A comprehensive guide to studying inflammasome activation and cell death

Abstract

Inflammasomes are multimeric heterogeneous mega-Dalton protein complexes that play key roles in the host innate immune response to infection and sterile insults. Assembly of the inflammasome complex following infection or injury begins with the oligomerization of the upstream inflammasome-forming sensor and proceeds through a multistep process of well-coordinated events and downstream effector functions. Together, these steps enable elegant experimental readouts with which to reliably assess the successful activation of the inflammasome complex and cell death. Here, we describe a comprehensive protocol that details several in vitro (in bone marrow–derived macrophages) and in vivo (in mice) strategies for activating the inflammasome and explain how to subsequently assess multiple downstream effects in parallel to unequivocally establish the activation status of the inflammasome and cell death pathways. Our workflow assesses inflammasome activation via the formation of the apoptosis-associated speck-like protein containing a CARD (ASC) speck; cleavage of caspase-1 and gasdermin D; release of IL-1β, IL-18, caspase-1, and lactate dehydrogenase from the cell; and real-time analysis of cell death by imaging. Analyses take up to ~24 h to complete. Overall, our multifaceted approach provides a comprehensive and consistent protocol for assessing inflammasome activation and cell death.

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Fig. 1: Schematic of protocol workflow.
Fig. 2: Real-time cell death analysis in BMDMs following inflammasome activation.
Fig. 3: ASC speck formation in BMDMs following inflammasome activation.
Fig. 4: Caspase-1 cleavage in BMDMs following inflammasome activation.
Fig. 5: Gasdermin D cleavage in BMDMs following inflammasome activation.
Fig. 6: IL-1β and IL-18 ELISA results from the supernatant of BMDMs following inflammasome activation.
Fig. 7: Lactate dehydrogenase release results from the supernatant of BMDMs following inflammasome activation.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files. No datasets were generated or analyzed during the current study. All source data underlying the figures are available from the corresponding author upon request.

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Acknowledgements

We thank members of the Kanneganti lab, both past and present, for their comments and suggestions. Work from our laboratory was supported by the US National Institutes of Health (AI101935, AI124346, AR056296, and CA253095 to T.-D.K.) and the American Lebanese Syrian Associated Charities (to T.-D.K.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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R.E.T., R.K.S.M., and T.-D.K. conceptualized the manuscript. R.E.T. and R.K.S.M. wrote the first draft. All authors reviewed and approved the final draft of the manuscript.

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Correspondence to Thirumala-Devi Kanneganti.

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Key references using this protocol

Zheng, M., Karki, R., Vogel, P. & Kanneganti, T.-D. Cell 181, 674–687.e13 (2020): https://www.cell.com/cell/fulltext/S0092-8674(20)30333-0

Samir, P. et al. Nature 573, 590–594 (2019): https://www.nature.com/articles/s41586-019-1551-2

Man, S. M. et al. Cell 167, 382–396.e17 (2016): https://www.cell.com/cell/fulltext/S0092-8674(16)31245-4

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Tweedell, R.E., Malireddi, R.K.S. & Kanneganti, T. A comprehensive guide to studying inflammasome activation and cell death. Nat Protoc 15, 3284–3333 (2020). https://doi.org/10.1038/s41596-020-0374-9

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