Irgm2 and Gate‐16 cooperatively dampen Gram‐negative bacteria‐induced caspase‐11 response
2020; Springer Nature; Volume: 21; Issue: 11 Linguagem: Inglês
10.15252/embr.202050829
ISSN1469-3178
AutoresElif Eren, Rémi Planès, Salimata Bagayoko, Pierre‐Jean Bordignon, Karima Chaoui, Audrey Hessel, Karin Santoni, Miriam Pinilla, Brice Lagrange, Odile Burlet‐Schiltz, Jonathan C. Howard, Thomas Henry, Masahiro Yamamoto, Étienne Meunier,
Tópico(s)Antimicrobial Resistance in Staphylococcus
ResumoArticle30 October 2020free access Source DataTransparent process Irgm2 and Gate-16 cooperatively dampen Gram-negative bacteria-induced caspase-11 response Elif Eren orcid.org/0000-0002-0328-5609 Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Rémi Planès Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Salimata Bagayoko orcid.org/0000-0002-0956-4641 Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Pierre-Jean Bordignon Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Karima Chaoui Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Mass Spectrometry Core Facility, Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Audrey Hessel Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Karin Santoni Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Miriam Pinilla Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Brice Lagrange CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, CNRS, UMR5308, École Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Univ Lyon, Lyon, France Search for more papers by this author Odile Burlet-Schiltz Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Mass Spectrometry Core Facility, Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Jonathan C Howard Fundação Calouste Gulbenkian, Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Thomas Henry orcid.org/0000-0002-0687-8565 CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, CNRS, UMR5308, École Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Univ Lyon, Lyon, France Search for more papers by this author Masahiro Yamamoto Department of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Laboratory of Immunoparasitology, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan Search for more papers by this author Etienne Meunier Corresponding Author [email protected] orcid.org/0000-0002-3651-4877 Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Elif Eren orcid.org/0000-0002-0328-5609 Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Rémi Planès Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Salimata Bagayoko orcid.org/0000-0002-0956-4641 Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Pierre-Jean Bordignon Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Karima Chaoui Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Mass Spectrometry Core Facility, Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Audrey Hessel Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Karin Santoni Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Miriam Pinilla Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Brice Lagrange CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, CNRS, UMR5308, École Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Univ Lyon, Lyon, France Search for more papers by this author Odile Burlet-Schiltz Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Mass Spectrometry Core Facility, Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Jonathan C Howard Fundação Calouste Gulbenkian, Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Thomas Henry orcid.org/0000-0002-0687-8565 CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, CNRS, UMR5308, École Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Univ Lyon, Lyon, France Search for more papers by this author Masahiro Yamamoto Department of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan Laboratory of Immunoparasitology, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan Search for more papers by this author Etienne Meunier Corresponding Author [email protected] orcid.org/0000-0002-3651-4877 Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France Search for more papers by this author Author Information Elif Eren1,‡, Rémi Planès1,‡, Salimata Bagayoko1, Pierre-Jean Bordignon1, Karima Chaoui1,2, Audrey Hessel1, Karin Santoni1, Miriam Pinilla1, Brice Lagrange3, Odile Burlet-Schiltz1,2, Jonathan C Howard4, Thomas Henry3, Masahiro Yamamoto5,6 and Etienne Meunier *,1,† 1Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France 2Mass Spectrometry Core Facility, Institute of Pharmacology and Structural Biology (IPBS), CNRS, UMR5089, University of Toulouse, Toulouse, France 3CIRI, Centre International de Recherche en Infectiologie, Inserm, U1111, CNRS, UMR5308, École Normale Supérieure de Lyon, Université Claude Bernard Lyon 1, Univ Lyon, Lyon, France 4Fundação Calouste Gulbenkian, Instituto Gulbenkian de Ciência, Oeiras, Portugal 5Department of Immunoparasitology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan 6Laboratory of Immunoparasitology, WPI Immunology Frontier Research Center, Osaka University, Osaka, Japan †Present address: Institute of Pharmacology and Structural Biology (IPBS), CNRS, Toulouse, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 0 561175832; E-mail: [email protected] EMBO Rep (2020)21:e50829https://doi.org/10.15252/embr.202050829 See also: R Finethy et al and A Linder & V Hornung (November 2020) PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Inflammatory caspase-11 (rodent) and caspases-4/5 (humans) detect the Gram-negative bacterial component LPS within the host cell cytosol, promoting activation of the non-canonical inflammasome. Although non-canonical inflammasome-induced pyroptosis and IL-1-related cytokine release are crucial to mount an efficient immune response against various bacteria, their unrestrained activation drives sepsis. This suggests that cellular components tightly control the threshold level of the non-canonical inflammasome in order to ensure efficient but non-deleterious inflammatory responses. Here, we show that the IFN-inducible protein Irgm2 and the ATG8 family member Gate-16 cooperatively counteract Gram-negative bacteria-induced non-canonical inflammasome activation, both in cultured macrophages and in vivo. Specifically, the Irgm2/Gate-16 axis dampens caspase-11 targeting to intracellular bacteria, which lowers caspase-11-mediated pyroptosis and cytokine release. Deficiency in Irgm2 or Gate16 induces both guanylate binding protein (GBP)-dependent and GBP-independent routes for caspase-11 targeting to intracellular bacteria. Our findings identify molecular effectors that fine-tune bacteria-activated non-canonical inflammasome responses and shed light on the understanding of the immune pathways they control. Synopsis Caspase-11 targets cytosolic Gram-negative bacteria, inducing pyroptosis and IL-1 maturation. IFN-inducible GTPases promote caspase-11 targeting to bacterial membranes, whereas Irgm2 and the non-canonical autophagy protein Gate-16 restrain unnecessary caspase-11 targeting. Irgm2 and Gate16 cooperatively inhibit Gram-negative bacteria-induced non canonical inflammasome activation. Irgm2/Gate16 deficiency drives exaggerated caspase-11 response in a GBP-dependent and -independent manner. Irgm2 deficiency enhances endotoxemia susceptibility of mice. Introduction Inflammasomes are cytosolic innate immune complexes that initiate inflammatory responses upon sensing of microbe- and damage-associated molecular patterns (MAMPs and DAMPs, respectively) (Hayward et al, 25). Specifically, the rodent caspase-11 (and its human orthologs caspase-4 and caspase-5) detects the presence of the Gram-negative bacterial cell wall component lipopolysaccharide (LPS) within the host cell cytosol (Kayagaki