The Inflammasome: Methods and Protocols (Methods in Molecular Biology, 2459) 1071621432, 9781071621431

Table of contents :
Preface
Contents
Contributors
Chapter 1: Assessment of ASC Oligomerization by Flow Cytometry
1 Introduction
2 Materials
2.1 Solutions and Stimulations
2.2 Materials and Equipment
3 Methods
3.1 Peripheral Blood Mononuclear Cell Isolation
3.2 Cell Stimulation
3.3 Assessing Inflammasome Activation by Flow Cytometry
4 Notes
References
Chapter 2: In Vitro Assays to Study Inflammasome Activation in Primary Macrophages
1 Introduction
1.1 ASC Oligomerization
1.2 Caspase-1 Activation
1.3 IL-1 Family Cytokine Substrates
1.4 Gasdermin-D and Pyroptosis
1.5 The Inflammasome Assay: Rationale
2 Materials
2.1 Primary Murine BMDMs
2.2 Cell Stimulations and Infection
2.3 ELISA and Immunoblot
2.4 Cell Death Assays
2.5 ASC Speck Microscopy
3 Methods
3.1 Differentiation of Primary BMDMs
3.2 Cell Stimulations and Infection
3.3 ELISA and Immunoblot
3.4 Cell Death Assays
3.4.1 LDH Assay
3.4.2 Intracellular ATP Measurement
3.5 ASC Speck Microscopy
4 Notes
References
Chapter 3: Measuring Non-canonical Inflammasome Activation in Neutrophils
1 Introduction
2 Materials
2.1 Isolation of Murine Neutrophils
2.2 Activation of Non-canonical Inflammasome in Murine Neutrophils
2.3 Isolation of Neutrophil Nucleus
2.4 Imaging of Neutrophil Extracellular Traps
2.5 Equipment
3 Methods
3.1 Non-canonical Inflammasome Activation in Murine Neutrophils
3.2 Activation of the Non-canonical Inflammasome in Mouse Neutrophils
3.3 Isolation of Neutrophil Nuclei
3.4 Imaging of Neutrophil Extracellular Traps
4 Notes
References
Chapter 4: Gasdermin D Cleavage Assay Following Inflammasome Activation
1 Introduction
2 Materials
2.1 Generation of Murine Primary Cells
2.2 Human Cell Line Culture
2.3 Canonical Inflammasome Activation
2.4 Immunoblotting
2.5 Equipment
3 Canonical Inflammasome Activation in Murine Macrophages and Neutrophils
3.1 Purification of Murine Primary Neutrophils and Macrophages
3.1.1 Purification of Murine Primary Neutrophils
3.1.2 Generation of Primary Murine Bone Marrow Macrophages
3.2 Activation of Murine Inflammasome
3.2.1 Activation of the NLRC4 Inflammasome
3.2.2 Activation of the NLRP3 Inflammasome
4 Activation of the Canonical Inflammasome in Human Cell Lines
4.1 Preparation of THP-1 Cells
4.2 Activation of the NLRP3 Inflammasome in THP-1
5 Measuring GSDMD Cleavage
5.1 GSDMD Immunoblotting
6 Notes
References
Chapter 5: Activation of the Non-canonical Inflammasome in Mouse and Human Cells
1 Introduction
2 Materials
2.1 Generation of Primary Mouse Macrophages
2.2 Human Cell Line Culture
2.3 Non-canonical Inflammasome Activation
2.4 Non-canonical Inflammasome Measurement
3 Activation of the Non-canonical Inflammasome in Mouse Macrophages
3.1 Production of Primary Mouse Bone Marrow-Derived Macrophages
3.2 LPS Transfection in Mouse Cells
4 Activation of NCI in Human Cell Lines
4.1 Preparation of THP-1 Cells
4.2 Preparation of HeLa Cells
4.3 LPS Transfection in Human Cells
5 Analysis of NCI Activation
5.1 Immunoblotting for Inflammatory Caspase Cleavage
5.2 Detecting Pyroptosis Using LDH Release Assay (See Note 8)
6 Notes
References
Chapter 6: Measurement of Inflammasome-Induced Mitochondrial Dysfunction by Flow Cytometry
1 Introduction
2 Materials
2.1 Generation of Bone Marrow-Derived Macrophages (BMDM)
2.2 Cell Stimulations
2.3 Flow Cytometry Staining
3 Methods
3.1 Generation of Primary Mouse Bone Marrow-Derived Macrophages (BMDM)
3.2 Stimulation of BMDMs to Activate NLRP3 Inflammasome
3.3 Staining and Measurement of NLRP3 Inflammasome-Induced Mitochondrial Damage by Flow Cytometry
4 Notes
References
Chapter 7: Detection of ASC Oligomerization by Western Blotting
1 Introduction
2 Materials
2.1 Solutions and Stimulations
2.2 Materials and Equipment
3 Methods
3.1 Cell Stimulations
3.2 Cell Lysis and ASC Oligomerization
4 Notes
References
Chapter 8: Reconstitution System of NLRP3 Inflammasome in HEK293T Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Plasmid Transfection
2.3 Detection of NLRP3 Inflammasome Activation
3 Methods
3.1 Cell Preparation
3.2 Plasmid Preparation and Transfection
3.3 Detection of NLRP3 Inflammasome Stimulation
4 Notes
References
Chapter 9: Intracellular Potassium Ion Measurements by Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES)
1 Introduction
2 Materials
2.1 Cell and Medium
2.2 Reagents
2.3 Equipment
3 Method
3.1 Cell Stimulation Assays
3.2 Cell Lysis Preparation and Intracellular K+ Content Measurement
3.3 Remaining Adherent Cell Numbers and Cell Death Analysis
4 Notes
References
Chapter 10: NLRP3 Phospho-residue Mapping by Phospho Dot Blots
1 Introduction
1.1 Inflammasomes and Posttranslational Modification of NLRP3
1.2 Principle of Phospho Dot Blot
1.3 Advantages of Phospho Dot Blot
1.4 Disadvantages of Phospho Dot Blot
2 Materials
2.1 Kinase Reaction
2.2 Kinase Removal Using Magnetic Beads
2.3 Dot Blot Analysis of the Phospho Peptides and Chemiluminescent Detection
3 Methods
3.1 Kinase Reaction
3.2 Kinase Removal Using His-Tagged Beads
3.3 Dot Blot
3.4 Control Total Peptide Staining
3.5 Incubation with Phospho-Specific Antibodies
3.6 Detection of Chemiluminescence
4 Notes
References
Chapter 11: Analysis of Activity and Expression of the NLRP3, AIM2, and NLRC4 Inflammasome in Whole Blood
1 Introduction
2 Materials
2.1 Inflammasome-Specific Stimulation
2.1.1 NLRP3 Stimulation
2.1.2 AIM2 Stimulation
2.1.3 NLRC4 Stimulation
2.2 RT-qPCR and Cytokine Measurement
3 Methods
3.1 Inflammasome-Specific Stimulation
3.1.1 NLRP3 Stimulation
3.1.2 AIM2 Stimulation
3.1.3 NLRC4 Stimulation
3.2 RNA Isolation and Gene Expression Analysis
3.3 Cytokine Measurement
4 Notes
References
Chapter 12: Measurement of Cytosolic Mitochondrial DNA After NLRP3 Inflammasome Activation
1 Introduction
2 Materials
2.1 Cell Culture and Inflammasome Induction
2.2 Cell Fractionation
2.3 Other Items
3 Methods
3.1 NLRP3 Inflammasome Stimulation
3.2 Cell Fractionation
3.3 Isolation of Mitochondrial DNA
3.4 Amplification of mtDNA by Quantitative PCR
4 Notes
References
Chapter 13: Inflammasome-Derived Exosomes Isolation and Imaging by Transmission Electron Microscopy
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Activation of the NLRP3 Inflammasome
2.3 Isolation of Exosomes (Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids)
2.4 Staining and Observation of Exosomes
3 Methods
3.1 Preparation of BMDMs (Bone Marrow-Derived Macrophages)
3.2 Activation of the NLRP3 Inflammasome
3.3 Isolation of Exosomes
3.4 Exosomes Imaging by Transmission Electron Microscopy
4 Notes
References
Chapter 14: In Vitro and In Vivo Model for Sepsis Through Non-canonical NLRP3 Inflammasome Activation
1 Introduction
2 Materials
2.1 Biological Materials and Buffers
2.2 Bone Marrow Cell Isolation and Differentiation
2.3 In Vitro Non-canonical NLRP3 Inflammasome Activation
2.4 Measurement of Pyroptosis and IL-1β Release
2.5 Caspase-1 and IL-1β Western Blot Analysis
2.6 General Equipment
3 Methods
3.1 Differentiation of Murine BMDMs
3.2 Induction of Non-canonical Inflammasome Activation in BMDMs
3.3 Measurement of Pyroptotic Cell Death and IL-1β Release
3.4 Analysis of Caspase-1 and IL-1β Maturation by Western Blotting
3.5 Bacteria Cultivation and Septic Shock Induction in Mice
3.6 LPS-Induced Septic Shock in Mice
4 Notes
References
Chapter 15: Inflammasome Activation in Gingival Epithelial Cells
1 Introduction
2 Materials
2.1 Sandwich ELISA
2.2 Immunofluorescence Staining
2.3 Western Blot Analysis
3 Methods
3.1 Sandwich ELISA
3.2 Immunofluorescence Staining
3.3 Western Blot
3.3.1 Sample Preparation and SDS-PAGE
3.3.2 Wet Transfer and Staining
4 Notes
References
Chapter 16: Imaging of Inflammasome Speck Formation in Living Cells
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Counterstaining
2.3 Stimulation, Live Imaging, and Analysis
3 Methods
3.1 Cell Culture and Seeding
3.2 Microscope Preparation and Live Imaging
4 Notes
References
Index

Citation preview

Methods in Molecular Biology 2459

Ali Abdul-Sater Editor

The Inflammasome Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-bystep fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

The Inflammasome Methods and Protocols

Edited by

Ali A. Abdul-Sater School of Kinesiology and Health Science, York University, Toronto, ON, Canada

Editor Ali A. Abdul-Sater School of Kinesiology and Health Science York University Toronto, ON, Canada

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-2143-1 ISBN 978-1-0716-2144-8 (eBook) https://doi.org/10.1007/978-1-0716-2144-8 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.

Preface Antigens constantly challenge the immune system prompting activation of both innate and adaptive immunity. Innate immunity provides the first line of defense against foreign antigens and contributes to containment of the pathogen while adaptive immunity eliminates antigens in a specific and sustained manner. Cells of the innate immune system recognize molecules that are unique to pathogens, called pathogen-associated molecular patterns (PAMPs) and/or danger-associated molecular patterns (DAMPs) through various germline-encoded receptors known as pattern recognition receptors (PRRs), such as tolllike receptors (TLR), RIG-I-like receptors (RLR), and Nod-like receptors (NLR). Stimulation of these receptors leads to activation of several signaling pathways culminating in the release of various inflammatory cytokines (e.g., IL-1β, IL-6, TNF-α, and type-I interferons) and/or activation of pathways involved in clearing dead cells and initiation of tissue repair. Of these cytokines, interleukin-1β (IL-1β) and IL-18 are unique in that their release is tightly regulated and requires the assembly of a multiprotein complex, called the inflammasome, typically composed of a sensor NLR or ALR protein, the adapter protein apoptosisassociated speck-like protein containing a CARD (ASC), and the inactive zymogen caspase-1. Following activation, the NLR sensor oligomerizes and forms a platform that recruits ASC subunits, which, in turn, form long filaments that nucleate the oligomerization of ASC and formation of large specks. Caspase-1 can then be recruited to the ASC oligomers, which induces the autoproteolytic cleavage and activation of caspase-1. Active caspase-1 then cleaves pro-IL-1β and pro-IL-18 into their biologically active forms, IL-1β and IL-18, respectively. The canonical inflammasome is typically nucleated by the NLR members NLRP1, NLRP3, and NLRC4 or by the ALR member AIM2. Recently, closely related caspases, caspase-11 (in mouse) and caspase-4/5 (in humans), were shown to sense the presence of lipopolysaccharides (LPS) in the cytosol resulting in the activation of the non-canonical inflammasome. After recognizing LPS, caspase-11 is activated and results in pyroptotic cell death. Recent advances have shed light on how caspase-1, IL-1β, and IL-18 are released from the cell following inflammasome activation. Active caspase-1 or caspase-11 cleave gasdermin D (GSDMD) to generate pore-forming N-terminal fragments that oligomerize and target the plasma membrane. In addition to allowing pro-inflammatory cytokines to be released, these pores eventually lead to a form of programmed cell death, called pyroptosis, as they overwhelm the cell’s ability to repair the damaged membrane. Of the inflammasomes that have been described, the NLRP3 inflammasome stands out for the large number of “molecular patterns” it responds to and the large number of human diseases to which it has been linked. Although hotly debated, several mechanisms have been proposed to activate the NLRP3 inflammasome. These include potassium efflux, mitochondrial dysfunction, production of reactive oxygen species (ROS), and lysosomal rupture. For example, extracellular ATP binds to the P2X7R ATP-gated ion channel and triggers potassium efflux and formation of pores in the membrane, mediated by pannexin-1 channels, and thereby activation of the NLRP3 inflammasome. Similarly, pore-forming toxins like nigericin act as a potassium ionophore that disrupts the membrane potential causing potassium efflux, which leads to NLRP3 inflammasome activation. Moreover, mitochondrial reactive

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oxygen species (mROS) generated in spatial and temporal proximity have been shown to contribute to the activation of the NLRP3 inflammasome. Unwarranted, excessive, or chronic activation of the inflammasome and secretion of IL-1β and IL-18 are associated with chronic inflammatory diseases, autoimmune disorders, Alzheimer’s disease, metabolic diseases, and sepsis. Therefore, it is not surprising that inflammasome activation is tightly regulated. These include transcriptional, translational, and posttranslational control of inflammasome components. Reflecting the importance of inflammasomes in health and disease, research assessing mechanisms of inflammasome activation, and its function continue to emerge. To this end, researchers have established specific protocols to study various aspects of inflammasome activation and regulation. These protocols can sometimes be technically challenging or difficult to reproduce without a fully detailed and standardized protocol. Therefore, in this volume, worldwide experts in inflammasome research have provided step-by-step instructions and specific tips and notes to study different aspects of inflammasome biology, including how to assess canonical and noncanonical inflammasome activation in vitro as well as in immune and non-immune cells or in whole blood. These protocols encompass biochemical assays to evaluate ASC oligomerization, gasdermin D cleavage, and mapping phosphorylated residues of NLRP3. Furthermore, the techniques include quantifying key mechanistic processes that lead to inflammasome activation, like mitochondrial dysfunction and ROS production, measurement of cytosolic mitochondrial DNA, and intracellular potassium levels. Recent advances in visualization techniques of inflammasome activation by electron microscopy and live-cell imaging are also described here to assess ASC speck formation and inflammasome-derived exosomes. I would like to thank Dr. John Walker for identifying the need to produce this up-todate and important resource for inflammasome researchers, and for his guidance and feedback during the editorial process. Of course, none of this would have been possible without the phenomenal contributions from all the authors who worked tirelessly despite the Covid-imposed shutdowns and delays to their research and to the editing process. I hope that the protocols provided in this volume will serve as the definitive tool for current and aspiring researchers interested in studying inflammasomes by employing cutting-edge techniques in a reproducible and reliable manner. Toronto, ON, Canada

Ali A. Abdul-Sater

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1 Assessment of ASC Oligomerization by Flow Cytometry . . . . . . . . . . . . . . . . . . . . 1 Laura Hurtado-Navarro, Alberto Baroja-Mazo, Helios Martı´nez-Banaclocha, and Pablo Pelegrı´n 2 In Vitro Assays to Study Inflammasome Activation in Primary Macrophages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 Ishita Banerjee 3 Measuring Non-canonical Inflammasome Activation in Neutrophils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 Mercedes Monteleone and Dave Boucher 4 Gasdermin D Cleavage Assay Following Inflammasome Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Louisa Janice Kamajaya and Dave Boucher 5 Activation of the Non-canonical Inflammasome in Mouse and Human Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Jelena S. Bezbradica, Rebecca C. Coll, and Dave Boucher 6 Measurement of Inflammasome-Induced Mitochondrial Dysfunction by Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Mayoorey M. Thasan and Ali A. Abdul-Sater 7 Detection of ASC Oligomerization by Western Blotting . . . . . . . . . . . . . . . . . . . . . 73 Safoura Zangiabadi, Ali Akram, and Ali A. Abdul-Sater 8 Reconstitution System of NLRP3 Inflammasome in HEK293T Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Sheng Chen, Zhexu Chi, and Di Wang 9 Intracellular Potassium Ion Measurements by Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES). . . . . . . . . . . . . . . . . . . . . . . . . . 85 Yifei Zhang and Yan Shi 10 NLRP3 Phospho-residue Mapping by Phospho Dot Blots . . . . . . . . . . . . . . . . . . . 93 Sangeetha Shankar, Zsofia A. Bittner, and Alexander N. R. Weber 11 Analysis of Activity and Expression of the NLRP3, AIM2, and NLRC4 Inflammasome in Whole Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105 Lev Grinstein and Stefan Winkler 12 Measurement of Cytosolic Mitochondrial DNA After NLRP3 Inflammasome Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117 Olga M. Ant
on and Javier Traba 13 Inflammasome-Derived Exosomes Isolation and Imaging by Transmission Electron Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Yuehui Zhang and Jian Wang

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Contents

In Vitro and In Vivo Model for Sepsis Through Non-canonical NLRP3 Inflammasome Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137 Xiao Sun, Tae-Bong Kang, and Kwang-Ho Lee Inflammasome Activation in Gingival Epithelial Cells. . . . . . . . . . . . . . . . . . . . . . . . 149 ¨ zlem Yilmaz Kalina R. Atanasova and O Imaging of Inflammasome Speck Formation in Living Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 Lucas Secchim Ribeiro

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Contributors ALI A. ABDUL-SATER • School of Kinesiology and Health Science, Muscle Health Research Centre (MHRC), York University, Toronto, ON, Canada ALI AKRAM • School of Kinesiology and Health Science, Muscle Health Research Centre (MHRC), York University, Toronto, ON, Canada OLGA M. ANTO´N • Lymphoid Malignancies Branch, Center for Cancer Research, National Cancer Institute (NIH), Bethesda, MD, USA KALINA R. ATANASOVA • Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL, USA ISHITA BANERJEE • Pandion Therapeutics – a wholly-owned subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA; Merck & Co., Inc., Kenilworth, NJ, USA ALBERTO BAROJA-MAZO • Biomedical Research Institute of Murcia (IMIB), Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain JELENA S. BEZBRADICA • Medical Sciences Division, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, Kennedy Institute of Rheumatology Research, University of Oxford, Oxford, UK ZSOFIA A. BITTNER • Department of Immunology, University of Tu¨bingen, Tu¨bingen, Germany DAVE BOUCHER • Department of Biology, York Biomedical Research Institute, University of York, Heslington, York, UK SHENG CHEN • Institute of Immunology, and Department of Orthopaedic Surgery of the Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China; Department of Colorectal Surgery and Oncology, Key Laboratory of Cancer Prevention and Intervention, Ministry of Education, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China ZHEXU CHI • Institute of Immunology, and Department of Orthopaedic Surgery of the Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China REBECCA C. COLL • The Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast, UK LEV GRINSTEIN • Department of Pediatrics, University Medical Centre HamburgEppendorf, Hamburg, Germany LAURA HURTADO-NAVARRO • Biomedical Research Institute of Murcia (IMIB), Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain LOUISA JANICE KAMAJAYA • Department of Biology, York Biomedical Research Institute, University of York, Heslington, York, UK TAE-BONG KANG • Department of Applied Life Science, Graduate School, Konkuk University, Chungju, South Korea; Department of Biotechnology, College of Biomedical and Health Science, Research Institute of Inflammatory Diseases, Konkuk University, Chungju, South Korea KWANG-HO LEE • Department of Applied Life Science, Graduate School, Konkuk University, Chungju, South Korea; Department of Biotechnology, College of Biomedical and Health Science, Research Institute of Inflammatory Diseases, Konkuk University, Chungju, South Korea

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Contributors

HELIOS MARTI´NEZ-BANACLOCHA • Biomedical Research Institute of Murcia (IMIB), Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain MERCEDES MONTELEONE • Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD, Australia PABLO PELEGRI´N • Biomedical Research Institute of Murcia (IMIB), Clinical University Hospital Virgen de la Arrixaca, Murcia, Spain; Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Medicine, University of Murcia, Murcia, Spain LUCAS SECCHIM RIBEIRO • Department of Microbiology and Immunology, University of Bonn, Bonn, Germany SANGEETHA SHANKAR • Department of Immunology, University of Tu¨bingen, Tu¨bingen, Germany YAN SHI • Tsinghua Institute for Immunology and Department of Basic Medical Sciences, Beijing Key Lab for Immunological Research on Chronic Diseases, School of Medicine, Tsinghua University, Beijing, China; Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China; Department of Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, AB, Canada; Snyder Institute, University of Calgary, Calgary, AB, Canada XIAO SUN • Department of Applied Life Science, Graduate School, Konkuk University, Chungju, South Korea; Department of Pathophysiology, Academy of Medical Sciences, Zhengzhou University, Zhengzhou, Henan, People’s Republic of China MAYOOREY M. THASAN • School of Kinesiology and Health Science, Muscle Health Research Centre (MHRC), York University, Toronto, ON, Canada JAVIER TRABA • Departamento de Biologı´a Molecular, Centro de Biologı´a Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientı´ficas-Universidad Aut
onoma de Madrid (CSIC-UAM), Madrid, Spain DI WANG • Institute of Immunology, and Department of Orthopaedic Surgery of the Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China JIAN WANG • State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China ALEXANDER N. R. WEBER • Department of Immunology, University of Tu¨bingen, Tu¨bingen, Germany STEFAN WINKLER • Department of Pediatrics, University Hospital Carl Gustav Carus, Technische Universit€ a t Dresden, Dresden, Germany ¨ ZLEM YILMAZ • Department of Oral Health Sciences, College of Dental Medicine, Medical O University of South Carolina, Charleston, SC, USA; Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA SAFOURA ZANGIABADI • School of Kinesiology and Health Science, Muscle Health Research Centre (MHRC), York University, Toronto, ON, Canada YIFEI ZHANG • Academy of Medical Engineering and Translational Medicine, Tianjin University, Tianjin, China YUEHUI ZHANG • State Key Laboratory of Proteomics, Beijing Proteome Research Center, National Center for Protein Sciences (Beijing), Beijing Institute of Lifeomics, Beijing, China

Chapter 1 Assessment of ASC Oligomerization by Flow Cytometry Laura Hurtado-Navarro, Alberto Baroja-Mazo, Helios Martı´nez-Banaclocha, and Pablo Pelegrı´n Abstract Inflammasomes are multiprotein complexes that critically control different aspects of innate and adaptive immunity. Upon activation, inflammasome proteins oligomerize forming scaffolds to nucleate the apoptosis-associated speck-like protein containing a CARD (ASC) in filaments that will finally result in large ASC oligomers that are commonly named as ASC specks. In this chapter, we present a method to monitor NLRP3 or pyrin inflammasome activation in human monocytes upon extracellular ATP or Clostridium difficile toxin B treatment, respectively, by detecting intracellular oligomers of ASC by flow cytometry. This method could be used to evaluate the degree of inflammasome activation in blood samples from patients suffering from different chronic inflammatory diseases. Key words NLRP3, Pyrin, Flow cytometry, ASC, Monocyte, Inflammasome, ATP, Toxin B of Clostridium difficile

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Introduction Inflammasomes are multiprotein complexes that critically control different aspects of innate and adaptive immunity. The nucleotidebinding domain and leucine-rich repeat containing receptor with a pyrin domain 3 (NLRP3) inflammasome is the most studied among several known inflammasome sensors [1]. High concentrations of extracellular ATP (in the mM range) activates the purinergic P2X7 receptor [2, 3]. Upon activation, NLRP3 oligomerizes forming scaffolds to nucleate the apoptosis-associated speck-like protein containing a CARD (ASC) in filaments that will finally result in large ASC oligomers that are commonly named as ASC specks [4, 5]. This ASC oligomerization is common in other described inflammasomes activated with a wide variety of pathogenic ligands and intracellular mediators, including the pyrin inflammasome which is induced by bacterial toxins that modify RhoA GTPases, as Clostridium difficile toxin B (TcdB) [4]. After oligomerization,

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Laura Hurtado-Navarro et al.

ASC interacts with the CARD domain of pro-caspase-1 inducing its activation within the inflammasome [6]. Caspase-1 will now execute a specific type of cell death called pyroptosis upon gasdermin D cleavage and plasma membrane pore formation [7]. Active caspase1 also cleaves the inactive precursors of the cytokines interleukin (IL)-1β and IL-18 into their mature forms [8] and is able to process other cytosolic substrates to induce the release of different intracellular proteins [9]. The detection of IL-1β processing and release by Western blot or ELISA is one of the hallmarks of the inflammasome activation. Here, we describe the use of a flow cytometry technique to determine intracellular ASC speck formation [10] in human monocytes after ATP or TcdB stimulation.

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Materials Prepare all solutions using ultrapure water and analytical grade reagents. Prepare and store all reagents at room temperature (unless indicated otherwise). Diligently follow all waste disposal regulations when disposing waste materials.

2.1 Solutions and Stimulations

1. Sterile Ficoll solution (see Notes 1 and 2). 2. Sterile phosphate-buffered saline (see Note 3). 3. Culture media: Opti-MEM Reduced Serum Media (see Note 4). 4. ATP dilution buffer: 147 mM NaCl, 10 mM HEPES, 13 mM D-(+)-Glucose, 2 mM KCl, 2 mM CaCl2, and 1 mM MgCl2, pH 7.4. After preparation, sterilize the buffer through 0.22-μm filters (see Note 5). 5. 100 ng/ml ultrapure lipopolysaccharide from Escherichia coli 0111:B4 (LPS) solution in culture media (see Notes 6 and 7). 6. 100 mM ATP stock solution in ATP dilution buffer pH 7.4 (see Note 8). 7. 0.2 mg/ml toxin B from Clostridium difficile, strain VPI10463 (TcdB) solution in sterile double-distilled water (see Note 9). 8. Flow cytometry staining buffer: phosphate-buffered saline with 1% fetal calf serum (FCS) and 0.1% sodium azide (see Note 10). 9. Cell permeabilization buffer: phosphate-buffered saline with 3% FCS, 0.1% sodium azide, and 0.1% saponin (see Note 10). 10. Cell fixation buffer: 4% paraformaldehyde in phosphatebuffered saline (see Note 11).

2.2 Materials and Equipment

1. Cell culture CO2 incubator set at 5% CO2 and 37  C. 2. Biological Safety Cabinets Class II.

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3

3. Flow cytometer equipped with argon laser and filter settings for the employed fluorophores (see Note 12). 4. Flow cytometry data analysis software, such as FCS express (De Novo Software), but other similar software is also suitable. 5. Vortex. 6. Centrifuge. 7. Micro-pipettes and pipette-tips of 10, 100, 200, and 1000 μl. 8. 15-ml sterile conical centrifuge tubes. 9. SepMate™-15 tubes for density gradient centrifugation (see Note 13). 10. Plastic sterile Pasteur pipettes. 11. 10-ml sterile glass pipettes and pipette vacuum cleaner. 12. Flow cytometry polypropylene tubes. 13. Specific antibodies for the detection of ASC (see Note 14). 14. Goat or donkey anti-rabbit Alexa Fluor 488 secondary antibody (see Note 15). 15. Mouse anti-human CD14 fluorophore-conjugated antibodies for the detection of monocytes within peripheral blood mononuclear cells (PBMCs) (see Note 16). 16. Peripheral blood human sample (see Notes 17 and 18). 17. Ice.

3

Methods

3.1 Peripheral Blood Mononuclear Cell Isolation

1. Add 3 ml of anticoagulated blood to a conical centrifuge tube and then add 3 ml of sterile phosphate-buffered saline. 2. Mix the blood and buffer by inverting the tube several times or by gently pipetting the mixture in and out of a pipette. 3. Agitate by inversion the sterile Ficoll solution bottle several times to ensure thorough mixing. 4. Add 4.5 ml of sterile Ficoll media to a SepMate™-15 tube by carefully pipetting it through the central hole (see Note 19). 5. Keeping the SepMate™ tube vertical, add the diluted blood sample by pipetting it down the side of the tube (see Note 20). 6. Centrifuge at 1200  g for 10 min at room temperature with centrifuge brake turned on. 7. Carefully recover the layer of mononuclear cells using a sterile pipette and transfer to a sterile conical centrifuge tube. 8. Add at least 3 volumes of phosphate-buffered saline to the mononuclear cells in the centrifuge tube.

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9. Suspend the cells by gently pipetting repeatedly in and out of a pipette. 10. Centrifuge at 400  g for 8 min at 18  C (see Note 21). 11. Remove the supernatant. 12. Resuspend the mononuclear cells in 8–10 ml of phosphatebuffered saline. 13. Centrifuge at 400  g for 8 min at 18  C (see Note 21). 14. Remove the supernatant. 15. Resuspend the mononuclear cells in 1 ml of culture media. 16. Count the cells (see Note 22). 3.2

Cell Stimulation

1. Seed 5  105 cells in 500 μl of culture media per duplicate and per condition to test in sterile flow cytometry tubes. 2. Incubate the cells with 100 ng/ml LPS (final concentration) for 2 h at 37  C and 5% CO2 (see Note 7). For negative control, use two replicates incubated with culture media without LPS. 3. Add 3 mM of ATP (final concentration) to the cultured cells in the presence of LPS and incubate for 15 min at 37  C and 5% CO2 to activate the NLRP3 inflammasome through the purinergic P2X7 receptor (see Notes 23 and 24). 4. Add 1 μg/ml of TcdB (final concentration) to the cultured cells in the presence or absence of LPS and incubate for 30 min at 37  C and 5% CO2 to activate the pyrin inflammasome (see Notes 23 and 25).

