ZBP1 promotes fungi-induced inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis)

Candida albicans and Aspergillus fumigatus are dangerous fungal pathogens with high morbidity and mortality, particularly in immunocompromised patients. Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is an integral part of host defense against pathogens. Inflammasomes, which are canonically formed upstream of pyroptosis, have been characterized as key mediators of fungal sensing and drivers of proinflammatory responses. However, the specific cell death pathways and key upstream sensors activated in the context of Candida and Aspergillus infections are unknown. Here, we report that C. albicans and A. fumigatus infection induced inflammatory programmed cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis). Further, we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as the apical sensor of fungal infection responsible for activating the inflammasome/pyroptosis, apoptosis, and necroptosis. The Zα2 domain of ZBP1 was required to promote this inflammasome activation and PANoptosis. Overall, our results demonstrate that C. albicans and A. fumigatus induce PANoptosis and that ZBP1 plays a vital role in inflammasome activation and PANoptosis in response to fungal pathogens. Candida albicans and Aspergillus fumigatus are dangerous fungal pathogens with high morbidity and mortality, particularly in immunocompromised patients. Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is an integral part of host defense against pathogens. Inflammasomes, which are canonically formed upstream of pyroptosis, have been characterized as key mediators of fungal sensing and drivers of proinflammatory responses. However, the specific cell death pathways and key upstream sensors activated in the context of Candida and Aspergillus infections are unknown. Here, we report that C. albicans and A. fumigatus infection induced inflammatory programmed cell death in the form of pyroptosis, apoptosis, and necroptosis (PANoptosis). Further, we identified the innate immune sensor Z-DNA binding protein 1 (ZBP1) as the apical sensor of fungal infection responsible for activating the inflammasome/pyroptosis, apoptosis, and necroptosis. The Zα2 domain of ZBP1 was required to promote this inflammasome activation and PANoptosis. Overall, our results demonstrate that C. albicans and A. fumigatus induce PANoptosis and that ZBP1 plays a vital role in inflammasome activation and PANoptosis in response to fungal pathogens. Mycotic diseases pose a significant global health burden, particularly among immunocompromised patients, and they are under studied compared with other infectious diseases (1Benedict K. Richardson M. Vallabhaneni S. Jackson B.R. Chiller T. Emerging issues, challenges, and changing epidemiology of fungal disease outbreaks.Lancet Infect. Dis. 2017; 17 (28774697): e403-e41110.1016/S1473-3099(17)30443-7Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Candida albicans and Aspergillus fumigatus infections often result in invasive candidiasis and aspergilliosis if not cleared early, and they are among the most dangerous fungal pathogens with high morbidity and mortality rates (2Kullberg B.J. Arendrup M.C. Invasive Candidiasis.N. Engl. J. Med. 2015; 373 (26444731): 1445-145610.1056/NEJMra1315399Crossref PubMed Scopus (565) Google Scholar, 3Segal B.H. Aspergillosis.N. Engl. J. Med. 2009; 360 (19403905): 1870-188410.1056/NEJMra0808853Crossref PubMed Scopus (529) Google Scholar). The host innate immune system is critical for recognizing fungal particles and mediating their clearance (4Lionakis M.S. Iliev I.D. Hohl T.M. Immunity against fungi.JCI Insight. 2017; 210.1172/jci.insight.93156Crossref PubMed Scopus (47) Google Scholar). Innate immune-mediated programmed cell death (pyroptosis, apoptosis, necroptosis) is also an essential part of this host defense (5Jorgensen I. Rayamajhi M. Miao E.A. Programmed cell death as a defence against infection.Nat. Rev. Immunol. 