Fig 1: Pregnanolone and etiocholanolone specifically trigger pyrin inflammasome step 2(A) Pregnanolone (blue) and etiocholanolone (red) were added at different concentrations on cells expressing (in the presence of doxycycline, +Dox) or not (No Dox) p.S242R MEFV. Cell death was determined at 3 h post-addition. The concentration triggering 50% cell death (horizontal dotted line) determined the EC50 (half-maximal effective concentration, which is inversely correlated to the activity of the tested molecule).(B–D) (B) Structure activity of various steroid hormones, their catabolites, (C) of etiocholanolone (also known as 3a-hydroxy 5ß-androstan-17-one (3a,5ß)) and its two stereoisomers 3ß-hydroxy-5ß-androstan-17-one (3ß,5ß) and androsterone (3a,5a), (D) of pregnanolone and molecules closely related. EC50 values were calculated as in (A).(E) Structures of selected compounds. All compounds can be found in Figure S1.(F) Primary monocytes from healthy donors (HDs, n = 3) were pre-treated with pregnanolone (6 µM) or etiocholanolone (12 µM) for 1 h followed by addition of the PKC superfamily inhibitor, UCN-01. Cell death was monitored every 5 min for 2 h.(G) The area under the curve (AUC) was computed for each HD.(H) Primary monocytes from HDs (n = 3–4) were treated with LPS for 2 h, then pre-treated with pregnanolone (6 µM) or etiocholanolone (12 µM) for 1 h followed by UCN-01 addition. IL-1ß concentration in the supernatant was quantified at 3 h post-UCN-01 addition.One experiment representative of three (A) or two (B–D) independent experiment is shown. Mean and SEM of triplicates are shown. Sulfate, 5ß-pregnan-3a-ol-20-one sulfate; 11aOH, 5ß-pregnan-3a,11a-diol-20-one; 11-one, 5ß-pregnan-3a-ol-11,20-dione; 11ßOH, 5-ß-pregnan-3-a,11ß-diol-20-one; 17OH, 5-ß-pregnan-3-a,17-diol-20-one; 20H, 5-ß-pregnan-3-a-ol; 21OH, 5-ß-pregnan-3-a,21-diol-20-one; hemisuccinate, 5-ß-pregnan-3-a,21-diol-20-one 21 hemisuccinate. (B–D) One-way ANOVA with Dunn’s correction was applied. ***p < 0.001, **p = 0.007. (F) Each point corresponds to the mean ± SEM of three HD values, each one being the mean of a triplicate. (F) Each point corresponds to the mean ± SEM of three HD values, each one being the mean of a triplicate. (G) Each point corresponds to the mean AUC of kinetics of one HD performed in triplicate, the bar represents the mean ± SEM of three HD values. AUC are expressed as arbitrary units (a.u.). Friedman test with Dunn’s correction for multiple analysis was performed in comparison with untreated cells. *p = 0.023 (Etio + UCN); p = 0.011 (Pregna + UCN). (H) Each point corresponds to the mean IL-1ß concentration of one HD calculated from a triplicate, the bar represents the mean ± SEM of three to four HDs. Kruskal-Wallis test with Dunn’s correction for multiple analysis was performed in comparison with LPS-treated cells. *p = 0.011, **p = 0.002.
Fig 2: Nocodazole and colchicine, the latter in a dose-dependent manner, inhibit UCN-01-mediated responses APrimary human monocytes from FMF patient were primed with LPS and stimulated as indicated with UCN-01 in the presence of paclitaxel (Taxol, 5 µM), nocodazole (5 µM), or colchicine (1 µM).BPropidium iodide incorporation was monitored every 5 min post-UCN-01 addition in the presence of Taxol (5 µM), nocodazole (5 µM), or colchicine (1 µM). PI incorporation was normalized using TX-100 cells (total PI incorporation). (A, C, D) IL-1ß concentration in the supernatant was quantified by ELISA.C, DPrimary human monocytes from the indicated healthy donor (HD) or FMF patient were primed with LPS and stimulated as indicated with (C) UCN-01 or (D) TcdA (1 µg/ml) in the presence of the indicated concentration of colchicine.Data information: (A) Each symbol corresponds to the mean of a biological triplicate for one FMF patient (square, triangle, and round, patients #35, 36, 37 (all M694V/M694V), respectively), and the bar shows the median ± interquartile range. (B) Each symbol represents the mean (± SD) of a biological triplicate for one FMF patient. (C, D) Each dot represents one biological replicate, and the bar shows the mean of a biological triplicate from one individual.
