Figure 1.

Pseudomonas aeruginosa biofilm persistence is associated with attenuated activation of host proinflammtory responses and decreased bacterial eradication ability, whereas increased programming of macrophages toward M2 phenotype. (A) P. aeruginosa biofilm formation on catheters was observed by scanning electron microscopy (SEM; 5000X magnification). Bacterial loads (B), production of tumor necrosis factor (TNFα) and IL12 p40/70 (C), and F4/80⁺/iNOS⁺ and F4/80⁺/Arg1⁺ macrophages (D,E) in planktonic P. aeruginosa (1 day)‐ or catheter biofilm (7 days)-infected tissues were determined by colony forming unit (CFU) enumeration, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining, respectively. For immunofluorescence staining, M1 macrophages were counterstained with fluorescein isothiocyanate (FITC)-conjugated anti-F4/80 and AF647-conjugated anti-inducible nitric oxide synthase (iNOS) antibodies. M2 macrophages were counterstained with FITC-conjugated anti-F4/80 and AF647-conjugated anti-Arg1 antibodies; cellular nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The static P. aeruginosa biofilms were co-cultured with Raw264.7 at 37°C, with 5% CO₂ for 3 h (MOI = 10), and relative gene expression levels were analyzed by real-time quantitative PCR (RT-qPCR) using mouse GAPDH as a reference gene (F). To generate M1 and M2-polarized macrophages, Raw264.7 cells were pretreated with 100 ng/ml lipopolysaccharide (LPS) + 50 ng/ml IFNγ (named as LPS group) or 20 ng/ml IL4 for 6 h, respectively. The cells were then co-cultured with static P. aeruginosa biofilms at a ratio of 1:10 at 37°C, 5% CO₂ for 30 min. Phagocytosis was detected by immunofluorescence staining (G) and CFU enumeration (H). Cytoskeleton was labeled with phalloidin (red); PAO1 was visualized by FITC anti-PAO1 antibody (green); cellular nuclei were stained with DAPI (blue). Error bars indicate the means ± standard errors of the means (SEM). An unpaired Student’s t test was used for statistical analysis (B,C,F,H). ^*p < 0.05; ^**p < 0.01; ^***p < 0.001. Planktonic (Plank.), biofilm (Bio.), sterile catheter (steri cath).