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Methods in Molecular Biology 1417 Francesco Di Virgilio Pablo Pelegrín Editors NLR Proteins Methods and Protocols M M B ETHODS IN OLECULAR IOLOGY Series Editor: John M. Walker University of Hertfordshire School of Life and Medical Sciences Hatfield, Hertfordshire, AL10 9AB, UK For further volumes: http://www.springer.com/series/7651 NLR Proteins Methods and Protocols Edited by Francesco Di Virgilio Department of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara, Italy Pablo Pelegrín Molecular Inflammation Group, Murcia Biomedical Research Institute (IMIB-Arrixaca), Hospital Virgen de la Arrixaca, Murcia, Spain Editors Francesco D i Virgilio Pablo P elegrín Department of Morphology Molecular Inflammation Group Surgery and Experimental Medicine Murcia Biomedical Research Institute (IMIB- University of Ferrara Arrixaca), Hospital Virgen de la Arrixaca Ferrara, Italy Murcia, S pain ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-3564-2 ISBN 978-1-4939-3566-6 (eBook) DOI 10.1007/978-1-4939-3566-6 Library of Congress Control Number: 2016935501 © Springer Science+Business Media New York 2 016 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifi cally the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfi lms 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 specifi c 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, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. Printed on acid-free paper This Humana Press imprint is published by Springer Nature The registered company is Springer Science+Business Media LLC New York Prefa ce Infl ammation is the most fundamental defense mechanism developed by multicellular organ- isms [1]. Central to infl ammation is the ability to discriminate noxious from non-n oxious agents and to detect signs of tissue damage or cellular distress that might signal an impend- ing danger. Given the multiplicity of foreign microorganisms which our body gets in contact with throughout its life, the ability to tell “dangerous” from “non-dangerous” is crucial because we do not want to rouse a potentially destructive response, such as infl ammation, if not absolutely necessary. The immune system selected the capability to cause cell or tissue damage as an unequivocal proof of a given microorganism “dangerousness.” Furthermore, as it is well known to clinicians, harmful agents are very often of endogenous origin (e.g., misfolded proteins or products of abnormal metabolic pathways); thus, an effi cient defense mechanism should also be able to detect the presence of these agents. Thus, as pointed out by Carl Nathan, in order to start infl ammation in response to a foreign microorganism, our body needs to detect the guest (the pathogen) and have evidence of its “dangerousness,” i.e., detect the damage [2]. This “two-hit” system is based on the ability to identify on one hand molecular signs of the presence of the pathogen, i.e., pathogen-a ssociated molecular patterns, PAMPs, and on the other molecular signs of possible cell damage or distress, i.e., damage-associated molecular patterns, DAMPs [3]. Very interestingly, DAMPs released as a consequence of sterile tissue damage, as, for example, in the case of closed trauma, autoim- mune diseases, or metabolic stress, are themselves suffi cient to trigger infl ammation (sterile infl ammation), in the absence of PAMPs. This self-suffi ciency of endogenous factors may tell us something of the evolutionary driving forces behind the infl ammatory response. Immune cells have developed a sophisticated array of receptors to monitor the extracellular and intracellular environment for the presence of PAMPs and DAMPs. At least four different families of receptors are known: (1) C-type lectin receptors (CLRs), (2) retinoid- acid inducible gene (RIG)-I-like receptors (RLRs), (3) toll-like receptors (TLRs), and (4) nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). TLRs are specialized to detect pathogens in the extracellular space or in the endosome lumen that can be equated to the extracellular space. NLRs are specialized to sense pathogen presence in the cytoplasm; thus, they can be considered the prototypic intracellular pathogen/danger- sensing receptors. Over the last 10 years, our knowledge of NLRs, as well as the number of diseases in which these key defense molecules are involved, has increased exponentially. We now know