fungal taxa (Cui et al., 2013). Nevertheless, care must be taken when selecting the target for rRNA sequencing, as it has been shown that some commonly used primer sets are biased towards Ascoymycota or Basidomycota, the two most abundant fungal phyla in humans (Bellemain et al., 2010). Metagenomic approaches have the advantage over rRNA sequencing, in that they sequence all prokaryotic and eukaryotic genes present in an environmental sample, allowing for the taxonomic, genotypic, and functional (e.g., metabolic) community profiling of the entire sample. The disadvantage however, is that they are currently economically and computational more expensive, and require in-depth sequencing followed by complex bioinformatics techniques to uncover less abundant genomes. However, as fungi account for only a small proportion of

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the entire microbiome, and requiring expensive deep sequencing to capture and identify fungal species of low abundance, metagenomic approaches are not currently routinely used for the characterization of the mycobiome.

22.4. THE MYCOBIOME 22.4.1. A Healthy Mycobiome In the early 2000’s the Human Microbiome Project was established to help define the “core” human microbiome. Although, this project continues to generate meaningful microbiome insights, most of the data concerns the bacterial microbiome at various body sites in states of health, and limited work has been done on characterizing the mycobiome. To date, the studies that have looked at characterizing the mycobiome in states of health, have focused on several body niches; oral cavity (Ghannoum et al., 2010), lungs (Nguyen et al., 2015), skin (Zhang et al., 2011), and gastrointestinal tract (Hoffmann et al., 2013; Iliev et al., 2012; Ott et al., 2008). Similar to what has been reported in microbiome studies, the diversity and richness of the human mycobiome varies significantly by individual and anatomical site (Fig. 22.1). As shown in Fig. 22.1, most human body sites are predominated by members of the Ascomycota (e.g., Saccharomyces cerevisiae (baker’s yeast), Candida spp., Cladosporium spp.)

FIGURE 22.1 The relative proportions of fungal genera reported at different body sites. Distribution of fungal genera identified by targeted rRNA sequencing in human samples isolated from different body sites. The charts depict the proportion of fungal genera identified in samples taken from the oral cavity (oral rinse), the lung (sputum), skin (swab), and GIT (stool). Only fungal genera present at a relative abundance of greater than 1%, in at least 1 sample are shown. The term other refers to all the remaining fungal genera that had an abundance of less than ,1%, nonculturable refers to those genera that could not be taxonomically identified by the reference database, other Ascomycota refers to members of the Ascomycota phyla that could not be identified down to genera level, whilst other Basidiomycota refers to members of the Basidiomycota phyla that could not be identified down to genera level. The data for this illustration was derived from a study of the gastrointestinal tract by Hoffmann et al. (2013), a study on oral wash samples by Ghannoum et al. (2010), a study on sputum samples from healthy adults by Van Warden et al. (2013), and a study on skin by Zhang et al. (2011).

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and Basidiomycota (e.g., Cryptococcus spp., Filobasidium spp., Malessezzia spp.) phyla (Ghannoum et al., 2010; Hoffmann et al., 2013; van Woerden et al., 2013; Zhang et al., 2011). Interestingly, the number of fungal genera present at a given body site or in an individual has been shown to vary significantly, with individuals harboring anywhere between 5 and 39 fungal genera (average 15 genera) in the oral cavity (Ghannoum et al., 2010), and between 1 and 19 in the gastrointestinal tract (Hoffmann et al., 2013). Perhaps the most abundantly studied niche for mycobiome studies is the gastrointestinal tract, with a number of authors profiling the fungal community of stools in health (Dollive et al., 2012; Hoffmann et al., 2013; Iliev et al., 2012), as well as in disease using case-control models (Iliev et al., 2012; Mar Rodriguez et al., 2015; Ott et al., 2008; Sokol et al., 2016). In 2012, Dollive et al., investigated the fungal composition of stools from 10 healthy adults by both ITS1 and 18S rRNA targeted sequencing and identified a high abundance of the fungal phyla Ascomycota and Basidiomycota irrespective of the sequencing method employed (Dollive et al., 2012). The major differences between the 18S and ITS sequencing methods were seen at the genus level, where the majority of the sequencing reads belonging to the Saccharomycetaceae family mapped to the Saccharomyces genera by 18S, compared to both Saccharomyces spp. and Candida spp. by ITS, highlighting the poor discriminatory power of 18S rRNA primers for these taxa (Dollive et al., 2012). Interestingly, irrespective of the sequencing method employed, a small number of reads were assigned to plant taxa, reiterating findings from other authors, that fungal rRNA from food sources (such as Agaricus bisporus the common button mushroom) can be easily identified in stool samples as food contaminants (Dollive et al., 2012; Sokol et al., 2016). In separate studies Hoffmann and colleagues investigated the influence of diet on shaping the gut microbiome (Wu et al., 2011) and gut mycobiomes (Hoffmann et al., 2013) in 96 healthy adults, and found that both gut biomes were modulated by diet (Hoffmann et al., 2013; Wu et al., 2011). The authors found that the bacterial profiles in stools could be clustered into enterotypes, based on the relative abundance of Bacteroides spp. and Prevotella spp., similar to reports from other authors (Arumugam et al., 2011). A high animal fat and protein diet correlated with a high abundance of Bacteroides spp., while a high carbohydrate diet positively correlated with Prevotella spp. (Wu et al., 2011). Similar correlations were observed in the mycobiome, where a high abundance of Candida spp. strongly correlated with the presence of Prevotella spp. and the recent consumption of carbohydrates, while negatively correlating with diets rich in fats and protein (Hoffmann et al., 2013). Irrespective of diet, the Ascomycetes, Saccharoymces spp. (present in 89% of samples), Candida spp. (57%), and Cladosporium spp. (42%) and the Basidiomycete Cystofilobasidium spp. (24%) were the most prevalent fungal genera identified in the gut of the study participants (Hoffmann et al., 2013). Similar findings were reported in a 2012 study by Iliev et al., who described the presence of a commensal fungal community, dominated by Candida spp., Saccharomyces spp., and Trichosporon spp. in the intestinal tract of mice, humans and other mammals (Iliev et al., 2012).

22.4.2. The Diseased Microbiome and Mycobiome With so much effort being expended to characterize the microbiome in healthy individuals, it was perhaps inevitable that attention would turn to the microbiome in different disease conditions. With the advent of metagenomics techniques, we are beginning to see differences in microbial communities and genes associated with specific conditions. These differences have been associated with a variety of conditions ranging from relatively minor (such as dandruff (Park et al., 2012)) to serious (such as immune-mediated inflammatory disease (Forbes et al., 2016)). While some of these conditions are preexisting (such as cystic fibrosis), and thus are not caused by dysbiosis (an imbalance in microbial composition), there is intense debate whether other conditions (such as IBD) are driven by dysbiosis or whether the changes in microbiome seen in these diseases are simply a result of the underlying condition. Given the microbial loads in the gut, it is, perhaps, not surprising that many of the conditions associated with alterations in the core microbiome have so far been gut-related, such as diabetes, obesity and IBDs. 22.4.2.1. Obesity and the Mycobiome In the Western world, one in four adults is considered obese, and are at increased risk of developing chronic diseases such as type 2 diabetes, coronary heart disease, stroke or metabolic syndrome. Researchers have tried to better elucidate what predisposes an individual to obesity, with numerous authors evaluating the relationship between the bacterial microbiome, metabolic syndrome and obesity (Mar Rodriguez et al., 2015; Tilg and Kaser, 2011). Using both mouse models and human samples, authors have identified a shift in the relative abundance of

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the dominant bacterial phyla Bacteroidetes and Firmicutes in the distal gut between obese and nonobese subjects (Tilg and Kaser, 2011; Turnbaugh et al., 2006). Where an increase in the relative abundance of Firmicutes and a proportional reduction in Bacteroidetes, in obese individuals, has been linked with changes in metabolic function of the gut microbiome (Tilg and Kaser, 2011; Turnbaugh et al., 2006). In contrast only a single study has evaluated the relationship between obesity and the fungal constituents of the microbiome. Mar Rodriguez, in 2015, characterized the mycobiome in 27 obese (BMI 45.36 6 5.52) and 12 nonobese (BMI 22.66 6 3.34) individuals, and identified no significant differences in the richness of the mycobiome between obese and nonobese individuals (Mar Rodriguez et al., 2015). The most abundant fungal phyla in all cases were Ascomycota (Candida spp., Penicillium spp. And Aspergillus spp.), Basidiomycota (Rhodotorula spp., Wallemia spp.) and Zygomycota (Mucor spp., in nonobese individuals only), and there was no statistical difference in the relative abundance of Ascomycota and Basidiomycota between obese and nonobese individuals. Despite these extensive similarities, however, obese and nonobese individuals could be distinguished from each other based on principle component analysis (PCA) at the phyla level, as the relative abundance of the Zygomycota phyla (due to the relative abundance of the Mucor spp.) was significantly reduced in the obese population. Unfortunately, the specific role members of the Zygomycota phyla, and Mucor genus (particularly, M. racemosus and M. fuscus) play in obesity have not yet been elucidated. The reduction of Mucur spp. seen in obese individuals is accompanied by a relative increase in the Nakaseomyces spp. (a member of the Ascomycota phylum), highlighting a loss of abundance of the Zygomycota phylum and increase in the Ascomycota phylum in obese individuals. This is not the only time this negative correlation has been seen—in HIV patients, Pichia spp. antagonize the growth of Candida spp. (Mukherjee et al., 2015), although these species are both from the Ascomycota phylum. We can draw some direct analogies here with bacterial species. It would appear that the Mucor genus is analogous to the bacterial Bacteoidetes, with both showing a correlation between abundance and host weight loss (Ley et al., 2006; Mar Rodriguez et al., 2015; Turnbaugh et al., 2006). Given that the Mucor genus is the most abundant in nonobese individuals, questions arise as to why, and whether these species play a role in obesity (Mar Rodriguez et al., 2015). One possible explanation is provided by the observation that the polysaccharide chitosan has a multifunctional role as a protective agent against obesity (Walsh et al., 2013). Given that the cell wall of Mucor spp. has a large chitin-chitosan polysaccharide component (Karimi and Zamani, 2013), it is unsurprising that members of this genus have been described as a source of chitosan (Tajdini et al., 2010). However, there is, as yet, no definitive proof that this fungal genus plays any role in obesity. Interestingly, comparison of relative abundance patterns with metabolic profiles identified a negative correlation between body fat parameters such as BMI, and biochemical analytes (cholesterol, LDL, and fasting triglycerides) with the relative abundance of the Zygomyocata phylum, specifically Mucor spp., as well as the Penicillium genera (Ascomycota phylum). In contrast, the relative abundance of the Aspergillus genus (Ascomycota phylum member) was positively associated with markers of adiposity (BMI, and fat mass), as well as markers for glucose metabolism (Mar Rodriguez et al., 2015). Perhaps most surprisingly from studies carried out to date is the finding that the relative abundance of Candida spp. does not change in obese relative to nonobese individuals. This genus is well known for its significant presence in the gut mycobiome, being identified in both humans and mice (Ott et al., 2008; Scanlan and Marchesi, 2008), so it is unexpected that the abundance of this pathobiont does not change, particularly given that some studies have demonstrated an association between Candida spp. and both forms of diabetes (Type I and Type 2) (Gosiewski et al., 2014), as well as the known associations with gut inflammation (Cui et al., 2013; Iliev et al., 2012; Ott et al., 2008). The association of the different fungal species and genera with obesity and related conditions is interesting, given what we know about the effects of different metabolites and fungi. There are some metabolites that are known to exert effects that are significant in obesity and health. For example, N-acetyl-L-glutamic acid (metabolized by members of the Ascomycota phylum) exerts natriuretic effects, decreasing blood pressure (Hofbauer et al., 1985). However, little is currently known about the interactions between fungi/host and host or fungal derived metabolites. This is a new area of research, and one that in the coming years should reveal rich information regarding how we interact with our fungal communities. 22.4.2.2. IBD and the Mycobiome IBD refers to a group of chronic inflammatory diseases of the gastrointestinal tract that are characterized by recurrent periods of abdominal cramping, and diarrheal remission and relapse. The two most common manifestations of IBD are Crohn’s disease (CD) and ulcerative colitis (UC), and the major difference between CD and UC relates to the specific sites of inflammation; UC is restricted to the colon, while CD can affect the entire

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gastrointestinal tract. The exact etiology of IBD, and IBD relapse is still unclear, although both inflammatory and autoimmune involvement have been highlighted as being important. Nevertheless, IBD is currently reported to occur in genetically susceptible individuals, due to a dysregulated immune response to resident gastrointestinal microbiota, in the presence of adverse external environmental factors (Morgan et al., 2012; Sokol et al., 2016). In 2001, a genetic link to IBD was reported, when the NOD2 locus on chromosome 16 was characterized and identified as important genetic loci influencing the pathogenesis and susceptibility to IBD. Subsequent genetic studies have identified another 163 IBD risk-associated loci such as ATG16L1, IRGM, LRRK2, and Card9 and although the vast majority are shared between CD and UD disease phenotypes; some are specific for the manifestation of either phenotype alone (Jostins et al., 2012). The presence of genetic variants at any of the IBDassociated loci, are not causative, however. Instead they increase the relative risk of an individual to develop the disease. Interestingly, some of the genetic risk-markers identified in IBD are involved in the regulation of the host immune response to microbial cells, highlighting the important role the microbiome might play in the development of this disease (Sokol et al., 2016). The role that immune genes, and through them the microbiome, might play in IBD was recently highlighted by a study characterizing a knockout of the NLR receptor NLRP6 in colonic epithelial cells in mice (Elinav et al., 2011). These mice proved to be more susceptible to experimental colitis. However, a critical observation was made that wild-type pups reared with nlrp/ mothers also demonstrated an increased susceptibility to experimental colitis. Further investigation documented that the nlrp6/ mice had an altered microbiota, and their coreared wild-type pups also shared this altered microbiota. Thus, it would appear that NLRP6 increases susceptibility to IBD by altering the resident microbiota, rather in a direct effect. Given that many of the IBD susceptibility genes are also immunological, it is conceivable that this may be a major phenomenon in both etiology and progression of IBD. In an attempt to further elucidate the role that the microbiome plays in the etiology and progression of IBD, numerous authors have used case-control models to study the microbial communities of the intestinal tract in disease. Although a causative organism has not yet been elucidated, most authors report a dysbiosis in the gut microbiota of patients suffering with IBD, which is characterized by a loss of diversity and richness within the microbial community (Ananthakrishnan, 2015). More specifically, this perturbation seen in IBD is characterized by a general reduction in biodiversity (Ott et al., 2008), categorized by a reduction in the proportion of Firmicutes, and an increase in the Proteobacteria and Bacteroidetes bacterial phyla (Ott et al., 2008; Sokol et al., 2016). These changes in the bacterial component of the gut microbial community do not happen in isolation. These changes are coupled with an increase in the proportion of the fungal Basidiomycota phyla at the expense of Ascomycota (Fig. 22.2) (Sokol et al., 2016). Fig. 22.2 explores this relationship in more detail, and highlights that the increased Basidiomycota/Ascomycota ratio observed in IBD patients is characterized by a reduction in the proportion of Saccharomyces spp., and Penicillium spp., and an increase in the proportion of Malasseziales, Filobasidiaceae and Candida spp., compared to healthy controls (Sokol et al., 2016). Similar dysbiosis patterns have been observed in both stool and mucosal biopsy samples from CD patients by other authors. Examination of ileal mucosal biopsies from individuals suffering with CD showed an increase in the proportion of Candida spp., Cryptococcus neoformans and Gibberella moniliformis, and a reduction in the abundance of S. cerevisiae compared to healthy controls (Li et al., 2014). While in a separate study, Ott and colleagues reported that although the Ascomycota and Basidiomycota phyla dominated both colonic biopsies and stool samples, the body sites harbored significantly different fungal OTU profiles (Ott et al., 2008). Perhaps most interesting is the report that stool samples were dominated by the presence of Penicillium spp. and Saccharomyces spp., compared to healthy controls, while neither Saccharomyces spp. nor Penicillium spp. were detectable in the colonic-biopsy samples. This is of interest, as it suggests that the microbiota profile at the site of inflammation differs from that of the noninflamed mucosa and fecal communities (Li et al., 2014; Ott et al., 2008).

22.5. CONCLUDING REMARKS One of the big knock-on effects of the completion of the HGP was the development of the “superorganism” theory, and the realization of the important role that host microbial communities play in health and disease. However, although we are developing an ever-increasing appreciation of the pivotal role that the gut microbiota plays in these processes, we are also becoming more aware of just how complex these microbial communities are. The development of affordable high throughput sequencing technologies and analytical tools has seen a quantum leap in our understanding of these communities. Despite this, knowledge of the fungal component of these communities is lacking. What is clear, though, is that these fungal communities vary between different sites

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FIGURE 22.2 Fungal taxa identified in the Gastrointestinal Tract of Healthy and IBD suffering individuals. The figure depicts the incidence of fungal taxa reported in healthy controls, and IBD cases, including (UC) Ulcerative Colitis and (CD) Crohn’s Disease as described in the case-control study by Sokol et al. (2016). For each condition, the key fungal phyla, and the key fungal species present are noted. In states of disease, arrows indicate either an increase or decrease in the relative abundance of the phyla relative to the control group used in the study (case-control study). The pie charts depict the key fungal taxa (present at . 1% abundance) for each condition, where the prefixes g_, o_, f_, designate the genera, order and family taxonomy identified. Other signifies all remaining fungal genera present at less than 1% relative abundance, unassigned, refers to those sequences that could not be assigned to a fungal taxonomic rank, and unidentified, refers to sequences that could be assigned to the fungal kingdom, but could not be identified at lower dominations of fungal taxonomy.

and that these variations in their makeup play a significant role in a series of host homeostatic processes as well as being impacted by or impacting on disease. It will be interesting in the future to see how these fungal communities are shaped by and conversely shape the bacterial and viral communities, as well as specifically how the host interacts with the mycobiome as its constituents change. Given that the responses at specific mucosal sites are likely tailored to interact with fungi in different ways, it is probable that these interactions will be key in our understanding of host-mycobiome interactions and their impact on health and disease. Thus, future studies are required to analyze the composition of the mycobiome more completely, and to determine how the mucosal immune system interacts with both these unique mycobiomes as well as the combination of fungi and bacteria that make up the complete microbiome. How we can then exploit these interactions will determine the next generation of therapies that enable us to treat dietary and inflammatory diseases at these sites, including the gut.

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C H A P T E R

23 Flavonoids in Treating Psoriasis Marco Bonesi, Monica R. Loizzo, Francesco Menichini and Rosa Tundis University of Calabria, Rende, Italy

23.1. INTRODUCTION Psoriasis is a common skin disorder that is associated with both a physical and psychological burden. The etiology remains unclear, although there is evidence for genetic predisposition. The role of the immune system in psoriasis causation is also a major topic of research. Although there is a suggestion that psoriasis could be an autoimmune disease, no autoantigen that could be responsible has been defined yet. External and internal triggers, including mild trauma, sunburn, infections, systemic drugs and stress (Boehncke and Scho¨n, 2015) can provoke psoriasis. Psoriasis occurs worldwide. It affects both sexes of all ages, regardless of ethnic origin, in all countries. Published data on the prevalence of psoriasis in countries vary between 0.09% and 11.4%. In most developed countries, prevalence is between 1.5% and 5%. There is also evidence to suggest that the prevalence of psoriasis may be increasing (Danielsen et al., 2013). Recently, one study demonstrated that men have more severe forms of the disease than women (Ha¨gg et al., 2013). On May 24, 2014, the 67th World Health Assembly of the World Health Organization (WHO) passed a resolution on psoriasis (WHA 67.9). WHO recognizes the urgent need to follow multilateral efforts to raise awareness regarding the disease and to fight stigmatization suffered by those with psoriasis. It can manifest in many different forms. Based on the type of skin lesions, location, age of onset and course of disease, five types of psoriasis have been described: psoriasis vulgaris (also known as plaque psoriasis), which is the most prevalent form of the disease, affecting approximately 80% of individuals with psoriasis; eruptive psoriasis (droplet), which is characterized by scaly teardrop-shaped spots and is the second most common form of the disease; inverse psoriasis, also called intertriginous or flexural psoriasis, that is usually more prevalent in obese and overweight individuals and those with deep skin folds; pustular psoriasis, which can either take the form of palmoplantar pustulosis, or generalized pustular psoriasis; and erythrodermic psoriasis, which is a rare but very serious complication of psoriasis that can manifest as a result of the progression of chronic plaque psoriasis. Several triggers are associated with psoriasis outbreaks. Psoriasis triggers can vary from person to person. Established triggers of psoriasis include obesity, infections, skin injuries, stress, and the use of drugs like nonsteroidal anti-inflammatory agents, β-blockers, lithium and antimalarials. Allergies, diet, weather, tobacco smoking, and alcohol use may also be psoriasis triggers (Kimball et al., 2008; Wolk et al., 2009). The course of psoriasis is characterized by periods of exacerbations and remissions with wide inconsistencies in the length of time of exacerbations and remissions (Langley et al., 2005). Severe forms of the disease affect about 10% of patients; psoriatic arthritis can be present in 20%30% of cases. About 70%80% of patients have mild psoriasis, controlled by topical therapies alone (Scho¨n and Boehncke, 2005). In addition to ethnicity, the prevalence of psoriasis may also be affected by exposure to the sun and the climate. However, recently, Jacobson et al. (2011) revealed a weak correlation between psoriasis prevalence and latitude in which patients live. This evidence suggests that other factors might be involved. Several studies have reported the coexistence of psoriasis and other serious systemic diseases, including cardiovascular diseases, metabolic syndrome, including hypertension, dyslipidemia and diabetes mellitus, nonalcoholic fatty liver disease, multiple sclerosis, Crohn’s disease, anxiety, depression and lymphoma (Augustin et al., 2010). The incidence of chronic obstructive pulmonary

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disease is also significantly higher in those individuals who have psoriasis, independent of other risk factors (Dreiher et al., 2008). These comorbidities are likely to influence patients’ health and quality of life, and contribute to the 34-year reduction in life expectancy in patients of severe psoriasis (Gelfand et al., 2007). Plant products have been used with varying success to cure and prevent diseases throughout history. The strong historic bond between plants and human health began to unwind in 1897, when Friedrich Bayer and Co. introduced synthetic acetyl salicylic acid in commerce. However, the benefits of modern drugs are felt primarily in developed countries, and developing countries continue to rely on ethnobotanical remedies as their primary medicines. Until now, plants were an important source for the drugs, with many blockbuster drugs being derived directly or indirectly from plants (Cordell, 2000; Newman et al., 2000). During the 21th century, the emphasis gradually moved from extracting bioactive compounds from plants to making these compounds synthetically. Natural products were widely considered as leading compounds for structure optimization programs designed to make more active and less toxic drugs. About 50% of patients affected by psoriasis use complementary and alternative medicine, including plant-based medicines to treat the disease (Smith et al., 2009). Flavonoids are a large family of over 5000 hydroxylated polyphenolic compounds that have an important role in plants, including attracting pollinating insects; combating environmental stresses, such as microbial infection; and regulating cell growth (Kumar and Pandey, 2013). Their chemical nature depends on their structural class, degree of hydroxylation, other substitutions and conjugations, and degree of polymerization. The bioactivities of flavonoids and their metabolites depend on their chemical structure and the relative orientation of various moieties in the molecule. Flavonoids have been shown to exhibit antioxidant and anti-inflammatory activities through different mechanisms of action in vitro and in vivo models (Manach et al., 2004). A large number of flavonoids have been shown to be potential immunomodulatory, anti-inflammatory, antistress, and anticancer agents. Several recent research papers investigated the use of these phytochemicals for the treatment of psoriasis (Deng et al., 2014; Bonesi et al., 2016). Herein, we report and discuss the role of flavonoids as bioactive compounds useful in treating psoriasis.

23.2. PSORIASIS: A CHRONIC IMMUNEMODULATED INFLAMMATORY DISEASE Psoriasis is one of the most common chronic, inflammatory, T-cell-mediated autoimmune diseases marked by increased amounts of phosphorylated NF-κB, which is postulated to drive altered keratinocyte and immune cell behavior. Translational research studies strongly evidenced that psoriasis is actually caused by a combination of both a primary defect in keratinocytes and an inappropriate innate and adaptive immune response-driven type I interferon (IFN) and that it is mediated mainly by resident and infiltrating T-cells (Flatz and Conrad, 2013). In patients with psoriasis, IFN production by plasmacytoid dendritic cells is the first event in disease development by driving autoimmunity. IFN induces the activation and maturation of conventional dendritic cells, which are stimulators of T-cells, thus joining the gap between innate and adaptive immunity (Nestle et al., 2005). Crosstalk between the innate and the adaptive immune system is mediated by cytokines including tumor necrosis factor (TNF)-α, interferon γ, and interleukin (IL) 1. Successively, autoreactive T cells proliferate and migrate into the epidermis. This event is controlled by the expression of alpha 1 beta 1 integrin on effector T-cells. T- cells involved in psoriasis are IFN-γ-secreting type 1T helper (Th1) cells and Th17/Th22 cells producing IL-17 and IL-22 (Flatz and Conrad, 2013). Another cytokine involved in the pathogenesis of psoriasis is IL-9 that support Th17-related inflammation (Singh et al., 2013). Researchers drew attention to Th17/Th22 cells and IL-23, which induces IL-17 and IL-22 production by T-cells (Tonel and Conrad, 2009). In fact, IL-22 induces keratinocytes hyperproliferation, and both cytokines increase the production of the peptide LL-37 called also cathelicidin, which continues the activation of the immune system, by the stimulation of dermal plasmacytoid dendritic cells to produce interferon α (TNA-α). TNF-α induces secondary mediators and adhesion molecules, all of which have been implicated in psoriatic disease (Locksley et al., 2001). In psoriatic skin, IL-17 is produced by CD41 T cells, epidermal CD81 T cells, neutrophils, mast cells and macrophages (Scho¨n, 2014). Considering that IFN could upregulate the expression of IL-22-receptor on keratinocytes, its role in psoriasis pathogenesis is multiple. In fact, it drives immune activation with the induction of IL-17, 22, and 23; provides the interface between immune activation and epidermal remodeling by increasing keratinocyte IL-22 responsiveness; and enhances expression of major histocompatibility complex class I, which may promote presentation of putative autoantigens to intraepidermal T cells (Lang et al., 2005).

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Recently, the implication of innate gamma-delta (γδ) T-cells to psoriasis pathogenesis was described since these cells are present in psoriasis plaques. Dermal γδ T-cells produce large amounts of IL-17 upon stimulation with IL-23 (Cai et al., 2011). Therefore, γδ T cells may amplify Th17 responses and induce autoimmunity. Similarly, natural killer (NK) cells and natural killer T- (NKT) cells have been proposed as essential players in psoriasis pathogenesis. Both NK and NKT cells are able to produce TNF-α and IFN-γ in psoriasis plaques (Bos et al., 2005). The presence of NK and NKT cells was demonstrated by both immunohistochemical and flow cytometric studies in the mid and papillary dermis of psoriasis patients (Cameron et al., 2002; Liao et al., 2006). Moreover, it is evidenced that NK cells may be necessary for the induction of psoriatic lesions in animal models, and that there is a strong genetic association between genes and single nucleotide polymorphisms of NK cell biology and pathology. Although the role of these cells is not entirely clear to date, and some questions are still open. One of the most important questions: is NK activation and migration to the skin pivotal to the development of psoriasis plaques or does it represent a secondary phenomenon? Completely understanding the role of immune systems in psoriasis pathogenesis may open new possibilities therapeutic targeting (Tobin et al., 2011).

23.3. CONVENTIONAL THERAPIES TO TREAT PSORIASIS Psoriasis treatment options are often based on the severity of the psoriasis. Patients with psoriasis on less than 3% of the body are considered to have a mild case. Psoriasis on 3%10% of the body surface is moderate and greater than 10% is a severe case. Three different treatment options, namely topical therapy (ointments, creams, lotions, gels, or foams applied to the skin), phototherapy (UV-light therapy), and systemic therapy (tablets or injections/infusion) are used to treat psoriasis. Mild psoriasis usually is treated with topical therapy, progressing to phototherapy in the case of insufficient response. Moderate to severe psoriasis requires systemic therapy. The first line drugs include acitretin, ciclosporin, etretinate and methotrexate. Other systemic therapies are biologic agents and fumaric acid esters (Mansouri et al., 2013). Except retinoids like tazarotene or soriatane, all treatments are primarily anti-inflammatory, and subsequently lead to slowed epidermal keratinocyte turnover and flattening of plaques. Among topical drugs, glucocorticosteroids and vitamin D3 derivatives or their combinations are frequently used while calcineurin inhibitors (tacrolimus and pimecrolimus) are used for the treatment of intertriginous areas or the face. Patients affected by moderate and severe psoriasis generally use a combination of phototherapy and systemic therapy. A variety of phototherapy treatments is available for individuals with psoriasis. The use of lasers allows for targeted phototherapy that can more effectively limit the treatment areas to the location of psoriatic lesions. Excimer and pulsed dye lasers are two lasers approved by the Food and Drug Administration (FDA) for treating chronic, localized plaques in mild to moderate psoriasis. PUVA (Psoralen Plus Ultraviolet Light Therapy) is a combination of ultraviolet A (UVA) light therapy and a psoralen medication. Unfortunately, the carcinogenic effects of PUVA limit its long-term use. Among biological agents, several TNF-α inhibitors namely adalimumab, etanercept, infliximab golimumab and are used together with inhibitors of IL 12 and 23 (ustekinumab). Other biological drugs are alefacept that act by blocking the activation of T-cells, and apremilast that is recently approved by FDA as NK cells blocker. All these drugs are used when conventional systemic therapies have failed. Biological drugs are characterized by a good safety profile, a low level of toxicity but are more expensive (WHO, 2015).

23.4. FLAVONOIDS IN TREATING PSORIASIS Many phytochemicals, particularly polyphenols, are now being used to treat psoriasis (Bonesi et al., 2016). Polyphenols are recognized as multifunctional compounds that can act as antioxidants, anti-inflammatory and antiproliferative agents through the modulation of multiple signaling pathways (Gupta et al., 2014). These properties could be beneficial for the treatment of multicausal diseases, including psoriasis. Flavonoids are the main polyphenols class. Over 4000 flavonoids have been isolated from plants (Harborne, 1994). Flavonoids have been shown potent antioxidant effects, to inhibit cycloxygenase, lipoxygenase, microsomal monooxygenase, glutathione S-transferase, mitochondrial succinoxidase and NADH oxidase (Odontuya et al., 2005). Herein, we report the potential of some flavonoids, namely amentoflavone, apigenin, baicalin, delphinidin, genistein, luteolin, and quercetin, in treating psoriasis. Table 23.1 summerizes the in vitro and in vivo anti-inflammatory effects of these flavonoids as promising antipsoriatic agents.

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TABLE 23.1

The main anti-inflammatory effects of selected flavonoids as potential agents in treating psoriasis

Flavonoid

Activity

References

Amentoflavone

Reduction of ear fold thickness and skinfold, inhibition of the IMQinduced increase of mRNA expression, inhibition of HaCaT cells proliferation, promotion of apoptosis, and inhibition of the increase of IL17A, IL-22, and NF-κB expression

An et al. (2016)

Topical anti-inflammatory activity with reduction of edema

Sosa et al. (2007)

Inhibition of TNF-α-induced NF-κB-mediated transactivation of a luciferase reporter gene

Vogl et al. (2013)

Decrease of H5N1-induced production of IL-6, IP-10, and TNF-α

Sithisarn et al. (2013)

Reduction of TNF-α

Drummond et al. (2013)

Inhibition of expression of TNF-α and IL-6, iNOS mRNA and protein

Sae-Wong et al. (2011)

Suppression of the expression of TNF-α, IL-8, IL-6, GM-CSF, and COX-2 by decreasing intracellular Ca21 level and inhibiting NF-κB activation

Kang et al. (2011)

Inhibition of production of TNF-α, IL-1β, IL-6, and IL-33

Borghi et al. (2013)

Reduction of the levels of TNF-α, IL-1β, and IL-6

Rithidech et al. (2012)

Attenuation of TNF-α-induced VCAM-1 mRNA and protein expression

Yamagata et al. (2010)

Inhibition of TNF-α and IL-6 by inhibiting NF-κB activation

Xie et al. (2012)

Astilbin

Amelioration of keratinocyte proliferation, elevations in circulating CD4 and CD81 T cells and inflammatory cytokines

Di et al. (2016)

Baicalin

Anti-inflammatory activity and keratinocyte differentiation-inducing activity

Wu et al. (2015)

Delphinidin

Abrogation of the histological characteristics of psoriasis lesions and great reduction of infiltration of neutrophils and macrophages

Pal et al. (2015)

Genistein

Reduction of the expression of TNF-α, IL-1 β, IL-6, and RANKL

Karieb and Fox (2013)

Inhibition of TNF-α-induced endothelial inflammation

Jia et al. (2013)

Inhibition of the LPS-induced overproduction of TNF-α and IL-6; activation of NF-κB

Ji et al. (2012)

Downregulation of TNF-α and IL-6 production and expression

Gao et al. (2013)

Reduction of IL-1β, IL-6, TNF-α, and neuropathic pain

Valsecchi et al. (2011)

Inhibition of LPS-induced anorexia

Tsushima and Mori (2001)

suppressed the expression of VCAM-1 and E-selectin on activated human umbilical vein endothelial cells (HUVEC), markedly interfered with THP monocyte adhesion to TNF-α-activated endothelial cells and attenuated platelet endothelial cell adhesion molecule-1 expression induced by TNF-α

Kwon et al. (2007)

Antinephritic effects

Hattori et al. (2007)

Inhibition of the activation of NF-κB p65 and AP-1

Feldman et al. (2011)

Apigenin

Isoliquiritigenin

The mechanism was that ISL could induce HO-1 expression through the ERK 1/2 pathway in RAW264.7 macrophages, with the inhibition of LPSinduced NO, IL-1β, and TNF-α production, and HO-1 could mediate the anti-inflammatory effects of ISL through LPS-induced NO and TNF-α production reversedly Meanwhile, due to the ability to affect Th2-mediated inflammation, ISL could be served as antiasthma herbal medicine with inhibitory effects on memory Th2 responses in vitro and antigen-induced Th2 inflammation in vivo via significant suppressing IL-4 and IL-5 production

Yang et al. (2013)

(Continued)

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TABLE 23.1

(Continued)

Flavonoid

Activity

References

Significant inhibition of LPS-induced IL-6 and IL-12 p40 production, moderate inhibition of LPS stimulated production of TNF-α

Li et al. (2014)

Reduction of the expression of IL-6 Decrease of IL-6 and IL-8 levels, CD4, CD8, CD11b/c, F4/80, and VEGF, suppression of NF-κB expression and reduction of IL-6 and IL-8

Wu et al. (2016)

Luteolin

Inhibition of human keratinocyte activation and decrease of NF-κB induction

Weng et al. (2014)

Quercetin

Significant orthokeratosis, change in epidermal thickness, reduction of granular layer of the epidermis

Vijayalakshmi et al. (2012)

FIGURE 23.1 Chemical structure of Amentoflavone.

23.4.1. Amentoflavone The biflavonoid amentoflavone (Fig. 23.1) demonstrated to possess interesting properties, including antioxidant activity, inhibition of cyclooxygenases, inducible nitric oxide synthase and phospholipase A2, and inhibition of degranulation and arachidoinic acid release from neutrophils (Banerjee et al., 2002a,b; Garcia-Lafuente et al., 2009; Huang et al., 2012; Kim et al., 1998, 2001, 2004; Mora et al., 1990; Tsai et al., 2012; Tordera et al., 1994; Woo et al., 2005). Recently, the effects of amentoflavone on psoriasis in imiquimod (IMQ) psoriasis-like lesions in mice were investigated (An et al., 2016). The IMQ-treated murines are in vivo models widely used for the study of psoriasis-like lesions (Nadeem et al., 2015). The keratinocyte proliferation in HaCaT cells was also studied. Amentoflavone considerably decreased ear fold thickness and skinfold in a dose-dependent manner in comparison to the IMQ group. The biflavonoid enhanced histological injuries in IMQ-treated mice and showed anti-inflammatory effects by influencing several proinflammatory cytokines. It dose-dependently inhibited the IMQ-induced increase of mRNA expression, upregulated protein levels, and serum levels of IL-17A, IL-22, IL-23, and TNF-α in skin lesion. The effects of amentoflavone on M5-induced alterations in keratinocytes were also examined. M5 is a mix of IL-1α, IL-17A, IL-22, oncostatin M and TNF-α that promoted the proliferation of HaCaT cell line. Moreover, M5 decreased apoptosis in HaCaT cells. Amentoflavone demonstrated to inhibit HaCaT cells proliferation, to promote apoptosis, and to inhibit the increase of IL-17A, IL-22, and NF-κB expression. Previously, Sosa et al. (2007) investigated amentoflavone for its topical anti-inflammatory activity evaluated as inhibition of Croton-oil-induced ear edema in mice. Amentoflavone reduced edema by 36% and 94% at doses of 0.1 and 1.0 μmol/cm2, respectively.

23.4.2. Apigenin Apigenin (Fig. 23.2) is a flavone abundantly present in fruits and vegetables that has shown to possess several interesting biological properties including anti-inflammatory and antioxidant activities. Apigenin, together with

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FIGURE 23.2 Chemical structure of apigenin.

FIGURE 23.3 Chemical structure of astilbin.

luteolin, was reported to be among the most potent compounds in inhibiting inflammatory cytokines production in different cell lines (Comalada et al., 2006; Hougee et al., 2005; Shanmugam et al., 2008; Xagorari et al., 2001; Ueda et al., 2004). The flavone showed significant inhibition of NF-κB and a strong downregulation of both E-selectin (a proinflammatory adhesion molecule) and IL-8 proteins (Vogl et al., 2013). In particular, apigenin inhibited E-selectin expression with an IC50 value of 17.7 μM after TNF-α stimulation and an IC50 value of 13.6 μM after LPS stimulation. IC50 values of 31.7 and 18.9 μM in TNF-α- and LPS-stimulated cells, respectively, were found about IL-8 expression. The inhibitory activity of apigenin of TNF-α and IL-6 by inhibiting NF-κB activation was previously investigated in mouse peritoneal macrophages (Xie et al., 2012). Kang et al. (2011) investigated the effects of apigenin on the production of inflammatory cytokines such as IL6, IL-8, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) in HMC-1 cells. Apigenin inhibited the inductive effect of phorbol 12-myristate 13-acetate (PMA) plus A23187 on the production of IL-6, IL-8, TNF-α, and GM-CSF. Additionally, apigenin was able to attenuate the expression of cyclooxygenase (COX)-2. In activated HMC-1 cells, apigenin inhibited the PMA plus A23187-induced activation of nuclear factor (NF)-κB, IκB degradation, and luciferase activity. Furthermore, the flavone suppressed the expression of COX-2, IL-6, IL-8, GM-CSF, and TNF-α by decreasing the intracellular Ca21 levels and inhibiting NF-κB activation.

23.4.3. Astilbin The flavonoid astilbin (Fig. 23.3) is the major active component extracted from the rhizome of Smilax glabra. S. glabra is widely used in traditional Chinese medicine for the treatment of inflammatory and autoimmune diseases (Di et al., 2016). Several studies reported the ability of astilbin to downregulate T-cell activity by inducing apoptosis, stimulating negative regulatory cytokine (IL-10) and suppressing activated T-cell adhesion and migration (Cai et al., 2003a,b; Zou et al., 2010). Astilbin inhibited migration and antigen presenting of dendritic cells, reduced the activation of both T- and B-cells in lupus-prone mice, and inhibited T lymphocyte functions in acute heart allograft rejection (Guo et al., 2015; Xu et al., 2015). Zou et al. (2010) demonstrated as astilbin showed downregulation of T-cell activity, induction of apoptosis, a negative stimulation of regulatory cytokine expression, suppression of activated T-cell adhesion and migration. Recently, Di et al. (2016) studied the potential role of astilbin as antiposoriatic agent by using an IMQ-induced psoriasis-like mouse model. Doses of 2550 mg/kg of astilbin were used. Astilbin ameliorated keratinocyte proliferation, elevations in circulating CD4 and CD81 T cells and inflammatory cytokines such as IFN-γ, IL-17A, IL-2, IL-6, and TNF-α. The flavonoid is able to inhibit in vitro IL-17 secretion, Th17 cell differentiation, and Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) signaling in Th17 cells, and to upregulate SCOSE3 expression in

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psoriatic lesions, suggesting that astilbin directly affects multiple processes involved in Stat3 signaling in IMQinduced psoriasis-like inflammation. Jak/Tyk proteins have recently been proposed as potential targets for the treatment of psoriasis (Andre´s et al., 2013). In psoriasis, these proteins signal through STAT molecules, including STAT3 expression and activation, and been augmented in psoriatic lesions (Lu et al., 2013). Di et al. (2016) found that the levels of both the phosphorylated and nonphosphorylated forms of Stat3 and jak3 proteins decreased by using high dose of astilbin in treated mice. Taking into account all of the reported studies, astilbin appears to target multiple processes and may be useful in a variety of inflammatory diseases with fewer associated toxicities and adverse effects than conventional treatments.

23.4.4. Baicalin Baicalin (Fig. 23.4) is one of the main constituents responsible for the pharmacologic actions of Scutellaria baicalensis, a Chinese herbal medicine used for centuries to treat psoriasis. It is found in other Scutellaria species, including S. lateriflora and S. galericulata. This compound exhibited many different pharmacological activities, including antiviral activity and photoprotective effects against acute and multiple UVB-induced photodamage associated with reduction of oxidative stress (Nayak et al., 2014; Wang et al., 2013a,b; Zheng et al., 2014). Moreover, baicalin has been investigated as anti-inflammatory and antiallergic agent for the treatment of diseases such as asthma, bronchitis, hepatitis, nephritis, and atopic dermatitis (Kubo et al., 1984; Huang et al., 1986). Several studies documented the antiinflammatory properties of baicalin (Lee et al., 2015; Li et al., 2000; Lin and Shieh, 1996). Recently, Wu et al. (2015) evaluated the anti-inflammatory activity and the keratinocyte differentiationinducing activity of baicalin in vivo by using the 2,4-dinitrofluorobenzene-induced contact hypersensitivity model and mouse-tail test for psoriasis, demonstrating that topical application of a cream with baicalin alleviates inflammatory reaction and stimulates normal keratization. Baicalin cream at concentrations of 1% and 3% showed epidermis differentiation, although with less magnitude than 5% baicalin cream. In psoriatic patients, under the influence of proinflammatory cytokines, keratinocytes express several growth factors and cytokines involved in the immune responses that in turn drive angiogenesis. Therefore, drugs able to block the pathological changes of keratinocytes will be useful for the treatment of patients affected by psoriasis. Generally, results obtained in this study suggested that 5% baicalin cream was a promising agent for psoriasis treatment. However, more in vivo studies are needed.

23.4.5. Delphinidin Delphinidin is an anthocyanidin abundantly identified in pigmented vegetables and fruits, particularly blueberry, characterized by interesting antioxidant and anti-inflammatory properties (Fig. 23.5). The effects of delphinidin on psoriatic epidermal keratinocyte differentiation, proliferation and inflammation by using a reconstructed human psoriatic skin equivalent (PSE) model were recently investigated (Chamcheu et al., 2013, 2015).

FIGURE 23.4 Chemical structure of baicalin.

FIGURE 23.5 Chemical structure of delphinidin.

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The treatment of PSE with delphinidin improved keratinocytes differentiation. Delphinidin induced cornification (without affecting apoptosis) and the protein expression and mRNA of markers of differentiation such as caspase-14, filaggrin, involucrin and loricrin. It also decreased the expression of markers of inflammation often induced in psoriatic skin such as antimicrobial peptides S100A7-psoriasin and S100A15-koebnerisin. Pal et al. (2015) evaluated the ability of delphinidin topically applied to modulate pathological markers of psoriasis lesions in the flaky skin mouse model. The anthocyanidin significantly reduced the infiltration of macrophages and neutrophils and abolished the histological characteristics of psoriatic lesions. The treatment with delphinidin restored in flaky skin mice the differentiation of markers including keratin-1, keratin-10, loricrin, caspase-14, and filaggrin. Loricrin is a marker of epidermal differentiation and exerts a significant role in the formation of epidermal skin barrier. In addition to the structural function, keratins regulate differentiation, proliferation and migration of epithelial cells, contributing to maintain the structural integrity of the epidermis. Caspase-14 is an important marker that is involved in the cleavage of profilaggrin into filaggrin, which is a major structural protein in the terminal differentiation and formation of cornified envelops in the stratum corneum. The treatment with delphinidin also modulated the expression of tight junction proteins that is altered in inflammatory diseases such as psoriasis, and increased the protein expression of JunB that is greatly reduced in psoriatic skin. The interesting antioxidant and anti-inflammatory properties, the high bioavailability, the low molecular size and the expected good skin tissue distribution of delphinidin makes this anthocyanidin a promising candidate for the treatment of psoriasis.

23.4.6. Genistein Genistein (Fig. 23.6), an isoflavone abundant in soybeans, has demonstrated promising biological effects. It is a potent antioxidant and anti-inflammatory agent and a specific inhibitor of protein tyrosine kinase. The isoflavone exerts antitumor effects, especially against breast and prostate cancers, chemoprevention against photocarcinogenesis, prevention of photoaging and antiangiogenesis (Kang et al., 2003; Gupta et al., 2014; Valsecchi et al., 2011; Yu et al., 2010). Recently, Terra et al. (2015) showed that genistein could prevent a nitrosative event, which leads to tissue protection and cell proliferation. These results reveal the importance of UVB radiation-induced nitrosative damage and help to clarify the mechanism of the photoprotective effect of genistein. Previously, Ji et al. (2012) studied the effects of genistein on the inflammatory response in LPS-treated RAW264.7 macrophages. Authors showed that concentrations of genistein of 1, 5, and 10 μM decrease proinflammatory responses in LPS-treated macrophages by inhibiting the activation of NF-κB following adenosine monophosphate-activated protein kinase stimulation. These results are of interest for the implications in the treatment of chronic low-grade inflammatory diseases.

23.4.7. Isoliquiritigenin Isoliquiritigenin (Fig. 23.7) is a chalcone that has been demonstrated to possess interesting biological properties, including antioxidant, anti-inflammatory, antiviral, antidiabetic, antispasmodic, and antitumor activities

FIGURE 23.6 Chemical structure of genistein.

FIGURE 23.7 Chemical structure of isoliquiritigenin.

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(Peng et al., 2015; Yamamoto et al., 1991; Yadav et al., 2011). Due to the ability to affect Th2-mediated inflammation, the chalcone showed antiasthma properties with inhibitory effects in vitro on memory Th2 responses and antigen-induced Th2 inflammation in vivo via suppressing the production of IL-4 and IL-5 (Yang et al., 2013). Moreover, isoliquiritigenin exhibited an inhibitory activity on LPS-stimulated production of TNF-α, IL-6 and IL-12 p40 production (Li et al., 2014). Focusing especially on the anti-inflammatory activity, isoliquiritigenin suppressed expression and mRNA accumulation of E-selectin and (vascular cell adhesion molecule) VCAM-1. Moreover, the chalcone attenuated the expression of platelet endothelial cell adhesion molecule-1 induced by TNF-α and downregulated cell adhesion molecule proteins in TNF-α-activated cells by blocking degradation of IkBα and nuclear translocation of NF-κB at the transcriptional levels (Kwon et al., 2007). As reported by Hattori et al. (2007), isoliquiritigenin exerted anti-inflammatory effects also through antinephritic effects. Isoliquiritigenin reduced the inflammatory response of macrophages via the inhibition of the activation of NF-κB p65 and AP-1 (Feldman et al., 2011). In RAW264.7 macrophages, isoliquiritigenin demonstrated to induce the expression of HO-1 through the ERK 1/2 pathway, with inhibition of LPS-induced NO, IL-1β, and TNF-α production. Chen et al. (2012a,b) has shown that isoliquiritigenin is able to reduce the expression of cytokine IL6, suggesting its potential use in inflammatory disorders. Based on these results, Wu et al. (2016) examined the effects of the chalcone in some psoriasis models such as the human keratinocytes HaCaT and NHEK in vitro, the keratin 14/vascular endothelial growth factor (VEGF) transgenic mouse, and the IMQ-induced psoriasis-like mouse. In vivo isoliquiritigenin has been shown to improve psoriatic lesion and to slow down the pathologic process of psoriasis by decreasing IL-6 and IL-8 levels and by reducing CD4, CD8, CD11b/c, F4/80, and VEGF expression in the ear and back skin. Additionally, isoliquiritigenin suppressed NF-κB expression and inhibited its activation by downregulation of phosphorylated levels of NF-κB, which consequently resulted in the reduction of proinflammatory cytokines IL-6 and IL-8. Similar results were obtained in vitro. Isoliquiritigenin reduced NF-κB at both protein and mRNA levels. This result suggests isoliquiritigenin as a promising NF-κB inhibitor and a potential agent for treatment of psoriasis.

23.4.8. Luteolin Recently, the effects of the flavone luteolin (Fig. 23.8) on human cultured keratinocytes were investigated (Weng et al., 2014). Authors showed that TNF, at the concentration of 50 ng/mL, produced significant production of IL-6, IL-8 and VEGF from primary keratinocytes and human HaCaT cells. Luteolin pretreatment (at concentrations of 10100 μM) inhibited in a concentration-dependent manner both mRNA expression and release of all mediators. Luteolin reduced also the TNF-induced mRNA expression of RELA gene that encoding the NF-κB subunit NF-κB p65. The gene expression of RELA is increased in human psoriatic skin. Moreover, the flavone decreased TNF-induced phosphorylation, DNA binding and nuclear translocation of NF-κB. A previous work demonstrated the ability of luteolin to decrease TNF-triggered phosphorylation, nuclear translocation and DNA-binding activity of NF-κB (Zbytek et al., 2003). Of interest is the inhibition of IL-6 gene expression and secretion by luteolin, since IL-6 is a requisite to drive maturation of Th17 cells, also involved in the pathogenesis of psoriasis and the significant decrease of HaCaT, but not normal keratinocyte proliferation. The flavone is lipid-soluble. This characteristic may be useful for the development of topical formulations that easily penetrate the skin.

FIGURE 23.8 Chemical structure of luteolin.

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FIGURE 23.9 Chemical structure of quercetin.

23.4.9. Quercetin Quercetin (Fig. 23.9) is a flavonol found in various fruits and vegetables, in particular in capers, blueberries, broccoli, lovage, sorrel, dill, plums, cranberries, apples, sweet potatoes, and many other commonly eaten foods. Quercetin showed interesting biological properties including anti-inflammatory, antiviral, antibacterial, and antitumor effects (Kaur and Kapoor, 2001; Materska, 2008). The flavonol is considered a strong antioxidant due to its ability to scavenge free radicals, to bind transition metal ions, and to inhibit lipid peroxidation. Chen et al. (2005) demonstrated that quercetin inhibits IFNγ-induced STAT-1 activation in mouse BV-2 microglia. Quercetin also inhibited LPS-induced STAT-1 activation, iNOS expression and NF-κB activation (Ha¨ma¨la¨inen et al., 2007). The ability of quercetin to suppress ultraviolet irradiation-induced expression of cytokines IL-1β, IL-6, IL-8 and TNF-α was demonstrated by Vicentini et al. (2011) in human keratinocytes. Moreover, the flavonol inhibited contact dermatitis and photosensitivity in humans (Weng et al., 2012). Successively, Vijayalakshmi et al. (2012) investigated the antipsoriatic activity of quercetin isolated from the rhizome of Smilax china by using the mousetail test. The in vitro antiproliferative activity on HaCaT cells was also studied. In the mouse-tail test, quercetin at doses of 25 and 50 mg/kg produced remarkable orthokeratosis and important change in epidermal thickness in comparison to the control. Psoriatic lesions, in fact, are characterized by a granular layer of the epidermis seriously reduced or absent. Quercetin, able to act by multiple mechanisms, may be proposed as a potential agent in treating psoriasis that is a chronic inflammatory skin disease with multiple etiologies.

23.5. CONCLUSION The quest for agents in treating psoriasis with a low profile of adverse effects and manageable cost is ongoing. In this regard, natural compounds hold promise. Some classes, in particular flavonoids, have been identified mainly for their interesting and promising antioxidant and anti-inflammatory effects. Generally, the researches herein reported showed significant biological properties of plant-derived flavonoids in vitro and in vivo, especially about anti-inflammatory activity, which could be beneficial for both the prevention and the treatment of psoriasis. Flavonoids can modulate action and production of inflammatory mediators, not only directly but also indirectly, by modulating the action of other molecules. Appropriate formulations of these phytochemicals may be promising candidates for the development of agents for local and systemic treatment of psoriasis and other inflammatory skin diseases. However, further studies should focus more on understanding the efficacy of flavonoids in humans and bringing them to the forefront of treatment of human diseases such as psoriasis.

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Hougee, S., Sanders, A., Faber, J., Graus, Y.M., van den Berg, W.B., Garssen, J., et al., 2005. Decreased pro-inflammatory cytokine production by LPS-stimulated PBMC upon in vitro incubation with the flavonoids apigenin, luteolin or chrysin, due to selective elimination of monocytes/macrophages. Biochem. Pharmacol. 69, 241248. Huang, N., Rizshsky, L., Hauck, C.C., Nikolau, B.J., Murphy, P.A., Birt, D.F., 2012. The inhibition of lipopolysaccharide-induced macrophage inflammation by 4 compounds in Hypericum perforatum extract is partially dependent on the activation of SOCS3. Phytochemistry. 76, 106116. Huang. In: Huang, X., Tu, C.H.L., Zhang, L.X., Dai, J.R., Jin, Y.D., Huang (Eds.), Immunopharmacology. Shanghai Science and Technology Press, Shanghai. Jacobson, C.C., Kumar, S., Kimball, A.B., 2011. Latitude and psoriasis prevalence. J. Am. Acad. Dermatol. 65, 870873. Ji, G., Zhang, Y., Yang, Q., Cheng, S., Hao, J., Zhao, X., et al., 2012. 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24 Probiotics and Anti-inflammatory Processes in HIV Infection: From Benchside Research to Bedside Marcella Reale and Katia Falasca University “G.D’Annunzio” Chieti-Pescara, Chieti, Italy

24.1. INTRODUCTION Probiotics are nonpathogenic microorganisms that confer a number of beneficial effects on the host’s health (Marteau, 2006). The Food and Agricultural Organization (FAO) and the World Health Organization (WHO) have recognized the ability of probiotic foods to provide health benefits and their use is considered safe for human consumption. Foods containing probiotic bacteria range from dairy products (such as fermented milk), to drinks and supplements containing freeze-dried probiotics. Recent clinical trials on the use of probiotics in the prevention and treatment of human disorders have evidenced that probiotics have multiple and diverse influences on the host and that their use is particularly helpful in diseases or conditions characterized by altered processes of intestinal mucosa interaction with gut microbiota, such as inflammatory bowel diseases or AIDS (Falasca et al., 2015). The stimulating signals conferred by the gut flora are dynamically affected by the microbial composition, and dietary components may influence various elements of immune responses such as inflammation. Also nutrition, that may play a role in inflammatory conditions, may be useful in the therapy of several inflammatory diseases. This chapter will discuss the anti-inflammatory and immunomodulation of probiotics, the mechanism of their action and finally the impact of probiotic therapy on the progression of HIV.

24.2. OVERVIEW OF PROBIOTICS AND THEIR MODE OF ACTION The most important characteristics for the selection of probiotics are a high survival rate through the digestive tract (low pH, digestive enzymes, bile toxicity), and the ability to bind to the gut mucosal surface. These characteristics allow probiotics to effectively compete against pathogenic bacteria in terms of adhesion to mucosal intestinal wall and nutrition, resulting in potential prevention against the effects of pathogenic bacteria invasion. The rationale behind the study of the influence of probiotics in a healthy population lies in the research of a possible general improvement of health and long-term prevention of diseases. Some probiotics are used to prevent or treat infections, whereas others are of value in the prophylaxis or treatment of allergic or inflammatory disorders. Species of Lactobacilli and Bifidobacteria are prominent probiotics with immuno-modulator properties (Fig. 24.1). Probiotics can affect the composition of the resident gut microbiota, may compete with diarrheal pathogens for adhesion sites, strengthen the mucosal barrier, tighten junctions between enterocytes, and/or enhance the mucosal IgA-mediated immune responses to pathogens (Mantis et al., 2011). The secretion of antimicrobial substances and the induction of intestinal mucin production may also contribute to the beneficial effects of probiotics. Different probiotic strains may differently modulate the immune system, depending on host Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00024-X

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FIGURE 24.1 Anti-inflammatory activity of multifunctional probiotics bacteria modulate the Th1/Th2 balance, suppress pathogenic Th17 and induces steady-state Th17, to prevent the development of inflammatory diseases. Probiotic strain such as Lactobacillus casei Shirota (LcS) exert multifunctional activities and is efficacius in diseases caused by compromised or iperactivated immune functions. Inducing macrophages functions by IL-12 production, LcS promote NK activation and Th1 polarization, while by induction of IL-10 production promotes Treg developments with consequent control of inflammatory responses.

characteristics, genetic background, pathogenesis of diseases, as well as on their ability to survive in the gastrointestinal tract, dose ranges and treatment periods. The precise molecular mechanisms of probiotic strains are not completely known. Probiotic effects can be direct or indirect through modulation of the endogenous intestinal microflora or immune system. The evidence of probiotics direct benefit to human health is given by their ability to adhere to the intestinal mucosa wall, decrease the intestinal permeability, and enhance the intestinal epithelial resistance (Madsen et al., 2001). Actin and occludin phosphorylation is also evidence of probiotics driven direct benefit to human health, since they contribute to the formation of strengthened tight junctions by which the protective function of the epithelial barrier is enhanced. Probiotics adhere to mucosal tissue in a strain-dependent manner, thus transiently enhancing intestinal persistence and limiting pathogens access to the epithelium. The findings that emerge from studies of probiotics use on immune, infectious and inflammatory conditions in humans are heterogeneous as a consequence of species-dependent and strain-dependent effects. Probiotics are able to modulate the composition of intestinal microbiota by lowering the pH and competing for nutrients, and for binding to specific receptors on host epithelial cells (Caballero-Franco et al., 2007). Several studies on allergic children have reported that supplementation with a probiotic can reduce pathogenic bacteria and enhance levels of beneficial bacteria (Klewicka et al., 2011). Probiotics also promote the production of significant quantities of short-chain fatty acids (SCFAs) deriving from fermentation of dietary fiber, and exert an anti-inflammatory activity (Hemarajata and Versalovic, 2013; Puddu et al., 2014), such as the reduction of inflammatory lesions in animal models of asthma and colitis. Probiotics also promote neutrophils anti-inflammatory effects by binding to the G protein-coupled receptors GPR41 and GPR43 (Maslowski et al., 2009). Tedelind et al. demonstrated that SCFAs act through modulation of nuclear factor κB (NF-κB) and cytokine activities (Tedelind et al., 2007). Several studies have reported that probiotic exert their effects on cells involved in the innate and adaptive immune responses, such as epithelial cells, dendritic cells, monocytes/macrophages, B- cells, T-cells and natural killer (NK) cells. Probiotic bacteria modulated dendritic cells (DCs) maturation (Borchers et al., 2009) Humanmonocyte-derived DCs (MoDCs) treated with a probiotic culture supernatant released IL-10 that enabled the differentiation and survival of regulatory T (Treg) cells (Rimoldi et al., 2005). Bifidobacterium animalis and Bifidobacterium longum were shown to induce IFN-γ and tumor necrosis factor-α (TNF-α) release by DCs, while only Bifidobacterium bifidum could induce Th17 cell activation through the release of IL-1 by DCs (Lopez et al., 2010). Lactobacilli attenuated proinflammatory responses by regulating NF-κB

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activity (Hwang et al., 2013), while other probiotics reduced TNF-α induced NF-κB activation in a TLR9dependent manner (Ghadimi et al., 2008). Clinical approaches with probiotics are appealing because of their lack of toxicity and the desire of patients to use natural physiological treatments. Studies that have evaluated the benefits of many probiotic strains are still too few and recent, and many of them have been carried out on animal models and must be confirmed in humans (Rijkers et al., 2010). Thus, the optimal strains, composition, dose and length of probiotic treatment in various clinical settings should be examined by conducting large, placebo-controlled, prospective trials. Therefore, we will continue to study the effects of probiotics for long time.

