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. 2017 Aug 28:8:1036.
doi: 10.3389/fimmu.2017.01036. eCollection 2017.

The Microbial Metabolite Butyrate Induces Expression of Th1-Associated Factors in CD4+ T Cells

Meike Kespohl  1 , Niyati Vachharajani  1 , Maik Luu  1 , Hani Harb  2 , Sabine Pautz  1 , Svenja Wolff  1 , Nina Sillner  3   4 , Alesia Walker  3 , Philippe Schmitt-Kopplin  3   4   5 , Thomas Boettger  6 , Harald Renz  2 , Stefan Offermanns  7 , Ulrich Steinhoff  1 , Alexander Visekruna  1
Affiliations

The Microbial Metabolite Butyrate Induces Expression of Th1-Associated Factors in CD4+ T Cells

Meike Kespohl et al. Front Immunol. .

Abstract

Short-chain fatty acids (SCFAs), which are generated by the bacterial fermentation of dietary fibers, promote expansion of regulatory T cells (Tregs). Potential therapeutic value of SCFAs has been recently highlighted in the experimental models of T cell-mediated autoimmunity and allergic inflammation. These studies suggest that physiological intestinal concentrations of SCFAs within the millimolar range are crucial for dampening inflammation-mediated processes. Here, we describe opposing effects of SCFAs on T cell-mediated immune responses. In accordance with published data, lower butyrate concentrations facilitated differentiation of Tregs in vitro and in vivo under steady-state conditions. In contrast, higher concentrations of butyrate induced expression of the transcription factor T-bet in all investigated T cell subsets resulting in IFN-γ-producing Tregs or conventional T cells. This effect was mediated by the inhibition of histone deacetylase activity and was independent of SCFA-receptors FFA2 and FFA3 as well as of Na+-coupled SCFA transporter Slc5a8. Importantly, while butyrate was not able to induce the generation of Tregs in the absence of TGF-β1, the expression of T-bet and IFN-γ was triggered upon stimulation of CD4+ T cells with this SCFA alone. Moreover, the treatment of germ-free mice with butyrate enhanced the expression of T-bet and IFN-γ during acute colitis. Our data reveal that, depending on its concentration and immunological milieu, butyrate may exert either beneficial or detrimental effects on the mucosal immune system.

Keywords: butyrate; inhibition of histone deacetylase activity; interferon-gamma; regulatory T cells; short-chain fatty acids.

