The Complex Interplay Between Gut-Brain, Gut-Liver, and Liver-Brain Axes [1 ed.] 0128219270, 9780128219270

Table of contents :
Front Cover
The Complex Interplay Between Gut–Brain, Gut–Liver, and Liver–Brain Axes
Copyright Page
Dedication
Contents
List of contributors
Preface
Acknowledgments
Introduction
I. Gut-brain axis
1 The pathophysiology of gut–brain connection
1.1 Introduction
1.2 The anatomical entity
1.3 The functional entity and the role of microbiota
1.4 The pathological entity
1.5 Conclusion
References
2 The interactions between gut and brain in gastrointestinal disorders
2.1 Introduction and anatomo-physiological background
2.2 Clinical presentation of the disorders of the gut–brain interaction
2.2.1 Gastroduodenal motility
2.2.2 Impaired gastric accommodation
2.2.3 Gastroduodenal sensitivity
2.2.4 Gastroduodenal inflammation and mucosal permeability
2.2.5 Visceral hypersensitivity in irritable bowel syndrome
2.2.6 Altered gastrointestinal motility in irritable bowel syndrome
2.2.7 Gut microbiota dysbiosis in irritable bowel syndrome
2.2.8 Immune activation and alteration in mucosal permeability in patients with irritable bowel syndrome
2.2.9 Postinfection irritable bowel syndrome
2.3 Therapy of main disorders of the gut–brain axis
2.3.1 Treatment of functional esophageal disorders
2.3.1.1 Mechanism of action of pain modulators
2.3.1.2 Pain modulators in the treatment of functional esophageal disorders
2.3.1.3 Other pain modulators
2.3.1.4 Psychological interventions in functional esophageal disorders
2.3.2 Treatment of functional dyspepsia
2.3.2.1 General and dietary recommendations
2.3.2.2 Pharmacological treatment of functional dyspepsia
2.3.2.3 Antisecretory drugs and antacids in the treatment of functional dyspepsia
2.3.2.4 Centrally active neuromodulators in the treatment of functional dyspepsia
2.3.2.5 Use of prokinetics in functional dyspepsia
2.3.2.6 Fundus relaxing drugs
2.3.2.7 Alternative and nonpharmacological treatment
2.3.3 Treatment of irritable bowel syndrome
2.3.3.1 Diet and physical activity in irritable bowel syndrome
2.3.3.2 Modulators of gut microbiota in irritable bowel syndrome
2.3.3.3 Treatment of irritable bowel syndrome with predominant constipation
2.3.3.4 Treatment of irritable bowel syndrome with predominant diarrhea
2.3.3.5 Treatment of abdominal pain in irritable bowel syndrome
2.3.3.6 Treatment of bloating
2.3.4 Alternative therapies and psychological interventions
References
3 The interactions between gut and brain in psychiatric and neurological disorders
3.1 Introduction
3.2 Development of gut microbiota
3.3 Pathways of brain–gut–microbiota interaction
3.3.1 Immune pathway
3.3.2 Vagus nerve
3.3.3 Microbial metabolites
3.4 Tryptophan metabolism
3.5 Endocrinologic pathway
3.6 Microbial neural substrates
3.7 Neuropsychiatric disorders affected by brain–gut interplay
3.7.1 Autism spectrum disorder
3.7.2 Schizophrenia
3.7.3 Depression
3.7.4 Parkinson’s disease
3.7.5 Alzheimer’s disease
3.8 Psychiatric disorders in gastrointestinal diseases
3.9 Conclusion
References
4 The role of serotonin and its pathways in gastrointestinal disorders
4.1 Role of serotonin in the physiology of digestive and extradigestive systems
4.1.1 Main functions of 5-HT in extradigestive systems
4.1.1.1 Central nervous system
4.1.1.2 Platelets
4.1.1.3 Blood vessels
4.1.2 Serotonin in gut physiology
4.1.2.1 Secretory functions
4.1.2.2 Emesis
4.1.2.3 Gut motility
4.1.2.4 Immune system
4.2 5-HT receptors, serotonin transporter, and their polymorphisms
4.2.1 5-HT receptors and serotonin transporter
4.2.1.1 5HT1 receptor
4.2.1.2 5-HT2 receptor
4.2.1.3 5-HT3 receptor
4.2.1.4 5-HT4 receptor
4.2.1.5 5-HT5 receptor
4.2.1.6 5-HT6 receptor
4.2.1.7 5-HT7 receptors
4.2.2 Serotonergic system, polymorphic variants and functional gastrointestinal diseases
4.3 Serotonin in functional gastrointestinal disorders and the brain–gut axis
4.3.1 Serotonin in functional gastrointestinal disorders
4.3.2 Serotonin in the brain–gut axis
4.4 Serotonin, psychological/psychiatric and extra-gastrointestinal comorbidities
4.5 Serotonin and microbiota, the brain–gut axis and the psychobiota
4.5.1 Serotonin and microbiota
4.5.2 Serotonin in the brain–gut axis and the psychobiota
4.6 Conclusion
References
II. Gut-liver axis
5 The pathophysiology of gut–liver connection
5.1 Regulation of intestinal permeability
5.1.1 The intestinal mucosa structure
5.1.2 The “leaky gut”
5.1.3 Assessment of epithelial barrier dysfunction
5.1.4 Therapeutic potential of intestinal mucosa regulation
5.1.5 Regulation of intestinal permeability by inflammatory cytokines
5.1.6 Regulation of intestinal permeability by gut microbiota
5.1.7 Regulation of intestinal permeability by diet
5.2 Role of altered intestinal permeability in the pathogenesis of NAFLD
5.2.1 Gut microbiota and NAFLD
5.2.2 Role of the leaky gut in the progression of NAFLD
5.2.3 Influence of liver homeostasis on intestinal barrier function
5.3 Role of incretins in NAFLD
5.3.1 Physiological effects of incretins
5.3.2 Effects of incretins on hepatic glucose and lipid metabolism
5.3.3 Effects of incretins on NAFLD and NASH
5.3.4 The role of inhibitors of dipeptidyl peptidase-4 activity
5.3.5 The role of incretin co-agonists
5.4 Alteration of bile acid pathways in NAFLD
5.4.1 Bile acid synthesis and metabolism
5.4.2 Effects of the FXR/FGF-19 pathway on NAFLD and glucose and lipid metabolism
5.4.3 Bile acid receptors as therapeutic targets in NAFLD
References
6 The role of gut microbiota in chronic liver diseases
6.1 Introduction
6.2 Metabolic liver disease
6.3 Primary sclerosing cholangitis
References
7 Gut-liver The role of serotonin and its pathways in hepatic fibrogenesis
7.1 The state of the art
7.2 Serotonin synthesis and metabolism
7.3 Serotonin receptors
7.3.1 5-HT1A
7.3.2 5-HT1B
7.3.3 5-HT1D
7.3.4 5-HT2A
7.3.5 5-HT2B
7.3.6 5-HT2C
7.3.7 5-HT3
7.3.8 5-HT5
7.3.9 5-HT7
7.4 The natural course of chronic liver disease
7.5 Serotonin and liver fibrogenesis
7.6 Serotonin and hepatocellular carcinoma
7.7 Gut microbiota
7.8 Serotonin and gut microbiota
7.9 Conclusions
Acknowledgment
References
III. Liver-brain axis
8 Gut–liver–brain axis in chronic liver disease with a focus on hepatic encephalopathy
8.1 Introduction
8.2 Gut–brain axis in health and liver disease
8.2.1 Changes of gut–liver–brain axis in progression of liver disease
8.3 Pathogenesis of hepatic encephalopathy
8.3.1 Dysbiosis
8.3.2 Neurotoxins
8.3.3 Impairment of neurotransmission
8.3.4 Systemic response to infections and neuroinflammation: proinflammatory mechanisms
8.3.5 Shunting
8.3.6 Sarcopenia
8.4 Clinical relevance and presentation of hepatic encephalopathy
8.4.1 Classification
8.4.2 Diagnostic tests
8.4.3 Relevance of hepatic encephalopathy
8.4.3.1 Hepatic encephalopathy and fitness to drive
8.4.3.2 Hepatic encephalopathy and quality of life
8.4.3.3 Hepatic encephalopathy and sleep disorders
8.4.3.4 Hepatic encephalopathy in clinical scoring systems
8.5 Treatment options
8.5.1 General management
8.5.2 Diet
8.5.3 Treatment of precipitating factors of hepatic encephalopathy
8.5.4 Shunting
8.5.5 Differentiated step-by-step pharmacotherapy
8.5.5.1 Lactulose (nonabsorbable disaccharides)
8.5.5.2 Rifaximin
8.5.5.3 Branched-chain amino acids
8.5.5.4 L-ornithine L-aspartate
8.5.6 Intensive care aspects of hepatic encephalopathy
8.5.7 Treatment of posttransjugular intrahepatic portosystemic shunts hepatic encephalopathy
8.5.8 Prevention of overt hepatic encephalopathy
8.6 Recent advances
8.6.1 Medications in cirrhosis and microbial changes
8.6.1.1 Probiotics
8.6.1.2 Fecal or intestinal microbiota transplantation
8.7 Outlook
References
9 The gut microbiota in hepatic encephalopathy
9.1 Introduction
9.2 Defining hepatic encephalopathy
9.3 Ammonia as a driver of hepatic encephalopathy
9.4 Classifying HE by cause of hyperammonemia
9.4.1 Hyperammonaemia in the brain
9.5 The role of inflammation
9.6 The gut microbiome in health and disease
9.7 Gut dysbiosis in cirrhosis
9.8 Drug use in cirrhosis contributes to dysbiosis
9.9 How dysbiosis drives systemic inflammation and HE
9.10 Directly altering the gut microbiome improves outcomes in HE
9.11 Conclusion and future directions
References
10 The gut–liver–brain axis: dietary and therapeutic interventions
10.1 Introduction
10.2 Routes of communication between periphery and brain
10.3 Gut–brain axis dysregulation in liver disease
10.4 Changes in brain function and behavior in liver disease
10.5 Therapeutics targeting gut–liver–brain axis
10.5.1 Therapeutics targeting the gut microbiome
10.5.1.1 Diet
10.5.1.1.1 Polyunsaturated fatty acids
10.5.1.1.2 Polyphenols
10.5.1.1.3 Dietary patterns
10.5.1.1.4 Prebiotics
10.5.1.2 Probiotics
10.5.1.3 Fecal microbial transplantation
10.5.2 Therapeutics targeting antiadhesion molecules and cytokines
10.5.2.1 Antiadhesion therapies
10.5.2.2 Anticytokine therapies
10.5.3 Therapeutics targeting neural transmission
10.5.3.1 Antidepressants
10.6 Concluding remarks
References
Index
Back Cover

Citation preview

THE COMPLEX INTERPLAY BETWEEN GUT
BRAIN, GUT
LIVER, AND LIVER
BRAIN AXES

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THE COMPLEX INTERPLAY BETWEEN GUT
BRAIN, GUT
LIVER, AND LIVER
BRAIN AXES Edited by

CRISTINA STASI MASVE Interdepartmental Hepatology Center, Department of Experimental and Clinical Medicine, University of Florence and CRIA-MASVE Center for Research and Innovation, Careggi University Hospital, Florence, Italy; Epidemiology Unit, Regional Health Agency of Tuscany, Florence, Italy

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2021 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: 978-0-12-821927-0 For Information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Editorial Project Manager: Mona Zahir Production Project Manager: Sreejith Viswanathan Cover Designer: Mark Rogers Typeset by MPS Limited, Chennai, India

Dedication To my mom for her love and all the opportunities she gave me. To the memory of my father and my aunt Maria. To my brother Luigi and his wife Pina, and my sister Maria and her husband Pietro for their continuous support that has made possible the fruition of my efforts. To my nephews Vincenzo, Domenico, Vincenzo C., and Bruno and my nieces Caterina and Gemma for their nice encouragement. To my closest friends.

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Contents List of contributors ix Preface xi Acknowledgments xiii Introduction xv

3.3 Pathways of brain
gut
microbiota interaction 50 3.4 Tryptophan metabolism 53 3.5 Endocrinologic pathway 53 3.6 Microbial neural substrates 54 3.7 Neuropsychiatric disorders affected by brain
gut interplay 55 3.8 Psychiatric disorders in gastrointestinal diseases 58 3.9 Conclusion 59 References 59

Section I Gut-brain axis 1. The pathophysiology of gut
brain connection 3

4. The role of serotonin and its pathways in gastrointestinal disorders 67

Giulia Scalese and Carola Severi

1.1 Introduction 3 1.2 The anatomical entity 4 1.3 The functional entity and the role of microbiota 5 1.4 The pathological entity 10 1.5 Conclusion 14 References 14

Massimo Bellini, Matteo Fornai, Paolo Usai Satta, Francesco Bronzini, Gabrio Bassotti, Corrado Blandizzi and Rocchina Colucci

4.1 Role of serotonin in the physiology of digestive and extradigestive systems 67 4.2 5-HT receptors, serotonin transporter, and their polymorphisms 71 4.3 Serotonin in functional gastrointestinal disorders and the brain
gut axis 79 4.4 Serotonin, psychological/psychiatric and extra-gastrointestinal comorbidities 82 4.5 Serotonin and microbiota, the brain
gut axis and the psychobiota 83 4.6 Conclusion 85 References 86

2. The interactions between gut and brain in gastrointestinal disorders 17 Teodora Surdea Blaga, Dan L. Dumitrascu, Andrei V. Pop and Simona Grad

2.1 Introduction and anatomo-physiological background 17 2.2 Clinical presentation of the disorders of the gut
brain interaction 19 2.3 Therapy of main disorders of the gut
brain axis 28 References 39

Section II Gut-liver axis 5. The pathophysiology of gut
liver connection 97

3. The interactions between gut and brain in psychiatric and neurological disorders 49

Luca Maroni, Francesca Fianchi, Luca Miele and Gianluca Svegliati Baroni

Cheolmin Shin and Yong-Ku Kim

3.1 Introduction 49 3.2 Development of gut microbiota

5.1 Regulation of intestinal permeability 97 5.2 Role of altered intestinal permeability in the pathogenesis of NAFLD 101

50

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5.3 Role of incretins in NAFLD 106 5.4 Alteration of bile acid pathways in NAFLD 112 References 116

6. The role of gut microbiota in chronic liver diseases 123 Stefano Brillanti and Giada Alterini

8.6 Recent advances 8.7 Outlook 176 References 176

174

9. The gut microbiota in hepatic encephalopathy 187 Sandip Samanta and Debbie L. Shawcross

6.1 Introduction 123 6.2 Metabolic liver disease 123 6.3 Primary sclerosing cholangitis References 127

125

7. Gut-liver The role of serotonin and its pathways in hepatic fibrogenesis 129 Cristina Stasi, Stefano Milani and Andrea Galli

7.1 The state of the art 129 7.2 Serotonin synthesis and metabolism 130 7.3 Serotonin receptors 133 7.4 The natural course of chronic liver disease 136 7.5 Serotonin and liver fibrogenesis 140 7.6 Serotonin and hepatocellular carcinoma 141 7.7 Gut microbiota 143 7.8 Serotonin and gut microbiota 145 7.9 Conclusions 148 Acknowledgment 148 References 149

Section III Liver-brain axis 8. Gut
liver
brain axis in chronic liver disease with a focus on hepatic encephalopathy 159 Anna-Lena Laguna de la Vera, Christoph Welsch, Waltraud Pfeilschifter and Jonel Trebicka

8.1 Introduction 159 8.2 Gut
brain axis in health and liver disease 160 8.3 Pathogenesis of hepatic encephalopathy 162 8.4 Clinical relevance and presentation of hepatic encephalopathy 165 8.5 Treatment options 169

9.1 Introduction 187 9.2 Defining hepatic encephalopathy 189 9.3 Ammonia as a driver of hepatic encephalopathy 190 9.4 Classifying HE by cause of hyperammonemia 190 9.5 The role of inflammation 193 9.6 The gut microbiome in health and disease 195 9.7 Gut dysbiosis in cirrhosis 196 9.8 Drug use in cirrhosis contributes to dysbiosis 197 9.9 How dysbiosis drives systemic inflammation and HE 198 9.10 Directly altering the gut microbiome improves outcomes in HE 199 9.11 Conclusion and future directions 200 References 200

10. The gut
liver
brain axis: dietary and therapeutic interventions 205 Charlotte D’Mello and Mark G. Swain

10.1 Introduction 205 10.2 Routes of communication between periphery and brain 206 10.3 Gut–brain axis dysregulation in liver disease 210 10.4 Changes in brain function and behavior in liver disease 213 10.5 Therapeutics targeting gut–liver–brain axis 216 10.6 Concluding remarks 227 References 228

Index 237

List of contributors Giada Alterini Dipartimento delle Mediche, AOU Senese, Siena, Italy

Matteo Fornai Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy

Scienze

Gabrio Bassotti Gastroenterology and Hepatology Section, Department of Medicine, University of Perugia Medical School, Perugia, Italy

Andrea Galli Gastroenterology Unit, Department of Experimental and Clinical Biomedical Sciences, Florence, Italy Simona Grad 2nd Department of Internal Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

Massimo Bellini Gastrointestinal Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy

Yong-Ku Kim Department of Psychiatry, Korea University Ansan Hospital, Ansan, Kyunggido, Republic of Korea

Teodora Surdea Blaga 2nd Department of Internal Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

Anna-Lena Laguna de la Vera Department of Internal Medicine I, J.W. Goethe University Hospital, Frankfurt, Germany

Corrado Blandizzi Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy

Luca Maroni Clinic of Gastroenterology and Hepatology, Universita` Politecnica delle Marche, Ancona, Italy

Stefano Brillanti Dipartimento di Scienze Mediche, Chirurgiche e Neuroscienze (DSMCN), Universita’ di Siena, Siena, Italy

Luca Miele Dipartimento di Scienze Mediche e Chirurgiche, Fondazione Policlinico Gemelli IRCCS, Universita` Cattolica del Sacro Cuore, Roma, Italy

Francesco Bronzini Gastrointestinal Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy

Stefano Milani Gastroenterology Unit, Department of Experimental and Clinical Biomedical Sciences, Florence, Italy

Rocchina Colucci Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy

Waltraud Pfeilschifter Department of Neurology, J.W. Goethe University Hospital, Frankfurt, Germany

Dan L. Dumitrascu 2nd Department of Internal Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

Andrei V. Pop 2nd Department of Internal Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

Charlotte D’Mello Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada

Sandip Samanta Department of Inflammation Biology, Faculty of Life Sciences and Medicine, School of Immunology and Microbial Sciences, Institute of Liver Studies, King’s College London, London, United Kingdom

Francesca Fianchi Dipartimento di Scienze Mediche e Chirurgiche, Fondazione Policlinico Gemelli IRCCS, Universita` Cattolica del Sacro Cuore, Roma, Italy

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Giulia Scalese Department of Translational and Precision Medicine, Sapienza University of Rome, Rome, Italy Carola Severi Department of Translational and Precision Medicine, Sapienza University of Rome, Rome, Italy Debbie L. Shawcross Department of Inflammation Biology, Faculty of Life Sciences and Medicine, School of Immunology and Microbial Sciences, Institute of Liver Studies, King’s College London, London, United Kingdom Cheolmin Shin Department of Psychiatry, Korea University Ansan Hospital, Ansan, Kyunggido, Republic of Korea Cristina Stasi MASVE Interdepartmental Hepatology Center, Department of Experimental and Clinical Medicine, University of Florence and CRIA-MASVE Center for Research and Innovation, Careggi University Hospital, Florence, Italy;

Epidemiology Unit, Regional Health Agency of Tuscany, Florence, Italy Gianluca Svegliati Baroni Clinic of Gastroenterology and Hepatology, Universita` Politecnica delle Marche, Ancona, Italy Mark G. Swain Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada; Calgary Liver Unit, Division of Gastroenterology and Hepatology, University of Calgary, Calgary, AB, Canada Jonel Trebicka Department of Internal Medicine I, J.W. Goethe University Hospital, Frankfurt, Germany; European Foundation for Study of Chronic Liver Failure, Barcelona, Spain Paolo Usai Satta Department of Internal Medicine, Division of Gastroenterology, Brotzu Hospital, Cagliari, Italy Christoph Welsch Department of Internal Medicine I, J.W. Goethe University Hospital, Frankfurt, Germany

Preface reflect the progress of our knowledge concerning the pathophysiology of gastrointestinal and liver diseases, also taking into consideration the interactions between them and those with the brain. The brain
gut, gut
liver, and liver
brain interactions represent a relatively new field that combines research and clinical issues that outlined the concept of a rapidly growing discipline. This area has become too big to be covered all in depth by a single book. Some topics are discussed in more than one chapter of the book because they belong to more than one aspect considered. We have attempted to be accurate as much as possible, but I recognize that it may contain some omissions. This book is divided into three sections and resolves some important clinical issues concerning gut, liver, and brain interactions. This has been possible thanks to the direct involvement of international experts with a large scientific production and with great teaching experience. They have been able, on the basis of current scientific evidence, to synthesize the translational effects, starting from the pathophysiological mechanisms to the clinical practice. This fascinating revolution in gastroenterology has changed the management of gastrointestinal and liver disease. The huge effort together with a strong cooperation among the editorial team and authors has been considerable in this moment of public health emergency of international importance due to SARS-CoV-2, which emphasizes that passion can ignite enthusiasm even in moments of great difficulty such as the one we are currently facing.

When the publisher invited me to write a book regarding the gastroenterology field, including an overview of the scientific background, the involved mechanisms, and the translational effect of the research on clinical practice, I was very excited. I felt very happy to show the new research areas that look toward the future. This book represents to me an opportunity to find a way to reassemble the research interests I have been carrying out for over 20 years in the different fields of gastroenterology, but all are strongly connected to each other. The aim of this book is to reach an audience of researchers, with crossover for clinicians. I have always loved the translational research and this book seemed to be a great idea. At the beginning, when I started studying medicine at the Catholic University of Rome, the main part of international scientific articles could be found within the medical library. Gradually everything became much simpler, so that I could download many articles from my personal computer while staying home. Nowadays, even medicine is highly accelerated by the huge flow of information provided by a computerized society. However, I have always loved medical books, which I have gradually collected over the years, because they give in most cases, a multidisciplinary approach to the patient. The primary goal of this book is to collect in one volume the most current and wideranging understanding about gastrointestinal, liver, and brain interactions. In fact, the information explosion related to that field, caused the need of an urgent update, in order to

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Acknowledgments My special thanks go to Elsevier publisher Stacy Masucci for her invitation to assume the role of editor for a book project about innovative and focused topics spanning gastroenterology. I would like to thank the editorial project manager Mona Zahir for her continuous support and encouragement to achieve the goal of concluding our project, and especially for her valuable and pleasant interactions during the last year. I also thank Sreejith Viswanathan (production project manager) and the entire Elsevier team for their kind and useful support. I owe my gratitude to all authors who have prepared their own chapter during these difficult times of the coronavirus outbreak. This is also the time to thank all my professors and colleagues from Florence (particularly those of CRIA-MaSVE Center), Pisa, and Rome, for their long support of my clinical research projects, which have represented the scientific basis for this book. Many thanks

to all the staff of the Regional Agency of Tuscany, in particular, Caterina with whom I have been sharing the arduous work on the coronavirus epidemic since this unprecedented outbreak began. My thanks also go to my family, especially to my mum Caterina for her constancy and for being my source of unwavering and loving support; to my dad Vincenzo, and my aunt Maria, who from up there in heaven have always been a light even in the darkest days of existence. I thank my brother Luigi and his wife Pina, and my sister Maria and her husband Pietro, for always supporting me in my choices, even the most difficult and painful ones. I thank my nephews Vincenzo, Domenico, Vincenzo C., Bruno, and my nieces Caterina and Gemma for their gentle and sunny presence in my life. Finally, I thank all my relatives and my close friends, together with Don Vittorio, who with me have shared joys and hardships of my life.

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Introduction In the first section, it was outlined that the gut
brain axis encompasses two-way communication between the enteric nervous system and the central nervous system, with the parallel involvement of multiple interacting brain and gut networks although the homeostasis of the whole system is highly dependent on gut microbiota. Alterations at any level of this complex communication system play a key pathogenic role in several digestive diseases. The body’s response to stress is one of the main factors that can modulate motility and visceral perception through the brain
gut axis. The complexity of these pathophysiological mechanisms would be linked to the role of integrative brain structures. The role of psychological factors, emotional state, and modulation of the central nervous system in the pathophysiology of some gastrointestinal diseases has been confirmed. The most common disorders of the gut
brain interaction are the functional gastrointestinal ones, diagnosed and classified according to the outcomes of the working committees of the Rome Foundation. Moreover, a key role in the modulation of gut
brain communication may be played by serotonin. In this context, the role of the intestinal microbiota, with its ability of both producing 5-HT and being modulated by 5-HT, is considered a field of high interest. The second section has reviewed some of the pieces of evidence about the bidirectional connection between the physiological functions of the liver and the intestine. The bile acids are involved in the lipid digestion but secreted by the liver in the intestine, they

During my university studies, the scientific and medical world had already taken an ultra-specialized connotation, I have always thought that an organ affection couldn’t be considered as something separated from the rest of the body, but those organs and organ systems were closely interrelated and interdependent. In fact, without losing sight of the main objective of diagnosis and treatment, I have always been fascinated with the idea that there could be close connections between the diseased organ and the apparently healthy organs that could certainly be explained based on etiopathogenetic and physiopathological mechanisms. With this conviction, during my university and specialization studies, I tried to deepen the new theories of psycho-neuroimmuno-endocrinology, and I consequently became aware of some publications that were among the first on the gut
brain axis and later on the liver
brain axis, with the construction of pathophysiological models some of which were connected to the onset of the disease. Currently, I am mainly concerned with liver diseases and the attention of the scientific world is particularly drawn to the intestinal microbiota and its interactions with the liver. This book is divided into three sections: Gut
Brain axis, Gut
Liver axis, and Liver
Brain axis. It deals with the advances in our understanding of the pathophysiology of gastrointestinal and liver diseases, taking into consideration the interactions between them and even those with the brain.

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Introduction

also regulate the composition of the gut microbiota. In turn, the intestine regulates the synthesis of bile acids generating a feedback loop based on the bile acid-farnesoid X receptor
fibroblast growth factor 19 axis. Moreover, the secretion of incretins into the bloodstream modulates hepatic functions. Alteration of the gut’s permeability influences the quantity and quality of metabolites and bacterial products that, reaching the liver via portal blood, can modulate hepatic molecular pathways. The dysregulation of the gut
liver axis plays a crucial role in the pathogenesis of many liver diseases, such as nonalcoholic fatty liver disease and primary sclerosing cholangitis. On the other hand, some evidence suggests that there is a correlation between gut microbiota and liver diseases via neuroendocrine markers. Recent studies have highlighted the role of serotonin in the development of liver fibrogenesis. It has also been shown that 5HT can influence the proliferation and apoptosis of activated hepatic stellate cells. The intestinal microbiota plays a key role in the intestinal homeostasis of 5-HT. Local serotonin concentrations in the portal blood and then in the serotonin outflow can be regulated by the changes of the intestinal microbiota through the intestine
liver axis. The third section has discussed the importance of the gut microbiome in the pathogenesis of hepatic encephalopathy through an evolution from a healthy gut

microbiome to one characterized by a rise of pathogenic resident species. Hepatic encephalopathy or brain dysfunction due to liver insufficiency and/or portosystemic shunting is one of the determinants of prognosis and outcome of patients with liver cirrhosis. Hyperammonemia is still considered the crucial factor in the pathogenesis of hepatic encephalopathy leading to astrocyte swelling and glial oedema, which drive to psychological and psychomotor abnormalities through impaired glioneuronal communication. Hepatic encephalopathy remains a clinical diagnosis, and its assessment is performed by specific tests. From the therapeutic point of view, beneficial effects have been shown by using strategies to target the gut microbiome with antibiotic treatment or experimental therapies based on faecal microbiota transplantation. Moreover, this section deals with the changes occurring within the brain, which result in sickness behaviours and altered mood, highly prevalent in liver diseases of different etiologies, and with a special focus on diet. A significant impact on the gut
liver
brain axis has been reached by treatments able to target the gut microbiome or inflammatory modulators, including specific diets or biologics targeting cytokines or cell adhesion molecules. Future basic and clinical trials are needed to firmly establish the complex interplay between gut
brain, gut
liver, and liver
brain axes.

S E C T I O N

I

Gut-brain axis

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C H A P T E R

1 The pathophysiology of gut
brain connection Giulia Scalese and Carola Severi Department of Translational and Precision Medicine, Sapienza University of Rome, Rome, Italy

1.1 Introduction The gut
brain axis (GBA) is a bidirectional “double-track” system that contributes both to the proper coordination as well as the maintenance of gastrointestinal functions and to link emotional and cognitive centers of the brain with the gut. Reciprocal communications between the brain and gut are facilitated by microbiota whose key regulatory role is exponentially emerging to a point that the concept of microbiome-GBA is now widely accepted [1]. Alterations in GBA function appear to have a key role in several digestive and notdigestive diseases. In the gut, most evidence is available on the pathogenesis of functional gastrointestinal disorders, a group of disorders classified by gastrointestinal symptoms related to any combination of the following: motility disturbance, visceral hypersensitivity, altered mucosal and immune function, altered gut microbiota, and altered central nervous system (CNS) processing. The key role of GBA alterations in their pathogenesis has meant that, since 2016, these disorders are defined as disorders of gut
brain interactions [2]. Prototypes of GBA dysfunction are irritable bowel syndrome (IBS) [3] and functional abdominal pain [4]. However, evidence in GBA dysfunction has been hypothesized also to contribute to exacerbation of inflammatory bowel diseases (IBD) [5]. In an extradigestive setting, evidence is increasing on the role of GBA dysfunctions in neurological diseases (i.e., Alzheimer’s disease, autism, epilepsy) [6,7] and psychiatric disorders (i.e., depression) [8]. Increased rates of comorbidities exist between psychiatric and gastrointestinal diseases: mood disorders affect more than half of patients with IBS, and gastrointestinal symptoms are common among patients suffering from anxiety and depressive disorders. The unifying pathogenetic element associated with GBA dysfunction is the presence of perturbations in the gut affecting brain homeostasis that can be driven by direct intestinal damage or indirect damage through central alterations. The primary defendant is frequently

The Complex Interplay Between Gut
Brain, Gut
Liver, and Liver
Brain Axes DOI: https://doi.org/10.1016/B978-0-12-821927-0.00001-2

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an alteration in the composition and/or function of the microbiota, which leads to a dysbiosis triggered by nonspecific environmental factors, such as chronic infections and/or unhealthy diet. Dysbiosis causes an inflammatory-driven increased intestinal permeability (“leaky gut”) which induces a systemic subclinical inflammatory state. The weakening of integrity and function of the gastrointestinal barrier provokes an increased permeability of blood-brain barrier (BBB) that, in genetically susceptible individuals, can promote neuroinflammation, neuroinjury, and degeneration [6,9]. This “common ground” oversimplified hypothesis is largely based on preclinical studies on animal models or correlational observational clinical studies that have demonstrated, in patients affected by specific neurological disorders, alterations in microbiota composition versus healthy age-matched individuals. Even if this evidence seems promising, it is now necessary to move to causative studies to confirm the role of the gut in the pathogenesis of these disorders. Interactions between disease processes and the microbiome may be bidirectional and, in the meanwhile, the possibility that the disease drives changes in the microbiota and not the other way around must remain open to the possibility that any changes observed in the microbiota is secondary. Evidence on the multitude of molecular pathways potentially involved in GBA bidirectional flow of communication represents today an attractive target for the development of novel therapies considering that perturbations at any level of this complex communication system can propagate dysregulation throughout the circuit.

1.2 The anatomical entity GBA encompasses bidirectional communication between the enteric nervous system (ENS) and the CNS. Communication and interaction in the GBA occur through efferent and afferent neural pathways and neuroendocrine and metabolic signals from the gut to the brain. The neuroanatomical substrate for the GBA consists of gut intrinsic innervation, namely the ENS, and extrinsic innervation, the autonomic nervous system (ANS), that includes the vagus nerve (VN), the parasympathetic pelvic nerves, and the splanchnic nerves, containing both afferent and efferent fibers. Afferent fibers reach the higher brain centers in two main nuclei: the nucleus of the solitary tract, which receives vagal afferents, and the thoracolumbar and sacral spinal cord, which receives splanchnic and pelvic nerves, respectively. Even if both branches of the ANS regulate gut functions, to simplify this elaborated system it can be said that VN typically transmit information regarding mucosal mechanical changes and luminal gut ecosystem, whereas the sympathetic afferents transmit visceral sensitivity [10]. Enteroceptive signals allow the brain to monitor the physiological status of the gut, its luminal composition, and the eventual presence of inflammation. Efferent fibers from the cortex reach the subcortical contributors of the circuit, namely the limbic system with hypothalamus and amygdala, from which then they depart through the autonomic nervous efferent nerves and reach the gut [11]. The essential component of the axis is the hormonal hypothalamic-pituitary-adrenal (HPA) axis, the core efferent axis that provides the main regulation of various body processes in response to psychological and physical stressors and the coordination of the

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adaptive response [12,13]. Moreover, this axis supports emotional response and memory. Its activation by environmental or inflammatory stress is mediated by the secretion of corticotropin-releasing factor from the hypothalamus that stimulates adrenocorticotropic hormone secretion from pituitary gland that, in turn, leads to cortisol release from the adrenal glands. Finally, the gut microbiota is the third functional entity that participates actively to the homeostasis of the GBA. Its key role in facilitating the reciprocal communications between the gut and brain bolsters the concept of a “Microbiome-GBA.” Microbiota produces messages for the brain and vice versa replies to different signals originated in the brain, preserving a complex mechanism essential for the normal feedback between brain and gut. Preclinical studies on germ-free rodents (devoid of any microbes), pending confirmation to be translatable to human physiology, have demonstrated that gut microbiota is fundamental for the development and maturation of both ENS and CNS. Preclinical studies conducted on germ-free animals or by the use of antibiotics have highlighted the bidirectional interplay between microbiota and the nervous systems. Germ-free animals have both an altered expression of neurotransmitters in the nervous system and an impairment in gut sensory and motor functions, all anomalies that are restored after animal microbiota colonization [11]. Neural processes such as development, myelination, neurogenesis, and microglia activation have been shown to be crucially dependent on microbiota composition [7]. There is also evidence of analogies between microbiota modifications and dynamic periods in brain development [14]. Finally, microbiota influences the development of emotional behavior, stress-, and pain-modulation systems [1]. Given that the brain is dependent on gut microbes for essential metabolic products, it is not surprising that dysbiosis can have serious negative consequences for brain function, both from neurologic and mental health perspectives [15]. Several neurological disorders are now characterized by an imbalance in gut flora and cross-sectional clinical studies are bolstering the concept of altered microbial composition contributing to the pathophysiology of Alzheimer’s disease and autism spectrum disorders [16]. However, at the moment, it still remains to be clarified whether alterations observed in the microbiota of patients with these disorders arise from primary alterations at the gut microbial interface (bottomup effects) and/or from changes in brain-to-gut signaling (top-down effects) [17].

1.3 The functional entity and the role of microbiota The parallel involvement of multiple interacting brain and gut networks has led to the concept of considering the system composed not as single entities, namely individual brain regions and cell types in the gut, but as “brain connectome” and “gut connectome” [18]. This integrative model better elucidates the functions of the entire districts due to dynamic interactions between the single components of the GBA. Indeed, from structural and functional magnetic resonance imaging (MRI) studies and biochemical positron emission tomography ligand imaging, new data are emerging on brain networks, distributed in different areas of the cortex and limbic system, whose interaction might contribute to the clinical disease presentation. In IBS, for example, structural and functional alterations have been reported for default mode, emotional arousal, central autonomic, sensorimotor,

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central executive, and salience networks that results are all activated by enteroceptive signals [1]. The gut connectome is composed by the ENS, the different types of tissues/cells (enterochromaffin cells, enteric neurons, epithelial cells, muscle cells, and interstitial cells of Cajal and immune cells) present in gut wall and the microbiota (Fig. 1.1). The main role of ENS, in association with microbiota, is to coordinate motility, visceral sensation, secretion, mucosal transport and blood flow [10]. Brain modulation of regional gut transit and secretions can affect the community structure and function of the gut microbiota. From the gut connectome to brain, communication is mediated by neural, endocrine, inflammatory, and microbial metabolites pathways while, from brain to gut, communication mainly relies on ANS-driven neurotransmission and on the endocrine HPA axis. Both hormonal and neural communication lines converge both to send enteroceptive signals from gut to brain and to enable the human brain to directly influence the activities of various intestinal functional effector cells. IBS might represent a model of dual alterations in neuroendocrineimmune pathways in that symptoms may be caused by alterations either primarily in the CNS (top-down model), or in the gut (bottom-up model), or a combination of both [19]. The homeostasis of the whole system is highly dependent on gut microbiota metabolites, consisting in fermentation end-products and bioactive metabolites that preserved

FIGURE 1.1 The gut connectome and the role of dysbiosis. From the lumen to the deepest layer gut connectome is composed by gut microbiota, the mucus layer, the columnar epithelium composed by different specialized cell types (intestinal epithelial cells, goblet cells and enteroendocrine cells), gut-associated lymphoid tissue, the enteric nervous system, and the two muscle layers. Gut microbiota alterations (dysbiosis) drive an increase in the epithelial permeability, a chronic activation of the mucosal immune system, activation of nociceptive sensory pathways, and dysregulation of motility.

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brain and gut homeostasis by maintaining the intestinal barrier protection and the tight junction integrity, the mucosal immune regulation, and the modulation of enteric sensory afferents. Of note is that mucus thickness, the first mechanism of antimicrobial protection, is inversely proportional to bacteria concentration in the gut lumen. Mucus is organized in two overlapping layers, while the inner layer is denser and does not contain any organism, the outer layer contains microbes and provides glycans as a source of nutrition for the microorganisms [20]. Whereas in the colon the primary defensive role is exerted by the mucus layer, in the small intestine, where the mucus layer is discontinuous and inadequate, antimicrobial proteins play the larger defensive role. Thus mucus thickness changes along the gut, and dysbiosis, through a modification in bacteria strains and concentration, may damage the mucus layer that represents the first defensive structure of the human gut [21]. Gut microbiota is currently viewed as a bioreactor that, through the anaerobic fermentation of dietary carbohydrates and proteins components and the biotransformation of endogenous compounds, produces an extraordinarily diverse molecular repertoire of bioactive metabolites otherwise unavailable to the host [22,23], essential to maintain homeostasis of digestive, endocrine, metabolic, and immune and neural functions [24,25] (Fig. 1.2). Of note, microbiota appropriate biodiversity and redundancy represents a prerequisite for the maintenance of these metabolic functions essential for GBA health [24].

FIGURE 1.2 Microbiota bioconversion of dietary products into active metabolites. Intestinal microbes produce a wide range of bioactive small molecules otherwise unavailable to the host. In particular, the catabolism of indigestible polysaccharides leads to the production of short-chain fatty acids whereas the catabolism of aminoacids produces neurotransmitters, such as serotonin, gamma aminobutyric acid and catecholamines. Microbiota metabolized also polyphenols, phytochemical compounds with antioxidant, antiinflammatory, and neuroprotective properties, making them potentially more biologically active.

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Two main groups of metabolites directly affect the GBA, namely short-chain fatty acids (SCFAs) and tryptophan-derived metabolites, in particular serotonin. SCFAs comprise mostly of acetate, propionate, and butyrate, produced by the microbiota in the large intestine, that represent the end-product of anaerobic fermentation of indigestible polysaccharides, such as dietary fiber and resistant starch [26]. Another less important source of SCFAs production is the amino acid metabolism. Approximately 500
600 mmol of SCFAs are produced in the gut per day, depending on the fiber content in the diet, microbiota composition, and gut transit time. SCFAs concentration varies along the gut, showing a higher value in the proximal colon (70
140 mM), which progressively declines toward the distal colon (20
40 mM) because of the gradual absorption of SCFAs by colonocytes. Colonocytes absorb SCFAs and among them butyrate, being the preferred energy source for colonocytes, is mostly consumed locally. SCFAs are then release in the portal bloodstream and reach the liver, where they are used as an energy substrate for hepatocytes, except for acetate, that is not oxidized in the liver. The concentrations of butyrate and propionate dramatically decrease in the systemic venous system, while that of acetate abounds [27]. Acetate is considered the most “systemic” of the SCFAs. However, despite their low peripheral concentration, propionate and butyrate retain the potential to control distant organs by activating hormonal and nervous systems. In the gut connectome, SCFAs act as energy substances to protect intestinal barrier [28] and are potent immune regulators exerting their action locally and systematically on both innate and adaptive components of immune response [29]. SCFAs influence the fate of immune cells through direct epigenetic modification of their metabolism [30] and also modulate cytokines production by increasing the production of antiinflammatory cytokines (IL-18 by intestinal epithelial cells, IL-10 by dendritic cells) and suppressing that of proinflammatory cytokines (TNFα, IL-6, IFNγ) [31]. Note that germ-free animals present an underdeveloped intestinal immune system [32]. Furthermore, SCFAs fortify the innate immunity of the intestinal mucosa, reinforce the intestinal epithelial cell barrier, increase mucus production by goblet cells, and strengthen the tight junctions. By functioning as a source of oxidative energy for epithelial cells, butyrate is also fundamental to maintain anaerobiosis in the lumen and thus to contain the aerobic expansion of potential pathogens in the gut [22]. Evidence is emerging that SCFAs exert their beneficial role also by acting over more components of the nervous system such as the BBB, microglia, astrocytes, and neurons [26]. Brain endothelial cells abundantly expressed SCFAs membrane transporters that might facilitate their crossing of the BBB, favoring the maintenance of barrier integrity by upregulating the expression of tight junction proteins [33]. SCFAs seem also to keep a pivotal role in the development of the nervous system, particularly in the maturation and refinement of circuits and connections through the reduction of neuroinflammation driven by a decrease in proinflammatory cytokines (IL-1β, IL-6, and TNF-α) [34]. Lastly, SCFAs influence neural functions by the modulation of neurotransmitters synthesis. In animal models, SCFAs regulate the expression levels of tyrosine hydroxylase involved in the biosynthesis of dopamine, noradrenaline, and adrenaline [35], induce the synthesis of brain-derived neurotrophic factor that promotes neurogenesis, neural proliferation, and long-term memory consolidation [36]. Further, acetate alters the levels of glutamate, glutamine, and gamma aminobutyric acid (GABA) in the hypothalamus and increases the anorexigenic neuropeptide expression [37].

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Through the action of tryptophan hydroxylases and decarboxylases or tryptophanases, the microbiota produces also a wide range of tryptophan metabolites, the most important being serotonin (5-hydroxytryptamine, 5-HT) [38]. Central serotonin only accounts for a small proportion of the body’s total serotonin, while over 90% of serotonin is mainly produced from enterochromaffin cells [39] Serotonin functions as a key neurotransmitter in the modulation of central neurotransmission and enteric physiological function, in the regulation of visceral pain, secretion, and peristaltic reflex [40]. Besides, tryptophan is also metabolized in indole, indole acetic acid, indole propionic acid, indole-3-acetaldehyde and tryptamine, which all together can be defined aryl hydrocarbon receptor (AhR) agonists. These products, as a whole, engage the gene expression for the modulation of important immune homeostatic factors. Their receptor is a transcription factor, expressed by intestinal immune and epithelial cells, that induces an increased production of IL-22, which regulates the epithelial cell repair, the transcription of antimicrobial peptides, the activation of adaptive and innate lymphoid cells, and the reinforcement of mucosal tight junctions [17,22]. Further, indole metabolites have been detected in human cerebrospinal fluid and hippocampus, suggesting its possible role in microglial control and behavioral responses [17]. Gut microbiota produces also numerous other small molecules that can affect GBA homeostasis, both by preserving locally gut connectome functions and by affecting other districts by hormonal and neural signaling. Among the former, polyamines represent important factors involved in the maintenance of gut connectome homeostasis. Spermine and spermidine, the principal polyamines, are synthetized by gut bacteria through the action of constitutive or inducible forms of amino acid decarboxylases. These molecules are mostly linked to cell growth, survival, and proliferation and result to be essential to maintain enterocytes turnover and to enhance the integrity of the intestinal barrier [41]. Particularly, in animal models, spermine can inhibit macrophage polarization, decreasing the production of TNF-α and IL-6, and stimulate mucin and IgA production ending in an overall antiinflammatory activity [22]. The microbiota has also an important role in production, expression, and turnover of neurotransmitters (Fig. 1.2). Through the enzymatic activities of glutamic acid decarboxylase, gut microbiota has the capacity to convert the amino acid glutamate into GABA, the main inhibitory neurotransmitter of the nervous system and through phenylethanolamine N-methyltransferase, some microbiota species, such as Escherichia and Bacillus, are able to convert biologically inactive noradrenaline and dopamine produced by enteric nerves into active epinephrine [17]. Some microbiota strains are able to synthetize nitric oxide and hydrogen sulfide [42,43], two important neurotransmitters for gut motility modulation. Finally, gut microbiota operates as an endocrine organ producing and metabolizing numerous chemicals that act as hormones. Indeed, microbial communities carries clusters of genes encoding enzymes that metabolize sex hormones through hydrolytic, reductive, and oxidative reactions and converts glucocorticoids into androgens. Enzymes encoded by bacterial microbiome, such as hydroxysteroid dehydrogenases, regulate the balance between active and inactive steroids influencing host hormones levels, included sex hormones [24]. This peculiar function of microbiota on sex hormones might have a clinical role on the effect of gender in GBA physiology. In clinical practice in the management of functional gastrointestinal disorders (FGIDs), a biopsychosocial approach is recommended

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in order to consider separately sex and gender influences, sex referring to the biological make-up of the individual’s reproductive anatomy whereas gender to an individual’s lifestyle or personal identity [44]. Sex hormones may influence peripheral and central regulatory mechanisms of the GBA affecting visceral sensitivity, motility, intestinal barrier function, and immune activation of intestinal mucosa as well as pain modulation, stress responsiveness, and neuronal excitability in the brain, and contribute to the higher prevalence of FGIDs in women [45]. Of note is that androgens, mainly testosterone, exert a protective role over inflammation and pain modulation, inhibiting the development of visceral hyperalgesia [46].

1.4 The pathological entity Perturbations in the GBA function can arise both in the gut and brain connectomes. Enteric acute infections and enteric inflammation (interoceptive stress factors) may persistently induce a low-grade mucosal inflammation that might affects the neuronal enteric network activating ascending pathways (bottom-up model) [19]. The brain might perturbate the normal luminal/mucosal habitat by altering intestinal motility and secretions and regional and global changes in gastrointestinal transit can have profound effects on the delivery of important nutrients, mainly prebiotics and dietary fibers, to the enteric microbiota [11]. Further, different types of chronic psychological stressors (exteroceptive stress factors), acting on the cortex, can cause abnormal activation of brain areas (particularly the amygdala, hippocampus, locus coeruleus) and a decreased HPA axis response, resulting in reduced cortisol release ending in increased immune response toward luminal antigens and consequent low-grade inflammation (top-down model) [19]. These latter factors are known to modulate the composition and total biomass of the enteric microbiota, probably due to the presence of neurotransmitter receptors on bacteria [47]. Studies on the risk factors to develop postinfective IBS have shown the importance of both local and microbiological factors, as well as of psychological factors, including adverse life events, anxiety, and depression [48]. Among the various mechanisms involved in the pathophysiology of GBA connections, the larger amount of recent available data concerns dysbiosis. Certain circumstances can rupture the mutualistic pact between the host and microbiota, pushing the gut microbiome toward a dysbiotic layout, where microbiome-derived molecules may contribute to a disease state. The common traits of dysbiosis are an imbalance in the microbiota community with reduced diversity, loss of beneficial species, and pathobiont expansion [9]. This imbalance causes a reduction of SCFAs producing bacteria (the butyrate producers such as Faecalibacterium, Roseburia, Lachnospiraceae, and Eubacterium), an increased mucus degradation by abnormal mucin degraders species that displace Akkermansia, a reduced hydrogen and methane production combined with an increased production of hydrogen sulfide that is toxic for the epithelium, and an increase in abundance of bacteria with lipopolysaccharide endotoxins (Proteobacteria) that can drive inflammation-oxidative stress that can allow microbes to proliferate in close vicinity to the epithelium. Dysbiosis can be driven by several factors that have been identified as possible modulators of gut microbiota (Fig. 1.3). Among these factors, age, environment, diet, lifestyle, sex,

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FIGURE 1.3 Modulating factors of gut microbiota. Different factors have been identified to influence the composition of gut microbiota. These variables play a crucial role in the maintenance of the microbial homeostasis (eubiosis) or in the induction of imbalance (dysbiosis) that can drive the onset of gut
brain axis disorders.

and drugs play a crucial role on gut microbiota composition. Each of these variables may have a positive or a negative effect, contributing to maintaining the homeostasis (eubiosis) or promoting the imbalance (dysbiosis) of the system and the possible onset of GBA disorders. Considering the possible influence of GBA dysfunctions in neurological disorders mainly occurring in elderly people, age is the first influencing factor that needs to be considered. In elderly people, gut microbiota becomes less variable and instable. Aging is the period of lifespan characterized by a gradual deterioration of homeostasis, organ dysfunctions, and a likewise increase in pathological conditions. It is not a case that aging is also associated with changes in gut physiology, including hypochlorhydria, gastric motility disorders, and degenerative changes in the ENS, yielding dramatic effects on the composition and function of the gut microbiome [49]. Senescent gut microbiota has also been proposed to contribute to “inflammaging” [50], a term that refers to the simultaneous chronic proinflammatory state and the decline in adaptive immunity progressively expressed in older age [51]. It has been proposed that this state may be the possible cause of progression and worsening of many age-related diseases, such as neurological and metabolic disorders, like Alzheimer’s disease, type II diabetes, heart diseases, osteoporosis, and many others [17]. The link among aging, gut microbiota, inflammation, and even neuronal diseases should depend on the weakening of integrity and function of the intestinal barrier, which in turn provokes an increased permeability of BBB that potentially accelerates inflammaging. Furthermore, factors such as diet, sleep patterns, and exposure to antimicrobials have proven to alter brain function through changes in the gut microbiome. Any pathological process that either increases or decreases food intake can affect the microbiome and compromises brain function. Diet is perhaps one of the greatest factors influencing microbiota composition that can acquire a further importance in neurological disorders affecting

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appetite, swallowing, and nutrition, considering, for example, people in nursing homes eating processed and bland food [6]. Another important factor to consider in elderly people is the relevant role of polypharmacy, namely the concurrent use of multiple medications. A clinical study indeed has demonstrated the negative correlation between the number of different drugs consumed and microbial diversity [52]. During life, local environmental factors can also strongly influence microbiota composition, since several chemicals have a detrimental effect on bacteria survival. Different microbes of the gut microbiota metabolize more than 40 different environmental chemicals utilizing the enzymes azoreductase, nitroreductase, glucuronidases, and sulfatases [53]. Other factors that must be taken into consideration in the context of microbiota-driven GBA dysfunction are the effects of stress. Acute stress activates the HPA axis, resulting in an immediate release of cortisol. This instantaneous reaction allows the individual to defend against or escape from threat. When the stressful event is over, the system tends to return to the homeostasis, but in the case of a chronic activation of the stress response, a dysregulation of the HPA axis results influencing microbiota composition and facilitating pathogens colonization [7]. Finally, reduced physical exercise has been demonstrated to impact brain health, supporting the idea that exercise may improve nervous structures and their functions. In this context, recent human clinical studies have attempted to elucidate if a positive connection exists for exercise and gut microbiota even if at the moment results are still scanty. However, it has been proven that regular physical activity increases microbiota diversity and enhances the level of some members of Firmicutes phylum [54]. Other influence in the pathophysiology of GBA disorders might derive from intrinsic alterations in the gut connectome that can influence its homeostasis. This aspect is emerging from clinical observations of a possible involvement of GBA in the context of IBD [5]. Evidence from preclinical models indicates that intestinal inflammatory mediators alter brain function and modify behavior and that preexisting behavioral disturbance increases susceptibility to inflammatory stimuli or exacerbates quiescent inflammation. The possible presence of disturbed bidirectional brain-gut interactions in IBD might then open new holistic therapeutic scenario in their management. In humans, the influence of gut inflammation on GBA is supported by different indirect evidences. Brain MRI studies of patients with Crohn’s disease and no psychiatric comorbidity has demonstrated alterations in both global network organization and regional connectivity that involved emotion and visceral pain networks [55,56]. Clinical studies also suggest that effective antiinflammatory therapies improve psychological distress and quality of life in IBD patients [57]. Interestingly, subjective improvements were associated with posttreatment changes in the limbic system assessed by MRI [58]. By the other side, clinical evidence shows that emotional, stress, and mood disorders might influence the clinical course of IBD, a history of depression presenting an increased risk of developing Crohn’s disease [59]. However, clinical data on the effects of interventions on psychiatric comorbidities, namely depression and anxiety, in IBD patients are controversial [60]. Preclinical studies, however, have demonstrated protective effects of vagus efferent nerve fibers in a model of intestinal inflammation providing a potential new therapeutic target in the management of IBD [61,62]. Again, the question remains as to whether behavioral changes in IBD patients are due to the physical impact of a chronic active

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disease or whether behavioral disposition plays a role in the development of these diseases. However, the actual evidence provides additional grounds for examining braingut interactions in the natural history of IBD. Of note is that patients with IBD during clinical remission phases experience IBS-like symptoms, suggesting that IBS and IBD may present common alterations of the GBA, particularly in ANS mechanisms [63]. Another aspect in GBA alterations that mainly contributes to the clinical scenario of abdominal pain is visceral hypersensitivity, namely an increase sensation or response to visceral stimuli. Visceral pain negatively impacts the overall patient quality of life with a tremendous pressure on the health care system. Visceral hypersensitivity occurs via a disturbance of the sensitization pathways that might be located at different levels [64]. The anatomical substrate is represented by intestinal chemical, mechanical, and thermal nociceptors on the primary afferent nerve terminations, whose cell bodies are located in the dorsal root ganglia for spinal afferents that sense peripheral painful stimuli and project them onto the spinal nociceptor neurons (secondary afferent neurons) located in the dorsal horn of the spinal cord. The information is then conveyed to supraspinal centers. Vagal primary afferent neurons have in turn cell bodies in vagal ganglia. Anyway, vagal afferents are thought to encode nonnoxious sensations arising from the GI tract, such as satiety and nausea while spinal afferents are thought to be responsible for visceral pain transduction. In the brain connectome, different cerebral areas involved in the somatosensory sensation of pain elaborate the afferent information (i.e., thalamus, hypothalamus, limbic system, and cortex) and an efferent signal is sent back to the periphery. The descending pathways (opioidergic and adrenergic nerves) modulate neuronal activity, exerting either an inhibition or a facilitation of the pain sensation. Visceral hypersensitivity can occur due to an altered peripheral neural activation of the ascending visceral pain pathways through peripheral of sensory ending in the gut wall or to an hyperexcitability of spinal ascending neurons (central sensitization) with increased flow of nociceptive information traveling through the sensory afferents at the level of the dorsal root ganglia. Peripheral sensitization of nerve ending occurs in the presence of repeated nociceptors’ activation due to chronic release of inflammatory mediators and pain signals following tissue injury [65]. However, hypersensitivity can also occur due to a dysregulation of descending pathways through a central amplification of afferent signals (anticipation and hypervigilance) or a reduced antinociceptive effect of descending inhibitory pathways acting in the spinal cord [66]. Modulation of visceral pain represents another suitable example of the GBA bidirectional networks, pain perception being the result of the interaction between peripheral receptors that activate afferent fibers and central regulatory processes. Pain is a characteristic symptom of FGIDs that in IBS is principally ascribed to enhanced peripheral stimulation from abdominal viscera, while in Chronic Abdominal Pain Syndrome (CAPS), it is caused primarily by central sensitization, with disinhibition of pain signals [67]. Trigger factors in IBS are represented by inflammation, infection, diet, abdominal surgery, and menses, while that in CAPS by anxiety disorders, depression, somatization, poor coping skills, maladaptive cognitions, and abuse. This different pathophysiology implies different therapeutic approaches, namely the management of CAPS relying on a strong patient
physician relationship and early incorporation of nonpharmacological therapies [4].

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1.5 Conclusion Increased evidence, mainly derived from animal preclinical studies and human association studies, is highlighting a dysfunction in GBA in an ever-growing list of disorders related to cognitive function, mental health, endocrine, and metabolic dysfunctions. The correct comprehension of GBA physiology and of the mechanisms involved in its homeostasis is actually opening new therapeutic perspectives toward a personalized medicine, at least for FGIDs, as recently suggested [68]. However, to reach this target it is necessary to move from the actual simple association to causality studies.

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C H A P T E R

2 The interactions between gut and brain in gastrointestinal disorders Teodora Surdea Blaga, Dan L. Dumitrascu, Andrei V. Pop and Simona Grad 2nd Department of Internal Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj-Napoca, Romania

2.1 Introduction and anatomo-physiological background The gastrointestinal (GI) tract is connected to the central nervous system (CNS) by nervous and endocrine connections. One can consider that both systems create a couple. The term gut
brain axis defines a bidirectional association between the gut and the brain, playing an important role in the understanding of the GI disorders [1]. It has been described as an interconnection between the gut functions and the emotional part of the brain [2]. Two neurological pathways are involved in the exchange of information between the brain and the gut: a direct one consisting of vagus nerve (VN) and autonomic nervous system (ANS); and a bidirectional one which represents a bicommunication between enteric nervous system (ENS) in the gut; ANS and VN, in the spinal cord [3]. The importance of bidirectional brain
gut interactions (“gut
brain axis”) in GI illness is increasingly recognized, most prominently in the area of functional GI syndromes such as irritable bowel syndrome (IBS), functional dyspepsia (FD), and functional chest pain (FCP). Bidirectional brain
gut interactions play an important role in the regulation of many vital functions in health and disease. In health, brain
gut interactions are crucial in the regulation of digestive processes (including appetite and food intake), in the modulation of the gut associated immune system, and in the coordination of the overall physical and emotional state of the organism (sleep, stress, anxiety) with activity in the GI tract. In disease, peripheral and central alterations in brain
gut interactions are likely to underlie symptoms of chronic abdominal pain and associated GI dysfunction.

The Complex Interplay Between Gut
Brain, Gut
Liver, and Liver
Brain Axes DOI: https://doi.org/10.1016/B978-0-12-821927-0.00008-5

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© 2021 Elsevier Inc. All rights reserved.

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2. The interactions between gut and brain in gastrointestinal disorders

Fig. 2.1 represents the pathogenesis of Disorder of the Gut
Brain Interaction (DGBI). The brain and gut communicate continuously through a number of complex pathways involving the ENS, the ANS, the hypothalamus
pituitary axis (HPA), and the CNS. Each pathway is highly integrated and regulated by interrelations of neuronal and neurohumoral factors. The ENS is modulated by the extrinsic innervation coming from the ANS and the CNS. The ANS consists of sympathetic (splanchnic) nerves and parasympathetic (vagal-sacral) nerves. The somatic nervous system controls the striated muscles of the proximal esophagus and the external anal sphincter [4]. Gut functions are being controlled by neural anatomical pathways that develop a hierarchic integrative system. ENS (gut glial cells, submucous ganglion, myenteric ganglia) constituting the first level and prevertebral ganglia constituting the second level [3]. The afferent and efferent nerve fiber of the VN have the origin in the dorsal motor nucleus of VN and in the brainstem nucleus tractus solitarius and along with ANS (the sympathetic nervous system (T2-L5) and parasympathetic nervous system (S2-S4) in the spinal cord) form the next level. Higher brain centers represent the fourth level [5]. In the CNS, the highly integrated gut
brain communication axis is mainly controlled by the limbic system, which receives input from the ENS through the ANS and is modulated by higher cortical areas [4]. The CNS modulates the perception of the stimuli from the gut and is influenced by every person’s emotional state. Gut functions are modulated by the limbic system and prefrontal cortex, hypothalamus, and amygdala that all form the emotional motor system. These are playing an important role in the emotional changes from the ANS to the gut [5]. The limbic system consists of the amygdala, hypothalamus, medial thalamus, and the anterior cingulate cortex (ACC). It is primarily concerned with the regulation of behavior, emotions, arousal, memory, and motivation [6]. The visceral region, also known as the “visceral brain,” lies specifically in the hypothalamus which is a central component of the limbic system [6]. The amygdala is responsible for emotional and stressful responses and drives other areas such as the prefrontal cortex for execution of complex functions. Functional brain imaging using magnetic resonance imaging has shown that damage to the amygdala results in an alteration in stress responses such as flat affect seen in schizophrenia patients.

FIGURE 2.1 Pathogenic mechanisms of the functional gastrointestinal disorders (disorders of the brain
gut interaction). CNS, central nervous system; ENS, enteric nervous system; HPA, hypothalamus
pituitary axis.

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2.2 Clinical presentation of the disorders of the gut
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The damage may also lead to inhibition of social interaction and emotional conditioning. The amygdala also consolidates memories with the help of emotional stimuli [1,7]. Another important function of the gut
brain axis is played by the hormone release. Corticotrophin-releasing hormone (CRH) has an important role in the emotional nervous system, being part of the effector neurons of the paraventricular nucleus of the amygdala, hypothalamus, and the locus coeruleus complex, stimulating activation of HPA and ANS. The physiological reaction to stress is modulated by the activation of HPA axis and the adrenal cortex secretion of corticosteroid hormones (cortisol). Alteration of these two mechanisms are associated with GI symptoms, alteration of the gut motor function [8]. Patients with trauma or psychiatric conditions can experience an increased response to painful stimuli transmitted by the CNS [9]. For the gut
brain axis to function properly it must have a good activity of the intestine as well as a normal behavior of the brain, these being translated by signals received from the brain, the intestine, or from both [10]. Any disturbance in one of these systems can cause a malfunction of both [2]. It is already known that microbiota plays an important role in the gut
brain axis, this synergy being also referred as microbiota
gut
brain axis. Besides the nervous system (through VN and ANS) in the interaction between gut microbiota and the brain, a key role is also played by the gut production of metabolites, hormones (Table 2.1) [11
16], and immune factors [3]. For example gamma-amino butyrate can be produced by Lactobacillus species and Bifidobacterium species; serotonin can be produced by Escherichia species, Streptococcus species, and Enterococcus species; dopamine can be produced by Bacillus species; acetylcholine can be produced by Lactobacillus; and norepinephrine can be produced by Bacillus, Proteobacteria, and Escherichia coli [11,17]. Gut microbiota can be influenced, both directly by CNS, producing catecholamines, being affected by physiological and physical stress, and indirectly by modification in the gut habitation, influenced by ANS. Microbial structure is affected by the changes in the gut physiology, changes which are modulated by ANS, altering both functions and content [18]. The dysfunction of the gut
brain communication leads to clinical complaints labeled as functional gastrointestinal disorders (FGIDs) [19].

2.2 Clinical presentation of the disorders of the gut
brain interaction FGIDs represent the most common diagnoses in gastroenterology. According to Rome IV criteria, the term “functional” has been replaced largely by “disorders of gut
brain interaction” (DGBIs) [20]. DGBIs are defined as a group of disorders classified by the presence of GI symptoms related to any combination of motility disturbance, visceral hypersensitivity, altered mucosal and immune function, altered gut microbiota, and altered CNS processing. Common DBGIs include IBS, FD, chest pain of esophageal origin, and functional heartburn. There is considerable overlap of the DGBIs with each other, with other visceral and somatic “functional” pain syndromes (including urological pelvic pain syndromes, vulvodynia, fibromyalgia, and chronic back pain, and with psychiatric disorders, in particular, anxiety and depression).

