Impaired NLRP3 inflammasome activation/pyroptosis leads to robust inflammatory cell death via caspase-8/RIPK3 during coronavirus infection

Coronaviruses have caused several zoonotic infections in the past two decades, leading to significant morbidity and mortality globally. Balanced regulation of cell death and inflammatory immune responses is essential to promote protection against coronavirus infection; however, the underlying mechanisms that control these processes remain to be resolved. Here we demonstrate that infection with the murine coronavirus mouse hepatitis virus (MHV) activated the NLRP3 inflammasome and inflammatory cell death in the form of PANoptosis. Deleting NLRP3 inflammasome components or the downstream cell death executioner gasdermin D (GSDMD) led to an initial reduction in cell death followed by a robust increase in the incidence of caspase-8– and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)–mediated inflammatory cell deathafter coronavirus infection. Additionally, loss of GSDMD promoted robust NLRP3 inflammasome activation. Moreover, the amounts of some cytokines released during coronavirus infection were significantly altered in the absence of GSDMD. Altogether, our findings show that inflammatory cell death, PANoptosis, is induced by coronavirus infection and that impaired NLRP3 inflammasome function or pyroptosis can lead to negative consequences for the host. These findings may have important implications for studies of coronavirus-induced disease. Coronaviruses have caused several zoonotic infections in the past two decades, leading to significant morbidity and mortality globally. Balanced regulation of cell death and inflammatory immune responses is essential to promote protection against coronavirus infection; however, the underlying mechanisms that control these processes remain to be resolved. Here we demonstrate that infection with the murine coronavirus mouse hepatitis virus (MHV) activated the NLRP3 inflammasome and inflammatory cell death in the form of PANoptosis. Deleting NLRP3 inflammasome components or the downstream cell death executioner gasdermin D (GSDMD) led to an initial reduction in cell death followed by a robust increase in the incidence of caspase-8– and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)–mediated inflammatory cell deathafter coronavirus infection. Additionally, loss of GSDMD promoted robust NLRP3 inflammasome activation. Moreover, the amounts of some cytokines released during coronavirus infection were significantly altered in the absence of GSDMD. Altogether, our findings show that inflammatory cell death, PANoptosis, is induced by coronavirus infection and that impaired NLRP3 inflammasome function or pyroptosis can lead to negative consequences for the host. These findings may have important implications for studies of coronavirus-induced disease. Coronaviruses are single-stranded positive sense RNA viruses with a wide range of hosts (1MacLachlan N.J. Dubovi E.J. Chapter 24 Coronaviridae.Fenner's Veterinary Virology. 5E. Academic Press, Boston2017: 435-46110.1016/B978-0-12-800946-8.00024-6Google Scholar). Four coronavirus genera (alpha, beta, gamma, and delta) have been identified based on their genetic and serologic properties. Two members of the betacoronavirus genera, severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and Middle East respiratory syndrome-associated coronavirus (MERS-CoV), have caused zoonotic events in the recent past (2Chan J.F.W. Lau S.K.P. To K.K.W. Cheng V.C.C. Woo P.C.Y. Yuen K.