Butyrate directly decreases human gut lamina propria CD4 T cell function through histone deacetylase (HDAC) inhibition and GPR43 signaling

Abstract

An important function of the gut microbiome is the fermentation of non-digestible dietary fibers into short chain fatty acids (SCFAs). The three primary SCFAs: acetate, propionate, and butyrate, are key mediators of metabolism and immune cell function in the gut mucosa. We previously demonstrated that butyrate at high concentrations decreased human gut lamina propria (LP) CD4 T cell activation in response to enteric bacteria exposure in vitro. However, to date, the mechanism by which butyrate alters human gut LP CD4 T cell activation remains unknown. In this current study, we sought to better understand how exposure to SCFAs across a concentration range impacted human gut LP CD4 T cell function and activation. LP CD4 T cells were directly activated with T cell receptor (TCR) beads in vitro in the presence of a physiologic concentration range of each of the primary SCFAs. Exposure to butyrate potently inhibited CD4 T cell activation, proliferation, and cytokine (IFNγ, IL-17) production in a concentration dependent manner. Butyrate decreased the proliferation and cytokine production of T helper (Th) 1, Th17 and Th22 cells, with differences noted in the sensitivity of LP versus peripheral blood Th cells to butyrate's effects. Higher concentrations of propionate and acetate relative to butyrate were required to inhibit CD4 T cell activation and proliferation. Butyrate directly increased the acetylation of both unstimulated and TCR-stimulated CD4 T cells, and apicidin, a Class I histone deacetylase inhibitor, phenocopied butyrate's effects on CD4 T cell proliferation and activation. GPR43 agonism phenocopied butyrate's effect on CD4 T cell proliferation whereas a GPR109a agonist did not. Our findings indicate that butyrate decreases in vitro human gut LP CD4 T cell activation, proliferation, and inflammatory cytokine production more potently than other SCFAs, likely through butyrate's ability to increase histone acetylation, and potentially via signaling through GPR43. These findings have relevance in furthering our understanding of how perturbations of the gut microbiome alter local immune responses in the gut mucosa.

Keywords: Butyrate; CD4; Human gut T cell; SCFA; T cell activation; T helper cell.

Fig. 1.

Butyrate reduces TCR-mediated proliferation and activation LP CD4 T cells in a concentration dependent manner. (A-C) Lamina propria mononuclear cells (LPMC; N = 3) or (D-F) purified LP CD4 T cells (N = 3) were pre-labelled with CFSE and cultured with or without TCR-activating beads (TCR beads) and exogenous butyrate (0.0625–0.5 mM) for four days and percentages of LP CD4 T cells that (A,D) proliferated (CFSE^dim) or expressed (B, E) CD25 or (C, F) HLA-DR⁺CD38⁺ determined using multi-color flow cytometry. FM2 (Fluorescence minus two; HLA-DR and CD25) and isotype control (CD38) values have been subtracted. Values are shown as mean ± SEM. Statistical analysis: Paired t tests were conducted to determine differences in proliferation or activation between unstimulated and TCR bead stimulated conditions and between each butyrate concentration versus no butyrate conditions. *P < 0.05, *P < 0.01, ^#P < 0.06.

Fig. 2.

Butyrate decreases proliferation of LP and PB T helper cells. CFSE-labelled (A) LPMC (N = 6) or (B) PBMC (N = 6) were cultured with TCR-activating beads and exogenous butyrate (0.0625–0.5 mM) for four days with mitogenic stimulation during the last 4 h of culture. Percentages of proliferating (A, D) Th1, (B, E) Th22 or (C, F) Th17 cells were determined using multi-color flow cytometry. Isotype control values have been subtracted. Values are shown as mean ± SEM of net proliferation (TCR-stimulated minus unstimulated). Statistical analysis: Paired t tests were conducted to determine differences in Th subset proliferation between butyrate concentrations versus no butyrate conditions. Percentages represent the average percentage decrease in Th cell proliferation versus no butyrate. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 3.