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TABLE 2.1 The effect of hormones on gut motility. Hormone

Main action

Vasoactive intestinal polypeptide (VIP)

Substance P

Inhibits gastrointestinal motility Stimulates fluid secretion Inhibits gastrointestinal motility Stimulates fluid secretion Stimulates motility

Neurokinin A

Stimulates motility

Neurokinin B

Stimulates motility

Motilin

Stimulates motility (Contracts gastrointestinal smooth muscles)

Calcitonin gene-related peptide (CGRP)

Modulates motility, blood flow and secretion

Galanin Orexin

Stimulates motility Stimulates luminal secretion Stimulates gut motility

Gastric inhibitory peptide (GIP)

Higher concentrations - inhibit gastrointestinal motility

Glucagon like-peptide 1 (GLP-1)

Higher concentrations - inhibit gastrointestinal motility Inhibits colonic transit Inhibits gastric emptying Inhibits food intake Triggers gallbladder emptying Accelerates small intestinal transit Leads to increased gastrointestinal motility

Peptide histidine isoleucine (PHI)

Cholecystokinin

Ghrelin Peptide YY (PYY)

Serotonin

Slows gastric emptying Slows small intestinal transit Inhibits colonic transit Accelerates gastric emptying Initiates peristaltic reflex Promotes colonic motility

The current diagnostic criteria for DGBIs, as well as illness severity, frequency, duration, and treatment efficacy all rely exclusively on subjective patient reports and not on objective biomarkers [20,21]. The FGID representing in fact disorders of the gut
brain axis are classified according to the work of the Rome IV expert as in Table 2.2 [22]. We will present the main FGID. Functional esophageal disorders (FEDs) consist of a disease category that presents with esophageal symptoms (heartburn, chest pain, dysphagia, globus) that are not explained by mechanical obstruction (stricture, tumor, eosinophilic esophagitis), major motor disorders (achalasia, esophagogastric junction outflow obstruction, absent contractility, distal esophageal spasm, jackhammer esophagus), or gastroesophageal reflux disease (GERD). Although mechanisms responsible are unclear, it is theorized that visceral hypersensitivity and hypervigilance play an important role in symptom generation, in the context of normal or borderline function.

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TABLE 2.2 Rome IV classification of the functional gastrointestinal disorders
disorders of gut
brain interaction. A. Esophageal disorders A1. Functional chest pain

A4. Globus

A2. Functional heartburn

A5. Functional dysphagia

A3. Reflux hypersensitivity B. Gastroduodenal disorders B1. Functional dyspepsia B1a. Postprandial distress syndrome B1b. Epigastric pain syndrome

B3. Nausea and vomiting disorders B3a. Chronic nausea vomiting syndrome B3b. Cyclic vomiting syndrome B3c. Cannabinoid hyperemesis syndrome

B2. Belching disorders B2a. Excessive supragastric belching B2b. Excessive gastric belching

B4. Rumination syndrome

C. Bowel disorders C1. Irritable bowel syndrome IBS with predominant constipation IBS with predominant diarrhea IBS with mixed bowel habits IBS unclassified

C2. Functional constipation C3. Functional diarrhea C4. Functional abdominal bloating/distension C5. Unspecified functional bowel disorder C6. Opioid-induced constipation

D. Centrally mediated disorders of gastrointestinal pain D1. Centrally mediated abdominal pain syndrome

D2. Narcotic bowel syndrome/opioid-induced gastrointestinal hyperalgesia

E. Gallbladder and sphincter of Oddi disorders E1. Biliary pain E1a. Functional gallbladder disorder E1b. Functional biliary Sphincter of Oddi disorder

E2. Functional pancreatic sphincter of Oddi disorder

F. Anorectal disorders F1. Fecal incontinence F2. Functional anorectal pain F2a. Levator ani syndrome F2b. Unspecified functional anorectal pain F2c. Proctalgia fugax

F3. Functional defecation disorders F3a. Inadequate defecatory propulsion F3b. Dyssynergic defecation

The mechanisms by which visceral hypersensitivity of the esophagus occurs are incompletely understood, but some studies have demonstrated the potential importance of both peripheral sensitization, central sensitization, and viscero-visceral hyperalgesia [23].

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The peripheral sensitization refers to pain hypersensitivity at the site of injury or inflammation and is also known as primary hyperalgesia. Repetitive of peripheral nociceptors induces changes at the spinal dorsal horn (central sensitization), which may lead to the amplification of afferent signals to the brain in response to both noxious and innocuous stimuli that can outlast the initial injury, leading to an allodynic state. Viscero-visceral hyperalgesia is an epiphenomenon characterized by the intensification of pain severity due to the interaction of algogenic visceral pain syndromes from organs that share at least part of the innervation level at the spinal cord [24]. Given that the esophagus contains afferents capable of transducing many different types of noxious stimuli, including mechanical, thermal, electrical, and chemical, many of these have been used as provocative techniques to delineate the presence or absence of esophageal hypersensitivity. There are five currently accepted FEDs (ROME IV classification): A1. A2. A3. A4. A5.

FCP Functional heartburn Reflux hypersensitivity Globus Functional dysphagia

Functional chest pain is defined as recurring, unexplained, retrosternal chest pain of presumed esophageal origin, not explained on the basis of reflux disease, other mucosal or motor processes, and representing pain different from heartburn. FCP is a subset within the broad umbrella of noncardiac chest pain (NCCP) [25]. The major physiologic mechanisms that underlie FCP focus on hypersensitivity from peripheral and/or central sensitization, altered central processing of visceral stimuli, and altered autonomic activity. Studies consistently have shown altered pain perception and heightened visceral sensitivity in FCP. Autonomic dysregulation could reflect another subcategory within FCP [26]. In FCP, it has been proposed that symptoms arise as a result of a variable combination of afferent nociceptive pathway hypersensitivity, aberrant cortical processing of nociceptive signaling, or indeed hypervigilance to pain [27]. Furthermore, the stress responsive physiological systems, including the ANS, have also been implicated as a pathophysiological feature in a number of functional disorders [28]. Various central aspects and psychological factors can influence esophageal pain perception. For instance, the personality trait of neuroticism, referring to the long-term stable trait of negative emotional state, has been shown to be associated with heightened emotional arousal and cognitive pain processing in response to the anticipation of esophageal balloon distension [29]. These data suggest that neuroticism may cause a maladaptive response to esophageal pain. Moreover, the induction of anxiety can facilitate central sensitization, and thus esophageal hyperalgesia, in response to distal esophageal acidification [30]. Up to 60% of those with FCP have psychological comorbidity, which itself may enhance esophageal perception possibly through hypervigilance [31,32]. FH is defined as retrosternal burning discomfort or pain refractory to optimal antisecretory therapy in the absence of GERD, histopathologic mucosal abnormalities, major motor disorders, or structural explanations [26].

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The mechanism of symptom generation in FH is based on altered esophageal perception as a major factor. Also, a role for abnormal central processing of esophageal signals could support symptom generation without a reflux event trigger [26]. After analyzing the symptoms reflux correlation, 40% of patients have a positive correlation, so they have reflux hypersensitivity, while at 60% this correlation is not observed, being finally classified as FH [33]. Reflux hypersensitivity identifies patients with esophageal symptoms (heartburn or chest pain) who lack evidence of reflux on endoscopy or abnormal acid burden on reflux monitoring, but show triggering of symptoms by physiologic reflux [26]. Pathophysiologic mechanisms underlying reflux hypersensitivity are similar to those underlying FCP and FH [26]. FD is characterized by one or more of the following symptoms: postprandial fullness, early satiation, epigastric pain, and epigastric burning that are unexplained after a routine clinical evaluation. It includes two subcategories: • postprandial distress syndrome (PDS) that is characterized by meal-induced dyspeptic symptoms, and • epigastric pain syndrome (EPS) that does not occur exclusively postprandially; the two subgroups can overlap [34]. FD is considered a multifactorial disorder in which different pathophysiological mechanisms play a part, and each one could contribute to all subtypes [35]. Communication between the CNS and the ENS has been recognized for over a century, but the fact that brain
gut communications are bidirectional has only been appreciated more recently [36]. Innervation of the GI tract regulates secretions, sphincter control, motility, blood flow, and enteroendocrine function, and the ENS also communicates with the intestinal barrier via neuroendocrine and mucosal immune cells.

2.2.1 Gastroduodenal motility Abnormal motility patterns in FD occur in the stomach, pylorus, and small intestine during digestive or interdigestive periods as well as during triggered reflex activity [37]. The pathological consequences of motility disturbances range from altered gastric emptying (GE) and intestinal reflex activity to increased chemosensitivity or mechanosensitivity. GE is delayed in about 25%
35% of unselected FD patients, while rapid GE is uncommon probably occurring in under 5% of cases [38].

2.2.2 Impaired gastric accommodation Patients with FD have impaired accommodation of the proximal stomach in response to gastric balloon distension in the fasted state and after meal ingestion. This impaired accommodation results in disproportional volume distribution, with a larger than normal antral volume but smaller fundus volume. A reduced gastric relaxatory response to

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ingestion of a meal has been seen in about one-third of FD patients. Impaired gastric accommodation is associated with early satiety [39,40].

2.2.3 Gastroduodenal sensitivity Even patients with normal accommodation report discomfort after gastric distension. Some patients are also hypersensitive to duodenal, jejunal, or rectal distension, which suggests a more-generalized sensitization in the central and autonomic (vagal, spinal, and enteric) nervous systems [41]. The finding that intraduodenal lipid infusion sensitizes the stomach to distension in patients with FD, but not in controls, suggests a cross-sensitization between mechanosensors and chemosensors and could explain why fatty meals can exaggerate symptoms related to gastric filling [40]. Lipids in the duodenum can provoke symptoms through different possible mechanisms: direct neuronal stimulation, higher lipid sensitivity of enteroendocrine cells or nerves, increased levels of systemic or local cholecystokinin (which is secreted by lipidactivated enteroendocrine cells, stimulates the release of digestive enzymes and bile, and induces satiety) and/or increased sensitivity to cholecystokinin receptors [42]. In the duodenal bulb, the number of chromogranin A-positive enteroendocrine cells was reduced in FD, mostly in patients with EPS [43]; this could contribute to the development of abnormal sensations in FD.

2.2.4 Gastroduodenal inflammation and mucosal permeability Some studies show that gastric disturbances in FD could be secondary to duodenal inflammation. Eosinophil and mast cell numbers are increased in the submucosal plexus of the duodenum in patients with FD, and this finding was accompanied by a clear impairment of nerve excitability in the duodenal submucosal plexus [44]. Duodenal eosinophilia has been reported in FD patients and is apparently related to early satiety [45]. Acute psychological stress can also increase duodenal mucosal permeability via mast cell activation mediated by CRH [46]. Psychosocial factors constitute integral components of a biopsychosocial model of FD as a disorder of the brain
gut axis. The association between dyspepsia and psychiatric disorders, especially anxiety, depression, and neuroticism is commonly recognized. A bidirectional relationship probably exists between gut and psyche [47]. IBS is a common pathological condition, being the prototype of FGID. IBS is a functional bowel disorder in which recurrent abdominal pain is associated with defecation or a change in bowel habits. Bowel habits disorders are typically present (i.e., constipation, diarrhea, or a mix of constipation and diarrhea), as well as symptoms of abdominal bloating/distention [48]: IBS has multifactorial pathogenesis [49
51]. These factors may be broadly classified into: 1. Peripheral factors: altered GI motility, GI inflammation and permeability alteration, altered luminal microenvironment including gut microbiota dysbiosis and small-

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intestinal bacterial overgrowth (SIBO), host-microbe interaction, bile acid recirculation defect, pathogenic infection, dietary factors, neurohumoral dysregulation including altered serotonergic transmission (see Chapter 3, where Bellini et al. provide an extensive review on neurobiological effects of serotonin outflow), and visceral hypersensitivity [52]. 2. Central factors: psychological stress [53], abnormal emotional arousal system response, cognitive dysfunction, and sleep dysfunction [54]. Genetic factors may underlie peripheral and central pathophysiological mechanisms [55].

2.2.5 Visceral hypersensitivity in irritable bowel syndrome Hypersensitivity to peripheral stimulation is one of the common features of functional disorders. Visceral hypersensitivity can be observed in 30%
40% of IBS patients [56] and also in FD [57], fibromyalgia [58], and chronic fatigue syndrome [59]. Studies using positron emission tomography and functional magnetic resonance imaging increased our understanding of how the brain encodes noxious colorectal distention [60]. Similarly to somatic noxious stimulation, visceral stimulation activates a set of brain areas involved in processing ascending nociceptive input and correlated with pain experience across studies, including the insula (posterior/middle/anterior), ACC, midcingulate cortex (anterior/posterior) thalamus, and primary somatosensory cortex. Other areas that are not known to encode nociceptive input are also activated, including prefrontal cortex and posterior partial cortex [61]. When comparing brain activity between healthy controls and IBS patients at similar levels of colorectal distention, the intensity-coding areas discussed earlier, particularly the insula, thalamus, somatosensory cortex, and cingulate cortex are hyperactivated in patients with IBS [62]. Larsson et al. divided IBS patients into two groups: normosensitive (similar sensitivity to healthy controls) and hypersensitive patients [63]. Visceral hypersensitivity is defined as an enhanced perception of mechanical triggers applied to the bowel, which seems as pain and discomfort. Allodynia refers to pain sensitization to normally nonpainful stimuli, and hyperalgesia refers to increased sensitivity to painful stimuli. Frequency of visceral hypersensitivity among patients with IBS has been reported to vary widely between 33% and 90%. Visceral hypersensitivity may be at the local level in the gut wall, or at the level of posterior column of the spinal cord, subcortical thalamic level, at the cerebral cortex, and due to reduced descending inhibition of pain sensation or due to inappropriate exaggerated activation of emotional arousal system by visceral sensory stimuli [64]. Visceral hypersensitivity may determine occurrence of symptoms and of FGID phenotype. For example, it has been found that patients with IBS-C often have visceral hypersensitivity as compared with the patients with functional constipation suggesting that the pain symptom might result from visceral hypersensitivity [65]. The mediators of visceral sensation include serotonin, cannabinoid, tachykinin, histamine 1, tyrosine kinase, protease activated receptors, voltage-gated sodium and calcium channels, acid-sensing ion channel, and transient receptor potential vanilloid-1. Some of these have been found to be the therapeutic targets for treatment of patients with IBS [64].

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Serotonin is an important chemical that promotes gut motility, secretion, and sensation. 95% of body serotonin is in the gut and only 5% is in the brain. Serotonin released from enteroendocrine cells acts as a mucosal signaling molecule and that released from enteric nerves in the myenteric and submucosal plexus as a neurotransmitter. Serotonin is synthesized from dietary tryptophan in the gut by tryptophan hydroxylase (TpH) 1 and in the brain by TpH-2. Serotonin synthesized in the gut cannot enter into brain due to blood-brain barrier. Serotonin in the brain also gets converted into melatonin, which is the normal sleepinducing chemical. Since indoleamine 2, 3 dioxygenase can shunt tryptophan in the brain to Kynurenine, it may lead to less serotonin production and its conversion to melatonin leading to sleep disorder [66,67]. Some microbes such as Clostridium sporogens, Lactobacilli and Ruminococcus gnavus express tryptophanase and hence help in serotonin biosynthesis [68]. As excess serotonergic function is associated with diarrhea and its reduction is associated with constipation, modulation of serotonin by its antagonists and agonists are useful in treatment of IBS-D and IBS-C. Brain activity during rectal distention can also be modified by psychological factors such as depression or anxiety. Hence, despite similar symptoms, brain activity in IBS patients can be different depending on a variety of factors, including visceral hypersensitivity or psychological factors. Thus, brain activity may be different in IBS patients not specifically because of gut pathology, but because of patients “physiological or psychological characteristics” [61]. One of the common cognitive and emotional features among patients with IBS is fear of the unpredictable worsening of symptoms such as diarrhea, bloating, or abdominal pain [69]. Patients with IBS have a tendency to overestimate the potential for physical harm, and imagine worsening trajectories of symptoms and dysfunction (i.e., catastrophizing) [69,70]. They become hypervigilant about their food, eating at restaurants, or anything that might exacerbate symptoms. This anticipatory concern may itself trigger their symptoms [69]. The brain of IBS patients may be hypervigilant even in relatively safe conditions [61]. The amygdala is one of the key descending pain control pathways and the fear of pain may disrupt the pathway. Cognitive and emotional factors, particularly fear toward the symptoms, may change brain processing of nociceptive signals from the body, as well as their endogenous modulation [71]. Stress has been reported to change GI motility, including findings of delayed GE, impaired gastric accommodation [72], prolonged small bowel motility, and increased colonic motility and secretion. Stress is also known to increase visceral perception, intestinal permeability, and emotional responses to abdominal events, and influence gut microbiota in healthy people and patients with IBS. The main neuronally activated stressresponse systems regulating peripheral function under these stress responses are the hypothalamic
autonomic nervous system and the HPA axes, as well as aspects of the immune system [73,74]. These systems mediate the bidirectional communication between the brain and peripheral organs including the gut [75,76]. CRH is the key hormone in the body’s response to stress. The amygdala regulates the release of CRH that stimulates the cleavage of the precursor peptide proopiomelanocortin and the production of beta-endorphin and adrenocorticotropic

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hormone from the pituitary gland [74] that in turn regulates cortisol secretion from the adrenal gland along the HPA axis. However, it is also a neurotransmitter that stimulates the neurons in medial prefrontal areas, in the hippocampus, and in the hypothalamic nuclei [77,78].

2.2.6 Altered gastrointestinal motility in irritable bowel syndrome Whereas patients with constipation-predominant IBS (IBS-C) have a slow gut, particularly colonic transit, diarrhea-predominant IBS (IBS-D) patients have faster motility. Multiple studies showed that Bristol stool form is determined by colonic motility; slower colon transit is associated with harder stools [79,80]. Subsets of patients with IBS-C as well as functional constipation have slow gut motility with or without associated fecal evacuation disorder. Moreover, drugs that increase gut motility are known to improve chronic constipation in general and slow-transit constipation in particular, and the drugs that reduce gut motility often improve chronic diarrhea in these patients [81].

2.2.7 Gut microbiota dysbiosis in irritable bowel syndrome Recently, studies have shown that one of the main inputs to the gut
brain axis comes from microbiota, leading to the coining of the term “microbiome
gut
brain axis” [82]. At the interface between the microbiota and the host lies a network of neurons known as the ENS, positioned to respond either directly or indirectly to the microbiota and its metabolites. In the context of gut
brain signaling, the ENS communicates with the CNS via intestine neurons to sympathetic ganglia with sensory information traveling via extrinsic primary afferent neurons that follow spinal and vagal afferent routes. These intrinsic and afferent neural pathways provide opportunities for factors derived from the gut lumen, and therefore potentially the microbiota, to influence not only gut function but also the CNS [83]. The HPA axis is one of the main neuroendocrine systems in the human body and one of the key routes of communication within the microbiota
gut
brain axis. It is best known as the principal neuroendocrine coordinator of the response to stress [84]. There are many factors that have been shown to have a modulating effect on both the brain and microbiota, including socioeconomic status, host diet, congenital factors, environmental factors, exercise and level of host activity, medications, and mode of delivery at birth [11]. The microbiota
gut
brain axis is affected by many factors, including social and cognitive behavior, fear, stress, and food intake. Most studies found an association between SIBO and IBS. In a metaanalysis of 23 case-control studies on 1340 participants, patients with IBS had lower levels of good bacteria (Lactobacillus and Bifidobacterium) and marginally higher levels of bad bugs (Enterobacter) compared to the controls [85]. The other sets of evidences on relationship between dysbiosis and IBS come from studies on patients with functional constipation, IBS-C and FD, which are often associated with IBS. Methanogenic microbes, particularly Methanobravibacter smithii, produce methane in the gut, which slows gut transit causing constipation [86].

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2.2.8 Immune activation and alteration in mucosal permeability in patients with irritable bowel syndrome During the last decades, several studies showed that colonic biopsies in patients with IBS, particularly those with IBS-D, often show mononuclear inflammatory infiltrates including mast cells in the duodenum, jejunum, ileum, cecum, and rectum suggesting a low-grade inflammatory basis of a subset of these patients [87,88]. Mast cell is an important cell of innate immune response that on degranulation releases histamine, tryptase, and chymase. T lymphocytes are important in adaptive immune response to luminal antigen including microbes and quite a few studies reported increased mucosal T cells in duodenum, jejunum, ileum, cecum, rectum, and recto-sigmoid region in patients with IBS as compared with controls [88]. Of 15/82 patients with IBS having SIBO on quantitative upper gut aspirate culture, mucosal IL-1 α and β (proinflammatory cytokine) levels were higher than those without SIBO and higher IL-1 β levels were associated with loose stool and abdominal bloating [89]. Number of gene copies of the good bugs such as Lactobacillus and Bifidobacteria showed correlation with antiinflammatory cytokine IL-10 [90]. Most of the evidences mentioned above suggest that patients with IBS, particularly those with IBS-D, have immune activation in the gut suggesting the condition to be a lowgrade inflammatory state [91].

2.2.9 Postinfection irritable bowel syndrome Following acute infectious gastroenteritis, 7% to 9% subjects continue to experience GI dysfunction fulfilling the Rome criteria for IBS or FD or both though they did not have these disorders earlier [92,93]. These conditions are called PI-IBS and postinfection functional dyspepsia (PI-FD). In a metaanalysis of 45 studies including 21.421 subjects with acute gastroenteritis, a pooled prevalence at 12 months following the episode was 10.1% (95% confidence interval, 7.2
14.1) [52]. Mechanism of development of PI-IBS includes alteration in gut microbiota, immune activation, low-grade inflammation, alteration in mucosal permeability, neuro-hormonal dysfunction, and alteration in GI motility [94]. Taken together, these findings indicate that patients with IBS and FD are not only characterized by abnormal brain responses to visceral pain stimuli, but also by abnormal brain activity and connectivity at rest. These abnormalities seem to be at least partly related to comorbid anxiety and depression. The different factors that may influence the gut
brain axis, can be used in the future as possible therapeutic targets.

2.3 Therapy of main disorders of the gut
brain axis 2.3.1 Treatment of functional esophageal disorders There is no definite treatment for FEDs, and several approaches are usually considered in the same patient. There is no dietary advice in FEDs, but some patients might identify

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triggers for their symptoms, and eliminate them. Psychological features such as anxiety should be identified and treated, as it might influence therapeutic responses. Proton pump inhibitors (PPIs) are the first treatment option in FEDs, even if in some cases (like in FH), there is no evidence of pathological reflux or association with reflux events. However, some patients with FEDs respond to PPIs, partly due to a placebo effect [95]. In case of failure, one important armamentarium for all FEDs are pain modulators. Some cases with motility changes might respond to smooth muscle relaxants. If medical treatment fails, psychological interventions might provide symptom improvement [96]. 2.3.1.1 Mechanism of action of pain modulators Pain modulators include several antidepressants like tricyclic antidepressants (TCAs), trazodone (serotonin 5-HT2 antagonist and reuptake inhibitor), selective serotonin reuptake inhibitors (SSRIs) and serotonin and noradrenaline reuptake inhibitors (SNRI) [20,26], but also pregabalin and theophylline [97]. These drugs act by modulating central hyperalgesia and have some effect on peripheral hyperalgesia and visceral hypersensitivity. The mechanism of action of antidepressants in chronic pain disorders are based on neuroplasticity [98]. Chronic pain disorders, depression, abuse, and war determine death of nerve cells [99]. Antidepressants increase the concentration of Brain Derived Neurotrophic Factor (BDNF) that supports the survival of neurons and favors the growth and differentiation of new neurons and synapses. Therefore, antidepressants determine regeneration of neurons in the affected regions, reversing the effect of chronic pain [98]. Antidepressants increase the amount of norepinephrine and serotonin in the synaptic cleft, resulting in enhancement of the descending inhibitory pathways of pain transmission [97]. Moreover, TCAs improve sleep quality, resulting in increased tolerance to visceral sensations, including pain [100]. The visceral analgesic effect of SSRIs is related to their 5-HT reuptake inhibitory property, in the descending serotonergic systems, which are involved in antinociception. SSRIs bind to serotonin transporter and inhibit the serotonin reuptake, increasing the serotonin level in the synapse, in both supraspinal and spinal pathways [101]. SSRIs also stimulate adult neurogenesis in areas like hippocampus, an area involved in pain and analgesia processing [102]. 2.3.1.2 Pain modulators in the treatment of functional esophageal disorders Three decades ago, Clouse reported that trazodone improved esophageal symptoms in more than half of patients with esophageal motility changes [103]. Afterwards, the role of antidepressants (TCAs and SSRIs) as pain modulators was studied mostly in NCCP. Imipramine 50 mg daily for three weeks, reduced the frequency of chest pain [104], while adding 10 mg of amitriptyline at bedtime was more effective than doubling PPI dose in patients with FCP nonresponsive to single dose PPI [105]. The long-term use of TCAs has beneficial effects on chest pain episodes [106]. Among SSRIs, sertraline (50 mg, gradually increased to 200 mg daily) also has beneficial effects on FCP [107]. For paroxetine existing data is conflicting [108]. In FCP patients (Rome III), venlafaxine (SNRI) 75 mg at bedtime (4 weeks) improved chest pain in 52% of patients compared to 4% in the placebo group, with 1 in 10 patients reporting complete symptom resolution [109]. The study group included mainly young males (, 25 years), and no data regarding response to PPIs were provided,

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making hard to estimate the real benefit of SNRIs against PPIs [110]. For patients with FH or hypersensitive esophagus (HE) fluoxetine (20 mg daily) and Citalopram (20 mg daily) can reduce heartburn episodes [111,112]. Imipramine (25 mg daily) although improved quality of life (QoL), was not superior to placebo on FH and HE symptoms [113]. One metaanalysis concluded that pain modulators improved QoL, depression and anxiety scores, and the frequency and severity of symptoms in NCCP patients [114]. We recommend starting treatment with low doses of antidepressants, and if necessary, to increase doses after 2
4 weeks, because antidepressants have a delayed onset of action. If no beneficial effect is observed after 12 weeks, the treatment should be stopped. Antidepressants have sedative side effects, and therefore are administered at bedtime. Side effects (see Table 2.3) reduce adherence to treatment and also limit the long-term administration of these drugs. TABLE 2.3 Pain modulators and their dosing in patients with functional esophageal disorders.

Medication Tricyclic antidepressants (TADs)

Maximal therapeutic dose

Initial dose Amitriptyline 10 mg at bedtime Imipramine

50
150 mg

25
50 mg

Nortriptyline 25 mg

Selective serotonin reuptake inhibitors (SSRIs)

Main side effects urinary retention, dry mouth, drowsiness, blurred vision, confusion, mental status change, constipation, hypotension, sexual dysfunction, tachycardia

Studied in following disorders All functional esophageal disorders

desipramine has the lowest anticholinergic and sedative adverse effects

Desipramine

25
50 mg

Sertraline

25
50 mg

50
200 mg

Paroxetine

10
20 mg

20
60 mg

Fluoxetine

10
20 mg

20
80 mg

Citalopram

20 mg

Venlafaxine

Digestive symptoms: nausea, gastric discomfort, vomiting, anorexia, and diarrhea

Functional chest pain, functional heartburn, hypersensitive esophagus

75 mg at bedtime

sleep disturbance, nausea, dizziness, diarrhea, increased liver enzymes, hypertension, risk of upper gastrointestinal bleeding

Functional chest pain

Trazodone

100
150 mg, qid

dry mouth, dizziness, blurred vision, nausea, vomiting, insomnia

Functional chest pain

Theophylline

200 mg bid

headache, insomnia, irritability, seizures, tremor, tachycardia, miocardial infarction

Functional chest pain

Gabapentin

300 mg tid

dizziness, drowsiness, viral infections, tremors

Globus

Tegaserod

6 mg bid

headache, dizziness, abdominal pain, nausea, vomiting, bloating, diarrhea, fatigue

Functional heartburn

Serotonin and noradrenaline reuptake inhibitors (SNRI)

qid, four times a day; bid, twice a day; tid, three times a day.

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2.3.1.3 Other pain modulators Three decades ago, esophageal motility changes were considered an important mechanism for NCCP, and smooth muscular relaxants like calcium channel blockers were studied. These studies included patients with major motility disorders, like esophageal spasm and achalasia, therefore the results cannot be generalized to FEDs defined based on Rome IV criteria. Nifedipine (10
30 mg tid) [115] and diltiazem (90 mg qid) [116] showed inconsistent results when compared to placebo. There is no evidence to support the use of toxin botulin injection or nitrates [96]. In other attempts to influence functional esophageal pain, ondansetron (5-HT3 antagonist) and pregabalin were used, but the results of pilot studies were not reproduced by further research [97]. Tegaserod (5-HT4 receptor agonist, with prokinetic effect) 6 mg bid decreased the severity of heartburn compared to placebo and increased esophageal pain threshold to mechanical distention, in patients with FH [117,118]. In patients with FCP, theophylline (adenosine receptor antagonist) improved duration and severity of chest pain episodes, with an overall improvement of symptoms in 58% of cases, compared to 6% in the placebo group. Intravenous administration of theophylline relaxes the esophageal wall and decreases hypersensitivity [119]. The rationale of using theophylline in FCP comes from the observation that adenosine is a mediator of visceral pain and when infused induces angina-like pain [120]. There are no recent studies to support the use of these drugs in FEDs. Patients with globus can benefit also from PPIs and pain modulators (amitriptyline 25 mg) [121]. As almost half of patients with globus have esophageal motility changes, like ineffective esophageal motility, prokinetics would be the right choice [122]. However, few prokinetics are efficient in the esophagus. Itopride added to PPI reduced the recurrence of globus sensation in one study [123]. Good results were observed using gabapentin (a drug used for neuropathic pain) 300 mg tid, with two-thirds of patients reporting improvement of globus sensation after 2 weeks [124]. Studies regarding the treatment of functional dysphagia are lacking, and in expert’s opinion neuromodulators and psychological interventions can be used [26]. 2.3.1.4 Psychological interventions in functional esophageal disorders Among psychological interventions, cognitive behavioral therapy (CBT) and hypnotherapy were most studied in patients with FEDs. Patients with FCP interpret the pain as a signal of cardiac disease, leading to anxiety and more attention to bodily sensations. During CBT sessions patients are taught breathing and relaxation techniques, catastrophic misinterpretations are identified and “replaced” with rational beliefs, with a positive impact on their emotional, cognitive, and behavioral response [125]. Hypnotherapy uses suggestibility to “disable” analytical aspects of the patient’s mind, while aspects of the normally subconscious mind are activated [126]. Both CBT and hypnotherapy are efficient in NCCP [108,127]. Hypnotherapy is also efficient in FH [128] and globus [129]. An algorithm for the treatment of FEDs is summarized in Fig. 2.2.

2.3.2 Treatment of functional dyspepsia 2.3.2.1 General and dietary recommendations Patients with FD should be reassured about the benign course of this disorder, and about its chronic nature, with reoccurrence and exacerbation of symptoms over time. Patients are

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FIGURE 2.2 Treatment options in patients with functional esophageal disorders. PPIs represent the firstline treatment, followed by pain modulators. Psychological interventions were studied only in some of the functional esophageal disorders, and come as third line treatment. For functional chest pain, both cognitive behavioral therapy and hypnotherapy were effective, while for heartburn and globus there are positive results with hypnotherapy. In the end, are mentioned drugs for which only limited data is available. FCP, functional chest pain; FH, functional heartburn; PPIs, proton pump inhibitors; RH, reflux hypersensitivity; bid, twice a day; tid, three times a day.

advised to eat small amounts, frequently, as this pattern is associated with lower odds of postprandial fullness and early satiety [130]. Foods that trigger symptoms (high fat content, sour foods, citrus, coffee, alcohol) should be avoided [40], as well as nonsteroidal antiinflammatory drugs. Evidence is accumulating regarding the use of yogurt enriched with probiotics as “functional food” in FD. Strains of Lactobacillus (Lactobacillus rhamnosus, Lactobacillus reuteri, Lactobacillus gasseri) and Bifidobacterium bifidus were studied. Lactobacillus gasseri administered for 12 weeks, was superior to placebo on postprandial fullness [131]. Patients positive for Helicobacter pylori should receive eradication therapy to exclude H. pylori-associated dyspepsia [132]. The number needed to treat (NNT) for one positive response on dyspepsia symptoms is 8 [133]. 2.3.2.2 Pharmacological treatment of functional dyspepsia First-line treatment in patients with EPS is represented by gastric antisecretory drugs, followed by drugs that reduce visceral perception, like TCAs. Patients with PDS receive first prokinetics followed by fundus relaxing drugs, and centrally active neuromodulators (mirtazapine) as third line treatment. In case of failure, psychological interventions (CBT or hypnotherapy) might be tried [40]. A treatment algorithm for FD is presented in Fig. 2.3 in the spirit of other guidelines [40].

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FIGURE 2.3 Treatment algorithm in functional dyspepsia. CBT, cognitive behavioral therapy; EPS, epigastric pain syndrome; Hp, Helicobacter pylori; H2RA, H2 receptor antagonists; PDS, postprandial distress syndrome; PPIs, proton pump inhibitors; TCAs, tricyclic antidepressants; 1 , a favorable response to treatment; 2 , no response to treatment.

2.3.2.3 Antisecretory drugs and antacids in the treatment of functional dyspepsia Two metaanalyses confirmed that both H2 receptor antagonists (H2RA) and PPIs are more effective than placebo in improving global symptoms in patients with FD [134,135]. H2RAs improve both epigastric pain and postprandial fullness, but tolerance develops quickly, after 1
2 weeks of treatment [134]. PPIs ameliorate mainly epigastric pain and reflux symptoms [135]. There is no added benefit to increase the dose of PPIs in nonresponsive patients [40]. PPIs are rather safe drugs. However, when used for a long period of time, the risk of side effects (fracture, Clostridium difficile infection, etc.) increases [136]. Antacids and sucralfate administered alone are not effective in treating FD [137]. 2.3.2.4 Centrally active neuromodulators in the treatment of functional dyspepsia Antidepressants have a visceral analgesic effect by modulating central hyperalgesia. In FD, small studies attributed their efficacy to an increased tolerance to aversive visceral sensations. Amitriptyline is one of the most studied drugs in FD patients, and the best results were observed in patients with epigastric pain, at a dose of 50 mg daily [138]. Mirtazapine (15 mg daily) was superior to placebo in patients with FD, early satiation and weight loss. QoL, GI-specific anxiety, weight, and nutrient tolerance significantly

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improved in mirtazapine group [139]. Mirtazapine can be recommended as a third line treatment in PDS [40]. Nortriptyline, fluoxetine, venlafaxine, and sertraline failed to show superiority compared to placebo in the treatment of FD [140]. 2.3.2.5 Use of prokinetics in functional dyspepsia Prokinetics are a very heterogenous group of drugs that influence the gastric dysmotility like delayed GE and impaired accommodation to food. Some of them also alleviate visceral hyperalgesia. Acting on GI smooth muscle, prokinetics enhance contractility and increase GE rate [137]. Classic prokinetics are dopamine D2 receptor antagonists (itopride, metoclopramide, domperidone) and serotonin 5HT4 receptor agonists (metoclopramide, mosapride, tegaserod). Mosapride and tegaserod failed to show convincing benefits in FD. Other prokinetics are motilin receptor agonists (erythromycine), opioid receptor agonists (trimebutine), and acetylcholinesterase inhibitors (acotiamide) [137,141] (Table 2.4). A recent metaanalysis showed that prokinetics reduce global symptoms of FD, but the evidence regarding symptomatic benefit is of low quality [142]. 2.3.2.6 Fundus relaxing drugs Fundus relaxing drugs are efficient mainly in patients with PDS. Acotiamide is an acetylcholinesterase inhibitor and an antagonist of inhibitory muscarinic type 1 and type 2 (M1/M2) auto-receptors on cholinergic nerve endings. It has both fundus relaxing properties and gastroprokinetic properties and improves postprandial symptoms of PDS. Longterm use (up to 1 year) is safe and efficient [143]. Acotiamide is currently available in many countries for the treatment of FD (Table 2.4). Buspirone and tandospirone are anxiolytics and 5-HT receptor 1A agonists. The activation of 5-HT1A receptors relaxes the proximal stomach through inhibition of cholinergic tone [141]. Buspirone reduced PDS symptoms and increased gastric accommodation, after 4 weeks of treatment [144]. Currently, these drugs are used only in clinical trials.

TABLE 2.4 Prokinetics and fundus relaxing drugs in the treatment of functional dyspepsia. Prokinetic/fundus relaxing drugs

Dosing

Domperidone

10 mg tid QT interval prolongation- recommendation to limit its use (,1 week)

Metoclopramide

10 mg tid Dyskinesia and hyperprolactinemia, anxiety, agitation, somnolence, insomnia

Itopride

50 mg tid Hyperprolactinemia

Acotiamide

100 mg tid

Buspirone

10 mg tid Sedation and dizziness

Side effects

Comparable to placebo

tid, three times a day.

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2.3.2.7 Alternative and nonpharmacological treatment Herbal preparations were studied both in FD and in IBS. Their mechanisms of action are not completely understood but seemed related to essential oils, known for centuries for their spasmolytic, carminative, and local anesthetic actions [145]. Rikkunshito, a standardized Japanese herbal medicine, improved epigastric pain, postprandial fullness and global FD symptoms. The presumed mechanism of action is the acceleration of GE and improved gastric accommodation [146]. Another well studied herbal preparation is STW 5 (Iberogast), who was superior to placebo in decreasing the most bothersome GI symptoms. STW 5 relaxes the fundus and corpus, while increasing antral contractions [147]. Acupuncture also showed some benefits on QoL, postprandial fullness and early satiation [148]. Psychological interventions can also be effective in some patients with FD. The techniques for coping with FD, CBT, and gut-oriented hypnoses were effective in the management of FD [149,150]. Hypnotherapy can reduce medication use and consultation rate because it has long-term effects [150,151].

2.3.3 Treatment of irritable bowel syndrome Patients with IBS consult very often, therefore a good physician-patient relationship is of utmost importance, leading to better outcomes. The patient’s education and understanding of the disease, as well as his/her involvement in treatment decisions, are part of a good relationship. The reason for the current visit should always be determined. Some patients fear that cancer has developed. They should be reassured about the benign course of IBS. Others seek care for worsening symptoms. The most bothersome symptom should be identified each time and severity should be established, to see if pharmacological treatment is necessary [141]. 2.3.3.1 Diet and physical activity in irritable bowel syndrome IBS patients often report exacerbation of their symptoms after food ingestion, and therefore they institute dietary changes. Patients exclude foods that they observe they cannot tolerate or they suppose are allergic to [141]. Research in the last years focused on a low FODMAPs (Fermentable Oligo-, Di-, Mono-saccharides, and Polyols) diet (LFD), which consists in eliminating fermentable foods for a period of 4
8 weeks, and then gradually reintroducing them to determine personal tolerances. FODMAPs include fruits, vegetables, legumes, cereals, gluten, honey, milk and dairy products, and sweeteners. All FODMAPs can trigger IBS symptoms, but only a few of them will exacerbate abdominal symptoms in the same patient [152]. Low FODMAP diet reduces bowel distention because it determines less water in the small bowel, less gas production by bacteria, and decreased production of short-chain fatty acids, which can increase visceral sensitivity [153] and colonic contractions [154]. Some studies showed favorable results of LFD on abdominal pain, bloating and IBS-QoL when compared to unrestricted diets, and the favorable results were confirmed by a metaanalysis [155]. Patients with IBS should be encouraged to perform a physical activity because there is evidence that 20
60 minutes of moderate to vigorous activity, three to five times weekly

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decreases IBS symptoms severity score, compared to usual activity [156]. The beneficial effects might be due to changes in the ratio of anti- to proinflammatory cytokines [157]. 2.3.3.2 Modulators of gut microbiota in irritable bowel syndrome This category includes probiotics and antibiotics (riaximin). Lately, the role of fecal transplantation was assessed, but the results are inconsistent. Some strains of probiotics, like Bifidobacterium infantis, determine a global improvement of IBS symptoms [158], others improve bloating or flatulence (i.e., VSL#363 and Bifidobacterium animalis [159]) or change stool consistency (Lactobacillus plantarum) [160]. The positive effects are thought to be determined by antiinflammatory effects, changes in motility, in gut permeability and in visceral hypersensitivity [161]. Which strain would be most efficient in reducing symptoms in IBS-C or IBS-D, is not yet determined [162]. Rifaximine is a nonabsorbable intestinal antibiotic, efficient on symptoms mainly in IBS-D with SIBO [163], but also in IBS-C by diminishing the population of methanogenic bacteria, which slows colonic transit [164]. Pharmacological treatment in IBS is targeted against the main abdominal complaint: diarrhea, constipation, pain, or bloating (Fig. 2.4). 2.3.3.3 Treatment of irritable bowel syndrome with predominant constipation First-line treatment in IBS-C includes fibers and laxatives. Vegetables and fruits might accelerate colonic transit but can increase bloating, flatulence, and abdominal pain, because of a high FODMAPs content. Therefore, soluble fibers, such as psyllium, are

FIGURE 2.4 Treatment algorithm in irritable bowel syndrome. CBT, cognitive behavioral therapy; IBS-C, IBS with predominant constipation; IBS-D, IBS with predominant diarrhea; LFD, low FODMAP diet; SSRIs, selective serotonin re-uptake inhibitors; TCAs, tricyclic antidepressants.

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preferred [165], which also reduces the number of pain episodes, irrespective of IBS subtype [166]. Laxatives in IBS treat constipation but have less effect on abdominal pain. Among osmotic agents, polyethylene glycol (PEG 3350 1 E) can be used up to three times a day [141]. Linaclotide is safe even when administered for months, and increases the number of complete spontaneous bowel movements/week. Diarrhea is the most frequent side effect of prosecretory laxatives [167]. The laxatives used in IBS-C, their mechanism of action and daily doses, are presented in Table 2.4. Rifaximine (400 mg tid, 14 days) might also increase weekly stool frequency [164]. In order to reduce the severity of abdominal pain, antispasmodic drugs are prescribed (options, doses, in Table 2.5). Simethicone and probiotics can be added for bloating. 2.3.3.4 Treatment of irritable bowel syndrome with predominant diarrhea Antidiarrheal dugs represent the first-line treatment in IBS-D. Many patients respond to low-potency antidiarrheals, such as loperamide, but the existing evidence for loperamide in IBS is scarce, therefore American College of Gastroenterology gave a strong recommendation against its use [165]. Serotonin receptor 5HT3 antagonists are potent antidiarrheal drugs that reduce stool frequency and consistency, fecal urgency, and incontinence [168]. Only alosetron and ramosetron are commercially available. Given the risk of ischemic colitis, their use is limited to severe refractory cases of IBS-D. Eluxadoline is a new drug, approved by FDA and the European Medical Agency, with mixed opioid effects. It reduces abdominal pain severity in IBS-D, and improves stool consistency. As side effects nausea, constipation, abdominal pain and pancreatitis (0.3%) were observed [169]. Patients TABLE 2.5 The pharmacological treatment used in irritable bowel syndrome. Drug

Mechanism of action

Dose

Main effects

Polyethylene glycol

Osmotic agent

13,8 g, tid

Changes stool consistency

IBS-C

Lubiprosone

chloride channel (ClC-2) activator, increases intestinal secretion

8 mcg, bid

Changes stool consistency

IBS-C

Linaclotide

Guanylate cyclase C agonist, increases luminal secretion of chloride and bicarbonate

290 mcg daily

Changes stool consistency

IBS-C

Laxatives

Antidiarrheal drugs Loperamide

acts on μ opioid receptor on intestinal smooth muscle and inhibits peristalsis, prolonging transit time

Alosetron, ramosetron

Antagoni¸stii receptorilor 0.5
1 mg serotoninergici—5HT3, reduce bid motility and intestinal secretion, and decrease visceral perception

Reduces stool frequency, and increases consistency; No effect on pain

(Continued)

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TABLE 2.5 (Continued) Drug

Mechanism of action

Dose

Main effects

5 mcg daily Eluxalodine

μ- and κ-opioid receptor agonist and 100 mg bid δ-opioid receptor antagonist

Refractory IBS-D, females Reduces abdominal pain and improves stool consistency

IBS-D

Antispasmodics Scopolamine (hyoscine Anticholinergic butylbromide)

10 mg tid

Improves abdominal pain

All IBS subtypes

Otilium bromide

40 mg tid

Improves abdominal pain, reduces stool frequency

All IBS subtypes

Alverine citrate 60 mg Calcium channel blocker, selective 5- 3 times daily Improves abdominal 1 simethicone 300 mg HT1A receptor antagonist 1 pain and bloating antifoaming agent

All IBS subtypes

Peppermint oil

Calcium channel blocker, antimuscarinic and, a tachykinin NK2 receptor antagonist

Calcium channel blocker

200 mg tid

Improves abdominal pain and global IBS symptoms

All IBS subtypes

Pinaverium bromide 1 Calcium channel blocker simethicone (300 mg)

50
100 mg tid

Improves abdominal pain, stool frequency, and bloating

IBS-C, IBSM

Trimebutine

100
200 mg tid

Improves abdominal pain

All IBS subtypes

Agonist of opioid receptors, parasympatholytic

Antidepressants TCAs (amitriptyline, nortriptyline, imipramine, desipramine)

10
200 mg daily

All IBS subtypes

SSRIs (sertraline, paroxetine, fluoxetine, citalopram)

10
100 mg daily

All IBS subtypes

tid, 3 times a day; IBS-C, irritable bowel syndrome with predominant constipation; bid, twice a day; TCAs tricyclic antidepressants, SSRIs, selective serotonin reuptake inhibitors, IBS-D, IBS with predominant diarrhea, IBS-M, mixed form of IBS.

with IBS-D and SIBO might benefit from treatment with rifaximine, with an NNT of 9 [165]. 2.3.3.5 Treatment of abdominal pain in irritable bowel syndrome Most drugs used to treat abdominal pain in IBS target peripheral mechanisms. Antispasmodics are superior to placebo for improvement of abdominal pain, with an NNT of 7 [170]. The highest benefits were obtained with cimetropium/dicyclomine, peppermint

I. Gut-brain axis

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oil (NNT 5 3 [171], small-intestinal relief formulation being preferable), pinaverium, and trimebutine. Some antispasmodics are not available in all countries, and the date we have in some cases is from the 1990s. Their effect is of short duration, therefore are beneficial in reducing postprandial IBS symptoms. Some antispasmodics improve also stool frequency and consistency. The main antispasmodics, the mechanism of action and doses are presented in Table 2.5 [170,172]. The so-called pain modulators (TCAs, SSRIs, SNRIs) act also on central mechanisms of IBS and were renamed visceral neuromodulators [91]. Antidepressants reduce abdominal pain and global IBS symptoms and improve the psychiatric disorders that frequently coexist. Not all studies showed the superiority of SSRIs or TCAs compared to placebo. However, a recent metaanalysis [173] concluded that antidepressants are effective in symptom relief in IBS with an NNT of 4.5. When using TCAs, the number needed to harm was 8.5, drowsiness and dry mouth being frequent adverse events. 2.3.3.6 Treatment of bloating Some lifestyle and diet changes can reduce bloating by influencing mechanisms responsible for bloating and distention: eating slowly, avoiding chewing-gum or sparkling water to reduce aerophagia; reducing lipids (absorbable lipids increase visceral hypersensitivity), and fermentable foods (lactose or fructose if intolerant, or via an LFD); exercise and maintaining a normal colonic transit are useful [141]. Not always symptoms are corelated with the volume of intestinal gas. If bloating persists, simethicone can be used. Simethicone is an antifoaming agent that reduces the surface tension of gas bubbles, which combine into larger bubbles that are more easily eliminated. It is administered alone or combined with antispasmotics (Table 2.4) [174]. There is low evidence to support the use of charcoal for bloating in IBS. Probiotics and rifaximine can reduce bloating in IBS patients [165].

2.3.4 Alternative therapies and psychological interventions STW5 is a combination of nine herbal extracts. Data showed that STW 5 reduces abdominal pain in all IBS subtypes. STW 5 has antiinflammatory effects, reduces visceral hypersensitivity and has a dual action on motility, depending on the basal tone [175]. Several psychological interventions were studied in IBS. A recent metaanalysis [173] concluded that taken as a whole, these strategies are efficient, with an NNT of 4, and a relative risk of IBS symptoms not improving with these techniques of 0.69. The best results were obtained with CBT, relaxation therapy, multicomponent psychological therapy (a combination of relaxation therapy, thermal biofeedback, education, and stress coping strategies), hypnotherapy, and dynamic psychotherapy.

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C H A P T E R

3 The interactions between gut and brain in psychiatric and neurological disorders Cheolmin Shin and Yong-Ku Kim Department of Psychiatry, Korea University Ansan Hospital, Ansan, Kyunggido, Republic of Korea

3.1 Introduction The gut and brain are involved in a complex bidirectional communication, beyond sensing hunger and regulating eating habits, which can affect emotions, thinking, and behaviors. This interaction between the gut and brain is mediated by the abundant microbiome in the gut. The microbiome is essential and aids in maintenance of health by producing short-chain fatty acids (SCFAs), digesting carbohydrates, synthesizing vitamins, and metabolizing toxins. Recent advances in metagenomics technology have enabled in-depth analysis of gut microbial genes [1,2], which have demonstrated closer interactions between the host and microbiome via more diverse pathways that are involved in brain development and function, mood, and cognition. Sudo et al. were the first to establish the mechanism of interaction between the microbiome and the brain
gut axis. The purpose of their study was to determine the possible influence of postpartum microbial colonization in sterile cats on the development of brain plasticity and physiological responses [3]. The study compared the hypothalamus
pituitary
adrenal (HPA) axis response to stress in germ-free (GF), specific pathogen-free (SPF), and nonliving mice to test the hypothesis that the intestinal microbiome may affect the development of the nervous system, which controls the endocrine response to stress. The study identified that the HPA response of GF mice was more sensitive to restraint stress than that of SPF mice, whereas the two groups showed no difference in sensitivity to ether stress. In addition, GF mice showed lower expression of brain-derived neurotrophic factor in the cortex and hippocampus than SPF mice.

The Complex Interplay Between Gut
Brain, Gut
Liver, and Liver
Brain Axes DOI: https://doi.org/10.1016/B978-0-12-821927-0.00010-3

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Recent systematic reviews have reported on alteration of the gut microbiome in various neuropsychiatric illnesses. Furthermore, several meta-analyses have reported on the efficacy of microbiota modulation in neuropsychiatric disorders. In this chapter, we studied the role of the gut microbiome in the network between the gut and brain, immune, endocrine, and metabolic pathways, and neuropsychiatric disorders.

3.2 Development of gut microbiota The majority of the microbiome in the human body is present in the intestine. Human microbiota develops after birth, with major contributions from the maternal microbial community, contact with microbial communities present in other proximal individuals, and the environment that causes a stochastic change in colonization. The composition of microbial colonies in the intestine is variable in early infancy, and only some strains that reach the gut of the infant colonize successfully [4]. The predominant genera of gut microbiota acquired by the newborn during vaginal delivery include Bifidobacterium, Lactobacillus, and Prevotella [5]. Staphylococcus and Corynebacterium are the main genera acquired following delivery via cesarean section [6]. Microbial diversity was found to be lower in infants born via cesarean section, and a study reported that this effect lasted more than 2 years [7]. Based on development of the gut microbiota, cesarean section was not associated with certain long-term outcomes including overweight status [8,9]. Moreover, no causal effect was reported even in cases of any association [10]. The proportion of anaerobic bacteria classified as Firmicutes begins to increase [11], and the microbiome becomes similar to that of an adult gut by 3 years of age [12].

3.3 Pathways of brain
gut
microbiota interaction 3.3.1 Immune pathway Activation of the inflammatory pathway appears to play an important role between microbiome and neonatal neural development. The gut microbiota can directly affect the immune system by activating the vagus nerve, causing bidirectional communication with the central nervous system (CNS) [13
15]. Indirect effects of the gut microbiome on the innate immune system alter the levels of circulating proinflammatory cytokines that directly affect brain function. Additionally, effects of the gut microbiome on the acquired immune system can affect the CNS through the blood
brain barrier (BBB). Innate immunity is the connatural immune system that can eliminate microorganisms without any prior sensitization to an antigen. Immune cells involved in innate immunity are distributed in the mucosal region and form the first line of defense against infections. These cells detect the components or products of microorganisms and elicit a reaction against them [16]. Microglia are the major innate immune cells of the CNS, and they play an important role in CNS immune defense. Microbiota may contribute to microglia homeostasis mediated by signal transduction through SCFA [17]. Since GF mice exhibit a marked defect in the function of microglia, microbiota are thought to have a significant effect on the

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development of microglia [18]. Even from the prenatal stage, the maternal microbiome appears to affect microglia maturation in a sex- and time-specific manner [19]. The host detects components of the microbiome through pattern recognition receptors including Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) [20]. Studies on mouse models reported dysbiosis in cases of innate immune deficiency, wherein TLR 5 and NOD-containing protein 2 genes were removed [21,22]. These innate immune receptors are essential for defense against pathogens. As an example, a study reported that mice lacking TLRs developed characteristics of metabolic syndrome, which correlated with altered microbiota [21]. Likewise, deficiency of NLRs caused dysbiosis in the form of expansion of Bacteroides vulgatus, which subsequently induced colitis and colorectal cancer in mice [23]. Inflammasomes are multiprotein complexes that are a component of innate immunity. Inflammasomes secrete interleukin (IL)-1β and IL-18 on activation, which in turn promote Th1 and Th17 immune responses [24]. Microbiome-derived metabolites, such as histamine, spermine, and taurine, can induce activation of inflammasomes and subsequent production of antimicrobial peptides [25]. A study reported remodeling of the gut microbiota through activation of inflammasomes in a mouse model of colitis, which induced regulatory T (Treg) cells and ultimately conferred resistance against colitis and colon cancer [26]. Macrophages are important cells related to innate immunity. Recent research has focused on the interaction between macrophages and symbiotic microbiome in immune regulation. A study observed that the antiinflammatory gene was activated in the macrophages of mice by a polysaccharide derived from Helicobacter hepaticus [27]. The microbiota-derived metabolite butyrate induced macrophage differentiation from monocytes through inhibition of histone deacetylase (HDAC) 3 [28]. Recently identified neutrophil extracellular traps (NETs) are web-like DNA structures composed of antimicrobial proteins and proteolytic enzymes [29], which entangle with and eradicate bacteria. There is evidence of a link between NETs and dysbiosis. Enteropathogenic Escherichia coli exacerbated antibiotic-induced dysbiosis and activated NETs [30]. In contrast, Lactobacillus species, which are known probiotics, underactivated neutrophils, and resulted in lesser formation of NETs [31]. Adaptive immunity refers to the immunity acquired by lymphocytes such as T cells and B cells through a pathogen-specific process. In adaptive immunity by T cells, in particular, Treg cells play an important role in suppressing immune reactions against body tissues as a negative regulator for adaptive immunity. GF and antibiotic-treated mice are partially deficient in adaptive immunity, presenting a lack of Th17 and Treg cells and skewed Th2 in the intestine [32]. Treg cells halt the initiation of immunopathology by blocking abnormal immune responses to dietary antigens and the commensal microbiome. Intestinal Treg cells are induced by a specific microbiome. For example, Bacteroides fragilis-derived polysaccharide was able to restore immune deficiency in GF mice [33]. Treg can be induced by SCFAs through HDACs inhibition and consequent histone H3 acetylation of the resulting Foxp3 gene [34] or by activating G-protein-coupled receptors [35]. SCFAs obtained from the diet have been reported to promote Treg cells through the suppression of the c-Jun N-terminal kinase 1 and p38 pathways to regulate autoimmunity in the CNS [36]. Commensal gut microbiota have been shown to be important in the regulation of meningeal IL-17 1 γδ T cells that affect the development of ischemic brain injury [37].

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B cells are also a major part of adaptive immunity through the production of antibodies that protect the host from microorganisms [38]. Immunoglobulin A (IgA), a type of antibody, coats and captures microorganisms and plays a fundamental role in mucosal defense [39]. IgA coating facilitates the translocation of noninvasive microorganisms, promoting antigen presentation and the production of antigen-specific IgA [40]. Therefore IgA plays a role in controlling the microbial community of symbiosis [41]. In mice with low IgA, intestinal microbes degrade the secretory component of IgA and IgA itself, leading to susceptibility to colitis [42]. The effects of adaptive immunity on neuro-immunity in the brain
gut axis are being explored through several studies. Depletion of intestinal commensal bacteria by antibiotic treatment exacerbates the progression of experimental autoimmune encephalomyelitis, which is mediated by the induction of IL-10-producing Treg cells [43]. Intestinal microbiota are required for the induction and acceleration of encephalomyelitis through the activation of myelin oligodendrocyte glycoprotein-specific T cells and recruitment of autoantibodyproducing B cells [44]. Maternal gut bacteria colonized with segmented filamentous bacteria that induce a T helper 17 response have an increased risk of developing neurogenic abnormalities in offspring mice [45].

3.3.2 Vagus nerve The vagus nerve is a component of the parasympathetic nervous system and is the major route of neural communication between the CNS and gut. The vagus nerve actively participates in the bidirectional interaction between the gut microbiome and the brain to maintain homeostasis in both organs. Lipopolysaccharide (LPS) activates cytokines, such as IL-1β, causing disease through the vagus nerve. The induction of such cytokines is blocked in the CNS after vagotomy [46,47]. Modulation of the vagus nerve by intestinal microbiota was supported by the observation that the administration of Campylobacter jejuni activated neurons of the nucleus tractus solitarius, the first intracranial gateway to the vagal afferent nerve [48]. Another study showed that vagotomized mice treated with Lactobacillus rhamnosus showed improvement in anxiety- and depression-related behaviors without altering the expression of gamma aminobutyric acid (GABA) receptors in the brain [49]. A population-based cohort study conducted in Denmark revealed that truncal, but not selective, vagotomy reduced the hazard ratio of Parkinson’s disease (PD) [50]. A Swedish cohort human study also provided suggestive evidence of the potential protective effects of truncal, but not selective, vagotomy in the development of PD, suggesting that PD pathologically originates in peripheral tissues and later spreads through the vagus nerve to the CNS [51].

3.3.3 Microbial metabolites SCFAs that are produced by bacterial fermentation of dietary carbohydrates are the most important among all the bacterial metabolites. SCFAs are best known to affect gut cell nutrition and exert considerable hormone-like activity, have immunomodulatory properties, and interact with nerve cells by stimulating the sympathetic branch of the autonomic nervous system. In addition, microbial-derived SCFAs can cross the BBB, directly

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influence DNA expression in brain cells, and regulate microglial signaling involved in proper brain development and regulation of behavior. SCFAs also regulate the release of intestinal peptides and the synthesis of serotonin by enteroendocrine cells (EEC) [52]. Microbial metabolites with confirmed bioactivity against EEC include SCFAs, indole, secondary bile acid, and LPS [53
56]. Serotonin plays an essential role in gut and peripheral metabolism but can locally activate afferent nerve terminals that are directly connected to the CNS [57]. An observational study in humans and rats demonstrated that gut microbiota promoted colonic tryptophan hydroxylase 1, which is a rate-limiting enzyme for serotonin biosynthesis and production following stimulation of EC cells by SCFAs such as butyrate and acetate [58]. LPSs are bacterial metabolites largely derived from the cell wall of gram-negative bacteria in the gut. LPS enter the systemic circulation in leaky gut syndrome where the tight junction of the gut epithelium is impaired due to defects in permeability. The human body recognizes LPS as a pathogen and produces antibodies through an immune reaction; however, studies have shown that antibody levels against LPS are higher in patients with major depression than in controls [59].

3.4 Tryptophan metabolism Gut microbiota can contribute to the regulation of brain function through tryptophan metabolism. Tryptophan is an essential amino acid that is a precursor to serotonin and kynurenine synthesis in the CNS [60]. In the process of absorption from the gut, tryptophan can participate in serotonin synthesis across the BBB [60]; however, there are several other pathways that involve tryptophan. Availability of tryptophan is partly controlled by the gut microbiota. GF mice showed an increased plasma tryptophan concentration, which was normalized following colonization [61]. Intestinal bacteria affect the availability of tryptophan by utilizing the amino acid for growth [62], catabolizing it to indole, tryptamine, or indolelactic acid [63,64], or influencing the host enzyme responsible for the degradation of tryptophan [61]. Limiting the availability of tryptophan can affect serotonergic neurotransmission in the CNS. A decrease in circulating concentrations of tryptophan affected mood in depressed patients and administration of antidepressant was shown to restore depressive symptoms [65] In addition, the gut microbiome was shown to affect the production of neuroprotective and neurotoxic components in the kynurenine pathway, which is related to degradation of tryptophan [60].

3.5 Endocrinologic pathway The gut is the largest endocrine organ and EEC are the most important despite comprising less than 1% of the endothelial cells in the intestinal tract [66]. There are over 20 different types of EEC, which secrete serotonin, regulatory peptides [e.g., glucagon-like peptides, pancreatic peptides (YY, PYY), cholecystokinin (CCK), and secretin], and bioactive molecules. EEC regulate digestive function through the enteric nervous system circuit and communicate with the hypothalamus through vagal afferents via the endocrine

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pathway. The production of hormones such as serotonin, CCK, and peptide YY are stimulated by bacteria-derived metabolites through TLRs expressed on the surface of EEC [67]. Previously, EEC and afferent neurons were thought to communicate indirectly through neuropeptides released by these cells. However, a recent study demonstrated a direct synaptic contact between the EEC and neurons through the rabies virus experiment in the intestinal lumen [68]. These hormonal mediators are involved in additional activation of afferent nerve fibers by binding to chemoreceptors. During signal transduction from the intestine, EEC form a connection with the brain through synapses with a particular subset of vagal afferent nerves [69]. Here, the synapse between EEC of the intestinal lumen and the vagal neurons deliver sensory information to glutamate as the main neurotransmitter. The gut microbiota transmits the stimuli to the EEC, leading to physiological and pathological responses of the peptide [66,70]. Interaction between the microbiota and the brain
gut axis can occur through the release of biologically active peptides by EEC [71]. For example, the neuropeptide, galanin, is thought to be involved in many important neurobiological functions, including nociception, regulation of circadian rhythm and blood pressure, lactation, mood, and cognition. Galanin stimulates activity of the HPA axis, releasing key substances such as corticotropin-releasing factor and adrenocorticotropic hormone (ACTH), thereby enhancing glucocorticoid secretion from the adrenal cortex. In the absence of stimulation of the HPA axis, galanin can stimulate cortisol secretion directly from adrenal cortex cells and stimulate norepinephrine release from the adrenal medulla [72]. Another hormone, ghrelin, has a pronounced effect on the release of ACTH and cortisol in humans and may be involved in the regulation of the HPA response to stress and changes in nutrition/metabolism [73]. Ghrelin production takes places in the gastrointestinal tract with the greatest concentration in the fundus of the stomach. The release of ghrelin modulates the production of neuropeptide Y by arcuate nuclei thus influencing appetite regulation and emotional homeostasis [74].

3.6 Microbial neural substrates Specific gut bacteria can produce neurotransmitters or similar substances such as GABA, glutamate, serotonin, catecholamines, and histamine. These bacteria-derived neurotransmitters can transmit signals to the CNS through enterochromaffin cells and receptors in the enteric nerve. Glutamate and GABA are major excitatory and inhibitory neurotransmitters [75]. Coryneform bacteria, such as Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum, have been utilized for the industrial fermentative production of glutamate [76]. GABA is also produced by gut microbiota such as Lactobacillus brevis and Bifidobacterium dentium. An animal study reported that GABA produced by gut bacteria may act through the BBB and enter the CNS [77]. In other studies, L. rhamnosus reduced anxiety- and depression-related behavior in mice and increased GABA levels in the hippocampus [49,78]. Dopamine and norepinephrine act as neurotransmitters, both of which are synthesized from catecholamine. Environments rich in dopamine and norepinephrine can help bacterial growth. For example, E. coli O157:H7 increases growth in the presence of dopamine

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and norepinephrine [79]. On the other hand, they can be produced by the gut microflora. They are relatively low in the cecum lumen of GF mice, suggesting that gut microbiota is a potential source of catecholamine [80]. Some bacteria species have a gene that expresses a transcript with a sequence similar to tyrosine hydroxylase, which is a rate-limiting enzyme for norepinephrine and dopamine synthesis [81]. However, there is still insufficient evidence that catecholamines produced by microorganisms affect the CNS, because dopamine synthesized at the periphery cannot cross the BBB. Nevertheless, in GF mice, tyrosine levels are low compared to ex-GF mice, which is indirect evidence that gut microbiota increase dopamine levels in the brains of GF mice [82]. This was reproduced by a study that reported that the level of catecholamine was increased more in the brains of GF mice than ex-GF mice, but after the microorganism was colonized, the level was regulated by the turnover of dopamine and norepinephrine in the brain [83]. Histamine modulates the immune response and is a neurotransmitter that regulates activities such as arousal, cognition, circadian rhythm, and neuroendocrine regulation [84,85]. Intestinal microbes can produce histamine and related compounds under physiological conditions [86]. For example, Lactobacillus reuteri expresses a gene for histidine decarboxylase to synthesize histamine [87]. When L. reuteri culture was supplemented with histidine, histamine production was increased as well as histidine decarboxylase expression. L. reuteri also inhibited the production of tumor necrosis factor (TNF)-α, a proinflammatory cytokine, in myeloid progenitor cells through histamine production. In mice, histamine was able to exert antiinflammatory effects on the host by inhibiting the production of IL-18 in the intestine [25]. In contrast to the potential role of histamine in the intestinal tract in the regulation of intestinal immunity, the role of histamine as a neurotransmitter is not well understood.