-Y. Middle East respiratory syndrome coronavirus: another zoonotic betacoronavirus causing SARS-like disease.Clin. Microbiol. Rev. 2015; 28 (25810418): 465-52210.1128/CMR.00102-14Crossref PubMed Scopus (610) Google Scholar). Now, a new member of the betacoronavirus genera, SARS-CoV-2, which was first isolated in Wuhan, China in 2019, has emerged and is causing a pandemic (3Jaimes J.A. André N.M. Chappie J.S. Millet J.K. Whittaker G.R. Phylogenetic analysis and structural modeling of SARS-CoV-2 spike protein reveals an evolutionary distinct and proteolytically sensitive activation loop.J. Mol. Biol. 2020; (32320687)10.1016/j.jmb.2020.04.009Crossref PubMed Scopus (275) Google Scholar). SARS-CoV-2 infection leads to coronavirus disease 2019 (COVID-19), which is most often a mild to moderate respiratory illness with symptoms including fever, fatigue, and dry cough (4Zhou M. Zhang X. Qu J. Coronavirus disease 2019 (COVID-19): a clinical update.Front. Med. 2020; 14 (32240462): 126-13510.1007/s11684-020-0767-8Crossref PubMed Scopus (259) Google Scholar). However, patients can develop severe COVID-19 with acute respiratory distress syndrome and present with acute lung damage (5Fox S.E. Akmatbekov A. Harbert J.L. Li G. Brown J.Q. Heide R.S.V. Pulmonary and cardiac pathology in Covid-19: the first autopsy series from New Orleans. 8. 2020: 681-68610.1016/S2213-2600(20)30243-5Google Scholar, 6Luo W. Yu H. Gou J. Li X. Sun Y. Li J. Liu L. Clinical pathology of critical patient with novel coronavirus pneumonia (COVID-19).Preprints. 2020; Google Scholar, 7Xu Z. Shi L. Wang Y. Zhang J. Huang L. Zhang C. Liu S. Zhao P. Liu H. Zhu L. Tai Y. Bai C. Gao T. Song J. Xia P. et al.Pathological findings of COVID-19 associated with acute respiratory distress syndrome.Lancet Respir. Med. 2020; 8 (32085846): 420-42210.1016/S2213-2600(20)30076-XAbstract Full Text Full Text PDF PubMed Scopus (5614) Google Scholar). Furthermore, cytokine storm has been suggested to be involved in the pathogenesis of severe and critical cases (8Hadjadj J. Yatim N. Barnabei L. Corneau A. Boussier J. Pere H. Charbit B. Bondet V. Chenevier-Gobeaux C. Breillat P. Carlier N. Gauzit R. Morbieu C. Pene F. Marin N. et al.Impaired type I interferon activity and exacerbated inflammatory responses in severe Covid-19 patients.medRxiv. 2020; (32661059)10.1101/2020.04.19.20068015Google Scholar, 9Huang C. Wang Y. Li X. Ren L. Zhao J. Hu Y. Zhang L. Fan G. Xu J. Gu X. Cheng Z. Yu T. Xia J. Wei Y. Wu W. et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; 395 (31986264): 497-50610.1016/S0140-6736(20)30183-5Abstract Full Text Full Text PDF PubMed Scopus (28324) Google Scholar, 10Moore J.B. June C.H. Cytokine release syndrome in severe COVID-19.Science. 2020; 368 (32303591): 473-47410.1126/science.abb8925Crossref PubMed Scopus (1188) Google Scholar). Many cytokines, including proinflammatory cytokines and chemokines, have been found to be increased after SARS-CoV-2 infection (9Huang C. Wang Y. Li X. Ren L. Zhao J. Hu Y. Zhang L. Fan G. Xu J. Gu X. Cheng Z. Yu T. Xia J. Wei Y. Wu W. et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; 395 (31986264): 497-50610.1016/S0140-6736(20)30183-5Abstract Full Text Full Text PDF PubMed Scopus (28324) Google Scholar, 11Blanco-Melo D. Nilsson-Payant B.E. Liu W.-C. Uhl S. Hoagland D. Møller R. Jordan T.X. Oishi K. Panis M. Sachs D. Wang T.T. Schwartz R.E. Lim J.K. Albrecht R.A. tenOever B.R. Imbalanced host response to Sars-CoV-2 drives development of COVID-19.Cell. 2020; 181 (32416070): 1036-1045.e910.1016/j.cell.2020.04.026Abstract Full Text Full Text PDF PubMed Scopus (2374) Google Scholar). Some of them, such as tumor necrosis factor (TNF) and IL-6, are correlated with disease development (8Hadjadj J. Yatim N. Barnabei L. Corneau A. Boussier J. Pere H. Charbit B. Bondet V. Chenevier-Gobeaux C. Breillat P. Carlier N. Gauzit R. Morbieu C. Pene F. Marin N. et al.Impaired type I interferon activity and exacerbated inflammatory responses in severe Covid-19 patients.medRxiv. 