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• W3039784919 abstract "•Mice lacking pyroptosis and apoptosis cannot control Salmonella infection•Macrophages lacking pyroptosis and apoptosis resist Salmonella-induced killing•Caspase-1 kills Salmonella-infected cells by activating GSDMD, BID, or other caspases•Caspase-1 and -8 act as cell death executioners when all cell death effectors are lost Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections. Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections. Metazoans employ different types of programmed cell death (PCD), including apoptosis, necroptosis, and pyroptosis, for the removal of unwanted cells, such as those infected with pathogens (Green, 2019Green D.R. The Coming Decade of Cell Death Research: Five Riddles.Cell. 2019; 177: 1094-1107Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Apoptosis is executed by so-called effector caspases (caspases-3 and -7, and possibly caspase-6) (Salvesen and Dixit, 1997Salvesen G.S. Dixit V.M. Caspases: intracellular signaling by proteolysis.Cell. 1997; 91: 443-446Abstract Full Text Full Text PDF PubMed Scopus (1917) Google Scholar) that promote cellular fragmentation into apoptotic bodies and engulfment of dying cells by neighboring cells, thus preventing release of intracellular content causing inflammation (Nagata, 2018Nagata S. Apoptosis and Clearance of Apoptotic Cells.Annu. Rev. Immunol. 2018; 36: 489-517Crossref PubMed Scopus (309) Google Scholar). Apoptosis can be induced by death receptors such as FAS or TNFR1, which activate caspase-8, or in response to diverse cellular stresses via the intrinsic pathway, which involves BH3-only protein-initiated and Bcl-2-associated protein (BAX) and BCL-2 homologous antagonists killer (BAK)-mediated mitochondrial outer membrane permeabilization (MOMP) (Czabotar et al., 2014Czabotar P.E. Lessene G. Strasser A. Adams J.M. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy.Nat. Rev. Mol. Cell Biol. 2014; 15: 49-63Crossref PubMed Scopus (1860) Google Scholar). This causes activation of the initiator caspase, caspase-9, and subsequent proteolytic triggering of the effector caspases. Pyroptosis is induced through nucleotide-binding oligomerization domain and leucine-rich repeat-containing receptor (NLR)-dependent activation of caspase-1 or LPS-induced activation of caspase-11 (Lamkanfi and Dixit, 2014Lamkanfi M. Dixit V.M. Mechanisms and functions of inflammasomes.Cell. 2014; 157: 1013-1022Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar; Zhao and Shao, 2016Zhao Y. Shao F. Diverse mechanisms for inflammasome sensing of cytosolic bacteria and bacterial virulence.Curr. Opin. Microbiol. 2016; 29: 37-42Crossref PubMed Scopus (38) Google Scholar). Ligation of tumor necrosis factor receptor-1 (TNFR1) or Toll-like receptors (TLRs) causes phosphorylation of receptor interacting serine/threonine kinase (RIPK) 1 and RIPK3 to initiate necroptosis when caspase-8 activity is absent (Ofengeim and Yuan, 2013Ofengeim D. Yuan J. Regulation of RIP1 kinase signalling at the crossroads of inflammation and cell death.Nat. Rev. Mol. Cell Biol. 2013; 14: 727-736Crossref PubMed Scopus (338) Google Scholar; Vandenabeele et al., 2010Vandenabeele P. Galluzzi L. Vanden Berghe T. Kroemer G. Molecular mechanisms of necroptosis: an ordered cellular explosion.Nat. Rev. Mol. Cell Biol. 2010; 11: 700-714Crossref PubMed Scopus (1530) Google Scholar). Pyroptosis and necroptosis are both executed through lysis of the plasma membrane that releases cellular content into the extracellular space, which can elicit pro-inflammatory responses priming the innate as well as the adaptive immune systems. One important biological function of cellular suicide is to control intracellular pathogens (Jorgensen et al., 2017Jorgensen I. Rayamajhi M. Miao E.A. Programmed cell death as a defence against infection.Nat. Rev. Immunol. 