Evolutionary loss of inflammasomes in the Carnivora and implications for the carriage of zoonotic infections

•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 driven by caspase-8, rather than Zoonotic pathogens, such as COVID-19, reside animal hosts before jumping species to infect humans. The Carnivora, like mink, carry many zoonoses, yet how diversity host immune genes across affect pathogen carriage poorly understood. Here, we describe progressive evolutionary downregulation pathogen-sensing pathways Carnivora. This includes the loss nucleotide-oligomerization domain leucine-rich repeat receptors (NLRs), acquisition effector fusion that processes gasdermin D pore formation without inducing rapid lytic cell death, caspase-8 containing inefficiently interleukin-1β. Inflammasomes regulate gut immunity, but carnivorous diet has antimicrobial properties could compensate for these pathways. We speculate consequences systemic downregulation, however, can impair sensing specific pathogens they undetected Viral bacterial zoonotic coronavirus disease 2019 (COVID-19) Salmonella species, an asymptomatic or symptomatic manner, which may facilitate transmission Pathogen genomics have yielded important discoveries about different microorganisms context (Weinert et al., 2015Weinert L.A. Chaudhuri R.R. Wang J. Peters S.E. Corander Jombart T. Baig A. Howell K.J. Vehkala M. Välimäki N. al.BRaDP1T ConsortiumGenomic signatures human Streptococcus suis.Nat. Commun. 2015; 6: 6740Crossref PubMed Scopus (84) Google Scholar). Comparative biology systems their links infection susceptibility are less well partly due tools, example, antibodies other resources make studies tractable, use CRISPR-Cas9 gene editing universal technique be applied cells from animals. Approximately 49% all carnivore (e.g., dogs), highest proportion any mammal order including bats, one more (Han 2016Han B.A. Kramer A.M. Drake J.M. Global Patterns Disease Mammals.Trends Parasitol. 2016; 32: 565-577Abstract Full Text PDF (210) Whether this because Carnivora large group animals harboring so proportionally zoonoses (Mollentze Streicker, 2020Mollentze Streicker D.G. risk homogenous among taxonomic orders mammalian avian reservoir hosts.Proc. Natl. Acad. Sci. USA. 2020; 117: 9423-9430Crossref (129) Scholar), factors differences system remains determined. central importance protection against viral diseases drive inflammation control infections humans mice (Broz Dixit, 2016Broz P. Dixit V.M. Inflammasomes: mechanism assembly, regulation signalling.Nat. Rev. Immunol. 16: 407-420Crossref (1537) Canonical inflammasomes multi-protein complexes composed pathogen-recognition receptor, receptor (NLR; NLRP1, NLRP3 NLRC4), pyrin absent-in-melanoma 2 (AIM2), adaptor (apoptosis-associated speck-like [ASC]), (caspase-1; CASP1). role pro-inflammatory complex process immature cytokines pro-interleukin-1β (IL-1β) pro-IL-18 into mature, active forms cleave pyroptotic death its forming N-terminal fragment Non-canonical also formed cytosolic delivery toxin lipopolysaccharide (LPS), activates caspase-11 caspase-4 -5 D, turn Scholar; Broz 2020Broz Pelegrín Shao F. gasdermins, family executing inflammation.Nat. 20: 143-157Crossref (473) Lieberman 2019Lieberman Wu H. Kagan J.C. Gasdermin defense.Sci. 2019; 4: eaav1447Crossref (74) There wide AIM-2 (ALRs), with AIM2 being non-functional species. Evolutionary analysis reveals considerable plasticity ALR genes, no single preserved mammals. Instead, undergone extensive, species-specific diversification, suggesting pressures shaped sequences functions throughout mammals (Brunette 2012Brunette R.L. Young Whitley Brodsky I.E. Malik H.S. Stetson D.B. Extensive functional AIM2-like receptors.J. Exp. Med. 2012; 209: 1969-1983Crossref (153) comparing distribution evolution find profound compromise functionality, caspase-dependent pathways, critical NLR genes. A caspase-1/caspase-4 found despite functionally capable processing