2020; (32661059)10.1101/2020.04.19.20068015Google Scholar, 9Huang C. Wang Y. Li X. Ren L. Zhao J. Hu Y. Zhang L. Fan G. Xu J. Gu X. Cheng Z. Yu T. Xia J. Wei Y. Wu W. et al.Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China.Lancet. 2020; 395 (31986264): 497-50610.1016/S0140-6736(20)30183-5Abstract Full Text Full Text PDF PubMed Scopus (28324) Google Scholar, 12Yang Y. Shen C. Li J. Yuan J. Yang M. Wang F. Li G. Li Y. Xing L. Peng L. Wei J. Cao M. Zheng H. Wu W. Zou R. et al.Exuberant elevation of IP-10, MCP-3 and IL-1ra during SARS-CoV-2 infection is associated with disease severity and fatal outcome.medRxiv. 2020; 10.1101/2020.03.02.20029975Google Scholar). Despite these findings, little is known about what cellular and molecular mechanisms contribute to the lung damage and determine the onset of cytokine storm in response to SARS-CoV-2 infection. With other infectious respiratory RNA viruses such as influenza A virus (IAV) and MERS, studies have shown that aberrant programmed cell death may be a critical driver of organ failure and overt cytokine release in severe cases (13Fujikura D. Miyazaki T. Programmed cell death in the pathogenesis of influenza.Int. J. Mol. Sci. 2018; 19 (30012970): 206510.3390/ijms19072065Crossref Scopus (35) Google Scholar, 14Herold S. Becker C. Ridge K.M. Budinger G.R.S. Influenza virus-induced lung injury: pathogenesis and implications for treatment.Eur. Respir. J. 2015; 45 (25792631): 1463-147810.1183/09031936.00186214Crossref PubMed Scopus (248) Google Scholar, 15Yeung M.-L. Yao Y. Jia L. Chan J.F.W. Chan K.-H. Cheung K.-F. Chen H. Poon V.K.M. Tsang A.K.L. To K.K.W. Yiu M.-K. Teng J.L.L. Chu H. Zhou J. Zhang Q. et al.MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2.Nat. Microbiol. 2016; 1 (27572168): 1600410.1038/nmicrobiol.2016.4Crossref PubMed Scopus (114) Google Scholar, 16Yue Y. Nabar N.R. Shi C.-S. Kamenyeva O. Xiao X. Hwang I.-Y. Wang M. Kehrl J.H. SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death.Cell Death Dis. 2018; 9 (30185776): 1-1510.1038/s41419-018-0917-yCrossref PubMed Scopus (28) Google Scholar). Programmed cell death is a genetically encoded, actively controlled cellular process for the targeted removal of redundant, irreversibly damaged, and/or potentially deleterious cells (17Galluzzi L. Vitale I. Aaronson S.A. Abrams J.M. Adam D. Agostinis P. Alnemri E.S. Altucci L. Amelio I. Andrews D.W. Annicchiarico-Petruzzelli M. Antonov A.V. Arama E. Baehrecke E.H. Barlev N.A. et al.Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.Cell Death Differ. 2018; 25 (29362479): 486-54110.1038/s41418-017-0012-4Crossref PubMed Scopus (2752) Google Scholar). Among the programmed cell death pathways, apoptosis, pyroptosis, and necroptosis have been the best characterized and function during organismal development and infection. Apoptosis is a caspase-8/10– or -9–dependent form of cell death that is executed by caspase-3 and -7 (18Elmore S. Apoptosis: a review of programmed cell death.Toxicol. Pathol. 2007; 35 (17562483): 495-51610.1080/01926230701320337Crossref PubMed Scopus (8668) Google Scholar, 19Kesavardhana S. Malireddi R.K.S. Kanneganti T.-D. Caspases in cell death, inflammation, and pyroptosis.Annu. Rev. Immunol. 