et al, 26, 27; Broz et al, 6; Aachoui et al, 1; Hagar et al, 22; Yang et al, 75). LPS interaction with the caspase activation and recruitment domain (CARD) of caspase-11 promotes its oligomerization and activation, which triggers the activation of the non-canonical inflammasome (Yang et al, 75). Upon activation (Lee et al, 36), caspase-11 cleaves and activates the pyroptosis executioner gasdermin-D (gsdmD) into the p30 active fragment (Kayagaki et al, 28; Shi et al, 65). Cleaved gsdmD then forms a pore into phosphatidylinositol-4,5-bisphosphate (PIP2)-enriched domains at the plasma membrane, which triggers pyroptosis, a pro-inflammatory form of cell death (Shi et al, 65; Aglietti et al, 2; Liu et al, 39; Sborgi et al, 63). In parallel, gsdmD pore-induced ionic perturbations also trigger activation of the canonical NLRP3 inflammasome, which results in the caspase-1-dependent maturation of the pro-inflammatory cytokines interleukins (IL)-1β and IL-18 (Kayagaki et al, 26; Rühl & Broz, 57; Schmid-Burgk et al, 64). Although caspase-11 confers host protection against intracellular Gram-negative bacteria (Aachoui et al, 1; Cerqueira et al, 7; Chen et al, 8), its unrestrained activation provokes irreversible organ failure, blood clothing and sepsis (Kayagaki et al, 26, 27, 28; Napier et al, 50; Cheng et al, 9; Deng et al, 13; Rathinam et al, 56; Yang et al, 76). This suggests that host regulators might fine-tune the non-canonical inflammasome in order to optimize caspase-11-dependent response. To date, only few of them were described including SERPINB1-inhibited caspase-11/-4/-1 activation in resting cells or ESCRT-mediated plasma membrane repair (Rühl et al, 58; Choi et al, 10). Crucial at regulating the activation the non-canonical inflammasome pathway are the IFN-inducible GTPases, the so-called guanylate binding proteins (GBPs) and the immunity-related GTPase (Irg) Irgb10 (Meunier et al, 48, 49; Pilla et al, 54; Finethy et al, 15; Man et al, 41, 42; Wallet et al, 70; Zwack et al, 78; Cerqueira et al, 7; Costa Franco et al, 12; Lagrange et al, 33; Liu et al, 40). Specifically, GBPs (1, 2, 3, 4 and 5) are recruited on LPS-enriched structures such as cytosolic Gram-negative bacteria and their derived products outer membrane vesicles (OMVs) (Meunier et al, 48; Man et al, 41; Finethy et al, 16; Lagrange et al, 33; Santos et al, 60; Fisch et al, 19). As such, these GBPs then engage caspase-11 that will bind the LPS moiety lipid A, hence promoting the non-canonical inflammasome pathway (Fisch et al, 19). Beyond their role at triggering GBP expression, IFNs induce more than 2,000 antimicrobial genes (Green et al, 20). Among them, many IFN-inducible regulatory genes also counter-balance overactivation of the cells (Green et al, 20). For instance, SOCS1 and USP18 are ISGs that balance the level of the host cell response (Basters et al, 4; Liau et al, 38). In this context, we hypothesized that IFNs, in addition to their ability to promote GBP expression, might also induce negative regulators of the non-canonical inflammasome. In this regard, Irgm proteins belong to the IFN-inducible immunity-related GTPase (Irg) family proteins (Kim et al, 30, 31; Pilla-Moffett et al, 55). Human being possess one IRGM protein, with various spliced variants, that is not IFN-inducible but that requires IFN signalling to be functional (Kim et al, 31). By contrast, mice display three different Irgms, namely Irgm1, Irgm2 and Irgm3 (Kim et al, 30). All Irgms lack the ability to hydrolyse the GTP due to a mutation in their catalytic domain (GMS), whereas other Irgs are GTPase active (GKS) (Coers, 11). Previous studies underscored an inhibitory role of Irgm1 and Irgm3 on the recruitment and/or activation of the GBPs and Irg-GKS on microbial membranes although independent processes can also occur (Haldar et al, 23, 24; Feeley et al, 14). In addition, recent