Abstract
Pyroptosis is a highly regulated inflammatory form of cell death that plays a role in many different diseases, including cancer. Pyroptosis was initially described to be mediated by caspase-1, which is activated by innate immune signaling complexes called inflammasomes. Inflammasomes trigger caspase-dependent activation of the pore-forming protein, gasdermin D, and plasma membrane disruption. In this protocol, we describe a method to simultaneously detect two hallmarks of inflammasome-mediated pyroptosis. Using a fluorescently tagged inflammasome adaptor protein (ASC-Citrine) and membrane-impermeable nuclear dyes, we can track inflammasome formation and plasma membrane disruption over time in the same cell population.
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References
Cookson BT, Brennan MA (2001) Pro-inflammatory programmed cell death [2]. Trends Microbiol 9:113–114. https://doi.org/10.1016/s0966-842x(00)01936-3
Fink SL, Cookson BT (2005) Apoptosis, pyroptosis, and necrosis: mechanistic description of dead and dying eukaryotic cells. Infect Immun 73:1907–1916. https://doi.org/10.1128/IAI.73.4.1907-1916.2005
Broz P, Dixit VM (2016) Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol 16:407–420
Zhao Y, Yang J, Shi J et al (2011) The NLRC4 inflammasome receptors for bacterial flagellin and type III secretion apparatus. Nature 477:596–602. https://doi.org/10.1038/nature10510
Malik A, Kanneganti TD (2017) Inflammasome activation and assembly at a glance. J Cell Sci 130:3955–3963. https://doi.org/10.1242/jcs.207365
de Alba E (2019) Structure, interactions and self-assembly of ASC-dependent inflammasomes. Arch Biochem Biophys 670:15–31
Tzeng TC, Schattgen S, Monks B et al (2016) A fluorescent reporter mouse for Inflammasome assembly demonstrates an important role for cell-bound and free ASC specks during in vivo infection. Cell Rep 16:571–582. https://doi.org/10.1016/j.celrep.2016.06.011
Jorgensen I, Zhang Y, Krantz BA, Miao EA (2016) Pyroptosis triggers pore-induced intracellular traps (PITs) that capture bacteria and lead to their clearance by efferocytosis. J Exp Med 213:2113–2128. https://doi.org/10.1084/jem.20151613
Davis MA, Fairgrieve MR, den Hartigh A et al (2019) Calpain drives pyroptotic vimentin cleavage, intermediate filament loss, and cell rupture that mediates immunostimulation. Proc Natl Acad Sci U S A 116:5061–5070. https://doi.org/10.1073/pnas.1818598116
Bergsbaken T, Fink SL, den Hartigh AB et al (2011) Coordinated host responses during Pyroptosis: Caspase-1–dependent lysosome exocytosis and inflammatory cytokine maturation. J Immunol 187:2748–2754. https://doi.org/10.4049/jimmunol.1100477
Fink SL, Cookson BT (2006) Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages. Cell Microbiol 8:1812–1825. https://doi.org/10.1111/J.1462-5822.2006.00751.x
Liu X, Zhang Z, Ruan J et al (2016) Inflammasome-activated gasdermin D causes pyroptosis by forming membrane pores. Nature 535:153–158. https://doi.org/10.1038/nature18629
Shi J, Zhao Y, Wang K et al (2015) Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death. Nature 526:660–665. https://doi.org/10.1038/nature15514
Evavold CL, Ruan J, Tan Y, et al. (2018) The pore-forming protein Gasdermin D regulates Interleukin-1 secretion from living macrophages. Immunity 48:35–44.e6. https://doi.org/10.1016/j.immuni.2017.11.013
Kovacs SB, Miao EA (2017) Gasdermins: effectors of Pyroptosis. Trends Cell Biol 27:673–684
Galluzzi L, Vitale I, Aaronson SA et al (2018) Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. Cell Death Differ 25:486–541
Wei Q, Mu K, Li T et al (2014) Deregulation of the NLRP3 inflammasome in hepatic parenchymal cells during liver cancer progression. Lab Investig 94:52–62. https://doi.org/10.1038/labinvest.2013.126
Gao J, Qiu X, Xi G et al (2018) Downregulation of GSDMD attenuates tumor proliferation via the intrinsic mitochondrial apoptotic pathway and inhibition of EGFR/Akt signaling and predicts a good prognosis in non-small cell lung cancer. Oncol Rep 40:1971–1984. https://doi.org/10.3892/or.2018.6634
Zhang Z, Zhang Y, Lieberman J (2021) Lighting a fire: can we harness pyroptosis to ignite antitumor immunity? Cancer Immunol Res 9:2–7. https://doi.org/10.1158/2326-6066.CIR-20-0525
Xia X, Wang X, Cheng Z et al (2019) The role of pyroptosis in cancer: pro-cancer or pro-“host”? Cell Death Dis 10:650. https://doi.org/10.1038/s41419-019-1883-8
Karki R, Kanneganti TD (2019) Diverging inflammasome signals in tumorigenesis and potential targeting. Nat Rev Cancer 19:197–214
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den Hartigh, A.B., Fink, S.L. (2022). Simultaneous Detection of Inflammasome Activation and Membrane Damage During Pyroptosis. In: Barcenilla, H., Diaz, D. (eds) Apoptosis and Cancer. Methods in Molecular Biology, vol 2543. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2553-8_15
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DOI: https://doi.org/10.1007/978-1-0716-2553-8_15
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