2020; 38 (32017655): 567-59510.1146/annurev-immunol-073119-095439Crossref PubMed Scopus (262) Google Scholar). Pyroptosis is executed by gasdermin family members (20Shi J. Gao W. Shao F. Pyroptosis: gasdermin-mediated programmed necrotic cell death.Trends Biochem. Sci. 2017; 42 (27932073): 245-25410.1016/j.tibs.2016.10.004Abstract Full Text Full Text PDF PubMed Scopus (1351) Google Scholar), which can be activated through inflammasome activation-mediated caspase-1 cleavage of gasdermin D (GSDMD); caspase-11/4/5– or caspase-8–mediated cleavage of GSDMD; caspase-3–mediated cleavage of GSDME; or granzyme A-mediated cleavage of GSDMB (21He W.T. Wan H. Hu L. Chen P. Wang X. Huang Z. Yang Z.H. Zhong C.Q. Han J. Gasdermin D is an executor of pyroptosis and required for interleukin-1β secretion.Cell Res. 2015; 25 (26611636): 1285-129810.1038/cr.2015.139Crossref PubMed Scopus (1195) Google Scholar, 22Kayagaki N. Stowe I.B. Lee B.L. O'Rourke K. Anderson K. Warming S. Cuellar T. Haley B. Roose-Girma M. Phung Q.T. Liu P.S. Lill J.R. Li H. Wu J. Kummerfeld S. et al.Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.Nature. 2015; 526 (26375259): 666-67110.1038/nature15541Crossref PubMed Scopus (1927) Google Scholar, 23Orning P. Weng D. Starheim K. Ratner D. Best Z. Lee B. Brooks A. Xia S. Wu H. Kelliher M.A. Berger S.B. Gough P.J. Bertin J. Proulx M.M. Goguen J.D. et al.Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death.Science. 2018; 362 (30361383): 1064-106910.1126/science.aau2818Crossref PubMed Scopus (456) Google Scholar, 24Sarhan J. Liu B.C. Muendlein H.I. Li P. Nilson R. Tang A.Y. Rongvaux A. Bunnell S.C. Shao F. Green D.R. Poltorak A. Caspase-8 induces cleavage of gasdermin D to elicit pyroptosis during Yersinia infection.Proc. Natl. Acad. Sci. U. S. A. 2018; 115 (30381458): E10888-E1089710.1073/pnas.1809548115Crossref PubMed Scopus (382) Google Scholar, 25Shi J. Zhao Y. Wang K. Shi X. Wang Y. Huang H. Zhuang Y. Cai T. Wang F. Shao F. Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death.Nature. 2015; 526 (26375003): 660-66510.1038/nature15514Crossref PubMed Scopus (2907) Google Scholar, 26Wang Y. Gao W. Shi X. Ding J. Liu W. He H. Wang K. Shao F. Chemotherapy drugs induce pyroptosis through caspase-3 cleavage of a gasdermin.Nature. 2017; 547 (28459430): 99-10310.1038/nature22393Crossref PubMed Scopus (1209) Google Scholar, 27Zhou Z. He H. Wang K. Shi X. Wang Y. Su Y. Wang Y. Li D. Liu W. Zhang Y. Shen L. Han W. Shen L. Ding J. Shao F. Granzyme A from cytotoxic lymphocytes cleaves GSDMB to trigger pyroptosis in target cells.Science. 2020; 368 (32299851): eaaz754810.1126/science.aaz7548Crossref PubMed Scopus (431) Google Scholar). Necroptosis is characterized by RIPK3-activated MLKL oligomerization to mediate cell death (28Galluzzi L. Kepp O. Kroemer G. MLKL regulates necrotic plasma membrane permeabilization.Cell Research. 2014; 24 (24418759): 139-14010.1038/cr.2014.8Crossref PubMed Scopus (60) Google Scholar). Numerous pathogens induce at least one of these three programmed cell death pathways during infection. Some pathogens, such as IAV and vesicular stomatitis virus (VSV), can even induce all three in the same cell population through a process termed PANoptosis (29Christgen S. Zheng M. Kesavardhana S. Karki R. Malireddi R.K.S. Banoth B. Place D.E. Briard B. Sharma B.R. Tuladhar S. Samir P. Burton A. Kanneganti T.-D. Identification of the PANoptosome: a molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis).Front. Cell. Infect. Microbiol. 2020; 10 (32547960): 23710.3389/fcimb.2020.00237Crossref PubMed Scopus (129) Google Scholar, 30Kuriakose T. Man S.M. Malireddi R.K. Karki R. Kesavardhana S. Place D.E. Neale G. Vogel P. Kanneganti T.D. ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways.Sci. Immunol. 