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Inflammasome Activation and Inhibition in Primary Murine Bone Marrow-Derived Cells, and Assays for IL-1α, IL-1β, and Caspase-1

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The Inflammasome

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1040))

Abstract

Through its ability to control the proteolytic maturation and secretion of interleukin-1 family cytokines, the inflammasome occupies a central role in the activation of inflammation and also influences the shaping of adaptive immunity. Since it affects a multitude of different immune responses from autoinflammatory diseases to host defense, vaccine efficacy, and even cancer, it has become of interest to many researchers. Here, we describe a straightforward method for inflammasome assays in primary murine bone marrow-­derived myeloid cells. The protocol encompasses cell handling, inflammasome activation and inhibition, as well as the detection of IL-1β, caspase-1, and IL-1α by ELISA and Western blot.

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References

  1. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J (2006) Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature 440(7081):237–241. doi:nature04516 [pii], 10.1038/nature04516

    Article  PubMed  CAS  Google Scholar 

  2. Fettelschoss A, Kistowska M, LeibundGut-­Landmann S, Beer HD, Johansen P, Senti G, Contassot E, Bachmann MF, French LE, Oxenius A, Kundig TM (2011) Inflammasome activation and IL-1beta target IL-1alpha for secretion as opposed to surface expression. Proc Natl Acad Sci USA 108(44):18055–18060. doi:10.1073/pnas.1109176108

    Article  PubMed  CAS  Google Scholar 

  3. Gross O, Yazdi AS, Thomas CJ, Masin M, Heinz LX, Guarda G, Quadroni M, Drexler SK, Tschopp J (2012) Inflammasome activators induce interleukin-1alpha secretion via distinct pathways with differential requirement for the protease function of caspase-1. Immunity 36(3):388–400. doi:10.1016/j.immuni.2012.01.018

    Article  PubMed  CAS  Google Scholar 

  4. Keller M, Ruegg A, Werner S, Beer HD (2008) Active caspase-1 is a regulator of unconventional protein secretion. Cell 132(5):818–831. doi:10.1016/j.cell.2007.12.040

    Article  PubMed  CAS  Google Scholar 

  5. Kuida K, Lippke JA, Ku G, Harding MW, Livingston DJ, Su MS, Flavell RA (1995) Altered cytokine export and apoptosis in mice deficient in interleukin-1 beta converting enzyme. Science 267(5206):2000–2003

    Article  PubMed  CAS  Google Scholar 

  6. Dinarello CA (2009) Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol 27:519–550. doi:10.1146/annurev.immunol.021908.132612

    Article  PubMed  CAS  Google Scholar 

  7. Sims JE, Smith DE (2010) The IL-1 family: regulators of immunity. Nat Rev Immunol 10(2):89–102. doi:10.1038/nri2691

    Article  PubMed  CAS  Google Scholar 

  8. Nickel W, Seedorf M (2008) Unconventional mechanisms of protein transport to the cell surface of eukaryotic cells. Annu Rev Cell Dev Biol 24:287–308. doi:10.1146/annurev.cellbio.24.110707.175320

    Article  PubMed  CAS  Google Scholar 

  9. Carruth LM, Demczuk S, Mizel SB (1991) Involvement of a calpain-like protease in the processing of the murine interleukin 1 alpha precursor. J Biol Chem 266(19):12162–12167

    PubMed  CAS  Google Scholar 

  10. Kobayashi Y, Yamamoto K, Saido T, Kawasaki H, Oppenheim JJ, Matsushima K (1990) Identification of calcium-activated neutral protease as a processing enzyme of human interleukin 1 alpha. Proc Natl Acad Sci USA 87(14):5548–5552

    Article  PubMed  CAS  Google Scholar 

  11. Schroder K, Tschopp J (2010) The inflammasomes. Cell 140(6):821–832. doi:S0092-­8674(10)00075-9 [pii], 10.1016/j.cell.2010.01.040

    Article  PubMed  CAS  Google Scholar 

  12. 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. doi:S1097276502005993 [pii]

    Article  PubMed  CAS  Google Scholar 

  13. Dostert C, Petrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J (2008) Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science 320(5876):674–677. doi:1156995 [pii], 10.1126/science.1156995

    Article  PubMed  CAS  Google Scholar 

  14. Henderson C, Goldbach-Mansky R (2010) Monogenic autoinflammatory diseases: new insights into clinical aspects and pathogenesis. Curr Opin Rheumatol 22(5):567–578. doi:10.1097/BOR.0b013e32833ceff4, 00002281-201009000-00017 [pii]

    PubMed  CAS  Google Scholar 

  15. Ghiringhelli F, Apetoh L, Tesniere A, Aymeric L, Ma Y, Ortiz C, Vermaelen K, Panaretakis T, Mignot G, Ullrich E, Perfettini JL, Schlemmer F, Tasdemir E, Uhl M, Genin P, Civas A, Ryffel B, Kanellopoulos J, Tschopp J, Andre F, Lidereau R, McLaughlin NM, Haynes NM, Smyth MJ, Kroemer G, Zitvogel L (2009) Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors. Nat Med 15(10):1170–1178. doi:nm.2028 [pii], 10.1038/nm.2028

    Article  PubMed  CAS  Google Scholar 

  16. Masters SL, Dunne A, Subramanian SL, Hull RL, Tannahill GM, Sharp FA, Becker C, Franchi L, Yoshihara E, Chen Z, Mullooly N, Mielke LA, Harris J, Coll RC, Mills KH, Mok KH, Newsholme P, Nunez G, Yodoi J, Kahn SE, Lavelle EC, O’Neill LA (2010) Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1beta in type 2 diabetes. Nat Immunol 11(10):897–904. doi:ni.1935 [pii], 10.1038/ni.1935