2017; 17: 151-164Crossref PubMed Scopus (382) Google Scholar; Kayagaki et al., 2015Kayagaki N. Stowe I.B. Lee B.L. O’Rourke K. Anderson K. Warming S. Cuellar T. Haley B. Roose-Girma M. Phung Q.T. et al.Caspase-11 cleaves gasdermin D for non-canonical inflammasome signalling.Nature. 2015; 526: 666-671Crossref PubMed Scopus (1395) Google Scholar; Shi et al., 2015Shi 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: 660-665Crossref PubMed Scopus (1926) Google Scholar). The killing of infected cells is thought to remove a replicative niche, re-expose the pathogen to extracellular immune effector mechanisms, and make antigens available for triggering pathogen-specific adaptive immune responses. Salmonella has been widely used as a model for studying the role of programmed cell death in host defense (Broz et al., 2012Broz P. Ruby T. Belhocine K. Bouley D.M. Kayagaki N. Dixit V.M. Monack D.M. Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1.Nature. 2012; 490: 288-291Crossref PubMed Scopus (362) Google Scholar; Franchi et al., 2009Franchi L. Eigenbrod T. Muñoz-Planillo R. Nuñez G. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.Nat. Immunol. 2009; 10: 241-247Crossref PubMed Scopus (1152) Google Scholar). This intracellular pathogen can cause typhoid fever, a systemic infection that affects 10−20 million people worldwide and kills >135,000 individuals per annum (Browne et al., 2020Browne A.J. Kashef Hamadani B.H. Kumaran E.A.P. Rao P. Longbottom J. Harriss E. Moore C.E. Dunachie S. Basnyat B. Baker S. et al.Drug-resistant enteric fever worldwide, 1990 to 2018: a systematic review and meta-analysis.BMC Med. 2020; 18: 1Crossref PubMed Scopus (42) Google Scholar). The disease can be modeled by infecting mice with S. enterica serovar Typhimurium (Kupz et al., 2014Kupz A. Bedoui S. Strugnell R.A. Cellular requirements for systemic control of Salmonella enterica serovar Typhimurium infections in mice.Infect. Immun. 2014; 82: 4997-5004Crossref PubMed Scopus (24) Google Scholar), where spleen and liver are major sites of replication of these bacteria. The primary target of Salmonella spp. are phagocytes in which the bacteria survive by repurposing a host-cell-derived membrane compartment into a specialized niche. Phagocytes, such as macrophages, respond to Salmonella infection through inflammasome formation involving NLR family apoptosis inhibitory proteins (NAIP)2 or NAIP, and NLRs such as NLRC4 and NLRP3 (Franchi et al., 2009Franchi L. Eigenbrod T. Muñoz-Planillo R. Nuñez G. The inflammasome: a caspase-1-activation platform that regulates immune responses and disease pathogenesis.Nat. Immunol. 2009; 10: 241-247Crossref PubMed Scopus (1152) Google Scholar; Miao et al., 2010Miao E.A. Leaf I.A. Treuting P.M. Mao D.P. Dors M. Sarkar A. Warren S.E. Wewers M.D. Aderem A. Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria.Nat. Immunol. 