3.3 Assessing Inflammasome Activation by Flow Cytometry

1. After inflammasome activation, centrifuge tubes at 600  g for 5 min to pellet floating cells and carefully remove the supernatants (see Note 26). 2. Resuspend the cells in 100 μl of staining buffer containing phycoerythrin (PE)-conjugated anti-CD14 antibody to stain the surface of monocytes. 3. Incubate cells during 30 min at room temperature in the dark. 4. Wash the cells with 2 ml of staining buffer and centrifuge cells at 600  g for 5 min at room temperature. 5. Resuspend the cells in 500 μl of cell fixation buffer. 6. Incubate cells for 10 min on ice. 7. Wash the cells two times with 2 ml of staining buffer and centrifuge cells at 400  g for 5 min at room temperature. 8. Add 250 μl of permeabilization buffer to the cells. 9. Add 250 μl of a 1:500 dilution of anti-ASC antibody in staining buffer to achieve a final dilution of 1:1000 in the desired tubes (see Note 27). 10. Incubate for 45 min at room temperature in the dark.

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Fig. 1 Staining of intracellular ASC in human monocytes by flow cytometry. (a) Peripheral blood mononuclear cells (PBMCs) were gated based on SSC and FCS dot plot. (b) Monocytes were gated based on SSC and CD14PE dot plot. (c) Histogram showing the mean fluorescent intensity of ASC-FITC in lymphocytes (gray) and monocytes (black). Cells were stained with rabbit anti-ASC antibody clone N-15-R from Santa Cruz followed by the staining with secondary antibody directly conjugated with Alexa Fluor 488

11. Wash cells with 2 ml of staining buffer and centrifuge at 600  g during 5 min at room temperature. 12. Add 100 μl of a 1:1000 dilution of donkey anti-rabbit IgG Alexa Fluor 488 antibody in cell permeabilization buffer per tube and incubate tubes for 30 min at room temperature in dark (see Note 27). 13. Wash cells with 2 ml of staining buffer at 600  g for 5 min at room temperature. 14. Resuspend the cells in 500 μl of staining buffer. 15. Acquire the cells in a flow cytometer and analyze the staining of ASC in monocytes (Fig. 1), as well as the distribution of ASC FITC width vs ASC FITC area in CD14+ monocytes to determine and quantify the percentage of ASC specking monocytes (Fig. 2).

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Fig. 2 Staining and quantification of intracellular ASC specks in human monocytes by flow cytometry after P2X7 receptor triggering NLRP3 inflammasome or Pyrin inflammasome stimulation. Dot plots showing intracellular ASC specks in (a) resting, (b) LPS, (c) LPS + ATP, and (d) LPS + TcdB treated monocytes; black dots represent monocytes not forming intracellular ASC specks; red dots represent monocytes forming intracellular ASC specks. Cells were fixed and stained with rabbit anti-ASC antibody clone N-15-R from Santa Cruz followed by the staining with secondary antibody directly conjugated with Alexa Fluor 488

4

Notes 1. If you have Ficoll solution stored at 4  C, it must be allowed to warm at room temperature prior to its use. 2. Ficoll® is a neutral, highly branched, high-mass, hydrophilic polysaccharide, patented by GE Healthcare. It can be found commercialized by different companies as Ficoll-Paque®. Histopaque®-1077 can be used. 3. For peripheral blood mononuclear cell isolation, a sterile 0.9% NaCl solution could replace the use of phosphate-buffered saline during the isolation steps.

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4. Other culture media can be used, i.e., Roswell Park Memorial Institute (RPMI) 1640 medium with 10% of heatinactivated FCS. 5. For long storage periods, keep the buffer at 4  C. 6. Prepare 0.2 mg/ml stock of LPS solution in sterile Opti-MEM media, aliquot, and store at 20  C. 7. If desired, cells could be primed with 1 or 2 μg/ml of LPS to obtain higher inflammasome activation rates. 8. This is the stock ATP solution, aliquot and store at not refreeze aliquots.

20  C, do

9. Prepare 0.2 mg/ml stock of TcdB solution in sterile doubledistilled water, aliquot, and store at 20  C. 10. Store the buffer at 4  C until use. Flow cytometry staining buffer and cell permeabilization buffer can be stored at 4  C for up to 6 months. 11. To prepare this buffer, we normally use a commercial 32% solution of paraformaldehyde from Electron Microscopy Sciences (#50-980-494), although paraformaldehyde from other companies is also suitable. Paraformaldehyde is toxic and carcinogenic. Wear protective equipment according to the local regulations. Paraformaldehyde solutions must be made fresh every time before use. 12. We normally use a flow cytometer equipped with a 488 nm laser with 505/45 nm emission optics. 13. With these tubes, defibrinated or anticoagulant-treated blood is carefully layered on a density gradient medium and centrifuged for a short period of time. However, conical centrifuge tubes can be used. In this case, 3 ml of sterile Ficoll media will be added to a conical centrifuge tube. After that, carefully layer the diluted blood sample on the top of Ficoll media solution and centrifuge at 400  g for 40 min at room temperature with centrifuge brake turned off (StemCell Technologies, #85415). 14. There are different anti-human ASC antibodies suitable to detect intracellular ASC specks by flow cytometry; here we use the rabbit polyclonal anti-ASC from Santa Cruz (clone N-15-R); however, this antibody is currently discontinued. Other antibodies or clones might be also suitable, such as the mouse monoclonal anti-ASC, clone HASC-71 (Biolegend, #653904). 15. These conjugated antibodies are exclusive of Life Technologies (#A32731 and #A32790, respectively), although other fluorophore-conjugated antibodies from other companies are also suitable.

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16. Alternatively to CD14, monocytes can also be identified in PBMCs with anti-CD33 staining. We use PE-conjugated mouse anti-human CD14 (clone 61D3) from Tonbo Biosciencies (#60-0149-T100) or APC-Vio770-conjugated mouse anti-human CD33 (clone AC104.3E3) from Miltenyi Biotech (#130-113-349). 17. Fresh blood should be used to ensure high viability of isolated mononuclear cells. Human samples should be obtained under specific consent from donors and according to specific local legislation. Blood could be collected in tubes containing either EDTA or sodium heparin as anticoagulating agents. 18. Other macrophages or monocytes types could also be used, i.e., THP-1 cells, mouse peritoneal macrophages or mouse bone marrow–derived macrophages. 19. The top of the density gradient medium will be above the insert. Small bubbles may be present in the density gradient medium after pipetting. These bubbles will not affect the performance. 20. The sample can be poured down to the side of the tube. Take care not to pour the diluted sample directly through the central hole. 21. A centrifugation at high speed (400–600  g) increases the mononuclear cell recovery. To remove platelets, lower centrifugation speed is recommended (60–100  g). In both cases, temperature could range from 18 to 20  C. 22. Make a dilution of obtained cells with trypan blue to exclude dead cells from the counting and put 10 μl of diluted cells in a Neubauer chamber. Count at least three large squares and calculate the concentration of cells using the following formula: (number of cells  1000)/number of squares counted  dilution factor). For a dilution of 1/10 with trypan blue, the dilution factor will be 0.1. 23. Inflammasome activation in cells usually results in disintegration of the plasma membrane due to gasdermin D pore formation and cell morphology changes, resulting in a loss of cell size (FSC) and granularity (SSC). This must be taken into account when analyzing inflammasome-activated cells by flow cytometry. 24. This method is also suitable to study and quantify ASC specking macrophages upon different stimuli activating the NLRP3 inflammasome (as nigericin), and other inflammasome activation (as AIM2 or NLRC4) in human PBMCs and other cell types. 25. TcdB is able to activate the pyrin inflammasome in LPS-primed and un-primed cells. However, a higher activation of pyrin inflammasome is found in LPS-treated cells.

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9

26. The cell supernatants could be centrifuged to remove detached cells and stored at 80  C for posterior analysis of other inflammasome activation parameters as the release of IL-1β or IL-18 by ELISA or the presence of extracellular lactate dehydrogenase as marker of pyroptotic cell death. 27. To optimize staining of ASC specks, it is important to titrate the antibodies to get the best signal. We found that the primary anti-ASC antibody from Santa Cruz works well at a final dilution of 1:1000. In the case of the fluorescently conjugated secondary antibody from Life Technologies, the optimal signal was obtained with a 1:1000 dilution. Unless stated otherwise, this concentration was used throughout in the experiments described here.

Acknowledgments This work was supported by grants to PP from the MCIN/AEI/ 10.13039/501100011033 (grant PID2020-116709RB-I00), the Fundacio´n Se´neca (grants 20859/PI/18, 21081/PDC/19 and 00003/COVI/20), and the European Research Council (ERC-2013-CoG grant 614578 and ERC-2019-PoC grant 899636). PP also receive support from the EU Horizon 2020 project PlasticHeal (grant 965196). LH-N was funded by the fellowship 21214/FPI/19 (Fundacio´n Se´neca). References 1. Zheng D, Liwinski T, Elinav E (2020) Inflammasome activation and regulation: toward a better understanding of complex mechanisms. Cell Discov 6:36 2. Pelegrin P (2011) Many ways to dilate the P2X7 receptor pore. Br J Pharmacol 163: 908–911 3. Young MT, Pelegrin P, Surprenant A (2006) Identification of Thr283 as a key determinant of P2X7 receptor function. Br J Pharmacol 149:261–268 4. de Torre-Minguela C, Mesa Del Castillo P, Pelegrin P (2017) The NLRP3 and Pyrin inflammasomes: implications in the pathophysiology of autoinflammatory diseases. Front Immunol 8:43 5. Schmidt FI, Lu A, Chen JW et al (2016) A single domain antibody fragment that recognizes the adaptor ASC defines the role of ASC domains in inflammasome assembly. J Exp Med 213:771–790

6. Boucher D, Monteleone M, Coll RC et al (2018) Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity. J Exp Med 215:827–840 7. Shi J, Gao W, Shao F (2017) Pyroptosis: gasdermin-mediated programmed necrotic cell death. Trends Biochem Sci 42:245–254 8. Compan V, Baroja-Mazo A, Bragg L et al (2012) A genetically encoded IL-1beta bioluminescence resonance energy transfer sensor to monitor inflammasome activity. J Immunol 189:2131–2137 9. de Torre-Minguela C, Barbera-Cremades M, Gomez AI et al (2016) Macrophage activation and polarization modify P2X7 receptor secretome influencing the inflammatory process. Sci Rep 6:22586 10. Sester DP, Thygesen SJ, Sagulenko V et al (2015) A novel flow cytometric method to assess inflammasome formation. J Immunol 194:455–462

Chapter 2 In Vitro Assays to Study Inflammasome Activation in Primary Macrophages Ishita Banerjee Abstract Inflammasomes are multimeric complexes that can sense pathogens and danger signals in the environment. Upon detection of stimuli, caspase-1 is recruited to the inflammasome complex that cleaves and activates pro-inflammatory cytokines, thus initiating a cascade of inflammatory events. While inflammasomes form a crucial component of the host response to pathogens and danger molecules, their unchecked activation can result in the development of autoimmune diseases, metabolic disorders, and pathological outcomes. This chapter describes some assays to detect the measurable outcomes of inflammasome formation and activation. The protocol describes the methods to study the inflammasome pathway using an in vitro assay in primary macrophages. It can be applied to studies investigating the pathway mechanisms and potential therapeutics in the form of inhibitors or activators. Key words Inflammasome, ASC speck, Caspase-1, IL-1β, Gasdermin-D, Bone marrow–derived macrophages, Immunoblot, Cell death assays, Confocal microscopy

1

Introduction Inflammasomes are cytosolic multimeric complexes that are assembled in response to microbial pathogens or danger signals by the host cells. Inflammasome complex formation has been extensively studied in immune cells of the myeloid lineage such as macrophages, dendritic cells, and neutrophils. Inflammasome complexes comprise a sensor, adapter, and an effector. The inflammasome sensor recognizes unique pattern recognition repeats and danger signals and distinguishes the inflammasome named after it. Members of the nucleotide binding and oligomerization leucine-rich repeat (NLR) and absent-in melanoma2 (AIM2)-like receptor (ALR) family are most commonly known to form the inflammasome sensor. The NLR family member, NLRP3 is activated in response to a variety of stimuli, including bacterial products like pore-forming toxins, particulate stimuli for

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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instance alum, silica, and mono-sodium urate (MSU) crystals, metabolites like extracellular ATP and cellular perturbations such as K+ efflux and lysosomal disruptions [1]. In contrast, the other inflammasome sensors detect fewer ligands, such as NLRC4, which is activated by the type-III secretion system and bacterial flagellin [2, 3] while AIM2 initiates inflammasome assembly upon binding to double-stranded (ds) DNA in the cytoplasm [4]. The activation of the inflammasome complex requires the transcriptional expression of its components, NLRP3 and precursor form of interleukin-1β (IL-1β), called priming. Priming is provided by the ligands of the toll-like receptor2 (TLR2)/TLR4 pathway that induce NF-κB signaling [5]. The sensors, NLRP1, NLRP3, NLRC4, and AIM2 contain the caspase activation and recruitment domain (CARD) or the pyrin (PYD) domain [6]. The CARD and PYD domains possess the ability to oligomerize upon recognition of stimuli and thereby induce inflammasome assembly [7–9]. The oligomerization of the inflammasome sensor recruits a bipartite protein, apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC) [10, 11]. 1.1 ASC Oligomerization

ASC filaments oligomerize and form micrometer-sized specks or foci that can be detected even by light microscopy. An ASC speck is a supra-molecular signaling platform in a cell and has the potential to greatly amplify the inflammasome signal [12–14]. The detection of ASC specks is an important measurement of inflammasome complex formation. Imaging techniques such as fluorescence and confocal microscopy are commonly used methods to visualize ASC specks. ASC is diffused in the cytoplasm and the nucleus of myeloid cells. Upon ligand stimulation, they aggregate into a single foci per cell. This can be visualized under a microscope. ASC speck formation can also be detected by a quantitative albeit sensitive method, using time-of-flight inflammasome evaluation by a flow cytometry assay. The formation of ASC foci can be detected by changes in the height and width of the pulse of emitted fluorescence.

1.2 Caspase-1 Activation

Caspase-1 is recruited by ASC into the inflammasome assembly by interaction with its CARD domain [12]. Its engagement in the ASC puncta increases the local availability of caspase-1, facilitating its dimerization and activation [15, 16]. The C-terminal catalytic domain is cleaved upon activation and self-processing of caspase-1. This releases two biologically active subunits, the p20 and the p10 fragments [16–18]. Caspase-1 activation is the gold standard read-out for inflammasome activation and is typically assayed by immunoblotting or using flow cytometry by detecting the binding of caspase-1 to a fluorochrome labeled inhibitor, FLICATM (ImmunoChemistry

Assessing Inflammasome Activation in Macrophages

13

Technologies, Minneapolis, MN, USA) [19, 20]. Both the p20 and p10 active fragments can be detected by immunoblot along with the 45 kDa pro-caspase-1. Active caspase-1 cleaves the precursor forms of pro-inflammatory cytokines IL-1β and IL-18 to their bio-active forms. Caspase-11, another member of the family of inflammatory caspases, can also trigger pyroptosis by cleaving GSDMD. However, unlike caspase-1, it cannot directly process the precursor forms of IL-1β and IL-18. Caspase-11 is a cytosolic lipopolysaccharide (LPS) sensor and is activated by directly binding to its lipidA component [21]. Caspase-11 responds to a variety of intracellular as well as extracellular gram-negative bacterial infections. Guanylate binding proteins (GBPs) facilitate the release of LPS in the cytosol in case of intracellular bacterial infections such as Salmonella typhimurium, Citrobacter rodentium, and Vibrio cholerae [22]. However, during infection with extracellular bacteria like Escherichia coli (E. coli), outer membrane vesicles (OMVs) secreted during infection can serve as a vehicle to deliver LPS to the cytosol when taken up by phagocytic cells [23]. 1.3 IL-1 Family Cytokine Substrates

Unrestricted IL-1 signaling has been implicated in many autoinflammatory disorders such as type 1 diabetes, gout, rheumatoid arthritis, and osteoarthritis [24]. IL-1β is present in the cells in its 31-kDa precursor form with little or no biological activity. Proteolytic processing of IL-1β is required to convert it to its 17-kDa active form that can bind the IL-1 receptor (IL-1R) and initiate pro-inflammatory signaling. Caspase-1 is an IL-1β-converting enzyme (ICE) that typically cleaves IL-1β into its mature form that is secreted from the cells [25]. This cleaved fragment can be detected by immunoblot or enzyme-linked immunosorbent assay (ELISA). Similarly, caspase-1 also cleaves another IL-1 family cytokine, IL-18 into its active fragment. The precursor form of IL-18 is constitutively expressed in most resting cells. This 24-kDa precursor is cleaved by caspase-1 into its 17.2-kDa active form [26, 27]. Detection of the active fragment by immunoblot or ELISA has limitations. Recent development of reporter cells has attempted to address this issue. HEK-Blue IL-1β cells developed by Invivogen is one such reporter cell system that quantitatively monitors the production of IL-1β by using NF-κB and AP-1 activation upon IL-1R signaling as a surrogate marker.

1.4 Gasdermin-D and Pyroptosis

Gasdermin-D (GSDMD), a member of the gasdermin family of proteins, has recently been identified as a substrate of the inflammasome complex that mediates pyroptotic cell death, another hallmark of inflammasome activation. GSDMD can be cleaved into its C-terminus and N-terminus domain by the inflammatory caspases1 and -11 in mouse and 4 and 5 in human [28–30]. GSDMD

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N-terminus binds to lipids in the plasma membrane. The size of the GSDMD pore formed is 10–16 nm wide. The pore is non-selective where molecules with a smaller size can be released through the pore, while higher molecular size proteins are restricted. The active forms of pro-inflammatory cytokines such as IL-1β and IL-18 have a molecular size of 4 nm and are hence released through the GSDMD pore [31–33]. The cell subsequently loses membrane integrity and undergoes lysis. Pyroptotic cell death can be used to measure inflammasome responses. Lactate dehydrogenase (LDH) secretion from cells into the extracellular media upon cell lysis is a typical assay to measure cell death. However, this assay does not distinguish pyroptosis from other forms of cell death. It cannot detect cells that have GSDMD pore formation but have not lost plasma membrane integrity. Other methods of assaying cell death include the measurement of ATP release in the extracellular media. ATP is a small molecule and can pass through GSDMD pores and can therefore measure pore formation in addition to cell lysis. Detection of DNA-binding fluorescent dyes such as propidium iodide or SYTOX green can also be a measure of both GSDMD pore formation and cell death. 1.5 The Inflammasome Assay: Rationale

The methods presented in this chapter utilize optimal parameters to study inflammasome responses. Primary bone marrow–derived macrophages (BMDMs) are used in this protocol. Although the components of the inflammasome are expressed in most healthy cells, their expression is significantly higher in cells of the myeloid lineage resulting in the most pronounced inflammasome responses in them. BMDMs have been the model of choice for defining the inflammasome pathway and its mechanisms. They are easily obtained from the femur and tibia of mice and can be cultured in vitro to give a good yield of primary macrophages. Although, primary cells are limited by their ability to be passaged, they are the most physiologically relevant in vitro model system. A variety of macrophage cell lines are available as research tools. One such prominent example is RAW264.7 murine macrophages. However, RAW264.7 cells are deficient in the inflammasome adapter, ASC. They are unable to recruit caspase-1 and process IL-1β even though they possess these components. Some laboratories use immortalized BMDMs for testing inflammasome responses. Although these cells may be a good tool given their ease of propagation yielding a high number of cells, they are immortalized in individual laboratories rather than being obtained from a standard commercial source. Therefore, the consistency of results varies between research groups along with the pathways that have been re-programmed during the immortalization process. While the mechanisms of the inflammasome complex have been largely studied in the murine system, their validation in human models is essential for application to human disease. Similar

Assessing Inflammasome Activation in Macrophages

15

to mouse, human primary macrophages can also be used for in vitro experiments. Human monocytes can be enriched from peripheral blood mononuclear cells (PBMCs) and differentiated into macrophages using recombinant human macrophage-colony stimulating factor (M-CSF). Human macrophages can be cultured for 7–10 days. However, their yield in culture is much lower when compared to murine primary macrophages. This is because unlike mouse bone marrow precursors, peripheral blood monocytes do not possess the ability to readily proliferate in culture. Their use is also limited by variability among human donors [34]. Another popular model for studying human inflammasome responses are THP-1 monocytes, which derived from acute monocytic leukemia. THP-1 monocytes can be differentiated to macrophages using phorbol-12-myristate-13-acetate (PMA) [35]. The inflammasome complex responds to diverse activators. However, not all activators have equal signal strength. For example, among the stimuli for the NLRP3 inflammasome, some ligands such as extracellular ATP activate the inflammasome complex strongly with copious amounts of secreted IL-1β accompanied by pyroptosis, while bacterial products like LPS do not activate the inflammasome complex with similar signal strength, producing only a fraction of active IL-1β compared to ATP. It is therefore advisable to optimize the duration of treatment for each inflammasome ligand. Detection of active fragments of cytokines IL-1β, IL-18, and cell death measurement (Fig. 1) are the most typical read-outs of inflammasome activation. However, the sample type used to assess these products should be taken into consideration. Secreted fragments of cytokines are analyzed in the supernatant of an in vitro experiment. The precursor forms are detected in the cell lysate. The analysis of both the active and the pro forms is required to measure processing. Although, processed caspase-1 fragments are largely found in the cell lysate along with its precursor form. Cleaved GSDMD (N-terminus) fragment binds to the lipids in the plasma membrane and can only be detected when gentle lysis buffers are used that does not destroy the lipid components of the lysate (Table 1). A graphical schematic of the inflammasome assay is depicted in Fig. 2.

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2.1 Primary Murine BMDMs

1. C57BL/6 mice. 2. Clean dissection tools (forceps, scissors). 3. 70% Ethanol. 4. Non-tissue culture–treated 10 cm petri dishes (n ¼ 4).

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Fig. 1 IL-1β and cell death measurement in BMDMs following inflammasome stimulation. BMDMs differentiated as described in Fig. 2 were primed with 400 ng/mL Pam3CSK4 for 3–4 h, infected with Enterohemorrhagic E. coli (EHEC) or Shigella flexneri at an MOI of 50 for 16 h. Cells were transfected with 2 mL/mL (of DNA) lipofectamine 2000 along with 1 μg/106 cells poly(dA:dT) for 8 h. Cells were stimulated with 10 μM Nigericin for 1 h. Supernatants were collected, and an IL-1β ELISA (a) or LDH release cell death measurement (b) was performed. Data are displayed as mean  SEM

5. 1 PBS (Sterile). 6. 10-mL syringes. 7. 25-G and 22-G needles. 8. 70-μm Cell Strainers. 9. Complete media [DMEM with 10% heat-treated fetal bovine serum (FBS) and 1% Penicillin/Streptomycin]. 10. 1 RBC Lysis Buffer. 11. L929 supernatant (filtered) or murine recombinant M-CSF. 12. Tissue-culture treated 15-cm culture dishes. 13. 50-mL Falcon tubes (Sterile). 14. Aspirator pipettes. 15. Cell scraper. 16. 37  C, 5% CO2 cell-culture incubator. 17. Laminar-flow Hood.

Assessing Inflammasome Activation in Macrophages

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Table 1 Measurable outcomes in inflammasome complex formation and activation Inflammasome read-outs

Cellular event

Assay

Cellular/ secreted

ASC speck formation

ASC oligomerization

Immunofluorescence microscopy; flow Intracellular and cytometry secreted

Active caspase-1

Self-processing of Immunoblot; flow cytometry effector Caspase-1

Active GSDMD

Activation and cleavage of the pore-forming effector

Immunoblot

Intracellular (lipid membrane)

Active IL-1β and IL-18

Cleavage of the precursor cytokines to their active form

Immunoblot; ELISA

Secreted (supernatant)

Precursor forms of caspase-1, GSDMD, IL-1β, and IL-18

Processing to release Immunoblot their active fragments

Cellular (lysate)

Cell death

LDH detection; intracellular ATP GSDMD poremeasurement; cellular metabolic formation leading status, propidium iodide staining, to cell lysis etc.

Secreted (LDH), Cellular (ATP and PrestoBlue)

Mostly secreted

ASC apoptosis-associated speck-like protein containing a caspase activation and recruitment domain, GSDMD gasdermin-D, IL-1β interleukin-1β, IL-18 interleukin-18, ELISA enzyme-linked immunosorbent assay, LDH lactate dehydrogenase, ATP adenine triphosphate

2.2 Cell Stimulations and Infection

1. Tissue culture–treated 96-well plate. 2. BMDMs (Days 7–12) 3. Trypan Blue Solution 0.4%. 4. Hemocytometer (Neubauer modified). 5. Bacterial stock of Enterohemorrhagic E. coli. 6. Poly(dA:dT). 7. Pam3CSK4. 8. Ultrapure E. coli LPS O111:B4. 9. ATP. 10. Nigericin. 11. Clostridium difficile Toxin-B. 12. Lipofectamine 2000. 13. Optimem. 14. Media (DMEM without phenol red with 2.5% heattreated FBS).

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Fig. 2 Graphical schematic of an inflammasome assay. (a) Differentiation of bone marrow cells into BMDMs. Bone marrow cells isolated from the femur and tibia of a mouse are cultured in the presence of M-CSF for 6 days with media replacement on day 3 and cell splitting on day 5. Differentiated macrophages are collected and plated followed by inflammasome stimulation between days 7 and 12. (b) In vitro inflammasome assay. This assay is usually performed in a 96-well or 12-well format. The 96-well format is best suited for cytokine measurement by ELISA (supernatant) and cell death assay (supernatant or cells). The 12-well format is commonly done for measuring active and precursor inflammasome components by immunoblot (supernatant and cell lysate). Cells can also be plated on an imaging dish for microscopic visualization of ASC specks. (Created with BioRender.com)

15. Triton X-100. 16. Multichannel pipette. 2.3 ELISA and Immunoblot

1. IL-1β ELISA kit. 2. IL-18 ELISA kit. 3. High protein binding plates flat bottom. 4. Plate Reader.

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5. Nonidet P(NP)-40 lysis buffer [150 mM sodium chloride (NaCl); 1% NP-40; 50 Mm Tris (pH 8.0)]. 6. Radioimmunoprecipitation assay (RIPA) lysis buffer [150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate (DOC); 0.1% sodium dodecyl sulfate (SDS); 50 mM Tris (pH 8.0). RIPA buffer can be made as a 10 stock solution and stored in 4  C for 1 month]. 7. Protease inhibitor cocktail. 8. Phosphatase inhibitor cocktail. 9. Western Blot equipment. 10. Trans-Blot Turbo Transfer System. 11. ECL HRP Substrate. 12. Nitrocellulose membranes. 13. Milk Powder. 14. Chemiluminescence Imager. 2.4 Cell Death Assays

1. 96-well non-tissue culture–treated assay plate. 2. LDH detection kit. 3. Cell Titer Glo detection reagent. 4. White flat-bottom plates.

2.5 ASC Speck Microscopy

1. 16% paraformaldehyde aqueous solution (stock): Make 4% solution at the time of the experiment by diluting with 1 PBS. 2. Triton X-100 detergent. 3. Goat serum. 4. Anti-ASC antibody. 5. Alexa fluor 488–conjugated anti-rabbit IgG. 6. Alexa fluor 647–conjugated cholera toxin subunit B. 7. 40 ,6-Diamidino-2-phenylindole (DAPI). 8. Cell culture imaging dishes. 9. Fluorescent microscope.

3

Methods

3.1 Differentiation of Primary BMDMs

Primary BMDMs are generated by culturing bone marrow cells from mice with recombinant M-CSF or the supernatant of L929 cells that secrete M-CSF. 1. Prepare four petri dishes, adding 10 mL sterile PBS in two of them and place them in the laminar flow hood. 2. Euthanize the mouse with CO2 or cervical dislocation. Spray 70% ethanol over the dead mouse carcass.

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3. Raise the skin over the belly with a forceps and make a cut with sharp dissection scissors. Remove the skin from the belly and the hind legs. Using the dissection scissors, make a cut at the ball of the femur where it is attached to the pelvic cavity such that the head of the femur remains intact. Place the hind legs in a 50-mL Falcon tube with DMEM in ice and take it inside the laminar flow hood. Remove the flesh from the hind legs and cut the joint between the femur and tibia. Make another cut between the tibia and the digits (see Note 1). 4. Put the two femur and two tibias in the first petri dish containing sterile PBS. Use the forceps, scissors, and paper towels to completely clean the bones. Add 2 mL 70% ethanol to the empty petri dish and place the bones in it for 30 s. Immediately transfer the bones to the third petri dish containing 10 mL sterile PBS. Wash the bones and cut the heads of the bones on both ends. 5. Using a 10-mL syringe and a 25-gauge needle, flush the bone marrow out from the open-ended bones and the heads of the bones into the fourth petri dish (see Note 2). Make a single cell suspension of the bone marrow cells by flushing the bone marrow cells through a 22-gauge needle and a 10-mL syringe. 6. Transfer the cell suspension to a 50-mL Falcon tube by filtration through a 70-μm cell strainer and spin to pellet the cells at 400  g for 5 min at room temperature (RT). 7. Resuspend the cells in 1 RBC lysis buffer and incubate the cells at RT for 2–3 min along with gentle tapping. Fill the tube with sterile PBS and spin to pellet the cells. 8. Resuspend the cells in DMEM with 10% FBS and 1% Penicillin/Streptomycin. Culture the cells by dividing them equally into two 15 cm cell culture–treated dishes along with 25 mL of the above-described media with 20% filtered L929 supernatant or 20 ng/mL of recombinant murine M-CSF in each dish (see Note 3). 9. Replace the cell culture media on day 3 with fresh DMEM with 10% FBS and 20% L929 supernatant or recombinant M-CSF (see Note 4). 10. Split the cells on day 5 or whenever they achieve 70% confluency. 11. Harvest the cells and use for experiments between days 7 and 12 of culture (see Note 5). 3.2 Cell Stimulations and Infection

Inflammasome activation can be measured by in vitro experiments by plating the BMDMs in cell culture plates and stimulating them with ligands or infecting them with pathogens that activate the inflammasome complex (Fig. 3).

