Fig. 3

L.E.M. extract activates BMDMs and induces M1-like macrophages. L.E.M. extract activates BMDMs and induces M1-like macrophages. (a) IFN-γ (20 ng/mL) or IL-4 (20 ng/mL) were added to BMDMs to induce M1-like macrophages or M2-like macrophages, respectively, then stimulated with 10 µg/mL L.E.M. extract or 10 ng/mL LPS (a positive control). After culturing for three days, the expression levels of CD86 and CD80 were analyzed by flow cytometry (N = 3 per group). (b) Similarly, M1-like macrophages and M2-like macrophages were induced and stimulated with 0.1 or 10 µg/mL L.E.M. extract. After culturing for three days, the macrophages were stimulated with 10 ng/mL LPS for 6 h, and the level of IL-12p70 production in the M1-like macrophage culture supernatant and level of IL-10 and TGF-β production in the M2-like macrophage culture supernatant were measured by ELISA assay (N = 3 per group). (c) Similarly, M1-like macrophages and M2-like macrophages were induced, IL-4 (20 ng/mL) was added to M1-like macrophages to induce M2-like macrophages, and the cells were cultured for three days after stimulating with 10 µg/mL L.E.M. extract (M1 to M2). IFN-γ (20 ng/mL) was added to M2-like macrophages to induce M1-like macrophages, whereas 10 µg/mL L.E.M. extract was added to stimulate the cells, which were then cultured for three days (M2 to M1). After culturing, each set of cells were stimulated for 6 h with 10 ng/mL LPS, and the level of IL-12p70 production in the culture supernatant was measured by ELISA analysis (N = 3 per group). The median value is shown in the figure. The data are representative of two independent experiments and were statistically analyzed by one-way ANOVA (*, p < 0.05; **, p < 0.01; N.S., not significant)