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• W3194589294 abstract "•Carnivorans lack key NLRs and express a unique caspase-1/-4 hybrid protein•This protein is defective in mediating activation of common inflammasome pathways•What little activity occurs is driven by caspase-8, rather than caspase-1/-4 Zoonotic pathogens, such as COVID-19, reside in animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity in host immune genes across species affect pathogen carriage is poorly understood. Here, we describe a progressive evolutionary downregulation of pathogen-sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing rapid lytic cell death, and the formation of a caspase-8 containing inflammasome that inefficiently processes interleukin-1β. Inflammasomes regulate gut immunity, but the carnivorous diet has antimicrobial properties that could compensate for the loss of these immune pathways. We speculate that the consequences of systemic inflammasome downregulation, however, can impair host sensing of specific pathogens such that they can reside undetected in the Carnivora. Zoonotic pathogens, such as COVID-19, reside in animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity in host immune genes across species affect pathogen carriage is poorly understood. Here, we describe a progressive evolutionary downregulation of pathogen-sensing inflammasome pathways in Carnivora. This includes the loss of nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition of a unique caspase-1/-4 effector fusion protein that processes gasdermin D pore formation without inducing rapid lytic cell death, and the formation of a caspase-8 containing inflammasome that inefficiently processes interleukin-1β. Inflammasomes regulate gut immunity, but the carnivorous diet has antimicrobial properties that could compensate for the loss of these immune pathways. We speculate that the consequences of systemic inflammasome downregulation, however, can impair host sensing of specific pathogens such that they can reside undetected in the Carnivora. Viral and bacterial zoonotic pathogens, such as coronavirus disease 2019 (COVID-19) and Salmonella species, can infect animal hosts in an asymptomatic or symptomatic manner, which may facilitate the transmission to humans. Pathogen genomics have yielded important discoveries about the diversity of different microorganisms in the context of disease (Weinert et al., 2015Weinert L.A. Chaudhuri R.R. Wang J. Peters S.E. Corander J. Jombart T. Baig A. Howell K.J. Vehkala M. Välimäki N. et al.BRaDP1T ConsortiumGenomic signatures of human and animal disease in the zoonotic pathogen Streptococcus suis.Nat. Commun. 2015; 6: 6740Crossref PubMed Scopus (84) Google Scholar). Comparative biology of animal immune systems and their links to infection susceptibility are less well understood. This is partly due to a lack of tools, for example, antibodies or other resources that make immune studies tractable, but the use of CRISPR-Cas9 gene editing is a universal technique that can be applied to cells from many animals. Approximately 49% of all carnivore species (e.g., mink, dogs), the highest proportion of any mammal order including bats, carry one or more unique zoonotic pathogens (Han et al., 2016Han B.A. Kramer A.M. Drake J.M. Global Patterns of Zoonotic Disease in Mammals.Trends Parasitol. 2016; 32: 565-577Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). Whether this is because Carnivora are a large group of animals harboring many pathogens, so they carry proportionally more zoonoses (Mollentze and Streicker, 2020Mollentze N. Streicker D.G. Viral zoonotic risk is homogenous among taxonomic orders of mammalian and avian reservoir hosts.Proc. Natl. Acad. Sci. USA. 2020; 117: 9423-9430Crossref PubMed Scopus (129) Google Scholar), or due to other factors such as differences in the immune system remains to be determined. Inflammasomes are of central importance in host protection against viral and bacterial diseases and drive inflammation to control infections in humans and mice (Broz and Dixit, 2016Broz P. Dixit V.M. Inflammasomes: mechanism of assembly, regulation and signalling.Nat. Rev. Immunol. 