24.2.1. Probiotics and Anti-inflammatory Activity The body has the capability to recognize pathogens from the external environment and to act in order to remove them by innate, humoral and cellular immune system responses. Inflammation represents an essential component of immune-mediated protection against pathogens and also against tissue damage. Factors such as age and diet, in addition to genetic predisposition, trauma, physical and emotional stress contribute to inflammation. Acute inflammatory reactions are usually self-limiting and resolve rapidly by a negative feedback mechanism. Thus, regulated inflammatory responses are essential to remain healthy and maintain homeostasis. Typical characteristics of inflammatory responses include loss of barrier function, infiltration of inflammatory cells into compartments where they are not normally found, and overproduction of proinflammatory cytokines such as those of the interleukine (IL) family; IL-1β, IL-6, and IL-18, tumor necrosis factor alpha (TNF-α), chemokines such as IL-8, IP-10, monocyte chemoattractant protein (MCP-1), macropage inflammatory protein (MIP-1), RANTES (regulated on activation, normal T cell expressed and secreted), matrix metalloproteases (MMPs) such as MMP-1, -3, -9 and -13, eicosanoids such as Cox-1, Cox-2, and iNOS (Nitric oxide synthase 2) that, by interacting with each other in a complex manner, trigger a vicious cycle of pro-inflammatory signals resulting in ongoing inflammation. The transcription NF-κB is considered an important mediator of inflammation. Following stimulation of cells with proinflammatory cytokines, bacteria or bacterial cell products, inhibitor of κB (IκB) is phosphorylated and then degradated, while NF-κB translocates into the nucleus and activates transcription of numerous proinflammatory genes. The nuclear protein high mobility group box-1 (HMGB1) also known as a danger signal, is one of the important mediators in infection, injury and inflammation that act by activation of receptor for advanced glycation end-products (RAGE) and Toll-like receptors (TLRs) such as TLR2, TLR4, or TLR9. Activation of these receptors will ultimately result in activation of NF-κB, known to induce upregulation of leukocyte adhesion molecules and production of proinflammatory cytokines and angiogenic factors in both hematopoietic and endothelial cells, thereby promoting inflammation (Kawai and Akira, 2007). NF-κB can activate antiapoptotic genes, such as TNF-receptor-related genes, and reduce the apoptosis of inflammatory cells elongating and worsening tissue inflammatory injury. Unfortunately, when inflammatory responses fail to regulate themselves, danger signals can become chronic and contribute to the perpetuation and progression of disease, and irreparable damage to host tissues can occur. Long-term inflammation increases the risk for cardiovascular diseases, cancer and dementia; moreover, at the moment, it is still unclear if the inflammation is the cause or a consequence of type 2 diabetes and obesity. Typically, diseases or conditions with a well-recognized inflammatory component are treated with general or specific anti-inflammatory drugs. However, a nonpharmacologic strategy is the stimulation of gut microorganisms; indeed several studies have reported that stimulating signals from gut microorganisms can mitigate inflammation and maintain the immune function (Cebra, 1999; Holt and Jones, 2000). In patients with ulcerative colitis, where NF-κB p65 protein is highly activated, it was observed that after probiotic ingestion, NF-κB p65 level was lowered. These data are in accord with the finding of Shi et al. showing that Bifidobacterium longum downregulates TNF-α and IL-8 production and inhibits NF-κB activation in inflamed mucosa of active ulcerative colitis (Shi et al., 2003). Probiotics modulate both DCs and Treg cells, whereby DCs exposed to some probiotics induce anti-inflammatory responses and generate IL-10-producing Treg cells. Probiotics inhibit the production of IL-8 and TNF-α by intestinal epithelial cells exposed to pathogen microorganisms and induce the production of anti-inflammatory mediators such as TGF-β, which can stimulate the differentiation of immature DCs in regulatory dendritic cells (DCregs). Probiotics may determine and lead the differentiation of DCregs, followed by the induction of Treg cells. They exert an anti-inflammatory function by controlling type 1 T helper (Th1), type 2 Thelper (Th2) and probably type 17 T helper (Th17) cells. In animal models used to study intestinal inflammation, a favorable action of probiotics has been shown (Dieleman et al., 2003; McCarthy et al., 2003; Asahara et al., 2004). Lactobacillus casei prevents the development of

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acute dextran sodium sulfate-induced colitis in TLR-4 mutant (lps/lps) mice by inhibiting myeloperoxidase activity and IL-12p40, and increasing TGF-β and IL-10 mRNA. These effects suggest that the mechanism of action of Lactobacillus casei largely depends on TLR-4 status (Chung et al., 2008). The intestine usually produces small quantities of IL-12, but probiotics have the potential to increase this production dependent on the local environment. By this mechanism, it is possible to stimulate the differentiation of CD41, and increase the activity of NK cells, resulting in a local intestinal defense by cytolysis. Intestinal inflammation is associated to imbalance of the intestinal microflora and ingestion of probiotics help to stabilize the gut immunologic barrier, thus reducing the generation of inflammatory mediators such as IL-1, TNF, and IFNg. The immunoregulation and suppression of Th17 activity and IL-17 production can be regarded as a possible mechanism to explain the protective anti-inflammatory effects of probiotics. Probiotics may determine an increased production of IL-10, limit the incidence of tumors, and inhibit the production of IgE that mediates allergic reaction (So et al., 2008; Sudo et al., 2002). It was recently reported that probiotic bacteria differently modulate phagocytosis in healthy and allergic subjects: in healthy subjects probiotics have an immunostimulatory effect, while in allergic subjects they downregulate the inflammatory response (Pelto et al., 1998). Borruel et al. have suggested that the anti-inflammatory effects of probiotic bacteria could be systemic, at least in part, rather than localized, as evidenced by a significant reduction in proinflammatory cytokines, including TNF-α and a reduction in the number of CD4 cells (Borruel et al., 2002). In general, the effect of probiotics on in vitro and in vivo models has been the suppression of proinflammatory signaling cascades. 24.2.1.1. In Vitro Studies In vitro studies have been performed to understand the effects of probiotics on the immune system; however, as these studies may not accurately reflect the events in vivo, the mechanism of immune responsiveness modulation has not been completely understood yet (Ibnou-Zekri et al., 2003). The prototype of in vitro model to study the effects of probiotics in intestinal inflammation is performed by intestinal epithelial cell lines and proinflammatory stimuli, such as cytokines, bacterial cell wall, as well as lipopolysaccharides (LPS), flagellin, muramyl dipeptide, or intact bacterial cells. Probiotics suppress the production of TNF-α in intestinal epithelial cells (IECs) exposed to pathogenic stimuli and induce production of TGF-β, which promotes the differentiation of immature dendritic cells (iDCs) to DCregs. Also, probiotics decrease IL-6 and increase IL-10 production by macrophages in the inflamed mucosa. Probiotics may affect the expression level of TLR-4 and other Toll-like receptors on mucosal and systemic cells, increase Toll-like receptors responsiveness to bacterial stimuli and modulate the cytokine production capacity towards LPS stimulation. Culture of probiotic bacteria with either mouse or human colon cells activated antiapoptotic Akt/protein kinase B and inhibited activation of the proapoptotic p38/mitogen-activated protein kinase by TNF-α, IL-1b, or IFNg. Inhibition of apoptosis may enhance survival of intestinal cells and promote proliferation during recovery from epithelial injury. The mechanism of anti-inflammatory effects of Bifidobacteria has not been identified yet. Some results suggest that Bifidobacterium strains, by suppressing the secretion of IL-8, inhibit NF-κB signal pathway activity, which can be due to downregulation of IκB phosphorylation, decrease of nuclear translocation of NF-κB, or inhibition of pro-inflammatory ligand binding to its cellular receptor (Heuvelin et al., 2009). Khokhlova et al. reported that conditioned media of all Bifidobacterium strains studied are able to attenuate TNF-α and LPS-induced inflammatory responses in the HT-29 cell line, while neither killed bifidobacterial cells, nor cell-free extracts showed such abilities (Khokhlova et al., 2010). Other studies suggest that anti-inflammatory activity is associated to either intact bacterial cells or bacterial DNA. In any case, most studies agree on the Bifidobacteria strain-specific immunomodulatory ability. In general, gut flora-derived Bifidobacteria are able to inhibit production of proinflammatory cytokines, including the INF-g-inducing IL-12, but not the production of the anti-inflammatory cytokine IL10. Stimulation of murine splenocytes with TGF-β plus IL-6 induced high IL-17 production that was suppressed by several Bifidobacteria. In contrast, other bacteria tested did not exert the suppressive effect on IL-17 production (Tanabe et al., 2008; Miyauchi et al., 2013; Ogita et al., 2011). The IL-23/IL-17-mediated inflammatory axis has recently been implicated in the pathogenesis of inflammatory bowel disease (IBD) but our knowledge on how probiotics influence the differentiation of Th17 cells is not complete. The observation that different probiotic strains have the ability to modulate Treg and/or Th1 subsets and to inhibit the pro-inflammatory Th17/IL-17 activity boosts knowledge of immunomodulatory function of commensal Bifidobacteria and creates new perspectives on the possibility of their use in the treatment of Th17-related diseases, such as IBD. Th1 and Treg cells induced by probiotics could inhibit Th17 activity involved in inflammatory pathologies. Evidence has been

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provided that probiotics favor the conversion of immature DCs to DCregs, thus leading to the induction of IL-10 producing Treg cells (Smits et al., 2005; Foligne et al., 2007). Probiotic bacteria, by induction of T-cells with regulatory properties, can lead the mucosal immune system toward a noninflammatory pattern. Probiotics can downregulate Th1 response and inhibit the production of proinflammatory cytokines, IL-12, TNF-α, and IFN-γ, and alter the TNF-α /IL-10 balance in vitro (Pena and Versalovic, 2003). Several probiotic strains demonstrated the ability to potently suppress TNF production by LPSactivated monocytes and primary monocyte-derived macrophages from children with Crohn’s disease. The study of Lin et al. reported that human TNF and MCP-1 suppression by probiotic Lactobacillus reuteri was straindependent, and the activation of c-Jun and AP-1 represents primary targets for probiotic-mediated suppression of TNF transcription (Lin et al., 2008). Probiotics are able to modulate the Th1/Th2 balance to prevent the development of inflammatory diseases. Since in humans it would be difficult to directly study interactions between mucoid immune system cells and lactic acid bacteria (LAB), the use of peripheral blood mononuclear cells (PBMCs) is a good alternative to study in vitro the effects of LAB-immune cells interactions. Pochard et al. Pochard et al. (2002) have reported that PBMCs from allergic patients had reduced Th2 responses characterized by lower IL-4 and IL-5 secretion when pre-treated in vitro with several LAB and then exposed to house dust mite. Other studies have shown similar effects on IL-1b, IL-6, IL-8, and TNF-α production from PBMCs treated with Lactobacillus bulgaricus (Niers et al., 2005), or opposite effects on IFN-g, TNF-α, and IL-10 when treated with different LAB (Miettinen et al., 1998; Veckman et al., 2004). The species-specific effects of probiotics was determined by studying the ability to induce functional FoxP31 Treg from CD251 cells in PBMCs from healthy adults (de Roock et al., 2010; Ashraf and Shah, 2014). Several Lactobacillus species and Streptococcus thermophilus induce a Th1-type cytokine profile, increasing IL-12 and IFN-γ and reducing IL-10 production. In contrast, Bifidobacteria induce Th2-cytokines, such as IL-10 and IL-6, and less IL-12 and IFN-γ from NK cells (Wiese et al., 2012; Gackowska et al., 2006). Fink et al. recently demonstrated that different gut-derived probiotic bacteria can distinctly imprint monocytederived dendritic cell functions in initiating T or NK cell responses (Fink et al., 2007; Rizzardini et al., 2012). Bui et al. published in Frontiers in Immunology a study showing that probiotics induce decreased NK cell cytotoxicity but increased secretion of IFN-γ and TNF-α: they have defined this effect as “split anergy” (Bui et al., 2015). Probiotic bacteria, inducing split anergy in NK cells, promote differentiation of target cells, increase key differentiation receptors on tumor cells, induce tumor cell resistance to NK cell-mediated cytotoxicity, and inhibit inflammation due to a decrease of cytokine release. In addition, probiotic bacteria induce significant expansion of NK cells (Tseng et al., 2015). 24.2.1.2. In Vivo Studies Studies with probiotics, in animal models, are characterized by a high level of heterogeneity due to the wide variety of animal models used. The most well documented effects of probiotics have been evidenced by studies on several diseases, such as infantile diarrhea (Guandalini et al., 2000; Rosenfeldt et al., 2002; Szajewska and Mrukowicz, 2005), atopic eczema in children (Isolauri et al., 2012; Kalliomaki et al., 2010; Harata et al., 2016; Rosenfeldt et al., 2003) and IBD (Soo et al., 2008; Floch et al., 2006; Do et al., 2010). Clinical and experimental evidence has demonstrated the potential role of probiotics in the prevention or treatment of IBD. Probiotics may exert an immunomodulatory activity that may have anti-inflammatory effects in the intestine. In the study of Pena et al., Lactobacillus paracasei/Lactobacillus reuteri-treated animals demonstrated a partial elimination of intestinal inflammation and significantly reduced mucosal IL-12 mRNA levels. Possibly, Lactobacilli inhibit NF-κB activation in the intestinal mucosa, resulting in diminished expression of IL-12 (Pena et al., 2005). Clinical trials of probiotics in patients with IBD have recently been conducted, with encouraging preliminary data in children and in adults with Crohn’s disease (Gupta et al., 2000). Several studies showed that a probiotic mixture of different strains in pouchitis disease reduced the Activity Index score, increased the percentage of infiltrating CD4 1 CD25 high and CD4 1 LAP-positive cells to the lamina propria, reduced IL-1β mRNA expressions, and increased Foxp3 mRNA expression. During mild inflammation, this expansion of regulatory cells seems to be adequate to dampen inflammation leading to pouchitis (Bibiloni et al., 2005; Sood et al., 2009). In patients with IBD, after consumption of a probiotic mixture, a significantly increased proportion of CD4 1 CD25 high cells, decreased concentrations of IL-12 in serum as well as decreased percentages of TNF-α- and IL-12-producing monocytes and myeloid dendritic cells were also detected (Lorea Baroja et al., 2007). In a pilot study on elderly people, the intestinal load of Lactobacilli

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was linked to all risk markers in the pathogenesis of inflammation, metabolic syndrome and cardiovascular disease, and a significant increase of phagocytic activity and NK cells activity was observed (Mikelsaar et al., 2010). The study of Senol et al. Senol et al. (2011) demonstrated that a pre-treatment with a mixture of 13 different probiotic bacteria attenuated the aspirin-induced gastric lesions by reducing the proinflammatory cytokines and the lipid peroxidation, enhancing mucosal sIgA production and stabilizing mucosal mast cell degranulation into the gastric mucosa. The efficacy of probiotics in the treatment of inflammation-based GI diseases is founded on the inhibition of pathogenic bacteria and inhibition of inflammatory processes (Yoshimura et al., 2003). Helicobacter pylori infection increases expression of inflammatory mediators (e.g., TNF-α, IL-8, inducible NOS and COX-2), and pretreatment of cells with Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus acidophilus inhibited these mediators, probably by enhancing the expression and signaling of SOCS-2 and SOCS-3. SOCS-2/-3 expression is mediated by phosphorylation of signal transducers and activation of transcription (STAT)-1 and STAT-3, and by simultaneous inhibition of Janus kinase (JAK) 2 phosphorylation, that negatively signals SOCS-2/-3 (Hanada et al., 2003). Several Lactobacillus strains can inhibit the proliferation of CD41 lymphocytes without affecting the production of TNF-α, IL-4, IL-5, and IL-10, both in healthy individuals and in patients with IBD (Schultz et al., 2003). In a recent study in patients with ulcerative colitis, psoriasis and chronic fatigue syndrome (Groeger et al., 2013), researchers measured inflammatory cytokines (C-reactive protein, TNF-α and IL-6) before and after administration of Bifidobacterium infantis. After taking the probiotic for 68 weeks, all inflammatory markers were substantially reduced in all conditions, with an average of 70% of participants experiencing positive results. Conditions such as heart disease, arthritis, type 2 diabetes, depression, cancer, obesity and many others show increased levels of C-reactive protein, IL-6 and TNF-α. In obesity, fat accumulation represents a high risk for type 2 diabetes, chronic inflammation, and hyperlipidemia, predisposing to coronary heart disease, cardiac arrest, and heart failure. The use of probiotics to reset the dysbiotic obese gut microbiome is a proposed approach to reduce the risk. Probiotics may reduce adipocyte cell size in high-fat diet mice, which can improve oxidative stress and the subsequent chronic inflammation (Lee et al., 2006). Beneficial effects in inflammatory and atopic diseases, probably by suppressing the production of IL-5, IL-6, and IFN-γ, were observed in adults affected by seasonal allergic rhinitis (Wichers, 2009). There are very limited data, however, regarding the effect of probiotics on acquired immunity, in particular in older people. It is well known that a wide variety of reactive oxygen species are continuously produced in the human body, and play an important role in the pathogenesis of cancer, cardiovascular diseases, allergies and atherosclerosis. Thus, the antioxidative effect of LAB, reported by several studies conducted in healthy volunteers (Wang et al., 2006; Kullisaar et al., 2003), may have an important impact on human health (Terahara et al., 2000). The ability of cyclooxygenase enzyme (COX) inhibitors, such as nonsteroidal anti-inflammatory drugs (NSAIDs), to delay or prevent the development and metastatic spread of certain cancers, underlines the relationship between inflammation and tumors. De Moreno de LeBlanc A et al. suggested that LAB present in the yogurt may exert an antitumor activity by an anti-inflammatory action. The authors suggest that yogurt could modulate the immune response by stimulating cytokine production when this is required, by increasing the IL-10-mediated down-regulation of the immune cells to avoid an exacerbated immune response, and by immune cells infiltration observed in the large intestine of long-term yogurt-fed mice. When the yogurt feeding was stopped, the animals did not develop tumors (de Moreno de LeBlanc et al., 2007; Perdigon et al., 1998). There are many studies relating to periodontal disease and systemic inflammation, and probiotics showed a positive impact on the affection of oral cavity, such as dental caries and halitosis (Burton et al., 2006). Several studies in healthy volunteers have reported that probiotic strains boost NK cell activity and phagocytic activity (Klein et al., 2008). A controlled, randomized, double-blind trial on smokers, with increased risk of developing systemic inflammation (Tappia et al., 1995), showed that consuming Lactobacillus plantarum strain 299 v for 6 weeks affected several systemic parameters, such as reduction of systolic blood pressure, blood concentration of leptin, fibrinogen, F2-isoprostanes (marker of oxidative stress), and IL-6 (Naruszewicz et al., 2002), and 3 weeks of daily Lactobacillus casei Shirota intake in Italian male smokers was associated with improved NK activity and increased the number of CD161 cells (Reale et al., 2012). A recent research of The Arthritis Foundation revealed the anti-inflammatory properties of Bifidobacterium infantis. This research was performed in lab animals and needs to be replicated in human studies before any firm conclusion can be drawn. In autism spectrum disorders, an intestinal inflammation and alterations of function linked to gut dysbiosis were observed, where the gut dysbiosis represents the phenotype known as gut-brain

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axis disruption and is associated with increased IL-6, TNF-α, and adipokines levels (Theoharides et al., 2013). Preliminary results showed a significant behavioral improvement by probiotics treatment. Even in healthy subjects, there was a slight modification of TNF-α and IL-6 inflammatory markers, although most clinical studies were performed in the elderly or in young adults (Gill et al., 2001a; Gill et al., 2001b; Ahmed et al., 2007; Schiffrin et al., 2010; McKenzie et al., 2016; Mortaz et al., 2015). All in vivo studies on animal models and in vitro studies suggest that many disorders associated with inflammation could benefit from taking probiotics and that the effects of probiotics are strain-specific, highlighting the variability of mechanisms of action across the multitude of probiotic strains and species.

24.3. PROBIOTICS AND HIV 24.3.1. HIV Since the original description in 1981 of an unusual cluster of cases of Pneumocystis carinii pneumonia and Kaposi’s sarcoma in previously healthy men who had sex with men (MSM), substantial advances in our understanding of the acquired immune deficiency syndrome (AIDS) have been achieved. The identification of a cytopathic retrovirus in 1983 and the development of a diagnostic serologic test for HIV in 1985 have served as the basis for developing improvements in diagnosis. In addition, therapy was dramatically altered with the introduction of antiretroviral drugs in 1987, and revolutionized by combination antiretroviral therapy (ART) in 1996. In the three years following the introduction of ART, in mortality, there was a 60%80% decline in AIDS, AIDSdefining diagnoses, and hospitalizations. The EuroSIDA study, comparing this early ART period to pre-ART and later-ART (19982002) treatment periods, observed a sustained decrease in mortality and progression to AIDS with ongoing ART (Mocroft et al., 2003). Despite the absence of a cure, the natural history of the disease was radically changed, and nowadays patients with HIV infection without other significant comorbidities who are appropriately treated may have the same life expectancy as the general population (Samji et al., 2013). The Center for Disease Control (CDC) and the World Health Organization (WHO) definition of HIV, which is the cornerstone for diagnosing HIV and AIDS includes: (1) a positive result on an HIV antibody test confirmed by a positive result on a second, different HIV antibody test and/or (2) a positive virological test confirmed by a second virological test (UNAIDS/WHO, 2007). The WHO classification system for staging established HIV infection uses both immunological and clinical criteria. HIV infection can be divided into the following stages: • • • • • • •

viral transmission; acute HIV infection (also called primary HIV infection or acute seroconversion syndrome) and seroconversion; chronic HIV infection; asymptomatic; early symptomatic HIV infection (previously known as AIDS-related complex, or ARC, and Class B); AIDS characterized by a CD4 cell count ,200 cells/μL or by the presence of any AIDS-defining condition; advanced HIV infection characterized by a CD4 cell count ,50 cells/μL.

Viral transmission—HIV infection is usually acquired through sexual intercourse, exposure to infected blood, or perinatal transmission. The mode of acquiring HIV infection was undetermined in 4% of the cases originally reported to the CDCs. A careful review of 32,497 cases, however, revealed that only 0.2% had no clearly defined risk factor. The distribution of the modes of transmission of HIV infection varies in different countries. In resource-limited areas, vaginal sex is responsible for 70%80% of HIV infections, and perinatal transmission and injection drug use (IDU) for 5%10% each (Adler, 2001). In contrast, during the first 25 years of the epidemic in the US, male-to-male sexual contact and IDU accounted for about one-half of cases (Cohen et al., 2008). Risk factors—They include high viral load, certain sexual behaviors, presence of ulcerative sexually transmitted infections, lack of circumcision, as well as certain host and genetic factors (Quinn et al., 2000; Gray et al., 2001; Dorak et al., 2004).

24.3.2. Effects of Probiotics on HIV-Infected Subjects During HIV infection there is hyperactivation of the immune system that may persist over the course of the disease. In fact, one of the best correlations of disease progression is the expression of the activation markers

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CD38 and HLA-DR on T lymphocytes (Giorgi et al., 1999; Deeks et al., 2004). Additionally, the level of proinflammatory cytokines including TNF-α, IL-6, and IL-1β are elevated. In addition to excess antigen exposure (e.g., uncontrolled viral replication), the HIV proteins Env and Nef are able to directly stimulate T lymphocytes. Furthermore, the massive depletion of T lymphocytes that occurs in the gut-associated mucosa during acute infection is associated with loss of mucosal integrity and translocation of gut flora into the peripheral circulation (Brenchley et al., 2006). It is also possible that activation is induced by other pathogens (e.g., Cytomegalovirus and Epstein-Barr virus) that have been reactivated due to a decline in cell-mediated control as CD41 T-cells are lost. Treg cells are thought to be important for controlling immune activation. Two regulatory subsets that likely play a role in the pathogenesis of HIV infection are Tregs and Th17 cells. Studies suggest that the inflammatory milieu during HIV infection may promote the expansion of Tregs at the expense of Th17 cells (Tsunemi et al., 2005; Favre et al., 2009). The role of Tregs in the pathogenesis of HIV remains unclear. Tregs can suppress immune activation (studies suggest an inverse relationship between the proportion of Tregs and the proportion of activated CD81 T-cells (Tenorio et al., 2008; Jiao et al., 2009)) as well as HIV-specific cytolytic activity, cytokine and chemokine expression, and proliferation (Kinter et al., 2007). Th17 cells are critical for maintaining the integrity of the gut mucosa. It has been proposed that the loss of these cells in HIV-infected individuals may contribute to translocation of microbial products (e.g., a component of the bacterial cell wall and LPS) into the peripheral circulation (Hunt, 2010), thus perpetuating the hyperactivation of the immune system. The accepted hypothesis is that the systemic inflammation observed in virologically-suppressed HIV-infected subjects is partially due to the breakdown of microorganisms in the gut mucosa, together with increased translocation of LPS and dysfunction of immunoregulatory cytokine production (Brenchley et al., 2006; Mavigner et al., 2012; Katsikis et al., 2011). Microbial translocation is facilitated by HIV-induced depletion of CD41 T-cells from the gutassociated lymphoid tissue, and intestinal barrier dysfunction has been proposed as a potential cause of persistent immune activation (Hummelen et al., 2010). Even after initiation of ART, microbial translocation does not normalize and continues to be associated with T-cell activation (Cassol et al., 2010). However, ART intensification trials did not provide results that were sufficiently consistent to dismiss the role of HIV replication in persistent immune activation (Buzon et al., 2010). Recent studies have shown that probiotics may counteract the inflammatory process by stabilizing the gut microbial environment and the intestinal barrier, lowering systemic inflammation and stimulating NK cell

FIGURE 24.2 Local and systemic consequence of HIV-infection, and potential effect of probiotic diet supplementation.

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activity. The mechanisms by which probiotics modulate the immune system, however, are not entirely understood (Gori et al., 2011) (Fig. 24.2). It is well known that Lactobacillus casei Shirota (LcS), a commercial probiotic strain, increases the numbers of bacterial species in the gut that are considered beneficial, improves the balance between beneficial and potentially harmful intestinal bacteria and enhances NK cell activity (Dong et al., 2013). Several in vitro studies have also shown that LcS enhances NK cell activity and induces IL-12 production in human PBMCs from healthy subjects. Furthermore, heat-killed LcS has been shown to stimulate IL-10, IL-12, TNF-α, and IFN-production, to promote NK cell activity and to activate CD69 expression on NK cells (Takeda et al., 2006).

24.3.3. Safety, Efficacy/Caution in the Use of Probiotics Micronutrient supplements may be of benefit in some patients with HIV infection: • A randomized trial with vitamin supplements in 1078 pregnant women in Tanzania found out that women who received a multivitamin supplement (vitamins B, C, and E dosed at the recommended dietary allowance [RDA] level) had delayed progression of HIV disease compared with women receiving placebo; vitamin A supplementation was not beneficial (Fawzi et al., 2004). • Forty HIV-infected patients taking ART were randomly assigned to receive a micronutrient supplement (with 33 ingredients) or placebo twice daily for 12 weeks (Kaiser et al., 2006). The absolute CD4 count increased by an average of 24% in the micronutrient group versus no change in the placebo group. However, despite the observation that vitamin deficiencies persist in HIV-infected patients consuming micronutrients at the RDA level (Baum et al., 1992), administration of vitamins beyond standard dosing does not appear to offer incremental benefit in the setting of ART and may have greater adverse effects. In a trial of 3418 HIV-infected patients initiating ART in Tanzania, there was no difference in HIV disease progression or death from any cause between those randomly assigned to standard (at the RDA) versus high (2 to 21 times the RDA) dose vitamin B complex, E, and C supplementation (Isanaka et al., 2012). The study was halted prematurely due to a higher rate of abnormally elevated alanine aminotransferase levels among those taking high-dose vitamins. Further trials are needed to confirm these effects of standard-dose vitamin administration and to extend them to other populations before multivitamin supplementation can be recommended as routine therapy for the purpose of delaying HIV progression (Marston and De Cock, 2004). In conclusion, the data on the effect of supplementation of diet by vitamin or probiotics in improving the compromised immune system of HIV infected individuals are somewhat conflicting. Probiotics certainly improve the microorganism strength (which is suppressed in HIV-infected individuals) in the gut mucosa and studies also show that the addition of a probiotic Lactobacillus to a micronutrients fortified fermented milk drink translated into reduction in clinical symptoms of HIV-infected adults with a starting CD4 count $ 200 cells/μL. The micronutrient fortified probiotic fermented milk drink was well tolerated among the participants and was not associated with adverse events (Hummelen et al., 2011). In the other studies, the micronutrients supplementation has been shown to reduce the progression of HIV, but does not have an effect on the intestinal barrier or the intestinal microbiota of HIV patients. Thus probiotic supplementation may modulate certain immunological parameters and some of the cytokines serum as TGF-β, IL-10, IL-12, IL-1β, and IL-23 as reported elsewhere (Falasca et al., 2015), but its direct effect on HIV-infected individuals in terms of decrease in mortality or progression of the disease is unsubstantiated. Based on its modulation of the immune system, we propose that LcS and other probiotics may be an inexpensive and practical strategy to support the immune function of HIV-infected patients (Falasca et al., 2015).

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25 Chronic Inflammation in Asthma: Antimalarial Drug Artesunate as a Therapeutic Agent Thai Tran, Yongkang Qiao, Huihui You and Dorothy H. J. Cheong National University of Singapore, Singapore, Singapore

Asthma is a chronic inflammatory disorder of the airways associated with declining airway function and remodeling of the airway tissue structure. Inflammation of the airways produces the classical symptoms which include wheezing, coughing, chest-tightening, and breathing difficulty. Despite advances in effective therapy, the prevalence of asthma remains high, with approximately 334 million people suffering from asthma worldwide (Global Asthma Network, 2014). Moreover, the healthcare expenditure associated with asthma is enormous as disease control and morbidity continue to escalate. Airway inflammation is mediated by cells of the adaptive immune system, i.e., T-helper type 2 (Th2) cells, mast cells, eosinophils, B-cells and inflammatory cytokines and chemokines. B-cells are stimulated by IL-4 for isotype switching for the synthesis of IgE. IgE then binds to the high-affinity IgE receptors found abundantly on mast cells and basophils. This leads to the activation and degranulation of these cells to release inflammatory mediators, such as histamine, leukotrienes and various cytokines, that cause airway smooth muscle contraction and exudation of plasma into the airways. These compromise epithelial integrity and reduce the removal of mucus, worsening airway obstruction. Anti-inflammatory drugs used to treat asthma include corticosteroids and leukotriene inhibitors but use of these drugs are associated with either endocrine-related side effects with highdose long-term usage (corticosteroids) or of benefit to only a small group of asthma sufferers with allergy component (leukotriene inhibitors), respectively.

25.1. ARTESUNATE: A POTENT DRUG BEYOND ITS ANTIMALARIAL PROPERTY Recently in 2015, the Nobel Prize in Medicine was awarded to Youyou Tu, a Chinese scientist for her outstanding contribution to antimalarial therapy by discovering and purifying artemisinin, a natural product from the herb medicine Artemisia annua L in 1970s. Since the discovery of artemisinin, Tu’s group and others have produced various artemisinin derivatives, which were later applied as antimalarial drugs and these have saved millions of lives in tropical Africa, Asia, and South America (Klayman, 1985; Meshnick et al., 1996). Interestingly, beyond its direct effects in inducing toxicity to the malarial parasites, artemisinins have been found to have broader therapeutic applications including immune diseases, cancer and viral infection, via their emerging roles in regulating immune response and antitissue remodeling properties. In this review, we will specifically focus on artesunate, one of the semisynthetic derivatives of artemisinin recognized as the best drug in the treatment of severe and complicated malaria (Li and Weina, 2010), and provide an overview of the pharmacological action of artesunate in the remedies of diseases other than malaria infection. We hope this will facilitate both bench and clinical research in different diseases categories.

Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00025-1

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25.1.1. Artemisinin and Derivatives 25.1.1.1. Discovery of Artemisinin Malaria is the most prevalent parasitic disease transmitted by certain species of anopheline mosquitoes and killed over one million people worldwide annually (White, 2004; WHO, 2006). The antimalarial drug evolution can be traced back to the isolation of quinine from cinchona bark in 1820 by French chemists, which was then used as the main treatment for malaria from the mid-19th century to the 1940s. During World War II, the cutoff of the quinine supply catalyzed the synthesis of multiple antimalarial quinolone drugs including chloroquine, which later proved to be the most effective drugs and became the main choice in the 1950s and 1960s (Meshnick and Dobson, 2001). However, in the late 1950s, chloroquine resistance in Plasmodium falciparum, the parasite responsible for the majority of fatal malaria, first emerged in South Africa and soon spread worldwide. In this situation, development of new antimalarial drugs was urgently needed. In 1967, the Chinese government launched a program to discover novel drugs to treat malaria, particularly in those with drug resistance (Tu, 2004). In 1972, screening was done of more than 2000 Chinese herb preparations, with over 200 herbs and 380 extracts tested in malarial animal models. Lead Chinese scientist, Youyou Tu finally discovered and extracted artemisinin from the plant qinghao, also named Artemisia annua L, which has been used in China for over one thousand years as traditional remedies for fever and malaria (Tu, 2004; Tu, 2011). Subsequently, a group of artemisinin derivatives was generated for better chemical properties and effect (Fig. 25.1). After the discovery, artemisinins have been used as effective antimalarial drugs and intensively applied worldwide in the scenario of multidrug-resistant malaria. Although the early preclinical studies did not follow the stringent Western drug regulation, large-scale clinical data later did not show notable side effects and proved artemisinins to be generally safe and highly tolerated drugs (Meshnick et al., 1996). 25.1.1.2. Antimalarial Activity of Artemisinin and Artesunate Artemisinins are a group of endoperoxides that contain the endoperoxides bridge (COOC) that proved to be particularly important in their antimalaria activity. It makes artemisinins unique and limited cross-resistant with other antimalaria drugs. In clinical and animal model research, artemisinins could kill the parasites more rapidly than the preexisting drugs with only nanomolar concentrations in vitro. The mechanism for the

FIGURE 25.1 Structures of artemisinin and its representative derivatives (Ho et al., 2014)

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antimalarial activity of artemisinins remains to be fully elucidated. Nevertheless, it is believed to begin with the cleavage of the endoperoxides bridge by heme or molecular iron to produce the free radicals and alkylating intermediates, which then target specific malaria membrane-associated proteins and cause damage (Meshnick et al., 1996; O’Neill et al., 2010). Artesunate is the semisynthetic product of artemisinin. It is water-soluble and can be applied both by oral intake and intravenous/intramuscular injection. The chemical property and the pharmacokinetic profile of artesunate make it superior to artemisinin and other derivatives that are oil-based (Li and Weina, 2010), for it is almost immediately converted into dihydroartemisinin after intake, which accounts for the antimalarial activity. Furthermore, it has been shown to be more effective in parasite clearance and reducing mortality than quinine, the previous standard treatment for malaria (Noubiap, 2014; Sinclair et al., 2012). Based on this clinical evidence, the World Health Organization (WHO) revised the guidelines for malaria treatment in 2006 and recommended intravenous artesunate as the choice to treat severe malaria in both the first and second editions (WHO, 2006; Reyburn, 2010).

25.1.2. Immunosuppressive Activity of Artesunate Early in 1981, Chinese scientists suggested that artemisinin may have immunological and antiviral effects (Qian et al., 1982), which started a novel research field of immunological activity for artemisinins. Later in the 1980s, a series of research articles was reported in Chinese journals regarding the effects of artemisinins in the immune response, although the exact mechanism for its action was not clear (Lin et al., 1988; Lin et al., 1985; Li and Liang, 1986; Dan et al., 1989). For instance, the artemisinin derivative, hemisuccinate NA (QHS) is immunosuppressive in vitro and in vivo via the inhibition of mitogen-stimulated mouse spleen cells and human peripheral lymphocytes (Shen et al., 1984). In another report, artesunate ameliorated contact-hypersensitivity in a model of dermatitis (dinitrochlorobenzene (DNCB) challenged guinea pig model) if administrated after DNCB challenge (Chen and Maibach, 1994). Later, the dual effect of artesunate in suppressing the humoral immune response and enhancing the cell-mediated immunity in different animal models was also observed (Lin et al., 1995). Since then, increasing research has been conducted and elucidated the immunomodulatory function of artesunate in various directions, including autoimmune diseases, sepsis, lung diseases, cancer and viral infection, with more details of the underlying mechanism disclosed. These findings render artesunate as an attractive strategy in broader applications that are beyond its antimalaria activity. 25.1.2.1. Function of Artesunate in Immune System Experiments performed in various in vitro immune cell cultures suggested a direct modulation of the immune system by artesunate. For instance, in cultured peripheral blood mononuclear cells, researchers found that artesunate diminished the mitogen-induced lymphocyte proliferation and activation by reducing CD69 and CD25 positive CD41 and CD81 T cells (Veerasubramanian et al., 2006) but did not have any effect on the transcription levels of chemokines and cytokines such as interleukins (IL) IL-2, IL-12, IL-4, IL-15, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) . This study provides evidence that artesunate associates with the activation from naı¨ve T-cells (CD41) to the effector T-cells (CD81), but the exact events are not clear. Later in 2015, further investigations revealed that artesunate could suppress CD41 T-cell proliferation and activation with a subsequent reduction in IL-2 levels, while reinforcing or enhancing the function of both Type 1 and Type 2 T helper cells (Th1 and Th2) (Lee et al., 2015). This paradoxical result indicates the dual effect of artesunate in modulating immune function and suggests that the time of administration of artesunate may be critical in disease therapy. Besides the effects of artesunate in immune cell activation and cytokine release, the signaling pathways regulated by artesunate were also studied. In 2008, Efferth’s group performed the whole genome mRNA microarray analysis in mouse macrophage cells and linked artesunate to nitric oxide (NO)-related signaling pathway, as well as cAMP-mediated and Wnt/β-catenin signaling pathways (Konkimalla et al., 2008). In addition, dihydroartemisinin, the active metabolite of artesunate, was found to inhibit in vitro Th cell differentiation, while greatly promote regulatory T-cell (Treg) generation via TGF-β/Smad and mTOR pathways (Zhao et al., 2012). This finding was further supported by another report performed both in vitro and in vivo that artesunate could inhibit T-cell proliferation and meanwhile promote Treg differentiation and activation via NF-κB/p65 and Smad2/3dependent TGF-β signaling (Li et al., 2013). Notably, NF-κB signaling is one of the master regulators for inflammatory mediators secretion, and artesunate has been shown to inhibit NF-κB signaling in several studies of

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inflammatory disease models. For example, in an experimental model of colitis, the expression of NF-κB p65 and p-IκB-α were significantly reduced in artesunate-treated animals, with corresponding reductions in inflammatory cytokines, IFN-γ, IL-17, and TNF-α (Yang et al., 2012). This study indicates that NF-κB signaling may be one of the key targets of artesunate in inhibiting inflammation. Also interestingly, Treg cells are proven to be important in the maintenance of immune homeostasis (Sakaguchi et al., 2008). The positive regulation of Treg cells by artesunate may provide further evidence that artesunate could be applied as a safe remedy for the treatment of immune disorders by reducing the risk of systemic immunosuppression. The immunomodulatory function of artesunate was also demonstrated in other immune cell types such as microglial cells, the resident immune cells in the central nervous system that are responsible for the chronic inflammatory response in the brain. One study using the in vitro mouse microglia BV2 cells demonstrated that artesunate could inhibit lipopolysaccharide (LPS)-induced inflammatory in concomitant with reducing iNOS, COX-2, and IL-1β production. This effect was further proven to be mediated by ERK/Nrf2/ARE/HO-1 signaling. Since HO-1 is both anti-inflammatory and oxidative stress protective, artesunate was proposed to play dual roles in inhibiting inflammatory response as well as mediating antioxidant pathways (Lee et al., 2012). 25.1.2.2. Anti-inflammatory Effect of Artesunate in Diseases 25.1.2.2.1. MALARIA

Artesunate is recognized as the best drug for malaria treatment, albeit, there are surprisingly only a few papers mentioning the anti-inflammatory activity of artesunate in treating malaria. The fast action of artesunate in killing parasites may mask the effect of artesunate in the immune system. For example, artesunate treatment for cerebral malaria could efficiently rescue the mice from late-stage cerebral malaria by reducing the neurological signs with the leukocyte accumulation rapidly decreased in the brain in one study (Clemmer et al., 2011), and inhibits IL-2, IL-6, IL-10, IL-17, IFN-γ, and TNF in the serum (Miranda et al., 2013). However, the antiinflammatory function of artesunate was obscured since it could result from the rapid elimination of the parasites within 24 hours after treatment (Clemmer et al., 2011). Fortunately, more solid evidence was provided by Souza, M.C and coworkers in 2012, which suggested that artesunate treatment significantly reduced blood brain barrier breakdown and TNF -α transcriptional level as early as 12 h post treatment, while the parasites levels still maintained. Furthermore, pretreatment of artesunate may abrogate the adherence of parasitized red blood cells to endothelial cells by inhibiting NF-κB nuclear translocation and the subsequent ICAM-1 expression (Souza et al., 2012), which may partially explain the immunosuppressive function of artesunate. 25.1.2.2.2. AUTOIMMUNE DISEASES

Autoimmune diseases are characterized by the dysregulation of various aspects of immunity and inflammation, which includes abnormal T-cells and B-cells and aberrant secretion of inflammatory mediators. The current treatments include immunosuppressive drugs targeting immune cells or secreted mediators, which dramatically improved patient outcomes. However, despite their efficiency, these drugs have severe side effects since they also affect the normal immune system. Those adverse effects include systemic immunosuppression leading to infection, autoimmune diseases and cancer (Her and Kavanaugh, 2016; Vial and Descotes, 2003). In this regard, developing novel drugs with low side effects cannot be overlooked. Artemisinin and its derivatives proved to be safe drugs in malaria treatment and possess immunomodulatory function, and have been tested in various autoimmune disease models with strikingly promising results (Li et al., 2006; Wang et al., 2007). The first disease model that artesunate was tested on was human rheumatoid arthritis. The in vitro analysis conducted in human rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) showed that artesunate significantly suppressed IL-1β, IL-6, and IL-8 secretion stimulated by TNF-α through NF-κB signaling pathway by preventing NF-κB translocation and IκBα degradation (Xu et al., 2007). This in vitro effect of artesunate was further supported by in vivo collagen-induced arthritis rat model where artesunate attenuated inflammation by reducing IL-1β, TNF-α, IL-17α, and MMP-9, probably via suppression of NF-κB and MAPK signaling pathways (Li et al., 2013). Consistent with the protection afforded by artesunate in arthritis, another study of Freund’s complete adjuvant-induced monoarthritis rat model supports that artesunate could significantly ameliorate the function limitation and hyperalgesia through reducing levels of NO and COX-2, neutrophils influx, ROS generation and apoptosis (Guruprasad et al., 2015). Furthermore, besides targeting macrophages and T-cells, artesunate is also capable of inhibiting the proliferation of B-cells in the germinal center (GC), and subsequently suppressing the differentiation of GC B-cells and production of autoantibodies in the K/BxN mouse model of rheumatoid arthritis, which suggested versatile capacity of artesunate in treating autoimmune diseases (Hou et al., 2014).

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Besides arthritis, artesunate also exhibited its potency in systemic lupus erythematosus (SLE), colitis and uveitis. For instance, artesunate could inhibit the autoantibody generation and inflammation in the lupus-like disease of MRL/lpr mice, via reducing the B-cell activating factor (BAFF) and MCP-1 expression in kidney (Jin et al., 2009), which supports the therapeutic efficacy for artesunate in SLE. In the colitis model, artesunate protected the mice from sulfate sodium salt (DSS) and intrarectal delivery of 2,4,6-trinitrobenzene sulfonic acid (TNBS)induced colitis, including reduced weight loss and disease activity, as well as macroscopic and microscopic colonic injury in colitis mice. Meanwhile, the pro-inflammatory cytokines, IFN-γ, IL-17 and TNF-α, was suppressed by artesunate in vivo and in vitro. This effect was further suggested to associate with the inhibition of artesunate on NF-κB signaling (Yang et al., 2012). Finally, artesunate was proven to be protective in endotoxininduced uveitis rat model, with the effect to reduce cell infiltration as well as proinflammatory cytokine levels (Wang et al., 2011). 25.1.2.2.3. CHRONIC INFLAMMATORY LUNG DISEASES—ASTHMA AND COPD

Emerging evidence indicates that artesunate may be a potent treatment for chronic inflammatory lung diseases, in particular asthma and chronic obstructive pulmonary disease (COPD) that are characterized by airway hyperresponsiveness and chronic airway inflammation. This is encouraging in the scenario that current drugs like steroids are not always effective in reversing the syndromes (Reddy and Little, 2013). The failure of traditional drug treatment is probably due to the heterogeneity of the various cell types whose dysfunction at the molecular and cellular levels translates via a complex network of immune cells and structural cells into a clinical manifestation (Brusselle et al., 2013; Mannino, 2002). The application of artesunate in the murine model of asthma showed for the first time that the efficacy of artesunate to protect mice from experimental allergic asthma 2011 (Cheng et al., 2011). In this report, both ovalbumin (OVA)-induced murine model and more clinical relevant model induced by house dust mite (HDM) showed that artesunate treatment dose-dependently inhibited total and eosinophil counts, IL-4, IL-5, IL-13, and eotaxin levels in the bronchoalveolar lavage fluid (BALF), as well as lung tissue eosinophilia and airway mucus production, which are recognized as important clinical hallmarks in asthma patients. Meanwhile, the transcription levels of E-selectin, IL-17, IL-33, and Muc5ac in the lung tissues were concomitantly reduced. Furthermore, the reduction of airway hyperresponsiveness to methacholine, the clinical syndrome of asthma, in artesunate-treated mice suggests the efficiency of artesunate to relieve clinical asthma syndromes. This promising anti-inflammatory effect of artesunate in asthma may be explained by the suppression of PI3K/Akt/NF-κB pathway, as demonstrated in in vitro normal human bronchial epithelial cells (BEAS-2B) and in vivo asthma model lung tissues (Cheng et al., 2011). One year later, the same group uncovered that artesunate could also prevent oxidative stress and ameliorate oxidative lung damage in OVA-induced asthma model via activation of Nrf2 pathway, which provides another possible mechanism for the action of artesunate in asthma. Interestingly, this effect of artesunate is more potent than the traditional drug dexamethasone, which renders artesunate an alternative/supplementary treatment over traditional medicine (Ho et al., 2012). To further elucidate the antiallergic activity of artesunate, Wong and colleagues proceeded to examine the effect of artesunate in in vivo passive cutaneous anaphylaxis and passive systemic anaphylaxis mouse models, ex vivo bronchial rings isolated from sensitized guinea pig, as well as in vitro RBL-2H3 mast cell line and human mature cultured mast cells. Strikingly, they found that artesunate could significantly block the IgE-induced mast cell degranulation via Syk-PLCγ pathway in vitro, prevent IgEmediated cutaneous vascular hyperpermeability, hypothermia, plasma histamine elevation and bronchial smooth muscle contraction in vivo and ex vivo (Cheng et al., 2013). This study strongly supports the potency of artesunate in treating allergic diseases. Based on the previous work in the asthma model, it is tempting to speculate that artesunate possesses therapeutic potency for other lung diseases characterized by chronic inflammation and oxidative stress, such as COPD. One supportive study was conducted in the in vivo cigarette smoke-induced COPD mouse model by Wong and colleagues, which exhibited that artesunate protects against COPD hallmarks by suppressing both inflammatory and oxidative biomarkers. The subsequent in vitro study performed in cigarette smoke extract (CSE)-stimulated BEAS-2B further supports the involvement of PI3K/Akt, p44/42 MAPK, and Nrf2 pathways in mediating the effect of artesunate, which is consistent with what was observed in the asthma model (Ng et al., 2014). Interestingly, a more recent study went further by looking into the mixed eosinophilic and neutrophilic airway inflammation induced by concurrent exposure of cigarette smoke and OVA, which stands for a more complicated situation in severe asthma for which traditional drug glucocorticoid, e.g., dexamethasone, failed to manage. Strikingly, artesunate could significantly suppress the clinical and inflammatory hallmarks, including airway hyperresponsiveness, inflammatory cell infiltration in the BALF, as well as IL-4, IL-8, IL-13, and TNF-α

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levels. Again, the PI3K/Akt pathway was affected. Moreover, artesunate could reverse the glucocorticoid insensitivity in in vitro BEAS-2B cells stimulated by CSE, and that HDAC2 activity, which accounts for the glucocorticoid insensitivity in severe asthma, was reversed in artesunate treatment (Luo et al., 2015). The asthma and COPD models implicate artesunate as a potent treatment that targets various pathways in the lung diseases. Recently, a comprehensive study performed in BEAS-2B cells using untargeted proteomics approach and transcriptional factor array uncovered that artesunate binds to a number of mitochondrial enzymes responsible for glucose and energy metabolism, mRNA and gene expression, ribosomal regulation, stress responses and structural proteins, as well as regulates multiple transcriptional factors including FOXO1, AP-1, IRF, NFAT, NF-κB, Nrf2, SRF, and STAT3 (Ravindra et al., 2015). Within these transcriptional factors, some of them have been implicated in the disease models, while others need to be further elucidated. This study provides comprehensive information on the regulatory effect of artesunate in multiple pathways and would facilitate the research on artesunate activity. 25.1.2.2.4. PATHOGEN INFECTION DISEASES—SEPSIS AND VIRAL INFECTION

Pathogen infection, including bacterial and viral infection, is responsible for the majority of clinical cases. In most situations, it is self-limited, e.g., the common cold, or easy to control by incorporation of the innate immunity and antibiotics intake (Heikkinen and Ja¨rvinen, 2003). However, in some scenarios, it could develop into serious systematical diseases characterized by exaggerated inflammatory and immune response to infections, which may lead to death. Sepsis triggered by bacteria and lipopolysaccharide (LPS) is one example with a high mortality rate. Strikingly, in the sepsis murine models, artesunate exhibited protective effects by inhibiting proinflammatory cytokines release, enhancing antibiotics effect and LPS internalization with the subsequently reduced LPS levels in the serum. Multiple pathways, including TLR, NF-γB, Nod2, and scavenger receptors (SR) signaling, have been proposed to be regulated by artesunate (Li et al., 2008; Jiang et al., 2011; Li et al., 2014). Apart from its activity against bacterial infection, artesunate is also protective against viral infection. This antiviral activity of artesunate was demonstrated in various virus types, including human cytomegaloviruses (Efferth et al., 2002; Kaptein et al., 2006; Chou et al., 2011), Hepatitis B (Romero et al., 2005), Hepatitis C (Dai et al., 2016), Epstein-Barr (Auerochs et al., 2011), and Herpes simplex viruses (Canivet et al., 2015). More recently, a clinical study reported that the combination of artesunate and amodiaquine significantly improved the survival rate of ebola virus disease in West Africa (Gignoux et al., 2016), which strongly supports the potency of artesunate as the remedy of viral infection diseases. Unlike bacterial and parasites, viruses are required to interact with the host system to replicate themselves and survive, and the host immune response is closely related to the severity of viral infection. According to Ravindra and colleagues, artesunate could bind to multiple protein targets and regulate a wide range of transcription factors that are involved in metabolism and host immune response (Ravindra et al., 2015). Therefore, it is not surprising that artesunate may target and modulate critical cellular pathways and innate immunity, which counteract the viral infection. Indeed, studies on cytomegaloviruses support that artesunate could suppress cytomegaloviruses replication by targeting the PI3K/Akt/NF-κB and SP1 pathways (Efferth et al., 2002), and interestingly, this effect is somewhat similar to its antimalarial activity that ferrous iron combination strengthened this antiviral activity (Kaptein et al., 2006). Besides, the cell surface marker Thy-1, associated with the proinflammatory response, was also significantly suppressed (Kaptein et al., 2006), suggesting the immunosuppressive function of artesunate. As further evidence, the exaggerated innate immunity characterized by the inflammatory cytokine storm was significantly inhibited by artesunate treatment in a murine model of herpes simplex virus encephalitis (Canivet et al., 2015), thus, the protective effect of artesunate results from both inhibiting viral replication and suppressing the aberrant innate immunity.

25.1.3. Artesunate in Other Directions: Antiproliferative and Antitissue Remodeling Tissue remodeling in disease refers to the pathological change and reorganization of the tissues resulting from intrinsic functional change of the resident cells, e.g., hyperplasia and hypertrophy, and environmental changes, extracellular matrix deposition, hypoxia and inflammation (Markert et al., 2012; Gilkes et al., 2014). These pathological changes are typical characteristics for many diseases such as cancer and chronic inflammatory diseases. Artesunate’s antiproliferative activity in specific cell types, e.g., airway smooth muscle cells and carcinoma cells, both in vitro and in vivo, that occurs via induction of cell cycle arrest and apoptosis (Efferth et al., 2003; Efferth et al., 2008; Ma et al., 2011; Tan et al., 2014), can potentially target tissue remodeling. For example, our laboratory has shown that artesunate suppresses airway wall remodeling in the experimental asthma model, via inhibiting

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airway smooth muscle cells both in vivo and in vitro (Tan et al., 2014). Furthermore, this antiproliferative property of artesunate is selective so that carcinoma cells are more sensitive to artesunate treatment due to more reactive oxygen species (ROS) produced and higher iron levels (Zeng and Zhang, 2011; Beccafico et al., 2015; Eling et al., 2015; Holien et al., 2013), which is believed to mediate the cytotoxicity in the parasites. Interestingly, a recent report indicates that the biomechanical properties are also changed in artesunate-treated carcinoma cells, with increased adhesive force and less elasticity, which may provide another possible explanation for the mode of action of artesunate (Shi et al., 2015). The preferentially antiproliferative effect of artesunate renders artesunate an attractive drug to inhibit abnormal tissue growth with low side-effect towards normal tissues. Apart from antiproliferative effects, artesunate could inhibit angiogenesis, a critical characteristic for tissue remodeling, by inhibiting the endothelial cell proliferation and inducing apoptosis, as well as decreasing the vascular endothelial growth factor (VEGF) levels (Chen et al., 2004; Zhou et al., 2007; Dell’Eva et al., 2004). Additionally, the effect of artesunate on extracellular matrix deposition via matrix metalloproteinase (MMPs) signaling serves to be another possible mechanism that mediates the antiremodeling activity of artesunate (Willumsen et al., 2014; Lai et al., 2015).

25.1.4. Limitations of Artesunate-Drug Resistance Extensive research on the clinical and basic sciences has supported the potential of artesunate to act as a drug to treat different kinds of diseases. However, one limitation of artesunate is the short half-life time that corresponds with the fast elimination after a single treatment. This may lead to high recrudescence of artemisinins monotherapy in the early clinical reports of malaria (de Vries et al., 1999). As an improvement over this shortcut as well as to retard drug resistance, artemisinin combination therapies (ACTs) was recommended to be the firstline treatment for malaria, which includes artemisinins, e.g., artesunate, with fast action mode, and a partner drug with longer acting time that is responsible for the subsequent elimination of parasites (WHO, 2006; Reyburn, 2010). To date, ACTs are still the best treatments for parasite infection. However, concerns have been raised since scientists from Thailand reported the declining susceptibility of the parasites to ACTs treatment, which indicates the threat of artemisinins resistance (Wongsrichanalai and Meshnick, 2008). Following this report, increasing research has been conducted to elucidate whether resistance to artemisinins already exists. Although the preexisting multidrug resistance previous to ACT treatment may be misleading, evidence supports the presence of artesunate resistance in parasites isolated from malaria patients (Phompradit et al., 2014; Chaijaroenkul et al., 2010), and identified the molecular markers including multidrug resistance 1 (pfmdr1) and P. falciparum ATPase 6 (pfatp6) to be the key mediators and predictors for artesunate resistance (Chaijaroenkul et al., 2010; Na-Bangchang et al., 2013; Ferreira et al., 2007; Alker et al., 2007). The evidence for artesunate resistance was additionally observed in specific carcinoma cell lines, which are less sensitive to artesunate treatment. This may be induced by alternative mechanisms modulated by glutathione-related enzymes, the transferrin receptor (TfR) and NF-κB/AP-1 pathways that induced higher MMP-1 related to invasion and Bcl2/bax skewing leading to antiapoptosis (Efferth and Volm, 2005; Kelter et al., 2007; Bachmeier et al., 2011).

25.1.5. Summary Based on its versatile pharmacological actions, artesunate appears to be a powerful drug in the remedy of many different kinds of inflammatory diseases beyond its initial antimalarial application. However, there is still a long way to go from bench work to clinical trials. The administration time of artesunate seems to be critical since it may regulate the immune cell function at different stages (Lee et al., 2015). In addition, the dosage and application route would also affect the drug potency, which need to be further elucidated. Furthermore, in some of the models, the dosage of artesunate is relatively high, where drug safety should be more fully explored. Finally, more in-depth work is required to comprehensively investigate the pathways regulated by artesunate in disease models, which would pave the way for more specific targeting in the disease therapy. With the emerging notion of artesunate resistance to specific parasite stains and carcinoma types, further molecular analysis research is needed to identify potential molecular markers that could modulate the artesunate susceptibility. This could help to predict the outcome for artesunate treatment. In addition, artesunate-combination therapy may be recommended not only for the treatment of malaria but also for other disease applications like asthma. This would increase the genetic barrier for drug resistance and overcome the shortcomings for artesunate monotherapy.

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Anti-malarial drug artesunate ameliorates oxidative lung damage in experimental allergic asthma. Free Radic. Biol. Med. 53 (3), 498507. Ho, W.E., et al., 2014. Artemisinins: pharmacological actions beyond anti-malarial. Pharmacol. Ther. 142 (1), 126139. Holien, T., et al., 2013. Lymphoma and myeloma cells are highly sensitive to growth arrest and apoptosis induced by artesunate. Eur. J. Haematol. 91 (4), 339346. Hou, L., Block, K.E., Huang, H., 2014. Artesunate abolishes germinal center B cells and inhibits autoimmune arthritis. PLoS One. 9 (8), e104762. Chen, H.H, You, L.L, Li, S.B., 2004. Artesunate reduces chicken chorioallantoic membrane neovascularisation and exhibits antiangiogenic and apoptotic activity on human microvascular dermal endothelial cell. Cancer Lett. 211 (2), 163173. Jiang, W., et al., 2011. Artesunate in combination with oxacillin protect sepsis model mice challenged with lethal live methicillin-resistant Staphylococcus aureus (MRSA) via its inhibition on proinflammatory cytokines release and enhancement on antibacterial activity of oxacillin. Int. Immunopharmacol. 11 (8), 10651073. Jin, O., et al., 2009. A pilot study of the therapeutic efficacy and mechanism of artesunate in the MRL/lpr murine model of systemic lupus erythematosus. Cell Mol. Immunol. 6 (6), 461467. Kaptein, S.J., et al., 2006. The anti-malaria drug artesunate inhibits replication of cytomegalovirus in vitro and in vivo. Antiviral Res. 69 (2), 6069. Kelter, G., et al., 2007. Role of transferrin receptor and the ABC transporters ABCB6 and ABCB7 for resistance and differentiation of tumor cells towards artesunate. PLoS One. 2 (8), e798.

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C H A P T E R

26 Nutrition as a Tool to Reverse Immunosenescence? Anis Larbi1,2,3,4,5, Olivier Cexus1 and Nabil Bosco6 1

Agency for Science Technology and Research (ASTAR), Biopolis, Singapore 2University of Sherbrooke, Sherbrooke, Canada 3National University of Singapore (NUS), Singapore, Singapore 4Nanyang Technological University (NTU), Singapore, Singapore 5El Manar University Tunis, Tunis, Tunisia 6Nestle Research Centre, Singapore, Singapore

26.1. INTRODUCTION The past century has been the most exciting in the field of medicine and science, and one of the most important effects is the increased life expectancy in humans which has a significant impact on our societies and in the way in which life is managed. Developed countries display a life expectancy greater than 75 years and developing countries are catching up. Progress in medicine is not the only reason for this phenomenon, as improved hygienic conditions and wealth are also important factors that are connected to the quantitative and qualitative nutritional intake. Better access to food, improved food processing, conservation and storage conditions have reduced the proportion of malnourished individuals worldwide, although many population groups are still at risk in the developing as well as in developed countries. While developing countries face food supply challenges for the general population, especially infants who are often under/malnourished, older populations in developed countries are also susceptible to poor nutritional intake. This chapter will first describe problematic nutrition in older adults and how immunity is eroded in this population. The second part of the chapter will be dedicated to the concept and regulatory aspects of functional foods and how they can be used to ameliorate immunity in elderly.

26.2. EPIDEMIOLOGICAL, PHYSIOLOGICAL AND CLINICAL FEATURES OF AGING As we enter the 21st century, the aging of the population has appeared as a major demographic trend worldwide where the association of declining fertility and improved health and life expectancy has led to an important increase in the number of people reaching the age of 65 onward. Indeed, the UN assessed that the proportion of the world’s elderly population will more than double by the year 2050 from 7.6% today to 16.2% (http://www.un.org/en/development/desa/population/publications/pdf/ageing/WorldPopulationAgeing2013. pdf) (Lutz et al., 2008). This increase affects all countries but is especially pertinent in the case of regions such as China and India which are projected to present a 30% increase of the older population, bringing the number of elderly people to 350 million and 240 million respectively (from the current numbers of 109 million and 62 million). Other countries such as Japan are facing an important crisis whereby the percentage of people over 60 will explode from 27% of the whole population to 44% by 2050. This increase associated with a declining fertility rate below one child per family has led numerous countries to take this problem seriously and invest massively into research on aging (Beard et al., 2016; Kennedy, 2012). One such country, Singapore, has seen its proportion of elderly residents (65 years and above) soaring from 3.4% in the 1970s to nearly 12% in 2015. Indeed the proportion of 6574 and 75-year-old population, which comprised 2.6% and 0.8% of the total inhabitants in Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00026-3

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the 1970s, has now jumped to 7.3% and 4.5% respectively. In this context, the Singaporean government and other allied authorities have realized the seriousness of the situation and have introduced incentives not only aimed at increasing fertility rates of Singaporeans but also at improving aging and promoting “healthy aging” to ultimately reduce the economic, health and social burden of its aging population (http://www.singstat.gov.sg/statistics/browse-by-theme/elderly-youth-and-gender-profile) (Bloom et al., 2015). As life expectancy grows with advances in medical research, diagnosis and treatments, there in an increased risk of developing one or several chronic diseases and physiological limitations associated with an aging. Aging can therefore be described as a series of multicomorbidity processes where diabetes, respiratory diseases, cancer, cardiovascular diseases, arthritis and other types of chronic conditions accumulate to impact the elderly’s health status and general quality of life (Aarts et al., 2015; Kowal et al., 2015; Quinones et al., 2016). If a diversity of conditions appears during aging this is because many systems can be altered both dependently and independently of each other. The main chronic conditions in older adults are diabetes, hypertension, hypercholesterolemia, sarcopenia (and related frailty) and dementia (van den Bussche et al., 2011; Wu et al., 2013). While metabolic syndromes are quite well-controlled, other conditions still need improved healthcare as those conditions and complications often worsen with time. In older age the main causes of death are cardiovascular diseases, cancers and infections (Hunter and Reddy, 2013; Roth et al., 2015). By extension, the cardiovascular and immune systems are more susceptible to erosion during aging. It would seem that either these two systems are more fragile than the others and functional decline appears first, or the recent extension of lifespan has happened faster than their adaptation. It is also clear that if the disease history of an individual impacts his ability to “age better”; multiple studies have highlighted that previous stressors such as infections can hinder the capacity of an individual to counteract new infections while enhancing its likelihood to respond to common infections which are otherwise silent in young individuals (Beeson, 1985; Terpenning and Bradley, 1991). This suggests that a history of stressors will impact on different systems depending on the target cells or organs and the source of stress. Reducing the impact of the stressors could lead to reduced erosion of the systems and improved resilience (Miller, 1996). Overall, as life expectancy is increasing, it is important to compress the number of years of life with loss of autonomy. The longitudinal follow-up of the elderly is important in this case and implies that lifestyle factors must be taken into consideration along with efforts to tackle malnutrition, improve elderly diet, and restore food deficiencies. While it is difficult to characterize older adults beyond the sole status of clinical frailty (which is probably the first “disease” of the elderly), one must realize that as cancer patients are being stratified, aging must also go through a process of stratification and move to the area of personalized medicine. Geriatric assessment must therefore be extended from clinical assessments and include lifestyle, social, nutritional and biological data to better characterize the phenomenon of aging so as to improve the health of aging individuals.

26.3. NUTRITION AND WEIGHT LOSS IN OLDER ADULTS While several studies have highlighted positive associations between healthy lifestyle and healthy aging, it is clear that nutrition and the maintenance of a healthy gastrointestinal tract would contribute to a successful/ healthy aging (Hickson, 2006; Maijo et al., 2014). Older adults have been shown to be particularly vulnerable to malnutrition, while attempts to provide adequate nutritional supplements have encountered multiple practical problems as their nutritional requirements are not well defined (Wells and Dumbrell, 2006). This however poses important problems since the nutritional status can affect the risk of developing diseases while putting older adults at greater risks of developing infections (High, 2001). It has, for instance been shown that just a 10% loss of lean tissue in healthy individuals puts them at greater risk of infection and associated mortality, a phenomenon exacerbated during aging and associated with malnutrition (Broadwin et al., 2001; Landers et al., 2001). It is also evident that the burden of diseases during aging influences the nutritional status of elderly individuals, while their altered body composition during aging in turn impacts their nutritional status. One of the main changes is the reduction of the fat mass of elderly individuals where there is an accumulation in brown central visceral fat associated with a decrease of the appendicular fat mass (muscle, skin, etc.). This redistribution of fat has been associated with increased risks of developing strokes, diabetes, heart diseases and hypertension (Kuczmarski, 1989). This change in the overall composition of fat is accompanied by a reduction in muscle mass as it is overall linked with decreased physical function, strength and higher morbidity (Zamboni et al., 2008). This has been recently termed sarcopenic obesity, which is prevalent in the elderly population.

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TABLE 26.1

Etiology of Weight Loss in the Elderly Other diseases associated with this weight loss

Terminology Loss

Cause

Characteristics

Biological signatures

Wasting

Total weight

Reduced dietary intake

Etiology unknown

No specific signatures described

Trouble of appetite, depression, chronic diseases, cancer, infections

Cachexia

Fat Free Mass ( muscle, organs, skin and bone) or Body Cell Mass (muscle, viscera, immune system)

Altered catabolism/ metabolic rate; increased protein degradation

Acute immune response involving exacerbated production of IL-1 and subsequently of TNFα and then IL-6

Effect on hormone production, hormonal control of metabolism leading to an increase in resting energy expenditure, loss of amino acids from the muscle to the liver, increase in glucogenesis, shift from albumin production to production of acute phase protein such as CRP

Rheumatoid arthritis, congestive heart failure, HIV infection, cancer, trauma, infections

Sarcopenia

Muscle Mass

Altered metabolic rate and increased protein degradation

Etiology unknown

Decrease in levels of estrogen/ androgen, increase in levels of proinflammatory messengers (IL-6, IL-1, TNFα, IFNγ), increased inactivity and reduction in growth hormone secretion. Increased concentration of glucocorticoids and catecholamines

Physical disability, depression, pathology leading to the loss of neurons from spinal cord

PubMed literature search results done in September 2016 based at nutrition related keywords like “vitamin,” “mineral,” “antioxidant,” as well as “etiology of weight loss,” “Wasting,” “Cachexia” and “Sarcopenia.”

Several factors can explain the phenomenon of weight loss associated to malnutrition in aging (Table 26.1): 1. Involuntary loss of weight also known as wasting, primarily due to a reduced or inappropriate dietary intake. 2. Cachexia, which is an involuntary loss of weight caused by an altered catabolism in the elderly. This results in changes in body composition and is characterized by increased metabolic rates and protein degradations. 3. Sarcopenia which is an intrinsic part of the aging process characterized by the general loss of muscle mass. Causes of malnutrition in the elderly are frequent and extremely varied, including psychological factors (confusion, dementia, depression, bereavement and anxiety) as well as lifestyle and social factors (poverty, inability to shop or prepare food, isolation, loneliness and lack of knowledge about food, cooking or nutrition). Medical factors also play an important role in increasing the risks of malnutrition in older adults: poor appetite, oral/buccal problems (poor dentition, difficulty or discomfort in swallowing or dysphagia), loss of taste and smell, respiratory disorders (emphysema), gastrointestinal disorders (malabsorption), endocrine disorders (diabetes, thyrotoxicosis), neurological disorders (history of stroke, Parkinson’s disease), infections (urinary or respiratory tract infections), physical disorders (osteoporosis, rheumatoid arthritis, poor mobility), drug interactions (digoxin, metformin, antibiotics) or other pathologies (e.g., cancer) (Wells and Dumbrell, 2006; Sternberg and Roberts, 2006). Overall, the hallmarks of the physiological changes associated with aging are multiple and linked with an increased risk for the elderly to develop infections and other immuneassociated pathologies. If aging is associated with increased malnutrition, weight loss and fat redistribution, this can exacerbate a gradual decline in immune functions, which is described in the next section.

26.4. IMMUNOBIOLOGY OF AGING While aging mainly refers to chronological aging (time), each individual ages biologically (Belsky et al., 2015) at a different pace while this process starts well before entering the old age category. Adults in their fourth decade of life show rudimentary signs of aging that will very likely translate into diseases later in life.

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Additionally, not all biological systems and functions are eroded at the same time during the lifespan. As mentioned above, the cardiovascular and immune systems are more vulnerable during aging since they are not able to cope with the stressors as other systems may do. It is probable that the biological reserve of these systems is less likely to occur due to the continuous workload and the importance of these systems for survival. The gradual decline of immune functions is called immunosenescence. This functional decline of the immune system is associated with the increased inability for older adults to fight infections and respond to antigenic challenges, e.g., vaccination, while at the same time putting them at risk of developing more cancers and debilitating chronic inflammatory diseases. In humans immunosenescence affects both innate and adaptive immunity, as described below. Some physiological changes happen during the lifespan and directly or indirectly impact both adaptive and innate immunity. Changes of the immune system with age include the presence of dysfunctional immune cells among them lymphocytes (B-cells, T-cells), monocytes, natural killer cells and neutrophils. As short-lived cells such as neutrophils also show impaired functions, this suggests that other factors affect immune cell capacities beside peripheral stress. It has been shown that, already at the progenitor stage, cells are showing significant molecular and epigenetic profile alterations (Benayoun et al., 2015; Chambers et al., 2007) suggesting that all hematopoietic cells could be affected while there is a shift towards myeloid differentiation with aging (Muller and Pawelec, 2014). The next three subsections highlight three major alterations ultimately affecting both innate and adaptive immunity.

26.4.1. Impact of Aging of the Bone Marrow Composed of the red and yellow marrows, the bone marrow is the major source of numerous hematopoietic stem cells giving rise to a wide array of immune cells, a process called hematopoiesis (Morrison and Scadden, 2014; Wilson and Trumpp, 2006). While hematopoiesis takes place in the red marrow, the yellow marrow is composed of adipocytes. If the marrow of newborns is principally composed of red marrow, the proportion of adipocytes steadily increases with age. This increased adipogenesis is associated with increased destruction of the bone (by osteoclasts) and a reduced bone remodeling (orchestrated by osteoblasts), ultimately impacts the bone marrow’s hematopoietic functions, and is linked with various pathologies such as diabetes or osteoporosis in the elderly (Justesen et al., 2001; Rosen et al., 2009; Takeshita et al., 2014). Several hematopoietic processes are altered in the elderly such as erythropoiesis. Indeed, red blood cell production takes place in the bone marrow under the control of erythropoietin (EPO) and androgens (Price and Schrier, 2008; Price, 2008). Low levels of androgens associated with decreased EPO levels in the elderly can lead to anemia, and thus their treatment with androgenbased therapies (Bachman et al., 2014; Coviello et al., 2008; Guo et al., 2013). While aging is associated with hematopoietic failure, the number of hematopoietic stem cells in the bone marrow increases with age (Kuranda et al., 2011; Pang et al., 2011). They however present both an increased homogeneity in stem-cell marker expression and an epigenetic dysregulation, shedding light on their inability in completely repopulating the hematopoietic system (Chambers et al., 2007; Chambers and Goodell, 2007). It has thus been described that the increased inflammatory environment in the bone marrow as a result of bone degradation and adipogenesis impairs the development of B-cells and progenitor T-cells (Kennedy et al., 2016; Sun et al., 2012). Similarly various subsets of antigen-presenting cells such as macrophages see their functions altered (Linehan and Fitzgerald, 2015) which ultimately impacts not only on their ability to fight infections and orchestrate efficient T-cell responses, but also to contribute to wound repair and tissue regeneration (Danon et al., 1989; Swift et al., 1999, 2001). Indeed, macrophages in the elderly show reduced phagocytic activities, impaired differentiation characterized by a decrease in CD681 cells in the bone marrow of elderly individuals (Ogawa et al., 2000). These macrophages are also skewed towards the anti-inflammatory M2 phenotype associated with an increased expression of IL-10 but low levels of IL-12 and TNF-α (Jackaman et al., 2013; Kelly et al., 2007). The anti-inflammatory phenotype of macrophages in the elderly is consequently associated with an inability to respond to common pathogenic stressors such as bacterial lipopolysaccharide (LPS). While the TLR4 receptor for LPS sees its expression decreased (Chelvarajan et al., 2005), stimulated macrophages show reduced secretion of proinflammatory markers such as IL-6, TNF-α, IL-1β and IL-12 compared to macrophages from young individuals. However, the expression of anti-inflammatory molecules such as IL-10, prostaglandin E2 is increased (Chelvarajan et al., 2005; Boehmer et al., 2004; Wu et al., 1998). This state of non-responsiveness is associated with defective intracellular signaling as commonly phosphorylated p38 and JNK mitogen-activated protein kinases are under phosphorylated (Chelvarajan et al., 2006). Similarly, IFNγ, which plays an important role in macrophage

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activation, fails to efficiently promote the expression of MHC class II presenting molecules necessary for T-cell priming, while they present a drastic reduction in their capacity to produce superoxide anions essential to mediate their bactericide functions (Davila et al., 1990; Herrero et al., 2001).

26.4.2. Thymic Involution One of the major features characterizing the aging of the immune system is the slow shrinking of the thymus with age or thymic involution, which significantly starts at the third decade of life and alters the daily number of naı¨ve T-cells generated. This leads to a general decline in T-cell production and a loss of T-cell repertoire diversity (Aw and Palmer, 2011). Similarly to the bone marrow, aging is associated with an increase in adipose tissue in the thymus which is associated with factors detrimental for thymic maintenance such as Leukemia Inhibitory Factor, Oncostatin M, IL-6 and sex hormones (Rega et al., 2007; Sempowski et al., 2000; Trayhurn and Wood, 2004). The development of thymic adipocyte is also at the expense of thymic fibroblasts, which are key in the maintenance of thymic epithelial cells via the secretion of various mediators such as stem cell factor, fibroblast growth factors (FGF7, FGF10) and vascular endothelial growth factor (Yang et al., 2009). It has also been proposed that the reduction in sex hormones occurring in the elderly is aggravating this process (Sutherland et al., 2005). By acting on thymic epithelial cells expressing androgen/estrogen receptors, sex hormones are thought to be required for normal thymic development and ultimately thymic involution as their amounts are reduced during aging (Staples et al., 1999).

26.4.3. Alteration of the Cellular Components of the Adaptive Immune System While thymic involution impairs the output of T-cells from the thymus to ultimately alter the pool of circulating T-cells, other factors alter the adaptive immune system. Historically, T-cells have been very well studied in the context of immunosenescence. An alteration in CD41 T-helper 1/T-helper 2 (Th1/Th2) profile is associated with an immune risk profile characterized by an inverted CD4/CD8 ratio (Muller et al., 2015; Yan et al., 2010). The latter is commonly associated with a history of persistent infections such as cytomegalovirus (CMV), and sees an increase in the number of CD81CD282 cells (CD28 is one of the proteins expressed on T cells that provide co-stimulatory signals required for T cell activation) as well as CD31CD571 (CD57 is a marker of terminal differentiation on human CD81 T cells) cells and terminal effector T-cells (Pera et al., 2016; Pita-Lopez et al., 2009). The increased likelihood of an elderly individual getting infections is also characterized by a general antiinflammatory profile where decreased levels of cytokines such as interferon gamma (IFNγ) are associated with increased levels of immuneregulatory cytokines such as IL-4 and IL-10. The reduced levels of IFNγ are also linked with a decreased number of CD282 T-cell effector memory as well as CD81 cytotoxic T lymphocytes; both of which are being associated with poor vaccine response in the elderly (Lord, 2013; Nikolich-Zugich et al., 2012; Qin et al., 2016). Other proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukins (IL-6 and IL-1 family) linked with accelerated weight loss and malnutrition in the elderly (Table 26.1) also increase contributing to the general inflammation status observed during aging, and the occurrence of recurrent and persistent infections (Bruunsgaard et al., 2001; Michaud et al., 2013). The increased frequency and number of senescent T-cells can be explained by the various stressors encountered during lifespan, and recent evidence showed the mechanisms leading senescent T-cells to switch functionality and metabolism. Henson et al. (2014) have explained it by the reduced number of mitochondria in senescent T-cells and implication of the p38 pathway and autophagy. Other cells show significant alterations but not as well-defined as in the T-cell compartment. Nonetheless, one should not omit the importance of antigen-presenting cell as well as antibody-producing B-cell defects in the age-associated reduced immunity (Muller and Pawelec, 2014). Another key regulator of immune responses and tissue homeostasis is the pool of CD41 regulatory T-cells (Treg), which is altered during aging. If thymic involution is affecting the output of naturally occurring Treg selected in the thymus, the homeostasis of CD41 Treg is preserved via different mechanisms among which is an increase in the generation of Treg in the periphery associated with increased suppressive functions on T-cells proliferation and activities (VukmanovicStejic et al., 2006). The accumulation and increased suppressive activity of Treg in aging has been associated with the spontaneous reactivation of common chronic infectious diseases as well as the increased risk of developing cancer and autoimmune diseases (Dejaco et al., 2006; Lages et al., 2008). This gain of function is linked with an upregulation of FoxP3, the master regulator of their function, as well as their ability to secrete IL-10, suppress the expression of the costimulatory molecule CD86 on dendritic cells and deplete the microenvironment in key

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amino acids such as cysteine (Garg et al., 2014; Hwang et al., 2009). Interestingly, despite an accumulation of Treg in peripheral blood, the decreased ability of muscle repair during aging has been linked with the failure of Treg to accumulate in skeletal muscle following injury (Castiglioni et al., 2015; Kuswanto et al., 2016). Meanwhile, age-associated insulin-resistance, but not obesity associated insulin-resistance, is characterized by the accumulation of Treg in fat tissues with age (Bapat et al., 2015).

