Abstract
Inflammasomes are multiprotein complexes whose activity has been implicated in physiological and pathological inflammation. The hallmarks of inflammasome activation are the secretion of the mature forms of Caspase-1 and IL-1β from cells of the innate immune system. This protocol covers the methods required to study inflammasome activation using mouse bone marrow-derived dendritic cells (BMDCs) as a model system. The protocol includes the generation and handling of BMDCs, the stimulation of BMDCs with established Nlrp3 inflammasome activators, and the measurement of activation by both ELISA and western blot. These methods can be useful for the study of potential inflammasome activators, and of the signaling pathways involved in inflammasome activation. General considerations are provided that may help in the design and optimization of modified methods for the study of other types of inflammasomes and in other cell types.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Schroder, K., Tschopp, J. (2010) The inflammasomes, Cell 140, 821–832.
Martinon, F., Petrilli, V., Mayor, A. et al. (2006) Gout-associated uric acid crystals activate the NALP3 inflammasome, Nature 440, 237–241.
Dostert, C., Petrilli, V., Van Bruggen, R. et al. (2008) Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica, Science 320, 674–677.
Yazdi, A. S., Guarda, G., Riteau, N. et al. (2010) Nanoparticles activate the NLR pyrin domain containing 3 (Nlrp3) inflammasome and cause pulmonary inflammation through release of IL-1alpha and IL-1beta, Proc Natl Acad Sci USA 107, 19449–19454.
Henderson, C., Goldbach-Mansky, R. (2010) Monogenic autoinflammatory diseases: new insights into clinical aspects and pathogenesis, Curr Opin Rheumatol 22, 567–578.
Okamoto, M., Liu, W., Luo, Y. et al. (2010) Constitutively active inflammasome in human melanoma cells mediating autoinflammation via caspase-1 processing and secretion of interleukin-1beta, J Biol Chem 285, 6477–6488.
Ghiringhelli, F., Apetoh, L., Tesniere, A. et al. (2009) Activation of the NLRP3 inflammasome in dendritic cells induces IL-1beta-dependent adaptive immunity against tumors, Nat Med 15, 1170–1178.
Zhou, R., Yazdi, A. S., Menu, P. et al. (2010) A role for mitochondria in NLRP3 inflammasome activation, Nature doi:10.1038/nature09663
Masters, S. L., Dunne, A., Subramanian, S. L. et al. (2010) Activation of the NLRP3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced IL-1beta in type 2 diabetes, Nat Immunol 11, 897–904.
Duewell, P., Kono, H., Rayner, K. J. et al. (2010) NLRP3 inflammasomes are required for atherogenesis and activated by cholesterol crystals, Nature 464, 1357–1361.
Bauer, C., Duewell, P., Mayer, C. et al. (2010) Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome, Gut 59, 1192–1199.
Gross, O., Poeck, H., Bscheider, M. et al. (2009) Syk kinase signalling couples to the Nlrp3 inflammasome for anti-fungal host defence, Nature 459, 433–436.
Hornung, V., Bauernfeind, F., Halle, A. et al. (2008) Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization, Nat Immunol 9, 847–856.
Dinarello, C. A. (2009) Immunological and inflammatory functions of the interleukin-1 family, Annu Rev Immunol 27, 519–550.
Burckstummer, T., Baumann, C., Bluml, S. et al. (2009) An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome, Nat Immunol 10, 266–272.
Fernandes-Alnemri, T., Yu, J. W., Datta, P. et al. (2009) AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA, Nature 458, 509–513.
Hornung, V., Ablasser, A., Charrel-Dennis, M. et al. (2009) AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC, Nature 458, 514–518.
Roberts, T. L., Idris, A., Dunn, J. A. et al. (2009) HIN-200 proteins regulate caspase activation in response to foreign cytoplasmic DNA, Science 323, 1057–1060.
Poeck, H., Bscheider, M., Gross, O. et al. (2010) Recognition of RNA virus by RIG-I results in activation of CARD9 and inflammasome signaling for interleukin 1 beta production, Nat Immunol 11, 63–69.
Boyden, E. D., and Dietrich, W. F. (2006) Nalp1b controls mouse macrophage susceptibility to anthrax lethal toxin, Nat Genet 38, 240–244.
Mariathasan, S., Newton, K., Monack, D. M. et al. (2004) Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf, Nature 430, 213–218.
Martinon, F., Burns, K., and Tschopp, J. (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta, Mol Cell 10, 417–426.
Mariathasan, S., Weiss, D. S., Newton, K. et al. (2006) Cryopyrin activates the inflammasome in response to toxins and ATP, Nature 440, 228–232.
Bauernfeind, F. G., Horvath, G., Stutz, A. et al. (2009) Cutting edge: NF-kappaB activating pattern recognition and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression, J Immunol 183, 787–791.
Greten, F. R., Arkan, M. C., Bollrath, J. et al. (2007) NF-kappaB is a negative regulator of IL-1beta secretion as revealed by genetic and pharmacological inhibition of IKKbeta, Cell 130, 918–931.
Bauernfeind, F., Ablasser, A., Bartok, E. et al. (2011) Inflammasomes: current understanding and open questions, Cell Mol Life Sci 68, 765–83.
Bergsbaken, T., Fink, S. L., Cookson, B. T. (2009) Pyroptosis: host cell death and inflammation, Nat Rev Microbiol 7, 99–109.
Keller, M., Ruegg, A., Werner, S. et al. (2008) Active caspase-1 is a regulator of unconventional protein secretion, Cell 132, 818–831.
Lutz, M. B. (2004) IL-3 in dendritic cell development and function: a comparison with GM-CSF and IL-4, Immunobiology 209, 79–87.
Acknowledgments
I would like to thank Michael Bscheider, Manuel Ritter, and James Vince for helpful suggestions, and Christina Thomas for proofreading and correcting the manuscript. My work is funded by an EMBO long-term fellowship. I declare no competing financial interests.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Groß, O. (2012). Measuring the Inflammasome. In: Ashman, R. (eds) Leucocytes. Methods in Molecular Biology, vol 844. Humana Press. https://doi.org/10.1007/978-1-61779-527-5_15
Download citation
DOI: https://doi.org/10.1007/978-1-61779-527-5_15
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-61779-526-8
Online ISBN: 978-1-61779-527-5
eBook Packages: Springer Protocols