3.7 Neuropsychiatric disorders affected by brain
gut interplay 3.7.1 Autism spectrum disorder Autism spectrum disorder (ASD) is a group of complex neurodevelopmental disorders characterized by defects in social interaction, communication, and stereotyped behavior. Since gastrointestinal dysfunction is often accompanied by ASD [88], the potential role of the gut microbiota in the pathology of ASD has been suggested. Several recent studies have indicated that patients with ASD showed an altered gut microbiome. According to a recent systematic review, patients with ASD had a lower abundance of Akkermansia, Bacteroides, Bifidobacterium, E. coli, and Enterococcus, a slightly increased abundance of Ruminococcus and Clostridium, and a higher abundance of Faecalibacterium and Lactobacillus [89]. In an animal model of ASD exhibiting a gastrointestinal barrier defect and microbiota alterations, administration of B. fragilis led to the correction of gut permeability and amelioration of ASD symptoms, including defects in communicative, stereotyped, anxiety-like, and sensorimotor behaviors [90]. Therefore the gut microbiota is considered a novel target for the treatment of patients with ASD. Positive results have been reported, although currently inconsistent, based on the improvement in core symptoms of ASD as well as gastrointestinal symptoms [91].

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3.7.2 Schizophrenia Schizophrenia is a serious mental disorder in which patients experience delusions and hallucinations, demonstrate disorganized actions and behavior, and other negative symptoms. Patients with schizophrenia often have comorbidities related to gastrointestinal dysfunction including ulcerative colitis and celiac disease [92,93]. Moreover, an autopsy study of patients with schizophrenia showed an association between the condition and various inflammatory states in the gut that led to structural instability of the intestinal wall. Severance et al. conducted a cohort study and found that the presence of soluble CD14, a marker of the translocation of enteric bacteria, was associated with a threefold increase in the risk of schizophrenia [94]. A study found that gastrointestinal infection with Toxoplasma gondii led to dysbiosis of the intestinal microbiota and an inflammatory state [95]; and a cohort study reported that such an infection may be a risk factor for the development of early-onset schizophrenia [96]. A recent fecal microbiota analysis on a small cohort of first episode psychosis patients showed increased counts of Lactobacillus and Bifidobacterium, which was associated with the severity of psychosis symptoms [97]. Diversity of the gut microbiome was reduced and unique bacterial taxa—including Veillonellaceae and Lachnospiraceae—were associated with disease severity in patients with schizophrenia [98]. In the same study, transplantation of the microbiome extracted from the feces of patients with schizophrenia into mice modulated the glutamate
glutamine
GABA cycle and schizophrenia-related behaviors. In a randomized controlled trial (RCT) of patients with schizophrenia, a 12-week administration of L. rhamnosus and Bifidobacterium lactis Bb12 did not lead to changes in the Positive and Negative Syndrome Scale (PANSS) score, but showed reduction in gastrointestinal dysfunction [99]. In another clinical trial involving patients with schizophrenia, probiotics containing Lactobacilli and Bifidobacterium bifidum markedly improved the general and total PANSS scores, reduced inflammation, and increased the plasma antioxidant capacity as determined by circulating levels of C-reactive protein [100]. In another trial, consumption of Bifidobacterium breve A-1 for four weeks improved the PANSS and anxiety/depression scores, increased interferon-gamma, IL-1R1, IL-10, and IL-22 levels, and reduced the level of TNF-α [101]. IL-22 is associated with maintenance of the intestinal barrier and host defense against bacterial pathogens in the intestine [102], while TNF-β and IL-1R1 are markers of inflammatory response; therefore act in opposition [103]. However, an increase in the levels of IL-10 and a reduction in TNF-α are known to be associated with antiinflammatory effects [104].

3.7.3 Depression Although the causes of depression have not been clearly identified, dysregulation of the endocrine and immune systems have been accepted to play a major role in its pathogenesis. Since a preliminary study showed that regulation of the HPA axis was different in GF mice from that in wild-type mice, it has been widely accepted that the gut microbiota might have an impact on its effect [3,105]. Gastrointestinal dysfunctions such as inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) are often accompanied by depressive symptoms; therefore can serve as epidemiologic evidence pertaining to the influence of gut microbiota on depressive symptoms [106,107].

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gut interplay

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Underlying mechanisms may include the direct or indirect influences of microbial metabolites such as SCFA, or tryptophan on the CNS [108]. The leaky gut hypothesis suggests that infiltration of microbes due to changes in gut permeability causes inflammation, which in turn leads to depression-like symptoms [59]. A recent study demonstrated that markers reflecting gut permeability and inflammatory markers were increased in major depressive disorder (MDD) patients with recent suicidal behavior [109]. Alteration of the gut microbiome has also been reported in patients with depression, and a recent meta-analysis showed that microorganisms belonging to the family Prevotellaceae and genus Coprococcus and Faecalibacterium were reduced in patients with MDD compared to nondepressed controls [110]. In meta-analysis of three RCTs for MDD, limited evidence disallowed the conclusion of whether probiotics were efficacious in dealing with depression, but probiotics administered as an add-on antidepressant was suggested to have beneficial effects [111]. Fecal microbiota transplantation of the gut microbiome obtained from patients with depression into mice revealed more depressive and anxious behavior compared to mice that received the microbiota from healthy humans [105,112].

3.7.4 Parkinson’s disease PD is a neurodegenerative disease in which neuronal cell death is caused by the accumulation of α-synuclein. The cellular hallmarks of PD include the loss of nigral dopaminergic neurons and formation of α-synuclein-enriched Lewy bodies and Lewy neurites. Prior to the onset of neurological symptoms of PD, gastrointestinal dysregulation occurs that leads to a cascade of symptoms, which may result in changes in the relationship between the gut and brain. Braak et al. hypothesized that PD begins in the gut and spreads to the brain via the gut
brain axis [113]. Cell culture analysis has demonstrated that enteric neurons can secrete α-synuclein [114]. It has been confirmed that α-synuclein is transmitted in a time-dependent manner from the distal to the proximal vagus nerve [115]. Therefore it may be hypothesized that α-synuclein produced by any pathological process in the intestine results in spread of the disease to the brain over time. Aggregation of α-synuclein in rats containing bacteria that produce the extracellular bacterial amyloid protein curli can serve as indirect evidence for this hypothesis [116]. Additionally, a cohort study reported lower incidence of PD in patients with duodenal ulcers, in whom the communication pathway between the brain
gut axis was removed via vagotomy [117]. GF mice that overexpressed α-synuclein had relatively fewer toxic brain fibers and less motor symptoms, such as tremors, than mice that had gut microbiota [118]. In particular, a human study reported that the abundance of specific bacterial families was associated with the severity of several movement-related symptoms of PD [119]. Another human study reported that alteration of the gut microbiome in patients with PD was associated with the disease phenotype [22].

3.7.5 Alzheimer’s disease Alzheimer’s disease (AD) is a neurodegenerative disease involving extracellular plaques composed of amyloid-β peptides (Aβ) and intracellular neurofibrillary tangles composed

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of the hyperphosphorylated tau protein. AD is the most common type of dementia. In addition, neuroinflammation is known to play an important role in the pathophysiology of this disease. Infection with the gut bacterium Helicobacter pylori has been linked to the severity of cognitive decline and is considered to have an effect on the course of AD [120]. Antibiotic-treated gut microbiota was reported to influence neuroinflammation and amyloidosis in a mouse model of AD [121]. Microbiota dysbiosis-induced increase in the permeability of the gut and the BBB may mediate or affect the etiology of AD and other neurodegenerative disorders, especially those related to aging. Moreover, bacteria that constitute the gut microbiota can secrete large amounts of amyloid and LPS, which may contribute to the regulation of signaling pathways and production of proinflammatory cytokines associated with the development of AD. Higher levels of proinflammatory cytokines were found in individuals diagnosed with amyloidosis by amyloid positron emission tomography. The same study observed a reduction in the abundance of an antiinflammatory taxon Eubacterium rectale and an increase in a proinflammatory gut microbiota taxon Escherichia/Shigella in these individuals [122]. An observational study that analyzed feces of patients with AD found decreased diversity and different compositions of the gut microbiome [123]. This study identified phyla through genus-wide differences in bacterial abundance, including decreased Firmicutes, increased Bacteroidetes, and decreased Bifidobacterium in the microbiome of participants with AD. The study suggested that changes in the gut microbiome could be a target for therapeutic interventions, and a subsequent RCT in which patients with AD were administered probiotics showed that a 12-week administration of 200 mL/day probiotic milk containing Lactobacillus acidophilus, Lactobacillus casei, B. bifidum, and Lactobacillus fermentum improved cognitive function and metabolic profiles [124]. In another clinical trial, probiotics and selenium were coadministered, which improved cognitive function and metabolic profiles [125]. However, probiotics containing Lactobacillus and Bifidobacterium did not improve cognitive function in patients with severe AD [80]. A meta-analysis of the three aforementioned RCTs concluded that, despite a lack of evidence regarding the effectiveness of probiotics in improving cognitive function in patients with AD, probiotics may have a positive effect on metabolic profiles [126].

3.8 Psychiatric disorders in gastrointestinal diseases The gastrointestinal diseases were largely divided into functional gastrointestinal disorders, inflammatory bowel diseases, and malabsorption syndrome such as celiac disease. Functional gastrointestinal disorders, including IBS and functional dyspepsia, can occur in many cases after an infectious insult, which causes disruptions of gut barrier integrity. Thereafter, the microbiome may contribute to gastrointestinal symptoms by altering the brain
gut axis. In addition to previous studies showing that the gut microbiota of IBS patients exhibits dysbiosis, recent studies have shown that the fecal microbiota of IBS patients can be classified into a different cluster from that of healthy controls [127]. Shah et al. reported that depression and anxiety prevalence was 33% and 19%, respectively, in IBS patients in pooled data from seven case-control studies [128]. Several studies showed anxiety and depression symptoms in IBS patients [128
132]. In particular, a monocentric study showed that on 150 IBS patients, more

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than 50% showed “psychopathological features.” This group presented more severe gastrointestinal symptoms and worse quality of life than that without psychiatric comorbidity. Psychiatric patients also showed a significant impairment of physical state, subjective feeling of well-being, and leisure activities in comparison with no psychiatric patients [132]. Chronic exteroceptive, stress acting on the cortex, can cause abnormal activation of brain areas (particularly the amygdala, hippocampus, and locus coeruleus) and a decreased HPA response, resulting in reduced cortisol release. This results in an increased immune response toward luminal antigens and consequent low-grade inflammation (top-down model). In parallel, interoceptive stress factors such as infections and/or low-grade inflammation may activate ascending pathways (bottom-up model) [133]. IBD, including Crohn’s disease and ulcerative colitis, is characterized by chronic inflammatory reactions in tissues. Several lines of evidence suggest that chronic immune/inflammatory reaction in IBD may be originated from dysbiosis in the gut microbiota [134]. A systematic review reported that the prevalence of ulcerative colitis was 39% in depression and 42% in anxiety across three case-control studies and that it was higher than in the control group [128]. According to a cohort, IBD patients had incidence rates of depression, anxiety disorder, bipolar disorder, and schizophrenia of 1.61 (CI: 1.48
1.75), 1.37 (CI: 1.27
1.47), 1.95 (CI: 1.67
2.29), and 1.51 (CI: 0.99
2.30), respectively, relative to the healthy control group [135]. Besides, in a population-based study, the incidence of bipolar disorder was 2.1 times higher in IBD compared to the healthy control group [136]. Celiac disease is an autoimmune disease caused by an allergic reaction to the gluten proteins in wheat, oats, oatmeal, and barley as the cilia of the intestinal mucosa are lost or deformed. A high incidence of depression was reported in a meta-analysis [137], and a high incidence of suicide was reported in a population-based study [138]. The incidence of bipolar disorder is reported to be not significantly higher than that of the control group in one study [139] but significantly higher in another study [140]. Schizophrenia has been reported to be more prevalent in this disease [93,141].

3.9 Conclusion Mounting evidence indicates that the gut microbiome plays an important role in bidirectional interactions between the gut and the CNS. Brain
gut interaction affects the pathophysiology of neuropsychiatric illnesses. However, many observations of the interaction between the brain and gut have been obtained through animal experiments, and additional human studies are needed to reveal the effect of gut microbiota on the mechanism of human neuropsychiatric illness. Modulation of gut microbiota is in the spotlight for the treatment of neuropsychiatric illness, and the most research is being done on probiotics. According to recently published meta-analyses, probiotics for treatment of neuropsychiatric disorders still lack certain evidence in terms of efficacy. Perhaps there are certain indications that may require proper diagnostic refinement.

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C H A P T E R

4 The role of serotonin and its pathways in gastrointestinal disorders Massimo Bellini1, Matteo Fornai2, Paolo Usai Satta3, Francesco Bronzini1, Gabrio Bassotti4, Corrado Blandizzi2 and Rocchina Colucci5 1

Gastrointestinal Unit, Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy 2Department of Clinical and Experimental Medicine, University of Pisa, Pisa, Italy 3Department of Internal Medicine, Division of Gastroenterology, Brotzu Hospital, Cagliari, Italy 4Gastroenterology and Hepatology Section, Department of Medicine, University of Perugia Medical School, Perugia, Italy 5Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, Italy

4.1 Role of serotonin in the physiology of digestive and extradigestive systems Serotonin (5-hydroxytryptamine or 5-HT) is a monoamine localized mainly in serotonergic neurons of the central nervous system (CNS), enterochromaffin cells (ECs) of the digestive tract, and blood platelets. In 1937, the Italian scientist Vittorio Erspamer first isolated an indolalkylamine from intestinal tissues that he named “enteramine” since it was identified as the main compound released in the gastrointestinal (GI) tract by the ECs [1]. Later on, Page and Rapport isolated a compound from bovine serum, which they named “serotonin” due to its ability of eliciting contractile responses in isolated blood vessels [2]. The structure of 5-HT was characterized by Rapport and colleagues, while Erspamer demonstrated that enteramine was indeed 5-HT. Of the overall body 5-HT content, about 95% is found in the gut, whereas only 5% is distributed in the brain. Nevertheless, until recently, most of the studies on 5-HT were focused on its roles in the CNS, including the control of anxiety, mood, and behavior. At the digestive level, about 90% of 5-HT is localized in ECs, while the remaining 10% is produced by enteric neurons.

The Complex Interplay Between Gut
Brain, Gut
Liver, and Liver
Brain Axes DOI: https://doi.org/10.1016/B978-0-12-821927-0.00009-7

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4. The role of serotonin and its pathways in gastrointestinal disorders

ECs synthesize 5-HT from L-tryptophan (L-TRP), through a first rate-limiting step where the enzyme tryptophan hydroxylase (TPH) converts this amino acid into 5-hydroxy-L-tryptophan (5-HTP). Then, the enzyme L-amino acid decarboxylase generates 5-HT by 5-HTP decarboxylation. The enzyme TPH exists in two isoforms, TPH1, mainly present in ECs, and TPH2 expressed in the CNS and enteric neurons [3]. ECs express mechano- and chemo-sensitive ion channels, ligand-gated ion channels and G-protein coupled receptors, and release 5-HT in response to various mechanical and chemical stimuli. The 5-HT released from ECs takes part to the regulation of various digestive functions, as discussed below. Once released, 5-HT is transported into the surrounding epithelial cells by the serotonin reuptake transporter (SERT) and degraded to the inactive compound 5-hydroxyindoleacetic acid (5-HIAA). SERT is responsible also for the uptake of 5-HT by circulating platelets. The actions of 5-HT in the gut are mediated by several 5-HT receptor subtypes expressed on smooth muscle, enteric neurons, enterocytes, and immune cells [3].

4.1.1 Main functions of 5-HT in extradigestive systems 4.1.1.1 Central nervous system In the CNS, 5-HT is synthesized and released mainly from neurons originating from the raphe nuclei of the brainstem. These serotonergic neurons are widely distributed throughout the mammalian brain, making the serotonergic system the largest and most complex efferent system of the CNS [4]. Accordingly, the functions regulated by 5-HT in the CNS are very broad, affecting physiology, cognition, and behavior. The rostral, dorsal, and medial nuclei of the serotonergic system innervate the CNS diffusely, and their projections participate to the regulation of temperature, appetite, sleep cycles, emesis, and sexual behavior. The caudal nuclei send projections to the spinal cord and modulate the nociception and motor tone. Every cell in the brain is in close proximity to a serotonergic fiber, and all CNS regions express 5-HT receptors. Individual neurons have been shown to express different 5-HT receptors, which mediate distinct actions of 5-HT [4]. All serotonergic receptor subtypes have been found to be expressed in the CNS (Table 4.1). In addition, the serotonergic system interacts with other important neurotransmitter systems, such as the catecholaminergic system, influencing further a variety of functions. 5-HT has been implicated also in the development of the CNS, acting as a growth factor regulating the proliferation, organization and maturation of the developing brain. Despite the high number of functions regulated by 5-HT at the CNS level, the most clinically relevant aspect has been its involvement in the pathophysiology of neuropsychological disorders, including schizophrenia, depression, and alcoholism. 4.1.1.2 Platelets 5-HT is deeply involved in the regulation of various vascular functions, including the control of vascular resistance, blood pressure and platelet functions. The majority of circulating 5-HT in the bloodstream is stored in blood platelets. Despite platelets cannot synthesize 5-HT, due to the lack of the enzymes needed for its biosynthesis, circulating platelets can easily uptake 5-HT from the blood and store it in their δ-granules, ready to be released

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4.1 Role of serotonin in the physiology of digestive and extradigestive systems

TABLE 4.1 Main central and peripheral effects mediated by serotonergic receptors. System

Main receptor subtypes

Function

References

Central All 5-HT receptor nervous system subtypes

Regulation of body temperature, appetite, sleep cycles, emesis, sexual behavior, nociception, motor tone, cognition, mood, behavior

[4]

Platelet

5-HT2A

Platelet aggregation

[5]

Cardiovascular system

5-HT1B, 5-HT2A

Arterial contraction

[6]

5-HT1B, 5-HT2B, 5-HT4, 5-HT7

Arterial relaxation

5-HT1B, 5-HT2A

Vein contraction

5-HT2 5-HT1, 5-HT3, 5-HT4 5-HT3, 5-HT4

Mucus secretion, ion fluxes

Secretion/absorption

5-HT3

Emesis

[8]

5-HT3, 5-HT4

Peristaltic activity

[3,9
12]

5-HT3, 5-HT4, 5-HT7

Secretion of proinflammatory cytokines by activated monocytes

[4]

5-HT7

Proliferation and activation of T cells

5-HT7

Dendritic cell-mediated proinflammatory T cell response

5-HT1, 5-HT2, 5-HT3, 5-HT7

Proliferation and early phase activation of B cells

Gut

Immune system

[3,7]

in response to platelet activating stimuli [5]. Upon platelet activation, 5-HT released from dense granules can trigger the recruitment of circulating platelets, thereby enhancing thrombus formation and growth. This process is mediated by 5-HT2A receptors, expressed on the surface of circulating platelets [5] (Table 4.1). The activation of 5-HT2A receptors triggers the Gq protein signaling via the phospholipase C (PLC) β pathway, which, in turn, leads to intracellular Ca21 mobilization from the Ca21 stores through the inositol 1,4,5-triphosphate receptor (IP3R) and mediates diacylglycerol (DAG)-dependent protein kinase C (PKC) activation, thereby amplifying platelet reactivity. Following Ca21 release and PKC activation, the integrin αIIbβ3 is exposed and activated on the platelet surface, thus promoting platelet aggregation and thrombus formation [5]. 4.1.1.3 Blood vessels The human cardiovascular system is exposed to plasma 5-HT, usually released from platelets. In human arteries, the use of selective antagonists along with molecular biology investigations has allowed the identification of different 5-HT receptor subtypes that mediate vasoconstriction. Human arterial vasoconstriction elicited by 5-HT is mediated by 5-HT1B and 5-HT2A receptors that are coexpressed in various arterial districts, including coronary,

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pulmonary, temporal and occipital arteries. Moreover, 5-HT-induced contractions of human mesenteric, internal mammary, umbilical, omental and uterine arteries appear to be mediated also by 5-HT1B and 5-HT2A receptors (Table 4.1). One exception is represented by intracranial arteries, which appear to contract exclusively through 5-HT1B receptors [6]. In humans, arterial relaxation by 5-HT appears to be mediated mainly by endothelial 5-HT1B, 5-HT2B and possibly 5-HT4 receptors. Smooth muscle 5-HT7 receptors could be involved also in human vascular relaxation, although direct evidence from human blood vessels is scarce. Indeed, most of current data have been obtained from isolated human smooth muscle and endothelial cells [6] (Table 4.1). Data on the role of 5-HT in the control of vein contractions are scarce. Overall, it appears that 5-HT is able to elicit contractile responses in various venous districts, including pulmonary, hand, saphenous and umbilical veins. These actions are likely mediated mainly by 5-HT1B receptors and, in some cases, 5-HT2A receptors [6] (Table 4.1).

4.1.2 Serotonin in gut physiology 5-HT is deeply involved in the regulation of secreto-motor and sensory functions of the gut through different receptor subtypes expressed throughout the GI tract. The various responses, including nausea, vomiting, intestinal secretion, and peristaltic activity are regulated by 5-HT released from ECs and enteric neurons [3,7]. 4.1.2.1 Secretory functions Mucus secretion and ion fluxes in the GI tract are regulated by the release of 5-HT. These effects are mediated by epithelial 5-HT2 and neuronal 5-HT1, 5-HT3 and 5-HT4 receptors. 5-HT2 receptors, expressed at colonic mucosal level, are involved in the intestinal electrolyte transport. Moreover, 5-HT3 and 5-HT4 receptors regulate also 5-HT-mediated intestinal secretion and absorption [3,7] (Table 4.1). 4.1.2.2 Emesis Emesis is a complex process, which involves both peripheral and central sensory inputs coordinated with both somatic and visceral motor outputs. The main components of the emetic reflex include a portion of the area postrema, known as the chemoreceptor trigger zone, located in the medulla oblongata of the brainstem, as well as visceral and somatic motor nuclei. Among various mediators, 5-HT has been recognized as a central player in the induction of emesis. Indeed, several lines of evidence have shown that 5-HT3 receptors are abundantly expressed in the area postrema, and that selective 5-HT3 receptor antagonists can prevent and counteract vomiting [8] (Table 4.1). 4.1.2.3 Gut motility 5-HT plays a prominent role in the regulation of gut motility. Agonists of 5-HT3 and 5-HT4 receptors can enhance the peristalsis, while their antagonists can inhibit the mucosal stimuli-induced peristaltic reflex; 5-HT secreted by ECs stimulates both extrinsic and intrinsic enteric neurons, eliciting motor responses [9
12]. Bulbring and colleagues first proposed that 5-HT released from mucosal ECs plays a role in initiating the peristalsis. On

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the other hand, the idea of an involvement of 5-HT released from neurons, rather than from ECs, in the constitutive GI motility was suggested following the observation that the deletion of TPH1 exerted the same effect on GI motility as the simultaneous deletion of both TPH1 and TPH2. However, recent studies on TPH1-deficient mice showed that mucosal 5-HT plays a role in the onset of peristaltic motor responses elicited by mucosal stimulation [3]. 5-HT takes part also to the regulation of segmentation motor patterns, consisting of alternating contractions of the smooth muscular layers. Segmentation aids to mix small bowel contents and increase their exposure to digestive enzymes. 5-HT3 and 5-HT4 receptors appears to be mainly involved in the regulation of segmentation patterns, as selective receptor antagonists inhibited the motor responses induced by the SERT inhibitor fluoxetine [9
12] (Table 4.1). 5-HT can regulate indirectly enteric smooth muscle contractions via the stimulation of cholinergic neurons, and smooth muscle relaxations through the activation of inhibitory nitrergic neurons [3]. 4.1.2.4 Immune system 5-HT receptors are expressed on almost all immune cells of humans and rodents. During acute inflammation, 5-HT promotes the recruitment of innate immune cells, including immature dendritic cells (DCs), monocytes, mast cells, neutrophils, and eosinophils in the inflammation site. 5-HT regulates the innate immune response of colonic epithelial cells through the production of NADPH oxidase 2 (Nox2)-derived transient reactive oxygen species. Indeed, 5-HT was shown to upregulate inflammatory cytokines and adhesion molecules, while decreasing adherens junction molecules, such as E-cadherin, thus disrupting the epithelial barrier and favoring the translocation of luminal microbiota through the intestinal mucosa. In addition, the exposure of human blood monocytes to 5-HT resulted in a decrease in TNF production via 5-HT4 receptors [4]. However, once activated with lipopolysaccharide and stimulated with 5-HT, these cells increased the secretion of IL-1β, IL-6, IL-8 via 5-HT3, 5-HT4, and 5-HT7 receptors. 5-HT is able also to stimulate the proliferation of natural killer cells and to protect these cells from oxidative damage. DC are subjected also to control by 5-HT via 5-HT7 receptors. In particular, DC increase proinflammatory T cell responses and enhance the ability of macrophages in promoting the activation of T cells. 5-HT, via 5-HT7 receptors, phosphorylates extracellular signal-related kinase-1 and -2 (ERK1/2) and promotes the proliferation and activation of T cells. In mouse and rat models, 5-HT influences the proliferation and early phase activation of B cells, via the recruitment of 5-HT1, 5-HT2, 5-HT3 and 5-HT7 receptors [4] (Table 4.1). Taken together, these observations suggest a regulatory role of 5-HT in both innate and adaptive immune responses during gut inflammation.

4.2 5-HT receptors, serotonin transporter, and their polymorphisms 4.2.1 5-HT receptors and serotonin transporter In the GI tract, 5-HT regulates motor and sensory functions by seven families of 5-HT receptors, which differ on the basis of structure, function, and signaling mechanisms [13].

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The actions of 5-HT are terminated by its intracellular reuptake operated by the SERT, which is encoded by a single gene (SLC6A4) located on chromosome 17q11 and comprising 14 exons [14]. There is a 44-bp insertion/deletion in the 50 -flanking promoter region of the SERT gene, which generates a short and a long allele (rs4795541). The short (S) allele appears to account for lower transcriptional efficiency and lower reuptake of 5-HT than the long allele (L) [15]. Another region of interest in the SERT gene is a variable number of tandem repeat polymorphism in the second intron, designated as STin2. This polymorphism consists of a17-bp variable number of tandem repeats with four different alleles that correspond to the number of tandem repeats (12, 10, 9, or 7), the 12 and 10 being the most frequently observed [16]. The 12-repeat allele has been reported to be a transcriptional enhancer and is associated with increased SLC6A4 expression [17]. In subjects with low expression of SLC6A4, there may be a greater saturation of SERT when exposed to selective serotonin reuptake inhibitors (SSRIs), with a consequent increase in both central and peripheral 5-HT availability [18]. The current classification of 5-HT receptors [19], based on functional, structural, and transductional information, including data obtained by recombinant and native receptors, implies that the designation of receptor should be applied only to entities for which all three classes of information are available. Based on these criteria, currently seven families of 5-HT receptors have been recognized, and more than 14 subtypes have been identified in humans. Receptor subtypes labeled with lower case designate receptors that lack a conclusive demonstration of functioning in native systems (e.g., 5-ht1E, 5-ht5A, 5-ht5B). With the exception of 5-HT3 receptor, all other 5-HT receptors are members of the seven transmembrane domain G protein-coupled receptor family. The 5-HT3 receptor is a ligandgated ion channel belonging to the Cys-loop superfamily of pentameric proteins [20]. Overall, the 5-HT receptor subtypes presently included in the 2019 IUPHAR classification are: 5-HT1A, 5-HT1B, 5-HT1D, 5-ht1E, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3A, 5HT3B, 5-HT3C, 5-HT4, 5-ht5A, 5-ht5B, 5-HT6 and 5-HT7. In humans, the 5-HT7 receptor includes three subtypes, designated as 5-HT7(a), 5-HT7(b), and 5-HT7(c) (Table 4.2). 4.2.1.1 5HT1 receptor The 5-HT1 receptor family comprises five subtypes (5-HT1A, 5-HT1B, 5-HT1D, 5-ht1E, and 5-HT1F) coupled preferentially to Gi/o proteins leading to the inhibition of cyclic adenosine monophosphate (cAMP) formation. They are located mainly in the CNS and some on blood vessels (5-HT1B/D). They function mainly as inhibitory presynaptic TABLE 4.2 Current classification of 5-HT receptors. 5-HT1

5-HT2

5-HT3

5-HT4

5-HT5

5-HT6

5-HT7

5-HT1A

5-HT2A

5-HT3A

5-HT4

5-ht5A

5-HT6

5-HT7

5-HT1B

5-HT2B

5-HT3B

5-HT1D

5-HT2C

5-HT3C

5-ht5B

5-ht1E 5-HT1F

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receptors to induce neurotransmission inhibition and vasoconstriction through inhibition of adenylate cyclase. The 5-HT1A gene sequence is intronless and codes for a 422, 421, and 422 amino acid product in human, rat, and mouse, respectively. The human 5-HT1A receptor is located on chromosome 5q11.2
q13. No splice variants have been identified. Two polymorphisms, Gly22-Ser and Ile28-Val, have been found to modify the extracellular amino terminal region of the receptor [21]. The polymorphism Arg219-Leu has been associated with the Tourette’s syndrome. The polymorphism Ala50-Val, occurring in the transmembrane region 1, results in a loss of response to 5-HT [22]. The 5-HT1A subtype is largely distributed in the brain being expressed in limbic brain areas, hippocampus, lateral septum, cortical areas, and mesencephalic raphe nuclei. It plays important roles in the control of mood and behavior. 5HT1 knockout (KO) mice display sleep dysregulation, reduced learning ability and increased responsiveness to stress [23]. GI 5-HT1A receptors were identified in the guinea pig myenteric plexus where they function as inhibitory modulators of fast excitatory postsynaptic potentials. The human 5-HT1B receptor gene is located on chromosome 6q13, codes for a protein of 390 amino acids and is coupled negatively to cAMP production or calcium stimulation [24]. There are no splice variants. Single nucleotide polymorphisms (SNPs) in position 861 have linked the 5-HT1B receptor with various disorders, such as alcoholism, substance abuse, attention deficit hyperactivity disorder, aggression, and depression [25,26]. At the central level, 5-HT1B receptors act mainly as terminal autoreceptors [26] being expressed abundantly in the basal ganglia, especially in the substantia nigra, globus pallidus, ventral pallidum, and entopeduncular nucleus, but also in a number of vessels. Indeed, together with the 5-HT1D subtypes, they are expressed on cerebral blood vessels and are the targets for tryptans, employed in the management of migraine [27]. Peripheral actions mediated by 5-HT1B receptors have been described, such as the inhibition of noradrenaline release in the vena cava and the contraction of rat caudal arteries. The 5-HT1D receptor consists of 377, 374, and 374 amino acids in humans, rats, and mice, respectively. There are no splice variants, and the human 5-HT1D gene is located on chromosome 1p36.3
p34.3. Its polymorphisms have been associated with an increased susceptibility to substance abuse and linked to anorexia nervosa [28]. 4.2.1.2 5-HT2 receptor This family includes the subtypes 5-HT2A, 5-HT2B, and 5-HT2C, which are expressed in the CNS and many peripheral sites, particularly blood vessels, platelets, and autonomic neurons. They are coupled preferentially to Gq/11 to increase inositol phosphate and cytosolic calcium [29]. On neurons and smooth muscle, 5-HT2 receptors mediate excitatory responses, but they can elicit also blood vessel dilation by nitric oxide release from endothelial cells. The 5-HT2A receptor gene is located on human chromosome 13q14
q21 and codes for a protein of 471 amino acids [30]. Nonfunctional splice variants have been reported. A polymorphism in the promoter region has been associated with the responses to clozapine in schizophrenic patients [31]. The 5-HT2A receptor KO mouse shows behavioral sensitization to amphetamine [32]. The 5-HT2B receptor mRNA is found in the rat fundus, gut, heart, kidney, lung and brain, and its activation has been implicated in the pathophysiology of hyperphagia. The suppression of 5-HT2B receptor in mice is lethal and

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results in severe embryonic defects [33]. By contrast, the overexpression of 5-HT2B receptors in mice causes abnormal mitochondrial function and cardiac hypertrophy [34]. The 5HT2C receptor consists of 458
460 amino acids coded by a gene, mapped to the human chromosome Xq24, endowed with a highly complex exon-intron structure. The 5-HT2C receptor, which is coupled to PLC activity, can yield 32 different mRNAs and 24 different proteins by means of mRNA editing or splicing. There are also two nonfunctional short splice variants in addition to the multiple RNA editing variants [35]. The different editing variants are characterized by decreasing degrees of affinity and intrinsic activity for 5-HT and other agonists, as well as reduced constitutive activities and altered coupling to G proteins. A -759T allele within the promoter region of the 5-HT2C gene has a higher transcriptional activity than the more frequent -759C allele, which may lead to a higher basal expression of 5-HT2C receptors and subsequent protection against antipsychotic-induced weight gain. The 5-HT2C receptor KO mouse is severely obese with defective food intake regulation and can experience fatal seizures. The expression of 5-HT2C subtype has been detected in some thalamic nuclei, lateral basal ganglia and particularly in the subthalamic nucleus and substantia nigra [36]. 4.2.1.3 5-HT3 receptor 5-HT3 receptors are ion channels, which allow the entry of cations, causing cell membrane excitation. They are located on nociceptive afferent neurons and enteric neurons in the peripheral nervous system. Once activated, they mediate strong excitatory responses and regulate both motility and secretion throughout the GI tract [20]. Excitation of many types of vascular, pulmonary and cardiac sensory nerves can take place following 5-HT3 receptor activation. They are expressed also in the CNS level, particularly in the area postrema, where they are involved in the initiation and coordination of the emetic reflex. 5-HT3 receptors are structured as a pentameric complex, with five different subunits (5-HT3A-E) [37], each encoded by specific subunit genes [38]. These subunits can form homomeric (all 3A subunits) or heteromeric (mixture of 3A and 3B, 3C, 3D, or 3E subunits) functionally active channels [39]. The 5-HT3A subunit seems to play a key role in the channel assembly, since it is the only subunit that can generate functional homopentameric channels. The other subunits can form only functional heteromeric channels containing the 5-HT3A subunit [37]. The 5-HT3A subunit is expressed throughout all colonic layers, whereas the 5-HT3E subunit is present predominantly in the mucosa, and the 5-HT3B in the muscularis [38]. SNPs of the 5-HT3 receptor subunits have been linked to some clinical disorders, such as chemotherapy-induced nausea and vomiting, irritable bowel syndrome (IBS), bipolar disorders, schizophrenia, and alcohol dependence [39]. 4.2.1.4 5-HT4 receptor They are expressed in the brain and peripheral organs, such as the GI system, bladder, and heart. The activation of these receptors causes excitatory responses, through the stimulation of adenylyl cyclase, resulting in an increased gut motility. The 5-HT4 receptor gene was mapped to chromosome 5q31
33, and initially two splice variants were identified. However, later on, a high complexity of the 5-HT4 receptor gene was highlighted. Indeed, the gene consists of 700 kb and 38 exons, which can give rise to a number of splice variants in the majority of mammalian species including humans. Various 5-HT4 variants,

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generated by alternative splicing, are widely distributed throughout the body and, at present, at least 11 human 5-HT4 receptor splice variants have been reported to be expressed in the digestive tract at level of both smooth muscle layers and mucosa [38]. Some of these receptor variants are endowed with high levels of constitutive activity, while others are coupled to different transduction mechanisms. For instance, besides the adenylate cyclase stimulation, a direct coupling to potassium channels and voltage-sensitive calcium channels has been proposed [20]. The 5-HT4(a) and 5-HT4(b) receptors are the only variants found to be expressed in the bladder and kidney, respectively. Apparently, the 5-HT4(d) receptor is the sole variant expressed in the intestine [40]. It has been suggested that most 5-HT4 receptor variants may display distinctive functional properties, and that changes in the splicing pattern could be associated with pathological conditions [39]. Since the 5-HT4 receptor undergoes desensitization through the GRK-arrestin system, it has been hypothesized that the functional diversity of 5-HT4 splice variants in different regions of the colon could arise from differences in the C-terminal domain of the receptor protein, that is deputed to interact with the GRKarrestin system during the desensitization process [41]. The 5-HT4 receptor KO mouse is characterized by a normal behavior in a standard environment, but displays a very low locomotor activity accompanied by hypophagia in response to situations of novelty and stress. Furthermore, the 5-HT4 KO mouse has increased sensitivity to the proconvulsant pentylenetetrazole, which might be related to the expression of 5-HT4 receptors on central gammaaminobutyric acid (GABA)ergic neurons [42]. There is evidence suggesting that gene polymorphisms could be important with regard to mRNA interactions with microRNAs (miRNAs), which are short noncoding RNA molecules of 18
25 nucleotides involved in the posttranslational regulation of mRNA expression, and that impaired miRNA-mRNA interactions could contribute to disease etiology [43]. In this regard, some interesting observations have been made on a SNP in the 5-HT4 receptor gene that has been identified in a small proportion of patients with diarrheapredominant IBS (D-IBS) [43]. Indeed, this SNP involves a binding site for the miRNA-16 family and miRNA-103/miR-107. In particular, the interactions of 5-HT4 receptor mRNA with miR-16 family and miRNA-103/miRNA-107 have been implicated in the control of gut barrier function and motility [43]. In further studies, the same authors showed a colocalization of miRNA-16 and 5-HT4 receptor mRNA in the colon, thus supporting a role for miRNA-16 in the regulation of this serotonergic receptor [43]. Since these miRNAs appear to downregulate the 5-HT4 receptor, which is known to act as a main regulator of gut motility, it is not surprising that the levels of miRNA-16 and miRNA-103/miRNA-107 correlated inversely with bowel frequency [43]. Overall, these findings suggest that specific miRNA, and not just the 5-HT4 receptor itself, could be explored as molecular targets for future drug discovery in IBS [43]. 4.2.1.5 5-HT5 receptor Together with 5-HT6 and 5-HT7 receptors, 5-HT5 receptors are currently among the less characterized 5-HT receptors. Since functional evidence in native systems is still sparse, the members of this receptor family are designated with lower cases. They include the 5-ht5A and 5-ht5B subtypes. The 5-ht5A subtype consists of 357 amino acids across the species. In humans, it is located on chromosome 7q36.1 and has no splice variants [44]. The human recombinant 5-ht5A receptors inhibits the forskolin-stimulated cAMP

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production, even though this receptor can couple also positively to cAMP and inositol phosphate production [45]. 5-ht5A mRNA expression in the human brain is localized mainly to the cerebral cortex, hippocampus and cerebellum. The 5-ht5A receptor KO mouse shows increased exploratory activity in a novel environment, and is less reactive to LSD, which has high affinity to this receptor. 5-ht5A receptors seem to play a role in the acquisition of adapted behavior under stressful situations [29]. 4.2.1.6 5-HT6 receptor The human 5-HT6 receptor gene has been mapped to the chromosome region 1p35
p36 and codes for 436
440 amino acids. The receptor is coupled positively to adenylate cyclase via a Gs protein. It is expressed mainly at central level, where moderate levels have been found in the hippocampal formation and cerebral cortex, thalamus, hypothalamus, and substantia nigra; very low levels in the globus pallidus, cerebellum, other mesencephalic regions, and the rhombencephalon [46]. A truncated, nonfunctional 5-HT6 receptor, with a 289 bp deletion of the region coding for the transmembrane IV domain and the third intracellular loop, has been identified in the caudate and substantia nigra of the human brain [47]. 5-HT6 receptor activation modulates glutamate, GABA, dopamine and noradrenaline release. Furthermore, the control exerted by 5-HT6 receptors on the cholinergic neurotransmission makes this receptor a putative target for the management of cognitive disorders, such as Alzheimer’s disease [29]. 4.2.1.7 5-HT7 receptors They are members of the G protein-coupled receptor family, which modulate positively cAMP formation [48] and are encoded by a gene located on the human chromosome 10q21
q24 [49]. The human 5-HT7 receptor consists of 479 amino acids, encoded by a cDNA containing two introns: one located in the second intracellular loop and the second one in the predicted intracellular carboxylic ending [49,50]. Alternate splicing of the latter intron has been reported to generate at least four 5-HT7 receptor variants (5-HT7a to 5-HT7d), which differ in their C-termini and vary among the species [51], while no differences have not been shown in their respective pharmacology, signal transduction or tissue distribution. A human 5-HT7(a) receptor polymorphism (Thr92-Lys) is associated with reduced agonist binding affinities. Since 5-HT7 receptors are highly expressed in the brain with high densities in the thalamic nuclei, limbic and cortical regions, and lower densities in the substantia nigra, hypothalamus, central grey, and dorsal raphe nuclei [52], it has been suggested that the receptor plays a role in the control of sleep, circadian rhythmic activity and mood [29]. 5-HT7 KO mice show reduced immobility in the forced swim test, suggesting an “antidepressant-like” phenotype [53]. 5-HT7 receptors share with 5-HT4 an identical sequence up to Leu 432 and differ C-terminal with the PDZ C-terminal domain ligand binding site, which is a common structural domain of 80
90 amino acids necessary for proper receptor protein anchoring to cell surface [54]. Therefore 5-HT7 may display similar characteristics with the 5-HT4 receptor in terms of desensitization, internalization, trafficking, and signaling. It has been proposed that an abnormal stimulation of 5-HT7 receptors might contribute to IBS-like symptoms. In addition, it has been suggested that the 5-HT7d splice variant is expressed at relatively

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low levels in tissues such as the brain and spleen, while being preferentially distributed in the intestine [55].

4.2.2 Serotonergic system, polymorphic variants and functional gastrointestinal diseases With regard for the 5-HT receptor polymorphic variants and functional GI diseases (FGIDs), the 5-HT3 receptor has gained a special interest among the seven families of 5-HT receptors, since 5-HT3 receptor antagonism can control dyspeptic symptoms and anxiety relief. It is located in the CNS and enteric neurons, as well as in the mucosal terminals of extrinsic primary afferents. A functional polymorphism, c.-42C . T (rs1062613), has been identified in the 5-HT3A gene. The T allele promotes an increased transcription of 5-HT3A mRNA, resulting in enhanced production of the 5-HT3A subunit [56]. Of note, the c.-42C . T polymorphism has been associated with depressive disorder, anxiety-related trait harm avoidance [57], and IBS, a FGID in which visceral hypersensitivity plays an important role and comorbidity with anxiety and depression is often present [58]. Mujakovic et al. [59] reported a possible relationship between the genetic variations of serotonergic system and the severity of functional dyspepsia. Patients were genotyped for the 5-HT3A c.-42C . T SNP and the 44 bp insertion/deletion polymorphism of the SERT promoter (5-HTTLPR). 5-HT3A c.-42 . T allele carriers were more prevalent in patients with severe dyspepsia. This association appeared to be stronger in females and homozygous patients for the long (L) variant of the 5-HTTLPR polymorphism. The 5-HT3A c.-42T allele was associated with severe dyspeptic symptoms. The stronger association among patients carrying the 5-HTTLPR L allele suggested an additive effect of the two polymorphisms with dyspeptic symptoms. These results support the hypothesis that a decrease in 5-HT3-mediated antinociception predisposes to increased visceral sensitivity in the GI tract. The C/C genotype of the c.-42C . T SNP has been found to be associated with amygdala responsiveness in IBS patients [58]. Moreover, the 5-HT3A c.-42C . T and 5-HTTLPR polymorphisms likely represent predisposing genetic variants in common to psychiatric morbidity and dyspepsia. Another study investigated some of polymorphisms in the untranslated regions of 5-HT3A [-42C . T; -25C . T (currently -7C . T); *70C . T (currently *70C . A); *503C . T; Caucasian patients; 99 with constipation predominant IBS (C-IBS) and 217 D-IBS patients; Rome II/III and 5-HT3E (*76G . A, *115T . G, *138C . T, *191T . C; Caucasian patients; 95 C-IBS, 143 D-IBS; Rome II/III). In this study, *76G . A and -42C . T were associated with D-IBS. However, *76G . A was positively associated only in female patients [60]. A recent study [61] found that D-IBS patients with a SNP in 5-HT3C (rs6766410) displayed a better response to treatment with ondansetron (5-HT3 receptor antagonist) and had increased mucosal concentrations of the 5-HT metabolite 5-HIAA along with an increased 5-HIAA/5-HT ratio, suggesting a greater turnover of mucosal 5-HT in D-IBS patients. Several studies have investigated the relationship between SERT polymorphisms, behavioral and psychological disorders [62,63], and IBS [64]. In a recent study, the association of polymorphisms in 5-HT-related genes with depressive or anxiety disorders in IBS

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patients was assessed. IBS patients with depressive disorders were characterized by higher frequency of the 5-HTTLPR L allele as compared to IBS patients with anxiety disorders. A lower frequency of the 1438A allele of the 5-HT2A gene was found in IBS patients with depressive disorders in comparison to IBS patients without psychopathological disorders. A lower G allele frequency of the 5-HT2C rs6318 polymorphism among IBS patients with anxiety disorders was also observed, suggesting that the 5-HT2A rs6311 polymorphism may be associated with susceptibility to anxiety disorders in IBS patients. Camilleri et al. [65] investigated a series of polymorphisms in 5-HT receptors and SERT, and found a lack of association for most of the candidate genes with FGID, including 5-HT1A Pro16Leu (47C . T), 5-HT1A Gly272Asp (818G . A), 5-HT2A -1438G/A (currently -998G . A), 5-HT2C Cys23Ser (68G . C), and 5-HTTLPR. Recently, Grzesiak et al. [66] assessed the association of HTTLPR (rs4795541), 5-HT1A (rs6295), 5-HT2A (rs6313 and rs6311), 5-HT2C (rs6318), and TPH1 (rs1800532) in patients with IBS and depressive or anxiety disorders. IBS patients with depressive disorders were characterized by higher frequency of the 5-HTTLPR L allele as compared to IBS patients with anxiety disorders. A lower frequency of the 1438A allele in the HTR2A gene was found in IBS patients with depressive disorders in comparison to IBS patients without psychopathological disorders. A lower G allele frequency of the HTR2C rs6318 polymorphism was observed also among IBS patients with anxiety disorders. A recent metaanalysis [67], including 25 studies on a total of 3443 IBS cases and 3359 controls (Rome I/II/III), concluded that the 5-HTTLPR L allele and L/L genotype have a significant impact on C-IBS development, and that this association is evident in the East Asian population, but not in Caucasian, Iranian, Turkish, and Indian populations. These results were in contrast with the analysis made in a previous meta-analysis by Areeshi et al. [68], who found no positive associations for the 5-HTTLPR variants. Some studies have examined the possible association of the SERT gene polymorphism STin2 with IBS. However, several groups [42,69] did not found any association of this polymorphism with IBS or its clinical variants (patients with undetermined ethnicity, 26 C-IBS, 18 D-IBS,10 mixed IBS, Rome I; mainly Caucasians, 42 C-IBS, 98 D-IBS, 36 mixed IBS, Rome II; Caucasians, 99 C-IBS, 97 D-IBS, Rome II). In addition, Kohen et al. [70] extended their search to 179A . G (currently-1936A . G), a SNP located immediately upstream of 5-HTTLPR with opposite effects on SERT expression, showing a positive association of the G allele with IBS. A SNP in the 5-HT3E subunit (c.76G . A) was found to be associated with D-IBS in females. Of interest, this variant was found to confer resistance to the inhibitory effect of miRNA-510 on 5-HT3 mRNA, leading to an increased expression of the 5-HT3E protein in D-IBS patients. This observation suggests that precursors of miRNA-510 might exert a protective effect against D-IBS in individuals without the variant allele of c.76G . A [60]. In another study, Zhou et al. [71] evaluated miRNA expression in blood microvesicles (circular membrane fragments shedding from cell surfaces) and gut tissues in D-IBS patients, who exhibit increased intestinal membrane permeability and glutamine synthetase deficiencies, and found that an increased expression of miRNA-29 leads to decreased glutamine synthetase levels which regulates intestinal membrane permeability resulting in a positive correlation with an increased permeability in D-IBS female patients.

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4.3 Serotonin in functional gastrointestinal disorders and the brain
gut axis 4.3.1 Serotonin in functional gastrointestinal disorders In the setting of inflammatory bowel disease [72,73], 5-HT is involved in the activation and maintenance of the inflammatory process exerting proinflammatory actions. Conversely, the inflammation increases the 5-HT activity, reduces its reuptake and leads to an increase in the number of ECs [74]. However, the digestive diseases where the role of 5-HT is fairly more evident are the FGIDs, which include a number of clinical conditions diagnosed on the basis of specific symptoms in the absence of tissue or biochemical abnormalities [75]. Several mechanisms have called into play in the pathophysiology of FGID, such as diet, genetic factors, infections, inflammation, immune system alterations, brain
gut axis disorders, changes in the gut microbiota and abnormalities of 5-HT metabolism [76
79]. Indeed, 5-HT homeostasis has been shown to be altered in patients with FGIDs [78]. In patients with gastroesophageal reflux disease, 5-HT appears to reduce the integrity of the barrier function of the esophageal squamous epithelium through a modulation of the levels of tight-junction proteins [80], making the esophagus more susceptible to mucosal damage. For this reasons, it has been suggested that patients with FGIDs and GERD could be treated with SSRIs [81,82]. 5-HT appears to play a pivotal role in the pathophysiology of IBS through a variety of actions exerted at the level of CNS and the enteric nervous system (ENS) [83,84], as suggested also by the therapeutic effects of tricyclic antidepressants and SSRIs in this setting [85,86]. Some evidence about the role of 5-HT in IBS stems from studies showing a persistent increase ECs in individuals with postinfectious (PI)-IBS, as compared to individuals exposed to acute enteric infection with subsequent full recovery [87]. A study that assessed 5-HT concentrations in platelet-depleted plasma showed reductions of 5-HT release in patients with C-IBS after a standard meal, as compared to PI-IBS and healthy individuals, and a higher peak of 5-HT postprandial levels in PI-IBS as compared to C-IBS or healthy controls. This study reported also a normal 5-HIAA/5-HT ratio in C-IBS, but a reduced ratio in D-IBS, suggesting that patients with D-IBS might have a reduced capacity of 5-HT reuptake, while those with C-IBS might have an impaired 5-HT release [88]. Similar results were obtained by other authors in D-IBS, C-IBS, and healthy controls [83]. More recently, it was observed that 5-HT reuptake was reduced in D-IBS patients with decreased SERT mRNA levels in the duodenal mucosa [89]. High plasma 5-HT levels have been reported in patients with D-IBS as compared to controls [90,91]. Spiller et al. [92] observed an increased density of ECs in rectal biopsies from D-IBS patients. In contrast, relatively low postprandial 5-HT plasma levels were reported in patients with C-IBS [88]. The different successful therapeutic interventions in IBS having different 5-HT receptor as a target add further support to the notion that 5-HT plays a relevant role in the pathophysiology and the clinical features of IBS, at least in specific subgroups of patients [9,93,94]. The involvement of serotonergic system in digestive diseases extends also to the role played by TPH and SERT. Mice deficient in TPH1 have been shown to be protected against several diseases, including hemostasis disorders, inflammatory, fibrotic, metabolic disorders and GI diseases. Nevertheless, no obvious GI phenotypes were observed in early

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studies on TPH1 deficient mice [95
98], and only in one study slight GI motility disorders were reported [99]. On the other hand, GI motility was clearly impaired in TPH2-deficient mice lacking 5-HT in the ENS [95,96]. Therefore 5-HT synthesis in the gut by both TPH isoforms could be an interesting target for treatment of GI diseases, and, in this respect, the lack of selectivity of most TPH inhibitors could be an advantage. Recently, the first nonselective TPH inhibitor, telotristat ethyl, was launched on the market for treatment of the carcinoid syndrome [100]. In some mouse models of bowel inflammation telotristat ethyl was shown to be effective [101,102]. Moreover, in a phase II clinical trial, LX-1031, a TPH inhibitor unable to cross the intestinal barrier, was shown to relieve abdominal symptoms in D-IBS [103,104]. The extent of 5-HT reuptake occurring in the extracellular space is genetically influenced and depend on the presence of polymorphisms in the promoter of SERT gene. Camilleri et al. [105] showed that these polymorphisms influence the colonic transit in response to treatment with the 5-HT3 receptor antagonist alosetron in D-IBS. They identified a greater colonic transit response in patients with L 5HTTLPR homozygous than heterozygous genotype. The overactivation of SERT can lead to an enhanced 5-HT reuptake, resulting in a reduction of its biological actions. On the other hand, a SERT hypofunction may lead to increased 5-HT concentrations in the extracellular space, leading to intestinal hypercontractility, diarrhea, visceral hypersensitivity, and abdominal pain [7,103]. Genetic and epigenetic changes in SERT may contribute to the pathogenesis and clinical presentation of IBS [106,107]. In this regard, Colucci et al. observed an association of 5HTTLPR with the severity of abdominal pain in IBS independently from the bowel habit [108]. Indeed, studies on the association of SERT polymorphisms with specific clinical features of IBS have reported conflicting results [109]. In an attempt of determining whether and how the intestinal serotonergic system is altered in patients with IBS, Coates et al. evaluated 5-HT, TPH1 mRNA, SERT mRNA and SERT immunoreactivity in patients with IBS and ulcerative colitis (UC) in comparison with healthy controls [72]. These parameters were all significantly reduced in UC, IBS-C, and IBS-D as compared to controls. When 5-HT release was studied under baseline conditions and mechanical stimulation, no changes were detected in any study group, thus suggesting that UC and IBS were associated with similar changes in the serotonergic signaling system. Although UC and IBS are different diseases with different pathogenesis, these data suggest that shared defects in serotonergic signaling may be the basis for altered motility, secretion and sensation in both disorders [110].

4.3.2 Serotonin in the brain
gut axis The CNS and ENS are closely connected through nervous and hormonal pathways, giving rise to an anatomo-functional entity known as the brain
gut axis. Brain
gut axis is characterized by bidirectional communications through various systems that include mainly the hypothalamic
pituitary
adrenal (HPA) axis, the limbic system, the autonomic nervous system, endogenous pain regulation systems and ascending aminergic pathways, the endocrine system and the gut microbiota. In this context, 5-HT plays a pivotal role in the regulation of GI motility by paracrine, endocrine and neurocrine mechanisms [111,112].

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It is known that the activity of HPA axis is regulated by 5-HT [113]. 5-HT stimulates the production of corticotropin releasing factor (CRF) in the hypothalamic paraventricular nucleus (PVN), promoting the production of ACTH by the pituitary gland [114]; it acts also with a paracrine mechanism on the cells of adrenal cortex, promoting the release of cortisol, adrenaline, and aldosterone into the circulatory stream [115]. Increased adrenaline secretion stimulates both ECs and ENS neurons. Neuronal 5-HT stimulates the GI motility and intestinal mucosa growth, and promotes the development of ENS [95,96,116]. When blood levels of CRF and ACTH increase (e.g., during emotional stress), the adrenal gland sends negative inputs to the pituitary gland and PVN nucleus to decrease their secretory activities (Fig. 4.1) [117]. It is presently acknowledged that psychological/psychiatric disorders, such as stress, anxiety, and depression, often coexist in patients with FGIDs, and this concept allows to frame these disorders within the so called “biopsychosocial model” [118
124]. There is also evidence that patients diagnosed with a FGID in concomitance with anxiety or depression often suffer from severe symptoms, longer recovery times, and a worse prognosis. Moreover, they need more health resources [76,125
127]. A large number of psychosocial factors have been recognized to affect the subjective experience of illness and influence treatment and outcomes. These factors can include a history of emotional, sexual, physical abuse, chronic social stress, anxiety disorder, or poor adaptation to life itself and other stressful life events. The current conceptual model

FIGURE 4.1 Nerve pathways in the GI tract and their modulation by emotional stress. ANS, Autonomic nervous system; CNS, central nervous system; ENS, enteric nervous system; GI, gastrointestinal tract; HPA, hypothalamic
pituitary
adrenal. Source: Modified from Lokesh Agrawal, Mustafa Korkutatab, Sunil Kumar Vimalc, Manoj Kumar Yadavd, Sanjib Bhattacharyyac, Takashi Shigaa. Therapeutic potential of serotonin 4 receptor for chronic depression and its associated comorbidity in the gut. Neuropharmacology 166; 2020; 107969.

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predicts that adverse events, both past and present, may influence the psychological response to stress and the susceptibility to develop and exacerbate symptoms through the amplification of brain
gut interactions. Characterizing these psychosocial factors is very important in FGIDs since they influence the perception of pain and therefore the choice of treatment [128,129]. A variety of stimuli defined as “stressful” can lead to the activation of the aforementioned brain
gut axis and, consequently, promote the onset of functional and behavioral disorders. The origin of stress origin be central (e.g., psychological distress) or peripheral (e.g., infections, surgery). Stress then leads to increased activation of the CRF and noradrenergic system, resulting in autonomic and behavioral responses [84,130
133] (Fig. 4.1). It has been hypothesized that, in predisposed subjects, a condition of sustained stress can lead to a persistent increase in the responsiveness of central stress circuits and vulnerability to develop emotional and functional disorders. The role of stress could be particularly important in the alterations of brain
gut interactions, thus promoting the development and/or exacerbation of the symptoms that characterize FGIDs [134]. There is also a growing body of evidence showing that the pathophysiology of FGIDs involves the abnormal processing of visceral nociceptive signals in the brain
gut axis, leading to hypersensitivity and hyperalgesia [135]. IBS patients, as compared to healthy subjects, seem to have a greater responsiveness to stressful stimuli in terms of gut motility, visceral and emotional sensitivity as well as higher HPA hormone levels [136
139]. Stressinduced changes in GI physiology have been reported in patients with IBS, including mainly delayed gastric emptying, increased intestinal motility, decreased perception threshold to both painful and nonpainful stimulation of rectum [140].

4.4 Serotonin, psychological/psychiatric and extra-gastrointestinal comorbidities Several studies have suggested the existence of common pathophysiological mechanisms in psychological/psychiatric disorders and FGIDs, involving 5-HT [98,125
127,141,142], and it appears that they could share a common genetic predisposition involving changes in 5-HT signaling [106,143
147]. Wohlfarth et al. [43] reported that SNPs in the 5-HT3 and 5-HT4 receptor genes are responsible for an increased severity of anxiety and IBS symptoms. In a more recent study on a murine model, the responsibility of neuronal 5-HT in the development of depression with associated GI comorbidities was confirmed. In this study, mice with low 5-HT levels in enteric neurons presented abnormalities of ENS development, reduced intestinal motility and reduced intestinal mucosal growth [112]. Lokesh et al. [117] proposed a model able to explain the role of 5-HT and 5-HT4 receptor in the comorbidity between depression and GI disorders. In particular, since 5-HT4 receptors are expressed both in the limbic system and GI tract, these authors suggested that this receptor is involved both in the pathogenesis of several neurological/psychiatric diseases and GI disorders, as in the case of depression-induced IBS. In addition, some authors suggested that, in patients with depression, 5-HT, via the activation of 5-HT4 receptors, can activate the HPA axis and alter GI functions, leading to the occurrence of IBS symptoms [117].

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Current data suggest that alterations of 5-HT production and neurotransmission may be involved in psychological changes and symptoms in IBS patients [148], and some studies have shown that patients with IBS report more stressful events than patients with organic GI diseases or healthy subjects [149]. Hyperactivity of the neuroendocrine system, induced by stressful psychological stimuli, may be responsible for visceral responses in the GI tract, such as stress-induced bowel movements in IBS [118,139,150]. This suggestion is supported by the finding of sensory-motor gastric dysfunctions in patients with functional dyspepsia [151]. In addition, the release of adrenaline from the adrenal gland in patients affected by depression has been documented to increase the activity of beta adrenergic receptors, which in turn induces visceral hypersensitivity and constipation symptoms in patients with C-IBS [152]. It is well known that drugs acting on the serotonergic system, such as tricyclic antidepressants and SSRIs, can exert beneficial effects in the treatment of FGIDss. These drugs increase 5-HT levels both in the CNS and the gut, with favorable effects on both visceral perception and mood. However, it should be noted that no effects of antidepressants have been observed on the physiology of the digestive system in subjects with functional disorders, but only improvements in terms of subjective global well-being [153
155]; moreover, some randomized controlled clinical studies have shown a comparable efficacy of psychotherapy in IBS patients [156,157]. Patients with FGIDs may complain frequently of extra-GI comorbidities. Associations have been reported with headache, migraine, fibromyalgia, temporo-mandibular joint disorder, back pain, chronic pelvic pain, dysuria, interstitial cystitis, sexual disorders, premenstrual syndrome, dysmenorrhea, asthma, bronchial hyperreactivity, palpitations, sleep disorders and chronic fatigue syndrome [128,158
160]. Most of these disorders are associated with a dysregulation of 5-HT pathways [161
169]. 5-HT could be relevant in the association of FGIDs with other chronic pain syndromes, such as migraine and fibromyalgia. Indeed, they are characterized by an altered perception and elaboration of nociceptive stimuli both in the CNS and peripheral nervous system [170
172]. 5-HT is one of the neurotransmitters involved in the inhibition of pain through descending and medullary nerve fibers. Therefore a decrease in 5-HT levels would explain the increase in pain in these syndromes [173
179]. In accordance with the hypothesis of a possible shared involvement of 5-HT in the pathophysiology of these disorders, a positive impact was reported when using antidepressant drugs acting on the serotonergic system [162,180
184].

4.5 Serotonin and microbiota, the brain
gut axis and the psychobiota 4.5.1 Serotonin and microbiota The gut is a complex ecosystem comprising a microbial community designated as gut microbiota, which has coevolved with the host to develop a mutualistic relationship [185]. The microbiota colonizing the GI tract is a complex, heterogeneous and dynamic microbial system. In humans, the GI tract contains up to 1 3 1014 colony-forming units of bacteria, this colonization occurring after the birth. These commensal microorganisms play crucial roles in the homeostasis of GI functions, provide competitive barriers against invasions by

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pathogens, and contribute to the development of the host immune system. In addition, the gut microbiota is sensitive to changes in response to environmental factors, such as diet and drugs. The composition of gut microbiota is very dynamic, varying from birth to adult life. It includes bacteria, archea, viruses, and protozoa. Bacteria can be classified into different phyla, orders, families, genuses, and species. Over 90% of the bacterial microbiota belong to two phyla, that is, Firmicutes and Bacteroidetes, followed by Actinobacteria and Verrucomicrobia. Abnormal changes in the composition of gut microbiota are termed dysbiosis, that is recognized as a predisposing factor for the development and progression of several chronic diseases. Recent research has increasingly investigated on TRP, the precursor of 5-HT, and the microbial regulation of TRP metabolism, with an emphasis on the host-microbe control. Although TRP is the least represented amino acid in proteins and cells, it is a biosynthetic precursor of a large number of microbial and host metabolites [186]. In the GI tract TRP metabolism follows three major pathways: (1) direct transformation into ligands of the aryl hydrocarbon receptor (AhR) by the gut microbiota [187]; (2) kynurenine pathway in both immune and epithelial cells via indoleamine 2,3-dioxygenase [188]; (3) 5-HT production pathway via TPH1in ECs. AhR signaling is regarded as a key component of the immune response and it is crucial for intestinal homeostasis by acting on barrier integrity and several immune cell types, such as intraepithelial lymphocytes, Th17 cells, innate lymphoid cells, macrophages, DCs, and neutrophils. Kynurenine pathways are implicated in the regulation of a number of host biological processes, involving neurotransmission, inflammation and immunity. The perturbation of these TRP pathways has been related to digestive diseases, such as inflammatory bowel diseases and IBS, metabolic syndrome and neuropsychiatric disorders. Recent studies highlighted the role of gut microbiota in the control of blood 5-HT levels. Serum 5-HT levels are substantially reduced in germ-free mice as compared to specific pathogen free controls [189]. In addition, the size of ECs is larger in germ-free than colonized rats [190]. This suggests that microbes could impact on the development and function of 5-HT-producing cells. There is evidence that the gut microbiota, and particularly some microbial metabolites produced by spore-forming bacteria, promote 5-HT biosynthesis from colonic ECs in an inducible and reversible manner [191]. According to several lines of evidence, the microbiota can produce relatively high levels of peripheral 5-HT, providing about 64% of colonic and 49% of serum concentrations [189]. Some bacteria, including Corynebacterium, Streptococcus, and Escherichia coli, are reported to synthesize 5-HT in culture [192] by decarboxylation of TRP to tryptamine [193]. In addition, a study in a mouse model, conducted by De Vadder et al. [194] showed that the microbiota is able to influence the development of ENS through the production and release of intestinal 5-HT and activation of 5-HT4 receptors. Conversely, 5-HT is reported to stimulate the growth of Enterococcus faecalis, E. coli and Rhodospirillum rubrum in culture [195,196]. The gut microbiota may influence also the intestinal homeostasis of 5-HT by affecting the SERT. Germ-free mice display an elevated colonic expression of SERT, synthetized by enterocytes to promote 5-HT uptake. This may represent a compensatory response to a deficient 5-HT synthesis by host ECs [191]. In addition, several studies have shown that a chlorogenic acid diet can alter the gut microbial composition, and Wu et al. [197] reported that this diet could change the colonic 5-HT levels, suggesting a specific role of this dietary regimen in regulating colonic 5-HT.

