Fig. 4.

Endogenous PGE₂ regulates S. aureus-induced TLR2/NLRP3 expression and cytokine secretion in macrophages but not bacterial phagocytosis or intracellular killing. Macrophages (untreated, pre-treated with 10 µM NS398 for 40 min, or pre-treated with 1 µM CAY10404 for 40 min) were infected with WT S. aureus at MOI 30:1 or not infected. NLRP3, TLR2, and TLR4 expression in macrophages was measured by flow cytometry and quantified as the median fluorescence intensity using Flowjo 10.0 software (a–c). TNF-α, IL-1β, and RANTES production in the supernatants of macrophages was analyzed by ELISA 9 h after infection (d–f). SR-A expression in macrophages was measured by flow cytometry and quantified as the median fluorescence intensity using Flowjo 10.0 software (g). Phagocytosis of Hoechst 33258 (blue)-labeled WT S. aureus within DiI-labeled macrophages (orange) was analyzed by microscopy (× 400, h, i). Bacterial intracellular killing was measured by tetrazolium dye reduction assay (j). The results are expressed as the mean ± SD of 3 independent experiments and were analyzed by one-way ANOVA followed by Tukey's multiple comparisons test. * p < 0.05; ** p < 0.01; *** p < 0.001. NLRP3, NOD-like receptor P3; TLR, toll-like receptors.