Figure 5. cAMP is involved in PGE₂-mediated NLRP3 inhibition.

(A, B) primary MDM were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by treatment for 1 h with KH7 (25 μM) or IBMX (200 μM) or forskolin (50 μM), and followed by PGE₂ (0.1 μM) or vehicle (EtOH) for 30 min and stimulated with alum for 5 h (400 μg/ml) in serum-free medium. Supernatants and lysates were collected for WB (A) or IL-1β ELISA (B). IL-1β release data represent the mean ± SEM from 3 independent experiments from 3 healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 76.7 to 562.8 pg/ml. ^# P < .05 as compared to LPS/alum treatment, * P < .05 as indicated, as assessed by one-way ANOVA, followed by Holm-Sidak post hoc test (B). (C) primary MDM were treated with IBMX (200 μM) with either vehicle (EtOH), PGE₂ (0.1 μM) or forskolin (50 μM) for 15 min. cAMP measured in cell lysates represents the mean ± SEM from 3 independent experiments from 3 healthy donors, each performed in triplicate. * P < .05 as indicated, as assessed by Kruskal-Wallis ANOVA on ranks, followed by Dunn's post hoc test. (D and E) MDM were treated with/without LPS (100 ng/ml) for 4 h in complete medium, followed by 30 min treatment with indicated doses of 8-Bromo-cAMP or vehicle (Tris) and then stimulated with alum for 5 h (400 μg/ml) in serum-free medium. Supernatants and lysates were collected for WB (D) or IL-1β ELISA (E). IL-1β release data represent the mean ± SEM from 3 independent experiments from 3 healthy donors, each performed in duplicate. Data are presented as the percentage of the response after LPS/alum treatment, which ranged from 70.2 to 1100.9 pg/ml.* P < .05 as compared to LPS/alum treatment, as assessed by Kruskal-Wallis ANOVA on ranks, followed by Dunn's post hoc test (E). The WB are representative of 3 independent experiments from 3 healthy donors, each showing similar results (A and D).