26.4.4. Nutrition and Immunity in the Elderly The dysregulation between pro- and anti-inflammatory molecules leads to a general state of low level of chronic inflammation called “inflammaging.” While it is undeniable that the genetic framework and previous medical history impact inflammaging, its bearing is detrimental during aging and is related to increased risk of developing multiple comorbidities and ensuing increased mortality. Inflammaging could therefore be linked to a general cumulative exposure to antigen load during the lifetime and caused both by external infections and clinical or subclinical chronic inflammatory diseases. Mounting evidence also highlights the important role played by the gastrointestinal tract in regulating multiple inflammatory diseases from conception to adulthood. Indeed, lifestyle factors such as the quality of the diet, nutritional status or physical activity have been shown to impact the immune system as it is now admitted that deficiencies in vitamins and minerals, malnutrition or diet excessively rich in saturated fatty acids can dramatically impair immune responses. Studies clearly showed how the microbiota shapes immunity and how immunity also shapes the gut microbiota. The metabolites produced by the gut microbiota during nutrient processing will directly affect the functionality of local as well as peripheral cells. Thus, the type of nutrient (food intake), the type of processor (bacteria and host cell) and how efficiently this is performed (e.g., gut physiology) will directly impact on bioavailability of nutrients for the organism. In many studies researchers have tried to modulate the immune system of elderly individuals or old animals using nutritional approaches (micronutrients (Bao et al., 2003; Belisle et al., 2008, 2009; Corridan et al., 2001; Cossack, 1989; Farges et al., 2012; Fortes et al., 1998; Kahmann et al., 2006; Prasad, 2003; Prasad et al., 2006; Watson, 1991; Watson et al., 1991), pre/probiotics (Arunachalam et al., 2000; Dong et al., 2013; Gill et al., 2001a,b; Guigoz et al., 2002; Ouwehand et al., 2009; Vulevic et al., 2008), (poly)-phenols (Currier and Miller, 2000; Monagas et al., 2009; Tasat et al., 2003; Yuan et al., 2012), macronutrients (Bechoua et al., 2003; Bouwens et al., 2009, 2010; Calder, 2010; Galli and Calder, 2009; Meydani et al., 1991; Pae et al., 2012) or Mediterranean diet (Azzini et al., 2011; Carluccio et al., 2003; Cortes et al., 2006; Dell’Agli et al., 2006; Feart et al., 2009; Lasheras et al., 2000; Osler and Schroll, 1997; Scarmeas et al., 2009; Tangney et al., 2011; Trichopoulou et al., 2003; Valls-Pedret et al., 2012)). Some examples are described in Table 26.2.

26.5. PREVENTION BY NUTRITION: THE GROWING IMPORTANCE OF FUNCTIONAL FOOD IN OLDER ADULTS The primary role of diet is to provide sufficient nutrients to meet individual daily nutritional requirements. There is now increasing scientific evidence to support that some foods and food components have beneficial physiological and perhaps psychological effects above the provision of the basic nutritional requirements. The concept of foods that were developed specifically to promote health or reduce the risk of diseases was introduced in Japan by health authorities in the mid-1980s under the term functional foods. Health authorities recognized that preventive nutrition-based approaches can improved the quality of life and reduce healthcare cost for the expanding number of elderly (Arai, 2000). There are many possible definitions for the term functional foods and no worldwide consensus on its meaning. Today five categories can be described: nonaltered products, fortified products, enriched products, altered products, and enhanced commodities. Functional foods represent a doubledigit annual growing market rate even if the exact size of the market is difficult to measure with imprecise definitions of the product categories. Product marketing is a driving force behind functional food sciences where health claims are a key target, shown to impact consumer attitudes (Ares and Gambaro, 2007; Ares et al., 2008; Tudoran et al., 2009; van Kleef et al., 2005; Verbeke et al., 2009). Understanding consumers’ health motivation, nutrition knowledge and perception of product effectiveness are key indicators dictating consumer behaviors toward purchase and consumption of functional food (Ares et al., 2008; Vella et al., 2014). Demographic characteristics seem to play a minor role in consumer acceptance even though they often highlight a gender bias with stronger purchase behavior by females (Ares and Gambaro, 2007; Ares et al., 2008; Vella et al., 2014). Regarding age groups, seniors perceive the importance of functional foods more than younger

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26.5. PREVENTION BY NUTRITION: THE GROWING IMPORTANCE OF FUNCTIONAL FOOD IN OLDER ADULTS

TABLE 26.2

Examples of Food Supplement and Dietary Interventions Affecting the Immune System in the Elderly

Nutrient/diet

General impact/features

Study

Bioactive compound tested

Vitamin E

Enhance T-cell function directly or indirectly by reducing production of suppressive factors (PGE2) by macrophages

Belisle 2008 and 2009

DL-α-tocopherol,

Deficiency leads to reduced immune cell proliferation, cytokine production and specific reduction in NK cell and neutrophil function

Cossack 1989; Prasad 2003 and 2006; Bao 2003

45 mg of zinc/day for 6 months (zinc sulfonate)

Increased expression of IL-2 and IL-2R mRNA in PBMC, Improvement of Delayed Type Hypersensitivity

Fortes 1998

25 mg/day for 3 months (Zinc sulfate)

Increased numbers of activated CD41 T-cells and CD81 cytotoxic lymphocytes

Kahmann 2006; Metz 2007

10 mg of pure zinc (50 mg zinc-aspartate) per day for 48 days

Reduction in CD41 CD251 activated T-cells; no difference in Th2/Th1 profile (CCR41/CCR51)

30 mg β-carotene, 15 mg lycopene, 9 mg lutein

Increased bactericidal activity, increased serum IgA, reduced B cell numbers, shift to T-cells expressing mature phenotype, increased NK cell population

13.3 mg Lycopene or 8.2 mg β-carotene

No change in lymphocyte subsets or expression of adhesion molecules. No change in terms of lymphocyte production or cytokine secretion

2060 mg/day carotenoids

Global improvement in immune functions including increases in NK cell numbers and Th cell functions

Bifidobacterium lactis (3 3 1011 CFUs/day) for 6 weeks

Significant increase in PMN phagocytic and bactericidal activity to Staphylococcus aurerus

Zinc

Carotenoids

multivitamin and mineral supplements for 1 year

Carotenoids are stored in tissue Farges 2012 and can be converted to vitamin A. They are linked to increased immune functions. Deficiency is associated with increased plasma levels of ROS, Corridan 2001 and sIL-2R

Watson 1991a and 1991b Probiotics

Prebiotics

200 IU/day of

Live microorganisms that when Arunachalam 2000 administrated in adequate amount, confer a health benefit to the host Gill 2001

Significant effects in supplemented group versus control or placebo Increased cytokine production of IL-1β, IL-6, IFNγ and TNFα. Response influenced by gene polymorphism and background cytokine profile

Lactobacillus rhamnosus Increase in blood NK cells and HN001 (5 3 1010 CFUs/day) tumoricidal activity or B. lactis NH019 (5 3 109 CFUs/day) for 3 weeks

Gill 2001

B. lactis NH019 (5 3 109 CFUs/day or 5 3 1010 CFUs/day) for 3 weeks

Increased NK frequency and tumoricidal activity, increased phagocytic activity of PBMC and PMN

Dong 2013

Lactobacillus casei Shirota (1.3 3 1010 CFU/day) for 4 weeks

Significant increase in NK cell activity. Significant decrease in CD25 expression by resting T lymphocytes, increase of UL10/IL-12 ratio

Galactooligosaccharides (5.5 g/day) for 4 weeks

Increased NK cell activity, reduction plasma levels of TNFα, IL-1β, and IL-6. Increased IL-10 production by PBMC, increase phagocytic activity of PMN and monocytes. Increased bacterial counts of bifidobacteria

Guigoz 2002

Fructooligosaccharides (8 g/day) for 3 weeks

Increased bacterial counts of bifidobacteria. Decreased phagocytic activity of granulocytes and monocytes, decreased IL-6 and TNFα mRNA in peripheral monocytes

Ouwehand 2009

Lactitol and L. acidophilus NCFM (2 3 109 CFU/g) for 3 weeks

Reduced fecal levels of PGE2, Increased bacterial counts of bifidobacteria. Increased IgA levels overtime

Prebiotics are substrates for gut Vulevic 2008 bacteria with the aim to improving their growth

(Continued)

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TABLE 26.2

(Continued)

Nutrient/diet

General impact/features

Study

Bioactive compound tested

(Poly)-phenols

Mainly found in red wine, fruits, vegetables, tea, coffee, cocoa and cereals

Tasat 2003

In vitro studies

Improve production of IL-12, IL-10, and IL-1β by MNC. Increase production of IgA and IgG by B cells

Currier 2000

Mouse study

Increased NK cell number and cytolytic activity

Yuan 2012

Rat study (resveratrol)

Increased T-helper cells

Monagas 2009

40 g/day Cocoa for 4 weeks. Individuals at high risk of cardiovascular diseases

Significant lower expression of cell adhesion molecules VLA-4, CD40 and CD36 on monocytes. Lower plasma levels of inflammatory markers P-selectin and ICAM-1

Pae 2012

long chain n-3 PUFA (polyunsaturated fatty acids)

Improve cardiovascular, degenerative neurological, inflammatory and autoimmune diseases

Galli 2009; Calder 2010

n-3 PUFA

Anti-inflammatory properties: inhibition formation of eicosanoids (Thromboxan A2) required for platelet aggregation; inhibition of proinflammatory cytokines IL-1β, TNFα, and IL-6. Reduction of IL-8, MCP-1, ROS, NOS and adhesion molecules (ICAM-1, VCAM-1 and selectins)

Meydani 1991

n-3 PUFA (1.68 g EPA and 0.72 g DHA/day) for 3 months

Reduction in cytokine production, inhibition in mitogen-induced PBMC proliferation

Bechoua 2003

low doses of PUFA (30 mg EPA and 150 mg DHA/day) for 6 weeks

Decrease in lymphocyte proliferation in response to mitogens. Reduction in the glutathione activity

Bouwens 2009 and 2010

high doses of EPA (1.8 g) and DHA (1.8 g) equivalent to ten portions of oily fish per week for 26 weeks

Decrease plasma levels of free fatty acids and triglycerides, reduction in proinflammatory genes including NF-κB target genes, proinflammatory cytokines and genes involved in eicosanoid synthesis

Osler 1997; Lasheras 2000; Trichopoulou 2003

MD

Association with a significant and substantial reduction in overall mortality

Carluccio 2003; Cortes 2006; Dell’Agli 2006

Olive oil consumption in patients at risk of coronary heart disease

Reduced expression of ICAM-1, VCAM-1, and E-selectins

Tangney 2011

MD

Improved cognitive performances in dementia patients. MD associated with slower cognitive decline, reduction of mild cognitive impairment, reduction of neurodegenerative disorders such as Parkinson and Alzheimer

MD

MD associated with down-regulation of CD49d and CD40 expression in monocytes. Reduced plasma expression of inflammatory markers such as sICAM-1, svCAM-1, CRP, IL-6, TNFα, IL-12. Higher levels of anti-inflammatory cytokine IL-10

Fatty Acids

Mediterranean Diet (MD)

Cellular fatty acids impact on gene expression and act as precursors of prostaglandins, leukotriens, lipoxins and resolvins

Characterized by dietary patterns found in olivegrowing regions of the Mediterranean: high consumption of olive oil, vegetable, fruits, nuts and cereals. Moderate intake of fish, poultry. Low intake of dairy product, red and processed meat (Gonzalez, 2000).

Valls-Pedret 2012; Feart 2009 Scarmeas 2009 Azzini 2011

Significant effects in supplemented group versus control or placebo

Decrease plasma concentration of sICAM1, sVCAM-1, sE-selectin, IL-6, and CRP

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consumers (Ares and Gambaro, 2007; Vella et al., 2014). Personal health history with more direct or indirect exposure to health problems may be an explanation. Regrettably, most of the consumer literature is based on Western societies with bias in nutrition culture and limited literature exists regarding the elderly per se. Recently, Vella et al. (2014) conducted a study on Canadian consumers’ perception of functional foods. The authors showed a high prevalence of functional food consumption (93%) among community dwelling older adults. Those consumers expressed high interest in nutrition to prevent chronic diseases and more than half of them reported to actively seek for information on functional food, with the most common source being food labels, press or discussion with their relatives. Also, knowledge and awareness as well as influence of health professionals were found to be the best promotors of senior consumers’ ability to purchase functional food products, whereas price ranked fifth. Among the health benefits frequently reported to be a driver of consumption was the one related to immunity, ranked high being desired by about 40% of senior consumers (Verbeke et al., 2009; Vella et al., 2014).

26.6. FUNCTIONAL FOOD TO IMPROVE IMMUNITY: A CLOSER LOOK AT CLAIMS AND CLINICAL REALITY In response to the growing demand for functional foods, regulatory bodies in many countries have developed policies and regulations governing health claims for foods and food supplements. Approval requires substantiation with strong scientific evidence mainly based on human data, while the level of evidence varies between countries with important economic interests at stake. We exemplified this information below with a snapshot of the European legislation on functional foods managed by the European Food Safety Authority (EFSA). EFSA is an independent body that works in close cooperation with various scientific agencies and institutions in European Union member states providing independent scientific assessments on all matters related to food from farm to fork including food safety and health assessments (risk assessment role). Each member state is then responsible for the implementation of adequate policies (risk management role). In Europe functional foods are considered as foods that are intended to be consumed as part of the normal diet and that contain biologically active components that offer the potential of maintaining health or reduced risk of disease. Food with a longterm history of safe usage (i.e., generally recognized as safe, GRAS) as well as novel food (i.e., recently identified safe food bioactives or extracts) will fall into well-defined processes, largely accepted across Europe, to document their safety and allow their introduction into specific food matrix. However, beyond safety, the next stages are difficult, particularly for the definition and acceptation of health-related functions. In early 2016, the consultative panel on Dietetic Products, Nutrition and Allergies from the EFSA has updated the so-called “Guidance on the scientific requirements for health claims related to the immune system, the gastrointestinal tract and defense against pathogenic microorganism” first published in 2011 (https://www.efsa. europa.eu/en/efsajournal/pub/4369). This guidance is mainly intended to assist applicants in preparing applications for health claims authorization. Claims under article 13.1 on the maintenance of unspecified functions of the immune system were based on the essentiality of a given nutrient often coming from biochemically previously well-established nutrient role and or reported undesirable related clinical symptoms in specific micronutrient deficiencies. So far, vitamins A, B6, B12, C, D as well as copper, folate, iron, selenium and zinc received a favorable opinion. Hence products that contain sources of these nutrients (usually .15% of the daily reference intake) are allowed to advertise for their contribution to the maintenance of a normal immune system. Claims other than those based on the essentiality of nutrients are still intensely debated, i.e., new function health claims under article 13.5, especially defense against pathogens or disease risk reduction claims under article 14. This may be linked to the lack of strong science and consensus on the relevant clinical evidence to provide outcome(s) to measure. So far, all modifications in immune or inflammatory markers heavily used by the scientific community as an indication of immune responsiveness were unacceptable as evidence. Indeed, not every change of these markers may be considered as beneficial to human health. However, it is accepted as supportive evidence regarding the mechanism(s) by which a food may exert the claimed effect. Thus, cell subset phenotype or distribution, proliferative, lytic or phagocytic activities as well as cytokine and immunoglobulin secretion, despite clear association with a disease onset or recovery are regulatory speaking not acceptable. The holy grail of immunerelated claim remains immune defense against pathogens. Classically incidence, severity and/or duration of symptoms only with validated questionnaires, and/or even better by physician diagnosis following well-defined criteria are requested in preferably repeated high standard randomized clinical trials. Hence, more recent nutritional interventions meet pharma industry standards, which we believe may lead to a general improvement of the quality of the study in the field.

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26.7. FUNCTIONAL FOOD WITH PROVEN CLINICAL EFFICACY TO AMELIORATE ELDERLY IMMUNITY Today, vaccination study results are acceptable regarding EFSA standards and often used as a faster and objective approach to demonstrate food derived bioactive with defense against pathogens properties. However, strict correlation between antibody titers in response to vaccination and protection against infection is not always evident or established for all vaccines. Influenza disease is perhaps one of the few exceptions explaining why it is often used in the nutritional immunology field. The flu disease is highly prevalent, the vaccine is safe, its efficacy proven supporting yearly recommendation by health authorities, the etiological agents of the disease well characterized with higher susceptibility in infants and elderly (Dominguez et al., 2016; Goodwin et al., 2006). In addition, molecular vaccine response features with expected flu-specific antibody titers to claim seroconversion and reach seroprotection are known and accepted by authorities. Hence, nutritional interventions conducted by academics and food industries to study immune system functionality frequently used the vaccine challenge model. Regarding elderly immunity, influenza and pneumococcal vaccine responses were chosen as relevant read-outs. Influenza virus, as well as (secondary) pneumococcal bacterial infections, significantly affects morbidity and mortality in aged individuals. Although immunization protocols have been shown to be effective in reducing respiratory illnesses, hospitalization and death in people aged 65 and older, the vaccinated elderly mount weaker antibody responses than that observed in young healthy individuals (Dominguez et al., 2016; Goodwin et al., 2006; Furman et al., 2013). Therefore, any alternative to improve vaccine responses and efficacy in the elderly is an area of active investigations with major public health impact. Introduction of adjuvant such as MF59 to influenza vaccine has been a major breakthrough in the field to increase immunogenicity of influenza vaccines (De Donato et al., 1999). However, a recent report by Goodwin et al. (2006) highlighted massive vaccine efficacy differences between young healthy adults (70%90%) and elderly individuals (17%53%). So alternatives or supportive strategies based on nutrition, and in particular functional food to improve vaccine response, are very welcome. There is long-lasting evidence supporting the role of nutrition for senior immunity. We can cite the link between single micronutrient deficiencies and alteration of vaccine responses in the 1950’s; total protein and albumin serum level decline as a primary reflection of poor nutritional status and prediction of vaccine responses and also association of undernutrition or poor nutritional status assessed subjectively (questionnaires) or objectively (biochemical measures) among elderly with diminished delayed type hypersensitivity responses as reviewed in Beisel (1982) or Chandra (2004). Thus, in the section below, we captured and discussed the existing nutritional interventions with vaccine response as an outcome for elderly only define as 65 years and above. We identified 25 studies that have attempted to improve vaccine responses in elderly individuals by nutrition using three main different approaches (Table 26.3): whole dairy-based nutritional supplementation or enteral formula (Akatsu et al., 2013b; Boge et al., 2009; Bunout et al., 2002, 2004; Freeman et al., 2010; Langkamp-Henken et al., 2004, 2006; Vidal et al., 2012; Wouters-Wesseling et al., 2002), single or mixture of micronutrient supplements (Allsup et al., 2004; Chandra, 1992; Chandra and Puri, 1985; Crogan et al., 2005; Girodon et al., 1999; Meydani et al., 1997; Provinciali et al., 1998; Schmoranzer et al., 2009) and more recently pre/probiotics (Akatsu et al., 2013a; Bosch et al., 2012; Maruyama et al., 2016; Namba et al., 2010; Przemska-Kosicka et al., 2016; Van Puyenbroeck et al., 2012) and few others (Gibson et al., 2012; Miyagawa et al., 2008) were reported. These studies more often resulted in improvement of immune responsiveness (15 studies). We cannot exclude potential existing publication bias with positive results. The earliest reports providing positive results explored the effects of trace elements like zinc or selenium, and often in very elderly nursing home residents (over 80-year-old) particularly in long-lasting interventions (from 6 months up to 2 years (Chandra, 1992; Meydani et al., 1997; Girodon et al., 1997)). However, this literature has to be taken with caution in the light of the existing negative reports presented in Table 26.3, as well as the lack of well-defined health conditions of the subjects recruited. The same is true with the reports using multivitamin, multivitamin and mineral supplementation, nutritional formula or promotion of fruits and vegetables in a daily intake (Gibson et al., 2012) (often containing a blend of minerals and vitamins) where reports found positive outcomes in both community and nursing home residents. More recently, some interventions with probiotics—live microorganisms that when administrated in adequate amount, confer a health benefit to the host—hold promising results in infants where daily ingestion enhances the response to vaccine and/or reduces the rate of infections, the mean duration of an episode of acute respiratory infections, antibiotic use, or absenteeism as shown in recent meta-analysis (Hao et al., 2015). However, in the elderly, less is known and among the eight reports found, only half were positive for vaccine outcomes (Bosch et al., 2012; Maruyama et al., 2016; Namba et al., 2010; Van Puyenbroeck et al., 2012). This is also perhaps due to strain-specific differences and numbers, as well as age and medical condition of the subjects followed in the studies.

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TABLE 26.3

Study Akatsu (2013)

Summary of Intervention Trials With Eligible Functional Food Reporting on Vaccine Responses Trial and population characteristics

Agea

Bioactive compound tested

Primary outcome

RCT

76 6 7

Heat killed 1010 probiotic containing jelly daily for 12 weeks (Lactobacillus paracasei MoLac-1)

Trivalent influenza vaccine response

82 6 8

Trivalent influenza 5.1010 probiotics (Bifidobacterium longum vaccine response BB536) twice daily for 12 weeks in enteral nutrition

Higher number of patients with H1N1 titers .20 at week 6

83 6 7

Complex micronutrient supplement twice daily to provide the reference nutrient intake for 4 weeks

Trivalent influenza vaccine response

None

n 5 15 Nursing home residents

Akatsu (2013)

R DB PC n 5 45 Tube fed patients

Allsup (2004) R DB PC n 5 164 Nursing home residents

Significant effects in supplemented group vs. control or placebob None

Boge (2009)

R DB PC n 5 86 (trial 1) and 241 (trial 2) Nursing home residents

82 6 8 (trial 1) 85 6 7 (trial 2)

Fermented dairy drink containing probiotic (Lactobacillus casei) twice daily for 7 or 13 weeks

Trivalent influenza vaccine response

Higher mean titers and seroconversion rate for influenza B Subanalysis showed higher H1N1 seroprotection rate found in non seroprotected patients at baseline

Bosch (2012)

R DB PC

651

5.108 or 5.109 probiotics (Lactobacillus plantarum CECT 7315/7316) daily for 3 months

Trivalent influenza vaccine response

Higher mean titers for influenza specific IgA and IgG

701

Nutritional complete formula of Trivalent influenza and None pneumococcal vaccine 240 Kcal daily with prebiotic responses (6 g/day fructooligosaccharides) for 28 weeks

74 6 5

Nutritional complete formula of Trivalent influenza and Lower respiratory self-reported 480 Kcal daily for 1 year pneumococcal vaccine infection rate responses

651

Iron supplement daily for 30 days

Influenza vaccine response

None (underpowered study)

701

Nutritional advice and oral dietary or medicinal supplements appropriate for the deficiency identified

Influenza H1N1 vaccine response

Higher mean fold increase titers and seroconversion rate

75

Complex trace elements 6 vitamins daily supplement for 1 year

Influenza vaccine (no precision)

Higher mean fold increase titers (no precision)

n 5 60 Nursing home residents Bunout (2002)

R PC n 5 66 Community dwelling

Bunout (2004)

 n 5 60 Community dwelling

Crogan (2005)

 n57 Nursing home residents

Chandra and  Puri (1985) n 5 30 Community dwelling with identified nutritional deficiencies Chandra (1992)

R PC n 5 96 Community dwelling

Rk: study recently retracted

(Continued)

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TABLE 26.3

Study Freeman (2010)

(Continued) Trial and population characteristics

Agea

Bioactive compound tested

Primary outcome

R DB PC

67 6 6

5 g whey supplements three times daily for 8 weeks

Pneumococcal vaccine response

None (underpowered study)

71 6 5

Increase from 2 to 5 fruit and vegetable daily portion intake for 16 weeks

Tetanus toxoid and Pneumococcal vaccine responses

Higher titers and seroconversion rate for pneumococcal capsular polysaccharides (more pronounced in vaccination-naı¨ve group)

84 6 7

Complex trace elements 6 vitamins daily supplement for 2 years with up to twofold reference nutrient intake

Trivalent influenza vaccine response

Higher mean fold increase titers for influenza A and seroprotection with trace elements alone or trace elements 1 vitamins

83 6 2

Nutritional formula of 360 Kcal daily for 6 months

Trivalent influenza vaccine response and self-reported day with URTI symptoms

Reduction in the numbers of days with URTI symptoms and higher seroconversion rate for influenza A

81 6 1

Nutritional formula of 360 Kcal daily for 10 weeks

Trivalent influenza vaccine response

Higher H1N1 seroprotection rate, less fever and newly prescribed antibiotics

89 6 5

Heat killed 1010 probiotic containing jelly daily for 6 weeks (Lactobacillus paracasei MoLac-1)

Trivalent influenza vaccine response

None

71 6 5

Vitamin E supplementation 60, 200, or 800 mg daily for 235 days

Hepatitis B, tetanus toxoid, diphtheria and pneumococcal vaccine responses

Higher titers for hepatitis B, tetanus toxoid and pneumococcal capsular polysaccharides

n 5 24

Significant effects in supplemented group vs. control or placebob

 Gibson (2012) RCT n 5 83 Community dwelling Girodon (1999)

R DB PC n 5 725 Nursing home residents

LangkampHenken (2004)

R DB PC n 5 66 Nursing home residents and community dwelling

LangkampHenken (2006)

R DB PC n 5 157 Nursing home residents

Maruyama (2016)

R DB PC n 5 45 Nursing home residents

Meydani (1997)

R DB PC n 5 88 Community dwelling

Only subanalysis showed higher response to H1N1 and B strains in the oldest old, i.e., 851

Miyagawa (2008)

PC n 5 67 Nursing home residents

76 6 9

700 mg L-cystine 1 280 mg L-Theanine daily for 14 days

Trivalent influenza vaccine response

Higher HI titers for influenza B Higher H1N1 seroconversion rate after stratified analysis in low serum Hb or total protein subjects

Namba (2010)

R DB PC

87 6 7

1011 probiotics daily (Bifidobacterium longum BB536) for 19 weeks

Trivalent influenza vaccine response

Less subjects contracted influenza or had fever episodes

82 6 7

Zinc supplementation 200 mg 6 2 g Arginine twice daily for 60 days

Trivalent influenza vaccine response

None

n 5 27 Nursing home residents

Provinciali (1998)

RCT n 5 384 Nursing home residents

(Continued)

V. NUTRACEUTICALS IN BOOSTING IMMUNE SUPPORT AND AS THERAPEUTICS FOR INFLAMMATORY DISEASES

26.7. FUNCTIONAL FOOD WITH PROVEN CLINICAL EFFICACY TO AMELIORATE ELDERLY IMMUNITY

TABLE 26.3

Study PrzemskaKosicka (2016)

(Continued) Trial and population characteristics

Agea

Bioactive compound tested

Primary outcome

R DB PC

69 6 5

Synbiotic (109 Bifidobacterium longum bv. Infantis CCUG 52486 and 8 g glucooligosaccharides) daily

Trivalent influenza vaccine response

85

Complex micronutrient Trivalent influenza supplement from wheat sprouts vaccine response for 3 months

None (even lower HI titers for influenza B)

84

1.3 3 1010 probiotics daily (Lactobacillus casei Shirota) for 176 days

Trivalent influenza vaccine response (secondary outcome)

None (with no difference in susceptibility to URTI)

67 6 2

Goji berry milk based extracts for 3 months

Trivalent influenza vaccine response

Higher HI titers for influenza A

84 6 8

Nutritional formula of 250 Kcal twice daily for 7 months

Trivalent influenza vaccine response

Higher mean fold increase in titers for influenza A

n 5 63 Community dwelling

Schmoranzer (2009)

331

R DB PC n 5 106

Significant effects in supplemented group vs. control or placebob None

Nursing home residents Van Puyenbroeck (2012)

R DB PC n 5 737 Nursing home residents

Vidal (2012)

R DB PC n 5 150 Community dwelling

WoutersWesseling 2002

R DB PC n 5 19 

Mean 6 SD of the supplemented group when available. Only significant effects related to vaccine response and or infection are given. PubMed literature search results done in August 2016 looking at nutrition related keywords like “vitamin,” “mineral,” “antioxidants,” “polyphenol,” “prebiotic,” “probiotic” and or “fibers” with “vaccine” or “influenza” or “pneumococcal” with filters applied being “human” and “clinical trial” in “age 651.” After elimination of reviews, duplicates, articles not in English or where vaccination results were not presented, 25 articles remained. R Randomized, DB Double blind, PC Placebo controlled, RCT Randomized controlled trial, HI hemagglutination inhibition, Hb hemoglobin, URTI upper respiratory tract infections and - stands for no information.

a

b

Overall, we noticed a lack of consistency between several studies where either mean titer values or seroconversion and sometimes seroprotection reached significance. Best results were often obtained with the oldest nursing home residents whereas many confounding factors and comorbidities in this institutionalized population may exist. Indeed, the elderly is nowadays recognized as a heterogeneous declining population from healthy/robust to frail individuals, according to a few validated geriatric evaluation tools like Fried’s score (Fried et al., 2001) or Rockwood’s index (Mitnitski et al., 2002). These tools confirmed that malnourished individuals with a set of comorbidities could predict weaker pneumococcal or influenza vaccine responses and subsequent related illnesses. This deserves to be readdressed properly with clinical settings paying better attention to age, nutritional and frailty status. We propose that the immune response in the elderly could be more sensitive to nutritional status/deficiencies than in young adults, and therefore aging and nutritional deficiency could exert a cumulative influence on immune responsiveness. Specific nutritional needs may exist for each elderly condition, understanding and addressing them may benefit globally each individual and in particular its immune system. It is noteworthy that, despite being out of the scope of our literature search focusing on the influence of nutrition on influenza or pneumococcal vaccine responses, a few studies have also investigated the direct reduction of infection rates or days of illness as primary outcomes and not vaccine responses as reviewed by Yaqoob (Yaqoob, 2014). Fermented milk consumption with either Lactobacillus casei, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus pentosus or Bacillus subtilis as well as symbiotic (probiotic plus prebiotic formulation) generate positive results for acute respiratory infections in the elderly and lower the risk of catching colds and their duration. (Fujita et al., 2013; Guillemard et al., 2010; Lefevre et al., 2015; Makino et al., 2010; Pregliasco et al., 2008; Shinkai

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26. NUTRITION AS A TOOL TO REVERSE IMMUNOSENESCENCE?

et al., 2013; Turchet et al., 2003). The difficulty and long nature of such studies may be reasons behind their scarcity. Their positive nature may also suggest that disease prevention may be achieved by different approaches. Vaccine challenge studies may mainly address adaptive immune system decline, and omit age-related alteration of innate immune system function to deal with certain infections.

26.8. CONCLUSIONS AND FUTURE DIRECTIONS Functional foods offer great potential to improve health and/or help prevent certain diseases when consumed as part of a balanced diet and healthy lifestyle. However, the research opportunities in nutrition to explore the relationship between a food or a food component and an improved immune system are very challenging. Although several epidemiological studies in the elderly highlighted the role of minerals and/or vitamins and probiotics to improve or prevent, for instance, respiratory infections, inconsistent results have been observed in intervention studies. Evaluation methods (titers, seroconversion or seroprotection), supplementation strategies used (dose and route), or even population characteristics (elderly heterogeneity) could be provided as reasons. In addition, we propose that deeper understanding of nutrition-related physiology (mastication, digestion, nutrient metabolism), elderly eating behavior, and identification of specific senior nutritional needs could help to rethink the rationale behind functional foods for elderly immunity.

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27 Therapeutic Interventions to Block Oxidative Stress-Associated Pathologies Nupoor Prasad, Prerna Ramteke , Neeraj Dholia and Umesh C.S. Yadav Central University of Gujarat, Gujarat, India

27.1. INTRODUCTION Oxidative stress occurs when the balance between generation of reactive oxygen species (ROS) and their destruction by cellular antioxidants and other scavenging mechanisms gets disturbed. During the evolution of life forms, aerobic organisms evolved the mechanism of metabolism that uses oxygen as an electron acceptor during ATP production. Many times, excessive oxygen utilization causes leakage of ROS, which are neutralized in the presence of cellular detoxification machinery including enzymatic and dietary nonenzymatic antioxidants. d d ROS include various reactive species such as superoxide (Od2 2 ), hydroxyl (OH ), alkoxyl (RO ) and peroxyl d (RO2 ) ions. Few other oxidizing agents or molecules which are easily convertible into free radicals such as singlet oxygen (1O2), hydrogen peroxide (H2O2), and hypochlorous acid (HOCl) are also included into ROS (Russell and Cotter, 2015; Woolley et al., 2013). If detoxification of ROS fails due to some reasons, it results in excessive buildup of ROS and oxidative stress (Isaksson et al., 2011). The increased ROS attack the biomolecules, i.e., nucleic acids, lipids and proteins, leading to their oxidative damage, which impacts cellular functions. The paradox of ROS is that while excessive ROS can cause indiscriminate damage to cells and tissues, regulated and controlled ROS production is a protective mechanism against pathogen infection and also plays role in growth and development. Indeed while low levels of ROS trigger specific physiologic signaling pathways, increased ROS damage DNA, protein and lipids due to their high chemical reactivity. The balanced cellular redox state confirms the proper functioning of cells; however when this balance is disturbed by exogenous factors such as γ-rays, UV-rays, pollutants, toxins, xenobiotics and foods or endogenous sources such as mitochondria, cytochrome P450, peroxisomes and activated inflammatory cells (Khansari et al., 2009), it leads to development of pathological condition or worsening of existing pathological conditions. Oxidative stress mediates activation of a few redox-sensitive transcription factors that lead to the expression of inflammatory cytokines, chemokines and growth factors. These inflammatory molecules together create a microenvironment that is characteristic of various inflammatory diseases (Srivastava et al., 2011; Reuter et al., 2010). Several inflammatory diseases such as cardiovascular diseases (CVDs), neurodegenerative diseases (NDs), diabetes, asthma and various cancers either developed or get strongly influenced by oxidative stress. Oxidative stress-induced inflammatory condition influences the activation of various transcription factors such as nuclear factor kappa B (NF-κB), NF-E2 related factor-2 (Nrf2), activator protein-1 (AP-1), signal transducer and activator of transcription-3 (STAT-3), hypoxia inducible factor-1α (HIF-1α) and nuclear factor of activated T cells (NFAT). Further, these transcription factors stimulate transcription of inflammatory markers including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), inflammatory cytokines (e.g., TNF-α, IL-1 and IL-6) and chemokines (IL-8 and CXCR4), which aggravate the inflammation. Furthermore, as recently reported, alterations in the



Equal contribution.

Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00027-5

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expression of some specific micro RNAs have also been shown to play an important part in oxidative stressinduced inflammation (Hussain and Harris, 2007). This chapter discusses oxidative stress-induced inflammatory pathologies such as asthma, cancer, CVDs, diabetes and neurodegenerative diseases and the selective targets with therapeutic potential to reduce oxidative stress in order to achieve the redox balance. The clear understanding of oxidative stress-induced pathologies requires modern techniques such as RNAi, computation-based approaches, stem cell therapy, etc. In numerous studies nutraceuticals have been found to play protective role in oxidative stress-induced pathologies. However, they are also associated with some paradoxes, which will be discussed here. In the end we have discussed some of the drugs which are in clinical trials for various oxidative stress-induced pathologies.

27.2. OXIDATIVE STRESS-INDUCED DISEASES A number of inflammatory diseases including asthma, cancers, CVDs, diabetes and neurodegenerative diseases are strongly influenced by oxidative stress. Normally, the ROS are scavenged by the antioxidant system of the body including molecules like ascorbic acid, tocopherols, reduced glutathione, thioredoxin, transferrin and vitamins; enzymes including superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and glutathione reductase (GR). However, under certain pathophysiological conditions these antioxidant defense systems are reduced or compromised thus leading to oxidative stress-induced disease. The mechanisms by which ROS and oxidative stress facilitate the development of inflammation and disease in humans are primarily via DNA damage and mutagenesis leading to cell death, unregulated cell growth, invasiveness, transdifferentiation and irregular wound healing. Additionally, a number of genes could get upregulated during oxidative stress that may result in altered cellular function and physiology leading to pathogenesis. Besides these, oxidative stress is responsible for upregulation of redox-sensitive signaling mechanisms that upregulate expression of several inflammatory genes, which depending on the target tissue could result in airway inflammation, cancers of different organs, cardiovascular and neurodegenerative diseases. Some of these diseases are discussed below.

27.2.1. Asthma Oxidative stress is involved in various airway inflammatory diseases such as chronic obstructive pulmonary disease (COPD), chronic bronchitis, respiratory distress syndrome (RDS) and asthma (Kottova et al., 2007). Asthma, the most common airway disease, is a major respiratory problem where oxidative stress plays an important role (Masoli et al., 2004). Asthma is characterized by inflammation of airways, thickening of basement membrane, mucus hypersecretion, increased vascular permeability, synthesis and release of chemo-attractants, narrowing of windpipe, airflow obstruction, whooping and coughing (Nadeem et al., 2014; Xiao et al., 2013). Oxidative stress is known to facilitate the advancement of existing lung inflammation as described by the exacerbation of the airway hyper-responsiveness, excessive mucus secretion and increased secretion of inflammatory markers. These events are known to aggravate the severity of asthma (Fig. 27.1). In the airway passage, innumerable factors may be responsible for the increased production of ROS. In T helper 2 (Th-2) mediated asthma, various allergens have been found to cause excessive ROS production. Indeed, the pollens have been shown to possess NOX (NADPH oxidase) activity in their outer membrane, which gets activated upon coming in contact with the humid environment in the air passage and produce ROS that facilitate airway inflammation. In response to allergens and other inducers, the immune cells such as lung macrophages also produce excessive ROS. Various studies have shown that inflammatory cells-induced oxidative stress leads to overproduction of free radicals, which contributes to tissue damage (Fujisawa, 2005). Additionally, the structural lung cells including epithelial cells also produce ROS (Rahman et al., 2006). Various catalytic proteins such as NADPH oxidase (NOX2, and its isoforms), inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO), mitochondrial oxidases, and eosinophil peroxidase (EPO) also produce ROS. Most of the superoxide (Od2 2 ) is produced through NOX2 as microbiocidal agent during infection, e.g., leukocytes produce ROS, which they use for destroying the invading pathogens. Macrophages, monocytes, neutrophils and eosinophils express high levels of NOX2. In addition, different homologs of NOX2 such as NOX1, NOX3, NOX4, NOX5, DUOX1 and DUOX2 have been identified in nonphagocytic cells including fibroblasts and endothelial cells (ECs) (Bedard and Krause, 2007). Most of the Od2 produced by these cells are processed by nonenzymatic or SOD-catalyzed 2

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FIGURE 27.1 Effect of oxidative stress on lung tissue leading to Asthma. Detailed explanation has been given in the text part.

reactions and results in its dismutation into hydrogen peroxide (H2O2). H2O2 can also be produced by DUOX enzymes in the respiratory tract from epithelial cells (Nadeem et al., 2014; Fischer, 2009). Increased expression of oxidative stress markers such as H2O2, nitric oxide, 8-isoprostane and carbon monoxide are found in exhaled breath of asthmatic patients (Rahman et al., 2006). Enhanced level of malondialdehyde (MDA) and protein carbonyls, decreased protein sulfhydryl and antioxidant activity were detected in plasma, bronchoalveolar lavage (BAL) fluid and respired air of asthmatic patients (Ozaras et al., 2000; Pobed’onna, 2005). This evidence indicates that oxidative stress plays a key role in lung inflammation and asthma; however, it is not clear whether it is the only factor in the development of the disease.

27.2.2. Cancer Warburg had suggested an association between oxidative stress and cancer in 1920s (Warburg, 1956); however, the real impetus for this link was provided in 1968 by McCord and Fridovich, who discovered superoxide dismutase (SOD) (McCord and Fridovich, 1988) that is well established to quench superoxide. Subsequently various studies have established the causative link between oxidative stress and cancer (Klaunig et al., 2010; Weinberg and Chandel, 2009). The accepted paradigm is that ROS and oxidative stress cause cancer by (1) DNA damage and epigenetic alterations, (2) low expression of antioxidants of the SOD family such as MnSOD (Church et al., 1993; Szatrowski and Nathan, 1991), (3) increase of the genomic instability and activate transcription factors such as NF-κB, and (4) increase in inflammation that presumably occurs via activation of inflammatory signaling cascades. Cancer cells with active oncogenes and loss of tumor suppressors produce higher levels of ROS compared to normal cells, and are more susceptible to mitochondrial dysfunction resulting in a higher metabolic rate of cancer cells (Acuna et al., 2012; Cairns et al., 2011). Increased level of ROS in the tumor cells can increase the genomic instability and activate transcription factors such as NF-κB, which causes tumor heterogeneity (Kohen and Nyska, 2002; Klaunig et al., 2010). ROS-induced genomic instability can be caused due to DNA damage by various mechanisms including base modifications, deletions, chromosomal rearrangements, strand breakage, and hyper- and hypo-methylation of DNA (Valko et al., 2004).

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The microenvironment of a tumor, which includes tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs) and the tumor itself produce ROS, reactive nitrogen species (RNS) and various inflammatory mediators that produce a chronic oxidative stress condition, which in turn drives cell proliferation and tumor development thus perpetuating a vicious cycle (Solinas et al., 2009; Khaled et al., 2013). Therefore, oxidative stress, inflammation and development of various cancers, as discussed below, are positively correlated and fuel each other (Fig. 27.2). In breast cancer, the breast epithelium damage is caused by ROS that activate fibroblast proliferation and conversion into myofibroblasts of mesenchymal nature, which secrete various inflammatory cytokines, growth factors, and metalloproteinase. Hyperplasia of epithelium leads to cellular atypia and finally breast cancer development. The myofibroblasts also generate various peroxides which further activate other epithelial cells to become cancerous. In most of the breast cancers, oxidative stress is induced by overexpression of the enzyme thymidine phosphorylase, which catabolizes thymidine into 2-deoxy-D-ribose-1-phosphate and thymine. The former one is a powerful reducing sugar which quickly glycate proteins, generating oxygen free radicals in the mammary epithelial cells (Brown et al., 2000). Phaniendra et al. (2015) recently reported that lactoperoxidase enzyme present in mammary gland catalyzes oxidation of 17-β-oestradiol to a reactive phenoxyl radical leading to neoplasia (Phaniendra et al., 2015). Further, it has been found that NOX4 overexpression in normal breast epithelial cells leads to cellular senescence, resistance to apoptosis, and oncogenic transformation. It also increases aggressiveness of breast cancer cells (Jezierska-Drutel et al., 2013; Graham et al., 2010). Increased oxidative stress is known to activate matrix metalloproteinases (MMPs) such as, MMP-2 and MMP-9 whose increased expression enhances extracellular matrix remodeling (Jezierska-Drutel et al., 2013). In bladder cancer the crucial oxidant factors are derived from cigarette smoking, exposure to industrial carcinogens (aromatic amines), arsenic intake and diet. Increased nitric (NO) level has also been observed in patients with bladder cancer (Eijan et al., 2002). NO stimulates MMPs, especially their protease activities that is important in the last step of collagen degradation. Significantly increased levels of protein carbonyl groups and lower levels of plasma protein, total thiol groups and protein bound thiol groups were detected in patients with bladder cancer as compared to healthy persons (Yilmaz et al., 2003). ROS generated due to these factors mobilize pro-form of

FIGURE 27.2 Pathophysiology of oxidative stress-induced cancer. Increased oxidative stress progressively causes conversion of normal cells to cancerous cells either through genetic modification or by activating inflammatory pathway.

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heparin-binding epidermal growth factor (HB-EGF) which act in an autocrine manner and activate EGFR/Akt1 pathway that add in bladder carcinogenesis (Afanas’ev, 2011). In colorectal cancer the epithelial cells that line the bowel are the site of cancer origin. The colon epithelium comes in contact with food-derived carcinogens and gut bacteria-derived oxidants, which cause redox imbalance, altered intestinal metabolic homeostasis and DNA damage leading to colon cancer development (Guz et al., 2008). Human colorectal cancers have been detected with increased NO (Haklar et al., 2001), lipid peroxides (Rainis et al., 2007) and the oxidatively modified nucleoside, 8-oxodG (Guz et al., 2008). Further, increased levels of oxidized low density lipoprotein in serum of patients with colorectal cancer have also been observed (Suzuki et al., 2004). The patients suffering from chronic inflammatory disease such as ulcerative colitis and Crohn’s disease have an increased risk of colorectal carcinogenesis. During chronic inflammation increased production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 are released which activate redox-sensitive pathways such as NF-κB and STAT-1. Activation of these pathways accelerates the tumor formation and progression by initiating proliferation and antiapoptosis property of epithelial cells (Viennois et al., 2013). Lung cancer, as studies suggest, is due to smoking with an almost 80% burden in males and 50% in females (Jemal et al., 2011). Cigarette smoke contains a complex mixture of various carcinogens and stable ROS that have very long half-lives. This leads to higher frequency of mutations in lung epithelial cells which accumulate over time. Further, ROS damage the lung epithelium resulting in transformation of airway epithelial cells into a malignant form (Hoagland et al., 2007). Tobacco smoke is reported to increase the expression of cytokines, such as IL-8 and TNF-α, in in vitro as well as in murine models in vivo (Mercer et al., 2006). These cytokines and chemokines attract neutrophils and resident macrophages, which secrete matrix metalloproteases and serine proteases that destroy extracellular matrix and create space for neoplastic cells growth (Goldkorn et al., 2014). Further, ROS activated NF-κB signaling promotes expression of various antiapoptotic proteins such as Bcl-XL, IAP-1 and IAP-2; mitogenic proteins such as myc, and cyclin D1; and inflammatory enzymes such as cyclooxygenase-2 (COX-2). These factors together promote cancer by increasing proliferation, loss of contact inhibition via reduction in cell contact proteins such as E-cadherin, and increased anchorage-independence of the epithelial cells leading to cancer development (Weinberg, 2006). In prostate cancer, overexpression of NOX1 has been identified as an early event (Lim et al., 2005). The NOX produced superoxides induce cancer-promoting effect through resistance to programmed cell death through p90 ribosomal S6 kinase (p90 RSK) and other redox-regulated transcription factors like NF-κB and AP-1 (Brar et al., 2003). An increased level of p66Shc has also been implicated in elevated levels of ROS in prostate cancer cells (Veeramani et al., 2008). Dihydrotestosterone (DHT) when bound with androgen receptor (AR), increases p66Shc protein expression and translocate it into mitochondria, where it oxidizes cytochrome c and activates superoxide production. Elevated ROS cause inactivation of phosphatases which results in activation of tyrosine kinases and finally endorses cell cycle progression through Cyclin D1 (Veeramani et al., 2008).

27.2.3. Cardiovascular Diseases CVD in the form of atherosclerosis, myocardial infarction (MI), hypertension and associated vascular diseases such as aneurysms (an abnormal bulge or ballooning in the vessel wall) and restenosis (blockage of stents inserted in patients with CVD to keep arteries open) have been attributed to oxidative stress and the subsequent inflammation induced thickening of the vascular wall or plaque formation or thrombosis. Outlined below are some of the signaling pathways that initiate CVD. Normally, low levels of ROS act as signaling molecules and regulate normal vascular functions such as growth of vascular smooth muscle cells (SMCs), and their contraction and relaxation. The different layers of vasculature such as endothelium, adventitia and smooth muscles produce ROS including superoxide, radicals, hydroxyl radicals, nitric oxide, peroxynitrite and hydrogen peroxide. The cardiomyocytes, when exposed to H2O2, expressed decreased levels of alpha-actin, troponin1, myosin light chain 2 and creatinine kinase resulting in their dysfunction (Torti et al., 1998). Similarly, vasculature is also impacted by circulating and localized ROS such as superoxide anions, oxidized low density lipoprotein (Ox-LDL), lipid aldehydes leading to complications such as hypertension, atherosclerosis and MI (Fig. 27.3). Atherosclerosis is characterized by blockage and hardening of the arteries. Increased level of oxidized low density lipoproteins (ox-LDL), free fatty acids and glucose are the causative factors that lead to atherosclerosis. ROS have been shown to play important role in endothelial dysfunction, and growth of atherosclerosis lesion (Papaharalambus

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FIGURE 27.3 Pathophysiology of oxidative stress-induced cardiovascular diseases. Oxidative stress leads to development of atherosclerosis, myocardial infarction and hypertension.

and Griendling, 2007). Macrophages, ECs and SMCs may be different sources of free radicals in the vessel wall. Cigarette smoke consists of a large amount of free radicals that can downregulate various antioxidants such as carotenes, Vitamin D, GPx and SOD in the vasculature, which can ultimately lead to the dysfunction of vascular cells (Barnoya and Glantz, 2005). Low bioavailability of NO is one of the important features in CVDs (Lakshmi et al., 2009). The production of NO, which regulates vasodilation, elasticity of vascular wall, vaso-reactivity and platelet aggregation, maintains a fine equilibrium between the growth of SMCs and their differentiation. A low accessibility of tetrahydrobiopterin (BH4) or L-Arg leads to uncoupling of eNOS from NOd, which then interacts with Od2 2 (Yang and Ming, 2006) and produces ONOO2. The latter is a cytotoxic free radical and disturbs cardiovascular function. Since most of NOd gets used up in ONOO2 formation, it results in a low level of NOd which leads to increased vascular dysfunction and reduced endothelial vascular regulatory capacity. Oxidative stress-mediated differentiation of foam cells also contribute to the development of atherosclerotic plaques. Theox-LDL plays important role in activating the ECs and attracting the circulating monocytes which get inflamed and accumulate increased amount of modified lipids and secrete excessive inflammatory cytokines, chemokines and growth factors. These factors, on the one hand further aggravate the oxidative stress and attract more inflamed cells to the site, and on the other hand cause growth of smooth muscle cells that contribute in the atherosclerosis plaque growth. Thus, ROS is at the core of the atherosclerosis pathogenesis and are also involved in the rupture of plaques leading to MI and heart failure. Hypertension, a risk factor of heart disease, stroke, kidney failure, and premature mortality also involves oxidative stress. Since increased ROS decreases NO level as described earlier, the unavailability of sufficient NO further decreases endothelium-dependent vasodilation and thus result in hypertension (Lakshmi et al., 2009; Ceconi et al., 2003). On the other hand, increased ROS leads to oxidative modification of many biomolecules such as LDL which further precipitate hypertension. Further, renin-angiotensin system, a vasoconstrictive and hypertensive peptide hormone system, is a key regulator of oxidative stress-induced vessel wall inflammation (Dzau and Lopez-Ilasaca, 2005). This system reveals its effect through two receptors, angiotensin II type-I receptor (AT1) and angiotensin II type-2 receptor (AT2). AT2 is expressed at very low level in adults and its higher expression is found in pathological conditions such as vascular injury, heart failure and cardiac hypertrophy (Ceriello, 2008). Angiotensin II induces cytokines and adhesion molecules in leukocytes that cause inflammation. Inflammatory cells themselves produce angiotensin II, which results in increased inflammation and affects

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endothelial functioning. Oxidative stress-induced lesser amount of NO also affects the angiotensin II activity. As NO is a vasodilator and angiotensin II is a vasoconstrictor, so a balance between these should be maintained for proper vascular tone. Oxidative stress decreases bioavailability of NO which alters this balance and increases vasoconstriction resulting in hypertension (Dzau, 2001). The role of elevated and oxidatively damaged molecules and tissues is evident in MI or heart failure. Oxidative stress actually contributes to the biochemical alterations that result in MI. Further, the level of 8-isoprostane in pericardial fluid and malondialdehyde (MDA), which is a lipid peroxidation-derived aldehyde, increases in patients with MI (Sorescu and Griendling, 2002) which points to a role for role of ROS in driving MI. The ROS is mostly produced by mitochondria of the stressed cardiomyocytes (Terman et al., 2004), which could decrease the bioavailability of NO affecting the diastolic function of heart (Lakshmi et al., 2009; Ferdinandy et al., 2000). Along with this, increased peroxinitrite formed by the reaction of superoxide with NO, can cause cytokineenabled myocardial contractile failure by inactivating Ca21-ATPase of sarcoplasm and thereby deregulating Ca12 homeostasis. Evidence also suggests the role of oxidases such as xanthine oxidase, NOX in ROS generation and heart failure (Saavedra et al., 2002; De Biase et al., 2003; Lakshmi et al., 2009).

27.2.4. Diabetes Mellitus (DM) Oxidative stress induces various complications in DM such as coronary artery disease, retinopathy, neuropathy, nephropathy, and stroke. DM patients have increased levels of glucose along with dyslipidemia that develop macroangiopathy, which further cause oxidative stress and lead to atherosclerosis (Asmat et al., 2015). Various studies have confirmed the role of oxidative stress in vascular diabetic complications in both type-1 and type-2 diabetes (Costacou et al., 2013; Broedbaek et al., 2013; Ceriello et al., 2016). Hyperglycemia (HG) during diabetes results in increased production of superoxides. Studies show that HG activates protein kinase-C (PKC)-mediated activation of NOX enzymes, which produce excessive ROS in tissues including vessels (Cosentino-Gomes et al., 2012). HG-induced free radicals activate redox pathways which lead to endothelial dysfunction (Ceriello et al., 2016). Hyperglycemic condition selectively increases iNOS expression followed by an increase in NO. In hyperglycemic conditions, ECs uptake excessive glucose through GLUT1 transporters. This amplified supply of glucose results in hyperactive mitochondria, which generates excessive energy and increased quantities of superoxide radical (Od2 2 ). These ROS at different concentrations display different molecular pathological state. Simultaneous increases in NO and Od2 2 produce peroxynitrite free radicals, which inactivate NO and cause endothelial dysfunction. Peroxynitrite species, being potent oxidants, have toxic effects on vasculature, especially endothelial lining of the vasculature. Therefore, they may contribute to myocardial damage in diabetic persons (Ceriello et al., 2002). In diabetes condition AGEs (advanced glycation end products)/RAGEs (AGE receptors), PKC and angiotensin II (AT-II) activate NOX enzymes leading to reactive species production that may cause cardiovascular complications as well as cardiac dysfunction accompanied by inflammation, fibrosis and apoptosis (Teshima et al., 2014). Several studies have shown that HG, particularly acute HG, results in increased ROS, inflammation and subsequently in endothelial dysfunction (Ceriello et al., 2013) (Fig. 27.4). During diabetes the free radical production occurs via four different methods as mentioned below: 1. Increased glycolysis causes increased oxidation ratio of glyceraldehyde 3-phosphate (G3-P) to 1,3-bisphosphoglycerate (1,3-DPG), followed by enhanced NADH/NAD1 ratio, i.e., disturbed redox balance 2. Activated sorbitol pathway during HG causes the increased sorbitol and fructose accumulation that results in decreased levels of GSH and augmentation of NADH/NAD1 ratio. 3. Autoxidation of glucose lead to the production of various free radicals including OHd, H2O2, Od2 2 and aldehydes. 4. Nonenzymatic glycation of proteins result in the generation of AGEs which generate oxidative stress upon interacting with RAGEs (Ahmed, 2005). These ROS lead to increased insulin resistance, damage to enzymes system and cellular components by creating oxidative stress (Maritim et al., 2003). Not only lipid but LDL is also responsible for generation of insoluble aggregates which causes oxidative stress-induced damages in diabetics. Apolipoprotein component of LDL such as apo-B monomers get crosslinked by hydroxyl radicals (Pham-Huy et al., 2008), which results in modified lipids that cause other diabetic complications.

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FIGURE 27.4 Routes of ROS and Pathological role of ROS in oxidative stress-induced diabetes. Exogenous and endogenous sources function for formation of free radical species impacts glycolysis, sorbitol pathway activation, autoxidation of glucose and protein glycation reactions. Dysfunction of these processes are directly related to insulin resistance and hence diabetes (A). The endothelial dysfunction is one of the major events in hyperglycemia as well as CVDs. The increased iNOS, advanced glycation end product (AGE), superoxides and peroxynitrites, hyperactive mitochondria assist in endothelial dysfunction and hence CVDs (B).

27.2.5. Neurodegenerative Disease The central nervous system is highly susceptible to oxidative stress caused due to high level of lipids, low levels of antioxidant enzymes and high oxygen consumption (Pollack and Leeuwenburgh, 1999). Some of the brain regions are more susceptible to oxidative stress, such as the hippocampus, striatum and the substantia nigra, than others. This phenomenon is known as selective neuronal vulnerability and it differentiates the identity of each neuronal disease, but each type of cells which are involved in the pathology of neurodegenerative disease may have enhanced vulnerability to oxidative stress (Niedzielska et al., 2015). Oxidative stress-induced peroxidation of lipids results in detrimental effects including decreased membrane potential, loss of membrane fluidity and greater permeability to ions such as Ca12 (Rivas-Arancibia et al., 2010; Uttara et al., 2009). Age factor is widely accepted in neurodegenerative diseases because as age increases the capacity of cells to maintain redox balance decreases, and as a result free radicals accumulate that cause mitochondrial dysfunction and neuronal damage (Niedzielska et al., 2015). Alzheimer’s disease (AD), a progressive neurodegenerative disease, generally starts with short-term memory loss and progresses towards severity. Symptoms of AD include difficulty in language, disorientation, mood swings, loss of self-care, and behavioural problems (Burns and Iliffe, 2009; WHO, 2016). Progressive damage of neurons and synapses in hippocampus, neocortex and other subcortical regions in brain are pathological features of AD (Niedzielska et al., 2015). This progressive loss of neurons is associated with protein aggregation, which are extracellular as Aβ-amyloid plaques, and intracellular as neurofibrillary tangles (Gandhi and Abramov, 2012). Increased levels of H2O2 and OHd radical are reported in AD. A high rate of lipid peroxidation causes death of neurons through various mechanisms, including impairment in functioning of glucose transporters, glutamate transporters and two ion pumps, i.e., Na1/K1-ATPase and Ca12-ATPase (Uttara et al., 2009). Oxidative damage through DNA oxidation is also exhibited in AD. Increased levels of 8-hydroxydeoxyguanosine are found in mitochondria and nucleus, which is a DNA oxidation product and causes oxidative damage. Likewise elevated levels of oxidized and glycated proteins are also found in AD patients. Proteins modified by oxidative stress have a tendency to aggregate such as Aβ-amyloid plaques and neurofibrillary tangles as observed in AD patients. Oxidized and modified proteins may act as inhibitor of proteasomal activity, which may result in accumulation of

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FIGURE 27.5 Pathology of oxidative stress-induced neurodegenerative diseases. Redox balance is required to maintain homeostasis of neuronal cells. Accumulation of free radical responsible for lipid peroxidation, DNA and protein oxidation causes oxidative stress-induced pathophysiological conditions called Alzhiemer’s disease. Hydroxyl radical generation, dysfunction of glucose transporters, altered mitochondrial function in neuronal cells leads to vulnerable neuronal cells due to oxidative stress; all are results in oxidative stress-induced Parkinson’s disease.

abnormal proteins. The aggregated proteins give positive feedback to decrease proteasomal activity, stimulate ROS generation and causes neural cytotoxicity in AD (Fig. 27.5). Parkinson’s disease (PD) is characterized by jerky movements and trembling of peripheral organs, especially the lips and hands. It is the second-most common neurodegenerative disease after AD. The oxidative stress increases with aging and it makes neuronal system more susceptible to neurodegenerative disease like PD. Pathologically PD involves progressive damage of dopaminergic neurons in substantia nigra region, and aggregation of the α-synuclein in intracellular inclusion bodies along with synphilin-1 and other compounds (Chen et al., 2012). PD is associated to increased levels of ROS, altered catecholamine metabolism, changes in electron transporter chain functioning and increased iron deposition in the substantia nigra pars compacta (Blesa et al., 2015). Impaired mitochondrial function, such as defective complex I activity, is found in the brain of PD, which leads to deposition of α-synuclein. Dopamine reduces the oxidation state of metals such as Cu12 and Fe13, which induces the production of H2O2. Oxidative stress also stimulates defective activity of ubiquitin-proteasome pathway that hampers the degradation of defective and aggregated α-synuclein causing their accumulation (Gandhi and Abramov, 2012; Chen et al., 2012). Hydroxyl radicals damage dopaminergic neurons, which are involved in memory, learning and motor processes (Eijan et al., 2002). Multiple sclerosis (MS) is an autoimmune disease of neurons in which demyelination of nerves results in impaired nerve conduction. ROS, including superoxide ions, peroxinitrite, hydrogen peroxide and nitric oxide, are produced as inflammatory response in MS (Witherick et al., 2010). ROS produced by activated macrophages induce lipid peroxidation that finally destroy neurons by demyelination. ROS-mediated dephosphorylation makes the axons more vulnerable to proteolytic degradation and axon degeneration. It is reported that inflammatory and oxidative agents such as NO dephosphorylate neurofilament and cause axon destruction (Wilkins and Compston, 2005). NO and its derivatives can block signal conduction in axons and demyelinated axons are susceptible to NO-mediated conduction block (Witherick et al., 2010). Increased TBARS and decreased SOD are reported in MS condition which makes the tissue more vulnerable towards oxidative stress-induced damages (Mitosek-Szewczyk et al., 2010; Eijan et al., 2002) (Fig. 27.6).

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FIGURE 27.6 Pathology of oxidative stress-induced multiple sclerosis.

27.3. TRADITIONAL AND NOVEL THERAPEUTIC TARGETS From the preceding section it appears that restoring the body’s redox balance could be an important strategy in combating the progression of oxidative stress-induced diseases, additionally blocking the activation of oxidative and inflammatory pathways (such as NF-κB and AP-1 that produce more proinflammatory cytokines and chemokines and further contribute to disease severity (Palanki, 2002)). However, since these redox-sensitive inflammation signaling pathways are also involved in immune response, resolution of inflammation, wound healing, and other important homeostasis processes, their inhibition has not been successful in bringing out beneficial clinical outcomes. Therefore, it is more effective to target the initiator of oxidative stress than these effectors. The preventive strategy in order to maintain the redox state may include lowering or avoiding the exposure to environmental pollutants and other chemicals with oxidizing properties, maintaining healthy BMI by physical activity and exercise, increasing intake of fruits and vegetables and reducing consumption of high fat and high carbohydrate food (Poljsak, 2011). Therapeutic potential to reduce oxidative stress could be achieved either by increasing levels of endogenous and exogenous antioxidants or by targeting the complexes that trigger ROS production such as NADPH oxidase, mitochondrial complexes, xanthine quinone reductase, etc. Further, targeting the inflammatory signaling pathways, which contribute towards oxidative and inflammatory conditions in microenvironment of the tissues and help in progression of disease, could be an important therapeutic approach.

27.3.1. Therapeutic Targets Which Can Increase the Endogenous Levels of Antioxidant 27.3.1.1. Superoxide Dismutase SOD exists in three different isoforms: SOD1, SOD2, and SOD3. SOD1 is distributed throughout the cell cytoplasm, nucleus and in the lumen between outer and inner membranes of mitochondria, SOD2 isoforms are located in matrix of mitochondria, while SOD3 is found mostly extracellularly. SOD is an enzymatic antioxidant that catalyzes the conversion of Od2 2 to H2O2 and helps maintain the redox balance by diffusing the superoxide. Therapeutically increasing the levels of SOD could be an important treatment strategy in oxidative stressinduced pathology. However, exogenous SODs administration may be problematic; the drawbacks of SOD therapy are hypersensitivity, low half-life and low uptake. Accordingly, several types of SOD mimetics have been constructed which may increase the level of endogenous SOD and act to catalyze the removal of superoxide species. Several classes of SOD mimetics have been found to be effective in animal models of various diseases. For example, SOD3 has been found to be more effective therapeutic target in emphysema than other isoforms of SOD

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(Sorheim et al., 2010). Studies have shown that administration of a SOD mimetic in SOD3 knockout mice resulted in a significant decrease in elastase-induced emphysema (Yao et al., 2010). SOD2 was found more physiologically important in CVDs. In a study, SOD2 silencing in apolipoproteindeficient mice resulted in endothelial dysfunction in carotid artery (Ohashi et al., 2006). Although SOD mimetics have displayed efficacy in diabetes experimental models, its role in improving vascular complication remains controversial in patients (Rochette et al., 2014). The reason behind this could be that SOD produces excessive hydrogen peroxide, which is known to cause irreversible endothelial damage. In such cases, a combination of SOD mimetics along with catalase mimetics could be helpful in patients with vascular complications. In diabetes, SOD mimetics have been developed as therapeutic alternatives. Metalloporphyrin-based SOD mimetics were found to be effective in T-cell mediated autoimmune diabetes (Piganelli et al., 2002). These findings suggest that elevating the endogenous levels of SOD can be effective therapeutic strategy in oxidative stress-induced pathology. SOD mimetics have also been tested along with radiotherapy in the cancer treatment. SOD could inhibit superoxide-induced DNA strand breaks, which suggests its importance in cancer therapy. SOD has also been used to ameliorate the ill effects of cancer radiotherapy. SOD also acts as anti-inflammatory due to its inhibitory effects on the release of lipid peroxidation-derived prostaglandins, thromboxane and leukotrienes, making it a potent traditional therapeutic target for the treatment of oxidative stress-induced pathologies. 27.3.1.2. Glutathione Peroxidase and Catalase GPx and catalase family of enzymes are involved in the termination reaction of ROS pathway. Different isoforms of GPx (1 through 8) are known to date, out of which GPx-1 is a widely available isoform present in cytoplasm of all mammalian cells (Vlahos and Bozinovski, 2013). Like SOD, increasing the endogenous levels of GPx could also be an important strategy to resolve oxidative stress-induced pathologies. Catalase or GPx mimetics can be used to treat many diseases such as diabetes, COPD and CVDs. Ebselen is one of the best known GPx mimetic and its therapeutic potential has been studied in various experimental models of diabetes (de Haan and Cooper, 2011). These studies have shown that Ebselen has the capacity to reduce diabetes associated atherosclerosis in apolipoprotein E/GPx-1 double knockdown mice (Chew et al., 2010). In COPD, GPx-1 knockout mice showed increased neutrophils and macrophages in BALF, elevated IL-17A and proteolytic burden in lungs as compared to wild-type mice (Duong et al., 2010). Supplementation of GPx mimetics, Ebselen reversed the inflammatory profile of COPD. GPx mimetics have also shown protective effects in atherosclerosis and cerebral ischemia reperfusion injury during diabetes (Wong et al., 2008). Thus, GPx could be an effective traditional therapeutic target in inflammation-induced diseases. 27.3.1.3. NRF2/ARE Pathway NRF2 is a leucine-zipper transcription factor. In the normal state, NRF2 remains bound to its inhibitory protein Keap1. As oxidative stress increases, the cysteine group of keap1 becomes oxidized, allowing NRF2 to become disassociated and enter inside the nucleus where it binds to antioxidant response element (ARE) on DNA. NRF2ARE interaction is known to regulate transcription of more than 200 antioxidant and anti-inflammatory genes such as glutathione, SOD, NOX and quinone reductase (Nguyen et al., 2009). Therefore, NRF2 could be an important therapeutic target in attaining redox homeostasis. In neurodegenerative diseases, due to the blood brain barrier it is difficult to increase the levels of exogenous antioxidants, and therefore elevating endogenous antioxidants could be a better strategy. Studies on PD experimental models have indicated that increasing expression of endogenous NRF2 has more neuroprotective effects as compared to conventional antioxidant therapy (Zhang et al., 2014). In vivo studies on genetically modified NRF22/2 mice has shown that astroglial and neuronal cultures from these mice are more susceptible to oxidative stress (Lee et al., 2003). Further, in mixed cultures of rat neuronal and glial cortical cells NRF2 overexpression has been shown to increase antioxidant capacity in both neuronal and astroglial cells and protected cortical neurons from excitotoxicity (Shih et al., 2003). NRF2 could also be an important therapeutic target in the management of lung inflammatory diseases. In COPD patients, decreased levels of NRF2 protein in the lungs have been observed (Nguyen et al., 2009). Studies have demonstrated that inactivation of NRF2 genes in mice model of asthma leads to severe allergen-associated airway inflammation and hyper-responsiveness (Rangasamy et al., 2004). Harvey et al. (2011) showed that NRF2 strengthen alveolar macrophages to clear bacteria easily, which is important in prevention and treatment of COPD (Harvey et al., 2011). In another study cigarette smoke-induced emphysema was attenuated by NRF2 activators such as CDDO-imidazolide (Sussan et al., 2009).

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A protective role of NRF2 has also been found in patients suffering from diabetes mellitus. Streptozotocininduced diabetes in NRF22/2 mice caused increased oxidative and nitrosative stress as well as elevated blood glucose level. Administration of CDDO (an NRF2 inducer) in db/db mice showed an antidiabetic effect (Uruno et al., 2013). These studies suggest that NRF2 can be an effective therapeutic target in diabetes mellitus patients as well. In cancer cells, a dual role of NRF2 has been discovered. On the one hand, NRF2 provides chemoprevention to normal cells and prevent their conversion to malignancy, and in another study NRF22/2 mice displayed increased sensitivity towards toxicants and carcinogens (Menegon et al., 2016). On the other hand, NRF2 not only protects normal cells but it also provides selective growth advantage and chemotherapy resistance to cancer cells (No et al., 2014). Due to this duality in the effects of NRF2, its usefulness as therapeutic target in cancer cells is still questionable.