2016; 16: 407-420Crossref PubMed Scopus (1537) Google Scholar). Canonical inflammasomes are multi-protein complexes composed of a pathogen-recognition receptor, such as a nucleotide-oligomerization domain leucine-rich repeat receptor (NLR; NLRP1, NLRP3 and NLRC4), pyrin or absent-in-melanoma 2 receptor (AIM2), an adaptor (apoptosis-associated speck-like protein [ASC]), and an effector protein (caspase-1; CASP1). The role of this pro-inflammatory protein complex is to process the immature cytokines pro-interleukin-1β (IL-1β) and pro-IL-18 into their mature, more active forms and to cleave the lytic pyroptotic cell death effector gasdermin D to its pore forming N-terminal fragment (Broz and Dixit, 2016Broz P. Dixit V.M. Inflammasomes: mechanism of assembly, regulation and signalling.Nat. Rev. Immunol. 2016; 16: 407-420Crossref PubMed Scopus (1537) Google Scholar). Non-canonical inflammasomes can also be formed by cytosolic delivery of the bacterial toxin lipopolysaccharide (LPS), which activates caspase-11 in mice or caspase-4 and -5 in humans to cleave gasdermin D, which in turn activates NLRP3 (Broz and Dixit, 2016Broz P. Dixit V.M. Inflammasomes: mechanism of assembly, regulation and signalling.Nat. Rev. Immunol. 2016; 16: 407-420Crossref PubMed Scopus (1537) Google Scholar; Broz et al., 2020Broz P. Pelegrín P. Shao F. The gasdermins, a protein family executing cell death and inflammation.Nat. Rev. Immunol. 2020; 20: 143-157Crossref PubMed Scopus (473) Google Scholar; Lieberman et al., 2019Lieberman J. Wu H. Kagan J.C. Gasdermin D activity in inflammation and host defense.Sci. Immunol. 2019; 4: eaav1447Crossref PubMed Scopus (74) Google Scholar). There is wide species diversity in AIM-2 like receptors (ALRs), with AIM2 being non-functional in many species. Evolutionary analysis reveals considerable plasticity in mammalian ALR genes, with no single ALR gene preserved among all mammals. Instead, the ALR genes have undergone extensive, species-specific diversification, suggesting that evolutionary pressures may have shaped ALR sequences and functions throughout the mammals (Brunette et al., 2012Brunette R.L. Young J.M. Whitley D.G. Brodsky I.E. Malik H.S. Stetson D.B. Extensive evolutionary and functional diversity among mammalian AIM2-like receptors.J. Exp. Med. 2012; 209: 1969-1983Crossref PubMed Scopus (153) Google Scholar). Here, by comparing the distribution and evolution of inflammasome and cell death genes across the order Carnivora, we find a profound compromise in inflammasome functionality, caspase-dependent lytic cell death pathways, and a critical loss of NLR genes. A caspase-1/caspase-4 fusion protein found in all Carnivora, despite being functionally capable of processing substrates in vitro, is inactive in cells from a model carnivore (dog). Caspase-8, which is conserved in the Carnivora, processes delayed pro-IL-1β and upregulates the expression of this protein. This compromised inflammasome activity, coupled to the absence of the necroptotic effector mixed-lineage kinase domain-like pseudokinase (MLKL) (Dondelinger et al., 2016Dondelinger Y. Hulpiau P. Saeys Y. Bertrand M.J.M. Vandenabeele P. An evolutionary perspective on the necroptotic pathway.Trends Cell Biol. 2016; 26: 721-732Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), suggests that the order Carnivora are immunologically challenged, particularly in gut mucosal immunity, but ecology studies suggest that a high-protein diet, such as that consumed by carnivores, has antimicrobial properties. This may explain why these innate immune pathways have been lost in the Carnivora, but the consequences for the carriage of zoonotic pathogens, particularly in organs other than the gut, may be detrimental. Carnivora, such as dogs, cats, and mink, through their close proximity with humans, can be susceptible to human pathogens. There