2010; 11: 1136-1142Crossref PubMed Scopus (803) Google Scholar), which activate caspase-1 (Zhang et al., 2015Zhang L. Chen S. Ruan J. Wu J. Tong A.B. Yin Q. Li Y. David L. Lu A. Wang W.L. et al.Cryo-EM structure of the activated NAIP2-NLRC4 inflammasome reveals nucleated polymerization.Science. 2015; 350: 404-409Crossref PubMed Scopus (216) Google Scholar). Caspase-1 then causes the proteolytic maturation of the inflammatory cytokines interleukin (IL)-1β and IL-18 and release of N-terminal fragments of gasdermin D (GSDMD) proteins that form pores in the cell membrane to elicit pyroptosis. Although these processes appear highly relevant in vitro, with caspase-1- or GSDMD-deficient phagocytes resisting Salmonella-induced killing (Franchi et al., 2006Franchi L. Amer A. Body-Malapel M. Kanneganti T.D. Ozören N. Jagirdar R. Inohara N. Vandenabeele P. Bertin J. Coyle A. et al.Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected macrophages.Nat. Immunol. 2006; 7: 576-582Crossref PubMed Scopus (871) Google Scholar; Mariathasan et al., 2004Mariathasan S. Newton K. Monack D.M. Vucic D. French D.M. Lee W.P. Roose-Girma M. Erickson S. Dixit V.M. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf.Nature. 2004; 430: 213-218Crossref PubMed Scopus (1321) Google Scholar), in vivo studies suggest that Salmonella can be controlled in the absence of inflammasome-driven pyroptosis (Broz et al., 2010Broz P. Newton K. Lamkanfi M. Mariathasan S. Dixit V.M. Monack D.M. Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella.J. Exp. Med. 2010; 207: 1745-1755Crossref PubMed Scopus (386) Google Scholar). This may reflect the capacity of the host to compensate for the lack of one type of cell death by using another. Such “fail-safe” systems have been hypothesized before (Jorgensen et al., 2017Jorgensen I. Rayamajhi M. Miao E.A. Programmed cell death as a defence against infection.Nat. Rev. Immunol. 2017; 17: 151-164Crossref PubMed Scopus (382) Google Scholar; Rauch et al., 2017Rauch I. Deets K.A. Ji D.X. von Moltke J. Tenthorey J.L. Lee A.Y. Philip N.H. Ayres J.S. Brodsky I.E. Gronert K. Vance R.E. NAIP-NLRC4 Inflammasomes Coordinate Intestinal Epithelial Cell Expulsion with Eicosanoid and IL-18 Release via Activation of Caspase-1 and -8.Immunity. 2017; 46: 649-659Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar; Van Opdenbosch et al., 2017Van Opdenbosch N. Van Gorp H. Verdonckt M. Saavedra P.H.V. de Vasconcelos N.M. Gonçalves A. Vande Walle L. Demon D. Matusiak M. Van Hauwermeiren F. et al.Caspase-1 Engagement and TLR-Induced c-FLIP Expression Suppress ASC/Caspase-8-Dependent Apoptosis by Inflammasome Sensors NLRP1b and NLRC4.Cell Rep. 2017; 21: 3427-3444Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and may represent the host’s response to offset a variety of evasion strategies employed by pathogens to prevent immune recognition (Bedoui et al., 2010Bedoui S. Kupz A. Wijburg O.L. Walduck A.K. Rescigno M. Strugnell R.A. Different bacterial pathogens, different strategies, yet the aim is the same: evasion of intestinal dendritic cell recognition.J. Immunol. 