Fig 2: High concentrations of etiocholanolone and pregnanolone trigger full activation of pyrin inflammasome(A and B) U937 cells expressing Dox (plain lines) or not (No Dox, dotted lines) p.S242R (red) or WT (black) MEFV were treated with various concentration of etiocholanolone (A) or pregnanolone (B). Cell death was determined at 3 h post-addition.(C) 3xFlag-WT pyrin from U937 cells treated with the indicated stimuli at the indicated concentrations was immunoprecipitated. Ser242 phosphorylation (P-pyr) and total pyrin levels were monitored by western blot. “*” indicates a cleaved form of pyrin.(D) ASC immunoblot from U937 cells expressing WT MEFV. Cells were treated with the indicated molecules. ASC oligomers in the insoluble fraction were treated with DSS (2 mM) cross linker after lysis. ASC monomers in the soluble fraction is shown. “*” and “**” correspond to the sizes of ASC dimer and trimer, respectively.(E) Monocytes from HDs (n = 3–12) were treated with low (black) or high concentrations of etiocholanolone (red) and pregnanolone (blue) or with LPS + nigericin (magenta). Propidium iodide (PI) incorporation was monitored every 5 min for 3 h.(F) The corresponding area under the curve (AUC) for each donor are shown.(G) Monocytes were primed with LPS for 3 h and exposed to the indicated stimuli at the indicated concentrations. IL-1ß concentration in the supernatant was quantified at 3 h post-addition.(H) Monocytes from one HD were primed or not with LPS for 3 h before addition of the indicated stimuli. Caspase-1, IL-1ß, and GSDMD processing were analyzed by western blot in the cell lysate and supernatant at 3 h post-stimuli addition.(I and J) (I) MEFV-expressing U937 monocytes or (J) PMA-differentiated U937 macrophages WT or knockout for CASP1 or GSDMD as indicated were treated with doxycycline during 16 h. (I) PI incorporation was monitored every 5 min for 3 h post stimuli addition. (J) Cells were primed with LPS for 3 h before addition of the indicated stimuli. IL-1ß concentration in the supernatant was quantified at 3 h post-addition.One experiment representative of three (A–C) to two (H and I) independent experiments is shown. Mean and SEM of triplicates are shown. (A and B) Non-linear regression curve computed using least squares fit method is shown. (G) Kruskal-Wallis test with Dunn’s multiple comparisons tests was performed to compare the different treatments with the LPS treatment. *p = 0.026, **p = 0.0011, ***p < 0.001. (J) One-way ANOVA analysis with Sidak’s multiple comparisons test was performed to compare WT U937 to CASP1KO or GSDMDKO cells. ***p < 0.001.

Fig 3: GSDMD is associated with the mitochondria and Mtb ESX-1 influences IL-1ß secretion in infected macrophages.a, b Immunofluorescence staining of GSDMD in uninfected (UI) and Mtb-infected primary human macrophages. a Cells were stained with DAPI (blue), GSDMD (green), and MitoTracker (red) as indicated. b Merge pictures of a. Images are representative of three individual experiments (scale bar: 50 µM). c Quantification of the Pearson correlation coefficient to determine overlapping signals from the GSDMD and MitoTracker staining from a and b. Standard deviation of the mean is indicated and T test with Welsh’s correction was used to determine statistical significance. **=0.01. d Detection of GSDMD in mitochondrial lysates of uninfected and Mtb-infected primary human macrophages by western blot analysis. The blot is representative of four individual experiments. e IL-1ß concentration (pg/ml) from THP-1 GSDMD knock-out cells infected with Mtb (MOI 1) and treated 5 h post infection with BTP-15 (10 µM), as indicated. IL-1ß was measured from total cell lysates and the supernatant at 5 h post infection and 24 h post BTP15 treatment. Results are expressed as mean ± SEM. Analysis was done using one-way ANOVA with Bonferroni post-test (ns, not significant; *=0.05, **=0.01, ****p = 0.0001). f Infection of macrophages by Mtb leads to an NLPR3 inflammasome formation and activation with consequent secretion of cleaved IL-1ß. Further, Mtb infection induces an accumulation of GSDMD-N at the mitochondria, which is associated with mitochondrial damage MPTP opening. BCL-2, as a mitochondrial protein, interferes with mitochondrial damage. Consequently, Mtb induces necrotic cell death in macrophages.