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85

4.5.2 Serotonin in the brain
gut axis and the psychobiota The brain
gut axis is a bidirectional communication network connecting the CNS and GI tract, where 5-HT acts as a key mediator. The relationship between the brain and the gut is primarily regulated at immune, neural and endocrine levels. Some clinical studies [198,199] have investigated the role played by hypothalamic
autonomic nervous system (HANS) and HPA axes in IBS patients through the assessment of the sympathetic nerve activity and plasma levels of neuroendocrine markers. In particular, in IBS patients elevated plasma levels of endothelin, neuropeptide Y, and 5-HT were found to be associated with some psychological disorders together with an altered cardiovascular autonomic reactivity to acute stressors compared to healthy volunteers [199]. Moreover, no correlation between cortisol (considered a marker of HPA) and neuropeptide Y (considered a marker of the HANS axis) levels was found, confirming the general uncoupling of the HPA and HANS axes in IBS patients, while D-IBS patients showed a substantial correlation between plasma 5-HT and cortisol levels [198]. It is hypothesized that alterations of gut microbiota in the early life acts as a factor which impacts on the endocrine and immune pathways of the gut
brain axis. These changes would then predispose the individuals to the development of stress related disorders. The interactions between gut microbiota and CNS are bidirectional as well. In detail, the brain can affect the composition of microbiota, as well as food preferences and appetite, by specific release of neurohormones into the gut lumen. Of note, the brain TRP levels of germ-free mice are lower than those of colonized mice [200]. By contrast, in the hippocampus of germ-free mice, the levels of 5-HT and its main metabolite, 5-HIAA, are higher than in colonized mice. A recent study [201] investigated the interactions between intestinal microbiota, gut physiology and social behavior in an animal model. The authors found a significant reduction of gut mucosal 5-HT, with a downregulation of TPH1 and an upregulation of the SERT gene expression. Finally, indirect evidence on the relationship between microbiota and brain
gut axis come from therapeutic experiences. Recently the “psychobiotics” have been characterized as a subclass of probiotics and defined as live organisms which, when ingested in sufficient amounts, produce health benefits in patients with psychiatric disorders [202]. These “psychobiotics” could be capable of producing neuroactive substances (such as GABA and 5-HT), thereby acting on the gut-brain axis. In addition, olanzapine, a second-generation antipsychotic drug that binds 5-HT2A, 5-HT1A, and 5-HT2C receptors with high affinity, was found to promote relevant changes in gut microbiota, along with an associated body weight gain [203].

4.6 Conclusion 5-HT plays an important role both in the CNS and digestive system, regulating motor, secretory and sensory functions through seven families of receptors and SERT, which modulates its reuptake and hence its activity. The serotonergic system is involved in the pathogenesis of several GI diseases, particularly the FGIDs, and the pathophysiology of some frequently associated psychiatric and psychological comorbidities. The continuous bidirectional dialog between brain and gut (brain
gut axis), in which 5-HT is deeply

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involved, can explain the multifaceted actions of this mediator. Moreover, the role of gut microbiota, with its ability of producing 5-HT and being modulated by 5-HT, is a field of high interest deserving extensive investigations.

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C H A P T E R

5 The pathophysiology of gut
liver connection Luca Maroni1, Francesca Fianchi2, Luca Miele2 and Gianluca Svegliati Baroni1 1

Clinic of Gastroenterology and Hepatology, Universita` Politecnica delle Marche, Ancona, Italy 2 Dipartimento di Scienze Mediche e Chirurgiche, Fondazione Policlinico Gemelli IRCCS, Universita` Cattolica del Sacro Cuore, Roma, Italy

5.1 Regulation of intestinal permeability 5.1.1 The intestinal mucosa structure The intestinal mucosa is a semipermeable barrier that serves as the first line of defense between the host and the luminal environment [1]. This complex system includes, from lumen to the basolateral surface, the mucus layer, the epithelial cell monolayer, the lamina propria, the muscolaris mucosae, and the submucosa [2]. Apart from physical barriers, an important defensive role is exerted by immune cells of the lamina propria, which are crucial to maintain tolerance against commensal microorganisms, while simultaneously keeping away pathogens and reducing bacterial translocation. The selective permeability is guaranteed by the epithelial cell monolayer, which allows essential nutrients and fluid absorption, while preventing the passage of potentially harmful molecules from the intestinal lumen to host systemic circulation [3]. The intestinal epithelium is composed of several different cell types, such as enterocytes, Paneth cells, and goblet cells, which form a tight barrier toward the luminal milieu. The transepithelial permeability is modulated by tight junctions (TJs), multiprotein complexes that mechanically link epithelial cells together, exerting a crucial role for the integrity of the epithelial layer [4]. TJs are located on the lateral membrane of intestinal cells, forming a network of linking strands. They are not visible by light microscopy and a freeze fracture technique is necessary for examination through transmission electron microscope. Due to their

The Complex Interplay Between Gut
Brain, Gut
Liver, and Liver
Brain Axes DOI: https://doi.org/10.1016/B978-0-12-821927-0.00002-4

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location on epithelial cell membranes, TJs regulate the paracellular passage of small molecules and solutes, and a constant remodeling of these complexes is essential in maintaining intestinal barrier functions. TJs appear like three-dimensional structures composed of various transmembrane and cytosolic proteins, including occludin, large families of claudin proteins, tricellulin, cingulin, TJ-associated MARVEL domain-containing proteins (TAMPs) and junctional adhesion molecules (JAMs), as well as a variety of cytoplasmic adaptor and scaffolding proteins [5]. Combination of these proteins is highly dynamic and is able to adapt to the changing environment in order to modulate the transepithelial permeability. Indeed, TJ composition and structure can modify in response to various extracellular stimuli. Moreover, the tight junction complex is connected to the cytoskeleton of the adjacent cells via the scaffolding proteins—such as zonula occludens (ZO)-1, ZO-2, and ZO-3-, and several peripheral proteins like cingulin and symplekin. This interaction is crucial to maintain gut epithelial integrity in response to mechanical stress: indeed, TJs can modulate their protein expression and assembly according to various mechanical stimuli. Cytoskeleton-dependent regulation of TJs is mainly mediated via phosphorylation of myosin light chain (MLC) by myosin II kinase (MLCK). Apart from mechanical stimuli, inflammatory mediators—such as tumor necrosis factor (TNF)-α and interferon (IFN)-γ—and enteropathogenic Escherichia coli are able to induce an MLCK-dependent increase in paracellular permeability [6].

5.1.2 The “leaky gut” As previously outlined, the intestinal barrier constitutes the interface between the outside and the internal milieu of the body, and for this reason it exerts an essential role in human health. A reduced integrity of this barrier results in a condition commonly known as “leaky gut,” in which luminal contents translocate from the intestinal lumen into the host systemic circulation. Several different stimuli—such as dietary components, psychological stressors, intensive exercise, or conditions causing a dysbiosis of the gut microbiota—can disrupt the intestinal epithelial barrier leading to an increased permeability. As a result, an elevated number of harmful molecules (bacteria, bacterial products, and antigens) enter the systemic circulation, causing systemic inflammation and potentially leading to remote organ injury [7]. One of the most important bacterial product translocating across the epithelium is lipopolysaccharide (LPS), which is present in the outer membranes of gram-negative bacteria. Increased levels of serum-LPS originating from the intestinal microbiota are responsible for the socalled “endotoxemia.” Indeed, even though bacterial translocation is a physiological process that is crucial for host immunity and tolerance, and small amounts of LPS have been detected in the blood stream of healthy individuals, in case of an altered gut permeability translocation is enhanced and a high amount of bacteria and bacterial endotoxins reach the systemic circulation, being able to trigger numerous pathologic conditions. Moreover, the altered permeability induces the activation of the mucosal immune system of the lamina propria, with consequent development of local inflammation and increased susceptibility to systemic infection and malabsorption.

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As above described, TJs are the main responsible for gut epithelial permeability, and for this reason an alteration of TJs structure has been implicated in the pathogenesis of a variety of local and systemic diseases [8], including not only diseases originating in the gastrointestinal tract but also metabolic disorders and various other conditions.

5.1.3 Assessment of epithelial barrier dysfunction Due to the central role of the intestinal mucosa in the pathogenesis of diseases, during the last decade great efforts have been put in finding new methods to detect mucosal dysfunction. Techniques to investigate the gut barrier permeability are rapidly expanding and new methodological approaches are under study [9]. One of the most promising strategies is the identification of biomarkers of intestinal permeability in patient’s bloodstream. Among these biomarkers, the most investigated were Zonulin, fatty acid binding proteins (FABP), Citrulline, Glucagon-Like Peptide (GLP)-2. However, up to now the most reliable tool is the measurement of endotoxin levels in patient’s blood: in particular LPS has been identified as a potential marker of an increased intestinal permeability, and more specifically a marker of bacterial translocation [7]. The association between endotoxin levels to certain diseases, such as diabetes, has recently gained a large interest. Interestingly, a high-fat diet (HFD) has also been shown to temporarily increase LPS levels in blood of healthy individuals [10].

5.1.4 Therapeutic potential of intestinal mucosa regulation Given the important role of the intestinal epithelial barrier in human disease, improvement of its integrity and function by regulation of TJs protein expression has been proposed as a promising treatment strategy and is currently the field of intense research. In order to identify potential targets of therapy, several different actors implicated in TJs remodeling have been investigated: among them, inflammatory cytokines, gut microbiota and diet are the most important and studied. For this reason, dietary modifications, probiotics, and other interventions intended to increase “gut integrity” are under study as therapeutic options for a large number of diseases.

5.1.5 Regulation of intestinal permeability by inflammatory cytokines The role of inflammatory cytokines in regulation of intestinal permeability was initially highlighted by in vitro models of inflammatory bowel disease (IBD), showing epithelial responses to exogenous TNF and interleukin (IL)-13. These molecules are able to enhance the paracellular permeability modulating tight junction’s protein expression via distinct mechanisms. TNF is able to induce epithelial barrier dysfunction by enhancing transcription of MLCK, multiple claudins, MARVEL proteins (TAMPs; occludin triceullin, and marvelD3), and ZO-1 [6,11]. IL-13 augments paracellular permeability by increasing claudin-2 expression, which can create paracellular cation-selective pores [12].

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5.1.6 Regulation of intestinal permeability by gut microbiota There is considerable evidence that the gut microbiota—the trillions of microorganisms inhabiting the gut—is involved in intestinal barrier homeostasis. Indeed, gut dysbiosis, characterized by an imbalance in the composition of gut microbial communities, has been described in many conditions harboring a leaky gut, suggesting an intimate link between microbiome shifts and alteration of the gut barrier permeability. Among these diseases, the most studied were IBDs, asthma, depression, and Alzheimer’s disease [13]. Interestingly, dysbiosis and altered gut permeability have been recently described as characteristic features of many metabolic disorders: obese and diabetic individuals, as well as patient affected by nonalcoholic fatty liver disease (NAFLD), showed an increased intestinal permeability in association with drastic differences in their gut microbiota when compared to healthy counterparts. Subsequent studies with microbiota-directed interventions confirmed the causal relationship between gut microbiota community shifts and metabolic disorders development: for example, in a study conducted in mice, gut microbiota transfer from mice with obesity into germ-free mice induced significant weight gain in the latter, which was not reproducible in those receiving microbiota transfers from lean individuals [14]. As for diabetes, in a study conducted in humans, gut microbiome transfer from healthy subjects into patients with type 2 diabetes (T2D) resulted in a significant improvement in insulin sensitivity [15]. Moving from this evidence, studies on characterization of the gut microbiota community allowed the identification of a bacterial signature for each one of these metabolic conditions. Disruption of gut barrier homeostasis has been associated with a reduced number of strains belonging to the commensal flora and able to exert beneficial effects. Apart from compositional changes, quantitative changes of the microbiome have also been described in metabolic disorders: for example, there is some evidence for a connection between small intestine bacterial overgrowth (SIBO) and metabolic disease such as T2D and NAFLD, while association with obesity is not completely clear. Similar to what happens in IBDs, the link between dysbiosis and the pathogenesis of metabolic diseases seems to be represented by an alteration of gut barrier permeability. In fact, increased circulation of surrogate markers of intestinal permeability has been linked to enhanced systemic inflammation observed in metabolic diseases. The connection between dysbiosis and metabolic disorders is consistent with the emerging evidence on the fundamental roles exerted by gut microbiota for the host metabolism: indeed, commensal bacteria are involved in breaking down of otherwise indigestible polysaccharides, production of vitamins, and transformation of several xenobiotics. Moreover, commensal bacteria are involved in the maintenance of barrier integrity by several different mechanisms. First of all, they are involved in modulation of TJs [2,16], and at the same time they are able to induce the physiological paracellular permeability essential for nutrient uptake [17,18]. Faecalibacterium prauznitzii plays another important role in maintaining the intestinal barrier function: indeed, it is essential for the fermentation of nondigestible substrates and supports the growth of short chain fatty acids (SCFAs) producer bacteria [19]. SCFAs, and in particular butyrate, are the main energy source for intestinal epithelial cells and are essential for human gut homeostasis [20]. Likewise, enteric pathogens cause an alteration of mucosal barrier function by disrupting TJs through different mechanisms, including degradation of specific TJ proteins,

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reorganization of the cell cytoskeleton, and utilization of TJs proteins as receptors for internalization [21,22]. Hence, assessing the gut microbiota composition, microbial metabolites, as well as inflammatory markers can be important to map the mechanisms behind a perturbed intestinal barrier. Nevertheless, the precise molecular basis of gut microbiota-epithelial barrier interaction remains unclear due to the inability of in vitro models to recreate the differentiated tissue structure and components observed in the normal intestinal epithelium.

5.1.7 Regulation of intestinal permeability by diet Diet seems to have an impact on intestinal barrier function, and several different nutrients have shown to exert beneficial effects on intestinal permeability, such as casein, vitamins (such as vitamin D and retinol), polyphenols, and minerals such as zinc. On the other hand, deleterious effects have described for other dietary elements such as alcohol and medium chains fatty acids. Trials of specific amino acids administration have been conducted, some of them showing promising results: for example, glutamine and tryptophan administration showed to decrease intestinal permeability via direct effects on tight junction expression. These results were also confirmed in a human study, in which supplementation of glutamine reduced intestinal permeability and endotoxin levels [23].

5.2 Role of altered intestinal permeability in the pathogenesis of NAFLD NAFLD represents the hepatic manifestation of the metabolic syndrome and it can be considered, nowadays, the predominant chronic liver disease worldwide, affecting up to one-third of the global population. Its prevalence is continuously rising along with the incidence of obesity and other metabolic disorders, both in the pediatric and in the adult population [24]. Indeed, NAFLD should be considered as a spectrum of chronic liver diseases, ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis [25]. However, despite the high prevalence of these conditions, therapeutic options are still limited, and there is a critical need to develop novel and effective therapeutic approaches for NAFLD related disorders. The pathogenesis of NAFLD is multifactorial, involving genetic and epigenetic factors, insulin resistance, hormones secreted from the adipose tissue, nutritional factors and gut microbiota [26]. The result of this multiple-hit pathogenesis is a lipid accumulation in the hepatocytes with consequent oxidative stress, lipid peroxidation, adipokine signaling, and proinflammatory cytokine expression. In addition to the well-known risk factors for the disease, such as high-fat content diets (HFCD) and sedentary lifestyles of Western countries, emerging data suggest that alterations of intestinal permeability can contribute to liver injury in NAFLD. For example, in a study conducted in mice fed with a HFCD, animals with defects of the epithelial barrier

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developed a more severe steatohepatitis after diet administration when compared to control mice [27]. Indeed, in the past decade a strict relationship between gut homeostasis and liver function has been described as a result of intense research. In fact, the liver receives the great part of its blood supply from the intestinal portal circulation and provides first-pass metabolism for the intestinal luminal contents, including dietary nutrients, xenobiotics [28], as well as metabolites of the gut microbiota that translocate across the intestinal epithelium. Several different studies highlighted how integrity of the gut epithelial barrier is crucial in order to maintain liver homeostasis and prevent liver injury [29,30]. In fact, as previously described, in presence of an altered endothelial barrier there is an enhanced permeability to luminal contents such as microbes and microbial products (endotoxins, LPS, peptidoglycan). These molecules enter the portal circulation and reach the liver, with consequent hepatic exposure to injurious substances that can be responsible for liver damage. Translocated bacteria and their products bind to immune receptors expressed on hepatocytes, with consequent activation of the innate immune system cascade and development of liver inflammation and, eventually, fibrosis [31]. This inflammatory milieu represents the first line of defense against the invading pathogens, but a sustained elevation of cytokines and other immune mediators exerts a detrimental effect for the host and is involved in the pathogenesis of NAFLD. Evidence in the literature highlighted, for example, the role of LPS signaling in experimental models of NAFLD. Indeed, the abundance of this microbial product in the portal circulation elicits host innate immune responses through its binding with toll-like receptors (TLRs) on Kupffer cells and hepatic stellate cells, with consequent activation of the inflammatory cascade and production of proinflammatory cytokines such as TNF-α, IL-1β, and interferons [32]. TLRs are a family of pattern-recognition receptors that play a key role in host defense against invading pathogens by activation of the innate immune system and induction of inflammatory signaling. Among TRLs, various receptors have been implicated in the pathogenesis of NAFLD: in particular TLR2, TLR4, TLR5, and TLR9 were demonstrated to bind intestinal ligands and to induce liver inflammation through different molecular pathways [33]. For example, LPS binding to TLR4 expressed on Kupffer cells induces the release of numerous proinflammatory mediators such as TNFα, with consequent inflammation, hepatic steatosis, and fibrosis [34]. In fact, the expression of proinflammatory cytokines is suppressed in TLR4 mutant mice, even in presence of equivalent LPS levels. As a result, TLR4 mutant mice are resistant to liver injury and fibrosis [35], as well as being protected against diet-induced obesity and insulin resistance [36]. TLR9 was demonstrated to promote steatohepatitis in mice by induction of IL-1β [37]. The molecular mechanisms underlying the increased gut permeability observed in NAFLD involve a disruption of intercellular TJs. In a study conducted in mice, for example, it has demonstrated that disruption of the gene encoding for junctional adhesion molecule A, a component of TJs, results in altered intestinal barrier function that contributes to the development of NASH [27]. In human models of NAFLD, enhanced intestinal permeability seems to be related to a decreased expression of zonula occludens-1 (ZO-1) [38].

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Another mechanism that has been suggested to promote gut permeability is a reduction in intestinal mucus layer, caused by lower amount of Akkermansia muciniphila in the commensal microbiota of NAFLD patients [39].

5.2.1 Gut microbiota and NAFLD Gut microbiota plays a crucial role in human health, being involved in the digestion of food, in the protection of mucosal surfaces and in modulation of the immune system. Besides these functions, this population of microbes can interfere with the intestinal barrier homeostasis and it has been described as the main responsible of the altered gut permeability and of the consequent systemic inflammation observed in liver diseases. Indeed, a disturbed imbalance of the endogenous gut microbiota, otherwise known as dysbiosis, has been implicated in the pathogenesis of a range of chronic liver conditions including NAFLD-spectrum diseases [40], alcoholic liver disease [41], as well as cirrhosis and its complications (hepatic encephalopathy, hepatocellular carcinoma) [42]. Interestingly, the altered gut permeability observed in NAFLD has been associated with both quantitative (overgrowth) and qualitative taxonomic changes in the intestinal microbiota, detected by analyses of its metagenome and metabolome [43]. As for quantitative changes in the microbiota population, a wide literature described for example an association between SIBO and enhanced intestinal permeability in NAFLD, as measured via dual sugar absorption test [44]. In another study conducted in obese patients, SIBO showed a high prevalence and was associated with severe hepatic steatosis [45]. This observation was then confirmed by several different studies having NAFLD patients as population of the study [46]. As regards qualitative modification of commensal microbiota, NAFLD patients are characterized by a reduced diversity of microbes compared with healthy individuals. Moreover, both animal and human studies aimed to characterize the fecal microbiota of patients with NAFLD-spectrum diseases, identified compositional shifts in association with these conditions, although some contrasting evidence have emerged (Table 5.1). In a study by Raman et al., for example, NAFLD patients showed increased Lactobacillaceae, Lachnospiraceae, and Veillonellaceae but decreased Ruminococcaceae in their microbiota population when compared to healthy controls [52]. Another study by Mouzaki et al. reported the differences in the gut microbiota of subjects with NAFLD, NASH and healthy controls, founding an inverse association between the presence of NASH and the percentage of Bacteroidetes, and association that continued to be significant even after adjusting for the body mass index and dietary fat intake [53]. In another study conducted in obese children, there were significant differences in gut microbiota composition of patients with biopsyproven NASH when compared to patients with obesity alone; in particular, authors found higher levels of ethanol-producing bacteria in NASH, even including increased levels of Bacteroidetes, in contrast to the finding by Mouzaki et al. [49]. In a recent cross-sectional study conducted in the adult population, NAFLD was associated with reduced abundance of several bacterial taxa (Ruminococcus, Coprococcus, and Faecalibacterium prausnitzii) independent of BMI and insulin resistance, and the result was also validated using quantitative PCR [50]. Moreover, selected bacterial products related to fermentation were higher in NAFLD patients.

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TABLE 5.1 Compositional shifts of gut microbiota community in NAFLD. Population of the study

Results

Raman et al. [47]

Obese patients with presumed nonalcoholic steatohepatitis (NASH) (n 5 30) and healthy controls (HC) (n 5 30)

Over-representation of Lactobacillus species and selected members of phylum Firmicutes (Lachnospiraceae; genera, Dorea, Robinsoniella, and Roseburia) and underrepresentation of one member of phylum Firmicutes in nonalcoholic fatty liver disease (NAFLD) patients (Ruminococcaceae; genus, Oscillibacter).

Mouzaki et al. [48]

Adults with biopsy-proven NAFLD [simple steatosis (n 5 11) or NASH (n 5 22)] and HC [n 5 17]

Patients with NASH had a lower percentage of Bacteroidetes compared to NAFLD patients and HC, and higher fecal Clostridium coccoides compared to those with NAFLD.

Zhu et al. [49]

Three groups of children and adolescents: NASH patients (N 5 22), obese patients (n 5 25), and healthy controls (n 5 16)

Proteobacteria/Enterobacteriaceae/Escherichia was similarly represented between healthy and obese microbiomes, but was significantly elevated in NASH.

Wong et al. [49]

Adults with histology-proven NASH (n 5 16) and HC (n 5 22)

NASH patients had lower fecal abundance of Faecalibacterium and Anaerosporobacter but higher abundance of Parabacteroides and Allisonella.

Jiang et al. [50]

NAFLD patients (n 5 53) and HC (n 5 32)

Escherichia, Anaerobacter, Lactobacillus and Streptococcus were increased in the gut microbiota of NAFLD patients compared to HC, while five genera (including Alistipes and Prevotella), were significantly less abundant, five genera, including Alistipes and Prevotella, were significantly more abundant.

Boursier et al. [51]

57 adult patients with biopsy-proven NAFLD and different degrees of fibrosis

Bacteroides abundance was significantly increased in NASH and F $ 2 patients, whereas Prevotella abundance was decreased. Ruminococcus abundance was significantly higher in F $ 2 patients.

De Silva et al. [50]

Adults with biopsy-proven NAFLD [simple steatosis (n 5 15) or NASH (n 5 24)] and HC (n 5 28)

NAFLD was associated with reduced abundance of several bacterial taxa (Ruminococcus, Coprococcus and Faecalibacterium prausnitzii) independent of BMI and IR.

However, examining all this evidence emerging from human studies, although certain taxonomic changes have been associated with NAFLD, there is still some controversy regarding the microbiome profile in these conditions, and a microbiota signature has not been identified yet [47]. Changes in bacterial taxonomy might not be as important as changes in bacterial genes expression (gut microbiome) in the pathogenesis of NAFLD, and metagenomics and metatranscriptomics studies are currently ongoing [48]. One of the biggest challenges of research in NAFLD has been establishing whether dysbiosis could play a causal role or rather represents an epiphenomenon of the disease. For this reason, numerous studies investigated the potential causal relationship between microbiota and NAFLD: animal studies in which the gut microbiota is manipulated and observational studies in patients with NAFLD suggest that dysbiosis plays an important role in the pathogenesis of NAFLD.

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An elegant study conducted in mice, for example, demonstrated that the risk of developing NAFLD is transmissible through fecal transplant: indeed, gut microbiota transfer from mice showing a metabolic response to HFD (in terms of hyperglycemia and proinflammatory cytokines) to germ-free mice produced hyperglycemia, hyperinsulinemia, and hepatic steatosis in the latter. On the other hand, mice accepting microbiota from a donor mouse that had not developed a metabolic response to HFD remained normoglycemic and did not develop steatosis. Interestingly, these results were independent from weight gain, which had been comparable between the two donors. The characterization of the transplanted microbiotas revealed significant differences at the phyla, genera, and species levels, highlighting the role of dysbiosis [54]. A similar investigation was also conducted in humans, demonstrating that transfer of intestinal microbiota from lean donors to patients affected by metabolic syndrome could increase insulin sensitivity in the latter, along with an increase in butyrate-producing intestinal bacteria [55]. Of course, once establishing this causal relationship, the most important observation is that restoration of intestinal barrier integrity and manipulation of gut microbiota may represent promising therapeutic strategies. In particular, microbiota-based treatment strategies have gained a great interest in the field of NAFLD: for example, animal and small human studies show that probiotics and prebiotics may reduce liver inflammation [56].

5.2.2 Role of the leaky gut in the progression of NAFLD Despite its high prevalence, only a minority of NAFLD patients progress to NASH and, eventually, fibrosis. Interestingly, the leaky gut is involved not only in the initiation but also in the progression of liver injury in NAFLD [57]. For example, in an experimental model of NASH, induction of intestinal inflammation by dextran sulfate sodium administration in mice, with consequent damage of the intestinal barrier, demonstrated to promote LPS translocation and to enhance hepatic inflammation and fibrogenesis [58]. These observation were also confirmed in human studies: for example, in a study conducted in the pediatric population, intestinal permeability measured through lactulosemannitol test appeared to be higher in children with NASH than in children with NAFLD [59]. Accordingly, in a meta-analysis of five human studies, 39.1% of NAFLD patients showed an increased gut permeability compared with 6.8% of healthy controls, and this percentage was as high as 49.2% when the NASH subgroup was analyzed [60]. Indeed, liver inflammation, once activated, can perpetrate the local damage, as demonstrated by the fact that portal inflammatory infiltrate contributes directly to fibrogenesis in NAFLD and is strongly related to disease severity in terms of fibrosis stage [61]. One of the reasons is that, even though gut microbiota represents a source of TLR ligands, in physiologic conditions there is a high tolerance to these ligands because hepatic cells express minimal TLRs. In contrast, in presence of local inflammation—such happens in NAFLD—there is an altered immunological tolerance and TLR signaling in hepatocytes is activated. Up to now, one of the most important clinical challenge in NAFLD is identification of patients who are more likely to develop NASH and fibrosis, as these patients are at greater risk for liver-related adverse events. Unfortunately, to date there is no reliable predictor of

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disease progression. For this reason, evidence that an altered intestinal permeability correlates with the severity of liver damage may have promising diagnostic implications. As previously outlined, the gut microbiota composition is one of the main modulators of intestinal permeability, and for this reason taxonomic changes of this population of microbes have been related to the severity of disease. For example, in a study examining the gut microbiota composition of patients with biopsy-proven NAFLD and different degrees of inflammation and fibrosis, results showed that Bacteroides were independently associated with NASH and Ruminococcus with significant fibrosis [51]. These results were compliant with the aforementioned study of 2013 [53], in which NASH patients showed higher amount of Bacteroidetes not only when compared to healthy controls, but also in comparison to NAFLD patients [47]. In a recent case series by Bastian et al., gut microbiota profiles in NAFLD seem to have an impact on disease progression in terms of degree of liver fibrosis, evaluated with transient elastography [62].

5.2.3 Influence of liver homeostasis on intestinal barrier function Interestingly, a wide literature described how liver homeostasis can influence the intestinal barrier function, in a mutual relationship in which each one can influence the other. For this reason, researchers have started to talk about the existence of a “gut
liver axis” [59], referring to the complex network of cross talking between the gut, the microbiome, and the liver through the portal circulation. For example, some evidence suggest that liver inflammation and early-phase hepatic injury may contribute to altered intestinal permeability in NAFLD, in a vicious circle in which enhanced permeability is then able to perpetrate the liver damage [63]. Indeed, some authors studied the modifications of intestinal barrier function in a dietinduced methionine-and-choline-deficient (MCD) murine model of NASH. They showed that liver injury arises early in the course of the MCD diet, before any change in intestinal permeability occurs, suggesting that early changes in liver physiology may affect intestinal homeostasis and contribute to the intestinal permeability. The mechanism by which hepatic injury can alter intestinal permeability, however, is not completely understood.

5.3 Role of incretins in NAFLD 5.3.1 Physiological effects of incretins Together with fundamental functions in food digestion and absorption, the digestive tract exerts a complex modulation of metabolic pathways and is recognized as an endocrine organ. In fact, the intestine secretes a number of hormones that contribute to regulate plasma glucose levels, gut motility, and appetite. In response to a meal, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP) are released in the blood stream by enteroendocrine L-cells and K-cells, respectively [64]. L-cells are located throughout the intestine with a concentration that progressively increases in the distal segments, and may therefore respond to digested

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nutrients and bacterial products. K-cells, on the other hand, predominantly reside in the upper small intestine [65]. GLP-1 and GIP are known as incretins since they both potentiate insulin release from pancreatic β-cells in response to glucose levels in healthy subjects. In patients affected by T2D, pancreatic sensitivity to GLP-1 is maintained while the pancreas cease to respond to GIP. The potency of insulin secretion induced by GLP-1 and GIP is nearly equal and the molecules have an additive effect [66,67]. In a euglycemic state, however, the two incretins exert opposite effects on glucagon secretion. Indeed, GLP-1 suppresses glucagon release while GIP induces its secretion [68]. Besides their insulinotropic effect, incretins have a number of additional biological effects, including modulation of caloric intake and body weight, regulation of gastric emptying and intestinal transit, beneficial effects of cardiovascular risk factors, and modulation of bone homeostasis [69]. After their release from enteroendocrine cells, incretins are rapidly degraded by the enzyme dipeptidyl peptidase-4 (DPP-4), which is expressed on the apical membrane of epithelial cell, endothelial cells, fibroblast and lymphocytes. DPP-4 is present also as a circulating soluble form in plasma [70]. To overcome the extremely short half-life of incretins (about 2 minutes), a number of synthetic analogues of GLP-1 resistant to DPP-4 degradation (i.e., exenatide, liraglutide, lixisenatide, albiglutide, etc.) have been developed for clinical use. Moreover, in an effort to potentiate the pathway, inhibitor of DPP-4 activity such as gliptins are now available for clinical use.

5.3.2 Effects of incretins on hepatic glucose and lipid metabolism The liver plays essential roles in maintaining both glucose and lipid homeostasis. In the postprandial state, glucose is actively transported into hepatocytes by the glucose transporter type 2 (GLUT2) and is phosphorylated to glucose-6-phosphate by the enzyme glucokinase. Depending on the metabolic state of the organism, glucose-6-phosphate is then further directed to glycolysis or utilized for glycogen synthesis in a series of processes tightly regulated by pancreatic insulin and glucagon. In the fasting state, the liver is responsible for maintaining glucose blood levels by breaking down of glycogen in the process of glycogenolysis. In case of prolonged fasting periods, the liver is also capable to actively produce glucose molecules via hepatic gluconeogenesis. Lipid metabolism is also regulated by hepatic functions. Intestinal lipid absorption is favored by bile acid-induced emulsification of luminal lipid droplets, which facilitates lipid hydrolysis by lipases. Once absorbed in the enterocytes, triglycerides are packaged with cholesterol and other lipids into particles called nascent chylomicrons, which are further metabolized by the adipose tissue and the muscle. Chylomicron remnants reenter the blood circulation and are recognized by the low density lipoproteins (LDL) receptor-related protein expressed on liver cells. In the hepatocytes, free fatty acids (FFA) that derived from chylomicrons or are actively absorbed from the circulation in case of lipolysis, may be oxidized as source of energy, stored in lipid droplets within the hepatocyte or processed to very low density lipoproteins (VLDL). Insulin promotes also de novo lipogenesis in the liver via the metabolization of acetyl-CoA that derives from glycolysis [71]. The expression of the GLP-1 receptor has been demonstrated in hepatocyte cell lines, primary human cultures and human hepatocytes [72,73]. It has to be mentioned, however, that

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not all studies found the expression of the GLP-1 receptor in the liver [74,75]. Recently, a marked uptake of radiolabelled GLP-1 and exendin-4 was demonstrated in liver, pancreas, and intestine of mice, with significant reduction after diet-induced obesity [76]. The positive influence of GLP-1 on glucose levels may be explained, at least in part, by indirect effects due to weight loss and reduced glucotoxicity in T2D. However, a recent study in healthy volunteers showed that GLP-1 directly inhibits glucose production in an experimental condition in which glucose, insulin and glucagon are unable to fluctuate, suggesting a direct effect on hepatocytes [77]. In vitro, the COOH-terminal fragment of GLP-1 has been shown to reduce glucose production in primary hepatocytes by down-regulation of a number of genes involved in gluconeogenesis (i.e., Pck1, G6pc, and Ppargc1a) by stimulation of the cAMP/PKA pathway. Similar effects were also noted in vivo, in mice fed a HFD and subjected to intraperitoneal injections of a GLP-1 derivative [78]. Together with direct and indirect glucose-lowering effects, GLP-1 agonists have been demonstrated to reduce lipid concentrations in T2D, both in fasting and postprandial states [79
82]. A recent study demonstrated that liraglutide, a long-acting GLP-1 analogue, improved the insulin sensitivity of both the liver and the adipose tissue, thereby reducing the overflow of FFA to the liver stimulated by a restored antilipolytic effect of insulin. Moreover, liraglutide reduced hepatic de novo lipogenesis, both in vitro and in vivo [83]. The molecular mechanisms that regulate hepatic lipid metabolism of GLP-1 are only partially understood (Fig. 5.1). GLP-1 incubations in hepatocytes induce an increase in cAMP intracellular level with subsequent activation of AMP-activated protein kinase, a suppressor of lipogenesis. A similar effect was also demonstrated in vivo, in DPP-4 deficient rats, which showed down-regulation of lipogenic genes and upregulation of carnitine palmitoyltransferase-1, a key enzyme in fatty acid β-oxidation [84]. DPP-4 inhibition was also demonstrated to decrease the expressions of sterol regulatory element-binding

cAMP

PKA

SREBP1c SLD-1 FAS

Lipogenesis

CPT1 GLP-1

PPAR

GPCR

PPAR

GLP-1RA

cAMP

PKA

ACOX1 CPT1

-oxidation

PCK1 G6PC PPARGC1a

Gluconeogenesis

FIGURE 5.1 Intracellular pathways activated in hepatocytes by GLP-1/GLP-1 receptor agonists and their effect on glucose and lipid metabolism.

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protein-1c (SREBP-1c), stearoyl-CoA desaturase-1 (SCD-1), and fatty acid synthase (FAS), enzymes involved in de novo lipogenesis and lipid metabolism [85]. The GLP-1 analogue exenatide has also been shown to induce the expression of peroxisome proliferator-activated receptor (PPAR)α and PPARγ and downstream target genes, such as ACOX1 (a ratelimiting enzyme in peroxisomal FFA β-oxidation) and CPT1A (a key enzyme in mitochondrial β-oxidation of FFA) [73]. At least in HepG2 and Huh7 cells, exenatide seems to modulate also a number of genes involved in the insulin signaling pathway, such 3-phosphoinositide-dependent kinase-1, AKT, and protein kinase C zeta [72].

5.3.3 Effects of incretins on NAFLD and NASH The positive effects of GLP-1 receptor agonists on hepatic steatosis have been observed in several mouse models [86
89]. Ding et al. showed that the administration of exendin-4 (10 or 20 μmg/kg) for 60 days to ob/ob mice (which develop obesity and hepatic steatosis due to a mutation in the leptin gene) resulted in reduced ALT level, improved insulin sensitivity and reduction of hepatic steatosis assessed by immunohistochemistry [86]. Similar effects were also obtained in HFD-induced obesity in mice subjected to exendin-4 gene therapy. Lipid and triglyceride content was significantly reduced in the liver of exending-4 treated mice compared to controls; moreover, the expression levels of key lipogenic genes were also decreased [87]. Treatment with liraglutide (200 μg/kg body weight for 4 weeks) reduced hepatic steatosis and endoplasmic-reticulum stress in mice fed with the American LifestyleInduced Obesity Syndrome (ALIOS) diet and high fructose corn syrup [88]. A possible efficacy of GLP-1 receptor agonists has been explored also in small clinical trials, usually involving patients with T2D, with promising results. In general, all GLP-1 receptor agonists have shown a reduction in serum transaminases and a reduction in hepatic triglycerides content. A previous study in 974 patients with T2D, demonstrated a reduction in ALT level in the subgroup of patients with elevated transaminases at baseline (53%), with 39% of the subjects achieving normal levels after 104 weeks of exenatide treatment [90]. A number of posthoc analyses and meta-analyses showed a reduction in liver enzymes in patients treated with liraglutide [91], lixisenatide [92], and exenatide [90,93,94]. The administration of dulaglutide, a long lasting GLP-1 analogue, which is administered only once a week, was associated with a dose-dependent reduction of ALT levels, especially in subjects with elevated ALT before treatment [95,96]. Liver transaminases were also reduced by semaglutide treatment in T2D patients, with a significant effect (9% reduction vs placebo) in the 1.0 mg/week group [97]. Together with amelioration of serum markers of liver damage, GLP-1 receptor agonists have shown promising effects also in reducing lipid accumulation in the liver parenchyma, and in the visceral and subcutaneous compartment. An ancillary study of the Liraglutide Effect and Action in Diabetes-2 (LEAD-2) and LEAD-3 evaluated the effect on body composition of liraglutide [98]. In the study, liver-to-spleen attenuation ratio was assessed by computed tomography (CT) scan to evaluate hepatic steatosis. Liraglutide treatment administered at the higher dose of the studies (1.8 mg/day) significantly reduced hepatic fat content [98]. Along with a reduction in liver steatosis, a number of studies also demonstrated positive effects of liraglutide on visceral and subcutaneous fat

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and waist circumference [98
101]. Liraglutide has also been specifically investigated in biopsy-proven NASH patients, in the LEAN randomized, placebo-controlled phase 2 study [102]. Liraglutide at a dose of 1.8 mg/day was subcutaneously injected in 26 patients for 48 weeks and compared to placebo. About 39% of liraglutide-treated patients versus 2% in the placebo arm (p 5 0.019) achieved the primary endpoint of resolution of definite nonalcoholic steatohepatitis assessed with liver histology. Liraglutide was well-tolerated and induced only mild or moderate gastrointestinal side effects [102]. Initial evidence for a positive effect on hepatic fat accumulation in T2D patients exists also for exenatide. In case series and small cohorts of T2D patients, exenatide treatment was associated with a reduction of liver fat evaluated by abdominal ultrasound, liver histology, or magnetic resonance spectroscopy [100,103,104]. Interestingly, the combination of pioglitazone (PPAR-γ agonist) and exenatide induced a significantly higher reduction in liver fat evaluated by magnetic resonance spectroscopy than pioglitazone alone in T2D, suggesting a possible additive effect [82]. A similar trend was demonstrated for the combination of exenatide with dapaglifozin, an inhibitor of the sodium-glucose transport proteins responsible for the majority of glucose reabsorption in the kidney [105]. GLP-1 receptor agonists seem to exert beneficial effects also on hepatic fibrosis, despite conclusive data in humans are still lacking. A previous retrospective study in T2D showed that the AST to platelet counts ratio (APRI index), an indirect fibrosis score, was reduced by administration of liraglutide and pioglitazone but not sitagliptin, an inhibitor of the DPP-4 enzyme [106]. Fibrosis was also reduced at histological evaluation in four out of eight patients in a small case series of T2D treated with exenatide [103]. In the LEAN study, fibrosis progression occurred in fewer patients treated with liraglutide compared to placebo, despite sample size was small (2 out of 23 patients in the liraglutide group vs 8 out of 22 patients in the control group) [102]. Altogether, despite the majority of study involved only limited amount of patients, were conducted in the setting of T2D and occasionally lacked a control group, available data suggest positive effects of GLP-1 receptor agonists on transaminases, hepatic fat accumulation, and possibly fibrosis. A recent meta-analysis involving six randomized controlled trials and observational studies with GLP-1 receptor agonists showed a reduction of NASH activity and stage of fibrosis, suggesting that the benefits of their use outweigh risk in both patients with and without T2D [107].

5.3.4 The role of inhibitors of dipeptidyl peptidase-4 activity The DPP-4 is the enzyme responsible for inactivation of incretins and other substrates. The expression of DPP-4 in the liver is increased in patients affected by NAFLD and is influenced by insulin resistance [108]. High serum DPP-4 activity in NAFLD patients is associated with alteration of liver tests and, more importantly, has been shown to have a positive correlation with the histologic grade of NASH [109,110]. Preliminary data showed that DPP-4 inhibition with sitagliptin in a nonobese T2D mouse model protected against diet-induced hepatic steatosis [85]. Moreover, DPP4deficient rats have a reduced triglyceride content in the liver paralleled with downregulation of lipogenic genes and upregulation of β-oxidation genes [84]. The effect of

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sitagliptin on hepatic steatosis in rat is due, at least in part, to the amelioration of insulin sensitivity and lipid profile. In fact, sitagliptin-treated rat subjected to HFD showed improved glucose tolerance and reduced serum lipid, together with reduction of hepatic steatosis with major effect on hepatic inflammation [111]. Few data on the effects of DPP-4 inhibitors in humans are also present in the literature. The administration of sitagliptin (50 mg/body/day) for 4 months to 30 NAFLD patients with T2D resulted in a significant decrease of both glucose and liver tests [112]. Improvement of histological parameters, such as ballooning and NASH score, was also reported in a small cohort of T2D patients treated with sitagliptin 100 mg/day [113]. However, these effects were not confirmed in other studies, in which neither an effect on serum transaminases nor on intrahepatic lipids evaluated with magnetic resonance spectroscopy was noted [114,115]. Results of randomized clinical trials are also contrasting. Administration of sitagliptin 100 mg/die for one year was shown to improve steatosis and hepatic ballooning, and to induce a higher NAFLD activity score amelioration in treated patients compared to controls [116]. On the contrary, Cui et al. did not find any significant effect of stiagliptin (100 mg/die for 24 weeks) versus placebo on hepatic fat evaluated by MRI-derived proton density-fat fraction [117]. No effect of sitagliptin versus placebo on liver fibrosis evaluated by liver histology was reported in a small trial involving NASH patients [118]. Altogether, available data suggest a mild effect of DPP-4 inhibitors on NAFLD and NASH and larger clinical trials are eagerly awaited.

5.3.5 The role of incretin co-agonists A number of new drugs that combine GLP-1 receptor agonists with other molecules are actively investigated, especially for the treatment of obesity and T2D. The rationale behind the development of co-agonists is to maximize the beneficial effects of the hormones while reducing the side effects of each drug. GLP-1 agonists have been combined with glucagon, an analogue of oxyntomodulin and GIP; moreover triple co-agonism with GLP-1/GIP/ glucagon has also been investigated in preclinical studies [119]. Data on a possible beneficial effects of co-agonist on hepatic steatosis are currently derived mainly from animal models. The administration of dual GLP-1/glucagon coagonists resulted in normalization of adiposity and glucose tolerance in diet-induced obesity in mice, with a parallel reduction of hepatic fat content [120,121]. Patel et al. specifically investigated the effects of GLP-1/glucagon co-agonist injections on the development of NAFLD induced by HFD in mice. The expression levels of SREBP-1C, SCD-1, acetylCoA carboxylase (ACC), and FAS were decreased by co-agonist treatment together with improvement of body weight gain and glucose intolerance. Moreover, at histological evaluation, hepatic steatosis, inflammation, and fibrosis were all decreased in treated animals compared to controls [122]. The dual GLP-1/GIP unimolecular co-agonist also showed promising effects on NAFLD in mice. The administration of the GLP-1/GIP co-agonist induced a significant reduction of serum transaminases and hepatic steatosis evaluated by histology when compared to controls and liraglutide-treated mice [123]. Interestingly, a recent study in T2D

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patients, showed that 24-week long administration of tirzepatide, a dual GLP-1/GIP coagonist, induced a significant decrease in markers of NASH and hepatic fibrosis compared to controls [124]. Finally, triple co-agonism of GLP-1, GIP, and glucagon receptors have been found to exert potent effects on metabolic parameter and hepatic steatosis in mice. The administration of a monomeric triple agonist has been shown to reduce body weight, improve glycemic control, and ameliorate liver steatosis in mice more efficiently than dual co-agonist. The metabolic efficacy of the triple co-agonist was dependent on GLP-1-related improvement of glucose homeostasis and food intake, GIP-related potentiation of incretin effects and protection against glucagone-induced iperglycemia, and glucagon-related increase in energy expenditure [125].

5.4 Alteration of bile acid pathways in NAFLD 5.4.1 Bile acid synthesis and metabolism The synthesis of bile acids in hepatocytes represents the major metabolic pathway of cholesterol catabolism. The enzymatic reactions responsible for cholesterol conversion take place in the endoplasmic-reticulum, mitochondria, cytoplasm, and peroxisomes, and involve a number of different enzymes. Primary bile acids (i.e., cholic acid and chenodeoxycholic acid) are directly synthesized in the liver, while secondary bile acids (mainly deoxycholic acid and lithocholic acid) are formed in the intestine via metabolization by gut bacteria. The biosynthesis of bile acids involves two major pathways: the classical pathway and the alternative pathway [126]. The classical pathway is responsible for about 90% of bile acid synthesis. The cholesterol 7 alpha-hydroxylase (CYP7A1) is the rate-limiting enzyme of the pathway, responsible for the hydroxylation of cholesterol to 7α-hydroxycholesterol and for maintaining the bile acid pool. The alternative pathway is more active during childhood and mediates the formation of chenodeoxycholic acid via the action of CYP8B1 among others. Primary bile acids are then conjugated with either taurine or glycine, in order to increase hydrophilicity of the molecules and reduce their potential cellular toxicity, and excreted in the bile canaliculus by the bile salt export pump [127]. After ingestion of a meal, bile is released in the intestine from the gallbladder. In the digestive tract, bile acids help the emulsification and digestion of lipids by participating in the formation of mixed micelles, which also contain cholesterol and phospholipids. At this stage, gut bacteria extensively metabolize primary bile acids. Bacterial hydrolases are responsible for deconjugation of primary bile acids, which results in the formation of the two secondary bile acids lithocolic acid and deoxycholic acid. Moreover, intestinal bacteria can carry out a variety of additional modifications of bile acids such as amidation, oxidation, epimerization, esterification, and desulfatation [128]. The vast majority of intestinal bile acids (about 95%) are reabsorbed, mainly in the terminal ileum, and form the bile acid pool. The apical sodium
bile acid transporter (ASBT) is responsible for the uptake of bile acids in the enterocytes, while the organic solute transporter α/β (OST α/β) on the basolateral membrane mediates the efflux in the portal

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system. The sodium/bile acid cotransporter also known as the Na 1 -taurocholate cotransporting polypeptide (NTCP) finally mediated the uptake of bile acids from the portal blood into the hepatocytes [129]. Once inside the enterocytes, bile acids bind to the farnesoid X receptor (FXR), which is considered a master regulator of bile acid homeostasis and also deeply influences glucose and lipid metabolism [130]. The most potent FXR agonist is chenodeoxycholic acid, followed by lithocholic acid and deoxycholic acid. In contrast, more hydrophilic bile acids, such as ursodeoxycholic acid, do not activate FXR. Upon bile acid-inducted activation, FXR induces the expression of small heterodimer partner (SHP), which in turn suppresses the expression of ASBT and induces the expression of OST α/β in order to limit ileal uptake and increase portal excretion of bile acids [131,132]. Moreover, FXR activation in the intestine induce the expression of fibroblast growth factor (FGF)-19 in human and FGF-15 in mice, which are secreted in the portal blood, reach the liver and bind to the FGF receptor 4 (FGFR4). As a result of FGFR4 activation, FGF-19 down-regulates the expression of both CYP7A1 and CYP8B1, thereby exerting a negative feedback loop on bile acid synthesis [133].

5.4.2 Effects of the FXR/FGF-19 pathway on NAFLD and glucose and lipid metabolism Systemic activation of FXR has been shown to exert protective effects on insulin sensitivity, hepatic lipid accumulation, liver inflammation, and lipid metabolism in different animal models. Activation of FXR seems to improve glucose tolerance in both in vitro and in vivo models. FXR stimulation by synthetic agonist GW4064 administration or adenovirus-mediated gene transfer significantly reduced glucose levels and increase glycogen synthesis in diabetic db/db mice. Accordingly, FXR knockout mice showed reduced glucose tolerance and insulin sensitivity [134]. Bile acids have been shown to inhibit in a SHP-dependent fashion the expression of a variety of genes involved in gluconeogenesis, such as glucose-6-phosphatase, phosphoenolpyruvate carboxykinase (PCK), and fructose 1,6-bis phosphatase [135]. Moreover, FXR-independent repression of PCK expression by bile acids is also reported in the literature [136]. It has to be underlined, however, that opposite effects of FXR on glucose metabolisms have also been described. Stayrook et al. showed that GW4064 administration significantly increase PCK expression and glucose output in C57BL6 mice [137]. Important effects on lipid metabolism of FXR activation have also been described. The administration of GW4064, a synthetic FXR agonist, to mice fed an HFD resulted in significantly lower levels of triglycerides and free fatty acid in the liver as compared to control mice [138]. Similar effects on hepatic triglycerides and cholesterol levels were reported for the administration of an alternative FXR agonist (i.e., WAY-362450) in LDL receptor knockout mice fed with a Western diet [139]. The same FXR agonist also reduced gene expression of fibrosis markers and hepatic collagen deposition in mice fed with MCD diet as a model of NASH [140]. To confirm these data, FXR knockout mice have been shown to have a proatherogenic serum lipoprotein profile and increased hepatic levels of cholesterol and triglycerides [141]. The effect of FXR activation of hepatic fat is mediated by decreased lipogenesis and increased fatty acid oxidation. In fact, natural or synthetic FXR agonists

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induce the down-regulation of SREBP-1c in a SHP-dependent fashion, with consequent decreased expression of genes involved in lipogenesis [142]. Moreover, activation of the FRX pathway induces also the upregulation of PPARα, which contains a FXR response element in its promoter region, and downstream response genes that favor fatty acid β-oxidation [143]. Many effects of FXR activation on metabolic pathways are mediated by the secretion of FGF-19. Interestingly, in pediatric or adolescent forms of NAFLD, circulating levels of FGF-19 have been found reduced, suggesting a possible role on disease pathogenesis [144,145]. FGF-15 knockout mice showed worsened hepatic steatosis when fed on a HFD [146]. On the other hand, FGF19 transgenic mice are resistant to the development of obesity or diabetes when fed on a HFD [147]. The effects of FGF-19 on hepatic lipid metabolism are mediated by the down-regulation of a number of genes involved in lipid synthesis and lipid uptake [148]. Moreover, mice treated with FGF-19 have significantly reduced levels of toxic lipid species (i.e., diacylglycerols, ceramides, and free cholesterol), which may contribute to a reduced hepatic inflammation. Mice fed with a high-fat, highfructose, high-cholesterol diet (HFFCD) and treated with FGF-19 had reduced levels of hepatic steatosis, inflammation and fibrosis [149]. However, a potential use of FGF-19 analogues in the clinical setting is currently hindered by a potential protumorigenic effect. Indeed, mice with transgenic expression of FGF-19 in the skeletal muscle have been shown to develop hepatocellular carcinoma by 10 months of age [150].

5.4.3 Bile acid receptors as therapeutic targets in NAFLD In light of the modulation of multiple pathways involved in glucose and lipid metabolism, a number of bile acid receptor ligands are actively been investigated in clinical trials for the treatment of NASH. The best-studied bile acid derivative in NAFLD is obeticholic acid (OCA), a semisynthetic chenodeoxycholic acid analogue with potent FXR agonistic activity [151]. In the first phase 2 trial, OCA was administered at a dose of 25 or 50 mg for 6 weeks to patients with NAFLD and T2D mellitus. Interestingly, insulin-sensitivity improved by 24.5% in the combined OCA groups as compared to placebo-treated patients. In parallel, OCA-treated patients showed reduced levels of transaminases and liver fibrosis evaluated by the ELF score [152]. In the FLINT study, 141 noncirrhotic NASH patients were treated with 25 mg OCA for 72 weeks and compared to 142 patients receiving placebo. The primary outcome (i.e., improvement in NAFLD activity score by at least 2 points without worsening of fibrosis at liver histology) was achieved in 45% of OCA-treated patients compared to 21% in the control group (p 5 0.0002). NASH resolution occurred in 22% of patients treated with OCA, without a significant difference compared to placebo [153]. Recently, a phase 3 trial confirmed the antifibrotic effect of OCA in adult patients with NASH. The primary endpoint of fibrosis improvement ($1 stage) with no worsening of NASH was achieved in 18% of patients in the 10 mg OCA group (p 5 0.045) and in 23% in the 25 mg OCA (p 5 0.0002) compared to 12% in controls. On the other hand, there was again no significant difference in NASH resolution (8%, 11%, and 12% in the placebo, 10 mg OCA and 25 mg OCA, respectively) [154]. Despite promising results, the safety profile of OCA has

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raised some concerns among clinicians. In OCA-treated patients, all reports consistently show a substantial occurrence of pruritus, which ranges from 25% to 50%, is generally mild to moderate in grade and has a correlation with the dosage of the drug. Moreover, significant increases in total serum cholesterol and LDL-cholesterol have been described and may not be unimportant in a population at increased metabolic and cardiovascular risk. Finally, special attention has to be paid in patients with decompensated liver disease. In fact, a dose-related hepatotoxicity with severe cases of liver injury has been reported for OCA, especially in cirrhotic patients [155]. A number of alternative FXR agonists are actively studied in preclinical and clinical trial. The newly discovered FXR agonist tropifexor showed promising efficacy in reversing fibrosis, reducing NAFLD activity score and hepatic triglycerides accumulation in animal models [156]. A recent interim analysis of the FLIGHT-FXR phase 2 trial (NCT02855164) reported that administration of tropifexor to NASH patients resulted in decrease of transaminases, hepatic fat fraction, and body weight, in a dose-dependent fashion and with good safety and tolerability after 12 weeks of treatment. Interesting data are also emerging for the newly discovered nonsteroidal FXR agonist cilofexor (formerly known as GS-9674). Cilofexor is a selective agonist that primarily activates the intestinal FXR and does not undergo enterohepatic circulation, which may reduce side effects of systemic FXR agonism such as dyslipidemia, pruritus and hepatotoxicity. The administration of cilofexor (100, 30 mg or placebo) in a phase 2 trial in 140 patients with noncirrhotic NASH resulted in a reduction of hepatic fat measured by magnetic resonance imaging
proton density-fat fraction of 222.7% in the 100 mg group and 21.8% in the 30 mg group compared to an increase of 1.9% in those receiving placebo. Interestingly, no major variations were reported for serum cholesterol levels, while pruritus remained the most frequent side effect [157]. Despite the results of the study are promising, the lack of improvement of transaminase levels and the short duration of the trial (24 weeks) warrant further investigations [158]. A number of different FXR agonists (e.g., nidufexor, MET409, EDP305) are actively been investigated in preclinical and clinical studies and represent possible future alternatives for the treatment of NAFLD patients [159]. Collectively, the efficacy of FXR agonists seems limited in NASH resolution and partial in improvement of liver fibrosis and transaminase levels. Moreover, class-related side effects limit the clinical applicability of bile acid receptor agonist, especially at the higher doses needed to achieve clinical significant results. In an effort to achieve better results in terms of fibrosis regression and NASH resolution, combination therapies with different drug classes are currently under investigation. In fact, response rates of monotherapy trials do not exceed 32% when compared to placebo, irrespective of the mechanism of action of the selected drug [160]. Preliminary data on the combination of cilofexor 30 mg plus firsocostat 20 mg (an inhibitor of the ACC) for 12 weeks in 20 NASH patients showed a reduction in hepatic fat .30% in 74% of the cohorts. Liver stiffness, transaminases, and serum markers of hepatic fibrosis were also improved by the combination treatment (NCT02781584). The ATLAS study evaluated the efficacy of monotherapy or dual combination regimens of cilofexor 30 mg, firsocostat 20 mg, and selonsertib 18 mg (a apoptosis signal-regulating kinase 1 inhibitor). In the treatment arm with combination of cilofexor plus firsocostat, 20.9% of patients achieved a reduction in hepatic fibrosis of $ 1 stage without worsening of NASH after 48 weeks,

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compared to 10.5% in the placebo group. Despite the difference was not statistically significant, possibly due to small sample size, these data support the concept that combination therapies may be an attractive future possibility in the treatment of NASH patients. A number of other trials evaluating combination therapies with FXR agonists are underway. A phase 2 evaluating the combination of cilofexor, firsocostat and semaglutide (GLP-1 receptor agonist) has been recently completed (NCT03987074). The ELIVATE phase 2 trial is actively recruiting patients for testing the efficacy and safety of the combination of tropifexor and licogliflozin (SGLT1/2 inhibitor) (NCT04065841). Tropifexor is also being evaluated in combination with the leukotriene A4 hydrolase inhibitor LYS006 (NCT04147195). GLP-1 and GLP-1RA bind to G protein-coupled receptors (GPCRs) and activate intracellular pathways that ultimately influence lipogenesis, β-oxidation and gluconeogenesis. Reduced lipogenesis occurs as a consequence of down-regulation of SREBP1c, SLD-1, and FAS due to cAMP-mediated PKA activation. The activation of PKA determines also the down-regulation of a number of genes involved in gluconeogenesis. Increased β-oxidation is dependent on the upregulation of CTP-1, which is mediated by both the activation of PKA and also by the induction of PPARα and PPARγ and downstream target genes.

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C H A P T E R

6 The role of gut microbiota in chronic liver diseases Stefano Brillanti1 and Giada Alterini2 1

Dipartimento di Scienze Mediche, Chirurgiche e Neuroscienze (DSMCN), Universita’ di Siena, Siena, Italy 2Dipartimento delle Scienze Mediche, AOU Senese, Siena, Italy

6.1 Introduction In the previous chapter, the authors elegantly addressed the composition of the human intestinal microbiota and the pathophysiology of dysbiosis in the gut
liver connection. In the present chapter, we address the role of gut microbiota in chronic liver disease. From a clinical point of view, two major questions need to be answered: (1) Do changes in gut microbiota contribute to liver disease development, or are they simply a consequence of the disease? (2) May altering gut microbiota prevent the onset or influence the outcome of liver diseases? These two fundamental issues need to be solved to translate the amount of pathophysiologic data into clinical practice. Changes in microbiota may contribute to liver diseases through several mechanisms that can be influenced by bacterial composition, metabolism of bile acids, diet, environmental factors, and genetics, with the possibility of bacteria and bacterial products and metabolites to translocate through the intestinal barrier into the portal vein system, and then reaching the liver. Even though all these possible mechanisms may contribute to the pathogenesis of liver disease, strong evidence of changes in intestinal microbiota as the causal mechanism of liver disease is still elusive. This chapter aims to highlight the best possible evidence of a causal relationship between dysbiosis and the best-studied types of chronic liver diseases. If a causal relationship is found, altering the gut microbiota to ameliorate liver disease evolution is discussed.

6.2 Metabolic liver disease Metabolic associated fatty liver disease (MAFLD), also known as nonalcoholic fatty liver disease (NAFLD), is a common cause of chronic liver disease [1]. The disease

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Brain, Gut
Liver, and Liver
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spectrum ranges from a potentially nonevolving fat deposition in hepatocytes (steatosis) to necroinflammation (steatohepatitis) to liver fibrosis and cirrhosis [2]. The prevalence of this condition is progressively increasing in Western countries, affecting 15%
30% of the general population [3]. MAFLD is often observed in patients affected by metabolic syndrome, diabetes, and obesity, representing a phenotype with complex and disparate causes and multiple underlying subtypes [4]. One initial question is whether changes in intestinal microbiota, eventually found in patients with MAFLD, are distinctive of this general condition or reflect changes associated with the underlying predisposing conditions. For example, gut microbiota dysbiosis has been repeatedly observed in obesity, and type 2 diabetes mellitus, two metabolic diseases strongly connected with MAFLD. Microbiota alterations have been repeatedly described in patients with MAFLD. Some consistent microbiome signatures discriminating healthy individuals from those with MAFLD have been found, but whether these alterations are independent of the concomitant presence of obesity or type 2 diabetes is not clear in many studies [5
7]. We do not intend to enumerate and summarize the long list of published papers on these intriguing aspects, but we prefer to focus on the results of a few prominent studies. In an elegant study of 2017, Rohit Loomba and coworkers analyzed the gut microbiome compositions using whole-genome shotgun sequencing of DNA extracted from stool samples in patients with clinical and biopsy-proven MAFLD [8]. Gut microbiomes were dominated by members of Firmicutes and Bacteroidetes, followed by Proteobacteria and Actinobacteria in much lower abundances. However, as the disease progresses from mild/moderate MAFLD to advanced fibrosis, the Proteobacteria phylum had a statistically significant increase in abundance while the Firmicutes phylum decreased. This decrease of Grampositive Firmicutes and an increase of Gram-negative Proteobacteria in patients with advanced MASH fibrosis suggests that the microbiota shifts toward more Gram-negative microbes as the liver disease progresses. At the species level, Eubacterium rectale (2.5% median relative abundance) and Bacteroides vulgatus (1.7%) were the most abundant organisms in mild/moderate MAFLD, while B. vulgatus (2.2%) and Escherichia coli (1%) were the most abundant in advanced fibrosis. These data suggest that E. coli dominance occurs earlier in the stage of fibrosis progression and support the hypothesis that dysbiosis may precede the development of portal hypertension, even if it does not imply causality, and dysbiosis with predominant Gram-negative bacteria might contribute to liver fibrosis. The results of this work confirm and evolve our knowledge of the association between gut dysbiosis and advanced fibrosis in MAFLD, as assessed in other seminal studies [9,10]. Even if the association between alterations in gut microbiota and liver fibrosis stages has been repeatedly assessed, fundamental questions remain to be answered. These findings may simply reflect differences in age and may not be specific to the fibrosis stage, and, based on our knowledge, these data do not prove causality. If dysbiosis plays a role in the pathogenesis of steatosis and fibrosis, one should expect some modification in these parameters if the alterations in gut microbiota are corrected. A recent study from Scorletti et al. addressed this clinical important point [11]. Patients with steatohepatitis were given synbiotic agents orally for about one year. Synbiotic agents successfully altered gut microbiota: fecal samples from patients who received the synbiotic had higher proportions of Bifidobacterium and Faecalibacterium species and reductions in

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Oscillibacter and Alistipes species, compared with the baseline. Unfortunately, even if the authors successfully induced a change in gut microbiota with synbiotic administration, changes in the gut microbiota’s diversity did not translate into a reduction in liver fat composition or liver fibrosis changes. Interestingly, an overall reduction in liver fat content was independently associated only with weight loss. We do not intend to dismiss the role of gut microbiota in the pathogenesis of MAFLD, steatohepatitis, and metabolic cirrhosis, as suggested by many well-performed studies, and negate the possibility of therapeutically intervene with effective changes in gut microbiota. Available data strongly indicate a possible causal role of gut microbiota in the development of MAFLD, but the definite proof and the efficacy of therapeutic interventions have not arrived yet.