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Fig. 3 Cell stimulations and infection. BMDMs from two genotypes (C57BL/6 wild-type and C57BL/6 NLRP3/) are plated in replicates. BMDMs are primed with Pam3CSK4 for 3–4 h and treated with the indicated stimuli, as described in Subheading 3.2. The inflammasome pathway activated by each stimulus is denoted in brackets. Triton-X is added to the cells 30 min before the endpoint to serve as a positive control for LDH release cell death assay (Created with BioRender.com)

1. Count and plate the cells at a concentration of 106 cells/mL. Use a 96-well plate, if the purpose of the experiment is cytokine analysis by ELISA or cell death assays. For immunoblot experiments, use a 12-well plate. Let the cells adhere to the plate for at least 4 h before stimulation (see Note 6). 2. Prime the cells in antibiotic-free media with 400 ng/mL Pam3CSK4 for 3–4 h before stimulations to assess inflammasome responses (see Note 7). 3. For infection with bacteria such as Enterohemorrhagic E. coli, pellet the required number of bacterial cells from an overnight broth culture. Resuspend in media without antibiotics. Infect the cells at an MOI of 50–100 for 1–2 h. Replace the media with 100 mg/mL gentamicin containing medium (see Note 8). Collect the supernatant and perform cell-based assays 4–16 h post stimulation. 4. For ligand stimulations, transfect cells with 2 mL/mL (of DNA) lipofectamine 2000 complexed with 1 μg/106 cells poly(dA:dT) or 1 μg/106 cells LPS. Collect the supernatant and perform cell-based assays 6–8 h post stimulations. 5. For stimulation with particulate stimuli and toxins, resuspend the cells in media containing stimuli such as 5 mM ATP or 10 μM Nigericin. Collect the supernatant and perform cellbased assays 1–4 h post stimulation, depending on the individual stimulus (see Note 9).

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3.3 ELISA and Immunoblot

1. Collect 50–100 μL of the supernatant for cytokine (IL-1α, IL-1β, IL-18, IL-6, and/or TNF) measurement by ELISA according to the manufacturer’s instructions. Technical replicates should be used to maintain accuracy (see Notes 10–12). 2. For immunoblotting, both supernatant and cell lysate can be assessed. 3. To process supernatant samples, concentrate at least 600 μL of supernatant sample for immunoblot to detect cleaved IL-1β, IL-18, caspase-1, or GSDMD fragment (see Note 13). 4. For cell lysate preparation, detach the cells by mechanical scraping. Use 1% NP-40 lysis buffer [or RIPA buffer if blotting for GSDMD (see Note 14)] to lyse the cells. Add 1 protease and/or phosphatase inhibitor cocktail to the lysis buffer before use. Rotate the samples for 30 min at 4  C in lysis buffer. Spin at 24,000  g for 10 min. Discard the pellet. Transfer the supernatant to a new tube for analysis. 5. Mix the samples with 4 loading dye, boil for 10 min, and run on 12.5% polyacrylamide gel (see Note 15). 6. Transfer the proteins onto a nitrocellulose membrane and block with 2% milk for 1 h at RT. 7. Probe with appropriate primary antibody overnight at 4  C and secondary antibody for 2 h at RT. 8. Visualize the protein bands with a chemiluminescence HRP substrate (see Note 16).

3.4 Cell Death Assays

1. Measure the release of LDH from the cell to extracellular media upon cell lysis.

3.4.1 LDH Assay

2. Subject 50 μL of fresh supernatant for LDH detection, according to manufacturer’s instruction (see Notes 17–19).

3.4.2 Intracellular ATP Measurement

1. Remove the supernatant from the wells. Rinse the well containing adherent cells with PBS. Add luciferase reagent and incubate at RT with gentle shaking for 10 min in dark. The reagent lyses the cells and reacts with the endogenous ATP to produce light signal. 2. Read the luminescence signal on a plate reader, according to manufacturer’s instructions (see Note 20).

3.5 ASC Speck Microscopy

1. Plate the cells in a cell culture–treated imaging dish and treat the cells with inflammasome stimuli as described above (see Note 21). 2. Wash the adherent cells in the well with 1 PBS once (see Note 22). 3. Fix the cells with 4% paraformaldehyde for 30 min at RT.

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4. Permeabilize the cells with 0.1% Triton-X for 20 min at RT. 5. Block with 10% goat serum for 1 h at RT. 6. Incubate overnight at 4  C with primary anti-ASC antibody. 7. Wash with 3% FBS in PBS twice, followed by gentle shaking on the plate-shaker for 5 min. 8. Stain with fluorescent-labeled secondary antibody for 1 h at RT (see Note 23). 9. Stain the plasma membrane with cholera toxin B Alexa fluor 647 conjugate (CTB) and the nucleus with DAPI. 10. Visualize the cells using a confocal microscope (see Note 24).

4

Notes 1. While extracting bone marrow cells from a mouse, make a snip over the belly and tear the skin all around it and over the legs. The use of scissors is not required, unless necessary. Make the cut above the head of the femur and the pelvic cavity to separate the leg. A common mistake at this stage is to cut the head of the femur. This can result in the loss of many bone marrow cells. Place the legs in a 50-mL sterile Falcon tube with DMEM and take the tubes inside the laminar flow hood for subsequent processing. 2. The heads of the bones are rich in bone marrow cells. Therefore, flushing the heads can increase the yield of cells. 3. L929 cells secrete M-CSF, and their supernatant can be collected and filtered, with aliquots stored in 80  C for culturing BMDMs. Using L929 supernatants can be much more economical than recombinant M-CSF. 4. BMDMs are adherent, spindly, long cells with projections. At Day 3 of the culture, few adherent cells are expected. The floating cells are undifferentiated or dying cells. They should be removed during media replacement. On Day 5 of the culture, the cells are expected to be more confluent. They can sometimes appear round due to high confluency. 5. A quick flow experiment to check the purity of the BMDM culture is advisable, if optimizing the culture for the first time. 6. Primary macrophages are adherent in nature. Plate the macrophages at least 4 h before stimulation to allow them to adhere to the plate surface. It is ideal to leave the cells plated overnight. 7. Priming the cells for 3–4 h before stimulation is required for the transcription of the precursor form of IL-1β, NLRP3, and caspase-11. Pam3CSK4, a TLR2 ligand, signals through the MyD88 pathway and causes transcription of pro-IL-1β and

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NLRP3 but not caspase-11. For measuring responses dependent on caspase-11, priming with poly(I:C) or LPS that activate TRIF signaling through TLR3 and 4 respectively is necessary for its transcription [36]. 8. Spin the plate after adding the bacteria, to pellet the bacteria over the cells. Replace the media with gentamicin containing media after 1–2 h of the infection to kill all the extracellular bacteria. While replacing media post infection, use colorless DMEM without phenol red as the LDH assay is a colorimetric assay. 9. The strength of the inflammasome stimuli determines the duration of the treatment. ATP and nigericin are very strong inducers of inflammasome activation and lead to the production of large amounts of IL-1β within 30 min to 1 h. Particulate stimuli like alum, monosodium urate crystals, silica; nucleic acids like poly(dA:dT), and intracellular bacterial infections like Francisella or Salmonella sp. are moderately strong inducers and require 5–8 h of treatment; extracellular bacterial infection like Enterohemorrhagic E. coli requires 8–16 h of treatment to produce a detectable quantity of active cytokines. Adding stimuli to the cells at different times, starting with the stimulus of the longest duration and ending with the shortest, keeping a common endpoint for supernatant collection, adds efficiency while conducting experiments with numerous different conditions. 10. As discussed above, the stimulus strength is directly proportional to the amount of IL-1β and IL-18 secreted. The supernatants for ELISA should be diluted accordingly to prevent saturation of the ELISA signal. For example, when performing inflammasome stimulation with 1 μg/106 cells transfected poly (dA:dT) the following ELISA dilutions are suggested: 1 in 5 diluted supernatant for IL-1 measurement, undiluted for IL-18, 1 in 50 dilution for IL-6, and 1 in 25 dilution for TNF. 11. It is advisable to use fresh supernatants for ELISA, especially for the weaker inflammasome stimuli. Most ELISA kits require coating of plates overnight before adding the supernatant sample. Therefore, coating the plates one day prior to performing the experiment is helpful. 12. Measurement of cytokines produced upon TLR stimulation such as TNF and IL-6 that are independent of inflammasome activation can serve as good controls for equal cell number across genotypes. 13. While probing for secreted proteins such as caspase-1 or IL-1β cleaved fragment, concentration of the supernatant is required to detect measurable quantities of the proteins.

Assessing Inflammasome Activation in Macrophages

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14. To probe immunoblots for GSDMD cleaved fragment, use a gentle lysis buffer, such as RIPA buffer. 15. As equal cells are plated and treated across genotypes, protein estimation is not required at the time of sample loading is not required. However, to account for experimental errors during cell counting, a constitutive gene like β-actin or GAPDH should be probed for. 16. While developing immunoblots, the heavy chain of immunoglobulin forms a band around 60 kDa and adds considerable background to the blots. Using antibody probes with only the light chain can address this issue. 17. Perform the LDH assay immediately after harvest with fresh supernatants. 18. Keep extra wells of unstimulated cells. Add 10% Triton X-100 to them for 30 min before supernatant collection. Triton-X being a detergent causes rapid cell lysis. This can serve as the positive control for the assay. For negative control, use unstimulated cells with culture media only. 19. The plate is read by a spectrophotometer to assay the absorbance (A) at 490 nm. To calculate the percentage cell death, subtract the average A490 of the technical replicates of the negative control from the A490 of each well (adjusted A490). Calculate the percent cell death as follows: Percent cell death ¼

Adjusted A 490 of experimental well  100 Adjusted A490 of positive control

20. The metabolic state of the cell can also serve as a measurement of cell death. PrestoBlue assay (Invitrogen) employs a resazurin-based reagent that can detect the reducing environment of the cell as a measure of viability by a colorimetric assay. Perform the viability assay as per the manufacturer’s instructions. 21. For optimum level of ASC speck detection, the duration of treatment should allow maximum speck formation without pyroptosis initiation. Therefore, shorter stimulation periods are suggested for this assay. 22. Washing of the wells should be very gentle as cells tend to get washed away, especially when they become pyroptotic. 23. This method describes the use of an unconjugated primary antibody. The primary and secondary antibodies should be titrated, and an isotype control should be used to maximize the signal to noise ratio. 24. For quantitative results, the ASC specks within an average of 20 fields should be counted per sample. As this can be quite labor intensive, a flow cytometry–based assay can serve as an alternative method.

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Acknowledgments I would like to thank Vijay Rathinam, D.V.M., Ph.D. for permitting the use of representative experimental data generated in his laboratory, proofreading the manuscript, and providing his helpful feed-back. I declare no competing financial interests. References 1. Swanson KV, Deng M, Ting JP (2019) The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol 19(8):477–489. https://doi.org/10. 1038/s41577-019-0165-0 2. Miao EA, Mao DP, Yudkovsky N, Bonneau R, Lorang CG, Warren SE, Leaf IA, Aderem A (2010) Innate immune detection of the type III secretion apparatus through the NLRC4 inflammasome. Proc Natl Acad Sci U S A 107(7):3076–3080. https://doi.org/10. 1073/pnas.0913087107 3. Zhao Y, Yang J, Shi J, Gong Y-N, Lu Q, Xu H, Liu L, Shao F (2011) The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477(7366): 5 9 6 – 6 0 0 . h t t p s : // d o i . o r g / 1 0 . 1 0 3 8 / nature10510 4. Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G, Caffrey DR, Latz E, Fitzgerald KA (2009) AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature 458(7237):514–518. https://doi.org/10. 1038/nature07725 5. Yang Y, Wang H, Kouadir M, Song H, Shi F (2019) Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors. Cell Death Dis 10(2):128. https://doi. org/10.1038/s41419-019-1413-8 6. Lamkanfi M, Dixit VM (2014) Mechanisms and functions of inflammasomes. Cell 157(5): 1013–1022. https://doi.org/10.1016/j.cell. 2014.04.007 7. Cai X, Chiu YH, Chen ZJ (2014) The cGAScGAMP-STING pathway of cytosolic DNA sensing and signaling. Mol Cell 54(2): 289–296. https://doi.org/10.1016/j.molcel. 2014.03.040 8. Lu A, Magupalli VG, Ruan J, Yin Q, Atianand MK, Vos MR, Schro¨der GF, Fitzgerald KA, Wu H, Egelman EH (2014) Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell 156(6): 1193–1206. https://doi.org/10.1016/j.cell. 2014.02.008

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Chapter 3 Measuring Non-canonical Inflammasome Activation in Neutrophils Mercedes Monteleone and Dave Boucher Abstract Neutrophils are innate immune cells that play critical functions during infections through diverse mechanisms. One such mechanism, the generation of extracellular traps (NETs), enables direct bacterial killing during infections. We recently reported that the activation of the non-canonical inflammasomes in neutrophils allows for the generation of NETs and is an important host defence mechanism in vivo in response to intracellular Gram-negative bacterium. This process is dependent on inflammatory caspases and the cell death effector Gasdermin D. Here, we describe a simple approach to study the functions of the non-canonical inflammasome in murine neutrophils using microscopy and cellular fragmentation assays. Key words Neutrophils, Non-canonical inflammasome, Gasdermin D, Pyroptosis, Caspase-4, Caspase-11, Neutrophil extracellular traps

1

Introduction Neutrophils are key sentinel cells that protect against infections through a wide range of antimicrobial mechanisms including antimicrobial peptide and specialized cell death modalities [1]. Recognition of intracellular Gram-negative pathogens like Salmonella activate inflammasomes, a signaling pathway that allows the activation of proteases called inflammatory caspases [2, 3]. Salmonella activates the canonical inflammasome following the detection of various ligands including flagellin and type III secretion system proteins [3, 4] and the non-canonical inflammasome upon detection of lipopolysaccharides (LPS) by guanylate-binding proteins and caspase-4/11 [5–7]. Inflammasome activation triggers cytokine maturation and secretion from various cell type and a form of program cell death called pyroptosis [8, 9]. Pyroptosis is executed by a caspase substrate called Gasdermin D (GSDMD) [10]. Upon cleavage by inflammatory caspases, GSDMD forms pores at the plasma membrane and into various organelles, leading to cell

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_3, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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swelling and ultimately loss of cellular integrity [10, 11]. However, certain cell types, like neutrophils, do not undergo pyroptosis. Instead, intracellular LPS detection in neutrophils generate DNA structures called neutrophil extracellular traps (NETs) [12]. NETs are complex antibacterial structures that can trap and kill extracellular bacteria [13]. Upon cleavage, GSDMD will form pores in neutrophil’s nuclear membrane, an event that allows the generation of NETs [12]. This protocol describes how to study neutrophil extracellular trap formation in neutrophils downstream of non-canonical inflammasome using microscopy and simple cellular fragmentation assays.

2

Materials

2.1 Isolation of Murine Neutrophils

1. Mouse (C57BL/6). 2. Low endotoxin fetal calf serum (see Note 1). 3. RPMI 1640, endotoxin tested. 4. Opti-MEM media. 5. 1 M HEPES (tissue culture grade). 6. Red blood cell lysis buffer: 150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA in distilled water, final pH adjusted to 7.2. 7. Neutrophil media: RPMI 1640, 10% heat-inactivated fetal calf serum, 5 mL TC grade 1 M HEPES. 8. Neutrophil enrichment or isolation kit. 9. D-PBS (without Ca2+ Mg2+). 10. Aprotinin: prepare stock at 4 mg/mL in ultrapure sterile water, store at 4
C. 11. 70% ethanol (diluted from 100% ethanol in distilled water). 12. Tissue culture-coated flat bottom 24-well plate. 13. 70-μm pore size cell strainers or mesh. 14. 21-gauge and 27-gauge needles. 15. 10-mL sterile pipettes. 16. 15-mL sterile conical tubes. 17. 12-mm round poly-L-lysin-coated coverslips.

2.2 Activation of Non-canonical Inflammasome in Murine Neutrophils

1. Opti-MEM media. 2. Pam3CSK4: prepare stock at 1 mg/mL in ultrapure water. 3. FuGENE HD transfection reagent. 4. Ultrapure LPS (see Note 2).

Measuring Non-canonical Inflammasome Activation in Neutrophils

2.3 Isolation of Neutrophil Nucleus

31

1. Magnesium chloride (MgCl2). 2. Potassium chloride (KCl). 3. Phenylmethanesulfonyl fluoride (PMSF): prepare 0.2 M stock solution in ethanol. 4. EDTA-free protease inhibitor tablets. 5. IGEPAL CA-630: prepare 10% solution in distilled water. 6. ATP sodium: prepare 1 M stock solution distilled water, aliquot, and store at 80
C. 7. Dithiothreitol (DTT): prepare 1 M stock in distilled water, aliquot, and store at 20
C. 8. Hypotonic buffer: 10 mM HEPES (pH 8.0), 10 mM KCl, 3 mM NaCl, 3 mM MgCl2, 1 mM EDTA (ethylenediaminetetraacetic acid), 1 mM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid), 2 mM DTT, 2 mM PMSF, 2 cOmplete mini tabs/10 mL buffer. Add DTT, PMSF, and cOmplete mini tabs just before use. 9. Horse radish peroxidase (HRP)-coupled secondary antibodies. 10. Mouse GSDMD antibody (Abcam, Cat. No. ab209845). 11. Mouse H3 antibody (Cell Signaling Technology. Cat. No. 96C10). 12. GAPDH antibody. 13. Western blot running buffer 10: 30 g Tris Base, 144 g Glycine, 1% SDS (sodium dodecyl sulfate). 14. 4–20% Tris–glycine precast protein gels. 15. LDS loading buffer (4): 988 mM Tris, 2.04 mM EDTA, 8% LDS (lithium dodecyl sulfate), 40% glycerol, 0.88% Coomassie Brilliant Blue G250, 0.7 mM Phenol red. 16. Whole cell lysis buffer: 66 mM Tris pH 8.0, 2% sodium dodecyl sulfate. 17. Nitrocellulose membrane. 18. 1.5-mL microcentrifuge tubes. 19. Cell scrapers.

2.4 Imaging of Neutrophil Extracellular Traps

1. Tissue culture-coated 24-well plate. 2. Ultra pre DNase/RNase-free distilled water. 3. PBS. 4. 8% Paraformaldehyde solution (PFA; prepared in PBS and stored at 20
C). 5. 0.1% Triton X-100 (prepared in PBS). 6. 1 M NH4Cl solution.

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7. 1% bovine serum albumin (BSA Fraction V; freshly prepared in PBS by diluting 1 g of BSA into 100 mL of PBS). 8. Anti-myeloperoxidase antibody (R&D Systems AF3667). 9. Anti-neutrophil elastase antibody (Abcam, ab131260). 10. Anti-histone H3 antibody (citrulline R2 + R8 + R17, ab5103). 11. Secondary antibodies, fluorescently labeled. 12. Diamidino-2-phenylindole (DAPI) dye: prepare 1 mg/mL stock in distilled water. 13. Mounting media. 14. Microscope slides. 15. Parafilm M flexible film. 16. Aluminum foil. 17. Clear nail polish. 18. Microscopy slides storage box. 2.5

Equipment

1. Imaging system for Western blotting. 2. Dissection scissors. 3. Microscopy tweezers set. 4. Vortex mixer. 5. Inverted fluorescence microscope.

3

Methods

3.1 Non-canonical Inflammasome Activation in Murine Neutrophils

1. Euthanize the mouse in a CO2 gas chamber (or according to the recommendation of your ethics committee (see Note 3). 2. Using 21-gauge needle, pin down the legs of the mice (on its back) on a Styrofoam board. 3. With dissecting scissors, cut the posterior legs at the hip level. Avoid damaging the bone or exposing the bone marrow. Remove the skin and muscle around the bones with the dissection scissors and Kimwipes tissues and put the bones in 70% ethanol. Quickly transfer the bones to a tissue culture hood. 4. Incubate the bones for 15 s in 70% ethanol and transfer the bones into a six-well plate containing 5 mL neutrophil media per well. 5. Cut the end joints of each bone with scissors to gain access to the bone marrow. Cut as little as possible. With a 27-gauge needle, flush the bones using 5 mL neutrophil media into a 50-mL tube through a 70-μM cell strainer to remove aggregates and pieces of tissue. Handle the cells carefully (see Note 4).

Measuring Non-canonical Inflammasome Activation in Neutrophils

33

6. Rinse the cell strainer with 5 mL of neutrophil media. 7. Centrifuge the 50-mL tube containing the flushed bone marrow for 5 min at 500  g at room temperature (RT). 8. Discard the supernatant, resuspend gently the pellet in 2 mL of red blood cell lysis buffer, incubate 3 min at RT, and then fill up to 50 mL with neutrophil media. Centrifuge again for 5 min at 500  g at RT. 9. Purify neutrophils using the Mouse Neutrophil Enrichment or isolation Kit according to the manufacturer instructions. 10. Count the neutrophils and resuspend by pipetting gently to a concentration of 10  106cells/mL, in tepid Opti-MEM supplemented with 4 μg/mL aprotinin. 11. In a TC-coated 24-well plate, seed 300 μL of cells per well to fractionate neutrophils (Subheading 3.3). For microscopy (Subheading 3.4), dilute the neutrophils to 1  106 cells/mL in tepid Opti-MEM supplemented with 4 μg/mL aprotinin and plate 500 μL of cells/well in a 24-well plate containing poly-L-lysin-coated coverslips. Neutrophils can now be treated to activate the non-canonical inflammasome (Subheading 3.2) (see Note 5). 3.2 Activation of the Non-canonical Inflammasome in Mouse Neutrophils

1. To each well, add 30 μL of 10 μg/mL Pam3CSK4 (diluted from 1 mg/mL stock in Opti-MEM). The final concentration of Pam3CSK4 in each well is 1 μg/mL. Put the plate back in the cell culture incubator for 4 h. 2. Preheat the Opti-MEM media (37
C). 3. Following priming, remove the cell media from all the wells, and gently add 270 μL of pre-heated Opti-MEM (supplemented with 4 μg/mL aprotinin) to each well. 4. Prepare the LPS transfection mix as follows: In a 1.5-mL microcentrifuge tube, combine 262.5 μL of Opti-MEM and 30 μL of 1 mg/mL ultrapure LPS stock. Vortex vigorously for 30 s. 5. Add 7.5 μL of FuGENE HD. Mix by flicking the tube. Incubate at RT for 15 min. 6. Prepare a no FuGENE HD and a no LPS transfection control by omitting each respective component. 7. At the end of the incubation, add 30 μL of the transfection mix (or control) to each well accordingly. The final concentration per well is 0.25% FuGENE and 10 μg/mL LPS. Try to add the transfection mix over the whole well. 8. Centrifuge the 24-well plate at 500  g for 5 min at RT (see Note 6).

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9. Place the plate at 37
C in a cell culture incubator. 10. After 4 h, proceed to the isolation of neutrophil nucleus (Subheading 3.3) or to the staining of the cells for microscopy (Subheading 3.4). 3.3 Isolation of Neutrophil Nuclei

1. Following treatment (see Subheading 3.2), remove the cell culture media and add 300 μL of ice-cold hypotonic buffer. Incubate on ice for 15 min. 2. Add 15 μL of a 10% IGEPAL solution and incubate on ice for 2 min. 3. Using a cell scraper, gently detach the cells. Transfer the content of the well into a 1.5-mL microcentrifuge tube. Vortex at slow speed for 15 s. 4. Centrifuge for 10 min at 4
C at 500  g. 5. Transfer the supernatant to a new tube. This contains the cytoplasmic fraction. Add 100 μL of 4 LDS loading buffer containing 40 mM DTT. Keep on ice or freeze at 20
C until usage. 6. Wash the pellet once with 300 μL of hypotonic buffer. Centrifuge for 10 min at 4
C at 500  g. 7. Discard the supernatant. 8. Resuspend the pellet into 30 μL of 1 LDS loading buffer containing 10 mM DTT and prepared in whole cell lysis buffer. This contains the nuclear fraction. Keep on ice or freeze at 20
C until usage. 9. Heat the samples for 3 min at 100
C. 10. On a 4–20% SDS PAGE gel, load 20 μL of cytosolic and nuclear fraction. 11. Run the gels in 1 Western blot running buffer at 150 V until the dye front exits the gel. 12. Transfer proteins from the gel to a nitrocellulose membrane using your preferred transfer method and probe by immunoblot using standard methods to detect GAPDH (1:3000), Histone H3 (1:3000), and GSDMD (1:1000) using appropriate primary and secondary antibodies (see Note 7).

3.4 Imaging of Neutrophil Extracellular Traps

1. Add 500 μL of pre-warmed (37
C) 4% PFA to each well. Add directly to the cell culture media (for a final concentration of 2%). Incubate for 15 min at RT. 2. Discard the PFA/media mix using a P1000 pipette. Wash the coverslips with 500 μL PBS with care. Discard the PBS. 3. Repeat step 2 two more times.

Measuring Non-canonical Inflammasome Activation in Neutrophils

35

4. After the washes, keep the coverslips in PBS to avoid drying. The cells are now ready for immunostaining. All staining steps are performed at RT. 5. Using curved end tweezers, carefully remove the coverslips from the plate and place them onto labeled parafilm in a humidified chamber (e.g., cover the bottom of a tray with a wet paper towel with parafilm placed on top to avoid wrinkles. Cover the tray lid with aluminum foil). Wash the coverslips three times in PBS. 6. Permeabilize the cells by incubating the coverslip (facing upward) with 250 μL 0.1% Triton X-100 for 10 min. Aspirate the Triton X-100 solution and wash the coverslips three times with 250 μL of PBS. Discard PBS in between washes. All further steps are carried out by dispensing reagents onto the parafilm sheet and inverting the coverslips face-down onto the reagent droplets (50 μL per drop). 7. Quench PFA using a freshly prepared 50 mM NH4Cl (diluted in PBS) for 10 min. 8. Invert the coverslip and wash three times with 250 μL PBS. Discard PBS in between washes. 9. Prepare a fresh blocking buffer solution by diluting the stock solution of BSA in PBS to a final concentration 0.5% (w/v). Primary and secondaries antibodies are also incubated in this buffer (see Note 8). 10. Add 50 μL blocking buffer and incubate the coverslip facedown for 30 min. 11. Prepare primary antibody solution in blocking buffer (see Note 8). Dispense a minimum of 30 μL per coverslip and incubate for 1 h at RT. 12. Invert the coverslip and wash four times with 250 μL of PBS, 5 min per wash. 13. Dilute the fluorescent secondary antibodies (see Note 8) and DAPI (1 μg/mL final concentration) in blocking buffer and incubate together for 1 h in the dark (tray lid covered with aluminum foil) at RT. 14. Invert the coverslip and wash four times in PBS, 5 min per wash. 15. To mount the coverslip on a slide, place a drop of mounting media onto a labeled microscope slide. Lower the coverslip face-down onto the drop of mounting media. Remove the excess of media and any air bubbles by pressing down firmly onto the coverslip with squared forceps. Aspirate off excess mounting media around the edge of the coverslip. Seal with clear nail polish and store at 4
C until image acquisition.

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Mercedes Monteleone and Dave Boucher

16. Use an inverted fluorescence microscope to acquire at least five images of random fields (avoiding the coverslip edges) with the adequate filters based on the fluorophores selected (see Note 9). 17. Image processing is made using ImageJ (e.g., % of H3citrullination) or Imaris (e.g., nucleus volume) (see Note 10).

4

Notes 1. The quality of the fetal calf serum is important for this type of experiment. The use of low endotoxin fetal calf serum is essential. We recommend testing the endotoxin level of each fetal calf serum batch before purchasing a large amount. We used Thermo Fisher Cat 16000044. 2. LPS quality is another critical determinant in the success of the experiments. LPS with lower purity can contain impurities that can also activate the canonical inflammasome. We highly recommend ultrapure LPS-EK from Invivogen (Cat tlrl-3pelps or Cat tlrl-peklps) to efficiently and specifically activate the NCI in mouse cells. 3. Before beginning this protocol, it is essential that you have obtained ethical approval for this procedure and that your euthanasia protocol conforms to your institutional guidelines. 4. Neutrophils are very fragile cells. Resuspend them with care and avoid brusque movement and vigorous pipetting. 5. Neutrophils are short-lived cells and need to be used immediately after purification for experiment. They cannot be frozen and experiments must be planned accordingly. 6. The temperature of centrifugation is a key element for the success of this procedure. Centrifugation at temperature colder than RT will lead to an unsuccessful transfection and will fail to activate the non-canonical inflammasome. 7. We routinely used horse radish peroxidase-coupled secondary antibody in 5% fat-free milk/ TBST at a 1:5000 dilution. Transfer methods, the Western blot membrane, and the technique used to image the Western blot are among the parameters that will affect the final detection. 8. We routinely uses secondary antibodies (coupled to Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647) from Molecular Probes, according to manufacturer suggestions. We recommend to optimize secondary antibodies concentration if necessary.

Measuring Non-canonical Inflammasome Activation in Neutrophils

37

9. We use a 40 and a 60 objectives to calculate the percentages of NETosis or citrullination. We use a 100 objective to create representative images for each condition. 10. Image quantification can be done as described previously by the Zychlinsky lab [14].

