Short-chain fatty acids induce both effector and regulatory T cells by suppression of histone deacetylases and regulation of the mTOR-S6K pathway

Abstract

Microbial metabolites, such as short-chain fatty acids (SCFAs), are highly produced in the intestine and potentially regulate the immune system. We studied the function of SCFAs in the regulation of T-cell differentiation into effector and regulatory T cells. We report that SCFAs can directly promote T-cell differentiation into T cells producing interleukin-17 (IL-17), interferon-γ, and/or IL-10 depending on cytokine milieu. This effect of SCFAs on T cells is independent of GPR41 or GPR43, but dependent on direct histone deacetylase (HDAC) inhibitor activity. Inhibition of HDACs in T cells by SCFAs increased the acetylation of p70 S6 kinase and phosphorylation rS6, regulating the mTOR pathway required for generation of Th17 (T helper type 17), Th1, and IL-10(+) T cells. Acetate (C2) administration enhanced the induction of Th1 and Th17 cells during Citrobacter rodentium infection, but decreased anti-CD3-induced inflammation in an IL-10-dependent manner. Our results indicate that SCFAs promote T-cell differentiation into both effector and regulatory T cells to promote either immunity or immune tolerance depending on immunological milieu.

Figures

Fig. 1

SCFAs promote the generation of Th17 and Th1 effector cells. (a)) Effects of C2 and C3 on naïve CD4+ T cell differentiation into Th17 and Th1 cells were determined. Naive CD4+ T cells, depleted of Treg (CD25+) and memory T (CD44+ CD69+) cells, were activated with anti-CD3/CD28 in a Th17 (IL-1β, IL-6, IL-21, IL-23, TGFβ1, anti-IL-4, and anti-IFN-γ) or Th1 (IL-12, IL-2, and anti-IL-4) condition for 5-6 days. C2 (0, 1, 10, and 20 mM) or C3 (0, 0.1, 0.5, and 1 mM) was added as indicated. (b) Expression patterns of T helper signature genes for Th1 and Th17 cells, generated in the presence of SCFAs, were determined by qRT-PCR. (c) Expression of FoxP3 by T-bet+ or RORγt+ T cells, generated in Th1 or Th17 condition in the presence of SCFAs. Cells were prepared in a Th17 or Th1 condition with C2 (10 mM) or C3 (1 mM). *Significant differences from blank groups (no treatment with C2 or C3) (P≤0.05).

Fig. 2

The colitogenic activity of C2-treated T cells in Rag1(−/−) mice. (a) Cytokine profiles of cultured T cells in the presence or absence of C2 in a Th17 cell condition. Also shown is the weight change of Rag1(−/−) mice following injection of the cultured T cells. (b) Histological changes in the distal colon of Th17-transferred Rag1(−/−) mice were examined (×100 original magnification; scale bar = 200 μm), and histological scores indicating tissue inflammation are shown. (c) Frequencies of Th1, Th17, and FoxP3+ T cells in the colon of Th17-transferred Rag1(−/−) mice were determined by flow cytometry. Similarly, effector T cells induced in the absence or presence of C2 in a Th1 condition were assessed for their effects on (d) body weight change, (e) histological changes, and (f) frequencies of Th subsets. Representative or pooled data from 2-3 experiments are shown (n=5 for a; 10 for b; 9 for c; 6-7 for d; 4 for e; 6-7 for f). *Significant differences from the control group or between indicated groups (P≤0.05).

Fig. 3

SCFAs induce IL-10-producing CD4+ or CD8+ T cells admixed with effector T cells. (a) The cytokine phenotype of CD4+ T cells cultured in Tnp, Th17, or Th1 condition with indicated SCFA was determined by flow cytometry. (b) Dose-dependent effects of C2 and C3 on the induction of IL-10+ CD4+ T cells. Conditioned media were examined for secreted IL-10 (c) and IL-10 mRNA (d). (e) The suppressive activity of control and C2-treated T cells on the proliferation of responder T cells was examined. Suppressor T cells were co-cultured with responder cells (CFSE-labeled CD4+CD25− T cells) in the presence of anti-CD3 and irradiated T-cell-depleted splenocytes as antigen presenting cells for 3 days before flow cytometric analysis. C2-treated suppressor T cells were prepared from the culture of naïve CD4+ T cells, isolated from WT or IL-10(−/−) mice, for 5-6 days in the presence of C2 (10 mM) in a Th1 cell-polarization condition. (f) SCFAs also induce expression of IL-10 in CD8+ T cells. Total CD8+ T cells were activated with anti-CD3/28 in a Tc1 (IL-2, IL-12, and anti-IL-4) or Tc17 (TGFβ1, IL-6, IL-1β, IL-23, IL-21, TNFα, anti-IL4, and anti-IFN-γ) polarization condition for 5-6 days. If not indicated, the concentrations of SCFAs were 10 mM (C2) or 1 mM (C3) for all experiments. Representative or pooled (b-f) data obtained from 3-4 experiments are shown. *Significant differences from blank groups or between indicated groups (P≤0.05).