2016; (27917412)10.1126/sciimmunol.aag2045Crossref PubMed Scopus (335) Google Scholar). The concept of PANoptosis was established based on our studies that have identified cross-talk between the inflammasome/pyroptosis and apoptosis and necroptosis (29Christgen S. Zheng M. Kesavardhana S. Karki R. Malireddi R.K.S. Banoth B. Place D.E. Briard B. Sharma B.R. Tuladhar S. Samir P. Burton A. Kanneganti T.-D. Identification of the PANoptosome: a molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis).Front. Cell. Infect. Microbiol. 2020; 10 (32547960): 23710.3389/fcimb.2020.00237Crossref PubMed Scopus (129) Google Scholar, 30Kuriakose T. Man S.M. Malireddi R.K. Karki R. Kesavardhana S. Place D.E. Neale G. Vogel P. Kanneganti T.D. ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways.Sci. Immunol. 2016; (27917412)10.1126/sciimmunol.aag2045Crossref PubMed Scopus (335) Google Scholar, 31Lamkanfi M. Kanneganti T.D. Van Damme P. Vanden Berghe T. Vanoverberghe I. Vandekerckhove J. Vandenabeele P. Gevaert K. Núñez G. Targeted peptidecentric proteomics reveals caspase-7 as a substrate of the caspase-1 inflammasomes.Mol. Cell. Proteomics. 2008; 7 (18667412): 2350-236310.1074/mcp.M800132-MCP200Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 32Malireddi R.K.S. Gurung P. Mavuluri J. Dasari T.K. Klco J.M. Chi H. Kanneganti T.-D. TAK1 restricts spontaneous NLRP3 activation and cell death to control myeloid proliferation.J. Exp. Med. 2018; 215 (29500178): 1023-103410.1084/jem.20171922Crossref PubMed Scopus (118) Google Scholar, 33Malireddi R.K.S. Kesavardhana S. Kanneganti T.-D. ZBP1 and TAK1: master regulators of NLRP3 inflammasome/pyroptosis, apoptosis, and necroptosis (PAN-optosis).Front. Cell. Infect. Microbiol. 2019; 9 (31850239)10.3389/fcimb.2019.00406Crossref PubMed Scopus (131) Google Scholar, 34Malireddi R.K.S. Gurung P. Kesavardhana S. Samir P. Burton A. Mummareddy H. Vogel P. Pelletier S. Burgula S. Kanneganti T.-D. Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity–independent pyroptosis, apoptosis, necroptosis, and inflammatory disease.J. Exp. Med. 2020; 217 (31869420)10.1084/jem.20191644Crossref PubMed Google Scholar, 35Gurung P. Burton A. Kanneganti T.-D. NLRP3 inflammasome plays a redundant role with caspase 8 to promote IL-1β-mediated osteomyelitis.Proc. Natl. Acad. Sci. U. S. A. 2016; 113 (27071119): 4452-445710.1073/pnas.1601636113Crossref PubMed Scopus (74) Google Scholar, 36Gurung P. Anand P.K. Malireddi R.K. Vande Walle L. Van Opdenbosch N. Dillon C.P. Weinlich R. Green D.R. Lamkanfi M. Kanneganti T.D. FADD and caspase-8 mediate priming and activation of the canonical and noncanonical Nlrp3 inflammasomes.J. Immunol. 2014; 192 (24453255): 1835-184610.4049/jimmunol.1302839Crossref PubMed Scopus (368) Google Scholar, 37Zheng M. Karki R. Vogel P. Kanneganti T.-D. Caspase-6 is a key regulator of innate immunity, inflammasome activation, and host defense.Cell. 2020; 181 (32298652): 674-68710.1016/j.cell.2020.03.040Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 38Samir P. Malireddi R.K.S. Kanneganti T.-D. The PANoptosome: a deadly protein complex driving pyroptosis, apoptosis, and necroptosis (PANoptosis).Front. Cell. Infect. Microbiol. 2020; 10 (32582562): 23810.3389/fcimb.2020.00238Crossref PubMed Scopus (99) Google Scholar, 39Malireddi R.K.S. Ippagunta S. Lamkanfi M. Kanneganti T.-D. Cutting edge: proteolytic inactivation of poly(ADP-ribose) polymerase 1 by the Nlrp3 and Nlrc4 inflammasomes.J. Immunol. 2010; 185 (20713892): 3127-313010.4049/jimmunol.1001512Crossref PubMed Scopus (95) Google Scholar, 40Lukens J.R. Gurung P. Vogel P. Johnson G.R. Carter R.A. McGoldrick D.J. Bandi S.R. Calabrese C.R. Walle L.V. Lamkanfi M. Kanneganti T.