27.3.2. Strategy to Decrease Oxidative Stress by Targeting the Sources of ROS Production 27.3.2.1. NADPH Oxidase NOXs are a family of enzymes that generate ROS in many cell types. NOX-1 and NOX-2 are primary generators of Od2 and are a major source of ROS during hypertension, diabetes, hypercholesterolemia and COPD 2 (Drummond et al., 2011). In an excessive oxidative stress condition inhibition of NOX could decrease production of singlet oxygen species resulting in decreased H2O2 and ONOO2 generation, and subsequent reduction of OHd generation and increased NO bioavailability. Apocyanin, the most common NOX inhibitor, preferentially blocks NOX-2 at lower doses. When administered to mice with cigarette smoke-induced lung inflammation apocyanin decreased airway inflammation (Bernardo et al., 2015). NOX inhibition has also been found useful in experimental models of vascular diseases, cerebral ischemia and diabetes-related complications (Drummond et al., 2011). Thus, targeting the NOXs may ameliorate excessive oxidative stress-induced pathologies. 27.3.2.2. P66shc Mitochondria are an important source of ROS production in many tissues including heart and neurons and are implicated in cardiovascular and neurodegenerative diseases. Studies have revealed that p66shc, a 66 kDa Src homologous-collagen homologue (Shc) proto-oncogene, is involved in redox balance of mitochondria (De Marchi et al., 2013), thus acting as an important redox enzyme and ROS (H2O2) producer within mitochondria. Increased levels of p66shc result in the inhibition of FOXO transcription factor that decreases ROS scavenging enzymes synthesis (Nemoto and Finkel, 2002). In p66shc2/2 diabetic mice enhanced antioxidant defense and lower ROS generation was observed (Camici et al., 2007). Moreover, p66shc has also been implicated in CVDs including endothelial and vascular dysfunction, plaque formation, myocardial remodeling, atherosclerosis and ischemia (De Marchi et al., 2013). This evidence indicates that mitochondrial p66shc could be an effective novel therapeutic target in oxidative stress-induced pathology.

27.4. MODERN APPROACHES TO UNDERSTAND OXIDATIVE STRESS-INDUCED PATHOLOGIES Research in oxidative stress-induced pathologies is continuously evolving, and in the past decade or so a number of new techniques have emerged which have helped tremendously in enhancing the understanding of oxidative pathogenesis. In order to study the role of oxidative stress in different inflammatory pathologies, the following modern tools and techniques can be utilized.

27.4.1. RNA Interference Approach RNA interference (RNAi) is posttranscriptional silencing of genes in the cells by either endogenously produced or artificially introduced small interfering double-stranded RNA, which possess sequence complementarity with target genes (Bosher and Labouesse, 2000). In this approach gene transcription is usual; however, its translation into protein is blocked because mRNA is selectively degraded. RNAi is an effective tool to study functions of individual proteins separately or a set of proteins (Milhavet et al., 2003). RNAi provides for a novel gene knockdown approach that allows targeted and precise suppression of genes. Through RNAi-mediated specific

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knockdown of a protein involved in oxidative stress, a specific function of a given protein can be analyzed. Accordingly, in the oxidative stress-driven pathologies such as CVDs, diabetes, cancers and neurodegenerative diseases, specific knockdown of genes involved in oxidative stress may provide insight on the role of those genes in causing disease severity. For example, specific knockdown of genes such as NOX, catalase, SOD, Nrf2/keap-1, GPx, p66shc and NOS has established their role in the development and severity of several diseases. This method has thus identified and ascertained these enzymes as effective therapeutic targets. Further, research on RNAi therapy in which the key mediating gene is transiently silenced by small interfering RNA could establish siRNA as interventional drug for clinical use in various types of diseases (Milhavet et al., 2003). Thus, RNAi may become an effective tool in establishing function of genes involved in oxidative stress-mediated diseases as well as in their treatment.

27.4.2. Stem Cell Approach Stem cells are specialized and naı¨ve cells that possess self-renewing and differentiation capacity into multiple cell lineages (Weissman, 2000). Stem cells can be categorized into two different categories, embryonic stem cells and adult stem cells. Embryonic stem cells have the ability to generate any type of terminally differentiated cells in the body, whereas adult stem cells can produce tissue-specific differentiated cells (Blau et al., 2001). Stem cells have increased resistance to different types of stress that protects them from apoptosis. Stem cells express a set of genes including SOD, catalase and GPx that protect them against oxidative stress (Sattler et al., 1999). It has been reported that ROS plays an important role in deciding the balance between self-renewal and differentiation of stem cells. In neural progenitor cells factors causing self-renewal are expressed more in reduced redox state, and exposure to oxidative molecules promote differentiation in the progenitor cells. Oxidative stress has been reported to shorten the average life span of stem cells (Noble et al., 2003). CVDs are characterized by endothelial dysfunction and apoptosis. Endothelial progenitor cells (EPCs) play an important role in neovascularization and regeneration of ECs (Tousoulis et al., 2008). Evidence suggests that during vascular injury, EPCs derived from bone marrow are routed into the peripheral blood where they differentiate into mature ECs (Urbich and Dimmeler, 2004). However, in the presence of excessive oxidative stress and inflammation, mobilization of EPCs gets hampered leading to complications such as atherosclerosis. Maintaining intact and functional endothelium is an important target in the prevention and treatment of atherosclerosis. To achieve this, the use of drugs that could decrease oxidative stress may be helpful in improving the redox state that may enhance the quantity and quality of EPCs. Further, ex vivo expansion of EPCs could be a promising therapeutic strategy. Excessive oxidative stress alters EPCs function, which indicates that antioxidant therapeutic strategy may be helpful in the management of various CVDs (Case et al., 2008). In alloxan-induced diabetic rats increased triglyceride, serum glucose and oxidative stress in pancreatic tissue was ameliorated by treatment with mesenchymal stem cells, which prevented these alterations and attenuated oxidative stress. This evidence suggests that mesenchymal stem cells could convert into functional insulin cells and reduced HG and oxidative stress (El-Tantawy and Haleem, 2014). However, further studies on the effect of oxidative stress on stem cells are needed to establish stem cells as a modern therapeutic approach for oxidative stress-induced pathologies.

27.4.3. Computational and In Silico Approach Computational biology through pragmatic remodeling and theoretical exploration has helped in addressing critical scientific questions (Kitano, 2002). Such in silico studies are recently being used widely in studying the complexities in oxidative stress-induced pathology, e.g., the role of oxidative stress in the regulation of insulin signaling has been explored through computational modeling. Construction of computational model consists of constructing a topological model by integrating pooled genes and their interactions based on comprehensive data available from pubmed database. This assists in obtaining a global picture of the various genes, proteins and their interaction in the development of particular disease. The computational data obtained from such projections can be experimentally validated by evaluating protein or gene interactions by wet lab techniques such as immunoprecipitation, yeast 2 hybrid system, etc. Using this computational approach link between type-2 diabetes, hypertension, obesity and inflammation has been established (Jesmin et al., 2010). Further, in silico studies can help in understanding drug-target molecule interaction and in designing multiple drug therapies. In a recent in silico study, curcumin was shown to be effective in PD (Jagatha et al., 2008). In this study a dynamic model was prepared where bioenergetics of mitochondria and glutathione metabolism were regulated by oxidative stress

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and curcumin. Curcumin elevated the expression of GSH, which had been depleted during PD, that helped prevent protein oxidation and preserved activity of mitochondrial complex-I (Jagatha et al., 2008). In another study, a mathematical and computational model of oxidative as well as nitrosative stress was prepared in which interac2 tion of reactive species such as NO, O2 2 , and ONOO was found to be an important player in CVDs, hypertension and diabetes. The mathematical modeling thus helps in identifying the intermediate products involved in reactions that cannot be studied through in vitro and in vivo models (Kavdia, 2011). Since the true potential of this approach is yet to be utilized, future in silico studies should be further explored in oxidative stress-induced pathologies for therapeutic drug discovery and designing therapeutic gene circuits (Chan and Loscalzo, 2012).

27.4.4. Phytochemicals and Synthetic Inhibitors Although phytochemicals have been used in the traditional medicine systems, in recent times studies on the role of phytochemicals and synthetic inhibitors in oxidative stress-induced various pathologies has been reinvigorated. Phytochemicals are antioxidant in nature endowed with ability to scavenge intracellular ROS, which help in understanding the role of oxidative stress in complexity of diseases. Many synthetic as well as natural molecules including tocopherol, ascorbate, lipoic acid, carotenoids, flavonoids and polyphenols are being investigated for their antioxidants and free radical scavenging properties (Carocho and Ferreira, 2013). Among the natural inhibitor of ROS, vitamins C and E have potent antioxidant properties. Ascorbic acid is a reducing agent and neutralizes H2O2, therefore decreases the oxidative stress. Vitamin E, a lipid soluble antioxidant, prevents lipid peroxidation by scavenging the lipid free radicals and protects the membrane (Lobo et al., 2010). These compounds can be used to understand as well as ameliorate oxidative stress-induced diseases. Among synthetic inhibitors N-acetyl cysteine (NAC) is widely used in the research to study oxidative stress-induced pathogenesis. NAC is an aminothiol and synthetic precursor of intracellular antioxidants cysteine and GSH (Sun, 2010). Edaravone is also a potent synthetic phenolic compound that can also be utilized as an inhibitor to prevent lipid peroxidation. Edaravone has been studied in streptozotocin-induced diabetes in mice where it has been shown to decrease lipid peroxidation and counteract elevated HG (Fukudome et al., 2008). Hence, natural and synthetic inhibitors and quenchers of ROS are being used in recent studies to develop potential approaches for the prevention and therapy of oxidative stress-induced pathologies.

27.4.5. Advantages and Limitations of These Approaches RNAi method has proven to be a powerful tool for the study of specific gene involvement as well as protein’s function in disease development and thus has opened new area of basic investigation. However, major hurdles remain to be crossed in the progression of RNAi approach is its application in in vivo study models and in clinical studies. Moreover, suppression of gene expression by RNAi is usually a transient phenomenon. Gene expression usually regains after 96120 h or 35 cell divisions after transfection (Mocellin and Provenzano, 2004). Another limitation of RNAi method is that all sequences do not work, and there is usually increased cell death due to toxicity. Further, in some cases even after silencing by siRNA due to the redundancy of genes there is a compensatory effect in which other similar proteins take over the function of silenced protein. Furthermore, knockdown efficiency does not reach 100% and off target effects remain doubtful (Sledz and Williams, 2005). As regenerative medicine, stem cell therapy has provided some benefits in therapeutics of degenerative problems such as neurodegenerative disease, e.g., PD, and other oxidative stress-induced diseases such as diabetes and cancers. Another advantage of stem cell therapy is low risk of rejection due to use of the patient’s own body cells. However, stem cell therapy includes ethical issues since it involves destruction of blastocysts that are formed from laboratory fertilization of human eggs. Since this area is still developing, the long-term consequences and side-effects of stem cell therapy are still unknown. Computational and in silico studies are good tools in finding new antagonists and agonists as drug targets. Further, it helps in understanding underlying biology using networks based on annotated data and also helps in understanding the mechanism of action of drug virtually. However, in silico-based studies have several disadvantages as well. Due to inbuilt flexibility and molecular conformations of various proteins, accurate predictions remain hindered. Further, for validation of computational data in vitro experiments and in vivo testing are compulsory. The fact remains that no computer program, however accurate, will ever fully mimic the complexity of a biological system.

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The use of phytochemicals as a tool to study oxidative stress-induced pathology is due to their antioxidative potential. However, besides acting as ROS scavengers they also act on multiple intracellular signaling pathways. Hence, it is difficult to predict the result that actual inhibition in diseased condition is through ROS-mediated mechanism or through mediation by some other pathway. Therefore, selectivity of inhibition is questioned when phytochemicals are used as inhibitors. Synthetic inhibitor such as NAC is an effective scavenger of total intracellular ROS, hence the study requiring the source of ROS generation cannot be fulfilled using NAC.

27.5. REGULATORY ROLE OF NUTRACEUTICALS AND THE PARADOXES Nutraceuticals, often referred as functional foods or phytochemicals, have selective physiological function and valuable biological activities. The term “nutraceutical” was coined by merging the words “nutrition” and “pharmaceutical” by Stephen DeFelice in 1989 (Kalra, 2003). Nutraceuticals came to be known for reducing the risks of cancer and CVDs and also for prevention or treatment of oxidative pathologies such as hypertension, osteoporosis, diabetes, arthritis (Stamler, 1994; Halliwell, 2013), high cholesterol, excessive weight, macular degeneration (leading to irreversible blindness), cataracts, menopausal symptoms, insomnia, diminished memory and concentration, digestive upsets and constipation (reviewed in Srivastava et al., 2011; Prakash and Gupta, 2009). Although nutraceuticals are known for their traditional nutritional benefits, still there is a lot that needs to be understood about the detailed regulatory role and molecular target of these biologically active compounds (discussed elsewhere in this chapter) (Tsunoda et al., 2012; Iuchi et al., 2007; Wang et al., 2011; Aqil et al., 2013; Biswas, 2016). Nutraceuticals are nutritionally-oriented pharmaceutical molecules, whereas their antioxidant property is itself called the antioxidant paradox. This paradox refers to the observation that, although ROS are pivotal in onset of several diseases, giving large doses of dietary antioxidant supplements to human subjects has not demonstrated any preventative or therapeutic effect. (Allen, 2010; Biswas, 2016; Reuter et al., 2010). Studies showed the lack of effectiveness of ascorbate, vitamin E and carotenoids in cancer and dementia (Halliwell, 2013). Ascorbate sometimes acts as an antioxidant and other times as pro-oxidants in various cancer models (Du et al., 2012). The controversial role of vitamin E as a prophylactic agent in CVD is also reported (Halliwell, 2013). Therefore, more studies need to focus on both the types of reactions involved in oxidative damage, and consequences of nutraceutical treatment. These reactions are: (1) reaction of ROS with proteins, lipid and nucleic acids that causes the pathology, (2) reaction between ROS and individual phytochemical used as a drug to understand the paradoxes of functions of nutraceuticals/phytochemicals (Cerella et al., 2014; Rafieian-Kopaei et al., 2013; Rahman et al., 2012; Lee et al., 2004; Busuttil et al., 2005; Peled-Kamar et al., 1995; Nadeem et al., 2014). The antioxidant paradox implies that the total antioxidant capacity of the body may not be linked. Thus, the amount of oxidative damage to key biomolecules (lipids, proteins, nucleic acids) is least affected despite pharmacological high doses of dietary antioxidant. The external supply of weak pro-oxidant (minerals, vitamins, etc.) in the form of dietary supplement may be helpful to the endogenous antioxidant system and this could be better approached in treatment and prevention of diseases. Another paradox we observed throughout the literature is the biochemical activity of phytochemicals or nutraceuticals (Das et al., 2012; Chen et al., 2015), i.e., although antioxidants can scavenge ROS and protect against oxidative stress, reactivity of end-product formation of reaction between ROS and phytochemical cannot be predicted in in vivo and in orally consumed foods. Therefore, measurement of “total antioxidant activity/capacity” of a nutraceutical/phytochemical should be included as an important aspect of research both in lab and clinical research. It can be measured in the form of ferric reducing antioxidant power, oxygen radical absorbing capacity, etc. Certainly, the antioxidant compounds generated after food or nutrient consumption may not be able to decrease ROS-induced oxidative damage. Therefore external supplementation of nutraceutical antioxidant would presumably be a more beneficial approach (Halliwell, 2013; Prakash and Gupta, 2009; Prior et al., 2005). A more detailed mechanistic approach is required to study these paradoxes in in vitro, in vivo and clinical applications in humans. Such studies may help design and/or select target- and site-specific phytochemicals. Further, directly targeting the source of free radicals such as H2O2 could minimize or inhibit the free radical generation. Studies to target the subcellular localization and activities of enzymes involved in natural antioxidant defense system such as NADPH oxidases that act in both beneficial as well as hazardous way to cause disease. Additionally, cellular organelles that are known sources of H2O2 such as mitochondria can also be considered while addressing this paradox (Singh and Gujar, 2013).

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27.6. DRUGS IN CLINICAL TRIALS FOR OXIDATIVE STRESS-INDUCED PATHOLOGIES Nutraceuticals, medicines of new era, critically found to be important in life style-related diseases such as obesity, metabolic syndrome, diabetes, CVDs, and aging. Their potential benefits will ensure their efficacy and safety as drugs. Therefore, regulations on formulations, production and clinical research of nutraceuticals and functional foods will lead to their appropriate clinical applications. On the other hand, use of one nutrient or antioxidant for one particular disease will achieve only symptomatic relief and not cure the disease unless they have target- and site-specific therapeutic properties. Thus it is required to target the inter-connected metabolic abnormalities associated with many diseases. Therefore, clinical studies should be designed on the basis of clinical consequences such as purity of compounds, dosage requirement as well as age and gender of patients. Lack of quality control and safety maintenance regulatory bodies are two major limitations in achievement of nutraceutical as a therapeutics and preventive healthcare. Regulatory bodies working in a collaborative manner with scientific research can be helpful in achieving the preventive healthcare targets. Following are a few such agencies that regulate different aspects of nutraceuticals development as drugs. 1. Food and Drug association (FDA): controls good manufacturing practices. 2. Dietary supplements health and education act: regulates manufacturing, importing and marketing of dietary supplements or healthy foods. 3. Central drug control department (India): controls the rules and regulations, also conflict resolution in manufacturing, marketing and transport of nutraceuticals within states and country. Along with these regulatory bodies, supporting scientific studies in the form of new independent association in countries like India, China, Brazil, and other traditional medicine hubs are required to discuss questions related to clinical trials of nutraceuticals. Also, international alliance of dietary supplements association and government health departments could work in tandem to share information, experiences and perspectives on the use and regulation of nutraceuticals as dietary supplements or drugs (Woolley et al., 2013; Stamler, 1994; Wink and Mitchell, 1998; Gupta et al., 2010, 2013; Rafieian-Kopaei et al., 2013) (Table 27.1).

27.7. PROBLEMS AND LIMITATIONS ASSOCIATED WITH NUTRACEUTICALS For oxidative stress-induced diseases, blocking the origin of oxidative damage and the dysfunction of antioxidant defense system in aerobes could be an appropriate target for the nutraceutical-based preventive and therapeutic approaches. However, the wide variation in the levels of oxidative biomarkers such as F2-isoprostanes (biomarker of lipid peroxidation) and urinary 8-hydroxydeoxyguanosine (marker of DNA damage) levels have been observed between different individuals. This could result in confusion and difficulty in selection of appropriate phytochemical as a drug against a specific disease. The wide-ranging library of polyphenols such as flavonoids is largely used in preventive healthcare. In in vitro studies, polyphenols quickly oxidize in cell culture media, which limits their antioxidant property through generating mild stress. Although, polyphenols are less effective in in vivo studies, the pro-oxidants generated in cell culture media enhance the endogenous glutathione level and sometimes they influence life span in a better way, when involved in the aging process. The phenomenon of scavenging radicals and thereby reducing cell death or apoptosis as exhibited by nutraceuticals gets altered when tissues are in a highly oxidized state. Cells that escape from necrotic cell death can release metal ions (e.g., ferric ions) and destructive free radicals into the surrounding area of tissues and lead to severe injury. This renders the cellular systems to become more resistant towards natural antioxidant defense (Chen et al., 2015; Singh and Gujar, 2013; Fischer, 2009; Masoli et al., 2004; Rahman et al., 2006). Therefore, strict and careful monitoring of a patient’s state of body is required before prescribing any nutraceuticals. Glucoraphanin a bio-precursor of sulforaphane is a chemo-preventive agent found in broccoli. It is responsible for the activation of a variety of carcinogens and is also involved in oxidative stress-induced DNA damage (Paolini et al., 2004). Thus its consumption may not necessarily scavenge ROS and promote health. Similarly, consumption of vitamins D, B1, and calcium and zinc when in excess, may not promote health, and their supplementation in diet may not be beneficial (Umegaki, 2016). Further, despite successful in vitro and in silico approaches, researchers are still struggling with bioavailability of flavonoids because of their high molecular weight. They

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TABLE 27.1

Different Classes of Nutraceuticals/Phytochemicals Involved in Pathophysiologies

Sr. Phytochemical/ No. nutraceutical

Disease

Effects

Age/ gender

1

β-thalassemia major

Biological effects: cytoprotective, antioxidant, anti-inflammatory, and especially as hepatoprotective agents

12 years Phase 1 and older/ both

Silymarin

Phase

Immunomodulatory actions: lymphocyte proliferation, interferon gamma, interleukin (IL)-4 and IL-10 secretions by stimulated lymphocytes 2

Purple Grape Juice, Apple Juice

Vascular Health in Childhood Cancer Survivors

Higher reactive Hyperemia Peripheral Arterial Tonometry 1030 Phase 1 (RH-PAT) Index Score indicates better endothelial years/both function

3

Blueberry Tea

Type-2 Diabetes

Improve glucose metabolism, reduce blood lipids, reduce oxidative stress and improve vascular function

4

Quercetin

COPD

5

Quercetin

Sarcoidosis

antioxidative and anti-inflammatory capacities

Not provided/ both

5

Flavonoid-rich dark chocolate

Chronic Heart Failure

Effect on oxidative stress, platelet adhesion as well as systemic inflammatory response such as CRP and proinflammatory cytokines

3080 Not years/both provided

6

Milk Thistle

Hemodialysis

Age 1860 Phase 2

7

Milk Thistle Extract 1 Vit E

End Stage Renal Disease

Effect against oxidative stress-induced antioxidant, anti-inflammatory, cell regenerating, and antifibrotic action.

8

Grape Seed Extract

Diastolic Heart Failure

Improve heart and blood vessel function

50 years Phase 1 older/both

9

Vegetable Consumption Health, Obesity, Oxidative Stress

Vegetables contain a range of vitamins, minerals, dietary fibers & phytochemicals including potassium, flavonoids, carotenoids, and Vit C

1845 years/ Male

10

Almonds

Endothelial Function In Patients With Coronary Artery Disease

Favorable effects on cardiovascular disease by improving endothelial function

2180 Not years/both provided

11

Pomegranate

Hemodialysis



1885 Not years/both provided

1875 Not years/both provided 4080 Phase 1 years/both Phase 2 Not provided

Not provided

Data modified from the US clinical trials website clinicaltrails.gov.com.

have to undergo several reactions (deglycosylation, esterification, etc.) to form low molecular weight derivatives. Even when flavonoid consumption is high, the quantity observed in blood stream has been reported to be quite low and not sufficient for efficacy of the compounds (Carocho and Ferreira, 2013). Over 50% of drugs failed in phase III clinical trials just because of a lack of efficacy as compared to placebo. Until recently approximately 1500 drugs were approved by FDA to target over 400 proteins involved in various pathologies (Gupta et al., 2013; Vinod et al., 2013). Thus researchers have a long way to go to until the drugs derived from nutraceuticals can cure diseases and promote health.

27.8. CONCLUSION AND FUTURE DIRECTIONS In conclusion, oxidative stress is an important factor in health as well as in causing various inflammatory diseases. These diseases arise either due to elevated production of ROS in cells and tissues, or due to downregulation of antioxidant machinery of the body. In addition, ROS and ROS-induced oxidative stress can activate a

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number of proinflammatory transcription factors that trigger inflammation-induced pathologies like CVD and cancer. Hence, targeting the source of ROS production and elevating the endogenous antioxidant potential of the cells and tissues could be helpful in curbing oxidative stress-mediated diseases. Various traditional and modern approaches could be useful in not only understanding the links between oxidative stress and diseases, but also to provide novel preventive and therapeutic strategies against these pathologies. In this direction, nutraceuticals could be an important source of a new regimen of preventive and clinical interventions in oxidative stressinduced diseases. However, nutraceuticals also have side-effects if used at higher doses, which represents the paradox associated with the use of these compounds. New therapeutic targets also need to be discovered that may potentially help decrease ROS production or their effects. Although several antioxidants and nutraceuticals have been successful in in vitro and in vivo studies of oxidative stress-induced pathology, they have measurably failed in clinical trials on various counts. Therefore, future studies should focus more on clear target identification, increasing bioavailability by modifying structure of nutraceuticals and decreasing the doses to minimize side-effects.

Acknowledgments Dr. Umesh C.S. Yadav acknowledges the award of Ramanujan Fellowship and financial support from Department of Science and Technology (DST), government of India.

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Further Reading Fiaschi, T., Chiarugi, P., 2012. Oxidative stress, tumor microenvironment, and metabolic reprogramming: a diabolic liaison. Int. J. Cell Biol. 2012, 762825. Scandalios, J.G., 2005. Oxidative stress: molecular perception and transduction of signals triggering antioxidant gene defenses. Braz. J. Med. Biol. Res. 38, 9951014.

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28 Phytochemicals as Anti-inflammatory Nutraceuticals and Phytopharmaceuticals Melanie-Jayne R. Howes Royal Botanic Gardens, Kew, Surrey, United Kingdom

28.1. INTRODUCTION Chronic inflammation has been associated with the pathology of a range of diseases, including atherosclerosis and cardiovascular disease, cancer, dementia, inflammatory bowel diseases, and metabolic diseases linked with diabetes and obesity (Howes and Houghton, 2009; Wu and Schauss, 2012; Siriwardhana et al., 2013). Numerous studies, reporting on findings ranging from in vitro studies and in vivo tests to clinical and epidemiological data, associate some dietary foods and their constituents with effects relevant to maintaining health or preventing disease (Howes and Simmonds, 2014). Many of these dietary components considered as relevant for human health include fruit and vegetables or other plant-derived components, and a diverse array of phytochemicals continue to emerge as important to prevent or delay the onset of chronic diseases. In this context, there has been a growing interest over recent decades in functional foods and nutraceuticals, which contain food or plant-derived constituents that are often associated with health benefits, although the scientific evidence to support many of their claims for health is often variable. In contrast to the use of phytochemicals as dietary components or as nutraceuticals to maintain health, is the use of plants or their constituents to treat or alleviate symptoms of disease. Indeed, plants and their constituents have been used for centuries for their reputed medicinal properties. One of the most widely used anti-inflammatory (and antiplatelet) pharmaceuticals is the cyclooxygenase (COX) inhibitor aspirin, which was originally developed from a natural product. It is almost 250 years since it was first discovered that willow (Salix species) bark could treat symptoms of the ague, now known as malaria. This discovery stimulated further research and eventually the active component salicin was isolated from willow bark in 1828 and soon after, it was isolated from another plant, meadowsweet (Filipendula ulmaria). Throughout the 19th century, research continued to improve the pharmaceutical properties of salicin, with the successful synthesis of acetyl salicylic acid in 1899, which was marketed as aspirin (Sutton, 2013). Since this discovery, numerous plant species and their constituents have been shown to mediate anti-inflammatory effects, although none have yet matched the wonder-drug status of aspirin, perhaps partly due to the cost of extensive clinical trials and by potential issues with patenting natural products to achieve exclusivity. Plants in this form are often referred to as herbal medicines or phytopharmaceuticals and there have been numerous studies to investigate the scientific basis to explain their traditional and potential uses for medicinal applications. The boundaries between phytopharmaceuticals, nutraceuticals and functional foods are often blurred and some plants and their constituents may be used in each of these circumstances, depending on the preparation, dose or other factors. This chapter describes some of the phytochemicals that have been associated with antiinflammatory effects, and discusses their relevance to attenuate inflammation for use as nutraceuticals and phytopharmaceuticals.

Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00028-7

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28.2. FLAVONOIDS Flavonoids are a large group of natural phenolic compounds that occur as different subclasses (including flavones, flavonols, flavanones, isoflavones, flavan-3-ols, and anthocyanidins); they exist as free aglycones or with sugars attached to the chemical structures to form glycosides. A wide range of flavonoids with various chemical structures have been associated with different anti-inflammatory mechanistic effects (Gomes et al., 2008), although their efficacy against specific inflammatory diseases has not been extensively investigated. The chemical structures of some flavonoids associated with anti-inflammatory activity are shown in Figs. 28.128.4. However, since flavonoids occur in numerous medicinal plants used as phytopharmaceuticals and have been considered as the active components of many nutraceuticals, they have been of much interest to maintain human health,

FIGURE 28.1 Chemical structures of some flavones, flavonols and a flavanone, associated with anti-inflammatory activity.

FIGURE 28.2 Chemical structures of some isoflavones associated with anti-inflammatory activity.

FIGURE 28.3 Chemical structures of some catechins associated with anti-inflammatory activity.

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FIGURE 28.4 Chemical structures of some anthocyanidins associated with anti-inflammatory activity.

although their oral bioavailability and metabolism may influence their efficacy. Indeed, deglycosylation of flavones increases their anti-inflammatory activity and absorption (Hostetler et al., 2012).

28.2.1. Flavones, Flavonols and Flavanones One of the most common flavones, occurring in a wide range of plant species, is apigenin, which suppresses nitric oxide (NO) and prostaglandin production via inhibition of inducible nitric oxide synthase (iNOS) and COX-2, respectively; and it inhibits phosphorylation of signal transducer and activator of transcription (STAT)-1 to decrease interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) levels, it suppresses p38 mitogen-activated protein kinase (MAPK), c-Jun N-terminal kinase (JNK) phosphorylation and inactivates nuclear factor κB (NF-κB) (Venigalla et al., 2016). It is suggested that an apigenin-rich diet could mediate anti-inflammatory effects in vivo via regulation of gene expression, perhaps explaining the epidemiological correlations linking apigenin with a reduced risk of some diseases (Arango et al., 2015). The C-glucosides of apigenin, vitexin, and isovitexin, were considered to contribute to the anti-inflammatory effects of mung bean extracts via inhibition of COX-2 mRNA expression (Xiao et al., 2016), thus plants containing these flavone glycosides may also be of interest as nutraceuticals. Another common plant flavone is luteolin, which occurs in numerous edible plants, such as pomegranate (Punica granatum), artichoke (Cynara scolymus) and herbs such as rosemary (Rosmarinus officinalis), and it is of interest from the dietary and nutraceutical perspectives. Luteolin has been investigated for mechanistic effects relevant to a range of diseases including those associated with inflammatory processes such as neurodegenerative diseases (Nabavi et al., 2015). Luteolin inhibits chronic inflammation in an in vitro coculture of adipocytes and macrophages and inhibits the phosphorylation of JNK in macrophages (Hirai et al., 2010), thus may be a useful nutraceutical for some metabolic disorders. Isoorientin, the 6-C-glucoside of luteolin, inhibits thromboxane (TX)-B2 synthesis in rat peritoneal leukocytes in vitro (Xiao et al., 2016), while a mixture of flavone C-glycosides (vicenin-2, spinosin, isovitexin, swertisin, isoswertisin) was anti-inflammatory in vivo and in vitro, which may be due to reduced iNOS and COX-2 expression (Xiao et al., 2016). Ac¸aı´ (Euterpe oleracea) fruit, which contains flavones in addition to other phytochemical classes (PacheoPalencia et al., 2009) was anti-inflammatory in studies in vitro, in vivo and in some clinical trials (Wu and Schauss, 2012), although all of the compounds responsible for these effects have not been fully characterized. Some studies suggest the flavone velutin may contribute to the anti-inflammatory effects of this fruit, since it was discovered as more potent than other flavones from ac¸aı´ fruit in reducing TNF-α and IL-6 production in macrophages, and it was effective in blocking the degradation of IκB and could inhibit MAPK p38 and JNK phosphorylation, the signaling pathways involved in the production of TNF-α and IL-6 (Wu and Schauss, 2012). Other flavones from medicinal plants have been of interest for use as phytopharmaceuticals to alleviate pain and inflammation. The flavone glycoside, lonicerin (luteolin 7-neohesperidoside) occurs in Veronicastrum species and has antiarthritic activity (Laev and Salakhutdinov, 2015). Baicalein from the traditional Chinese medicine (TCM) Scutellaria baicalensis root, was anti-inflammatory in vitro, since it inhibited IL-1β-induced rheumatoid arthritis (RA) fibroblast-like synoviocyte proliferation via inactivation of NF-κB and inhibition of the macrophage migration inhibitory factor signaling pathway (Laev and Salakhutdinov, 2015). Baicalin is the 7-glucuronide of baicalein, which inhibits IL-17-mediated joint inflammation in murine adjuvant-induced arthritis (Laev and Salakhutdinov, 2015). A combination of baicalin with catechin (see 28.2.3) inhibits COX and 5-lipoxygenase (5LOX), iNOS expression, NF-κB binding activity and TNF-α in lipopolysaccharide (LPS)-stimulated macrophages (Altavilla et al., 2009). A proprietary blend (‘flavocoxid’) of flavonoids, including baicalin and catechin, was approved by the US Food and Drug Administration as a prescription product for osteoarthritis (Laev and Salakhutdinov, 2015).

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One of the most common flavonols distributed in the plant kingdom is quercetin, which occurs in various vegetables and fruits, such as apples (Malus x domestica). Apple flavonoids, which include flavonol glycosides and flavan-3-ols, have been associated with anti-inflammatory effects (Simmonds and Howes, 2016). Furthermore, supplementation with quercetin alone (2 g in 24 h) reduced markers of oxidative stress and inflammation in sarcoidosis patients (Boots et al., 2011). In addition to the nutraceutical role of flavonols, they may also be of value as phytopharmaceuticals. For example, when administered via i.p. (80 mg/kg daily) quercetin, the 3rutinoside (rutin) and the flavanone glycoside hesperidin (hesperetin 7-rutinoside) inhibited acute and chronic inflammation in an animal model of arthritis, with rutin the most active against chronic inflammation (Laev and Salakhutdinov 2015). Rutin may also be of value against inflammatory bowel disease as it suppressed inflammation and colonic damage in animal models of colitis when administered orally, via attenuation of proinflammatory (IL-1β, IL-6) gene expression (Hur et al., 2012). Quercetin has also been explored for potential to alleviate inflammatory airways disease, as when formulated as a microemulsion to improve oral bioavailability, it reduced inflammation in an animal model of allergic airway inflammation (Rogerio et al., 2010). Flavonoids from Citrus species inhibit a range of proinflammatory mediators, including those derived from the arachidonic acid cascade (Benavente-Garcı´a and Castillo, 2008). Although Citrus flavanones are more abundant than flavones, the flavones are suggested to exhibit higher biological activity (Laev and Salakhutdinov, 2015). However, the combination of flavanones and flavones in Citrus-based nutraceuticals may contribute to their overall benefits for health. Indeed, Citrus flavanones also mediate anti-inflammatory effects; naringenin is suggested to modulate neuroinflammation via interaction with p38 signaling cascades and STAT-1 (Vafeiadou et al., 2009), while the 7-neohesperidoside, naringin, suppressed the inflammatory response in an animal model of arthritis when administered orally (Laev and Salakhutdinov, 2015). Other studies have revealed that orange (Citrus x aurantium) juice reduces inflammatory stress induced by a high-fat and -carbohydrate meal, so may have benefits against insulin resistance and atherosclerosis (Ghanim et al., 2010). Red orange juice intake also reduced inflammatory markers and improved endothelial function in nondiabetic subjects with increased cardiovascular risk (Buscemi et al., 2012). However, one of the limitations of studies investigating the effects of fruit products on inflammatory parameters in humans is that in many cases, the chemical composition of the fruit product/juice has not been characterized, so conclusions on the most active or relevant constituents for health cannot be fully understood. Although, one study did conclude that regular consumption of orange juice for 4 weeks could alter leukocyte gene expression to an anti-inflammatory and antiatherogenic profile, and that the flavanone hesperidin was important for this effect (Milenkovic et al., 2011). Supplementation with hesperidin (600 mg/day) for 4 weeks also reduced inflammatory markers and improved lipid profile in myocardial infarction patients (n 5 75) in a randomized controlled trial (RCT) (Haidari et al., 2015), thus providing further evidence for the anti-inflammatory role of this flavanone to maintain health.

28.2.2. Isoflavones Isoflavones occur in a range of plants, particularly those in the Leguminosae family, which includes legumes such as soya (Glycine max) and medicinal plants such as red clover (Trifolium pratense). Isoflavones have been extensively investigated and display a myriad of pharmacological activities (Howes and Houghton, 2009; Howes and Simmonds, 2014) that have been supported by clinical and epidemiological studies, particularly estrogenic effects, but some have also shown anti-inflammatory properties. Soya isoflavones such as genistein have been associated with potential health benefits in relation to metabolic disorders such as diabetes. Genistein may inhibit inflammation to ameliorate the endothelial dysfunction implicated in insulin resistance, since it inhibits NF-κB activation, it downregulates TNF-α and IL-6 expression and production, and it restores insulin-mediated Akt and endothelial NO synthase phosphorylation; it also inhibits inflammation associated with the mitogenic actions of insulin by downregulating endothelin-1 and vascular cell adhesion molecule-1 (VCAM-1) overexpression (Howes and Simmonds, 2014). In addition, soya isoflavones suppress adipose tissue inflammation and improve insulin sensitivity, as in an animal model of obesity, they decreased adiposity and serum levels of adipokines, including resistin, IL-6 and TNF-α, and increased the level of the anti-inflammatory adipokine, adiponectin (Siriwardhana et al., 2013). Other studies suggest soya isoflavones may have anti-inflammatory benefits beyond their function as nutraceuticals. For example, genistein mediates mechanistic anti-inflammatory effects relevant to alleviating arthritis (Li et al., 2013) and in rats (20 mg/day for 50 days) it delayed the onset of RA symptoms, while also reducing disease symptoms and serum levels of TNF-α, IL-6 and leptin (Laev and Salakhutdinov, 2015). The isoflavone

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367

daidzein has been investigated for its potential to reduce lung inflammation, as it modulated the NF-κB pathway in an animal model of acute lung injury (Feng et al., 2015). Isoflavones such as genistein and daidzein also occur in some medicinal plants and may explain some of their uses as phytopharmaceuticals. Other anti-inflammatory isoflavones include puerarin and related isoflavones from the TCM Pueraria montana var. lobata (Lim et al., 2013); and biochanin A from alfalfa (Medicago sativa) and red clover (Ming et al., 2015).

28.2.3. Catechins In plants, flavan-3-ols can occur as monomers (e.g., catechin and epicatechin), as oligomers (dimers to decamers) or as polymers (.decamers), with oligomers and polymers described as proanthocyanidins or condensed tannins. Flavan-3-ols occur in some dietary plants, including tea (Camellia sinensis) and cocoa (Theobroma cacao), that have been associated with maintaining health and preventing disease, thus are of interest as nutraceuticals. Epidemiological studies suggest that chronic tea consumption may inhibit low-grade inflammation and benefit human health (Wu and Schauss, 2012; Vuong, 2014), while monomeric and oligomeric flavan-3-ols from grape (Vitis vinifera) seeds reduced inflammatory gene expression in leukocytes and improved vascular health in smokers when taken as a supplement (200 mg daily for 8 weeks) in an intervention study (Weseler et al., 2011). Flavan-3-ols from cocoa reduce biomarkers of inflammation, particularly in relation to cardiovascular health (Goya et al., 2016). Furthermore, regular consumption of dark chocolate has been linked with low serum C-reactive protein (CRP) concentrations in two separate clinical trials (Wu and Schauss, 2012), which may be due to the flavan-3ol and procyanidin constituents. Catechins interfere with the inflammatory processes that contribute to atherosclerosis progression (Wu and Schauss, 2012) and epigallocatechin gallate (EGCG) in particular could modulate glucose-induced vascular inflammatory pathways via inhibition of protein kinase C (PKC) and NF-κB activation in vitro, thus may have some relevance for prevention of vascular complications in diabetes (Howes and Simmonds, 2014). Mechanistic effects of cocoa flavan-3-ols include inhibition of eicosanoid production, reduction of platelet activation and modulation of NO-dependent mechanisms (Selmi et al., 2006). Different cocoa flavan-3-ols and their procyanidin oligomers also modulate cytokine production including Il-2, IL-1β, IL-4 and IL-5, and TNF-α, with varying potency. Flavan-3-ol monomers through to the procyanidin tetramers suppressed IL-1β mRNA expression, while larger procyanidins (pentamers to decamers) stimulated IL-1β production (Selmi et al., 2006). Thus, the degree of polymerization of these flavan-3-ols appears to affect their ability to modulate inflammatory processes to different extents. EGCG inhibits IκB kinase (IKK) activity to block phosphorylation-dependent degradation of IκBα, which decreases nuclear localization of p65 protein, resulting in inhibition of NF-κB activation (Surh and Na, 2008). EGCG also inactivates NF-κB and inhibits COX-2 induction by suppressing signal transduction by MAPKs and P13K-Akt (Surh and Na, 2008), and it has neuroprotective effects via modulation of inflammation (Venigalla et al., 2016), it decreases iNOS activity in peritoneal macrophages, and COX-2 expression in colon cancer cells (Hur et al., 2012), thus may have other potential anti-inflammatory applications, such as in neurodegeneration and carcinogenesis. The therapeutic potential of EGCG to alleviate pain and inflammation in some other disease states has also been explored. EGCG could dose-dependently suppress IL-1β-induced activity of COX-2 and iNOS in human chondrocytes (Laev and Salakhutdinov, 2015), while in an animal model of arthritis (20 μg/g body weight i.p. for 15 days) it suppressed osteoclast differentiation and ameliorated RA symptoms (Morinobu et al., 2008). Another study revealed that EGCG blocks NF-κB activation in intestinal epithelial cells and ameliorates mucosal inflammation, while catechin reduces myeloperoxidase (MPO) activity and reduces colitis severity (Hur et al., 2012), thus suggesting these catechins may have potential applications as nutraceuticals or as phytopharmaceuticals for inflammatory bowel disorders. It is also suggested that poor intestinal absorption of anti-inflammatory procyanidins enables adequate concentrations in the intestine to accumulate and counteract inflammation specifically in this region (Mena et al., 2014).

28.2.4. Anthocyanidins In common with other flavonoids, anthocyanidins can occur as glycosides and these are known as anthocyanins. They are responsible for the blue, purple and red pigments of many plants and although they have been associated with many biological activities, they are not frequently used as phytopharmaceuticals. Clinical studies investigating anthocyanins have yielded inconsistent results, which may be associated with biaoavailability

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issues. Although plasma concentrations of anthocyanins have been reported as low following ingestion, more recent research suggests anthocyanins may undergo extensive presystemic metabolism and that some can be effectively absorbed from the gastrointestinal (GI) lumen and enter the systemic circulation as metabolites (Howes and Simmonds, 2014). While intake of anthocyanin (300 mg/day for two weeks) beverages derived from red grapes and bilberres (Vaccinium species) showed no changes in inflammatory parameters (Mena et al., 2014), another study concluded that a bilberry extract (300 mg anthocyanins/day) could inhibit NF-κB transactivation and decrease plasma concentrations of proinflammatory chemokines, cytokines, and inflammatory mediators [IL8, interferon (IFN)-α, IL-4, IL-13] when ingested by 118 healthy volunteers for 3 weeks (Mena et al., 2014). Other clinical studies suggest anthocyanin-containing bilberry extracts may reduce CRP levels, although not all controlled trials concluded this effect (Mena et al., 2014). Bilberries and their anthocyanins also ameliorate experimental colitis (Piberger et al., 2011), while an anthocyanin-rich bilberry preparation improved disease activity in ulcerative colitis patients in an open pilot study (Biedermann et al., 2013), suggesting that anthocyanins may directly act in the GI tract to exert anti-inflammatory effects. Blackcurrants (Ribes nigrum) also contain anthocyanins (in addition to flavonols and phenolic acids) and a purified anthocyanin extract improved serum lipid profiles and decreased inflammatory markers in hypercholesterolemic individuals (Gopalan et al., 2012). An anthocyanin-rich elderberry (Sambucus nigra) extract could protect against inflammatory impairments related to atherosclerosis progression in an animal model of hyperlipidemia (Farrell et al., 2015), providing further evidence for the potential value of anthocyanins against inflammatory pathways in cardiovascular disease. Blackberries (Rubus species), which also contain anthocyanins, have also been associated with anti-inflammatory activity to mediate health benefits (Kaume et al., 2012). While small clinical studies report a lack of any anti-inflammatory effects with purple carrot (Daucus carota) (119 mg anthocyanins; 259 mg phenolic acids daily) or wild blueberry (Vaccinium species) (375 mg anthocyanins; 128 mg chlorogenic acid daily) products (measured by inflammatory markers), other clinical studies suggest pomegranate (Punica granatum) juice and cornel (Cornus mas) berries may ameliorate vascular inflammation, although the role of anthocyanins in these effects was not determined (Mena et al., 2014). Pomegranate extracts can also reduce gastric and intestinal inflammation via different mechanisms (Colombo et al., 2013), which may be due to different phytochemical constituents, including anthocyanins. Anthocyanins modulate different signaling pathways to mediate their anti-inflammatory effects, particularly via the MAPK pathway (Vendrame and Klimis-Zacas, 2015). Mechanistic studies report the glycosides of malvidin, delphinidin, cyanidin, petunidin, and peonidin to dose-dependently reduce IL-1β-activation of NF-κB in vitro (Mena et al., 2014). Malvidin also reduces TNF-α-induced inflammatory responses in endothelial cells so is of interest to manage inflammation in chronic diseases (Huang et al., 2014). Other studies show that delphinidin downregulates COX-2 and targets Fyn kinase to down-regulate TNF-α-stimulated COX-2 expression, while cyanidin inhibits UVB-induced COX-2 expression (Murakami and Ohnishi, 2012). Interestingly, delphinidin inhibits histone acetyltransferase and suppresses inflammatory signaling by preventing NF-κB acetylation in fibroblast-like synoviocytes (Seong et al., 2011), thus may be useful to explore further for potential to counteract inflammatory arthritis but may also have relevance for use in other diseases such as cancer. One of the major grape anthocyanins, malvidin 3-O-glucoside, has been of interest to target inflammation in arthritis, as it decreased transcription of genes encoding inflammatory mediators and reduced inflammation in an animal model of RA (Decendit et al., 2013). The antioxidant and anti-inflammatory effects of the anthocyanidin malvidin are also suggested to contribute to the positive effects on health of moderate red wine consumption (Howes and Simmonds, 2014).

28.3. TERPENOIDS 28.3.1. Monoterpenoids and Sesquiterpenoids Plant-derived volatile oils consist of highly complex mixtures of phytochemicals and main components include monoterpenoids (monoterpenes) and sesquiterpenoids (sesquiterpenes). Volatile oils (also known as essential oils) are distributed in around 50 different plant families, including the Asteraceae, Rutaceae, and Lamiaceae, with the latter including many herbs that are used for culinary and medicinal purposes. A range of volatile oil constituents have been associated with mechanistic effects associated with anti-inflammatory activity (examples are presented in Table 28.1 and Fig. 28.5). It should also be considered that many

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28.3. TERPENOIDS

TABLE 28.1 Activity

Mechanistic Effects of Monoterpenoids and Sesquiterpenoids, and Their Derivatives, Relevant to Anti-inflammatory

Terpenoid

Plant sourcea

Mechanistic effects

References

Salvia lavandulaefolia

Inhibits LTB4 generation in vitro

Howes et al. (2003)

(synonym of S. officinalis subsp. lavandulifolia)

Inhibits NF-κB signaling via increased expression of IκBα

Salminen et al. (2008)

Eucalyptus species

Reduces TNF-α and IL-1β levels in airways (bronchoalveolar fluid) after inhalation and in an animal model of pancreatitis

Bastos et al. (2011)

Increases IL-10 and suppresses toll-like receptor (TLR)-4 and NF-κB expression in lung tissue to inhibit acute pulmonary inflammation

Lima et al. (2013a)

Reduces brain p38 MAPK, iNOS, COX-2, IL-1β and microgliosis in a transgenic animal model of Alzheimer’s disease

Sabogal-Gua´queta et al. (2016)

Reduces carrageenan-induced edema in vivo

Peana et al. (2002)

Suppresses iNOS and IL-1β in a rat model of colitis associated colon cancer

Arigesavan and Sudhandiran (2015)

Attenuates paw edema; associated with reduced TNF-α, IL-1β and PGE2 and COX-2 expression, and increased IL-10

Lima et al. (2013b)

MONOTERPENOIDS AND DERIVATIVES α-Pinene

1,8-Cineole

Linalool

Carvacrol

Lavandula angustifolia

Oreganum species

Zhao et al. (2014)

Thymol

Thymus species

Inhibits TNF-α and IL-6 production; reduces iNOS and COX-2 Liang et al. (2014) expression; and inhibits phosphorylation of IκBα, NF-κB p65, ERK, JNK, and p38 MAPKs in LPS-stimulated mammary epithelial cells

Citronellal

Cymbopogon winterianus

Inhibits carrageenan- and arachidonic acid-induced rat paw edema Melo et al. (2011)

Citronellol

Cymbopogon winterianus

Inhibits neutrophil infiltration and reduces TNF-α in an animal model of pleurisy; reduces inflammatory pain in vivo

Brito et al. (2012)

Limonene

Anethum graveolens

Inhibits NF-κB signaling

Salminen et al. (2008)

Perillyl alcohol

Perilla frutescens

Suppresses IL-1β, TNF-α and IL-6; downregulates COX-2, iNOS and NF-κB expression in middle cerebral artery occlusion

Tabassum et al. (2015)

α-Terpineol

Melaleuca alternifolia

Inhibits carrageenan-, TNF-α- and PGE2-induced hypernociception, de Oliveira et al. and neutrophil influx in pleurisy (2012)

Menthol

Mentha x piperita

Inhibits NF-κB signaling

Sabinene

Myristica fragrans

Inhibits NO production in LPS- and IFN-γ-stimulated macrophages Valente et al. (2013)

Myrtenol

Myrtus communis

Inhibits inflammatory mediator release (including IL-1β), cell migration and signaling pathways involved in pain

Silva et al. (2014a)

Geraniol

Cymbopogon winterianus

Decreases myeloperoxidase activity, iNOS and COX-2 expression, TNF-α, IL-1β and IL-6, and NF-κB signaling, in an animal model of colitis

Medicherla et al. (2015)

p-Cymene

Trachyspermum ammi

Reduces carrageenan-, TNF-α- and PGE2-induced hyperalgesia; associated with reduced leukocyte migration, neutrophils, TNF-α and NO

De Santana et al. (2015)

Borneol

Salvia officinalis subsp. lavandulifolia

Reduces carrageenan-induced leukocyte migration

Almeida et al. (2013)

Isoespintanol

Oxandra xylopioides

Reduces carrageenan-induced paw edema, IL-1β production and mRNA synthesis

Rojano et al. (2007)

Nocellaralactone

Olea europaea

Inhibits IFN-γ- and histamine-induced ICAM-1 and iNOS expression; increases IL-10 release in vitro

Serrilli et al. (2013)

Salminen et al. (2008)

(Continued)

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28. PHYTOCHEMICALS AS ANTI-INFLAMMATORY NUTRACEUTICALS AND PHYTOPHARMACEUTICALS

(Continued)

Terpenoid

Plant sourcea

Mechanistic effects

References

Paeoniflorin

Paeonia species

Protects against LPS-induced liver inflammation

Kim and Ha (2010)

Inhibits NO, TNF-α and IL-1β in microglia

Nam et al. (2013)

Downregulates IL-1β and TNF-α expression, inhibits phosphorylation of p38 MAPK and JNK and inhibits NF-κB activation in lung tissue

Zhou et al. (2011)

Inhibits NO, PGE2, TNF-α and IL-6 production

Wang et al. (2014)

Albiflorin

Paeonia species

Reduces COX-2, iNOS, IL-6 and IL-6 gene expression Paeonidanins 13

Paeonia species

Inhibit TNF-α and NO

Fu et al. (2015)

Aucubin

Plantago lanceolata

Inhibits NF-κB signaling via inhibition of IκBα degradation

Salminen et al. (2008)

Catalposide

Catalpa ovata

Inhibits NF-κB signaling via inhibition of IκBα degradation

Salminen et al. (2008)

Attenuates TNF-α induced p38 and ERK phosphorylation Genipin

Gardenia jasminoides

Inhibits NF-κB signaling via inhibition of IκBα degradation Inhibits expression of iNOS and NO production

Salminen et al. (2008)

SESQUITERPENOIDS AND DERIVATIVES Farnesol

Citrus x aurantium

Increases IL-10 in an animal model of asthmatic inflammation

Ku and Lin (2015)

α-Bisabolol

Matricaria chamomilla

Reduces carrageenan-induced paw edema and associated leukocyte migration and TNF-α

Rocha et al. (2011)

Nerolidol

Citrus x aurantium

Reduces IL-13 and TNF-α in an animal model of colitis

Gonza´lez-Ramı´rez et al. (2016)

Patchouli alcohol

Pogostemon cablin

Suppresses the ERK-mediated NF-κB pathway

Jeong et al. (2013)

α-Humulene

Humulus lupulus

Inhibits NF-κB signaling and the LPS-induced inflammatory response in paw edema

Salminen et al. (2008)

Reduces carrageenan-induced inflammatory nociception and edema; inhibits IL-1β, TNF-α and PGE2, and expression of COX-2 and iNOS

Guimara˜es et al. (2014)

β-Patchoulene

Pogostemon cablin

Suppresses carrageenan-induced paw edema via suppression of TNF-α, IL-1β, IL-6, PGE2 and NO; and inhibition of NF-κB

Zhang et al. (2016)

Matricine

Matricaria recutita

Inhibits NF-κB transcriptional activity and signaling

(synonym of M. chamomilla)

Inhibits LPS- and TNF-α-induced ICAM-1 expression

Flemming et al. (2015)

7-Hydroxy-14cadalenal

Heterotheca inuloides

Inhibits ear edema in vivo

Egas et al. (2015)

Elemene

Curcuma species

Downregulates IL-17, IFN-γ and TNF-α

Aggarwal et al. (2013)

Curdione

Curcuma species

Inhibits PGE2 via COX-2 suppression

Aggarwal et al. (2013)

Turmerone

Curcuma species

Suppresses COX-2 and iNOS expression; inhibits MMP-9, COX-2 and NF-κB

Aggarwal et al. (2013)

Bisacurone

Curcuma species

Downregulates TNF-α-induced VCAM-1 expression

Aggarwal et al. (2013)

Germacrone

Curcuma species

Inhibits NO in vitro; anti-inflammatory against carrageenaninduced paw edema

Aggarwal et al. (2013) (Continued)

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28.3. TERPENOIDS

TABLE 28.1

(Continued)

Terpenoid

Plant sourcea

Mechanistic effects

References

Curcumolide

Curcuma wenyujin (synonym of C. aromatica)

Suppresses LPS-induced NF-κB activation; decreases TNF-α, IL-6, IL-1β and NO

Dong et al. (2015)

Aremisinin

Artemisia annua

Inhibits NF-κB signaling

Salminen et al. (2008)

DSF-52

Artemisia argyi

Inhibits microglia-mediated neuroinflammation via suppression of NF-κB, JNK/p38 MAPKs and STAT-3 pathways

Zeng et al. (2014)

Costunolide

Saussurea costus

Inhibits NF-κB signaling via inhibition of IκBα phosphorylation

Salminen et al. (2008)

Inhibits iNOS and COX-2 expression, and NO and PGE2 production

Park et al. (2014)

Dehydrocostuslactone Saussurea costus

Reduces IL-1β and TNF-α Pulchellamin G

Saussurea pulchella

Inhibits iNOS and COX-2 expression, and PGE2 production

Lee et al. (2013)

Reduces TNF-α and IL-1β production Alantolactone

Aucklandia lappa

Suppresses NF-κB activation and MAPKs phosphorylation

Chun et al. (2005)

Suppresses TNF-α- and IFN-γ-induced production of IL-8 via inhibition of STAT-1 phosphorylation

Lim et al. (2015)

Chlorojanerin

Saussurea heteromalla (synonym of Himalaiella heteromalla)

Inhibits TNF-α and IL-6 production and activation of NF-κB

Saklani et al. (2012)

Parthenolide

Tanacetum parthenium

Modulates TLR-4-mediated MAPK and NF-κB pathways

Salminen et al. (2008)

Inhibits IL-6, IL-1β, IL-8, IL-18, TNF-α and NO production

Li et al. (2015)

Valerenic acid

Valeriana officinalis

Inhibits NF-κB signaling

Salminen et al. (2008)

Zerumbone

Zingiber species

Inhibits NF-κB signaling via inhibition of IκBα degradation

Salminen et al. (2008)

8α-Hydroxyhirsutinolide

Vernonia cinerea (synonym of Cyanthillium cinereum)

Inhibits TNF-α-induced NF-κB activity

Youn et al. (2012)

8α-Tigloyloxyhirsutinolide13-O-acetate

Vernonia cinerea (synonym of Cyanthillium cinereum)

Inhibits TNF-α-induced NF-κB activity

Youn et al. (2012)

Vernolide A

Vernonia cinerea

Inhibits TNF-α-induced NF-κB activity

Youn et al. (2012)

(synonym of Cyanthillium cinereum) Caruifolin

Artemisia absinthium

Inhibits neuroinflammatory mediator release from microglia

Zeng et al. (2015)

Lychnopholide

Lychnophora trichocarpha

Reduces carrageenan-induced paw edema via topical application

Ferrari et al. (2013)

Increases IL-10 and inhibits NO production Eremantholide

Lychnophora trichocarpha

Reduces carrageenan-induced paw edema via topical application

Ferrari et al. (2013)

Increases IL-10 and inhibits TNF-α production Ziniolide

Xanthium spinosum

Inhibits 5-LOX and NF-κB activation

Bader et al. (2013)

Teuclatriol

Salvia mirzayanii

Inhibits NF-κB signaling and reduces TNF-α production

Ziaei et al. (2015)

Tussilagone

Tussilago farfara

Suppresses iNOS and COX-2 expression and NO and PGE2 production

Lee et al. (2016)

Inhibits phosphorylation and degradation of IκBα and nuclear translocation of NF-κB (Continued) VI. NEW PERSPECTIVES AND FUTURE DIRECTIONS

372 TABLE 28.1

28. PHYTOCHEMICALS AS ANTI-INFLAMMATORY NUTRACEUTICALS AND PHYTOPHARMACEUTICALS

(Continued)

Terpenoid

Plant sourcea

Mechanistic effects

References

Neurolobatin B

Neurolaena lobata

Downregulates LPS-and TNF-α-induced IL-8 expression

Lajter et al. (2014)

3-epi-Desacetylisovaleroylheliangine

Neurolaena lobata

Downregulates LPS-and TNF-α-induced IL-8 expression

Lajter et al. (2014)

Neurolenins

Neurolaena lobata

Reduce LPS-induced TNF-α production

Walshe-Roussel et al. (2013)

Neurolaena lobata

Reduces LPS-induced TNF-α production

Walshe-Roussel et al. (2013)

B, C and D Lobatin B a

Examples of plant species that the terpenoid occurs in are presented; terpenoids may also be distributed in other plant species; in the case of a plant name described by a synonym in the source reference, the currently accepted taxonomic name is also provided.

FIGURE 28.5 Chemical structures of some monoterpenoids and sesquiterpenoids associated with anti-inflammatory activity.

monoterpenes and sesquiterpenes may occur in nutraceuticals, phytopharmaceuticals or culinary herbs as a mixture with other plant constituents, and their relevance to health via these routes of administration requires further understanding. Of the volatile monoterpenes investigated for anti-inflammatory effects, 1,8-cineole (also referred to as cineole), has shown some promising results in respiratory disorders. The anti-inflammatory properties of cineole (Table 28.1) may have contributed to the reported efficacy of this monoterpene against bronchitis when administered (3 3 200 mg/day) to 242 patients in a RCT (Fischer and Dethlefsen, 2013). In another RCT involving 247 patients with asthma, administration of cineole at the same dose for 6 months was associated with improved lung function and reduced dyspnea (Worth and Dethlefsen, 2012). These findings are in agreement with an earlier controlled trial that concluded an equivalent dose of cineole to enable a reduced steroid dose to control asthma symptoms, due to its anti-inflammatory effects (Juergens et al., 2003). Of the volatile sesquiterpenes with anti-inflammatory mechanistic effects (Table 28.1), α-humulene has been of particular interest for its potential development as a phytopharmaceutical, since it displayed a range of activities indicating modulation of inflammatory pathways and has been described in a patent for use against inflammation and associated pain (Guimara˜es et al., 2014).

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Monoterpenes can also occur as nonvolatile derivatives in plants and many such compounds have also displayed mechanistic effects against inflammatory processes (Table 28.1; Fig. 28.5). Monoterpene glycosides, including paeoniflorin and albiflorin, from the roots of Paeonia species, which are used in TCM, have shown a range of mechanistic effects relevant to modulating inflammation (Table 28.1) and extracts from the roots of these species are of interest as potential phytopharmaceuticals. In a study that directly compared the antiinflammatory effects of paeoniflorin and the isomer albiflorin, both compounds were concluded to have similar effects (Wang et al., 2014). Extracts prepared from Paeonia lactiflora root have demonstrated anti-inflammatory effects in animal models of acute and sub-acute inflammation, mechanistically associated with inhibition of prostaglandin (PG)-E2, leukotriene (LT)-B4 and NO production, while RCTs indicate benefits in RA (He and Dai, 2011). Paeoniflorin is also suggested to be neuroprotective via modulation of the microglial anti-inflammatory response in the brain (Nam et al., 2013), thus may have therapeutic relevance for neurodegenerative disorders such as Alzheimer’s disease. Dimeric monoterpene glycosides (paeonidanins 13) from P. lactiflora are also reported as anti-inflammatory (Fu et al., 2015). From the root of P. suffruticosa, the monoterpene glycosides, paeonides A and B, paeoniflorin, benzoylpaeoniflorin and 4-O-methyl-paeoniflorin could inhibit COX-1 and COX-2 in vitro (Zhu and Fang, 2014). Other monoterpene derivatives of interest for use in inflammatory disorders are the monoterpene glycosidegallic acid conjugates, globulusin A, and eucaglobulin from Eucalyptus globulus, which suppressed IL-1β and TNF-α production in vitro (Hasegawa et al., 2008). From a pharmaceutical development perspective, the monoterpene alkaloid, incarvillateine, from the aerial parts of Incarvillea sinensis, and the monoterpene iridoid, sweroside, have been patented as potential new analgesic and anti-inflammatory drugs (Guimara˜es et al., 2014). Other monoterpene derivatives have been of interest as nutraceuticals from dietary plants, including auraptene from the fruit of Citrus species, which regulates transcription of peroxisome proliferator activated receptor-γ (PPAR-γ) target genes and inhibits expression and secretion of monocyte chemoattractant protein-1 (MCP-1) in adipocytes, and it suppresses inflammatory processes in adipose tissue (Hirai et al., 2010), thus may be of value for use in metabolic disorders associated with obesity. One of the most investigated terpenoid classes against inflammation are the sesquiterpene lactones and many have relevant mechanistic effects (examples are presented in Table 28.1). Sesquiterpene lactones from Saussurea species, used in TCM, have been of interest for use as phytopharmaceuticals in inflammatory disorders. Studies show Saussurea costus root extracts to inhibit IL-8 induction (Lee et al., 1995) and TNF-α (Cho et al., 1998), with the latter effect due to cynaropicrin (Fig. 28.5), which also dose-dependently suppressed lymphocyte proliferation and IL-2 in separate studies (Cho et al., 1998, 2000). The sesquiterpene lactone costunolide inhibited IL-1β gene expression (Kang et al., 2004), NO production and activation of NF-κB in vitro; dehydrocostuslactone and the sesquiterpene-amino acid adducts, saussureamines A and B, also inhibited these effects (Matsuda et al., 2003). Dehydrocostuslactone decreased TNF-α in vitro and in vivo (Lee et al., 1999; Zhao et al., 2008); costunolide, α-cyclocostunolide, alantolactone and isoalantolactone produced this effect in vitro (Zhao et al., 2008). Another sesquiterpene from S. costus is santamarin, which inhibited iNOS protein, reduced iNOS-derived NO, suppressed COX-2 protein and reduced COX-derived PGE2 production in vitro, in addition to reducing TNF-α and IL-1β production (Choi et al., 2012). Furthermore, a root extract of S. costus and the sesquiterpene lactone fraction, could reduce inflammation in vivo (Gokhale et al., 2002). Costunolide and dehydrocostuslactone are of particular therapeutic relevance, since they could ameliorate inflammatory processes in an animal model of pleurisy (Butturini et al., 2014) and counteract the proinflammatory effects of IFN-γ and IL-22 in keratinocytes (Scarponi et al., 2014), thus are suggested as potential new phytopharmaceuticals for use in lung inflammation and psoriasis. Feverfew (Tanacetum parthenium) is a phytopharmaceutical used for RA and for prevention of migraine, with some evidence for clinical efficacy in the latter; the sesquiterpene lactone, parthenolide (Fig. 28.5) is considered to be the main active constituent (Herbal Medicines, 2013), which has shown a range of anti-inflammatory mechanistic effects (Table 28.1). Feverfew extracts standardized to contain parthenolide at specified levels are used as phytopharmaceticals for some inflammatory disorders and may also have potential for use against inflammatory bowel disease, since parthenolide reduced TNF-α and IL-1β and produced beneficial effects in an animal model of colitis (Zhao et al., 2012). Another known phytopharmaceutical that may have similar applications is burdock (Arctium lappa), as a fraction enriched with the sesquiterpene lactone onopordopicrin reduced edema, MPO activity, TNF-α levels and COX-2 expression in an animal model of colitis (de Almeida et al., 2013). Brazilian arnica (Lychnophora trichocarpha) extracts and sesquiterpene lactone constituents have also been associated with antiinflammatory effects (Table 28.1) and are of interest as a potential phytopharmaceuticals for topical application to reduce inflammatory pain.

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Although the phytopharmaceutical ginger (Zingiber officinale) has been associated with anti-inflammatory effects (Herbal Medicines, 2013), another species in this genus, Zingiber zerumbet has been of interest for its potential to prevent cancer, as the sesquiterpene zerumbone from this species reduced PGE2 and COX-2 expression in colonic mucosa (Murakami and Ohnishi, 2012) and may modulate inflammation relevant to chemoprevention (Murakami and Ohnishi, 2012). From the pharmaceutical perspective, sesquiterpenes from Artemisia species (a source of useful pharmaceuticals against malaria) have shown activities against inflammatory and immune responses (Ho et al., 2014; Table 28.1), thus may have potential for drug repositioning.

28.3.2. Diterpenoids Diterpenoids (diterpenes) occur in many plants used for their reputed medicinal properties and have been of interest as potential phytopharmaceuticals in the form of standardized plant extracts. One of the most widely investigated phytopharmaceuticals is Ginkgo biloba, which has produced promising outcomes on memory and cognitive functions in numerous clinical studies (Howes and Perry, 2011). The pharmacological effects of G. biloba leaf have been attributed to the flavonoid and terpene constituents, which include a series of unusual diterpenes, the ginkgolides, which have a range of biological effects relevant to cognitive and circulatory disorders. Ginkgolides A, B, and C (Fig. 28.6) are platelet activating factor antagonists, which is considered to contribute to their effects on cognitive disorders (Howes and Houghton, 2012) and they inhibit NF-κB signaling and iNOS activation (Salminen et al., 2008). Further evidence for their relevance for CNS disorders was revealed in a study that concluded ginkgolide B could inhibit brain edema and neurological deficits in an animal model of cerebral ischemia, effects that were linked with inhibition of NF-κB activation and proinflammatory cytokine production (Gu et al., 2012). Ginkgolide A could be explored for a potential role in managing vascular complications in diabetes, since it improved high glucose-induced vascular inflammation via modulation of the STAT-3 pathway (Zhao et al., 2015). Another herbal medicine (marketed as a dietary supplement in the United States) that contains biologically active diterpene constituents is Andrographis paniculata. Extracts standardized to contain labdane diterpenes reduced liver inflammation (Chua, 2014). The diterpenes that occur in the leaves of this species include andrographolide (Fig. 28.6), which suppresses receptor activator of NF-κB ligand-induced osteoclastogenesis via attenuation of NF-κB and extracellular signal-regulated kinase/MAPK signaling pathways in vitro (Laev and Salakhutdinov, 2015); and it inhibits TNF-α release (Low et al., 2015) and Il-6 and IL-17 production (Kou et al., 2014). Andrographolide, dehydroandrographolide, and neoandrographolide also interfere with COX activity and inflammatory cytokine release, and it is suggested that the anti-inflammatory effects of andrographolide may be

FIGURE 28.6 Chemical structures of some diterpenoids associated with anti-inflammatory activity.