that some NLRs are intracellularly assembled together with other proteins (i.e., ASC and caspase-1) in a macromolecular complex named the infl ammasome and that this complex undergoes a complex molecular rearrangement during immune cell activation [4]. Such changes (translocation to different intracellular compartments, shift in the affi nity for intracellular nucleotides, conformational changes) are intimately linked to immune cell effector responses. The tumultuous growth of interest on the NLRs requires a parallel increase in the tech- nical weaponry for the molecular and biochemical investigation. This is the need that this book aims to satisfy. In the fi rst chapter (by Edward Lavelle), we provide a succinct albeit authoritative appraisal of current knowledge of innate immune receptors. In the second chapter Fayyaz Sutterwala reports on the so-called “atypical” infl ammasomes. Then four chapters, by Isabelle Couillin, David Brough, Pablo Pelegrin, and Veit Hornung, follow on v vi Preface classical biochemical and novel bioluminescence techniques for the measurement of IL-1β release as a readout of infl ammasome activation. Francesco Di Virgilio describes a novel bioluminescent probe for in vivo imaging of extracellular ATP, the prototypic DAMP. The following fi ve chapters, by Christian Stehlik, Bernardo Franklin, Fatima Martin, Monica Comalada, and Ming-Zong Lai, reports on different biochemical and microscopy techniques that can be used to monitor NLR oligomerization. The following chapters focus on the consequences of infl ammasome activation. Virginie Petrilli describes techniques to measure caspase-1 activation. Fabio Martinon reports on cell-f ree systems for the study of infl ammasome function. On the other hand, Vincent Compan details the protocols for NLR reconstitution in a cell model such as HEK293 cells. Gloria Lopez-Castejon describes the procedure to investigate posttranscriptional NLR modifi cations. Lorenzo Galluzzi describes the protocols for the study of one of the most unfavorable consequences of infl ammasome activation, i.e., cell death. F inally, Marco Gattorno and Anna Rubartelli give an update appraisal of the application of infl ammasome studies to the clinic . We hope that this book will provide a sound basis for the molecular investigation of NLR function in health and disease and will sparkle interest in these fascinating molecules by investigators from many different and faraway disciplines. Ferrara, Italy Francesco Di Virgilio Murcia, Spain Pablo Pelegrín References 1 . Medzhitov R (2008) Origin and physiologi- 3 . S chroder K, Tschopp J (2010) The infl amma- cal roles of inflammation. Nature 454: somes. Cell 140:821–832 428–435 4 . Gross O, Thomas CJ, Guarda G, Tschopp J 2 . Nathan C (2002) Points of control in infl amma- (2011) The infl ammasome: an integrated view. tion. Nature 420:846–852 Immunol Rev 243:136–151 Contents Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i x 1 I nnate Immune Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Natalia Muñoz-Wolf and E d C. L avelle 2 A typical Inflammasomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 5 Ann M . J anowski and F ayyaz S. Sutterwala 3 A ssessment of Inflammasome Activation by Cytokine and Danger Signal Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 3 Nicolas R iteau , A urélie G ombault , and Isabelle Couillin 4 I nvestigating IL-1β Secretion Using Real-Time Single-Cell Imaging. . . . . . . . . . . . 7 5 Catherine D iamond , James Bagnall , D avid G. Spiller , Michael R. White , A lessandra M ortellaro , P awel Paszek , and David Brough 5 M easuring IL-1β Processing by Bioluminescence Sensors I: Using a Bioluminescence Resonance Energy Transfer Biosensor . . . . . . . . . . . . . . . 8 9 Vincent Compan and P ablo P elegrín 6 Measuring IL-1β Processing by Bioluminescence Sensors II: The iGLuc System . . . 9 7 Eva B artok , Maria Kampes , and Veit Hornung 7 Assessing Extracellular ATP as Danger Signal In Vivo: The pmeLuc System . . . . . . 1 15 Francesco D i Virgilio , P aolo Pinton , and Simonetta F alzoni 8 Measuring NLR Oligomerization I: Size Exclusion Chromatography, Co-immunoprecipitation, and Cross-Linking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 31 Sonal K hare , Alexander D . Radian , Andrea Dorfleutner , and C hristian Stehlik 9 M easuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence . . . . . . . . . . . . . . . . 1 45 Michael B eilharz , Dominic D e Nardo’s , E icke L atz , and Bernardo S. Franklin 10 M easuring NLR Oligomerization III: Detection of NLRP3 Complex by Bioluminescence Resonance Energy Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 59 Fátima M artín-Sánchez , V incent Compan , and Pablo Pelegrín 11 M easuring NLR Oligomerization IV: Using Förster Resonance Energy Transfer (FRET)-Fluorescence Lifetime Imaging Microscopy (FLIM) to Determine the Close Proximity of Inflammasome Components . . . . . . . . . . . . . 1 69 Catrin Youssif , B árbara F lix , O livia Belbin , and Mònica C omalada 12 Measuring NLR Oligomerization V: In Situ Proximity Ligation Assay. . . . . . . . . . . 1 85 Yung-Hsuan Wu and M ing-Zong Lai 13 Assessing Caspase-1 Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 97 Baptiste G uey and V irginie P etrilli 14 Cell-Free Assay for Inflammasome Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 07 Yvan J amilloux and F abio Martinon vii viii Contents 15 F unctional Reconstruction of NLRs in HEK293 Cells . . . . . . . . . . . . . . . . . . . . . . 2 17 Vincent C ompan and Gloria López-Castejón 16 M ethod to Measure Ubiquitination of NLRs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 23 Pablo P alazón-Riquelme and Gloria López-Castejón 17 Cytofluorometric Quantification of Cell Death Elicited by NLR Proteins . . . . . . . . 2 31 Valentina Sica , Gwenola M anic , G uido K roemer , Ilio Vitale , and L orenzo G alluzzi 18 NLR in Human Diseases: Role and Laboratory Findings. . . . . . . . . . . . . . . . . . . . . 2 47 Sonia C arta , M arco G attorno , and Anna Rubartelli Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Contributors JAMES BAGNALL • Faculty of Life Sciences, U niversity of Manchester , M anchester, U K EVA B ARTOK • Institute of Molecular Medicine, University Hospital, U niversity of Bonn , Bonn, G ermany; Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn, Bonn, Germany MICHAEL BEILHARZ • Institute of Innate Immunity, University Hospitals, University of Bonn , B onn , Germany OLIVIA BELBIN • Memory Unit, Neurology Department, H ospital de la Santa Creu i Sant Pau , B arcelona, Spain ; C entro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERned) , M adrid, Spain DAVID BROUGH • Faculty of Life Sciences, U niversity of Manchester , Manchester, U K SONIA CARTA • Cell Biology Unit, I RCCS Azienda Ospedaliera Universitaria San Martino-IST , G enoa, Italy MÒNICA COMALADA • Institute for Research in Biomedicine (IRB Barcelona) , B arcelona, Spain VINCENT COMPAN • Institut de Génomique Fonctionnelle, Labex ICST, Centre National de la Recherche Scientifi que, Unité Mixte de Recherche 5203, U niversité Montpellier , Montpellier, F rance ; I nstitut National de la Santé et de la Recherche Médicale Unité 1191 , M ontpellier, F rance ISABELLE COUILLIN • INEM, CNRS, UMR7355, University of Orleans , Orleans, F rance; Molecular and Experimental Immunology and Neurogenetics, Orleans, France CATHERINE DIAMOND • Faculty of Life Sciences, U niversity of Manchester , M anchester, U K; Singapore Immunology Network (SIgN), Agency for Science Technology and Research (A*STAR) , S ingapore, Singapore ANDREA DORFLEUTNER • Division of Rheumatology, Department of Medicine, Feinberg School of Medicine, Northwestern University , C hicago, IL, U SA SIMONETTA FALZONI • Department of Morphology, Surgery and Experimental Medicine, University of Ferrara , F errara , I taly BÁRBARA FLIX • Memory Unit, Neurology Department, H ospital de la Santa Creu i Sant Pau , B arcelona, Spain BERNARDO S. FRANKLIN • Institute of Innate Immunity , U niversity Hospitals, University of Bonn , B onn , G ermany LORENZO G ALLUZZI • Gustave Roussy Cancer Campus , V illejuif, F rance ; I NSERM, U1138 , Paris , F rance; ’ E quipe 11 labellisée par la Ligue Nationale contre le Cancer, C entre de Recherche des Cordeliers , P aris , France ; U niversité Paris Descartes/Paris V, Sorbonne Paris Cité , P aris , France; Université Pierre et Marie Curie/Paris VI, Paris, France MARCO GATTORNO • UO Pediatria 2, Istituto G. Gaslini , G enoa , I taly AURÉLIE G OMBAULT • INEM, CNRS, UMR7355, U niversity of Orleans , O rleans , F rance BAPTISTE GUEY • INSERM U1052, Centre de Recherche en Cancérologie de Lyon , Lyon, France ; C NRS UMR5286, C entre de Recherche en Cancérologie de Lyon , L yon, France ; Université de Lyon , L yon, France ; Centre Léon Bérard , L yon, France ix