6.3 Primary sclerosing cholangitis Primary sclerosing cholangitis (PSC) is a relatively rare, immune-related cholestatic liver disease, characterized by chronic biliary inflammation and fibrosis of the biliary tree, leading to end-stage liver disease, possibly cholangiocarcinoma [12]. PSC is strongly associated with a phenotype of another immune-mediated condition, inflammatory bowel disease (IBD), especially ulcerative colitis [13,14]. To date, no medical therapies have proved effective, and the only available treatment for end-stage PSC is liver transplantation, but disease recurrence is a significant complication. PSC is a complex disease involving both genetic and environmental factors. During recent years, the potential relationship between the gut microbiota and PSC has been extensively studied. In general, patients with PSC have a marked decrease in gut microbiota diversity and dysbiosis, and a substantial overrepresentation of Enterococcus, Escherichia, Fusobacterium, Lactobacillus, Veillonella, Blautia, Lachnospiraceae, Barnesiellacea, and Megasphaera genera compared with healthy controls and patients with IBD alone. In contrast, patients with PSC have marked reductions in Clostridiales II, Prevotella and Roseburia, and Bacteroides, compared to patients with IBD alone and healthy controls. Among the published studies, we like to focus on the work of 2016 by Sabino et al. [15]. In this study, the fecal microbiota of patients with PSC was characterized by decreased microbiota diversity and a significant overrepresentation of Enterococcus, Fusobacterium, and Lactobacillus genera. This dysbiosis was present in patients with PSC with and without concomitant IBD, was distinct from IBD, and independent of treatment with ursodeoxycholic acid. One operational taxonomic unit belonging to the Enterococcus genus was associated with increased serum alkaline phosphatase (ALP) levels, a marker of disease severity. These data indicate that fecal microbiota in patients with PSC is significantly different from that of healthy volunteers and IBD patients. Furthermore, samples from PSC patients and concomitant IBD clustered together with PSC only and apart from patients with IBD without PSC. Therefore PSC seems to shape the microbiota composition stronger than IBD, allowing for a clear separation of samples of patients with PSC according to PSC diagnosis and regardless of concomitant IBD. In a subsequent elegant study of 2017, Kummen and coworkers confirmed that patients with PSC have reduced bacterial diversity in stools compared with healthy controls and

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found a different global microbial composition than both controls and patients with ulcerative colitis [16]. As in the previous study, the microbiota of patients with PSC with and without IBD was similar. Twelve genera separated PSC and healthy controls, out of which 11 were reduced in PSC. However, the Veillonella genus showed a marked increase in PSC. Again, patients with PSC exhibited a distinct gut microbial signature irrespective of the concomitant presence or absence of IBD, and ursodeoxycholic acid therapy did not alter gut microbiota in PSC patients. As mentioned in MAFLD, one crucial question is whether the microbiota alterations represent an actual link between the gut and the liver in PSC or secondary to the advanced liver disease. The lack of a correlation between PSC duration or biochemical parameters and microbiota diversity seems to be against the latter, although a link between the particularly high prevalence of Veillonella and more severe liver disease cannot be excluded. Therefore the observed microbiota alterations in PSC may be involved in disease development. In addition to our knowledge, a recent elegant study from Liwinski and coworkers [17]. The authors studied the microbiome composition of the oral cavity, duodenal fluid, and bile duct fluid in patients with PSC and controls. The bile fluid harbored a diverse microbiome distinct from that in the oral cavity, the duodenal fluid, and duodenal mucosa. The upper alimentary tract microbiome differed between PSC patients and controls. However, the most significant differences were observed in the ductal bile fluid, including reduced biodiversity and increased pathogen Enterococcus faecalis in PSC. Enterococcus abundance in ductal bile was strongly correlated with the noxious secondary bile acid taurolithocholic acid concentration. Therefore PSC seems characterized by an altered microbiome of the upper alimentary tract and bile ducts, and biliary dysbiosis appears to be linked with increased concentrations of the proinflammatory and potentially cancerogenic agent taurolithocholic acid. These findings support the hypothesis that changes in the biliary microbiome may contribute to PSC pathogenesis by enhancing bile duct mucosa damage and potentially by effects on the noxious bile acid concentration lithocholic acid, even if these novel results cannot prove causality. Differently than for metabolic liver disease, some evidence exists on the potential beneficial effects of altering gut microbiota on markers of liver disease severity in PSC [18]. In a recent paper, Shah et al. conducted a systematic review and metaanalysis to assess the effect of antibiotic therapy in PSC [19]. Effect of antibiotic therapy on Mayo PSC Risk Score (MRS), serum ALP, total serum bilirubin (TSB), and adverse events (AEs) rates were analyzed. Five studies, including 124 PSC patients who received antibiotics, were included. Overall, antibiotic treatment was associated with a significant reduction in ALP, MRS, and TSB. ALP reduction was most significant for vancomycin than for metronidazole. Overall, less than 10% of patients had AEs severe enough to discontinue antibiotic therapy. Therefore in PSC patients, antibiotic treatment resulted in a significant improvement in cholestasis markers and MRS markers. These data suggest that antibiotics may be an effective treatment option for PSC, especially in those patients with IBD. However, the exact mechanism of action of antibiotic therapy remains unclear. In addition to the antimicrobial and subsequent antiinflammatory effects of antibiotics, antibiotic therapy almost certainly alters the gut microbiome. Several antibiotics have been used so far without major adverse side effects; however, antibiotic drug resistance always remains a concern. Oral vancomycin

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seems to be the most promising antibacterial pharmacotherapy for PSC that has been evaluated in RCTs. However, most studies are limited by the sample sizes of the clinical trials. Interestingly, oral vancomycin has also been used to treat recurrent PSC after orthotopic liver transplant, suggesting that the disease mechanism is not confined to the liver and involves the intestine, for example, the backflow of gut bacteria or gut bacteremia [20]. Thus it can be hypothesized that oral vancomycin not only specifically suppresses Grampositive bacteria involved with primary bile acid metabolism but might also be directly or indirectly involved with attenuating the inflammatory response underpinning periportal inflammation and hepatic damage. However, even if antibiotic therapy seems beneficial in improving PSC patients’ disease activity, the ideal antibiotic, dose, and regimen remain mostly unknown. Also, the use of definite primary endpoints rather than surrogate markers to define the natural history of PSC needs to be studied. Finally, molecular technologies should be applied to properly explore the interdependence between the gastrointestinal microbiome, immune system, genetic profile, and response to antibiotic therapy in PSC.

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610. Available from: https://doi.org/10.1053/j.gastro.2020.01.031. e7. [12] Lindor KD, Kowdley KV, Harrison ME. ACG clinical guideline: primary sclerosing cholangitis. Am J Gastroenterol 2015;110(5):646
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[13] Loftus EV, Harewood GC, Loftus CG, Tremaine WJ, Harmsen WS, Zinsmeister AR, et al. PSC-IBD: a unique form of inflammatory bowel disease associated with primary sclerosing cholangitis. Gut 2005;54(1):91
6. Available from: https://doi.org/10.1136/gut.2004.046615. [14] Weismu¨ller TJ, Strassburg CP, Trivedi PJ, Hirschfield GM, Trivedi PJ, Bergquist A, et al. Patient age, sex, and inflammatory bowel disease phenotype associate with course of primary sclerosing cholangitis. Gastroenterology 2017;152(8):1975
84. Available from: https://doi.org/10.1053/j.gastro.2017.02.038. e8. [15] Sabino J, Vieira-Silva S, Machiels K, Joossens M, Falony G, Ballet V, et al. Primary sclerosing cholangitis is characterised by intestinal dysbiosis independent from IBD. Gut 2016;65(10):1681
9. Available from: https://doi.org/10.1136/gutjnl-2015-311004. [16] Kummen M, Holm K, Anmarkrud JA, Nyga˚rd S, Vesterhus M, Høivik ML, et al. The gut microbial profile in patients with primary sclerosing cholangitis is distinct from patients with ulcerative colitis without biliary disease and healthy controls. Gut 2017;66(4):611
19. Available from: https://doi.org/10.1136/gutjnl-2015310500. [17] Liwinski T, Zenouzi R, John C, Ehlken H, Ru¨hlemann MC, Bang C, et al. Alterations of the bile microbiome in primary sclerosing cholangitis. Gut 2020;69(4):665
72. Available from: https://doi.org/10.1136/gutjnl2019-318416. [18] Damman JL, Rodriguez EA, Ali AH, Buness CW, Cox KL, Carey EJ, et al. Review article: the evidence that vancomycin is a therapeutic option for primary sclerosing cholangitis. Aliment Pharmacol Ther 2018;47 (7):886
95. Available from: https://doi.org/10.1111/apt.14540. [19] Shah A, Crawford D, Burger D, Martin N, Walker M, Talley NJ, et al. Effects of antibiotic therapy in primary sclerosing cholangitis with and without inflammatory Bowel disease: a systematic review and meta-analysis. Semin Liver Dis 2019;39(4):432
41. Available from: https://doi.org/10.1055/s-0039-1688501. [20] Hey P, Lokan J, Johnson P, Gow P. Efficacy of oral vancomycin in recurrent primary sclerosing cholangitis following liver transplantation. BMJ Case Rep 2017;2017. Available from: https://doi.org/10.1136/bcr-2017221165.

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C H A P T E R

7 Gut-liver The role of serotonin and its pathways in hepatic fibrogenesis Cristina Stasi1,2, Stefano Milani3 and Andrea Galli3 1

MASVE Interdepartmental Hepatology Center, Department of Experimental and Clinical Medicine, University of Florence and CRIA-MASVE Center for Research and Innovation, Careggi University Hospital, Florence, Italy 2Epidemiology Unit, Regional Health Agency of Tuscany, Florence, Italy 3Gastroenterology Unit, Department of Experimental and Clinical Biomedical Sciences, Florence, Italy

7.1 The state of the art The crucial point of chronic hepatopathies is specifically represented by their capacity for fibrotic evolution. Liver fibrosis is the result of the imbalance between extracellular matrix (ECM) production and its degradation, mediated by the balance between the matrix metalloproteinases and their inhibitors (TIMPs). This process may evolve on one hand into cirrhosis [1], but on the other hand retrace the reverse pathway, leading to fibrosis regression [2,3]. The excessive wound healing response that occurs in most forms of chronic liver disease results in hepatic fibrosis that is characterized by the deposition of ECM components such as collagens, proteoglycans, and glycoproteins [4
6]. Quantitative changes are associated with qualitative alterations in the composition of matrix, resulting in a predominance of type I and III fibrillar collagens, which accumulate up to 10-fold over time and build up a network resistant to fibrolysis following crosslinking of collagen bundles. Hepatic stellate cells (HSCs) are the main cellular population involved in ECM deposition. During liver injury, HSCs undergo a process of phenotypical transformation into activated myofibroblast-like cells that synthesize proinflammatory/proangiogenic cytokines and a large amount of ECM components. Cytokines and growth factors act with autocrine and paracrine effects coordinating the interaction between the different neighbouring cell populations and the activation of specific networks that direct the wound healing reaction in the liver and influence its progression. The death of hepatocytes leads to the release of cellular

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contents such as damage-associated molecular patterns that activate resident macrophages (Kupffer cells) to release proinflammatory molecules like tumour necrosis factor alpha (TNFα), interleukin 1 beta (IL-1β), and IL-6, monocyte chemoattractant protein-1 (MCP-1) and profibrogenic factors, especially transforming growth factor-beta (TGF-β) and connective tissue growth factor. However, synthesis of TGF-β maintain the HSC activated phenotype, whereas hepatocyte growth factor stimulates regeneration of adjacent hepatocytes and MCP-1 and as well as other chemokines such as platelet-derived growth factor (PDGF), regulated upon activation, normal T cell expressed and secreted, IL-8, IL-6 are involved in the recruitment of leukocytes, neoangiogenesis, and HSC proliferation [7
9]. In addition, the production of free radicals generates oxidative stress that contributes to all fibrogenic disorders characterized by chronic tissue damage and to the overexpression of critical genes related to ECM synthesis and inflammation. Liver fibrosis has been considered traditionally as an irreversible process, but several data suggest that the removal of the etiological agent or condition can result in significant regression of liver fibrosis. For instance, positive results in terms of fibrosis regression have been described in chronic hepatitis C virus (HCV) infected patients who have achieved sustained virological response after antiviral therapy [10,11]. Fibrosis regression is associated with HSC apoptosis and activation of ECM degradation systems. Serotonin (5-HT) has a pleiotropic function in gastrointestinal, neurological/psychiatric, and liver diseases [12]. Nowadays, several studies have demonstrated the profibrogenic role of 5-HT in the liver, showing that it works synergistically with PDGF in stimulating HSCs proliferation. This biogenic amine exerts its activity through a family of seven receptors (from 5-HT1 to 5-HT7) and it regulates many key factors involved in the tissue repair process. It has also been shown that 5-HT can influence the proliferation and apoptosis of activated HSC and the selective antagonists of the 5-HT receptors’ family may have potential antifibrotic effects. Moreover, activated HSCs express a 5-HT transporter protein (SERT) [13]. Probably, the SERT (in the L/L variant) could play an antiproliferative role that could partly explain the limited effects of 5-HT in the proliferation of HSCs. It has also been suggested that there is a correlation between the gut-microbiota, the liver and the 5-HT metabolism. The intestinal microbiota plays an important role in the intestinal homeostasis of 5-HT, affecting the SERT and/or the expression of the 5-HT receptors. In fact, the changes in the composition of the commensal bacteria subsequently change 5-HT concentrations associated with specific bacteria. Moreover, the decreased SERT expression was associated with a shift in gut microbiota from homeostasis to inflammatory type microbiota [14]. Currently, the major research efforts are to find pharmacological targets for therapies aimed at inhibition of fibrosis progression or induction of fibrosis reversal [15
17].

7.2 Serotonin synthesis and metabolism Serotonin is a monoamine neurotransmitter that regulates important functions in the gastrointestinal tract and the liver. The gastrointestinal tract comprises about 95% of the body’s 5-HT. Enterochromaffin cells (ECs) contain about 90% of 5-HT and release it following mechanical or chemical stimuli [18,19]. This neurotransmitter released in the bloodstream is captured by the SERT and stored in the granule cargo of platelets [20].

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FIGURE 7.1 Tryptophan metabolism. Tryptophan contains an indole functional group in its structure and it is metabolized through several different biochemical pathways, but the main are the serotonin and kynurenine pathways. L-tryptophan is converted to serotonin (5-hydroxytryptamine or 5-HT) via 5-hydroxytryptophan. The enzyme indoleamine 2,3-dioxygenase (IDO), a heme enzyme, catalyzes the first and rate-limiting step in tryptophan catabolism into the kynurenine-pathways.

The tryptophan (TRP), precursor of 5-HT, is an essential amino acid and it contains an indole functional group. Irreversible cleavage of the indole ring catalyzed by tryptophandioxygenase (TDO) leads to the formation of 5-HT (Fig. 7.1). The TRP is the precursor of many physiologically important metabolites produced during the course of its degradation along four pathways, one of which is of quantitatively major significance, the kynurenine pathway, accounting for B95% of overall TRP degradation [21]. The kynurenine pathway is rate-limited by its first enzyme, tryptophandioxygenase, which is expressed in the liver and has a very strict substrate specificity acting only on L-TRP, on the contrary indolamine 2,3-dioxygenase 1 or 2 (IDO-1 or IDO-2) has a lower substrate specificity and has a much wider tissue distribution, including peripheral blood and immune cells [21,22]. Only small amounts of TRP are metabolized into the 5-HT pathway, while most TRP is processed along the kynurenine pathway. The kynurenine pathway produces many biologically active metabolites, including the important redox cofactors [21]. The increased TRP degradation into kynurenine by IDO can induce a reduction of 5-HT production [23,24]. In human exist two major reservoirs of 5-HT: brain and peripheral 5-HT. Peripheral 5-HT is synthesized from the TRP by two enzymes: tryptophan hydroxylase 1 (TPH1) and

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aromatic acid decarboxylase (AADC). Only 5% of 5-HT is present in the brain and its synthesis is controlled by TPH2 and AADC [25,26]. Peripheral 5-HT plays a key role in glucose metabolism, gluconeogenesis, and glycolysis in the liver [27,28]. Some studies conducted in mice demonstrated that an increased level of 5-HT in the blood induced hepatic steatosis [29,30]. HSCs are also able to store 5-HT to an internal compartment from which it can be released thereby initiating a possible feedback loop sustaining both hepatocyte and activated HSC proliferation. Both cell types are known to contribute to the regeneration of hepatic mass following liver injury [31]. Serotonin has been recently shown to mediate the pathology of many liver diseases, including steatohepatitis, chronic cholestasis, and cirrhosis [32]. The endogenous activity and availability of 5-HT are controlled by a specific SERT that mediates the intracellular reuptake of 5-HT and can be specifically blocked by selective 5-HT reuptake inhibitors (SSRIs) [33]. The biogenic amine transporters are membrane proteins that carry the neurotransmitter into the cell. Along with the transporters of biogenic amine (including transporters for dopamine, noradrenaline, glycine, γ-aminobutyric acid, proline, creatine and betaine), SERT is involved in the uptake of the 5-HT biogenic amine. SERT is very similar to the catecholamine transporters [34] and it is classified as a membrane Na1/K1 pump dependent neurotransmitter transporter [35]. The SERT is encoded in humans by a single gene (SLC6A4) that has been cloned and mapped on chromosome 17q11.1-q12 [36,37]. SERT gene codes for a polypeptide of 630-amino acids, which crosses the membrane 12 times [38]. These transmembrane domains likely contain amino acid residues related to the binding site for 5-HT. Specifically, the major determinants of substrate binding are the residues of the first and third transmembrane domains [39]. The 5-HT is involved in SERT regulation affecting activity and cell surface expression [40]. SERT is widely expressed in multiple tissues [41,42] including platelet membranes [43,44], placenta [45], hepatocytes, bile duct epithelial cells, and HSCs [31]. SERT ensures the basal levels of 5-HT by 5-HT uptake through sodium and chloride cotransport coupled to potassium efflux. SSRIs prevent uptake of 5-HT resulting in its accumulation and prolonging its effects [46
48]. Polymorphisms of the SERT (5-HTTLPR) result in a short (S) and a long (L) allele. Genotypes S/S and L/S have a reduced expression and low efficacy of 5-HT reuptake [49]. The monoamine oxidases (MAO) are the main enzymes inducing the catabolism of 5HT and catalyze the oxidative deamination of 5-HT into 5-hydroxy-acetaldehyde. MAO-A isoform is mainly located in fibroblasts and placental tissue, while MAO-B is the only isoform found in platelets and lymphocytes [50]. The intermediate 5-hydroxyindole-3acetaldehyde (5-HIAL) is either oxidized into 5-hydroxyndolacetic acid (5-HIAA) or reduced into 5-hydroxytryptophol (5-HTOL) by aldehyde dehydrogenase and aldehyde reductase, respectively [51,52]. 5-HIAA can be excreted in the urine in a free form, while 5-HTOL is mainly excreted after either glucuro- or sulfo-coniugation. Another biotransformation pathway of 5-HT is the acetylation by N-acetyltransferase. The N-acetylserotonin is converted into melatonin by hydroxyindole O-methyltransferase [51]. In fact, in the pineal gland, thyroid, and gastrointestinal tract, 5-HT may also serve as a substrate for the synthesis of melatonin [53].

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7.3 Serotonin receptors Serotonin performs its biological function through interactions with distinct receptor subtypes, currently classified according to their structures, molecular mechanisms and pharmacological profiles. The availability of 7 receptor classes including 14 subtypes of 5-HT receptors reflects the diversity of the serotonergic actions [54]. Serotonin acts predominantly via G-protein coupled receptors, except for the 5-HT3 receptor, a ligand-gated ion channel, which activate an intracellular second messenger cascade to produce an excitatory or inhibitory response. Most of 5-HT receptor subtypes are widely expressed along the gut and regulate a variety of digestive functions [51]. Within these subtypes, 1A, 1B, 2A, 2B, and 7 5-HT receptors are involved in normal liver function and disease. On the contrary, 5-HT receptors 4 and 5 are probably not or poorly expressed in the liver [55]. Several researches have demonstrated the key role played by 5-HT in gastrointestinal disorders [19,56] and the involvement of SERT in the pathophysiology of these disorders [57
64]. Briefly, in the digestive tract, 5-HT released from EC cells is involved in sensory, motor and secretory functions by both the extrinsic nervous system (provided by the parasympathetic and sympathetic nervous systems) and the intrinsic innervation (provided by neurons of the enteric nervous system, ENS). The ENS resides in the myenteric and submucosal plexus and it can work independently from the central nervous system, owing to the presence in the bowel of intrinsic primary afferent neurons (IPANs) [65], although bowel can affect the brain through extrinsic primary afferent neurons located in cranial nerves and dorsal root ganglia and the brain, in turn, can affect functions of the ENS via the parasympathetic nervous system. In particular, the peristaltic and secretory reflexes are regulated by the release of 5-HT from EC and depend on the stimulation of 5-HTR1P and 5-HTR4 on submucosal IPANs, while 5-HTR3 receptors on IPANs may play a role in the initiation of other types of intestinal reflexes. In addition, postsynaptic 5-HTR3 receptors are present in both plexuses, including the motor neurons that innervate smooth muscle [65]. 5-HT can induce the release of acetylcholine by cholinergic neurons, producing smooth muscle contraction. It can also stimulate nitrergic neurons to release nitric oxide causing smooth muscle relaxation [18]. In the liver, 5-HT released from platelets mediates hepatic wound healing and hepatocyte regeneration [20]. Serotonin promotes liver tissue repair after ischemia/reperfusion injury. Indeed, several 5-HT receptor subtypes have been identified in the liver of naı¨ve mouse, including 5-HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT3A, and 5-HT3B in addition to the mRNA expression of 5-HT2A and 2B receptors that are associated with abnormal development and mitogenic stimulation. Some studies have demonstrated that 5-HT increases after partial hepatectomy, while liver regeneration is suppressed by 5-HT antagonists [32]. Platelets represent the main source of 5-HT at the level of the hepatic sinusoids in the course of liver damage and they are capable to interact with sinusoidal endothelial cells and also to move into the sinusoidal and perisinusoidal Disse spaces. In this way, upon platelets activation, the released 5-HT is free to bind its receptors expressed by hepatocytes and both quiescent and activated HSC. The binding of 5-HT to its receptors expressed on endothelial cells and HSCs can regulate sinusoidal blood flow [31]. Some functions of these receptors are summarized below and in Fig. 7.2.

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FIGURE 7.2 The actions of serotonin (5-HT) in liver disease. Platelets represent the main source of sinusoidal serotonin (5-HT) during liver injury. 5-HT stimulates hepatocyte proliferation via 5-HT2A receptors. Moreover, the activation of 5-HT receptors in hepatic stellate cells (HSCs) enhances TGFβ1 production, promoting liver fibrosis. In normal liver, the local production of 5-HT by cholangiocytes inhibits their proliferation, but during liver injury, this autocrine negative feedback is suppressed by TGFβ1. In response to proinflammatory mediators, 5-HT is released from dense granules of platelets in the space of Disse and it is free to bind 5-HT receptors expressed by hepatocytes, and by both quiescent and activated HSC. The binding of 5-HT to its receptors expressed on endothelial cells and HSCs can regulate sinusoidal blood flow. Therefore serotonin induces fibrosis, lipogenesis, liver tumorigenesis, proliferation and regeneration of hepatocytes and cholangiocytes. CCA, Cholangiocarcinoma; HCC, hepatocellular carcinoma; HSC, hepatic stellate cell; TGFβ1, transforming growth factor-beta 1; 5-HT, serotonin.

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7.3.1 5-HT1A This receptor stimulates hepatic glucose uptake. The involvement of 5-HT1 and 5-HT2A receptors in the 5-HT stimulatory glycogen synthesis response is supported by the inhibitory effects of 5-HT1 and 5-HT2A receptor antagonists such as olanzapine, an antagonist of 5-HT1B and 5-HT2A receptors [66]. Moreover, a decreased hepatic 5-HT1A receptor function is associated with hepatocyte regeneration and neoplasia, on the contrary stimulation of 5-HT1A receptor inhibited hepatocyte DNA synthesis [67,68].

7.3.2 5-HT1B The study of Soll et al. [69] demonstrated that HT1B and HT2B are overexpressed in tumour tissue and the expression of these receptors correlated with a higher proliferation index. Specific HT1B and HT2B antagonists inhibited the proliferation of cells in two human hepatocellular carcinoma (HCC) cell lines. The expression of 5-HT1B, 5-HT2A, and 5-HT2B being induced on rat and human HSC activation [70]. HT1B and HT2B are expressed on cholangiocytes and their activation markedly inhibits the growth and choleretic activity of the biliary tree in the bile duct-ligated rat, a model of chronic cholestasis [71].

7.3.3 5-HT1D Zuo et al. [72] demonstrated that the 5-HT1D expression level was significantly upregulated in HCC tissues and cell lines. The 5-HT1D expression level predicts poor overall survival and high recurrence probability in HCC patients, promoting HCC proliferation, epithelial-mesenchymal transition, and metastasis in vitro and in vivo. 5-HT1D could stabilize phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) by inhibiting its ubiquitinmediated degradation.

7.3.4 5-HT2A Ruddel et al. [31] reported that 5-HT2A receptors were more weakly expressed than 5-HT2B receptors in a CCl4-induced rat model of hepatic fibrosis. Moreover, antagonists of 5-HT2A and 7 diminished liver fibrosis through a reduction in oxidative stress/TGF-β1induced HSCs activation pathway [73].

7.3.5 5-HT2B Fibrogenesis due to the activation of HSCs in the liver is a negative regulator of hepatocyte regeneration. This negative regulatory function is induced by stimulation of the 5-HT2B on HSCs by 5-HT, which activates expression of TGF-β1, a potent suppressor of hepatocyte proliferation, through signaling by mitogen-activated protein kinase 1 (ERK) and the transcription factor JunD [74]. Moreover, pharmacological targeting of these receptors may be therapeutic in chronic liver disease [75]. 5-HT2 antagonists inhibited the proliferation and increased the apoptosis of HSCs [70].

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7.3.6 5-HT2C In vitro, cell lines of murine cholangiocytes and human HSCs express 5HT2C, 5HT2A, and 5HT2B; the treatment of these cell lines with antagonists or TPH1 inhibitor decreased 5-HT levels and subsequently the fibrosis and inflammation genes expression [76].

7.3.7 5-HT3 Within the other 5-HT receptors, 5-HT3 receptor is a ligand-gated, nonspecific cation channel, which is modulating ion flux [32]. The intestinal 5-HT content increased in obese (ob/ob) mice and the treatment with a 5-HT3 receptor antagonist induced the reduction of the 5-HT levels and an increase in SERT in the duodenum. In these obese mice, the 5-HT3 receptor antagonist induced diminished fat content, inflammation, and necrosis in the liver [77].

7.3.8 5-HT5 In HCC tumour tissues, 5-HT5 and 5-HT2A receptors were reduced, on the contrary, the 5-HT1D, 5-HT2B and 5-HT7 receptors were overexpressed [78].

7.3.9 5-HT7 In vitro and in vivo, Polat et al. [79] showed 5-HT7 expression in CCl4-induced liver damage. 5-HT7 agonist protected liver tissue from oxidative stress and fibrosis, inducing anti-inflammatory, antifibrotic, and anti-cytokine actions [79]. Moreover, 5-HT7 receptors could be a new potential target to inhibit hepatitis B virus (HBV) infection by activating adenylyl cyclase/protein kinase A/extracellular signal-regulated kinase pathways by phosphorylating p53 via the 5-HT agonist response [80]. Tzirogiannis et al. [81] showed that 5-HT7 receptor is involved in liver regeneration after partial hepatectomy. Serotonin through 5-HT7 receptor could induce its auxiliary proliferative effect close to G1/S transition point and during the S phase. Therefore the results identify a novel type of 5-HT receptor that mediates the proliferative effect of the 5-HT in the liver [81].

7.4 The natural course of chronic liver disease Liver fibrosis occurs in response to chronic damage and is characterized by intrahepatic deposition of ECM. This process involves several cell types and mediators. The initial immune-inflammatory response plays different roles in liver damage progression and evolution and it engages different cell populations that produce and release cytokines. In particular, T and B lymphocytes, macrophages, natural killer cells, neutrophils, HSCs, dendritic cells, and mast cells are involved in the maintenance of the chronic inflammatory process. Different receptors of 5-HT are express on immune cells: the 5-HT1 receptors are expressed on mast cells, macrophages, and dendritic cells; 5-HT2 receptors are expressed on macrophages, dendritic cells, and eosinophils. The 5-HT3 receptors are expressed on B cells, and 5-HT4 and 7 on T-cells and dendritic cells [82]. Dendritic cells

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are able to pick up 5-HT released from activated T-cells that synthesize 5-HT, through the SERT and store it in vesicles and subsequently release it via Ca21 sensitive exocytosis to induce T-cell proliferation and differentiation of naı¨ve T-cells [83]. Chronic HBV or HCV infections are characterized by intrahepatic cellular infiltrates represented by immune effectors. Viruses are recognized by Toll-like 3- and 7-receptors, with the subsequent induction of the secretion of type I interferon (α and β), together with cytokines and other molecules that inhibit viruses replication [84]. In HSCs lipopolysaccharide (LPS) activates Toll-like receptor 4-, which in turn induces fibrosis through the involvement of TGFβ [85]. This growth factor is capable to induce fibrosis through increased Th17 and activation of HSCs, in parallel with the natural killer (NK) cell activity reduction [86]. Evans et al. [87] demonstrated that SSRIs enhanced the cytosolic functions of NK cells in vitro. Moreover, TGF-β can induce recruitment of macrophages, NK cells, and dendritic cells [88]. Mapping studies demonstrated that 5-HT2 receptors activated mitogenic signaling components such as protein kinase C, extracellular signal-regulated kinase, and oxidants, directly linked to the regulation of TGF-β1 [89]. A mitogenic circuit has been identified linking serotonin receptor 5HT2B and the receptor of PDGF and similarly linking SERT and PDGF receptor [90]. Both NK cells and cytotoxic T lymphocytes CD81 play immunoregulatory roles through direct mechanisms and production of cytokines [91]. These two cell populations produce IFNγ and TNFα, which in turn are capable to inhibit viruses and induce liver damage, through TNFrelated apoptosis-inducing ligand (TRAIL)-mediated death of hepatocytes [92]. Activation of 5-HT4 and 5-HT7 decreased TNFα release by monocytes in both baseline and LPS stimulation conditions [93]. Chemokine ligands, induced by IFNγ, especially, CXCL9, CXCL10, and CXCL11, cause migration of mononuclear cells into the liver, which then can be responsible for additional liver damage [94]. The antigen-presenting cell function of T-cells during chronic viral infection could be impaired; moreover, altered T-cells can express the inhibitory receptor programmed death-1 (PD-1) with immune activity inhibition and induction of apoptosis of T-cells [95]. Several 5-HT receptors were expressed on T-cells [96,97]. In fact, SSRIs can reverse 5-HT-driven modulation of lymphoid-cell function at therapeutically concentrations suggesting that SSRIs directly impact immune function in a different way than 5-HT [96]. The study by Stefulj et al. [97] provides evidence on the presence of mRNAs for six out of 13 receptor subtypes, such as 5-HT1B, 1F, 2A, 2B, 6, and 7, in resting cells of three different immune compartments. The same mRNAs were found in mitogen-activated spleen cells, and 5-HT3 mRNA was also found. Nordlind et al. [98] showed that low concentrations of ketanserin eliminated the inhibiting effect of 5-HT on mercuric chloride induced proliferation of T lymphocytes probably mediated by 5-HT1c or 5-HT2 receptors [98]. T-cell function is then impaired during the chronic phases of the disease and this function can be associated with the inhibition of other immune cells, together with altered regulation of Th1/Th2 cells, neutrophils/lymphocytes, neutrophils/CD8 1 T-cells, and Th1/Th2 cytokines, which characterizes the biological basis for the immune dysregulation of chronic viral hepatitis and subsequent evolution towards liver cirrhosis [99]. Many several inflammatory signaling pathways are involved in chronicization of viral inflammation, in particular, NF-κB activation and the consequent chemokines secretion [100,101]. Some evidence highlighted the impact of 5-HT on liver regeneration, indicating that 5-HT2 receptor is the main receptor involved in liver regeneration after partial

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hepatectomy. In a series of experiments using agonists and antagonists of 5-HT receptors, it was demonstrated that liver regeneration is predominantly mediated by 5-HT2A and 5-HT2B receptors [90]. Lesurtel et al. [102] demonstrated that knockout mice for TPH1 display a normal phenotype except for a dramatic reduction in 5-HT content in platelets. In fact, after hepatectomy, these mice failed to induce liver regeneration as measured by proliferative activity. After treatment of these mice with 5-HTP, a 5-HT precursor, in parallel with detectable 5-HT levels in the blood, the regenerative potential was rescued. Liver fibrogenesis can also be modulated through stimulation by IL-6, directly released by activated macrophages [103]. Mu¨ller et al. [104] showed that 5-HT induces migration of immature dendritic cells by the activation of the 5-HT1 and 5-HT2 receptors. They also demonstrated that the binding of 5-HT to 5-HT3, 5-HT4, and 5-HT7 receptors upregulates the production of IL-6. HSCs are especially involved in fibrogenesis through the activation of PI3K-AKT/PKB and Ras-MAPK pathways [105]. The PDGF is able to activate RasMAPK pathway and elicit migration and proliferation of HSCs [106]. Fibrosis induced by PDGF-B activation is relatively independent from TGF-β upregulation, which suggests that the fibrogenic effect of PDGF might be a TGF-β-independent mechanism [107]. HSC activation is a pivotal event in the initiation and progression of hepatic fibrosis and the principal contributor to collagen deposition into the liver parenchyma. Activation of these cells, during the ongoing progressive liver fibrosis and cirrhosis, is characterized by cell proliferation, migration, contraction production of ECM components and angiogenesis [108]. Both the initial phase of HSC activation and the persistence of the activated phenotype involve subtle variations of regulatory intracellular mechanisms, which are orchestrated by an elaborated network of transcription factors coordinating a complex reprogramming of gene expression [109]. Interestingly, quiescent HSCs express adipocytic markers and the transcriptional program required for maintaining their fat-storing phenotype has striking similarities with that of adipocyte differentiation [110]. In particular, the peroxisome proliferator-activated receptor gamma (PPARγ) and liver X receptor alpha (LXRα), the master regulators of adipocyte differentiation [111], are expressed in HSC and their expression and activity are reduced during in vivo and in vitro HSC activation [112]. Other cell populations within the liver involving in liver fibrosis are liver sinusoidal endothelial cells (LSECs). They are present in the sinusoidal wall, or the endothelium. Endothelial cells express 5-HT1B and accordingly 5-HT1Dβ, 5-HT2A, 5-HT2B, 5-HT2C, and 5-HT4 receptor mRNA [113]. Serotonin regulates either vasoconstriction or vasorelaxation in a complex way involving the interaction of different 5-HT receptor subtypes. In particular, a constrictor activity in blood vessels is exerted by 5-HT2A receptors located on smooth muscle cells, while 5-HT2B receptors expressed on the endothelium relaxes the vessels. 5-HT4 receptors are potentially expressed on endothelial cells and the positive coupling of this receptor type to adenylate cyclase suggests that counteracts the effects of 5-HT1Dβ receptors in endothelial cells. 5-HT1Dβ receptors contribute to vessel relaxation. 5-HT7 receptors were shown to stimulate adenylate cyclase activity with subsequent increase of cAMP in smooth muscle cells that inhibits myosin light chain kinase, causing direct relaxation [113]. LSECs structure is fenestrated and these fenestrae act as a dynamic filter facilitating exchange of fluids, solutes, and particles between the bloodstream and liver parenchyma [114]. Chronic alcohol abuse induces a defenestration of these cells, and this process,

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together with the ongoing capillarization of liver sinusoids, plays a major role for hepatic endothelial dysfunction in liver cirrhosis [115,116]. On the contrary, the complete differentiation of LSECs can induce the dedifferentiation of activated HSC towards a quiescent state, thus contributing to prevent the progression of fibrosis, through overproduction of nitric oxide (NO), induced by vascular endothelial growth factor (VEGF) [117]. There is important crosstalk between HSC and LSEC during fibrogenesis. HSCs release potent angiogenic molecules and promote LSEC migration and tubulogenesis via expression of the chicken ovoalbumin upstream promoter transcription factor II (COUP-TFII) [9]. COUP-TFII is essential for fibrosis and cancer-associated angiogenesis through regulation of pericyte-derived paracrine signals that target endothelium. COUP-TFII regulates expression of VEGF-C [118], which is one of the main angiogenic factors produced by COUPTFII-expressing HSC and recognizes VEGFR-2 and VEGFR-3 receptors that are expressed in LSEC [119]. COUP-TFII positively controls NF-κB transcriptional activity in HSC and NF-κB silencing abrogates COUP-TFII-regulated expression of IL-8 and VEGF-C, interrupting the proangiogenic cross-interaction between HSC and LSEC [120]. Endothelial dysfunction is characterized by insufficient release of vasodilators, of which the most important is NO. Release of NO is inhibited by a low activity of endothelial NO synthase (eNOS) and corresponding increased production of vasoconstrictors (such as thromboxane A2, the renin-angiotensin-aldosterone system, endothelins, and antidiuretic hormone) [121]. Kupffer cells (KC) are also involved in liver fibrosis and the transition from chronic liver disease to cirrhosis. These cells, present in the sinusoidal wall together with pericytes, and constituting part of the reticuloendothelial system, can undergo activation through different stimuli, such as alcohol exposure, viral infections, high-fat diet, and iron overload. These activated cells are particularly harmful, either such as antigen-presenting cells during viral infections or such as toxic mediator producing cells [122]. Many different signaling pathways are induced through KC activation, either for enhanced inflammation or increased fibrogenesis; furthermore, these cells contribute to increase portal pressure via overproduction of thromboxane A2 [123]. Finally, hepatocytes themselves can play a role in inducing liver fibrosis through different mechanisms, including the release of reactive oxygen species (ROS) and fibrogenesisinducing molecules, activation of HSCs, and induction of fibrogenesis via activated myofibroblasts [124]. Apoptosis is another frequent mechanism present in liver fibrosis that involves HSCs and hepatocytes. Steatohepatitis enhances Fas-mediated hepatocyte apoptosis, so as chronic HCV infection and alcoholic liver disease, especially through downregulation of Bcl-2 signaling [125]. Niture et al. [126] in liver cancer cell lines demonstrate that 5-HT positively modulates cell proliferation/survival and cell steatosis in liver cancer cells by inducing autophagy and activating Notch signaling. Another two interesting mechanisms operating during the long course of liver cirrhosis, and possibly contributing to perpetuate the injury, are the increased production of TGF-β1 by hepatocytes during hypoxia and, furthermore, the role of telomere shortening and senescence in aggravating fibrotic scarring [127,128]. Treatment of HSCs with 5-HT2 antagonists inhibited their proliferation and increased their apoptosis [70,129]. Serum 5-HT levels are increased in cirrhotic patients in comparison with chronic hepatitis, and HCC cirrhotic patients in comparison with cirrhotic patients without HCC [67]. Cirrhosis is characterized by enhanced resistance to portal blood flow, and then by portal hypertension.

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Both architectural or structural mechanisms and functional or vasoactive ones contribute to the onset and the perpetuation of the disease. Increased portal pressure could be aggravated by splanchnic vasodilation, which in turn is considered such as a counteracting response to the increased intrahepatic vascular tone. In an advanced stage of decompensated liver cirrhosis, characterized by the so-common complications of the disease, such as ascites, portal hypertensive gastropathy, gastroesophageal varices with the impending risk of bleeding, hepatic encephalopathy and liver failure, increased splanchnic circulation leads to hyperdynamic circulation, and huge splanchnic and systemic vasodilation. All these processes play pivotal pathogenetic roles in the onset and the maintenance of ascites and hepatorenal syndrome, the onset of hepatopulmonary syndrome, the development of collateral blood flow and portosystemic shunts, and eventually gastroesophageal bleeding varices.

7.5 Serotonin and liver fibrogenesis The assessment of liver fibrosis is of fundamental importance in the management and therapeutic approach of patients with chronic liver disease. In the case of chronic HCV- or HBV-related hepatitis the severity of the liver disease is represented by its capacity for fibrotic evolution. Currently, several drugs are available to treat more than 90% of HCVinfected patients, and the cure rates are continuously improving. However, the fibrosis regression is still a matter of discussion as regards the natural history of cirrhosis [10]. The assessment of liver fibrosis is still useful to predict prognosis in patients with PBC [15]. Fibrogenesis in NASH correlates with inflammation since fibrosis progression is driven by repetitive periods of repair. In fact, the hepatocyte lipoapoptosis plays a key role in fibrosis progression, thus activating HSCs, portal fibroblasts, cholangiocytes, macrophages, and components of the pathological ECM. Recent studies have highlighted the role of 5-HT in the development of liver fibrogenesis (Fig. 7.2). Serotonin regulates many key factors involved in the tissue repair process. Five members of the serotonin receptors’ family were identified in HSCs: 5-HT1B, 5-HT2A, 5-HT2B, and 5-HT7 [70]. Both 5-HT2A and 5-HT2B receptors are upregulated in response to activation, suggesting an important role of these receptors in the function of HSCs [70,130]. The study of El-Tanbouly et al. [131] evaluated the effects of mirtazapine, a 5-HT2A antagonist, in a mouse model of liver fibrosis. The experimental mice were divided into four groups, six animals each. The first group served as control and received normal saline by intraperitoneal injection (ip), twice weekly, for 9 weeks; the experimental mice received thioacetamide (TAA, 150 mg/kg/biweekly, ip) for nine successive weeks for induction of liver fibrosis. The results of this study showed that the administration of mirtazapine significantly improved plasma aminotransferases (AST and ALT) levels, reduction of liver procollagen I content and alpha-smooth muscle actin expression, reduction of collagen accumulation between hepatic lobules and attenuation of portal hypertension. The reduction of hepatic collagen deposition was histologically demonstrated using Masson’s trichrome staining. Moreover, the increment of TGF-β1 and PKC levels due to the TAA-induced liver fibrosis and consequently HSCs activation markers (α-SMA and procollagen type I) were significantly inhibited in mirtazapine groups, indicating that

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mirtazapine suppressed 5-HT
mediated TGF-β1/Smad3 and ERK1/2 signaling pathways as well as oxidative stress that contribute to the progression of liver fibrosis. The activated HSCs express SERT and they are able to release 5-HT. It has also been shown that 5-HT can influence the proliferation and apoptosis of activated HSCs and the selective antagonists of the 5-HT receptors’ family may have potential antifibrotic effects [130,132]. Serotonin’s reuptake is mediated by SERT and its alteration may affect serotoninergic transmission. SERT is present with the same morphological and functional characteristics at the level of the intestinal epithelium, serotonergic neurons and platelets. Insertion or deletion of a 44 bp long region gives rise to short “S” and long “L” forms. The S/S and L/S genotypes result in decreased expression and low efficacy of 5-HT reuptake [64]. Only few studies so far have examined the role played by SERT in the regulation of HSCs, while some others have already been conducted onto nonhepatic pathologies [133]. Probably, the SERT (in the L/L variant) could play an antiproliferative role (similar to that performed in the mesangial cells [134]) that could partly explain the limited effects of 5-HT in the proliferation of HSCs [70]. Ruddell et al. [70] emphasized the profibrogenic role of 5-HT in the liver, demonstrating that it works synergistically with PDGF in stimulating HSC proliferation. They also demonstrated that stem cells activated in culture express the SERT and can release 5-HT into the culture medium. Reverse transcription polymerase chain reaction (RT-PCR) and immunoblot analysis revealed that rat HSCs express SERT, which appears to be substantially induced by culture activation. Besides, same lipid-related effects of same antipsychotics such as clozapine and olanzapine have been attributed to drug-mediated blockade or antagonism of 5-HT2 receptors and the consequent inhibited expression of PPARγ and LXR [135] suggesting a direct involvement of 5-HT pathway in HSC transcriptional network during myofibroblastic transdifferentiation. Cholangiocytes variably express markers of neuroendocrine cells [136], in particular, they are known to release 5-HT [136]. Ometti et al. [137] examined adult cholangiocytes for expression of the enzymes, receptors, and transporters that control 5-HT homeostasis, and their ability to produce 5-HT when cultured alone or with liver myofibroblasts. The direct effects of 5-HT on cholangiocyte proliferation were characterized and a myofibroblasts-derived factor that regulated cholangiocyte 5-HT production was identified. The results of this study showed that 5-HT was an important modulator of fibroductular response by demonstrating that bile duct ligationinduced ductular cell proliferation and liver fibrosis are both exacerbated significantly in TPH2 (neuron-specific TPH isoform) knockin mice that have an inactivating mutation of TPH2 and reduced biliary 5-HT content. In fact, cholangiocytes express both TPH1 (the typical TPH isoform, expressed by nonneuronal cells) and TPH2 and they are able to synthesize 5-HT de novo. The results also indicate that 5-HT directly represses cholangiocyte proliferation and demonstrate that 5-HT cholangiocyte production is influenced by TGF-β1, a myofibroblasts-derived factor that selectively downregulates cholangiocyte expression of TPH2.

7.6 Serotonin and hepatocellular carcinoma The fibrosis stage represents the most important predictor of disease progression and conditions therapeutic choices in many chronic liver diseases. In the management of

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patients with chronic HBV infection, the assessment of liver fibrosis and the degree of necro-inflammation are of fundamental importance for identifying the subjects at the greatest risk of hepatic damage evolution into advanced fibrosis and HCC for formulating the therapeutic indication [138]. Regarding patients with chronic HCV-related liver disease progression of hepatic fibrosis appears to be strongly related to the incidence of HCC despite direct-acting antiviral treatment leads to a sustained virological response in over 90% of patients. According to the guidelines of the European Association for the Study of the Liver, all patients with primary sclerosing cholangitis should be evaluated for their risk of developing complications (such as advanced fibrosis and HCC) in order to assess the need for additional treatments [139]. Recently, Chang et al. [140] compared a cohort of newly diagnosed chronic HBV-infected patients with SSRI use with a cohort consisting of an equal number of HBV patients (N 5 1,380) without SSRI use matched by age, sex, liver cirrhosis, and index year, followed from 2000 to 2012 to identify the incidence of HCC. In the cohort with SSRI use, the incidence rate of HCC was 1.28 per 1000 person-years and in the cohort of patients without SSRI use, the incidence was 3.51. This study indicated that SSRIs use could decrease the risk of HCC in HBV-infected patients in a dose-responsive manner. Aryal et al. [141] in a cohort of forty patients diagnosed with HCC undergoing partial hepatectomy evaluated the 5-HT levels in serum, plasma and intraplatelet preoperatively and four weeks after liver resection. The patients were followed every three months after the surgery. The results of this study demonstrated that patients with recurrence had significantly reduced serum and intraplatelet 5-HT levels at 4 weeks of liver resection and they were able to independently predict the recurrence. The optimal cut-off value of 42.77 ng/mL for serum [area under the curve (AUC): 0.78, P 5 0.003] and 0.3117 ng per 106 platelets (AUC: 0.733, P 5 0.015), on receiver operating characteristic (ROC) curve corresponded to maximum sensitivity and specificity of prediction. Abdel-Razik et al. [67] have investigated whether 5-HT is a useful marker for the diagnosis of HCC in cirrhotic patients compared to α-fetoprotein protein and prothrombin induced by vitamin K absence-II (PIVKA-II). The authors found a significant positive correlation between serum 5-HT and α-fetoprotein protein and serum 5-HT and PIVKA-II among the patient groups. The ROC showed a higher area under the curve for 5-HT than α-fetoprotein and PIVKA-II (0.942, 0.824, and 0.921, respectively). According to these data, several studies indicate that 5-HT promotes the proliferation and invasion of liver cells. A recent study has shown that serum 5-HT levels are elevated in patients with rapid progression to HCC and poor prognosis [142]. In fact, it has already been shown that 5-HT is a strong mitogen, and its concentration is closely associated with hepatic regeneration and hepatocarcinogenesis [143,144]. In particular, Zuo et al. [72] investigated the clinicopathological significance of 5-HT1D in HCC, through the mRNA expression level of 5-HT1D in 96 human HCC tissues and their adjacent peritumor specimens by quantitative RT-PCR (qRT-PCR). The results revealed that 5-HT1D mRNA level was markedly upregulated in HCC tissues. The study of Niture et al. [62] analyzed the effects of 5-HT on liver cancer cell growth and survival. The data demonstrate that 5-HT positively modulates cell proliferation/survival and cell steatosis in liver cancer cells by inducing autophagy and activating Notch signaling. Moreover, mice fed with chronic ethanol showed elevated levels of 5-HT in serum. These were associated with increased hepatic steatosis and autophagy in mice liver.

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Yang et al. [145] investigated the role of 5-HT and its contribution to sex discrepancy during HCC. To study sex disparity in HCC initiation and progression, this group previously generated several inducible HCC models by transgenic expression of an oncogene in hepatocytes in zebrafish [146]. In particular, given that Ras signaling is frequently activated by major HCC etiological factors, a transgenic zebrafish constitutively expressing the krasV12 oncogene in the liver was generated for this study. Serotonin levels resulted significantly increased in inflammation and cirrhosis, and marginally in HCC samples as compared with normal liver control samples. This zebrafish study showed that the sex disparity of 5-HT accumulation can be attributed to the difference in the expression and phosphorylation of rate-limiting enzyme TPH1A. Moreover, increased HTR2b activation in HSCs resulted in overexpression of TGFß1. Both inactivation of HSCs and depletion of TGFß1 signaling significantly relaxed the rate of carcinogenesis in male krasV12-expressing livers. Therefore these data suggest the differential accumulation of 5-HT during liver disease progression between sexes, which could be attributed to the disparity of phosphorylation of TPH level. The antagonist of HT2B receptors and inhibition of TGFß signaling in krasV12-expressing livers of male zebrafish inhibited phosphorylated-TPH1 and 5-HT expression, suggesting that the activation of HSCs is mediated by TGFß signaling. The consequent high levels of 5HT accelerate carcinogenesis via the multifunctional cytokine, TGFß1. This cytokine showed a similar sex-biased expression in inflammation, cirrhosis, and HCC of patients. However, a significant correlation between 5-HT and TGF-ß1 was observed only in HT2B-activated HSCs expressed TGF-ß1 that promoted HCC progression and concomitantly increased 5-HT synthesis. Therefore HSCs promote liver tumour progression and maintenance the sex disparity observed in HCC patients [145].

7.7 Gut microbiota The normal human intestine is colonized by a large number of microorganisms, at least 100 billion, which maintain symbiotic relationships with the host and contribute to various functions including digestion, vitamin synthesis, and release of neuroendocrine factors and resistance to colonization of the intestine by pathogens [147]. Recently, several studies have shown that changes in the intestinal microbiota, through the intestine
liver axis, can influence the health or disease status of the individual. In fact, the “normal” intestinal microbiota has been shown to confer various physiological benefits for the host, including the development of the immune system, protection against pathogens, regulation of intestinal homeostasis, and metabolic functions. The qualitative or quantitative modification of the intestinal microbiota is called dysbiosis, and can be considered a predisposing factor for the development and progression of many chronic diseases. The intestinal microbiota is defined as the complex of microorganisms hosted by each person and it is characterized by a high number of genes collectively called microbiome. The microbiome is not equally distributed along the intestine since the number of species increases from the esophagus to the rectum [148]. The composition of the intestinal microbiota is very dynamic: immediately after birth, when the first colonization of the gastrointestinal tract occurs, its composition is strongly influenced by several factors, including environmental hygiene. This composition tends to stabilize

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towards the third year of life. Bacteria are present with relative abundance such as phylum, order, family, genus or species. Over 90% of the microbiota belongs to two phyla, that is, Firmicutes and Bacteroidetes, followed by Actinobacteria and Verrucomicrobia [149]. In particular, the gut-microbiota is constituted by 79.4% of Firmicutes (Ruminococcus, Clostridium, and Eubacteria), 16.9% of Bacteroidetes (Porphyromonas, Prevotella), 2.5% Actinobacteria (Bifidobacterium), 1% Proteobacteria, and 0.1% Verrumicrobia [150]. During adult life, many conditions can influence the composition of the intestinal microbiota, such as ageing, diet and improper use of antibiotics. The intestinal
liver axis is essential for the regulation of systemic metabolism, for the release of intestinal hormones and the immune response. Several chronic liver diseases such as chronic HBV, chronic HCV, alcoholic liver disease (ALD), NAFLD and their evolution toward liver cirrhosis, and HCC are associated with gut dysbiosis. Zeng et al. [151] characterized the gut microbiota composition in faecal sample, using 16S ribosomal RNA gene sequencing of 50 healthy subjects and 21 patients with chronic HBV, 25 with liver cirrhosis and 21 with HBV-related HCC. They found a lower abundance for Firmicutes and a higher abundance for Bacteroidetes in patients with chronic HBV than in healthy controls; they also observed similar findings in patients with liver cirrhosis and HBV-related HCC compared with the healthy controls. Inoue et al. [152], using 16S ribosomal RNA gene sequencing, analyzed faecal samples from 23 healthy subjects and 18 chronic HCV patients with persistently normal serum alanine aminotransferase without evidence of liver cirrhosis, 84 patients with chronic HCV, 40 with liver cirrhosis, and 24 with HCC. This study showed that gut bacterial diversity significantly decreases in patients compared with healthy individuals in association with the severity of the clinical stage. The healthy group was highly abundant in class Clostridia (Lachnospiraceae and Ruminococcaceae). The chronic HCV patients with persistently normal serum alanine aminotransferase without evidence of liver cirrhosis group showed an increased family Enterobacteriaceae and genus Bacteroides. In liver cirrhosis group was significantly abundant the class Bacilli, mainly including genus Streptococcus and Lactobacillus. Moreover, Streptococcus salivarius and some other minor species belonging to viridans streptococci or genus Lactobacillus increased in association with chronic HCV progression. This study also evaluated the effect of possible confounding factors on the microbiota and it demonstrated that lactulose, histamine H2-receptor antagonists, ursodeoxycholic acid, and probiotics have no significant influence; on the contrary, proton-pump inhibitor increased Streptococcus. Therefore in chronic viral hepatitis, the liver injury originates from both the cellular immune response caused by virus and by the natural immune response after intestinal dysbiosis. A prospective study by Loomba et al. [153] included 86 patients with biopsy-proven NAFLD: 72 patients had stage 0
2 fibrosis and were classified as mild/moderate NAFLD and 14 patients had stage 3
4 fibrosis and were classified as advanced NAFLD. In this study, the composition of gut microbiome of the patients was assessed using wholegenome shotgun sequencing of DNA extracted from their faecal samples. At the phylum level, in both mild/moderate and advanced fibrosis groups Firmicutes and Bacteroidetes were predominant, followed by Proteobacteria and Actinobacteria. In particular,

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Firmicutes were higher in mild/moderate NAFLD and Proteobacteria were higher in advanced fibrosis. At the species level, Eubacterium rectale and Bacteroides vulgatus were the most abundant organisms in mild/moderate NAFLD, while B. vulgatus and Escherichia coli were the most abundant in advanced fibrosis. Ruminococcus obeum CAG: 39, R. obeum, and E. rectale were significantly lower in advanced fibrosis than mild/ moderate NAFLD. In ALD patients, Addolorato et al. [154] identified a reduction of Akkermansia and the increase of Bacteroides with an accuracy of 93.4%. Their gut microbiota showed an increased expression of gamma-aminobutyric acid metabolic pathways and energy metabolism. Moreover, the authors found an increment of serum levels of LPS and proinflammatory mediators (TNFα, IL-1β, MCP-1, IL-6) patients compared to controls and in cirrhotic patients compared to noncirrhotic ones.

7.8 Serotonin and gut microbiota The intestinal microbiota plays an important role in the intestinal homeostasis of 5-HT, affecting the SERT and/or the expression of the receptors of 5-HT [12]. In fact, 5-HT levels are reduced in mice in the absence of microbial colonization (germfree, GF), compared to colonized (specific pathogen-free, SPF) controls [155,156]. Recently, Yano et al. [156] have studied potential host
microbial interactions that modulate peripheral 5-HT by surveying microbial influences on the faecal metabolome. The authors demonstrated that germ-free mice present significantly decreased levels of colonic and faecal 5-HT compared to specific pathogen-free controls. These decreased 5-HT levels are observed broadly across the distal, medial, and proximal colon, but not in the small intestine, suggesting a specific role for the microbiota in regulating colonic 5-HT. Gut microbiota can also impact 5-HT intestinal homeostasis by acting on 5-HT transporter (SLC6A4, mouse ortholog of human SERT) and/or 5-HT receptor expression (Fig. 7.3). The study found that germ-free colon exhibits reduced expression of nonneuronal tryptophan hydroxylase (TPH1). Germ-free mice also display a high colonic expression of the 5-HT transporter SLC6A4, synthesized broadly by enterocytes to enable 5-HT reuptake. Serotonin plays a key role in the control of intestinal permeability [157] and subsequently in the translocation of intestinal endotoxin in mice chronically exposed to fructose [157,158]. Haub et al. [159], using a mouse model of sugar-induced steatosis, investigated the role of 5-HT and SERT in the onset of fructose-induced NAFLD. The results of this study indicated that chronic fructose feeding induced the early phase of NAFLD, characterized by hepatic steatosis, neutrophil infiltration, increased ROS, and induction of TNF-expression in the liver. In this study, hepatic steatosis was associated with the mice exposition of 30% fructose that induced a marked reduction of SERT protein in the duodenum of these animals. These data suggest that the hepatic triglyceride accumulation was associated with an almost complete loss of SERT in the duodenum, in fact, hepatic steatosis was markedly increased in SERT 2/2 mice compared to wild-type mice. Moreover, these data suggest that the markedly greater damaging effect of fructose compared with glucose on the liver found in wild-type mice under the present conditions can be the result of a loss of SERT in the duodenum, although mechanisms by which fructose, but not

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L

glucose, leads to the loss of SERT in the small intestine need to be clarified. In a subsequent study, Haub et al. [77] showed that the 5-HT3 antagonists (tropisetron and palonosetron) ameliorating NAFLD in a genetic mouse model of obesity with a marked decrease in the intestinal translocation of bacterial endotoxin. These results were probably associated with the effects of 5-HT3 antagonists on the intestinal barrier integrity with a consequent decrease in hepatic inflammation and fat accumulation. Choi et al. [160] demonstrated that local 5-HT levels in the portal blood are selectively augmented by high-fat diet feeding in mice inducing endocrine effects on the liver. The liver is the first organ to encounter gut-derived 5-HT through the portal vein. Both gutspecific TPH1 knockout mice and liver-specific 5-HT2A knockout mice are resistant to high-fat diet-induced hepatic steatosis, without influencing systemic energy homeostasis. Additionally, selective HT2A antagonists prevent high-fat diet-induced hepatic steatosis. Thus, the gut TPH1-liver 5-HT2A axis shows promise as a drug target to ameliorate NAFLD with minimal systemic metabolic effects. Therefore the authors demonstrate that the gut
liver axis regulates hepatic steatosis via peripheral gut-derived 5-HT concentrations in the portal blood. In particular, inhibition of gut-derived 5-HT synthesis ameliorates hepatic steatosis through a reduction in liver 5-HT2A signaling. Crane et al. [161] demonstrated that reducing circulating 5-HT by inhibition of TPH1 or deletion of the TPH1 gene increases the sensitivity of brown adipose tissue cells to noradrenaline and β-adrenergic receptor signaling, effects that depend on mitochondrial uncoupling protein 1 (UCP1)-mediated thermogenesis. This protein is found in the mitochondria of brown adipose tissue that uncouples the respiratory chain from ATP production, with subsequent rapid substrate oxidation and high heat generation. Sustained thermogenesis recruits additional fat and glucose from peripheral tissues for oxidation by brown adipose tissue cells, which reduces fat storage in white adipose tissue, skeletal muscle, and liver. This process protects against obesity, skeletal muscle insulin resistance, and NAFLD. Serotonin signaling might also potentially be involved with glucometabolic changes and hepatic steatosis associated with the use of some antidepressants. In fact, some 5-HT reuptake inhibitors, which inhibit SERT function and prolong serotonin neurotransmission cause alteration in lipid metabolism pathways, hepatic steatosis, and insulin resistance in mice lacking of SERT [162,163]. In particular, Singhal et al. [163] demonstrate that SERT deletion is associated with dysbiosis similar to that observed in obesity. Moreover, they FIGURE 7.3 Liver homeostasis and disease. Serotonin (5-HT) has a pleiotropic function in gastrointestinal, and liver diseases. Approximately 90% of 5-HT is stored in enterochromaffin cells (ECs), which release the transmitter in response to mechanical or chemical stimuli. After its release into the bloodstream, serotonin is taken up by a specialized SERT and stored in the dense granules of platelets. In normal liver (histology of normal liver), quiescent stellate cells (HSCs) play a key role in the storage and transport of retinoids (vitamin A compounds). During liver injury (histology of liver cirrhosis), HSCs undergo a process of phenotypical transformation into activated myofibroblast-like cells that synthesize proinflammatory/proangiogenic cytokines and a large amount of ECM components (yellow lines). HSCs is also able to store 5-HT to an internal compartment from which it can be released thereby stimulating both hepatocytes and activated HSC proliferation. Serotonin exerts its biological function through interactions with distinct receptor subtypes, in particular, the activation of HSCs is mediated by 5-HT1B, 5HT2A, and 5-HT2B. The intestinal microbiota affects the SERT and/or the expression of the 5-HT receptors. Modifications in the composition of the commensal bacteria subsequently change serotonin levels that correlate with specific bacteria. SERT, serotonin transporter protein; HSC, hepatic stellate cell; ECM, extracellular matrix.

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showed that differential abundance of bacteria was correlated with changes in host gene expression. Bifidobacterium and Bacilli species exhibited significant associations with host genes involved in lipid metabolism pathways. El Aldy et al. [14] demonstrated that the 5HT transporter genotype, mainly when associated with early life stress, such as maternal separation, induces a state of microbiota dysbiosis. In fact, rats with decreased expression of 5-HT transporter exposed to maternal separation showed a high presence of Desulfovibrio, Mucispirillum, and Fusobacterium, indicating the shift of gut-microbiota from homeostasis to inflammatory type microbiota. According to these studies, De Long et al. [164] assessed glucometabolic changes and hepatic pathophysiology in the offspring of female nulliparous Wistar rats were given vehicle or fluoxetine hydrochloride orally for 2 weeks prior to mating until weaning. Fluoxetine (a selective 5-HT reuptake inhibitor) exposed offspring demonstrated altered glucose homeostasis with mild to moderate NASH and dyslipidemia. Of these, the males showed increased levels of TNFα, IL-6, MCP-1, while female offspring had higher expression of TNFα, and increased MCP-1. In a subsequent study, De Long et al. [165] demonstrated that elevated hepatic triglyceride levels observed in the SSRI-exposed offspring may be due, in part, to activation of the NLRP3 inflammasome (apoptosis-associated speck-like protein containing a caspase activation recruitment domain and caspase-1) and augmentation of de novo lipogenesis. It is expectable that changes in glucose metabolism and hepatic triglyceride levels are related to increased intestinal availability of 5-HT due to the use of SSRIs, with subsequent increased 5-HT concentrations in the portal blood via the gut
liver axis. Therefore, the severity of altered glucose levels and dyslipidemia might be related to the degree of altered 5-HT uptake. As mentioned in the previous paragraph, all the above alterations in 5-HT levels and its transporter may induce liver fibrogenesis.