2017; 17 (28138137): 151-16410.1038/nri.2016.147Crossref PubMed Scopus (342) Google Scholar). Pathogen-associated molecular patterns and damage-associated molecular patterns (PAMPs and DAMPs) are recognized through pattern recognition receptors (PRRs) and can cause the formation of cytosolic multimeric protein complexes known as inflammasomes (6Man S.M. Kanneganti T.D. Regulation of inflammasome activation.Immunol. Rev. 2015; 265 (25879280): 6-2110.1111/imr.12296Crossref PubMed Scopus (397) Google Scholar). The inflammasome drives the activation of the inflammatory caspase-1 (CASP1), which further proteolytically processes cytokines including interleukin (IL)-1β and IL-18 and the executioner molecule gasdermin D (GSDMD), resulting in pyroptotic cell death (6Man S.M. Kanneganti T.D. Regulation of inflammasome activation.Immunol. Rev. 2015; 265 (25879280): 6-2110.1111/imr.12296Crossref PubMed Scopus (397) Google Scholar, 7Karki R. Kanneganti T.D. Diverging inflammasome signals in tumorigenesis and potential targeting.Nat. Rev. Cancer. 2019; 19 (30842595): 197-21410.1038/s41568-019-0123-yCrossref PubMed Scopus (100) Google Scholar, 8Man S.M. Kanneganti T.D. Gasdermin D: the long-awaited executioner of pyroptosis.Cell Res. 2015; 25 (26482951): 1183-118410.1038/cr.2015.124Crossref PubMed Scopus (63) Google Scholar). Previously, we have characterized inflammasome sensors, including NLRP3 and AIM2, to mediate innate immune responses against fungal pathogens, C. albicans and A. fumigatus (9Briard B. Karki R. Malireddi R.K.S. Bhattacharya A. Place D.E. Mavuluri J. Peters J.L. Vogel P. Yamamoto M. Kanneganti T.D. Fungal ligands released by innate immune effectors promote inflammasome activation during Aspergillus fumigatus infection.Nat. Microbiol. 2019; 4 (30510167): 316-32710.1038/s41564-018-0298-0Crossref PubMed Scopus (30) Google Scholar, 10Karki R. Man S.M. Malireddi R.K.S. Gurung P. Vogel P. Lamkanfi M. Kanneganti T.D. Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection.Cell Host Microbe. 2015; 17 (25704009): 357-36810.1016/j.chom.2015.01.006Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 11Lamkanfi M. Malireddi R.K. Kanneganti T.D. Fungal zymosan and mannan activate the cryopyrin inflammasome.J. Biol. Chem. 2009; 284 (19509280): 20574-2058110.1074/jbc.M109.023689Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Fungi-mediated inflammasome activation causes cytokine release and tissue damage because of inflammatory cell death (12Caffrey A.K. Obar J.J. Alarmin(g) the innate immune system to invasive fungal infections.Curr. Opin. Microbiol. 2016; 32 (27351354): 135-14310.1016/j.mib.2016.06.002Crossref PubMed Scopus (11) Google Scholar). In addition to pyroptosis, other forms of programmed cell death important for host defense include apoptosis and necroptosis. Apoptosis is induced on activation of apical caspases caspase-8 (CASP8) or caspase-9 (CASP9) that results in activation of executioner caspases, including caspase-3 (CASP3) and caspase-7 (CASP7) (13Kesavardhana 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 (51) Google Scholar, 14Tummers B. Green D.R. Caspase-8: regulating life and death.Immunol. Rev. 2017; 277 (28462525): 76-8910.1111/imr.12541Crossref PubMed Scopus (231) Google Scholar). The apoptotic caspase-3 can also activate gasdermin E to induce a lytic form of cell death (15Rogers C. Erkes D.A. Nardone A. Aplin A.E. Fernandes-Alnemri T. Alnemri E.S. Gasdermin pores permeabilize mitochondria to augment caspase-3 activation during apoptosis and inflammasome activation.Nat. Commun. 