-D. Dietary modulation of the microbiome affects autoinflammatory disease.Nature. 2014; 516 (25274309): 246-24910.1038/nature13788Crossref PubMed Scopus (212) Google Scholar, 94Karki R. Sharma B.R. Lee E. Banoth B. Malireddi R.K.S. Samir P. Tuladhar S. Mummareddy H. Burton A.R. Vogel P. Kanneganti T.-D. Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer.JCI Insight. 2020; 5 (32554929): e13672010.1172/jci.insight.136720Crossref PubMed Scopus (72) Google Scholar). PANoptosis is a unique inflammatory programmed cell death regulated by the PANoptosome, which provides a molecular scaffold that allows for interactions and activation of the machinery required for inflammasome/pyroptosis (such as NLRP3, ASC, caspase-1), apoptosis (caspase-8), and necroptosis (RIPK3/RIPK1) (29Christgen S. Zheng M. Kesavardhana S. Karki R. Malireddi R.K.S. Banoth B. Place D.E. Briard B. Sharma B.R. Tuladhar S. Samir P. Burton A. Kanneganti T.-D. Identification of the PANoptosome: a molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis).Front. Cell. Infect. Microbiol. 2020; 10 (32547960): 23710.3389/fcimb.2020.00237Crossref PubMed Scopus (129) Google Scholar, 34Malireddi R.K.S. Gurung P. Kesavardhana S. Samir P. Burton A. Mummareddy H. Vogel P. Pelletier S. Burgula S. Kanneganti T.-D. Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity–independent pyroptosis, apoptosis, necroptosis, and inflammatory disease.J. Exp. Med. 2020; 217 (31869420)10.1084/jem.20191644Crossref PubMed Google Scholar, 37Zheng M. Karki R. Vogel P. Kanneganti T.-D. Caspase-6 is a key regulator of innate immunity, inflammasome activation, and host defense.Cell. 2020; 181 (32298652): 674-68710.1016/j.cell.2020.03.040Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 38Samir P. Malireddi R.K.S. Kanneganti T.-D. The PANoptosome: a deadly protein complex driving pyroptosis, apoptosis, and necroptosis (PANoptosis).Front. Cell. Infect. Microbiol. 2020; 10 (32582562): 23810.3389/fcimb.2020.00238Crossref PubMed Scopus (99) Google Scholar). The ability of these molecules to interact allows for intricate coregulation between cell death pathways that had previously been thought to be independent. PANoptosis has been implicated in infectious and autoinflammatory diseases, cancer, and beyond (29Christgen S. Zheng M. Kesavardhana S. Karki R. Malireddi R.K.S. Banoth B. Place D.E. Briard B. Sharma B.R. Tuladhar S. Samir P. Burton A. Kanneganti T.-D. Identification of the PANoptosome: a molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis).Front. Cell. Infect. Microbiol. 2020; 10 (32547960): 23710.3389/fcimb.2020.00237Crossref PubMed Scopus (129) Google Scholar, 30Kuriakose T. Man S.M. Malireddi R.K. Karki R. Kesavardhana S. Place D.E. Neale G. Vogel P. Kanneganti T.D. ZBP1/DAI is an innate sensor of influenza virus triggering the NLRP3 inflammasome and programmed cell death pathways.Sci. Immunol. 2016; (27917412)10.1126/sciimmunol.aag2045Crossref PubMed Scopus (335) Google Scholar, 32Malireddi R.K.S. Gurung P. Mavuluri J. Dasari T.K. Klco J.M. Chi H. Kanneganti T.-D. TAK1 restricts spontaneous NLRP3 activation and cell death to control myeloid proliferation.J. Exp. Med. 2018; 215 (29500178): 1023-103410.1084/jem.20171922Crossref PubMed Scopus (118) Google Scholar, 34Malireddi R.K.S. Gurung P. Kesavardhana S. Samir P. Burton A. Mummareddy H. Vogel P. Pelletier S. Burgula S. Kanneganti T.-D. Innate immune priming in the absence of TAK1 drives RIPK1 kinase activity–independent pyroptosis, apoptosis, necroptosis, and inflammatory disease.J. Exp. Med. 2020; 217 (31869420)10.1084/jem.20191644Crossref PubMed Google Scholar, 35Gurung P. Burton A. Kanneganti T.