Acknowledgments M.M. is supported by a Discovery Early Career Research Award (DE200101300). The Boucher lab is supported by a Springboard award (SBF006\1025) from the Academy for Medical Science and institutional funding from the University of York. References 1. Lawrence SM, Corriden R, Nizet V (2020) How neutrophils meet their end. Trends Immunol 41(6):531–544 2. Schroder K, Tschopp J (2010) The inflammasomes. Cell 140(6):821–832 3. Bierschenk D, Boucher D, Schroder K (2017) Salmonella-induced inflammasome activation in humans. Mol Immunol 86:38–43 4. Vance RE (2015) The NAIP/NLRC4 inflammasomes. Curr Opin Immunol 32:84–89 5. Shi J, Zhao Y, Wang Y, Gao W, Ding J, Li P et al (2014) Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514(7521):187–192 6. Santos JC, Boucher D, Schneider LK, Demarco B, Dilucca M, Shkarina K et al (2020) Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria. Nat Commun 11(1):3276 7. Wandel MP, Kim B-H, Park E-S, Boyle KB, Nayak K, Lagrange B et al (2020) Guanylatebinding proteins convert cytosolic bacteria into caspase-4 signaling platforms. Nat Immunol 21(8):880–891 8. Chan AH, Schroder K (2020) Inflammasome signaling and regulation of interleukin-1 family cytokines. J Exp Med 217(1):e20190314

9. Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol 16(7):407–420 10. Broz P, Pelegrı´n P, Shao F (2020) The gasdermins, a protein family executing cell death and inflammation. Nat Rev Immunol 20(3): 143–157 11. de Vasconcelos NM, Van Opdenbosch N, Van Gorp H, Parthoens E, Lamkanfi M (2019) Single-cell analysis of pyroptosis dynamics reveals conserved GSDMD-mediated subcellular events that precede plasma membrane rupture. Cell Death Differ 26(1):146–161 12. Chen KW, Monteleone M, Boucher D, Sollberger G, Ramnath D, Condon ND et al (2018) Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps. Sci Immunol 3(26): eaar6676 13. Burgener SS, Schroder K (2019) Neutrophil extracellular traps in host defense. Cold Spring Harb Perspect Biol 12(7):a037028 14. Gonzalez AS, Bardoel BW, Harbort CJ, Zychlinsky A (2014) Induction and quantification of neutrophil extracellular traps. Methods Mol Biol 1124:307–318

Chapter 4 Gasdermin D Cleavage Assay Following Inflammasome Activation Louisa Janice Kamajaya and Dave Boucher Abstract Gasdermin D (GSDMD) is a recently identified pore-forming protein that is crucial for the execution of pyroptosis, a highly inflammatory form of cell death. GSDMD contains an N-terminal and a C-terminal domain that are separated by a proteolysis-sensitive linker. Upon cleavage of this linker by inflammasomeactivated caspases, the N-terminal domain of GSDMD oligomerizes and forms pores at the plasma membrane, allowing cell swelling and subsequently membrane rupture to mediate pyroptosis. GSDMD is a key substrate of inflammatory caspases downstream of inflammasome activation and is driving various pathologies. Here, we describe a simple method to study GSDMD cleavage following canonical inflammasome activation in murine primary macrophages and neutrophils and human cell lines using immunoblotting. Key words Gasdermin D, Proteolytic cleavage, Caspase-4, Caspase-11, Non-canonical inflammasome, Neutrophils, Macrophages

1

Introduction Gasdermin D (GSDMD) is a 484 amino acid cytoplasmic protein and a member of the Gasdermin [1] protein family. All members of this protein family contain conserved N- and C-terminal domains separated by a flexible linker sensitive to proteolysis by various proteases [1]. GSDMD is expressed in various cell types and tissues [2]. GSDMD is a key executor of a form of cell death called pyroptosis, acting as the final executioner downstream of canonical and non-canonical inflammasomes [2–4]. Recently, GSDMD pores were shown to allow terminal cell lysis through a plasma membrane protein called NINJ1 [5]. GSDMD is a substrate of various inflammatory caspases such as caspase-1, -4, -5, -8, and -11 [4, 6– 8]. Cleavage occurs at D275 (human) or D276 (mouse) within the linker between the C- and N-terminal domains, resulting in an

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_4, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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N-terminal 32 kDa fragment (GSDMD-NT) and a C-terminal 20 kDa fragment (GSDMD-CT) [3]. On the other hand, cleavage of GSDMD by caspase-3/-7 at D87 results in a 43 kDa fragment, which block the pore-forming activity of GSDMD [7, 9]. When liberated from GSDMD-CT, the GSDMD-NT fragment forms pores in various membranes. GSDMD-NT binds strongly to membrane lipids, cardiolipin, phosphoinositide, phosphatidylserine (PS), and phosphatidylinositol phosphates (PIPs), oligomerizes, and forms pores [3, 10]. These pores disrupt electrochemical gradients in the cell, causing pyroptosis and releasing intracellular components such as IL-1β, IL-18, and lactate dehydrogenase (LDH) [11, 12]. GSDMD-NT itself is sufficient to trigger pyroptosis [1, 3, 10]. GSDMD also plays an important role in the secretion, but not the maturation of IL-1β [12–14]. IL-1β along with IL-18 are pro-inflammatory cytokines that are crucial for the innate immune response following an infection [15]. GSDMD cleavage is required for lipopolysaccharide (LPS)induced pyroptosis through the activation of caspase-4/-11 by intracellular LPS independently of Toll-like receptor 4 (TLR4) [4, 6, 16, 17]. This results in the activation of non-canonical NLRP3 inflammasome due to potassium efflux caused by GSDMD-NT fragment [18, 19]. GSDMD cleavage also allows the generation of neutrophil extracellular traps (NETs) upon non-canonical inflammasome activation in neutrophils [20]. In this chapter, we will present protocols that allow for the detection of GSDMD cleavage in murine macrophages and neutrophils and in human cell lines in response to canonical inflammasome activation.

2

Materials

2.1 Generation of Murine Primary Cells

1. Mouse (C57BL/6). 2. Recombinant human macrophage colony-stimulating factor 1 (hM-CSF) (see Note 1). 3. Low-endotoxin fetal calf serum (see Note 2). 4. RPMI 1640, endotoxin tested. 5. DMEM. 6. Opti-MEM media. 7. Penicillin/Streptomycin-Glutamine (100 stock; 10,000 U/ mL Penicillin, 10 mg/mL Streptomycin, 29.2 mg/mL L-Glutamine in 10 mM citrate buffer). 8. Complete mouse bone marrow–derived macrophage media (cBMM media): RPMI 1640, 10% heat-inactivated fetal calf serum, 1% Penicillin/Streptomycin-Glutamine, and 100 ng/ mL hM-CSF freshly added to media.

Gasdermin D Cleavage Assay Following Inflammasome Activation

41

9. Red blood cell lysis buffer (home-made 150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA in H2O, final pH adjusted to 7.2 or commercial). 10. Neutrophil media: RPMI 1640, 10% heat-inactivated fetal calf serum, 5 mL TC grade 1 M HEPES. 11. EasySep neutrophil enrichment kit. 12. DPBS. 13. TC grade 1 M HEPES. 14. Aprotinin (4 mg/mL, in ultrapure sterile water). 15. EtOH (70%, in distilled water). 16. TC-coated flat bottom 96-well plate. 17. Square 100-mm petri dishes, Sterilin. 18. 70-μm pore size cell strainers or mesh. 19. 18-G blunt needles. 20. 21-G needles. 21. 27-G needles. 22. 10-mL sterile pipettes. 23. 15-mL sterile conical tubes. 2.2 Human Cell Line Culture

1. THP-1 cells (ATCC TIB-202). 2. Phorbol 12-myristate 13-acetate (resuspended in DMSO at 10 μg/mL). 3. Low-endotoxin fetal calf serum (see Note 2). 4. RPMI 1640, endotoxin tested. 5. Penicillin/Streptomycin-Glutamine (100 stock; 10,000 U/ mL Penicillin, 10 mg/mL Streptomycin, 29.2 mg/mL L-Glutamine in 10 mM citrate buffer). 6. Complete THP-1 media: RPMI 1640, 10% heat-inactivated fetal calf serum, 1% Penicillin/Streptomycin-Glutamine. 7. Trypsin/EDTA solution. 8. T75 Tissue culture flask. 9. TC-coated flat bottom 96-well plate. 10. 10-mL sterile pipettes. 11. 15-mL sterile conical tubes.

2.3 Canonical Inflammasome Activation

1. Opti-MEM media. 2. Pam3CSK4 (in ultrapure water at 1 mg/mL). 3. Nigericin (5 mM stock in EtOH). 4. Recombinant 100 μg/mL).

flagellin

(prepared

in

ultrapure

water,

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Louisa Janice Kamajaya and Dave Boucher

5. FuGENE HD transfection reagent. 6. Lipofectamine LTX transfection reagent. 7. VX765 (prepared at 10 mM, in DMSO). 2.4

Immunoblotting

1. Western blot running buffer 10 (30 g Tris Base, 144 g Glycine, 1% SDS). 2. MeOH. 3. Chloroform. 4. Mouse Caspase-1 p20 (Casper-1) antibody (Adipogen, Cat AG-20B-0042-C100). 5. Human Caspase-1 antibody clone D7F10 (Cell Signaling Technology, Cat 3866S). 6. Human GSDMD antibody CSB-PA009956LA01HU).

(Cusabio,

Cat

7. Mouse GSDMD antibody (Abcam, Cat ab209845). 8. Human/mouse tubulin antibody (Sigma-Aldrich, Cat T5168). 9. 12% Mini-PROTEAN® TGX™ Precast Protein Gels. 10. NuPAGE LDS loading buffer (4) (Thermo Fisher, Cat NP007). 11. Dithiothreitol (prepared at 1 M in distilled water). 12. Whole-cell lysis buffer: 66 mM Tris pH 8.0, 2% sodium dodecyl sulfate. 13. Nitrocellulose membrane. 14. 1.5-mL microcentrifuge tubes. 2.5

Equipment

1. Imaging system. 2. Dissection scissors.

3

Canonical Inflammasome Activation in Murine Macrophages and Neutrophils This section of the protocol will describe how to activate the NLRP3 and NLRC4 inflammasomes in primary macrophages and neutrophils.

3.1 Purification of Murine Primary Neutrophils and Macrophages 3.1.1 Purification of Murine Primary Neutrophils

1. Euthanize the mouse in a CO2 gas chamber (or according to the recommendation of your ethics committee; see Note 3). 2. Using 21-G needle, pin down the legs of the mice (on its back) on a Styrofoam board. 3. With dissecting scissors, cut the posterior legs at the hip level. Do not damage the bone or expose the bone marrow. Remove the skin and muscle around the bones with the scissors and

Gasdermin D Cleavage Assay Following Inflammasome Activation

43

Kimwipes tissues and put the bones in ethanol 70%. Quickly transfer the bones to a tissue culture hood. 4. Incubate the bones 15 s in ethanol 70% and transfer the bones in a six-well plate containing 5 mL RPMI media (unsupplemented) per well. 5. Cut the end joints of each bone with scissors to gain access to the bone marrow. With a 27-G needle, flush the bones using 5 mL RPMI media into a 50-mL tube through a 70-μM cell strainer to remove aggregates and tissue. Treat the cells with care (see Note 4). 6. Rinse the cell strainer with 5 mL of RPMI media. 7. Centrifuge the 50-mL tube containing the flushed bone marrow for 5 min at 500  g at room temperature (RT). 8. Discard the supernatant, resuspend gently the pellet in 2 mL of red blood cell lysis buffer, incubate 3 min at RT, and then fill up to 50 mL with neutrophil media. Centrifuge again at 5 min at 500  g at RT. 9. Purify neutrophils using the EasySep™ Mouse Neutrophil Enrichment Kit according to the manufacturer instructions. 10. Count the neutrophils and resuspend them at a concentration of 10  106 cells/mL in tepid Opti-MEM supplemented with aprotinin (4 μg/mL). 11. In a 96-well plate, seed 100 μL of cells. Neutrophils can then be used for inflammasome assay (Subheading 3.1.2) (see Note 5). 3.1.2 Generation of Primary Murine Bone Marrow Macrophages

1. Euthanize the mouse in a CO2 gas chamber (or according to the recommendation of your ethics committee; see Note 3). 2. Using 21-G needle, pin down the legs of the mice (on its back) on a Styrofoam board. 3. With dissecting scissors, cut the posterior legs at the hip level. Do not damage the bone or expose the bone marrow. Remove the skin and muscle around the bones with the scissors and Kimwipes tissues and put the bones in ethanol 70%. Transfer the bones to a cell culture hood. 4. Cut the end joints of each bone with scissors to gain access to the bone marrow. With a 27-G needle, flush the bones using 5 mL RPMI media (unsupplemented) into a 50-mL tube through a 70-μM cell strainer to remove aggregates and tissue. 5. Rinse the cell strainer with 5 mL of RPMI media. 6. Centrifuge the 50-mL tube containing the flushed bone marrow for 5 min at 500  g at room temperature (RT).

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Louisa Janice Kamajaya and Dave Boucher

7. Discard the supernatant, resuspend the pellet in 2 mL of red blood cell lysis buffer, incubate 3 min at RT, and then fill up to 50 mL with cBMM media. Centrifuge again at 5 min at 500  g at RT (see Note 6). 8. Resuspend the cells in cBMM media and plate into 6 square Sterilin dishes to allow cell differentiation for 6 days. 9. On the fourth day of differentiation, supplement the media with 5 mL of cBMM media (with MCSF-1). 10. On the sixth day of differentiation, remove the media and add 10 mL of DPBS to the plate. Incubate 2 min at RT. 11. Using a 18-G blunted needle and a 10-mL syringe, gently detach the BMM and transfer into a 50-mL Falcon tube. Rinse the plate with an additional 5 mL of DPBS to ensure that you are taking out all the cells. 12. Using a hemocytometer, count the cells and resuspend them at 1  106 cells/mL in cBMM (supplemented with MCSF-1). 13. Plate 300 μL of cells in a 24-well TC-treated plate and put back the plate overnight. 3.2 Activation of Murine Inflammasome 3.2.1 Activation of the NLRC4 Inflammasome

1. After purification of your primary cells (as described in Subheading 3.1), prime your cells by adding 1 μg/mL of Pam3CSK4. Put the cell back in the incubator for 4 h. 2. 1 h after the end of the priming, add 10 μM of VX-765 to the caspase control well. 3. Preheat Opti-MEM media (37  C). 4. Remove the media in each well and add 90 μL of pre-heated Opti-MEM. Where appropriate, add VX-765. 5. 30 minutes before the end of the priming incubation, start preparing the transfection mix in a 1.5-mL microcentrifuge tube. (a) To 100 μL of tepid Opti-MEM, add 1 μL of flagellin (stock at 100 ng/μL). (b) Add 2.5 μL of FuGENE HD and incubate 20 min at RT. Also prepare a control reaction that only contain FuGENE HD. (c) Add 10 μL/well of the transfection mix to a 96-well plate (neutrophil) or 30 μL/well to a 24-well plate (macrophage). (d) Centrifuge the plate at RT for 5 min (see Note 7). 6. Put back the plate in the incubate for 5 h. 7. At the end of the incubation, pull together the supernatant of your triplicates in a fresh microcentrifuge tube. Add 30 μL (96-well) or 100 μL (24-well) of boiling lysis buffer per well.

Gasdermin D Cleavage Assay Following Inflammasome Activation

45

From this step, you can freeze your samples in a 20  C or 80  C (cell extract and supernatant) or proceed immediately to immunoblotting (see Subheading 5). 3.2.2 Activation of the NLRP3 Inflammasome

1. After purification of your primary cells (as described in Subheading 3.1), prime your cells by adding 1 μg/mL of Pam3CSK4. Put the cell back in the incubator for 4 h. 2. 1 h after the end of the priming, add 10 μM of VX-765 to the caspase control well. 3. Preheat Opti-MEM media (37  C). 4. Remove the media in each well and add 90 μL of preheated Opti-MEM. Where appropriate, add VX-765. 5. In a 1.5-mL microcentrifuge tube, add 2 μL of Nigericin (stock at 5 mM, final concentration in the well at 5 μM) to 198 μL of warm Opti-MEM. 6. Where indicated, add 10 μL of the solution prepared in step 4 to the 90 μL of Opti-MEM already in the well. 7. Put the cells back in the incubator for 2 h (macrophages) or 5 h (neutrophils). 8. At the end of the incubation, pull together the supernatant of your triplicates in a fresh microcentrifuge tube. Add 30 μL of boiling lysis buffer per well. From this step, you can freeze your samples in a 20  C or 80  C (cell extract and supernatant) or proceed immediately to immunoblotting (see Subheading 5).

4

Activation of the Canonical Inflammasome in Human Cell Lines This section of the protocol will describe how to differentiate THP1 cells and how to activate the NLRP3 inflammasome.

4.1 Preparation of THP-1 Cells

1. Culture THP-1 cells in THP-1 media (see details in Subheading 2) in T75 flasks. When the cells reach a density of 1,000,000 cells/mL, dilute them in fresh media to a density of 0.2  106 cells/mL. 2. Using a sterile pipette, centrifuge 15 mL of cells at 500  g for 5 min at RT. 3. Discard the supernatant and resuspend the cells in 1 mL of THP-1 media. 4. Count the cells and adjust the total volume to a concentration of 700,000 cells/mL. 5. Add PMA to a final concentration of 10 ng/mL and mix gently using a sterile pipette. 6. Plate 100 μL of the cell suspension (70,000 cells) per well, into a flat bottom 96-well cell culture plate.

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7. Put the cells back in a cell culture incubator and allow to differentiate for 48 h. 8. After differentiation, remove the media and replace with fresh THP-1 media without PMA. Put the cells back in the incubator overnight. The cells are now ready to be used for inflammasome activation (see Subheading 4.2). 4.2 Activation of the NLRP3 Inflammasome in THP-1

1. After purification of your primary cells (as described in Subheading 4.1), prime your cells by adding 1 μg/mL of Pam3CSK4. Put the cell back in the incubator for 4 h. 2. 1 h after the end of the priming, add 10 μM of VX765 to the caspase control well. 3. Preheat Opti-MEM media (37  C). 4. Remove the media in each well, and add 90 μL of pre-heated Opti-MEM. Where appropriate, add VX765. 5. In a 1.5-mL microcentrifuge tube, add 4 μL of Nigericin (stock at 5 mM, final concentration in the well at 10 μM) to 198 μL of warm Opti-MEM. 6. Where indicated, add 10 μL of the solution prepared in step 4 to the 90 μL of Opti-MEM already in the well. 7. Put the cells back in the incubator for 3 h. 8. At the end of the incubation, pull together the supernatant of your triplicates in a fresh microcentrifuge tube. Add 30 μL of boiling lysis buffer per well. From this step, you can freeze your samples in a 20  C or 80  C (cell extract and supernatant) or proceed immediately to immunoblotting (see Subheading 5).

5

Measuring GSDMD Cleavage Immunoblotting is the easiest method to monitor GSDMD cleavage in cells treated with inflammasome triggers.

5.1 GSDMD Immunoblotting

1. Combine cell lysis extracts from triplicate wells. To 75 μL of cell lysate, add 25 μL of 4 LDS loading dye, to mouse and human lysates. Add DTT to a final concentration 10 mM. 2. Heat samples for 3 min at 100  C. 3. Combine cell supernatants and precipitate using methanolchloroform extraction, as previously described [21] and as follows: (a) To 300 μL of supernatants, add 300 μL of MeOH and 100 μL of Chloroform. Vortex vigorously for 15 s. (b) Centrifuge at 17,000  g for 15 min at RT.

Gasdermin D Cleavage Assay Following Inflammasome Activation

47

Table 1 Recommended antibodies to study GSDMD cleavage Target

Antibody

Dilution

Expected molecular weight (kDa)

hCasp1

D7F10

1/1000

45 (FL); 33 (active); 20 (active)

mCasp1

casper-1

1/1000

45 (FL); 33 (active); 20 (active)

hGSDMD

4B9

1/1000

52(FL); 30 (cleaved)

mGSDMD

EPR18628

1/1000

52(FL); 30 (cleaved)

Tubulin

B-5-1-2

1/5000

52

Human (h) and mouse (m) antibodies recommended to detect full-length proteins (FL) and their active cleaved species (cleaved)

(c) Discard the upper phase carefully (do not disturb the protein interphase). (d) Add 500 μL of MeOH and vortex 1 s. (e) Centrifuge at 17,000  g for 15 min at RT. (f) Discard the soluble phase and let the protein pellet dry 15 min. (g) Resuspend the protein pellet in 30 μL of lysis buffer. Add 10 μL of 4 LDS loading buffer. Add DTT to final concentration 10 mM. 4. On a 4–20% SDS PAGE gel, load 15 μL of cell extract or supernatant fraction. 5. Run the gels in the Tris–glycine buffer at 150 V until the dye front exits the gel. 6. Transfer proteins from the gel to a nitrocellulose membrane using your preferred transfer method and probe by immunoblot using standard methods to detect caspase-1 and GSDMD cleavage. Note that cleaved forms can be detected in both the cell lysate and in the supernatant as a result of pyroptosis (see Notes 8 and 9). The expected molecular weights for each protein are presented in Table 1.

6

Notes 1. We recommend optimizing the concentration of hM-CSF-1 for bone marrow macrophage differentiation. While the concentration recommended here works well with hM-CSF-1 from Immunotools (Cat 11343118), hmCSF-1 from other suppliers might differentiate cells with a different efficiency. 2. The quality of the fetal calf serum is important for this type of experiment. The use of low endotoxin fetal calf serum is

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essential. We recommend testing the endotoxin level of each fetal calf serum batch before purchasing a large amount. We used Thermo Fisher, Cat 16000044. 3. Before beginning this protocol, it is essential that you have obtained ethical approval for this procedure and that your euthanasia protocol conforms to your institutional guidelines. 4. Neutrophils are very fragile cells. Resuspend them with care and avoid brusque movement and vigorous pipetting. 5. Neutrophils are short-lived cells and need to be used immediately for experiment. They cannot be frozen, and experiments must be planned accordingly. 6. If needed, the bone marrow can be frozen after flushing the bones. Red blood cells do not need to be lysed for this procedure. We routinely resuspend bone marrow from one mouse in 2 mL of freezing media (10% DMSO, 90% FCS). When replating frozen bone marrow, replate one vial of cells in two Sterilin plates. 7. The temperature of centrifugation is a key element for the success of this procedure. Centrifugation at temperature colder than RT will lead to an unsuccessful transfection and will fail to activate the NLRC4 inflammasome. 8. To facilitate detection of cleaved GSDMD, concentrated cell supernatant can be resuspended in 30 μL of sample buffer (1 + DTT) and loaded separately from the cell extract fraction. 9. Neutrophils cleave less GSDMD following canonical inflammasome activation potentially because of smaller canonical inflammasome [22]. Non-canonical inflammasome activation (as described in this book by Bezbradica and colleagues, Chapter 5) enables higher GSDMD cleavage.

Acknowledgments L.J.K. was supported by a Laidlaw Scholarship from the Laidlaw Foundation. The Boucher lab is supported by a Springboard award (SBF006\1025) from the Academy for Medical Science and institutional funding from the University of York. References 1. Broz P, Pelegrı´n P, Shao F (2020) The gasdermins, a protein family executing cell death and inflammation. Nat Rev Immunol 20(3):143–157 2. Feng S, Fox D, Man SM (2018) Mechanisms of Gasdermin family members in inflammasome signaling and cell death. J Mol Biol 430(18 Pt B):3068–3080

3. Ding J, Wang K, Liu W, She Y, Sun Q, Shi J et al (2016) Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535(7610):111–116 4. Kayagaki N, Stowe IB, Lee BL, O’Rourke K, Anderson K, Warming S et al (2015) Caspase11 cleaves gasdermin D for non-canonical

Gasdermin D Cleavage Assay Following Inflammasome Activation inflammasome signalling. Nature 526(7575):666–671 5. Kayagaki N, Kornfeld OS, Lee BL, Stowe IB, O’Rourke K, Li Q et al (2021) NINJ1 mediates plasma membrane rupture during lytic cell death. Nature 591:131–136 6. Shi J, Zhao Y, Wang K, Shi X, Wang Y, Huang H et al (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526(7575):660–665 7. Chen KW, Demarco B, Heilig R, Shkarina K, Boettcher A, Farady CJ et al (2019) Extrinsic and intrinsic apoptosis activate pannexin-1 to drive NLRP3 inflammasome assembly. EMBO J 38(10):e101638 8. Orning P, Weng D, Starheim K, Ratner D, Best Z, Lee B et al (2018) Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death. Science 362(6418):1064–1069 9. Rogers C, Fernandes-Alnemri T, Mayes L, Alnemri D, Cingolani G, Alnemri ES (2017) Cleavage of DFNA5 by caspase-3 during apoptosis mediates progression to secondary necrotic/pyroptotic cell death. Nat Commun 8:14128 10. Liu X, Zhang Z, Ruan J, Pan Y, Magupalli VG, Wu H et al (2016) Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535(7610):153–158 11. Kovacs SB, Miao EA (2017) Gasdermins: effectors of pyroptosis. Trends Cell Biol 27(9):673–684 12. Monteleone M, Stanley AC, Chen KW, Brown DL, Bezbradica JS, von Pein JB et al (2018) Interleukin-1β maturation triggers its relocation to the plasma membrane for GasderminD-dependent and -independent secretion. Cell Rep 24(6):1425–1433 13. Evavold CL, Ruan J, Tan Y, Xia S, Wu H, Kagan JC (2018) The pore-forming protein Gasdermin D regulates interleukin-1 secretion

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from living macrophages. Immunity 48(1):35–44.e6 14. Heilig R, Dick MS, Sborgi L, Meunier E, Hiller S, Broz P (2018) The Gasdermin-D pore acts as a conduit for IL-1β secretion in mice. Eur J Immunol 48(4):584–592 15. Chan AH, Schroder K (2020) Inflammasome signaling and regulation of interleukin-1 family cytokines. J Exp Med 217(1):e20190314 16. Kayagaki N, Wong MT, Stowe IB, Ramani SR, Gonzalez LC, Akashi-Takamura S et al (2013) Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341(6151):1246–1249 17. Santos JC, Boucher D, Schneider LK, Demarco B, Dilucca M, Shkarina K et al (2020) Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria. Nat Commun 11(1):3276 18. Baker PJ, Boucher D, Bierschenk D, Tebartz C, Whitney PG, D’Silva DB et al (2015) NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5. Eur J Immunol 45(10):2918–2926 19. Ru¨hl S, Broz P (2015) Caspase-11 activates a canonical NLRP3 inflammasome by promoting K(+) efflux. Eur J Immunol 45(10):2927–2936 20. Chen KW, Monteleone M, Boucher D, Sollberger G, Ramnath D, Condon ND et al (2018) Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps. Sci Immunol 3(26): eaar6676 21. Gross O (2012) Measuring the inflammasome. Methods Mol Biol 844:199–222 22. Boucher D, Monteleone M, Coll RC, Chen KW, Ross CM, Teo JL et al (2018) Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity. J Exp Med 215(3):827–840

Chapter 5 Activation of the Non-canonical Inflammasome in Mouse and Human Cells Jelena S. Bezbradica, Rebecca C. Coll, and Dave Boucher Abstract The non-canonical inflammasome is a signaling platform that allows for the detection of cytoplasmic lipopolysaccharides (LPS) in immune and non-immune cells. Upon detection of LPS, this inflammasome activates the signaling proteases caspase-4 and -5 (in humans) and caspase-11 (in mice). Inflammatory caspases activation leads to caspase self-processing and the cleavage of the pore-forming protein Gasdermin D (GSDMD). GSDMD N-terminal fragments oligomerize and form pores at the plasma membranes, leading to an inflammatory form of cell death called pyroptosis. Here, we describe a simple method to activate the non-canonical inflammasome in myeloid and epithelial cells and to measure its activity using cell death assay and immunoblotting. Key words Non-canonical inflammasome, Caspase, Caspase-11, Caspase-4, Gasdermin D, Pyroptosis, Lactate dehydrogenase, Lipopolysaccharides, Macrophages, Monocytes, Epithelial cells

1

Introduction The inflammasome [1, 2] is an immune signaling platform that activates a family of proteases called the inflammatory caspases [3]. Canonical inflammasomes are activated by cytosolic sensors that recognize a wide range of pathogen-derived and self-derived molecules, which generally indicate the loss of cellular homeostasis [3, 4]. Canonical inflammasomes activate the effector enzyme caspase-1, which cleaves the proinflammatory cytokines Interleukin (IL)-1β and IL-18 into their bioactive forms that are then secreted [5, 6]. Caspase-1 also cleaves the pore-forming protein Gasdermin D (GSDMD). The cleaved N-terminus of GSDMD oligomerizes to create large pores in the plasma membrane, leading to cell swelling and an inflammatory form of lytic cell death called pyroptosis [7]. Recently, a novel inflammasome that recognizes the presence of cytosolic bacterial lipopolysaccharide (LPS) from Gram-negative bacteria was identified: the non-canonical inflammasome (NCI)

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_5, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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Fig. 1 The non-canonical inflammasome pathway. Cytosolic LPS is sensed by GBPs and caspase-4/11. Active caspase-4/11 cleaves GSDMD causing membrane pore formation. GSDMD pores allow the efflux of cellular contents such as LDH and potassium ions ultimately causing pyroptotic cell death. Potassium efflux from the cell also triggers NLRP3 activation and formation of the inflammasome which activates caspase-1. Caspase-1 cleaves pro-IL-1β and pro-IL-18 to their bioactive forms that are secreted

[8–11]. The NCI is assembled upon the detection of LPS by members of the guanylate-binding protein (GBP) family, which allows the recruitment of inflammatory caspases to the surface of cytosolic Gram-negative bacteria or to outer membrane vesicles secreted by these bacteria. The expression of GBPs is typically induced by TLR ligands or interferon-γ [12–15]. The NCI activates caspase-11 in mice and caspase-4 and caspase-5 in human cells. The activation of these caspases is often associated with selfprocessing [16–18]. Caspases-4/-5/-11 cleave the pore-forming protein GSDMD, to induce pyroptotic death of infected cells. GSDMD pores also drive potassium efflux, which indirectly activates the canonical NLRP3 inflammasome and NLRP3-dependent secretion of IL-1β and IL-18 (Fig. 1) [19, 20]. NCI activity can be inhibited using the inflammatory caspase inhibitor VX-765 [21], whereas cytokine secretion can be inhibited using the NLRP3specific inhibitor MCC950 [22]. Much of what we know about NCI is derived from studies of macrophages and epithelial cells, but the NCI can have specialized functions in other cell types such as neutrophils [23]. In this chapter, we will describe how to activate the NCI in primary mouse macrophages and human monocytic and epithelial cell lines. We will also detail how to monitor NCI activation by using immunoblotting to assess caspase activation and by measuring cell death (LDH release). GSDMD cleavage can also be used to monitor NCI activation, and a protocol to measure this is

Activation of the Non-canonical Inflammasome in Mouse and Human Cells

53

described in this volume (Kamalaya and Boucher, Chapter 4). The whole protocol should be executed within 2 weeks for mouse cells and within 1 week for human cell lines.

