Figure 6. Silencing miR-155 promotes macrophage polarization toward M2 phenotype in vitro.

Bone marrow cells were obtained by flushing the femurs from miR-155^−/− and WT mice, and bone marrow–derived macrophages (BMDMs) were prepared. Macrophage polarization was obtained by removing the culture medium and culturing cells for an additional 48h in RPMI 1640 supplemented with 5% FCS and 100 ng/ml LPS plus 20 ng/ml IFN-γ (for M1 polarization) or 20 ng/ml IL-4 (for M2 polarization). Then M1 and M2 macrophage–associated markers were analyzed, respectively (A–D) for M1, (E–G) for M2). (A) TNF-α and (B) IL-12 in the supernatant were assayed by ELISA. (C) The levels of NOS2 were determined by real-time PCR. (D) The surface levels of MHCII and CD16/32 were determined by FACS. (E) Arg1, FIZZ1, and YM-1 were determined by real-time PCR. (F) The surface levels of CD206 and CD301 were determined by FACS. (G) BMDMs from miR-155^−/− and WT mice were stimulated with IL-4 and collected after 30 min, 1 hour and 2 hour and subjected to Western blotting. Cells lysates were used to determine total STAT6 and p-STAT6 levels normalized against β-actin. Experiments were repeated three times in triplicate with 8 mice per group. Bar graph data are presented as mean ± SD; *P < 0.05 and ***P < 0.001 as compared with WT.