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. 2022 Nov 7;20(11):e3001351. doi: 10.1371/journal.pbio.3001351

Fig 6. Increased Pyrin expression is required for Pyrin activation in human macrophages.

Fig 6

(A) IL-1β 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-1β 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-1β 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-1β. (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.