The Prostaglandin E2-EP3 Receptor Axis Regulates Anaplasma phagocytophilum-Mediated NLRC4 Inflammasome Activation
Fig 7
PGE2 activates the A. phagocytophilum-induced NLRC4 inflammasome.
(A-C) Ptgs2-/- BMDMs (1 x106 cells) were infected with A. phagocytophilum (MOI50) for 4 hours followed by addition of PGE2 (10 μM), PGD2 (10 μM) or TBXA2 (10 μM) for 18 hours. (A) Levels of IL-1β and (B) IL-18 in supernatants were measured by ELISA. (C) Caspase-1 autoproteolysis was measured in supernatants of infected cells. pro-IL-1β, pro-IL-18 and β-actin were detected in cell lysates with SDS-PAGE immunoblot (IB). (D-H) Wildtype (WT) BMDMs (1 x106 cells) were pre-treated with the specific inhibitor of mPGES1 (CAY10526–1 μM) for 30 minutes followed by A. phagocytophilum infection (MOI50) for 18 hours. The levels of (D) PGE2, (E) PGD2, (F) IL-1β and (G) IL-18 in the culture supernatants were measured by ELISA. (H) Caspase-1 p20 autoproteolysis in culture supernatants. pro-IL-1β, pro-IL-18 and β-actin were detected in cell lysates with a SDS-PAGE immunoblot. (I-J) WT and mPGES1-/- BMDMs (1 x106 cells) were infected with A. phagocytophilum (MOI50) for 18 hours. The levels of (I) PGE2 and (J) PGD2 were measured in culture supernatants by ELISA. (K-M) mPGES1-/- BMDMs (1 x106 cells) were infected with A. phagocytophilum for 4 hours followed by addition of PGE2 at indicated concentrations for 18 hours. (K) IL-1β and (L) IL-18 levels in cell culture supernatants were measured by ELISA. (M) Caspase-1 autoproteolysis in cell culture supernatants. pro-IL-1β, pro-IL-18 and β-actin in cell lysates were detected with SDS-PAGE immunoblot (IB). One-way ANOVA-Tukey and Student’s t test; *P < .05; NS, not significant. (-) non-stimulated.