studies identified Irgm1 and its human homologous IRGM, as being critical for the NLRP3 canonical inflammasome regulation by modulating the autophagy pathway, suggesting a close link between Irgm proteins and inflammasomes (Pei et al, 53; Mehto et al, 46,b). In this context, we hypothesized that Irgm proteins might be IFN-inducible regulators of the non-canonical inflammasome activation threshold. Here, we report that IFN-inducible Irgm2 and the non-canonical autophagy effector Gate-16 indirectly fine-tune non-canonical inflammasome activation by intracellular bacteria, which protects against endotoxemia. Results and Discussion IFN-inducible protein Irgm2 restrains Caspase-11-dependent responses to Gram-negative bacteria IFN-inducible Irgms control Irg and GBP microbicidal activity against intracellular pathogens (Pilla-Moffett et al, 55). In this context, we sought to determine whether Irgms might also modulate the non-canonical inflammasome response. Using an RNA interference approach (siRNA), we silenced the three murine Irgms in primary murine bone marrow-derived macrophages (BMDMs) and measured their ability to undergo caspase-11-dependent cell death and IL-1β maturation upon Salmonella Typhimurium challenge. To ensure that the inflammasome response in macrophages is caspase-11-dependent, we used an isogenic mutant of Salmonella (orgA−) lacking expression of SP1-encoded T3SS secretion system (Broz et al, 6). As previously published, Casp11 and Gbp2 silencing reduced macrophage death (LDH release) and IL-1β release after 16 h of infection (Figs 1A and EV1A; Meunier et al, 48). Importantly, Irgm2-silenced BMDMs had higher levels of cell death and IL-1β release than the wild-type (WT) macrophages (Figs 1A and EV1A). Such process was specific to Irgm2 given that Irgm1- and Irgm3-targeted siRNAs did not induce significant variation in macrophage death and IL-1β release upon Salmonella (orgA−) infection, despite the fact that their mRNA levels were efficiently reduced (Figs 1A and EV1A). To further validate that Irgm2 is a regulator of the non-canonical inflammasome response, we challenged WT, Irgm2−/−, Casp11−/− and GBPChr3−/− BMDMs with a panel of Gram-negative bacteria all known to activate the non-canonical inflammasome. Immunoblotting experiments in WT and Irgm2−/− BMDMs showed that Irgm2 is IFN-inducible and that Irgm2 deficiency does not lead to a defect in caspase-1, caspase-11, GBP2 or GBP5 expression, all involved in the non-canonical inflammasome pathway (Fig EV1B and C). Yet, when challenged with various Gram-negative bacteria, Irgm2−/− macrophages showed an exacerbated cell death, IL-1β release and gasdermin-D p30 (active) and processed caspase-1 p20 (inactive) fragments compared with their WT counterparts (Fig 1B and C). In addition, Irgm2-regulated cell pyroptosis upon Gram-negative bacterial challenge was independent of NLRP3 as the use of the NLRP3 inhibitor MCC950 or Nlrp3−/− BMDMs did not drive any defect in cell death but significantly reduced NLRP3-dependent IL-1β release (Fig EV1D). As expected, both Casp11−/− and GBPChr3−/− BMDMs were protected against Gram-negative bacteria-induced non-canonical inflammasome response (Fig 1B). Importantly, CRISPR-deleted Irgm2 gene expression in immortalized (i) Casp11−/− BMDMs (referred as Casp11−/−sgIrgm2) did not reinduce pyroptosis and IL-1β release upon Gram-negative bacterial infections (S. Typhimurium orgA− and Escherichia coli) or E. coli-derived OMVs exposure, thus confirming that Irgm2 negatively regulated caspase-11-dependent response (Figs 1D and EV1E). Next, we assessed whether Irgm2 directly or indirectly regulates the non-canonical inflammasome response. To this end, we electroporated LPS into the host cell cytosol of IFNγ-primed WT, Irgm2−/− and Casp11−/− BMDMs and evaluated their ability to undergo pyroptosis. Surprisingly, we observed that WT and