Figure 8. cPLA₂α is involved in endogenous NLRP3 inflammasome regulation.

(A) THP-1 cells were treated with cPLA₂ inhibitor (pyrrolidone derivative, 2 μM, for 30 min) or vehicle (DMSO) then primed with LPS (100 ng/ml) for 4 h in serum-free medium, followed by treatment with nigericin (4 μM for 45 min), ATP (1 mM, for 1 h), monosodium urate crystals (MSU, 100 μg/ml, for 5h) or alum (200 μg/ml, for 5h). Supernatants and lysates were collected for WB. The WB are representative of 2 independent experiments, each showing similar results. (B) THP-1 cells were transfected with cPLA₂α siRNA or control siRNA (1 μM). After 48 h transfected cells were primed with LPS (100 ng/ml) for 4 h in serum-free medium, followed by stimulation with alum (200 μg/ml, for 5h). Supernatants and lysates were collected for WB. The WB are representative of 2 independent experiments, each showing similar results. (C, D and E) human primary MDM were treated with/without LPS (100 ng/ml) with cPLA₂ inhibitor (2 μM) or DMSO for 4 h in complete medium, followed by subsequent treatment with cPLA₂ inhibitor or DMSO for 30 min, and then stimulated with alum (400 μg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for PGE₂ ELISA (C), WB (D) or IL-1β ELISA (E). PGE₂ (C) and IL-1β (E) release data represent the mean ± SEM from 3 independent experiments from 3 healthy donors, each performed in duplicate. IL-1β data are presented as the percentage of the response after LPS/alum treatment, which ranged from 409.4 to 647.6 pg/ml (E). (F, G and H) human primary MDM were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by treatment with cPLA₂ inhibitor or DMSO for 30 min, and then stimulated with alum (400 μg/ml for 5 h) in serum-free medium. Supernatants and lysates were collected for PGE₂ ELISA (F), WB (G) or IL-1β ELISA (H). PGE₂ (F) and IL-1β (H) release data represent the mean ± SEM from 3 independent experiments from 3 healthy donors, each performed in duplicate. IL-1β data are presented as the percentage of the response after LPS/alum treatment, which ranged from 261.2 to 516.7 pg/ml (E). The WB are representative of 3 independent experiments from 3 healthy donors, each showing similar results (D, G). * P < .05 as indicated, as assessed by Kruskal-Wallis ANOVA on ranks, followed by Dunn's post hoc test. b.p. (before priming), a.p. (after priming)