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Figures

Figure 1
Figure 1
Butyrate promotes the expansion of Foxp3+ regulatory T cells (Tregs). (A,B) Wild-type (WT) mice were orally treated with 100 mM sodium butyrate for 3 weeks. The frequency of Foxp3+ Tregs was measured in colonic lamina propria, mesenteric lymph nodes, and spleen by FACS analysis. Bars represent mean ± SEM of two independent experiments; **P = 0.001–0.01, n.s., not significant. (C) The percentage of Th1 and Th17 cells within colonic CD4+ T lymphocytes was analyzed after 3 weeks of oral treatment of WT mice with 100 mM sodium butyrate. Data are shown as mean ± SEM; n.s., not significant. Two experiments were performed. (D) The relative expression of Foxp3, Il10, and Tbx21 was analyzed by qRT-PCR in the colonic tissue of WT mice after oral treatment with butyrate (3 weeks, 100 mM sodium butyrate). Two similar experiments were performed. Data are the mean ± SEM; *P = 0.01–0.05, **P = 0.001–0.01, and n.s., not significant. (E,F) CD4+ T cells were cultured under Treg-inducing conditions in the presence of indicated concentrations of sodium butyrate and TGF-β1 for 5 days. Bars represent the mean ± SEM of three independent experiments; *** indicates P < 0.001, n.s., not significant.
Figure 2
Figure 2
Butyrate induces the expression of IFN-γ and T-bet during the differentiation of regulatory T cells (Tregs). (A,B) The impact of increasing butyrate concentration on the Foxp3 expression was analyzed in T cells cultured under Treg-inducing conditions (1 ng/ml TGF-β1 and 100 U/ml rhIL-2) on day 6 of cell culture. The bars (B) represent mean ± SEM of three independent experiments; **P = 0.001–0.01, ***P < 0.001. (C,D) CD4+ T cells were differentiated into Tregs under optimal Treg-inducing conditions with 2 ng/ml TGF-β1 and 100 U/ml rhIL-2 in the presence of increasing concentrations of acetate, propionate, or butyrate. At day 6 of the cell culture, cells were restimulated for 4 h with PMA/ionomycin in the presence of Brefeldin A, stained for IFN-γ and IL-17A, and analyzed by flow cytometry. Bars (D) represent the mean ± SEM of IFN-γ+ cells. Two independent experiments were performed. *P = 0.01–0.05, ***P < 0.001, n.s., not significant. (E) CD4+ T cells isolated from LNs and spleens of Ffar2−/−Ffar3−/− and Slc5a8−/− mice were cultured under optimal Treg conditions for 6 days. The frequency of IFN-γ+ cells in the absence or presence of 1 mM sodium butyrate is shown. ***P < 0.001. (F) CD4+ T cells purified from WT mice were cultured under optimal Treg conditions for 3 days with or without 1 mM butyrate. The expression of Ifnγ, Tbx21, Rorγt, and Gata3 was analyzed by qRT-PCR. Data are the mean ± SEM of two independent experiments. ***P < 0.001, n.s., not significant. (G) CD4+ T cells were differentiated under optimal Treg conditions for 3 days and subsequently histone deacetylase (HDAC) inhibition assay in the presence of acetate, propionate, or butyrate was performed. Bars represent the mean ± SEM; **P = 0.001–0.01, ***P < 0.001, n.s., not significant.
Figure 3
Figure 3
Butyrate induces changes in H3 acetylation at the Ifng and Tbx21 locus during the differentiation of regulatory T cells (Tregs). (A) Western blot analysis showing the acetylation of histones H3 and H4 in the presence of 1 mM butyrate in CD4+ T cells cultured under Treg-inducing conditions for 3 days. Three similar experiments were performed. (B) Analysis of acetylated state of H3 at the promoter region of Ifng, Tbx21, and Il17a in T cells cultured under Treg-inducing conditions and treated with 1 mM butyrate for 3 days. Chromatin immunoprecipitation assay was performed using an anti-acetyl-H3 antibody. Data are average of two independent experiments; *P = 0.01–0.05, **P = 0.001–0.01, and n.s., not significant. (C) CD4+ T cells were differentiated into Tregs with 1 ng/ml TGF-β1 in the presence or absence of 1 mM butyrate. On day 3 of the cell culture, T-bet and Foxp3 expression was measured by FACS analysis. (D) Representative dot blots showing IFN-γ and IL-17A expression in wild-type (WT) and Tbx21−/− CD4+ T cells cultured under Treg- or Th1-inducing conditions for 3 days in the presence of 1 mM of sodium butyrate. One of three similar experiments is shown. (E) Representative dot blots showing T-bet and Foxp3 expression in Tbx21−/− CD4+ T cells cultured under Treg-inducing conditions with 1 ng/ml TGF-β1 in the presence 1 mM butyrate. (F,G) The frequency of IFN-γ+ and IL17A+ cells was determined on day 3 during the differentiation of Tregs in the presence of indicated TSA concentrations. Data (G) represent the mean ± SEM; ***P < 0.001. Two experiments were performed. (H,I) The percentage of Foxp3+CD4+ T cells was determined on day 3 during the differentiation of Tregs in the presence of indicated TSA concentrations. Results in (I) represent the mean ± SEM of two experiments; *P = 0.01–0.05, **P = 0.001–0.01.
Figure 4
Figure 4
Butyrate induces IFN-γ expression in unpolarized CD4+ T cells. (A,B) FACS analysis of IFN-γ and IL-17A expression in CD4+ T cells cultured under unpolarized conditions in the presence of indicated short-chain fatty acid concentrations. Results in (B) represent the mean ± SEM; *P = 0.01–0.05, ***P < 0.001, n.s., not significant. Three independent experiments were performed. (C) CD4+ T cells were left unpolarized for 3 days in the presence of increasing TSA concentrations. Frequency of IFN-γ+ and IL-17A+ cells was determined using flow cytometry. Dot plots are representative of three similar experiments. (D) CD4+ T cells were left undifferentiated for 3 days in the presence of sodium butyrate and TSA, respectively. Intracellular staining for Foxp3 was performed by FACS analysis. Data are displayed as the mean ± SEM; n.s., not significant. Two experiments were performed.
Figure 5
Figure 5
Impact of butyrate on Th17 and Th2 cells. (A–D) FACS analysis showing IFN-γ and IL-17A expression in CD4+ T cells cultured under Th17 conditions (A,B) or IFN-γ and IL-4 expression under Th2-polarizing conditions (C,D) for 6 days in the presence of indicated butyrate concentrations. Results in (B,D) represent the mean ± SEM of two experiments; *P = 0.01–0.05, ***P < 0.001, and n.s., not significant. (E,F) CD4+ T cells isolated from LNs and spleens of wild-type (WT) and Tbx21−/− mice were cultured under Th17- or Th2-polarizing conditions in the absence or presence of 1 mM butyrate. After 6 days of the cell culture, the FACS analysis for IFN-γ and IL-17A (Th17 cells) or IFN-γ and IL-4 (Th2 cells) was performed. Data (F) are displayed as the mean ± SEM obtained from two independent experiments; ***P < 0.001.
Figure 6
Figure 6
Impact of butyrate treatment on colonic acute inflammation in germ-free (GF) mice. (A) Short-chain fatty acid concentrations in colonic luminal content of GF and specific pathogen-free mice measured with ultra-high performance liquid chromatography–mass spectrometry. (B,C) GF mice were orally pretreated with 100 mM sodium butyrate for 3 weeks. Afterward, 1.5% DSS was given into drinking water for 5 days in the presence or absence of 100 mM butyrate. Changes in initial body weight (B) and determination of colitis score (C) on day 8 after DSS treatment are shown (n = 6 mice per group). Bars (C) represent the mean ± SEM; *P = 0.01–0.05, ***P < 0.001. (D) Acute colitis was induced in GF mice as described above. The colonic mRNA expression of Tbx21, Ifnγ, and Foxp3 was analyzed on day 8 after colitis induction by qRT-PCR (n = 6 mice per group). Results are displayed as the mean ± SEM; *P = 0.01–0.05, n.s., not significant. (E,F) The percentages of T-bet+ cells within colonic CD4+ T lymphocytes on day 8 after induction of colitis in the indicated groups. Histograms (E) are representatives of two similar experiments showing the frequency of T-bet+ in CD4+ gate. Data (F) are presented as mean ± SEM, **P = 0.001–0.01.
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