2

Materials

2.1 Generation of Primary Mouse Macrophages

1. Mouse (C57BL/6). 2. Recombinant human macrophage colony-stimulating factor 1 (hM-CSF) (see Note 1). 3. Low-endotoxin fetal calf serum (see Note 2). 4. RPMI 1640, endotoxin tested. 5. DMEM. 6. Penicillin/Streptomycin-Glutamine (100
stock; 10,000 U/ mL Penicillin, 10 mg/mL Streptomycin, 29.2 mg/mL L-Glutamine in 10 mM citrate buffer). 7. Complete mouse bone marrow–derived macrophage media (cBMM media): RPMI 1640, 10% heat-inactivated fetal calf serum, 1% Penicillin/Streptomycin-Glutamine, and 100 ng/ mL hM-CSF freshly added to media. 8. Red blood cell lysis buffer (home-made 150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA in H2O, final pH adjusted to 7.2 or commercial). 9. DPBS. 10. Ethanol (70%, in distilled water). 11. TC-coated flat bottom 96-well plate. 12. Square 100-mm petri dishes, Sterilin. 13. 70-μm pore size cell strainers or mesh. 14. 18-G blunt needles. 15. 21-G needles. 16. 27-G needles. 17. 10-mL sterile pipettes. 18. 15-mL sterile conical tubes. 19. Cell counter or hemocytometer.

2.2 Human Cell Line Culture

1. HeLa cells (ATCC CCL2). 2. THP-1 cells (ATCC TIB-202). 3. Phorbol 12-myristate 13-acetate (resuspended in DMSO at 10 μg/mL). 4. Low-endotoxin fetal calf serum (see Note 2). 5. RPMI 1640, endotoxin tested.

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6. DMEM. 7. Penicillin/Streptomycin-Glutamine (100
stock); 10,000 U/ mL Penicillin, 10 mg/mL Streptomycin, 29.2 mg/mL L-Glutamine in 10 mM citrate buffer). 8. Complete THP-1 media: RPMI 1640, 10% heat-inactivated fetal calf serum, 1% Penicillin/Streptomycin-Glutamine. 9. Complete HeLa media: DMEM, 10% heat-inactivated fetal calf serum, 1% Penicillin/Streptomycin-Glutamine. 10. Trypsin/EDTA solution. 11. T75 Tissue culture Flask. 12. TC-coated flat bottom 96-well plate. 13. 10-mL sterile pipettes. 14. 15-mL sterile conical tubes. 2.3 Non-canonical Inflammasome Activation

1. Opti-MEM media. 2. Pam3CSK4 (in ultrapure water at 1 mg/mL). 3. Ultrapure EK-LPS (prepared in ultrapure water, 5 mg/mL) (see Note 3). 4. Human Interferon-γ (see Note 4). 5. FuGENE HD transfection reagent. 6. Lipofectamine LTX transfection reagent. 7. VX765 (prepared at 10 mM, in DMSO). 8. MCC950/CP-456,773 sodium salt (prepared at 10 mM in water).

2.4 Non-canonical Inflammasome Measurement

1. Western blot running buffer 10
(30 g Tris Base, 144 g Glycine, 1% SDS). 2. MeOH. 3. Chloroform. 4. CytoTox 96® Non-Radioactive Cytotoxicity Assay, LDH assay. 5. Human IL-1β ELISA kit. 6. Mouse IL-1β ELISA kit. 7. Mouse Caspase-1 p20 (Casper-1) antibody (Adipogen, Cat AG-20B-0042-C100). 8. Human Caspase-1 antibody clone D7F10 (Cell Signaling Technology, Cat 3866S). 9. Mouse Caspase-11 antibody (Abcam, Cat EPR18628). 10. Human Caspase-4 antibody clone 4B9 (Santa Cruz Biotechnology, Cat sc56056). 11. Human/mouse T5168).

Tubulin

antibody

(Sigma-Aldrich,

Cat

Activation of the Non-canonical Inflammasome in Mouse and Human Cells

55

12. 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels. 13. NuPAGE LDS loading buffer (4
). 14. Dithiothreitol (prepared at 1 M in distilled water). 15. Whole-cell lysis buffer: 66 mM Tris pH 8.0, 2% sodium dodecyl sulfate.

3

Activation of the Non-canonical Inflammasome in Mouse Macrophages

3.1 Production of Primary Mouse Bone Marrow-Derived Macrophages

1. Euthanize the mouse in a CO2 gas chamber (or according to the recommendation of your ethics committee; see Note 5). 2. Put the mouse on its back and pin down the legs on a board using a 21-G needle. 3. With dissecting scissors, cut the posterior legs at the hip level, carefully to not damage the bone or expose the bone marrow. Remove the skin and muscle around the bones with the scissors and put bones in ethanol 70%. Transfer the bones to a cell culture hood. 4. Cut the end joints of each bone with scissors to gain access to the bone marrow. With a 27-G needle, flush the bones using 5 mL RPMI media (unsupplemented) into a 50-mL tube through a 70-μM cell strainer to remove aggregates and tissue. 5. Rinse the cell strainer with 5 mL of RPMI media. 6. Centrifuge the 50-mL tube containing the flushed bone marrow for 5 min at 500
g at room temperature (RT). 7. Discard the supernatant, resuspend the pellet in 2 mL of red blood cell lysis buffer, incubate 2–3 min at RT, and then fill up to 50 mL with cBMM media. Centrifuge again at 5 min at 500
g at RT (see Note 6). 8. Plate the bone marrow cells in four Sterilin non-TC treated plates (15 mL per plate). Put the cells in a cell culture incubator at 37  C, 5% CO2. This is about seven million cells per plate when working from freshly isolated bone marrow (see Note 6). The plating day is counted as day zero of differentiation. 9. On day 4 of the differentiation, add 5 mL of fresh cBMM media to each plate, without removing any media, and put back the cells in the incubator. 10. On day 6 of the differentiation, remove the media and add 10 mL of cold sterile DPBS. Leave the cold DPBS on cells for 3–5 min. 11. Using an 18-G blunt syringe, flush cells to detach them from the plates and transfer to a 50-mL Falcon tube. 12. Rinse each plate with 5 mL of cold sterile DPBS.

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Fig. 2 Schematic 96-well plate layout for a non-canonical inflammasome experiment

13. Centrifuge for 5 min at 500
g at RT. 14. Count the cells (using a cell counter or hemocytometer). 15. Add 100,000 cells per well of a flat bottom 96-well cell culture plate in 100 μL of cBMM media and place in the cell culture incubator overnight. For a properly controlled experiment, we suggest plating cells as illustrated in Fig. 2. The cells are now ready to be used for non-canonical inflammasome activation. 3.2 LPS Transfection in Mouse Cells

1. To each well, add 10 μL of Pam3CSK4 (10 μg/mL, diluted in cBMM media). The final concentration of Pam3CSK4 in the well is 1 μg/mL. Put the plate back in the cell culture incubator for 4 h. 2. Preheat Opti-MEM media (37  C). 3. Remove cBMM media from all the wells, and add 90 μL of preheated Opti-MEM to each well. 4. Prepare the LPS transfection mix as follows. In a 1.5-mL microcentrifuge tube, combine 95.5 μL of Opti-MEM and 2 μL of ultrapure LPS stock (stock concentration is 1 mg/ mL). Vortex vigorously for 30 s. 5. Add 2.5 μL of FuGENE HD. Mix by flicking the tube. Incubate at RT for 15 min. 6. Prepare a no FuGENE HD and a no LPS transfection control by omitting each respective component.

Activation of the Non-canonical Inflammasome in Mouse and Human Cells

57

7. At the end of the incubation, add 10 μL of the transfection mix (or control) to each well. The final concentration in each well is 0.25% FuGENE and 2 μg/mL LPS. 8. Centrifuge the 96-well plate at 500
g for 5 min at RT (see Note 7). 9. After 4 h, collect supernatants, and lyse cells in 50 μL of wholecell lysis buffer per well. 10. Measure cell death using LDH release assay (see Note 8), as described in the protocol in Subheading 5. 11. Measure IL-1β and IL-18 secretion using ELISA, according to the manufacturer’s recommendation. 12. Measure cleavage of Caspase-11 and Caspase-1 by immunoblotting, in cell lysates and supernatants, as described in the protocol in Subheading 5.

4

Activation of NCI in Human Cell Lines This section of the protocol will describe how to activate the NCI in human cell lines.

4.1 Preparation of THP-1 Cells

1. Culture THP-1 cells in THP-1 media (see details in Subheading 2) in T75 flasks. When the cells reach a density of 1,000,000 cells/mL, dilute them in fresh media to a density of 0.2
106 cells/mL (see Note 9). 2. Using a sterile pipette, centrifuge 15 mL of cells at 500
g for 5 min at RT. 3. Discard the supernatant and resuspend the cells in 1 mL of THP-1 media. 4. Count the cells and adjust the total volume to a concentration of 700,000 cells/mL. 5. Add PMA to a final concentration of 10 ng/mL and mix gently using a sterile pipette. 6. Plate 100 μL of the cell suspension (70,000 cells) per well, into a flat bottom 96-well cell culture plate. 7. Put the cells back in a cell culture incubator and allow to differentiate for 48 h. 8. After differentiation, remove the media and replace with fresh THP-1 media without PMA. Put the cells back in the incubator overnight. The cells are now ready to be used for inflammasome activation (see Subheading 4.3).

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4.2 Preparation of HeLa Cells

1. Culture HeLa cells in complete HeLa medium in a T75 flask. Passage the cells when they are 70–75% confluent. 2. The day prior to the experiment, remove the cell media and gently rinse the cells with 5 mL of prewarmed (37  C) DPBS. Do not detach the cells. 3. Remove the DPBS and add 2 mL of Trypsin/EDTA (37  C). Incubate for 3 min at RT. 4. Add 5 mL of complete HeLa medium. With a serological pipette, detach the remaining cells and transfer into a 15-mL conical tube. 5. Centrifuge the cells at 500
g for 5 min at RT. 6. Discard the media and resuspend the cell pellet in 1 mL of complete HeLa media. 7. Using a hemocytometer, count the cells and resuspend to 200,000 cells/mL. 8. Plate 100 μL of cells (20,000 cells) per well, into a flat bottom 96-well cell culture plate. 9. Where indicated, add 5 μL of Interferon-γ (stock 200 ng/mL; final concentration 10 ng/mL). 10. Put the cells back in the cell culture incubator for 16 h.

4.3 LPS Transfection in Human Cells

1. To each well, add 10 μL of Pam3CSK4 (diluted in complete THP-1 or HeLa media at a concentration of 10 μg/mL). The final concentration of Pam3CSK4 in the well is 1 μg/mL. Put the plate back into the cell culture incubator for 4 h (see Note 10). 2. Preheat Opti-MEM media (37  C). 3. Remove the media in each well and add 90 μL of preheated Opti-MEM. 4. Prepare the transfection mix as follows. To 87.5 μL of OptiMEM, add 10 μL of ultrapure LPS (stock concentration is 1 mg/mL). Vortex vigorously for 30 s. 5. Add 2.5 μL of Lipofectamine LTX. Mix by flicking the tube. Incubate at RT for 15 min. 6. Prepare a no lipofectamine and a no LPS transfection control by omitting each respective component. 7. At the end of the incubation, add 10 μL of the transfection mix (or control) to each well. The final concentration in each well is 0.25% Lipofectamine LTX and 10 μg/mL LPS. 8. Centrifuge the 96-well plate at 500
g for 5 min at RT (see Note 7). 9. Put the plate back in the cell incubator for 5 h.

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10. After 5 h, collect supernatants, and lyse cells in 25 μL whole cell lysis buffer per well. 11. Measure cell death using LDH release assay, as described in the protocol in Subheading 5. 12. Measure IL-1β and IL-18 secretion using ELISA, according to the manufacturer’s recommendation. For HeLa cells, we recommend measuring only IL-18 as those cells produce only little IL-1β. 13. Measure cleavage of Caspase-4 and Caspase-1 in lysates and supernatants by immunoblotting, as described in the protocol in Subheading 5. The cleavage of Gasdermin D can also be monitored, as described by Kamalaya and Boucher in this volume (Chapter 4).

5

Analysis of NCI Activation NCI activation can be assessed using several assays. In this section, we will describe how to monitor NCI activation by detecting the autoprocessing of Caspase-11 (in mice) and Caspase-4 (in humans) and by measuring pyroptosis. IL-1β and IL-18 secretion can be measured by ELISA following manufacturer’s protocol.

5.1 Immunoblotting for Inflammatory Caspase Cleavage

1. Combine cell lysis extracts from triplicate wells. Total volume is 150 μL from mouse cells and 75 μL from human cells. Add 50 μL or 25 μL of 4
LDS loading dye, to mouse and human lysates, respectively. Add DTT to a final concentration 10 mM. 2. Heat samples for 3 min at 100  C. 3. Combine cell supernatants and precipitate using methanol– chloroform extraction, as previously described [24] and as follows: (a) To 300 μL of supernatants, add 300 μL of MeOH and 100 μL of Chloroform. Vortex vigorously for 15 s. (b) Centrifuge at 17,000
g for 15 min at RT. (c) Discard the upper phase carefully (do not disturb the protein interphase). (d) Add 500 μL of MeOH and vortex 1 s. (e) Centrifuge at 17,000
g for 15 min at RT. (f) Discard the soluble phase and let the protein pellet dry 15 min. (g) Resuspend the protein pellet in 30 μL of lysis buffer. Add 10 μL of 4
LDS loading buffer. Add DTT to a final concentration 10 mM.

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Table 1 Recommended antibodies for NCI activation analysis Target

Antibody

Dilution

Expected molecular weight (kDa)

hCasp1

D7F10

1/1000

45 (FL); 33 (active); 20 (active)

mCasp1

casper-1

1/1000

45 (FL); 33 (active); 20 (active)

hCasp4

4B9

1/750

43 (FL); 32 (active); 24 (active)

mCasp11

EPR18628

1/1000

43 (FL); 38 (ALT), 32 (active)

Tubulin

B-5-1-2

1/5000

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Human (h) and mouse (m) antibodies recommended to detect full length (FL), alternative caspase splicing (ALT), and their active cleaved species

4. On a 4–20% SDS PAGE gel, load 15 μL of cell extract or supernatant fraction. 5. Run the gels in the Tris–glycine buffer at 150 V until the dye front exits the gel. 6. Transfer proteins from the gel to a nitrocellulose membrane using your preferred transfer method and probe by immunoblot using standard methods to detect full-length caspases and their cleaved forms. Note that cleaved forms can be detected in both the cell lysate and the supernatant as a result of pyroptosis. The expected molecular weights are presented in Table 1. 5.2 Detecting Pyroptosis Using LDH Release Assay (See Note 8)

1. At the end of the cell stimulation, add 1 μL of 10% Triton X-100 solution to each well that will serve as 100% lysis control (see plate layout in Fig. 2). Mix and incubate for 5 min at 37  C. 2. Spin the 96-well plate for 5 min at 500
g at RT to reduce floating cells. 3. Transfer 20 μL of cell-free supernatants and the 100% lysis controls into a new clear 96-well plate (non-sterile). 4. Add 20 μL of media to the blank control wells. 5. Using a multi-channel pipette, add 20 μL of LDH substrate (reconstituted as described in the manufacturer’s instructions) to each well. 6. Incubate 15–30 min at RT in an opaque container or wrapped in foil to protect from light (see Note 11). 7. Add 20 μL of Stop solution (provided with the LDH kit) to stop the reaction. 8. Make sure there are no bubbles in the wells. Use a 27-G needle to gently remove them. 9. Measure absorbance at 490 nM in a spectrophotometer plate reader.

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10. Determine the level of cell death as follows: Cell death ð%to 100%controlÞ ¼


Sample OD490nM  Blank OD490nM
100%OD490nM  Blank OD490nM
100

11. Plot your data using GraphPad Prism or other data analysis software.

6

Notes 1. We recommend optimizing the concentration of hM-CSF-1 for bone marrow macrophage differentiation. While the concentration recommended here works well with hM-CSF-1 from Immunotools (Cat 11343118), hmCSF-1 from other suppliers might differentiate cells with a different efficiency. 2. The quality of the fetal calf serum is important for this type of experiment. The use of low-endotoxin fetal calf serum is essential. We recommend testing the endotoxin level of each fetal calf serum batch before purchasing a large amount. We used Thermo Fisher, Cat 16000044. 3. LPS quality is another critical determinant in the success of the experiments. LPS with lower purity can contain impurities that can also activate the canonical inflammasome. We highly recommend ultrapure LPS-EK from Invivogen (Cat tlrl-3pelps or Cat tlrl-peklps) to efficiently and specifically activate the NCI in mouse and human cells. 4. Human and murine interferon-γ are not cross-reactive. Human M-CSF is cross reactive with mouse and human cells. We used interferon-γ from Biolegend, Cat 570204. 5. Before beginning this protocol, it is essential that you have obtained ethical approval for this procedure and that your euthanasia protocol conforms to your institutional guidelines. 6. If needed, the bone marrow can be frozen after flushing the bones. Red blood cells do not need to be lysed for this procedure. We routinely resuspend bone marrow from one mouse in 2 mL of freezing media (10% DMSO, 90% FCS). When replating frozen bone marrow, replate one vial of cells in two Sterilin plates. 7. The temperature of centrifugation is a key element for the success of this procedure. Centrifugation at temperature colder than RT will lead to an unsuccessful transfection and will fail to activate the NCI.

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8. Cell supernatants must be used fresh for LDH assays. Frozen supernatants lose LDH activity and cannot be used for this assay. 9. The density of cells plated in this experiment is critical for transfection success. We recommend plating cells to a density of 70–80% at the time of transfection. 10. HeLa cells do not require further priming as they are already primed with IFN-γ. 11. Certain cell types contain less LDH and will develop slower. It is recommended to incubate the LDH reaction for a longer period of time.

Acknowledgments Figures were created with BioRender (www.biorender.com). J.S.B. is supported by Kennedy Trust, KTRR start-up fellowship (KENN 15 16 06), and M.R.C. New Investigator Grant (MR/S000623/1). R.C. is supported by an Academy of Medical Sciences Springboard Award (SBF005\1104), a Royal Society Research Grant (RGS\R1\201127), and institutional funding from the Wellcome-Wolfson Institute for Experimental Medicine and Queen’s University Belfast. D.B. is supported by a Springboard award (SBF006\1025) from the Academy for Medical Science and institutional funding from the University of York. References 1. Schroder K, Tschopp J (2010) The inflammasomes. Cell 140(6):821–832 2. Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol 16(7):407–420 3. Man SM, Karki R, Kanneganti T-D (2017) Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol Rev 277(1):61–75 4. Liston A, Masters SL (2017) Homeostasisaltering molecular processes as mechanisms of inflammasome activation. Nat Rev Immunol 17(3):208–214 5. Boucher D, Monteleone M, Coll RC, Chen KW, Ross CM, Teo JL et al (2018) Caspase-1 self-cleavage is an intrinsic mechanism to terminate inflammasome activity. J Exp Med 215(3):827–840 6. Chan AH, Schroder K (2020) Inflammasome signaling and regulation of interleukin-1 family cytokines. J Exp Med 217(1):e20190314

7. Broz P, Pelegrı´n P, Shao F (2020) The gasdermins, a protein family executing cell death and inflammation. Nat Rev Immunol 20(3): 143–157 8. Kayagaki N, Warming S, Lamkanfi M, Vande Walle L, Louie S, Dong J et al (2011) Non-canonical inflammasome activation targets caspase-11. Nature 479(7371):117–121 9. Shi J, Zhao Y, Wang Y, Gao W, Ding J, Li P et al (2014) Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514(7521):187–192 10. Kayagaki N, Wong MT, Stowe IB, Ramani SR, Gonzalez LC, Akashi-Takamura S et al (2013) Noncanonical inflammasome activation by intracellular LPS independent of TLR4. Science 341(6151):1246–1249 11. Hagar JA, Powell DA, Aachoui Y, Ernst RK, Miao EA (2013) Cytoplasmic LPS activates caspase-11: implications in TLR4-independent endotoxic shock. Science 341(6151): 1250–1253

Activation of the Non-canonical Inflammasome in Mouse and Human Cells 12. Santos JC, Boucher D, Schneider LK, Demarco B, Dilucca M, Shkarina K et al (2020) Human GBP1 binds LPS to initiate assembly of a caspase-4 activating platform on cytosolic bacteria. Nat Commun 11(1):3276 13. Wandel MP, Kim B-H, Park E-S, Boyle KB, Nayak K, Lagrange B et al (2020) Guanylatebinding proteins convert cytosolic bacteria into caspase-4 signaling platforms. Nat Immunol 21(8):880–891 14. Kutsch M, Sistemich L, Lesser CF, Goldberg MB, Herrmann C, Coers J (2020) Direct binding of polymeric GBP1 to LPS disrupts bacterial cell envelope functions. EMBO J 39(13): e104926 15. Fisch D, Clough B, Domart M-C, Encheva V, Bando H, Snijders AP et al (2020) Human GBP1 differentially targets salmonella and toxoplasma to license recognition of microbial ligands and caspase-mediated death. Cell Rep 32(6):108008 16. Ross C, Chan AH, Von Pein J, Boucher D, Schroder K (2018) Dimerization and autoprocessing induce caspase-11 protease activation within the non-canonical inflammasome. Life Sci Alliance 1(6):e201800237 17. Casson CN, Yu J, Reyes VM, Taschuk FO, Yadav A, Copenhaver AM et al (2015) Human caspase-4 mediates noncanonical inflammasome activation against gramnegative bacterial pathogens. Proc Natl Acad Sci U S A 112(21):6688–6693 18. Lee BL, Stowe IB, Gupta A, Kornfeld OS, Roose-Girma M, Anderson K et al (2018) Caspase-11 auto-proteolysis is crucial for

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noncanonical inflammasome activation. J Exp Med 215(9):2279–2288 19. Baker PJ, Boucher D, Bierschenk D, Tebartz C, Whitney PG, D’Silva DB et al (2015) NLRP3 inflammasome activation downstream of cytoplasmic LPS recognition by both caspase-4 and caspase-5. Eur J Immunol 45(10):2918–2926 20. Ru¨hl S, Broz P (2015) Caspase-11 activates a canonical NLRP3 inflammasome by promoting K(+) efflux. Eur J Immunol 45(10): 2927–2936 21. Wannamaker W, Davies R, Namchuk M, Pollard J, Ford P, Ku G et al (2007) (S)-1((S)-2-{[1-(4-amino-3-chloro-phenyl)-methanoyl]-amino}-3,3-dimethyl-butanoyl)-pyrrolidine-2-carboxylic acid ((2R,3S)-2-ethoxy-5oxo-tetrahydro-furan-3-yl)-amide (VX-765), an orally available selective interleukin (IL)converting enzyme/caspase-1 inhibitor, exhibits potent anti-inflammatory activities by inhibiting the release of IL-1beta and IL-18. J Pharmacol Exp Ther 321(2):509–516 22. Coll RC, Hill JR, Day CJ, Zamoshnikova A, Boucher D, Massey NL et al (2019) MCC950 directly targets the NLRP3 ATP-hydrolysis motif for inflammasome inhibition. Nat Chem Biol 15(6):556–559 23. Chen KW, Monteleone M, Boucher D, Sollberger G, Ramnath D, Condon ND et al (2018) Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps. Sci Immunol 3(26): eaar6676 24. Gross O (2012) Measuring the inflammasome. Methods Mol Biol 844:199–222

Chapter 6 Measurement of Inflammasome-Induced Mitochondrial Dysfunction by Flow Cytometry Mayoorey M. Thasan and Ali A. Abdul-Sater Abstract A growing body of work has recently highlighted the pivotal role of mitochondria in the initiation and modulation of inflammasome activation. Specifically, mitochondrial dysfunction can induce NLRP3 inflammasome activation, where loss of mitochondrial potential leads to production of reactive oxygen species (ROS) and release of Ca2+, which in turn trigger inflammasome assembly. Therefore, several measures of mitochondrial parameters and components are routinely utilized in studies assessing mechanisms of inflammasome activation. In this chapter, we show detailed protocols on how to employ flow cytometry using three distinct mitochondria-specific dyes to measure mitochondrial ROS (MitoSOX), mitochondrial respiration (Mitotracker deep red), and total mitochondria (Mitotracker green), as well as a dye that measures reduced glutathione (mBBr). Key words Inflammasomes, Mitochondria, Reactive oxygen species (ROS), Glutathione (GSH), Mitotracker, MitoSOX, Monobromobimane (mBBr), Bone marrow–derived macrophages (BMDM), Flow cytometry

1

Introduction Inflammasomes are multimeric protein complexes that form following infection and/or cellular damage [1]. Thus, they play an important role in innate and adaptive immune responses. The nucleotide-binding oligomerization domain-like receptors or NOD-like receptors (NLRs) are one of the main groups of damage sensors that oligomerize to form a platform for the recruitment of an adaptor protein, called apoptosis-associated speck-like protein containing a CARD (ASC). Subsequently, ASC proteins form large filaments followed by oligomerization and recruitment of the effector zymogen, caspase-1 [2]. This triggers the autocleavage and activation of caspase-1, which can then cleave the inactive proinflammatory cytokines, pro-interleukin (IL)-1β, and pro-IL-18 into their biologically active forms, IL-1β and IL-18 followed by their release into the extracellular space [3].

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_6, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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NLRP3 is the best characterized inflammasome, largely because of the wide array of pathogens and pathogen-derived molecules, host-derived molecules, and environmental triggers that induce its activation. These molecules trigger inflammasome activation by a variety of mechanisms, including potassium efflux, lysosomal damage, and mitochondrial damage [4]. The latter has been widely recognized as a key event that can directly or indirectly activate NLRP3 inflammasomes [5]. For example, release of oxidized mitochondrial DNA and cardiolipin from damaged mitochondria has been shown to be sensed by NLRP3 directly followed by oligomerization and inflammasome activation [6–9]. On the other hand, reactive oxygen species (ROS) and calcium (Ca2+), released following loss of membrane potential of damaged mitochondria, can indirectly activate the NLRP3 inflammasome [10–12]. Thus, numerous studies probing the mechanistic details of inflammasome inducers employ various techniques to assess if these inducers cause mitochondrial dysfunction, including mitochondrial (mt) damage, mt membrane potential, mtROS production, and mtDNA. Moreover, bone marrow–derived macrophages are one of the best models to gain mechanistic insights into inflammasome activation. Here, we describe how to prepare bone marrow–derived macrophages, induce NLRP3 inflammasome activation, and assess mitochondrial dysfunction using flow cytometry by measuring mitochondrial ROS (MitoSOX), mitochondrial respiration (Mitotracker deep red), and total mitochondrial content (Mitotracker green), as well as a dye that measures reduced glutathione (mBBr). Combined, these stains distinguish healthy from damaged mitochondria and evaluate the levels of cellular oxidative stress.

2

Materials

2.1 Generation of Bone Marrow-Derived Macrophages (BMDM)

1. Cell culture media: RPMI-1640 supplemented with 10% fetal calf serum (FCS), 1 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 55 μM 2-mercaptoethanol, 0.1 mM Nonessential amino acids. 2. 70% ethanol. 3. L929 Conditioned Media (LCM) (see Note 1). 4. Red blood cell lysis buffer (commercially available). 5. Trypan blue dye. 6. Sterile 10-ml syringes. 7. Sterile 23- and 27-gauge needles. 8. 100-mm petri dishes. 9. 60-mm tissue culture dishes. 10. 25-cm cell scrapers. 11. Serological pipettes (5 ml, 10 ml, and 25 ml).

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12. Cell culture incubator set at 37
C and 5% CO2. 13. Hemocytometer. 14. Micropipettes and tips for 1000 μl. 15. 50-ml conical tubes. 16. Centrifuge for 50-ml conical tubes. 17. Styrofoam (or surgical) board. 18. Surgical scissors. 19. Tweezers. 20. Biosafety cabinet, Class A2. 2.2

Cell Stimulations

1. Six-well sterile tissue culture plates. 2. Cell culture incubator set at 37
C and 5% CO2. 3. Micropipettes and tips for 1000 μl, 200 μl, and 10 μl. 4. 5 mg/ml Lipopolysaccharide (LPS) from E. coli serotype 055: B5 stock dissolved in sterile PBS (see Note 2). 5. 5 mM Nigericin stock dissolved in DMSO or ethanol (see Note 3). 6. Phosphate-buffered saline without Ca2+ and Mg2+ (D-PBS). 7. FACS buffer: D-PBS containing 2% FCS.

