Figure 5.

PcrV-primed macrophages display increased phagocytosis, bacterial killing efficacy, and induction of proinflammtory cytokines. Raw264.7 cells pretreated with or without PcrV (10 μg/ml) for 6 h were co-cultured with P. aeruginosa biofilms (MOI = 10) for the indicated time point. The phagocyted bacteria were detected by immunofluorescence staining (A) and CFU enumeration (B). Cytoskeleton was labeled with phalloidin (red); PAO1 was visualized by FITC anti-PAO1 antibody (green); cellular nuclei were stained with DAPI (blue). BMDMs primed with or without PcrV (10 μg/ml) for 24 h were daily injected into the tissues surrounding biofilm-infected catheters after biofilm infection for 4 days. The infected tissues and catheters were harvested after the injection of PcrV for 3 days. F4/80⁺/iNOS⁺ macrophages in infected tissues (C), bacterial burdens (D), and production of TNFα and IL12 p40/70 (E) were analyzed by immunofluorescence staining, CFU enumeration, and ELISA, respectively. For immunofluorescence staining, M1 macrophages were counterstained with FITC-conjugated anti-F4/80 and AF647-conjugated anti-iNOS antibodies. M2 macrophages were counterstained with FITC-conjugated anti-F4/80 and AF647-conjugated anti-Arg1 antibodies; cellular nuclei were stained with DAPI. One-way ANOVA (Tukey’s post hoc, B) or unpaired Student’s t test (D,E) was used for statistical analysis. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.