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fundamentally due to down-expression of genes in the inflammatory cascade (Parichatikanond et al., 2010). Both andrographolide and the 14-deoxy-11,12-didehydro-derivative, have been of interest for asthma and other inflammatory airway disorders and synthetic derivatives of the latter diterpene have been patented as potential new anti-inflammatory pharmaceuticals (Aromdee, 2014). In a RCT in RA patients, A. paniculata extract (containing 30 mg andrographolide; three times daily for 14 weeks) was concluded to significantly reduce RA symptoms (Laev and Salakhutdinov, 2015), thus may also of value to explore in further RCTs. The diterpene triptolide from Tripterygium wilfordii inhibits the production and gene expression of a range of cytokines and chemokines, including IL-1β, IL-6, TNF-α, and IFN-γ, in vitro, while it also decreases PGE2 production via COX-2 gene suppression (Yang et al., 2013). Other mechanistic effects of triptolide include inhibition of NF-κB signaling via suppression of IκBα phosphorylation (Salminen et al., 2008) and it may modulate the T-cell response as it could also inhibit IL production in vitro (Ku and Lin, 2013). Furthermore, triptolide suppressed production and mRNA levels of promatrix metalloproteinase (MMP)-1 and -3 on human synovial fibroblasts induced by IL-1α, and cytokine-induced MMP-3, MMP-13 and aggrecanase-1 gene expression in chondrocytes and synovial fibroblasts (Yang et al., 2013). In animal models of arthritis, triptolide decreased the arthritic score and incidence, while also delaying injury onset and it increased production of the anti-inflammatory cytokine transforming growth factor (TGF)-β (Yang et al., 2013). However, triptolide is associated with hepatotoxicity (Jin et al., 2015), so chemical derivatives of this diterpene may need to be developed to reduce such toxic effects. Other diterpenes investigated as anti-inflammatory phytopharmaceuticals include abietic and dehydroabietic acids, which occur in the oleoresin of the grand fir (Abies grandis) and the lodgepole pine (Pinus contorta) as they were anti-inflammatory in macrophages, via PPAR-γ activation, while dehydroabietic acid also suppressed proinflammatory mediators, including TNF-α, and reduced macrophage infiltration in adipose tissue in a mouse model of diabetes and obesity (Hirai et al., 2010), thus may be of interest to modulate inflammatory processes in metabolic disorders. Salvia miltiorrhiza root extract was active against leukocyte eicosanoid formation in vitro (Howes et al., 2000) and the tanshinone diterpene constituents were revealed as the constituents that inhibit prostaglandin and leukotriene production (Howes and Houghton, 2009). Tanshinone IIA (Fig. 28.6) also inhibits NF-κB signaling via inhibition of IκBα phosphorylation (Salminen et al., 2008). Also from the genus Salvia is the diterpene carnosol, which inhibits NF-κB signaling via inhibition of IκBα phosphorylation, and reduces iNOS expression and NO production (Salminen et al., 2008); the related diterpene carnosic acid also modulates the NF-κB pathway and is of interest to control skin inflammation (Oh et al., 2012). The diterpene, tehuanine G, from another Salvia species, S. herbacea, showed comparable anti-inflammatory activity to indomethacin in an ear edema model (Bautista et al., 2012). Salvinorin A from S. divinorum inhibits leukotriene synthesis in experimental models of inflammation (Rossi et al., 2016), however it also has psychoactive effects, which may restrict its therapeutic potential. In general however, the genus Salvia appears to be a useful source of anti-inflammatory diterpenes. The steviol glycosides, diterpenes that occur in Stevia rebaudiana leaves, have been developed as a source of natural sweeteners in the food industry, although have also been investigated for their potential health benefits beyond their role as sweetening agents. Both steviol (Fig. 28.6) and the glycoside stevioside attenuated proinflammatory cytokine (TNF-α, IL-1β, IL-6) production via modulation of the IκBα/NF-κB pathway (Boonkaewwan and Burodom, 2013). Stevioside also inhibited proinflammatory cytokines, COX-2 and iNOS expression and the NF-κB pathway following lung injury in vivo (Yingkun et al., 2013); its ability to modulate the NF-κB pathway following muscle injury (Bunprajun et al., 2012) suggests it may have potential for use as a dietary supplement to aid recovery after injury. Other diterpenes from S. rebaudiana, austroinulin and the 6-O-acetyl derivative, block NF-κB activation, inhibit iNOS expression and NO production (Cho et al., 2013), providing further evidence that diterpenes from Stevia species may be useful anti-inflammatory compounds for therapeutic applications. The Stevia diterpenes may have advantages over other diterpenes for development as potential therapeutic agents, as in addition to their documented mechanistic effects, they have been evaluated for their toxicological effects in humans for their role as sweeteners in foods. Other food-derived diterpenes include those in Curcuma rhizomes and coffee (Coffea species), although they have not been extensively explored for use as phytopharmacuticals or nutraceuticals. There has been much research on the anti-inflammatory effects of curcumin from Curcuma species (see 28.5.), yet diterpenes (labda-8(17)-12-diene-15,16-dial, calcaratarin A, communic acid and copallic acid) from C. manga inhibit COX, with some selectivity against COX-2 (Liu and Nair, 2011). Kahweol, a diterpene from coffee, inhibits COX-2 expression and MCP-1 secretion (Ca´rdenas et al., 2011), and it inhibits NF-κB/STAT-1 pathways (Shen et al., 2010). However, any relevance to human health, either via dietary consumption or as a potential nutraceutical, requires investigation. Other diterpenes associated with anti-inflammatory effects via inhibition of cytokine release include grandiflorolic, kaurenoic, and trachylobanoic acids from sunflower (Helianthus annuus) heads (Dı´az-Viciedo et al., 2008);

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goshonoside F5 from Rubus chingii fruit (He et al., 2015); and acanthoic acid from Eleutherococcus divaricatus (Qiushi et al., 2015). The latter diterpene also inhibited NF-κB activation in gingival fibroblasts (Wei et al., 2015), thus has been of some interest for use against periodontitis and gingivitis.

28.3.3. Triterpenoids Triterpenoids (triterpenes) are widely distributed in nature and can occur as the C30 aglycones or as esters or glycosides (Fig. 28.7). Triterpenes from liquorice (Glycyrrhiza species) root include glycyrrhizin (a triterpene glycoside) and glycyrrhetinic acid, which have been investigated for their therapeutic potential to manage inflammatory lung diseases such as asthma, as they act via the P13K/Akt/GSK3-β pathway to reduce cytokine production, while 18β-glycyrrhetinic acid also blocks inflammation by causing dissociation of the glucocorticoid receptor (Kao et al., 2010). A study investigating G. uralensis root hot water extract showed it could suppress NF-κB activation and upregulation of IL-8 and intercellular adhesion molecule (ICAM)-1 in human pulmonary epithelial cells in vitro (Lee et al., 2009a), which is further evidence for its potential role in managing asthma. It is considered that glycyrrhizin inhibits NF-κB signaling via inhibition of IκBα phosphorylation (Salminen et al., 2008). Glycyrrhizin has also been of interest for inflammatory joint diseases, as in rat paw edema, it showed antiarthritic and anti-inflammatory effects (Yang et al., 2013). One of the major challenges with the use of natural product glycosides to manage inflammatory conditions, is that they may be hydrolyzed in the gut and the original biologically active constituent may not be adequately absorbed in this form. Indeed, glycyrrhizin is reported to have low oral bioavailability and poor mucosal permeability (Wang et al., 1994; Yamamura et al., 1995) and it is hydrolyzed by intestinal microflora to the aglycone, glycyrrhetinic acid (Shin et al., 2007; Wang et al., 1994). The phytopharmaceutical Centella asiatica, a traditional Ayurvedic medicine, contains triterpenes that are considered to be principally responsible for the anti-inflammatory effects of this plant. The triterpene glycoside madecassoside reduced NO, PGE2, TNF-α, IL-1β, and IL-6 in vitro via inhibition of protein and mRNA levels of iNOS and COX-2, in addition to NF-κB and DNA binding (Yang et al., 2013). In a mouse model of arthritis, madecassoside suppressed inflammatory cell infiltration, T-cell proliferation and reduced levels of PGE2, TNF-α and IL-6, while increasing anti-inflammatory cytokine levels, including that of IL-10; these effects were accompanied by reductions in paw edema, the arthritic score and pathological damage to joint tissue (Yang et al., 2013). Asiatic acid also occurs in C. asiatica and is anti-inflammatory via inhibition of iNOS, COX-2, IL-6, IL-1β and TNF-α expression through downregulation of NF-κB activation (Yun et al., 2008) and it suppresses inflammation and nociception in vivo (Huang et al., 2011). Thus, C. asiatica triterpenes are also of interest to manage inflammatory conditions. Other triterpene glycosides that have been associated with many biological activities, including anti-inflammatory effects, are the ginsenosides that occur in ginseng (Panax species) root (Lee and Lau 2011). However, ginseng is more widely used as an adaptogen rather than for a specific role against inflammatory conditions. Frankincense (Boswellia serrata) resin has been widely used in the perfume industry, but its constituent triterpenes show anti-inflammatory effects, so are potentially useful phytopharmaceuticals. An extract containing the triterpene 3-O-acetyl-11-keto-β-boswellic acid from B. serrata has been patented as a treatment for inflammatory pain, since it inhibits inflammation in vivo and suppresses NO and IL-1β release and 5-LOX activity; the inventors suggest a combination of this extract with curcuminoids may be formulated as a dietary supplement for inflammatory joint pain (Guimara˜es et al., 2014). The triterpene boswellic acid inhibits NF-κB signaling via

FIGURE 28.7 Chemical structures of some triterpenoids associated with anti-inflammatory activity.

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inhibition of IKK-α and -β (Salminen et al., 2008), and when taken for six months with methylsulfonylmethane, this formulation improved symptoms in a RCT with 120 arthritis participants (Notarnicola et al., 2016). Ursolic acid is widely distributed in many plants and has been of interest to manage inflammatory joint disease. This triterpene (50 mg/kg orally, daily for 10 days) suppressed acute inflammation and adjuvant-induced chronic arthritis in vivo (Laev and Salakhutdinov, 2015). Ursolic acid inhibits activation of NF-κB signaling (Salminen et al., 2008), it attenuates the expression of iNOS and COX-2 and suppresses activation of PKC, extracellular signal-regulated kinase (ERK)-1/2, JNK1/2 and p38 MAPK, and the binding of activator protein-1 (AP-1) to the COX-2 promoter (Murakami and Ohnishi, 2012). However a different study concluded that this triterpene may have proinflammatory effects, since it induced NO and TNF-α production in mouse macrophages, and further research revealed that ursolic acid stimulates macrophage migration inhibitory factor protein and induces activation of ERK1/2 (Murakami and Ohnishi, 2012). Therefore more research is needed to evaluate the anti- or proinflammatory effects of ursolic acid, particularly in vivo. Other triterpenes of therapeutic interest for their ability to inhibit the NF-κB pathway include betulinic acid from birch (Betula species) and lupeol, which is widespread in the plant kingdom (Salminen et al., 2008). Lupeol (50 mg/kg daily for 4 weeks) could ameliorate paw edema and reduce proinflammatory cytokine (TNF-α, IL-1β and IL-6) levels, while increasing the anti-inflammatory cytokine IL-10 in a rat model of RA (Laev and Salakhutdinov, 2015). Another triterpene, celastrol from Celastrus species, significantly reduced levels of chemokines as well as cytokines (TNF-α, IL-1β) in a rat model of RA, and it suppressed inflammatory arthritis and reduced bone and cartilage damage in joints (Laev and Salakhutdinov, 2015). Triterpenes that have been of interest for development as pharmaceuticals include the crossoptines A and B, from Crossopteryx febrifuga root, which were anti-inflammatory in vivo and have been patented as potential new pharmaceuticals for oral and topical use (Guimara˜es et al., 2014). Pulchinenoside triterpenes, which occur in Pulsatilla species, and synthetic derivatives are subject to a patent for controlling pain and inflammation, while triterpene glycosides from a species of holly from South America (Ilex paraguariensis), often prepared as a caffeinated tea (Mate´) have been patented for similar applications (Guimara˜es et al., 2014). A range of other triterpenes derived from natural products (including maslinic and oleanolic acids) and their synthetic derivatives have also been patented as potential new analgesic and inflammatory pharmaceuticals or dietary supplements (Guimara˜es et al., 2014).

28.4. STEROIDAL AGLYCONES AND SAPONINS Steroidal saponins are less widely distributed in the plant kingdom, compared to terpenoids, although their chemical structures have been useful for the development of pharmaceuticals with steroidal activity, such as diosgenin (Fig. 28.8) from yams (Dioscorea species) and hecogenin from sisal leaves (Agave sisalana), with saponins derived from the latter useful for partial synthesis of anti-inflammatory corticosteroid drugs. Steroidal saponincontaining plants have therefore been of greater interest for use as phytopharmaceuticals, rather than as nutraceuticals. Steroidal saponins that have shown anti-inflammatory activity against ear edema in vivo, include dioscin, gracillin (from Dioscorea species) and smilaxins A and B from Smilax species, although they were less potent than indomethacin and hydrocortisone (Kim et al., 1999). However, cantalasaponin-1 from Agave americana and a

FIGURE 28.8 Chemical structures of some steroidal compounds associated with anti-inflammatory activity.

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withanolide, virginol A, from Physalis virginiana were more or equally potent compared to indomethacin, respectively, against ear edema in vivo (Maldonado et al., 2010; Monterrosas-Brisson et al., 2013). Dioscin is suggested to mediate anti-inflammatory effects by suppressing TNF-α-induced VCAM-1, ICAM-1 and endothelial lipase expression via blocking the NF-κB pathway (Wu et al., 2015). The aglycone of dioscin, diosgenin, attenuates inflammatory mediators in macrophages and adipocytes, thus has been suggested as useful against inflammatory changes in obese adipose tissues (Hirai et al., 2010a). Diosgenin also inhibited IL-1β-induced NO and PGE2 production and expression of MMP-3, MMP-13, iNOS and COX-2 in osteoarthritis chondrocytes, so is of interest for use in osteoarthritis (Wang et al., 2015). Sarsaparilla (Smilax species) root was used traditionally against inflammatory joint and skin conditions and extracts have shown anti-inflammatory effects in vivo (Herbal Medicines 2013). Steroidal saponins from S. china inhibit COX-2 activity in vitro, while (25R) 26-O-β-D-glucopyranosyl3β,20α,26-trihydroxyfurostan-5,22-diene 3-O-α-L-rhamnopyranosyl-(1-2)-(α-L-rhamnopyranosyl-(1-4))-O-β-Dglucopyranoside also inhibited TNF-α production in macrophages (Shao et al., 2007). The traditional Ayurvedic medicine Withania somnifera (ashwagandha; also known as Indian ginseng) contains a series of steroidal saponins known as withanolides and root extracts have shown anti-inflammatory effects in several studies, including in animal models of colitis, systemic lupus erythematosus and arthritis and they reduce markers of inflammation (including IL-1 and TNF-α) in vivo, while the dimeric withanolide, ashwagandhanolide, could inhibit COX-2 activity (Herbal Medicines, 2013; Howes and Houghton, 2009). Another steroidal compound from W. somnifera, withaferin A (Fig. 28.8), inhibits NF-κB activation in response to different stimuli and is antiinflammatory in animal models of inflammation and in in vitro models of cystic fibrosis and vascular inflammatory disease (Lee et al., 2012; Maitra et al., 2009; Vanden Berghe et al., 2012). Ophiopogon japonicus root, used in TCM for inflammatory conditions, also contains steroidal constituents with anti-inflammatory effects in vitro and in vivo, including ruscogenin (Fig. 28.8) and ophiopogonin D (Kou et al., 2005). Ruscogenin could also suppress inflammation and renal abnormalities in an animal model of diabetes via inhibition of NF-κB-mediated inflammatory gene expression (Lu et al., 2014) and it downregulated NF-κB-stimulated inflammatory responses in experimental animal models of stroke (Gua et al., 2013) and acute lung injury (Sun et al., 2012), thus may be relevant to explore further for its role in managing diabetes complications, stroke and lung injury. Other steroidal saponins that have shown anti-inflammatory effects, so could be of interest as phytopharmaceuticals for inflammatory disorders, include anemarsaponin B from the TCM Anemarrhena asphodeloides rhizome (Kim et al., 2009), and minutoside B from fenugreek seeds (Trigonella foenum-graecum) (Kawabata et al., 2011).

28.5. CURCUMIN Curcuminoids such as curcumin (Fig. 28.9) occur in turmeric (Curcuma longa) root and their biological activities have been extensively investigated, including for their role as nutraceuticals with potential against cancer, diabetes and inflammation; the mechanistic and clinical effects of curcuminoids have been reviewed previously (Hatcher et al., 2008; Schaffer et al., 2011; Shehzad et al., 2011; Howes and Simmonds, 2014; Lu and Yen, 2015; Ghosh et al., 2015; Pulido-Moran et al., 2016). Curcumin is widely documented as anti-inflammatory with relevance for chemoprevention and has been suggested to modulate eicosanoid biosynthesis and to inhibit COX-1, COX-2 and LOX (Howes and Houghton, 2003) and in response to a tumor promoter (12-O-tetradecanoylphorbol13-acetate), curcumin inhibits expression of COX-2 via inactivation of NF-κB, which may be mediated by it blocking ERK1/2 and p38 MAPK (Surh and Na, 2008). Modulation of inflammatory processes has also been suggested to explain the potential role of curcumin as a nutraceutical to manage obesity and metabolic disorders such as diabetes. In an animal model of obesity, curcumin reduced macrophage infiltration into adipose tissue and decreased hepatic NF-κB activation; effects on adipose tissue are suggested to be due to curcumin activating

FIGURE 28.9 Chemical structures of curcumin and resveratrol, nutraceuticals associated with anti-inflammatory activity.

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Wnt/β-catenin signaling and suppressing MAPK and NF-κB activation pathways, with the latter via suppression of IκB (Siriwardhana et al., 2013). In adipocytes, curcumin also decreased TNF-α, IL-1β, IL-6 and COX-2 gene expression (Siriwardhana et al., 2013). In patients with metabolic syndrome, those receiving curcuminoids (n 5 59) in a RCT at a dose of 1 g daily (with 10 mg piperine to improve bioavailability), had reduced serum CRP levels (Panahi et al., 2015), indicating curcuminoids may have clinical benefits on inflammatory status in metabolic disorders. Epidemiological data associates consumption of curry, a source of turmeric that contains curcumin, with a lower prevalence of Alzheimer’s disease (Howes and Perry, 2011). Although curcumin has been associated with numerous mechanistic effects (including anti-inflammatory activity) that suggest it may improve cognitive functions in dementia patients, in a small RCT, a daily dose (1 g or 4 g) of curcumin did not protect against cognitive decline in 27 patients with Alzheimer’s disease (Howes and Perry, 2011). A separate RCT with 36 dementia patients (2 g or 4 g curcuminoids daily for 48 weeks) also concluded a lack of efficacy (Venigalla et al., 2016). However, plasma levels of curcumin were below therapeutic concentrations in RCTs that report a lack of effects on cognitive functions (Venigalla et al., 2016). In contrast, positive outcomes for cognitive functions were reported in healthy elderly volunteers administered with a higher bioavailability preparation of curcumin (400 mg/day for four weeks) in a RCT (Venigalla et al., 2016), although inflammatory markers were not assessed. Curcumin has also been considered as a nutraceutical to maintain joint health, as indicated from a range of studies (Mobasheri et al., 2012). Curcumin (110 mg/kg daily for 28 days) delayed and improved joint abnormality and injury in rodents with collagen-induced arthritis and it was suggested that these effects may be mediated by suppression of B cell-activating factor (Laev and Salakhutdinov, 2015). In two RCTs, participants with knee osteoarthritis (n 5 19 for both studies) administered 1500 mg curcuminoids daily showed improvements in clinical symptoms and in one study, significantly lower serum levels of IL-4, IL-6 and high-sensitivity C-reactive protein were also observed; however, other inflammatory parameters were not significantly affected, thus the improvements in clinical symptoms were not attributed to the anti-inflammatory effects of the curcuminoids (Panahi et al., 2014; Rahimnia et al., 2015). One of the major challenges for the use of curcumin as an anti-inflammatory nutraceutical, is that it has low oral bioavailability (Hatcher et al., 2008) although it may still mediate anti-inflammatory effects in the GI tract. Indeed, curcumin is suggested to have potential therapeutic relevance against inflammatory bowel disease (Brumatti et al., 2014) and a Cochrane Database review in 2012 concluded that curcumin may be a safe and effective therapy for maintenance of remission in ulcerative colitis as adjunctive therapy with conventional antiinflammatory drugs, although larger scale RCTs are needed to confirm these benefits (Garg et al., 2012).

28.6. STILBENES One of the most common dietary stilbenes that has been the subject of numerous studies for its potential health benefits is resveratrol (Fig. 28.9), which occurs in wine and grape (Vitis vinifera) juice at concentrations of 0.0525 mg/L, but also occurs in a range of other dietary sources, including peanuts (Arachis hypogaea), pistachios (Pistacia vera) and blueberries (#5.1 μg/g) (Howes and Perry, 2011). Resveratrol may therefore be cumulatively consumed from different dietary sources but is also of interest as a nutraceutical with a myriad of biological activities and potential health benefits, including those targeting inflammatory pathways. Resveratrol modulates the arachidonic acid and NF-κB pathways and its anti-inflammatory mechanistic effects and clinical efficacy to maintain health or manage diseases have previously been reviewed, with the overall conclusion that ˇ resveratrol is generally well-tolerated in humans but bioavailability is low (Svajger and Jeras, 2012; Singh et al., 2015; Smoliga et al., 2011). Resveratrol inhibits TNF-α, IL-1β and IL-6 expression (Venigalla et al., 2016) and its ability to suppress NF-κB activation, perhaps via activation of Sirt1, is suggested to be important in counteracting microglial inflammation, of relevance in some neurological disorders (Zhang et al., 2010). Indeed resveratrol and piceatannol (occurs in many resveratrol-containing species at lower concentrations and is a metabolite of resveratrol) have been associated with benefits against Alzheimer’s disease pathology (Howes and Perry, 2011; Williams et al., 2011). Numerous RCTs have investigated resveratrol for managing diabetes or metabolic syndromes (Fu¨rst and Zu¨ndorf, 2014) and mechanistically, it targets inflammatory processes in adipose tissue, in insulin resistance and in arterial wall inflammation (Siriwardhana et al., 2013; Howes and Simmonds, 2014). Other potential uses of resveratrol and piceatannol include management of inflammatory bowel conditions, as they both reduce inflammatory mediators and symptoms in animal models of colitis (Martin and Bolling, 2015; Kim et al., 2008). Piceatannol

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also inhibits mast cell-mediated allergic inflammation (Ko et al., 2013), thus may have potential for use against allergy-induced inflammatory diseases. Resveratrol suppresses TNF-α-induced activation of NF-κB and when injected (intra-articular; 10 μM/kg daily for 2 weeks) in an animal model of osteoarthritis, it reduced the severity of cartilage lesions and was suggested to protect cartilage (Laev and Salakhutdinov, 2015). Interestingly, the 3,5-dimethyl ether derivative of resveratrol, pterostilbene, is reported to have superior anti-inflammatory potency compared to resveratrol (Choo et al., 2014), thus is of interest to investigate further for use against inflammatory diseases.

28.7. PHENOLIC ACIDS Phenolic acids are widely distributed in many plants, including those used for dietary applications, such as some herbs, fruit and vegetables. Rosmarinic acid (Fig. 28.10) is a phenolic acid that occurs in some members of the Lamiaceae family, including the herbs rosemary (Rosmarinus officinalis), sage (Salvia officinalis) and lemon balm (Melissa officinalis). Oral administration of rosmarinic acid or an extract of Perilla frutescens enriched with rosmarinic acid inhibited seasonal allergic rhinoconjunctivitis in RCTs via inhibition of the inflammatory response (Takano et al., 2004; Osakabe et al., 2004), while a spearmint (Mentha spicata) tea containing a high concentration of rosmarinic acid improved symptoms in osteoarthritis patients in a double blind study (Connelly et al., 2014). Topical application of a rosmarinic acid (0.3%) emulsion reportedly improved symptoms in atopic dermatitis patients (Lee et al., 2008). Therefore plants containing rosmarinic acid at sufficient concentrations may have a number of therapeutic applications. Caffeic acid is a dihydroxycinnamic acid and is widespread in many plants. It inhibited Fyn kinase to attenuate COX-2 expression in JB6 P1 cells and in mouse skin (Murakami and Ohnishi, 2012) and caffeic acid derivatives have been the subject of different patents to develop new anti-inflammatory compounds for therapeutic use (Silva et al., 2014b). Natural product derivatives of caffeic acid include the caffeoylquinic acids that occur in many plants such as coffee and have been associated with health benefits mediated by both antioxidant and antiinflammatory effects (Liang and Kitts, 2016). Ferulic acid (Fig. 28.10), the 3-methyl ester of caffeic acid, is also widely distributed among many plants and it inhibits TNF-α and macrophage inflammatory protein-2 production, while isoferulic acid potently inhibits COX expression; and both ferulic and isoferulic acids inhibit the production of IL-8 in cells in response to the influenza virus (Yang et al., 2013). Ferulic acid also reduces inflammation in carrageenan-induced paw edema in vivo (Yang et al., 2013) and is considered as one of the principal anti-inflammatory constituents of the TCM Angelica sinensis, since it modulated NF-κB transactivation activity in macrophages, and inhibited the expression of IL-1β, TNF-α, MMP-1 and MMP-13 in H2O2-induced chondrocytes (Yang et al., 2013). The anti-inflammatory effects of caffeic acid, ferulic acid and some other common plant hydroxycinnamic acid derivatives are also suggested as relevant in obesity and adipocyte dysfunction (Alam et al., 2016), thus have potential for use as nutraceuticals in obesity and associated metabolic disorders. Protocatechuic acid (Fig. 28.10) occurs in plants used as nutraceuticals and phytopharmaceuticals, such as olives (Olea europaea), Hibiscus sabdariffa and grapes, and it suppresses TNF-α, IL-1β, iNOS, and COX-2 expression

FIGURE 28.10 Chemical structures of some phenolic acids associated with anti-inflammatory activity.

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via modulation of the NF-κB and MAPK pathways and reduces inflammatory mediator production in vivo (Semaming et al., 2015) including pulmonary inflammation in animal models of acute lung injury (Wei et al., 2012). A separate study concluded that protocatechuic acid may activate PPAR-γ, as was observed in gingival fibroblasts in vitro (Wang et al., 2015a), thus this phenolic acid may have potential to alleviate gingivitis. A role for protocatechuic acid against inflammatory brain disorders has been suggested, since it inhibited NF-κB and MAPK signaling pathways in microglia (Wang et al., 2015b). A derivative of protocatechuic acid is vanillic acid, which occurs in some edible plants and is used as a flavoring agent. Vanillic acid reduced inflammatory pain in vivo via inhibition of proinflammatory cytokine production and NF-κB activation (Calixto-Campos et al., 2015) thus may have a potential role as a phytopharmaceutical or nutraceutical, beyond its use for flavoring. Gallic acid occurs in many plants including some fruit and it can suppress NF-κB activation and mast cellderived inflammatory allergic reactions via blocking histamine release and proinflammatory cytokine expression; thus it has been investigated for its ability to reduce inflammation in ulcerative colitis and Crohn’s disease, and in allergic rhinitis and asthma (Pandurangan et al., 2015; Kim et al., 2006). Ellagic acid (Fig. 28.10) is the depside of gallic acid, which can also occur in plants in the form of hydrolysable tannins, such as the gallotannins and ellagitannins. Ellagic acid and related hydrolysable tannins occur in various fruit, including pomegranates (Punica granatum), which have been considered to have potential health benefits against some chronic diseases such as cancer and have demonstrated a range of biological activities in vitro and in vivo, including antioxidant and anti-inflammatory effects (Landete, 2011; Lansky and Newman, 2007). Mechanistically, ellagic acid reduces COX expression, IL-1β- and TNF-α-induced activation of MAPK and modulates the NF-κB pathway (Usta et al., 2013). In animal studies, pomegranate juice/extracts decreased expression of vascular inflammation markers in response to an atherogenic diet, reduced inflammation in an animal model of arthritis and were anti-inflammatory in the GI tract, although clinical studies are lacking to demonstrate such effects in humans (Colombo et al., 2013; Landete, 2011). Acute consumption of pomegranate extract (800 mg) could not reduce IL-6 4 hours after consumption; and other studies suggest that ellagic acid and ellagitannins have low bioavailability and their metabolites (the urolithins) have different biological effects (Landete, 2011). It is therefore apparent that more research is needed to understand the role of ellagic acid and ellagitannins in human health, as indicated by epidemiological studies.

28.8. CONCLUSION A diverse array of phytochemical classes (as illustrated in Figs. 28.128.10) have been associated with a myriad of mechanistic effects that target inflammatory processes, although the evidence for their efficacy is often variable. Although the use of phytochemicals as single chemical entities (e.g., curcumin) as nutraceuticals or phytopharmaceuticlas, or indeed as pharmaceuticals, may be one approach to maintain health or alleviate disease symptoms, the use of phytochemicals as mixtures in the form of extracts is still valuable. For example, apple peel extracts containing a mixture of phytochemical classes (phenolic acids, flavonol glycosides, flavan-3-ols, and procyanidins) could downregulate inflammatory mediators, (Simmonds and Howes, 2016), suggesting they may act polyvalently to produce an overall anti-inflammatory effect. Furthermore, if more than one phytochemical in a plant is responsible for the clinical effects, it may be too simplistic to conceive that a single phytochemical has the same effect or efficacy. Indeed, more research is needed to identify the active constituent(s) of anti-inflammatory plants, their modes of action, their polyvalent effects, while also understanding more about their pharmacokinetic and pharmacodynamic properties. The issue of bioavailability is also essential to understand when evaluating and selecting the most relevant anti-inflammatory phytopharmaceuticals and nutraceuticals. Also key for the safety, quality and efficacy of phytopharmaceuticals or nutraceuticals is their authentication and standardization so that all those investigated in clinical trials to assess their anti-inflammatory effects are phytochemically-characterized and standardized to contain known levels of the active constituents. Indeed, one of the main challenges for the use of plants and their constituents therapeutically is their standardization to contain relevant concentrations of known active constituents that correlate with the appropriate clinical doses. In conclusion, plants and their phytochemical constituents have shown numerous biological activities that indicate relevance to maintain health via modulation of inflammatory processes; although in general, studies in humans (clinical and epidemiological) that associate phytochemicals with health and reductions in risk for some diseases appear promising but are not yet conclusive; thus the most promising anti-inflammatory phytochemicals must be scrutinized more extensively.

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ACKNOWLEDGMENTS The author would like to thank the late Dr Nigel C. Veitch, Royal Botanic Gardens, Kew, for expertly drawing some of the chemical structures.

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29 Fermented Milk in Protection Against Inflammatory Mechanisms in Obesity Ramesh Pothuraju1,2, Vengala Rao Yenuganti1, , Shaik Abdul Hussain1, and Minaxi Sharma3 1

National Dairy Research Institute, Karnal, Haryana, India 2University of Nebraska Medical Center, Omaha, Nebraska, United States 3ICAR-Central Institute of Post-Harvest Engineering and Technology (CIPHET), Ludhiana, Punjab, India

29.1. INTRODUCTION Nowadays, obesity is considered to be an epidemic and is an important challenge in the realm of public health. In the United States alone, an estimated one-third of children and adolescents are overweight and obese (Ogden et al., 2014). According to the World Health Organization (WHO) , the number of obese adults has more than doubled since 1980. In 2014, almost 13% of adults (aged 18 years) were considered to be obese (WHO, 2015). Obesity is defined in terms of body mass index (BMI), a person’s weight in kilograms divided by the square of height in meters. Obesity predominantly occurs due to sedentary life style, environmental and genetic factors. Excess caloric intake combined with less calorie expenditure contributes towards development of metabolic diseases such as insulin resistance (IR), type-2 diabetes mellitus (T2DM), retinopathy, nephropathy, cardiovascular disease and even cancer (Reaven, 2002). In normal physiological conditions, excessive dietary calories accumulate in the adipose tissue (AT) and are stored in the form of lipid droplets. Lipid droplet formation inside the AT involves multiple steps viz., (1) After absorption of lipid molecules (triglycerides, TG), are incorporated into the chylomicrons and enters into the circulation where degraded into free fatty acids (FFAs) and glycerol by circulatory lipoprotein lipase (LPL), (2) FFAs along with glucose enter into the AT and are converted into lipid droplets, (3) Lipids droplets are protected by the protein perilipin from the hormone sensitive lipase. AT is classified as white and brown adipose tissue. White AT is either subcutaneous or visceral. The visceral adipose tissue surrounds organs and can be subdivided into omental, mesenteric, and retroperitoneal; whereas brown AT is present in the neck and supraclavicular area, and along the spinal cord. An increase in lipid content of AT results in increased size (hypertrophy) and number (hyperplasia) of droplets. Hypertrophy in AT is closely linked to metabolic disease. In hypertrophic condition, less vascularization (indicating lower number of blood vessels) results in hypoxia that leads to recruitment of macrophages, T lymphocytes and other immune cells. The resultant inflammation leads to release of various proinflammatory cytokines. Under conditions of obesity, AT also stores fat molecules and some of the FFAs are redirected to the liver causing dyslipidemia that is characterized by high levels of TG molecules. Further, circulatory FFAs are accumulated in other organs such as muscle, pancreas, liver, heart and kidney. Increased levels of FFAs in the circulation lead to the inhibition of insulin action which results in IR. IR is a hallmark of T2DM in which less or abnormal secretion of insulin occurs from the pancreas (Arner and Langin, 2014). In the IR state, serine phosphorylation (instead of tyrosine) of insulin receptor substrate impairs the downstream signaling pathways event like translocation of glucose transport (GLUT) receptors to the cell membrane for the uptake of glucose molecules (Arner et al., 2011; Ryden et al., 

Both the authors have contributed equally.

Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00029-9

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2013). Current treatments for obesity and its associated disorders include diet control, physical activity, surgery and pharmacological interventions, are often partially effective, and may have observed side effects (Yin et al., 2010). Another approach is the use of alternative therapies in the form of changing the microbiota in the gut to alter inflammation and associated pathologies. This has led to an interest in fermented milks (functional foods) by the microorganisms (lactobacilli) as a tool for obesity management.

29.2. IMPORTANCE OF FUNCTIONAL FOODS Functional foods or health foods are dietary substances that provide some health benefit beyond basic nutrition. Health benefits associated with functional foods depend on the bioactive components they contain (Shortt et al., 2004). With an annual average growth rate of about 8.5%, the global functional food market is expected to exceed $305.4 billion by 2020 (www.prnewswire.com). Functionality in foods is either inherent or can be incorporated through various means like fortification, enrichment, alteration and enhancement. Functionality in foods has been developed or enhanced in almost all food categories; foremost among these are fermented milks (FMs) that dominate the commercial market. FMs contain several health-promoting components including beneficial bacteria and their metabolites, bioactive peptides and other functional molecules derived during fermentation. The technique of fermenting the milk dates back to time immemorial and is regarded as the best way to preserve milk while improving its nutritional status. Lactic acid bacteria (LAB) species belonging to the genera Leuconostoc, Lactobacilli, Streptococci, and Lactococci are predominantly used for fermenting milk. In some cases, a few yeast species are also used in combination with LAB to ferment milk. The current tendency is to supplement starter cultures with probiotics to improve the therapeutic potential of the FMs. Health benefits like enhancement of immunity (Meng et al., 2016), lowering of cholesterol (Malpeli et al., 2015), regulation of blood pressure (Ivey et al., 2015), cancer prevention (Ghoneum and Felo, 2015), antidiabetic effects (Ostadrahimi et al., 2015), alleviation of lactose intolerance (de et al., 2015) and constipation (Liu et al., 2015) were reported upon consumption of fermented milk products (de et al., 2015; Ghoneum and Felo, 2015; Zhu et al., 2000; Douglas et al., 2013). With increasing health awareness among consumers coupled with advancements in food manufacturing technology and processes, the future for functional fermented foods appears to be very promising. The only challenge in positioning these products in the commercial markets is the taste and basic composition of the fermented milk, besides the price; indeed, functional fermented foods should be designed by keeping these factors in view.

29.3. ROLE OF GUT MICROBIOTA IN HEALTH In the body, gut mucosa is a protective layer that keeps out invading organisms, referred as gut microbiota (Caesar et al., 2010). Healthy human gut microbiota comprise around 1012 microbes in which Bacteroidetes are the major phyla and with fewer Firmicutes. Dysbiosis (alteration of gut microflora) of aforementioned phyla occurs in obese and lean subjects (Ley et al., 2005, 2006) and their role in controlling or regulating weight is still controversial (Arumugam et al., 2011). Several studies demonstrated that probiotics, which are “live microorganisms that when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2002), play a significant role in overall health of both humans and animals. The health-promoting effects of probiotics such as lactobacilli and bifidobacteria species are associated with their viability during storage, interaction through the gastro-intestinal (GI) tract and their growth and metabolic activity by modulation of functional ingredients/ additives. Because of these properties, functional dairy products are quite frequently enriched with probiotics (Saxelin et al., 2003). Several probiotic lactic acid organisms either individually or as cocktails have been studied for their antiobesity, IR, inflammation and/or hepatic steatosis (Pothuraju et al., 2016). Though the consumption of probiotics has been suggested to improve gut health and host metabolic health, the underlying mechanisms are poorly understood. Several milk products fermented by probiotics (Lactobacillus rhamnosus NCDC17, Lactobacillus plantarum NCDC625, Lactobacillus gasseri SBT2055, Lactobacillus plantarum KY1032, Lactobacillus rhamnosus PL60, and Lactobacillus rhamnosus GG) have shown different effects on gut microbiota, obesity, IR and inflammation (Hamad et al., 2009; Lee et al., 2006; Park et al., 2013; Pothuraju et al., 2015, 2016; Sakai et al., 2013). Indeed, the role of gut microbiota in obesity seems inconclusive and often contradictory. In a study, Backhed et al. (2004) reported that the colonization of germ-free (GF) mice with conventional gut microbiota results to a significant increase in the fat mass accumulation. Another study by the same group demonstrated that the feeding of a high

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fat diet (HFD) to GF mice protected them from diet-induced obesity (DIO). The reason may be a decrease in the glucose absorption with increase in the fatty acid oxidation resulting in accumulation of TG levels in the AT (Backhed et al., 2004; Backhed et al., 2007; Turnbaugh et al., 2006; Velagapudi et al., 2010). Contrary to the above study, Fleissner et al. (2010) observed that the absence of gut microbiota was not protective against DIO in C3H mice. This study concluded that HFD alone cannot contribute to DIO in mice and obesity may be due to the alteration of metabolic pathways by the specific dietary components in GI tract. Therefore, confounding results were obtained possibly due to the different types of diets (Western diet), animal models (mice and rats) and treatment length. Energy imbalance is a contributor to the obesity epidemic that results in a net accumulation of positive energy (Zemel, 2004). One of the strategies to prevent net positive energy is using milks fermented by probiotics for weight management. In the following sections we will focus milk fermented by probiotics with special reference to lactobacilli in obesity-induced inflammation.

29.4. EFFECT OF FERMENTED MILK (FM) BY PROBIOTICS ON OBESITY AND INFLAMMATION Consumption of milk predates modern civilization and a major form of its use worldwide has been as FMs. The potential health benefits of FMs include ease of digestion, besides an increased and unique flavor. Major probiotic microorganisms in FMs are lactobacilli and bifidobacteria in the GI tract (Delzenne et al., 2011; Gerritsen et al., 2011) known to confer antiobesity effects on animals (An et al., 2011; Ji et al., 2012; Park et al., 2013; Pothuraju et al., 2015, 2016; Rather et al., 2014), and on humans (Kadooka et al., 2010; Kadooka et al., 2013). Thus FMs, due to their probiotic strains, can have distinct effects in obesity and associated metabolic diseases.

29.4.1. Effect of Lactobacillus plantarum on Adipocyte Regulation and Inflammation Dairy products (milk fat, cheese, yogurt) that are a major source of conjugated linoleic acids (CLAs) from linoleic acid (Alonso et al., 2003; Coakley et al., 2003; Van Nieuwenhove et al., 2007) have potential antiobesity, anticarcinogenic (Ha et al., 1990) and antiatherogenic effects (Lee et al., 1994; Park et al., 1997). As CLA is both a cis fatty acid and a trans fatty acid, other isomers of CLA have been put to use. Lee et al. (2007b) studied the effect of trans-10, cis-12 CLA produced by Lactobacillus rhamnosus PL60 (109 CFU/mouse) in DIO C57BL/6J mice model and observed a significant reduction in body weight, epididymal fat mass, and plasma glucose levels, primarily due to an increased expression of an uncoupling protein in brown adipose tissue. In nature, this uncoupling protein generates heat by thermogenesis. Thus, its upregulation is accompanied by dissipation of metabolic stores and a subsequent loss in body weight. In a separate study the same group examined the effect of L. plantarum PL62 in mice and observed an antiobesity effect; this strain also produced the trans-10, cis-12-CLA isomer. They thus concluded that trans-10, cis-12-CLA could replace CLA for the treatment of obesity. In the gut, there are several structural entities that are protective against inflammation. The intestinal epithelial layer provides a barrier from the external environment including pathogens and toxins. Inflammatory bowel diseases such as Crohn’s disease and ulcerative colitis lead to damage of the intestinal barrier (Barbara, 2006; Bruewer et al., 2006). These barriers are maintained by complexes comprising tight junction (TJ) proteins (that are formed by protein dimers), adherens junctions, desmosomes and gap junctions (Farquhar and Palade, 1963). Of these, TJ proteins maintain the barrier between adjacent cell membranes (Farquhar and Palade, 1963); their disruption, often caused by detrimental bacterial strains, is the cause of progression of inflammation in the gut. Thus populating the intestines with health beneficial micro-organisms (probiotics) can, via modulation of harmful bacteria, protect and enhance the intestinal barrier layer. As shown in Fig. 29.1, the increase in the size of the AT that is caused by the consumption of HFD, leads to less vascularization which results in infiltration of the immune cells (macrophages). Previously, Cani et al. (2007) observed that lipopolysaccharide (LPS) (released from the Gram negative bacteria) levels were significantly higher in DIO mice. The increased circulatory LPS levels damage tight junction proteins (occludin and ZO-1) of the intestinal epithelial barrier. Increased LPS levels further promote binding to Toll-like/CD14 receptors (located on monocytes, macrophages and neutrophils) (Goyert et al., 1988; Haziot et al., 1988), which in turn trigger the secretion of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukins (IL-6 and IL-1).

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FIGURE 29.1 (A) High fat diet intake contributes alteration of gut microbiota. Due to alteration in the Firmicutes/Bacteroidetes ratio, lipopolysaccharides (LPS) released from the gram-negative bacteria damage the intestinal tight junction proteins and the increased circulatory LPS levels activate proinflammatory cytokines. (B) Modulation of gut microbiota by fermented milk with lactobacilli inhibits the plasma LPS to decrease inflammation. In addition, fermented products (probiotics, kefir, and yogurt) also showed less adiposity in animal and humans.

One of the probiotics, Lactobacillus plantarum MB452, isolated from VSL#3 (is a mixture of L. plantarum, L. casei, L. acidophilus, L. delbrueckii subspecies bulgaricus, B. longum, B. breve, B. infantis and Streptococcus thermophilus) was observed to have an enhanced effect on tight junction integrity as determined by trans-epithelial electrical resistance in Caco-2 cell layers (Anderson et al., 2010). The researchers observed that occludin and its associated protein (ZO-1, ZO-2, and cingulin) expression was higher in the presence of L. plantarum MB452. In another study, L. plantarum KY1032 (2 3 107 to 2 3 1010 cfu/day, isolated from Kiimchi, Korean traditional fermented cabbage) administered for 8 weeks to HFD fed obese mice showed a significant reduction in the body weight (35% reduction) and white fat mass (31%) compared with the control group. Further, L. plantarum KY1032 also showed a positive effect in terms of reduction in proinflammatory cytokines (TNF-α, IL-6, and IL-1β) of both liver and adipose tissue (Park et al., 2013). The same group also studied the probiotic effect of L. plantarum KY1032 in reducing fat accumulation in both liver and AT in high-fat high-cholesterol-fed mice for 9 weeks (Yoo et al., 2013). They observed a decrease in the fat accumulation that might be caused by downregulation of lipid metabolism genes peroxisome proliferator-activated receptor alpha (PPAR-α), LPL levels in AT and colon. Another possible reason for reduction of fat is that these micro-organisms are actively metabolizing carbohydrates from the diet to increase competition for nutrients in the gut or releasing antimicrobial proteins. Moreover, L. plantarum KY1032 supplementation also showed lower plasma and liver cholesterol levels by preventing intestinal cholesterol absorption which was excreted into feces. In addition, KY1032 inhibited the liver acyl-CoA:cholesterol acyltransferase activity to increase the storage of cholesterol. Another organism L. plantarum LG42 (1 3 107 and 1 3 109 cfu/mL, GLAB) isolated from gajami sik hae, a type of Korean traditional fermented seafood produced by fermentation of flat fish meat, was administered to C57BL/6J mice fed on HFD for a period of 12 weeks. Oral administration of both low and high doses of LG42 showed reduction in body weight and epididymal fat along with downregulation of acetyl-co-A carboxylase and upregulation of PPAR-α and carnitine palmitoyltransferase I levels (Park et al., 2014), all of which contribute to reduction in indices of metabolic syndrome. In our recent study, milk fermented by indigenous probiotic L. plantarum (LP625) alone or in combination of herbs (Aloe vera and Gymnema sylvestre) fed to HFD mice for 12 weeks, caused a significant reduction in the epididymal fat mass, fasting blood glucose, and serum insulin levels. Further, proinflammatory cytokines (TNF-α and IL-6) at mRNA levels were significantly downregulated (Pothuraju et al., 2016).

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29.4.2. Effect of Lactobacillus gasseri on Carbohydrate/Lipid Metabolism and Inflammation The micro-organism L. gasseri (genus of L. acidophilus) is considered to be a dominant lactobacilli species in the human intestine. It has potential immunomodulatory properties (Morita et al., 2006), is antihypercholesterolaemic (Usman and Hosono, 2000), prevents pathogen interaction (Zhu et al., 2000), and enhances intestinal functions (Olivares et al., 2006). Survival of L. gasseri SBT2055 cells (LG2055, 1 to 5 3 106 colony formation units, cfu/ml) in the human GI tract with low or no lactobacilli number was initially identified by the Takahashi group in 2006. Administration of yogurt with LG2055 showed increase in lactobacilli counts in feces indicating that LG2055 survived in the GI tract (Takahashi et al., 2006). According to the latest information, LG2055 was high in milk fermented by probiotic lactobacilli. Sato et al. (2008) first reported that LG2055 (6 3 107 cfu/g) administered to Sprague Dawley (SD) rats for 4 weeks resulted in a significant reduction in the adipocyte size of mesenteric white adipose tissue (Sato et al., 2008). In addition, they observed lower leptin (a cytokine produced in proinflammatory response) levels and concluded that LG2055’s antiobesity effects should be observed in other animal models. So, in a featured study, the same group chose Zucker lean/obese rats (that show extreme obesity owing to leptin receptor deficiency) for studying LG2055 effects. Administration of LG2055 (6 3 107 cfu/g) to Zucker lean/obese rats for 4 weeks led to reduction in fat weight, size and leptin levels, whereas for lean rats there was an increase of fatty acids in the feces. On the other hand, LG2055 (5 3 1010 cfu/100 g of fermented milk), when administered over 12 weeks, was also found to reduce obesity in adults. The results indicate that oral administration of LG2055 significantly reduced abdominal, visceral and subcutaneous fat besides body weight, BMI, waist and hip circumferences and body fat mass. The positive effects of LG2055 were presumably due to secretion of its metabolites, suggesting that additional research on the metabolites released by bacilli is required to understand the molecular mechanisms of LG2055 (Kadooka et al., 2010). The antiobesity effect of LG2055 could be either from the LG2055 alone or from non-fermented milk, i.e., skim milk (because of branched-chain amino acids and angiotensin-converting enzyme inhibitors). To evaluate this hypothesis, Kadooka et al. (2011) performed the experiment on SD rats with different dietary interventions (nonfermented milk, yogurt fermented with yogurt cultures, and yogurt fermented with LG2055) for 4 weeks. Interestingly, LG2055 alone showed a significant reduction in the mesenteric adipocyte cell size as compared to the remaining two groups, strongly suggesting that the antiadiposity effect was attributed by LG2055. Further, the anti-inflammatory role of LG2055 alone was confirmed as it inhibits the upregulation of soluble intercellular adhesion molecule-1 (sICAM-1). sICAM-1 is blood inflammatory marker and its levels are usually elevated in the obese condition (Kadooka et al., 2011). A general recommendation for probiotic dairy products is that the concentration of viable cells should be 1 3 106 cfu/g to compensate for the decline in viable probiotic cells upon storage (Sanders, 2003; Vasiljevic and Shah, 2008). Kadooka et al. (2013) studied different concentrations of LG2055 (1 3 106 and 1 3 107 cfu/g) in a double-blind randomized control trial of obese adults for 12 weeks. The study demonstrated that consumption of LG2055 (106 and 107 cfu/g) showed reduction in abdominal visceral fat, BMI, waist and hip circumference, and body fat mass. These are similar to the results they observed in their mice experiments (where mice were fed LG2055 at around 108 cfu/g). The reduction of abdominal fat in this study seems to be through the interaction between LG2055 and intestinal epithelial cells (IEC, a major constitute of intestinal wall). One function of IEC is binding of microbial components through their receptors and controlling intestinal inflammation and integrity (Ey et al., 2009; Rakoff-Nahoum et al., 2004; Cario, 2008; Lee et al., 2007a). Oral supplementation of LG2055 reportedly prevented intestinal inflammation through the interaction of IEC during abdominal adiposity (Kadooka et al., 2013). As stated above, obesity is linked to low-grade inflammation in the adipose tissue due to the size of AT with HFD intake. Miyoshi et al. (2014) studied the effect of LG2055 in mice fed with 10% fat (lard rich in saturated fatty acids to contribute obesity) for 24 weeks to evaluate the anti-inflammatory action. Body weight, visceral fat and proinflammatory chemokine (CC motif) ligand 2 (CCL2, also known as monocyte chemotactic protein 1, MCP1), chemokine (CC motif) receptor 2 (CCR2) and TNF-α were downregulated in the AT by LG2055. The inhibition of CCL2 and its receptor (CCR2) was attributed to LG2055, because it is mainly released from the AT to recruit macrophages. In addition, LG2055 administration reduced the expression of fatty acid synthase (FAS, lipogenic activity) and increased PPAR-α (lipolytic activity) in the liver (Miyoshi et al., 2014). The inhibitory effects of intestinal lipid absorption by administration of LG2055 (5 3 1010 cfu/g) in Japanese hypertriacylglycerolemic (induced by oral fat meal) were studied in human subjects for 4 weeks. At the end of treatment, a significant reduction in the post-prandial serum TG and non-esterified fatty acids was observed in LG2055 group by inhibiting the pancreatic LPL activity (Ogawa et al., 2014). A recent study by the same group studied the effect of

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LG2055 in SD rats to evaluate the metabolism of carbohydrates and fats as well as serum amyloid P component (SAP, is an acute phase reactant), because there is no direct report on how probiotic lactobacilli mediated energy expenditure and inflammatory markers (cytokines and acute phase reactants). They observed that administration of LG2055 for 4 weeks enhanced energy expenditure (oxidation of carbohydrates) and decreased glucose intolerance. Further, LG2055 also contributed toward increase of caecal butyrate (produced from the intestinal gut microbiota from dietary fiber) for the activation of G-protein coupled receptor 41 for enhancing energy expenditure. Finally, proinflammatory cytokines (SAP) levels were downregulated in LG2055 group suggesting that probiotic LG2055 can prevent low-grade inflammation which is associated with obesity and its metabolic disorders (Shirouchi et al., 2016).

29.5. FERMENTED MILK PRODUCTS According to the revised definition (Standard No. 163 and 164) of FMs, the International Dairy Federation’s guidelines are as follows: FMs are prepared from milk and/or milk products like one or combinations of whole, partially or fully skimmed, concentrated or powdered milk, buttermilk powder, concentrated or powdered whey, milk protein (whey proteins, whey protein concentrates, soluble milk proteins, edible casein and caseinates), cream, butter or milk fat by the action of specific microorganisms, which results in a reduction of the pH and coagulation. Several research groups have classified FMs into different categories based on their distinguished characteristics. Kurmann (1984) classified FMs into a family tree, which was based primarily on the optimum growth requirements of the starter cultures (i.e., mesophilic and thermophilic microflora). Marshall (1984) has classified FMs based on the dominating microflora of the product, which distinguishes four types of traditional FMs, given in Table 29.1. Another classification based on the metabolite production of principal microflora of the product is given by Robinson and Tamime (1995). They classified FMs as lactic FMs (dahi, cultured buttermilk, raabaadi, acidophilus milk and yakult) yeast-lactic FMs (kefir and koumiss) and mold-lactic FMs (villi).

29.5.1. Manufacturing Procedures for Selected FMs 29.5.1.1. Yogurt Yogurt or the yogurt-manufacturing technique is an ancient one, dating back to thousands of years. Symbiotic thermophilic starter cultures, namely Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus salivarius subsp. thermophilus, were the principal starter strains used in the yogurt preparation. The symbiotic blend of yogurt starter cultures helps in fermentation. Ultimately, the typical yogurt texture and flavor are due to the starter culture combination and acetaldehyde is the principal flavor compound in yogurt (Vinderola et al., 2002). Yogurt is available in several physical forms namely set, stirred or fluid (drinking yogurt), frozen and dried yogurt. In some countries, namely USA and European countries, the presence of live yogurt culture (.106 cfu/mL) is made essential in yogurt preparations (Yildiz, 2010). The addition of probiotic organisms as adjunct cultures to improve the therapeutic value of yogurt has been a recent trend. Yoghurt is prepared from both cow and buffalo milks. The general method of its preparation involves standardization of milk to meet the minimum legal requirements as laid down by local food regulatory authorities. Standardized milk is pasteurized (90 C95 C for 1020 min) to kill all the pathogenic and almost all the spoilage-causing organisms. After heat treatment, milk is quickly TABLE 29.1

Types of Fermented Milks (FM)

Type Cultures used

Examples

I

Mesophilic lactococci and Leuconostoc strains

Buttermilk, Cultured cream, Sour milk, Scandinavian buttermilks, Taetmjolk, Kjadermilk, Villi, Smetanka, Aerin, Dahi

II

Lactobacillus strains

Bulgarian buttermilk, yakult

III

Thermophilic streptococci and lactobacilli

Yogurt, Prostokvasha, Ryazhenka, Varenets, Skyr, Ayran, Gioddu, Tan, Tulum, Torba, Kurut, Gruzovina, Leben, Ayran, Lassi, Snezhanka

IV

Mixed populations of different lactic acid bacteria and yeasts; sometimes micrococci and acetic acid bacteria

Kefir, Koumys, Brano, Hooslanka, Zhentitsa, Maconi

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cooled to 42 C followed by the addition of yogurt starter culture at 2% level. After inoculation, milk is incubated at 42 C for 46 h or up to a period until the pH reaches 4.6, followed by cooling to stop the fermentation. Yogurt obtained in this process can also be added with various fruits and flavors to enhance its aesthetic appeal. 29.5.1.2. Cheese Cheese is obtained by the combined action of rennet and starter cultures. According to literature, there exist more than a thousand varieties of cheese. It was accidentally made around 7000 years BC by an Arabian merchant during his long day’s journey as he put his supply of milk into a pouch made of a sheep’s stomach containing rennet, which became cheese and whey. Historical records pertaining to the manufacture of cheese in India (60004000 BC), Egypt (4000 BC), and Babylonia (2000 BC) indicate its widespread popularity. It has high nutritional value due to its rich content of protein, calcium, riboflavin, and vitamins A and D3. Cheese manufacture involves standardization of milk followed by pasteurization and fermentation with selected starter cultures,or renneting, leading to curd formation. The curd is cut into several portions to facilitate the drainage of whey, followed by salting. Then it is ripened at specified time temperature and humidity conditions to get the desired final cheese variety. The different varieties of cheese are manufactured by altering different aspects of cheese manufacture including starter type, fermentation conditions, renneting, cutting the curd, draining of whey, salting, ripening period and conditions, additives like herbs, spices and beneficial flora, etc. (Heller et al., 2008). 29.5.1.3. Dahi and Lassi Dahi and lassi are popular fermented milk products of the South Asian region, which resemble plain yogurt in their appearance and consistency. They are prepared by fermentation of both cow and buffalo milk using a mixed culture combination of mesophilic bacteria (Hussain et al., 2016). Diacetyl is the principal flavor component in dahi. Dahi is consumed as such or can be utilized in various forms in culinary preparations. Highly regarded for its recuperative effects by Ayurveda (Indian medicinal literature), dahi is classified into two types, which include a sweet/mildly acidic variety with a pleasant flavor, and a sour variety with a sharp, acidic flavor. Dahi preparation resembles yogurt preparation except the starter culture used and the incubation conditions employed. Generally, dahi preparation involves the use of mesophilic mixed strain starter culture containing Lactococcus and Leuconostoc species. After standardization, milk is pasteurized followed by inoculation with starter cultures and incubation at 30 C37 C for 812 h. Further it is cooled to stop the fermentation process and refrigerated until consumption. Lassi is prepared from dahi by mixing it with sugar with the help of a stirrer. Salted and spiced lassi (called chhash) can be made by adding salt and spices like ginger, coriander and mint in the form of a paste in place of sugar; the desired consistency is attained with the addition of cold water (Hussain et al., 2015). Fruits and their preparations were generally added to dahi and lassi to increase their health status. Due to their characteristic taste and color, fruits also enhance the palatability of the FMs. 29.5.1.4. Raabadi Raabadi is a cereal-based fermented milk beverage, popular in rural parts of Northern India. Raabadi is served as a refreshing and thirst-quenching beverage during hot summer seasons in these regions. The preparation of raabadi includes cereal flour (germinated/non-germinated) for its manufacture, which makes use of underutilized cereals in an effective manner. The cereals used for this purpose are barley, wheat, maize, pearl millet and sorghum. In the traditional method, raabadi is prepared by adding the cooked and cooled cereal flour to buttermilk and allowing the mixture to ferment overnight. The resultant raabadi is consumed as such. Traditional process of raabadi-making yields a product with limited shelf life (one to two days) with unpredictable quality. In the new method of raabadi manufacture, germinated cereal flour is mixed with milk (standardized to a certain ratio of fat and solid not fat) and heated to pasteurization temperature, then the mix is cooled and inoculated with suitable starter culture and incubated for sufficient time to obtain 0.9 to 1% lactic acidity. The curd obtained is broken down and mixed thoroughly with pasteurized water to which pectin, spices and salt have been added (Hussain et al., 2014). 29.5.1.5. Viili Viili, a mesophilic fermented milk, is native to Scandinavia. It is popularly known as viilia in Finland (Chen et al., 2011) and is characterized by a pleasant sharp taste and a good diacetyl aroma linked to a stringy texture. Viiliculture consists of a combination of mesophilic LAB and yeast. Yeast provides a unique flavor to viili besides promoting the LAB to produce more exopolysaccharides. The manufacturing procedure for viili involves

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standardization of milk to a fat content in the range of 1.0%3.5% followed by pasteurization and subsequent cooling to 20 C. The milk is then inoculated with a culture combination comprising Lactococcus spp. and yeast spp. (Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. lactis, and Geotrichum candidum), followed by incubation for 20 h at 20 C. After fermentation, the viili is cooled to below 5 C and the shelf life is about three weeks at refrigeration temperature. 29.5.1.6. Kefir Kefir, a fermented milk beverage obtained by fermenting milk with kefir grains, originated in the Caucasus Mountains. The word kefir is derived from the Turkish word “keyif” which means “good feeling.” Its grains can be characterized as small cauliflower florets or cooked rice, having a length of 1030 mm, irregularly shaped, white to yellowish in color, lobed, having firm texture and slimy appearance. Kefir bacteria contain several species of lactic acid bacteria, yeast, and acetic acid bacteria. The traditional method of kefir preparation involves pouring milk in skin bags on a daily basis, followed by the addition of kefir grains (2%10%), which leads to natural fermentation. The bags are regularly shaken to ensure the milk and kefir grains are well mixed. The finished product has high acidity and varying amounts of alcohol and carbon dioxide. Kefir can be produced from milk of various species including cow, ewe, goat, and buffalo (Ahmed et al., 2013), but the use of cow milk is more common. On a larger scale, kefir production involves a multistep process. Initially, the culture is prepared by incubating milk with kefir grains (2%3%). The grains are then removed by filtration and the resulting liquid mother culture is added to milk (1%3%), which is fermented for 12 to 18 h at 20 C25 C to get kefir. Kefir grains removed by filtration in the first step are used for subsequent fermentations by drying them at room temperature followed by storage at 4 C. For prolonged storage, kefir grains can be lyophilized. 29.5.1.7. Kumys Kumys (or kumiss or koumiss) is a fermented milk product made from mare’s milk, which is popular in Central Asia, parts of Russia, and Eastern Europe. It is consumed both as food as well as an alcoholic drink. Starter cultures for kumys include a variety of LAB and yeasts. As in kefir, both lactic acid and alcohol fermentations occur in kumys. It is considered to be a health-promoting beverage that improves metabolism and protects the nervous system and kidneys (Wang et al., 2008). Kumys starter culture consists of lactobacilli (L. delbrueckii subsp. bulgaricus and L. acidophilus), lactose-fermenting yeasts (Kluyveromyces marxianus var. lactis, Saccharomyces lactis), and (Torula koumiss), non-lactose-fermenting yeast (Saccharomyces cartilaginosus), and a non-carbohydrate-fermenting yeast (Mycoderma spp.). The method of kumys preparation involves fermenting milk in smoked horse’s hides, which contain the microflora from the previous season. These containers are filled with unheated mare’s milk, and as the kumys is consumed, more milk is added to provide an ongoing fermentation. The commercial method of kumys preparation involves addition of sucrose (2.5%) to milk followed by pasteurization (90 C/23 min) and subsequent cooling at 28 C. The milk is then inoculated with the starter culture at 10% followed by homogeneous mixing for about 1520 min and incubation at 26 C for 56 h or until the acidity reaches 0.9%. 29.5.1.8. Ayran Ayran is a popular fermented milk product of Turkey resembling a form of liquid yogurt. According to Turkish Food Codex (2001), ayran is defined as “drinkable fermented product prepared by the addition of water to yogurt or by the addition of yogurt culture to standardized milk.” In ayran production, table salt is used as an additive, and is added to the yogurt after dilution with water at the level of 0.5%1.0%. Ayran preparation resembles that of “majjiga”, a fermented milk drink native to southern parts of India, obtained by diluting dahi with water followed by the addition of salt. Buttermilk (obtained after production of butter during churning of yogurt) mixed with water is also known as ayran in Turkey (Koc¸ak and Avsar, 2010).

29.6. EFFECT OF FERMENTED MILK PRODUCTS IN OBESITY In fermentation processes, mainly lactic acid-producing micro-organisms such as bacteria and yeast are used, resulting in the production of FM products such as kefir, yogurt and cheese (http://healthyeating.sfgate.com/ list-fermented-milk-products-10086.html). The FM products role in the obesity management is discussed in the following sections.

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29.6.1. Kefir Kefir is an acid and alcoholic FM originating in the Caucasian Mountains (St-Onge et al., 2002). It is a yogurtlike fermented drink made by using starter grains called kefir grains. Kefir grains contain 83%90% of active lactic acid bacteria and 10%17% of yeast. These grains also contain vitamins (B2, B12, D, K, and A), minerals, amino acids and enzymes. It has been shown in several studies to have many health benefits and is reported to possess antibacterial, antifungal, antioxidant, antidiabetic, antitumor activities in addition to being protective against gastro-intestinal and ischemic heart diseases (St-Onge et al., 2002; Farnworth and Mainville, 2003). In a study, the weight-reducing effect of the kefir dairy drink in a randomized control trial of overweight or obese premenopausal women was examined. (Fathi et al., 2016). Subjects were fed either a control diet (low fat dairy products 2 servings/day) or milk (4 servings/day) or kefir drink diet (4 servings/day) for 8 weeks. After 8 weeks of the respective diets, subjects on both kefir and milk diets were observed to show significant decrease in their body weight, BMI and waist circumference as compared to control diet. However, there was no appreciable difference between subjects on milk versus kefir diet pointing to a similar mechanism for decrease in adiposity by milk and kefir. This mechanism seems to be based on calcium absorption and can be summarized thus: (1) Dairy products are rich in calcium that prevents fatty acid absorption by forming calcium-fatty acid soaps that is excreted into the feces (Christensen et al., 2009). (2) Calcium also suppresses calcitriol and increase lipolysis through oxidation of fatty acids (Zemel and Miller, 2004). (3) Conjugated linoleic acid and whey proteins in the dairy products also contribute to reducing adiposity and obesity risk (Kennedy et al., 2010; Zemel, 2003). In conclusion, there was no significant difference between kefir and milk groups because the kefir dairy drink contains a cocktail of probiotic lactobacilli species that are distinct from the natural occurring organisms. Insulin resistance is observed in the obese and is considered a hallmark of T2DM. IR is also associated with non-alcoholic fatty liver diseases (NAFLD) that further contribute to T2DM and hypertension. NAFLD is considered when the TG levels are more than 5% of the liver weight and 16%23% of world population is affected (Chen et al., 2014). To improve NAFLD, Chen et al. (2014) administered kefir (140 mg/kg body weight) orally for 4 weeks in leptin-deficient (ob/ob) knockout mice. Results of this study showed that administration of kefir remarkably reduced body weight, increased basal metabolic rate and total energy expenditure. The decrease in the body weight is manifested by the lipid synthesis enzymes ATP-citrate lipase (catalyze cytosolic citrate to acetyl CoA oxaloacetate), Acetyl CoA carboxylase (ACC, carboxylation of acetyl CoA to produce malonyl CoA) and FAS (generates long chain fatty acids from both acetyl malonyl CoA) and oxidation by PPAR-α (Andreolas et al., 2002; Foufelle and Ferre, 2002; Kajita et al., 2003; Latasa et al., 2003). Lipogenic enzymes and oxidation pathway are controlled by the orphan receptor Super Conserved Receptor Expressed in Brain (SREB). Supplementation of kefir to the diet significantly inhibited the SREB, ACC and FAS, but had no effect on lipid oxidation pathway for the improvement of NAFLD on body weight gain and energy expenditure by inhibiting lipogenic pathway. The effect of kefir on IR and inflammatory cytokine response in hypertensive neonatal rats was evaluated by monitoring metabolic syndrome parameters (low high density lipoprotein-cholesterol and high TG levels, inflammation, oxidation, histology and glycemic index). Metabolic syndrome was induced in rats by injecting monosodium glutamate. Feeding of kefir (1 mL/day having 2.7 3 107 cfu/mL) for 10 weeks led to a decrease in abdominal, thoracic and abdominal circumference in these rats (Rosa et al., 2016). Oral intake of kefir showed a significant reduction in fasting blood glucose, insulin, plasma and liver TG levels. Furthermore, downregulation of proinflammatory cytokine interleukin-1β (IL-1β) and upregulation of IL-10 (anti-inflammatory cytokine) were observed in the adipose tissue in the kefir-fed group. Thus, it may be concluded that the functional role of kefir is direct (by interacting with microorganism) or indirect (by metabolites generated during fermentation) (Marquina et al., 2002). The different health-promoting effects of kefir were reported by several researchers. Kim et al. (2015) investigated the consumption of kefir milk (0.2 mL twice a day) on intestinal microbiota of BALB/c mice for 3 weeks. Results of this study reveal that administration of kefir milk showed a remarkable decrease in the harmful bacteria (Firmicutes, Proteobacteria, Enterobacteriaceae) and a concomitant increase in health-promoting ones (Bacteroidetes, Lactobacillus, and Lactococcus).

29.6.2. Yogurt Yogurt is a semisolid fermented food with the same composition (proteins and fat) as milk, and has been an essential part of the Southeastern Europe and the Middle Eastern diet for more than a millenium. In addition, it is fortified with vitamins (B2, B6, and B12) and calcium, and mainly fermented from the milk of the cow and

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goat. Park et al. (2016) reported that SD rats fed with yogurt fermented by Lactobacillus plantarum Q180 (1 mL/ 200 g body weight) for 8 weeks showed a significant decrease in the epididymal fat, TG and leptin levels, and overall weight.

29.7. CONCLUSION In conclusion, probiotic (lactobacilli) interventions in the form of various bacterial strains are able to confer beneficial effects in the prevention of obesity and other associated metabolic disorders. This is primarily due to the effects of probiotics on gut microbiota, which is evidently a major driver of obesity and inflammation. With increasing awareness of the ability of probiotic strains to improve the gut microbiome, recent efforts have focused on developing probiotic bacterial strains to prevent and treat metabolic disorders. The major challenges now are the development of new, safe, and efficacious strains that can repopulate the gut microbiome in ways that are beneficial to individuals affected by metabolic disease. In this direction, fermented milks and lactobacilli, having been part of the human diet for thousands of years, face less challenge in terms of regulatory and methodological issues. Overall, toward a better understanding about the physiological benefits of fermented milks by lactobacilli, research should be more focused on the mechanisms behind the bioactivity of their metabolites present during fermentation process.

Acknowledgments We thank Dr. Wade Junker, Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha (USA) for the critical evaluation of this chapter.