are, however, marked differences in Carnivora inflammasome effector caspases compared to humans and mice (Figure S1A). A unique caspase-1/-4 fusion protein is present in all cats and dogs (Figure S1B) (Eckhart et al., 2008Eckhart L. Ballaun C. Hermann M. VandeBerg J.L. Sipos W. Uthman A. Fischer H. Tschachler E. Identification of novel mammalian caspases reveals an important role of gene loss in shaping the human caspase repertoire.Mol. Biol. Evol. 2008; 25: 831-841Crossref PubMed Scopus (79) Google Scholar). This protein has the equivalent of the CARD1 domain of caspase-1, which should render it unable to respond to cytosolic LPS, while its catalytic domain is most closely related to that of mouse caspase-11 (caspase-4 or -5 in humans), being only distantly related to caspase-1 in terms of sequence identity (Figure S1B). This caspase-1/-4 fusion is conserved across all Carnivora (Figure 1A) and constitutively expressed in dog cells as shown by mass spectrometry analysis (Figures S1D and S1E). The absence of individual caspase-1 and caspase-4/-5/-11 genes in Carnivora suggests that there will be differences in how inflammasomes function in species of this order. The structure of the caspase-1/-4 fusion (Figure S1C) suggests it should be able to process gasdermin D in response to canonical inflammasome stimulation, but have a limited/no capacity to process IL-1β and IL-18. Analysis of dog gasdermin D shows that both domains are conserved and, although the linker region is more divergent, the aspartate cleavage site is present (Wang et al., 2020Wang K. Sun Q. Zhong X. Zeng M. Zeng H. Shi X. Li Z. Wang Y. Zhao Q. Shao F. Ding J. Structural Mechanism for GSDMD Targeting by Autoprocessed Caspases in Pyroptosis.Cell. 2020; 180: 941-955.e20Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), consistent with full functionality of this protein in the Carnivora. To characterize inflammasome functionality in Carnivora, we compared the kinetics and magnitude of cell death between immortalized bone marrow-derived macrophages (iBMDM) from wild-type (WT) mice and the DH82 dog macrophage-like cell line (used as a model for the Carnivora). Cells were infected with Salmonella enterica serovar Typhimurium (S. Typhimurium), which activates NLRC4 and NLRP3 canonical inflammasome formation (Man et al., 2014Man S.M. Hopkins L.J. Nugent E. Cox S. Glück I.M. Tourlomousis P. Wright J.A. Cicuta P. Monie T.P. Bryant C.E. Inflammasome activation causes dual recruitment of NLRC4 and NLRP3 to the same macromolecular complex.Proc. Natl. Acad. Sci. USA. 2014; 111: 7403-7408Crossref PubMed Scopus (212) Google Scholar). All of the mouse cells lysed within the first 2 h post-infection, while dog cells were more resistant and survived well beyond 12 h (Figure 1B). Mouse casp1−/−/11−/− cells, as expected, were also resistant to rapid cell death, but, unlike dog cells, started to lyse at 6 h post-infection (Figure 1C). Interestingly, dog cells showed clear processing of pro-IL-1β (Figure 1D) accompanied by the release of large amounts of IL-1β at 24 h post-infection (Figure 1E). Experiments in primary monocyte-derived macrophages (MNCs) isolated from dog peripheral blood mononuclear cells infected with S. Typhimurium for 24 h also induced IL-1β production with delayed lytic cell death (Figures 1F and 1G). This is unexpected because, based on the structure, this hybrid protein was predicted to process gasdermin D to lyse cells, but not cleave IL-1β in response to canonical inflammasome activity (Kayagaki et al., 2011Kayagaki N. Warming S. Lamkanfi M. Vande Walle L. Louie S. Dong J. Newton K. Qu Y. Liu J. Heldens S. et al.Non-canonical inflammasome activation targets caspase-11.Nature. 2011; 479: 117-121Crossref PubMed Scopus (1609) Google Scholar). To test this predicted activity, a novel mouse strain that carries a caspase-1/-11 fusion, equivalent to the one found in the Carnivora, was generated. Caspase-1 and caspase-11 are so close to each other in the mouse genome (Kayagaki et al., 2011Kayagaki N. Warming S. Lamkanfi M. Vande Walle L. Louie S. Dong J. Newton K. Qu Y. Liu J. Heldens S. et al.Non-canonical inflammasome activation targets caspase-11.Nature. 