2019; 10 (30976076)168910.1038/s41467-019-09397-2Crossref PubMed Scopus (143) Google Scholar). During necroptosis, proteins containing a receptor interacting protein (RIP) homotypic interaction motif (RHIM), including RIP kinase 1 (RIPK1) and RIP kinase 3 (RIPK3), play crucial roles in the phosphorylation of the mixed lineage kinase domain-like pseudokinase (MLKL), which executes cell death (13Kesavardhana 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 (51) Google Scholar, 16Weinlich R. Oberst A. Beere H.M. Green D.R. Necroptosis in development, inflammation and disease.Nat. Rev. Mol. Cell Biol. 2017; 18 (27999438): 127-13610.1038/nrm.2016.149Crossref PubMed Scopus (350) Google Scholar). The identification of extensive cross-talk between pyroptosis, apoptosis, and necroptosis has led to the establishment of the concept of "PANoptosis" (17Christgen 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 (38) Google Scholar, 18Gurung P. Burton A. Kanneganti T.D. NLRP3 inflammasome plays a redundant role with caspase 8 to promote IL-1beta-mediated osteomyelitis.Proc. Natl. Acad. Sci. U S A. 2016; 113 (27071119): 4452-445710.1073/pnas.1601636113Crossref PubMed Scopus (50) Google Scholar, 19Karki 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; 510.1172/jci.insight.136720Crossref PubMed Scopus (20) Google Scholar, 20Kesavardhana S. Malireddi R.K.S. Burton A.R. Porter S.N. Vogel P. Pruett-Miller S.M. Kanneganti T.D. The Zalpha2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development.J. Biol. Chem. 2020; 295 (32350114): 8325-833010.1074/jbc.RA120.013752Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 21Kuriakose 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; 1 (27917412)aag204510.1126/sciimmunol.aag2045Crossref PubMed Scopus (207) Google Scholar, 22Lamkanfi M. Kanneganti T.D. Van Damme P. Vanden Berghe T. Vanoverberghe I. Vandekerckhove J. Vandenabeele P. Gevaert K. Nunez 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 (204) Google Scholar, 23Lukens J.R. Gurung P. Vogel P. Johnson G.R. Carter R.A. McGoldrick D.J. Bandi S.R. Calabrese C.R. Vande Walle L. Lamkanfi M. Kanneganti T.D. Dietary modulation of the microbiome affects autoinflammatory disease.Nature. 2014; 516 (25274309): 246-24910.1038/nature13788Crossref PubMed Scopus (162) Google Scholar, 24Malireddi R.K. 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 (62) Google Scholar, 25Malireddi 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)e2019164410.1084/jem.20191644Crossref PubMed Google Scholar, 26Malireddi 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 (69) Google Scholar, 27Malireddi 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: 40610.3389/fcimb.2019.00406Crossref PubMed Scopus (49) Google Scholar, 28Samir 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 (35) Google Scholar, 29Zheng 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): 674-687.e1310.1016/j.cell.2020.03.040Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 30Zheng M. Williams E.P. Malireddi R.K.S. Karki R. Banoth B. Burton A. Webby R. Channappanavar R. Jonsson C.B. Kanneganti T.D. Impaired NLRP3 inflammasome activation/pyroptosis leads to robust inflammatory cell death via caspase-8/RIPK3 during coronavirus infection.J. Biol. Chem. 2020; 295: 14040-14052Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). PANoptosis ('P', Pyroptosis; 'A', Apoptosis; 'N', Necroptosis; and 'optosis', a form of programmed cell death) is defined as "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)" (17Christgen 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 (38) Google Scholar, 25Malireddi 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)e2019164410.1084/jem.20191644Crossref PubMed Google Scholar, 28Samir 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 (35) Google Scholar, 29Zheng 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): 674-687.e1310.1016/j.cell.2020.03.040Abstract Full Text Full Text PDF PubMed Scopus (50) 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 (17Christgen 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 (38) Google Scholar, 18Gurung P. Burton A. Kanneganti T.D. NLRP3 inflammasome plays a redundant role with caspase 8 to promote IL-1beta-mediated osteomyelitis.Proc. Natl. Acad. Sci. U S A. 2016; 113 (27071119): 4452-445710.1073/pnas.1601636113Crossref PubMed Scopus (50) Google Scholar, 19Karki 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; 510.1172/jci.insight.136720Crossref PubMed Scopus (20) Google Scholar, 20Kesavardhana S. Malireddi R.K.S. Burton A.R. Porter S.N. Vogel P. Pruett-Miller S.M. Kanneganti T.D. The Zalpha2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development.J. Biol. Chem. 2020; 295 (32350114): 8325-833010.1074/jbc.RA120.013752Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 21Kuriakose 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; 1 (27917412)aag204510.1126/sciimmunol.aag2045Crossref PubMed Scopus (207) Google Scholar, 23Lukens J.R. Gurung P. Vogel P. Johnson G.R. Carter R.A. McGoldrick D.J. Bandi S.R. Calabrese C.R. Vande Walle L. Lamkanfi M. Kanneganti T.D. Dietary modulation of the microbiome affects autoinflammatory disease.Nature. 2014; 516 (25274309): 246-24910.1038/nature13788Crossref PubMed Scopus (162) Google Scholar, 25Malireddi 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)e2019164410.1084/jem.20191644Crossref PubMed Google Scholar, 26Malireddi 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 (69) Google Scholar, 29Zheng 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): 674-687.e1310.1016/j.cell.2020.03.040Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 30Zheng M. Williams E.P. Malireddi R.K.S. Karki R. Banoth B. Burton A. Webby R. Channappanavar R. Jonsson C.B. Kanneganti T.D. Impaired NLRP3 inflammasome activation/pyroptosis leads to robust inflammatory cell death via caspase-8/RIPK3 during coronavirus infection.J. Biol. Chem. 2020; 295: 14040-14052Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar), and the molecular details and phenotypic outcomes of the cross-talk and coregulation among pyroptosis, apoptosis, and necroptosis are dependent on the stimulus provided. In this study, we show that C. albicans and A. fumigatus infection elicited inflammasome activation and pyroptosis, apoptosis, and necroptosis (PANoptosis). We found that the innate immune sensor Z-DNA binding protein 1 (ZBP1) functioned as the apical sensor to activate the C. albicans and A. fumigatus-induced inflammasome/pyroptosis, apoptosis, and necroptosis. Further, we demonstrate that the nucleic acid binding domain Zα2 of ZBP1 is crucial for C. albicans- and A. fumigatus-induced inflammasome activation and PANoptotic cell death. Collectively, our results establish that C. albicans and A. fumigatus induce PANoptosis and that ZBP1 is critical for inflammasome activation and PANoptosis in response to fungal pathogens. Our previous studies characterized inflammasomes as key players in sensing fungal infection and activating proinflammatory responses (9Briard B. Karki R. Malireddi R.K.S. Bhattacharya A. Place D.E. Mavuluri J. Peters J.L. Vogel P. Yamamoto M. Kanneganti T.D. Fungal ligands released by innate immune effectors promote inflammasome activation during Aspergillus fumigatus infection.Nat. Microbiol. 2019; 4 (30510167): 316-32710.1038/s41564-018-0298-0Crossref PubMed Scopus (30) Google Scholar, 10Karki R. Man S.M. Malireddi R.K.S. Gurung P. Vogel P. Lamkanfi M. Kanneganti T.D. Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection.Cell Host Microbe. 