7.9 Conclusions Peripheral 5-HT has established effects on liver fibrogenesis and it regulates many key factors involved in the tissue repair process. This was confirmed by the expression of 5HT1A, 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT2C, 5-HT3A, and 5-HT3B receptors identified in the animal liver. Moreover, several studies have demonstrated that there is an association between gut-microbiota, 5-HT metabolism, and its transporter. These studies provided a more complete understanding of the mechanisms underlying some hepatic diseases. Although some studies have already been conducted in humans, particularly in patients with NASH, CBP, and HCC, chronic viral hepatitis, future human data on the gut
liver axis effects are needed to define the role of peripheral 5-HT to firmly established the role of 5-HT and its pathways in hepatic fibrogenesis.

Acknowledgment We wish to thank Vincenzo Caputo for his precious help in graphical support to provide Figures 1
3.

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58. [159] Haub S, Kanuri G, Volynets V, Brune T, Bischoff SC, Bergheim I. Serotonin reuptake transporter (SERT) plays a critical role in the onset of fructose-induced hepatic steatosis in mice. Am J Physiol Gastrointest Liver Physiol 2010;298(3):G335
44. [160] Choi W, Namkung J, Hwang I, Kim H, Lim A, Park HJ, et al. Serotonin signals through a gut-liver axis to regulate hepatic steatosis. Nat Commun 2018;9:4824. [161] Crane JD, Palanivel R, Mottillo EP, Bujak AL, Wang H, Ford RJ, et al. Inhibiting peripheral serotonin synthesis reduces obesity and metabolic dysfunction by promoting brown adipose tissue thermogenesis. Nat Med 2015;21:166
72. [162] Chen X, Margolis KJ, Gershon MD, Schwartz GJ, Sze JY. Reduced serotonin reuptake transporter (SERT) function causes insulin resistance and hepatic steatosis independent of food intake. PLoS One 2012;7 1
1. [163] Singhal M, Turturice BA, Manzella CR, Ranjan R, Metwally AA, Theorell J, et al. Serotonin transporter deficiency is associated with dysbiosis and changes in metabolic function of the mouse intestinal microbiome. Sci Rep 2019;9:2138. [164] De Long NE, Barry EJ, Pinelli C, Wood GA, Hardy DB, Morrison KM, et al. Antenatal exposure to the selective serotonin reuptake inhibitor fluoxetine leads to postnatal metabolic and endocrine changes associated with type 2 diabetes in Wistar rats. Toxicol Appl Pharmacol 2015;285:32
40. [165] De Long NE, Hardy DB, Ma N, Holloway AC. Increased incidence of nonalcoholic fatty liver disease in male rat offspring exposed to fluoxetine during fetal and neonatal life involves the NLRP3 inflammasome and augmented de novo hepatic lipogenesis. J Appl Toxicol 2017;37:1507
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C H A P T E R

8 Gut
liver
brain axis in chronic liver disease with a focus on hepatic encephalopathy Anna-Lena Laguna de la Vera1, Christoph Welsch1, Waltraud Pfeilschifter2 and Jonel Trebicka1,3 1

2

Department of Internal Medicine I, J.W. Goethe University Hospital, Frankfurt, Germany Department of Neurology, J.W. Goethe University Hospital, Frankfurt, Germany 3European Foundation for Study of Chronic Liver Failure, Barcelona, Spain

8.1 Introduction Hepatic encephalopathy (HE) is a complication of liver disease with brain dysfunction caused by liver insufficiency and/or portosystemic shunting. Its manifestations vary considerably and include a wide range of cognitive, emotional, intellectual psychological, and psychomotor abnormalities that may range from subclinical alterations to coma [1
3]. The pathogenesis of hepatic encephalopathy is not yet fully understood. Hyperammonemia remains the key player in the pathogenesis of HE leading to astrocyte swelling and a glial edema [4,5], which accounts for psychological and psychomotor abnormalities through impaired glioneuronal communication [4]. Besides that, patients with liver cirrhosis show a chronic proinflammatory milieu [6]. Recently, the intestinal microbiota has gained attention as a pivotal player in the development of HE [4]. Certain microbial abundances that might lead to HE were identified [7] and linked to characteristic magnetic resonance imaging (MRI) spectroscopy and diffusion tensor imaging (DTI) patterns [8]. Yet it is still not known if dysbiosis is a cause or a result of liver damage (“chicken-egg problem”) [9]. HE remains a clinical diagnosis, yet there are forms of HE where mental or motor defect is not recognizable by the clinician or patient [2], namely covert HE. These forms of HE as well as the clinically noticeable HE (overt HE) episodes have a relevant impact on healthrelated quality of life (HRQOL) and prognosis [10,11].

The Complex Interplay Between Gut
Brain, Gut
Liver, and Liver
Brain Axes DOI: https://doi.org/10.1016/B978-0-12-821927-0.00004-8

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This chapter describes the physiopathological bases of the gut
liver
brain connection with a special focus on HE in chronic liver disease because 97% of HE episodes occur in patients with cirrhosis [3]. Herein, we also discuss the diagnosis and treatment of HE that includes the management of triggering causes (e.g., hyponatremia, infections, gastrointestinal bleeding) and the use of several drugs aiming at a reduction of ammonia levels as well as recent approaches to target the fecal microbiota [2], for example, fecal microbiota transplantation (FMT) [7].

8.2 Gut
brain axis in health and liver disease There is ample evidence of a bidirectional neuronal, endocrine, immune, and humoral communication between the central and enteric nervous system (CNS and ENS) [10]. The CNS modulates the ENS via the autonomous nervous system regulating intestinal motility, digestive functions, permeability, and immune responses. This supports a role of physical and psychological stressors in gastrointestinal disorders such as peptic ulcers and inflammatory bowel disease [12]. Reversely, well-characterized neurodegenerative diseases caused by protein misfolding and subsequent aggregation such as Parkinson’s disease [13] start with an early affection of the ENS preceding the neurological symptoms and seem to self-propagate trans-synaptically to the CNS [14]. In recent years, the intestinal microbiota has gained increasing interest as a modulating factor in psychiatric [15], neuroimmunologic [16], and neurodegenerative [17] diseases. Besides the brain, the gut microbiota also modulates the function of other extraintestinal organs, especially the liver [18], mainly via bacterial metabolites and signaling molecules [19]. Several factors can affect the composition of the microbiota and increase the risk of dysbiosis, including diet, stress, use of antibiotics, aging, and comorbid conditions [20]. An altered microbiota is thought to be associated with numerous conditions and the impact of the gut microbiota on human health and disease is a rapidly evolving field of research (this topic is discussed in detail in the book Section 3, Liver
Brain Axis, Chapter: The Gut Microbiota in Hepatic Encephalopathy). Proinflammatory microbiota such as Bacteroidetes seem to have harmful effects on the gut-barrier and result in low-grade inflammation of the intestinal mucosa [21].

8.2.1 Changes of gut
liver
brain axis in progression of liver disease The basis for the gut
liver
brain axis is the physiological enterohepatic circulation. The portal vein closely connects the function of the gut and the liver. Bile acids (BAs) are released by the liver and interact with gut mucosa and the microbiome [22,23]. It is determined by products from digestion and absorption as well as bacteria and bacterial components [22]. Dysbiosis may lead to a damaged intestinal barrier with the liver exposed to toxic factors and bacteria from the intestine [24]. Exposure of the liver to bacterial components, pathogen-associated molecular patterns can cause hepatic injury and release of damage-associated molecular patterns from injured cells that can drive inflammation via proinflammatory and antiapoptotic pathways [23].

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The BAs are not only digestive surfactants but also important cell signaling molecules, stimulating several signaling pathways to regulate biological processes. The bile acidactivated nuclear receptor, farnesoid X receptor (FXR), plays an important role in regulating bile acid, lipid, and glucose homeostasis as well as inflammatory responses, barrier function, and the prevention of bacterial translocation from the intestinal tract [25]. The pathophysiological concept for the development of alcoholic liver disease (ALD) is based upon increased gut permeability due to the toxic effects of alcohol on epithelial cells that result in an elevation of endotoxin plasma levels that may cause hepatic injury. It has been suggested that an altered gut microbiota can cause individual susceptibility to ALD [26]. Experimental and clinical data indicate that the intestinal microbiota might also play an important role in the pathogenesis of hepatic injury in ALD [27]. It has been shown that patients suffering from alcoholic cirrhosis frequently show small intestinal bacterial overgrowth compared to healthy controls [28,29]. Furthermore an increase in proinflammatory Bacteroidetes has been described [30]. The hypothesis of dysbiosis as a key driver of individual susceptibility to ALD is supported by animal studies showing an alcohol-induced inflammation in germ-free mice after microbiota transplantation from alcoholic patients [31]. A recent study analyzing gastrointestinal disturbances in alcoholdependent patients during alcohol withdrawal detected increased intestinal permeability in 43% of patients. This was associated not only with gut dysbiosis but also with depression, anxiety, and alcohol craving. This may represent the first hint in the involvement of the gut
liver
brain axis in the perpetuation of alcohol dependence [32]. Nonalcoholic fatty liver disease (NAFLD), which includes simple steatosis, nonalcohol steatohepatitis (NASH), fibrosis, and cirrhosis, has been studied in detail from a microbiota point of view. It is characterized by the accumulation of triglycerides in the absence of alcohol consumption [33]. A particular challenge with NAFLD and microbiota is the overlay of diabetes, metabolic syndrome and obesity [34], which often makes it difficult to differentiate between the impact of the liver disease versus other coexisting factors. Altered microbiota and increased permeability of the gut-barrier can cause steatohepatitis via hepatic inflammation from metabolic endotoxemia and toll-like receptor mediated cytokine production, as well as changes in BA metabolism. Typical compositional changes in gut microbiota are an increase in proinflammatory Bacteroidetes and a decrease in Firmicutes [35]. Several association studies have shown alterations of the gut microbiota in the early course of obesity and type 2 diabetes mellitus (T2DM), possibly also via changes in brain function and behavior [36] supporting its role as a causative cofactor. This has not yet been consistently proven by intervention studies but there are first randomized-controlled trials showing beneficial effects of probiotics on weight loss and abdominal adiposity in humans [37]. How far these are merely metabolic or mediated by effects on behavioral aspects [38] warrants further investigations. Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) represent the major clinical entities of chronic cholestatic liver diseases. Both disorders are characterized by portal inflammation. Increasing evidence support that dysbiosis might play a role in the pathogenesis of PBC and PSC. Putative triggers for the development of PSC or PBC are intestinal dysbiosis, changes in the composition of BAs and changes in the composition of biliary microbiota, and various adverse bacterial products and metabolites leading to inflammation [39]. Ursodeoxycholic acid (UDCA) is the first-line treatment in PBC that

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influences the enterohepatic circulation of BAs. In mice, oral administration of UDCA lead to a unique BA profile with high BA abundance and accelerated enterohepatic circulation of BAs through inhibition of intestinal FXR signaling, which in turn induced the expression of BA transporters in the liver [40]. Liver cirrhosis is characterized by destruction of liver cells and scarring of the liver. It represents the end-stage of all chronic liver diseases. Dysbiosis in cirrhotic patients has been shown to alter the composition of gut microbiota toward proinflammatory taxa, resulting in an impaired gut-barrier function and pathological bacterial translocation. Endotoxemia adds to the existing hepatic injury and accelerates systemic inflammatory responses [41,42]. Etiology of cirrhosis varies but microbiome-liver interaction is etiologyindependent and bacterial translocation is a frequent finding in decompensated cirrhosis [43,44]. During progression of cirrhosis, the blood pressure in the portal vein increases (portal hypertension) which leads to further complications such as bleeding and ascites. Portal hypertension facilitates microbial translocation from the gut to the liver through the angiogenesis of intestinal vessels which lead to an impaired microcirculation and an increased permeability of the gut-barrier [45,46]. It might predispose for the development of acute-on-chronic liver failure (ACLF) [47] and overt HE. Specific microbiome patterns in HE are found to be diagnostic and predictive [48]. Ascites in patients with decompensated liver cirrhosis determines the composition of the circulating microbiota. Here, several circulating microbiome markers correlate with inflammatory markers [46] and the development of ACLF [49].

8.3 Pathogenesis of hepatic encephalopathy 8.3.1 Dysbiosis Cirrhotic patients with liver synthetic failure show a decreased bile acid secretion, as well as an impaired immune response and an intestinal barrier dysfunction that lead to systemic inflammation and neuroinflammation [7]. Thus it has been proposed that the pathophysiology of HE is likely related to the activity of gut microbiota through hyperammonemia and systemic inflammation resulting from gut dysbiosis. Recent works have shown specific effects of different microbial families on HE pathology. It has been shown that HE patients have a significantly worse cognitive performance correlating with hyperammonemia, systemic inflammation, and dysbiosis compared to healthy controls and cirrhotic patients not suffering from HE. Specific microbial taxa could be correlated with ammonia associated astrocyte swelling via MRI spectroscopy with autochthonous taxa being negatively and Enterobacteriaceae being positively correlated with astrocyte swelling. Thus HE seems to be associated with decreases in relative abundances of Lachnospiraceae and Ruminococcacae and increases in relative abundances of Enterobacteriaceae, Streptococcaceae, Lactobaciallacea, and Peptostreptococcaceae [8]. Porphyromonadaceae by contrast were associated with neuronal white matter injury in MRI DTI providing a first clue that distinct microbial families have specific effects on different aspects of HE pathophysiology. The assumption that Streptococcaceae may be associated with the presence of HE is supported by another study showing that the abundance of the ammonia-increasing bacteria

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Streptococcus salivarius in the gut was significantly higher in cirrhotic patients with covert HE (cHE) than in those without [50]. Furthermore, patients with overt HE (oHE) have distinct changes in microbiota during the hospitalization that can predict recurrence of HE in these patients [51].

8.3.2 Neurotoxins Ammonia plays a key role in the pathogenesis of HE and is the best characterized neurotoxin linked to HE (Fig. 8.1A). It is produced by enterocytes from glutamine and by colonic bacterial catabolism of nitrogenous sources (such as blood after GI bleeding). Elimination of ammonia is primarily based on its conversion to urea via the urea cycle in periportal hepatocytes as well as on the formation of glutamine in perivenous hepatocytes. Cirrhotic patients often develop portal-systemic shunting combined with an impaired hepatocyte metabolic capacity resulting in hyperammonemia [52]. The classification of HE by cause of hyperammonemia is more extensively described in the chapter “The Gut Microbiota in Hepatic Encephalopathy.” It is believed that HE is precipitated by a heterogeneous panel of factors and conditions such as protein excess, GI bleeding, dehydration from diuretics and infections that lead to accumulation of ammonia and other HE-driving components, for example, metabolites, inflammatory cytokines, and hyponatremia [4]. In the brain ammonia is detoxified by the astrocytes. Hyperammonemia and excess of other relevant components can lead to swelling of astrocytes and induction of oxidative stress resulting in a glial edema via RNA oxidation, gene expression changes, and protein modifications. This results in astroglia dysfunction and impaired glioneuronal

(A)

(B) Ala, Gln

Brain

Muscle

Cytokines

NH3 NH4

NH3

Blood

Liver

Astrocyte

Urea aminoacids Ala, Gln

Hyponatraemia

NH3

Large intestine

Urea

Urease

Small intestine Glutaminase

Kidney

Urea NH3

Dysfunctional astrocyte-neurone unit

FIGURE 8.1 (A) Flow of ammonia and its metabolism in the body. (B) Pathophysiological insults on astrocytes in hepatic encephalopathy.

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communication leading to a disturbance of synchronized oscillatory activity in the brain that plays a key role in the processing neural information and accounts for the initially subtle HE symptoms. HE is characterized by an abnormal, low-frequency coupling behavior of central neurons, and peripheral motor neurons, which explain the numerous different cognitive and motor disturbances. It is important to note that blood ammonia levels in cirrhotic patients do not necessarily correlate with the severity of HE. This may be explained by the fact that other conditions, already mentioned above, including hyponatremia, inflammatory cytokines, or comedication such as benzodiazepine-type sedatives, can precipitate or aggravate HE without further increase in ammonia levels [53].

8.3.3 Impairment of neurotransmission Several neurotransmitter systems have been studied in experimental models of (mostly) acute liver failure (ALF) describing changes in the γ-Aminobutyric acid (GABA)benzodiazepine-ergic, serotoninergic, dopaminergic, and glutamate-ergic neurotransmitter systems [54
56]. GABA is the principal inhibitory neurotransmitter and is synthesized by gut bacteria. In a rabbit model the development of HE was associated with increased levels of GABA in plasma. It is thought that in liver failure gut-derived GABA passes through a permeable blood-brain barrier and induces its own receptors on postsynaptic neural membranes resulting in a pattern of neural activity similar to that induced by drugs which cause GABA-ergic neural inhibition. Furthermore it is postulated that an increased number of receptors might enhance sensitivity to barbiturates and benzodiazepines in liver failure [54]. In vivo substances related to activation of the GABA-ergic neurotransmission have been isolated as benzodiazepine-like compounds in brain and cerebrospinal fluid from patients with HE in ALF and in cirrhosis. Pathophysiologically relevant concentrations of neurosteroid precursors, which are endogenous benzodiazepine-like compounds, have been identified in the brain of patients with coma due to HE [55]. Extrapyramidal symptoms in patients with HE may be caused by alterations in the dopamine-ergic function. An accumulation of manganese in basal ganglia was shown in a rat model of ALF. Manganese appears to normalize low striatal levels of dopamine and it is consequently thought that increased levels of manganese in basal ganglia may represent an attempt of the brain to compensate dopamine deficiency in liver disease [57,58].

8.3.4 Systemic response to infections and neuroinflammation: proinflammatory mechanisms Systemic inflammation resulting from infection is a major precipitant of HE. Patients with cirrhosis are known to be functionally immunosuppressed and are thus susceptible to developing infections. The development of a systemic inflammatory response syndrome results from the release and circulation of proinflammatory cytokines and mediators such as tumor necrosis factor-alpha (TNFα), interleukin (IL)-1β, and IL-6 [3,52]. It has been shown that increased TNFα levels in cirrhotic patients correlate with the grade of HE [59]. At the cellular level, human cerebrovascular endothelial cells exposed to TNFα show an increased capacity for the transport of ammonia [60].

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8.3.5 Shunting In particular in patients with advanced-stage liver disease and cirrhosis, toxic substances reach the systemic circulation as a result of portal-systemic shunting or reduced hepatic clearance and produce deleterious effects on brain function [61]. Portosystemic shunts can also cause HE by diverting venous blood from the portal system into systemic circulation. These shunts develop in the setting of advanced liver disease, but can also be present in the absence of liver disease. Shunts may also be iatrogenic, such as the placement of transjugular intrahepatic portosystemic shunts (TIPS) or surgical shunts [62].

8.3.6 Sarcopenia Sarcopenia is an independent predictor of complications in cirrhotic patients, including the development of HE. A link between sarcopenia and HE in cirrhosis has been reported in recent studies. Both complications are interrelated and often affect patients with advanced cirrhosis of the liver. They can have synergistic effects on the deterioration in patients’ outcome. HE episodes are more common in patients with muscle wasting [63]. Muscle wasting might contribute to higher levels of serum ammonia due to an impaired extra hepatic ammonia removal through the muscle itself [3]. On the other hand, and in addition to other factors, hyperammonemia might increase sarcopenia by various mechanisms including increased expression of myostatin, increased phosphorylation of eukaryotic initiation factor 2a, cataplerosis of α ketoglutarate, mitochondrial dysfunction, increased reactive oxygen species (ROS) that result in decrease of protein synthesis, and increased autophagy-mediated proteolysis [64] and thus presents a vicious circle. Taken together the astrocyte-neuron-unit is affected due to different insults in HE (Fig. 8.1B). The dysfunction of this unit can lead to impressive clinical symptoms as highlighted in the subsequent subchapter.

8.4 Clinical relevance and presentation of hepatic encephalopathy The cumulative prevalence of manifest HE in cirrhotic patients is around 30%
45% and profoundly affects quality of life due to numerous and initially often subtle cognitive and emotional alterations including concentration deficits, mood alterations, and reduced autonomy in the activities of daily living, including fitness to drive [1,65
67].

8.4.1 Classification HE should be categorized according to the following four factors. 1. According to the underlying disease HE is subdivided into Type A resulting from ALF and Type B resulting predominantly from portosystemic shunting and Type C resulting from cirrhosis [2]. 2. According to the severity of manifestations [2].

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The West Haven criteria are the most frequently used grading system for assessing HE in patients with cirrhosis. They are characterized by high practicality in the clinical context [1,53]. The West Haven criteria enable grading of changes in consciousness, intellectual functions, and behavior based on the severity of the individual symptoms in a categorical classification scheme from HE grades 1
4. Patients with grade 1 HE show less pronounced clinical symptoms such as a reduced cognitive performance, a reduced attention span and impairments of the ability to add or subtract, while patients with grade 2 HE show lethargy, confusion, inappropriate behavior, flapping tremor as well as slurred speech. In grade 3 HE there is also somnolence (only flight reflexes, lack of vestibulo-ocular reflex, weakened pupil reaction), disorientation, bizarre behavior patterns, muscular stiffness, and clones or hyperreflexia. With grade 4 HE (coma) the patient no longer reacts to any verbal, visual, or strong external stimuli (repeated pain stimuli); decerebration may be present [1,2,68,69]. In the current guidelines of the specialist societies the European Association for the Study of the Liver (EASL), the American Association for the Study of Liver Diseases (AASLD), and the International Society of Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN) the minimal HE and HE grade 1 together are classified as “covered HE” (covert HE, cHE), HE grade 2
4 summarized as “obvious HE” (overt HE, oHE) [2]. 3. According to its time course, HE is subdivided into episodic, recurrent (episodes that occur with a time interval of 6 months or less), and persistent HE (pattern of behavioral alterations that are always present) [2]. 4. According to the existence of precipitating factors, HE is subdivided into nonprecipitated or precipitated. Precipitating factors can be infections, gastrointestinal bleeding, electrolyte disorder, diuretic overdose, or constipation [2,70].

8.4.2 Diagnostic tests According to the ISHEN classification, the term “covert” is used to describe that the mental or motor defect is usually not yet recognizable by the clinician or patient [2]. By definition, the minimal HE (mHE) can only be assessed using validated neurophysiological or psychometric tests, while grade 1 HE is a clinical diagnosis according to the West Haven criteria. The tests for diagnosing mHE have not yet been formally validated for grade 1 HE [71]. In 20%
85% of clinically inapparent patients with cirrhosis, indicators for cognitive dysfunctions can be found. This minimal HE is therefore defined as a clinically unremarkable neurological condition, but with demonstrable abnormalities in apparative and psychometric tests [1,2,68,69,72]. Diagnosing low-grade HE can be difficult because subjective symptoms such as reduced quality of life, sleep disorders and increased daytime sleepiness are most common. Clinical indicators for cHE can be assessed from the medical history or the medical history by proxy. Studies have shown that HE does not lead to a general cerebral impairment, but that individual cognitive functions such as attention, working memory, the secondary memory, or the control of movement can be severely impaired at an early stage [71
75]. Due to the complex multifactorial pathophysiology of HE, which is often triggered by different precipitating factors, there is no diagnostic gold

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standard and therefore multimodal diagnostic test procedures are required for reliable detection, grading and follow-up. The critical flicker frequency analysis and/or the fully performed Psychometric Hepatic Encephalopathy Score should preferably be used to assess the mHE [76
80]. Detection and treatment of mHE can prevent and delay the development of manifest HE [81] (Table 8.1). Manifest HE is diagnosed and classified clinically based on the West Haven criteria [2,65,68]. Diagnosing cognitive dysfunction from clinical observation or from using neuropsychological tests is unequivocal. Yet there might be alternative causes and therefore the diagnosis of HE is through exclusion of other causes of brain dysfunctions [2] such as seizures, CNS infections, other metabolic disturbances, intoxications, or additional neurodegenerative disorders. Laboratory diagnostic tests such as the determination of ammonia levels in arterial or venous blood show insufficient sensitivity and specificity due to a variety of confounding factors (including sample collection and analysis) and do not allow reliable detection of TABLE 8.1 Diagnostic methods in mHE. Critical flicker frequency analysis

Psychometric hepatic encephalopathy score

The frequency at which a flickering light is perceived as continuous which is reduced in retinal pathology involved in hepatic encephalopathy secondary to swelling of retinal astrocytes and oligodendroglia but also cortical visual processing spatial and temporal resolution • Spatial resolution (ability to discriminate, in space, between two adjacent objects; determined by several factors, such as the eye optics, spatial organization of the photoreceptors cells, the degree of neural convergence in the retina and higher visual areas in the brain) • Temporal resolution (temporal features of the stimuli and is defined as the ability to discern luminance changes over time; the visual system’s temporal performance is limited by the finite time required for collecting and processing information and intermittent stimuli presented to the eye are perceived as separate only if the presentation rate is below a certain threshold) [82,83] Battery of neuropsychological tests composed of five neuropsychological tests: the digit-symbol test, number connection tests A and B the serial dotting test and the line-drawing test [84]

Electroencephalography

Electrophysiological monitoring method to record electrical activity of the brain; typically noninvasive, with the electrodes placed along the scalp measures voltage fluctuations resulting from ionic current within the neurons of the brain [85]. High voltage, low-frequency (1.5
3 Hz) waves with triphasic appearance have been considered characteristic for HE [61]

Animal naming test

Maximum number of animals listed in 1 min [86]

Stroop test/smartphone application

Tests users’ mental speed/response rate in identifying the color of printed text Neutral stimuli (a nonverbal cue in which the goal is to identify the color, for example), congruent stimuli (ink color and the word refer to the same color), incongruent stimuli: ink color and the word refer to the different colors

Number connection test

Connection of numbers from 1 to 25 in chronological order in a certain time frame to assess the grade of hepatic encephalopathy [79]

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HE or a reliable grading of severity of HE. In individual cases, a determination of ammonia concentration can be helpful in the differential diagnosis of HE [76,87,88]. Imaging diagnostics using computed tomography or MRI are necessary to exclude other causes of impaired brain function, in particular to rule out structural causes such as intracranial bleeding in somnolent or comatose patients. With special MRI techniques, an increase in cerebral water content and changes in brain water diffusion can be measured in HE [89,90].

8.4.3 Relevance of hepatic encephalopathy 8.4.3.1 Hepatic encephalopathy and fitness to drive Deficits in the areas of attention, vigilance, perception, and motor skills associated with HE can lead to an impairment of the fitness to drive. The test of driving suitability through a real test with an expert (driving instructor) remains the gold standard for the assessment of driving performance, since computerized tests do not seem to reliably predict driving ability [91]. Due to the overall heterogeneous data situation regarding the fitness to drive of cirrhotic patients ranging from no restrictions in the ability to drive [67] to a significantly impaired fitness to drive [91], no general recommendation can currently be given concerning a driving ban in patients with HE [71]. Nevertheless, patients with cirrhosis and HE should be informed about the potential impairment of their fitness to drive and, if necessary, the objectification should be initiated by a driving suitability test. As a sensitive screening question the patient should be asked about road accidents in the previous year [71,92]. 8.4.3.2 Hepatic encephalopathy and quality of life An impaired HRQOL is a major consequence of HE. While oHE seems to negatively affect both physical and mental aspects of HRQOL, subclinical HE affects mainly mental aspects independently of the severity of liver disease [93]. Patients with higher cognitive reserve seem to be able to tolerate HE and its impact on HRQOL better than those with a poor cognitive reserve [67,94]. The assessment of HRQOL changes in outpatients can help to predict clinically relevant outcomes such as HE recurrence, hospitalizations, and death [10,11]. 8.4.3.3 Hepatic encephalopathy and sleep disorders Sleep-wake cycle changes are common in cirrhotic patients and have a high impact on HRQOL [95]. Insomnia and fragmented sleep, with multiple night awakenings, have been reported in cirrhotic patients [96]. There is evidence of an association between daytime sleepiness and HE. The absence of excessive daytime sleepiness had a negative predictive value of 92% in relation to the development of HE during a follow-up period of 8 months [97]. Sleepiness has been shown to be more common in cirrhotic patients with a history of HE and documented portal-systemic shunt [97] and to be positively correlated with the amount of slow activity on the wake electroencephalography (EEG) [98]. Induced hyperammonemia is associated with increased subjective sleepiness in both patients with cirrhosis and controls. Amino acid challenge led to changes in the EEG architecture of a

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subsequent sleep episode in patients with cirrhosis, pointing to a reduced ability to produce restorative sleep [99]. 8.4.3.4 Hepatic encephalopathy in clinical scoring systems HE is associated with outcome and prognosis of cirrhotic patients. The Child-Pugh Score, also called Child-Turcotte-Pugh Score (CTP Score), is used to classify cirrhosis of the liver. At the same time, the prognosis of the patient can be estimated on the basis of these stages. A total number of points is created based on five criteria: three laboratory values (albumin, bilirubin, INR), and two clinical findings (ascites, HE), whereby 1
3 points are assigned for each parameter so that a score of 5
15 can be achieved [100,101]. The relevance of HE is also reflected in two scores that are used for organ allocation in ALF, namely the Clichy criteria and the King’s College criteria [102].

8.5 Treatment options 8.5.1 General management In every cirrhotic patient HE should be assessed clinically at presentation. Many patients present with mHE which at first glance cannot be diagnosed but only elaborated either by reports of family or psychometric tests. At the same time, it is recommended to search for precipitants of HE. Correction or treatment of precipitants or either HE treatment improve outcome in patients with HE. Below we have highlighted some aspects of HE management.

8.5.2 Diet More than 100 g of protein intake on at least one of the last 4 days before the occurrence of an HE episode was considered to be a possible trigger for an HE [103]. The intake of 30
40 g of vegetable protein proved to be harmless [96,104]. Protein intake should be 1.2
1.5 g/kg body weight per day [105]. Protein restriction is not recommended even in the presence of HE, since a protein intake of less than 0.8 g/kg body weight per day leads to a catabolic metabolic situation with an increase in nitrogen levels in the circulatory system [106,107]. HE episodes are significantly more common in cirrhosis and sarcopenia [108]. Small meals or liquid nutritional supplements evenly distributed throughout the day and a late-night snack should be offered [109,110].

8.5.3 Treatment of precipitating factors of hepatic encephalopathy A HE episode can be based on one or more possible triggering causes which should be looked for and treated specifically. Up to 90% of episodically occurring HEs have a triggering cause and the successful therapy of which almost always leads to an improvement or elimination of HE [24,111]. Infections have repeatedly been shown to trigger a HE episode and to significantly increase the mortality risk of HE [70,106,112,113]. Treatment of the underlying infection leads to an improvement in the HE episode. After an upper GI

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bleeding, further complications occurred in 44.7% of patients [114] with a first HE episode or the deterioration of a preexisting HE which could be demonstrated in more than 50% of these patients. The occurrence of HE after upper GI bleeding was an independent negative prognostic indicator. In a cohort of patients with cirrhosis, variceal bleeding was the second most common cause of oHE after infections (23.3%) [115]. In up to 40% of patients with cirrhosis and HE, the cause of the oHE could be attributed to constipation [115,116]. Another indication of the possible role of constipation in the development of HE is that laxative measures lead to the improvement of HE [117]. In up to 74% of patients with HE the cause was acute kidney failure [116]. Dehydration was seen in 46%
76% of patients with HE, which probably contributed to the acute kidney failure. Possible reasons for dehydration are diuretics, excessive dosing of lactulose, reduced fluid intake, largevolume paracentesis, and preexisting diabetes mellitus. An increased creatinine value proved to be an independent risk factor for the occurrence of HE [24,118]. Hyponatremia seems to be an independent precipitating factor for the occurrence of HE [24]. Disorders of the acid
base balance and hypokalemia are further risk factors for this complication [103,115,116,119] (Table 8.2).

8.5.4 Shunting Medically refractory HE should raise suspicion of a spontaneous splenorenal shunt [61]. It has been shown that transvenous obliteration of large spontaneous splenorenal shunts in patients with severe recurrent HE improved in all patients at the time of discharge as well as at 4 month follow-up [123]. A retrospective study showed that patients that experienced complete occlusion of portosystemic shunts compared to patients who did not undergo embolization showed some benefit in embolization [124].

8.5.5 Differentiated step-by-step pharmacotherapy In patients with overt HE lactulose is the drug of choice for the therapy of an HE episode. In addition to oral administration or if oral administration is not possible, lactulose can be administered as an enema (300 mL lactulose/700 mL water). Combination therapy with rifaximin can be considered in individual cases. Rifaximin as monotherapy should only be used if the patient suffers from lactulose intolerance. The intravenous administration of branched-chain amino acids (BCAA) as well as intravenously administered L-ornithine L-aspartate (LOLA) can additionally or alternatively be used in patients for the therapy of acute oHE who have not responded to therapy with lactulose alone. There is insufficient evidence for the clinical efficacy of orally administered LOLA. In patients with oHE, flumazenil can be used in addition to standard therapy to clarify the HE trigger after possible exposure to benzodiazepines [71]. In minimal HE, therapy should be carried out in patients if a reduced quality of life is stated, if there is an objective restriction when performing everyday tasks or if there are occupational risks. If therapy of mHE is considered necessary, lactulose should be used as the treatment of first choice [71].

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TABLE 8.2 Triggering causes and precipitating factors of hepatic encephalopathy [24,103,116]. Trigger

Clinical event

Treatment

Infections

Spontaneous bacterial peritonitis (SBP), pneumonia, urinary tract infection, etc.

Community-acquired uncomplicated SBP: group 3a cephalosporins (e.g., ceftriaxone) Hospital-acquired SBP and/or risk factors for treatment failure (local resistance, antibiotic pretreatment in the last 12 weeks) and additional individual factors (clinical severity, MRE carrier status): empirical therapy with carbapenems (e.g., imipenem) [71] Prophylaxis of SBP: primary prophylaxis if there is ascites with a reduced total protein content (,1.5 g/dL) 1 / 2 presence of one of the two criteria [(1) severe hepatic insufficiency, i.e., Child-Pugh score. 9 with bilirubin. 3 mg/dL or (2) renal insufficiency with serum creatinine. 1.2 mg/dL, urea. 25 mg/dL or sodium ,130 mEq/L] [71] Community-acquired pneumonia: • slight pneumonia without comorbidity: amoxicillin p.o. • slight pneumonia with comorbidity: amoxicillin/ clavulanic acid • moderate pneumonia: for example, amoxicillin/ clavulanic acid or ceftriaxone 1 macrolide (e.g., clarithromycin) until exclusion of atypical pathogens • severe pneumonia: intravenous therapy, for example, piperacillin/tazobactam or ceftriaxone 1 macrolide (e.g., clarithromycin) until exclusion of atypical pathogens [120] Hospital-acquired pneumonia: • without risk for multiresistant pathogens: for example, amoxicillin/clavulanic acid or ceftriaxone or moxifloxacin • risk for multiresistant pathogens: for example, piperacillin/tazobactam or cefepim or imipenem 1 / 2 ciprofloxacin or gentamicin [121]

Gastrointestinal bleeding

Variceal bleeding, ulcer bleeding, etc.

Before endoscopic treatment: Terlipressin (1
2 mg), Ceftriaxone 2 g i.v., Erythromycin 250 mg or other prokinetic drugs i.v.; endoscopic treatment (early in shock, otherwise ,12 h)Pantoprazole 80 mg, erythromycin 250 mg i.v., endoscopy ,12 h in shock, promptly (,24 h) with risk constellation, stable situation ,72 h [122]

Electrolyte disorders

Hypokalemia, hyponatraemia

Potassium substitution Depending on the underlying condition

Drugs

Benzodiazepines, diuretics, neuroleptics, opioids,

Flumazenil Pause diuretic treatment Pause or switch medication if possible Naloxone (Continued)

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TABLE 8.2 (Continued) Trigger

Clinical event

Treatment

Dehydration

Diuretics, paracentesis, laxative measures, reduced fluid intake

Fluid therapy (stop or reduce dosage of diuretics)

Constipation

Hyperammonemia

Laxative measures (lactulose, therapy goal: 2
3 loose stools per day) [71]

Acid
base disorders

Acidosis

Depending on the underlying condition

Protein excess

More than .100 g protein/day on at least 1 day in the last 4 days

Protein intake should be 1.2
1.5 g/kg body weight per day [105]

Severe medical stress

Trauma, operation

MRE-multiresistente Erreger.

8.5.5.1 Lactulose (nonabsorbable disaccharides) Lactulose or lactitol are disaccharides that are nonabsorbable in the small intestine and reach the colon without being split. They are osmotically active and therefore accelerate the intestinal passage. The intraluminal pH is lowered resulting in ammonia turning into ammonium ions that cannot be absorbed. The release of ammonia from glutamine is also reduced by lactulose due to the inhibition of glutaminase. The dysbiosis in the colon that is found in cirrhotic patients is also favorably influenced (role of disaccharides as a so-called prebiotic) [125]. Lactulose has been shown to increase beneficial taxa on culturebased assays and to improve HE-typical measures of MRI spectroscopy indicating a beneficial effect on astrocytic swelling [126]. Data showing its impact on the microbiota in culture-independent studies are heterogenous [125,126] so it remains unclear to what extend the effect of lactulose on HE is related to alterations in gut microbiota. In a meta-analysis [127] of 38 randomized studies with 1828 participants, nonabsorbable disaccharides showed a statistically significant improvement in acute HE. Even if the studies included were mostly of moderate quality, there is still a clear recommendation regarding their preferred use in the treatment of manifest HE and for primary and secondary prevention [127]. Lactulose leads to a decrease in all-cause mortality and has a positive effect on complications of chronic liver failure such as variceal bleeding, severe infections, spontaneous bacterial peritonitis and hepatorenal syndrome [128]. When compared to rifaximin, lactulose was shown to be equivalent in several meta-analyses [129
131]. Side effects of therapy with nonabsorbable disaccharides are flatulence, abdominal pain, and diarrhea. 8.5.5.2 Rifaximin Rifaximin is nonabsorbable and almost exclusively acts in the intestinal lumen changing the quantitative and qualitative composition of the intestinal microbiota. It can prevent and ameliorate episodes of HE. Rifaximin affects the intestinal flora by killing pathogenic intestinal bacteria with endotoxin or ammonia formation. It has a broad antimicrobial spectrum (intestinal Gram-positive and Gram-negative organisms), which includes both

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aerobes and anaerobes. It influences the function and activities of intestinal bacteria with an increase in long-chain fatty acids and metabolites of the carbohydrate metabolism [132]. Rifaximin may have impacts beyond its intraluminal antibiotic action. It has been shown to modulate microbial function rather than composition and change the expression of intestinal glutaminase in germ-free models, reducing ammonia production [133]. Concerning ultrastructural changes in the brain, a short course of rifaximin monotherapy has been shown to improve MRI measures of neuronal connectivity but not HE markers in MRI spectroscopy [134]. This argues for a rather neuron-specific alleviation of HE in contrast to the rather astrocyte-specific effects of lactulose which might be caused by differential effects on the gut microbiome [8]. The primary use of rifaximin in acute manifest HE was not recommended in the common guideline of AASLD and EASL [2]. Rifaximin is also not approved for the treatment of the first HE episode in Germany. According to the guideline commission of the Deutsche Gesellschaft fu¨r Gastroenterologie, Verdauungsund Stoffwechselkrankheiten (DGVS), monotherapy with rifaximin should not be given during initial treatment, but only in the few cases of lactulose intolerance “Off-Label Use.” If patients do not respond to lactulose alone, rifaximin can also be given [71]. 8.5.5.3 Branched-chain amino acids Valine, leucine, and isoleucine are BCAA, which are required for cellular protein synthesis. In patients with cirrhosis of the liver, the relationship of BCAA to aromatic amino acids in serum is shifted in favor of BCAA. In these patients, BCAA administration seems to have a positive effect on albumin synthesis, insulin resistance and the synthesis of muscle proteins [135]. In patients with HE, BCAA treatment can cause nitrogen binding in proteins and a reduced release of ammonia with anabolic metabolism. Studies have shown that oral or intravenous BCAA-enriched formulations improve the manifestations of episodic HE whether oHE or mHE [128,136]. 8.5.5.4 L-ornithine L-aspartate Elevated serum ammonia levels can be reduced by exogenous intake of LOLA in patients with cirrhosis of the liver [137]. LOLA may stimulate the incorporation of ammonia into urea or glutamine through the increased range of precursors for urea synthesis and glutamine formation [138]. A positive effect of LOLA on the ammonia blood levels has been shown in some [139,140] but not in all studies [140]. The detection of ammonia reduction alone does not allow any conclusions on the clinical effectiveness of LOLA treatment [141]. All in all there is heterogenous data regarding the use of LOLA in patients with HE. The DGVS suggests that intravenously administered LOLA can additionally or alternatively be used in patients for the therapy of acute oHE who have not responded to therapy with lactulose alone. There is no reliable data for oral administration of LOLA [2,142].

8.5.6 Intensive care aspects of hepatic encephalopathy Patients with HE and ALF and ACLF or with decompensated cirrhosis and grade 3 or 4 HE should be monitored intensively by medical personnel. In comatose patients (grade 3 and above), intubation and ventilation should be initiated early [109,143]. It is suggested

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to place a gastric tube if oral feeding is not possible and after larger esophageal varices have been ruled out to avoid aspiration and to allow administration of medication and enteral nutrition [2,71]. With ALF, further specific measures are required for HE, since increased cerebral pressure can develop rapidly with progressive cerebral edema and fatal brain herniation [143,144]. A liver transplant (LT) is often indicated in this situation so that the patients should be transferred to a transplant center at an early stage. HE alone is not considered an indication for liver transplantation unless associated with poor liver function. Yet there are cases where HE severely impairs the quality of life and cannot be improved despite maximal medical therapy. These patients might be LT candidates despite otherwise good liver status [2].

8.5.7 Treatment of posttransjugular intrahepatic portosystemic shunts hepatic encephalopathy After TIPS, the median cumulative one-year incidence of oHE is 10%
50% [145,146]. The incidence of HE is greatly influenced by the patient selection criteria. Patients with HE before TIPS or higher Child-Pugh score have an increased risk of post-TIPS HE [141]. Primary drug prophylaxis of HE after TIPS implantation proved to be ineffective in a study with rifaximin or lactitol in comparison to no therapy, so this should be avoided [147]. HE occurring after TIPS is treated as in patients with cirrhosis without TIPS [148,149]. A triggering cause must first be sought and treated. Shunt diameter reduction can reverse HE [150]. If HE does not improve after stent reduction in TIPS patients liver transplantation should be considered [71].

8.5.8 Prevention of overt hepatic encephalopathy Therapy studies for secondary prevention of HE showed a recurrence rate of 47%
57% for a second HE episode [151
153]. Measures for secondary prophylaxis of HE prevent the occurrence of a repeated HE episode that is associated with rehospitalization, morbidity, mortality, and health costs [151
153]. In cirrhotic patients who had an HE episode, relapse prevention should be carried out. Lactulose should be used as a medication for relapse prevention. Rifaximin should be used in addition to lactulose in secondary prophylaxis of HE grade higher than 1 according to West Haven criteria if a relapse has occurred with the sole administration of lactulose. Monotherapy with rifaximin should only be given if lactulose therapy is not possible [151]. Routine prophylactic therapy (lactulose or rifaximin) is not yet recommended for the prevention of post-TIPS HE [2,147]. Nevertheless, recent reports have shown that preventive rifaximin is associated with a lower risk of HE and a higher rate of transplant free survival during the six-month period of treatment after the procedure [154].

8.6 Recent advances Patients with chronic liver disease and cirrhosis show a global mucosal immune impairment, which has been associated with altered gut microbiota composition and

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functionality. These changes seem to be linked with hepatic encephalopathy and infections as one of the important triggers of HE and represent a target for pharmaceutical interventions [155
157].

8.6.1 Medications in cirrhosis and microbial changes Cirrhotic patients often receive medications that either target the microbiota specifically or have an impact on it. Current therapeutic strategies targeting microbiota in liver disease include probiotics, prebiotics, antibiotics, laxatives and fecal or intestinal microbiota transplantation (FMT/ IMT). There seem to be differential effects of different therapies (e.g., lactulose, rifaximin, and LOLA) on different microbiota families that have been shown to be associated with specific ultrastructural changes in multimodal MRI of the brain [8]. However, there is currently no specific adaptation of these therapies based on the specific composition or function of the microbiota in patients to ensure the maximum benefit for this therapy [7]. 8.6.1.1 Probiotics Over a six-month period, the daily intake of the probiotic VSL#3 has shown to significantly reduce the risk of hospitalization for HE in patients with cirrhosis [158]. In another study 25 patients with cirrhosis of the liver and mHE confirmed by psychometric tests received probiotic yogurt (n 5 17) or no therapy (n 5 8) for 60 days. With yogurt consumption, the mHE (three psychometric tests) improved in 71% compared to 0% in the controls. Two of the patients in the control group and none in the “verum” group receiving a commercially available yogurt with Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus acidophilus, bifidobacteria, and Lactobacillus casei developed an HE episode [159]. Another study showed that probiotics were just as effective as LOLA or lactulose [160]. In contrast, Saji et al. observed no effect of probiotics on mHE in cirrhosis of the liver [161]. A meta-analysis of nine studies that used probiotics (living bacteria), prebiotics (lactulose) or synbiotics (both forms of therapy) found positive effects for the use of these therapies in mHE [162]. Improvement of mHE and fewer transitions into a clinically manifest HE under probiotics were also the result of a further meta-analysis, which, however, could not show any difference to lactulose, rifaximin or LOLA [163]. 8.6.1.2 Fecal or intestinal microbiota transplantation The published experience with FMT/IMT in cirrhosis include case reports, randomized-controlled trials and open-label studies which focus largely on hepatic encephalopathy and the altered activity of the gut
liver
brain axis [164
167]. The routes have ranged from colonoscopy to enema and capsules. It has been shown that cognitive function improved in the FMT arm at day 20 after FMT and remained significantly better [166]. One has to note that there are confounders related to use of rifaximin and lactulose as well as the broad use of proton pump inhibitors in these patients which have an impact on the microbial composition as well [166]. Another case report has shown that following FMT, the recipient’s fecal microbiota composition moved toward the donor’s composition, and that this was maintained through a follow-up of 7 weeks. The patient clinically improved in terms of attention, concentration and sleep-wake-cycle. Objectively, his

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inhibitory control test and Stroop test scores normalized by week 4 [165]. Beneficial taxa, such as Ruminococcaceae, Verrucomicrobiaceae, and Lachnospiraceae, were correlated with cognitive improvement and decrease in inflammation after FMT [168]. In-depth analyses of microbial engraftment, composition and function have been performed in some studies. The use of antibiotics pre-IMT was associated with a decimation of microbial diversity which was restored post-IMT [167].

8.7 Outlook The role of the gut microbiome in human health and disease has become a major point of interest linking dysbiosis to the development of several conditions such as HE. However, important confounders and only limited knowledge on the underlying mechanisms make it difficult to implement these findings into clinical practice [169]. Since liver cirrhosis presents a major interaction of the gut microbiota with the host, it seems that microbiome diagnostics and treatment are necessary to treat the progression of the disease. However, this is not yet reflected neither in the currently used diagnostic tools nor are specific microbiota-directed therapies currently being used in standard therapies. Future studies are needed to deepen the understanding of the mechanisms of dysbiosis affecting cirrhosis and its progression. Only with a deeper understanding of the underlying mechanisms and dynamics of gut microbiome changes it will be possible to specifically target dysbiosis as a possible treatment to prevent progression and decompensation of cirrhosis to prevent HE.

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91, 891. e1. Available from: https://doi.org/10.1053/j.gastro.2009.05.056. [153] Agrawal A, Sharma BC, Sharma P, Sarin SK. Secondary prophylaxis of hepatic encephalopathy in cirrhosis: an open-label, randomized controlled trial of lactulose, probiotics, and no therapy. Am J Gastroenterol 2012;107:1043
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288). Hepatology 2019;70:1
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C H A P T E R

9 The gut microbiota in hepatic encephalopathy Sandip Samanta and Debbie L. Shawcross Department of Inflammation Biology, Faculty of Life Sciences and Medicine, School of Immunology and Microbial Sciences, Institute of Liver Studies, King’s College London, London, United Kingdom

9.1 Introduction There is increasing interest in the gut microbiome and its impact on health and disease. The vast ecosystem of organisms housed within our digestive system is as complex and influential as our genes in everything from mental health to obesity. The gut microbiome propagates a myriad of influences impacting on the maintenance of health and, in turn, the development of disease in all ages [1]. Comprised of bacteria, archaea, viruses, and fungi, Qin et al. [2] first described the gut microbiome as a separate genomic entity behaving as an organ with a central role in endogenous metabolism with downstream effects on nutritional status and immune regulation. It is widely accepted that a functional gut flora is crucial to the maturation of a healthy immune system, [3] and as such, perturbations in the commensal gut ecosystem, often termed “dysbiosis,” act as a precursor to dysregulated immune activity, and thus may become a determinant in the development and progression of chronic liver disease (CLD) [4]. CLD has a high morbidity and mortality largely due to complications relating to cirrhosis, where long-term damage causes scarring of the liver, disrupting its architecture and therefore its function. Cirrhosis is initially asymptomatic as the body “compensates”; the onset of episodes of “decompensation” through ascites, jaundice, bleeding, or encephalopathy is associated with a reduction in median survival from 12 years to 2 years [5], showing a progression to more advanced disease. Worldwide, cirrhosis is the cause of over a million (1.32 million in 2017), deaths per year [6]. Though mortality has fallen overall over the last 30 years reflecting improvements in treatment and the adoption of liver transplantation, there has been a significant increase in the prevalence of decompensated cirrhosis [6]. Despite the threat of CLD to global health and the increasing burden on finite

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Liver, and Liver
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resources, there is little new focus on early or prophylactic intervention, with few studies identifying new therapeutic targets or opportunities for intervention. Hepatic encephalopathy (HE) represents one of the most severe and debilitating complications of CLD and is a manifestation of decompensated cirrhosis. A diagnosis of HE in a CLD patient reduces survival to between 0.95 and 2 years depending on age [7], with some data showing that HE is the most potent risk factor for repeated hospitalization in this patient cohort. Additionally, HE is a key feature in acute liver failure (ALF). This is a rare syndrome, where there is an acute liver insult or injury in the absence of a previous history of CLD. It can be subcategorized by onset of HE within 7 days, between 8
28 days, or 5
12 weeks of onset of jaundice being hyperacute, acute, and subacute ALF, respectively. These categories are correlated with survival, with the best outcomes in hyperacute ALF [8]. The main causes of acute and CLD are shown in Table 9.1. The clinical entity of acute-on-chronic liver failure (ACLF) has also been described, which differs from acutely decompensated chronic liver failure in severity and outcomes: it features intense systemic inflammation, has a clear precipitant temporarily, and occurs in association with single or multiorgan failure [9]. HE is a central feature of ACLF as well. In the context of cirrhosis development and progression, gut dysbiosis is now considered a crucial mediator, [10] highlighting a number of potential opportunities for therapeutic intervention in reducing the progression and downstream sequelae of end-stage CLD. There is a growing evidence base to support gut dysbiosis being a key determinant in the development of HE and the mainstay of therapy has historically always been targeted on the gut. In this chapter, we will focus on how the gut microbiome changes in HE and how this may play a role in the evolution of the clinical manifestations observed. We aim to demonstrate how manipulation of the gut microbiome in HE could favorably impact on outcomes for these patients.

TABLE 9.1 Major causes of acute liver failure (ALF) and chronic liver disease (CLD). Cause

Examples in acute liver failure

Examples in chronic liver disease

Viral

Hepatitis A, B, E

Hepatitis B, C, D

Drugs/ substance abuse

Paracetamol, flucloxacillin, anti-TB drugs

Alcohol

Vascular

Hypoxic liver injury Budd-Chiari syndrome

Budd-Chiari syndrome

Pregnancy

Preeclampsia, HELLP, fatty liver of pregnancy

None

Autoimmune

Autoimmune hepatitis

Autoimmune hepatitis, primary sclerosing cholangitis, primary biliary cholangitis

Metabolic and other

Wilson’s disease

Metabolic-associated fatty liver disease, Wilson’s disease, hemochromatosis, alpha-1 antitrypsin deficiency

Note: HELLP, Hemolysis, elevated liver function tests, low platelets syndrome; TB, tuberculosis.

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9.2 Defining hepatic encephalopathy HE is a term used to describe “brain dysfunction caused by liver insufficiency and/or porto-systemic shunting (PSS)” [11]; the latter plays a role in HE either alone or in conjunction with liver disease through the same underlying pathophysiology. The West Haven classification, formulated by Harold Conn and colleagues while investigating the therapeutic efficacy of lactulose, is currently the most utilized method to classify the degree of HE [12]. As mentioned in the previous chapter, the West Haven criteria classify patients from having no clinically distinguishable features of HE, through a spectrum that includes dysphoria, disorientation, sleep-wake reversal, confusion, stupor and coma (Table 9.2). More subtle changes in higher executive functioning may only be detected with neuropsychological function testing but may still significantly impair daily functioning, quality of life, and the ability to safely drive a car. HE is a clinical diagnosis but is often supported by investigations such as serum ammonia (not diagnostic, but helpful to exclude HE), neuropsychological testing, and electroencephalogram abnormalities, as well as excluding other causes of confusion and coma. The management options vary according to the type (A
C) of HE (see below), but the nonabsorbable disaccharide lactulose (or lactitol) and the nonabsorbable antibiotic rifaximin now form the mainstay of treatment for the most common type C. Diagnostic tests and treatment options are discussed in detail in the book Section 3 “Liver
brain axis,” in the chapter “Gut
Liver
Brain Axis in chronic liver disease with a focus on hepatic encephalopathy.”

TABLE 9.2 West Haven criteria for the diagnosis of hepatic encephalopathy. Stage

Distinguishing features

0

No overt abnormality detected

1

Trivial lack of awareness Euphoria or anxiety Shortened attention span Impaired performance of addition

2

Lethargy or apathy Minimal disorientation for time or place Subtle personality change Inappropriate behavior Impaired performance of subtraction

3

Somnolence to semistupor, but responsive to verbal stimuli Confusion Gross disorientation

4

Coma (unresponsive to verbal or noxious stimuli)

See Atterbury CE, Maddrey WC, Conn HO. Neomycin-sorbitol and lactulose in the treatment of acute portal-systemic encephalopathy. A controlled, double-blind clinical trial. Am J Dig Dis. 1978;23(5):398
406 [12].

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9.3 Ammonia as a driver of hepatic encephalopathy In health, nitrogenous byproducts are generated by both the consumption of amino acids and proteins by enterocytes and other body cells, as well as by the endogenous gut bacteria that make up the enteric microbiome. These are transported via the portal vein to the liver, where most are metabolized into less toxic products and/or excreted. These waste products include mercaptans, short chain fatty acids, phenol, and importantly, ammonia. Specifically, ammonia is produced via bacterial degradation of urea, amino acids, amines, and purines in the intestine. In the liver, the majority of this ammonia is converted to urea in the Krebs cycle, which is excreted by the kidneys. Additionally, ammonia is used to convert glutamate into glutamine via glutamine synthase. Glutamine can in turn be used to regenerate ammonia in the enterocytes by the action of glutaminase, beginning the cycle again; it can also be excreted via the kidneys. A small amount of ammonia is present in the systemic circulation and the glutamine pathway also occurs in muscle tissue and within astrocytes in the brain. Nencki, Pavlov, and Zaweski’s seminal experiments in the late 1890s demonstrated that the creation of a portacaval shunt in dogs led to behavioral symptoms, ataxia, convulsions, and coma; these symptoms were exacerbated by feeding the dogs meat, but not milk or bread. Their discovery of increased ammonia salts in urine first led to the theory that reduced hepatic ammonia metabolism caused this neurotoxicity [13]. Acute, chronic, and acute-on-chronic liver dysfunction all lead to reduced hepatic ammonia metabolism; this is demonstrated in that serum ammonia levels are significantly raised in compensated cirrhosis alone and increase further in ALF and ACLF [14]. This systemic hyperammonemia occurs via several different pathways and is reflected in the classification of HE as type A (acute), B (bypass), and C (cirrhosis).

9.4 Classifying HE by cause of hyperammonemia Type A HE occurs in the context of ALF, where widespread hepatocyte death leads to a failure to metabolize nitrogenous waste, resulting in increased ammonia levels in the systemic circulation. In the absence of the liver, the muscle becomes the main organ for ammonia detoxification and metabolism via the glutamate-glutamine pathway (Fig. 9.1). It is rich in the enzyme glutamine synthetase, but is much less efficient than a healthy liver; thus systemic levels remain pathologically high. This, in conjunction with additional factors detailed below, exerts a neurotoxic effect on the brain, resulting in the clinical phenotype of HE. In Type B HE, nitrogenous waste bypasses the liver (Fig. 9.1). This is usually due to congenital or spontaneous PSS, or the insertion of a transjugular intrahepatic portosystemic shunt, created to manage portal hypertension in CLD. The latter procedure is only conducted after assessing the postprocedure risk of HE. Type C HE accounts for the majority of cases and occurs in CLD in a cirrhotic liver. Replacement of functional hepatocytes with scar tissue leads to a reduced capability to metabolize ammonia overall. Furthermore, distortion of the liver structure by this scar tissue leads to vascular “back pressure” and increased portal venous hypertension, leading to nitrogenous waste bypassing the liver via PSS (e.g., esophageal varices) into the systemic circulation.

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FIGURE 9.1 Differentiating between different types of hepatic encephalopathy (HE): green arrows indicate transport of nitrogenous waste including ammonia. In health, nitrogenous waste is processed in the liver into urea, which is excreted by the kidney. Small amounts are metabolized via the glutamate-glutamine pathway in muscle and brain tissue. In Type A HE, acute liver failure (ALF) reduces hepatic ammonia metabolism, leading to increased ammonia uptake by the brain leading to HE. In Type B HE, porto-systemic shunting (PSS) leads to nitrogenous waste bypassing the liver to the brain. In type C HE, cirrhosis causes portal hypertension and PSS, as well as reducing hepatic ammonia metabolism. This is the most common type.

As noted above, cirrhosis is “compensated” via a variety of physiological adaptations. Type C HE arises due to decompensation and is often caused by a precipitant. Common precipitants are shown in Table 9.3 and all generally overwhelm the compensatory physiological mechanisms, worsening existing shunting, and further reducing ammonia metabolism, leading to systemic hyperammonemia.

9.4.1 Hyperammonaemia in the brain Ammonia can cross the blood
brain barrier (BBB), and its effects on the brain are complex and not entirely characterized. In postmortem human and animal studies of ALF, electron microscopy showed that the BBB remained anatomically intact, but capillary endothelial cell swelling was observed with increased vesicles and vacuoles. Notably, there was marked swelling of astroglial foot processes [15,16], implicating changes in astrocytes as being responsible for the neurological effects of hyperammonemia in ALF. Increased intracellular ammonia is thought to underlie this cytotoxicity. As detailed earlier, ammonia is metabolized within astrocytes via the conversion of glutamate to glutamine. Correspondingly, animal models of ALF show decreased brain glutamate and increased glutamine content, seen via imaging (1H-nuclear magnetic resonance spectroscopy) and via postmortem biochemical tissue analysis [17]. One theory is that this increased intracellular glutamine concentration causes an osmotic imbalance, increasing fluid shift into the cells and leading to cerebral edema. This is known as the “osmotic gliopathy” theory [18].

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TABLE 9.3 The major precipitating factors for hepatic encephalopathy in chronic liver disease. Precipitant

Key example(s)

Infection

Pneumonia, urinary tract infection, spontaneous bacterial pneumonitis (SBP)

Excessive nitrogen load

Upper gastrointestinal bleed Constipation

Electrolyte disturbances

Hyponatremia Metabolic alkalosis

Drugs

Sedatives, diuretics, alcohol

Iatrogenic

Post-TIPSS (transjugular intrahepatic porto-systemic shunt) procedure

However, there is poor correlation between intracellular glutamine levels and astrocyte swelling which this theory cannot explain. A rat model of ALF shows a significant 2
4.5-fold increase in total brain glutamine in the precoma stage of HE, but no further increase in the coma stage versus baseline [19]. Additional studies have shown that astrocytes in cell culture, exposed to toxic levels of ammonia, develop changes in their mitochondrial permeability in addition to cell swelling; this phenomenon is known as the mitochondrial permeability transition (MPT). Blocking the MPT with cyclosporin A leads to an inhibition in astrocyte swelling [20]. This led to a theory that glutamine may be transported into the mitochondria and converted back to ammonia and glutamate via phosphate-activated glutaminase. Ammonia and glutamine trigger the MPT through the production of reactive oxygen species, resulting in astrocyte swelling, cerebral edema, and HE. As ammonia gains access to the mitochondria in the form of glutamine, this is known as the “Trojan Horse” theory [21]. It is likely both mechanisms play a role in causing astrocyte swelling, leading to the cytotoxic cerebral edema seen in imaging studies of human patients; a small degree of interstitial edema is also seen in ALF, and is likely mediated by the vascular changes seen microscopically (vasogenic edema) [22]. Together, this degree of edema leads to the clinical phenotype described in Table 9.2 in ALF. However, the pathophysiology underlying type C HE differs in several ways. Microscopically, animal studies of astrocytes in CLD show changes concurrent with a microscopic diagnosis of Alzheimer’s type II astrocytosis: notably enlargement of the cytoplasm and nucleus [23] compared to podocyte swelling in ALF. Furthermore, electron microscopy shows an intact BBB and only minimal cerebral edema [24]. This is echoed in magnetic resonance imaging studies of cirrhotic patients, whose low-grade cerebral edema normalized after liver transplantation [25]. The neurotoxic effects of ammonia remain in CLD but other factors play more of a role. Induced hyperammonemia in cirrhotic patients leads to a deterioration in neuropsychiatric test scores in some patients but not others. These patients who deteriorate in a process analogous to HE have a significantly greater glutamine to creatine ratio seen via proton MR spectroscopy. Additionally, the same patients had a significantly lower myo-inositol to creatine ratio [26]; myo-inositol is a key osmolyte in the brain, indicating that these patients had a reduced ability to buffer increases in intracellular ammonium. Thus it appears that some cirrhotic patients are more susceptible to the effects of hyperammonemia than others.

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Furthermore, whilst the arterial ammonia concentration directly correlates with severity of HE in ALF [27,28], there is a less than direct correlation between serum ammonia levels and severity of HE in cirrhosis. Notably, cirrhotic patients with no neurocognitive or overt clinical changes can have elevated ammonia [27], but some with grade 4 HE can have normal ammonia [29] indicating the impact of additional factors in the pathophysiology of this type of HE.

