The Inflammasome: Methods and Protocols

228 Pages·2013·5.011 MB·English

by  Christine M. De Nardo, Eicke Latz

Methods in
Molecular Biology 1040
Christine M. De Nardo
Eicke Latz Editors
The
Infl ammasome
Methods and Protocols
M M B ™
ETHODS IN OLECULAR IOLOGY
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
For further volumes:
http://www.springer.com/series/7651

The Inflammasome
Methods and Protocols
Edited by
Christine M. De Nardo and Eicke Latz
Institute of Innate Immunity, University of Bonn, Bonn, Germany
Editors
Christine M. De Nardo, PhD Eicke Latz, MD, PhD
Institute of Innate Immunity Institute of Innate Immunity
University of Bonn University of Bonn
Bonn , G ermany Bonn , G ermany
ISSN 1064-3745 ISSN 1940-6029 (electronic)
ISBN 978-1-62703-522-4 ISBN 978-1-62703-523-1 (eBook)
DOI 10.1007/978-1-62703-523-1
Springer New York Heidelberg Dordrecht London
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Preface
Cells of the immune system, such as macrophages and dendritic cells, continuously survey
host tissue integrity. Immune cells can quickly respond to invading pathogens or tissue
damage by mounting an immune response geared to the reconstitution of tissue homeosta-
sis. Immune cells have the ability to distinguish foreign molecules from self-molecules,
detect altered self-molecules, or respond to metabolic derangements via the expression of
families of signaling receptors that can recognize such danger signals. Most of these immune
signaling receptor families, including Toll-like receptors, Rig-I like receptors or C-type
lectin receptors, as well as a number of Nod-like receptors (NLRs), induce signaling cas-
cades that eventually culminate in a transcriptional response of the cell and the production
of infl ammatory mediators. The activated factors act in concert with many cell types that
help in the defense against the invading pathogen or lead to the repair of damaged tissues.
Infl ammasomes, which are the main topic of this volume, have a seemingly simple
architecture. A sensor molecule of the NLR or PYHIN protein family recruits the common
adapter molecule apoptosis-associated speck-like protein containing a CARD (ASC), which
then leads to the approximation of pro-caspase-1 molecules inducing their autoproteolytic
activation. Intriguingly, following activation, ASC forms a large protein aggregate, termed
“ASC speck,” which is thought to provide a platform for the activation of caspase-1. Active
caspase-1, in turn, cleaves the inactive precursor molecules of the IL-β cytokine family
(IL- 1β and IL-18) into their biologically active forms. In addition, infl ammasomes control
the release of mature IL-1β cytokine family members, demonstrating that infl ammasomes
are key gatekeepers for these highly pro-infl ammatory cytokines. Indeed, uncontrolled
production and activation of IL-1β cytokines can lead to signifi cant infl ammatory reactions
that contribute to a range of diseases. For example, the NLRP3 infl ammasome, which
recognizes a range of microbes as well as many sterile danger signals, can contribute to
common infl ammatory pathologies, including gout, atherosclerosis, type 2 diabetes, and
Alzheimer’s disease.
While we have learned a great deal about the mechanisms leading to the production of
IL-1β family cytokines, more precise details of how infl ammasomes are activated, remain to
be elucidated. Elaborate mechanisms have evolved that control the activity of infl amma-
somes and we are just beginning to understand the upstream mechanisms that lead to the
formation of infl ammasomes by the many reported triggers. Future work in this area will
reveal novel targets for pharmacological interference and could lead to more specifi c anti-
infl ammatory interventions that are urgently needed.
Protocols used in the study of infl ammasomes can be diffi cult and intricate to master in
the beginning and thus, detailed protocols with tips from the experts can be of great value.
A number of expert labs have developed specifi c techniques to study many aspects of infl am-
masome function in human or mouse immune cells. Furthermore, some benchmark proto-
cols, such as the analysis of caspase-1 cleavage by immunoblotting, have evolved individually
in different labs around the world. We have the unique opportunity to present these proto-
cols in one volume, which will help broaden the infl ammasome fi eld by allowing others to
more easily and hopefully more successfully perform these assays.
v
vi Preface
Protocols are provided that detail the generation of infl ammasome stimuli, such as the
NLRP3-stimulating amyloid beta and islet amyloid amylin peptides. Other protocols
describe how perturbance of cellular homeostasis by crystalline materials, infectious agents,
or certain adjuvants can be used to activate infl ammasomes. Infl ammasome activation can
be studied at several levels, hence, detailed protocols including the assessment of ASC speck
formation, the monitoring of caspase-1 activity and processing, and the activation of IL-1β
cytokines are presented. In addition, protocols for the analysis of infl ammasome assembly
and ATP binding or ATPase activity of infl ammasomes are described. Another hallmark of
infl ammasome activation is the induction of a specialized form of cell death called “pyrop-
tosis.” Methods to quantify this process as well as the assessment of consequences of infl am-
masome activation for the cell and host are also detailed in this volume. These chapters will
be a useful resource for investigators who seek a better understanding of infl ammasome
activation pathways and would like to master techniques optimized by experts in the fi eld.
We hope this collection will form the defi nite lab protocol source for infl ammasome
research.
This project would not have been possible without the help of some key people. Thank