-D. NLRP3 inflammasome plays a redundant role with caspase 8 to promote IL-1β-mediated osteomyelitis.Proc. Natl. Acad. Sci. U. S. A. 2016; 113 (27071119): 4452-445710.1073/pnas.1601636113Crossref PubMed Scopus (74) Google Scholar, 37Zheng M. Karki R. Vogel P. Kanneganti T.-D. Caspase-6 is a key regulator of innate immunity, inflammasome activation, and host defense.Cell. 2020; 181 (32298652): 674-68710.1016/j.cell.2020.03.040Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 40Lukens J.R. Gurung P. Vogel P. Johnson G.R. Carter R.A. McGoldrick D.J. Bandi S.R. Calabrese C.R. Walle L.V. Lamkanfi M. Kanneganti T.-D. Dietary modulation of the microbiome affects autoinflammatory disease.Nature. 2014; 516 (25274309): 246-24910.1038/nature13788Crossref PubMed Scopus (212) Google Scholar, 94Karki R. Sharma B.R. Lee E. Banoth B. Malireddi R.K.S. Samir P. Tuladhar S. Mummareddy H. Burton A.R. Vogel P. Kanneganti T.-D. Interferon regulatory factor 1 regulates PANoptosis to prevent colorectal cancer.JCI Insight. 2020; 5 (32554929): e13672010.1172/jci.insight.136720Crossref PubMed Scopus (72) Google Scholar). This process can trigger the release of proinflammatory cytokines and damage-associated molecular patterns, which lead to robust inflammation (29Christgen S. Zheng M. Kesavardhana S. Karki R. Malireddi R.K.S. Banoth B. Place D.E. Briard B. Sharma B.R. Tuladhar S. Samir P. Burton A. Kanneganti T.-D. Identification of the PANoptosome: a molecular platform triggering pyroptosis, apoptosis, and necroptosis (PANoptosis).Front. Cell. Infect. Microbiol. 2020; 10 (32547960): 23710.3389/fcimb.2020.00237Crossref PubMed Scopus (129) Google Scholar, 38Samir P. Malireddi R.K.S. Kanneganti T.-D. The PANoptosome: a deadly protein complex driving pyroptosis, apoptosis, and necroptosis (PANoptosis).Front. Cell. Infect. Microbiol. 2020; 10 (32582562): 23810.3389/fcimb.2020.00238Crossref PubMed Scopus (99) Google Scholar). Therefore, dysregulation of these pathways can be detrimental to the host during infection. Sporadic studies have demonstrated that betacoronavirus proteins or infection with full virus can induce apoptosis, pyroptosis, and necroptosis in specific cell types. In HEK293T cells, ORF-3a of SARS-CoV activates necroptosis (in RIPK3-reconstituted cells) (16Yue Y. Nabar N.R. Shi C.-S. Kamenyeva O. Xiao X. Hwang I.-Y. Wang M. Kehrl J.H. SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death.Cell Death Dis. 2018; 9 (30185776): 1-1510.1038/s41419-018-0917-yCrossref PubMed Scopus (28) Google Scholar) or caspase-1 (in inflammasome component–reconstituted cells) (16Yue Y. Nabar N.R. Shi C.-S. Kamenyeva O. Xiao X. Hwang I.-Y. Wang M. Kehrl J.H. SARS-Coronavirus Open Reading Frame-3a drives multimodal necrotic cell death.Cell Death Dis. 2018; 9 (30185776): 1-1510.1038/s41419-018-0917-yCrossref PubMed Scopus (28) Google Scholar, 41Siu K.-L. Yuen K.-S. Castaño-Rodriguez C. Ye Z.-W. Yeung M.-L. Fung S.-Y. Yuan S. Chan C.-P. Yuen K.-Y. Enjuanes L. Jin D.-Y. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.FASEB J. 2019; 33 (31034780): 8865-887710.1096/fj.201802418RCrossref PubMed Scopus (322) Google Scholar), suggesting the capacity of SARS-CoV to activate pyroptosis. Furthermore, transfection assays indicate that ORF-8b (42Shi C.-S. Nabar N.R. Huang N.-N. Kehrl J.H. SARS-Coronavirus Open Reading Frame-8b triggers intracellular stress pathways and activates NLRP3 inflammasomes.Cell Death Discov. 2019; 5 (31231549): 10110.1038/s41420-019-0181-7Crossref PubMed Scopus (295) Google Scholar) and ORF-3a (41Siu K.-L. Yuen K.-S. Castaño-Rodriguez C. Ye Z.-W. Yeung M.-L. Fung S.-Y. Yuan S. Chan C.-P. Yuen K.