Figure 3. Diabetic wound macrophages exhibit increased EP2 and EP4, resulting in increased inflammation and impaired phagocytosis.

(A) Wound monocyte/macrophages from DIO and control mice were sorted on day 5 postwounding and analyzed for Ep2 and Ep4 receptor expression (n = 3/group, repeated in triplicate). (B) Relative expression of Ep2 versus Ep4 in day 5 wound monocyte/macrophages (MФs) (n = 3/group, repeated in triplicate). (C) BMDMs from controls were stimulated with media, PGE₂ (1000 nM), or PGE₂ (1000 nM) + AH6809 (10 μM), and expression of Il1b, Il12, and Nos2 was analyzed by qPCR (n = 3/group, run in triplicate). (D) Wound monocyte/macrophages from DIO mice or controls were incubated with fluorescently labeled P. aeruginosa for 2 hours, and the percentage of cells ingesting bacteria was determined in relative fluorescence units (RFU) (n = 4/group, repeated twice). (E) Wound monocyte/macrophages from DIO and control mice were isolated on day 5 postwounding and analyzed for Marco receptor expression (n = 3/group, run in triplicate). (F) Wound monocyte/macrophages from controls were incubated with media, PGE₂ (1000 nM), or PGE₂ (1000 nM) + AH6809 (10 μM) for 6 hours and then were exposed to fluorescently labeled P. aeruginosa for 2 hours, and the percentage of cells ingesting bacteria was determined in RFU (n = 3/group, repeated twice). (G) BMDMs from DIO mice or controls were infected with P. aeruginosa for 30 minutes before extracellular bacteria were removed. Cells were allowed to kill ingested bacteria for 2 hours before cells were lysed and CFU determined. Higher CFU/mL indicate impaired intracellular killing (n = 3/group, repeated twice). Data are presented as the mean ± SEM. Data were first analyzed for normal distribution, and if data passed normality test, 2-tailed Student’s t test for 2 groups and 2-way ANOVA for multiple groups was used.