you to Dr. John Walker and David Casey for identifying a niche and providing support
along the way. We are also very grateful to Dr. Dominic De Nardo for critically reviewing
all the manuscripts. Most importantly, we would like to thank all the authors for their excel-
lent contributions, enthusiasm, and kindness throughout this project. These protocols and
the personal tips and notes shared by each author will form an invaluable tool for current
and future generations of scientists interested in infl ammation and infl ammasome research.
Bonn, Germany Christine M. De Nardo
Eicke Latz
Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Assessing β-Amyloid-Induced NLRP3 Inflammasome Activation
in Primary Microglia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Mareike Schnaars, Hannes Beckert, and Annett Halle
2 Activating the NLRP3 Inflammasome Using the Amyloidogenic
Peptide IAPP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Clara Westwell-Roper, Aisling Dunne, Man Lyang Kim,
C. Bruce Verchere, and Seth L. Masters
3 Assessment and Quantification of Crystal-Induced Lysosomal Damage. . . . . . 19
Peter Duewell and Eicke Latz
4 Assessment of Inflammasome Activation in Primary Human Immune Cells. . . 29
Theo S. Plantinga, Leo A.B. Joosten, and Mihai G. Netea
5 NLRP3 Inflammasome Activation and Cytotoxicity Induced
by Particulate Adjuvants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Marie Yang, Claire H.A. Hearnden, Ewa Oleszycka, and Ed C. Lavelle
6 Measuring Inflammasome Activation in Response to Bacterial Infection . . . . . 65
Petr Broz and Denise M. Monack
7 Detection of Pyroptosis by Measuring Released Lactate
Dehydrogenase Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Manira Rayamajhi, Yue Zhang, and Edward A. Miao
8 ASC Speck Formation as a Readout for Inflammasome Activation. . . . . . . . . . 91
Andrea Stutz, Gabor L. Horvath, Brian G. Monks, and Eicke Latz
9 Immunoblotting for Active Caspase-1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
Christopher Jakobs, Eva Bartok, Andrej Kubarenko, Franz Bauernfeind,
and Veit Hornung
10 Inflammasome Activation and Inhibition in Primary Murine Bone
Marrow-Derived Cells, and Assays for IL-1α, IL-1β, and Caspase-1. . . . . . . . . 117
Katharina S. Schneider, Christina J. Thomas, and Olaf Groß
11 Reconstituting the NLRP1 Inflammasome In Vitro. . . . . . . . . . . . . . . . . . . . . 137
Benjamin Faustin and John C. Reed
12 Assessing ATP Binding and Hydrolysis by NLR Proteins. . . . . . . . . . . . . . . . . 153
Jinyao Mo and Joseph A. Duncan
13 Blue Native Polyacrylamide Gel Electrophoresis to Monitor Inflammasome
Assembly and Composition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Eric M. Kofoed and Russell E. Vance
vii
viii Contents
14 Analysis of Microbiota Alterations in Inflammasome- Deficient Mice . . . . . . . . 185
Eran Elinav, Christoph A. Thaiss, and Richard A. Flavell
15 Quantification of Adipose Tissue Leukocytosis in Obesity . . . . . . . . . . . . . . . . 195
Ryan Grant, Yun-Hee Youm, Anthony Ravussin, and Vishwa Deep Dixit
16 In Vivo Evaluation of Neutrophil Recruitment in Response
to Sterile Particulates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211
Tamiko Yanagida, Kaoru Orihashi, and Hajime Kono
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
Contributors
EVA BARTOK • Institute for Clinical Chemistry and Pharmacology, Unit for Clinical
Biochemistry , University Hospital, University of Bonn , Bonn , Germany
FRANZ BAUERNFEIND • Institute for Clinical Chemistry and Pharmacology,
Unit for Clinical Biochemistry , University Hospital, University of Bonn ,
Bonn , Germany
HANNES BECKERT • Max-Planck Research Group Neuroimmunology ,
Center of Advanced European Studies and Research (caesar) , Bonn , Germany
PETR BROZ • Department of Microbiology and Immunology, Stanford School of Medicine,
Stanford University , Stanford , CA , USA
VISHWA DEEP DIXIT • Immunobiology Laboratory, Pennington Biomedical
Research Center , Louisiana State University System , Baton Rouge, LA , USA
PETER DUEWELL • Medizinische Klinik und Poliklinik IV, Klinikum der Universität
München, Munich, Germany
JOSEPH A. DUNCAN • Division of Infectious Diseases, Department of Medicine,
University of North Carolina School of Medicine , Chapel Hill, NC , USA
AISLING DUNNE • Immunology Research Centre, School of Biochemistry
and Immunology, Trinity College , Dublin 2, Ireland
ERAN ELINAV • Department of Immunobiology , Yale University School of Medicine ,
New Haven, CT , USA ; Immunology Department, W eizmann Institute of Science ,
Rehovot , Israel
BENJAMIN FAUSTIN • Sandford-Burnham Institute for Medical Research ,
La Jolla , CA , USA
RICHARD A. FLAVELL • Department of Immunobiology , Yale University School of Medicine ,
New Haven, CT , USA ; Howard Hughes Medical Institute , Chevy Chase, MD , USA
RYAN GRANT • Immunobiology Laboratory, Pennington Biomedical Research Center,
Louisiana State University System , Baton Rouge, LA , USA
OLAF GROß • Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts
der Isar, Technische Universität München , Munich, Germany
ANNETT HALLE • Max-Planck Research Group Neuroimmunology , Center of Advanced
European Studies and Research (caesar) , Bonn , Germany
CLAIRE H.A. HEARNDEN • Adjuvant Research Group, School of Biochemistry
and Immunology, Trinity Biomedical Sciences Institute, Trinity College ,
Dublin 2, Ireland
VEIT HORNUNG • Institute for Clinical Chemistry and Pharmacology,
Unit for Clinical Biochemistry , University Hospital, University of Bonn , Bonn , Germany
GABOR L. HORVATH • Institute of Innate Immunity, University Hospital,
University of Bonn , Bonn , Germany
CHRISTOPHER JAKOBS • Institute for Clinical Chemistry and Pharmacology,
Unit for Clinical Biochemistry , University Hospital, University of Bonn , Bonn , Germany
ix