2011; 479: 117-121Crossref PubMed Scopus (1609) Google Scholar) that it is possible to delete the catalytic domain of caspase-1 and the N-terminal caspase recruitment domains (CARDs) of caspase-11 to make a mouse fusion protein equivalent to that found in the Carnivora. We therefore used a novel approach of CRISPR-Cas9 gene deletion to generate a mouse that expresses a fusion protein consisting of the caspase-1 CARD1 and the caspase-11 catalytic domain (DogMo) and confirmed expression via western blot analysis (Figures S2C and S2D). BMDMs from DogMo infected with S. Typhimurium, as expected, showed canonical inflammasome-driven cell lysis and gasdermin D processing, but no IL-1β production (Figures 1H–1K). Cell lysis in DogMo BMDMs was, interestingly, reduced very early during infection when compared to infected WT BMDM (Figure 1H). DogMo cells also showed no cell lysis or IL-1β production in response to non-canonical inflammasome activation induced by cytosolic LPS (Figures 2A and 2B ). Similarly, DH82 dog cells and primary dog mononuclear cells showed no cell lysis (Figures 2C and S3A), and the amount of IL-1β produced was the same whether cells were primed with the Toll-like receptor 2 (TLR2) ligand Pam3CSK4 alone (as a control) or after priming with Pam3CSK4 and then transfected with LPS (Figures 2B, 2D, and S3B). This suggests that this cytokine was induced by priming rather than non-canonical inflammasome activation, which was confirmed by western blot analysis (Figure 2E). To determine whether the enzymatic properties of the caspase-1/-4 hybrid could account for IL-1β processing in the absence of lytic cell death by inflammasomes in dog cells, we expressed the catalytic domain of this protein and tested its ability to process substrates in vitro (Figure 2F). Caspase-1/-4 processed both gasdermin D and IL-1β to their biologically active forms in vitro. Caspase-11, as expected, cleaved gasdermin D but not IL-1β (Figure 2F). We also used synthetic peptidyl substrates optimized, based on specificity screens, to improve selectivity for caspase-1 or caspase-11 (Ramirez et al., 2018Ramirez M.L.G. Poreba M. Snipas S.J. Groborz K. Drag M. Salvesen G.S. Extensive peptide and natural protein substrate screens reveal that mouse caspase-11 has much narrower substrate specificity than caspase-1.J. Biol. Chem. 2018; 293: 7058-7067Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Dog caspase-1/-4 cleaved these synthetic substrates at superior rates compared to caspase-11 and cleaved the caspase-1 optimum substrate at rates comparable to caspase-1 (Figure 2F). These data reveal that the substrate specificity of dog caspase-1/-4 resembles that of caspase-1 more closely than caspase-11 in vitro. This suggests that the defective canonical and non-canonical inflammasome responses in dog cells are not caused by an intrinsic loss of enzymatic activity of the caspase-1/-4 fusion protein, but most likely because of an alternative regulatory mechanism. An alternative explanation for our data is that species-specific differences in NLRs may account for the weak activation of canonical inflammasomes that we see in dog cells. AIM2, for example, is absent in many species, including Carnivora (Brunette et al., 2012Brunette R.L. Young J.M. Whitley D.G. Brodsky I.E. Malik H.S. Stetson D.B. Extensive evolutionary and functional diversity among mammalian AIM2-like receptors.J. Exp. Med. 2012; 209: 1969-1983Crossref PubMed Scopus (153) Google Scholar). Analysis of the repertoire of NLR genes across the Carnivora identified that the bacterial sensors NLR family of apoptosis inhibitory proteins (NAIPs) and NLRC4 are predominantly missing or are pseudogenes in the Canidae, whereas the Felidae lack another bacterial sensor NLRP1 (Figure 3A) (Eckhart et al., 2009Eckhart L. Ballaun C. Uthman A. Gawlas S. Buchberger M. Fischer H. Tschachler E. Duplication of the caspase-12 prodomain and inactivation of NLRC4/IPAF in the dog.Biochem. Biophys. Res. Commun. 