Fig. 4

Impact of infection on the SCFA effect on effector versus IL-10+ T cells. (a) The concentrations of SCFAs in cecal contents and intestine tissues of C2-fed mice were determined by LC-MS. (b and c) Some of the C2-fed mice were infected with C. rodentium. Changes in Th17, Th1, and IL-10+ T cells in indicated tissues 14 days after oral infection with C. rodentium were examined by flow cytometry. Pooled data (b; n=7-9) and representative dot plots (c) are shown. *Significant differences (P≤0.05).

Fig. 5

C2 feeding suppressed anti-CD3 induced inflammation in the intestine. (a-c) Mice were fed with C2 for 6-8 weeks, and anti-CD3 antibody (clone 145-2C11) was injected i.p. to stimulate the production of IL-10+ T cells (n=6-9/group). Mice were sacrificed 52 h after the injection of anti-CD3, and the frequencies and numbers of IL-10+ (a), Th17 (b), and Th1 (c) cells were examined by flow cytometry (6-9/group). (d) The inflammatory response in the terminal ileum of the mice fed with C2 and injected with anti-CD3 was examined. Tissue sections were stained with hematoxylin and eosin (×100 original magnification; scale bar= 200 μm). Pooled histological scores of the ileum of control and C2-fed mice (n=6-9/group) obtained from two experiments are shown. *Significant differences from the control group (regular water) or between indicated groups (P≤0.05).

Fig. 6

T cells poorly express GPR41 and GPR43, and GPR41−/− or GPR43−/− T cells normally respond to C2 or C3. (a and b) Cultured CD4+ T cells, CD326+ colonic epithelial cells, colonic lamina propria CD4+ or CD4− (colonic CD4-neg) cells, bone marrow Gr-1+ cells, and in vitro differentiated BM-DCs were compared for expression of GRP41 and GPR43 mRNA with qRT-PCR (A) and conventional PCR (B). (c and d)In vitro differentiation of GPR41−/− or GPR43−/− CD4+ T cells in response to C2 or C3. Naive CD4+ T cells were cultured in Th1 or Th17 cell condition for 5-6 days with C2 (10 mM) or C3 (1 mM). Frequencies of indicated T cell subsets were determined by flow cytometry. Representative (c) and pooled (d) data obtained from 3 experiments are shown. *Significant differences (P≤0.05) from control groups.

Fig. 7

Normal production of effector and IL-10+ T cells in mice deficient in GPR41 or GPR43 with or without C2 feeding. (a) Mice were sacrificed at 6-9 weeks of age. Indicated T cell subsets in the spleen, MLN, and various compartments of the intestine were examined by flow cytometry. (b) Mice were fed with regular or C2 water from 3 weeks of age for 6-8 weeks. IFN-γ+, IL-17+, and IL-10+ CD4+ T cells in the colon were examined. Pooled data (n=7-10 for a; 5 for b) are shown. *Significant differences (P≤0.05) from control groups.

Fig. 8

SCFAs suppress HDACs in a GPR41 or GPR43-independent manner. (a) In-cell HDAC inhibitor activity. T cells were activated for 48 h, pre-incubated with SCFAs or TSA for 2 h, and then assayed for HDAC activity. (b) Comparison of TSA and SCFAs in regulation of T cell differentiation into effector and IL-10+ T cells. Naïve CD4+ T cells were cultured for 5 days in the presence of indicated SCFAs or TSA. Pooled data from three independent experiments are shown. *Significant differences (P≤0.05) from control groups.

Fig. 9

SCFAs enhance mTOR activity. (a) Activation of the mTOR pathway based on rS6 phosphorylation in CD4+ T cells. T cells were activated with anti-CD3/CD28 and IL-2 in the presence or absence of C2 or C3 and examined by flow cytometry. (b) Acetylation of S6K in activated T cells in the presence of C2, C3 or TSA. The antibodies used for immunoprecipitation (IP) and western blotting (WB) were anti-acetylated-lysine and anti-S6K respectively. (c) The effect of rapamycin on SCFA-dependent generation of Th cell subsets was assessed. Naïve CD4+ T cells were cultured in a Tnp, Th1, or Th17 polarization condition for 5-6 days in the presence of C2 (10 mM), C3 (1 mM), and/or rapamycin (25 nM), and the cytokine phenotype of cultured T cells was examined by flow cytometry and ELISA (for IL-10). (d) AMPK activation suppressed the SCFA effect on T cell differentiation. Naïve CD4+ T cells were cultured with SCFAs and/or metformin in indicated polarization conditions for 5-6 days, and frequencies of indicated T cell subsets were examined. The dot plots were from the data obtained in a Th1 condition. (e) Activation of STAT3 by C2 or C3 was examined. CD4+ T cells were activated for 3 days with anti-CD3/28 and then examined for phosphorylation of STAT3. Representative and pooled data obtained from at least three experiments are shown. *Significant differences from blank or control groups (P≤0.05).

Fig. 10

Effects of C4 on rS6 phosphorylation (A) and T cell differentiation (B). Naive CD4+ T cells, depleted of Treg (CD25+) and memory T (CD44+ CD69+) cells, were activated with anti-CD3/CD28 for 3 days for rS6 phosphorylation and for 5-6 days in a Tnp, Th17, or Th1 condition for T cell differentiation at indicated concentrations of C4. Representative and pooled data obtained from at least three experiments are shown. *Significant differences from blank or control groups (P≤0.05).