Regulation of the effector function of CD8+ T cells by gut microbiota-derived metabolite butyrate

Abstract

The gut microbiota produces metabolites such as short-chain fatty acids (SCFAs) that regulate the energy homeostasis and impact on immune cell function of the host. Recently, innovative approaches based on the oral administration of SCFAs have been discussed for therapeutic modification of inflammatory immune responses in autoimmune diseases. So far, most studies have investigated the SCFA-mediated effects on CD4⁺ T cells and antigen presenting cells. Here we show that butyrate and, to a lesser degree, propionate directly modulate the gene expression of CD8⁺ cytotoxic T lymphocytes (CTLs) and Tc17 cells. Increased IFN-γ and granzyme B expression by CTLs as well as the molecular switch of Tc17 cells towards the CTL phenotype was mediated by butyrate independently of its interaction with specific SCFA-receptors GPR41 and GPR43. Our results indicate that butyrate strongly inhibited histone-deacetylases (HDACs) in CD8⁺ T cells thereby affecting the gene expression of effector molecules. Accordingly, the pan-HDAC inhibitors trichostatin A (TSA) and sodium valproate exerted similar influence on CD8⁺ T cells. Furthermore, higher acetate concentrations were also able to increase IFN-γ production in CD8⁺ T lymphocytes by modulating cellular metabolism and mTOR activity. These findings might have significant implications in adoptive immunotherapy of cancers and in anti-viral immunity.

Figure 1

Treatment of CD8⁺ T cells with butyrate results in preferential increase of IFN-γ production. (a,b) Frequency of IFN-γ⁺ cells cultured under sub-optimal CTL-inducing conditions and treated with SCFAs (1 mM). (c) Relative mRNA expression of Ifnγ in CTLs treated with 1 mM of various SCFAs. (d–f) Frequencies of IFN-γ⁺ and IL-17A⁺ Tc17 lymphocytes upon treatment with SCFAs (1 mM). (g,h) Frequency of IFN-γ⁺ CTLs in mLNs and spleen four weeks after oral treatment of WT mice with 150 mM sodium butyrate. Two experiments with five mice per group were performed. (b,c,e,f) Data are pooled from three independent experiments. Results are expressed as mean ± SEM. n. s. = not significant, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 2

Effects of butyrate on the expression of CTL-related effector molecules. (a,b) Percentages of granzyme B⁺ IFN-γ⁺ CTLs in the presence of various SCFAs (1 mM). (c,d) Expression of granzyme B in Tc17 cells treated with 1 mM sodium butyrate for three days. (e) Quantitative RT-PCR analysis of Rorγt and CTL-associated genes Tbx21, Eomes and Prf1 in Tc17 cells treated with 1 mM sodium butyrate for two days. (b,d,e) Three independent experiments were performed. Results are shown as mean ± SEM. n. s. = not significant, ^*p < 0.05, ^***p < 0.001.

Figure 3

Impact of butyrate on T-bet-deficient CTLs and Tc17 cells. (a,b) Percentages of IFN-γ⁺ and IL-17A⁺ cells within CTLs and Tc17 cells derived from WT and Tbx21^−/− mice. Cells were treated with 1 mM sodium butyrate or left untreated for three days. Results (b) are displayed as mean ± SEM. ^*p < 0.05, ^**p < 0.01, ***p < 0.001. (c) CTLs were cultured for two days in the presence or absence of 1 mM butyrate. Immunoblot analysis was performed by using the antibody specific for acetylated histone H4. Three independent experiments were performed. (d) ChIP analysis for acetylation of histone H4 at the promoter region of Ifnγ and Tbx21 genes after 24 hours of treatment of CTLs with 1 mM butyrate.

Figure 4

HDAC-inhibitory activity of butyrate promotes IFN-γ production in CTLs and Tc17 cells. (a) Impact of SCFAs on HDAC enzymatic activity in CTLs. 5 mM SCFAs or 500 nM TSA were added to CTL-derived cell lysates for 15 minutes. Fluorometric HDAC activity assay was performed as described in the section Methods. (b,c) Frequency of IFN-γ⁺ CTLs treated with indicated concentrations of TSA or sodium butyrate (1 mM). (d,e) CD8⁺ T cells were cultured under Tc17-inducing conditions and treated with increasing concentrations of TSA. Tc17 cells treated with 1 mM butyrate served as a control. (f,g) CTLs were treated with increased valproate concentrations (in g, 0.5 mM valproate is shown) for three days and the percentage of granzyme B⁺ IFN-γ⁺ cells was determined by flow cytometry. (h) Tc17 cells treated with 1 mM valproate for three days were analyzed for IL-17A and IFN-γ expression. The percentage of IFN-γ⁺ and IL-17A⁺ cells was measured by FACS analysis. (a,c,e,g,h) Results are pooled from three experiments. Data are expressed as mean ± SEM. n. s. = not significant, *p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 5

Dose-dependent impact of SCFAs on IFN-γ expression in CTLs. (a,b) CTLs were treated with increasing concentrations of SCFAs for three days. Flow cytometry analysis was used to determine percentages of IFN-γ⁺ and IL-17A⁺ cells within CTLs. Three experiments were performed. (c,d) CTLs were treated with 25 mM acetate or 1 mM butyrate in the presence or absence of rapamycin (25 nM). The production of IFN-γ is analyzed by flow cytometry. Three experiments were performed. (e) CTL-derived cell lysates were assayed for HDAC activity in the presence of acetate (10, 15 and 25 mM) or butyrate (1 mM). The data were compared to the HDAC activity of untreated CD8⁺ T cells, which was arbitrary set at 100 percent. Three experiments were performed. Data (b,d,e) are expressed as mean ± SEM. n. s. = not significant, *p < 0.05, **p < 0.01, ***p < 0.001.