2010; 184: 2237-2242Crossref PubMed Scopus (40) Google Scholar). However, very little is known about the organization, regulation, and kinetics of such functional backup in the use of different programmed cell death pathways during host defense against pathogens in vivo. Here, we investigated the relative contributions of all initiator and executioner caspases and the cell death effectors they activate to host defense against systemic Salmonella infections. To determine which cell death pathways the host requires for control of intracellular pathogens, we infected C57BL/6 (wild-type: WT) mice with a growth-attenuated strain of S. Typhimurium that mirrors the systemic phase of typhoid fever (Kupz et al., 2013Kupz A. Scott T.A. Belz G.T. Andrews D.M. Greyer M. Lew A.M. Brooks A.G. Smyth M.J. Curtiss 3rd, R. Bedoui S. Strugnell R.A. Contribution of Thy1+ NK cells to protective IFN-γ production during Salmonella typhimurium infections.Proc. Natl. Acad. Sci. USA. 2013; 110: 2252-2257Crossref PubMed Scopus (66) Google Scholar, Kupz et al., 2014Kupz A. Bedoui S. Strugnell R.A. Cellular requirements for systemic control of Salmonella enterica serovar Typhimurium infections in mice.Infect. Immun. 2014; 82: 4997-5004Crossref PubMed Scopus (24) Google Scholar). This infection follows a classical pattern where bacterial growth initially outpaces host defense. By about week 3, bacterial titers reach a peak that is followed by dropping titers and eventual clearance of the bacteria from the host. This type of infection thus allows detailed in vivo investigations into the mechanisms that enable Salmonella control by innate immune mechanisms over the first 3 weeks of the infection (Kupz et al., 2012Kupz A. Guarda G. Gebhardt T. Sander L.E. Short K.R. Diavatopoulos D.A. Wijburg O.L. Cao H. Waithman J.C. Chen W. et al.NLRC4 inflammasomes in dendritic cells regulate noncognate effector function by memory CD8+ T cells.Nat. Immunol. 2012; 13: 162-169Crossref PubMed Scopus (121) Google Scholar, Kupz et al., 2013Kupz A. Scott T.A. Belz G.T. Andrews D.M. Greyer M. Lew A.M. Brooks A.G. Smyth M.J. Curtiss 3rd, R. Bedoui S. Strugnell R.A. Contribution of Thy1+ NK cells to protective IFN-γ production during Salmonella typhimurium infections.Proc. Natl. Acad. Sci. USA. 2013; 110: 2252-2257Crossref PubMed Scopus (66) Google Scholar) and T-cell-mediated immune clearance thereafter (Benoun et al., 2018Benoun J.M. Peres N.G. Wang N. Pham O.H. Rudisill V.L. Fogassy Z.N. Whitney P.G. Fernandez-Ruiz D. Gebhardt T. Pham Q.M. et al.Optimal protection against Salmonella infection requires noncirculating memory.Proc. Natl. Acad. Sci. USA. 2018; 115: 10416-10421Crossref PubMed Scopus (22) Google Scholar). Consistent with earlier reports using WT strains of S. Typhimurium (Broz et al., 2012Broz P. Ruby T. Belhocine K. Bouley D.M. Kayagaki N. Dixit V.M. Monack D.M. Caspase-11 increases susceptibility to Salmonella infection in the absence of caspase-1.Nature. 2012; 490: 288-291Crossref PubMed Scopus (362) Google Scholar), we observed slightly elevated bacterial titers in Casp1–/–;Casp11–/– mice 3 weeks post-infection compared to WT controls (Figure 1A), but the lack of pyroptosis did not affect their capacity to clear the bacteria by 12 weeks post-infection. This indicated a minor defect in bacterial control. Exploiting this in vivo model of caspase-1 and -11 independent bacterial control, we explored the role of other cell death pathways and their key constituents. We first investigated whether the lack of caspases-1 and -11 was compensated for by caspase-12, given their substantial amino acid similarity and chromosomal co-localization. However, at week 3 post-infection, Casp1–/–;Casp11–/–;Casp12–/– and Casp1–/–;Casp11–/– mice presented with similar bacterial titers that were slightly higher compared to those observed in WT controls (Figure 