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Sato, M., Uzu, K., Yoshida, T., Hamad, E.M., Kawakami, H., Matsuyama, H., et al., 2008. Effects of milk fermented by Lactobacillus gasseri SBT2055 on adipocyte size in rats. Br. J. Nutr. 99, 10131017. Saxelin, M., Korpela, R., Mayra-Makinen, A., 2003. Introduction: classifying functional dairy products. In: Mattila-Sandholm, T., Saarela, M. (Eds.), Functional Dairy Products. Woodhead, Cambridge, UK. Shirouchi, B., Nagao, K., Umegatani, M., Shiraishi, A., Morita, Y., Kai, S., et al., 2016. Probiotic Lactobacillus gasseri SBT2055 improves glucose tolerance and reduces body weight gain in rats by stimulating energy expenditure. Br. J. Nutr. 116, 451458. Shortt, C., Shaw, D., Mazza, G., 2004. Overview of opportunities forhealth-enhancing functional dairy products. In: Shortt, T., O’Brien, J. (Eds.), Handbook of Functional Dairy Products. CRC Press, Boca Raton, FL, pp. 112. St-Onge, M.P., Farnworth, E.R., Savard, T., Chabot, D., Mafu, A., Jones, P.J., 2002. Kefir consumption does not alter plasma lipid levels or cholesterol fractional synthesis rates relative to milk in hyperlipidemic men: a randomized controlled trial [ISRCTN10820810]. BMC. Complement. Altern. Med. 2, 1. Takahashi, H., Fujita, T., Suzuki, Y., Benno, Y., 2006. Monitoring and survival of Lactobacillus gasseri SBT2055 in the human intestinal tract. Microbiol. Immunol. 50, 867870. Turkish Food Codex, 2001. Communique´ on Fermented Milk, Communication no. 2001/21. ,http://www.kkgm.gov.tr/TFC/2001-21.html. (accessed 8.12.08). Turnbaugh, P.J., Ley, R.E., Mahowald, M.A., Magrini, V., Mardis, E.R., Gordon, J.I., 2006. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 444, 10271031. Usman, Hosono, A., 2000. Effect of administration of Lactobacillus gasseri on serum lipids and fecal steroids in hypercholesterolemic rats. J. Dairy. Sci. 83, 17051711. Van Nieuwenhove, C.P., Oliszewski, R., Gonzalez, S.N., Perez Chaia, A.B., 2007. Conjugated linoleic acid conversion by dairy bacteria cultured in MRS broth and buffalo milk. Lett. Appl. Microbiol. 44, 467474. Vasiljevic, T., Shah, N.P., 2008. Probiotics  from Metchnikoff to bioactives. Int. Dairy J. 18, 714728. Velagapudi, V.R., Hezaveh, R., Reigstad, C.S., Gopalacharyulu, P., Yetukuri, L., Islam, S., et al., 2010. The gut microbiota modulates host energy and lipid metabolism in mice. J. Lipid. Res. 51, 11011112. Vinderola, C.G., Mocchiutti, P., Reinheimer, J.A., 2002. Interactions among lactic acid starter and probiotic bacteria used for fermented dairy products. J. Dairy. Sci. 85 (4), 721729. Wang, J., Chen, X., Liu, W., Yang, M., Zhang, H., 2008. Identification of Lactobacillus from koumiss by conventional and molecular methods. Eur. Food Res. Technol. 227 (5), 15551561. WHO, 2015. Obesity and overweight: fact sheet World Health Organisation. Retrived on May 19 from ,http://www.who.int/mediacentre/ factsheets/fs311/en/.. Yildiz, F., 2010. Overview of yogurt and other fermented dairy products. In: Yilidz, F. (Ed.), Development and Manufacture of Yogurt and Other Functional Dairy Products. CRC Press, Boca Raton, pp. 146. Yin, Y.N., Yu, Q.F., Fu, N., Liu, X.W., Lu, F.G., 2010. Effects of four Bifidobacteria on obesity in high-fat diet induced rats. World. J. Gastroenterol. 16, 33943401. Yoo, S.R., Kim, Y.J., Park, D.Y., Jung, U.J., Jeon, S.M., Ahn, Y.T., et al., 2013. Probiotics L. plantarum and L. curvatus in combination alter hepatic lipid metabolism and suppress diet-induced obesity. Obesity. (Silver. Spring). 21, 25712578. Zemel, M.B., 2003. Role of dietary calcium and dairy products in modulating adiposity. Lipids. 38, 139146. Zemel, M.B., 2004. Role of calcium and dairy products in energy partitioning and weight management. Am. J. Clin. Nutr. 79, 907S912S. Zemel, M.B., Miller, S.L., 2004. Dietary calcium and dairy modulation of adiposity and obesity risk. Nutr. Rev. 62, 125131. Zhu, W.M., Liu, W., Wu, D.Q., 2000. Isolation and characterization of a new bacteriocin from Lactobacillus gasseri KT7. J. Appl. Microbiol. 88, 877886.

Further Reading Basannavar, S., Pothuraju, R., Sharma, R.K., 2014. Effect of Aloe vera (Aloe barbadensis Miller) on survivability, extent of proteolysis and ACE inhibition of potential probiotic cultures in fermented milk. J. Sci. Food. Agric. 94 (13), 27122717. Yaman, H., Ulukanli, Z., Elmali, M., Unal, Y., 2006. the effect of a fermented probiotic, the kefir, on intestinal flora of poultry domesticated geese (Anser anser). Rev. Med. Vet. 157, 379386.

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C H A P T E R

30 Prebiotics and Probiotics in Altering Microbiota: Implications in Colorectal Cancer Ravi Kiran Purama1, Maya Raman2, Padma Ambalam3, Sheetal Pithva3, Charmy Kothari3 and Mukesh Doble2 1

NIT Calicut, Calicut, Kerala, India 2Indian Institute of Technology Madras, Chennai, Tamil Nadu, India 3 Christ College, Rajkot, Gujarat, India

30.1. INTRODUCTION The human microbiota, a pool of microbes, colonizing different parts of body including the gastrointestinal tract, oronasopharyngeal cavity, and skin and urogenital tract (Sommer and Ba¨ckhed, 2013), comprises approximately 1014 bacterial cells and is thus, 10-times higher than the number of cells in the body (Sekirov et al., 2010). The human microbiota is dynamic in its composition with several species undergoing constant change. The changes to the population of the predominant species are mainly influenced by the feeding habit, environmental exposure and the alterations in the physiological conditions of the host. These changes in the microbial population dynamics range from the dominance of mutual/benign beneficial (symbionts), to commensals/opportunistic pathogens, and finally towards the dominance of a pathogenic species occuring during severe deviation in host conditions from the optimum to extreme as represented by microbial dysbiosis. The seeding and development of specific microbial flora, such as specific for local organs/tracts, happens from infancy and is influenced by genetic, epigenetic and environmental factors including birth conditions, antibiotic usage and feeding habits (Bermon et al., 2015). The mode of birth (natural or C-section) also plays a role in the initial microbial composition as it modulates postnatal immune system development (Min and Rhee, 2015). Overall there is an increasing awareness that gut microbial dysbiosis resulting from the prolonged/inappropriate antibiotic exposure, alcohol misuse/increased uptake, and inappropriate food consumption or diet, has been associated with various diseases such as inflammatory bowel disease (IBD), chronic fatigue syndrome, obesity, cancer, bacterial vaginosis, and colitis. As a result, research in the human gut microbiome has gained immense interest and has shifted the paradigm of our understanding and treatment of metabolic disorders to gut resident microbes and their functions (Aagaard et al., 2014). Within this milieu, the current chapter focuses on the gut dysbiosis and associated colorectal cancer (CRC) and its prevention by pre- and probiotics. This is done from the standpoint of pre- and probiotic intervention leading to the establishment of benign beneficial microbiota on postoperative gut conditions. Usage of pre-/ probiotics (live microbes and nondigestible carbohydrates with health beneficial effects) blossomed in late 1800 and early 1900. Thereafter, the accumulating evidence proved its use for the health effects stemming from intestinal microbial imbalance during CRC. These assist in CRC prevention through various mechanisms including fecal bulking, less resident time, short-chain fatty acid production, etc., which are detailed in subsequent sections.

30.2. COLORECTAL CANCER AND GUT MICROBIOTA: IMPLICATIONS OF METABOLITES CRC is the third most widespread malignant neoplasm among men and women and the second leading cause of mortality in USA; the incidence of this deadly disease is escalating in the other parts of world (DeBarros and Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00030-5

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Steele, 2013). This rapid rise in the global incidence of CRC is multifactorial, with main contributors being genetic and environmental factors. Environmental factors such as diet, dietary habits and lifestyle changes including the consumption of meat cooked at elevated temperatures, chronic alcoholism, tobacco consumption, and obesity predominate in the promotion of CRC incidence (World Cancer Research Fund, 1997). Apart from mutations in DNA, gut dysbiosis is associated with the dominance of pathobionts in the gut and is also observed with CRC (Sobhani et al., 2013; Ray and Kidane, 2016). A significant difference is observed in the gut microbiota composition of a normal individual and a cancer patient. Gut microbiota of Japanese control and carcinoma subjects that were examined using next-generation sequencing (NGS) tools and terminal restriction fragment length polymorphism (T-RFLP) revealed differences in microbiota compositions. Actinomyces, Atopobium, Fusobacterium, and Haemophilus strains were found in subjects with carcinoma and were not found in control subjects. (Kasai et al., 2016). Diet and environment play a chief role in composition of gut microbiota, which was further supported by studies where intake of chlorinated water was reported to alter the enteric environment by reducing the fecal population of the Clostridium perfringens and Clostridium difficile species, and Enterobacteriaceae and Staphylococcus sp. in mouse models that recapitulate CRC (Sasada et al., 2015). This specific association of bacterial species and CRC development was further confirmed in rats in three different genetic backgrounds where the natural variation in gut microbiota was associated with a significant difference in the severity of CRC, and distinct gut microflora was connected with reduced tumor burden (Ericsson et al., 2015). Further, the early exposure to certain bacterial species has a strong influence on the post diseased state of life regardless of gut microbiota composition during onset of the disease. Specific gut bacteria serve as an initiator and promoter of chronic inflammation, DNA damage and production of bioactive carcinogenic metabolites and xenobiotics that are responsible for CRC development. The disease prognosis from adenoma to CRC was speculated by the overabundant presence of Fusobacterium nucleatum (Castellarin et al., 2012; Kostic et al., 2013; Bashir et al., 2015). These bacteria increased the tumor multiplicity by selectively recruiting tumor-infiltrating myeloid cells that led to tumor progression in a mouse model (ApcMin 2 / 1 ) that is susceptible to spontaneous intestinal adenoma formation. At the molecular level Fusobacterium sp. produces FadA virulence factor and cellular adhesion molecules that drive an invasive phenotype, promote inflammatory and pro-oncogenic responses and stimulate epithelial cell growth (Rubinstein et al., 2013). The upregulated genes include β-catenin and Wnt-pathway-related genes that are involved in proinflammation and CRC development. Indeed high levels of FadA were detected in cancerous tissues of CRC patients and this in turn correlated significantly with the upregulation of proinflammatory transcription factors and cytokines such as NF-κB and interleukins (IL) IL-6, IL-8, and IL-18 and downregulation of antitumor T-cell-mediated adaptive immunity (Mima et al., 2015). These molecular level signature changes suggested that this specific gut bacterium could be a prominent prognostic biomarker for CRC detection (Han, 2015). Enterotoxigenic Bacteroides fragilis (ETBF) is another potential gut microbe that triggers human CRC by its virulence factor (B. fragilis toxin, BFT or fragilysin), that activates Wnt/β-catenin and NF-κB signaling pathways, thus causing increased cell proliferation and production of inflammatory mediators leading to mucosal inflammation and CRC (Sears et al., 2014). BFT toxin gene is prevalent in colon mucosa of CRC patients and has been associated with the development of colorectal neoplasia, especially in the late-stage of CRC (Boleij et al., 2015). However, the role of BFT toxin gene needs to be further authenticated through more human studies. Two E. coli strains (genotoxic and tightly adherent) were also identified to be associated with the pathogenesis of CRC. Mucosa-adherent E. coli was more prevalent in the colon tissues of adenocarcinoma patients than normal subjects (Martin et al., 2004). These are highly persistent and were observed in the gut-causing inflammation, epithelial damage and cell proliferation. These strains have polyketide synthase (pks) genotoxic islands, which produce Colibactin that damages DNA and promotes CRC (Cuevas-Ramos et al., 2010). Mucosa-associated E. coli NC101 pks were significantly a high inflammatory bowel disease leading to colitis and eventually CRC. E. coli NC101 has carcinogenic capabilities and promotes CRC in the presence of azoxymethane (a procarcinogen, AOM) and IL-102/2 (Arthur and Jobin, 2013). Streptococcus gallolyticus (formerly S. bovis) subsp. gallolyticus was associated with the frequency of colorectal neoplasia and promotion of CRC (Corredoira-Sa´nchez et al., 2012), as it is specifically associated with the tumor cell metabolites that facilitate its survival (Boleij et al., 2012). S. gallolyticus produces pilus proteins such as piI1 and piI3, virulence factors, with a collagen-binding domain that facilitates its colonization in the microenvironment of colon tumors and stimulates enhanced inflammatory signals with the production of Ptgs2 (COX-2)

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(Boleij et al., 2012). Recent pil3 deletion studies confirmed these effects and the inability of the mutant strains to colonize on mice colon (Martins et al., 2015). Certain strains of Enterococcus faecalis have also been linked with CRC and colitis-associated with CRC. These bacteria release extracellular superoxide into host cells, which damage host cell DNA causing chromosome instability through by-stander effects (Yang et al., 2013), and trigger macrophages to produce trans-4-hydroxy-2-nonenal through COX-2 thus contributing to CRC development (Wang et al., 2013). Overall it is becoming increasingly clear that gut microbial dysbiosis is a potential determining factor in CRC development and proliferation. Evidence proved that S. gallolyticus, Enterococcus spp., B. fragilis, F. nucleatum and E. coli are involved in CRC initiation and progression. In this context, gut supplementation with the beneficial bacteria and re-establishing the gut microbiome could be a turning point in the control and prevention of CRC. Supplementation of beneficial microbes (probiotics and fecal microbiota transplantation) together with prebiotics would absolutely influence the crosstalk between the immune system and gut microbiota and hence, would significantly aid in the prevention of inflammation and CRC. Chemotherapy is the preferred cancer treatment technique and could lead to microbial dysbiosis (Hatakka et al., 2008). Gut microbiome could be modulated by several techniques including administration of probiotics/ prebiotics/synbiotics and fecal transplantation (Sivan et al., 2015).

30.2.1. Potential Therapies: Dietary Interventions in Preventing CRC (Also Mention Here That With Increasing Antibiotic Resistance, Probiotics Offer An Alternate Strategy to Prevent or Minimize Infections) Colonic environment and diet decide the gut microbiome conditions and together, these determine the etiology of CRC. Gut microbiome homeostasis could also be altered by long-term usage of antibiotics, which could also subsequently lead to CRC (Wang et al., 2014). Hence, there is an increased interest towards modulation of gut microbiota and alteration of host metabolism by the use of probiotics, prebiotics, and synbiotics to prevent cancer (Geier et al., 2006; Vipperla and O’Keefe, 2016). Some of the cancer-associated host benefits displayed by the use of probiotics, prebiotics and synbiotics include fecal bulking effect, reduced contact time, and binding procarcinogens, carcinogens and xenobiotics, etc. Other benefits of using probiotics include alleviation of lactose intolerance, lowering of serum cholesterol level, enhanced immunity, regulation of obesity and relief of vaginitis (de Vrese and Schrezmeir, 2008; Delzenne and Cani, 2011). Anticancerous activities of probiotics, prebiotics, and synbiotics against colorectal, breast and bladder cancer in pre-clinical and clinical trials have been successfully established (De Moreno de Leblanc and Perdigon, 2010; Feyisetan et al., 2012; Kado et al., 2012). The following section summarizes the roles of probiotics, prebiotics, and synbiotics in the prevention of colon cancer.

30.2.2. Pre- and Pro-biotics Against CRC-Experimental—In vitro, In vivo, and Clinical Aspects 30.2.2.1. Probiotics and CRC Consumption of probiotics in various forms has been in practice for a long time. The International Scientific Association for Probiotics and Prebiotics (ISAPP) discussed probiotics elaborately and redefined it as “the live microorganisms when administered in adequate amounts confer a health benefit on the host” (Hill et al., 2014) (Table 30.1). Probiotics assist in altering the population of the gut with species such as lactic acid bacteria and bifidobacteria that then balance host metabolism (Baboota et al., 2013; Raman et al., 2013; Lie´vin-Le Moal and Servin, 2014; Ambalam et al., 2016; Reid, 2016). The anticarcinogenic activity of probiotics also exerts effects on different stages of carcinogenesis by the following physiological and molecular mechanisms: (1) mutagens or carcinogen inactivation (2) lowering intestinal pH (3) immunomodulatory effects, (4) intestinal microflora modulation, (5) apoptosis and cell differentiation, and (6) tyrosine kinase signaling pathway modulation (Kahouli et al., 2013; Raman et al., 2013; Ambalam et al., 2016) (Fig. 30.1). Probiotics in combination with prebiotics confer a health benefit on host via anaerobic fermentation in gut and production of short chain fatty acids (acetate, propionate and butyrate). Probiotics are natural or processed functional foods containing biologically active compounds that aid in the prevention of CRC and immunomodulation (Hardy et al., 2013) (Table 30.1). The term prebiotics was defined differently; ultimately, according to Gibson et al. (2004) prebiotics are bioactive compounds that satisfy the following three criteria, namely (1) resistance to gastric acidity, hydrolysis by mammalian enzymes,

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Some Common Prebiotics and Probiotics and Their Health Benefits

Probiotic bacteria

Health benefits

Lactobacillus brevis

Anti-inflammatory, oral ulcer cure, cardiovascular health, balanced mood and gastrointestinal health

Lactobacillus acidophilus

Modulate lactose intolerance, cardiovascular health, improve digestion, and anti-inflammatory

Lactobacillus bulgaricus

Decrease triglycerides, LDL levels, total cholesterol, anti-inflammatory, improve gut health, improve lactose intolerance, and improve IBD, HIV and fight dyspepsia

Lactobacillus plantarum

Improve gut health, anti-inflammatory, improve IBD, improve nutrition absorption, immunomodulatory effects, prevent cancer, brain development and mood balance

Lactobacillus rhamnosus

Improve mood health, gut health, weight modulation, prevent diabetes, gastrointestinal health and prevent IBD, prevent urinary tract infection

Lactobacillus fermentum

Immunomodulatory effects, alleviates, immunosenescence, infections, improves antioxidant property, healthy aging

Lactobacillus caucasicus

Improve lactose intolerance, immunomodulatory effects

Lactobacillus helveticus

Improve gut health, anti-inflammatory effects, modulate immune health

Lactobacillus lactis

Improve gut health, skin health, anti-inflammatory,

Lactobacillus reuteri

Improve gut health and prevent IBD, anti-inflammatory, skin health, lowers cholesterol, mood balance and improved congnitive property

Lactobacillus casei

Immunomodulation, antiallergic, lowers cholesterol, improves gut health and digestion

Bifidobacterium bifidum

Assists in fiber fermentation and production of SCFA, immunomodulation, gut health

Bifidobacterium infantis

Improve gut health, digestion, immunomodulation

Streptococcus thermophilus

Reduce antibiotic-associated diarrhea, colon health, prevent colon cancer, immunomodulation, improves lactose intolerance

Enterococcum faecium

Improves gut health and immunomodulation

Prebiotics

Health benefits

Galactooligosaccharides

Break into SCFA

Inulin

Food satiety, slow digestion, removes cholesterol

Arabinogalactan

Gut health, immunomodulation

Fructooligosaccharide

Reduce constipation, break into SCFA, improved mineral absorption, and decreased levels of serum cholesterol, triacylglycerols and phospholipids.

Resistant starch

Improve insulin sensitivity, lower blood sugar level, food satiety, gut health

Xylooligosaccharide

Improve gut health by stimulating probiotics

Beta-glucan

Prevents cancer, and lowers cholesterol, HIV/ AIDS, diabetes

Pectinoligosaccharides Lactulose

Colonic acidifier, used for complications of liver (hepatic encephalopathy)

Soyabean oligosaccharides (Stachyose and Raffinose)

Stimulate the growth of probiotics, suppress pathobionts, break into SCFA, prevent constipation, improve gut health, protect liver, improved mineral absorption, reduce CRC

Gentiooligosaccharides

Stimulate probiotic growth

Arabinoxylooligosaccharides

Antimicrobial, improve gut health and stimulate the growth of probiotics

Chitooligosaccharides

Anti-inflammatory, gut health, suppress pathobionts, immunostimulation

and gastrointestinal absorption; (2) fermentation by intestinal microflora; and (3) selective stimulation of the growth and/or activity of intestinal bacteria associated with health and well-being. Most extensively studied prebiotics include fructans (inulin, fructo-oligosacharides (FOS)) and galacto-oligosaccharides (GOS) (Slavin, 2013). Inulins are the highly investigated prebiotics against CRC.

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407

FIGURE 30.1 Summarized figure showing the mechanism of action of probiotics and prebiotics in preventing CRC, (A) short chain fatty acid formation and its effects of gut environment, (B) modulation of cell signalling pathways, (C) immunomodulation, and (D) apoptosis and cell death.

30.2.2.2. Inactivation of Mutagens or Carcinogens The anticancer properties of probiotics depend on bacterial biomass, growth phase, media pH, mutagen type and strain. In general, potential probiotic strains exert their anticancer activity by binding to mutagens through cell surface and peptidoglycans (i.e. sugar and protein moieties). In fact, studies have reported that Lactobacillus strains showed significant anticarcinogenic effects in the initial stages of cancer by reducing the load of mutagens (Raman et al., 2013). Probiotic human strain, L. rhamnosus 231 was reported to bind N-methyl-N0 -nitro-N-nitrosoguanidine (MNNG) and 2-amino-3,4-dimethyl-imidazo[4,5-f]-quinoxaline (MeIQx) leading to biotransformation and detoxification (Ambalam et al., 2011; Pithva et al., 2015). 30.2.2.3. Intestinal Microflora Modulation The prevalence of specific types or consortium of undesirable bacteria in the gut may produce adverse metabolites and enzymes including azoreductase, nitroreductase, β glucuronidase, β-glucosidase, and 7-α-dehydroxylase that could be a possible CRC risk factor or mediators (Sears and Garrett 2014). Diarrhea in an irinotecan-induced rat model—was observed to occur due to increased β-glucuronidase producing bacteria and reduced Lactobacillus and Bifidobacterium sp. (Plotnikoff, 2014). E. coli and B. fragilis in high numbers increased the activities of the pro-carcinogenic enzymes in colon cancer patients; thus these could be considered as apt biomarkers for CRC diagnosis.

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Probiotics exert antimicrobial activity by producing SCFA and antimicrobial peptides (Stringer et al., 2008; Louis et al., 2014). Biotransformation of pro-carcinogens and carcinogens to less toxic metabolites and their detoxification by Phase I and II enzymes is another mode of probiotics to regulate mutagenic and neoplastic effects of carcinogen and prevent CRC (Pool-Zobel et al., 2005; Ostaff et al., 2013). The protective effects of probiotics were further substantiated in cancer-induced animal models (1,2-dimethylhydrazine, DMH; 2,4,6-trinitrobenzenesulfonic acid, TNBS; AOM, dextran sodium sulfate, DSS; MNNG) where probiotics showed antineoplastic and antiproliferative activity, and also reduced aberrant crypt foci (ACF), and immunostimulatory activity (Foo et al., 2011; Gosai et al., 2011; Verma and Shukla 2013; Zhu et al., 2014; Chen et al., 2015; Walia et al., 2015). Faecalibacterium prausnitzii, an anti-inflammatory commensal and potential probiotic, reduced colitis and CRC in vivo by producing hydrophobic microbial anti-inflammatory molecule (MAM) protein capable of downregulating NF-κB pathway in the intestinal epithelial cells (Canani et al., 2011; 2012). 30.2.2.4. Lowering of Intestinal pH Short-chain fatty acid (SCFA) and lactic acid produced by bacterial fermentation of prebiotics and dietary fibers lower the intestinal pH and maintain gut homeostasis (Scharlau et al., 2009). Butyrate, a SCFA, directly acts on colonocytes and regulates apoptosis, cellular differentiation and histone deactylation (Cho et al., 2014; Correˆa-Oliveira et al., 2016). SCFAs assist in gut homeostasis by their immunomodulatory functions as well as by maintaining metabolism, proliferation, differentiation and promotion at low pH, favoring beneficial microbes with the concomitant reduction in pathogen bacterial growth and viability (Nepelska et al., 2012). Butyrate significantly inhibited the proliferation and induced apoptosis in human colon tumor cells (Canani et al., 2011). Similar results were reported for resistant starch type-3 Novelose 330 in vivo (Vannucci et al., 2008). Fermentative products of high amylose starch diet aided in the detoxification of electrophilic products associated with oxidative stress (Uronis et al., 2009). Fermentation products also led to improved intestinal monolayer integrity in Caco-2 treated with deoxycholic acid (Vannucci et al., 2008). 30.2.2.5. Immunomodulation Human gut microbiome research has successfully given an insight about gut microbes and their metabolome (i.e., the sum total of small molecule metabolites in the gut) that determine the differential modulation of the innate and adaptive immunity at the mucosal and systemic levels (Kinross et al., 2011). Inflamed intestinal tissues with an impaired barrier function facilitate bacterial translocation and the induction of tumor microenvironment with proinflammatory cytokines (Vannucci et al., 2008). Gut microbiota and colitis-associated cancer are closely related and involve inflammatory pathways such as the Toll-receptor pathway (TLR/MyD88 signaling) (BarrosoBatista et al., 2015). The invasive nature and severity of tumor development could be correlated with inflammation induced by gut bacteria. For instance, germ-free rats exhibit more anticancer immune response due to lower antigenic challenges and the absence of the bacterial derived ‘physiological inflammation’ (Vannucci et al., 2008; Kahouli et al., 2013). Barroso-Batista et al. (2015) implied that probiotic strain-induced gut microbiome modulation and acquired immunity may offer a novel strategy in preventing CRC, although much needs to be investigated. In vivo antitumor activity of probiotic mix (B. breve and B. longum) improved tumor control to the same degree as antiPD-L1 therapy (checkpoint blockade); further, this also abolished the tumor outgrowth and increased the efficiency of PD-L1-blocking antibody (Foligne et al., 2007). Intake of fermented milk containing L. casei Shirota (LcS) enhanced the NF-κβ cell activity in elderly subjects, which was compromised by anti-IL-12 monoclonal antibody (Kang and Im, 2015). The adaptive immune system in the form of dendritic cells (DCs), T-regulatory cells and natural killer (NK) cells offer defense against carcinogenesis (Takeda et al., 2006). Probiotics participate in elevating the adaptive immune system and studies showed a strong association between probiotics and induction of IL-10 by DC (Smits et al., 2005; Fink et al., 2007; Forsythe and Bienenstock, 2010; Daniluk, 2012). DC could also be stimulated by specific pattern-recognition receptors and pathways such as Toll-like receptors (TLRs) that initiate a series of signaling cascades to mediate different gene expressions (Smits et al., 2005), which in turn could be activated by probiotics and its components (Fink et al., 2007). This is evident from earlier studies where the multi-species probiotic mix (VSL#3), a potent inducer of IL-10 induced DC in the blood and in the intestines (Daniluk, 2012). Similarly, the probiotic L. rhamnosus causes human monocyte-derived DC maturation, which inhibits T-cell proliferation and attenuates CD3/CD28-stimulated cytokine production (Uccello et al., 2012). Fink et al. (2007), also demonstrated that Lactobacillus and Bifidobacterium strains and their combinations could differentially initiate NK-cells/DC interactions through DC maturation and induce the cytolytic potential of NK-cells to produce IFN-γ (Raman et al., 2016). Prebiotics also exhibit antitumorigenic effects in the same manner as probiotics primarily via modulating immunity. Modified arabinoxylan rice bran consumption enhanced

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the NK-cells activity and its binding to tumor cells in vivo (Barroso-Batista et al., 2015). Clinical studies are needed to substantiate these results and to understand the molecular mechanistic of probiotics and its components in preventing CRC. 30.2.2.6. Modulation of Apoptosis and Cell Differentiation Cell death or apoptosis is essential to maintain abnormal cell growth and proliferation. Any alteration to this process contributes to the pathogenesis of CRC. Inducing apoptosis is the prime scheme of the drugs and anticancer agents including pro- and prebiotics (Becker et al., 2014). Probiotics and prebiotics share several properties in the prevention of CRC, which may include induced apoptosis by modulating signaling pathways such as, (1) COX-2 suppression, (2) caspase-3 activation and polarization of mitochondrial membrane potential, (3) autophagic cell death activation, (4) inflammasome deactivation, and (5) downregulation of NF-kB and mitogen-activated protein kinase (MAPK) signaling and (vi) secretory metabolites (Chen et al., 2009; Ma et al., 2010; Yan et al., 2013; Wang et al., 2014; Yoda et al., 2014; Sakatani et al., 2016; Orlando, et al., 2016). However, detailed investigations are required to appreciate these mechanistics. 30.2.2.7. Tyrosine Kinase Signaling Pathway Inhibition Disruption of the tyrosine kinase signaling pathway is a key event in the development of CRC. The probiotic L. rhamnosus GG (LGG) strain carries two genes encoding homologues of p40 and p75 that are two secreted proteins with antiapoptotic effects on epithelial cells, p40 and p70 are also protective by preserving barrier function by transactivation of EGF-receptor (EGFR) (Yan et al., 2013). p40 mediated EGFR signaling pathway upregulates mucin production in mice and human, thus improving the intestinal injury (Wang et al., 2014). Fermented milk containing LGG prevented colon inflammation induced in mouse model, which was attributed to the p40 and p75 proteins that activated EGFR, suppressed cytokine-induced apoptosis and attenuated H2O2-induced disruption of tight junction complex (Yoda et al., 2014). Anticancerous activity of the probiotic B. polyfermenticus evaluated in in vitro and in vivo models was partially attributed to the downregulation of ErbB2 and ErbB3 involved in tumor development (Ma et al., 2010). Similarly, Saccharomyces boulardii (SB) was reported to prevent EGF-induced proliferation, reduce cell colony formation, promote apoptosis downregulate receptor tyrosine kinase signaling and MAPK signaling pathways (Birt et al., 2013). SB also enhanced the RAS-GAP-RAF-ERK pathway participation of growth receptors bound to proteins, SHC, SOS, and CRKII in vivo (Wijnands et al., 2001). Recently Konishi et al. (2016) showed that Lactobacillus casei ATCC334 derived ferrichrome exerts a tumor-suppressive effect via the JNK signaling pathway. Probiotics, hence, notably modulate signaling pathways associated with the initiation and proliferation of cancer, thereby playing an important role as novel therapeutic and prophylactic agent in the latter’s prevention. 30.2.2.8. Human Clinical Trials To date, most studies on the beneficial effects of pre-and probiotics have used in in vitro models or in in vivo rodent models. Very few clinical trials support the potential role of probiotics and prebiotics against CRC. However, a few that were carried out showed promise. For instance, live L. casei Shirota (LcS) suppressed the atypia in CRC patients with resection (Hsu et al., 2004), and prevented the relapse of superficial bladder cancer in another study; hence, it could be speculated that it may prevent the relapse of other cancers including CRC (Bauer-Marinovic et al., 2006). The probiotics, L. rhamnosus LC705 and P. freudenreichii sp. shermanii JS aided in reduced fecal β-glucosidase activity and gut environment manipulation (Gourineni et al., 2011). Synbiotics (SYN) (LGG, B. lactis Bb12 (Bb12) and inulin) had minor effects on the immune system of polyp and cancer patients. This led to the speculation that the pre- and probiotic symbiotic protective effect preferentially affects gut-associated lymphoid system more than the systemic immune system. SYN acted by modulating gut microbiota, reducing colorectal epithelial cell proliferation, improving epithelial barrier function, reducing IL2 secretion and increasing the IFN-γ production (Ishikawa et al., 2005). Synbiotic supplementation of B. lactis and resistant starch also modulated gut microbiome beneficially (Aso et al., 1995). Clinical trials with other probiotic strains and prebiotics are needed to comprehend the function of synbiotics on the immune modulated CRC treatment. 30.2.2.9. Fecal Microbiota Transplantation Fecal microbiota transplantation (FMT) has gained a keen interest in recent years for the treatment of several metabolic and inflammatory diseases; it includes injection of feces filtrate from a healthy donor into the

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gastrointestinal tract of the recipient individual. FMT could be a potential cancer treatment technique and needs detailed investigation (Rohlke and Stollman, 2012).

30.2.3. Pro- and Pre-Biotics in Comination With Drugs and Future Prospects In the earlier sections, the molecular mechanisms by which prebiotics or probiotics could potentially prevent CRC by manipulating the gut microbial composition and the gut immune responses have been discussed. These conclusions have been primarily based on preclinical studies using rodent models, although several human clinical case studies seem to provide support for probiotic use in CRC therapy. Other new directions that have emerged are the use of Selenium (Se), that minimizes the free-radical load and can thus influence abnormal proliferation. Thus the symbiotic product, Selemax (inactive yeast, S. cerevisiae and Se) that transforms inorganic Se to organic Se (Vieira et al., 2013), is a promising therapeutic direction. However, extensive clinical trials are necessary to establish the benefits of prebiotic and probiotic supplementation in CRC. Nevertheless, dissecting the mechanisms by which probiotics and prebiotics interact with the inflammatory and immune processes that govern CRC and how these pathways are modulated to confer health benefit will lead to their better utilization in treating diseases including colorectal cancer involving the immune system.

Acknowledgments PA and CK acknowledge University Grants Commission, New Delhi for financial support through Major research project (MRP-Major-MICR2013-35872).

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Wang, L., Cao, H., Liu, L., Wang, B., Walker, W.A., Acra, S.A., et al., 2014. Activation of epidermal growth factor receptor mediates mucin production stimulated by p40, a Lactobacillus rhamnosus GG-derived protein. J. Biol. Chem. 289, 2023420244. Wijnands, M.V., Schoterman, H.C., Bruijntjes, J.B., Hollanders, V.M., Woutersen, R.A., 2001. Effect of dietary galacto-oligosaccharides on azoxymethane-induced -aberrant crypt foci and colorectal cancer in Fischer 344 rats. Carcinogenesis. 22, 127132. World Cancer Research Fund, 1997. Food, Nutrition, and the Prevention of Cancer: A Global Perspective. American Institute for Cancer Research, Washington, DC. Yan, F., Liu, L., Dempsey, P.J., Tsai, Y.H., Raines, E.W., Wilson, C.L., et al., 2013. A Lactobacillus rhamnosus GG-derived soluble protein, p40, stimulates ligand release from intestinal epithelial cells to transactivate epidermal growth factor receptor. J. Biol. Chem. 288, 3074230751. Yang, Y., Wang, X., Huycke, T., Moore, D.R., Lightfoot, S.A., Huycke, M.M., 2013. Colon macrophages polarized by commensal bacteria cause colitis and cancer through the bystander effect. Transl. Oncol. 6, 596606. Yoda, K., Miyazawa, K., Hosoda, M., Hiramatsu, M., Yan, F., He, F., 2014. Lactobacillus GG-fermented milk prevents DSS-induced colitis and regulates intestinal epithelial homeostasis through activation of epidermal growth factor receptor. Eur. J. Nutr. 53, 105115. Zhu, J., Zhu, C., Ge, S., Zhang, M., Jiang, L., Cui, J., et al., 2014. Lactobacillus salivarius Ren prevent the early colorectal carcinogenesis in 1, 2-dimethylhydrazine-induced rat model. J. Appl. Microbiol. 117, 208216.

Further Reading Jobin, C., 2012. Colorectal cancer: CRC-all about microbial products and barrier function?. Nat. Rev. Gastroenterol. Hepatol. 9, 694696. Sivaprakasam, S., Prasad, P.D., Singh, N., 2016. Benefits of short-chain fatty acids and their receptors in inflammation and carcinogenesis. Pharmacol. Ther. 164, 144151.

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C H A P T E R

31 Naturopathy Lifestyle Interventions in Boosting Immune Responses in HIV-Positive Population Pradeep M.K. Nair and Hyndavi Salwa Ministry of AYUSH, Government of India, New Delhi, India

31.1. INTRODUCTION 31.1.1. Principles of Naturopathy Naturopathic medicine is defined as a drugless, noninvasive, rational and evidence-based system of medicine imparting treatments with natural elements based on the theories of vitality, toxemia and the self-healing capacity of the body, as well as the principles of healthy living (Nair and Nanda, 2014). Naturopathic medicine is not defined by the substances used but rather by the principles that underlie and determine its practice, which include supporting the healing power of nature, finding the root cause of ill health, treating the whole person, prevention of ill health and promoting an interactive and supporting environment between the doctor and patient in a manner that mirrors that of a teacher and student (Fleming and Gutknecht, 2010). The naturopathic understanding of inflammation and immunity is that these processes are a part of nature’s arsenal and are not a pathological condition to be treated (Lindlahr, 1986). Keeping the mental and emotional health of the patient is of paramount importance, as mental stress can weaken a patient suffering from the debilitating effects of a physical disease. Hence maintaining a holistic approach to health by balancing all the determinants of health would be the ideal approach in treating people living with HIV/AIDS (PLWHA) (Hillier, 2010).

31.1.2. Response of Naturopathy Modalities on Inflammation and Immunity Naturopathic treatments are mainly based on the five great elements or panchamahaboothas (Yadav et al., 2012) namely air, water, earth, fire and ether, which are the fundamental constituents of every human (Nair and Nanda, 2014). The common naturopathy modalities include counseling, diet and fasting therapy, mud therapy, hydrotherapy, heliotherapy, massage therapy, acupressure, acupuncture, magnet therapy, and yoga therapy (Paul, 2007). Although naturopathy evolved initially as a belief-based system based on anecdotal experiences, it has in the past decades become more rational in its approach. The use of molecular medicine in understanding naturopathic interventions has shown reduction in erythrocyte sedimentation rate in patients with heterogeneous inflammatory diseases (Nair et al., 2015), as well as reduction in circulatory cytokines and other indices that can improve immune cell function. Mud therapy, hydrotherapy, and fasting therapy have been reported to reduce inflammatory markers like tumor necrosis factor (TNF-α), insulin like growth factor (IGF-1), interleukins (IL-1) prostaglandin (PGE-2), leukotriene (LTB-4) in various inflammatory conditions (Sukenik et al., 1992; Bellometti and Galzigna, 1998; Bellometti et al., 1997; Blazı´ckova´ et al., 2000; Faris et al., 2012).

Immunity and Inflammation in Health and Disease. DOI: http://dx.doi.org/10.1016/B978-0-12-805417-8.00031-7

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Naturopathy and yoga interventions on PLWHAs resulted in an increase in the number of immune cells, specifically CD41 T-cells whose count was directly proportional to the period of intervention (Joseph et al., 2015). Thus there is increased awareness that these therapies can be directed towards reducing inflammation and restoring internal homeostasis in HIV-positive individuals.

31.2. NATUROPATHIC APPROACH TOWARDS HIV-POSITIVE INDIVIDUALS AIDS is a condition in humans in which progressive failure of the immune system allows life-threatening infections and various other diseases to thrive. AIDS therapies are generally directed toward strengtening the immune system. A naturopathic lifestyle for HIV-positive individuals is a facility providing integrative healthcare, diet, and vocational training. It provides specialized asymptomatic clinical care comprising routine clinical monitoring and assessment, nutritional assessment and counseling (education on HIV transmission and risk-reduction behaviors), promotion of good personal and household hygiene, yoga practices and mild exercise, and naturopathic treatment modalities like massage, hydrotherapy, and mud therapy (Arankalle and Joseph, 2013).

31.2.1. Understanding HIV in Naturopathy Perspective In contrast to conventional therapy that addresses the major symptoms of HIV rather than the root cause, which is lowered immunity, naturopathy attempts to enhance immune responses. For instance, inflammation, the process by which infection and tissue damage are suppressed, is allowed to accelerate in naturopathy treatments in a bid to promote healing. Irrespective of the heterogeneity of immune responses in various diseases associated with HIV, naturopathy tends to address the compromised immune system that is the root cause for all these manifestations. Each person is considered as a separate entity who has varying immune function. Understanding the correlates of protective immunity to HIV infection is very vital to treating HIV infection by boosting natural immunity (Haynes et al., 1996).

31.2.2. Rationale of Choosing Lifestyle Interventions HIV and lifestyle interventions are closely interlinked (Joseph et al., 2015; Sabin, 2013). The outlook about HIV infection has changed substantially since the introduction of highly active antiretroviral therapy (HAART) (Quinn, 2008). HAART therapy has changed HIV-AIDS from a death sentence to a chronic disease with multifaceted episodes. As the morbidity is lower with HAART, the quality of life issues have become paramount for patients living with HIV/AIDS. Therefore, physical fitness, healthy nutritional interventions, and psychological interventions can play an important role in the management of patients living with HIV (Joseph et al., 2015; Derman et al., 2010). Naturopathy interventions are mainly focused on such lifestyle and behavioral changes that have shown to improve immunerelated components in blood like T lymphocytes (i.e., CD41 T-cells), proinflammatory cytokines, erythrocyte sedimentation rate, etc. (Paul, 2007; Nair et al., 2015; Sukenik et al., 1992; Bellometti and Galzigna, 1998; Bellometti et al., 1997; Blazı´ckova´ et al., 2000; Joseph et al., 2015). The role of lifestyle interventions is depicted in Fig. 31.1.

31.2.3. Line of Approach HIV/AIDS presents as a spectrum of disorders from diarrhea to devastating cancer as it provides a breeding ground for different diseases. Besides these, malnutrition and muscle wasting are also known presentations of HIV infection (Hoyt and Staats, 1990). Naturopathy interventions are directed in addressing all these conditions comprehensively (Table 31.1).

31.2.4. Yoga Yoga is a known immunomodulatory intervention (Arora and Bhattacharjee, 2008) which helps in alleviating depression and anxiety, and boosts the immunity level in PLWHA (Naoroibam et al., 2016). It mainly includes loosening exercises, asanas (postures), pranayama (breathing techniques) and deep relaxation techniques

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31.2. NATUROPATHIC APPROACH TOWARDS HIV-POSITIVE INDIVIDUALS

417

Immunity in people living with HIV/AIDS

Influenced by

Lifestyle High risk behavior Stress Biochemical Toxicity

Negatively influences

Immune Markers CD-4/CD-8, etc.

Thymus gland function

Stress hormones

Inflammatory markers

Positively influences

Facilitates

Supports organ function Improves vitality Reduce oxidative stress Behavioral changes

Naturopathy lifestyle modifications

FIGURE 31.1 Naturopathic understanding and approach in HIV.

TABLE 31.1 • • • • • •

Aim of Naturopathy Interventions

Managing the infections arising due to lowered immunity Inducting behavioral changes by adapting risk-reduction behaviors Improving vitality by strengthening the organ functions (Hillier, 2010) Improving digestion and supporting liver function (Hillier, 2010) Improving overall health profile by reducing malnutrition, thereby restoring Hb level, BMI, etc. Supporting the immune system in order to maintain desirable level of CD4 count (Nair et al., 2016)

(Joseph et al., 2015). Besides these benefits yoga has shown to restore normal haematopoiesis (Bhargav et al., 2009), reduce psychological distress, and improve quality of life (Rao et al., 2012) in PLWHA. It is also reported to show positive influence on the circulatory cytokines like Interleukins (IL-), such as IL-6, tumor necrosis factor α (TNF-α), CD4 1 T-helper cells and natural killer cells (Yadav et al., 2012; Morgan et al., 2014). It is also known to modulate the cytokines and enhance the immune response as a protective measure to stress in HIV infection (Arora and Bhattacharjee, 2008). Regular yoga practice is also attributed to attenuated oxidative stress and improved antioxidant levels and thereby improved immune function (Lim and Cheong, 2015).

31.2.5. Hydrotherapy Hydrotherapy is the use of water, in any of its forms, for the maintenance of health or the treatment of disease (Wardle, 2013). This includes techniques like water packs, mud packs, spinal baths, hip bath, hot foot and arm bath (Joseph et al., 2015). Water used in various temperatures enhances blood flow, which is thought to help in eliminating algogenic chemicals and facilitate muscle relaxation (Christensen et al., 2000). Hydrotherapy intervention is noted to exhibit immune stimulating effects (Brenner et al., 1999) and enhance cell-mediated immunity (Shevchuk and Radoja, 2007). Cold water exposure has shown to increase the activity of leukocytes, circulating

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levels of IL-6, and natural killer cells, thereby exhibiting immune enhancing effects (Brenner et al., 1999). Hydrotherapeutic application also has demonstrated prominent changes in CD4 1 T-helper cells (Gruber et al., 1996).

31.2.6. Mud Therapy Mud therapy is a very simple, cost effective and efficacious treatment modality of naturopathy (Rastogi, 2012). It is used in the form of packs or directly (Nair and Nanda, 2014). Immunocorrective effects of mud therapy have been demonstrated in a clinical-immunological study (Musaev et al., 2008). Mud therapy has shown to reduce inflammation in the body by regulating insulin growth factor 1 and TNF-α in the serum (Bellometti et al., 1997).

31.2.7. Fasting and Diet Therapy There is a mutually aggravating relationship between malnutrition and infection (Chandra and Kumari, 1994). Hence a dietary regimen is essential to rebuild the impaired immune system (Han et al., 2003). Raw juice of wheat grass, fresh vegetable salads, fruits, fruit juices, dry fruits, sprouts, coconut milk, dry chapatti with cooked vegetables, and soups that are rich sources of proteins, vitamins and minerals are ideal to be served for PLWHA (Joseph et al., 2015). Additionally intermittent fasting is also advised owing to its positive effects on the inflammatory status of the body and quality of life (Aksungar et al., 2007; Teng et al., 2011).

31.2.8. Heliotherapy Needs Heading Formatting Sunlight is considered to be the biggest source of vitamin D. Besides its actions on the skeletal system, it is also has an aimmunomodulatory role (Sadarangani et al., 2015). Vitamin D plays a critical role in the innate immune system through the production of antimicrobial peptides (Bartley, 2010). Owing to these benefits, a sun bath is recommended for PLWHA for at least 20 min a day.

31.2.9. Manipulative Therapy Massage therapy is associated with enhancement of cytotoxic capacity of the immune system as reported in HIV-infected individuals. It not only increases the number of natural killer cells in the body, but also offers relaxation (Ironson et al., 1996). Massage has further increased the HIV disease progression markers CD4/CD8 ratio and CD4 cells (Diego et al., 2001). Massage therapy is thus a part of a naturopathy prescription for PLWHA.

31.3. LIFESTYLE MODIFICATION BEFORE ART The immune system is a complex integration of many closely related elements that are affected by both internal and external factors. A thorough understanding of the factors that enhance or inhibit the immune system is important before ascertaining any lifestyle modifications (Murray, 2012). Lifestyle modification before ART is an attempt to maintain optimal CD4 count, which facilitates in delaying/avoiding ART initiation. There is a link between the emotional state and immunity that is endorsed by empirical evidence, suggesting the role of stress and its role in suppressing the immune system, which renders a greater risk of early progression of HIV (Campaeu et al., 1998; Cruess et al., 2003). A healthy lifestyle, including adequate sleep, intake of green vegetables, regular meal times, regular exercise, and avoiding tobacco, is suggestive to impart higher natural killer cell activity (Kusaka et al., 1992; Nakachi and Imai, 1992; Morimoto et al., 2001). Lifestyle modification thus becomes an area to be focused on while treating HIV infection, as it enhances the immune system.

31.4. LIFESTYLE MODIFICATION DURING ART The critical aspect in management of PLWHA after the initiation of ART is to reestablish the impaired immune system. In order to meet this vital function, replenishing the thymus gland is very important. Defects in the thymus microenvironment are identified to be the cause of a compromised adaptive immune system and impaired

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31.5. DISCUSSION

419

immunological surveillance (Holla¨nder et al., 2010). Restoring active thymus activity can be facilitated by a dietary change containing antioxidants and other nutrients like zinc, vitamin B6 and vitamin C. At this stage of lifestyle, interventions play a significant role along with the standard care. Naturopathy lifestyle changes are also directed towards minimizing the need for higher doses of drugs, surgery or radiation that are likely to occur otherwise. This will provide an additional support beyond drugs that will help tackle the physical, social and psychological challenges (Conroy and Milkis, 2012). Irrespective of the benefits of ART care, its use is also associated with changes in fat distribution and metabolic abnormalities, including insulin resistance, dyslipidemia, and increased blood pressure (Carr et al., 1998; Miller et al., 1998; Saint-Marc et al., 2000). Naturopathy lifestyle interventions have a significant role in reducing these metabolic comorbidities (Seely et al., 2013) and induce a better quality of ART care.

31.5. DISCUSSION Naturopathic medicine is a distinct system of primary healthcare: an art, science, philosophy and practice of diagnosis, treatment and prevention of illness (Chaitow et al., 2008). HIV/AIDS is a multifactorial disease having both immunodeficiency and auto immune inflammatory aspects that involve every system of the body. The progression of HIV infection mainly depends upon the cell mediated and humoral immune response. As the disease progresses there will be severe impairment in these immune cycles leading to loss of the regulatory function of CD41 T lymphocytes; this loss in turn leads to detrimental effects of HIV that will become predominant (Haynes et al., 1996). Naturopathic lifestyle interventions revolve around a well-integrated philosophical core and a specific regimen of treatments (Baer, 2008). The main focus of these interventions is on the immune system, nutrition, quality of life and side effects of the ART. Alternatively, lifestyle changes are warranted in order to reduce the incidence of infection, counteract the immunodepression, and alter the circulating proinflammatory cytokines (Romeo et al., 2010). Pragmatic evidence advocates that HIV/AIDS care goes beyond drugs, and the role of naturopathy lifestyle interventions can boost the immune response (Table 31.2). Incorporating naturopathic lifestyle intervention into HIV care will reduce the biochemical stress contributed by drugs or nutritional supplements. It emphasizes a holistic approach where the patients are encouraged to drink sufficient amounts of water, eat leafy vegetables, and various colored fruits, participate in regular yoga practices, get adequate sleep for 810 h, and engage in prayer. These practices have been demonstrated to reduce oxidative stress, increase self-confidence, enhance the circulating NK cells and lymphocytes, decrease plasma cortisol, and enhance several immune markers (Conroy and Milkis, 2012; Lule et al., 2005; Ichimura et al., 1999; Hendricks et al., 2003; Rehse, 1992; Lange et al., 2010; Moldofsky, 1995; Goodkin et al., 1998, 1999).

TABLE 31.2

Lifestyle Modifications and Immunity

Author

Studies on lifestyle interventions

Outcomes

Gil et al. (2005)

Dietary micronutrient intake and oxidative stress indicators

Beneficial effects on nutrition, systemic redox balance, and immune parameters

Bopp et al. (2004)

Physical activity and immunity

Increased physical activity will positively influence viral load

Joseph et al. (2015)

Naturopathy and yoga on CD4 counts

Increased CD4 counts were observed post interventions

Diego et al. (2001)

Massage and Immune function

Increased CD4/CD8 ratio and CD4 cells in massage group

Naoroibam et al. (2016)

Integrated yoga, depression and immunity

One month practice of yoga may reduce depression and improve immunity

Blazı´ckova´ et al. (2000)

Hyperthermic water bath on selected immune parameters

Increase in CD8 lymphocytes and NK cells activity

Nair et al. (2016)

Naturopathy and yoga on ART care

Naturopathy and yoga augment the efficiency of ART care

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31.6. CONCLUSION Lifestyle modification plays a major role in the rehabilitative and alterative efforts of PLWHA. These practices augment the immune response in HIV-positive individuals to a greater extent, and need to be considered while treating HIV infection. In the light of current evidence, lifestyle modification should be strongly recommended to enhance the quality and outcomes of HIV care.

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C H A P T E R

32 Eating Habits in Combating Disease: Nutraceuticals and Functional Foods at the Crossroads of Immune Health and Inflammatory Responses Shampa Chatterjee1 and Debasis Bagchi2 1

University of Pennsylvania School of Medicine, Philadelphia, PA, United States 2University of Houston College of Pharmacy, Houston, TX, United States

32.1. INTRODUCTION Since the last millennium, the composition of the human diet and the pattern of physical activities have undergone a major shift. However, these changes have not been uniform and vary largely depending on the region of the world. The poorer and disadvantaged regions face chronic hunger and malnutrition along with high levels of activity that are needed for survival, while high-income societies (and some parts of low-income societies) have diets that are high in total fat, cholesterol, sugar, carbohydrates and low in polyunsaturated fatty acids and fiber, and are accompanied by a sedentary lifestyle. Both these scenarios lead to diseased states. Malnourished individuals, specifically children, are highly susceptible to infections that impair physical growth and cognitive development. These infections are mostly in the form of enteric infections arising from attack by bacteria such as Escherichia coli, Vibrio cholerae, and several species of Salmonella, Shigella, and anaerobic streptococci. Infections are characterized by diarrhea, nausea, abdominal discomfort and vomiting; these in turn continue the vicious cycle of malnutrition (Hughes and Kelly, 2006). It is now well recognized that the immune dysfunction that arises from malnutrition gives rise to high incidences of communicable diseases. The consequences of overnutrition are also somewhat similar in that overnutrition also facilitates immune dysfunction, manifested in the form of a chronic inflammatory state. Inflammation is characterized by elevated levels of proinflammatory cytokines, chemokines and aggravated immune cell recruitment that collectively lead to early stages of disease development. The consequences of both undernutrition and overnutrition are thus the dysfunction in inflammatory activity (Soeters et al., 2008); therefore, controlling them is crucial to human health. Indeed a growing body of evidence shows that the nutrition-inflammation link is multifactorial and possibly reflects the effects of overnutrition in a population that has previously been exposed to malnutrition. In the final chapter of this book, we seek to reinforce the immune-inflammation-health-disease link by showcasing undernutrition and overnutrition from an “immune dysfunction perspective” and by elaborating on the link between an individual’s nutritional status and the onset of various inflammatory pathologies (Fig. 32.1). Studies showing the regulation of these diseases by fortifying our diet with functional foods and specific nutrients are also reviewed. Personalized diets that meet the challenge of human genetic diversity (nutrigenomics) that makes individuals respond differently to the same dietary intake are also discussed.

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FIGURE 32.1 The complex link between nutrition and disease. On the one hand, malnutrition is associated with a compromised immune-inflammatory system, while on the other, excessive calorie intake activates an inflammation cascade that leads to metabolic disorders. The microbiota too plays a pivotal role in the “malnutrition-decreased immune function” cycle as delay in development of gut microbiota causes childhood malnutrition. Meanwhile gut permeability, which is linked to the microbiota, is increasingly associated with metabolic disorders such as insulin resistance, type-2 diabetes, inflammatory bowel diseases, IBS, celiac diseases, etc.

32.2. INFLAMMATORY AND IMMUNE RESPONSES IN THE PATHOLOGY OF COMMUNICABLE AND NONCOMMUNICABLE DISEASES Both undernutrition and overnutrition related diseases arise from disordered nutrient assimilation that leads to recurrent infections and chronic inflammation respectively (Rahman and Adjeroh, 2015; Black et al., 2013; van der Klaauw and Farooqi, 2015). This implies an underlying immune defect. Malnutrition is well established in causing a large immunological deficit as a result of which individuals become susceptible to infections, perpetuating the malnutrition cycle as parasitic infection leads to increased inflammatory activity in the intestine, diarrhea and insufficient intake of food (Bourke et al., 2016; Bryce et al., 2005). Overnutrition also leads to increased inflammation, whereby an initial stimulus (such as injury, denudation of vascular layer, or hormonal imbalance, accumulation of fat cells, i.e., adipocytes, pathogenic attack) causes the induction of inflammation moieties such as cytokines and chemokines (interleukins (IL-), tumor necrosis factor (TNF-α), monocyte chemoattractant protein-1 (MCP-1)), that in turn lead to recruitment of immune cells (neutrophils, macrophages, monocytes) (Roubenoff, 2008; Lionetti et al., 2009). These are the hallmarks of acute inflammation that lead to oxidative damage to the tissue. Acute inflammation is often followed by chronic inflammation where inflammation, injury, and repair occur almost simultaneously within the tissue. For instance, in the case of the lung, chronic inflammation-induced structural changes cause airway remodeling via alterations in the airway epithelium, lamina propria and submucosa, leading to thickening of the airway wall (Fahy et al., 2000). Inflammation-induced thickening of the smooth muscle layer (in the medial layer of a blood vessel) is a major feature of vascular proliferative disorders encompassing cardiovascular disease (Sprague and Khalil, 2009). Eventually this process leads to alteration in the organ function (Sprague and Khalil, 2009). The onset of several noncommunicable diseases begins with a low grade inflammation that eventually leads to remodeling and altered tissue function. Indeed chronic inflammation is the common pathological basis for diseases such as cardiovascular disease, Alzheimer’s disease, systemic inflammatory state, metabolic disease, diabetes (inflammatory signals interfere with insulin signaling) (Sprague and Khalil, 2009; Yeh et al., 2016) and cancer (Coussens and Werb, 2002).

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The nutrition status of the individual plays a major role in immune dysfunction, either in the form of immune deficit or exaggerated immune response. Furthermore, deficiency of micronutrients such as deficiency or excess of certain micronutrients (e.g., folate, vitamin B12, vitamin B6, vitamin 1, vitamin E, zinc) may lead to an ineffective or excessive inflammatory response, respectively (Shenkin, 2006). Additionally, studies have shown that high consumption of fat and glucose may induce postprandial inflammation, which may have consequences for the development of diabetes and cardiovascular diseases (Giugliano et al., 2006).

32.3. DIET MICROBIOTA AND IMMUNE RESPONSES The microbiota composition in the gut is an important component of human metabolism, and diet plays a major role in the composition of the gut microbiota (Turnbaugh et al., 2009; De Filippo et al., 2010). The diet microbiota composition in turn affects immune and inflammatory responses (Wen et al., 2008; Maslowski et al., 2009). Microbiota in the gut is determined by various factors ranging from the maternal diet at the fetal stage and the mode of birth, as well as from postbirth factors such as diet, genetics and environmental stimuli. The mode of birth, i.e., normal or cesarean, decides the strain of microbiota that will populate the gut. In infants born normally, the microbiota is largely comprised of Lactobacillus, Prevotella or Sneathia spp. from the maternal vaginal tract; in infants born by cesarean section, bacteria from mothers’ skin (or the skin of participants in delivery procedure), i.e., Staphylococcus, Corynebacterium, and Propionibacterium spp., predominate the gut (Dominguez-Bello et al., 2010). It is well established that bacteria in gut and intestines participate in vitamin synthesis, the digestion of dietary fiber, and the regulation of inflammatory responses. Epidemiological data show a correlation between obesity and immune dysfunction-related diseases and as gut microbiota are known for their immunomodulatory properties (Maslowski et al., 2009); this implies a role for gut microbiota in driving obesity and other metabolic diseases. Indeed, studies have shown that composition of the gut microbiota during early life predicts the subsequent development of overweight and obesity. In a study using fecal samples from infants, it was found that those that had significantly higher bifidobacterial numbers during infancy had normal weight at 7 years of age, but those that had greater numbers of Staphylococcus aureus in infancy subsequently became overweight (Kalliomaki et al., 2008). Studies show that a low intake of fiber (complex plant polysaccharides) and high intake of carbohydrates and sugars affect the makeup of the intestinal microbiota, which require plant polysaccharides to produce immunomodulatory products such as short-chain fatty acids (SCFA) (Brestoff and Artis, 2013). SCFA acts as a ligand and binds to numerous receptors that participate in glucose homeostasis, appetite regulation, lipid metabolism and inflammatory response. A study comparing urban European and rural African children shows significantly less SCFA in the feces of European children (De Filippo et al., 2010). Of note is the fact that autoimmune disorders such as allergies and asthma are rarely observed in these African communities. Probiotic organisms in the gastrointestinal tract facilitate the production of SCFA; thus lactobacilli, bifidobacteria, enterococci in the gut may be important for maintaining the necessary amount of SCFA. In a trial carried out on 85 patients with irritable bowel syndrome (IBS), an inflammatory disorder of the gut, live combined Bifidobacterium, Lactobacillus and Enterococcus capsules, when administered for 4 weeks, significantly improved symptoms associated with IBS (Fan et al., 2014). Microbiota is altered in people with obesity, diabetes, allergies, or asthma (Sin and Sutherland, 2008; Patterson et al., 2009). Studies using humanized gnotobiotic mice showed that when these mice are switched from a diet low in fat and rich in plant polysaccharides to a Western diet high in fat and sugar and low in plant polysaccharides, they developed more adiposity within 2 weeks (Turnbaugh et al., 2009). Further, this change in their diet also altered their microbial composition, metabolic pathways and gene expression such that expression of proinflammatory moieties was facilitated. Immune signaling and gut microbiota are reported to play a role in development of type 1 diabetes. Nonobese diabetic mice deficient in the innate signaling molecule MyD88 (MyD882/2 NOD) are protected from the development of type 1 diabetes (Wen et al., 2008), implying that innate immunity plays a role in insulin secretion. But this protection is lost when MyD882/2 NOD mice are devoid of gut microbiota. The absence of MyD88 in NOD mice leads to an over-representation of bacteria of the Bacteroidetes phylum and this microbiota can, via production of an immunomodulatory product, prevent metabolic diseases such as diabetes (Wen et al., 2008). Overall, the gut microbiome is well accepted to communicate via this gut-brain axis, stimulating consumption of foods that promote their own growth. A growing body of evidence indicates that there is a crucial role for the

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microbiota in regulating different aspects of eating-related behavior, as well as behavioral comorbidities of eating and metabolic disorders (van de Wouw et al., 2017).

32.4. FUNCTIONAL FOODS IN REVERSING METABOLIC SYNDROME AS WELL AS IN IMPROVING MALNUTRITION-INDUCED IMMUNE IMPAIRMENT The manifestations of the metabolic syndrome in the form of obesity and cardiovascular disease, insulin resistance, dyslipidemia, enhanced proinflammatory state, and hypertension arise from high calorie intake and major lifestyle disorders. Currently, therapy involves myriad drug treatments for lowering cholesterol, controlling obesity, increasing insulin secretion, and surgical treatments. At the same time, malnutrition is a major driver of infection arising from reduced capacity of the immune system. Indeed studies have shown that nutritional intervention (micronutrients, aminoacids, simple lipids, etc.) helps in complete recovery from pathogen-induced infections even though the bacteria or protozoa were present (Amadi et al., 2005). There is now an increased awareness on a complementary approach to regulation of immunity in the form of lifestyle and diet modification. Functional foods such as fruits (berries, vegetables), and fiber-enriched grains are being reported to regulate immune function. There is also good evidence of links between micronutrient deficiencies such as zinc deficiency and the impairment of both innate (inborn) and adaptive (acquired) immune responses (Prasad, 2008). Exaggerated forms of inflammation are central to metabolic syndrome as several inflammatory mediators, such as cytokines like TNF-α, IL-1 and IL-6, and C-reactive protein that are the hallmark of inflammation, have been reported to promote signaling processes that lead to atherosclerosis, to insulin resistance, and to muscle protein catabolism (Klasing, 1988). In normal physiology, infection activates an immune response that manifests itself in the form of inflammation, whereby inflammatory mediators produced in the region of infection/injury attract multiple immune cells that inhibit pathogens. During this process, metabolic changes occur that ensure a rapid supply of aminoacids and glucose to fuel the immune response and enable rapid immune protein synthesis. Concurrently, fat oxidation is inhibited by inflammatory mediators leading to a state of hyperlipidemia, which neutralizes viruses, bacteria and other pathogen infectivity. However, once the pathogen is cleared from tissues, the inflammatory mediators decrease, leaving tissues free of both inflammation and infection. But in the presence of external agents (i.e., nonpathogens), ranging from cigarette smoke, to adipocytes, elevated blood cholesterol or glucose concentrations and obesity, the inflammatory mediators never leave and the cells/tissue are chronically stimulated leading to lasting insulin resistance, and hyperlipidemia, and ironically, chronic disease. Therefore, these diseases need strategies that focus on minimizing external factors (e.g., smoking cessation, weight loss, and stress management) as well as in reducing the inflammation response in general, to reduce disease risk. This can be done by supplementing our diet with nutraceuticals and functional foods that have bioactive ingredients with anti-inflammatory properties that are mediated by functional enzymes, probiotics, prebiotics, fibers, phytosterols, peptides, proteins, isoflavones, saponins or phytic acid, oligomeric proanthocyanidins, phytoalexins, and other bioactive substances (Abuajah et al., 2015).

32.5. BETWEEN DEPRIVATION AND OVERCONSUMPTION: MAINTAINING A BALANCED DIET TO COMBAT DISEASE Overconsumption refers to a condition where intake of food exceeds nutrient and energy requirements of the individual. Overconsumption is a major cause of overweight and obesity, with over one-third of the global adult population—1.9 billion people—now found to be either overweight or obese. Meanwhile, malnutrition that relates to deficiencies in energy/nutrient intake also occurs with overconsumption when micronutrients are lacking in the diet. Therefore, overconsumption can also result in a form of malnutrition. In poorer regions, malnutrition arises from low calorie intake that compromises immune function (Black et al., 2013; Bourke et al., 2016; Roubenoff, 2008), resulting in infectious diseases that in turn keep the subjects malnourished. In contrast, in developed countries, malnutrition can arise from overconsumption as well, as diets are based on relatively cheaper energy-dense convenience food. The WHO/FAO Expert Consultation on Diet, Nutrition and the Prevention of Chronic Diseases that met in Geneva in 2002 has issued guidelines based on the studies and data that showed a correlation between diet,

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physical activity and chronic inflammation-related diseases (WHA53.23, 2002). These recommendations in the form of both food intake and physical activity are under the following subheadings: 1. Food intake a. Replacing saturated and trans fats with unsaturated fats, including sources of omega-3 fatty acids. Replacing saturated fats with unsaturated fats will reduce the risk of coronary artery disease (CAD) (Hu and Willett, 2002) by reducing serum low-density lipoprotein (LDL) cholesterol. b. Addition of polyunsaturated fats (long-chain omega-3 fish oils, alpha-linoleic acid) to the diet (Baylin et al., 2003). c. Increasing consumption of fruits, vegetables and folic acid intake: high intake of fruits and vegetables has been found to relate to reduced indices of CAD and stroke (Conlin, 1999). d. Reduced consumption of sugar and sugar-based beverages. Sugar has no nutritional value and contributes to the dietary glycemic load which is linked to metabolic syndrome and thus to diabetes and artery diseases (Hu et al., 2001). e. Fortification of food with micronutrients: Food fortification has eliminated iodine deficiency, pellagra, and beriberi in much of the world. Grain products (flour, rice, and pasta) are fortified with B vitamins in several countries. Since 1998, grain products in the United States have been fortified with folic acid, which has almost eliminated folate deficiency, and reduced rates of neural tube defect pregnancies (Honein et al., 2001). In regions where intake of vitamins B12 and B6 are low as among vegetarian populations in India, simultaneous fortification of food with these vitamins is also being considered. 2. Physical activity as a key determinant of energy expenditure is fundamental to weight control. The beneficial effects of physical activity on the metabolic syndrome are mediated by mechanisms beyond controlling excess body weight. Walking, cycling, running can be an effective and practical means of engaging both in physical activity and in commuting and travel. Obesity connotes excessive energy intake, and hunger reflects an inadequate food supply; therefore, it seems paradoxical that these should prevail in the same population. However, it is possible that either a number of environmental, social, behavioral, or physiologic mechanisms could cause both hunger and obesity problems independently or these two could be causally related. For instance, past food deprivation may lead individuals to adopt harmful eating behaviors. It has been observed that World War II prisoners of war who experienced the trauma and food deprivation indulged in a very high rate of binge-eating behavior even 50 years later (Polivy et al., 1994). Refugees faced with past food deprivation also show similar eating behavior when food is plentiful (Peterman et al., 2010). Uneven access to food is associated with higher rates of overweight and obesity and weight gain in the United States (Wilde and Peterman, 2006), possibly because it may lead to excessive consumption of food in times of plenty (Toward et al., 2012). There are increased rates of chronic disease in these people that are possibly related to changes in food consumption in a postconflict environment with plentiful food (Dietz, 1995). Appetite is an important factor in guiding our food consumption that occurs via a complex physiological signaling system linking the brain and gut and hormones released from both the gut and the body’s fat deposits, indicating available energy levels to the brain, which in response triggers the feeling of either hunger or satiety. The consumption of foods and drinks high in sugar, fat, salt and calories are shown to activate the pleasure centers of the brain by stimulating increased dopamine production. The resulting rewarding effect can encourage emotional or comfort eating thus increasing the risk of overconsumption (Singh, 2014). Individual gene expression can be influenced by the mother’s diet, poorer diets increasing any offspring’s susceptibility to overconsumption and obesity (Drummond and Gibney, 2013).

