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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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
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
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):596–600. https://doi.org/10.1038/nature10510
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
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
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
Cai X, Chiu YH, Chen ZJ (2014) The cGAS-cGAMP-STING pathway of cytosolic DNA sensing and signaling. Mol Cell 54(2):289–296. https://doi.org/10.1016/j.molcel.2014.03.040
Lu A, Magupalli VG, Ruan J, Yin Q, Atianand MK, Vos MR, Schröder 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
Sborgi L, Ravotti F, Dandey VP, Dick MS, Mazur A, Reckel S, Chami M, Scherer S, Huber M, Böckmann A, Egelman EH, Stahlberg H, Broz P, Meier BH, Hiller S (2015) Structure and assembly of the mouse ASC inflammasome by combined NMR spectroscopy and cryo-electron microscopy. Proc Natl Acad Sci U S A 112(43):13237–13242. https://doi.org/10.1073/pnas.1507579112
Fernandes-Alnemri T, Wu J, Yu JW, Datta P, Miller B, Jankowski W, Rosenberg S, Zhang J, Alnemri ES (2007) The pyroptosome: a supramolecular assembly of ASC dimers mediating inflammatory cell death via caspase-1 activation. Cell Death Differ 14(9):1590–1604. https://doi.org/10.1038/sj.cdd.4402194
Huang MT, Taxman DJ, Holley-Guthrie EA, Moore CB, Willingham SB, Madden V, Parsons RK, Featherstone GL, Arnold RR, O’Connor BP, Ting JP (2009) Critical role of apoptotic speck protein containing a caspase recruitment domain (ASC) and NLRP3 in causing necrosis and ASC speck formation induced by Porphyromonas gingivalis in human cells. J Immunol 182(4):2395–2404. https://doi.org/10.4049/jimmunol.0800909
Broz P, Newton K, Lamkanfi M, Mariathasan S, Dixit VM, Monack DM (2010) Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella. J Exp Med 207(8):1745–1755. https://doi.org/10.1084/jem.20100257
Man SM, Hopkins LJ, Nugent E, Cox S, Glück IM, Tourlomousis P, Wright JA, Cicuta P, Monie TP, Bryant CE (2014) Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex. Proc Natl Acad Sci U S A 111(20):7403–7408. https://doi.org/10.1073/pnas.1402911111
Karki R, Man SM, Malireddi RKS, Gurung P, Vogel P, Lamkanfi M, Kanneganti TD (2015) Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection. Cell Host Microbe 17(3):357–368. https://doi.org/10.1016/j.chom.2015.01.006
Schroder K, Tschopp J (2010) The inflammasomes. Cell 140(6):821–832. https://doi.org/10.1016/j.cell.2010.01.040
Datta D, McClendon CL, Jacobson MP, Wells JA (2013) Substrate and inhibitor-induced dimerization and cooperativity in caspase-1 but not caspase-3. J Biol Chem 288(14):9971–9981. https://doi.org/10.1074/jbc.M112.426460
Ramage P, Cheneval D, Chvei M, Graff P, Hemmig R, Heng R, Kocher HP, Mackenzie A, Memmert K, Revesz L et al (1995) Expression, refolding, and autocatalytic proteolytic processing of the interleukin-1 beta-converting enzyme precursor. J Biol Chem 270(16):9378–9383. https://doi.org/10.1074/jbc.270.16.9378
Walsh JG, Logue SE, Lüthi AU, Martin SJ (2011) Caspase-1 promiscuity is counterbalanced by rapid inactivation of processed enzyme. J Biol Chem 286(37):32513–32524. https://doi.org/10.1074/jbc.M111.225862
Martinon F, Burns K, Tschopp J (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell 10(2):417–426. https://doi.org/10.1016/s1097-2765(02)00599-3
Sokolovska A, Becker CE, Ip WK, Rathinam VA, Brudner M, Paquette N, Tanne A, Vanaja SK, Moore KJ, Fitzgerald KA, Lacy-Hulbert A, Stuart LM (2013) Activation of caspase-1 by the NLRP3 inflammasome regulates the NADPH oxidase NOX2 to control phagosome function. Nat Immunol 14(6):543–553. https://doi.org/10.1038/ni.2595
Shi J, Zhao Y, Wang Y, Gao W, Ding J, Li P, Hu L, Shao F (2014) Inflammatory caspases are innate immune receptors for intracellular LPS. Nature 514(7521):187–192. https://doi.org/10.1038/nature13683