substrates vitro, inactive model (dog). Caspase-8, conserved delayed pro-IL-1β upregulates expression protein. compromised activity, coupled absence necroptotic mixed-lineage kinase domain-like pseudokinase (MLKL) (Dondelinger 2016Dondelinger Y. Hulpiau Saeys Bertrand M.J.M. Vandenabeele An perspective on pathway.Trends Cell Biol. 26: 721-732Abstract (85) suggests immunologically challenged, particularly mucosal ecology suggest high-protein diet, consumed carnivores, properties. explain why innate been lost organs gut, detrimental. dogs, cats, through close proximity humans, susceptible pathogens. are, marked caspases compared (Figure S1A). present cats dogs S1B) (Eckhart 2008Eckhart L. Ballaun C. Hermann VandeBerg J.L. Sipos W. Uthman Fischer Tschachler E. Identification novel shaping caspase repertoire.Mol. Evol. 2008; 25: 831-841Crossref (79) equivalent CARD1 caspase-1, should render it unable respond LPS, while catalytic most closely related mouse (caspase-4 humans), only distantly caspase-1 terms sequence identity S1B). 1A) constitutively expressed dog shown mass spectrometry (Figures S1D S1E). individual caspase-4/-5/-11 there will function order. structure S1C) able response canonical stimulation, limited/no capacity IL-1β IL-18. Analysis shows both domains and, although linker region divergent, aspartate cleavage site (Wang 2020Wang K. Sun Q. Zhong X. Zeng Shi Li Z. Zhao Ding Structural Mechanism GSDMD Targeting Autoprocessed Caspases Pyroptosis.Cell. 180: 941-955.e20Abstract (202) consistent full functionality To characterize kinetics magnitude between immortalized bone marrow-derived macrophages (iBMDM) wild-type (WT) DH82 macrophage-like line (used Carnivora). Cells were infected enterica serovar Typhimurium (S. Typhimurium), NLRC4 (Man 2014Man S.M. Hopkins L.J. Nugent Cox S. Glück I.M. Tourlomousis Wright J.A. Cicuta Monie T.P. Bryant C.E. Inflammasome causes dual recruitment same macromolecular complex.Proc. 2014; 111: 7403-7408Crossref (212) All lysed within first h post-infection, resistant survived beyond 12 1B). Mouse casp1−/−/11−/− cells, expected, but, unlike started lyse at 6 post-infection 1C). Interestingly, showed clear 1D) accompanied release amounts 24 1E). Experiments primary monocyte-derived (MNCs) isolated peripheral blood mononuclear induced production 1F 1G). unexpected because, based structure, was predicted not (Kayagaki 2011Kayagaki Warming Lamkanfi Vande Walle Louie Dong Newton Qu Liu Heldens al.Non-canonical targets caspase-11.Nature. 2011; 479: 117-121Crossref (1609) test strain carries caspase-1/-11 fusion, generated. Caspase-1 each genome Scholar) possible delete (CARDs) therefore used approach deletion generate expresses consisting (DogMo) confirmed via western blot S2C S2D). BMDMs DogMo Typhimurium, inflammasome-driven lysis processing, 1H–1K). was, interestingly, reduced very early during when WT BMDM 1H). non-canonical LPS 2A 2B ). Similarly, 2C S3A), amount produced whether primed Toll-like (TLR2) ligand Pam3CSK4 alone (as control) after priming then transfected 2B, 2D, S3B). cytokine activation, 2E). determine enzymatic account tested ability vitro 2F). Caspase-1/-4 processed biologically vitro. Caspase-11, cleaved synthetic peptidyl optimized, specificity screens, improve selectivity (Ramirez 2018Ramirez M.L.G. Poreba Snipas S.J. Groborz Drag Salvesen G.S. peptide natural substrate screens reveal much narrower caspase-1.J. Chem. 2018; 293: 7058-7067Abstract (52) Dog superior rates optimum comparable These data resembles responses caused intrinsic protein, likely alternative regulatory mechanism. explanation our weak see cells. AIM2, absent repertoire identified sensors apoptosis inhibitory proteins (NAIPs) predominantly missing pseudogenes Canidae, whereas Felidae another sensor NLRP1 3A) 2009Eckhart Gawlas Buchberger Duplication caspase-12 prodomain inactivation NLRC4/IPAF dog.Biochem. Biophys. Res. 2009; 384: 226-230Crossref (9) occurred