32.6. DIET, IMMUNITY AND ADVANCING AGE Although the rate and progression of cellular aging varies among individuals, it eventually affects the cells of every major organ and reduces their function in terms of their involvement in immune function, inflammation, proliferation, apoptosis, cell cycle regulation, etc. The past paradigm of advancing age causing a functional decline due to accumulation of noxious biomolecular fragments has given way to the “hyperfunction theory” which describes advancing age as a phase of continuous growth, regulated by signaling pathways such as TOR (target of Rapamycin). Indeed TOR analogs have been found to be effective in treating advancing age-induced diseases and in extending and maintaining a healthy life span. Specifically, Everolimus, a rapamycin analog, has

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been reported to significantly improve immunity and prevent diseases in the elderly pointing to a novel therapeutic regimen that can potentially establish an era of “Human Immortality” (Blagosklonny, 2015). Another strategy of minimizing age-related changes is to target or boost genes that are associated with longevity. The gene forkhead box O-3 (FOXO3), encoding the transcription factor FOXO3, for which genetic polymorphisms (in the form of single nucleotide polymorphisms or SNP) has been reported to correlate with longevity in diverse human populations (Willcox et al., 2008). FOXO3 is a central transcription hub for multiple cellular stimuli including metabolic signals (via its role in insulin/insulin like growth factor signaling (Peterman et al., 2010; Willcox et al., 2008)). Oxidation of FOXO3 boosts the expression of genes that encode proteins that participate in DNA repair and suppresses members of the progrowth mechanistic mTOR kinase pathway (Morris, 2005). Thus agents that boost FOXO3 can potentially be effective in extending lifespan. Phytochemicals show promise via their regulation of FOXO3; treatment of rats by oral delivery of green tea phytochemical epigallocatenin gallate was found to increase lifespan (by 14%), and to reduce inflammation signaling pathways (NF-κB, IL-6, TNF-α) (Niu et al., 2013). Studies on human populations showed that there was a correlation between tea drinking and risk for cognitive disability in the elderly but this was dependent upon FOXO genotype, with carriers of FOXO1 SNP rs17630266 and FOXO3 SNPs rs2253319 and rs2802292 displaying significantly reduced risk for cognitive disability at advanced ages (Zeng et al., 2015). Overall, natural compounds such as resveratrol, curcumin, astaxanthin, among others can, by stimulating the effect of FOXOs on numerous genes important for cellular health and amelioration of diseases of aging, help improve health and boost lifespan (Eijkelenboom and Burgering, 2013). Aging also alters the gut microbiota and causes an increase in harmful bacteria such as clostridia and enterobacteria (Hopkins et al., 2002), thus negatively impacting the immune system of the elderly (Claesson et al., 2012). One of the interventions to improve gut functions is administration of prebiotics, oligosaccharides such as fructooligosaccharides, and galactooligosaccharides. Several studies found that administration of prebiotics (Toward et al., 2012; Bindels and Delzenne, 2013) in older individuals improves the levels of fatigue and muscle strength. Prebiotics could therefore be included in the treatment protocol of people suffering especially from frailty, and as a preventive intervention in general.

32.7. GOOD EATING HABITS Healthy eating and drinking behaviors are closely associated to healthy lifestyles, while overindulgence of high calorie foods, smoking, and consumption of alcoholic beverages are related to metabolic disorders that can lead to cardiovascular diseases and diabetes. It has been well demonstrated that healthy eating practices, maintaining a normal body weight, controlled blood pressure, and regular physical activity could prevent up to 80% of coronary heart disease, 90% of type-2 diabetes and one-third of all cancers. Food research institutions and agro-based, food and nutraceutical industries have invested much effort in developing novel, healthier, more nutritious and fortified functional foods. Collectively called functional foods, these contain nutritional components or nutraceuticals, which have been observed to protect or delay the onset of several metabolic and associated diseases. Functional foods contain bioactive compounds from plant extracts and these bioactive phytoconstituents have antioxidant, antimicrobial, immunomodulatory, hypocholesterolemic, anti-inflammatory or anticancer activities. The following are some of the examples of nutraceuticals or functional foods conferring health benefits in the form of: Antioxidant and anticancer effects: Grape seed extracts containing oligomeric proanthocyanidins, berry fruits containing anthocyanin antioxidants, green tea epigallocatechin gallate, polygonum cuspidatum-derived transresveratrol, turmeric and curcumin extract, vitamins C, E and beta-carotene, and several other functional foods been reportedly extensively to function as antioxidants (Lobo et al., 2010). Additionally many of these have been found to possess noticeable anticancer effects with mechanisms of action varying from antimetastatic, antiproliferative, antiangiogenic, etc. Perez-Gregorio and Simal-Gandara (2017). Weight management supplements: Herbs such as Garcinia cambogia, Garcinia mangostana fruit, green coffee bean extract, Citrus aurantium, Coleus forskohlii, Lagerstroemia speciosa, curcumin and turmeric, marine lipids, conjugated linolenic acid, chromium (III) supplements, Sphaeranthus indicus flower, Piper betle leaf and Dolichos biflorus seed extracts have been found to be effective weight management supplements; these are

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backed by safety and efficacy studies in clinical studies and most of these are being sold as novel dietary weight management supplements in the marketplace (Rebello et al., 2014; Kovacs and Mela, 2006). Antiarthritic, joint-health and anti-inflammatory supplements: Traditional and natural medicines that contain n-3 and n-6 fatty acids, fish oil, seed oils, red ginger extract, bromelain from pineapple stem, turmeric and curcumin powder and extract, Boswellia serrata extract, several marine-based nutraceuticals, ginger, chicken cartilage-derived type-2 collagen, glucosamine hydrochloride, chondroitin sulfate, green lipid mussel, dehydroepiandrosterone, rosmarinic acid derived from rosemary plants, and several others have established their potential benefits in rheumatoid and osteoarthritis (Ameye and Chee, 2006; Akhtar and Haqqi, 2012). Antidiabetic supplements: Fenugreek seed, guava leaves, berries and cinnamon extracts, Gymnema sylvestre and Momordica charantia extracts and chromium (III) supplements have demonstrated significant benefits in attenuating healthy blood glucose level (Perera and Li, 2012; Mirmiran et al., 2014). Brain and ocular health supplements: Standardized Bacopa monniera, Huperzia serrata (huperzine A), Celastrus paniculatus, Convolvulus pluricaulis, Centenilla asiatica and Ginko biloba extracts, as well as ginseng, royal jelly, black pepper-derived bioperine, curcumin and turmeric powders, zinc supplements, berries and nuts, and St John’s wort are quite popular nutraceutical supplements for both brain and ocular health, while astaxanthin, zeaxanthin, zinc supplements and lutein are supplements that help maintain ocular health (Gomez-Pinilla, 2008; Bussel and Aref, 2014). Immune health supplements: Curcumin and turmeric extracts, probiotics and prebiotics, berry extracts, vitamin C, Andrographis paniculata, olive leaves extract, oregano oil, echnaceae, astragalus and blue green algae extracts are popular immune health supplements backed by credible scientific research (Lobo et al., 2010; Daily et al., 2016). Other benefits: Nutraceuticals and functional foods have exhibited several other benefits including aphrodisiac activity, sports nutrition, muscle building and exercise.

32.8. NUTRIGENOMICS: PERSONALIZED NUTRITION TO COMBAT DISEASE It is well known that individuals respond differently to the same diet. Differential dietary responsiveness among humans presumably stems from our unique genotype. Nutrigenomics, a branch of genomics that studies the genetic basis of our variable response to diet, is now an emerging field that can personalize diet in order to optimize health risk and identify an individual’s trajectories toward chronic inflammatory states (Sales et al., 2014). Nutrigenomics involves the study of (1) the direct interaction between nutrients and DNA to modify genetic expression, (2) epigenetic interactions between nutrients and DNA in which the latter modify the structure of DNA via methylation and chromatin remodeling, and (3) genetic variations within humans (SNPs) that create the variations in response to diet between individuals (Sales et al., 2014; Dang et al., 2014). For instance, plasma cholesterol levels are known to depend on dietary cholesterol intake (Miettinen and Kesaniemi, 1989); however this is dependent upon the individual (Glatz et al., 1993) indicating that besides the individual’s genetic background, epigenetic changes that can be modulated by nutritional input may potentially be useful in disease prevention via tailoring of our diets individually. To facilitate this, the “genetic landscape” of metabolic diseases will need to be better mapped out such that genetic variation or polymorphisms and/or epigenetic changes that relate to risks of metabolic disease are well established (Ferguson et al., 2010; Joffe et al., 2010). The metabolism of folic acid (Zeisel, 2007) is an example of the role of epigenetic changes in diet assimilation. A polymorphism in the methylene tetrahydrofolate reductase (MTHFR) gene is associated with a difference in nutrient folate uptake and has been recognized to correlate with heart disease (Gohil et al., 2009) and cancer (Galvan-Portillo et al., 2010). Folate supplementation in those carrying genetic risk due to MTHFR reduces the incidence of various health problems associated with low folic acid status (Galvan-Portillo et al., 2010). Thus, mapping the links between genetic variations and susceptibility or resistance to disease, and metabolic responses to diet, can lead to the discovery of associations between genetic variation, diet and phenotypes (Hindorff et al., 2009). This would enable dietary responsiveness based on the metabolic phenotype early in life.

32.9. THE TAKE-HOME MESSAGE Based on extensive studies both in rodents and human populations, it is evident that nutrition and the immune function are intertwined, but a clear picture of the dietary and calorie intake that would ensure good

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health has not emerged. This is partly because of genetic variations between populations and between individuals and partly because the various signaling pathways from nutrition to immune function have not been well studied. Nevertheless, the 2010 dietary guidelines in the United States advocate a diet that comprises 45%65% carbohydrates, 10%35% of proteins and 20%35% of fat. However, physical activity, age, genetic predisposition to metabolic disease, etc., also need to be factored into the diet.

32.10. SUMMARY AND CONCLUSIONS The human immune system that provides immunity to defend the body against injury and infection works by recruiting immune cells that trigger the onset of inflammation. Inflammation, while protective, is also a risk factor for various chronic illnesses, including cardiovascular disease, cancer, and diabetes. Recognizing the role of the inflammatory process in disease development, there has been a search for an optimal diet and the requisite nutrition that may either promote or inhibit the inflammatory processes. Malnutrition and overconsumption induce immune dysfunction; numerous studies on supplementation of diet with functional foods show an increased immune function in malnourished individuals, while downregulating genes that are pivotal in driving important in inflammatory pathways, lipid metabolism, and cancer. This interaction between nutrition and immune function is summarized in Fig. 32.1. Taken together, it is important to design diets where anti-inflammatory benefits are derived not merely from a single nutrient but also from the synergistic effect of foods eaten together. That, as well as focusing on personalized diets, is pivotal to combating inflammation and enhancing overall health.

Acknowledgments We thank Ms. Priyal Patel for her help with Figure 32.1 in this manuscript.

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Appendix

Abbreviations (PI(3)P) PI(3,5)P2 1,25D3 8-OHDG 8-OH-GUA AA ACAD Ach ADA AHL AICD AIDS AM AMP AMPK AOE AOM AP-1 APC APP ARDS ARE ART ASIC ASL AT ATF ATP BAL BaP BCG Bcl2 BET BFT BHT BMI BPI bZIP CAD CAM CAMP cAMP CARDs CAT CB CCL11 CCL14a(9-74) CCR4 CD CD CD26/DPP4

phosphatidylinositol 3-phosphate phosphatidylinositol 3,5-bisphosphate 1,25-dihydroxyvitamin D3 8-hydroxydeoxyguanosine 8-hydroxyguanine arachidonic acid activation-caused autonomous death acetylcholine adenosine deaminase acyl-homoserine lactone activation-induced cell death aquired immuno deficiency syndrome alveolar macrophages antimicrobial peptide; 50 adenosine monophosphate-activated protein kinase antioxidant enzymes azoxymethane activator protein-1 antigen presenting cell acute phase protein adult respiratory distress syndrome activated response elements antiretroviral therapy acid-sensing ion channel airway surface liquid adipose tissue activating transcription factor adenosine triphosphate bronchoalveolar lavage benzo[a]pyrene Bacillus Calmette Guerin B-cell lymphoma 2 BrunauerEmmettTeller B. fragilis toxin butylated hydroxytoluene body mass index bactericidal permeability increasing protein basic leucine zipper coronary artery disease cell adhesion molecule cathelicidin antimicrobial peptide cyclic adenosine monophosphate caspase activation and recruitment domains catalase cord blood eotaxin/CC motif chemokine 11 CC motif chemokine ligand 14 a (9-74) CC chemokine Receptor type 4 cluster of differentiation Crohn’s disease cluster of differentiation 26/dipeptidylpeptidase 4

433

434 CD41 , CD81 CDC CF CFTR CFU cGMP CLA CMV CNPY3 CO COAD COPD COX COX-2 CpG CpG CRC CREB CRP CRS CuZnSOD CVDs DAMP DB DC DEP DHA DHEA DIO DMH DNA DPPIV dsDNA DSS EBC e-cigarette EFSA EGFR EGFR ENaC EPA EPO EPO ER ERK Ero 1p ERS ESR ETBF ETC FadA FAEE FAO FcRN FCγR FDA FEV1 FFAs FGF FLT-3L FMs FMT FOS FOXO3 FOXP3

APPENDIX

cluster of differentiation 8 centers for disease control and prevention cystic fibrosis cystic fibrosis transmembrane conductance regulator colony forming unit cyclic guanosine monophosphate conjugated linoleic acid cytomegalovirus canopy 3 carbon monoxide chronic obstructive airway disease chronic obstructive pulmonary disease cyclooxygenase cyclooxygenase-2 cytosine-phosphate-guanine ODN-CpG oligodeoxynucleotides colorectal cancer cAMP response element-binding protein C-reactive protein chronic rhinosinusitis copper-zinc superoxide dismutase cardiovascular diseases damage-associated molecular pattern double blind dendritic cell diesel exhaust particles docosahexaenoic acid dehydroepiandrosterone diet induced obesity 1,2-dimethylhydrazine deoxyribonucleic acid dipeptidyl peptidase IV double stranded DNA dextran sodium sulfate exhaled breath condensate electronic cigarette European food safety authority EGF-receptor (epidermal growth factor receptor) epidemial growth factor receptor epithelial sodium channel eicosapentaenoic acid erythropoietin mitochondrial oxidases, and eosinophil peroxidase endoplasmic reticulum extracellular signal regulated kinases endoplasmic reticulum-resident protein (Ero1p) European respiratory society erthrocyte Sedimentation Rate enterotoxigenic bacteroides fragilis electron transport chain fad adhesion gene fatty acid ethyl esters food and agriculture organization neonatal fc receptor common γ chain of fcε receptor food & drug administration forced expiratory volume in 1 second free fatty acids fibroblast growth factor FMS-like tyrosine kinase 3 ligand fermented milks fecal microbiota transplantation fructooligosaccharides forkhead box O-3 forkhead box P3

APPENDIX

FRA GATA3 GBS GCL GCP-2/CXCL6 G-CSF GI GI GINA GIP GLP-1 GLUT1 GM-CSF GOLD GOS GPCR GPD2 GPR GPX GPx GRAS GRP94 GSH GSH GSPE GSSG GSSG GST GSTM1 GSTP1 GVHD H2O2 HA HAART Hb Hb HDAC HFD HGP HI HIF1-α HIV HLA-DR HMGB-1 HMGB1 HMOX HNE HO-1 HOBR HOCl HSV I/R-injury IBD IBS IC50 ICAM IDO IEC IFN IFN-γ IgA IgA/G IgCs IGF-1 IgG

Fos-related antigen trans-acting T-cell-specific transcription factor Group B streptococcus glutamate-cysteine ligase granulocyte chemotactic protein 2/CXC motif ligand 6 granulocyte colony stimulating factor gastrointestinal glycemic index global initiative for asthma glucose-dependent insulinotropic peptide glucagon-like peptide glucose transporter 1 granulocyte-macrophage colony-stimulating factor global initiative for chronic obstructive lung disease galactooligosaccharides G-protein-coupled receptor glycerol-3-phosphate dehydrogenase 2 G protein-coupled receptor glutathione peroxidase glutathione peroxidase generally recognized as safe glucose regulated protein glutathione reduced form glutathione grape seed-derived procyanidins disulfide form of glutathione glutathione disulfide glutathione S-transferase glutathione S-transferase Mu1 glutathione S-transferase P1 graft vs. host disease hydrogen peroxide hyaluronic acid highly active anti-retroviral therapy hemoglobin hemoglobin histone deacetylase high fat diet Human Genome Project hemagglutination inhibition hypoxia-inducible factor-1α human immuno deficiency virus human leukocyte—antigen D Related high mobility group box-1 high mobility group protein 1 heme oxygenase 4 hydroxy 2 nonenal heme-oxygenase-1 hypobromous acid hypochlorous acid herpes simplex virus ischemia/reperfusion-injury inflammatory bowel disease irritable bowel syndrome 50% inhibitory concentration intercellular adhesion molecule Indoleamine-pyrrole 2,3-dioxygenase intestinal epithelial cell compartment interferon interferon gamma immunoglobulin A immunoglobulin A/B IgG containing immune complexes insulin like growth factor-1 immunoglobulin G

435

436 IKK IL IL-1 IL-1β IL-6 ILCs IMQ iNKT cells iNOS IP-10/CXCL10 IP3 IP3R IR IRAK IRF IRI ISAPP I-TAC/CXCL11 ITAM ITS IUD JAK JNK KIR LAB LcS LDL LGG LIF LN LPL LPO LPS LTA LTB-4 MAGUK MAMP MAM MAPK MAS MBL MCC MCP-1 M-CSF MD MDA MDC/CCL22 MDSCs MeIQx MHC MHC-II MIF Mig/CXCL9 MIP MIP-1α MIP-1α/CCL3L1 MIP-1β/CCL4 MMP MMP-9 MNC MNNG MNP MoDCs MPO mROS

APPENDIX

IκB Kinase interleukin interleukin-1 interleukin 1 beta interleukin-6 innate lymphoid cells Imiquimod invariant natural killer T-cells inducible nitric oxide synthase interferon gamma-induced protein 10/CXC motif chemokine 10 inositol 3,4,5-triphosphate inositol trisphosphate receptor insulin resistance IL-1 receptor associated kinases interferon regulatory factor ischemia reperfusion injury International Scientific Association for Probiotics and Prebiotics interferon-inducible T-cell alpha chemoattractant/CXC motif chemokine 11 iimmunoreceptor tyrosine-based activation motif internal transcribed spacer injection drug use janus kinase jun amino terminal kinases iiller-cell immunoglobulin-like receptor lactic acid bacteria L. casei Shirota low density lipoprotein L. rhamnosus GG leukemia inhibitory factor lupus nephritis lipoprotein lipase lactoperoxidase lipopolysaccharides lipoteichoic acid leukotrine B-4 membrane-associated guanylate kinase-like microbial associated molecular pattern microbial anti-inflammatory molecule mitogen-activated protein kinase macrophage activation syn mannose binding lectin mucociliary transport monocyte chemoattractant protein-1 macrophage colony stimulating factor Mediterranean diet malondialdehyde macrophage-derived chemokine/CC motif chemokine 22 myeloid derived suppressor cells 2-amino-3,4-dimethyl-imidazo[4,5-f]-quinoxaline major histocompatibility complex major histocompatibility complex Class II macrophage migration inhibitory factor monokine induced by gamma interferon/CXC motif ligand 9 macrophage inflammatory protein macrophage inflammatory protein 1 alpha macrophage inflammatory protein-1α/CC motif ligand 3-like 1 macrophage inflammatory protein-1β/CC motif ligand 4 matrix metalloprotease matrix metalloproteinase-9 blood mononuclear cells N-methyl-N0 -nitro-N-nitrosoguanidine magnetic nanoparticles monocyte derived dendritic cells myeloperoxidase mitochondrial ROS

APPENDIX

mtDNA MTHFR mTOR MyD88 NAC NADPH NAFLD NC NCCD NCD ND NET NFAT NF-κB NGS NIH NK-cells NKT NLRP NO NOD NOS NOX NPY NQO1 Nrf2 OSM OVA-Ics PAFAH PAMP PBMC PC PCD PCL pDCs PDI PECAM PEG PEGDA PEG-PLGA PEI-PEG PGE2 PHOX PI3K PICD pks PLCγ1 PLCγ2 PLGA PLWHA PMA PMN PMN PPAR-α PROP PRRs PSE PTC Ptgs2 PUFAs PUVA PYY R R-848 RA

mitochondrial DNA methylene tetrahydrofolate reductase mechanistic target of rapamycin myeloid differentiation primary response gene 88 N-acetylcysteine Nicotinamide adenine dinucleotide phosphate non-alcoholic fatty liver diseases nanocarrier nomenclature committee on cell death noncommunicable diseases neurodegenerative disease Neutrophil extracellular trap nuclear factor of activated T-cells nuclear factor kappa B next-generation sequencing National Institute for Health natural killer cells natural killer T-cells NOD like receptor protein nitric oxide nonobese diabetic nitric oxide synthase NADPH oxidases neuropeptide Y NAD(P)H quinine oxidoreductase 1 nuclear factor erythroid 2-related factor 2 oncostatin M ovalbumin Immune complexes platelet-activating factor acetylhydrolase pathogen-associated molecular pattern peripheral blood mononuclear cells; placebo controlled primary ciliary dyskinesia periciliary layer plasmocytoid dendritic cells protein disulfide isomerase platelet-endothelial cell adhesion molecule-1 polyethylene glycol polyethylene diacrylate poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) polyethyleneimine-poly(ethylene glycol) Prostagladin E2 phagocytic NADPH oxidase Phosphoinositide 3 kinases phagocytosis induced cell death polyketide synthase Phospholipase C-gamma1 Phospholipase C gamma 2 poly(lactide-co-glycolide) people living with HIV/AIDS Phorbol myristate acetate polymorphonuclear leukocytes polymorphonuclear neutrophils peroxisome proliferator-activated receptor alpha propyl-2-thiouracil pattern recognition receptors psoriatic skin equivalent phenylthiocarbamde prostaglandin-endoperoxide synthase 2 polyunsaturated fatty acids psoralen plus ultraviolet light therapy peptide YY randomized resiquimod rheumatoid arthritis

437

438 RAGE RALDH RANTES/CCL5 RAR RARE RBC RCT RDA RES RLRs-RIG RNS ROCK ROS RSV RXR RXRE S aureus SARM SARs SB SCC SCF SCFA ScRs SD SDF1/CXCL12 SDF-1α sDPPIV/CD26 sIgA SLE SLGT SLN SMC Sn-G3PDH SOD SOCS SPRR SQR STAT STAT-3 SVCT Syk SYN T reg cells T1D T1R T2D T2DM T2R TCR TG TGF-β Th Th1 Th1 Th1/Th2 Th2 TIR TJ TLR TNBS TNFα TOR TRAIL TRAM

APPENDIX

receptor for advanced glycation end-products retinaldehyde dehydrogenase regulated on activation, normal T-cell expressed and secreted/CC motif chemokine ligand 5 retinoic acid receptor retinoid acid response element red blood cells randomized controlled trial recommended dietary allowance reticuloendothelial system 1 like receptors reactive nitrogen species Rho associated protein kinase reactive oxygen species respiratory syncytial virus retinoid X receptor retinoid X response element Staphylococcus aureus sterile α-and armadillo-motif-containing protein structure-activity relationships Saccharomyces boulardii solitary chemosensory cell stem cell factor short-chain fatty acid scavenger receptors Sprague Dawley stromal cell-derived factor 1/CXC motif chemokine 12 stromal cellderived factor-1α soluble form of DPPIV/CD26 secretory immunoglobulin A systemic lupus erythomatosus sodium-linked glucose transporter solid lipid nanoparticle smooth muscle cell sn-glycerol 3 phosphate dehydrogenase superoxide dismutase suppressor of cytokine signaling proteins Small proline-rich proteins Succinate-coenzyme Q reductase signal transducer and activator of transcription signal transducer and activator of transcription-3 sodium-vitamin C cotransporter spleen tyrosine kinase synbiotics T regulatory cells type 1 diabetes taste family type 1 receptor type 2 diabetes type 2 diabetes mellitus taste family type 2 receptor T-cell receptor triglycerides transforming growth factor beta helper T-cell helper T-cells type 1 T helper 1 cells T helper cells 1/2 helper T-cells type 2 toll/interleukin 1 (IL-1) receptor tight junction toll like receptors 2,4,6-trinitrobenzenesulfonic acid Tumor necrosis factor alpha target of Rapamycin TNF-related apoptosis-inducing ligand TRIF-related adaptor molecule

APPENDIX

Treg T-RFLP TRIF TRX TT Tx UC UNC93B1 URTI VCAM VDR VDRE VEGF VIP VLA-4 VLDL vRNA WHO XDH XO

CD4 1 regulatory T-cells terminal restriction fragment length polymorphism Toll-receptor-associated activation of interferon thioredoxin tetanus toxoid transplantation ulcerative colitis. Unc93 homolog B1 upper respiratory tract infections vascular cell adhesion molecule vitamin D receptor vitamin D response element vascular endothelial growth factor vasoactive intestinal peptide very late antigen-4 very low density lipoprotein viral RNA World Health Organization xanthine dehydrogenase xanthine oxidase

Amino acid codes Ala Arg Asn Asp Cys Gln Glu Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

alanine (A) arginine (R) asparagine (N) aspartic acid (D) cysteine (C) glutamine (Q) glutamic acid (E) glycine (G) histidine (H) isoleucine (I) leucine (L) lysine (K) methionine (M) phenylalanine (F) proline (P) serine (S) threonine (T) tryptophan (W) tyrosine (Y) valine (V)

439

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Index

Note: Page numbers followed by “f ” and “t” refer to figures and tables, respectively.

A AA. See Arachidonic acid (AA) Aberrant crypt foci (ACF), 408 Abietic acids, 375 ABs. See Apoptotic bodies (ABs) Abscesses, 168 Ac-SOD. See Acetylated hydrophobic derivative of SOD (Ac-SOD) ACAD. See Activation-caused autonomous death (ACAD) Ac¸aı´ fruit (Euterpe oleracea), 365 Acetyl CoA carboxylase (ACC), 397 Acetyl salicylic acid synthesis, 363 3-O-Acetyl-11-keto-β-boswellic acid, 376377 Acetylated hydrophobic derivative of SOD (Ac-SOD), 71 N-Acetylmuramic acid residues, 176, 181 ACF. See Aberrant crypt foci (ACF) Achillea, 243, 246247 Achillea “Moonshine”, 245246 comparison of antiacne sources to novel alkamide from, 247 isolation of antiacne compounds from, 246 Achillea asiatica, 245 Achillea Clypeolata, 245 Achillea lanulosa, 244245 Achillea millefolium, 243244, 243f, 244f anti-inflammatory and antiacne effects, 245 bioactivity, 245 identification and characterization of novel alkamide, 246247 inflammation, 241 molecular basis for antiacne activity, 246 phytochemistry, 244245 Acid-sensing ion channels (ASICs), 107 Acidic environment, 72 Acitretin, 261262 Acne, 242 Acne vulgaris, 241 etiology and pathology, 242 management, 242245 Acoustic enhancement, 72 Acquired immune deficiency syndrome (AIDS), 295, 301, 416 Acrolein, 86 Actin, 295296 Activated NOX, 120 Activating transcription factor (ATF), 8788 Activation-caused autonomous death (ACAD), 57 Activation-induced cell death (AICD), 57

Activator protein-1 (AP-1), 3940, 8788, 120, 204205, 341342, 377 ACTs. See Artemisinin combination therapies (ACTs) Acute HIV infection, 301 Acute inflammation, 424 Acute phase proteins (APPs), 67 Acute rhinosinusitis, 110 Acute seroconversion syndrome. See Acute HIV infection N-Acystelyn, 89 AD. See Alzheimer’s disease (AD) AD-EDA-ID. See Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD-EDA-ID) ADA. See Adenosine deaminase (ADA) Adalimumab, 283 Adaptive immune responses, 258, 296 cell types in, 8 crosstalk between innate and, 89 Adaptive immune system, 134, 165166 alteration of cellular components, 323324 cells, 4f chronic diseases, 10 components of immune response, 5 improving immunity for prevention and care, 1011 induction of adaptive immunity, 8 vascular endothelium, 910 Adaptive immunity, 249250, 257, 260, 264, 273, 282 Adaptive responses, 225226 Adaptor proteins, TLR, 226227, 227f Adenosine deaminase (ADA), 135, 165 50 Adenosine monophosphate-activated protein kinase (AMPK), 229 Adipocyte regulation, L. plantarum effect in, 391392 Adipose tissue (AT), 389 ADP-dependent glucokinase (ADPGK), 61 Adult respiratory distress syndrome (ARDS), 65 Advanced glycation end products (AGEs), 347 Aerobic exercise, 48 Agaricus bisporus, 275 Agave americana, 377378 AGEs. See Advanced glycation end products (AGEs) Aging age factor, 348

441

epidemiological, physiological and clinical features of, 319320 immunobiology of, 321324 AICD. See Activation-induced cell death (AICD) AIDS. See Acquired immune deficiency syndrome (AIDS) AIM2-like receptors (ALRs), 175176 Air pollution, 82 Airborne PM, 82 Airway inflammation, 10, 309 Airway surface liquid (ASL), 101103 Alanine (A), 107108 Alarmins, 56, 224 Albiflorin, 373 Alkamides, 245 comparison of antiacne sources, 247 identification and characterization, 246247 Alogliptin, 139t Alpha 2-Heremans-Schmid glycoprotein (AHSG). See Fetuin-A α-tocopherol, 85 ALRs. See AIM2-like receptors (ALRs) Alveolar macrophages (AMs), 16 Alzheimer’s disease (AD), 348349 Amentoflavone, 285, 285f Amino acids, 49 2-Amino-3,4-dimethyl-imidazo(4,5-f)quinoxaline (MeIQx), 407 AMPK. See 50 Adenosine monophosphateactivated protein kinase (AMPK) AMPs. See Antimicrobial peptides (AMPs) AMs. See Alveolar macrophages (AMs) Anagliptin, 137, 139t Androgen receptor (AR), 345 Andrographis paniculata, 374375 Andrographolide, 374375 Anemarsaponin B, 378 Angiogenesis, 315 Angiotensin II type-I receptor (AT1), 346347 Angiotensin II type-2 receptor (AT2), 346347 Ankyloses protein homolog (ANKH), 197 Anoikis, 51 Anthocyanidins, 365f, 367368 Anthocyanins, 367368 Anti-BP180 autoantibodies, 215 Anti-Candida immunity, 273 Anti-Saccharomyces cerevisea antibodies (ASCA), 182

442 Antiacne comparison of antiacne sources to novel alkamide, 247 effects, 245 isolation of antiacne compounds, 246 molecular basis for antiacne activity, 246 Antiapoptotic proteins, 5152, 345 Antiarthritic supplements, 429 Antibiotics, 242 exposure, 16 Anticancer effects, 428 Antidiabetic supplements, 429 Antigen presenting cells (APC), 6, 8, 57, 120, 223226, 231, 322323 interaction with T lymphocyte, 4f Antigens, 134 antigen-bearing DCs, 8 antigen-specific immune response, 257 presentation, 8 Anti-inflammatory activity, 285, 297301 in vitro studies, 298299 in vivo studies, 299301 drugs, 309 effects, 245, 283, 284t of artesunate in diseases, 312314 autoimmune diseases, 312313 chronic inflammatory lung diseases, 313314 malaria, 312 pathogen infection diseases, 314 pharmaceuticals, 363 of probiotics, 295 redox control of anti-inflammatory transcription factors, 3940 supplements, 429 Antimicrobial peptides (AMPs), 103105, 258 Antiobesity effect of LG2055, 393 Antioxidant enzymes (AOE), 65, 6768 Antioxidant response element (ARE), 39, 151, 351 Antioxidant(s), 59, 65 antioxidant-based therapeutic approaches for COADs, 8890 enzymatic antioxidant mimetics, 90 nonenzymatic antioxidants, 8990 defense system in lung, 8385 enzymatic antioxidants, 8385 nonenzymatic antioxidant, 85 effects, 428 enzymes, 229 nanocarrier-mediated delivery, 6974 lipid nanoparticles and complexes, 74 liposomes, 7072 MNPs, 7374 polymer nanocarriers, 7273 nanoparticle types, 70f paradox, 355 redox control, 3940 scavenging, 89 SLNs, 74 targeting strategies, 6869 as therapeutics, 6768 AntiPD-L1 therapy, 408409

INDEX

Antiproliferative remodeling, 314315 Antiretroviral therapy (ART), 301 Antitissue remodeling, 314315 Antitrypsin, 103 Antiviral activity by LAB, 252253, 252f AOE. See Antioxidant enzymes (AOE) AP-1. See Activator protein-1 (AP-1) APAF1. See Apoptotic protease-activating factor 1 (APAF1) APC. See Antigen presenting cells (APC) Apigenin, 285286, 286f, 364f, 365 Apo-B monomers, 347 Apoptosis, 50 modulation, 409 Apoptotic bodies (ABs), 193 Apoptotic protease-activating factor 1 (APAF1), 175 Appetite, 427 Apple flavonoids, 366 APPs. See Acute phase proteins (APPs) AR. See Androgen receptor (AR) Arachidonic acid (AA), 47 ARDS. See Adult respiratory distress syndrome (ARDS) ARE. See Antioxidant response element (ARE) Arginase-2, 28 ART. See Antiretroviral therapy (ART) Artemisia annua L, 309310 Artemisia species, 374 Artemisinin, 310f antimalarial activity, 310311 derivatives, 309311 discovery, 310 Artemisinin combination therapies (ACTs), 315 Artesunate, 309315 anti-inflammatory effect in diseases, 312314 antimalarial activity, 310311 function in immune system, 311312 immunosuppressive activity, 311314 limitations of artesunate-drug resistance, 315 in other directions, 314315 Arthritis, 168 ASCA. See Anti-Saccharomyces cerevisea antibodies (ASCA) Ascomycota phyla, 274275 Ascorbic acid, 68, 354 Ashwagandha. See Withania somnifera (Ashwagandha) ASICs. See Acid-sensing ion channels (ASICs) ASL. See Airway surface liquid (ASL) Aspergillus sp., 272, 275276 Aspirin, 363 Asthma, 79, 81, 271272, 313314, 342343, 343f chronic inflammation in, 309 artesunate, 309315 Astilbin, 286287, 286f AT. See Adipose tissue (AT) AT1. See Angiotensin II type-I receptor (AT1)

ATF. See Activating transcription factor (ATF) Atherosclerosis, 189, 345346 atherosclerotic lesion, 189190, 191f calcification associated with, 191192 inflammation in, 190 vitamin K as potential therapeutic target, 195196 Atopic dermatitis, 262, 265266, 266f ATP-citrate lipase, 397 AU-enriched elements. See AUUUA Autoantibodies, 213214 autoantibodies-associated humoral response, 215 BP180 NC16A serum titer, 215 Autoimmune diseases, 312313 DPPIV/CD26-inhibitor effects in, 136138 clinical studies, 137 immunological side effects, 137138 preclinical studies, 137 role, 228229 Autoimmune responses, 224 Autoimmune-mediated T1D, 150 Autoimmunity, 233, 234f to inflammation and blister formation, 215217 inflammation to autoimmunity and therapeutic response, 217218 Autophagy, 50 Autosomal dominant anhidrotic ectodermal dysplasia with immune deficiency (AD-EDA-ID), 171 AUUUA, 205 Ayran, 396 Azulenes, 245

B B cells, 8 B lymphocytes, 258 B-cell activating factor (BAFF), 313 B-cell receptors, 8 B-lymphocytes, 89 Bach1, 151 Bacteria, 178181, 272 bacterial substrates, 249 bacterialfungal interactions, 272 cell wall, 176 effects and metabolites on host, 180 innate immune receptor recognition, 175178 invasive bacteria modifying core peptidoglycan structure, 181 Bactericidal permeability increasing protein (BPI), 1718 Bacteroides. fragilis toxin (BFT), 404 Bacteroidetes, 390 Bacteroidetes bacterial phyla, 277 Bacteroidetes phylum, 425 BAFF. See B-cell activating factor (BAFF) Baicalin, 287, 287f, 365 BAL. See Bronchoalveolar lavage (BAL) Balanced cellular redox state, 341 BALF. See Bronchoalveolar lavage fluid (BALF) BaP. See Benzo[a]pyrene (BaP)

INDEX

Bardoxolone methyl, 154155 Bardoxolone Methyl Analog (dh404), 156157 Basal cell carcinoma, 262264, 263f Basic leucine zipper (bZIP), 8788, 152 Basidiomycota phyla, 274275 BBB. See Blood brain barrier (BBB) Bckdh. See Branched-chain oxo-acid dehydrogenase phosphatase (Bckdh) BEAS-2B. See Bronchial epithelial cells (BEAS-2B) Benzo[a]pyrene (BaP), 48 Benzoyl peroxides, topical, 243 Berberine, 138140 β-catenin, 403404 BFT. See Bacteroides. fragilis toxin (BFT) BHT. See Butylated hydroxytoluene (BHT) Bifidobacteria species, 295, 298, 405406 Bifidobacterium sp., 16 B. animalis, 296297 B. bifidum, 16, 296297 B. infantis, 300 B. longum, 296297 capsules, 425 Bilberries, 367368 Bioactivity of A. millefolium, 245 Biomarkers, 182 Biotherapies, 218 Birch (Betula species), 377 1,3-Bisphosphoglycerate (1,3-DPG), 347 Blackberries (Rubus species), 368 Blackcurrants (Ribes nigrum), 368 Bladder cancer, 344345 Blister formation, 215218 Blood brain barrier (BBB), 312 Blood flow, 417418 Body mass index (BMI), 389 Bone marrow impact of aging of, 322323 derived cells, 133134 Boswellia serrata extract, 429 BP. See Bullous pemphigoid (BP) BP180 antigen, 213214 BP230 antigen, 213214 BPI. See Bactericidal permeability increasing protein (BPI) Brain natriuretic peptide, 127128 Brain supplements, 429 Branched-chain oxo-acid dehydrogenase phosphatase (Bckdh), 59 Brazilian arnica. See Lychnophora trichocarpha extracts Breast epithelium damage, 344 Bric-a-brac domain (BTB domain), 153 3-Bromotyrosine, 88 Bronchial asthma, 81 Bronchial epithelial cells (BEAS-2B), 313314 Bronchoalveolar lavage (BAL), 86, 343 Bronchoalveolar lavage fluid (BALF), 313 BTB domain. See Bric-a-brac domain (BTB domain) Bullous pemphigoid (BP), 213 autoimmunity to inflammation and blister formation, 215217

clinical and biological aspects, 213215, 214f inflammation to autoimmunity and therapeutic response, 217218 Butylated hydroxytoluene (BHT), 70 N-Butyryl-L-homoserine lactone (C4HSL), 107108 Butyrate, 408 BXT-51072, 90 BXT-51077, 90 bZIP. See Basic leucine zipper (bZIP)

C C4HSL. See N-Butyryl-L-homoserine lactone (C4HSL) C5a. See Complement component 5a (C5a) C12HSL. See N-3-Oxo-dodecanoyl-Lhomoserine lactone (C12HSL) CAC. See Coronary artery calcium (CAC) Cachexia, 321 CAD. See Coronary artery disease (CAD) Caffeic acid, 380 Calcification, 193194 with atherosclerosis, 191192 calcifying extracellular vesicles, 193194 Calciprotein particle (CPP), 194195 Calcium, 257 CAM. See Cellular adhesion molecules (CAM) CAMP. See Cathelicidin antimicrobial peptide (CAMP) Cancer, 203204, 343345, 344f COX-2 in pathogenesis of inflammationassociated, 204 nutritional/dietary manipulation of aberrant inflammatory signaling, 208 Candida albicans, 272 Canonical pathway, monogenetic primary immunodeficiencies of, 167169 Canonical signaling, 166167 Canopy 3 (CNPY3), 228 Cantalasaponin-1, 377378 Cap “n” collar protein (CNC protein), 152 Carbocysteine, 8990 Carbohydrate moieties, 272273 Carbohydrate/lipid metabolism, L. gasseri effect in, 393394 Carbon monoxide (CO), 39, 79 Carbonyl adducts, 88 Carcinogen inactivation, 407 Carcinogenesis, 204 Carcinoma cell lines, 315 Cardiac valve calcification (CVC), 192 Cardiomyocytes, 345 Cardiovascular diseases (CVDs), 189, 341342, 345347, 346f, 353 CARDs. See Caspase activation and recruitment domains (CARDs) Carmegliptin, 140t Carnosol, 375 Carotenoids, 68 Caspase cascade. See Intrinsic pathways Caspase-1, 52 Caspase 1/11, 18 Caspase-14, 288

443 Caspase activation and recruitment domains (CARDs), 176 CAT. See Catalase (CAT) Catalase (CAT), 73, 83, 85, 149150, 232, 351 immune-conjugates, 69 Catalytic proteins, 342343 Catechins, 364f, 367 Cathelicidin. See LL-37 Cathelicidin AMP, 258 Cathelicidin antimicrobial peptide (CAMP), 264 CBC. See Complete blood count (CBC) CC motif. See Chemokine motif (CC motif) CC motif chemokine 11 (CCL11), 135136 CC motif chemokine ligand 2 (CCL-2), 149, 190, 393394 CC motif ligand 3-like 1 (CCL3L1), 135 CC motif ligand 4 (CCL4), 135 CCAAT/enhancer binding protein (CEBP), 204205 CCL-2. See CC motif chemokine ligand 2 (CCL-2) CCL3L1. See CC motif ligand 3-like 1 (CCL3L1) CCL4. See CC motif ligand 4 (CCL4) CCL11. See CC motif chemokine 11 (CCL11) CCR2. See Chemokine (CC motif) receptor 2 (CCR2) CD. See Crohn’s disease (CD) CD1d, cell surface receptor, 262 CD3-T-cell receptor (CD3-TCR), 231 CD41 T-cells, 134 global hypomethylation in SLE, 232233 CD1411CD162monocytes, 20 CD141CD161 monocytes, 20 CD26. See Cluster of differentiation 26 (CD26) CD26/DPP4, 127 clinical implications for, 130131 gliptins in experimental models, 129f modulation against lymphocytic inflammation, 130 as therapeutic target in experimental models against I/R-injury, 127130 CD44-ERM/ROCK pathway, 231232 CD64 expression, 20 CD711 erythroid cells, 28 CD95L gene, 6061 CDC. See Center for Disease Control (CDC) cDCs. See Conventional DCs (cDCs) CDDO-Me, 154155 CEBP. See CCAAT/enhancer binding protein (CEBP) Celastrol, 377 Cells, 134 cell-specific role of NADPH oxidases in pulmonary inflammation, 121122 death, 5052 occurring during metaphase, 51 differentiation, 409 Cellular adhesion molecules (CAM), 38, 67, 403404 Cellular debris in SLE pathogenesis, 229230

444 Cellular signal transduction pathways, 208 Cellular signaling, 5859 Centella asiatica, 376 Center for Disease Control (CDC), 301 Cerium oxide nanoparticles, 73 CF. See Cystic fibrosis (CF) CF transmembrane conductance regulator (CFTR), 104 CFTR. See CF transmembrane conductance regulator (CFTR) CG. See Cytosine and guanine (CG) CGD. See Chronic granulomatous disease (CGD) γ Chain of Fcε receptor (FCγR), 231 Chamazulene, 244 Cheese, 395 Chemokine (CC motif) receptor 2 (CCR2), 393394 Chemokine (CC motif) receptor 6 (CCR6), 156 Chemokine motif (CC motif), 393394 Chemokines, 135, 297, 341342 Chemoprevention, 203, 208 Chemosensory GPCRs, 105106 Chemotherapy, 405 Chhash, 395 Chloroquine, 310 Chromium (III) supplements, 429 Chronic diseases of innate and adaptive immune systems, 10 Chronic granulomatous disease (CGD), 37 Chronic immunemodulated inflammatory disease, 282283 Chronic inflammation, 363, 424 Chronic inflammatory diseases, 276277 lung diseases, 313314 skin disease, 290 Chronic microbiome dysbiosis, 179, 179t Chronic mucocutaneous candidiasis, 273 Chronic obstructive airway diseases (COADs), 79, 84t antioxidant-based therapeutic approaches for, 8890 mechanism of oxidative stress mediated COAD pathogenesis, 86 redox imbalance in, 80f ROS-mediated effects in, 88 ROS-regulated downstream signaling pathways in, 8788 Chronic obstructive pulmonary disease (COPD), 66, 79, 81, 84t, 313314, 342 Chronic oxidative stress condition, 344 Chronic rhinosinusitis (CRS), 101, 110 Cigarette smoke, 79, 82, 345346 Cigarette smoke extract (CSE), 313314 Cineole. See 1,8-Cineole 1,8-Cineole, 369t, 372 Cirsimaritin, 138140 Citrus species, 366, 373 c-Jun N-terminal kinase (JNK), 87, 365 CLAs. See Conjugated linoleic acids (CLAs) Classical immunodeficiency syndromes, 165 Clostridia, 428 Clostridium difficile species, 403404 Clostridium perfringens species, 403404

INDEX

CLRs. See C-type lectin receptors (CLRs) Cluster of differentiation 26 (CD26), 133 CMV. See Cytomegalovirus (CMV) CNC protein. See Cap “n” collar protein (CNC protein) CNPY3. See Canopy 3 (CNPY3) CO. See Carbon monoxide (CO) COADs. See Chronic obstructive airway diseases (COADs) Cocoa (Theobroma cacao), 367 Coenzyme Q10 (CoQ10), 68 Cold water exposure, 417418 Colitis model, 311313 Colitis-associated cancer, 408409 Collectin (Collagen-lectin), 103104 Colon epithelium, 345 Colorectal cancer (CRC), 403 and gut microbiota, 403410 potential therapies, 405 pre-and pro-biotics against CRCexperimental, 405410 pro-and pre-biotics in combination with drugs and future prospects, 410 Comedogenesis, 241 Commensal microbiota, 233, 234f Communicable diseases, 424425 Complement component 5a (C5a), 58 Complete blood count (CBC), 182 Computational approach, 353354 Conjugated linoleic acids (CLAs), 391 Conventional DCs (cDCs), 2223 Conventional therapies to treating psoriasis, 283 COPD. See Chronic obstructive pulmonary disease (COPD) CoQ10. See Coenzyme Q10 (CoQ10) Cornel berries (Cornus mas), 368 Corneocytes, 51 Cornification, 51 Coronary artery calcium (CAC), 191, 196 Coronary artery disease (CAD), 427 Corticosteroids, 215 Cosignaling molecules, 135136 Costimulation, 130 Costunolide, 373 COX. See Cyclooxygenase (COX) CpG. See Cytidine-phosphate-guanosine (CpG) CpG-ODN. See Cytidine-phosphateguanosine ligonucleotides (CpGODN) CPP. See Calciprotein particle (CPP) CRC. See Colorectal cancer (CRC) C-reactive protein (CRP), 11, 182, 196, 367 CREB. See Cyclic AMP response element binding protein (CREB) Crohn’s disease (CD), 175, 179, 179t, 276277 bacteria and human microbiome, 178181 diagnosis and treatment, 182183 biomarkers, 182 current treatments, 182 proposed pharmacoperones, 182183 innate immune receptor recognition of bacteria, 175178 NOD2 mutations correlate with, 176177

Crossopteryx febrifuga root, 377 Crossoptines A and B, 377 CRP. See C-reactive protein (CRP) CRS. See Chronic rhinosinusitis (CRS) Cryptococcus, 272 C. neoformans, 277 CSE. See Cigarette smoke extract (CSE) C-terminal region (CTR), 153 CTL. See Cytotoxic T lymphocytes (CTL) CTR. See C-terminal region (CTR) C-type lectin receptors (CLRs), 5, 175176, 272273 Cul3-E3-ligase. See Cullin3-Rbx1 E3 ubiquitin ligase (Cul3-Rbx1-E3-ligase) Cullin3-Rbx1 E3 ubiquitin ligase (Cul3-Rbx1E3-ligase), 151, 153 Curcuma rhizomes, 375 Curcumin (CUR), 69, 71, 154155, 157, 353354, 378379, 378f ameliorates macrophage, 154155 Curcuminoids, 378379 CVC. See Cardiac valve calcification (CVC) CVDs. See Cardiovascular diseases (CVDs) CX3CR1-CX3CL1 interactions, 22 C-X-C motif ligand 1 (CXCL1), 151 CXCL1. See C-X-C motif ligand 1 (CXCL1) Cyclic AMP response element binding protein (CREB), 153, 204205 Cyclooxygenase (COX), 204 COX-1, 204 COX-2, 155, 204, 286, 341342, 345, 367 intracellular signaling cascades in aberrant COX-2 induction, 205206 oncogenic potential of COX-2-drived PGE2, 206207, 207f in pathogenesis of inflammationassociated cancer, 204 transcriptional and posttranscriptional regulation of COX-2 expression, 204205 inhibitors, 300, 363 CYPs. See Cytochrome P450 (CYPs) Cysteine proteases, 133134 Cystic fibrosis (CF), 104, 110, 271272 Cytidine-phosphate-guanosine (CpG), 225226 Cytidine-phosphate-guanosine ligonucleotides (CpG-ODN), 17, 225226 Cytochrome c, 50 Cytochrome P450 (CYPs), 4647 CYP1A1, 4647 CYP3A4, 4647 Cytokines, 67, 149, 155, 218, 297298 expression, 216217 Cytomegalovirus (CMV), 323324 Cytomegaloviruses, 314 Cytosine and guanine (CG), 228229 Cytosol, ROS production in, 4546 Cytotoxic T lymphocytes (CTL), 8

D Daidzein, 364f, 366367 Damage associated molecular patterns (DAMPs), 56, 38, 166, 224

INDEX

DAMPs. See Damage associated molecular patterns (DAMPs) Danger-associated molecular patterns. See Damage associated molecular patterns (DAMPs) DCregs. See Regulatory dendritic cells (DCregs) DCs. See Dendritic cells (DCs) DD. See Death domain (DD) Death domain (DD), 227 Defensins, 103, 258 Dehydroabietic acids, 375 Dehydroandrographolide, 374375 Dehydrocostuslactone, 373 Delphinidin, 287288, 287f, 368 Denagliptin, 140t Dendrimers, 72 Dendritic cells (DCs), 6, 8, 16, 257, 296, 408409 DEP. See Diesel exhaust particles (DEP) Deprivation, 426427 Dermis, 257 Detoxifying enzymes, 149150, 155 Detrimental bacterial strains, 391392 Dexamethasone, 313 Dextran sodium sulfate (DSS), 408 DGR. See Double glycine repeats (DGR) dh404. See Bardoxolone Methyl Analog (dh404) DHT. See Dihydrotestosterone (DHT) Di-peptides, 140141 Diabetes islet inflammation in, 150151, 150f NF-κB in, 155 Nrf2 in, 154155 Diabetes mellitus (DM), 111, 149, 347 Diacetyl, 395 Diesel exhaust particles (DEP), 81 Diet, 233, 234f, 427428 dietary chemopreventive agents, 208 dietary components, 295 dietary stilbenes, 379 microbiota, 425426 therapy, 418 Diet-induced obesity (DIO), 390391 Dietary intervention COX-2 in pathogenesis of inflammationassociated cancer, 204 intracellular signaling cascades in aberrant COX-2 induction, 205206 nutritional/dietary manipulation of aberrant inflammatory signaling, 208 oncogenic potential of COX-2-drived PGE2 and metabolic inactivation, 206207, 207f transcriptional and posttranscriptional regulation of COX-2 expression, 204205 Dihydrotestosterone (DHT), 345 1,25-Dihydroxyvitamin D3 (1,25D3), 264 1,2-Dimethylhydrazine (DMH), 408 Diminished fetal immune system, 15 Dimyristoyl phophatidylcholine (DMPC), 71 Dinitrochlorobenzene (DNCB), 311

2,4-Dinitrofluorobenzene-induced contact hypersensitivity model, 287 DIO. See Diet-induced obesity (DIO) Dioscin, 377378 Dipeptidyl peptidase IV (DPPIV), 133134 in immune system, 134136 Dipeptidyl peptidases (DPPs), 127 Diphenyleneiodonium, 123 Diprotin A, 138 Diprotin B, 138 Disruption, 391392 Diterpenes. See Diterpenoids Diterpenoids, 374376, 374f DM. See Diabetes mellitus (DM) DMH. See 1,2-Dimethylhydrazine (DMH) DMPC. See Dimyristoyl phophatidylcholine (DMPC) DNA, 49 binding nuclear protein, 6 damage, 86 methylation, 232233 modification, 229 oxidation, 348349 DNA methyl transferases, 228230 DNCB. See Dinitrochlorobenzene (DNCB) Dopaminergic neurons, 349 Double glycine repeats (DGR), 153 Double-stranded DNA (dsDNA), 228 Double-stranded ribonucleic acid (dsRNA), 167 Doxycycline, 242 1,3-DPG. See 1,3-Bisphosphoglycerate (1,3DPG) DPPIV. See Dipeptidyl peptidase IV (DPPIV) DPPIV/CD26, 133134 components derived from plants, 138140 DPPIV in immune system, 134136 DPPIV/CD26-inhibitors, 138141 effects in autoimmune and inflammatory disease, 136138 effects on immune cells migration, 136 gliptins, 138 naturally occurring DPPIV/CD26inhibitors, 138 oral bioavailability, 140141 as T-cells costimulatory molecule, 135136 DPPs. See Dipeptidyl peptidases (DPPs) Drosophila melanogaster, 166, 223, 271 dsDNA. See Double-stranded DNA (dsDNA) dsRNA. See Double-stranded ribonucleic acid (dsRNA) DSS. See Dextran sodium sulfate (DSS) Dual oxidases (DUOX), 121 DUOX-1, 58 DUOX-2, 58, 122 Dutogliptin, 140t Dysbiosis, 253 Dysfunction of endothelium, 66 Dyslipidemia, 151

E E3ubiquitin ligase (Ub ligase), 227 Eating habits in combating disease complex link between nutrition and disease, 424f

445 deprivation and overconsumption, 426427 diet, immunity and advancing age, 427428 diet microbiota and immune responses, 425426 functional foods, 426 good eating habits, 428429 inflammatory and immune responses, 424425 nutrigenomics, 429 EBC. See Exhaled breath condensate (EBC) Ebselen, 90, 351 E-cigarettes. See Electronic cigarettes (Ecigarettes) EC-immune signaling, 10 ECM. See Extracellular matrix (ECM) ECs. See Endothelial cells (ECs) Ectodomain, 213214, 224225 Edaravone, 354 Effector T cells, 57 EFSA. See European Food Safety Authority (EFSA) EGCG. See Epigallocatechin gallate (EGCG) EGF. See Extracellular growth factor (EGF) EGF-receptor (EGFR), 409 EGFR. See EGF-receptor (EGFR) Elasticity, 314315 Electron transport chain (ETC), 58, 229 Electron-transfer flavoprotein:ubiquinone oxidoreductase (ETFQO), 59 Electronic cigarettes (E-cigarettes), 82 Electrons, 47 Electrophilic response element (EpRE). See Antioxidant response element (ARE) Eleutherococcus divaricatus, 375376 Ellagic acid, 380f, 381 ENaC. See Epithelial sodium channel (ENaC) Endocytosis, 56 Endogenous factors, 81 sources, 83 Endolysosome-specific function, 228 Endoperoxides bridge (COOC), 310311 Endoplasmic reticulum (ER), 47, 228 Endoplasmic reticulum-resident protein (Ero1p), 47 Endothelial abnormalities, 66 Endothelial cells (ECs), 9, 190 Endothelial P-selectin, 67 Endothelial progenitor cells (EPCs), 353 Endothelin-1, 366 Endothelium, 9 targeted delivery of antioxidant nanocarriers to, 71f Energy imbalance, 391 Enterobacteria, 428 Enterobacteriaceae sp., 403404 Enterococcus capsules, 425 Enterococcus faecalis, 405 Enterotoxigenic Bacteroides fragilis (ETBF), 404 Entosis, 52

446 Enzymatic antioxidant(s), 8385 CAT, 85 deficiencies, 88 GPx, 85 HMOX, 85 mimetics, 90 SOD, 8384 Enzyme mimetics, 90 Eosinophil peroxidase (EPO), 83, 342343 Eosinophilic airway inflammation, 83 Eotaxin, 135 Eotaxin-1, 136 EP receptors, 206207 EPCs. See Endothelial progenitor cells (EPCs) Epidermis, 257 Epigallocatechin gallate (EGCG), 364f, 367 Epithelial barrier function, 409 Epithelial cells, 345 as immune effectors, 103105 Epithelial sodium channel (ENaC), 107 EPO. See Eosinophil peroxidase (EPO); Erythropoietin (EPO) EpRE. See Electrophilic response element (EpRE) ER. See Endoplasmic reticulum (ER) Erdosteine, 8990 ERK. See Extracellular signal regulated kinases (ERK) Ero1p. See Endoplasmic reticulum-resident protein (Ero1p) Erythrocyte sedimentation rate (ESR), 182 Erythrodermic psoriasis, 281 Erythropoietin (EPO), 322 Escherichia coli, 272 E-selectins, 67, 190 ESR. See Erythrocyte sedimentation rate (ESR) Essential oils. See Volatile oils Etanercept, 283 ETBF. See Enterotoxigenic Bacteroides fragilis (ETBF) ETC. See Electron transport chain (ETC) ETFQO. See Electron-transfer flavoprotein: ubiquinone oxidoreductase (ETFQO) Eucalyptus globulus, 373 European Food Safety Authority (EFSA), 250, 327 Everolimus, 427428 Evogliptin, 139t EVs. See Extracellular vesicles (EVs) Exaggerated forms of inflammation, 426 Excessive ROS production, 59 Excitotoxicity, 51 Exhaled breath condensate (EBC), 88 Exogenous pathogenic agents, 5 ROS production, 4748 sources, 82 External elements, immune system, 5 Extracellular growth factor (EGF), 206 Extracellular matrix (ECM), 189 Extracellular peroxiredoxins, 4041 Extracellular proteins collagen, 86 Extracellular signal regulated kinases (ERK), 87 ERK-1/2, 205206, 377

INDEX

Extracellular vesicles (EVs), 192193 Extrinsic pathways, 50

F FadA virulence factor, 403404 FAEE. See Fatty acid ethyl ester (FAEE) Fas activation pathway, 50 receptor, 50 signaling, 151 Fasting therapy, 415, 418 Fatty acid ethyl ester (FAEE), 22 FcRn. See Neonatal Fc receptor (FcRn) FcRn receptor, 2324 FCγR. See γ Chain of Fcε receptor (FCγR) FDA. See U.S. Food and Drug Administration (FDA) Fecal microbiota transplantation (FMT), 409410 Fenton reaction, ROS production, 46f Fermented milk products (FM products), 390, 394396, 394t Ayran, 396 Cheese, 395 Dahi, 395 Kefir, 396 Kumys, 396 Lassi, 395 manufacturing procedures for selected FMs, 394396 Raabadi, 395 Viili, 395396 Yogurt, 394395 Ferulic acid, 380, 380f Fetuin-A, 194195 Fetuin-mineral complex (FMC), 194195 FEV1. See Forced expiratory volume in 1 s (FEV1) Feverfew (Tanacetum parthenium), 373 FFAs. See Free fatty acids (FFAs) Fibroblasts, 170, 257 Fibronectin, 86 Ficolins, 28 Filaggrin, 288 Filomicelles, 72 Flagellin, 168 Flavan-3-ols, 367 Flavanones, 364f, 365366 Flavocoxid, 365 Flavones, 364f, 365366 Flavonoids, 138140, 363368. See also Terpenoids anthocyanidins, 367368 catechins, 367 flavanones, 365366 flavones, 365366 flavonols, 365366 isoflavones, 366367 in treating psoriasis, 282290 amentoflavone, 285 apigenin, 285286 astilbin, 286287 baicalin, 287 chronic immunemodulated inflammatory disease, 282283

conventional therapies to treating psoriasis, 283 delphinidin, 287288 genistein, 288 isoliquiritigenin, 288289 luteolin, 289 quercetin, 290 Flavonols, 364f, 365366 Flexural psoriasis, 281 FLICE inhibitory protein (FLIP), 1819 Flu disease, 328 FM products. See Fermented milk products (FM products) FMC. See Fetuin-mineral complex (FMC) FMT. See Fecal microbiota transplantation (FMT) Folate biosynthesis, 232233 Folic acid, 427, 429 Food proteins, 138 Forced expiratory volume in 1 s (FEV1), 8384 Forkhead box O-3 (FOXO3), 428 FOXO3 SNPs rs2253319, 428 FOXO3 SNPs rs2802292, 428 FOS. See Fructo-oligosacharides (FOS) Fos related antigen-1 (FRA1), 8788 FRA1/AP1 transcription factor, 8788 Fos related antigen-2 (FRA2), 8788 FOXO1 SNP rs17630266, 428 FOXO3. See Forkhead box O-3 (FOXO3) FRA1. See Fos related antigen-1 (FRA1) Fragilysin. See Bacteroides. fragilis toxin (BFT) Frankincense resin (Boswellia serrata), 376377 Free fatty acids (FFAs), 151, 389 Free radicals, 341343 Freund’s complete adjuvant-induced monoarthritis rat model, 312 Fried’s score, 331 Fructo-oligosacharides (FOS), 405406 Fudosteine, 8990 Functional foods, 324, 363, 390, 426, 428 importance of, 390 to improving immunity, 327 with proven clinical efficacy to ameliorating elderly immunity, 328332 Fungal communities, 276 Fungal interactions bacterialfungal interactions, 272 hostfungal interactions, 272273 Fungi, 3, 166, 271274 Fusobacterium nucleatum, 403404

G G3-P. See Glyceraldehyde 3-phosphate (G3P) Galacto-oligosaccharides (GOS), 405406 Gallic acid, 381 γ-carboxylation, 196 Gamma-delta T-cells (γδ T-cells), 283 Gastrointestinal tract (GI tract), 15, 273277, 278f, 390 GC. See Germinal center (GC) GCL. See Glutamate-cysteine ligase (GCL)

INDEX

GCP-2/CXCL6. See Granulocyte chemotactic protein 2/CXC motif ligand 6 (GCP-2/CXCL6) G-CSF. See Granulocyte colony stimulating factor (G-CSF) Gemigliptin, 139t Gene array expression, 241 Generalized pustular psoriasis, 281 Generally recognized as safe (GRAS), 327 Genetic factors, 175 “Genetic landscape” of metabolic diseases, 429 Genetic polymorphisms, 88 Genetic predisposition, 90 Genetic theory of infectious disease, 165 Genistein, 288, 288f, 364f, 366 Germ-free mice (GF mice), 390391 Germinal center (GC), 312 GF mice. See Germ-free mice (GF mice) GI tract. See Gastrointestinal tract (GI tract) Gibberella moniliformis, 277 Ginger (Zingiber officinale), 374 Ginkgo biloba, 374 Ginkgolides, 374 Ginkolides A, B, and C, 374 GIP. See Glucose-dependent insulinotropic peptide (GIP) Gla-rich protein (GRP), 194195 Gliptins, 127, 129f, 131, 133, 137138, 139t, 140t Global hypomethylation in SLE CD41 Tcells, 232233 Global initiative for chronic obstructive lung disease (GOLD), 81 Glucagon-like peptide 1 (GLP-1), 127128, 134135 Glucocorticoids, 8889 Glucoraphanin, 356357 Glucose transport receptors (GLUT receptors), 389390 Glucose uptake, 61 Glucose-dependent insulinotropic peptide (GIP), 134 Glucose-regulated protein 94 (GRP94), 228 GLUT receptors. See Glucose transport receptors (GLUT receptors) Glutamate activation, 51 Glutamate-cysteine ligase (GCL), 149150 Glutathione (GSH), 68, 83, 85, 353354 Glutathione peroxidase (GPx), 85, 149150, 232, 342, 351 GPx-1, 351 mimetics, 90, 351 Glutathione reductase (GR), 342 Glutathione S transferase mu1 (GSTM1), 83 Glutathione S-transferase (GST), 149150 Glutathione to oxidized/disulphide form (GSSG), 85 Glutathionylation, 5960 Glyceraldehyde 3-phosphate (G3-P), 347 Glycerol-3-phosphate, 46 Glycerol-3-phosphate dehydrogenase 2 (GPD2), 61 Glycyl-prolyl-β-naphthylamidase, 133 Glycyrrhetinic acid, 376

Glycyrrhizin, 376 GM-CSF. See Granulocyte macrophage colony stimulating factor (GM-CSF) GOLD. See Global initiative for chronic obstructive lung disease (GOLD) Good eating habits, 428429 GOS. See Galacto-oligosaccharides (GOS) Gosogliptin, 139t GPCRs. See G-protein-coupled receptors (GPCRs) GPD2. See Glycerol-3-phosphate dehydrogenase 2 (GPD2) G-protein-coupled receptors (GPCRs), 105106 GPx. See Glutathione peroxidase (GPx) GR. See Glutathione reductase (GR) Graft versus host disease (GVHD), 119, 130 Grand fir (Abies grandis), 375 Granulocyte chemotactic protein 2/CXC motif ligand 6 (GCP-2/CXCL6), 135 Granulocyte colony stimulating factor (GCSF), 16 Granulocyte macrophage colony stimulating factor (GM-CSF), 16, 58, 286 Grape (Vitis vinifera), 367 seed extracts, 428 Grape seed-derived procyanidins (GSPE), 138140 GRAS. See Generally recognized as safe (GRAS) GRP. See Gla-rich protein (GRP) GRP94. See Glucose-regulated protein 94 (GRP94) GSH. See Glutathione (GSH) GSPE. See Grape seed-derived procyanidins (GSPE) GSSG. See Glutathione to oxidized/ disulphide form (GSSG) GST. See Glutathione S-transferase (GST) GSTM1. See Glutathione S transferase mu1 (GSTM1) Gut bacteria-derived oxidants, 345 Gut dysbiosis, 403 Gut inflammation, 276 Gut microbial dysbiosis, 405 Gut microbiome, 425426 Gut microbiota, 295, 390391, 403410, 425 Gut mucosa, 390 GVHD. See Graft versus host disease (GVHD) Gymnema sylvestre extracts, 429

H HAART. See Highly active antiretroviral therapy (HAART) Haemophilus influenzae, 103 HAT. See Histone acetyltransferases (HAT) HB-EGF. See Heparin-binding epidermal growth factor (HB-EGF) HCAM. See Homing cell adhesion molecule (HCAM) HDAC2. See Histone deacetylase 2 (HDAC2) HDM. See House dust mite (HDM) Health foods. See Functional foods

447 Healthy aging, 319320 mycobiome, 274275, 274f nutritional interventions, 416 Heat, 241 Helicobacter pylori, 300 Heliotherapy, 418 Helper T cells, 258 Th1, 232 Th2, 232 Hemagglutination inhibition (HI), 250 Hematopoiesis, 322 Hematopoietic stem-cell transplantation (HSCT), 172 Heme oxygenase-1 (HO-1), 39, 149150, 156 Hemeoxygenase (HMOX), 85 HMOX1, 83, 85 HMOX2, 85 Hemidesmosome, 213214 Hemisuccinate NA, 311 Heparin-binding epidermal growth factor (HB-EGF), 344345 Herbal medicines. See Phytopharmaceuticals Herbs, 428429 Herpes encephalitis, 169170 Herpes simplex-1 viral encephalitis (HSE), 169 Hesperidin, 366 HFD. See High fat diet (HFD) HG. See Hyperglycemia (HG) HGP. See Human Genome Project (HGP) HI. See Hemagglutination inhibition (HI) HIF-1α. See Hypoxia inducible factor-1α (HIF-1α) High fat diet (HFD), 390391 High mobility group box-1 (HMGB-1), 6, 297 Highly active antiretroviral therapy (HAART), 416 Hispidulin, 138140 Histone acetyltransferases (HAT), 155 Histone deacetylase 2 (HDAC2), 86, 313314 Histone deacetylase 3 (HDAC3), 155 HIV, 295 naturopathic approach towarding HIVpositive individuals, 416418 fasting and diet therapy, 418 heliotherapy needs heading formatting, 418 hydrotherapy, 417418 line of approach, 416 manipulative therapy, 418 mud therapy, 418 rationale of choosing lifestyle interventions, 416 understanding HIV in naturopathy perspective, 416 yoga, 416417 probiotics effects on HIV-infected subjects, 301303, 302f and HIV, 301303 and mode of action, 295301 HLE. See Human leukocyte elastase (HLE) HMGB-1. See High mobility group box-1 (HMGB-1)

448 HMOX. See Hemeoxygenase (HMOX) 4-HNE. See 4 Hydroxy 2 nonenal (4-HNE) HO-1. See Heme oxygenase-1 (HO-1) Homing cell adhesion molecule (HCAM), 231232 Host immune responses, 273 Host microbiota, 271272 Hostfungal interactions, 272273 House dust mite (HDM), 313 HPLC, 246 HSC-5. See Human skin cancer-5 (HSC-5) HSCT. See Hematopoietic stem-cell transplantation (HSCT) HSE. See Herpes simplex-1 viral encephalitis (HSE) HSV-1 infection, 1819, 169170 Human bronchial epithelial cells, 107 Human clinical trials, 409 Human colon cancer, 205 Human Genome Project (HGP), 271 Human immortality, 427428 Human immune response, 249250 Human leukocyte elastase (HLE), 217 Human microbiome, 178, 271 Crohn’s disease and chronic microbiome dysbiosis, 179 effects of bacterial cell wall fragments and metabolites on host, 180 to host, 178179 invasive bacteria modifying core peptidoglycan structure, 181 N-substitution on MDP effects NOD2 signaling and stability, 181 Human microbiota, 403 Human monocyte-derived DC maturation, 408409 Human rheumatoid arthritis, 312 Human skin, 257, 258f Human skin cancer-5 (HSC-5), 206 Human Toll-like receptor 4, 165166 Humoral response, 217218 Hydrogen peroxide (H2O2), 5860, 6567, 82, 341, 351 Hydrophobic antioxidants, 70 Hydrotherapy, 415, 417418 4 Hydroxy 2 nonenal (4-HNE), 86, 88 8 Hydroxy 20 deoxyguanosine (8-OHdG), 86 N-(21-Hydroxy-21-(piperidin-1-yl)henicosa17,19-diyl-1-yl) acetamide, 243, 244f 8-Hydroxydeoxyguanosine, 348349 8 Hydroxyguanine (8-OH-Gua), 86 Hydroxyl radicals (_OH), 47, 58, 68, 82 15-Hydroxyprostaglandin dehydrogenase (15-PGDH), 207 Hyper-keratinization, 246 Hyperactive mitochondria, 347 Hyperfunction theory, 427428 Hyperglycemia (HG), 154155, 347 Hyperlipidemia, 426 Hypertension, 71, 346347 Hypertensive neonatal rats, 397 Hypertensive peptide hormone system, 346347 Hypochlorous acid (HOCl), 58, 68, 82, 341 Hypomethylation in SLE CD41 T-cells, global, 232233