2.3 Flow Cytometry Staining

1. FACS buffer (DPBS containing 2% FBS (heat-inactivated)). 2. 5 mM MitoSOX Red stock dissolved in DMSO and stored at 20
C (see Note 4). 3. 1 mM Mitotracker deep red stock dissolved in DMSO and stored at 20
C (see Note 4). 4. 1 mM Mitotracker green stock dissolved in DMSO and stored at 20
C (see Note 4). 5. 40 mM Monobromobimane (mBBr) stock dissolved in DMSO and stored at 20
C. 6. Round bottom 96-well plates (Untreated). 7. 1.5-ml microcentrifuge tubes. 8. Centrifuge for 1.5-ml microcentrifuge tubes. 9. Micropipettes and tips for 1000 μl, 200 μl, and 10 μl. 10. Multichannel pipette for 200 μl. 11. Multichannel pipette basins. 12. Vacuum aspirator. 13. Flow cytometer equipped with 488 nm and 637 nm lasers. 14. Flow cytometry data analysis software (e.g., FlowJo, De Novo Software, FCS express).

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Methods All procedures are performed in the biosafety cabinet according to institutionally approved safety protocols (dissection of bones can be done on the bench if desired). Cell culture media should be pre-warmed to 37
C before use.

3.1 Generation of Primary Mouse Bone Marrow-Derived Macrophages (BMDM)

1. Euthanize the mouse according to institutionally approved animal protocols. 2. While lying on its back, pin all four mouse paws below the ankle joints on a Styrofoam or dissection board using 23-gauge needles. 3. Douse the abdomen and the hind legs with 70% ethanol. 4. With a dissecting scissors, make an incision in the middle of the abdomen and dissect the skin from the abdomen and legs. Without damaging the bones, remove the muscles attached to the hind legs and the pelvis. Remove the leg by cutting right after the pelvic joint. Make sure you don’t expose the bone marrow to maintain sterility of the cells. 5. Dip the bones in 70% ethanol for 15 s and transfer into a 6-cm dish or 15-ml conical tube containing 5 ml of cell culture media. 6. Separate the tibia and femur by cutting the knee joint, then cut the end joints of each bone to expose the bone marrow (see Note 5). 7. Flush each bone with 2.5 ml cell culture media and collect in a 50-ml conical tube (see Note 6). 8. Centrifuge the 50-ml conical tube at 350  g for 5 min at 4
C, then aspirate the supernatant. 9. Resuspend the cell pellet in 2 ml red blood cell Lysis buffer and mix gently by pipetting. 10. Incubate for 5 min at room temperature and then add 5 ml cell culture media to stop the lysis (see Note 7). 11. Centrifuge at 350  g for 5 min at 4
C. 12. Discard the supernatant and resuspend the cell pellet in 40 ml cell culture media supplemented with 25% LCM then transfer into eight 100-mm petri dishes (5 ml per dish). 13. Incubate for 3 days in a 37
C and 5% CO2 incubator. 14. On day 3, discard the media and add 3 ml of fresh cell culture media. 15. Using a cell scraper, scrape the cells and transfer into a 50-ml conical tube (see Notes 8 and 9). 16. Count the cells under the microscope using a hemocytometer. Use trypan blue dye to identify dead cells (see Note 10). 17. Seed 5  105 cells in six-well plates in cell culture media containing 25% LCM.

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18. Incubate for an additional two days in a 37
C and 5% CO2 incubator (see Note 11). 3.2 Stimulation of BMDMs to Activate NLRP3 Inflammasome

1. Wash the differentiated (adherent) pre-warmed (37
C) D-PBS.

macrophages

with

2. Add 1 ml of fresh cell culture media per well. 3. To activate NLRP3 inflammasome, prime the cells with 1 μg/ ml LPS for 4 h and then add 10 μM Nigericin for 1 h. Include appropriate controls (untreated and LPS-only treatments). 4. After treatments, wash the cells immediately with ice-cold D-PBS. 5. Remove D-PBS and add 500 μl of FACS buffer per well. 6. Gently scrape the cells using a cell scraper and transfer into 1.5ml microcentrifuge tubes. 7. Centrifuge at 350  g for 5 min and remove the supernatant. 8. Resuspend the cell pellet in 250 μl of FACS buffer and transfer to round bottom 96-well plates. 9. Centrifuge the plate at 350  g for 3 min at 4
C. 10. Proceed to staining steps.

3.3 Staining and Measurement of NLRP3 InflammasomeInduced Mitochondrial Damage by Flow Cytometry

1. For oxidative stress stain, resuspend cells in 100 μl staining buffer containing 2.5 μM MitoSOX red and 40 μM mBBr diluted in FACS buffer (see Note 12). 2. For mitochondrial health stain, resuspend cells in 100 μl staining buffer containing 50 nM Mitotracker deep red and 50 nM Mitotracker green diluted in FACS buffer. 3. Incubate for 10 min at 37
C in the cell culture incubator with 5% CO2. 4. Centrifuge at 350  g for 3 min and aspirate the supernatant. 5. Resuspend the cells in 200 μl FACS buffer (see Note 13). 6. Acquire the samples in a flow cytometer using the appropriate fluorescent channels for each stain as follows (see Note 14): (a) For oxidative stress stain, MitoSOX Red is excited by the blue laser (488 nm), and emission is detected either with a 585 nm or 675 nm filter; whereas mBBr is excited by the violet laser (405 nm), and emission is detected with the 450 nm filter (Fig. 1). (b) For the mitochondrial health stain, Mitotracker deep red is excited by the red laser (637 nm), and emission is detected with the 665 nm filter; whereas Mitotracker green is excited by the blue laser (488 nm) and detected with the 516 nm filter (Fig. 2). 7. Analyze and plot the data using a flow cytometry data analysis software.

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Control

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Fig. 1 Oxidative stress stain following NLRP3-inflammasome activation of BMDMs. Cells were stimulated to activate NLRP3 inflammasome (LPS + Nig), then stained with MitoSOX Red and mBBr, as described in Subheadings 3.2 and 3.3. Data was acquired by flow cytometry (Attune NxT), and mitochondrial reactive oxygen species (ROS)-producing cells (mtROS) were gated on the MitoSOX Red high, mBBr low population, while cells with low oxidative stress (Reduced Glutathione) were gated on the MitoSOX Red low, mBBr high population. Data were analyzed and plotted using FlowJo software

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Fig. 2 Mitochondrial health stain following NLRP3-inflammasome activation of BMDMs. Cells were stimulated to activate NLRP3 inflammasome (LPS + Nig), then stained with Mitotracker deep red and Mitotracker green, as described in Subheadings 3.2 and 3.3. Data was acquired by flow cytometry (Attune NxT) and gated on cells with depolarized mitochondria (damaged mitochondria). Data were analyzed and plotted using FlowJo software

Measuring Mitochondrial Damage by Flow Cytometry

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Notes 1. L929 conditioned media (LCM), which contains macrophage colony-stimulating factor (CSF), can be prepared from L929 murine fibroblast cell line. L929 cells are cultured in DMEM (supplemented with 10% FCS) in 10-cm tissue culture plates at 37
C and 5% CO2 until they reach confluency. 10 ml of fresh media is then added, and cells are incubated for 7 days. The conditioned media is then collected, centrifuged at 350  g for 5 min and filtered through 0.2 μm PES filter then stored at 80
C. Alternatively, recombinant mouse M-CSF is available commercially and can be used in lieu of LCM. 2. LPS 5 mg/ml stock solution was prepared in D-PBS and stored at 80
C in 500 μl aliquots. Once thawed, aliquots can be stored at 4
C for up to 4 weeks to avoid multiple freezethawing. Working dilution was made in cell culture media. 3. 5 mM Nigericin was prepared in 100% ethanol and stored at 20
C in 50 μl aliquots. 4. MitoSOX Red and Mitotracker dyes are packaged in lyophilized forms and stored at 20
C. Allow vials to warm to room temperature before opening them and reconstitute with anhydrous dimethyl sulfoxide (DMSO). 5. Separate/open the bones inside the biosafety cabinet to avoid contamination. 6. The reddish bones should turn white when the entire bone marrow has been flushed. 7. Do not incubate for a prolonged period to avoid lysis of leukocytes. 8. Cells from all eight petri dishes can be combined into one conical tube (24 ml total). 9. The percentage of LCM used to supplement the cell culture media should be optimized by counting the number of differentiated macrophages on day 3. The optimal percentage of LCM is generally between 15% and 25%. 10. Count four large squares using a cell counter and use the following formula to calculate the cell concentration (here we used a twofold dilution with trypan blue dye).   ðNumber of cells  10, 000Þ  dilutionð2Þ cells Cell concentration ¼ ml number of squares countedð4Þ 11. Cell density may vary with different cell types. 12. Prepare working staining solutions fresh every time at the latest within 2 h of use and keep protected from light.

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13. MitoSOX and mBBr stained cells must be kept on ice and protected from light. It is important to run the assay shortly after staining. 14. We used the ThermoFisher Attune NxT flow cytometer, equipped with BL1–530/30 nm, Bl2–574/26 nm, VL1–450/40 nm, and RL1–670/14 nm. No compensation is necessary with these dye combinations since there is minimal spectral overlap. References 1. Abdul Sater A, Philpott D (2016) Inflammasomes. In: Encyclopedia of immunobiology. Elsevier Science 2. Lu A, Magupalli VG, Ruan J et al (2014) Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell 156:1193–1206 3. Tweedell RE, Kanneganti T-D (2020) Advances in inflammasome research: recent breakthroughs and future hurdles. Trends Mol Med 26:969–971 ˜ ez G (2016) Mechanism 4. He Y, Hara H, Nu´n and regulation of NLRP3 inflammasome activation. Trends Biochem Sci 41:1012–1021 5. Liu Q, Zhang D, Hu D et al (2018) The role of mitochondria in NLRP3 inflammasome activation. Mol Immunol 103:115–124 6. Bordon Y (2018) mtDNA synthesis ignites the inflammasome. Nat Rev Immunol 18:539–539 7. Shimada K, Crother TR, Karlin J et al (2012) Oxidized mitochondrial DNA activates the

NLRP3 inflammasome during apoptosis. Immunity 36:401–414 8. Pizzuto M, Pelegrin P (2020) Cardiolipin in immune signaling and cell death. Trends Cell Biol 30:892–903 9. Iyer SS, He Q, Janczy JR et al (2013) Mitochondrial cardiolipin is required for Nlrp3 inflammasome activation. Immunity 39: 311–323 10. Zhou R, Yazdi AS, Menu P et al (2011) A role for mitochondria in NLRP3 inflammasome activation. Nature 469:221–225 11. Horng T (2014) Calcium signaling and mitochondrial destabilization in the triggering of the NLRP3 inflammasome. Trends Immunol 35:253–261 12. Murakami T, Ockinger J, Yu J et al (2012) Critical role for calcium mobilization in activation of the NLRP3 inflammasome. Proc Natl Acad Sci U S A 109:11282–11287

Chapter 7 Detection of ASC Oligomerization by Western Blotting Safoura Zangiabadi, Ali Akram, and Ali A. Abdul-Sater Abstract Apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) is an adaptor protein that is essential for the activation of several inflammasome complexes. Activation of inflammasomes leads to pathogenic clearance and inflammatory cell death called pyroptosis. Upon inflammasome activation, ASC oligomerization leads to the recruitment and activation of caspase-1, which in turn converts pro-inflammatory cytokines (e.g., pro-IL-1β, pro-IL-18) to their mature active form. Given its central role in inflammasome activation, ASC oligomerization is used as an indicator of inflammasome activation. Here we describe how ASC oligomerization can be detected by Western blotting. Key words ASC oligomerization, Inflammasomes, Cross-linking, Western blotting, THP-1 cells

1

Introduction Inflammasomes are multimeric protein complexes that are critical for the production of the potent pro-inflammatory cytokines interleukin (IL)-1β and IL-18. There are different kinds of inflammasomes, and apart from a few examples, they are comprised of three components: (1) a danger sensing protein, which is typically a NOD-Like receptor (e.g., NLRP3) or Absent in Melanoma 2 (AIM2); (2) an adaptor protein called apoptosis-associated speck-like protein containing CARD (ASC); and (3) an effector enzyme called pro-caspase-1 [1]. Inflammasome activation is triggered when a danger signal is sensed, leading to the oligomerization of NLRP3 or AIM2, which nucleates ASC oligomerization and filament formation [2]. ASC filaments then serve as a platform for pro-caspase-1 recruitment followed by autoproteolytic cleavage of pro-caspase-1 into active caspase-1. Finally, caspase-1 mediates the proteolytic cleavage of the biologically inactive pro-IL-1β and pro-IL-18 into IL-1β and IL-18, respectively. This is followed by caspase-1-mediated cleavage of the pore-forming protein, gasder-

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_7, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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min D (GSDMD), where the N-terminal domain oligomerizes and forms pores in the plasma membrane leading to the secretion of IL-1β and IL-18, and eventually a form of programmed cell death, called pyroptosis [3]. ASC oligomerization is a critical and unifying step in inflammasome activation [2]. Therefore, assays that measure oligomerization of ASC have been widely used to study inflammasome activation. These include, among others, measurements of ASC speck formation by microscopy, ASC oligomerization by Western blotting or flow cytometry. Here, we describe the stimulation and detection of ASC oligomerization in THP-1-derived macrophages. We describe how to differentiate THP-1 cells into macrophages followed by inflammasome stimulation and measurements of ASC oligomers by Western blotting.

2

Materials Prepare all stocks and solutions at room temperature unless otherwise indicated. In cases where solutions/stocks and specific cell lines need to be kept sterile, work in the biosafety cabinets (BSC). Use double-distilled water for making solutions and buffers unless otherwise instructed. Use your institutionally approved waste disposable methods to discard any waste products/solutions.

2.1 Solutions and Stimulations

1. THP-1 cell line. 2. Cell culture media: RPMI-1640 supplemented with 10% fetal bovine serum (FBS), 1 mM glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 55 μM 2-mercaptoethanol, 0.1 mM nonessential amino acids. 3. Sterile phosphate-buffered saline (PBS). 4. 5 mg/ml Lipopolysaccharide (LPS) from E. coli serotype 055: B5 dissolved in sterile PBS (see Note 1). 5. Opti-MEM I reduced serum media. 6. 1 mM Phorbol 12-myristate 13-acetate (PMA) stock dissolved in DMSO. 7. 200 mM Adenosine triphosphate (ATP) diluted in H2O (see Note 2). 8. 5 mM Nigericin stock dissolved in DMSO or ethanol (see Note 3). 9. 100 mM disuccinimidyl suberate (DSS) stock dissolved in DMSO (see Note 4). 10. Prestained protein marker.

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11. ASC polymerization lysis buffer: 20 mM HEPES (pH 7.4), 150 mM potassium chloride (KCl), 1% Igepal CA-630 (NP40), protease inhibitor cocktail (commercially available; must be added freshly before use). 12. 4 Laemmli sample buffer: 62.5 mM Tris–HCl (pH 6.8), 10% glycerol, 1% lithium dodecyl sulfate (LDS), 0.005% Bromophenol Blue. 13. 10% and 13.5% SDS-PAGE gels. 2.2 Materials and Equipment

1. 12-well tissue culture-treated plates. 2. 1.5-ml microcentrifuge tubes. 3. Cell scrapers. 4. 21-gauge needle. 5. 1-ml syringe. 6. Water bath. 7. Refrigerated microcentrifuge. 8. Rotating shaker. 9. Micropipette and pipette tips for 10, 100, 200, and 1000 μl. 10. Tissue culture CO2 incubator. 11. Biological safety cabinet. 12. Vortex. 13. β-Actin antibody (see Note 5). 14. ASC/TMS1 antibody (see Note 6). 15. Nitrocellulose membrane. 16. Vertical electrophoresis tank. 17. Semi-dry transfer apparatus. 18. Heat block. 19. Chemiluminescent imager. 20. High-sensitivity chemiluminescent substrate.

3

Methods All procedures should be performed at room temperature unless otherwise directed. Use aseptic techniques to obtain and prepare cells for Western blotting.

3.1

Cell Stimulations

1. Seed out 1.5  106 THP-1 cells in 1 ml of cell culture media per well of a 12-well plate. 2. Add 100 nM PMA and incubate overnight in tissue culture incubator at 37  C and 5% CO2.

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3. Remove cells from the incubator and wash them twice with sterile PBS (see Note 7). 4. Add 1 ml Opti-MEM per well and prime the cells with 500 ng/ ml LPS. Incubate for 4 h at 37  C and 5% CO2 (see Note 8). 5. Activate an ASC-dependent inflammasome to trigger ASC oligomerization with the appropriate inducers. For example, 5 mM ATP or 10 μM Nigericin for 30–60 min (see Note 9). 3.2 Cell Lysis and ASC Oligomerization

1. Following inflammasome activation, wash cells twice with ice-cold PBS. 2. Add 500 μl ice-cold ASC polymerization lysis buffer. 3. Using cell scrapers, detach the cells from the plate, then transfer to 1.5-ml microcentrifuge tube and keep on ice. 4. Pass the cell lysate 10 times through a 21-gauge needle to further shear the cells and complete the lysis process. 5. Save 30 μl of the lysate as “input control” of ASC isolation and keep on ice until gel loading (see Note 10). 6. Centrifuge the remaining 470 μl lysate at 3400  g in a microcentrifuge for 15 min at 4  C. 7. Carefully aspirate the supernatant and wash the pellet three times with 1 ml PBS at 3400  g for 10 min at 4  C. 8. Remove supernatant and resuspend the pellet in 500 μl PBS and add 10 μl of freshly prepared 100 mM DSS solution to obtain a final concentration of 2 mM (see Note 11). 9. Cross-link samples by incubation on a rotating shaker for 30 min at room temperature. 10. Centrifuge the cells for at 6000  g for 10 min at 4  C to separate unbound DSS from the pellets. 11. Using a 200-μl micropipette, carefully remove the supernatants and avoid any disturbance to cross-linked pellets. Repeat this step 3–4 times, as necessary, to ensure complete removal of supernatant from pellets (see Note 12). 12. Resuspend each pellet in 35 μl of 2 Laemmli buffer. 13. Using a heat block, incubate samples for 5 min at 95  C. 14. Load the input control samples on a 13.5% SDS-PAGE and the cross-linked samples on a 10% SDS-PAGE gel and transfer into nitrocellulose membranes (see Note 13). 15. Probe with ASC/TMS1 antibody and β-Actin (loading control) followed by the appropriate secondary antibody. 16. Image on a chemiluminescent imager.

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Notes 1. The quality of the fetal calf serum is critical for the success of the experiment. Use ultralow endotoxin FBS is highly recommended. LPS should be aliquoted and stored at 80  C. Make fresh LPS of desired concentration (e.g., 100 ng/ml) every time and store at 4  C or on ice until ready to use. 2. This is the stock concentration, which must be aliquoted and stored at 80  C. Adjusting pH with 1 N NaOH to 7.4 is optional but recommended. Discard unused ATP after thawing. 3. Aliquot Nigericin and freeze at avoid freeze/thaw.

20  C in small volumes to

4. Prepare fresh DSS solution before each experiment. Using an old DSS solution is not optimal. 5. We used ASC/TMS Rabbit PolyAb (Proteintech, Cat. 105001-AP) at 1:1000 dilution. Antibodies from other vendors should be tested for specificity and optimal dilution. 6. Other antibodies or methods can be used to determine equal protein loading. 7. Following PMA differentiation, THP-1 cells will become adherent and assume an elongated, macrophage-like shape. 8. All stimulations are performed in Opti-MEM without serum. Presence of serum in culture media will dramatically interfere with Western blotting of proteins precipitated from cell culture supernatants. 9. It is highly recommended that you collect supernatants to assess the release of active IL-1β and caspase-1 by Western blotting or ELISA. 10. The input control can be combined with 10 μl of 4 Laemmli buffer and stored at 20  C until the gel loading step. 11. DSS is a cross-linker that will stabilize ASC complexes. 12. It is critical to separate unbound DSS from pellets. Therefore, you may have to repeat the centrifugation step a few times to achieve complete removal of the supernatant from the pellets. 13. Proteins from cell culture supernatants can be precipitated and loaded on different percentile SDS-PAGE gels (i.e., 13.5% vs. 10%) before and after cross-linking. Before crosslinking, these ASC complexes have lower weight and should be run at higher (13%) SDS-PAGE gel. Following cross-linking, higher molecular weight is achieved as a result of oligomerization. These oligomers can be detected by 10% SDS-PAGE gels. Alternatively, a 4–16% gradient gel can be used throughout the assay.

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Acknowledgments This work was supported by grants received from the Arthritis Society and the Canadian Institute for Health Research (CIHR). References 1. Abdul Sater A, Philpott D (2016) Inflammasomes. In: Encyclopedia of immunobiology. Elsevier Science 2. Lu A, Magupalli VG, Ruan J et al (2014) Unified polymerization mechanism for the assembly of

ASC-dependent inflammasomes. Cell 156: 1193–1206 3. Tweedell RE, Kanneganti T-D (2020) Advances in inflammasome research: recent breakthroughs and future hurdles. Trends Mol Med 26: 969–971

Chapter 8 Reconstitution System of NLRP3 Inflammasome in HEK293T Cells Sheng Chen, Zhexu Chi, and Di Wang Abstract NLRP3 (NOD-, LRR-, and pyrin domain-containing protein 3) inflammasome is a cytosolic multimeric protein complex that plays key roles in the host innate immune response to both pathogenic and sterile insults. Here we describe a comprehensive guide to study NLRP3 inflammasome activation in HEK293T cell reconstitution system, which could provide direct biochemical evidence in protein interaction and posttranslational modification of the complex. Key words NLRP3 inflammasome, HEK293T reconstitution system, Innate immunity

1

Introduction NLRP3 inflammasome is a mega-Dalton cytosolic multimeric complex which may assemble in response to various pathogen- or damage-associated molecular patterns (PAMPs or DAMPs, respectively). This multi-protein complex consists of one sensor (NLRP3), two adaptors (ASC and NEK7), and one effector (pro-caspase-1) [1]. The past decade has witnessed a burgeoning appreciation of inflammasomes as critical innate immune components that orchestrate host immune defense and homeostasis, and a number of comprehensive guidelines to study inflammasomes and cell death have been published [2]. Among all these different approaches, NLRP3 inflammasome reconstitution system within HEK293 cell provide simple but robust biochemical proofs to investigate the fundamental mechanisms of NLRP3 inflammasome activation. Reconstitution system within HEK293T cells was first introduced by J-W Yu et al. in 2006 [3]. Though the reconstituted NLRP3 inflammasome complex does not recapitulate the normal NF-κB signaling cascade, which could not represent the full

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_8, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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spectrum of NLRP3 activation in primary cells (such as macrophage), it has been wildly adopted especially in studying protein– protein interactions [4–9], posttranslational modifications [10– 13], as well as evaluating potential therapeutic agents targeting NLRP3 inflammasome [14]. HEK293 reconstitution system provides a simplified platform to study the nature of NLRP3 inflammasome activation, and here we describe a comprehensive protocol to employ NLRP3 inflammasome reconstitution system.

2 2.1

Materials Cell Culture

1. HEK293T cells. 2. 12-well Clear TC-treated multiple well plates. 3. HEK293T cell culture medium: Dulbecco’s Modified Eagle Medium (DMEM) + 10% fetal bovine serum (FBS) + 100 U/ mL Penicillin-Streptomycin. 4. 0.25% Trypsin–EDTA. 5. Phosphate-buffered saline (PBS): 144.0 mg/L KH2PO4, 9000 mg/L NaCl, 795.0 mg/L Na2HPO4·7H2O, pH ¼ 7.4. 6. 37  C, 5% CO2 forced-air incubator. 7. 15-mL centrifuge tubes. 8. Hemacytometer. 9. Nigericin.

2.2 Plasmid Transfection

1. Lipofectamine 2000. 2. Opti-MEM-I Reduced Serum Medium. 3. 1.5-mL Eppendorf tubes. 4. Plasmid Midi Prep Kit. 5. Plasmid (see ref. 9). (a) pCMV-Tag2A-Flag-Nlrp3. (b) pcDNA3.1()B-Myc-Pro-Caspase1. (c) pcDNA3.1()B-HA-ASC. (d) pcDNA3.1()B-V5-Pro-Caspase1.

2.3 Detection of NLRP3 Inflammasome Activation

1. IL-1β ELISA kit. 2. Western blotting antibodies. (a) Anti-NLRP3 (Adipogen, Cat# AG-20B-0014, RRID: AB_2490202). (b) Anti-Caspase1 (Adipogen, Cat# AG-20B-0042, RRID: AB_2490248).

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(c) Anti-ASC (Adipogen, Cat# AG-25B-0006, RRID: AB_2490440). (d) Anti-IL-1β (R&D Systems, Cat# AF-401-NA, RRID: AB_416684). (e) Anti-Flag (HuaAn biotechnology, Cat# 0912-1). (f) Anti- HA (Thermo Fisher, Cat# 26183, RRID: AB_10978021). (g) Anti-Myc (HuaAn Biotechnology, Cat# EM31105). (h) Anti-V5 (HuaAn Biotechnology, Cat# M1008-2). (i) Anti-β-actin (Daigebio, Cat# db10001). 3. 2 SDS loading buffer: 100 mM Tris–HCl, 4% SDS, 20% glycerol, 2% 2-mercaptoethanol, and 0.05% bromophenol blue. 4. Chemiluminescent substrate with pg/mL sensitivity (e.g., SuperSignal™ West Pico).

3 3.1

Methods Cell Preparation

1. Wash HEK293T cells with 1 mL pre-warmed PBS. 2. Dispense the PBS and add 1 mL trypsin to digest cells at 37  C for 3 min. 3. Stop the digestion process by adding 1 mL HEK293T cell culture medium. 4. Transfer the cells into a 15-mL centrifuge tube and centrifuge the cells at 600  g for 5 min. 5. Resuspend the cells in 1 mL HEK293T cell culture medium and count the cells using a hemocytometer. 6. Plate the HEK293T cells in 12-well microplates at a density of 5  105 per well in 1 mL DMEM culture medium at 37  C in a forced-air incubator overnight (see Note 1).

3.2 Plasmid Preparation and Transfection

1. Extract all the plasmid required from their respective bacterial cultures using Plasmid Midi Prep Kit (see Note 2). 2. Dilute the plasmid with ddH2O to a final concentration of 500 ng/μL. 3. Mix all the plasmids (for per well): Myc-pro-caspase-1 (250 ng), HA-ASC (75 ng), HA-NLRP3 (100 ng), and pro-IL-1β (500 ng) in 100 μL Opti-MEM in a 1.5 mL Eppendorf tube (see Note 3). 4. Add 3 μL Lipofectamine 2000 in 100 μL Opti-MEM in a 1.5mL Eppendorf tube.

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5. Mix the two portions of diluted DNA and diluted Lipofectamine 2000 (1:1 ratio), incubate for 15 min at room temperature. 6. Gently mix the DNA–lipid complex and add to the HEK293T cells (cultured in 1 mL DMEM culture medium), and gently shake the cell plate to mix the complex with culture medium (see Note 4). 7. Replace the medium with 1 mL prewarmed fresh DMEM culture medium 4–6 h later (see Note 5). 8. After 24 h, stimulate cells with 10 μg/mL Nigericin for 1 h. 9. Collect the supernatants in 1.5-mL Eppendorf tube (see Note 6). 10. Wash the cells with 1 mL chilled PBS, then dispense the PBS and add 80 μL 2 SDS loading buffer to lyse the cells (see Note 7). 3.3 Detection of NLRP3 Inflammasome Stimulation

4

1. Detect the IL-1β concentration in the supernatant according to ELISA kit manufacturer’s instructions (see Note 8). 2. For cell lysates, typical Western blotting is performed. NLRP3 inflammasome activation is illustrated by cleaved Caspase-1 (could be blotted by either anti-Caspase-1 or anti-Myc) (see Note 9).

Notes 1. 5  105 per well of a 12-well plate is typically equal to 50–60% density. After overnight growth, the density could reach 70–80%, which is optimal for plasmid transfection. 2. Plasmid extraction is typically performed according to the manufacturer’s instructions. Complete plasmid sequencing is recommended before performing the experiments. 3. Though we have listed the amount of each plasmid (Myc-procaspase-1250 ng, HA-ASC 75 ng, HA-NLRP3 100 ng, and pro-IL-1β 500 ng), it is always recommended to optimize the concentration of each plasmid in your own system. 4. It is recommended to gently shake the cell plate when adding DNA–lipid complex to avoid sudden topical highconcentration of transfection regents, which may influence cell viability. 5. We recommend replacing the cell medium with fresh pre-warmed culture medium to minimize the influence of transfection regents.