Irgm2−/− macrophages engaged cell death to the same extent 4 h after LPS electroporation whereas Casp11−/− BMDMs were protected against LPS-induced cell death (Fig 1E). This suggests that Irgm2-inhibited caspase-11 response occurs upstream from LPS sensing by caspase-11. Figure 1. IFN-inducible protein Irgm2 restrains caspase-11-dependent response to Gram-negative bacteriaUnless otherwise specified, BMDMs were either infected with various Gram-negative bacterial strains (MOI25) or stimulated with outer membrane vesicles (OMVs) for 16 h. A. siRNA-treated BMDMs were infected for 16 h with S. Typhimurium (orgA−), and LDH and IL-1β release were measured. B. Cell death (LDH) and IL-1β release evaluation in WT, Irgm2−/−, GBPChr3−/− and Casp11−/− BMDMs infected for 16 h with different Gram-negative bacteria (MOI 25). C. Western blot examination of processed caspase-1 (p20) and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55) and GAPDH in cell lysates of WT and Irgm2−/− BMDMs infected for 16 h with different Gram-negative bacterial strains. D. IL-1β and cell death (% LDH) evaluation in immortalized WT, Irgm2−/−, Casp11−/− and Casp11−/−Irgm2−/− (referred as sgIrgm2) BMDMs after 16 h of Escherichia coli, S. Typhimurium orgA− and OMV treatment. E. Cell death (% LDH) evaluation in IFNγ-primed WT, Irgm2−/− and Casp11−/− BMDMs 4 h after electroporation or not with 1 μg of E. coli LPS. F–H. Survival of WT, Casp11−/− and Irgm2−/− mice primed with 100 μg poly(I:C) for 6 h and injected (i.p.) with 5 mg/kg LPS or 5 and 25 μg of OMVs (n = 6 animals per condition). Data information: Data shown as means ± SEM (graphs A, B, D and E) from n = 4 independent pooled experiments; **P ≤ 0.01, ***P ≤ 0.001 for the indicated comparisons using t-test with Bonferroni correction. Image (C) is representative of one experiment performed three times. (F–H) are representative of three independent experiments; *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001, log-rank Cox–Mantel test for survival comparisons (F–H). Source data are available online for this figure. Source Data for Figure 1 [embr202050829-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Irgm2 specifically controls the non-canonical inflammasome response qRT–PCR measurement of silencing efficacy on the mRNA levels of Irgm1-3, Gbp2 and Caspase-11 in BMDMs, prestimulated with 100 UI/ml of IFNγ for 16 h. N = 3 independent experiments normalized to β-actin mRNA levels. Data are expressed as mean ± SEM. ***P ≤ 0.001 for the indicated comparisons using t-test with Bonferroni correction. Immunoblotting of Irgm2, caspase-1, caspase-11 and GAPDH expression in IFNγ-primed WT or Irgm2−/− BMDMs. Image represents one experiment performed two times. Immunoblotting of GBP2, GBP5 and GAPDH expression in Salmonella (S. Tm)- or IFNγ-treated WT or Irgm2−/− BMDMs. Image represents one experiment performed two times. LDH and IL-1β release from WT, Irgm2−/−, Casp11−/− and Nlrp3−/− BMDMs treated for 16 h with 2.5 μg/2.105 cells of OMVs in the presence or not of 10 μM of MCC950 (NLRP3 inhibitor). Data are expressed as mean ± SEM from n = 4 independent pooled experiments. ***P ≤ 0.001 for the indicated comparisons using t-test with Bonferroni correction. Immunoblots of Casp11 or Irgm2 deletion efficacy in immortalized BMDMs. Image represents one experiment performed two times. Release of LDH and IL-1β from IFNγ- and PAM3CSK4-primed WT, Irgm2−/−, Casp11−/− or Casp1−/−Casp11−/− BMDMs transfected (using FuGeneHD) with flagellin or poly(dA:dT) or stimulated with either Nigericin or TcdB toxin for 6 h. Data are expressed as mean ± SEM from n = 4 independent pooled experiments. Western blot examination of processed caspase-1 (p20) and gasdermin-D (p30) in supernatants and pro-caspase-1 (p45), pro-gasdermin-D (p55) and GAPDH in cell lysates of WT and Irgm2−/− BMDMs infected for 4 h with S. Typhimurium and Pseudomonas