Microbial metabolite butyrate facilitates M2 macrophage polarization and function

Abstract

Metabolites from intestinal microbes modulate the mucosal immune system by regulating the polarization and expansion of T cells. Whether the microbial metabolites influence macrophage polarization, however, is poorly understood. Here, we show that the large bowel microbial fermentation product, butyrate, facilitates M2 macrophage polarization, in vitro and in vivo. The supernatant from butyrate-treated M2 macrophage increased the migration and enhanced the wound closure rate of MLE-12 cells. Butyrate attenuated intestinal inflammation in mice with dextran sulfate sodium (DSS)-induced colitis, with a significant increase in colonic expression of the M2 macrophage-associated protein, Arg1. M2 macrophage treated with butyrate, had increased activation of the H3K9/STAT6 signaling pathway, suggesting a mechanism for butyrate facilitated M2 macrophage polarization. Collectively, our study indicated that commensal microbe-derived butyrate is a novel activator of STAT6-mediated transcription through H3K9 acetylation driving M2 macrophage polarization, and delineated new insights into the immune interplay underlying inflammatory bowel disease.

Figure 1. Butyrate increases the expression of genes typical of M2 macrophage.

(a) M0-BMDMs were directly exposed in series concentrations of butyrate for 24 h. Expression of Arg1, Fizz1, Ym1, and MR was measured by quantitative RT-PCR. (b) The relative quantity of Arg1, Fizz1, Ym1, and MR mRNA transcribed by nonpolarized (M0-BMDMs, gray bars) or M2-BMDM after 3, 6, 12, and 24 h with IL-4 in the absence (white bars) or presence (black bars) of 50 μg/mL butyrate. (c) Arg1 protein levels in the absence or presence of 50 μg/mL butyrate in M2-BMDMs after 24 h treatment. Data are mean ± SD for at least three independent experiments. Comparisons between means used t tests (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2. Wound healing and cell migration in MLE-12 cell cultures.

(a) Wounded MLE-12 cells were treated with supernatants conditioned for 24 h by non-polarized (M0) or M2-BMDMs cells in the absence (M2) or presence (+But) of butyrate. Photographs (×100) were taken at 0 and 12 h, after the wound was made. The extent of wound closure of MLE-12 was measured and expressed as a percentage. (b) Transwell filter assay was used to measure chemoattraction and migration of MLE-12 cells to lower wells containing conditioned media from non-polarized (M0) or M2-BMDM cells in the absence (M2) or presence (+But) of butyrate. Migrated cells at the lower surface of the transwell filter were stained with DAPI and counted. (c) Total RNA was isolated from M0-BMDMs, and M2-BMDMs, untreated or treated with butyrate, and analyzed by RT-PCR for the expression of representative M2 macrophage chemokine genes. Data are shown as mean ± SD for three independent experiments, Comparisons between means used t tests (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3. Effect of butyrate on dextran sulfate sodium (DSS)-induced colitis.

(a) Relative body weight change, (b) disease activity index, (c) and colon length of mice with colitis treated with butyrate (+But) or left untreated (DSS), and healthy control mice (control). (d) Histological appearance and histological scores of colons from control mice, and mice with colitis, either treated with butyrate or untreated. (e) IL-1β, IL-6, and TNF-α in the serum of mice with colitis, treated with butyrate or untreated, and control mice. (f) Quantitation of the colonic Arg1 protein expression in serial sections by immunohistochemistry. The mean integrated optical density (IOD) (IOD/total area) represents the Arg1 expression level. Data are representative of 6 mice/group, with similar staining. Data are shown as mean ± SD for three independent experiments, Comparisons between means used t tests (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4. Therapeutic effect of M2-BMDMs on DSS colitis.

DSS was administered to mice to induce colitis and M0 or M2-BMDMs were transferred intravenously. (a) Relative body weight change, (b) disease activity index, and (c) colon length of mice with colitis, treated with M0 or M2 BMDMs or untreated (DSS), and control mice. (d) Histological appearance and histological scores of colons. (e) IL-1β, IL-6, and TNF-α in serum. (f) Quantitation of Arg1 protein expression in serial sections by immunohistochemistry. The mean IOD (IOD/total area) represents the Arg1 expression level. Data are representative of 6 mice/group, with similar staining. Data are shown as mean ± SD for three independent experiments, Comparisons between means used t tests (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5. Butyrate promotes IL-4-induced STAT6 signaling pathways in M2 macrophage.

M2-BMDMs were incubated with IL-4 and IL-4 with butyrate for the indicated time periods. Cell lysates were prepared and blotted with anti-phospho-STAT6. Total STAT6 and β-actin were probed as quantitative controls. Data are representative of three independent experiments.

Figure 6. Butyrate increases acetylation of H3K9.

(a) M2-BMDMs cultured in the presence of butyrate for 6 h were examined for expression of HDAC 1, 2, 3, 6, 7, and 9 by quantitative RT-PCR. Data are the mean ± SD of three independent experiments. (b) M2-BMDMs were treated with butyrate for 12 h. The level of H3K9 acetylation were assessed by immunoblot. Total histone and β-actin levels were used as loading controls. Data are representative of three independent experiments.

Figure 7. Histone acetyltransferase (HAT) inhibition mediates butyrate-treated M2 macrophage polarization by inhibiting the expression of acetylated H3K9.

(a) Arg1 and Ym1 gene expression in butyrate-treated M2 macrophage treated with increasing concentrations (2–20 μM) of HAT inhibitor, C646. (b) Arg1 and Ym1 gene expression in the presence of butyrate (50 μg/ml) and C646 (5 μM). (c) Western blot analysis of Arg1 protein in butyrate-treated M2-BMDMs after 12 h treatment with C646 (5 μM). (d) Quantitative RT-PCR analysis of HDAC1 gene expression in butyrate-treated M2-BMDMs after 6 h treatment with C646 (5 μM). (e) Western blot analysis of acetylated H3K9, total histone H3, and β-actin proteins in butyrate-treated M2-BMDMs after 12 h treatment with C646 (5 μM). Cells were lysed and western blotting performed with the indicated antibodies. (f) Phosphorylation of STAT6 in butyrate-treated M2-BMDMs. Western blotting was performed with anti-phospho-STAT6, STAT6, and β-actin. Data are representative of three independent experiments.