2009; 384: 226-230Crossref PubMed Scopus (9) Google Scholar). The loss of these NLRs occurred early in the evolutionary trees of both the Canidae and Felidae (Figure S4A). This diversity in NLRs suggests that the lack of functional NAIP/NLRC4 in dogs would at least partially explain the altered inflammasome responses to S. Typhimurium we saw when comparing mouse and dog macrophages. Comparison of the cell death induced by S. Typhimurium in DH82 cells with mouse Nlrc4−/− BMDM showed that both cell types resist rapid cell death (1 and 2 h), but by 6 h, Nlrc4−/− BMDM are dying, whereas DH82 cells remain resistant (Figure 1C). NLRP3, a non-specific sensor of cellular insults (Swanson et al., 2019Swanson K.V. Deng M. Ting J.P. The NLRP3 inflammasome: molecular activation and regulation to therapeutics.Nat. Rev. Immunol. 2019; 19: 477-489Crossref PubMed Scopus (1499) Google Scholar), in contrast, is conserved across all Carnivora. We next stimulated mouse WT, mouse Casp1/11−/− iBMDM, and dog DH82 macrophages with increasing concentrations of the canonical NLRP3 activator nigericin. We saw that low concentrations of nigericin activated NLRP3-induced cell lysis in WT mouse iBMMs but not in DH82 cells (Figure 3B). This was not due to a slower induction of NLRP3-mediated inflammasome activation in DH82 cells, because when these cells were stimulated with this low concentration of nigericin over a period of 24 h, they remained viable, showing no cell death (Figure 3E). Cell lysis in dog cells could be induced by very high concentrations of nigericin, but this cell death was independent of both caspase-1 and -11 in mouse iBMDMs (Figure 3B). The failure of nigericin to cause inflammasome-induced cell lysis was not due to the inability of the caspase fusion protein to function per se because stimulation of the DogMo macrophages again showed cell lysis and gasdermin D cleavage, with minimal IL-1β production (Figures 3F–3I). Mouse WT BMDM produced IL-1β, as expected, in response to NLRP3-activating concentrations of nigericin, but dog cells produced no IL-1β until very high concentrations of nigericin were used (Figure 3C). Processed IL-1β was detected in the supernatant of DH82 cells stimulated with high concentrations of nigericin by western blot analysis, but only in relatively small amounts (Figure 3D). How is IL-1β processed in response to S. Typhimurium and nigericin in dog cells? Caspase-8 can be recruited to the inflammasome to process IL-1β and gasdermin D, particularly in the absence of caspase-1 (Lee et al., 2018Lee B.L. Mirrashidi K.M. Stowe I.B. Kummerfeld S.K. Watanabe C. Haley B. Cuellar T.L. Reichelt M. Kayagaki N. ASC- and caspase-8-dependent apoptotic pathway diverges from the NLRC4 inflammasome in macrophages.Sci. Rep. 2018; 8: 3788Crossref PubMed Scopus (80) Google Scholar; Man et al., 2013Man S.M. Tourlomousis P. Hopkins L. Monie T.P. Fitzgerald K.A. Bryant C.E. Salmonella infection induces recruitment of Caspase-8 to the inflammasome to modulate IL-1β production.J. Immunol. 2013; 191: 5239-5246Crossref PubMed Scopus (162) Google Scholar; Newton et al., 2019Newton K. Wickliffe K.E. Maltzman A. Dugger D.L. Reja R. Zhang Y. Roose-Girma M. Modrusan Z. Sagolla M.S. Webster J.D. Dixit V.M. Activity of caspase-8 determines plasticity between cell death pathways.Nature. 