Meunier E, Dick MS, Dreier RF, Schürmann N, Kenzelmann Broz D, Warming S, Roose-Girma M, Bumann D, Kayagaki N, Takeda K, Yamamoto M, Broz P (2014) Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases. Nature 509(7500):366–370. https://doi.org/10.1038/nature13157
Vanaja SK, Russo AJ, Behl B, Banerjee I, Yankova M, Deshmukh SD, Rathinam VAK (2016) Bacterial outer membrane vesicles mediate cytosolic localization of LPS and caspase-11 activation. Cell 165(5):1106–1119. https://doi.org/10.1016/j.cell.2016.04.015
Dinarello CA (2010) Why not treat human cancer with interleukin-1 blockade? Cancer Metastasis Rev 29(2):317–329. https://doi.org/10.1007/s10555-010-9229-0
Howard AD, Kostura MJ, Thornberry N, Ding GJ, Limjuco G, Weidner J, Salley JP, Hogquist KA, Chaplin DD, Mumford RA et al (1991) IL-1-converting enzyme requires aspartic acid residues for processing of the IL-1 beta precursor at two distinct sites and does not cleave 31-kDa IL-1 alpha. J Immunol 147(9):2964–2969
Ghayur T, Banerjee S, Hugunin M, Butler D, Herzog L, Carter A, Quintal L, Sekut L, Talanian R, Paskind M, Wong W, Kamen R, Tracey D, Allen H (1997) Caspase-1 processes IFN-gamma-inducing factor and regulates LPS-induced IFN-gamma production. Nature 386(6625):619–623. https://doi.org/10.1038/386619a0
Gu Y, Kuida K, Tsutsui H, Ku G, Hsiao K, Fleming MA, Hayashi N, Higashino K, Okamura H, Nakanishi K, Kurimoto M, Tanimoto T, Flavell RA, Sato V, Harding MW, Livingston DJ, Su MS (1997) Activation of interferon-gamma inducing factor mediated by interleukin-1beta converting enzyme. Science 275(5297):206–209. https://doi.org/10.1126/science.275.5297.206
He W-t, Wan H, Hu L, Chen P, Wang X, Huang Z, Yang Z-H, Zhong C-Q, Han J (2015) Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion. Cell Res 25(12):1285–1298. https://doi.org/10.1038/cr.2015.139
Kayagaki N, Stowe IB, Lee BL, O’Rourke K, Anderson K, Warming S, Cuellar T, Haley B, Roose-Girma M, Phung QT, Liu PS, Lill JR, Li H, Wu J, Kummerfeld S, Zhang J, Lee WP, Snipas SJ, Salvesen GS, Morris LX, Fitzgerald L, Zhang Y, Bertram EM, Goodnow CC, Dixit VM (2015) Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling. Nature 526(7575):666–671. https://doi.org/10.1038/nature15541
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 EM, 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
Ding J, Wang K, Liu W, She Y, Sun Q, Shi J, Sun H, Wang D-C, Shao F (2016) Pore-forming activity and structural autoinhibition of the gasdermin family. Nature 535(7610):111–116. https://doi.org/10.1038/nature18590
Liu X, Zhang Z, Ruan J, Pan Y, Magupalli VG, Wu H, Lieberman J (2016) Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535(7610):153–158. https://doi.org/10.1038/nature18629
Evavold CL, Ruan J, Tan Y, Xia S, Wu H, Kagan JC (2018) The pore-forming protein gasdermin D regulates interleukin-1 secretion from living macrophages. Immunity 48(1):35–44.e36. https://doi.org/10.1016/j.immuni.2017.11.013
Kelly A, Grabiec AM, Travis MA (2018) Culture of human monocyte-derived macrophages. Methods Mol Biol 1784:1–11. https://doi.org/10.1007/978-1-4939-7837-3_1
Odero MD, Zeleznik-Le NJ, Chinwalla V, Rowley JD (2000) Cytogenetic and molecular analysis of the acute monocytic leukemia cell line THP-1 with an MLL-AF9 translocation. Genes Chromosomes Cancer 29(4):333–338
Rathinam VA, Vanaja SK, Waggoner L, Sokolovska A, Becker C, Stuart LM, Leong JM, Fitzgerald KA (2012) TRIF licenses caspase-11-dependent NLRP3 inflammasome activation by gram-negative bacteria. Cell 150(3):606–619. https://doi.org/10.1016/j.cell.2012.07.007
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.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2022 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Banerjee, I. (2022). In Vitro Assays to Study Inflammasome Activation in Primary Macrophages. In: Abdul-Sater, A.A. (eds) The Inflammasome. Methods in Molecular Biology, vol 2459. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2144-8_2
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2144-8_2
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2143-1
Online ISBN: 978-1-0716-2144-8
eBook Packages: Springer Protocols