1B), revealing that caspase-12 did not play a critical role in bacterial clearance by compensating for the combined absence of caspases-1 and -11. Caspase-8 has been suggested to coordinate an alternative pathway toward pyroptosis that operates independently of caspases-1 and -11 (Mascarenhas et al., 2017Mascarenhas D.P.A. Cerqueira D.M. Pereira M.S.F. Castanheira F.V.S. Fernandes T.D. Manin G.Z. Cunha L.D. Zamboni D.S. Inhibition of caspase-1 or gasdermin-D enable caspase-8 activation in the Naip5/NLRC4/ASC inflammasome.PLoS Pathog. 2017; 13: e1006502Crossref PubMed Scopus (75) Google Scholar; Orning et al., 2018Orning P. Weng D. Starheim K. Ratner D. Best Z. Lee B. Brooks A. Xia S. Wu H. Kelliher M.A. et al.Pathogen blockade of TAK1 triggers caspase-8-dependent cleavage of gasdermin D and cell death.Science. 2018; 362: 1064-1069Crossref PubMed Scopus (285) Google Scholar). This prompted us to investigate the contribution of caspase-8-driven cell death to Salmonella control in mice. To prevent the necroptosis-driven embryonic lethality caused by loss of caspase-8, we used Casp8–/–;Ripk3–/– mice (Alvarez-Diaz et al., 2016Alvarez-Diaz S. Dillon C.P. Lalaoui N. Tanzer M.C. Rodriguez D.A. Lin A. Lebois M. Hakem R. Josefsson E.C. O’Reilly L.A. et al.The Pseudokinase MLKL and the Kinase RIPK3 Have Distinct Roles in Autoimmune Disease Caused by Loss of Death-Receptor-Induced Apoptosis.Immunity. 2016; 45: 513-526Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar; Kaiser et al., 2011Kaiser W.J. Upton J.W. Long A.B. Livingston-Rosanoff D. Daley-Bauer L.P. Hakem R. Caspary T. Mocarski E.S. RIP3 mediates the embryonic lethality of caspase-8-deficient mice.Nature. 2011; 471: 368-372Crossref PubMed Scopus (690) Google Scholar; Oberst et al., 2011Oberst A. Dillon C.P. Weinlich R. McCormick L.L. Fitzgerald P. Pop C. Hakem R. Salvesen G.S. Green D.R. Catalytic activity of the caspase-8-FLIP(L) complex inhibits RIPK3-dependent necrosis.Nature. 2011; 471: 363-367Crossref PubMed Scopus (819) Google Scholar). The combined lack of caspase-8-mediated apoptosis and RIPK3-driven necroptosis did not have significant impact on Salmonella titers 3 weeks post-infection (Figure 1B). Mice lacking necroptosis alone (Mlkl–/– mice) or those with combined deficiency in pyroptosis and necroptosis (Casp1–/–;Casp11–/–;Casp12–/–;Ripk3–/– mice) had no defects in bacterial control until at least 3 weeks post-infection (Figures 1B and S1A). These findings demonstrate that mice with defects in select types of programmed cell death only have minor impairments in their ability to control bacterial replication. These findings raised the possibility that in vivo control of Salmonella infection was safeguarded by extensive functional backup between several programmed cell death processes. To investigate this, we generated Casp1–/–;Casp11–/–;Casp12–/–;Casp8–/–;Ripk3–/– mice that are deficient for pyroptosis, death-receptor-induced apoptosis, and necroptosis. These mice had drastically elevated bacterial titers in liver and spleen at both week 2 and 3 post-infection compared to WT animals (Figures 1B and 1C) and had to be sacrificed in accordance with ethical guidelines between 4 and 5 weeks post-infection (Figure 1D). This showed that host defense against Salmonella necessitated the activity of at least one of these types of programmed cell death pathways and that none of the other known cell death pathways (e.g., intrinsic