Fig 2: NLRP3, not Pyrin, is the responding inflammasome sensor to TcdB in BLaER1 cells and THP1 macrophages.(A) Differentiated WT BLaER1 cells were primed with LPS (100 ng/ml, 3 h), preincubated with either CP-456,773 (2.5 μM, 15 min), then activated with nigericin (8 μM), TcdB (20 ng/ml), or needletox (25 ng/ml each) for 2 h. IL-1β and caspase-1 activity were assessed from the harvested supernatants. (B) ASC-mCherry transduced WT BLaER1 cells treated as in (A). ASC is in blue; nuclei are red. Cells were then fixed and the number of ASC specks quantified by microscopy. (C) NLRP3 expression in differentiated BLaER1 cells (+/− 100 ng/ml LPS, 3 h) was assessed by immunoblot. (D) Differentiated caspase-4, NLRP3 double deficient BLaER1 cells reconstituted with either NLRP3-Flag, the NLRP3 walker A/B mutant (NLRP3 WAB-Flag), or the vector control treated as in (A) and the supernatants assessed for IL-1β or TNFα. Mean and SD of 3 technical replicates shown, representative of 3 independent experiments. (E) Immunoblot of Pyrin expression in differentiated BLaER1 cells (+/− 100 ng/ml LPS, 3 h). The Pyrin-deficient cells were reconstituted with Pyrin-Flag. Representative of 3 independent experiments. (F) Differentiated Pyrin-deficient BLaER1 cells reconstituted with either Pyrin-Flag or the vector control treated as in (A) and the supernatants assessed for IL-1β or TNFα. Mean and SD of 3 technical replicates shown, representative of 3 independent experiments. (G) LPS-primed WT THP-1s or the listed KOs were activated with inflammasome activators for either 1.5 h (nigericin, needletox) or for 8 h (TcdB). Supernatants were assessed for IL-1β. Where used, CP-456,773 (2.5 μM) and VX-765 (40 μM) were preincubated with the cells for 15 min prior to addition of the inflammasome activators. Mean and SEM of 3 independent experiments shown, * p < 0.05, n.s. not significant. The underlying data can be found in the summary data file in the tab Fig 3A, 3B, 3D, 3F and 3G.

Fig 3: The B30.2 domain regulates Pyrin activation in human macrophages and is disrupted by FMF mutations.(A) LPS-primed (10 ng/ml, 3 h or 18 h) hMDM were treated with TcdB (20 ng/ml, 1 h), then lysed and assessed for phosphorylation of Pyrin (S242), Pyrin, or actin by immunoblot. Representative of 3 independent experiments. (B) FACS profile of mCherry induction following doxycycline incubation, percentage positive cells shown. Representative of 2 independent experiments. (C) LDH release from Pyrin-deficient THP1 cells reconstituted with doxycycline-inducible hPyrin, mPyrin, or the vector control and treated with TcdA (1 µg/ml), BAA-473, (10 µM), or nigericin (8 µM) for 2 h. Mean and SEM from 3 independent experiments shown. (D) Table of the genotypes of the different FMF donors. (E) IL-1ß release from monocytes from HDs or FMF donors (FMF, donors 2–6 used for the experiment) pretreated with Pam3CSK4 (25 ng/ml, 3 h), incubated with colchicine (2.5 µM) for 20 min, and stimulated with TcdA (1 µg/ml) for 3 h. IL-1ß release from hMDM from HDs or FMF donors (FMF) pretreated with Pam3CSK4 (25 ng/ml, 3 h); incubated with CP-456,773 (2.5 µM), VX765 (40 µM), colchicine (2.5 µM), or CP-456,773 and colchicine together (TcdB only) for 20 min; and stimulated with (F) TcdA (1 µg/ml, FMF donors 1–6), (G) TcdB (20 ng/ml, FMF donors 3–6), or nigericin (8 µM) for 2 h. (H) Immunoblot for Pyrin expression in Pam3CSK4 (25 ng/ml, 3 h) primed hMDM from HD or FMF (donors 2 and 3). For experiment in (C) mean and SEM from 3 independent experiments. For (E-G), mean and SEM shown for 3–6 independent donors. * p < 0.05, n.s. not significant. The underlying data can be found in the summary data file in the tab Fig 7C and 7E–7G. FMF, familial Mediterranean fever; HD, healthy donor; hPyrin, human Pyrin-flag; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; mPyrin, mouse Pyrin-flag.

Fig 4: TcdB, but not TcdA, triggers CP-456,773 sensitive IL-1ß release in hMDM.(A) hMDM incubated with conditioned supernatant from toxin-proficient (TcdA+,TcdB+) or toxin-deficient (TcdA-,TcdB-) C. difficile for 4 h, then the supernatant assessed for IL-1ß and IL-18. (B) LPS-primed (10 ng/ml, 3 h) monocytes or hMDM were treated with a dose titration of either TcdB or TcdA for 3 h. Supernatants were harvested and assessed for IL-1ß. (C) Immunoblot of lysate from LPS treated (10 ng/ml, 3 h) monocytes or hMDM probed for either Pyrin or actin. Representative of 3 independent experiments. (D) LPS-primed (10 ng/ml, 3 h) monocytes or hMDM were preincubated for 15 min with the vehicle alone, CP-456,773 (2.5 µM), VX-765 (40 µM), or colchicine (2.5 µM), then stimulated with TcdB (20 ng/ml), nigericin (8 µM), or needletox (25 ng/ml) for 2.5 h. The supernatant was assessed for IL-1ß and LDH activity or (E) IL-1ß and caspase-1 cleavage by immunoblot. Mean and SEM shown for 3 donors or immunoblots 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 1A, 1B and 1D.

Fig 5: LPS, type I and type II interferons increase Pyrin expression and enable Pyrin activation in human macrophages.(A) Pyrin and IL-1ß expression in hMDM treated with either IFN-? (200 U/ml), LPS (10 ng/ml), TNFa (50 ng/ml), IL-10 (100 ng/ml), IFN-ß (5,000 U/ml), IL-4 (1,000 U/ml), or Pam3CSK4 (20 ng/ml) for either 5 or 18 h. Representative of 3 independent experiments. (B) Pyrin (MEFV) or IL-1ß (IL1ß) transcript from hMDM-treated LPS (10 ng/ml) or Pam3CSK4 (20 ng/ml) for 12 h. Mean and SEM of the fold change of 3 experimental replicates shown. The underlying data can be found in the summary data file in the tab Fig 5B.