9.5 The role of inflammation The effects of systemic ammonia are not solely responsible for these clinical changes. In ALF, raised inflammatory markers (tumor necrosis factor-alpha, interleukin-8, and toll-like receptor-9 expression) all correlate with increased severity of HE with associated poor prognosis [30,31]. Clinically this is manifested in these patients having a greater number of derangements concurrent with the systemic inflammatory response syndrome (SIRS) [31]. Injection of the proinflammatory stimulus lipopolysaccharide (LPS) (derived from the cell wall of Gramnegative bacteria such as Escherichia coli) in a porcine model leads to increased cerebral edema independent of serum ammonia levels [32]. This systemic inflammatory state is likely in response to the high concentration of these inflammatory markers released from necrotic hepatocytes. Additionally, overlying sepsis in these patients is common and may further drive inflammation [31]. A large North American cohort study of 2810 cirrhotic patients with type C HE has shown that infection is a precipitant in around 17% of cases [33]; a smaller study in Nepal, a nation where healthcare is less accessible, revealed that infection is responsible for almost 50% of HE cases in cirrhotics [34]. Additionally, severity of delirium in general intensive care unit patients is associated with raised inflammatory markers, notably S-100 beta levels which are associated with astrocyte activation [35]. Furthermore, cirrhotic patients are predisposed to infection via a variety of disease-specific processes. Scarring within the liver leads to a systemic inflammatory state, caused by repeated activation of immune cells from damage-associated molecular patterns released from damaged hepatocytes. This is exacerbated by reduced synthesis of immune mediator proteins within the liver and a crippled reticulo-endothelial system. Translocation of bacterial and their degradation products, termed pathogen-associated molecular patterns (PAMPs), across the intestinal wall and into the systemic circulation leads to systemic endotoxemia, also driving this systemic inflammatory state. Collectively, these processes are termed “cirrhosisassociated immune dysfunction” [36]. Thus, it appears highly likely that there is interplay between infection, inflammation, and HE in CLD, and this is supported by a large body of evidence summarized below. A seminal study highlighted the key role of inflammation as a driver in HE when 10 patients with cirrhosis were given an amino-acid solution to induce hyperammonemia. These patients were admitted to hospital for treatment of low-grade infection and were given the solution whilst infection was present (in the presence of a proinflammatory plasma milieu), and then once again once the infection had resolved with treatment. These patients’ neuropsychological scores were significantly worse in the presence of active infection and hyperammonemia but were unimpaired following resolution of the infection and inflammatory response, showing that infective processes and the resulting inflammatory state modulate the cerebral

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effects of ammonia [37]. This study noted these effects were associated with raised serum levels of proinflammatory cytokines IL-6 and IL-1 beta, and TNF-alpha, an immuneregulating protein, during the active infection. This is supported by findings that in 100 cirrhotic patients admitted to liver intensive care, severity of HE was significantly associated with SIRS score (P 5 .03) and the more precise sequential organ failure assessment score (SOFA score, P 5 .006), but not serum ammonia levels, showing that the importance of these inflammatory markers in modulating severity of type C HE [38]. Furthermore, the impact of low-grade systemic inflammation may be important in clinically stable cirrhotics. These patients may have a degree of subclinical HE (often termed minimal or covert HE), detectable only by psychometric testing but without any clinical manifestations of overt HE as graded by the West Haven Criteria in Table 9.2. Minimal or covert HE may be present in 30%
84% of cirrhotics depending on the clinical definition used [39]. A study with 84 cirrhotic patients, either given an amino-acid challenge inducing hyperammonemia or placebo, demonstrated that hyperammonemia produced a significantly greater degree of neuropsychological deterioration in those with raised inflammatory markers at baseline, independent of the severity of liver disease or plasma ammonia level; these markers were also significantly higher in those with minimal HE at baseline [39]. This demonstrates that low-grade inflammation in cirrhosis is associated with neurocognitive changes, both alone and synergistically with ammonia. Evidence indicates that neutrophils in cirrhosis are also dysfunctional. Neutrophils from 29 patients with alcohol-related cirrhosis were compared to those from healthy controls; those from the former group showed “priming” in a resting state, with increased IL-8 (which induces chemotaxis of neutrophils and stimulates phagocytosis) and lactoferrin (which has antimicrobial activity) in their local environment. Stimulation with E. coli produced exaggerated degranulation compared to controls [40]. Furthermore, neutrophils from healthy volunteers incubated with ammonia in a concentration similar to that seen in a patient with cirrhosis show swelling, impaired phagocytic function, and increased spontaneous oxidative burst when compared to controls; these results are replicated in both rats fed either an ammoniagenic diet and in cirrhotic patients given an ammonia challenge. Activation of the p38 (MAPK) pathway was shown to be important in the development of neutrophil swelling and dysfunction [41]. This suggests that the potential combination of hyperammonemia and circulating dysfunctional neutrophils may predispose to infection and a systemic inflammatory state. There is also evidence that this interplay between ammonia and proinflammatory cytokines takes place within the brain. Rats given the proinflammatory LPS intraperitoneally showed an increase in TNF-alpha and IL-6 levels within the brain, which was associated with cerebral edema. However, only cirrhotic rats given LPS showed precoma signs analogous to HE in humans; these signs were not present in noncirrhotic rats fed a hyperammonemic diet, though plasma and brain ammonia levels were elevated in both these groups compared to control groups [24]. This supports ammonia and inflammation as being synergistic in the development of HE in cirrhosis, as well as providing a rationale for the propensity of infection to precipitate decompensation in CLD. In ALF, ammonia also interacts with components of the immune system in HE. Taylor and colleagues have found evidence that increased arterial ammonia concentration correlates with impaired neutrophil phagocytic activity [42]. Neutrophils express toll-like receptor-9 (TLR-9), which recognizes PAMPs. There is increased expression of TLR-9 receptors on neutrophils

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FIGURE 9.2 A general summary of processes potentially underlying hepatic encephalopathy. Systemic hyperammonemia leads to increased ammonia (NH3) within astrocytes, which is incorporated into glutamine. This may cause an osmotic imbalance and/or lead to mitochondrial dysfunction, leading to cerebral edema and HE in acute liver failure. Cirrhosis in chronic liver disease is associated with systemic inflammation. Damage-associated molecular patterns (DAMPs), released from damaged hepatocytes and pathogenassociated molecular patterns (PAMPs), due to translocation of bacteria and their byproducts across the gut wall stimulate production of proinflammatory tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), and interleukin-8 (IL-8); these contribute to neuroinflammation and astrocyte dysfunction. Neutrophils are dysfunctional in cirrhosis, being more sensitive to stimulation, further contributing to neuroinflammation through degranulation and the release of lactoferrin and reactive oxygen species (ROS) at the blood
brain barrier. HE, Hepatic encephalopathy.

with increased severity of HE [43]. Furthermore, injection of ammonium salts into a TLR-9 mouse knockout model prevented an increase in brain water seen in wild-type mice [44]. Thus in ALF, ammonia also acts in conjunction with the immune system to mediate HE. These processes are summarized in Fig. 9.2. A culmination of this evidence suggests that not only is it likely that ammonia synergistically interacts with background neuroinflammation to cause HE, but that high ammonia levels alone cannot explain HE in cirrhosis.

9.6 The gut microbiome in health and disease In health, the gut microbiome has been characterized by extracting and analyzing DNA from fecal samples of healthy patients from a variety of populations. The microbiome varies from person to person, and even differs between identical twins, but some microbial species

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are more predominant than others. One study found 75 species were common to .50% of individuals and 57 species were common to .90% [2]. Ninety-nine percent of the genes sequenced are bacterial in origin, with 0.1% being eukaryotic (notably fungi) or viral in origin and the rest archaeal. The most prominent species are generally Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria [2,45]. The gut microbiome has many critical roles in maintaining health. The best known is the fermentation of dietary fiber, producing gases and short chain fatty acids such as acetate, propionate, and butyrate. Acetate is used in lipogenesis and cholesterol metabolism pathways, as well as being a metabolite for bacterial growth. Propionate regulates gluconeogenesis in the liver, whilst both propionate and butyrate regulate gut hormones governing appetite, amongst other actions. Other microbial products include: trimethylamine, produced from dietary meat and dairy products, whose metabolite trimethylamine N-oxide is implicated in increasing cardiovascular risk; and indolepropionic acid, also produced from dietary fiber and which evidence suggests may reduce the incidence of type 2 diabetes mellitus [46]. Changes in the gut microbiome have been implicated in a number of chronic and autoimmune diseases. These include, but are not limited to: inflammatory bowel disease, celiac disease, obesity, type 1 and 2 diabetes mellitus, cardiovascular disease, and neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease. The precise characteristics of the gut microbiota in these chronic diseases differs, but all these diseases share the hallmark of a greater proportion of opportunistic and proinflammatory species such as Enterobacteriaceae and a reduced proportion of species with a net antiinflammatory action, such as Faecalibacterium and Akkermansia [47]. Overall the trend is that there is reduced diversity of species in patients with systemic disease [46]. Intestinal homeostasis is established and maintained by commensal microbiota, a functional barrier and a tolerant immune response. Traditionally, microbial “dysbiosis” has been proposed to refer to a change in the structural and/or functional configuration of gut microbiota, which causes disruption of gut homeostasis and has been associated with obesity and a variety of chronic diseases. Changes in the composition or density of the microbiota have been shown to increase the susceptibility for a variety of pathogens to “bloom” with abnormal mucosal immune responses in humans and murine models [48]. For the purpose of this chapter, we define microbiota “dysbiosis” to be a change in microbial populations that promote pathogen bloom and mucosal barrier damage, which adversely prime mucosal immunity.

9.7 Gut dysbiosis in cirrhosis The impact of the gut microbiome upon the pathophysiology of cirrhosis and HE is complex. Current understanding on the composition and function of the gut microbiome and how this relates to the progression and outcomes in patients with cirrhosis remains in its infancy and is based on descriptive snapshots afflicted by confounders and lacks robust clinical validation. Perturbations in the gut microbiome are a hallmark of cirrhosis, influence the rate of progression to liver failure [49], and drive the susceptibility to infection [50]. Cirrhosis is associated with gut dysbiosis, with a significant difference in gut microbiotic composition between patients with cirrhosis and healthy controls. A key difference is an increase in

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potentially opportunistic Enterobacteriaceae and Bacteroidaceae species and a reduction in autochronous and/or beneficial species such as Lachnospiraceae, Ruminococcaeae, Clostridium Cluster XIV, and Veillonellaceae [49,51]. This relationship can be quantified, with the latter group as the numerator and the former group as the denominator, as the cirrhosis dysbiosis ratio (CDR). One key finding is that the CDR decreases with liver disease severity. Bajaj et al. found that the CDR was higher in healthy control stool compared to patients with compensated cirrhosis, decompensated cirrhosis, and in those hospitalized. Correspondingly, a low CDR was shown to be associated with the development of organ failure within 30 days and increased mortality [49]. This observed dysbiosis can in part be explained by the finding that many of the underlying mechanisms that underpin the development of cirrhosis, notably with alcoholrelated and fatty liver disease, also lead to increased intestinal permeability and impaired mucosal homeostasis [52]. Dysbiosis generally develops in parallel with liver disease progression and the resultant development of PSS increases delivery of pathogenic bacteria and their products into the systemic circulation. This is supported by the fact that serum endotoxin levels are negatively correlated with CDR [49]. Patients with cirrhosis also have salivary dysbiosis associated with impaired salivary defenses and systemic inflammation. Salivary dysbiosis has also been shown to be greater in patients with cirrhosis who developed complications necessitating hospitalization within 90-days [53]. Treating the complications of cirrhosis with broad-spectrum antibiotics also contributes to gut dysbiosis, augmenting disruption to the normally symbiotic population of intestinal bacteria and potentially predisposing to further opportunistic infections and small bowel bacterial overgrowth [49,54,55]. Furthermore, Duan and colleagues have shown that E. faecalis can secrete a cytolytic exotoxin—cytolysin, which causes hepatocyte death. Fecal samples from patients with alcoholic hepatitis have about 2700-fold more E. faecalis than samples taken from healthy controls [56]. Eighty percent of patients with acute alcoholic hepatitis have increased abundance of E. faecalis and 30% produce cytolysin. A remarkable 89% of cytolysin-positive patients died within 180 days of admission, compared to only 3.8% of cytolysin-negative [56]. Bacteriophages (viruses which infect and replicate within bacteria and archaea) which specifically target cytolysin producing E. faecalis have been shown to abrogate alcohol-related liver injury in a mouse model [56]. This not only suggests a means of targeting pathogenic species within the enteric microbiome, but also indicates that the development of CLD, cirrhosis, and dysbiosis may be a reciprocal process, rather than a progression of dysbiosis, CLD, and cirrhosis along a set timeline.

9.8 Drug use in cirrhosis contributes to dysbiosis Cirrhotic patients’ predisposition to infection often leads to recurrent hospital admissions for antibiotic treatment. A single course of antibiotics can alter the composition of the microbiome, and in those with preexisting dysbiosis this effect is further exacerbated. One study showed that a single course of beta-lactam antibiotics significantly decreased microbial diversity by increasing the proportion of Bacteroidetes species, while doubling the microbial DNA load measured in fecal samples. Similarly, fluoroquinolone antibiotics appear to increase the proportion of Bacteroidetes, but do not affect microbial load [45]. Additionally, antibiotic-induced dysbiosis also leads to an increased presence of multidrug

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resistant organisms. One study of 252 fecal samples from three countries found an average of 21 resistance genes per sample; antibiotics used in animal husbandry and those in use for a greater number of years were associated with greater resistance [57]. The drastic impact of antibiotics on the gut microbiome leads to significantly reduced bacterial diversity, but also leads to a degree of fungal dysbiosis with more Candida species present [54]. Interestingly, the same study found that omeprazole, a widely used proton-pump inhibitor which reduces gastric acid secretion and is commonly prescribed to patients with cirrhosis, led to changes in bacterial but not fungal diversity, perhaps indicating symbiosis between bacterial and fungal species within the gut.

9.9 How dysbiosis drives systemic inflammation and HE There is much evidence examining how gut dysbiosis contributes to HE in CLD. Cirrhotic patients with HE have been found to have a greater abundance of Veillonellaceae in stool samples compared to cirrhotics without HE; an increased presence of this species was correspondingly associated with increased inflammatory markers IL-2, IL-13, and IL-23 [58]. Interestingly, the stool microbiota did not significantly differ between the HE and non-HE cirrhotic patients otherwise. The reasons for this were elucidated by a later study, where samples taken from the sigmoid mucosa showed a lower abundance of Roseburia and higher abundance of Enterococcus, Megasphaera, and Burkholderia as well as Veillonella species in patients with cirrhosis and HE compared to those without HE. Enterococcus, Megasphaera, and Burkholderia were also linked to raised serum cytokine and endotoxin levels. Additionally, Roseburia has been linked to maintenance of intestinal wall integrity [59]. Overall, this suggests that HE is at least in part driven by a further exacerbation of existing dysbiosis in these CLD patients; increased “leakiness” of the gut allows greater translocation of proinflammatory bacteria and their nitrogen containing products into the bloodstream, where PSS facilitates these reaching the brain. Lactulose has been shown to be effective for the prevention of recurrent episodes of overt HE. It is a nonabsorbable disaccharide and was initially introduced with the aim of speeding intestinal motility, aiming to reduce bacterial load within the gut. It passes unmetabolized to the colon where it is metabolized to acetate and lactate. This acidic environment promotes the conversion of ammonia (NH3) to ammonium (NH41), which is less readily reabsorbed from the colon and is excreted in the stool. There is also some evidence that acetate and lactate are used as an energy source by the microbiota resulting in an increased mass and capacity to metabolize ammonia; lactulose thus acts as a “prebiotic” by stimulating the growth of beneficial bacteria (c.f. probiotics, where a preparation of live microorganisms is delivered to confer a health benefit). Lactulose may also change the microbiome in a subgroup of patients with minimal HE who respond to treatment, with a lower abundance of proteobacteria [60], but does not appear to change in larger groups of cirrhotic patients [61]. A small meta-analysis of 9 trials by Zhao and colleagues shows that dietary probiotics can improve MHE and reduce progression from MHE to overt HE; notably 126 studies were excluded from this analysis due to poor or unclear methodology [62]. More specifically, Lactobacillus GG was well-tolerated and has been shown to increase CDR and reduce

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endotoxemia versus placebo in MHE patients; however, there was no significant difference in cognition between the two intervention groups [63]. This may be because probiotics cannot provide the mass of beneficial bacteria required to significantly alter dysbiosis for clinical benefit. Further research in this area is needed, as this therapeutic avenue may represent an acceptable and cost-effective means of reversing dysbiosis. The nonabsorbable antibiotic rifaximin has a robust evidence base to support an important role in manipulation of the gut microbiome. It has been shown to reduce systemic inflammation and therefore lowers morbidity and mortality not only in patients with HE, but also in those with advanced cirrhosis by impacting on other sequelae of cirrhosis such as susceptibility to infection, variceal bleeding and all-cause hospitalization [64]. Studies looking at changes in the gut microbiome pre- and pos trifaximin using 16s rRNA sequencing have failed to show dramatic changes in composition but rifaximin may change its function. It does significantly reduce serum endotoxin levels consequently improving cognitive function in minimal HE and significantly increases serum fatty acids and intermediates of carbohydrate metabolism [65,66]. This indicates that rifaximin, and by extension lactulose, may alter the mechanisms underpinning endotoxin production, reducing the pathogenicity of certain bacterial species. It is key to note that Type A HE has not yet been associated with dysbiosis and changes in the gut microbiome which may simply relate to the rate of onset of liver injury. The changes we have described so far entirely relate to decompensated cirrhosis. However, in ACLF there were similar changes to those described above for decompensated cirrhosis, with reduced abundances of Bacteroidaceae and Ruminococcaceae but increased abundances of Pasteurella and Enterococceae. Similarly, there was a reduced abundance of Lachnospiraceae in ACLF patients with HE, indicating similar mechanisms underlying the pathophysiology of these two conditions [67].

9.10 Directly altering the gut microbiome improves outcomes in HE It is therefore clear that existing treatments for HE, introduced entirely with the aim of reducing ammonia-producing gut bacteria, act in a more complex way by altering the gut microbiome in an antiinflammatory fashion. A more recent intervention, fecal microbiota transplantation (FMT), aims to target the gut microbiome directly. This involves the transplant of a filtered fecal sample from a vigorously screened healthy donor into the gastrointestinal tract of a patient. The aim of this is to correct dysbiosis and improve microbial diversity, and there is already evidence for its use in the treatment of severe and recurrent C. difficile resulting in it now being considered by many as standard of care [68], irritable bowel syndrome, and inflammatory bowel disease [69
71], and covered more extensively in the next chapter. Although only phase 1 pilot studies have been undertaken to date, FMT has shown great promise in treating HE. Bajaj et al. first showed that in a small trial of 20 patients, FMT improved cognitive function and led to less recurrence of HE, possibly by increasing butyrate-producing Lactobacillaceae and Bifidobacteriaceae. There was also an increase in Lachnospiraceaeae and Ruminococcaceae species post-FMT [72]. Furthermore, cirrhotic patients receiving FMT capsules enriched in the latter two species showed a resulting

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greater abundance in the two species in their own stool; this was correlated with reduced systemic inflammation (measured via IL-6 and LPS binding protein) and cognitive improvement [73]. Another case series conducted by Mehta and colleagues demonstrated a sustained clinical response after 20 weeks in 6 of 10 patients, with significantly lower arterial ammonia concentrations [74]. In a seminal study from Liu and colleagues, transplanting stool from human cirrhotics into cirrhotic mice led to significantly higher levels of inflammation in the brain, but transplantation of post-FMT human cirrhotic stool led to less neuroinflammation [75]. Interestingly, these clinical improvements were sustained, although changes in the gut microbiome were not long lasting, suggesting that the any reduction in neuroinflammation was driven by changes in the gut microbial function not composition [76]. As the composition of the donor sample varies, some donors (known as “Super donors”) have been found to produce FMTs with better outcomes than others [77,78], further showing that there is more to be elucidated about this means of treatment and the resident gut microbiome it acts upon.

9.11 Conclusion and future directions Overall there is a robust evidence base that the pathophysiology of HE is driven by changes in the intestinal microbiome in CLD. Current treatment of HE is very much focused on modulation of the gut microbiome, and this very likely underlies how these treatments function; future treatments may revolve around more targeted alteration of the gut microbiome. Additionally, this opens up an opportunity to explore changing the microbiome in other areas of the gastrointestinal tract in patients with cirrhosis and HE. For example, as salivary dysbiosis is associated with increased complications in cirrhosis [53], one potential therapeutic route may be altering this with better dental care; a small proof of concept study of 24 cirrhotic patients showed periodontal treatment improved systemic endotoxemia and cognitive function [79]. The biliary tree may also exhibit changes in the microbiome [80], potentially helping to explain the relatively poor outcomes post-liver transplantation for patients with primary sclerosing cholangitis and the favorable impact on post-transplant disease recurrence by performing pre-transplant colectomy [81]. It is clear that the microbiome is an area which shows great promise for improving outcomes in this patient group.

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C H A P T E R

10 The gut
liver
brain axis: dietary and therapeutic interventions Charlotte D’Mello1 and Mark G. Swain1,2 1

Snyder Institute for Chronic Diseases, University of Calgary, Calgary, AB, Canada 2Calgary Liver Unit, Division of Gastroenterology and Hepatology, University of Calgary, Calgary, AB, Canada

10.1 Introduction Chronic liver disease affects over 30 million people worldwide and can originate from a wide range of etiologies including viral hepatitis, autoimmune liver disease [primary biliary cholangitis (PBC), autoimmune hepatitis, and primary sclerosing cholangitis (PSC)], nonalcoholic fatty liver disease (NAFLD), and alcohol related liver disease. Regardless of etiology and disease stage, chronic liver disease is commonly associated with changes in brain function which manifest as behavioral alterations such as fatigue, cognitive dysfunction, sleep disturbances, loss of social interest (collectively termed sickness behaviors), and altered mood (i.e., depression and anxiety) [1,2]. These behavior alterations typically have no correlation with disease severity and can greatly impact patient quality of life and disease prognosis. Studies that have examined changes within the brain in chronic liver disease have primarily focused on the development of hepatic encephalopathy (HE), which is associated with cirrhosis and liver failure [3,4]. However, changes in brain structure and function are also evident in patients with chronic liver disease in the absence of cirrhosis [5,6]. Furthermore, since there are some common features thought to contribute to development of HE and central nervous system (CNS) changes in the absence of cirrhosis, such as increased microglial (resident CNS immune cells) activation and presence of systemic inflammation, including increased circulating cytokine [e.g., tumor necrosis factor α (TNFα) and interleukin 6 (IL-6)] levels [4,7], some similarity likely exists in the signaling pathways that contribute to cerebral changes in these patients. New insights continue to be gained from studies in animal models and patients with liver disease with regards to how the liver communicates with the brain to bring about changes in central neurotransmission and thereby behavior. Traditionally, neural and humoral pathways of communication have been most commonly implicated. However, in

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more recent years, a peripheral immune cell driven pathway mediated by monocytes has been highlighted in models of systemic inflammation including liver disease [8,9]. In addition, it is becoming increasingly evident that gut microbial dysbiosis can impact brain function and behavior [10]. The gut microbiome is a complex ecosystem, comprised mainly of bacteria but also viruses, protozoa and fungi, that can influence health and disease pathology. The microbial phyla dominant within the gut are Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Fusobacteria, and Verrucomicrobia. The Firmicutes and Bacteroidetes phyla represent 90% of gut microbiota, and beneficial health effects are associated with a decreased Firmicutes/Bacteroidetes ratio [11]. The gut microbial composition can vary along the intestinal tract. Factors such as environment, lifestyle, body mass index (BMI), age, ethnicity, medication, antibiotics, exercise, and diet can all modulate gut microbiome composition and/or function. Gut microbial species have multifaceted roles including digestion and metabolism, development and maintenance of the intestinal epithelial barrier and modulation of the host immune system. Growing evidence have outlined significant contributory roles for gut microbiota in the development and function of the CNS, including maintenance of blood
brain barrier (BBB) integrity, neurogenesis and microglia maturation and function [12]. Microbial metabolites such as short chain fatty acids (SCFAs), butyrate, propionate, acetate being the most abundant, which are derived from gut fermentation of dietary fibers, are important in maintaining intestinal integrity as well as in regulating inflammation and microglia homeostasis [13,14]. Deficits in serotonin (5-HT) neurotransmission, dysregulation of the hypothalamic-pituitaryadrenal (HPA) axis and intestinal barrier breakdown are some of the typical biological parameters associated with systemic inflammation and development of sickness behaviors and depression; all of which can be influenced by the gut microbiome [2,15]. Microbial dysbiosis has been implicated in the pathophysiology of several neurological conditions, including mood and anxiety disorders and chronic pain [5]. Changes in the composition or function of the gut microbiome resulting in altered metabolite levels of SCFAs, or alterations in bile acid (BA) metabolism have been documented in patients with chronic liver diseases and are observed in the precirrhotic stages, at a time when sickness behaviors or altered mood are already present [16]. In addition, the liver receives more than 70% of its blood supply from the gut via the portal vein and is constantly exposed to microbial metabolites, and thereby must balance maintaining tolerance to harmless antigens and mounting an immune response to harmful microbial molecules [3]. Therefore, gut microbial dysbiosis can impact gut-to-brain signaling and through alteration of the gut
liver axis could influence disease pathology during chronic liver diseases, which could also impact liver-to-brain signaling. This chapter will discuss how the gut
liver
brain axis influences brain function and behavior with a focus on observations made in patients without cirrhosis or liver failure, as well as some of the gut microbiomecentered and inflammation targeted therapeutic approaches that can influence this axis.

10.2 Routes of communication between periphery and brain Systemic inflammation has a profound role in modulating behavior during peripheral organ centered inflammatory diseases, and in the clinical expression of mood disorders [17].

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TNFα, IL-1β and IL-6 are three cytokines that have received considerable attention as mediators of this periphery-to-brain communication and elevated levels of these cytokines have been reported in patients with chronic liver disease [18]. They may affect the function of central adrenergic, serotonergic neurons (with altered metabolism of tryptophan), and the neuroendocrine system (mainly the HPA and hypothalamus-pituitary-thyroid axes). In particular, they are involved in the activation of the stress-related outflow: IL-1b can stimulate the secretion of corticotropin-releasing hormone (CRH), the key hormone in the body’s response to stress, and IL-6 and TNFα can up-regulate the HPA-axis function [19]. The communication pathway linking the liver, gut, and brain is thought to be generated via four main peripheral routes (Fig. 10.1). 1. Neural pathways: The visceral organs, via vagal neural pathways, can communicate interoceptive information regarding the body’s physiological state to the CNS, a process important for maintaining homeostasis. Vagal afferents can relay cytokine induced inflammatory signals to the CNS through cytokine receptor expression (e.g., IL-1R). In addition, macrophages are interspersed between distal vagal fibers and can be activated to produce cytokines during tissue injury [20]. Changes in activity in brain regions where afferent vagal and spinal interoceptive neural pathways converge have been observed, and correlate with adverse behavioral symptoms including fatigue [21]. However, since chronic inflammatory diseases are typically associated with prolonged low grade, fluctuating increases in tissue and circulating cytokine levels, it is likely that these neural communication routes play a more significant role in sickness behavior development in early or more acute and robust inflammatory responses. Furthermore, liver transplantation (denervates the liver) in patients with liver disease often does not significantly improve fatigue severity or health-related quality of life (HRQoL) in many patients [22]. 2. Humoral signaling via activation of cerebral endothelial cells (CECs): Chronic liver diseases are often accompanied by elevated levels of circulating cytokines such as TNFα, as well as increased numbers of circulating activated immune cells, including monocytes that produce TNFα. IL-1β and TNFα can interact with receptors expressed on CECs and activate signaling pathways that involve nuclear factor kappa B (NF-κB) activation and production of secondary messengers [e.g., prostaglandins (PGs) and nitric oxide (NO)] [23]. NO and PGs are capable of modulating behavior [24,25]. In mice with liver inflammation, inducible nitric oxide synthase (iNOS) expression was upregulated in CECs in response to systemic TNFα signaling, and was important in driving microglial activation and subsequently sickness behavior development [26]. Prostaglandin E2 (PGE2) is the PG subtype that has received the most attention for its capacity to mediate central effects in response to circulating cytokines [27]. PGE2 receptors are expressed in brain regions involved in emotional and behavioral control, including the hypothalamus and amygdala [28]. Moreover, therapeutic targeting of PGs and NOS have been explored in neuroinflammatory diseases, as well as psychiatric disorders [29,30]. Although CECs do not express IL-6 receptors (IL-6Rs), soluble IL-6Rs are generated and released into the circulation during many inflammatory conditions, including liver disease [31]. Importantly, sIL-6Rs and IL-6 can form complexes within the circulation. These circulating IL-6
IL-6R complexes can bind to glycoprotein 130, a

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FIGURE 10.1 There are four main peripheral pathways of communication that can relay inflammatory signals to the brain and promote changes in neural activity and behavior during liver inflammation. (1) Liver inflammation is associated with increased activation of circulating platelets and monocytes and increased circulating cytokine levels, including TNFα, IL-1β and IL-6. There are increased interactions between activated platelets (express P-selectin and TLR4) and monocytes to form monocyte-platelet aggregates (MPAs). Monocytes aggregating with platelets have enhanced TNFα production. (2) Vagal afferent nerves can relay interoceptive and inflammatory signals to the brain. (3) Circulating TNFα, IL-1 β, and IL-6
IL-6R complexes can interact with receptors on cerebral endothelial cells (CECs) and activate signaling pathways leading to the production of secondary messengers (e.g. NO). Circulating cytokines or immune cells can also propagate signals into the brain via circumventricular organs (CVOs), regions lacking an intact blood
brain barrier (BBB). (4) Inflammatory Ly6Chigh monocytes roll and adhere along activated CECs. Monocytes adherent to CECs play a key role in inducing signals that are propagated into the brain, promoting activation of microglia. (5) In response to enhanced CCL2/MCP-1 chemotactic signals, monocytes transmigrate into the brain. Infiltrating monocytes serve as a source of cytokines further contributing to elevated cytokine levels within the brain, and as a source of chemokines that drive further monocyte infiltration into the brain. (6) Neural activity changes occur in the basal ganglia, hippocampus, limbic structures and additional brain regions involved with interoception. Changes in neural activity can manifest as sickness behaviors and altered mood. (7) Microbial dysbiosis can lead to a leaky gut, thereby increasing circulating LPS or pro-inflammatory cytokine levels driving activation of circulating monocytes, platelets and subsequently microglial activation. Dysregulation of the gut
brain axis also leads to changes in brain function. BBB, Blood
brain barrier; CECs, cerebral endothelial cells; CVOs, circumventricular organs; IL-1β, interleukin-1β; IL-6, interleukin-6; LPS, lipopolysaccharide; MPAs, monocyte-platelet aggregates; NO, nitric oxide; TNFα, tumor necrosis factor-α.

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transmembrane component of the IL-6R complex found on CECs [32]. IL-6 signaling involves activation of Janus kinases—signal transducer and activator of transcription (STAT) signaling pathway which can induce adhesion molecule expression [23]. IL-6 signaling has been observed to mediate sickness behavior development in mice with liver inflammation [31]. 3. Circulating cytokine signaling via brain circumventricular organs (CVOs): CVOs are regions of the brain that lack an intact BBB and can thereby serve as an access point for circulating cytokines to induce changes within the brain as well as a route of leukocyte entry. Consistent with this, systemic IL-1β or TNFα administration induces expression of CCL2 (a potent monocyte chemoattractant), and TNFR within the CVOs [33,34]. Furthermore, in brain sections from mice with liver inflammation, increased numbers of circulating monocytes were found within the subfornical region (a CVO) of the brain [8]. 4. Monocyte-to-brain signaling: Under steady state conditions, monocytes can infiltrate the brain and influence brain function [35]. During pathological conditions, monocytes serve as a source of proinflammatory mediators that contribute to tissue damage but can also produce mediators that facilitate tissue repair and healing [36]. Systemic inflammatory pathologies, including liver disease, as well as mood disorders, are often accompanied by increases in circulating inflammatory monocytes and in numbers of monocyte-platelet aggregates (MPAs) [37,38]. Platelets can modulate monocyte phenotype and monocytes in aggregates with platelets show enhanced production of proinflammatory cytokines [39]. In mice with liver inflammation, a key role for circulating monocytes in relaying peripheral inflammatory signals to the brain and subsequent sickness behavior development has been demonstrated. In this model, along with a significant increase in numbers of circulating TNFα expressing inflammatory Ly6Chigh monocytes, there is a large proportion of Ly6Chigh monocytes rolling and adherent along CECs; however, this is not associated with transmigration of monocytes into the brain in earlier disease stages [26]. Importantly, the intimate interactions of monocytes with CECs played a key role in mediating the development of sickness behaviors, altered central neural excitability, activation of microglia and increased microglial expression of TNFα and chemokine (C-C motif) ligand 2 (CCL2) observed in these mice [26]. In addition, platelets were important for generation of the monocyte inflammatory phenotype since depletion of circulating platelets reduced numbers of circulating inflammatory Ly6Chigh monocytes, and decreased Ly6Chigh monocyte: CEC adhesive interactions in parallel with an improvement in sickness behaviors [40]. In mice with liver inflammation of longer duration, sickness behaviors were more pronounced and associated with further increases in monocyte: CEC adhesive interactions, as well as in numbers of activated CCL2 expressing microglia [8]. In addition, increased microglial activation and monocyte: CEC adhesive interactions were accompanied by enhanced infiltration of CCR2-(cognate receptor for CCL2) expressing monocytes into the brain [8,41]. Infiltrating monocytes expressed CCL2, which likely acts as a positive feedback mechanism to promote further monocyte recruitment into the brain, as well as local TNFα release, which in addition to being a neuromodulator, can also induce activation of microglia [42]. Furthermore, infiltrating monocytes were found predominantly in the motor cortex, hippocampus and basal ganglia regions; areas of the brain implicated in mediating sickness behaviors and altered mood [8]. The

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inhibition of microglial activation or cerebral monocyte infiltration resulted in significant improvement in sickness behaviors. A key role for monocytes in relaying peripheral inflammatory signals to the brain has also been reported in other systemic inflammatory settings. In a model evaluating behavior changes in response to stress, CCL2 driven cerebral monocyte infiltration was a central driver in the development of anxiety and depression-like behaviors [43,44]. Furthermore, in patients with major depressive disorder (MDD), where systemic inflammation is also felt to be a driver of altered mood, elevations in circulating TNFα levels and in numbers of IL-6 and IL-1β expressing inflammatory monocytes have been reported [45]. In addition, increased numbers of macrophages and CCL2 expression in the dorsal anterior cingulate cortex area (a brain region implicated in mood disorders) were documented in brain tissue obtained from suicide victims with depression [46].

10.3 Gut
brain axis dysregulation in liver disease Brain function and behavior can be greatly impacted by changes in composition or functioning of the gut microbiome resulting in gut dysbiosis (Fig. 10.2). Much of our understanding about how brain physiology can be modulated by gut microbiota has come from studies in germ-free animals (i.e., deficient in gut microbiome) [47]. Germ-free mice display learning and memory deficits as well as impaired BBB function. Gut microbiota can impact the gut
brain axis via neural, humoral, and immune-mediated routes [48]. Signaling via vagal afferents has been demonstrated in studies where behavior changes associated with microbial species ingestion is prevented in vagotomized mice. For example, Lactobacillus rhamnosus ingestion reduced stress-induced depressive-like behavior in mice; an effect prevented in vagotomized mice [49]. The gut microbiota can stimulate the release of peptides and hormones from gut enteroendocrine cells which can also have central effects. There are innate and adaptive immune cells present within the intestinal mucosa, as well as dendritic cells beneath the epithelium that continuously monitor gut luminal content [13]. Changes within the gut environment that result in activation of resident immune cells can lead to increased production of immune mediators (e.g., TNFα), which can decrease expression of tight junction proteins and increase intestinal permeability resulting in a “leaky gut” [50]. A “leaky gut” can facilitate passage of endotoxin and bacterial metabolites (e.g., SCFAs, which have neuroactive properties) into the circulation that can either modulate activity of circulating immune cells or affect CNS activity. In pathologies associated with altered mood, evidence for a “leaky gut” has been observed in parallel with activation of circulating mononuclear cells [51]. Moreover, monocytes have been highlighted as an important relay of gut microbiota-to-brain signaling [52]. Gut microbiota can influence monocyte numbers and function. For example, depletion of gut microbiota in adult mice resulted in reductions in circulating inflammatory Ly6Chigh monocytes and reduced hippocampal neurogenesis. Adoptive transfer of Ly6Chigh monocytes into gut microbiota depleted mice was sufficient to rescue the neurogenesis phenotype in the hippocampus [53]. Gut dysbiosis in liver disease can be facilitated by numerous factors, including impaired liver functioning resulting in increased circulating levels of metabolic byproducts such as ammonia that are typically cleared by the liver, or conditions such as reduced small intestinal transit time that can favor small intestinal bacterial overgrowth (SIBO) [3].

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FIGURE 10.2 Gut
liver
brain axis. In a healthy body, there is a balance between different gut microbial communities, contributing to physiological homeostasis between gut, liver, and brain. Age, body mass index, diet, medication, ethnicity, environment, and lifestyle can all impact gut microbiome composition and/or function. Gut microbiota produce metabolites such as SCFAs (e.g. butyrate) which are important for maintaining intestinal homeostasis and intestinal barrier integrity. Gut microbiota can also modulate tryptophan availability within the circulation, thereby altering brain 5-HT levels. Liver disease is associated with several changes that can result in microbial dysbiosis, including an increased risk of SIBO, decreased bile acid or SCFA production, decreased intestinal motility and decreased luminal pH. Changes within the gut environment can activate immune cells in the gut wall, leading to enhanced cytokine production (e.g. TNFα) and increased gut inflammation with an associated increase in gut barrier permeability. Decreased intestinal barrier integrity leads to a leaky gut which facilitates translocation of bacteria, bacterial products (e.g. LPS) and bacterial metabolites into the systemic circulation, contributing to systemic inflammation. Vagal afferents can relay interoceptive signals, regarding changes in gut and liver homeostasis, to the brain. Dysregulation of the gut microbiome
brain axis also leads to changes in BBB permeability and altered brain function. Common features seen within the brain in the setting of liver inflammation, as a result of gut
liver
brain dysregulation, include microglial activation, infiltration of monocytes and altered neurotransmission (5-HT, CRH, GABA/glutamate, dopamine). Microglial activation and infiltrating monocytes are a source of pro-inflammatory cytokines (e.g. TNFα, IL-1β, IL-6) within the brain that can mediate changes in neural activity. Treatments that target systemic inflammation (e.g. anti-TNFα therapy, anti-immune cell adhesion therapy), altered neural activity (e.g. antidepressants) or the gut microbiome (e.g. diet, probiotics, FMT) can result in significant improvement in sickness behaviors and mood. CNS, Central nervous system; CRH, corticotropin releasing hormone; FMT, fecal microbial transplantation; 5-HT, serotonin; LPS, lipopolysaccharide; SCFA, short chain fatty acids; SIBO, small intestinal bacterial overgrowth; TNFα, tumor necrosis factor-α.

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Patients with cirrhosis commonly show increased circulating ammonia levels, contributing to the development of HE. SIBO is not just a consequence of advanced liver disease since it is also evident in patients without cirrhosis [54]. For example, SIBO has been reported to occur in B33% of patients with early-stage PBC [55]. Cytokines can contribute to delayed intestinal motility which facilitates the development of SIBO, as reported in patients with ulcerative colitis where significant positive correlations between elevations in circulating inflammatory cytokines (including TNFα and IL-6) and SIBO were reported. This finding was likely due to delayed orocecal transit times seen in ulcerative colitis patients with elevated circulating inflammatory cytokine levels [56]. In the setting of increased intestinal permeability, the presence of SIBO can result in increased bacterial translocation across the intestinal barrier, which can promote systemic inflammation. In patients with mild HE, increased bacterial translocation, as measured by elevations in circulating bacterial DNA, was associated with increased circulating levels of TNFα and IL-6 in parallel with poor neurocognitive scores [57]. Toll-Like Receptor 4 (TLR4) is a pattern recognition receptor, expressed on a range of cells including monocytes and platelets, which is activated by bacterial ligands [e.g., lipopolysaccharide (LPS)] and endogenous ligands (e.g., heat shock proteins). In mice with liver inflammation, elevated circulating LPS levels have been documented [58]. In these mice, where platelets play a key role in inducing an inflammatory monocyte phenotype, TLR4 expression on platelets was important for mediating this effect. In addition, in mice deficient in TLR4, along with attenuations in monocyte: CEC adhesive interactions and sickness behaviors were also significantly improved [40]. BAs are heterogenous compounds, and alterations in BA synthesis or BA signaling can have consequences for host health. Dysregulation of BAs is evident in patients with the liver diseases PBC, PSC and NAFLD [59]. BAs are important regulators of the gut microbiome and can generate production of antimicrobial peptides via activation of the farnesoid X receptor (FXR) expressed on gut epithelium. Reductions in BAs can promote overgrowth of pathogenic bacteria including Enterobacteriaceae [60]. Furthermore, microbial species within the gut (i.e., 7-α-dehydroxylating bacteria) convert primary BAs into secondary BAs, which are more toxic to certain bacterial species. Patients with advanced cirrhosis exhibit reduced fecal BA levels in parallel with reductions in the presence of 7-α-dehydroxylating bacteria, including Ruminococcaceae and the Clostridium cluster XIV group, which normally drive SCFA production and maintain gut barrier integrity [3]. In treatment naı¨ve PBC patients, gut microbial dysbiosis was documented, with reduced species richness and alterations in abundances of 12 genera. Although treatment with Ursodeoxycholic acid for six months in PBC patients did not affect microbial diversity, it reversed the abundance of six genera [61]. In addition, the increased presence of two genera from the pathogenic Enterobacteriaceae family was associated with increased ability for bacterial invasion of epithelial cells. Bacteria from the Enterobacteriaceae family, including E. coli and K. pneumonia, are commonly reported to translocate into the mesenteric lymph nodes and cause bacterial infection in cirrhotic patients [57]. BA dysregulation has also been described in neurodegenerative diseases and could contribute to changes in brain function. Altered serum BA profiles were associated with changes in brain structure and glucose metabolism, as well as with biomarkers of Alzheimer’s disease pathology in a cohort of patients with early-stage Alzheimer’s disease or who were at risk for Alzheimer’s disease [62]. Furthermore, in patients with Alzheimer’s disease increased levels of the secondary BA deoxycholic acid were documented, along with a strong association of an increase in

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the ratio of deoxycholic acid: cholic acid with cognitive dysfunction [63]. In addition, patients that progressed from mild cognitive impairment to Alzheimer’s disease-associated dementia showed altered BA profiles. Significant associations were reported with BA profiles and select immune-related genes implicated in Alzheimer’s disease.

10.4 Changes in brain function and behavior in liver disease Microglia are a heterogenous brain cell population that can rapidly respond and adapt to changes in their environment. With the emergence of novel single-cell techniques such as cytometry by time-of-flight mass spectrometry (CyTOF) and single-cell RNA sequencing, the spatial, temporal and functional diversity of microglial subsets has become more evident. Specifically, microglia have been observed in both animal models and humans to take on different phenotypes/activation states during health, aging, and disease [64]. Different inflammatory settings have delineated the presence of microglia subsets with specific transcriptional profiles, as documented in models of neuroinflammatory pathologies and systemic inflammation [65,66]. Evaluating the functional importance of unique microglial subsets during systemic inflammatory diseases could have potential therapeutic implications for sickness behaviors. In addition to neuroinflammatory diseases, microglial activation is evident in patients with depression or chronic inflammatory diseases that are associated with a high incidence of mood disorders [67]. In mice with liver inflammation, increased microglial activation was evident in areas around blood vessels and ventricles (areas which serve as routes of leukocyte entry into the brain) and was also important for driving sickness behaviors. In patients with chronic liver diseases such as hepatitis C, and PBC with no HE, microglial activation has been documented using cerebral proton magnetic resonance spectroscopy (used to obtain information about cerebral biochemical metabolites), and positron emission tomography [68
70]. Furthermore, elevated choline levels (suggestive of cellular infiltration or glial proliferation) in the basal ganglia of patients with chronic hepatitis C virus (HCV) infection were associated with greater cognitive impairments [70]. Interoception refers to the sensing of the internal state of the body. How interoceptive information is communicated to the brain and subsequently integrated can have implications for behavioral processes and disorders [71]. The insula cortex and thalamus are involved in interoception processing. Altered neural activity within these regions has been reported during systemic inflammation and in patients with MDD, and correlated with fatigue levels and depression scores, respectively [21,72]. In a study with PBC patients with mild disease, reduced neural activity in the anterior insula was observed along with reductions in thalamic volume coupled with a trend toward reduced intrinsic regional neural activity [73]. These findings suggest a potential link between altered interoception in these patients and the high prevalence of cognitive dysfunction, fatigue, and altered mood states that are observed in these patients and are associated with dysfunction in these brain regions. Behavioral changes such as altered mood and cognitive impairment have also been linked to dysfunction of the brain’s limbic network. The hippocampus is a major component of the limbic system, and reductions in hippocampal volume are a common

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finding in patients with MDD, with recent studies highlighting more extensive changes within hippocampal substructures in patients with recurrent MDD [74]. The hippocampus is the only region of the adult brain involved in neurogenesis. It can be subdivided into subfields that perform specific functions, and reduced subfield volume has been reported in MDD patients [75]. Quantitative susceptibility mapping is an imaging technique that allows for in vivo quantification of tissue iron. Increased susceptibility is suggestive of increased iron deposition within a brain region (considered a marker of neuroinflammation), and this in turn has been linked to reduced cognitive ability [76]. Reduced hippocampal volume and increased hippocampal susceptibility were observed in PBC patients [77]. In addition, reductions in volume of specific subfields of the hippocampus, including the subiculum and dentate gyrus, regions involved in memory and/or regulation of the HPA axis, were also observed. Furthermore, these reductions in hippocampal volume in PBC patients were reported to be similar in magnitude to those documented in patients with MDD, however none of the PBC patients were depressed. Since the median disease duration was only six years for the group of PBC patients examined in this study, it is plausible that hippocampal changes occur in the earlier stages of the disease before the development of altered mood, which may be associated with more prolonged or advanced disease. Studies in PBC patients using cerebral magnetic resonance imaging (MRI) also demonstrated that changes within the brain are evident within 6 months of diagnosis [78]. Systemic inflammation is known to cause reductions in hippocampal volume. Since systemic inflammation is evident in both patients with PBC and MDD, this suggests a possible common underlying mechanism leading to reductions in hippocampal volume observed in these diseases. Dysfunction within the cortico-striatal network, as a result of changes in structure or neurotransmitter imbalance (e.g., dopamine), can lead to disruption of the normal balance linking motivational behavior/effort to perceived reward, and has been linked with development of central fatigue [79]. In support of this, a functional MRI (fMRI) study in patients with chronic fatigue syndrome demonstrated that reduced neural activation within the basal ganglia significantly correlated with increased fatigue [80]. Resting-state fMRI allows for the assessment of strength of functional neural connections between brain regions, whereby an increase in resting-state functional connectivity (rsFC) indicates stronger functional connections between brain regions. Using resting-state fMRI, altered functional connectivity between the basal ganglia, amygdala, and hippocampus was observed in PBC patients, compared to healthy controls [81]. Moreover, fatigued PBC patients exhibited stronger rsFC between the basal ganglia and the motor and premotor cortices, compared to nonfatigued PBC patients. The increased rsFC in fatigued PBC patients could suggest a compensatory mechanism of the brain in response to potential motor cortex dysfunction. Interestingly, structural changes in the basal ganglia of patients with PBC have also been linked with fatigue severity [82]. Although altered functional connectivity within the basal ganglia network has been documented in cirrhotic patients with HE [83]; only recently have such changes been investigated in PBC patients and are evident in the absence of cirrhosis. In addition to dopamine, the 5-HT and CRH neurotransmitter systems have also received much attention with regards to development of sickness behaviors and altered mood during systemic inflammatory diseases. Furthermore, 5-HT and CRH are closely

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intertwined with dopaminergic neurotransmission [84,85]. Low circulating levels of tryptophan, an essential amino acid required for the synthesis of 5-HT, is commonly associated with depression. Indoleamine 2, 3 dioxygenase (IDO) is an enzyme that can be activated by cytokines including TNFα and IFN-γ [86], and converts tryptophan to kynurenine (thereby making less tryptophan available for 5-HT synthesis). Kynurenine is further metabolized to kyneuric acid and quinolic acid. IDO, kynurenine and quinolic acid have all been implicated in the pathophysiology of fatigue and mood alterations [87,88]. In addition, robust associations between plasma TNFα levels and plasma and cerebrospinal fluid levels of kynurenine have been reported in patients with depression, and were related to depression severity [88]. Depression was a frequent side effect in the era of interferon-based treatment of patients with chronic HCV infection. Interferon may increase tryptophan degradation into kynurenine by increasing the activity of indoleamine 2, 3-dioxygenase, thus reducing the amount of tryptophan available for the synthesis of 5-HT by tryptophan hydroxylase and the aromatic amino acid decarboxylase [89]. Patients with chronic HCV infection show elevations in cerebrospinal fluid levels of kynurenine and quinolic acid which positively correlated with depressive symptoms [90]. Quinolic acid is mainly produced by microglial cells and can modulate glutamatergic neurotransmission. Increased density of quinolic acid positive microglia has been observed in the anterior cingulate cortex of depressed patients [91]. In mice with liver inflammation, changes in 5-HT neurotransmission are associated with a depressive phenotype. Mice with liver inflammation show altered central 5-HT1A receptor expression, an observation also seen in patients with MDD [92]. Clinically, altered 5-HT neurotransmission has been documented in chronic HCV infection patients with fatigue [93]. In addition, blockade of the 5-HT3 receptor with ondansetron improved fatigue in patients with chronic HCV infection [94]. However, 5-HT3 receptor blockade did not improve fatigue in PBC patients [95]. In addition to its role in modulating behavior, 5-HT has been reported to contribute to liver inflammation, including increased circulating TNFα levels [96], and is an important neurotransmitter within the gut where it regulates gut motility and gastrointestinal secretion [97]. Importantly, central 5-HT production accounts for just 5% of total 5-HT synthesis within the body, with the majority of 5-HT production occurring in enterochromaffin cells in the gastrointestinal epithelium. Gut microbiota plays a crucial role in tryptophan availability and metabolism, suggesting that alterations in the gut microbiota may contribute to the modulation of gut
brain signaling [98]. Male germ-free mice show elevated circulating tryptophan levels and increased hippocampal 5-HT turnover [99]. Tryptophan levels in germ-free mice are restored to baseline values following colonization with intestinal bacteria, demonstrating the influence of gut microbiota on tryptophan availability. Although CRH is classically associated with regulation of the HPA axis, it can modulate a broad spectrum of behaviors including anxiety, fatigue and appetite control [100]. Rats with liver inflammation show altered central CRH neurotransmission, as demonstrated by decreased hypothalamic CRH levels, increased CRH type 1 receptor expression and enhanced sensitivity to the behavioral activating effects of centrally infused CRH [101
103]. Polymorphisms in the CRHR1 and CRHR2 genes have been linked with MDD and are also associated with depression in patients with irritable bowel syndrome (IBS) [104,105]. Furthermore, CRH can regulate dopamine output within the brain resulting in alterations in motivational behavior and reward processing during stress [84].

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10.5 Therapeutics targeting gut
liver
brain axis Patients with liver diseases of different etiologies and at different disease stages are likely to experience different outcomes on disease pathology and extra-hepatic manifestations (e.g., sickness behaviors) with the use of therapeutics targeting inflammation (e.g., biologics targeting cytokines) or the gut microbiome. The following section discusses the beneficial impact of therapies targeting the gut microbiome, cytokines, and/or antiadhesion molecules, have had on sickness behavior development and altered mood in the setting of systemic inflammation and/or in patients with liver disease (mainly without cirrhosis).

10.5.1 Therapeutics targeting the gut microbiome 10.5.1.1 Diet The gut microbiome is significantly influenced by diet, which can also impact brain function [106]. The western diet, with high sugar and lipid content, is associated with increased risk for metabolic dysfunction and nonalcoholic steatohepatitis (NASH). The prevalence of NAFLD is estimated to be B20%
30% in western countries and is increasing worldwide, in parallel to the obesity epidemic and associated metabolic syndrome [107]. Mice fed a western diet showed increased liver inflammation, oxidative stress, and brain macrophage numbers, as well as decreased 5-HT transporter expression in the prefrontal-cortex area (a region associated with inhibitory behavior control), together with behavioral alterations including loss of motor coordination and increased depression-like behavior [108]. Diet can also influence the immune system. Specifically, diet was shown to induce distinct transcriptional responses in tissue-resident macrophages, including peritoneal and adipose tissue macrophages and hypothalamic microglia [109,110]. Typically, chronic inflammatory conditions associated with gut dysbiosis demonstrate reduced microbial diversity [111]. The effect of dietary changes on gut microbiota can occur in a matter of days. This was seen in subjects who consumed a plant- or animal-based diet for five days, where changes in microbial community structure and metabolic activity were evident within six days postdiet consumption [112]. Hence, several studies have examined the impact of nutritional supplements in ameliorating inflammation and improving behavior symptoms across a range of inflammatory diseases. 10.5.1.1.1 Polyunsaturated fatty acids

There is increasing evidence suggesting that supplementing diets with omega-3 (n-3) polyunsaturated fatty acids (PUFAs) has beneficial health effects [113]. Lipids are the building blocks of the CNS. PUFAs have multiple roles in brain structure and neurological functions, including regulation of food intake, analgesia and modulation of the endocannabinoid system (which has roles in appetite, pain, mood, and neuroinflammation) [114,115]. The two predominant PUFAs in the brain are n-6 arachidonic acid (AA) and n-3 docosahexaenoic acid (DHA). With regards to n-3 PUFAs, which also includes eicosapentaenoic acid (EPA), DHA makes up over 90% of the n-3 PUFAs in the brain and 10%
20% of total brain lipids. Linoleic acid (precursor of AA) and α-linolenic acid (precursor of DHA) are not synthesized de novo by mammals, and thereby are essential nutrients predominantly derived from diet. Sources of EPA and DHA include oils from fish, and sources of AA

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include dairy, eggs, and animal products. PUFAs and their lipid mediators can modulate inflammation and behavior. Lipid mediators derived from the C20 PUFAs, such as EPA, are called eicosanoids and include PGs, leukotrienes, and E-series resolvins. Those derived from the C22 PUFAs, docosapentaenoic acid (DPA), and DHA are called docosanoids and include D-series resolvins. Altered dietary intake of PUFAs and/or altered PUFA metabolism have been documented in a range of neuroinflammatory and mood disorders [114]. The mechanistic pathways that mediate the beneficial effects of PUFA supplementation are numerous and include reduction in proinflammatory cytokines (including TNFα, IL-1β, and IL-6), and modulation of neural activity. Fish oil supplementation in aged rodents attenuated inflammation-induced increase in IL-1β, IL-6, and TNFα levels in hippocampus, striatum, and prefrontal-cortex cerebral areas. In addition, fish oil supplementation reversed the changes in cerebral levels of 5-HT and kynurenine pathway derivatives, in parallel with improvements in sickness behaviors [116]. PUFA supplementation in patients with inflammatory bowel disease (IBD) reduces intestinal inflammation, including TNFα levels, decreases disease activity and can lead to clinical remission [117]. Clinical observations of an improvement in fatigue with n-3 PUFA supplementation have been documented in patients with chronic fatigue syndrome [118]. Deficiency in n-3 PUFAs results in a depressive phenotype in rodents [119]. In patients with MDD, n-3 PUFA adjuvant supplementation improved depression, anxiety, sleep, and regulation of emotions compared to treatment with selective serotonin reuptake inhibitor (SSRI) alone [120]. Roles for n-3 PUFAs in neurogenesis, learning, and memory have also been described. While studies in humans are limited and inconsistent, observations made in animal models have showed that supplementation with n-3 PUFAs (mainly DHA) improves aging-related decline in cognitive function as reflected by improvement in learning and memory function [121]. In addition, supplementation with EPA attenuated the age-related increase in hippocampal IL-1β levels and decreased microglial activation and neuronal death in parallel with restoration of long-term potentiation [122]. In adults in middle to late adulthood, four weeks of n-3 PUFA supplementation lowered circulating levels of cytokines including TNFα, IL-1β, and IL-6 [123]. Although most studies have reported combined effects of EPA and DHA supplementation, it is felt that some PUFAs might have more potent effects on sickness behaviors than others. In a subgroup of Alzheimer’s disease patients with very mild cognitive dysfunction, an improvement in cognitive function was attained with combined DHA and EPA supplementation, although DHA dosage levels used were higher than that for EPA [124]. In addition, in a study that directly compared the effects of DHA with EPA in participants with mild cognitive impairment (a prodromal state that could precede progression to Alzheimer’s disease), while both EPA and DHA supplementation improved depression, only DHA supplementation was associated with better cognitive outcomes [125]. Furthermore, a diet that provides a balanced ratio of n-6: n:3 PUFAs, felt to be around a 4:1 ratio of n-6: n-3 PUFAs, is associated with better health parameters. However, Western diets usually have an unbalanced n-6: n-3 PUFA ratio, with low levels of n-3 PUFAs and excessive levels of n-6 PUFAs (ratios as high as 15:1 for n-6: n-3 PUFAs), which is often associated with increased inflammation, obesity, diabetes, and atherosclerosis [115]. Interestingly, populations (e.g., Native Americans of Alaska) that primarily consume diets enriched in n-3 PUFAs have a lower incidence of these diseases [126]. N-3 PUFAs can also impact gut microbiome

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composition. High serum DHA levels positively correlated with increased presence of bacteria of the Lachnospiraceae family, which contain SCFA-producing species [127]. In drug naı¨ve type 2 diabetic patients, diets supplemented with sardines (as a source of n-3 PUFAs) resulted in beneficial changes in the gut microbiome with a decrease in the Firmicutes/Bacteroidetes ratio and an increase in the Prevotella genus [128]. Given the roles of PUFAs in modulating brain function (including documented effects on sickness behaviors such as fatigue) and impacting the gut microbiome and systemic inflammation, they could have a potential therapeutic role in symptom management in patients with liver disease. 10.5.1.1.2 Polyphenols

Since there are numerous reports of beneficial health outcomes with consuming diets rich in plant-based foods, there have been several studies investigating the effects of polyphenols on brain function. Polyphenols are a diverse family of phytochemicals that are characterized by their phenolic structural features and are abundant in vegetables, fruits, nuts, and seeds [129]. Flavonoids are a subgroup of polyphenols, comprising over 4000 different compounds, that have a broad range of properties including antiinflammation, antioxidation, and glucose regulation. Hence, flavonoid supplementation has been assessed in various disease models including diabetes, Alzheimer’s disease, obesity, aging, and IBD. Flavonoid use can also modulate the gut microbiome, gut
liver axis, and brain function [117,129
131]. Flavonoids, including curcumin and resveratrol, have improved disease activity, resulting in the achievement of clinical remission and improved quality of life in IBD patients [117]. Flavonoids can inhibit activation of transcription factors NF-kB and STAT-1, which are part of signaling pathways that regulate the activation of several inflammatory genes. In line with this, flavonoids have been documented to inhibit production of proinflammatory cytokines such as TNFα, IL-1β, and IL-6, chemokines like CCL2 and CCL5 (monocyte and T-cell chemoattractants) and enzymes such as iNOS, all of which have key contributory roles in mediating sickness behaviors during liver inflammation [130]. Anthocyanins are flavonoids that are mainly found in red and blue fruits and vegetables, and are associated with neuroprotection, including suppression of microglial activation, modulation of 5-HT neurotransmission, and reduction in inflammation [132]. Supplementation with anthocyanins improved anxiety, in parallel with a reduction in hippocampal levels of reactive oxygen species, TLR4 expression, and proinflammatory cytokine levels, including IL-1β [133]. With regards to sickness behaviors, anthocyanins have been primarily investigated for their effects on cognitive function. In older adults with cognitive issues or memory decline, supplementation with anthocyanins improved cognitive [134] and memory function [135]. In addition, supplementation with flavonoids also affects neural activity as demonstrated in patients with mild cognitive impairment, where pre- and postintervention fMRI during a working memory task was performed. Following 16 weeks of anthocyanin supplementation, increased blood oxygen level-dependent activity within the left prefrontal gyrus and left interior parietal lobe were observed, suggesting an enhanced flavonoid-induced cerebral effect [136]. Increases in cerebral perfusion and brain activity was also observed using fMRI (during cognitive task performance) 12 weeks postflavonoid supplementation, in areas such as the anterior cingulate and insula/thalamus (associated with cognitive processes including memory and executive function, as well as mood) [137]. Polyphenols can also affect the

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composition of the gut microbiome, with some being able to inhibit the growth of harmful bacteria such as E. coli and others being able to increase the presence of beneficial bacteria such as Lactobacillus spp., as well as improve the Firmicutes/Bacteroidetes ratio [129]. The effects of flavonoid-induced modulation of gut microbiome on inflammation and disease pathology was shown in a model of NAFLD, whereby mice on high fat diets were treated with the flavonoid quercetin for 16 weeks [131]. Quercetin treatment resulted in several positive outcomes on intestinal inflammation and liver inflammation. Specifically, quercetin treatment decreased liver steatosis and lipid peroxidation, and reduced hepatic levels of TNFα, IL-6, and TLR4. Quercetin also reduced the presence of harmful gut Proteobacteria, decreased the Firmicutes/Bacteriodetes ratio, and restored the high fat diet-induced decrease in production of SCFAs. In addition, quercetin restored gut barrier function with increases in intestinal tight junction proteins claudin-1 and occludin, and an increase in intestinal phosphatase alkaline. With restoration of gut barrier function, quercetin treatment also reverted the increase in levels of plasma LPS. Hence, the beneficial impact of polyphenols on the gut microbiome and intestinal barrier function as well as hepatic and systemic inflammation could have significant implications for the modulation of the gut
liver
brain axis and symptom management in patients suffering with liver disease. 10.5.1.1.3 Dietary patterns

Diets enriched in vegetable protein and plant fibers are associated with increased gut presence of beneficial bacterial species. The Mediterranean diet, characterized by high intake of fruits, vegetables, grains, fish, and low-to moderate intake of meat and dairy products, is the most extensively studied dietary pattern and has been described to have several beneficial health effects. The positive outcomes of Mediterranean diet adherence on management and severity of liver disease has been demonstrated in studies done with patients with NASH and NAFLD [138]. IBD patients that followed a Mediterranean diet for six months showed reductions in BMI, liver steatosis, disease activity, and levels of C-reactive protein in parallel with improvement in HRQoL [139]. Beneficial effects on behavior were reported in a metaanalysis of 22 studies, which showed that increased adherence to a Mediterranean diet reduced development of depression, cognitive impairment, and neuropathologies such as Alzheimer’s disease [140]. Patients with Crohn’s disease typically have reduced fecal microbial diversity, with reductions in species of Firmicutes and Bacteroidetes and corresponding increases in Proteobacteria and Bacillus species. In a pilot study in patients with Crohn’s disease, administration of a Mediterranean inspired antiinflammatory diet for six weeks resulted in trends toward reduced inflammation, as well as changes in the gut microbiome, as reflected by an increase in beneficial microbial species such as Bacteroidetes and Clostridium clusters, and a decrease in Proteobacteria and Bacillaceae populations [141]. In patients with liver disease, the impact of diet on disease related behavior symptoms has been mainly assessed in patients with HE. Given that our diet provides substrates that are metabolized by the gut microbiome, it would be of utmost importance to define a dietary intake containing the optimal balance of nutrients to be most beneficial to the patient. For instance, in cirrhotic patients with HE, low protein intake can have adverse effects and potentially result in protein breakdown from muscles, which could contribute to increased ammonia levels [4]. Studies conducted with diets enriched in dairy or vegetable or animal proteins have shown better outcomes with regards to symptom improvement with diets high in vegetable protein.