apoptosis or ferroptosis) were sufficient to ensure control of the infection in their absence. Of note, we observed similar defects in host defense in bone marrow chimeras in which pyroptosis, caspase-8-mediated apoptosis, and necroptosis were only missing from the immune cell compartment (Figure 1E). Therefore, we conclude that Salmonella control broke down in Casp1–/–;Casp11–/–;Casp12–/–;Casp8–/–;Ripk3–/– mice because phagocytes could no longer purge the bacteria from their vacuolar compartments by undergoing programmed cell death. Previous reports suggest that caspase-8 can induce pyroptosis through proteolytic activation of GSDMD (Mascarenhas et al., 2017Mascarenhas D.P.A. Cerqueira D.M. Pereira M.S.F. Castanheira F.V.S. Fernandes T.D. Manin G.Z. Cunha L.D. Zamboni D.S. Inhibition of caspase-1 or gasdermin-D enable caspase-8 activation in the Naip5/NLRC4/ASC inflammasome.PLoS Pathog. 2017; 13: e1006502Crossref PubMed Scopus (75) Google Scholar). To explore the nature of the cell death induced by caspase-8 upon infection in the absence of the initiators of pyroptosis, we used bone-marrow-derived macrophages (BMDMs) deficient for caspases-1 and -11, or caspases-1, -11, and -12, and infected them with Salmonella. As previously reported (Franchi et al., 2006Franchi L. Amer A. Body-Malapel M. Kanneganti T.D. Ozören N. Jagirdar R. Inohara N. Vandenabeele P. Bertin J. Coyle A. et al.Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected macrophages.Nat. Immunol. 2006; 7: 576-582Crossref PubMed Scopus (871) Google Scholar; Mariathasan et al., 2004Mariathasan S. Newton K. Monack D.M. Vucic D. French D.M. Lee W.P. Roose-Girma M. Erickson S. Dixit V.M. Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf.Nature. 2004; 430: 213-218Crossref PubMed Scopus (1321) Google Scholar), Casp1–/–;Casp11–/– BMDMs are protected from Salmonella-induced killing at early time points. However, 6 h after infection with Salmonella, a substantial fraction of Casp1–/–;Casp11–/– and Casp1–/–;Casp11–/–;Casp12–/– BMDMs had died (Figure 2A), reiterating that caspase-12 was not critical for the response to Salmonella infection. The delayed type of Salmonella-induced cell death in Casp1–/–;Casp11–/– and Casp1–/–;Casp11–/–;Casp12–/– BMDMs was unlikely to be due to necroptosis, as we could not detect changes in phosphorylation of MLKL, a hallmark of necroptosis (Figure S1B). Instead, Casp1–/–;Casp11–/– and Casp1–/–;Casp11–/–;Casp12–/– BMDMs displayed hallmarks of apoptosis, including cleavage of Poly (ADP-ribose) polymerase (PARP) as well as caspases-3, -7, -8, and -9 and BH3 interacting-domain agonist (BID) (Figure 2B). This extends a previous report showing that anthrax lethal toxin can induce a NLRP1-dependent form of cell death with features of apoptosis in cells lacking caspase-1 (Van Opdenbosch et al., 2017Van Opdenbosch N. Van Gorp H. Verdonckt M. Saavedra P.H.V. de Vasconcelos N.M. Gonçalves A. Vande Walle L. Demon D. Matusiak M. Van Hauwermeiren F. et al.Caspase-1 Engagement and TLR-Induced c-FLIP Expression Suppress ASC/Caspase-8-Dependent Apoptosis by Inflammasome Sensors NLRP1b and NLRC4.Cell Rep. 2017; 21: 3427-3444Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Lattice light-sheet microscopy revealed nuclear condensation and plasma membrane blebbing, which was consistent with apoptotic death of Salmonella-infected Casp1–/–;11–/–;12–/– BMDMs and contrasted with the pyroptotic death observed