Fig 1: Pyrin drives neutrophil caspase-1-dependent pyroptosis in response to Y. pseudotuberculosis ?yopM infectionIndicated BMMs (A–C) and BMNs (D–I) were primed for 16 h, then left non-infected (NI) or infected with either Yptb-WT or ?yopM at MOI 30 for 3 h. Cytotoxicity was determined by LDH release assay (A, D, and G), IL-1ß release was determined by ELISA (B, E, and H), and immunoblots were probed with the indicated antibodies (C, F, and I). Actin was used as a loading control. Data are mean ± SEM of three independent experiments. Cytotoxicity and IL-1ß levels were analyzed by two-way ANOVA followed by Bonferroni’s multiple comparison test (A, B, D, E, G, and H). **p = 0.01, ***p = 0.001, ****p = 0.0001, ns, not significant. Asterisk indicates a non-specific band (I).
Fig 2: The Inflammasome Is Degraded by TRIM20-Mediated Precision Autophagy in 7-Ketocholesterol–Treated RAW264.7 Macrophages(A) Lysates from RAW264.7 macrophages treated with or without 7-ketocholesterol (7KC) were subjected to immunoprecipitation with the anti-TRIM20 antibody. The immunoprecipitants and 5% inputs were immunoblotted with the anti-ULK1, anti-Beclin1, and anti-ATG16L1 antibodies. (B) Lysates from RAW264.7 macrophages treated with or without 7KC were subjected to immunoprecipitation with the anti–NLR family pyrin domain containing 3 (NLRP3) antibody. The immunoprecipitants and 5% inputs were immunoblotted with the anti-TRIM20 antibody. (C) Representative immunoblot images of p-ULK1(Ser317), p-ULK1(Ser757), and total ULK1. (D) The representative images of in situ proximity ligation assays. Green dots indicate the NLRP3-TRIM20 interaction in RAW264.7 macrophages. (E) A schematic representation of precision autophagy. NLRP3 inflammasome is degraded by precision autophagy through the TRIM20-mediated mechanism. IP = immunoprecipitation.
Fig 3: Inflammasome activation and pyroptosis are dispensable for C. difficile TcdA-induced IEC cytotoxicity. a–d Primary intestinal organoids from wild-type (WT) and a Mefv-/-, b Asc-/-, c Casp1/11-/- or d Gsdmd-/- mice were stimulated with TcdA and PI incorporation analyzed by live-imaging for 16 h. Graphs correspond to PI quantification plotted by organoid area. Scale bars: 30 µm. The data are representative of at least 3 independent experiments
Fig 4: IEC apoptosis protects mice during in vivo C. difficile infection. a Scheme of C. difficile infection protocol. b–d Mefv+/+ (n = 9) and Mefv-/- (n = 11) littermates mice were infected with C. difficile and monitored for body weight (b), stool score (c) and bacterial burden in the stool (d). e–g Casp3/7F/F (n = 7) and Casp3/7IEC-KO (n = 7) littermates mice were infected with C. difficile and monitored for body weight (e), stool score (f) and bacterial burden in the stool (g). Nonparametric Mann–Whitney U test was used to analyze the data. Error bars represent SD. *P < 0.05, **P < 0.01, and ***P < 0.001
Fig 5: Pyrin is not expressed in IECs. a Public microarray dataset of Mefv expression profile across different cell types and tissues. Expression values relate to fluorescence intensity from Affymetrix chips. b Real time qPCR of Mefv expression in different tissues and cell types. c Cell lysates from primary intestinal organoids of wild-type and Mefv--/- mice were prepared and immunoblotted for total Pyrin and ß-actin. d Primary intestinal organoids wild-type mice were stimulated with TcdA, TcdB or FlaTox and PI incorporation analyzed by live-imaging for 16 h. e Primary intestinal organoids wild-type mice were stimulated with TcdA, TcdB or FlaTox for 16 h and culture supernatant was analyzed for IL-18 secretion. Scale bars: 30 µm. Data are representative of 3 independent experiments
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