aeruginosa (NLRC4 inflammasome). Image represents one experiment performed two times. Cytokine levels in plasma from WT, Casp11−/− and Irgm2−/− (n = 6 mice per condition) primed with 100 μg poly(I:C) for 6 h and injected i.p with 25 μg of OMVs for 5 h. Graphs represent one experiment out of three independent experiments; *P ≤ 0.05; **P ≤ 0.01, ***P ≤ 0.001, Mann–Whitney analysis test. Source data are available online for this figure. Download figure Download PowerPoint Based on these results, we next determined whether Irgm2 also inhibited canonical inflammasomes. We treated WT, Irgm2−/−, Casp11−/− and Casp1−/−Casp11−/− BMDMs with various inflammasome activators, including flagellin (NLRC4), poly-dAdT (AIM2), Nigericin (NLRP3) and TcdB (PYRIN), and measured their ability to commit pyroptosis and to release IL-1β. Although all canonical inflammasome activators induced significant caspase-1-dependent response, cell death and IL-1β release levels remained similar in both WT and Irgm2−/− BMDMs (Fig EV1F). In addition, activation of the NLRC4 inflammasome by T3SS-expressing Pseudomonas aeruginosa and S. Typhimurium remained similar between WT and Irgm2−/− BMDMs (Fig EV1G), suggesting that Irgm2 specifically regulates the non-canonical inflammasome response to Gram-negative bacteria. As caspase-11 also drives mouse susceptibility to LPS-induced inflammatory-related damages, we also evaluated whether Irgm2 deficiency might sensitize mice to sepsis. We used two LPS-dependent sepsis models, where WT, Irgm2−/− and Casp11−/− mice were intraperitoneally injected with poly(IC) to induce ISG expression (Kayagaki et al, 27; Santos et al, 60). Then, mice were injected either with pure LPS (5 mg/kg) or with OMVs (25 μg/ml; Vanaja et al, 69; Santos et al, 60). Mouse survival showed that while Casp11−/− mice had resistance to LPS- and OMV-induced sepsis, WT mice succumbed faster, hence validating our sepsis model (Fig 1F and G). Noticeably, Irgm2−/− mice were even more susceptible than WT mice to both LPS- and OMV-induced sepsis (Fig 1F and G). Therefore, we used a sub-lethal model of OMV-induced sepsis by injecting 5 μg of OMVs in mice. In such model, both WT and Casp11−/− mice recovered from OMV injection whereas all Irgm2−/− mice did succumb (Fig 1H). Moreover, cytokine assays showed that Irgm2−/− mice had an exacerbated release of all pro-inflammatory and inflammasome-related cytokines tested upon OMV challenge, a phenotype that was reduced in Casp11−/− mice, hence confirming that Irgm2 expression is crucial to temperate the activation level of the non-canonical inflammasome (Fig EV1H). Altogether, our data suggest that Irgm2 indirectly inhibits caspase-11-dependent endotoxemia, which protects against sepsis. Irgm2 regulates GBP-independent caspase-11 targeting to Gram-negative bacteria IFN-inducible GBPs are important regulators of the non-canonical inflammasome response. Specifically, GBP-1 and GBP-2 regulate human caspase-4/-5 activation while GBP-2 and GBP-5 control mouse caspase 11. Therefore, we hypothesized that Irgm2 might control caspase-11 response through the modulation of the GBPs. To this end, we silenced Irgm2 in WT and GBPChr3−/− BMDMs (lacking 5 GBPs, 1-3, 5 and 7) and evaluated the caspase-11 response upon OMV stimulation (Fig EV2A). While OMV-induced both cell death and IL-1β release was strongly reduced in GBPChr3−/−, Irgm2-silenced GBPChr3−/− BMDMs partially recovered a caspase-11-dependent response, suggesting that Irgm2-inhibited caspase-11 response could occur independently of GBPs (Fig 2A). Other and we previously showed that GBPs also controlled canonical AIM2 inflammasome activation upon Francisella tularensis spp novicida infection. In this context, we evaluated the importance of Irgm2 at controlling AIM2 inflammasome response upon F. novicida infection. Surprisingly, IL-1β and cell death levels were not