in Salmonella-infected WT BMDMs (Figure 2C; Videos S1, S2, and S3). Notably, combined loss of caspase-8 plus RIPK3 did not impair Salmonella-induced cell killing, as in vitro-infected Casp8–/–;Ripk3–/– BMDMs died with kinetics that were indistinguishable from WT cells, with both undergoing pyroptosis (Figure 2A). This was consistent with the observation that the combined loss of caspase-8 and RIPK3 did not impair bacterial control in vivo until at least 3 weeks post-infection (Figure 1B). These findings indicate that although caspase-8 was dispensable for the early pyroptotic cell death upon Salmonella infection, caspase-8-driven apoptosis, rather than caspase-8-mediated pyroptosis or RIPK3- and MLKL-driven necroptosis, was responsible for the delayed type of cell death observed in Casp1–/–;Casp11–/–;Casp12–/– BMDMs. Casp1–/–;Casp11–/–;Casp12–/–;Casp8–/–;Ripk3–/– BMDMs were not only profoundly resistant to Salmonella-induced killing in vitro (Figure 2A), but also contained large numbers of bacteria (Figure 2C). This resistance to Salmonella-induced killing was not due to a general defect in cell death, as Casp1–/–;Casp11–/–;Casp12–/–;Casp8–/–;Ripk3–/– BMDMs could still be killed through the intrinsic pathway of apoptosis by treatment with BH3 mimetic drugs, as shown by lactate dehydrogenase (LDH) release and activation of the apoptosis effector BAX (Figures S2A and S2B). Collectively, these findings uncover a backup system that enables the host to flexibly deploy different types of programmed cell death for the control of the intracellular pathogen Salmonella. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIwNDM5MjllY2UyMGQ3ZTZkZWU5Mzk2MmE4N2MwODBkOCIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQ1NTMzNzc1fQ.Lf67MwgkhJlJ5reC0gHR0eoeJod9QbINocTmCdbl0U7YA7CQsvNEXEHhjTU4rE_v9h0Xr9okEME0WO8-6A2ikhvs0KPtxDKJIx7WUmLYWMzH4Xv1b4AdrCfSfsWOc0Moh-eN624SIm-mAKq1SgswjS0-Stte_3oAU3cln2HmbQ0pLfgnjNSmDivFuAdimCdcVmbmCMpSKsKN1jku7sIDx_HxIWu8DilV67gkRKlIXywnj2MDC82RjvmmXI6_vvHMeJLrKuOEKLC6bTdWHYstqM5X454dBfUz8BJozBvnpb6newwaB5pdh2yODUCx2DaGuBjR4GYk0c2QO5rDk9tSvQ Download .mp4 (12.97 MB) Help with .mp4 files Video S1. WT Primary BMDMs Die by Pyroptosis upon Infection with Salmonella, Related to Figure 2Lattice light-sheet imaging of WT primary BMDMs infected with Salmonella SPI-2 (GFP) (MOI = 50) at 2 h post-infection. Yellow, Membranes; Magenta, Salmonella; Cyan, PI. Scale bars: 10 μm. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI1OTBiMGU4MDY4Mjk3MGQ2NjI3MjkwODkzZWYzMWVlOSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQ1NTMzNzc1fQ.RZK1xWC6NAOX5k5iVnC4UxrZuRxlxQajROEHiSlZMXZkMAWD6pV7Xlhzr_Fzw3APxfcujsnEogJWQjBxviP0sCmsJNTlMnOTT8QO51h_TzBHocgsVJDpOh_zDjQRgFKAcDWClXgmra5fBFwH6fXmC__2xF3iOz5PYKYcJX7xkAqmPZcPYd-Me1UcDxyUU2reT37PllnYUxSFpvRD9Rvo4a8ifcTuVJngEodxcpq1dQ_xKzlJAeuU-9oDxglZ5OyJSkI-oNJN239gvWMcxpun7B8wckaGDMGxGkbt1gd2N5Hgbcdibzc0mtfyaDzeB09TFnmy-rATIKWxy3wDaYoK9A Download .mp4 (14.58 MB) Help with .mp4 files Video S2. Casp1–/–;Casp11–/–;Casp12–/– Primary BMDMs Die by Apoptosis upon Infection with Salmonella, Related to Figure 2Lattice light-sheet imaging of Casp1–/–;11–/–;12–/– primary BMDMs infected with GFP-expressing Salmonella (MOI = 50) at 6 h post-infection. Yellow, Membranes; Magenta, Salmonella; Cyan, PI. Scale bars: 10 μm. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIzY2UyMzc1ZTI1OGYyNzM1ZjYyMGNlMWI5ZDYwNTU5YyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjQ1NTMzNzc1fQ.YBTfKp62bmgZGcwV3DRnTJQdbxlC070MCiQw3qunJDypwtoexeMq3pCTKY_necisyJYrIZgWf4u40N683eS_5NmKXIAuZtawCyVMElzkGkDGzajAg36hiF3QWjzUpjCrKm73PCbJfwp2ZhpswIxyA0FRZ-wIbv-k5H3Z1i81tECBM4XlU9vS2lEGbe6FNvD_Z-FMWYzajqwgupIYs_oziQ50Tv8eRI8yJ8qjIPCjoSPuD-z2o5zngyyqdz7Sv7u5IN_YB-cryKsLSxxUtpi5sFPhymMTl1SbRCLkoLdla1J-30SM7ZS1xRn3v4Vk9dlykhM7tap-Ah6OSk3KngLKJw Download .mp4 (19.01 MB) Help with .mp4 files Video S3. Primary BMDMs with Combined loss of Caspases-1, -11, -12, and -8 (plus RIPK3) Cannot Undergo Cell Death upon Infection with Salmonella, Related to Figure 2Lattice light-sheet imaging of Casp1–/–;Casp11–/–;Casp12–/–;Casp8–/–;Ripk3–/– primary BMDMs infected with GFP-expressing Salmonella (MOI = 50) at 6 h post-infection. Yellow, Membranes; Magenta, Salmonella; Cyan, PI. Scale bars: 10 μm. To gain a deeper understanding of this complex system of functional backup between different cell death processes, we employed our CRISPR-Cas9 gene editing platform (Aubrey et al., 2015Aubrey B.J. Kelly G.L. Kueh A.J. Brennan M.S. O’Connor L. Milla L. Wilcox S. Tai L. Strasser A. Herold M.J. An inducible lentiviral guide RNA platform enables the identification of tumor-essential genes and tumor-promoting mutations in vivo.Cell Rep. 2015; 10: 1422-1432Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar) to identify the initiators and effectors critical for the respective types of cell death upon Salmonella infection. We used immortalized BMDMs (iBMDMs) for these experiments, which exhibited comparable responses to Salmonella infection as primary BMDMs (Figures 3A and 3B ). While the combined loss of caspases-1, -11, and -12 delayed Salmonella-induced killing of iBMDMs, the loss of both caspase-8 and RIPK3 had no impact, and cells died in a manner comparable to WT cells (Figure 3A). Only the combined absence of caspases-1, -11, -12, and -8 and RIPK3 completely blocked Salmonella-infection-induced killing of iBMDMs (Figure 3A). The Casp1–/–;Casp11–/–;Casp12–/–;Casp8–/–;Ripk3–/– iBMDMs still underwent cell death in response to treatment with combinations of BH3 mimetics or etoposide, and this was accompanied by activation of the apoptosis effector BAX (Figures S3A and S3B), as was the case for primary BMDMs (Figures S2A and S2B). These results validate iBMDMs as useful tools to unravel the molecular requirements of the diverse cell death pathways induced upon Salmonella infection. We also noted in these experiments that Casp1–/–;Casp11–/–;Casp12–/– iBMDMs showed more prominent processing of caspase-8 following Salmonella infection compared to WT iBMDMs and that this was accompanied by classical markers of apoptosis, such as cleavage of BID and caspases-3, -7, and -9 (Figures 2B and 3B). Casp1–/–;Casp11–/–;Casp12–/– cells infected with Salmonella in the absence or presence" @default.
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• W3039784919 date "2020-09-01" @default.
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• W3039784919 title "Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection" @default.
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• W3039784919 doi "https://doi.org/10.1016/j.immuni.2020.07.004" @default.
• W3039784919 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7500851" @default.
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• W3039784919 hasPublicationYear "2020" @default.
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