Fig 3: Increased Pyrin expression is required for Pyrin activation in human macrophages.(A) IL-1ß release from LPS-primed (10 ng/ml, 3 or 18 h) hMDM were preincubated with compounds as noted previously, then stimulated with either TcdA (200 ng/ml), BAA-473 (10 µM), or (B) nigericin (8 µM) for 2.5 h. (C) IL-1ß release from hMDM primed with either LPS (10 ng/ml) or Pam3Cys4K (20 ng/ml) for 3 h or 18 h and stimulated with TcdB (20 ng/ml). (D) hMDM were transfected with siRNA targeting Pyrin mRNA (MEFV#1 and MEFV#2) or the scrambled control and assessed for Pyrin expression by immunoblot or stimulated a previously following primed with LPS (10 ng/ml) for 18 h. (E) Expression of Pyrin in hMDM incubated with pepinhTRIF or the scrambled control peptide (25 µg/ml, 5 h), then treated with LPS (10 ng/ml, 18 h). Representative of 3 donors. IL-1ß release from cell pretreated with pepinhTRIF or the scrambled control peptide (25 µg/ml) in response to LPS priming (10 ng/ml, 18 h) followed by stimulation with either TcdA (200 ng/ml) or nigericin (8 µM) for 2.5 h. LPS-primed (100 ng/ml, 3 h) differentiated caspase-4, NLRP3 double-deficient BLaER1 cells reconstituted with either Pyrin or the vector control were (F) lysed and assessed for Pyrin expression by immunoblot or (G) incubated with nigericin (8 µM) TcdB (20 ng/ml) or needletox (25 ng/ml) for 2.5 h +/- CP-456,773 (2.5 µM, 15-min incubation), and the supernatants were assessed for IL-1ß. (H) LDH release from Pyrin or vector alone reconstituted BLaER1 cells primed with LPS (3 h, 100 ng/ml) or left unprimed and stimulated with TcdB (20 ng/ml) or TcdA (1 µg/ml) for 2 h. For (A-E) and (H) mean and SEM of 3–4 experimental replicates shown. For (G) mean and SD of 3 technical replicates shown, representative of 3 independent experiments. * p < 0.05, n.s. not significant. The underlying data can be found in the summary data file in the tab Fig 6A–6E, 6G and 6H. LDH, lactate dehydrogenase; LPS, lipopolysaccharide; siRNA, small interfering RNA.
Fig 4: 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 5: Pyrin inflammasome activation proceeds differently following TcdA/B and steroid catabolite addition(A) Monocytes from HDs (n = 2–12) were treated with colchicine or nocodazole and 30 min later with the indicated stimuli. Propidium iodide incorporation was monitored every 5 min for 3 h.(B) The area under the cure (AUC) is shown. For each HD, the AUC values in the presence of inhibitors were normalized to the AUC value obtained in the absence of inhibitor.(C) Monocytes from HDs (n = 8–14) were treated with colchicine and 30 min later with the indicated stimuli. IL-1ß concentration in the supernatant was quantified at 3 h post-addition.(D) RhoA activity was determined by G-LISA at different time post-treatment in the lysate of U937 cells. The activity of the different treatments at the indicated time is presented in Figure S4B.(E) Doxycycline-induced, PMA-differentiated U937 macrophages expressing MEFV Full-length (FL), deleted of the pyrin (?PYD), of the phosphorylated linker (?PLD), of the BBox (?Bbox), of the coiled-coil (?CC), or the B30.2 (?B30.2) domains were treated with LPS for 3 h and then with the indicated stimuli. IL-1ß concentration in the supernatant was quantified at 3 h post-addition.(F) Doxycycline-induced, U937 monocytes expressing WT or ?B30.2 MEFV were treated with the indicated stimuli for 90 min. Pyrin S242 phosphorylation was assessed by western blot analysis following immunoprecipitation. “*” indicates a cleaved form of pyrin.(A–E) Mean and SEM are shown. (B and C) Each dot represents the value for one HD. (D and E) One experiment with technical triplicates representative of two (D) to three (E) independent experiments is shown. (B–C) Wilcoxon matched-pairs signed rank tests were performed to compare values with or without colchicine/nocodazole. Two-tailed p values: (B) **p = 0.0078, ***p < 0.001; (C) ***p < 0.001, **p = 0.002; (D) ordinary one-way ANOVA with Holm-Sidak’s correction for multiple tests was performed. *p = 0.015, ***p < 0.001. (E) Ordinary one way ANOVA with Sidak’s correction for multiple tests was performed. *p = 0.011, ***p < 0.001.
Supplier Page from Abcam for Anti-Pyrin (phospho S241) antibody [EPR19570]