Research Article

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

Pamela V. Chang, Liming Hao, Stefan Offermanns, and Ruslan Medzhitov
PNAS February 11, 2014 111 (6) 2247-2252; https://doi.org/10.1073/pnas.1322269111

  1. Contributed by Ruslan Medzhitov, December 3, 2013 (sent for review October 13, 2013)

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  • 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 to Rpl13a). (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 to Rpl13a). 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's t 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 to Rpl13a. (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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Inhibition of macrophage responses by n-butyrate
Pamela V. Chang, Liming Hao, Stefan Offermanns, Ruslan Medzhitov
Proceedings of the National Academy of Sciences Feb 2014, 111 (6) 2247-2252; DOI: 10.1073/pnas.1322269111
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Proceedings of the National Academy of Sciences: 111 (6)
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