    Article  PubMed  CAS  Google Scholar 

  17. Duewell P, Kono H, Rayner KJ, Sirois CM, Vladimer G, Bauernfeind FG, Abela GS, Franchi L, Nunez G, Schnurr M, Espevik T, Lien E, Fitzgerald KA, Rock KL, Moore KJ, Wright SD, Hornung V, Latz E (2010) NLRP3 inflammasomes are required for ­atherogenesis and activated by cholesterol crystals. Nature 464(7293):1357–1361. doi:nature08938 [pii], 10.1038/nature08938

    Article  PubMed  CAS  Google Scholar 

  18. 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. doi:nature07725 [pii], 10.1038/nature07725

    Article  PubMed  CAS  Google Scholar 

  19. Poeck H, Bscheider M, Gross O, Finger K, Roth S, Rebsamen M, Hannesschlager N, Schlee M, Rothenfusser S, Barchet W, Kato H, Akira S, Inoue S, Endres S, Peschel C, Hartmann G, Hornung V, Ruland J (2010) Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production. Nat Immunol 11(1):63–69. doi:ni.1824 [pii], 10.1038/ni.1824

    Article  PubMed  CAS  Google Scholar 

  20. Boyden ED, Dietrich WF (2006) Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin. Nat Genet 38(2):240–244. doi:ng1724 [pii], 10.1038/ng1724

    Article  PubMed  CAS  Google Scholar 

  21. Mariathasan S, Newton K, Monack DM, Vucic D, French DM, Lee WP, Roose-Girma M, Erickson S, Dixit VM (2004) Differential ­activation of the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430(6996):213–218. doi:10.1038/nature02664, nature02664 [pii]

    Article  PubMed  CAS  Google Scholar 

  22. Gross O (2012) Measuring the inflammasome. Methods Mol Biol 844:199–222. doi:0.1007/978-1-61779-527-5_15

    Article  PubMed  Google Scholar 

  23. Lutz MB (2004) IL-3 in dendritic cell development and function: a comparison with GM-CSF and IL-4. Immunobiology 209(1–2):79–87

    Article  PubMed  CAS  Google Scholar 

  24. Manzanero S (2012) Generation of mouse bone marrow-derived macrophages. Methods Mol Biol 844:177–181. doi:10.1007/978-1-61779-527-5_12

    Article  PubMed  CAS  Google Scholar 

  25. Palleroni AV, Varesio L, Wright RB, Brunda MJ (1991) Tumoricidal alveolar macrophage and tumor infiltrating macrophage cell lines. Int J Cancer 49(2):296–302

    Article  PubMed  CAS  Google Scholar 

  26. Murphy MP, Holmgren A, Larsson NG, Halliwell B, Chang CJ, Kalyanaraman B, Rhee SG, Thornalley PJ, Partridge L, Gems D, Nystrom T, Belousov V, Schumacker PT, Winterbourn CC (2011) Unraveling the biological roles of reactive oxygen species. Cell Metab 13(4):361–366. doi:10.1016/j.cmet.2011.03.010

    Article  PubMed  CAS  Google Scholar 

  27. Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, Speert D, Fernandes-­Alnemri T, Wu J, Monks BG, Fitzgerald KA, Hornung V, Latz E (2009) Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression. J Immunol 183(2):787–791. doi:jimmunol.0901363 [pii], 10.4049/jimmunol.0901363

    Article  PubMed  CAS  Google Scholar 

  28. Wewers MD, Pope HA, Miller DK (1993) Processing proIL-1 beta decreases detection by a proIL-1 beta specific ELISA but increases detection by a conventional ELISA. J Immunol Methods 165(2):269–278

    Article  PubMed  CAS  Google Scholar 

  29. Dinarello CA, Goldin NP, Wolff SM (1974) Demonstration and characterization of two distinct human leukocytic pyrogens. J Exp Med 139(6):1369–1381

    Article  PubMed  CAS  Google Scholar 

  30. Fuhlbrigge RC, Sheehan KC, Schreiber RD, Chaplin DD, Unanue ER (1988) Monoclonal antibodies to murine IL-1 alpha. Production, characterization, and inhibition of membrane-­associated IL-1 activity. J Immunol 141(8):2643–2650

    PubMed  CAS  Google Scholar 

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Acknowledgments

OG is supported by the Bavarian Ministry of Sciences, Research and the Arts in the Framework of the Bavarian Molecular Biosystems Research Network. C.J.T. is supported by an NSERC postgraduate scholarship. K.S.S. is supported by a Ph.D. scholarship by the faculty of medicine.

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Authors and Affiliations

  1. Institut für Klinische Chemie und Pathobiochemie, Klinikum rechts der Isar, Technische Universität München, Munich, Germany

    Katharina S. Schneider, Christina J. Thomas & Olaf Groß

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  1. Katharina S. Schneider
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  2. Christina J. Thomas
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  3. Olaf Groß
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Editors and Affiliations

  1. , Institute of Innate Immunity, University of Bonn, Sigmund-Freud-Strasse 25, Bonn, 53105, Germany

    Christine M. De Nardo

  2. , Institute of Innate Immunity, University of Bonn, Sigmund-Freud-Str. 25, Bonn, 53105, Germany

    Eicke Latz

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Schneider, K.S., Thomas, C.J., Groß, O. (2013). Inflammasome Activation and Inhibition in Primary Murine Bone Marrow-Derived Cells, and Assays for IL-1α, IL-1β, and Caspase-1. In: De Nardo, C., Latz, E. (eds) The Inflammasome. Methods in Molecular Biology, vol 1040. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-523-1_10

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  • DOI: https://doi.org/10.1007/978-1-62703-523-1_10

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-522-4

  • Online ISBN: 978-1-62703-523-1

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