Fig 9. The PGE₂-EP3 axis regulates activation of the NLRC4 inflammasome upon A. phagocytophilum infection.

(A-B) Wildtype (WT) BMDMs (1 x10⁶ cells) were infected with A. phagocytophilum (MOI50) for 18 hours or Salmonella (MOI25) for 1 hour. RNA and protein levels of the PGE₂-EP3 receptor was measured by qRT-PCR and western blot (IB). β-actin was also detected in cell lysates. (C-E) WT BMDMs (1 x10⁶ cells) was pre-treated with the EP3 antagonist (L-798106) for 30 minutes at indicated concentrations followed by A. phagocytophilum colonization (MOI50) for 18 hours. The levels of (C) IL-1β and (D) IL-6 were measured in cell culture supernatants by ELISA. (E) Caspase-1 autoproteolysis in cell culture supernatants. pro-IL-1β, pro-IL-18 and β-actin were detected in cell lysates by SDS-PAGE immunoblots (IB). (F-H) WT and Ep3 ^-/- BMDMs (1 x10⁶ cells) were infected with A. phagocytophilum (MOI 10/50) for 18 hours. The levels of (F) IL-1β, (G) IL-18 and (H) IL-6 in culture supernatants were measured by ELISAs. (I) Caspase-1 autoproteolysis in cell culture supernatants. pro-IL-1β, pro-IL-18 and β-actin were detected in cell lysates by SDS-PAGE immunoblots (IB). (J-M) Naïve or LPS-primed (50 ng/ml) WT and Ep3 ^-/- BMDMs (1 x10⁶ cells) were infected with Salmonella (MOI25) for 1 hour. The levels of (J) IL-1β, (K) IL-18 and (L) IL-6 in cultured supernatants were measured by ELISA. (M) Caspase-1 autoproteolysis in cell culture supernatants. pro-IL-1β, pro-IL-18 and β-actin were detected in cell lysates by SDS-PAGE immunoblots (IB). Student’s t test and ANOVA-Tukey. *P < .05; NS, not significant. (-) non-stimulated.