FIGURE 6.

PGE2 signaling and effects on inflammasome activation in S. Typhimurium and Y. enterocolitica infections. THP-1 macrophages (2.5 × 10⁵) were pre-treated with combinations of PGE2 (2 μM), PF04418948 [EP2(–), EP2 antagonist, 200 nM], L-161,982 [EP4(–), EP4 antagonist, 200 nM], Butaprost [EP2(+), EP2 agonist, 10 μM], L-902,688 [EP4(+), EP4 agonist, 1 μM], or YVAD-CHO (caspase-1 inhibitor, 1 μM) at 2 h prior to infection. The levels of IL-1β (A–C) and TNF-α (D) in cell culture supernatant from S. Typhimurium- (A,C,D) or Y. enterocolitica- (B) infected macrophages were measured via ELISA 2 hpi. One-way ANOVA test with Tukey’s multiple testing correction was used to establish statistical significance. p-values were indicated as follows: ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001; ^∗∗∗∗p ≤ 0.0001. (E,F) PGE2 and IL-1β release from THP-1 macrophages infected with S. Typhimurium strains. THP-1 cells were infected with indicated strains (Table 1) of wild-type S. Typhimurium (MOI 50:1, 2 hpi). PGE2 in cell culture supernatant was measured by Prostaglandin E2 ELISA Kit (Cayman Chemical, United States) and the results were displayed in GraphPad. One-way ANOVA test with Tukey’s multiple testing correction was used to establish statistical significance. p-values were indicated as follows: ^∗p ≤ 0.05; ^∗∗p ≤ 0.01; ^∗∗∗p ≤ 0.001; ^∗∗∗∗p ≤ 0.0001. (E) Cell culture supernatant was resolved on SDS-PAGE; IL-1β and caspase-1 p10 active form were visualized by western blotting (F).