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Patients with overt HE (this form of HE was defined in chapter 8 and 9) that consumed a high-calorie, high-protein vegetable diet for a 14-day period showed improved cognitive performance and lower serum ammonia levels [142]. Furthermore, fungal dysbiosis is evident in patients with cirrhosis and impacts hospitalization risk [143]. Diet can greatly assist in rebalancing bacterial and fungal communities of the gut microbiota. A study that evaluated impact of a mycobiome diet (combines elements from Mediterranean, vegetarian, paleo, and low-carb diets that are associated with reduced pathogenic fungal growth) on the gut microbiome observed reduced pathogenic Candida species within two weeks and increase in beneficial bacterial species. In addition, the changes in gut microbial composition were also associated with improvement in digestive symptoms, fatigue levels, and sleep quality [144]. Diets supplemented with nutritional components such as vitamin-B have also been suggested to improve cognitive impairment [145]. Nonetheless, while we continue to gain a better understanding of how different nutrient sources modulate the gut microbiome and ultimately brain function, planning a diet that is tolerable by the patient and one that is palatable so as to increase patient’s compliance would be expected to be most effective. 10.5.1.1.4 Prebiotics

A prebiotic is a nondigestible dietary fiber selectively fermented by host microorganisms that cause alterations in the composition and/or activity of gastrointestinal microbiota that confer health benefits [4]. Prebiotics increase abundance of beneficial bacteria such as Bifidobacteria and butyrate-producing bacteria, including Roseburia inulinivorans and Fecalibacterium prausnitzii, which are beneficial in reducing gut luminal pH and maintaining intestinal integrity, intestinal motility, and sensitivity. High fiber diets have been reported to have numerous benefits in a variety of inflammatory disease settings, and one of the ways it is felt to mediate its beneficial effects is through increased production of butyrate [146]. Butyrate has also been shown to affect brain function and have immune regulatory roles. Butyrate induced an antimicrobial phenotype in intestinal macrophages as demonstrated by increased bacterial killing and reduced dissemination of pathogenic bacteria in mice treated with butyrate [147]. Furthermore, mice fed a diet high in soluble fibers showed reduced development of LPS-induced sickness behaviors in parallel with decreased cerebral IL-1β and TNFα production [148]. Different sources of fiber can result in different amounts of butyrate being produced, as seen when comparison of three high fiber diets demonstrated increased butyrate levels with diets high in fructooligosaccharides (FOS) or resistant starches [149]. Clinically, commonly used prebiotics include inulin, FOS, and lactulose. The use of lactulose in patients with HE has been extensively investigated and is associated with reduced progression to overt HE, improvement in cognitive function, reduction in blood ammonia levels, and improved HRQoL [3]. Lactulose treatment reduces circulating levels of TNFα, IL-6 and endotoxin in patients with mild HE [150]. In addition, lactulose-induced reductions in circulating bacterial-DNA levels in patients with mild HE improved neurocognitive scores [57]. In a multicenter trial with cirrhotic patients, lactulose treatment for 60 days modulated the gut microbiota to reduce gram negative species, including pathogenic Proteobacteria. In addition, the study also suggested that lactulose responders may have differences related to gut microbial composition and function [151].

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10.5.1.2 Probiotics Probiotics are live microorganisms that are commonly ingested to provide health benefits. Probiotic use has been described as a promising therapeutic for inflammatory gastrointestinal disorders such as IBD [117]. Studies evaluating the use of probiotics have reported beneficial effects on disease severity and/or behavior, either with the use of a specific microbial species or a combination of species. In the setting of liver diseases, probiotics have mainly been evaluated clinically for their effect on HE. Probiotic use has been reported to be beneficial in patients with mild HE, reducing hospitalization, improving cognitive dysfunction, and preventing progression to overt HE [152]. In addition to cognition, probiotic ingestion can have beneficial effects on a range of behaviors including mood, fatigue, and sleep quality, and can also affect neural activity within the brain [153,154]. Patients with chronic fatigue syndrome, a condition associated with an altered gut microbial profile, showed decreased anxiety after ingesting Lactobacillus casei for two months [155]. A pilot study done in patients with IBS found that within six weeks of Bifidobacterium longum ingestion, depression was reduced and accompanied with changes in activation in brain limbic regions (in amygdala and fronto-limbic regions) compared to placebo [156]. Changes in central neurotransmission and intestinal permeability, in parallel with a decrease in sickness behaviors, have been reported with probiotic usage, further strengthening the connection between gut microbiota and brain in the modulation of brain function. In a model of HE, Lactobacillus helveticus NS8 supplementation-mediated improvement in cognitive dysfunction was associated with reductions in cerebral levels of PGE2, IL-1β, and 5-HT [157]. In mice fed a high fat diet to induce NASH, treatment with Lactobacillus plantarum EMCC-1039 for two weeks improved cognitive deficits and decreased hepatic and hippocampal TLR4 expression [158]. In addition, a study examining the effect of a multiplex probiotic on cerebral changes associated with aging-related gut dysbiosis showed that probiotic treatment attenuated the decreases in intestinal barrier and BBB tight junction protein expression and decreased microglial activation, cerebral LPS levels and TLR4 expression [159]. Furthermore, along with improvements in fatigue and depression-like behavior in rodents after myocardial infarction, probiotic consumption also resulted in reversal of the increase in intestinal permeability seen in this model [160]. Probiotic use has been associated with changes in gut microbial diversity and reductions in system inflammation. For example, administration of Bifidobacterium infantis to patients with IBS resulted in symptom improvement, and a normalization of the IL-10 to IL-12 cytokine ratio in peripheral blood mononuclear cells [161]. VSL#3 is a probiotic preparation containing eight live, freeze dried bacterial species (Streptococcus salivarius subsp., thermophilis, Bifidobacterium [B. breve, B. infanti, B. longum], Lactobacillus acidophilis, L. planarum, L. casei, and L. delbrueeki subsp. Bulgaricus). In pouchitis patients, VSL#3 treatment which was effective in maintaining remission, was associated with increased and reduced bacterial and fungal diversity respectively, and shifted gut bacteria colonization toward anaerobic species (as documented by an increase in Lactobacillus and Bifidobacterium species) [162]. In association with its impact on gut microbial diversity, VSL#3 treatment also modulated gene expression in the brain, reversing long-term potentiation deficits associated with aging [163]. In the setting of liver disease, VSL#3 administration improves mild HE, enhances HRQoL, and reduces hospitalizations for HE recurrence in patients with cirrhosis [164,165]. Mice

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with liver inflammation show relative changes in beta diversity of the gut microbiome, as reflected by an increase in Bacteroidetes and a reduction in Firmicutes. VSL#3 treatment in mice with liver inflammation significantly improved fatigue-like behaviors [166]. This effect was independent of changes in the gut microbiota composition or in severity of liver injury, indicating that the reduction in sickness behaviors seen with VSL#3 treatment was not simply due to changes in liver inflammation. In addition, the improvement in sickness behaviors with VSL#3 treatment was associated with significantly reduced levels of circulating TNFα linked to decreased microglial activation, monocyte: CEC adhesive interactions and cerebral monocyte infiltration [166]. VSL#3 treatment-induced reductions in circulating TNFα levels and improved clinical neuropsychiatric outcomes have also been observed in patients with cirrhosis [165,167]. However, some studies fail to show any beneficial behavioral effect of probiotics possibly due to the strain and/or dosage used, or timing of intervention during disease course, highlighting the need for more randomized controlled studies to evaluate the behavior modulating effects of probiotic usage in liver disease. 10.5.1.3 Fecal microbial transplantation Clinically, fecal microbial transplantation (FMT) has primarily been used in and has been found to be effective for treatment of recurrent Clostridium difficile infection. However, FMT is being explored more widely as a potential therapy to normalize abnormal gut microbiome composition, including in IBD, neuroinflammatory diseases (e.g., multiple sclerosis) and in cirrhotic patients with HE [168]. Studies with FMT have provided further insights into contributory roles for gut microbial species in disease pathology and in the behavior manifestations of diseases. FMT from patients with depression into germ-free rats induced a depressive phenotype, in parallel to decreased hippocampal levels of neurotransmitters including 5-HT, dopamine, and norepinephrine, and increased levels of CRH and ACTH (suggesting HPA-axis hyperfunction), as well as an increase in serum levels of TNFα, IL-6, and IL-1β [169]. In addition, recipients of FMT from depressed patients showed increased mitochondrial damage in intestinal epithelial cells, consistent with observations that the prevalence of psychiatric illness is higher in diseases associated with mitochondrial dysfunction [170]. A link between gut microbiome, intestinal inflammation and behavioral manifestations in IBS patients was suggested in a study where colonization of germ-free mice with microbiota from IBS patients (with diarrhea) with high levels of anxiety, induced “anxiety-like” behavior in recipient mice. These changes were paralleled by intestinal barrier dysfunction and increased intestinal T-cell infiltration [171]. Recipients of FMT from healthy donors demonstrate changes in microbiome composition, modulation of inflammation and improvement in disease-associated behavioral symptoms. For example, FMT in patients with IBS improved quality of life and fatigue severity within six months [172]. In the setting of liver disease, FMT has not been widely explored, and challenging questions remain that need to be addressed, including determining the duration and number of required FMT administrations, as well as the optimal donor microbiome to treat different types of liver disease [16]. FMT has been shown to be beneficial in patients with recurrent HE, reducing hospitalizations, improving cognitive function, and increasing gut microbial diversity. Specifically, FMT increased the relative abundance of Lachnospiracea and Ruminococcaceae families which contain SCFA-producing bacterial species [173].

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10.5.2 Therapeutics targeting antiadhesion molecules and cytokines Biologic therapies that target antiadhesion molecules or cytokines, such as TNFα, have been extensively used to treat IBD and can reduce disease activity [174]. However, the impact of these therapies on brain function and behavior has received limited attention despite the debilitating effect symptoms such as fatigue and depression, have on patient quality of life. Moreover, the severity of symptoms such as fatigue often do not correlate with disease activity, and improvements in symptoms can be observed in the absence of a significant reduction in disease activity [175]. In patients with liver disease, studies examining treatment outcomes with antiadhesion or anticytokine therapies on HRQoL, sickness behaviors, and/or mood are very limited. 10.5.2.1 Antiadhesion therapies Immune cell: endothelium antiadhesion treatments improves symptoms and quality of life in patients with IBD, and in patients with neuroinflammatory diseases including multiple sclerosis [176,177]. Although there are limited clinical studies examining behavioral effects of systemic anti-P-selectin therapy, P-selectin mediates changes in brain function in animal models. In a mouse model of pilocarpine-induced seizures, P-selectin driven neutrophil: CEC adhesive interactions were important for mediating changes in central neural excitability [178]. In mice with liver inflammation, inhibition of P-selectin improves sickness behaviors while having no effect on liver disease severity. While antiadhesion treatments are typically used to block leukocyte infiltration into tissues, in mice with liver inflammation treatment with an anti-P-selectin antibody significantly inhibited monocyte: CEC adhesive interactions and monocyte-platelet interactions, leading to decreased microglial activation. Moreover, P-selectin targeting was also associated with a normalization of disease-associated changes in central neural excitability [26]. Furthermore, inhibition of P-selectin in mice with liver inflammation reduced the number of circulating MPA, and the induction of a proinflammatory monocyte phenotype as demonstrated by a reduction in circulating TNFα expressing inflammatory Ly6Chigh monocyte numbers. Increased circulating P-selectin positive platelets correlate with depressive symptoms in depressed patients and resolution of depression was associated with a significant decrease in circulating numbers of activated P-selectin positive platelets [179]. Very few studies examining the behavioral effects of systemic anti-P-selectin therapy have been reported in patients; however, antiadhesive therapies targeting α4 integrin have been employed to treat patients with a range of medical conditions. In mice with more pronounced liver inflammation accompanied by monocyte infiltration of the brain, peripheral treatment with a combination of anti-P-selectin and anti-α4-integrin antibodies prevented cerebral monocyte infiltration and attenuated sickness behavior development [41]. Natalizumab is a neutralizing antibody that targets the α4-integrin chain, blocking leukocyte: endothelial cell adhesive interactions in multiple organs, including the gut and brain. Patients with relapsing-remitting multiple sclerosis exhibit improvements in fatigue and cognitive function after treatment with natalizumab [176]. Moreover, natalizumab treatment reduces plasma levels of osteopontin, a multifunctional molecule found in high levels in cerebrospinal fluid of patients with cognitive impairment, that can stimulate production of proinflammatory cytokines and promote monocyte recruitment [180,181]. Elevated circulating osteopontin levels have been documented in liver diseases and contribute to liver injury and

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the development of cirrhosis [182]. In addition to fatigue improvement, natalizumab treatment has also been reported to improve depression and day-time sleepiness in patients with multiple sclerosis [183]. An in vivo translocator protein-PET imaging study in multiple sclerosis patients treated with natalizumab demonstrated reduced microglial activation one year after treatment in areas of the brain related to progressive multiple sclerosis pathology [184]. Natalizumab treatment is also beneficial in patients with IBD. Specifically, treatment of patients with Crohn’s disease resulted in improved HRQoL 12 weeks after starting therapy [177]. However, natalizumab treatment was associated with the development of a devastating neurological disease, progressive multifocal leukoencephalopathy. As a result of this severe side effect, antiadhesion antibodies targeting α4β7 integrin (i.e., Vedolizumab) were developed for IBD to capitalize on their gut specificity. Vedolizumab selectively targets the α4β7 integrin which binds to mucosal addressin cell adhesion molecule (MAdCAM)-1 selectively expressed on intestinal vascular endothelium. The efficacy and safety of Vedolizumab has been demonstrated in several clinical trials in patients with IBD. Vedolizumab treatment in IBD patients resulted in significant improvement in HRQoL within six weeks of treatment, at a time when disease activity scores had not significantly improved [185]. Vedolizumab treatment in IBD patients also improved sleep quality and depression within six weeks of therapy initiation, and these improvements were sustained for one year [174]. Vedolizumab treatment also mediates changes in the gut microbiome. In patients with Crohn’s disease, treatment with Vedolizumab for 14 weeks was associated with a decreased abundance of five gut bacterial taxa and striking differences in functional pathways associated with the microbiome (e.g., branched chain amino acid biosynthesis), consistent with Vedolizumab-induced reductions in colonic inflammation and oxidative stress [186]. Interestingly, in this study the presence/abundance of certain gut microbial species predicted clinical outcome in patients treated with Vedolizumab. In chronic liver diseases, the efficacy of Vedolizumab has been investigated only in patients with PSC and coexisting IBD, with mixed results [187]. A systematic review including 10 studies of patients with IBD, treated with Vedolizumab or biological agents targeting anti-TNFα or anti-IL12/IL23, found similar trends in gut microbiome changes. They reported that these treatments consistently increased microbiome diversity, with specific increases in SCFA-producing bacteria and decreases in relative abundance of Escherichia and Enterococcus species. In addition, IBD patients with higher microbial diversity at baseline typically showed increased rates of remission with biologic treatment [188]. Etrolizumab is a newer monoclonal antibody that is being tested in phase III trials in IBD patients [189,190]. It is also gut-selective, acting with a dual mechanism targeting both α4β7 and αEβ7 integrin. αEβ7 integrin is expressed mostly by gut mucosal lymphocytes and facilitates adhesion to E-cadherin on epithelial cells. It is also expressed on dendritic cells in the intestine. Hence, Etrolizumab can inhibit both lymphocyte recruitment and lymphocyte retention in the intraepithelial lining of the gut. The use of Etrolizumab in IBD patients achieves clinical remission. However, there are no reports to date of the impact of Etrolizumab on patient HRQoL. 10.5.2.2 Anticytokine therapies TNFα is a multifunctional cytokine that mediates a range of effects that can directly impact brain function (via modulating neurotransmission) and the gut (e.g., affecting intestinal barrier permeability) and modulate tissue inflammation (e.g., activate macrophages

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to induce cytokine and chemokine expression). Increased circulating levels of TNFα and elevated cytokine production (including TNFα, IL-1β, and IL-6) by circulating leukocytes are commonly documented in patients with chronic inflammatory conditions associated with a high prevalence of sickness behaviors and mood disorders [191]. Hence, blockade of TNFα signaling has been extensively evaluated in clinical studies across several different chronic inflammatory conditions [192]. In addition, improvement in symptoms by TNFα signaling inhibition are often evident in treated patients with chronic inflammatory diseases, prior to overt changes in disease activity [193]. In patients with IBD, anti-TNFα therapy was associated with significant improvements in sleep, depression, and anxiety within six weeks of initiation of therapy, and these improvements were sustained for one year [174]. Attenuation of peripheral TNFα signaling results in rapid effects on brain neural activity in parallel with improvement in symptoms. Twenty-four hours post anti-TNFα therapy, patients with rheumatoid arthritis reported reduced pain intensity mirrored by decreased activity in brain regions associated with pain perception (i.e., thalamus and somatosensory cortex), and those involved in interoception (i.e., insular cortex) [194]. In addition, changes in brain neural activity were evident in the absence of changes in clinical measures of disease activity. In another study, rheumatoid arthritis patients showed changes in neural activity in the prefrontal cortex and limbic areas of the brain within three days after anti-TNFα treatment [193]. Another study assessed the effect of anti-TNFα therapy on interoceptive signaling in patients with Crohn’s disease and demonstrated reductions in visceral sensitivity and improved cognitive-affective processing after antiTNFα administration, paralleled by improved sense of well-being [195]. Furthermore, changes in cognition were linked to changes in neural activity in prefrontal and limbic brain areas. Moreover, the rapid behavioral and neural changes observed were not associated with changes in fecal calprotectin levels (marker of gut inflammation) suggesting they were not likely related to reduced intestinal inflammation. Anti-TNFα therapy also alters the gut microbiome in patients with IBD. Specifically, anti-TNFα therapy in IBD patients increased gut microbial species richness and phylodiversity to levels similar to healthy controls [196]. Importantly, this increase occurred specifically in species that produce SCFAs. Furthermore, changes in inferred gut luminal bacteria metabolic interactions, involving butyrate and substrates involved in butyrate synthesis, were associated with therapeutic outcome following anti-TNFα therapy. Since not all patients respond favorably to treatment with biological agents, the above study suggests that metabolic profiling of fecal samples could potentially help identify patients who are more likely to achieve disease remission following anti-TNFα therapy [196]. For liver disease, beneficial effects of anti-TNFα therapy on markers of liver function have been observed in patients with autoimmune hepatitis, PSC-IBD, or PBC with concomitant rheumatoid arthritis [197
199]. In mice with liver inflammation, inhibition of peripheral TNFα signaled improved sickness behaviors. TNFα inhibition was also associated with blockade of key events, that is, monocyte: CEC adhesive interactions, microglial activation, and cerebral monocyte recruitment, important in driving sickness behavior development in mice with liver inflammation. Microglial activation and production of proinflammatory cytokines, including TNFα, are cardinal features in several neuroinflammatory diseases and therapies targeting microglial activation are associated with improvement in sickness behavior and depressive-like behaviors [200]. Minocycline is a broad-spectrum tetracycline antibiotic

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widely used clinically for its antiinflammatory and neuroprotective properties. In addition, minocycline has been reported to reduce sickness behavior development (e.g., fatigue) [201] and improve depressive symptoms [202]. Minocycline-induced improvement in depression was associated with changes in the gut microbiome. In a model of anxiety/depression, after 3 weeks of minocycline treatment, depressive-like behavior was attenuated and microglia density reduced in the prefrontal cortex, a brain region known to be affected in patients with MDD. In addition, minocycline treatment increased the relative abundance of Lachnospiraceae and Clostridiales Family XIII, which contain butyrate-producing bacterial species with a concomitant increase in plasma butyrate levels [203]. Interestingly, PSC patients treated with minocycline showed significant improvements in liver function as determined by serum alkaline phosphatase activity [204]. In mice with liver inflammation, minocycline treatment attenuated brain microglial activation in parallel with improved sickness behaviors and a reversal of altered central neural excitability exhibited by these mice [26]. In addition to TNFα, IL-6 is another multifunctional pleiotropic cytokine that can contribute to development of fatigue, sleep disturbances and altered mood. Increased circulating levels of IL-6 or increased numbers of inflammatory monocytes producing IL-6, are evident in patients with depression or fatigue [45]. Blockade of IL-6 signaling has been extensively explored in patients with rheumatoid arthritis where it has beneficial effects on sickness behaviors [205]. For example, anti-IL6R therapy in patients with rheumatoid arthritis resulted in a reduction of disease activity and improvements in fatigue, pain, and depression [206,207]. In mice with liver inflammation, IL-6 signaling also plays a key role in mediating sickness behaviors. In mice with liver inflammation with a genetic absence of IL-6, improvement in sickness behaviors were reversed after intravenous administration of recombinant IL-6 [31]. In addition, IL-6 deficiency prevented liver inflammation-related increases in hippocampal CEC p-STAT3 expression, highlighting an important role for circulating IL-6 in signaling the brain via CECs to generate sickness behaviors [31]. Since TNFα can induce IL-6 production, and activated macrophages are a source of both cytokines, interplay between these two cytokines likely contributes to the development of sickness behaviors/mood during liver disease.

10.5.3 Therapeutics targeting neural transmission 10.5.3.1 Antidepressants While the beneficial effects of antidepressants on mood are well recognized in patients with depression, they have also been reported to improve sickness behaviors (e.g., fatigue) [208]. A range of antidepressants, including selective 5-HT reuptake inhibitors, tricyclic, and monoamine oxidase inhibitors can reduce glial activation and levels of proinflammatory mediators, such as TNFα, IL-6, and iNOS [209,210]. In addition to modulation of cytokine production, antidepressants such as imipramine can also affect the HPA axis and hematopoiesis. In a model of stress where monocyte recruitment to the brain has a key role in promoting behavior changes, the observed increases in levels of plasma corticosterone, monocyte progenitors in the bone marrow and in circulating monocytes were prevented with imipramine treatment. In addition, imipramine administration also reduced microglial expression of TNFα and CCL2, as well as monocyte infiltration into the brain, in parallel with improved depression-like behavior [211]. Patients with depression show enhanced platelet activation (i.e., increase in P-selectin1 platelets)

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and increased numbers of platelet-leukocyte aggregates (including MPAs). Moreover, there was a positive correlation between severity of depression and level of platelet activation. After six months of treatment using psychotherapy and/or antidepressants, there was reduction in platelet activation that paralleled resolution of depressive symptoms [179]. Attenuations in microglial activation, platelet activation, and MPA formation reported in the aforementioned studies with antidepressant use also improves sickness behaviors in mice with liver inflammation. Antidepressants can also affect the gut microbiome. A study evaluating changes in the gut microbiome composition with a range of antidepressants indicated that antidepressant treatment in general reduced the abundance of Ruminococcus, Adlercreutzia, and an unclassified Alphaproteobacteria species. Furthermore, administration of a Ruminococcus species attenuated antidepressive effects in association with an upregulation of genes involved in mitochondrial oxidative phosphorylation, and downregulation of genes involved in neural plasticity in medial prefrontal cortical (brain region implicated in depression) region [212]. Patients with depression exhibit increased risk for developing IBD, and usage of antidepressants was found to protect against Crohn’s disease and ulcerative colitis development [213]. Depression is prevalent across a range of chronic liver diseases. In the setting of liver disease, antidepressant therapy has been mainly evaluated in cirrhotic patients where depression is associated with worse clinical outcomes and decreased HRQoL, compared to cirrhotic patients without depression [214]. Therapies that target 5-HT neurotransmission can be associated with improvements in both fatigue and mood. Administration of selective 5-HT reuptake inhibitor type drugs that increase cerebral 5-HT levels, improve depression in patients with chronic HCV infection [215]. A recent study in patients with PBC, identified that among all types of antidepressants assessed, mirtazapine (an atypical tetracyclic antidepressant) was the only antidepressant independently associated with decreased mortality and better hepatic outcomes [216]. Mirtazapine has antagonistic activity at a broad range of receptor subtypes including those involved in 5-HT and norepinephrine neurotransmission, making it widely used clinically to treat depression and other symptoms including anorexia and poor sleep quality [217]. In a model of concanavalin A induced liver injury, mirtazapine administration resulted in inhibition of early innate immune responses, including monocyte/macrophage activation and neutrophil recruitment, which are critical for inducing hepatitis in this model [218].

10.6 Concluding remarks Changes within the brain that manifest as sickness behaviors and altered mood are highly prevalent in liver diseases and greatly impact patient quality of life. Despite their prevalence, the pathways that lead to changes in brain function are poorly understood in the setting of liver disease and receive little attention. Moreover, these behavioral symptoms occur early in the disease stage, in the absence of cirrhosis, and have no correlation with disease activity. Systemic inflammation and microbial dysbiosis are common features shared between mood disorders, neuroinflammatory diseases, and peripheral organ inflammatory diseases including liver disease. In addition, microbial dysbiosis can also significantly impact liver disease pathology. Treatments that affect systemic inflammation, including biologics targeting the proinflammatory cytokines TNFα and IL-6, or gut specific

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antiadhesion molecules, are associated with improvement in quality of life and diseaseassociated behavior symptoms. There is a greater appreciation for the significant role of the gut
brain axis in modulating behavior during health and disease. Therapies that modulate the gut microbiome composition and/or functioning, including diet, FMT, and probiotics, can affect the development of sickness behaviors. Furthermore, treatments such as antidepressants that have been primarily associated with modulating neurotransmission and affecting mood, are also associated with an impact on systemic inflammation, gut microbiome, and sickness behaviors. However, most of the observations with regards to treatment-induced symptom improvement are made in nonliver disease patient settings, or in patients with severe liver function impairment and HE, stressing the need for more randomized controlled studies in patients with precirrhotic liver diseases. In addition, further studies are still required to gain a better understanding of patient disease characteristics that can guide therapy. For instance, the presence of certain biomarkers or microbial signatures might allow for better decision making in terms of which adjuvant therapies might result in improvement in sickness behaviors or mood in patients with different inflammatory liver diseases. With techniques such as high throughput 16SrRNA sequencing that allows for better characterization of the gut microbiome, it would be beneficial to identify microbial signatures that are associated with better outcomes with specific therapeutics. In addition, the identification of microbial signatures that are associated with an improvement in fatigue or depression may also aid in determining which dietary patterns could result in beneficial patient health outcomes. While it is evident that a balance between different gut microbial communities can be associated with better health outcomes, there are still gaps in our understanding of the contributory role of fungal communities within the gut microbiome to behavior. As we gain further insights into the intimate interactions between the gut, the liver, and the brain in patients with liver diseases, it will greatly assist in our understanding of symptom development and enable symptom management to be approached from a more holistic viewpoint.

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III. Liver-brain axis

Index Note: Page numbers followed by “f” and “t” refer to figures and tables, respectively.

A Abdominal pain, 12
13 in irritable bowel syndrome, 38
39 Acetate, 7
8, 196 ACLF. See Acute-on-chronic liver failure (ACLF) Acotiamide, 34 Acupuncture, 35 Acute liver failure (ALF), 188t Acute-on-chronic liver failure (ACLF), 162, 173
174, 188 Acute psychological stress, 24 Acute stress, 12 Adaptive immunity, 51 Adenylyl cyclase, 74
75 Adrenocorticotropic hormone (ACTH), 54 Afferent fibers, 4 Afferent nerve fiber, 18 Aging, 11 AhR agonists. See Aryl hydrocarbon receptor (AhR) agonists Akkermansia, 10, 196 Alcoholic liver disease (ALD), 161 ALF. See Acute liver failure (ALF) Algogenic visceral pain syndromes, 22 ALIOS diet. See American Lifestyle-Induced Obesity Syndrome (ALIOS) diet Alistipes species, 124
125 Allodynia, 25 Alosetron, 37
38 α 2 synuclein, 57 Altered gastrointestinal motility, in irritable bowel syndrome, 27 Altered 5-HT neurotransmission, 214
215 Alzheimer’s disease, 57
58, 219
220 American Lifestyle-Induced Obesity Syndrome (ALIOS) diet, 109 Amitriptyline, 33
34 Ammonia, 163, 163f, 191 of hepatic encephalopathy, 190 Amygdala, 18
19, 26, 207
209 Anatomical substrate, 12
13 Antacids, 33 Anthocyanins, 218
219

Anti-adhesion therapies, 223
224 Antibiotic therapy, 126
127 Antibiotic-treated gut microbiota, 57
58 Anticytokine therapies, 224
226 Antidepressants, 29
30, 33
34, 39, 83, 226
227 Antidiarrheal dugs, 37
38 Anti-inflammatory cytokines, 8 Antisecretory drugs, 33 Antispasmodics, 38
39 Anti-TNFα therapy, 223
224 Anxiety, 26, 28
29 Apical sodium
bile acid transporter (ASBT), 112
113 Area postrema, 70 Aryl hydrocarbon receptor (AhR) agonists, 9 ASBT. See Apical sodium
bile acid transporter (ASBT) ASD. See Autism spectrum disorder (ASD) Astrocyte-neuron-unit, 165 Astrocyte swelling, 162
163 ATLAS study, 115
116 Autism spectrum disorder (ASD), 55 Autonomic dysregulation, 22 Autonomic nervous system (ANS), 4, 17
18, 160

B Bacilli species, 147
148 Bacterial hydrolases, 112 Bacterial microbiome, 9
10 Bacteriophages, 197 Bacteroidaceae, 199 Bacteroides, 106 Bacteroides vulgatus, 51, 124 Bacteroidetes, 103, 161, 219
222 Bacteroidetes species, 197
198 Barbiturates, 164 Basal ganglia, manganese in, 164 BBB. See Blood-brain barrier (BBB) BCAA. See Branched-chain amino acids (BCAA) B cells, 52 Behavioral and psychological disorders, 77
78 Behavioral changes, 213
214 Beneficial taxa, 175
176 Benzodiazepines, 164, 170 Benzodiazepine-type sedatives, 163
164

237

238 Bidirectional brain
gut interactions, 17 Bifidobacteria, 28 Bifidobacteriaceae, 199
200 Bifidobacterium, 124
125, 147
148 Bifidobacterium bifidus, 31
32 Bifidobacterium dentium, 54 Bifidobacterium infantis, 36, 221
222 Bifidobacterium longum, 221 Bifidobacterium species, 19 Bile acids (BAs) receptors, 160
161 enterohepatic circulation of, 161
162 and metabolism, 112
113, 205
206, 212
213 as therapeutic targets in, 114
116 Biopsychosocial approach, 9
10 Biopsychosocial model, 81 Bloating, treatment of, 39 Blood
brain barrier (BBB), 3
4, 8, 191 Blood vessels, 69
70 Bowel habits disorders, 24 Brain, 5 ammonia, 163
164 connectome, 5
6 dysfunctions, 167 function, 210 modulation, 6 Brain Derived Neurotrophic Factor (BDNF), 29 Brain endothelial cells, 8 Brain
gut axis, 80 and psychobiota, 85 serotonin in, 80
82 Brain
gut
microbiota interaction, 50
53 immune pathway, 50
52 microbial metabolites, 52
53 microbial neural substrates, 54
55 microbiota and, 54 neuropsychiatric disorders by, 55
58 Alzheimer’s disease, 57
58 autism spectrum disorder, 55 depression, 56
57 Parkinson’s disease, 57 schizophrenia, 56 tryptophan metabolism, 53 vagus nerve, 52 Brain plasticity, 49 Branched-chain amino acids (BCAA), 170, 173 Brevibacterium lactofermentum, 54 Buspirone, 34 Butyrate, 8

C Campylobacter jejuni, 52 CAPS. See Chronic Abdominal Pain Syndrome (CAPS) Carbohydrate metabolism, 172
173

Index

Catecholaminergic system, 68 Caudal nuclei, 68 CBT. See Cognitive behavioral therapy (CBT) CECs. See Cerebral endothelial cells (CECs) Celiac disease, 59 Cell culture analysis, 57 Centrally active neuromodulators, 33
34 Central nervous system (CNS), 17, 50, 67
68, 80, 83, 160, 205 Central serotonin, 9 Cerebral endothelial cells (CECs), 207
209 Chemoreceptor trigger zone, 70 Child-Turcotte-Pugh Score (CTP Score), 169 Cholangiocytes, 141 Cholecystokinin receptors, 24 Cholesterol 7 alpha-hydroxylase (CYP7A1), 112 Chronic Abdominal Pain Syndrome (CAPS), 13 Chronic active disease, 12
13 Chronic cholestatic liver diseases, 161
162 Chronic exteroceptive, 59 Chronic fatigue syndrome, 214, 221 Chronic liver disease (CLD), 144, 187
188, 188t, 205, 207
209 and cirrhosis, 174
175 gut microbiota in metabolic liver disease, 123
125 on hepatic encephalopathy. See Hepatic encephalopathy natural course of, 136
140 Chronic liver failure, 172 Chylomicron, 107 Cilofexor, 115 Circumventricular organs (CVOs), 209 Cirrhosis, 160, 165, 169
170, 187
188 chronic liver disease and, 174
175 gut dysbiosis in, 196
197 medications in, 175
176 Cirrhosis dysbiosis ratio (CDR), 196
197 CLD. See Chronic liver disease (CLD) Clinical scoring systems, hepatic encephalopathy in, 169 Clostridium cluster XIV group, 212
213 Clostridium sporogens, 26 Cognitive and emotional factors, 26 Cognitive behavioral therapy (CBT), 31 Cognitive disorders, 76 Colonocytes, 7
8 Communication, 6 routes of, 206
210, 208f Computed tomography, 168 Connatural immune system, 50 Constipation-predominant IBS (IBS-C), 27 Cortico-striatal network, 214 Corticotrophin-releasing hormone (CRH), 19

Index

Corticotropin releasing factor (CRF), 4
5, 81 Cortisol, 85 Coryneform bacteria, 54 Covert HE (cHE), 162
163 CRF. See Corticotropin releasing factor (CRF) CRH. See Corticotrophin-releasing hormone (CRH) Crohn’s disease, 12
13, 219
220, 223
224, 227 CTP Score. See Child-Turcotte-Pugh Score (CTP Score) CyTOF. See Cytometry by time-of-flight mass spectrometry (CyTOF) Cytokines, 129
130, 210
212, 223
226 Cytometry by time-of-flight mass spectrometry (CyTOF), 213

D Daytime sleepiness, 168
169 Decarboxylases, 9 Dehydration, 169
170 De novo lipogenesis, 107
109 Deoxycholic acid, 112 Depression, 26, 56
57, 82 Depressive/anxiety disorders, 77
78 Deutsche Gesellschaft fu¨r Gastroenterologie, Verdauungsund Stoffwechselkrankheiten (DGVS), 172
173 DGBI. See Disorder of the Gut-Brain Interaction (DGBI) DGVS. See Deutsche Gesellschaft fu¨r Gastroenterologie, Verdauungsund Stoffwechselkrankheiten (DGVS) Diarrhea, 36
37 Diarrhea-predominant IBS (IBS-D), 27, 75 D-IBS. See Diarrhea-predominant IBS (D-IBS) Diet, 11
12, 169, 216
220 by intestinal permeability regulation, 101 in irritable bowel syndrome, 35
36 patterns, 219
220 polyphenols, 218
219 polyunsaturated fatty acids, 216
218 prebiotics, 220 probiotics, 221
222 Dietary fiber, 7
8 Differentiated step-by-step pharmacotherapy, 170
173 rifaximin, 170, 172
173 Diffusion tensor imaging (DTI), 159 Digestive and extradigestive systems, 67
71, 187 central nervous system, 68 5-HT receptors. See 5-hydroxytryptamine (5-HT) receptors serotonergic system in, 79
80 serotonin. See Serotonin Diltiazem, 31 Dipeptidyl peptidase-4 (DPP-4) activity, 107, 110
111 Direct intestinal damage, 3
4

239

Disorder of the Gut
Brain Interaction (DGBI), 18 Disorders of gut
brain interaction (DGBIs), 19 Docosahexaenoic acid (DHA), 216
218 Docosapentaenoic acid (DPA), 216
218 Dopamine, 54
55 “Double-track” system, 3 DPP-4 activity. See Dipeptidyl peptidase-4 (DPP-4) activity DTI. See Diffusion tensor imaging (DTI) Duodenal eosinophilia, 24 Dysbiosis, 3
4, 10
11, 100, 103
104, 124
125, 159
160, 162
163, 187, 196
197 in cirrhotic patients, 162 in colon, 172 drives systemic inflammation and, 198
199 drug use in, 197
198 of gut microbiota, 98 hypothesis of, 161 role of, 6f

E Efferent nerve fiber, 4, 18 Eicosanoids, 216
218 ELIVATE phase 2 trial, 116 Eluxadoline, 37
38 Emesis, 70 Emotional and functional disorders, 82 Emotional arousal system, 25 Emotional motor system, 18 Emotional nervous system, 19 Emotional stress, 81f Endocrinologic pathway, 53
54 Endogenous gut microbiota, 103 Endothelial cells, 138 Endothelial dysfunction, 139 Endotoxemia, 98, 162 Endotoxin, 99 ENS. See Enteric nervous system (ENS) Enteric nervous system (ENS), 4
5, 17
18, 79
80, 160 Enteric pathogens, 100
101 Enterobacteriaceae family, 162
163, 212
213 Enteroceptive signals, 4 Enterococcus faecalis, 126, 197 Enterococcus species, 223
224 Enterochromaffin cells (ECs), 6, 9, 54, 67, 130, 147f Enteroendocrine cells (EEC), 53 Enzymes, 9
10 Eosinophil, 24 Epigastric pain syndrome (EPS), 23 Epithelial barrier dysfunction, 99 Epithelium, 98 EPS. See Epigastric pain syndrome (EPS) Escherichia coli, 98

240 Escherichia species, 223
224 Esophageal hypersensitivity, 22 Ethanol-producing bacteria, 103 Etrolizumab, 223
224 Eubacterium rectale, 57
58, 124 European Medical Agency, 37
38 Excessive wound healing, 129 Extra-gastrointestinal comorbidities, 82
83 Extrapyramidal symptoms, 164

F FABP. See Fatty acid binding proteins (FABP) Faecalibacterium, 196 Faecalibacterium prauznitzii, 100 Faecalibacterium species, 124
125 Farnesoid X receptor (FXR), 113
114 Fatty acid binding proteins (FABP), 99 FCP. See Functional chest pain (FCP) FD. See Functional dysphagia (FD) Fecalibacterium prausnitzii, 220 Fecal/intestinal microbiota transplantation (FMT/ IMT), 175
176 Fecal microbial transplantation (FMT), 160, 199, 222 FEDs. See Functional esophageal disorders (FEDs) Fermentable Oligo-, Di-, Mono-saccharides, and Polyols (FODMAPs) diet, 35 FGIDs. See Functional gastrointestinal disorders (FGIDs) FH. See Functional heartburn (FH) Fibroblast growth factor (FGF)-19, 113
114 Fibrosis, 102, 105, 110, 124
125, 130, 141
142, 161 Fibrosis regression, 115
116 Firmicutes phylum, 124 First-line treatment, 32 in IBS-C, 36
37 5-HT receptors. See 5-hydroxytryptamine receptors 5-HTTLPR polymorphism, 77 5-hydroxy-L-tryptophan (5-HTP), 67
68 5-hydroxytryptamine (5-HT) receptors, 9, 67, 71. See also Serotonin arterial relaxation by, 70 classification of, 72t current classification of, 72 in extradigestive systems, 68
70 blood vessels, 69
70 central nervous system, 68 platelets, 68
69 5-HT1 receptor, 72
73 5-HT1A, 135 5-HT1B, 73, 135 5-HT1D, 73, 135 5-HT2, 73
74 5-HT2A, 135

Index

5-HT2B, 135 5-HT2C, 73
74, 136 5-HT3 receptors, 74, 136 5-HT3A c.-42T allele, 77 5-HT4 receptor, 74
75 5-HT5 receptor, 75
76, 136 5-HT6 receptor, 76 5-HT7 receptors, 76
77, 136 in gastrointestinal disorders, 133 in liver disease, 134f and serotonin transporter, 71
77 Flavonoids, 218
219 Flumazenil, 170 FMT. See Fecal microbiota transplantation (FMT) FMT/IMT. See Fecal/intestinal microbiota transplantation (FMT/IMT) FODMAPs diet. See Fermentable Oligo-, Di-, Monosaccharides, and Polyols (FODMAPs) diet Free fatty acids (FFA), 107 Freeze fracture technique, 97
98 Fructooligosaccharides (FOS), 220 Functional chest pain (FCP), 22 Functional dyspepsia (FD), 23, 31
35 alternative and nonpharmacological treatment, 35 alternative therapies and psychological interventions, 39 antisecretory drugs and antacids in, 33 centrally active neuromodulators in, 33
34 fundus relaxing drugs in, 34, 34t pharmacological treatment of, 32 prokinetics in, 34, 34t treatment algorithm in, 33f Functional esophageal disorders (FEDs), 20 pain modulators in treatment of, 29
31, 30t psychological interventions in, 31 treatment of, 28
31, 32f Functional food, 31
32 Functional gastrointestinal disorders (FGIDs), 3, 9
10, 19, 58
59, 77
78, 81 pathogenic mechanisms of, 18f Rome IV classification of, 21t serotonin in, 79
80 Functional GI syndromes, 17 Functional heartburn (FH), 22 Functional hepatocytes, 190 Functional magnetic resonance imaging, 25 Functional polymorphism, 77 Fundus relaxing drugs, 34 Fungal dysbiosis, 219
220

G GABA. See γ
Aminobutyric acid (GABA)

Index

GABA ergic neurons. See Gamma aminobutyric acid (GABA)ergic neurons GABA receptors. See Gamma aminobutyric acid (GABA) receptors Galanin, 54 γ 2 Aminobutyric acid (GABA), 164 Gamma aminobutyric acid (GABA)ergic neurons, 74
75 Gamma aminobutyric acid (GABA) receptors, 52 Gastric emptying (GE), 23 Gastroduodenal inflammation, 24
25 Gastroduodenal motility, 23 Gastroduodenal sensitivity, 24 Gastroesophageal reflux disease (GERD), 20, 79 Gastrointestinal disorders, 58
59, 160, 221 anatomo-physiological background, 17
19 clinical presentation of, 19
28 gastroduodenal inflammation and mucosal permeability, 24
25 gastroduodenal motility, 23 gastroduodenal sensitivity, 24 impaired gastric accommodation, 23
24 irritable bowel syndrome. See Irritable bowel syndrome depression and, 82 5-HT in, 133 functional. See Functional gastrointestinal disorders functional dyspepsia, 31
35 general and dietary recommendations, 31
32 functional esophageal disorders. See Functional esophageal disorders motility disorders, 79
80 other pain modulators, 31 serotonin and. See Serotonin therapy of, 28
39 functional esophageal disorders, 28
31 Gastrointestinal dysfunctions, 56 GBA. See Gut
brain axis (GBA) Genetic factors, 25 GERD. See Gastroesophageal reflux disease (GERD) Germ-free (GF), 5, 49 Ghrelin, 54 GIP. See Glucose-dependent insulinotropic peptide (GIP) GLP-1. See Glucagon-like peptide-1 (GLP-1) GLP-1/GIP unimolecular coagonist, 111
112 GLP-1 receptor agonists, 110 Glucagon-like peptide-1 (GLP-1), 106
108 Glucagon receptors, 112 Gluconeogenesis, 108 Glucose, 113
114 Glucose-dependent insulinotropic peptide (GIP), 106
107

241

Glucose transporter type 2 (GLUT2), 107 Glutamine, 190 Glycogenolysis, 107 GPCRs. See G protein-coupled receptors G protein-coupled receptors (GPCRs), 116 Gram-negative Proteobacteria, 124 Gram-positive bacteria, 126
127 GS-9674, 115 Gut
brain axis (GBA), 3, 80 alterations in, 3 anatomical entity, 4
5 dysfunction, 3, 19 functional entity, 5
10 functional gastrointestinal disorders of, 21t to function properly, 19 gastrointestinal disorders. See Gastrointestinal disorders microbiota role, 5
10 pathological entity, 10
13 peripheral and central alterations in, 17 perturbations in, 10 Gut connectome, 5
6, 6f Gut dysbiosis, in cirrhosis, 196
197 Gut
liver axis, 106, 147
148, 205
206 Gut
liver
brain axis, 211f dysregulation in liver disease, 210
213 in health and liver disease, 160
162 intestinal mucosa, 97
98 periphery and brain, 206
210 in progression of liver disease, 160
162 therapeutics targeting, 216
227 Gut microbiome, 205
206, 216 in health and disease, 195
196 therapeutics targeting, 216
222 diet, 216
220 fecal microbial transplantation, 222 target antiadhesion molecules, 223
226 Gut microbiota, 5, 6f, 7
9, 19, 50, 100, 143
145, 160
161 in chronic liver diseases metabolic liver disease, 123
125 primary sclerosing cholangitis, 125
127 dysbiosis, 98 in irritable bowel syndrome, 27 hormones on, 20t intestinal permeability regulation by, 100
101 in irritable bowel syndrome, 36 modulating factors of, 11f and nonalcoholic fatty liver disease, 103
105, 104t predominant genera of, 50 serotonin and, 145
148 Gut microbiota, in hepatic encephalopathy ammonia as driver of, 190

242 Gut microbiota, in hepatic encephalopathy (Continued) defining, 189 drug use in cirrhosis, 197
198 dysbiosis drives systemic inflammation and, 198
199 drug use in, 197
198 HE classification by cause of hyperammonemia, 190
193 outcomes in, 199
200 precipitating factors for, 192t processes potentially underlying, 195f role of inflammation, 193
195 types of, 191f West Haven criteria for, 189t Gut motility, 70
71 Gut physiology emesis, 70 gut motility, 70
71 immune system, 71 secretory functions, 70

H HANS. See Hypothalamic
autonomic nervous system (HANS) Health and liver disease, gut
brain axis in, 160
162 Health-related quality of life (HRQOL), 159 Helicobacter hepaticus, 51 Helicobacter pylori, 31
32, 57
58 Hepatic encephalopathy (HE), 159 aspects of, 162
163 astrocytes in, 163f in cirrhosis, 165 clinical relevance and presentation of, 165
169 classification, 165
166 diagnostic tests, 166
168 in clinical scoring systems, 169 differentiated step-by-step pharmacotherapy, 170
173 branched-chain amino acids, 173 lactulose (nonabsorbable disaccharides), 172 L-ornithine L-aspartate, 173 rifaximin, 170, 172
173 and fitness to drive, 168 in gut microbiota. See Gut microbiota, in hepatic encephalopathy intensive care aspects of, 173
174 lactulose, 170 pathogenesis of, 159, 162
165 dysbiosis, 162
163 neurotoxins, 163
164 neurotransmission, impairment of, 164 proinflammatory mechanisms, 164 sarcopenia, 165

Index

shunting, 165 posttransjugular intrahepatic portosystemic shunts, 174 prevention of overt, 174 primary drug prophylaxis of, 174 and quality of life, 168 relevance of, 168
169 secondary prophylaxis of, 174 and sleep disorders, 168
169 treatment options, 169
174 diet, 169 precipitating factors, 169
170 shunting, 170 triggering causes and precipitating factors of, 171t Hepatic fibrogenesis, state of art, 129
130 Hepatic glucose, 107
109 Hepatic injury, 160
161 Hepatic steatosis, 109
110 Hepatic stellate cells (HSCs), 129
130 Hepatocellular carcinoma, 141
143 Hepatocytes, 107, 108f Herbal preparations, 35 Heterogenous compounds, 212
213 HFCD. See High-fat content diets (HFCD) Hierarchic integrative system, 18 High-fat content diets (HFCD), 101
102 Hippocampus, 213
214 Homeostasis (eubiosis), 10
11 of whole system, 6
7 Hormones, 53
54 on gut motility, 20t HPA. See Hypothalamic-pituitary-adrenal (HPA) H2RA. See H2 receptor antagonists (H2RA) H2 receptor antagonists (H2RA), 33 HRQOL. See Health-related quality of life (HRQOL) HSCs. See Hepatic stellate cells Human arterial vasoconstriction, 69
70 Human cardiovascular system, 69
70 Human 5-HT1A receptor, 73 Human 5-HT7 receptor, 76 Human recombinant 5-ht5A receptors, 75
76 Hydroxysteroid dehydrogenases, 9
10 Hyperalgesia, 82 Hyperammonemia, 159, 163
164, 168
169, 190 in brain, 191
193 hepatic encephalopathy classification by cause of, 190
193 Hypnotherapy, 31, 35 Hypokalemia, 169
170 Hyponatremia, 169
170 Hypothalamic
autonomic nervous system (HANS), 26, 85 Hypothalamic
pituitary
adrenal (HPA), 4
5, 80

Index

Hypothalamus, 19, 53
54, 207
209 Hypothalamus
pituitary
adrenal (HPA), 18
19, 49

I IBS-C, first-line treatment in, 36
37 IL-6 signaling, 226 Immune pathway, 50
52 Immune system, 71 Immunoglobulin A (IgA), 52 Impaired gastric accommodation, 23
24 Incretins, in NAFLD, 106
112 co-agonists role, 111
112 on hepatic glucose and lipid metabolism, 107
109, 108f and NASH, 109
110 physiological effects of, 106
107 Indirect intestinal damage, 3
4 Indole metabolites, 9 Inflammasomes, 51 Inflammatory bowel diseases (IBD), 3, 12
13, 79 Inflammatory cytokines, intestinal permeability by, 99 Inhibitory control test, 175
176 Innate immunity, 50 Insomnia, 168
169 Insula cortex, 213 Insulinotropic effect, 106
107 International Society of Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN), 166 Interoception, 213 Intestinal barrier function, liver homeostasis on, 106 Intestinal commensal bacteria, 52 Intestinal epithelium, 97 Intestinal homeostasis, 196 Intestinal lipid absorption, 107 Intestinal microbiota, 123
124, 143, 159 Intestinal mucosa regulation, 97
98 therapeutic potential of, 99 Intestinal permeability regulation, 97
101 by diet, 101 epithelial barrier dysfunction, assessment of, 99 by gut microbiota, 100
101 by inflammatory cytokines, 99 intestinal mucosa, 97
98 leaky gut, 98
99 pathogenesis of NAFLD, 101
106 Intestinal serotonergic system, 80 Intracellular ammonia, 191 Irritable bowel syndrome (IBS), 3, 5
6, 24 abdominal pain in, 38
39 algorithm in, 36f altered gastrointestinal motility in, 27 diet and physical activity in, 35
36 gut microbiota dysbiosis in, 27

243

immune activation and mucosal permeability in, 28 modulators of gut microbiota in, 36 postinfection, 28 with predominant constipation, 36
37 treatment of, 35
39, 37t treatment of bloating, 39 trigger factors in, 13 visceral hypersensitivity in, 25
27 ISHEN. See International Society of Hepatic Encephalopathy and Nitrogen Metabolism (ISHEN)

J Junctional adhesion molecules (JAMs), 98

K Kupffer cells (KC), 102, 139 Kynurenine, 53, 131, 214
215

L Lachnospiraceae, 199 Lachnospiraceaeae species, 199
200 Lactitol, 172 Lactobacillaceae, 199
200 Lactobacillus, 28 Lactobacillus brevis, 54 Lactobacillus casei, 221 Lactobacillus gasseri, 31
32 Lactobacillus helveticus NS8, 221 Lactobacillus plantarum EMCC-1039, 221 Lactobacillus reuteri, 55 Lactobacillus rhamnosus, 52, 210 Lactobacillus species, 19, 51, 218
219 Lactulose (nonabsorbable disaccharides), 170, 172, 174
176 LEAD-2. See Liraglutide Effect and Action in Diabetes2 (LEAD-2) Leaky gut, 3
4, 57, 98
99, 210 in progression of NAFLD, 105
106 Licogliflozin, 116 Limbic system, 12
13, 18 Linaclotide, 36
37 Lipid metabolism, 107
109 glucose and, 113
114 Lipids, 24 and triglyceride, 109 Lipopolysaccharide (LPS), 52
53, 98 Lipopolysaccharide endotoxins (Proteobacteria), 10 Liraglutide Effect and Action in Diabetes-2 (LEAD-2), 109
110 Lithocolic acid, 112 Liver cirrhosis, 162, 176 Liver disease

244 Liver disease (Continued) changes in brain function and behavior in, 213
215 gut
liver
brain axis dysregulation in, 210
213 Liver fibrogenesis, 138, 140
141 Liver fibrosis, 129
130, 136 Liver homeostasis, on intestinal barrier function, 106 Liver inflammation, 105 Liver sinusoidal endothelial cells (LSECs), 138 Liver transaminases, 109 Liver transplant (LT), 173
174 LOLA. See L-ornithine L-aspartate (LOLA) Long-chain fatty acids, 172
173 Loperamide, 37
38 L-ornithine L-aspartate (LOLA), 170, 173 Low-grade systemic inflammation, 194 LT. See Liver transplant (LT) Lymphocytes, 51

M Macrophages, 51 MAFLD. See Metabolic associated fatty liver disease (MAFLD) Manganese, in basal ganglia, 164 MCD. See Methionine-and-choline-deficient (MCD) Mediators, 25 Medications, in cirrhosis and microbial changes, 175
176 fecal/intestinal microbiota transplantation, 175
176 probiotics, 175 Mediterranean diet, 219
220 Metabolic associated fatty liver disease (MAFLD), 123
124 Metabolic diseases, 100 Metabolic liver disease, 123
125 Metabolic syndrome, 101, 105 Methanobravibacter smithii, 27 Methanogenic microbes, 27 Methionine-and-choline-deficient (MCD), 106 Microbial diversity, 50 Microbial metabolites, 52
53, 205
206 Microbial neural substrates, 54
55 Microbial species, 219
220 Microbial structure, 19 Microbiome, 124, 126, 143, 160 Microbiome-derived metabo, 51 Microbiota, 5, 9, 19, 161 bioconversion of dietary products, 7f gut. See Gut microbiota role of, 5
10 serotonin and, 83
84 Microglia, 50
51 MicroRNAs (miRNAs), 75 Minimal HE (mHE), 166
167 diagnostic methods in, 167t therapy, 170

Index

Minocycline, 225
226 Mirtazapine, 33
34, 227 Mitochondrial permeability transition (MPT), 192 Monoamine oxidases (MAO), 132 Monocyte-to-brain signaling, 209
210 Monotherapy, 174 MPT. See Mitochondrial permeability transition (MPT) Mucosal addressin cell adhesion molecule (MAdCAM)-1, 223
224 Mucosal immune system, 98 Mucosal permeability, 24
25 in irritable bowel syndrome, 28 Mucus, 6
7 secretion, 70 Muscle wasting, 165

N NAFLD. See Nonalcoholic fatty liver disease (NAFLD) Natalizumab, 223
224 Na 1 -taurocholate cotransporting polypeptide (NTCP), 112
113 NCCP. See Noncardiac chest pain (NCCP) Necroinflammation, 123
124 Neural processes, 5 Neuroendocrine system, 83 Neuroinflammation, infections and, 164 Neurological disorders, 4
5 Neuropsychiatric disorders, 55
58 autism spectrum disorder, 55 depression, 56
57 Parkinson’s disease, 57 schizophrenia, 56 Neuroticism, 22 Neurotoxins, 163
164 Neurotransmission, 83 impairment of, 164 Neurotransmitter receptors, 10 Neurotransmitter systems, 164 Neutrophil extracellular traps (NETs), 51 Nifedipine, 31 Nitrogenous byproducts, 190 Nonabsorbable antibiotic rifaximin, 199 Nonalcoholic fatty liver disease (NAFLD), 100, 123
124, 161, 205 bile acid pathways in, 112
116 bile acid receptors as therapeutic targets in, 114
116 DPP-4 activity in, 110 FXR/FGF-19 pathway on, 113
114 gut microbiota and, 103
105, 104t human models of, 102 leaky gut in progression of, 105
106 liver injury in, 105 and microbiota, 161 pathogenesis of, 101
106 role of incretins in, 106
112

Index

on hepatic glucose and lipid metabolism, 107
109 physiological effects, 106
107 Nonalcoholic steatohepatitis (NASH), 105, 216 ballooning and, 111 fibrosis regression and, 115
116 incretins in NAFLD and, 109
110 and Ruminococcus, 106 Noncardiac chest pain (NCCP), 22 Nonspecific environmental factors, 3
4 Norepinephrine, 29, 54
55 neurotransmission, 227 NTCP. See Na 1 -taurocholate cotransporting polypeptide (NTCP)

O Obeticholic acid (OCA), 114
115 OCA. See Obeticholic acid (OCA) Omega-3 (n-3) polyunsaturated fatty acids, 216
218 Oral vancomycin, 126
127 Oscillibacter species, 124
125 “Osmotic gliopathy” theory, 191 Overt HE (oHE), 162
163

P Pain, 13 Pain modulators, 39 mechanism of, 29 Paraventricular nucleus (PVN), 81 Parkinson’s disease, 57, 160 Patient
physician relationship, 13 Pattern-recognition receptors, 102 PBC. See Primary biliary cirrhosis (PBC) PCK. See Phospho-enolpyruvate carboxykinase (PCK) PDS. See Postprandial distress syndrome (PDS) Peripheral hyperalgesia, 29 Peripheral nociceptors, 22 Peripheral sensitization, 22 Peripheral serotonin, 131
132 Permeability, 24
25, 53, 57
58, 160 Phenylethanolamine N-methyltransferase, 9 Phospho-enolpyruvate carboxykinase (PCK), 113 Physical activity, in irritable bowel syndrome, 35
36 Physical exercise, 12 Physician-patient relationship, 35 PI-FD. See Postinfection functional dyspepsia (PI-FD) PI-IBS. See Postinfection irritable bowel syndrome (PIIBS) Platelets, 68
69, 133, 209
210 Polymorphic variants, 77
78 Polymorphisms, 73
74, 77
78, 80, 215 Polyphenols, 218
219 Polyunsaturated fatty acids (PUFAs), 216
218 Population-based cohort study, 52 Porphyromonadaceae, 162
163 Portal-systemic shunting, 165

245

Portosystemic shunts, 165 Positive and Negative Syndrome Scale (PANSS) score, 56 Positron emission tomography, 25 Postinfection functional dyspepsia (PI-FD), 28 Postinfection irritable bowel syndrome (PI-IBS), 28 Postprandial distress syndrome (PDS), 23 Posttransjugular intrahepatic portosystemic shunts hepatic encephalopathy, 174 PPIs. See Proton pump inhibitors (PPIs) Prebiotics, 172, 220 Primary bile acids, 112 Primary biliary cirrhosis (PBC), 161
162 Primary defendant, 3
4 Primary drug prophylaxis, 174 Primary hyperalgesia, 22 Primary sclerosing cholangitis (PSC), 125
127, 161
162 antibacterial pharmacotherapy for, 126
127 Probiotics, 221
222 Proinflammatory cytokines, 8, 102 Proinflammatory mechanisms, 164 Proinflammatory microbiota, 160 Prokinetics in functional dyspepsia, 34, 34t Protein intake, 169 Proteobacteria phylum, 124 Proton pump inhibitors (PPIs), 28
29, 175
176 PSC. See Primary sclerosing cholangitis (PSC) P-selectin positive platelets, 223
224 Psychiatric and neurological disorders in gastrointestinal diseases, 58
59 immune pathway, 50
52 microbial metabolites, 52
53 neuropsychiatric disorders. See Neuropsychiatric disorders vagus nerve, 52 Psychiatric comorbidity, 12
13 Psychiatric disorders, 3, 39 Psychobiota, 85 Psychological interventions, 35, 39 Psychological/psychiatric disorders, 81 Psychological stressors, 10, 160 Psychometric Hepatic Encephalopathy Score, 167 Psychometric tests, 166, 169, 175 Psychomotor abnormalities, 159 Psychosocial factors, 24, 81
82 Psyllium, 36
37 PVN. See Paraventricular nucleus (PVN)

Q Quality of life, 168 Quantitative susceptibility mapping, 213
214 Quercetin, 218
219 Quinolic acid, 214
215

246 R Ramosetron, 37
38 Randomized controlled trial (RCT), 56 Reactive oxygen species (ROS), 139 Reciprocal communications, 3 Reflux hypersensitivity, 23 Resistant starch, 7
8 Resting-state functional connectivity (rsFC), 214 Rifaximine, 36
37, 39, 170, 172
173, 175
176 Rome criteria, 28 Rome IV classification, of FGIDs, 21t Roseburia inulinivorans, 220 Routine prophylactic therapy, 174 rsFC. See Resting-state functional connectivity (rsFC) Ruminococcaceae species, 199
200 Ruminococcus, 227 Ruminococcus gnavus, 26

S Salivary dysbiosis, 197, 200 Sarcopenia, 165 Scaffolding proteins, 98 SCFAs, 52
53. See also Short-chain fatty acids (SCFAs) Schizophrenia, 56 Secondary afferent neurons, 12
13 Secondary bile acids, 112 Secondary prophylaxis of HE, 174 Secretory functions, 70 Segmentation, 71 Selective 5-HT reuptake inhibitor, 227 Selective permeability, 97 Selective serotonin reuptake inhibitors (SSRIs), 39, 71
72, 83 visceral analgesic effect of, 29 Senescent gut microbiota, 11 Sensory-motor gastric dysfunctions, 83 Serotonergic neurons, 68 Serotonergic neurotransmission, 53 Serotonergic system, 68, 77
78, 83 in digestive diseases, 79
80 Serotonin, 9, 26, 29, 53, 82
83, 130 in brain
gut axis, 80
82 and psychobiota, 85 central and peripheral effects by, 69t of digestive and extradigestive systems, 67
71 in functional gastrointestinal disorders, 79
80 and gut microbiota, 145
148 in gut physiology, 70
71 emesis, 70 gut motility, 70
71 immune system, 71 secretory functions, 70 and hepatocellular carcinoma, 141
143

Index

and liver fibrogenesis, 140
141 and microbiota, 83
84 psychological/psychiatric and extra-gastrointestinal comorbidities, 82
83 receptors, 133
136 synthesis and metabolism, 130
132 transporter, 71
77 Serotonin transporter protein (SERT), 71
72, 130, 132, 141 inhibitor fluoxetine, 71 polymorphisms, 77
78 Serum ammonia, 189 Serum 5-HT levels, 139
140 Sex hormones, 9
10 Short-chain fatty acids (SCFAs), 7
8, 10, 100, 196 Shunting, 165, 170 SIBO. See Small intestinal bacterial overgrowth (SIBO) Sickness behavior, 205, 209
210, 213, 218
219, 224
225, 227
228 Sigmoid mucosa, 198 Simethicone, 36
37 Single nucleotide polymorphisms (SNPs), 73, 78 Sitagliptin, 111 SLC6A4 expression, 71
72 Sleep disorders, 168
169 Small intestinal bacterial overgrowth (SIBO), 100, 210
212 Smooth muscle 5-HT7 receptors, 70 SNPs. See Single nucleotide polymorphisms (SNPs) Sodium/bile acid cotransporter, 112
113 Soluble fibers, 36
37 Somatic nervous system, 18 Specific pathogen-free (SPF), 49 Spermine, 9 Spinal nociceptor neurons, 12
13 SSRIs. See Selective serotonin reuptake inhibitors (SSRIs) Steatosis, 105 Streptococcus salivarius, 162
163 Stress, 26, 82 Stress-induced bowel movements, 83 Stress, physiological reaction to, 19 Stress responsive physiological systems, 22 Stroop test, 175
176 Substantia nigra, 76 Super donors, 200 Surgical shunts, 165 Synthetic FXR agonist, 113
114 Systemic inflammation, 206
207, 213
214 Systemic inflammatory response syndrome (SIRS), 164, 193 Systemic venous system, 7
8

Index

T TAMPs. See TJ-associated MARVEL domain-containing proteins (TAMPs) Tandospirone, 34 Target antiadhesion molecules, 223
226 antiadhesion therapies, 223
224 Tegaserod, 31 Thalamus, 213 Theophylline, 31 Tight junctions (TJs), 97
98 cytoskeleton-dependent regulation, 98 TIPS. See Transjugular intrahepatic portosystemic shunts (TIPS) TJ-associated MARVEL domain-containing proteins (TAMPs), 98 TLR4. See Toll-Like Receptor 4 (TLR4) TLR4 mutant mice, 102 TLR-9. See Toll-like receptor-9 (TLR-9) TLRs. See Toll-like receptors (TLRs) T lymphocytes, 28 TNFα. See Tumor necrosis factor-alpha (TNFα) TNFα inhibition, 225
226 Toll-like receptors (TLRs), 51, 102 TLR4, 210
212 TLR-9, 194
195 TPH inhibitor, 79
80 Transepithelial permeability, 97 Transjugular intrahepatic portosystemic shunts (TIPS), 165 Translocated bacteria, 102 Treg cells, 51 Tricyclic antidepressants (TCAs), 29, 39, 83

Triglyceride, 109 Triple coagonism, 112 “Trojan Horse” theory, 192 Tropifexor, 116 Tryptophan (TRP), 9, 131, 214
215 metabolism, 131f Tryptophan-derived metabolites, 7
8 Tryptophan hydroxylase (TPH), 9, 26, 67
68 Tryptophan metabolism, 53 Tumor necrosis factor-alpha (TNFα), 164

U UDCA. See Ursodeoxycholic acid (UDCA) Ursodeoxycholic acid (UDCA), 161
162

V Vagus nerve (VN), 17
18, 52 Variceal bleeding, 172 Vedolizumab, 223
224 Visceral brain, 18 Visceral hypersensitivity, 12
13, 20
21 in irritable bowel syndrome, 25
27 Visceral hypervigilance, 20 Visceral pain, 12
13 Viscero-visceral hyperalgesia, 22

W West Haven criteria, 166
167

Z Zonula occludens, 98

247