-Y. Enjuanes L. Jin D.-Y. Severe acute respiratory syndrome coronavirus ORF3a protein activates the NLRP3 inflammasome by promoting TRAF3-dependent ubiquitination of ASC.FASEB J. 2019; 33 (31034780): 8865-887710.1096/fj.201802418RCrossref PubMed Scopus (322) Google Scholar) of SARS-CoV can activate the NLRP3 inflammasome in THP1 cells. In addition, the ion channel activity of the SARS-CoV envelope (E) protein has been shown to contribute to IL-1β release in vivo after SARS-CoV infection (43DeDiego M.L. Nieto-Torres J.L. Jimenez-Guardeño J.M. Regla-Nava J.A. Castaño-Rodriguez C. Fernandez-Delgado R. Usera F. Enjuanes L. Coronavirus virulence genes with main focus on SARS-CoV envelope gene.Virus Res. 2014; 194 (25093995): 124-13710.1016/j.virusres.2014.07.024Crossref PubMed Scopus (122) Google Scholar, 44Nieto-Torres J.L. DeDiego M.L. Verdiá-Báguena C. Jimenez-Guardeño J.M. Regla-Nava J.A. Fernandez-Delgado R. Castaño-Rodriguez C. Alcaraz A. Torres J. Aguilella V.M. Enjuanes L. Severe acute respiratory syndrome coronavirus envelope protein ion channel activity promotes virus fitness and pathogenesis.PLoS Pathogens. 2014; 10 (24788150): e100407710.1371/journal.ppat.1004077Crossref PubMed Scopus (344) Google Scholar, 45Nieto-Torres J.L. Verdiá-Báguena C. Castaño-Rodriguez C. Aguilella V.M. Enjuanes L. Relevance of viroporin ion channel activity on viral replication and pathogenesis.Viruses. 2015; 7 (26151305): 3552-357310.3390/v7072786Crossref PubMed Scopus (55) Google Scholar), suggesting an essential role for the E protein in regulating SARS-CoV–induced inflammasome activation. This role is further supported by work showing that transfection of the SARS-CoV E protein into Vero E6 cells reconstituted with inflammasome components leads to IL-1β release (46Nieto-Torres J.L. Verdiá-Báguena C. Jimenez-Guardeño J.M. Regla-Nava J.A. Castaño-Rodriguez C. Fernandez-Delgado R. Torres J. Aguilella V.M. Enjuanes L. Severe acute respiratory syndrome coronavirus E protein transports calcium ions and activates the NLRP3 inflammasome.Virology. 2015; 485 (26331680): 330-33910.1016/j.virol.2015.08.010Crossref PubMed Scopus (308) Google Scholar). Moreover, MERS and mouse hepatitis virus (MHV), another betacoronavirus, have also been shown to activate apoptosis (15Yeung M.-L. Yao Y. Jia L. Chan J.F.W. Chan K.-H. Cheung K.-F. Chen H. Poon V.K.M. Tsang A.K.L. To K.K.W. Yiu M.-K. Teng J.L.L. Chu H. Zhou J. Zhang Q. et al.MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2.Nat. Microbiol. 2016; 1 (27572168): 1600410.1038/nmicrobiol.2016.4Crossref PubMed Scopus (114) Google Scholar, 47An S. Chen C.J. Yu X. Leibowitz J.L. Makino S. Induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of E protein as an apoptosis inducer.J. Virol. 1999; 73 (10438879): 7853-785910.1128/JVI.73.9.7853-7859.1999Crossref PubMed Google Scholar) and induce IL-1β release (48Guo S. Yang C. Diao B. Huang X. Jin M. Chen L. Yan W. Ning Q. Zheng L. Wu Y. Chen Y. The NLRP3 inflammasome and IL-1β accelerate immunologically mediated pathology in experimental viral fulminant hepatitis.PLoS Pathog. 2015; 11 (26367131): e100515510.1371/journal.ppat.1005155Crossref PubMed Scopus (54) Google Scholar, 49Jiang Y. Li J. Teng Y. Sun H. Tian G. He L. Li P. Chen Y. Guo Y. Li J. Zhao G. Zhou Y. Sun S. Complement receptor C5aR1 inhibition reduces pyroptosis in hDPP4-transgenic mice infected with MERS-CoV.Viruses. 2019; 11 (30634407): 3910.3390/v11010039Crossref Scopus (54) Google Scholar), suggesting the activation of the inflammasome and cell death pathways. However, the mechanistic details of the cell death induced by coronaviruses and the functional consequences of this