2019; 575: 679-682Crossref PubMed Scopus (141) Google Scholar). We visualized ASC specks in DH82 cells stimulated with NLRP3-activating concentrations of nigericin and saw recruitment of both caspase-1 and caspase-8 fluorescent substrates, consistent with the presence of both caspase-1/-4 fusion protein and caspase 8 within the inflammasome complex (Figure 4A). We used CRISPR-Cas9 to delete the caspase-1/-4 gene from dog DH82 cells. Clones of DH82 cells lacking the caspase-1/-4 gene showed similar responses (cell death and IL-1β production) to WT DH82 cells after infection with S. Typhimurium (Figure S4B), or stimulation with NLRP3-activating concentrations of nigericin (Figure S4C). When, however, we used CRISPR-Cas9 to delete caspase-8 from dog DH82 macrophages, we saw impaired responses in Caspase-8−/− bulk cells and individual Caspase-8−/− clones infected with S. Typhimurium (Figures 4B and 4C). DogMo BMDM, but not their WT counterparts, also showed substantial resistance to early pyroptotic cell death induced by Salmonella when caspase-8 was pharmacologically inhibited (Figure S4D). These data collectively suggest that inflammasome formation in dog macrophages uses caspase-8, rather than the caspase-1/-4 fusion. Caspase-8 regulates the receptor-interacting serine/threonine-protein kinase 1 (RIPK1)/RIPK3 pathway that triggers necroptosis, but also regulates TLR4-dependent nuclear factor κ-light-chain enhancer of activated B cells (NF-κB)-driven transcription of genes such as pro-IL-1β. Carnivora cells cannot undergo necroptosis as the effector protein MLKL is missing from their genomes (Dondelinger et al., 2016Dondelinger Y. Hulpiau P. Saeys Y. Bertrand M.J.M. Vandenabeele P. An evolutionary perspective on the necroptotic pathway.Trends Cell Biol. 2016; 26: 721-732Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). When we used CRISPR-Cas9 to inactivate RIPK1 from dog cells, IL-1β production was abolished in response to inflammasome stimulation (Figures 4D and 4F) without affecting cell death (Figures 4E and 4G). This RIPK1-dependent effect is most likely due to the loss of pro-IL-1β transcription driven by TLR4 priming from the Salmonella LPS (Figure 4H). This was confirmed by using the selective TLR4 inhibitor TAK242 on WT DH82 cells infected with Salmonella, which reduced IL-1β production without affecting cell death (Figure S4E). Collectively, our data suggest that what little inflammasome activation occurs in the Carnivora is predominantly driven by caspase-8, with any role for caspase-1/-4 being very minor. Our data suggest that in dogs and presumably in other Carnivora, non-canonical inflammasome activation is absent and canonical inflammasome activation is limited. In Carnivora there is a loss, or modification, of genes important in lytic cell death pathways (necroptosis and pyroptosis; Figure 5A). Gasdermin D pores can form, however, without inducing lytic cell death (Evavold et al., 2018Evavold C.L. Ruan J. Tan Y. Xia S. Wu H. Kagan J.C. The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages.Immunity. 2018; 48: 35-44.e6Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar), so as there are no antibodies available that cross-react with the dog gasdermin D protein, we measured propidium iodide (PI) uptake and used live cell imaging of DH82 cells to determine whether pyroptotic cell death pathways are completely missing in dog cells. S. Typhimurium-induced inflammasome activity classically processes gasdermin D to induce rapid lytic cell death in mouse or human cells, yet infected dog cells took up PI (Figure 5B), but appeared to be locked in the swollen phase and only ruptured very late during infection, presumably when the cell membrane could no longer contain the enormous intracellular bacterial load (Figure 5C; Video S1). In response to nigericin stimulation, DH82 cells again took up PI, suggesting that gasdermin D pores are formed (Figure 5B), but cells from these animals have a markedly reduced capacity for pro-inflammatory lytic cell death (Figure 5D; Video S2). The appearance of the caspase-1/-4 fusion and the loss of MLKL occurred early in the Carnivora evolutionary tree (Figure S4A). Gasdermin E, a protein that drives pyroptosis in response to caspase-3 activation (Wang et al., 2017Wang 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: 99-103Crossref PubMed Scopus (1085) Google Scholar), is conserved. The apoptotic caspases -3, -7, -8, and -9 are fully conserved across all Carnivora, suggesting that caspase-dependent cell death may be limited primarily to apoptosis pathways in these animals (Figure 5A). We do see some lytic cell death at very high doses of nigericin in dog cells (Figure 5D; Video S2), which could be driven by, for example, gasdermin E, but this occurs under conditions of limited physiological relevance. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiZTZhYzg4YWFlNGZhNTI2YmRmODE1MjE0ZGNmM2IyNSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjcyNjQ0NjU2fQ.EGFvC0wP0gyHNt4_S7ZiCXqrXGSpxuzdwh0M6amSA9bE0RkJg86GucsmAUzKRdtUzXT5ewZ0vHYxYa-HB6_m-40hK2ZBjA4rdJFs14UKlKfyRM5TE8rsArq8Illabeh5SLxMLoeqRU2oA0818gYjAQkkwJa-mqT9dsIMzz0JMtIMLlRSxZQNdpxgdUBIWDCTgJZljZbGfz2QOdjiIv62UaWwbFpgOHq1HskWNy5-mGhOO4Ml2Ur4-pdfnsYI9mc4Gs7MXhOPl2cpRAjwc5XHSOE3uQHEKOR7UkwEVnkYophOnZr5qc4rI_f1XUkhpoS0E7d3n9kwXPueFuqipv6nxg Download .mp4 (45.52 MB) Help with .mp4 files Video S1. Dog DH82 cells are locked in the swollen phase and only rupture very late in infection with S. Typhimurium, related to Figure 5 eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI2NGE3ZmZhN2NlZmRmNzM3N2RiMTYyMzA5Njk4ODA0YSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjcyNjQ0NjU2fQ.BC-q9MJqwsRGsjwdu7jGOYTGNwezd6EFfe5-du8P4B4_Es_bYwuqnc62kuKTd8V2qQ7Zc9Xy-8s4T3x4EOI5INIyJvpW5mgpuSV9LtkO52rSqe86llm4I4wQkBNgWRKYBXDxUZsRe8KtU2JSeU9re7Pd5sOBAocfnWe8-jgvRsvi1zrXzbR2WmPUTG4PYe3pP6FGLMOh4o-5__vSecdxoYPKxPFKYgG-iFQ_IuVcMHQ90egudnnAgp7oBin41AKpPkiuh9jrjh7syXw6s8ovgEpEy_0t7M8jDwTAthuONTAqp4hwxK3UmSfYIjeLrBYQA7168ScZE82acZ7RbitjxA Download .mp4 (21.42 MB) Help with .mp4 files Video S2. Dog DH82 cells have a markedly reduced capability for pro-inflammatory lytic cell death in response to nigericin stimulation, related to Figure 5 Here, we show that key inflammasome lytic cell death pathways thought to be critical for gut health in mammals are either genetically and/or functionally missing from the Carnivora. PI uptake without cell lysis occurs in dog cells in response to inflammasome activation, suggesting a dissociation of gasdermin D pore formation from lytic cell death. This is similar to the phenotype seen when the NINJ1 protein is deleted from human or mouse cells (Kayagaki et al., 2021Kayagaki N. Kornfeld O.S. Lee B.L. Stowe I.B. O’Rourke K. Li Q. Sandoval W. Yan D. Kang J. Xu M. et al.NINJ1 mediates plasma membrane rupture during lytic cell death.Nature. 2021; 591: 131-136Crossref PubMed Scopus (145) Google Scholar), yet all Carnivora have NINJ1. Our data suggest that inefficient inflammasome activation occurs in dog cells such that insufficient gasdermin D pores form in the cell membrane to drive cell lysis, although the pores that are formed, as indicated by the PI uptake, should facilitate cytokine release (Evavold et al., 2018Evavold C.L. Ruan J. Tan Y. Xia S. Wu H. Kagan J.C. The Pore-Forming Protein Gasdermin D Regulates Interleukin-1 Secretion from Living Macrophages.Immunity. 2018; 48: 35-44.e6Abstract Full Text Full Text PDF PubMed Scopus (532) Google Scholar). Inflammasome-driven lytic cell death is, therefore, lost in Canidae, and this is particularly interesting because the Carnivora also lack the necroptotic effector MLKL, such that two of the critical inflammatory cell death pathways that are thought to be essential for host protection against infection are absent (Table 1). One of the key functions for inflammatory cell death is to protect the gut against infection (Crowley et al., 2020Crowley S.M. Han X. Allaire J.M. Stahl M. Rauch I. Knodler L.A. Vallance B.A. Intestinal restriction of Salmonella Typhimurium requires caspase-1 and caspase-11 epithelial intrinsic inflammasomes.PLoS Pathog. 2020; 16: e1008498Crossref PubMed Scopus (41) 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. 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Guy C.S. Heckmann B.L. Rodriguez D.A. Ruhl S. Moretti J. Crawford J.C. Fitzgerald P. Kanneganti T.D. et al.Caspase-8-Dependent Inflammatory Responses Are Controlled by Its Adaptor, FADD, and Necroptosis.Immunity. 2020; 52: 994-1006.e8Abstract Full Text" @default.
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