INDEX

Hypomorphic ectodermal dysplasia with immunodeficiency (HED-ID). See Xlinked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID) Hypothermic organ storage, 119 Hypoxia inducible factor-1α (HIF-1α), 341342

I IBD. See Inflammatory bowel disease (IBD) ICAM. See Intercellular adhesion molecule (ICAM) Ichthyosis, 262 iDCs. See Immature dendritic cells (iDCs) IDU. See Injection drug use (IDU) IECs. See Intestinal epithelial cells (IECs) I-ficolin, 28 IFN. See Interferon (IFN) Ig. See Immunoglobulins (Ig) IGF-1. See Insulin like growth factor (IGF-1) IgG containing immune complexes (IgCs), 2324 IκB. See Inhibitor of kappa B (IκB) IKBA. See Inhibitor of Kappa B-alpha (IKBA) IKBKG/NEMO, 171 IKK. See Inhibitor of Kappa B kinase (IKK) IL. See Interleukin (IL) IL-2 receptor (IL-2r2/2), 232 ILCs. See Innate lymphoid cells (ILCs) Ilex paraguariensis, 377 Imiquimod (IMQ), 285 Immature dendritic cells (iDCs), 298 Immotile cilia syndrome. See Primary ciliary dyskinesia (PCD) Immune cells, 86, 257, 259260, 342343 DPPIV/CD26 effects on migration, 136 Immune dysfunction perspective, 423 Immune genes, 277 Immune health supplements, 429 Immune phenotype, 168171 Immune responses, 424425 clinical intervention with probiotics to boosting clinical trial to enhanced immunity, 250, 251t enhancement of NK cell activity and antiviral activity by LAB, 252253 IgG profiles in response to LAB, 250252, 251f diet microbiota and, 425426 in pathology of communicable and noncommunicable diseases, 424425 regulation by sensory receptors in sinonasal cavity, 105109 other pattern recognition receptors, 105 taste receptors, 105109, 109t TLRs, 105 Immune signaling, 425 Immune skin dysregulation, 213214 Immune system, 4f, 257258, 281, 295296, 298, 418 cells, 134 DPPIV in, 134136 DPPIV/CD26 effects on immune cells migration, 136

as T-cells costimulatory molecule, 135136 interactions with other molecules, 135 probiotics clinical application of probiotics for disease prevention, 253f, 254 molecular mechanism by LAB potentiates immune response, 253254 substrates, 135 in vertebrates, 5 Immune-mediated inflammatory disease, 275 Immunity, 427428 clinical trial to enhanced immunity, 250, 251t in elderly, 324, 325t functional food to improving, 327 probiotics on, 249 response of naturopathy modalities on, 415416 in skin, 258 Immunization protocols, 328 Immunobiology of aging, 321324 impact of aging of bone marrow, 322323 alteration of cellular components of adaptive immune system, 323324 nutrition and immunity in elderly, 324 thymic involution, 323 Immunogenic role of probiotics against influenza virus infection, 249 Immunoglobulins (Ig), 8, 104, 231 IgA, 250 IgG, 16, 250 profiles in response to LAB, 250252, 251f Immunologic immaturity, 15 Immunomodulation, 295, 408409 Immunosenescence, 322 Immunosuppressive drugs, 215 IMQ. See Imiquimod (IMQ) In silico approach, 353354 In vitro analysis, 312 In vitro studies, 298299 In vivo studies, 299301 Incarvillea sinensis, 373 Indian ginseng. See Withania somnifera (Ashwagandha) Inducers, 56 inducible nitricoxide synthase (iNOS), 155, 341342, 365 Inducing apoptosis, 409 Induction of adaptive immunity, 8 Infantile diarrhea, 299 Infection, 320, 322323 Infectious disease, genetic theory of, 165 Inflammaging, 324 Inflammasomes, 7, 38 Inflammation, 9, 37, 81, 149, 154155, 203204, 241, 295, 297, 423 in atherosclerosis, 190 to autoimmunity and therapeutic response, 217218 autoimmunity to inflammation and blister formation, 215217

INDEX

COX-2 in pathogenesis of inflammationassociated cancer, 204 and disease, 6667 inflammatory agents, 6667 markers of oxidative stress and inflammation, 67 reactive species, 6667 vascular endothelium, 6667 fermented milk effect by probiotics in, 391394 inflammation-induced thickening of smooth muscle layer, 424 and innate immunity, 67 Lactobacillus gasseri effect in, 393394 Lactobacillus plantarum effect in, 391392 nutritional/dietary manipulation of aberrant inflammatory signaling, 208 response of naturopathy modalities on, 415416 vicious cycle of, 196197 Inflammatory agents, 6667 cells, 242 cytokines/chemokines, 171, 258, 261262 diseases, 3, 133, 175176, 341342 DPPIV/CD26-inhibitor effects in inflammatory disease, 136138 clinical studies, 137 immunological side effects, 137138 preclinical studies, 137 genes, 342 mediators, 149 process, 149 redox control of inflammatory mediators, 3839 responses, 424425 in pathology of communicable and noncommunicable diseases, 424425 signaling pathways, 350 Inflammatory bowel disease (IBD), 271272, 276277, 295, 298299, 403 Inflammatory cascade, onset of emerging role of peroxiredoxins, 4041 redox control of antioxidant and anti-inflammatory transcription factors, 3940 of inflammatory mediators, 3839 ROS generation during, 3738 Infliximab golimumab, 283 Influenza A virus infection, 22 immunogenic role of probiotics against, 249 Influenza disease, 328 Inhibitor of kappa B (IκB), 171, 297 IκBα deficiencies, 172, 367 Inhibitor of Kappa B kinase (IKK), 155, 166167, 367 IKK-β, 155 IKKε, 167 Inhibitor of Kappa B-alpha (IKBA), 166167 Injection drug use (IDU), 301 iNKT. See Invariant natural killer T (iNKT) Innate immune cells of newborn, 1727 Innate immune receptors, 175 bacterial cell wall, 176

NOD2 as, 176 mutations correlate with Crohn’s disease, 176177 receptor stabilized by proteins, 177178 response in mice, 178 N-substitution on MDP effects NOD2 signaling and stability, 181 recognition of bacteria, 175178 of MDP by innate immune system and NOD2, 177 reduced signaling of Crohn’s diseaseassociated NOD2 mutants, 177 Innate immune responses, 273, 296 Innate immune system, 3, 134, 223 cell types, 4f, 56 chronic diseases, 10 components of immune response, 5 crosstalk between innate and adaptive immune responses, 89 improving immunity for prevention and care, 1011 inflammation and innate immunity, 67 renaissance in innate immune system research, 165166 signaling, 56 vascular endothelium, 910 Innate immunity, 257, 260, 282 at birth, 27f innate immune cells of newborn, 1727 microbiome in shaping newborn immune system, 1516 mucosal immunity at birth, 1617 soluble plasma components, 2728 Innate lymphoid cells (ILCs), 16 Innate responses, 225226 iNOS. See inducible nitricoxide synthase (iNOS) Inositol 3,4,5-triphosphate (IP3), 57 Insulin like growth factor (IGF-1), 415 Insulin resistance (IR), 389390, 397 Interaction of microbiota, 249 Intercellular adhesion molecule (ICAM), 128129, 155 ICAM-1, 67, 376 Interferon (IFN), 224, 282 IFN-induced human MxA, 253 IFN-α, 233, 367368 IFNγ, 190, 223224, 261262, 273, 282, 322324 Interferon gamma-induced protein 10/ CXC motif chemokine 10 (IP-10/ CXCL10), 135 Interferon regulatory factor-3 (IRF-3), 21 Interferon-inducible T-cell alpha chemoattractant/CXC motif chemokine 11 (I-TAC/CXCL11), 135 Interleukin (IL), 182, 216, 225226, 249250, 261262, 282, 403404, 424 IL-1, 38, 155, 166, 224225, 391392, 415 IL-1β, 38, 149, 206, 397 IL-1R, 166 IL-2, 57, 134, 232 IL-6, 155, 289, 323324, 365, 391392 IL-12, 223224, 273

449 IL-17, 272273 IL-23, 17 Interleukin-1 receptor associated kinases (IRAKs), 227 IRAK-1, 135136 IRAK-4, 167168 Internal elements, immune system, 5 Internal transcribed spacer region (ITS region), 243, 273 International Scientific Association for Probiotics and Prebiotics (ISAPP), 405406 Intertriginous psoriasis, 281 Intervening region (IVR), 153 Intestinal epithelial cells (IECs), 17, 298 IEC-6, 206 Intestinal immune system, probiotics in, 249 Intestinal inflammation, 182, 298 Intestinal microbiota, 249, 407408, 425 Intracellular ROS, 354 Intracellular thiol, 68 Intravenous or IgG (IVIG), 172 Intrinsic pathways, 50 Invariant natural killer T (iNKT), 26, 122 Involucrin, 288 IP3. See Inositol 3,4,5-triphosphate (IP3) IP-10/CXCL10. See Interferon gammainduced protein 10/CXC motif chemokine 10 (IP-10/CXCL10) IR. See Insulin resistance (IR) IR injury. See Ischemia-reperfusion injury (IR injury) IRAK3. See IRAKM IRAKM, 227 IRAKs. See Interleukin-1 receptor associated kinases (IRAKs) IRF-3. See Interferon regulatory factor-3 (IRF3) Irinotecan-induced rat model, 407 ISAPP. See International Scientific Association for Probiotics and Prebiotics (ISAPP) Ischemia-reperfusion injury (IR injury), 119, 127 CD26/DPP4as therapeutic target in experimental models against, 127130 clinical implications for CD26/DPP4 as therapeutic target in, 127130 free radicals and oxidative stress in lung IR injury, 120 IR-induced lung injury, 119 NADPH oxidases in, 120 Islet inflammation, 149 in diabetes, 150151, 150f rescue from islet inflammation, 156157 Isoflavones, 366367 Isoliquiritigenin, 288289, 288f 8-Isoprostane, 88 Isoprostanes, 86 Isotretinoin, 261262 I-TAC/CXCL11. See Interferon-inducible Tcell alpha chemoattractant/CXC motif chemokine 11 (I-TAC/CXCL11) ITS region. See Internal transcribed spacer region (ITS region)

450 IVIG. See Intravenous or IgG (IVIG) IVR. See Intervening region (IVR)

J Janus kinase (JAK), 300 Jak/Tyk proteins, 287 Janus kinase/signal transducer and activator of transcription 3 signaling (Jak/Stat3 signaling), 286287 JNK. See c-Jun N-terminal kinase (JNK) Joint-health supplements, 429

K Kahweol, 375 Kartagener’s syndrome, 110111 Kefir, 396397 grains, 397 Kelch domain, 153 Kelch-like ECH-associated protein 1 (Keap1), 149150, 153 Keratinization. See Cornification Keratinocytes, 257, 282 Killer Ig like receptor (KIR), 26 Koumiss. See Kumys Kumiss. See Kumys Kumys, 396

L L. rhamnosus 231, 407 L. rhamnosus GG strain (LGG strain), 409 LAB. See Lactic acid bacteria (LAB) Laboratory of genetics and physiology-2 (LGP-2), 7 Lactic acid, 249, 396, 408 Lactic acid bacteria (LAB), 249, 299, 390 clinical trial to enhanced immunity with intake of, 250 enhancement of NK cell activity and antiviral activity by, 252253 IgG profiles in response to, 250252 molecular mechanism by LAB potentiates immune response, 253254 Lactobacilli species, 249, 295 Lactobacillus casei ATCC334 derived ferrichrome, 409 Lactobacillus casei Shirota (LcS), 252, 303, 408409 Lactobacillus gasseri effect, 393394 LG2055, 250, 252254, 393 potentiating immune response clinical intervention with probiotics to boost immune response, 250253 general aspects of probiotics on immunity, 249 immunogenic role of probiotics against influenza virus infection, 249250 perspectives for research on probiotics in immune system, 253254 Lactobacillus sp., 16 capsules, 425 L. acidophilus, 300 L. bulgaricus, 299 L. casei, 297298

INDEX

L. plantarum, 300 effect, 391392 KY1032 supplementation, 392 MB452, 392 L. rhamnosus, 300 Lactobacillus strains, 300, 407 Lactoferrin, 103 Lactoperoxidase (LPO), 104 L-arginine, 28 LcS. See Lactobacillus casei Shirota (LcS) LDL. See Low-density lipoprotein (LDL) Leguminosae family, 366 Lemon balm (Melissa officinalis), 380 Leptin, 151 Leucine rich repeat (LRR), 176, 224225 Leukotriene (LT-B4), 373, 415 LGG strain. See L. rhamnosus GG strain (LGG strain) LGP-2. See Laboratory of genetics and physiology-2 (LGP-2) Lifestyle modification before art, 418 during art, 418419 Linagliptin, 139t Lipid(s), 49 droplets, 389 enhanced aggregation of lipid rafts, 231 nanoparticles and complexes, 74 peroxidation, 86 peroxides, 58 Lipopeptides, 168 Lipopolysaccharide (LPS), 52, 103, 166168, 224225, 298, 314, 322323, 365, 391392 Lipoprotein lipase (LPL), 389 Lipoproteins, 151 Liposomes, 7072 Lipoteichoic acid (LTA), 2122 Lipoxin A4, 1718 5-Lipoxygenase (5-LOX), 365 LL-37, 103104, 282 LN. See Lupus nephritis (LN) Lodgepole pine (Pinus contorta), 375 Lonicerin, 365 Loricrin, 288 Low-density lipoprotein (LDL), 427 Lowering of intestinal pH, 408 5-LOX. See 5-Lipoxygenase (5-LOX) LPL. See Lipoprotein lipase (LPL) LPO. See Lactoperoxidase (LPO) LPS. See Lipopolysaccharide (LPS) LRR. See Leucine rich repeat (LRR) L-selectin, 26 LT-B4. See Leukotriene (LT-B4) LTA. See Lipoteichoic acid (LTA) Lung cancer, 345 inflammation NADPH oxidases in, 120 IR injury free radicals and oxidative stress in, 120 molecular signaling pathways in pathogenesis of, 121f NOX isoforms and pharmacological inhibitor role in, 122123

ischemia, 121122 transplantation, 119 Lupeol, 377 Lupus mouse models, 232 patients, 232 Lupus nephritis (LN), 230231 Luteolin, 289, 289f, 364f, 365 Lychnophora trichocarpha extracts, 373 Lymphocyte differentiation and proliferation, 8 Lymphocytic inflammation clinical implications for CD26/DPP4 as therapeutic target in, 127130 modulation of CD26/DPP4 against, 130 Lysosomes, ROS production by, 47 Lysozyme, 103

M Macroangiopathy, 347 Macrocyclic ligands, 90 Macropage inflammatory protein-1 (MIP-1), 297 Macrophage activation syndrome (MAS), 223224, 228229 Macrophage inflammatory protein (MIP), 135 MIP-1α, 1718, 151 Macrophage migration inhibitory factor (MIF), 2122 Macrophage-derived chemokine/CC motif chemokine 22 (MDC/CCL22), 135 Macrophages, 6, 21, 190, 196, 322323 Magnetic nanoparticles (MNPs), 7374 MAGUK molecule. See Membrane-associated guanylate kinase-like molecule (MAGUK molecule) “Majjiga”, 396 Major and minor histocompatibility complexes (MHC), 8 MAL. See MyD88-adapter-like (MAL) Malaria, 310, 312, 363 Malnutrition, 424, 426 Malnutrition-induced immune impairment improvement, functional foods, 426 Malondialdehyde (MDA), 49, 86, 343, 347 MALT. See Mucosa-associated lymphoid tissue (MALT) Malvidin, 368 Malvidin 3-O-glucoside, 368 MAM. See Microbial anti-inflammatory molecule (MAM) MAMPs. See Microbial-associated molecular patterns (MAMPs) Manganese-based SOD mimetics, types of, 90 Manipulative therapy, 418 Mannan binding lectins (MBLs), 28 MAPK. See Mitogen activated protein kinases (MAPK) Mare’s milk, 396 Markers of oxidative stress and inflammation, 67 MAS. See Macrophage activation syndrome (MAS)

INDEX

Mass spectrometry, 246 Massage therapy, 418 Mast cells, 216 Master transcription factors, 194 Matrix gla protein (MGP), 194195 Matrix metalloproteinases (MMPs), 128, 133134, 196197, 246, 297, 315, 344 MMP-9, 51, 217 Maximum expiratory flow at 25% volume (MEF25), 8384 MBLs. See Mannan binding lectins (MBLs) MCC. See Mucociliary clearance (MCC) MCP-1. See Monocyte chemoattractant protein-1 (MCP-1) MDA. See Malondialdehyde (MDA) MDA5. See Melanoma differentiation factor-5 (MDA5) MDC/CCL22. See Macrophage-derived chemokine/CC motif chemokine 22 (MDC/CCL22) MDP. See Muramyl dipeptide (MDP) MDSCs. See Myeloid-derived suppressor cells (MDSCs) Meadowsweet (Filipendula ulmaria), 363 Medical factors, 321 MEF25. See Maximum expiratory flow at 25% volume (MEF25) MeIQx. See 2-Amino-3,4-dimethyl-imidazo (4,5-f)-quinoxaline (MeIQx) Melanocytes, 257 Melanoma differentiation factor-5 (MDA5), 7 Melogliptin, 140t Membrane potential (Δψm), 59 Membrane-associated guanylate kinase-like molecule (MAGUK molecule), 136 Men who had sex with men (MSM), 301 Meningitis, 168 Metabolic inactivation, 206207, 207f Metabolic syndrome, 149 Metabolites, implications of, 403410 Metagenomic approaches, 273274 Metalloporphyrin-based SOD mimetics, 351 Metaloporphyrins, 90 N-Methyl-N0 -nitro-N-nitrosoguanidine (MNNG), 407 Methylene tetrahydrofolate reductase gene (MTHFR gene), 429 Methylsulfonylmethane, 376377 MGP. See Matrix gla protein (MGP) MHC. See Major and minor histocompatibility complexes (MHC) MI. See Myocardial infarction (MI) mi-RNAs. See MicroRNAs (mi-RNAs) MIC. See Minimum inhibitory concentration (MIC) Mice, NOD2 response in, 178 Microbial dysbiosis, 403 flora, 403 infection, 282

Microbial anti-inflammatory molecule (MAM), 408 Microbial-associated molecular patterns (MAMPs), 175176. See also Crohn’s disease Microbiome, diseased, 275277 IBD and mycobiome, 276277 obesity and mycobiome, 275276 Microbiome in shaping newborn immune system, 1516 Microbiota composition in gut, 425 Microcalcifications, 191192 Microcomedones, 242 Micronucleation, 51 Micronutrients in skin immunity, 259266 vitamin A, 259262 vitamin C, 262264 vitamin D, 264265 vitamin E, 265266 MicroRNAs (mi-RNAs), 205 Microsomes, ROS production by, 4647 MIF. See Macrophage migration inhibitory factor (MIF) Mig/CXCL9. See Monokine induced by gamma interferon/CXC motif ligand 9 (Mig/CXCL9) Mineralization-regulating proteins, 194195 Minimum inhibitory concentration (MIC), 245 MIP. See Macrophage inflammatory protein (MIP) MIP-1. See Macropage inflammatory protein1 (MIP-1) Mitochondria, 38, 348349, 352 damage, 50 dysfunction, 51, 232 inefficiency, 229, 230f membrane potential, 59 metabolism, 60 ROS production in, 46 Mitochondrial DNA (mtDNA), 60, 228229 Mitochondrial ROS (mROS), 57. See also Reactive oxygen species (ROS) regulation of, 59 sources of, 5859 in T cell activation, 6061 targets of, 5960 Mitogen activated protein kinases (MAPK), 87, 120, 166167, 204206, 409 Mitogenic proteins, 345 Mitotic catastrophe, 51 MMPs. See Matrix metalloproteinases (MMPs) MNNG. See N-Methyl-N0 -nitro-Nnitrosoguanidine (MNNG) MNPs. See Magnetic nanoparticles (MNPs) MoDCs. See Monocyte-derived DCs (MoDCs) Mode of birth, 403 Modified arabinoxylan rice bran consumption, 408409 Momordica charantia extracts, 429 Monocyte chemoattractant protein-1 (MCP1), 151, 297, 373, 424 Monocyte-derived DCs (MoDCs), 296

451 Monocytes, 6, 20 Monogenetic primary immunodeficiencies alternative pathway and susceptibility to herpes encephalitis, 169170 pathogenesis and immune phenotype, 169170 of canonical pathway and susceptibility to pyogenic bacterial infections, 167169 clinical phenotype, natural history and management, 168169 immune phenotype and laboratory diagnosis, 168 Monogenic defects genetic theory of infectious disease, 165 monogenetic primary immunodeficiencies alternative pathway and susceptibility to herpes encephalitis, 169170 of canonical pathway and susceptibility to pyogenic bacterial infections, 167169 in NF-κB signaling, 170172 renaissance in innate immune system research, 165166 TLR signaling networks, 166167, 166f TLR3 signaling, 167 toll, interleukin-1 and discovery of TLR superfamily, 166 Monokine induced by gamma interferon/ CXC motif ligand 9 (Mig/CXCL9), 135 Monoterpene glycosides, 373 Monoterpenes, 373 Monoterpenoids, 368374, 369t, 372f Moraxella catarrhalis, 103 Mounting evidence, 324 MPO. See Myeloperoxidase (MPO) mRNA expression, 285 mROS. See Mitochondrial ROS (mROS) MS. See Multiple sclerosis (MS) MSM. See Men who had sex with men (MSM) mtDNA. See Mitochondrial DNA (mtDNA) MTHFR gene. See Methylene tetrahydrofolate reductase gene (MTHFR gene) Mucin proteins, 103 MUC5AC, 8990, 101103 Muc5B, 101103 Mucociliary clearance (MCC), 101103 Mucor genus, 276 Mucosa-adherent E. coli, 404 Mucosa-associated E. coli NC101 pks, 404 Mucosa-associated lymphoid tissue (MALT), 104 Mucosal immunity at birth, 1617 Mud therapy, 415, 418 Multinucleation, 51 Multiple sclerosis (MS), 349 Muramyl dipeptide (MDP), 176f, 177 N-substitution on MDP effects NOD2 signaling and stability, 181 recognition by innate immune system and NOD2, 177

452 Mutagen inactivation, 407 Mxa gene. See Myxovirus resistance A gene (Mxa gene) Mycobacterium tuberculosis, 21 Mycobiome, 271272 diseased microbiome and, 275277 healthy, 274275, 274f studying, 273274 MyD88. See Myeloid differentiation factor 88 (MyD88) MyD88-adapter-like (MAL), 226227 Myddosome, 166167 Myeloid differentiation factor 88 (MyD88), 19, 166168, 226227 MyD88-dependent signaling, 227 MyD882/2NOD mice, 425 Myeloid-derived suppressor cells (MDSCs), 1819, 344 Myeloperoxidase (MPO), 6667, 83, 342343, 367 Myocardial infarction (MI), 22, 345 Myofibroblasts, 344 Myxovirus resistance A gene (Mxa gene), 250, 253

N NAC. See N-Acetylcysteine (NAC) N-acetylcysteine (NAC), 68, 7071, 89, 354 NAD(P)H quinine oxidoreductase 1 (NQO1), 149150 NADPH. See Nicotinamide adenine dinucleotide phosphate (NADPH) NADPH oxidase (NOX), 37, 66, 119, 352 cell-specific role in pulmonary inflammation, 121122 family members, 120 free radicals and oxidative stress in lung IR injury, 120 isoforms, 121 isoforms and pharmacological inhibitor role in lung IR injury, 122123 in lung inflammation and IR injury, 120 NOX2-derived ROS, 38 NOX2, 165, 342343 role of NOX isoforms and pharmacological inhibitors, 122123 NADPHdependent oxidases, 119 NAFLD. See Non-alcoholic fatty liver diseases (NAFLD) Naive T cells, 57 Nanocarrier-mediated delivery of antioxidants, 6974 lipid nanoparticles and complexes, 74 liposomes, 7072 MNPs, 7374 polymer nanocarriers, 7273 Nanocarriers (NCs), 69 Nanoparticles, 7273, 82 Nanostructured lipid carriers (NLCs), 74 Nanozymes, 73 Naringenin, 138140, 364f, 366 National Institute for Health (NIH), 66 Natural alkamides, 246247 Natural compounds, 428

INDEX

Natural killer cells (NK cells), 6, 134, 224225, 249250, 252253, 252f, 283, 296, 408409 Natural killer T-cells (NKT cells), 5, 9, 283 Naturally occurring DPPIV/CD26-inhibitors, 138 Naturopathic/naturopathy interventions, 416, 417t lifestyle modification before art, 418 during art, 418419 and immunity, 419t medicine, 415 naturopathic approach towards HIVpositive individuals, 416418 principles of, 415 response of naturopathy modalities on inflammation and immunity, 415416 treatments, 415 NCs. See Nanocarriers (NCs) NDs. See Neurodegenerative diseases (NDs) Necrosis, 50 Neh1 domain, 152 Neh16. See Nrf2-ECH homology domains (Neh16) Neh2 domain, 153 Neh3 domain, 153 Neh4 domains, 153 Neh5 domains, 153 Neh6 domain, 153 NEMO. See Nuclear factor kappa B essential modulator (NEMO) Neoandrographolide, 374375 Neonatal dendritic cells, 2225 macrophages, 2122 malnutrition, 16 monocytes, 2021 neutrophils, 1719 NK cells, 2627 pDCs, 24 period, 15 Neonatal Fc receptor (FcRn), 16, 2324 NET. See Neutrophil extracellular traps (NET) NETosis, 18 Neurodegenerative diseases (NDs), 341342, 348349, 349f Neuropeptide Y (NPY), 127128, 134 Neutrophil extracellular traps (NET), 18, 229230 Neutrophil(s), 18, 322, 345 activation, 83 cytoplasmic granules, 1718 Newborn(s), 16 immune system microbiome in shaping, 1516 innate immune cells of, 1727 diminished neutrophil functions in the neonatal period, 19f neonatal dendritic cells, 2225 neonatal macrophages, 2122 neonatal monocytes, 2021 neonatal neutrophils, 1719 neonatal NK cells, 2627

quantitative, phenotypic and functional characteristics of, 23t plasma, 2728 stimulants in modulating innate immune response, 25t Next Generation Sequencing technologies (NGS technologies), 271, 403404 NF-κB. See Nuclear factor kappa B (NF-κB) NF-κB-inducing kinase (NIK), 206 NFAT. See Nuclear factors of activated T cells (NFAT) NGS technologies. See Next Generation Sequencing technologies (NGS technologies) Nicotinamide adenine dinucleotide phosphate (NADPH), 119 NADH/NAD isopotential group, 59 Nicotine, 82 NIH. See National Institute for Health (NIH) NIK. See NF-κB-inducing kinase (NIK) Nitric oxide (NO), 86, 104105, 311312, 365 NO synthase (NOS), 104105 Nitrosative stress, 353354 NK cells. See Natural killer cells (NK cells) NKT cells. See Natural killer T-cells (NKT cells) NLCs. See Nanostructured lipid carriers (NLCs) NLRP3. See Nucleotide-binding oligomerization domain (NOD): NOD-like receptor containing pyrin domain 3 (NLRP3) NLRPs. See Nucleotide-binding oligomerization domain (NOD): NOD-like receptor proteins (NLRPs) NLRs. See Nucleotide-binding oligomerization domain (NOD): NOD-like receptors (NLRs) NMR, 246 NO. See Nitric oxide (NO) NOD. See Nucleotide-binding oligomerization domain (NOD) Non-alcoholic fatty liver diseases (NAFLD), 397 Noncommunicable diseases, 424425 Nonenzymatic antioxidants, 65, 6768, 85, 8990 deficiencies, 88 spin traps, 89 thiols, 8990 Noninvasive bacterial infections, 168169 Nonmelanoma skin cancer, 262 Nonsteroidal anti-inflammatory drugs (NSAIDs), 204, 300 Nontumorigenic keratinocytes, 206 NOS. See Nitric oxide (NO): NO synthase (NOS) Notch-dependent signaling pathway, 260 NOX. See NADPH oxidase (NOX) Noxious gases, 82 NPY. See Neuropeptide Y (NPY) NQO1. See NAD(P)H quinine oxidoreductase 1 (NQO1) Nrf2-ECH homology domains (Neh16), 152 Nrf2. See Nuclear factor erythroid-derived related factor 2 (Nrf2)

453

INDEX

NSAIDs. See Nonsteroidal anti-inflammatory drugs (NSAIDs) N-terminal region (NTR), 153 NTR. See N-terminal region (NTR) Nuclear factor erythroid-derived related factor 2 (Nrf2), 3940, 149150, 152153, 229, 341342 activators CUR, 157 dh404, 156157 RES, 156 vitamin D, 157 crosstalk between Nrf2 and NF-κB pathways in diabetes, 155 in diabetes and its complications, 154155 Nrf2-Keap1, 153 ARE, 151 Cul3-Rbx1-E3-Ligase, 151 mechanism and regulation of, 153, 154f Nrf2-Keap1-ARE pathway, 149150 sMaf proteins, 152 system, 149150 NRF2/ARE pathway, 351352 pathway, 313 Nuclear factor kappa B (NF-κB), 3839, 87, 120, 166167, 169, 204205, 226227, 286, 295296, 341344, 365 crosstalk between Nrf2 and NF-κB pathways in diabetes, 155, 156f in diabetes and complications, 155 induced proinflammatory cytokines, 149 monogenic defects in NF-κB signaling, 170172 clinical phenotype, natural history and management, 171172 NEMO management and IκBα deficiencies, 172 pathogenesis and immune phenotype, 171 signaling, 311312 Nuclear factor kappa B essential modulator (NEMO), 169172 Nuclear factors of activated T cells (NFAT), 57, 341342 Nucleotide-binding oligomerization domain (NOD), 176 NOD2, 175176 as innate immune receptor, 176 mutations correlate with Crohn’s disease, 176177 receptor stabilized by proteins, 177178 recognition of MDP by, 177 response in mice, 178 NOD-like receptor containing pyrin domain 3 (NLRP3), 3839 NOD-like receptor proteins (NLRPs), 5, 7, 52 NOD-like receptors (NLRs), 38, 175176 Nucleus, 348349 Nutraceuticals, 342, 357t, 363, 429 problems and limitations associated with nutraceuticals, 356357 regulatory role of, 355 Nutrient deficiency, 48 Nutrigenomics, 429

Nutrition, 259, 295, 319 in elderly, 324 epidemiological, physiological and clinical features of aging, 319320 functional food to improving immunity, 327 with proven clinical efficacy to ameliorating elderly immunity, 328332 immunobiology of aging, 321324 intervention trials, 329t prevention by, 324327 and weight loss in older adults, 320321 Nutritional interventions, 328

O Obesity, 271272, 275276, 389 fermented milk effect by probiotics in, 391394 fermented milk products, 394396 effect of fermented milk products in, 396398 functional foods, 390 role of gut microbiota, 390391 Occludin phosphorylation, 295296 Ocular health supplements, 429 Odh. See 2-Oxoglutarate dehydrogenase (Odh) 8-OH-Gua. See 8 Hydroxyguanine (8-OHGua) 8-OHdG. See 8 Hydroxy 20 deoxyguanosine (8-OHdG) Oil-soluble compounds, 68 Oligosaccharides, 428 Omarigliptin, 139t Oncogenic potential of COX-2-drived PGE2, 206207, 207f 1T helper cells (Th1 cells), 282 Ophiopogon japonicus root, 378 Ophiopogonin D, 378 Optimal CD4 count, 418 Oral bioavailability, 140141 Oral contraceptives, 242 Osteochondrogenic differentiation, VSMCs, 194 Osteomyelitis, 168 Outer membrane, 342343 Ovalbumin (OVA), 313 Overconsumption, 426427 Overnutrition, 424 Ox-LDL. See Oxidized low density lipoprotein (Ox-LDL) Oxidation of membrane phospholipids, 86 Oxidative damage, 49, 341 Oxidative modification, 232 Oxidative phosphorylation (OXPHOS), 60 Oxidative signaling in COADs antioxidant defense system in lung, 8385 antioxidant-based therapeutic approaches, 8890 mechanism of oxidative stress mediated COAD pathogenesis, 86 redox imbalance, 80f ROS in lung, 8283 ROS-mediated effects, 88

ROS-regulated downstream signaling pathways, 8788 Oxidative stress, 45, 4849, 65, 79, 81, 90, 149150, 154155, 229, 230f, 262, 341342, 346347 drugs in clinical trials, 356 in lung IR injury, 120 markers and inflammation, 67 mechanism of oxidative stress mediated COAD pathogenesis, 86 DNA damage, 86 lipid peroxidation, 86 protein nitration, 86 oxidative stress-induced diseases, 342349 oxidative stress-induced pathologies, 352355 parameters, 21 problems and limitations associated with nutraceuticals, 356357 regulatory role of nutraceuticals and paradoxes, 355 traditional and novel therapeutic targets, 350352 Oxidative stress-induced diseases, 342349 asthma, 342343, 343f cancer, 343345, 344f CVD, 345347, 346f DM, 347 neurodegenerative disease, 348349, 349f Oxidative stress-induced pathologies, 352355 advantages and limitations of approaches, 354355 computational and in silico approach, 353354 drugs in clinical trials for, 356 phytochemicals and synthetic inhibitors, 354 RNAi approach, 352353 stem cell approach, 353 Oxidized low density lipoprotein (Ox-LDL), 345 Oxidoreductases, 46 N-3-Oxo-dodecanoyl-L-homoserine lactone (C12HSL), 107108 2-Oxoglutarate dehydrogenase (Odh), 59 OXPHOS. See Oxidative phosphorylation (OXPHOS) Oxygen (O2), 45, 47, 58 radicals, 45 reduction, 66f Ozone (O3), 58, 82

P P13K/Akt/GSK3-β pathway, 376 p38 mitogen-activated protein kinase (p38 MAPK), 365 p38, 87 p53 gene, 204205 p53/56 lyn, 19 P66shc, 352 p90 ribosomal S6 kinase (p90 RSK), 345 Paeonia species, 373 Paeonia lactiflora root, 373 Paeoniflorin, 373

454 PAFAH. See Platelet activating factor acetyl hydrolase (PAFAH) PAH. See Polycyclic aromatic hydrocarbons (PAH) Pain, 241 PAMPs. See Pathogen-associated molecular patterns (PAMPs) Pancreatic β-cell, 149, 155 Paradoxes, regulatory role of, 355 Paraptosis, 51 Parasitized red blood cells (pRBCs), 312 Parkinson’s disease (PD), 349 PARP-1. See Poly (ADP-Ribose) Polymerase 1 (PARP-1) Particulate matter (PM), 79 Pathogen infection diseases, 314 Pathogen-associated molecular patterns (PAMPs), 56, 16, 38, 103, 105, 166, 175176, 223, 272273. See also Crohn’s disease Pathogenic attack, 5 Pathological mediators, 67 Pattern recognition receptors (PRRs), 5, 7, 15, 105, 165166, 175176, 223 PBMCs. See Peripheral blood mononuclear cells (PBMCs) PCA. See Principle component analysis (PCA) PCD. See Primary ciliary dyskinesia (PCD) PCL. See Periciliary layer (PCL) PD. See Parkinson’s disease (PD) PD-L1-blocking antibody, 408409 pDCs. See Plasmacytoid dendritic cells (pDCs) PDGF. See Platelet derived growth factor (PDGF) PDH. See Pyruvate dehydrogenase (PDH) PDI. See Protein disulfide isomerase (PDI) PECAM-1. See Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1) PEG. See Polyethylene glycol (PEG) PEG-PLGA. See Poly(ethylene glycol)-b-poly (lactic-co-glycolic acid) (PEG-PLGA) PEGDA. See Polyethylene diacrylate (PEGDA) PEI-PEG. See Polyethyleneimine-poly (ethylene glycol) (PEI-PEG) Peptide YY (PYY), 134 Peptide(s), 140141 inhibitors, 138 LL-37, 282 Peptidoglycan, 176, 176f, 181f Periciliary layer (PCL), 101103 Perilla frutescens, 380 Peripheral blood mononuclear cells (PBMCs), 169, 299 Peripheral T-cell tolerance in SLE, 232 Peroxiredoxin 6 (Prdx6), 40 Peroxiredoxins (PRX), 59 in onset of inflammatory cascade, 4041 PrxI, 149150 Peroxisome proliferator activator receptor gamma (PPAR-γ), 1718, 197, 373 Peroxisome proliferator-activated receptor alpha (PPAR-α), 392

INDEX

Peroxisomes, ROS production by, 46 Peroxynitrite (ONOO), 82 free radicals, 347 PGD. See Primary graft dysfunction (PGD) 15-PGDH. See 15-Hydroxyprostaglandin dehydrogenase (15-PGDH) PGs. See Prostaglandins (PGs) Phagocyte-induced cell death (PICD), 20 Phagocytes, 37 Phagocytic cells, 17, 58, 189190, 273 Phagocytic NADPH oxidase (PHOX), 58 Phagocytosis, 6, 18 Pharma industry standards, 327 Pharmacokinetics (PK), 65 Pharmacoperones, 182183 Phenolic acids, 380381, 380f Phenolic compounds, 138140 Phenylthiourea (PTU), 107108 Phorbol 12-myristate 13-acetate (PMA), 286 Phosphatidyl-inositol-3-kinases (PI3K), 204205, 231 Phosphoinositide 3 kinases. See Phosphatidyl-inositol-3-kinases (PI3K) Phospholipase A2 (PLA2), 40, 123 Phospholipase C gamma 2 (PLCγ2), 231 Phospholipase C isoform β2 (PLCβ2), 107 Phosphorylation, 205206, 231232 Phototherapy, 283 PHOX. See Phagocytic NADPH oxidase (PHOX) Physalis virginiana, 377378 Physiological inflammation, 203, 408409 Phytochemicals, 282283, 354, 363, 428 curcumin, 378379 flavonoids, 363368 phenolic acids, 380381 steroidal aglycones and saponins, 377378, 377f stilbenes, 379380 terpenoids, 368377 triterpenoids, 376377 Phytochemistry of A. millefolium, 244245 Phytopharmaceuticals, 363 PI3K. See Phosphatidyl-inositol-3-kinases (PI3K) PI3K/Akt/NF-κB pathway, 313 PICD. See Phagocyte-induced cell death (PICD) Piceatannol, 379380 PK. See Pharmacokinetics (PK) PKA. See Protein kinase A (PKA) PKC. See Protein kinase C (PKC) PKC-dependent ADPGK activity, 61 pks genotoxic islands. See Polyketide synthase genotoxic islands (pks genotoxic islands) PLA2. See Phospholipase A2 (PLA2) Plant(s) components derived from, 138140 plant-derived remedies, 247 plant-derived volatile oils, 368372 Plaque psoriasis. See Psoriasis vulgaris Plasma membrane, 47

Plasmacytoid dendritic cells (pDCs), 2223, 227229, 282 Plasmodium falciparum, 310 Platelet activating factor acetyl hydrolase (PAFAH), 22 Platelet derived growth factor (PDGF), 58 Platelet-Endothelial Cell Adhesion Molecule1 (PECAM-1), 69 PLCβ2. See Phospholipase C isoform β2 (PLCβ2) PLCγ2. See Phospholipase C gamma 2 (PLCγ2) PLGA. See Poly(lactide-co-glycolide) (PLGA) PM. See Particulate matter (PM) PMA. See Phorbol 12-myristate 13-acetate (PMA) PMN. See Polymorphonuclear neutrophils (PMN) Pneumocystis carinii, 301 Pollution, 48 Poly (ADP-Ribose) Polymerase 1 (PARP-1), 155 Poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) (PEG-PLGA), 7273 Poly(lactide-co-glycolide) (PLGA), 72 Polycyclic aromatic hydrocarbons (PAH), 79 Polyethylene diacrylate (PEGDA), 71 Polyethylene glycol (PEG), 65 Polyethyleneimine-poly(ethylene glycol) (PEI-PEG), 73 Polyketide synthase genotoxic islands (pks genotoxic islands), 404 Polymer nanocarriers, 7273 Polymeric micelles, 72 Polymorphism(s), 83 in HMOX1, 85 Polymorphonuclear neutrophils (PMN), 5, 1718 Polyphenol(s), 11, 356 antioxidants, 68 Polyunsaturated fatty acids (PUFAs), 49 Pomegranate (Punica granatum), 368, 381 Postprandial inflammation, 425 Posttranscriptional regulation of COX-2 expression, 204205 PPAR-α. See Peroxisome proliferatoractivated receptor alpha (PPAR-α) PPAR-γ. See Peroxisome proliferator activator receptor gamma (PPAR-γ) PPi. See Pyrophosphate (PPi) pRBCs. See Parasitized red blood cells (pRBCs) Prdx6. See Peroxiredoxin 6 (Prdx6) Pre-biotics, 405406, 428 in combination with drugs and future prospects, 410 against CRC-experimental, 405410 and health benefits, 406t mechanism of action, 407f Preterm infants, 18 Primary ciliary dyskinesia (PCD), 110111 Primary graft dysfunction (PGD), 119 Primary HIV infection. See Acute HIV infection

INDEX

Primary immunodeficiency, 166f, 167168. See also Monogenic defects Principle component analysis (PCA), 275276 Pro-apoptotic proteins, 50 Proazulenes, 244 Probiotic(s), 140, 249, 295, 328 and anti-inflammatory activity, 297301 bacteria, 249 clinical intervention with probiotics, 250253 in combination with drugs and future prospects, 410 and CRC, 405406 against CRC-experimentalinvitro, invivo, and clinical aspects, 405410 effects of probiotics on HIV-infected subjects, 301303, 302f and health benefits, 406t and HIV, 301 in immune system, 253254 immunogenic role of probiotics against influenza virus infection, 249 interaction of microbiota and probiotics in intestinal immune system, 249 in intestinal immune system, 249 lactic acid organisms, 390391 mechanism of action, 407f and mode of action, 295301, 296f safety, efficacy/caution, 303 Procyanidins, 140141 Product marketing, 324 Proinflammatory cytokines, 151, 155, 287 Proinflammatory signaling pathways, 149 Proline (P), 107108 Prolonged ROS production, 59 PROP. See 6-Propyl-2-thiouracil (PROP) Prophylactic antibiotic therapy s, 169 Propionibacterium acnes, 242, 246, 262 6-Propyl-2-thiouracil (PROP), 107108 Prostaglandins (PGs), 204 H2 synthase, 204 PGE2, 373, 415 formation, metabolism and oncogenic function of, 207f oncogenic potential of COX-2-drived, 206207 Proteasomal activity inhibitor, 348349 Protein disulfide isomerase (PDI), 47 Protein kinase A (PKA), 120 Protein kinase C (PKC), 120, 204205, 347, 367 Protein(s), 140141 aggregation, 348349 kinases, 205206 nitration, 86 NOD2 receptor stabilized by, 177178 oxidation, 49 phosphatases, 205206 Proteobacteria, 277 Proteoglycans, 257 Protocatechuic acid, 380381, 380f PRRs. See Pattern recognition receptors (PRRs) PRX. See Peroxiredoxins (PRX)

PSE model. See Psoriatic skin equivalent model (PSE model) P-selectins, 67 Pseudomonas aeruginosa, 103, 272 Pseudomonas cepacia, 103 Psoralen Plus Ultraviolet Light Therapy (PUVA), 283 Psoriasis, 133, 137, 261262, 261f, 281283 Psoriasis vulgaris, 281 Psoriatic skin equivalent model (PSE model), 287 Psychoactive effects, 375 Psychological interventions, 416 Pterostilbene (PTS), 154155, 157 PTS. See Pterostilbene (PTS) PTU. See Phenylthiourea (PTU) Puerarin, 364f, 366367 PUFAs. See Polyunsaturated fatty acids (PUFAs) Pulchinenoside triterpenes, 377 Pulmonary inflammation, cell-specific role of NADPH oxidases in, 121122 Purple carrot (Daucus carota), 368 Pustular psoriasis, 281 PUVA. See Psoralen Plus Ultraviolet Light Therapy (PUVA) Pyogenic bacterial infections, 167169 Pyrin domains, 176 Pyronecrosis, 52 Pyrophosphate (PPi), 197 Pyroptosis, 7, 52 Pyruvate dehydrogenase (PDH), 59 PYY. See Peptide YY (PYY)

Q QH2/Q isopotential group, 59 Quercetin, 290, 364f, 366

R R213G missense mutation in SOD3, 8384 RA. See Rheumatoid arthritis (RA) RA FLS. See Rheumatoid arthritis fibroblastlike synoviocytes (RA FLS) Raabadi, 395 Rac-2, 1718 Radiation, 48 RAG1. See Recombination-activating gene (RAG1) RAGE. See Receptor for advanced glycation end products (RAGE) RALDH. See Retinal aldehyde dehydrogenase enzyme (RALDH) Randomized controlled trial (RCT), 366 RANTES/CCL5. See Regulated on activation, normal T-cell expressed and secreted/ CaC motif chemokine ligand 5 (RANTES/CCL5) RAR. See Retinoic acid receptor (RAR) RARE. See Retinoid acid response elements (RARE) RCT. See Randomized controlled trial (RCT) RDA. See Recommended dietary allowance (RDA) RDS. See Respiratory distress syndrome (RDS)

455 Reactive aldehydes, 86 Reactive oxygen species (ROS), 6, 16, 37, 45, 48, 5758, 66, 79, 104, 119, 128, 224, 229, 262, 314315, 341, 353. See also Mitochondrial ROS (mROS) cell death, 5052 exogenous ROS production, 4748 Fenton reaction, 46f generation during onset of inflammatory cascade, 3738 myriad cell death pathways, 50f oxidative damage, 49 oxidative stress, 4849 physiological role of, 48 production in living system or endogenously, 4547 ROS level heightened in T-cells isolated from lupus patients, 232 ROS-induced genomic instability, 343344 ROS-mediated effects in COADs, 88 ROS-regulated downstream signaling pathways in COADs, 8788 sources in lung, 8283 endogenous sources, 83 exogenous sources, 82 Reactive species, 6667 Receptor for advanced glycation end products (RAGE), 6, 228229, 297 Recombination-activating gene (RAG1), 165 Recommended dietary allowance (RDA), 303 Red clover (Trifolium pretense), 366 Redox control of antioxidant and anti-inflammatory transcription factors, 3940 of inflammatory mediators, 3839 Redox-regulated events, 40f Redox sensitive transcription, 149150 crosstalk between Nrf2 and NF-κB pathways in diabetes, 155, 156f factors, 341 islet inflammation in diabetes, 150151, 150f mechanism and regulation of Nrf2-Keap1ARE pathway, 153, 154f NF-κB in diabetes and complications, 155 Nrf2 activators, 156157 Nrf2 in diabetes and complications, 154155 Nrf2-Keap1-ARE machinery components, 151153 perspectives, 157 Regulated on activation, normal T-cell expressed and secreted/CaC motif chemokine ligand 5 (RANTES/CCL5), 135 Regulatory bodies, 356 Regulatory dendritic cells (DCregs), 297 Regulatory T cells (Treg cells), 232, 253, 258, 260, 296, 302, 311312, 323324, 408409 Renaissance in innate immune system research, 165166 Renin-angiotensin system, 346347 RES. See Resveratrol (RES); Reticuloendothelial system (RES)

456 Respiratory distress syndrome (RDS), 342 Respiratory Synctial Virus (RSV), 21 Resveratrol (RES), 68, 71, 154156, 378f, 379 RET. See Reverse electron transport (RET) Retagliptin, 140t Retenoids, topical, 242 Reticuloendothelial system (RES), 69 Retinal aldehyde dehydrogenase enzyme (RALDH), 17 Retinoic acid, 260 Retinoic acid receptor (RAR), 259260 Retinoid acid response elements (RARE), 259260 Retinoid X receptor (RXR), 259260 Retinoid X response elements (RXRE), 259260 Retinoids, 68 Reverse electron transport (RET), 59 Reversing metabolic syndrome, functional foods, 426 Rheumatoid arthritis (RA), 133, 137, 365 Rheumatoid arthritis fibroblast-like synoviocytes (RA FLS), 312 Rho associated protein kinase (ROCK), 231232 RIG-like receptor (RLR), 5, 7, 175176 RNA interference approach (RNAi approach), 352354 RNAi approach. See RNA interference approach (RNAi approach) ROCK. See Rho associated protein kinase (ROCK) Rockwood’s index, 331 ROS. See Reactive oxygen species (ROS) Rosemary (Rosmarinus officinalis), 380 Rosmarinic acid, 380, 380f 18S rRNA gene, 273 RSV. See Respiratory Synctial Virus (RSV) Ruscogenin, 378 Rutin, 364f, 366 3-Rutinoside, 366 Rutinosyl, 364f RXR. See Retinoid X receptor (RXR) RXRE. See Retinoid X response elements (RXRE)

S Saccharomyces boulardii (SB), 409 Sage (Salvia officinalis), 380 Salens, 90 Salix species, 363 Salvia genus, 375 Santamarin, 373 SAP. See Serum amyloid P component (SAP) SAPKs. See Stress activated protein kinases (SAPKs) Sarcopenia, 321 Sarcopenic obesity, 320 SARM. See Sterile α-and armadillo-motifcontaining protein (SARM) Sarsaparilla root, 377378 Saturated fatty acids, 151 Saussurea costus, 373 Saxagliptin, 139t SB. See Saccharomyces boulardii (SB)

INDEX

Scavenger receptors (SR), 314 Scavenging enzymes, 59 SCCs. See Solitary chemosensory cells (SCCs) SCFAs. See Short-chain fatty acids (SCFAs) SCIG. See Subcutaneous IgG (SCIG) Scutellaria baicalensis, 287, 365 SD rats. See Sprague Dawley rats (SD rats) SDF-1α. See Stromal cell-derived factor-1α (SDF-1α) SDF1/CXCL12. See Stromal cell-derived factor 1/CXC motif chemokine 12 (SDF1/CXCL12) sDPPIV/CD26. See Soluble DPPIV/CD26 (sDPPIV/CD26) Segmented filamentous bacteria (SFB), 233 Selective neuronal vulnerability, 348 Self-tolerance, 224 Sensory receptors, immune responses regulation by, 105109 other pattern recognition receptors, 105 taste receptors, 105109, 109t TLRs, 105 Sepsis, 168, 314 Ser40, 153 Serine proteases, 133134 serine-threonine kinases, 204206 Serologic tests, 182 Serum amyloid P component (SAP), 393394 Sesquiterpene lactones, 373 Sesquiterpenes, 245 Sesquiterpenoids, 368374, 369t, 372f SFB. See Segmented filamentous bacteria (SFB) Shc. See Src homologous-collagen homologue (Shc) Short-chain fatty acids (SCFAs), 295296, 408, 425 SHP-1. See Src homology domain 2 containing tyrosine phosphatase-1 (SHP-1) Siglec-9, 1819 Signal transducers and activation of transcription (STAT), 300 STAT-1, 169, 365 STAT-3, 341342 Signal transduction, 226227, 227f Signal transmission, 223 Signaling, innate immune system, 56 Single nucleotide polymorphisms (SNP), 428 Singlet oxygen (1O2), 58, 341 Sinonasal cavity, 101 regulation of immune responses by sensory receptors in, 105109 other pattern recognition receptors, 105 taste receptors, 105109, 109t TLRs, 105 Sinonasal innate immunity, 102f MCC as foundation of, 101103 Sinonasal mucus, 104 Sisal leaves (Agave sisalana), 377 Sitagliptin, 137, 139t Skin, 257 diseases, 257, 259

vitamin A in, 261t vitamin C in, 263t vitamin D in, 265t vitamin E in, 266t immunity, 257258 micronutrients in, 259266 inflammation, 213214, 217 inflammatory diseases, 261262 structure, 257 SLE. See Systemic lupus erythematosus (SLE) SLGTs. See Sodium-linked glucose transporters (SLGTs) SLNs. See Solid lipid nanoparticles (SLNs) sMaf proteins. See Small Maf proteins (sMaf proteins) Small Maf proteins (sMaf proteins), 152 Small proline-rich proteins (SPRR), 51 SMCs. See Smooth muscle cells (SMCs) Smoked horse’s hides, 396 Smooth muscle cells (SMCs), 133134, 345 sn-glycerol-3-phosphate dehydrogenase (snG3PDH), 59 SNP. See Single nucleotide polymorphisms (SNP) SOD. See Superoxide dismutase (SOD) Sodium-linked glucose transporters (SLGTs), 109 Solid lipid nanoparticles (SLNs), 74 Solitary chemosensory cells (SCCs), 101, 108109, 108f Soluble DPPIV/CD26 (sDPPIV/CD26), 133134, 136 Soluble plasma components, 2728 Soriatane, 283 Soya (Glycine max), 366 Sp-1. See Specificity protein 1 (Sp-1) SP-A. See Surfactant protein A (SP-A) Spearmint (Mentha spicata), 380 Specificity protein 1 (Sp-1), 155 Spin traps, 89 Sprague Dawley rats (SD rats), 393 SPRR. See Small proline-rich proteins (SPRR) SQR. See Succinate-coenzyme Q reductase (SQR) Squamous cell carcinoma, 262264, 263f SR. See Scavenger receptors (SR) Src homologous-collagen homologue (Shc), 352 Src homology domain 2 containing tyrosine phosphatase-1 (SHP-1), 1819 SREB. See Super Conserved Receptor Expressed in Brain (SREB) Staphylococcus sp., 403404 S. aureus, 425 S. epidermidis, 242 STAT. See Signal transducers and activation of transcription (STAT) Stem cell approach, 353 Sterile infection, 224 Sterile α-and armadillo-motif-containing protein (SARM), 226227 Steroid hormones, 157 Steroidal aglycones, 377378 Steroidal saponins, 377378 Stevia diterpenes, 375

INDEX

Stevia rebaudiana leaves, 375 Stevia species, 375 Steviol glycosides, 375 Stevioside, 375 Stilbenes, 379380 Stimulants in modulating innate immune response, 25t Stratum corneum, 257 Streptococcus gallolyticus, 404405 Streptococcus pneumoniae, 16, 103 Streptococcus thermophilus, 299 Stress activated protein kinases (SAPKs), 87 Stromal cell-derived factor 1/CXC motif chemokine 12 (SDF1/CXCL12), 135 Stromal cell-derived factor-1α (SDF-1α), 127129, 131 Sub-cellular localization-specific function, 228 Subcutaneous IgG (SCIG), 172 Subcutis, 257 Substrates, 135 Succinate-coenzyme Q reductase (SQR), 59 Sulfenylation, 5960 Sulfhydryl groups, 68 Sulforaphane, 154155, 356357 Sunflower (Helianthus annuus), 375376 Super Conserved Receptor Expressed in Brain (SREB), 397 “Superorganism” hypothesis, 271272 Superoxide (O22_), 58, 66, 82 radicals, 37 superoxide-induced DNA strand breaks, 351 Superoxide dismutase (SOD), 46, 58, 65, 8384, 149150, 232, 342, 350351 mimetics, 90, 351 SOD-loaded polyketal particles, 72 SOD2, 351 SOD3, 350351 Surfactant protein A (SP-A), 71 Surfactants, 103 Swelling, 241 Symbiotic bacteria, 249 Symbiotic thermophilic starter cultures, 394395 Synbiotics (SYN), 409 Synthetic inhibitors, 354 Systemic lupus erythematosus (SLE), 223224, 230231, 313 breakdown in disposal of cellular debris in pathogenesis, 229230 CD3-TCR signaling in SLE T-cells, 231 enhanced aggregation of lipid rafts and TCR activation in SLE T-cells, 231 enhanced CD44-ERM/ROCK pathway in manifestation, 231232 global hypomethylation in SLE CD41 Tcells, 232233 lack of IL-2 and peripheral T-cell tolerance, 232 Systemic therapy, 283

T T cell receptor (TCR), 8, 57, 135136, 231 enhanced aggregation of lipid rafts and TCR activation, 231 T cell(s), 8, 296, 299, 323324 activation, 57 mROS in, 6061 CD3-TCR signaling in SLE, 231 DPPIV/CD26 as T-cells costimulatory molecule, 135136 enhanced aggregation of lipid rafts and TCR activation in SLE, 231 global hypomethylation in SLE CD41, 232233 lack of IL-2 and peripheral T-cell tolerance in SLE, 232 ROS level heightened in T-cells isolated from lupus patients, 232 T helper cells (Th cells), 253 Th1 cells, 297, 323324 Th1-type cytokines, 15, 217218 Th2 cells, 81, 297, 309, 323324 Th-2 mediated asthma various allergens, 342343 Th17 cells, 233, 264, 297 T1D. See Type 1 diabetes (T1D) T1R. See Type 1 receptor (T1R) T2D. See Type 2 diabetes (T2D) T2DM. See Type-2 diabetes mellitus (T2DM) T2R38, 107108 T2Rs. See Type 2 receptors (T2Rs) TAMs. See Tumor-associated macrophages (TAMs) TANK-binding kinase-1 (TBK1), 167 Tanshinone IIA, 375 Targeting, antioxidant, 6869 TAS1R2 genes, 111 TAS1R3 genes, 111 TAS2R38 gene, 107108 Taste receptors, 101, 105109 airway expression of, 109t Tazarotene, 261262, 283 TBK1. See TANK-binding kinase-1 (TBK1) TCA. See Tri-carboxylic acid (TCA) TCM. See Traditional Chinese medicine (TCM) TCR. See T cell receptor (TCR) Tea (Camellia sinensis), 367 Tehuanine G, 375 Teneligliptin, 139t Terminal restriction fragment length polymorphism (T-RFLP), 403404 Terpenoids, 368377. See also Flavonoids diterpenoids, 374376 monoterpenoids, 368374 sesquiterpenoids, 368374 Tetanus toxoid (TT), 134 TG. See Triglycerides (TG) TGF-β. See Transforming growth factor-β (TGF-β) Th cells. See T helper cells (Th cells) Th1 cells. See 1T helper cells (Th1 cells) Therapeutic targets, traditional and novel, 350352

457 increasing endogenous levels of antioxidant, 350352 strategy to decreasing oxidative stress, 352 Thin layer chromatography (TLC), 246 Thiols, 5960, 8990 Thioredoxin (TRX), 149150 Thromboxane-B2 (TX-B2), 365 Thymic adipocyte, 323 fibroblasts, 323 involution, 323 Thymidine phosphorylase, 344 TICAM-1. See Toll-IL-1 receptor domaincontaining adaptor molecule-1 (TICAM-1) Tight junction proteins (TJ proteins), 391392 TIR. See Toll receptor/Interleukin-1 receptor (TIR) TIR containing adapter-inducing interferon beta-related adapter molecule (TRAM), 7, 226227 TIR domain-containing adaptor-inducing interferon β (TRIF), 7, 7f, 105, 226227 TRIF-dependent pathway, 167 TRIF-dependent TLR signaling, 228 TIR receptor domain-containing adapter protein (TIRAP), 7 TIRF. See TIR domain-containing adaptorinducing interferon β (TRIF) Tissue damage, 229, 230f injury, 217 remodeling in disease, 314315 TJ proteins. See Tight junction proteins (TJ proteins) TLC. See Thin layer chromatography (TLC) TLR. See Toll-like receptor (TLR) T-lymphocyte(s), 6, 810, 15, 258 APC interaction, 4f T lymphocyte-mediated responses, 261262 TNBS. See 2,4,6-Trinitrobenzene sulfonic acid (TNBS) TNF. See Tumor necrosis factor (TNF) TNF receptor-associated factor 3 (TRAF3), 167 Tobacco, 79, 82 smoking, 48, 345 Tocopherol(s), 68, 265 Tocotrienols, 265 Toll gene product, 166 Toll receptor/Interleukin-1 receptor (TIR), 166167, 224225 Toll-IL-1 receptor domain-containing adaptor molecule-1 (TICAM-1), 167 Toll-like receptor (TLR), 5, 7, 15, 2122, 38, 105, 165166, 175176, 223, 246, 250252, 264, 297, 408409 activation, 223224 adaptor proteins and signal transduction, 226227, 227f agonists, 24 breakdown in disposal of cellular debris in SLE pathogenesis, 229230

458 Toll-like receptor (TLR) (Continued) CD3-TCR signaling in SLE T-cells, 231 diet, commensal microbiota and autoimmunity, 233 discovery of superfamily, 166 enhanced aggregation of lipid rafts and TCR activation in SLE T-cells, 231 enhanced CD44-ERM/ROCK pathway in SLE manifestation, 231232 family, 224225, 225t, 226f global hypomethylation in SLE CD41 T-cells, 232233 immunomodulate both innate and adaptive responses, 225226 lack of IL-2 and peripheral T-cell tolerance in SLE, 232 monogenic defects genetic theory of infectious disease, 165 monogenetic primary immunodeficiencies, 167170 monogenic defects in NF-κB signaling, 170172 renaissance in innate immune system research, 165166 TLR signaling networks, 166167, 166f TLR3 signaling, 167 toll, interleukin-1 and discovery of TLR superfamily, 166 MyD88-dependent signaling, 227 oxidative stress, mitochondrial inefficiency, tissue damage, and TLR9 activation, 229 ROS level heightened in T-cells isolated from lupus patients and lupus mouse models, 232 signaling networks, 166167, 166f SLE, 230231 and sub-cellular localization-specific function, 228 TLR-PAMP interaction, 223 TLR3, 228 signaling, 167 TLR4, 2021, 165166 TLR9, 228 activation, 229, 230f in autoimmune diseases, 228229 self-recognition and role in autoimmune diseases, 228229 TRIF-dependent TLR signaling, 228 Topical therapy, 283 Traditional Chinese medicine (TCM), 365 TRAF-6. See Tumor necrosis factor receptorassociated factor-6 (TRAF-6) TRAF3. See TNF receptor-associated factor 3 (TRAF3) TRAIL. See Tumor necrosis factor related apoptosis inducing ligand (TRAIL) TRAM. See TIR containing adapter-inducing interferon beta-related adapter molecule (TRAM) Trans-10, cis-12-CLA isomer, 391 Trans-3,40 ,5-trihydroxystilbene. See Resveratrol (RES) Transcriptional factors, 262

INDEX

Transcriptional regulation of COX-2 expression, 204205 Transforming growth factor-β (TGF-β), 262, 273, 375 TGF-β1, 1718 Transgenic mice, 16, 8384, 204 Transmembrane protein, 7, 120, 213214, 228 Treg cells. See Regulatory T cells (Treg cells) Trelagliptin, 139t T-RFLP. See Terminal restriction fragment length polymorphism (T-RFLP) Tri-carboxylic acid (TCA), 60 TRIF. See TIR domain-containing adaptorinducing interferon β (TRIF) Triglycerides (TG), 389 2,4,6-Trinitrobenzene sulfonic acid (TNBS), 313, 408 Tripeptides, 140141 Tripterygium wilfordii, 375 Triptolide, 375 Triterpenes. See Triterpenoids Triterpenoids, 376377, 376f TRX. See Thioredoxin (TRX) Trypsin, 140 TT. See Tetanus toxoid (TT) Tumor cells, 203 Tumor growth, 203 Tumor microenvironment, 344 Tumor necrosis factor (TNF), 134 TNF-α, 38, 58, 149, 155, 166168, 225226, 261262, 282, 296297, 323324, 365, 391392, 415, 424 Tumor necrosis factor receptor-associated factor-6 (TRAF-6), 166167, 227 Tumor necrosis factor related apoptosis inducing ligand (TRAIL), 26 Tumor-associated macrophages (TAMs), 344 Turmeric root (Curcuma longa), 378379 TX-B2. See Thromboxane-B2 (TX-B2) Type 1 diabetes (T1D), 133, 137, 149, 155 Type I IFNs, 21, 170, 249250 Type 1 receptor (T1R), 107 Type 2 diabetes (T2D), 134, 149 Type II epithelial cell activation, 122 Type 2 receptors (T2Rs), 107 Type-2 diabetes mellitus (T2DM), 389 Tyrosine kinase signaling pathway inhibition, 409

U Ub ligase. See E3ubiquitin ligase (Ub ligase) UC. See Ulcerative colitis (UC) ucGRP. See Undercarboxylated GRP (ucGRP) ucMGP. See Undercarboxylated MGP (ucMGP) Ulcerative colitis (UC), 276277 Ultraviolet A (UVA), 283 Umbilical cord blood, 1718 Unc93 homolog B1 (UNC93B1), 167, 228 UNC93B-deficiency, 169 UNC93B1. See Unc93 homolog B1 (UNC93B1) Uncoupled nitric oxide synthase, 38 Undercarboxylated GRP (ucGRP), 195196 Undercarboxylated MGP (ucMGP), 195196

Undernutrition, 424 30 -Untranslated region (UTR), 205 Upper respiratory immunity and disease, impairment of, 110111 acute rhinosinusitis, 110 chronic rhinosinusitis, 110 cystic fibrosis, 110 diabetes mellitus, 111 PCD, 110111 Upper respiratory physiology and innate immunity, 101105 epithelial cells as immune effectors, 103105 impairment of upper respiratory immunity and disease, 110111 MCC, 101103 regulation of immune responses by sensory receptors in sinonasal cavity, 105109 Ursolic acid, 377 U.S. Food and Drug Administration (FDA), 138, 283, 356 UTR. See 30 -Untranslated region (UTR) UV irradiation stimulates production, 262 UV light, 48 UVA. See Ultraviolet A (UVA)

V Vaccine, 250, 251t Vaccinium species, 368 Valine (V), 107108 Vanillic acid, 380381 Vascular calcification (VC), 192195 molecular mechanism of, 195f pathophysiological mechanisms of, 192195 calcifying extracellular vesicles, 193194 mineralization-regulating proteins, 194195 VSMCs osteochondrogenic differentiation, 194 types of, 192f vicious cycle of inflammation and, 196197 Vascular cell adhesion molecule, 289 VCAM-1, 67, 190, 366 Vascular disease, 65 Vascular endothelial growth factor (VEGF), 289, 315 Vascular endothelium, 910, 6667 Vascular smooth muscle cells (VSMCs), 189 osteochondrogenic differentiation, 194 Vasculature, 345 Vasoactive intestinal peptide (VIP), 128129 VC. See Vascular calcification (VC) VDR. See Vitamin D receptor (VDR) VDRE. See Vitamin D response element (VDRE) VEGF. See Vascular endothelial growth factor (VEGF) Velutin, 364f, 365 Veronicastrum species, 365 Vertebrate(s), 3 immune system, 3, 5

459

INDEX

Very low-density receptor (VLDL), 22 Vesicular stomatitis virus (VSV), 170 Vicious cycle of inflammation, 196197 of inflammation and VC, 196197 Viili, 395396 Viiliculture, 395396 Vildagliptin, 139t VIP. See Vasoactive intestinal peptide (VIP) Viral double-stranded RNA (vRNA), 105 Viral infection, 314 Visceral adipose tissue, 389390 Vitamin A, 259262, 259f, 260f, 261t Vitamin C, 85, 257, 262264, 263t intake, 11 Vitamin D, 19, 157, 264265, 264f, 265t, 418 Vitamin D receptor (VDR), 264 Vitamin D response element (VDRE), 264 Vitamin E, 85, 265266, 265f, 266t, 354 Vitamin K as potential therapeutic target for atherosclerosis, 195196 Vitamin K-dependent protein (VKDP), 194195 Vitamin K2, 195196 VKDP. See Vitamin K-dependent protein (VKDP)

VLDL. See Very low-density receptor (VLDL) Volatile oils, 368372 vRNA. See Viral double-stranded RNA (vRNA) VSMCs. See Vascular smooth muscle cells (VSMCs) VSV. See Vesicular stomatitis virus (VSV)

W Wallerian degeneration, 51 Warburg effect, 61 Water-soluble antioxidant, 68, 70 Weight loss etiology of weight loss in elderly, 321t in older adults, 320321 Weight management supplements, 428429 White blood cells, 20 Whole genome mRNA microarray analysis, 311312 Wild blueberry, 368 Willow bark, 363 Withaferin A, 378 Withania somnifera (Ashwagandha), 378 Withanolides, 378 Wnt-pathway-related genes, 403404

World Health Assembly (WHA), 281 World Health Organization (WHO), 189, 281, 295, 301, 311 Wound healing, 262

X Xanthine, 4546 Xanthine dehydrogenase (XDH), 4546 Xanthine oxidase (XO), 38, 4546 Xenobiotic metabolism, 47 X-linked anhidrotic ectodermal dysplasia with immunodeficiency (XL-EDA-ID), 171

Y Y1-receptor, 135 Yams (Dioscorea species), 377 Yarrow, 243 Yoga, 416417 Yogurt, 250, 394395, 397398

Z Zingiber zerumbet, 374 Zygomycota phyla, 275276