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6. When collecting cell supernatants, it is optimal to centrifuge the supernatants at 600  g for 5 min to get rid of cells and debris. Discard the pellet (usually ignorable) and transfer the centrifuged supernatants to a new 1.5-mL Eppendorf tube. Use the fresh supernatants for ELISA detection or freeze at 20  C for future use. 7. After the collection of supernatants, transfer the cell plates on ice and wash with chilled PBS for one time. After adding the 2 SDS lysis buffer, it is recommended to put the plate in RT and flap the plate to allow complete lysis. It is optimal to stir the lysis buffer with a 200 μL pipette tip. We typically heat the lysates at 100  C for 10 min for thorough lysis before following experiments. 8. ELISA is performed according to the manufacturer’s instructions. It is always recommended to perform preliminary experiments using your reconstitution system to see the concentration of IL-1β in the supernatant. Appropriate dilution is required for optimal detection. We also recommend incubating the plate overnight at 4  C after adding samples, and read the plate using 450/570 nm settings of the plate reader. 9. Typical procedure of Western blotting is described thoroughly elsewhere [15]. Cleaved caspase-1 could be blotted by both anti-caspase-1 and anti-Myc antibodies. Blotting of Pro-caspase-1 (P45) and Pro-IL-1β (P31) could be regarded as input, and the blotting of NLRP3 and β-actin are also recommended. References 1. Swanson KV, Deng M, Ting JP (2019) The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol 19(8):477–489. https://doi.org/10. 1038/s41577-019-0165-0 2. Tweedell RE, Malireddi RKS, Kanneganti T-D (2020) A comprehensive guide to studying inflammasome activation and cell death. Nat Protoc 15(10):3284–3333. https://doi.org/ 10.1038/s41596-020-0374-9 3. Yu J, Wu J, Zhang Z, Datta P, Ibrahimi IM, Taniguchi S, Sagara J, Fernandesalnemri T, Alnemri ES (2006) Cryopyrin and pyrin activate caspase-1, but not NF-κB, via ASC oligomerization. Cell Death Differ 13(2):236–249 4. Shi H, Wang Y, Li X, Zhan X, Tang M, Fina M, Su L, Pratt D, Bu CH, Hildebrand S, Lyon S, Scott L, Quan J, Sun Q, Russell J, Arnett S, Jurek P, Chen D, Kravchenko VV, Mathison JC, Moresco EMY, Monson NL, Ulevitch RJ, Beutler B (2016) NLRP3 activation and

mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component. Nat Immunol 17(3):250–258. https:// doi.org/10.1038/ni.3333 5. Brough D, Rothwell NJ (2007) Caspase-1dependent processing of pro-interleukin1beta is cytosolic and precedes cell death. J Cell Sci 120(5):772–781 6. Chae JJ, Wood G, Masters SL, Richard K, Park G, Smith BJ, Kastner DL (2006) The B30.2 domain of pyrin, the familial Mediterranean fever protein, interacts directly with caspase-1 to modulate IL-1beta production. Proc Natl Acad Sci U S A 103(26):9982–9987 7. Vajjhala PR, Mirams RE, Hill JM (2012) Multiple binding sites on the pyrin domain of ASC protein allow self-association and interaction with NLRP3 protein. J Biol Chem 287(50): 41732–41743 8. Hafner-Bratkovicˇ I, Susˇjan P, Lainsˇcˇek D, Tapia-Abella´n A, Cerovic´ K, Kadunc L,

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Angosto-Bazarra D, Pelegrin P, Jerala R (2018) NLRP3 lacking the leucine-rich repeat domain can be fully activated via the canonical inflammasome pathway. Nat Commun 9(1): 5182. https://doi.org/10.1038/s41467018-07573-4 9. Guo C, Chi Z, Jiang D, Xu T, Yu W, Wang Z, Chen S, Zhang L, Liu Q, Guo X, Zhang X, Li W, Lu L, Wu Y, Song B-L, Wang D (2018) Cholesterol homeostatic regulator SCAPSREBP2 integrates NLRP3 inflammasome activation and cholesterol biosynthetic signaling in macrophages. Immunity 49(5): 842–856.e847. https://doi.org/10.1016/j. immuni.2018.08.021 10. Guo C, Xie S, Chi Z, Zhang J, Liu Y, Zhang L, Zheng M, Zhang X, Xia D, Ke Y, Lu L, Wang D (2016) Bile acids control inflammation and metabolic disorder through inhibition of NLRP3 inflammasome. Immunity 45(4): 802–816 11. Hughes MM, Hooftman A, Angiari S, Tummala P, Zaslona Z, Runtsch MC, McGettrick AF, Sutton CE, Diskin C, Rooke M, Takahashi S, Sundararaj S, Casarotto MG, Dahlstrom JE, Palsson-McDermott EM, Corr SC, Mills KHG, Preston RJS, Neamati N, Xie Y, Baell JB, Board PG, O’Neill LAJ (2019) Glutathione transferase Omega-1 regulates NLRP3 inflammasome activation

through NEK7 deglutathionylation. Cell Rep 29(1):151–161.e155. https://doi.org/10. 1016/j.celrep.2019.08.072 12. Zhao K, Zhang Y, Xu X, Liu L, Huang L, Luo R, Li J, Zhang N, Lu B (2019) Acetylation is required for NLRP3 self-aggregation and full activation of the inflammasome. bioRxiv. 2019.2012.2031.891556. https://doi.org/ 10.1101/2019.12.31.891556 13. Song N, Liu Z-S, Xue W, Bai Z-F, Wang Q-Y, Dai J, Liu X, Huang Y-J, Cai H, Zhan X-Y, Han Q-Y, Wang H, Chen Y, Li H-Y, Li A-L, Zhang X-M, Zhou T, Li T (2017) NLRP3 phosphorylation is an essential priming event for inflammasome activation. Mol Cell 68(1): 185–197.e186. https://doi.org/10.1016/j. molcel.2017.08.017 14. Lo Y-H, Huang Y-W, Wu Y-H, Tsai C-S, Lin Y-C, Mo S-T, Kuo W-C, Chuang Y-T, Jiang S-T, Shih H-M, Lai M-Z (2013) Selective inhibition of the NLRP3 inflammasome by targeting to promyelocytic leukemia protein in mouse and human. Blood 121(16): 3185–3194. https://doi.org/10.1182/ blood-2012-05-432104 15. Kim B (2017) Western blot techniques. Methods Mol Biol 1606:133–139. https://doi.org/ 10.1007/978-1-4939-6990-6_9

Chapter 9 Intracellular Potassium Ion Measurements by Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) Yifei Zhang and Yan Shi Abstract Potassium ion (K+) efflux is often considered as an upstream signaling event of NLRP3 activation. The main evidence to demonstrate the importance of K+ efflux is that high concentration of extracellular K+ inhibits NLRP3 inflammasome assembly. However, the conditions used to prevent K+ flowing also breaks down a basic parameter of eukaryotic biology, leading to sustained membrane potential depolarization and affecting normal signal transduction in cells. Therefore, direct measurement of intracellular ion concentration can more truly reflect the role of K+ flow during the activation of NLRP3. In this chapter, we will provide the rationale and a method to evaluate intracellular K+ concentration by ICP-OES (Inductively Coupled Plasma Optical Emission Spectroscopy), which helps us understand how disturbances in intracellular K+ level orchestrates NLRP3 inflammasome activation. Key words NLRP3 inflammasome, Intracellular K+ content

1

Introduction The NLRP3 inflammasome, which has been linked to human inflammatory diseases, is activated by diverse stimuli [1]. It has been shown that almost all NLRP3 stimuli can increase the permeability of the cell membrane to K+, and the high-level extracellular K+ inhibits NLRP3 inflammasome activation [2], suggesting that efflux of intracellular K+ may orchestrate NLRP3 inflammasome assembly. Such a scenario was built upon the assumption that high extracellular K+ abolishes its extracellular/intracellular gradient, stopping its efflux. Under physiological conditions, the main extracellular cation is Na+ while the main cytosolic cation is K+. As the plasma membrane is more permeable for K+, the tendency of K+ exodus outwards sets up a positive charge above the outer membrane, intracellular Cl
is thus drawn to be under the inner membrane, creating a
30 to
90 mV charge differential over the

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_9, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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plasma membrane. This phenomenon, known as the membrane potential (MP), is essential for eukaryotic biology and affects many functions of the cell. Rise in extracellular K+, artificial or biological, disrupts the MP. While this may implicate the reduced efflux, the consequences of this disruption reaches far beyond K+ efflux [3]. In his discovery of inflammasome complexes [4], Jurg Tschopp noticed that increase in extracellular K+ blocked their activation. He attributed this effect to the result of K+ efflux blockage. Since then, this treatment has been used as the standard protocol to assert the rule of K+ efflux in inflammasome activation. However, as disruption of the MP has implications beyond this simple blockage. Other consequences that might modulate inflammasome activation have not been fully investigated. The common protocols such as high extracellular K+ used in NLRP3 research disrupts the basic baseline of membrane potential, causing sustained depolarization [5]. Subsequent research has found that sustained hyperpolarization (CFTRinh-172 or Glyburide treatment) also blocked its activation. This is remarkable as hyperpolarization is diametrically opposite of depolarization induced by increased extracellular K+ [6]. This raises the issue whether membrane potential disruption is the true reason for inflammasome inhibition. We found that the activation of Ca2+dependent calpain is a ubiquitous consequence of NLRP3 stimuli, and a relatively stable membrane potential is critically required for calpain activities, indicating the maintenance of membrane potential has great significance for NLRP3 inflammasome activation. Therefore, under physiological conditions, whether K+ efflux is truly central to inflammasome activation should be confirmed by via direct measurement. ICP-OES is a multiple element quantitation technique based on the measurement of excited atoms and ions at the wavelengths characteristic for the specific elements being measured. The lower detection limit for ICP-OES can extend to parts per billion, sufficient for intracellular ion concentration detection. We have attempted to measure the intracellular K+ over time to elucidate how K+ flows during the activation of NLRP3 inflammasome by ICP-OES. The results showed that the ion concentration drops rapidly in a short time by nearly a half (see Fig. 1), seemingly in agreement with the K+ efflux proposal [6]. As we noted, the establishment of membrane potential is usually caused by the asymmetric distribution and the different permeability of ions across plasma membrane. A small amount of ion flowing will have a dramatic effect on membrane potential. It is hard to imagine that such remarkable K+ efflux could be an initial signal of inflammatory response.

Intracellular Potassium Measurement

5 mM K+ 130 mM K+ 20 15 10 5

0 0

1

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120 100 80 60 40 20 0

6 4 2

IL-1β (ng/ml)

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K+ (%) of untreated)

B

K+ IL-1β

IL-1β (ng/ml)

K+ (%) of untreated)

A

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Fig. 1 Uncorrected K+ content of PMA-differentiated THP-1 cells and IL-1β secretion [6]. PMA-differentiated THP-1 cells were treated with silica under indicated conditions for different time, including (a) 5 mM K+ or 130 mM K+ buffer, and (b) DMSO, 200 μM Glyburide or 10 μM CFTRinh-172 in Opti-MEM. The percentage of intracellular K+ content and IL-1β secretion were plotted

Upon detailed analysis, it was noted that this assay compares total available K+ ions before and after the stimulation, without taking into account the cell number changes, which was in reality reduced nearly as much. In addition to the cell death caused by NLRP3 activation (see Fig. 2a), we also found some cells were not firmly attached to the plate during the ion concentration detection (see Fig. 2b), which could have not been used in final K+ quantitation. If the cell numbers are not adjusted, comparing total cell associated K+ from the beginning to the end could lead to the false impression of K+ leakage [6]. In order to reveal the real ion flow during the activation of NLRP3 inflammasome, here we describe an optimized measurement to detect the intracellular K+ concentration by ICP-OES. In the process of sample preparation, it is critical to remove the interference of extracellular fluid, and the operation should be gentle. Quality control group treated with the same condition should be used as test group. At different time points after NLRP3 inducer stimulation, it is necessary to count the living cells of quality control group and calculate the average ion concentration percentage of a single living cell corresponding to the measured value of the diluted intracellular fluid sample. Following this strategy, it will avoid the influence of cell loss and cell death on the detection of intracellular K+ concentration. In addition, ICP-OES can simultaneously detect the content of multiple elements including non-metal elements [7], so the role of other intracellular ions can be studied in the activation of NLRP3 inflammasome.

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A

B 30 20 10 0

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Fig. 2 The percentage of cell death and adherent cell during silica triggered NLRP3 Inflammasome activation [6]. PMA-differentiated THP-1 cells were labeled by CFSE and treated with silica under the indicated conditions same as Fig. 1, and then the cells were also washed as we did for the K+ content measurement assay. Cell death (a) was analyzed by propidium iodine (PI) staining, and the remaining adherent cell numbers (b) were counted by flow cytometric analysis

2 2.1

Materials Cell and Medium

1. THP-1 cell line. 2. Complete RPMI 1640 medium: RPMI 1640 with 10% FBS, 1% Penicillin-Streptomycin antibiotics, 10 mM (pH 7.4) HEPES, and 50 μM β-ME. 3. Normal K+ buffer: 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2.5 mM CaCl2, 10 mM HEPES, and 10 mM glucose (pH 7.4). 4. High K+ buffer is similar with the normal, but the equal amount of NaCl is replaced by KCl. 5. Opti-MEM reduced serum medium.

2.2

Reagents

1. Phorbol-12-myristate-13-acetate, PMA (10 μg/ml in DMSO). 2. Crystal silica (US Silica, #Min-U-Sil 15, 50 mg/ml in endotoxin-free water). 3. Ultrapure LPS (E. coli 0111:B4, 1 mg/ml in endotoxin-free water).

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4. Glyburide (100 mM in DMSO). 5. CFTRinh-172 (10 mM in DMSO). 6. Dimethylsulfoxide, DMSO. 7. Human IL-1β ELISA kit. 8. Standard solution of K+ for ICP-OES (National certified reference material). 9. 3% HNO3. 10. Propidium iodide (PI) staining solution (1 mg/ml). 11. 5(6)-Carboxyfluorescein (CFSE) (10 mM). 2.3

Equipment

diacetate

N-succinimidyl

ester

1. Inductively Coupled Plasma Optical Emission Spectrometer (VISTA-MTX). 2. Plate reader. 3. Flow cytometer.

3

Method

3.1 Cell Stimulation Assays

1. 2  106 THP-1 cells in each well of six-well plate are cultured in complete RPMI 1640 medium and differentiated into macrophages with 10 ng/ml PMA for 48 h before stimulation, and the addition of PMA results in suspension cells attaching to tissue culture dish and adopting a stellate morphology (see Note 1). 2. Replace the differentiation medium and prime cells with 200 ng/ml Ultrapure-LPS in fresh complete RPMI 1640 medium for 3 h. 3. Remove the medium containing LPS and perform the subsequent NLRP3 activation with 400 μg/ml crystal silica in fresh Opti-MEM medium or K+ buffer for 0–5 h. For the inhibitor pretreatments, glyburide (200 μM) or CFTRinh172 (10 μM) must be added to cells 30 min prior to adding crystals in Opti-MEM (see Note 2). 4. Collect the cell supernatant at different time points for IL-1β production analysis by ELISA (see Note 3). The cells will be used for intracellular K+ concentration detection or adherent living cell number analysis.

3.2 Cell Lysis Preparation and Intracellular K+ Content Measurement

1. PMA-differentiated THP-1 cells are stimulated as described in Subheading 3.1, and briefly washed with 3 ml K+-free buffer to eliminated extracellular K+ carryover (see Note 4). 2. Aspirate the extracellular fluid as completely as possible, and extract cells with 3 ml 3% HNO3 for 30 min (see Note 5).

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3. Measure the K+ content of cell extract samples, blank control (H2O), reagent control (3% HNO3), and several K+ standard solution by ICP-OES (see Note 6). 4. According to the standard curve (defined by K+ standard solution), the value of intracellular K+ concentration is calculated. 1. THP-1 cells are labeled with 10 μM CFSE at 37  C for 15 min and gently washed twice by PBS buffer before cell stimulation assay (see Note 1).

3.3 Remaining Adherent Cell Numbers and Cell Death Analysis

2. PMA-differentiated THP-1 cells are stimulated as described in Subheading 3.1, and briefly washed with 3 ml K+-free buffer. 3. Collect the adherent cells in FACS buffer. Spin down the cells at 4  C 500  g for 5 min. 4. Resuspend the cells in 10 μg/ml PI staining buffer at room temperature for 15 min. 5. Wash the cells twice with PBS and resuspend in 300 μl PBS for FACS analysis (see Note 7). 6. The percentage of remaining adherent living cells are calculated by the count of CFSE-positive and PI-negative cells. 7. Correct the results of intracellular K+ concentration according to the adjusted live cell number (see Fig. 3). Calculate the percentage of intracellular K+ content using the following formula: Corrected ½Kn ¼

½Kn Adherent cell%  PI negative cell% ½Kn% ¼

Corrected ½Kn Corrected ½K0

[Kn]—the value of K+ concentration measured by ICP-OES at different time point (n hours).

5 mM K+ 130 mM K+ 20

150

10 50 0

5 0

1

2

3 4 Time (h)

5

0

K+ IL-1β DMSO Glyburide CFTRinh-172

150

6 4

100

2

50 0

IL-1β (ng/ml)

15 100

Corrected K+ (%) of untreated)

B

K+ IL-1β

IL-1β (ng/ml)

Corrected K+ (%) of untreated)

A

0 0

1

2

3 4 Time (h)

5

Fig. 3 Corrected K+ content of PMA-differentiated THP-1 cells and IL-1β secretion. Recalculate the percentage of intracellular K+ content in Fig. 1 excluding the cell loss and cell death

Intracellular Potassium Measurement

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Notes 1. Set up the ion concentration detection and cell loss analysis side by side. One well of cells are prepared for intracellular potassium concentration measurement, and the other one is labeled by CFSE for the living cell number counting. 2. Physiological condition of intracellular K+ is about 5 mM. High-level extracellular K+ leads to sustained depolarization, while glyburide or CFTRinh-172 causes sustained hyperpolarization of membrane potential. 3. Quantitative detection of human IL-1β in supernatant should follow the user guide of ELISA kit. Read absorbance of each samples and standards on a plate reader at 450 nm wavelength. Create a standard curve by plotting the mean absorbance for each standard concentration and draw a best fit curve through the points of graph (a 4-parameter curve fit). Calculate the human IL-1β concentration of samples according to curve fit equation. 4. For accurate measurement of the intracellular ions, a control was performed in every experiment to determine the extracellular amount of the investigated ion remaining after aspiration, and this value was subtracted from every measurement. 5. Avoid using viscous sulfuric acid or phosphoric acid to treat the sample. 6. The certified reference solution of K+ is used as stock solution of single element and can be formulated into serials of working standard solutions by stepwise dilution with 3% HNO3. The standard curve should cover the entire range of expected concentrations. When the sample concentration is high, the appropriate dilution could inhibit physical interference. 7. CFSE is a green-fluorescent dye with excitation/emission at 492/517 nm. PI bound with DNA has excitation/emission maxima of 535/617 nm. The fluorescence of CFSE and PI is excited with 488 nm laser line and detected with FLA-1 and FLA-2 channel of the BD Accuri™ C6 Plus Flow Cytometer (or a similar instrument), respectively.

Acknowledgments We thank Dr. Hua Rong for help in experiments. Y.S. is supported by the joint Peking-Tsinghua Center for Life Sciences and the National Natural Science Foundation of China grants 81621002, 31630023, 31370878, and 20171312479.

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References 1. Swanson KV, Deng M, Ting JP (2019) The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol 19(8):477–489. https://doi.org/10.1038/ s41577-019-0165-0 2. Munoz-Planillo R, Kuffa P, Martinez-Colon G, Smith BL, Rajendiran TM, Nunez G (2013) K+ efflux is the common trigger of NLRP3 inflammasome activation by bacterial toxins and particulate matter. Immunity 38(6):1142–1153. https://doi.org/10.1016/j.immuni.2013. 05.016 3. Wright SH (2004) Generation of resting membrane potential. Adv Physiol Educ 28(1–4): 139–142. https://doi.org/10.1152/advan. 00029.2004 4. Petrilli V, Papin S, Dostert C, Mayor A, Martinon F, Tschopp J (2007) Activation of the NALP3 inflammasome is triggered by low

intracellular potassium concentration. Cell Death Differ 14(9):1583–1589. https://doi. org/10.1038/sj.cdd.4402195 5. Belhage B, Hansen GH, Schousboe A (1993) Depolarization by K+ and glutamate activates different neurotransmitter release mechanisms in GABAergic neurons: vesicular versus non-vesicular release of GABA. Neuroscience 54(4):1019–1034 6. Zhang Y, Rong H, Zhang FX, Wu K, Mu L, Meng J, Xiao B, Zamponi GW, Shi Y (2018) A membrane potential- and calpain-dependent reversal of caspase-1 inhibition regulates canonical NLRP3 inflammasome. Cell Rep 24(9): 2356–2369.e2355. https://doi.org/10.1016/ j.celrep.2018.07.098 7. Vandecasteele C, Block CB (1993) Modern methods for trace element determination. Wiley, New York. p. 1,168

Chapter 10 NLRP3 Phospho-residue Mapping by Phospho Dot Blots Sangeetha Shankar, Zsofia A. Bittner, and Alexander N. R. Weber Abstract When characterizing posttranslational modifications like phosphorylation, using efficient screening methods to map the phospho sites is essential, especially when dealing with large multi-domain proteins. NLRP3 (the NOD, LRR, and pyrin domain-containing protein 3), which initiates the formation of an NLRP3 inflammasome complex, is regulated posttranslationally by phosphorylation at several Ser and Tyr residues. However, determining sites of modification are not straightforward. For quick and reliable screening of the candidate phospho sites in NLRP3, we use a phospho dot blot assay which we describe here. This technique employs an in vitro kinase assay with a candidate kinase, Bruton’s Tyrosine Kinase (BTK), and peptides derived from the region of interest in the protein that contains the potential phosphorylation sites. The reaction containing the phosphorylated peptides is quickly screened by a dot blot where the peptides are blotted with a commercially available anti-phospho-tyrosine antibody. This method can also be adapted to detect modified Ser or Thr residues and is an ideal screening assay to map phospho residues in NLRP3 or other proteins. This can be an initial screening procedure or can be complemented by other approaches such as site directed mutagenesis and by generating phospho site-specific antibodies. Key words Dot blot, Phosphorylation, Tyrosine phosphorylation, Posttranslational modification, NLRP3 inflammasome, NLRP3, BTK

1

Introduction

1.1 Inflammasomes and Posttranslational Modification of NLRP3

Inflammasomes are multi-protein complexes formed in response to pathogenic and sterile insults to release a pro-inflammatory cytokine IL-1β [1]. NLRP3 inflammasome is the most studied inflammasome complex, consisting of NLRP3, ASC, and caspase-1 that processes pro-IL-1β to the mature IL-1β [2]. Pivotal in the regulation of the NLRP3 inflammasome are posttranslational modifications (PTMs), including, for example, phosphorylation, acetylation, ubiquitinylation, and SUMOylation. Out of these, phosphorylation of Tyr, Ser, and Thr residues has been found to have both activating and regulatory roles in inflammasome assembly and IL-1β production. Recently, several phosphatases and kinases have been discovered to be involved in inflammasome

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_10, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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regulation. Examples are the protein tyrosine phosphatase non-receptor 22 (PTPN22) that dephosphorylates NLRP3 and facilitates the inflammasome activation [3], and Protein kinase D (PKD), which phosphorylates NLRP3 at the Golgi apparatus, shifting its localization and promoting inflammasome assembly [4]. Recently, we and others have found Bruton’s Tyrosine Kinase (BTK) to be involved in the phosphorylation of Tyr residues in NLRP3, thereby activating it [5–7]. One important goal in elucidating the NLRP3 regulatory mechanism is to identify how modifications, such as phosphorylation, affect NLRP3 function. In order to do so and to carry out subsequent, more in-depth studies (such as site-directed mutagenesis or the generation of phosphospecific antibodies), a precise mapping of phospho-sites is necessary. NLRP3 being a protein containing 1036 aa, in which there are 86 Ser, 42 Thr, and 27 Tyr residues, site-directed mutagenesis as a preliminary approach to map phospho sites is often impractical, and mass spectrometric analysis of NLRP3 has been notoriously difficult. On the other hand, mapping of the modified residues is essential to study the impact of the modification mechanistically and to develop phospho- and site-specific antibodies. Here, we offer phospho dot blots as an alternative approach for mapping modified residues, as exemplified for the identification of phospho-Tyrosine residues (p-Tyr). 1.2 Principle of Phospho Dot Blot

We have used phospho dot blot to map the sites of modification and to identify the residues phosphorylated by BTK. For this, we have designed synthetic peptides covering an NLRP3 region of interest (ROI). The ROI was defined by immunoblotting (IB) with antiphospho-Tyr antibodies and truncated NLRP3 constructs encompassing only individual domains [8]. Dot blot is a simplified form of the immunoblot (IB) procedure initially used by Hawkes and colleagues, where the protein or peptide is blotted directly on the PVDF or nitrocellulose membrane and detected with a modification-specific antibody [9]. This is different from conventional IB combined with SDS-PAGE in that the samples are not separated according to their molecular weight first, and it is thus a simpler and higher throughput screening method to quickly and qualitatively determine whether multiple given peptides are phosphorylated by a candidate kinase or not. Here we have adapted this technique to identify 12–20 amino acids long Tyr-phosphorylated peptides derived from NLRP3. In principle, the method can also be adapted to detect Ser- or Thr-modified peptides or to whole-cell lysates to screen for phosphorylation [10]. The in vitro kinase assay represents a reaction between commercially available recombinant BTK (but possibly other candidate kinases could also be immunoprecipitated from cell lysates if not available commercially/recombinantly) and synthetic 15-mer NLRP3 peptides containing the proposed Tyrosine residues. As

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controls, we used conditions without kinase and with peptides in which the Tyr residues were changed to Phe. The reaction setup includes NLRP3 peptides and His-tagged BTK combined in kinase buffer containing ATP and incubation for 3 h which allows for phosphorylation of the Tyr residues. To stop the reaction by denaturing the kinase, the reaction mixture is incubated briefly at a high temperature (95
C). As BTK is auto-Tyr-phosphorylated, it had to be removed from the reaction prior to dot blotting using His-tagged beads; otherwise, the kinase phospho-Tyr-signal would overlay the signal from phosphorylated peptides. The remaining reaction containing the peptides was then spotted onto nitrocellulose membrane. Using anti-p-Tyr antibodies, phosphorylated peptides were identified in a quick and efficient manner. Omission of BTK, staining of total peptide, and use of the Phe-containing peptides confirmed specificity of the modification. Checking the phosphorylation of BTK acted as an additional control for the kinase’s activation and buffer conditions. Furthermore, peptides synthesized with phospho-Tyr could be used as positive controls. 1.3 Advantages of Phospho Dot Blot

Phospho dot blot is a quick and simple procedure to identify the specific residues phosphorylated in a protein. Compared to conventional IBs that are traditionally used, the SDS-PAGE separation and transfer are omitted, saving considerably on time and sample consumption. Furthermore, as peptides can be manually spotted with ease (although there is dot blotting equipment available, see below), more samples can be analyzed simultaneously, rendering the method suitable for medium (described here) or even highthroughput analysis. Potentially limiting will be the costs for obtaining synthesized peptides, so that it may be advisable to delineate the modified domain first (e.g., by using truncation constructs in conventional IB). Phospho dot blot thus provides an efficient initial screen to identify residues which could be further confirmed by IB or other techniques using site-specific phosphoAbs directed against each of the candidate residues. Since generating the latter is time consuming and costly, phospho dot blots provide a way to identify candidates for further study and/or antibody generation and could be combined with MALDI mass spectrometry for further confirmation of phosphate addition.

1.4 Disadvantages of Phospho Dot Blot

Dot blot is a qualitative analysis and therefore cannot be used for quantifying the amount of peptide or protein phosphorylated. Care should also be taken when using peptides that are highly polar as suboptimal binding to the nitrocellulose membrane could be a potential problem. As the 3D conformation of the analyzed peptide sequence in the natural context of an entire domain may differ from a 15-mer in solution, there is the potential for false negatives (peptide solution structure lacks native conformation required for

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phosphorylation by the kinase) and positives (linearized peptide phosphorylated by promiscuous kinase in solution but 3D epitope not permissive for phosphorylation). These disadvantages can be slightly minimized using Tyr to Phe (or Ser/Thr to Ala) substitution peptides as controls but nevertheless require further validation using additional techniques including, for example, site-directed mutagenesis and analysis in cell lysates, mass spectrometry analysis, generation of phospho-antibodies, or structural methods. Another factor to be considered is the cost and purity of the required synthetic peptides, although the highest purity levels are not necessary, at least for an initial screen.

2 2.1

Materials Kinase Reaction

1. Kinase buffer (1): 25 mM Tris–HCl (pH 7.5), 5 mM β-glycerophosphate, 2 mM dithiothreitol (DTT), 0.1 mM Na3VO4, 10 mM MgCl2. 10 Kinase buffer can be prepared or purchased and diluted to 1 with ultra-pure H2O, and DTT is added to 1 buffer before use. Store aliquots at 20
C and thaw at 25–30
C. 2. 100 mM ATP for a final concentration of 2 mM in each reaction. Dilute in ultra-pure H2O, adjust the pH to 7.4 and store at 20
C in aliquots. Do not re-use already opened aliquots. 3. Recombinant kinase (BTK): 0.29 mg/ml stock concentration, stored in aliquots at 80
C. 4. 12–20 mer peptides: stock concentration of 10 mg/ml dissolved in DMSO or ultra-pure water based on their solubility. Dilute the peptides to 1 mg/ml working concentration using freshly prepared 1 kinase buffer (see Note 1). Refer to Table 1 for the peptides used and their sequences. 5. Thermocycler (preferentially with heated lid) or a water bath set to 30
C and 95
C for reaction and denaturing conditions, respectively. 6. Pipettes and pipette tips: Volume 10 μl, 200 μl, and 1000 μl.

2.2 Kinase Removal Using Magnetic Beads

1. His-tagged beads (magnetized), store at +4
C. 2. Magnetic stand. 3. 0.2-ml PCR tubes.

2.3 Dot Blot Analysis of the Phospho Peptides and Chemiluminescent Detection

1. Nitrocellulose membrane 0.45 μm. 2. Pierce Reversible Protein Stain. Other stains (e.g., Ponceau S) that are used to non-specifically stain peptides on PVDF or nitrocellulose membranes may also work.

