Fig 1: An hFL-OPG-Fc treatment induced M2 macrophage polarization in vitroMurine macrophage cells were stimulated with LPS (1,000 ng/mL) followed by a 24-h treatment with either PBS, hFL-OPG-Fc (200 ng/mL), or both IL-4 and IL-13 (10 ng/mL of each) for M2 phenotype polarization. (A) To test whether hFL-OPG-Fc treatment induces M2 polarization, total macrophage and M2 phenotypes were labeled using F4/80 and CD206, respectively, and sorted by flow cytometry. (B–D) hFL-OPG-Fc treatment induced a slight increase in total macrophages (F4/80+ cells) (B), a marked decrease in M1 macrophages (F4/80+/CD206-) (C), and a significant increase in M2 macrophages (F4/80+/CD206+) (D) compared to PBS-treated cells. (E and F) IL-4/IL-13-treated cells were used as a positive control for M2 polarization. The experiment was performed four separate times. Western blot results show that hFL-OPG-Fc significantly decreased TNF-a (E) and iNOS (F) proteins levels, both M1 macrophage markers. (G) The expression of chitinase 3-like 3 and 4 proteins (Ym1/2), an M2 macrophage marker, was significantly increased in hFL-OPG-Fc-treated cells. Protein levels were normalized to GAPDH content and expressed in ratio from PBS (n = 7–15). Data are expressed as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 indicate significantly different from PBS using one-way ANOVA for multiple comparisons followed by a Dunnett’s post hoc test.