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.2014 Feb 11;111(6):2247-52.
doi: 10.1073/pnas.1322269111. Epub 2014 Jan 3.

The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition

Affiliations

The microbial metabolite butyrate regulates intestinal macrophage function via histone deacetylase inhibition

Pamela V Chang et al. Proc Natl Acad Sci U S A..

Abstract

Given the trillions of microbes that inhabit the mammalian intestines, the host immune system must constantly maintain a balance between tolerance to commensals and immunity against pathogens to avoid unnecessary immune responses against otherwise harmless bacteria. Misregulated responses can lead to inflammatory bowel diseases such as ulcerative colitis or Crohn's disease. The mechanisms by which the immune system maintains this critical balance remain largely undefined. Here, we demonstrate that the short-chain fatty acid n-butyrate, which is secreted in high amounts by commensal bacteria, can modulate the function of intestinal macrophages, the most abundant immune cell type in the lamina propria. Treatment of macrophages with n-butyrate led to the down-regulation of lipopolysaccharide-induced proinflammatory mediators, including nitric oxide, IL-6, and IL-12, but did not affect levels of TNF-α or MCP-1. These effects were independent of toll-like receptor signaling and activation of G-protein-coupled receptors, two pathways that could be affected by short-chain fatty acids. In this study, we provide several lines of evidence that suggest that these effects are due to the inhibition of histone deacetylases by n-butyrate. These findings elucidate a pathway in which the host may maintain tolerance to intestinal microbiota by rendering lamina propria macrophages hyporesponsive to commensal bacteria through the down-regulation of proinflammatory effectors.

Keywords: inflammation; microbiome.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
n-Butyrate inhibits secretion of proinflammatory mediators. BMDM were stimulated with LPS (100 ng/mL) for 24 h ± (A) n-butyrate (But, 100 μM–2 mM), (B) acetate (Ace, 100 μM–1 mM), or (C) n-propionate (Pro, 100 μM–1 mM). Cell supernatants were collected and analyzed by the Griess assay or ELISA. Data are representative of at least two independent experiments. Error bars represent mean ± SD.
Fig. 2.
Fig. 2.
n-Butyrate inhibits proinflammatory mediators in colon LP macrophages in vitro and in vivo. (A) Colon LP macrophages (CD11b+CD11c-/lowSiglecF−) were sorted from C57BL/6 mice and cultured with LPS (100 ng/mL) ± n-butyrate (But, 500 μM). Cell supernatants were collected and analyzed by ELISA after 24 h. In a separate experiment, RNA was isolated after 4 h, and cDNA was synthesized and analyzed by qPCR (data normalized toRpl13a). (B) C57BL/6 mice were treated with metronidazole (1 g/L) and vancomycin (0.5 g/L) (Abx) for 10 d ad libitum, followed in combination with n-butyrate (But, 300 mM) in the drinking water for 7 d. Colon LP macrophages were sorted and analyzed by qPCR (data normalized toRpl13a). Data are representative of two or three independent experiments. Error bars represent mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant (unpaired Student'st test).
Fig. 3.
Fig. 3.
n-Butyrate acts as a histone deacetylase inhibitor in BMDM. (A) Whole-cell lysates from BMDM treated with LPS (100 ng/mL) ± n-butyrate (But,1 mM) for the indicated times were probed by Western blot with the indicated antibodies. (B) BMDM were stimulated with LPS (100 ng/mL) ± TSA (10–50 nM) for 24 h. Cell supernatants were collected and analyzed by the Griess assay or ELISA. (C) BMDM were treated with LPS (100 ng/mL) ± But (1 μM–2 mM) or TSA (10–50 nM) for 8 h. Cell lysates were probed for acetylated histone H3 (Ac-H3) by immunoblotting. Total protein loading was assessed by immunoblotting for histone H3 (H3). (D) Quantification of H3 acetylation was determined by densitometry and is normalized to β-actin. Bars represent BMDM treated with LPS (–), LPS + n-butyrate (But, 2 mM), and LPS + TSA (TSA, 50 nM). Data are representative of three independent experiments. Error bars represent mean ± SD.
Fig. 4.
Fig. 4.
Inhibition of proinflammatory mediators by n-butyrate and TSA is transcriptionally controlled. (A) BMDM were stimulated with LPS (100 ng/mL) ± n-butyrate (But, 100 μM–2 mM) or TSA (10–50 nM) for 4 h. cDNA was analyzed by qPCR. Data are normalized toRpl13a. (B) BMDM were stimulated with LPS (100 ng/mL) ± But (1 mM) or TSA (10 nM) for 1 or 3 h. Samples were analyzed by ChIP using antibodies against RNA polymerase II (pol II, black bars) or phosphorylated serine 5 pol II (S5P, white bars). Purified DNA was analyzed by qPCR using primers specific to the promoters of the indicated genes. Normalized results are shown as a percentage of input values. Data are representative of three independent experiments. Error bars represent mean ± SD.
Fig. 5.
Fig. 5.
n-Butyrate does not affect the severity of DSS colitis. C57BL/6 mice were administered metronidazole (1 g/L) and vancomycin (0.5 g/L) (Abx) for 21 d ad libitum. From day 13 to 21, the mice were gavaged with ± n-butyrate (But, 0.1–10 mg/kg, i.g.). From day 14 to 21, mice were given DSS [3.5% (wt/vol)]. On day 21, the mice were euthanized, and colons were harvested. (●, black) Abx, (■, red) Abx + DSS, (▲, light blue) Abx + DSS + But (0.1 mg/kg), (▼, blue) Abx + DSS + But (1 mg/kg), (◆, dark blue) Abx + DSS + But (10 mg/kg). (A) Mice were weighed daily to monitor disease. (B) Distal colons were sectioned and stained with H&E. Histopathologic scores were determined in a blinded fashion. Error bars represent mean ± SD. (C) H&E staining of distal colon. (Scale bar, 25 μm.) Data are representative of four to five mice per group and three independent experiments. (D) Model of n-butyrate’s effect on intestinal macrophages.
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