different between WT and Irgm2−/−, although they were strongly reduced in Casp1−/−Casp11−/− and GBPChr3−/− BMDMs (Figs 2B and EV2B). In addition, we observed that Irgm2-silenced GBPChr3−/− BMDMs did not recover an inflammasome response upon F. novicida infection. Then, we generated iGBPChr3−/−Irgm2−/− (referred hereafter as iGBPChr3−/−sgIrgm2) by crispr Cas9 and evaluated their response upon S. Tm (orgA−) challenge. iIrgm2−/− BMDMs showed time-dependent increased cell death compared with iWT cells (Fig EV2C and D). While cell death in iGBPChr3−/− BMDMs was strongly impaired, it was partially reversed in iGBPChr3−/−sgIrgm2, alluding that Irgm2 deficiency was sufficient to specifically promote caspase-11-dependent response in the absence of GBPs (Fig EV2C and D). GBP enrichment on microbial ligand is of importance for efficient caspase-11 and human caspase-4 recruitment (Thurston et al, 68; Fisch et al, 19). However, monitoring for GBP loading on mCherry-expressing S. Typhimurim did not show a significant change in the percentage of bacteria targeted by GBP2 (10–15%) in WT and Irgm2−/− BMDMs (Fig 2C), which suggests that Irgm2-inhibited non-canonical inflammasome response does not involve GBP2 recruitment modulation. Click here to expand this figure. Figure EV2. Irgm2 deficiency drives GBP-independent caspase-11 targeting to Gram-negative bacteria Immunoblots of Irgm2 silencing efficacy in primary BMDMs. Image represents one experiment performed two times. *Non-specific. Cell death (LDH) and IL-1β release evaluation in WT, Irgm2−/−, GBPChr3−/−, Casp11−/− and Casp1−/−Casp11−/− BMDMs infected for 16 h with either S. Tm orgA− or F. novicida (MOI 25). Data are expressed as mean ± SEM from n = 3 independent pooled experiments. Immunoblots of Irgm2 deletion efficacy in immortalized GBPChr3−/− BMDMs. Image represents one experiment performed two times. Kinetic of S. Tm (orgA−)-induced cell death (% LDH release) in IFNγ-primed iWT, iIrgm2−/−, iGBPChr3−/− and iGBPChr3−/−sgIrgm2 BMDMs. Data are expressed as mean ± SEM from n = 4 independent pooled experiments. Confocal fluorescence microscopy images and associated quantifications of caspase-11-C254G-GFP (green) recruitment to S. Tm-mCherry (orgA−, red) in IFNγ-primed iWT, iIrgm2−/−, iGBPChr3−/− and iGBPChr3−/−sgIrgm2 BMDMs after 4 h of infection. Nucleus (blue) was stained with Hoescht, scale bar 5 μm. For quantifications, the percentage of bacteria positive for caspase-11-C254G-GFP was determined by combining the bacterial counts from n = 3 independent experiments and expressed as mean ± SEM. “n” refers to the number of bacteria counted. Data information: ***P ≤ 0.001 for the indicated comparisons using t-test with Bonferroni correction. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. Irgm2 regulates GBP-independent caspase-11 targeting to Gram-negative bacteriaUnless otherwise specified, BMDMs were treated with 2.5 μg/2 × 105 cells of OMVs or infected with either S. Typhimurium orgA− (S. Tm orgA−) or F. tularensis spp novicida (F. novicida) with an MOI of 25 for various times. Measure of LDH and IL-1β release in WT, GBPChr3−/− and Casp11−/− BMDMs were Irgm2 was knocked down 16 h after exposure to 2.5 μg/2 × 105 cells of OMVs. Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences. Cell death (LDH) and IL-1β release evaluation in Irgm2-silenced WT and GBPChr3−/− BMDMs infected for 16 h with either S. Tm orgA− or F. novicida (MOI 25). Si Scramble (siScr.) refers to RNAi pools with non-targeting sequences. Florescence microscopy and associated quantifications of GBP-2 (green) recruitments to intracellular S. Tm orgA−-mCherry (MOI 10, red) in IFNγ-primed WT and Irgm2−/− BMDMs. Nucleus was stained with Hoechst (blue). Confocal images shown are from one experiment and are representative of n = 3 independent experiments; scale ba
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