2015; 17 (25704009): 357-36810.1016/j.chom.2015.01.006Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), but the specific cell death pathways activated in response to C. albicans and A. fumigatus remain unknown. Therefore, we systematically analyzed the programmed cell death pathways activated by C. albicans and A. fumigatus. We infected WT BMDMs with varying doses of C. albicans and A. fumigatus and biochemically assessed markers associated with pyroptosis, apoptosis, and necroptosis (PANoptosis). Consistent with previous reports (10Karki R. Man S.M. Malireddi R.K.S. Gurung P. Vogel P. Lamkanfi M. Kanneganti T.D. Concerted activation of the AIM2 and NLRP3 inflammasomes orchestrates host protection against Aspergillus infection.Cell Host Microbe. 2015; 17 (25704009): 357-36810.1016/j.chom.2015.01.006Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), we found C. albicans and A. fumigatus elicited inflammasome activation and pyroptosis in a dose-dependent manner, as indicated by activation of CASP1 and GSDMD (Fig. 1A, Fig. S1). Further, characterization of cell death markers associated with apoptosis revealed increased activation of the apoptotic initiator CASP8 (p18) and executioners CASP3 (p17/19) and CASP7 (p20) in a dose-dependent manner (Fig. 1B, Fig. S1). The phosphorylated form of MLKL (pMLKL) is a marker of necroptosis activation (31Sun L. Wang H. Wang Z. He S. Chen S. Liao D. Wang L. Yan J. Liu W. Lei X. Wang X. Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3 kinase.Cell. 2012; 148 (22265413): 213-22710.1016/j.cell.2011.11.031Abstract Full Text Full Text PDF PubMed Scopus (1327) Google Scholar). We observed that infection of WT BMDMs resulted in dose-dependent necroptotic cell death as evidenced by the enhanced levels of pMLKL (Fig. 1C, Fig. S1). Further, we also observed a dose-dependent induction of cell death in response to C. albicans infection in primary human peripheral blood mononuclear cells (hPBMCs), corroborating our findings in primary BMDMs (Fig. 1D). Overall, these data indicate that fungal infection with C. albicans and A. fumigatus activates PANoptosis. Because we observed PANoptosis during fungal infection, we further evaluated the role of key molecular components of PANoptosis using a genetic approach. We infected murine BMDMs lacking crucial components of PANoptosis with C. albicans or A. fumigatus (Fig. 2, Fig. S2). Infection of WT BMDMs with C. albicans or A. fumigatus resulted in robust activation of proteins involved in pyroptosis, apoptosis, and necroptosis (Fig. 2A–C, Fig. S2A), as observed earlier (Fig. 1). Pyroptotic cell death is largely mediated by CASP1, CASP11, ASC, and GSDMD. Loss of these individual molecules led to reduced activation of pyroptotic proteins (CASP1 and GSDMD; Fig. 2A, Fig. S2A) and had minimal effect on the activation of apoptotic proteins (CASP8, CASP3, and CASP7; Fig. 2B, Fig. S2A); however, the expression of pMLKL was clearly increased during C. albicans infection and trended toward an increase during A. fumigatus infection (Fig. 2C, Fig. S2A), suggesting increased activation of necroptosis when pyroptosis is blocked in response to fungal pathogens. Deficiency of necroptotic mediators MLKL or RIPK3 had minor impacts on the activation of pyroptotic and apoptotic markers (Fig. 2A, B, Fig. S2A). CASP8 regulates both canonical and noncanonical NLRP3 inflammasome activation in bacterial and viral infections (17Christgen 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 (38) Google Scholar, 32Gurung 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 (284) Google Scholar), and it also negatively regulates the necroptotic pathway (14Tummers B. Green D.R. Caspase-8: regulating life and death.Immunol. Rev. 2017; 277 (28462525): 76-8910.1111/imr.12541Crossref PubMed Scopus (231) Google Scholar). BMDMs lacking both CASP8 and RIPK3 were largely protected from C. albicans- or A. fumigatus-induced PANoptosis, as evident by the reduced activation of pyroptotic (CASP1, GSDMD; Fig. 2A, Fig. S2A), apoptotic (CASP3, CASP7; Fig. 2B, Fig. S2A), and necroptotic (pMLKL) biochemical markers (Fig. 2C, Fig. S2A). BMDMs lacking critical components of PANoptosis (CASP1, CASP11, RIPK3, and CASP8 combined) are protected from bacteria and virus–induced PANoptosis (17Christgen 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 (38) Google Scholar). Similar to the Ripk3–/–Casp8–/– cells, Casp1/11–/–Ripk3–/–Casp8–/– BMDMs showed reduced activation of host cell death markers in response to C. albicans or A. fumigatus infection (Fig. 2A–C, Fig. S2A). However, although Ripk3–/–Casp8–/– cells showed residual levels of GSDMD activation, the activation of GSDMD was completely abolished in Casp1/11–/–Ripk3–/–Casp8–/– BMDMs (Fig. 2A, Fig. S2A). Combined with the reduced activation of caspase-3, -7 and MLKL, these findings suggest cells undergo reduced cell death when crucial components of PANoptosis are missing during fungal infection. Activation of the inflammasome and inflammatory cell death often results in the release of various cytokines, chemokines, and DAMPs which further amplify the inflammatory response and associated pathology (7Karki R. Kanneganti T.D. Diverging inflammasome signals in tumorigenesis and potential targeting.Nat. Rev. Cancer. 2019; 19 (30842595): 197-21410.1038/s41568-019-0123-yCrossref PubMed Scopus (100) Google Scholar, 33Place D.E. Kanneganti T.D. Cell death-mediated cytokine release and its therapeutic implications.J. Exp. Med. 2019; 216 (31186281): 1474-148610.1084/jem.20181892Crossref PubMed Scopus (22) Google Scholar). We assessed inflammatory cytokine IL-18 release after C. albicans or A. fumigatus infection as a measure of inflammasome activation and associated cell death. WT BMDMs released a significant amount of IL-18 after fungal infection, whereas the Casp1/11–/–Ripk3–/–Casp8–/– BMDMs released significantly less (Fig. 2D, Fig. S2B). Together, these findings suggest that the components of PANoptosis are crucial in mediating inflammatory cell death and cytokine release during fungal infection. The interferon (IFN)-inducible protein ZBP1, also known as DAI (DNA-dependent activator of IFN regulatory factors), is an innate immune sensor that mediates NLRP3 inflammasome activation in response to influenza A virus (IAV) infection (21Kuriakose 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; 1 (27917412)aag204510.1126/sciimmunol.aag2045Crossref PubMed Scopus (207) Google Scholar, 29Zheng 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): 674-687.e1310.1016/j.cell.2020.03.040Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). ZBP1 contains two N-terminal nucleic acid binding domains (Zα1 and Zα2) followed by a RHIM domain responsible for mediating homotypic interactions with cell death signaling proteins RIPK1 and RIPK3 (20Kesavardhana S. Malireddi R.K.S. Burton A.R. Porter S.N. Vogel P. Pruett-Miller S.M. Kanneganti T.D. The Zalpha2 domain of ZBP1 is a molecular switch regulating influenza-induced PANoptosis and perinatal lethality during development.J. Biol. Chem. 2020; 295 (32350114): 8325-833010.1074/jbc.RA120.013752Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 41Ishii K.J. Kawagoe T. Koyama S. Matsui K. Kumar H. Kawai T. Uematsu S. Takeuchi O. Takeshita F. Coban C. Akira S. TANK-binding kinase-1 delineates innate and adaptive immune responses to DNA vaccines.Nature. 2008; 451 (18256672): 725-72910.1038/nature06537Crossref PubMed Scopus (461) Google Scholar). In the context of IAV, ZBP1 sensing of the virus leads to PANoptosome assembly and drives pyroptosis through NLRP3 inflammasome activation, apoptosis via FADD-CASP8, and necroptosis through RIPK3-MLKL (17Christgen 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 (38) Google Scholar, 21Kuriakose 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; 1 (27917412)aag204510.1126/sciimmunol.aag2045Crossref PubMed Scopus (207) Google Scholar, 29Zheng M. Karki R. Vogel P. Kanneganti T.D. Caspase-6 is a key regulator of innate immunity, inflammasome activation, and host def