trees Canidae S4A). NAIP/NLRC4 would least partially altered saw macrophages. Comparison Nlrc4−/− types resist (1 h), h, dying, remain NLRP3, non-specific cellular insults (Swanson 2019Swanson K.V. Deng Ting J.P. inflammasome: molecular therapeutics.Nat. 19: 477-489Crossref (1499) contrast, next stimulated WT, Casp1/11−/− iBMDM, increasing concentrations activator nigericin. low nigericin activated NLRP3-induced iBMMs 3B). slower induction NLRP3-mediated concentration over period remained viable, showing 3E). high nigericin, independent -11 iBMDMs failure cause inflammasome-induced inability per se stimulation again cleavage, minimal 3F–3I). IL-1β, NLRP3-activating until 3C). Processed detected supernatant analysis, relatively small 3D). How cells? Caspase-8 recruited (Lee 2018Lee B.L. Mirrashidi K.M. Stowe I.B. Kummerfeld S.K. Watanabe Haley B. Cuellar T.L. Reichelt Kayagaki ASC- caspase-8-dependent apoptotic pathway diverges macrophages.Sci. Rep. 8: 3788Crossref (80) Man 2013Man Fitzgerald K.A. induces modulate production.J. 2013; 191: 5239-5246Crossref (162) 2019Newton Wickliffe K.E. Maltzman Dugger D.L. Reja R. Zhang Roose-Girma Modrusan Sagolla M.S. Webster J.D. Activity determines pathways.Nature. 575: 679-682Crossref (141) visualized ASC specks fluorescent substrates, presence 8 4A). Clones lacking similar (cell production) S4B), S4C). When, macrophages, impaired Caspase-8−/− bulk clones 4B 4C). BMDM, counterparts, substantial resistance pharmacologically inhibited S4D). collectively uses fusion. regulates receptor-interacting serine/threonine-protein 1 (RIPK1)/RIPK3 triggers necroptosis, TLR4-dependent nuclear factor κ-light-chain enhancer B (NF-κB)-driven transcription pro-IL-1β. cannot undergo necroptosis MLKL genomes When inactivate RIPK1 abolished 4D 4F) affecting 4E 4G). RIPK1-dependent effect TLR4 4H). using selective inhibitor TAK242 Salmonella, S4E). Collectively, what minor. Our presumably limited. In loss, modification, (necroptosis pyroptosis; Figure 5A). pores form, (Evavold 2018Evavold C.L. Ruan Tan Xia Pore-Forming Protein Regulates Interleukin-1 Secretion Living Macrophages.Immunity. 48: 35-44.e6Abstract (532) available cross-react measured propidium iodide (PI) uptake live imaging completely Typhimurium-induced classically induce took up PI 5B), appeared locked swollen phase ruptured late infection, membrane longer contain enormous intracellular load 5C; Video S1). PI, markedly 5D; S2). appearance tree E, drives pyroptosis caspase-3 2017Wang Gao He Chemotherapy drugs gasdermin.Nature. 2017; 547: 99-103Crossref (1085) conserved. -3, -7, -8, -9 fully limited primarily do some doses S2), by, under conditions physiological relevance. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiZTZhYzg4YWFlNGZhNTI2YmRmODE1MjE0ZGNmM2IyNSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjcyNjQ0NjU2fQ.EGFvC0wP0gyHNt4_S7ZiCXqrXGSpxuzdwh0M6amSA9bE0RkJg86GucsmAUzKRdtUzXT5ewZ0vHYxYa-HB6_m-40hK2ZBjA4rdJFs14UKlKfyRM5TE8rsArq8Illabeh5SLxMLoeqRU2oA0818gYjAQkkwJa-mqT9dsIMzz0JMtIMLlRSxZQNdpxgdUBIWDCTgJZljZbGfz2QOdjiIv62UaWwbFpgOHq1HskWNy5-mGhOO4Ml2Ur4-pdfnsYI9mc4Gs7MXhOPl2cpRAjwc5XHSOE3uQHEKOR7UkwEVnkYophOnZr5qc4rI_f1XUkhpoS0E7d3n9kwXPueFuqipv6nxg Download .mp4 (45.52 MB) Help files S1. rupture 5 eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI2NGE3ZmZhN2NlZmRmNzM3N2RiMTYyMzA5Njk4ODA0YSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjcyNjQ0NjU2fQ.BC-q9MJqwsRGsjwdu7jGOYTGNwezd6EFfe5-du8P4B4_Es_bYwuqnc62kuKTd8V2qQ7Zc9Xy-8s4T3x4EOI5INIyJvpW5mgpuSV9LtkO52rSqe86llm4I4wQkBNgWRKYBXDxUZsRe8KtU2JSeU9re7Pd5sOBAocfnWe8-jgvRsvi1zrXzbR2WmPUTG4PYe3pP6FGLMOh4o-5__vSecdxoYPKxPFKYgG-iFQ_IuVcMHQ90egudnnAgp7oBin41AKpPkiuh9jrjh7syXw6s8ovgEpEy_0t7M8jDwTAthuONTAqp4hwxK3UmSfYIjeLrBYQA7168ScZE82acZ7RbitjxA (21.42 S2. capability show thought health either genetically and/or dissociation death. phenotype seen NINJ1 deleted 2021Kayagaki Kornfeld O.S. 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