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Table 1 List of NLRP3 peptides used in the Fig. 2, containing Tyr phosphorylated by BTK or Y>F replacements as negative controls and a peptide synthesized with p-Y as a positive control Original Y containing NLRP3 peptides, Y > F containing or phospho-Y containing derivatives Sequence Y136 Y140 Y143

KKDYRKKYRKYVRSR

Y136

ICKMKKDYRKKYRKY

Y168

SVSLNKRYTRLRLIK

Y123

EWMGLLEYLSRISIC

p-Y

KKDpYRKKpYRKpYVRSR

3 Y > F

KKDFRKKFRKFVRSR

Y140 > F and Y143 > F

KKDYRKKFRKFVRSR

Y136 > F and Y143 > F

ICKMKKDFRKKYRKF

Y168 > F

SVSLNKRFTRLRLIK

Y123 > F

EWMGLLEFLSRISIC

3. Tris-buffered saline (TBS; 10): 1.5 M NaCl, 0.2 M Tris base, pH 7.4, stored at RT. 4. TBS-T: TBS with 0.1% (v/v) Tween 20, store at RT. 5. Blocking solution 1: 5% (w/v) Bovine Serum Albumin Fraction V in TBS-T, stored at +4
C. 6. Blocking solution 2: 5% (w/v) non-fat milk powder in TBS-T, stored at +4
C. 7. Rabbit Phospho-Tyrosine primary antibody, stored at 20
C, and diluted 1:1000 in blocking solution 1, stored at +4
C. 8. Anti-rabbit secondary antibody, stored at +4
C, diluted 1: 5000 in blocking solution 2, stored at +4
C. 9. Roller shaker. 10. Chemiluminescent substrate. 11. Chemiluminescent Imaging System.

3

Methods An overview of the workflow is given in Fig. 1.

3.1

Kinase Reaction

1. For setting up the kinase reaction in a total reaction volume of 25 μl, mix 10 μl of 1 kinase buffer (with DTT: refer to Subheading 2) and 10 μl of ATP (final concentration of 2 mM in the reaction).

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Fig. 1 Schematic representation of the work flow for the kinase assay and the dot blot

2. Add 4 pmol of the protein kinase and 2 nmol of the sample peptide. Calculate the peptide volume based on the molecular mass of each peptide such that 2 nmol of peptide are added to the each kinase reaction. For example, add 1 μl of 0.29 μg/μl BTK recombinant protein to 3–4 μl of 1 μg/μl sample peptide (see Notes 2 and 3). Include a control sample containing BTK only without peptides to assess the phospho-background signal after BTK depletion/control BTK-depletion efficiency. 3. Mix the reaction mixture well by pipetting the mixture 3–4 times. 4. Incubate in Thermocycler (recommended) or heat block for 3 h at 30
C. 5. Set the Thermocycler to 95
C and incubate the samples for 5 min to denature the protein (this allows the separation of BTK from the peptides, its denaturation and subsequent removal using His-tagged beads). 6. Chill the reaction by incubating at 4
C in Thermocycler or wet ice for 10 min. 3.2 Kinase Removal Using HisTagged Beads

1. Wash the beads once in 1 kinase buffer by placing a 1.5-ml reaction tube containing the required amount beads in a magnetic stand (2 μl per sample  number of samples, e.g., 2 μl  10 samples ¼ 20 μl). The beads should precipitate because of the magnetic field.

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2. Aspirate the supernatant with a pipette set to 20 μl. 3. Add 100–200 μl of 1 kinase buffer and mix the solution thoroughly by pipetting 5–7 times with a 200 μl pipette (see Note 4). 4. Place tubes back into magnetic stand and allow the beads to precipitate for 1–1.5 min. 5. Add equal amount of the kinase buffer and repeat steps 3 and 4 once more. 6. Now add 20 μl of 1 kinase buffer. 7. Mix the solution thoroughly using a 20-μl pipette and add 2 μl to the chilled samples placed on wet ice. 8. Incubate at 4
C for 1.5 h preferably in a Thermocycler and mix the reaction mixture every 20 min to prevent the beads from settling down. 9. Transfer the samples to 1.5-ml Eppendorf tube and place them in a magnetic stand. Transfer the supernatant containing the BTK-depleted peptides to a fresh tube. 3.3

Dot Blot

1. On a nitrocellulose membrane, pore size of 0.45 μm, add 2 μl of the supernatant from each sample by pipetting it on top of the membrane and allow it to dry completely. This should take approximately 3–4 min. Take care to not add the samples too close to each other to avoid mixing the samples. Alternatively, a dot blot apparatus could be used (see Note 5). Synthetic p-Ycontaining peptides may be spotted directly as positive controls for detection. 2. Before proceeding, check that all samples have completely dried by holding the membrane up in front of a light source, and checking whether any spotted samples are still visible as a wet (i.e., darker) dot/spot on the membrane. If so, wait longer and re-check (Fig. 2).

3.4 Control Total Peptide Staining

1. Reversible protein staining is used according to the manufacturer’s instructions to detect the presence of the peptide in the sample. 2. Briefly, wash the membrane with ultra-pure water followed by the pierce stain such that the membrane is completely submerged in the stain and shake the membrane in a shaker for maximum of 1 min. 3. Add the de-staining solution, making sure that the membrane is completely submerged by the solution and shake the membrane in a shaker for 5 min to remove the background stain. 4. Wash the membrane briefly for 1 min using 5–10 ml ultra-pure water (to completely submerge the membrane) three times and

-BTK +BTK

Y123>F

Y168>F

Y136>F Y143>F

Y140>F Y143>F

Y123>F

p-Y

3>F

BTK alone

phos. Peptide

Y168

p-Y

(d)

Y136

(c)

Y123

+BTK

Y123

+BTK

Y168>F

-BTK

Y136>F Y143>F

-BTK

Y140>F Y143>F

Total peptide stain

3>F

BTK alone

phos. Peptide

Y168

Total peptide stain

(b)

Y136

(a)

Y136 Y140 Y143

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Y136 Y140 Y143

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-BTK +BTK

Fig. 2 (a) and (b): Pierce total protein stain of the indicated peptides without or with BTK in the kinase reaction as indicated in the upper and lower lanes, respectively. The “BTK alone” lane indicates that recombinant BTK protein is added to the membrane directly, appearing as a p-Y signal. The presence of peptides in the reactions is visualized by the total protein stain. However, for certain peptides, such as a peptide including Y123 and Y123 > F, the staining is less prominent (see Note 6). (c) and (d): p-Y blot of the same samples as in (a) and (b). Y>F indicates that the tyrosine is mutated to phenylalanine which does not get phosphorylated and therefore acts as a control. In (c), all the tyrosine containing peptides are phosphorylated in the presence of BTK and the phosphorylated peptide shows a p-Y signal without BTK (positive control 1). BTK itself is also phosphorylated which usually is one of the markers for the kinase’s activation (positive control 2). In (d), the Y > F and 3  Y > F mutant peptides completely lack p-Y signals, whereas in the 2  Y > F mutant peptides the signal is recovered partially due to the presence of the third unmodified tyrosine residue

wash once again for 5 min. Now the presence of the protein can be detected by the appearance of turquoise blue color at the spot where the sample is added (see Note 6). 5. The membrane can be imaged in a scanner or in a CCD system for future reference. 6. To erase the stain, add the stain eraser to the membrane and shake for 2 min. This is followed by rinsing with ultra-pure water four times followed by 5 min washing (see Note 7). 3.5 Incubation with Phospho-Specific Antibodies

1. Block the membrane using the blocking solution 1: 5% BSA in TBS-T and shake in a roller shaker at room temperature for 1 h. 2. Discard the blocking solution and wash the membrane using 5 ml of TBS-T thrice for 5 min, changing the wash buffer after every wash.

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3. Add the primary p-Y antibody diluted 1:1000 in the blocking solution 1 and incubate it at 4
C overnight in a roller shaker (see Note 8). 4. The next day, repeat the step 2 at room temperature. The primary antibody can be stored at 4
C for 1–2 weeks, if desired, for re-using. 5. Add the secondary antibody (1:5000) diluted in 5% non-fat milk blocking solution 2 and incubate for 1 h in a roller shaker at room temperature. 3.6 Detection of Chemiluminescence

1. Wash the membrane by repeating the step 2 mentioned in Subheading 3.5. 2. Add at least 250 μl of the chemiluminescent substrate on to the membrane and incubate the membrane at room temperature for 1 min. 3. The chemiluminescence signal is acquired using the CCD camera system for 3 min in auto exposure settings. Alternatively, manual or signal accumulation can also be used and the acquisition timings needs to be optimized based on the CCD system used.

4

Notes 1. The 15-mer peptides are synthesized in house, checked for 95% purity by HPLC, lyophilized, and then resuspended to 10 mg/ ml stock concentration in DMSO. They were then, stored in aliquots at 20
C. Alternatively, synthetic peptides can be obtained from a commercial supplier. 2. The peptides are diluted in 1 kinase buffer to a working concentration of 1 μg/μl as described in Subheading 2. 3. A similar method as mentioned in step 2 of Subheading 3.1 can be used for proteins from immunoprecipitation or cell lysates. However, it is recommended to titrate and determine the best ratio of protein kinase used in such cases. 4. It is usually not recommended to vortex the magnetic beads. 5. Bio-Dot® and (Bio Rad).

Bio-Dot

SF

Microfiltration

Apparatus

6. In a few cases, the pierce stain does not stain or only weakly stains certain peptides. This is most probably due to the specific amino acid sequence of the peptides. Therefore, the intensity of staining is not a direct indication of the amount of peptide added. The stain is mostly to prove that in the absence of a phospho-Y signal, this was not due to omission of the peptide.

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7. If fluorescent detection is used for imaging (e.g., NIR imaging system), it is recommended to check for any interference or possible background fluorescence created by the protein stain. 8. If using a primary antibody from a different manufacturer or against another modification, dilutions and incubation conditions for both the primary and secondary antibodies need to be explored. CST Multimab P-Y1000, #8954 delivered very good results in our hands.

Acknowledgments We thank Xiaowu Zhang from Cell Signaling Technologies for helpful advice on the initial conceptual implementation of this method. The work was supported by the Else-Kro¨ner-Fresenius Stiftung (to A.W.), the Deutsche Forschungsgemeinschaft (German Research Foundation, DFG) grant We-4195/15-1 (to A.W.), and the University Hospital Tu¨bingen. Infrastructural funding was provided by the University of Tu¨bingen, the University Hospital Tu¨bingen, and the DFG Clusters of Excellence “iFIT—ImageGuided and Functionally Instructed Tumor Therapies” (EXC 2180, to AW), “CMFI—Controlling Microbes to Fight Infection” (EXC 2124, to AW), Gefo¨rdert durch die Deutsche Forschungsgemeinschaft (DFG) im Rahmen der Exzellenzstrategie des Bundes und der L€ander—EXC 2180 and EXC 2124. References 1. Broderick L, De Nardo D, Franklin BS, Hoffman HM, Latz E (2015) The inflammasomes and autoinflammatory syndromes. Annu Rev Pathol Mech Dis 10(1):395–424. https://doi. org/10.1146/annurev-pathol012414-040431 2. Mangan MSJ, Olhava EJ, Roush WR, Seidel HM, Glick GD, Latz E (2018) Targeting the NLRP3 inflammasome in inflammatory diseases. Nat Rev Drug Discov 17(8):588–606. https://doi.org/10.1038/nrd.2018.97 3. Spalinger MR, Schwarzfischer M, Hering L et al (2019) Loss of PTPN22 abrogates the beneficial effect of cohousing-mediated fecal microbiota transfer in murine colitis. Mucosal Immunol 12(6):1336–1347. https://doi.org/ 10.1038/s41385-019-0201-1 4. Zhang Z, Meszaros G, He W-T et al (2017) Protein kinase D at the Golgi controls NLRP3 inflammasome activation. J Exp Med 214(9): 2671–2693. https://doi.org/10.1084/jem. 20162040

5. Bittner ZA, Liu X, Tortola MM et al (2019) BTK operates a phospho-tyrosine switch to regulate NLRP3 inflammasome activity. J Exp Med 218(11):e20201656. https://doi.org/ 10.1084/jem.20201656 6. Liu X, Pichulik T, Wolz OO et al (2017) Human NACHT, LRR, and PYD domain–containing protein 3 (NLRP3) inflammasome activity is regulated by and potentially targetable through Bruton tyrosine kinase. J Allergy Clin Immunol 140(4):1054–1067.e10. https://doi.org/10.1016/j.jaci.2017.01.017 7. Ito M, Shichita T, Okada M et al (2015) Bruton’s tyrosine kinase is essential for NLRP3 inflammasome activation and contributes to ischaemic brain injury. Nat Commun 6:1–11. https://doi.org/10.1038/ncomms8360 8. Mayor A, Martinon F, De Smedt T, Pe´trilli V, Tschopp J (2007) A crucial function of SGT1 and HSP90 in inflammasome activity links mammalian and plant innate immune

NLRP3 Phospho Dot Blot responses. Nat Immunol 8(5):497–503. https://doi.org/10.1038/ni1459 9. Hawkes R, Niday E, Gordon J (1982) A dot-immunobinding assay for monoclonal and other antibodies. Anal Biochem 119(1): 142–147. https://doi.org/10.1016/00032697(82)90677-7

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10. Hantschel O, Rix U, Schmidt U, Bu¨rckstu¨mmer T, Kneidinger M, Schu¨tze G, Colinge J, Bennett KL, Ellmeier W, Valent P, Superti-Furga G (2007) The Btk tyrosine kinase is a major target of the Bcr-Abl inhibitor dasatinib. Proc Natl Acad Sci 104(33):13283. https://doi.org/10.1073/pnas.0702654104

Chapter 11 Analysis of Activity and Expression of the NLRP3, AIM2, and NLRC4 Inflammasome in Whole Blood Lev Grinstein and Stefan Winkler Abstract Pro-inflammatory caspase-1 is a key player in innate immunity. Following activation in heterogenic protein complexes called the inflammasome, caspase-1 processes IL-1β and IL-18 to their mature forms and triggers pyroptosis. Here, we describe a small-volume whole blood assay facilitating the measurement of caspase-1 activity and inflammasome-related gene expression following specific stimulation of either the NLRP3, NLRC4, or AIM2 inflammasome. Key words Whole blood assay, Inflammasome, Caspase-1, NLRP3, NLRC4, AIM2, MCC950

1

Introduction Pathogens, stress, and damage signals induce activation of caspase1, typically mediated by proximity-induced autoproteolysis in multimeric protein complexes called inflammasomes. Subsequently, caspase-1 activates pro-IL-1β and pro-IL-18 by proteolytic cleavage into their mature forms and triggers pyroptosis, a programmed pro-inflammatory cell death, thereby initiating a pro-inflammatory immune response [1]. Most inflammasomes consist of at least three components: an intracellular sensor (pattern recognition receptor) responsible for the recognition of specific pathogen- or dangerassociated molecular patterns (PAMPs, DAMPs), the adaptor protein apoptosis speck-like protein containing a CARD (ASC), and procaspase-1. The current model of inflammasome activation consists of two steps. First, transmembrane bound Toll-like receptors (TLR) located at the cell surface recognize extracellular DAMPs or PAMPs leading to enhanced transcription of proinflammatory target genes and priming of the inflammasome. A second signal is essential for inflammasome activation by triggering the intracellular sensor, leading to inflammasome assembly and caspase-1 activation

Ali A. Abdul-Sater (ed.), The Inflammasome: Methods and Protocols, Methods in Molecular Biology, vol. 2459, https://doi.org/10.1007/978-1-0716-2144-8_11, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2022

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[2]. Physiologically, inflammasome/caspase-1 signaling contributes to host defense against invading pathogens. Dysregulation of this pathway is involved in the pathogenesis of numerous diseases, from rare autoinflammatory syndromes to more common diseases like rheumatoid arthritis, gout, or sepsis [3]. In order to unveil new therapeutic targets against inflammasome-driven diseases, detailed understanding of pathways involved is crucial but functional analysis of specific inflammasomes in patients remains challenging. Typically, studies are performed using peripheral blood mononuclear cell (PBMC)-based assays [4–7]. Thus, soluble plasma factors as well as cellular interactions, both known to modulate innate immune responses, are lost within PBMC isolation. Furthermore, caspase-1 is expressed not only in monocytes, macrophages and dendritic cells, but also in PMNs representing the major leukocyte subset in peripheral blood, typically absent in PBMC preparations. Thus, analyzing inflammasome/caspase-1 activity in purified PBMCs seems to remain incomplete. Here, we present the detailed protocol of a validated low-volume human whole blood assay facilitating the measurement of caspase-1 activation and inflammasome-related gene expression upon specific stimulation of the NLRP3, AIM2, or NLRC4 inflammasome [8]. The whole blood assay utilizes the IL-1β concentration in stimulated samples as surrogate parameter for inflammasome/caspase-1 activity.

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Materials Prepare all solutions and cell culture plates using a laminar flow hood. Prepare all reagents before the start of the experiment in order to shorten handling time of blood samples. Follow all waste disposal regulations when discarding material.

2.1 InflammasomeSpecific Stimulation

1. Hirudin blood collection tubes (see Note 1). 2. Sterile 96-well plates (cell culture grade, flat-bottom). 3. Sterile phosphate-buffered saline without CaCl2 and MgCl2. 4. Shaker for well plates installed within a cell culture CO2-incubator set to 37
C, 5% CO2.

2.1.1 NLRP3 Stimulation

1. Ultra-pure LPS dissolved in sterile, deionized water as stock solution (1 mg/ml), stored in aliquots at 20
C (see Note 2). Dilute the upLPS stock solution (1 mg/ml) to the working solution (9 μg/ml) using PBS. 2. ATP dissolved in sterile, deionized water as stock solution (200 mM), stored in aliquots at 80
C (see Note 3). Dilute the ATP stock solution (200 mM) to the working solution (10 mM) using PBS.

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3. MCC950 dissolved in sterile, deionized water as stock solution (10 mM), stored in aliquots at 20
C or at 4
C for shortterm use (see Note 4). Dilute the MCC950 stock solution (10 mM) to the working solution (100 μM) using PBS. 2.1.2 AIM2 Stimulation

1. Ultra-pure LPS dissolved in sterile, deionized water (1 mg/ml) stored in aliquots at 20
C (see Note 2). Dilute the upLPS stock solution (1 mg/ml) to the working solution (9 μg/ml) using PBS. 2. Poly(dA:dT) dissolved in sterile, deionized water (1 mg/ml), stored in aliquots at 20
C. 3. Lipofectamine 2000 (ThermoFisher Scientific) stored at 4
C (see Note 5). 4. Opti-MEM medium. 5. MCC950 dissolved in sterile, deionized water as stock solution (10 mM), stored in aliquots at 20
C or at 4
C for shortterm use (see Note 4). Dilute the MCC950 stock solution (10 mM) to the working solution (100 μM) using PBS.

2.1.3 NLRC4 Stimulation

1. High-salt Lysogeny broth (LB) medium: Suspend the specified amount of LB medium powder (typically 20 g/l) and 17.5 g NaCl (final concentration 300 mM) in 1 l of deionized water. Mix well until fully dissolved. Sterilize in an autoclave at 121
C for 15 min. Cool down to room temperature and add Streptomycin to an aliquot (final concentration 200 μg/ml). LB medium should be stored at 2–8
C. Streptomycin-containing aliquots can be used for 1 week, whereas the stock solution can be stored for 4 weeks. 2. Salmonella enterica, Serovar Typhimurium (Strain SL1344) stored as bacterial glycerol stock at 80
C (see Note 6). 3. Photometer and cuvettes capable of measuring the optical density (OD) at 600 nm. Gentamicin dissolved in sterile, deionized water as stock solution (50 mg/ml), stored at 4
C. Dilute the gentamicin stock solution (50 mg/ml) to the working solution (360 μg/ ml) using PBS. 4. MCC950 dissolved in sterile, deionized water as stock solution (10 mM), stored in aliquots at 20
C or at 4
C for shortterm use (see Note 4). Dilute the MCC950 stock solution (10 mM) to the working solution (90 μM) using PBS.

2.2 RT-qPCR and Cytokine Measurement

1. Erythrocyte lysis buffer. 2. RNeasy Micro Kit (see Note 7). 3. Sensiscript® Reverse Transcriptase Kit (see Note 7). 4. qPCR Master Mix (e.g., SYBR® green based).

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Table 1 Name, primer sequences, and amplicon length of inflammasome-associated genes and one reference gene (HPRT1) analyzed in the study from Grinstein et al. [8] (bp, base pairs) Name

Sequence (50 ! 30 )

Amplicon (bp)

AIM2

F: TGG CAA AAC GTC TTC AGG AGG R: AGC TTG ACT TAG TGG CTT TGG

83

ASC

F: TGG ATG CTC TGT ACG GGA AG R: CCA GGC TGG TGT GAA ACT GAA

110

CASP1

F: TTT CCG CAA GGT TCG ATT TTC A R: GGC ATC TGC GCT CTA CCA TC

54

HPRT1

F: CCT GGC GTC GTG ATT AGT GAT R: AGA CGT TCA GTC CTG TCC ATA A

131

IL1B

F: CAC GAT GCA CCC TGT ACG ATC A R: GTT GCT CCA TAT CCT GTC CCT

122

NLRC4

F: GAA CTG ATC GAC AGG ATG AAC G R: ACC CAA GCT TGA CGA GTT GT

133

NLRP3

F: TAG CCA CGC TAA TGA TCG ACT R: TTG ATC GCA GCG AAG ATC CAC

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5. Benchtop cooling centrifuge. 6. qPCR Primer (see Table 1). 7. Real-Time PCR System. 8. Plate- or bead-based IL-1β immunoassay (see Note 8). 9. Flow cytometer or plate reader (dependent on the IL-1β assay used).

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Methods Use all reagents at room temperature for the stimulation of whole blood unless otherwise specified. The following protocols describe all steps on the single sample level. Prepare a master-mix wherever possible in order to reduce pipetting errors and intra-assay variance.

3.1 InflammasomeSpecific Stimulation 3.1.1 NLRP3 Stimulation

1. Collect blood samples in hirudin blood collection tubes, gently mix by inverting the tube several times. 2. Add 140 μl of hirudinized whole blood per well of the 96-well plate. The typical assay consists of mock, LPS, and LPS/ATP samples, each with and without MCC950 (see Note 9). All samples are run in triplicates. 3. Add 20 μl of 100 μM MCC950 working solution to mock/ MCC950, LPS/MCC950, and LPS/ATP/MCC950 samples (final concentration 10 μM). Add 20 μl PBS to mock, LPS, and

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LPS/ATP samples. Add 20 μl of 9 μg/ml upLPS working solution to LPS, LPS/MCC950, LPS/ATP, and LPS/ATP/ MCC950 samples (final concentration 1 μg/ml). Add 20 μl of PBS to mock and mock/MCC950 samples. 4. Place the plate on the shaker set to 450 rpm and incubate for 5.5 h (incubation is performed within a cell culture CO2incubator set to 37
C, 5% CO2 (see Note 10). Add 20 μl of 10 mM ATP working solution to the LPS/ATP and LPS/ATP/MCC950 samples (final concentration 1 mM). Add 20 μl of PBS to the remaining samples. 5. Mix samples by pipetting up and down five times. 6. Place the plate on the shaker set to 450 rpm and incubate for 30 min (incubation is performed within a cell culture CO2incubator set to 37
C, 5% CO2 (see Note 10). 7. Add 100 μl PBS to each sample and gently mix by pipetting up and down. 8. Centrifuge the plate at 300  g for 5 min and collect 150 μl of the plasma for analysis of the IL-1β concentration. Store the plasma at 80
C until analysis. Proceed with RNA isolation of the remaining cell pellet (see Subheading 3.2). 3.1.2 AIM2 Stimulation

1. Collect blood samples in hirudin blood collection tubes, gently mix by inverting the tubes several times. 2. Add 140 μl of hirudinized whole blood per well of the 96-well plate. The typical assay consists of mock, LPS/Lipofectamine, and LPS/Lipofectamine/poly(dA:dT) samples, each with and without MCC950 (see Note 11). All samples are run in triplicates. 3. Add 20 μl of 100 μM MCC950 working solution to mock/ MCC950, LPS/Lipofectamine/MCC950, and LPS/Lipofectamine/poly(dA:dT)/MCC950 samples (final concentration 10 μM). Add 20 μl PBS to mock, LPS/Lipofectamine and LPS/Lipofectamine/poly(dA:dT) samples. Add 20 μl of 9 μg/ml upLPS working solution to all LPS-based samples (final concentration 1 μg/ml). Add 20 μl of PBS to mock and mock/MCC950 samples. 4. Place the plate on the shaker set to 450 rpm and incubate for 5.5 h (incubation is performed within a cell culture CO2incubator set to 37
C, 5% CO2) (see Note 10). 5. Dilute 0.4 μg poly(dA:dT) in 10 μl Opti-MEM and 0.8 μg (¼ 0.8 μl) Lipofectamine 2000 in 10 μl Opti-MEM for all LPS/Lipofectamine/poly(dA:dT) samples (with and without MCC950). Performing this step using a master-mix is possible. 6. Mix diluted poly(dA:dT) and Lipofectamine 2000, incubate for 5 min at room temperature.

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7. Dilute 0.8 μg (¼ 0.8 μl) Lipofectamine 2000 in 20 μl OptiMEM for all LPS/Lipofectamine samples (with and without MCC950). 8. Add 20 μl of the poly(dA:dT)/Lipofectamine 2000 mixture to LPS/Lipofectamine/poly(dA:dT) and LPS/Lipofectamine/ poly(dA:dT)/MCC950 samples to reach a final concentration of 2 μg/ml poly(dA:dT) and 4 μg/ml Lipofectamine (see Note 4). Add 20 μl Opti-MEM to mock and mock/MCC950 samples and 20 μl Opti-MEM containing 0.8 μg Lipofectamine 2000 to LPS/Lipofectamine and LPS/Lipofectamine/ MCC950 samples. 9. Place the plate on the shaker set to 450 rpm and incubate for 6 h (incubation is performed within a cell culture CO2-incubator set to 37
C, 5% CO2) (see Notes 10 and 12). 10. Add 100 μl PBS to each sample and gently mix by pipetting up and down. 11. Centrifuge the plate at 300  g for 5 min and collect 150 μl of the plasma for analysis of the IL-1β concentration. Store the plasma at 80
C until analysis. Proceed with RNA isolation of the remaining cell pellet (see Subheading 3.2). 3.1.3 NLRC4 Stimulation

1. The day before the assay, prepare an overnight culture of Salmonella enterica by adding 5 μl of the bacterial glycerol stock to 5 ml of high-salt LB medium containing Streptomycin. 2. Incubate S. enterica at 37
C without shaking until an OD 600 of 1.0 is reached (typically 12–16 h) (see Note 13). 3. Centrifuge 2 ml of this over-night culture (13,000  g, 2 min). 4. Discard supernatant and wash with 1 ml of sterile PBS (see Note 14). 5. Centrifuge (13,000  g, 2 min). 6. Repeat steps 4 and 5. 7. Discard supernatant and resuspend in PBS until an OD 600 of 1.0 is reached, correlating with a concentration of 1  106 bacteria/μl. 8. Collect blood samples in hirudin blood collection tubes, gently mix by inverting the tube several times. 9. Add 140 μl of hirudinized whole blood per well of the 96-well plate. The typical assay consists of mock and S. enterica samples, each with and without MCC950 (see Note 15). All samples are run in triplicates. Add 20 μl of 90 μM MCC950 working solution to mock/MCC950 and S. enterica/ MCC950 samples (final concentration 10 μM). Add 20 μl PBS to mock and S. enterica samples.

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10. Add 14 μl of washed S. enterica from step 7 to S. enterica and S. enterica/MCC950 samples (see Note 13). Add 14 μl PBS to mock and mock/MCC950 samples. Mix well by pipetting up and down. 11. Incubate 4 h at 37
C and 5% CO2. 12. Add 10 μl of 360 μg/ml gentamicin working solution to all samples (final concentration 20 μg/ml). Mix well by pipetting up and down. 13. Incubate 2 h at 37
C and 5% CO2. 14. Add 116 μl PBS to each sample and gently mix by pipetting up and down. 15. Centrifuge the plate at 300  g for 5 min and collect 150 μl of the plasma for analysis of the IL-1β concentration. Store the plasma at 80
C until analysis. Proceed with RNA isolation of the remaining cell pellet (see Subheading 3.2). 3.2 RNA Isolation and Gene Expression Analysis

1. Perform inflammasome-specific stimulations as indicated above in technical triplicates. 2. Centrifuge the plate and discard the supernatant (see Subheading 3.1 for details). 3. Pool the cell pellets of the triplicates. 4. Perform erythrocyte lysis by adding 5 volumes of EL Lysis buffer to the pooled cell pellets. 5. Incubate for 10–15 min on ice. Mix by vortexing briefly 2 times during incubation. 6. Centrifuge at 400  g for 10 min at 4
C, and completely remove and discard supernatant. 7. Add 2 volumes of Buffer EL to the cell pellet. Resuspend cells by vortexing briefly. 8. Centrifuge at 400  g for 10 min at 4
C, and completely remove and discard supernatant. 9. Perform RNA-isolation using the RNeasy MicroKit (Qiagen) according to manufacturer’s protocol. Use an elution volume of 14 μl. Store isolated RNA at 80
C or directly proceed with step 10. 10. Determine RNA concentration using a highly sensitive RNA detection system. If the RNA concentration is below the detection limit or a RNA detection system is not available use the maximum RNA input for reverse transcriptase reaction. 11. Perform reverse transcriptase using the Sensiscript® Reverse Transcriptase Kit (Qiagen) with oligo-dT primer and