Fig 4: Deletion of the B30.2 domain suppresses pyrin regulation step 2.A, B U937 monocytes (A) or macrophages (B) incubated with doxycycline to induce pyrin expression were treated with UCN-01 for 3 h. IL-18 (A) and IL-1ß (B) concentration were determined in the supernatant by ELISA. C Cell death kinetics upon doxycycline addition followed by UCN-01 treatment were determined in U937 monocytes expressing the indicated pyrin variants. PI incorporation/fluorescence was monitored every 15 min for the indicated time. D U937 monocytes were incubated with doxycycline for 9 h, GSDMD cleavage was analyzed in the cell lysate (top panels) and the cell supernatant (bottom panels) by capillary-based Western blot analysis. * and ** indicate the full length and the cleaved GSDMD bands, respectively. E, F Areas Under the Curve (AUC) corresponding to the above kinetics are shown. G U937 monocytes were treated with doxycycline for 16 h followed by UCN-01 addition for 2 h, IL-18 concentration in the supernatant was measured by ELISA. Data information: Data from one experiment representative of one (D) to three (A–C; E–G) independent experiments. Mean, SD of triplicates and individual data points are shown. E a.u.: arbitrary units. A, B Kruskal–Wallis and Dunn’s multiple comparisons tests and (F, G) ordinary one-way ANOVA and Dunnett’s multiple comparisons tests were performed to compare U937 cells expressing WT pyrin to the other cell lines. E *: p = 0.0232; **: p = 0.0038; (F) ** (left to right): p = 0.0076, 0.0014; ***: p < 0.001; (G) ***: p < 0.001.

Fig 5: Specific mutations in the CHS domain render pyrin highly sensitive to steroid catabolites.U937 monocytes incubated with doxycycline (unless otherwise indicated (C)) to induce expression of the indicated pyrin variants, were treated (A, B) with decreasing doses of pregnanolone or (C–E) the indicated stimuli. A Cell death levels were measured at 3 h post-treatment in response to varying concentrations of pregnanolone. B EC50 was determined at 3 h post-treatment. C Cell death levels were measured at 3 h post-treatment in the absence of doxycycline or in the presence of VX-765. Results were normalized to the cell death value of cells treated with doxycycline and pregnanolone at 50 µM. D Caspase-1 (top panels) and GSDMD (bottom panels) cleavage were monitored in the cell lysate or the cell supernatant at 90 min post-UCN-01 addition or at 3 h post pregnanolone addition. E Cells were treated with UCN-01 for 15 min or with VX765 for 30 min followed by pregnanolone at the indicated dose for 30 min. Pyrin phosphorylation at the S242 site was assessed by Western blot following immunoprecipitation and quantified by densitometry. Data information: (A) Data from one experiment representative of three independent experiments. The curve was obtained by an ordinary (Least squares) fit using the log (agonist) vs. normalized response-variable slope model. B EC values were obtained from the above analysis using best-fit values. Each point represents the mean EC50 calculated from one biological triplicate. The bars represent the mean and SD of 3–7 independent experiments. Kruskal–Wallis and Dunn’s multiple comparisons tests for the panel on the left were performed. C Data from one experiment. Each point represents the value of one well, mean and SEM are shown. D Data from one experiment. (E-Top panel) Images from one experiment representative of three independent experiments are shown. (Bottom panel): Each point represents the densitometry quantification (normalized to the untreated sample) from one experiment. The bars represent the mean and SD of 3 independent experiments. B Ordinary one-way ANOVA and Dunnett’s multiple comparisons tests were performed. **: p = 0.0078; *: p = 0.0168. C One way Anova with Sidak’s correction for multiple test was applied. Two-tailed p values are shown. ***: p < 0.001. E * (left to right): p = 0.0198, 0.0104; ** (left to right): p = 0.0017, 0.0088; ***: p < 0.001.