Inhibition of LP CD4 T cell proliferation is dependent of the timing of butyrate exposure relative to TCR stimulation. LPMC (N = 3) were pre-labelled with CFSE and either (A) pre-treated with exogenous butyrate (0.5 mM) for 0, 2, 6 and 18 h before the addition of TCR-activating beads (TCR beads) or (B) exposed to exogenous butyrate 0, 6, 18 or 36 h after the addition of TCR beads. Percentages of proliferated (CFSE^dim) LP CD4 T cells was measured 4 days after the addition of TCR beads using multi-color flow cytometry. Values are shown as mean ± SEM. Statistical analysis: Paired t tests were conducted to determine differences in proliferation between TCR bead-stimulated LPMC in the absence of butyrate (open bar) versus the simultaneous addition of butyrate (filled bar; 0hrs) as well as comparisons between 0hrs butyrate and each time point either pre- or post-treatment. *P < 0.05, **P < 0.01.

Fig. 4.

Higher concentrations of acetate and propionate relative to butyrate are required to inhibit LP CD4 T cell proliferation and activation. LPMC were pre-labelled with CFSE and cultured with TCR-activating beads (TCR beads) and exogenous butyrate (0.5 mM) or (A-C) acetate (1‒10 mM; N = 3) or (D-F) propionate (0.25‒10 mM; N = 4) for four days and percentages of LP CD4 T cells that (A,D) proliferated (CFSE^dim) or expressed (B, E) CD25 or (C, F) HLA-DR⁺CD38⁺ determined using multi-color flow cytometry. FM2 (Fluorescence minus two; HLA-DR and CD25) and isotype control (CD38) values have been subtracted. Values are shown as mean ± SEM of net proliferation or activation (TCR-stimulated minus unstimulated). Statistical analysis: Paired t tests were conducted to determine differences in proliferation or activation between each SCFA concentration (filled bars) versus no SCFA condition (open bar). *P < 0.05, **P < 0.01, ***p < 0.01, ^#P ≤ 0.07.

Fig. 5.

HDAC inhibitors phenocopy butyrate’s ability to decrease LP CD4 T cell proliferation and activation. (A) Levels of acetylation of histone 3 lysine 9 (H3K9) were measured in LP CD4 T cells by multi-color flow cytometry 24hrs after culture of LPMC (N = 4) in the presence or absence of exogenous butyrate (0.5 mM) and TCR-activating beads (TCR beads). Values are shown as the mean fluorescence intensity (MFI) of acetylation of H3K9 (acetylated H3) (mean ± SEM). Statistical analysis: Paired t-tests were conducted to determine differences in acetylation of H3K9 levels in LP CD4 T cells in the presence or absence of butyrate in unstimulated or TCR bead-stimulated conditions. *P < 0.05. (B) CFSE-labelled LPMC (N = 3) were exposed to TCR beads and Class I specific HDAC inhibitors (HDACi’s; apicidin and MGCD) or pan HDACi’s (TSA and ITF2357) and levels of LP CD4 T cell proliferation (CFSE^dim) determined at 4 days using multi-color flow cytometry. Values are shown as mean ± SEM. Statistical analysis Paired t tests were conducted to determine differences in proliferation between TCR bead-stimulated LPMC in the absence of butyrate (open bar) and in the presence of butyrate or HDAC inhibitors (filled bars). *P < 0.05, #P ≤ 0.07. (C) LPMC (N = 3) or (D) purified LP CD4 T cells (N = 3) were cultured with or without TCR beads and Apicidin (25‒100 nM) for four days and percentages of LP CD4 T cells that proliferated (CFSE^dim) or expressed CD25 or HLA-DR⁺CD38⁺ determined using multi-color flow cytometry. FM2 (Fluorescence minus two; HLA-DR and CD25) and isotype control (CD38) values have been subtracted. Values are shown as mean ± SEM. Statistical analysis: Paired t tests were conducted to determine differences in proliferation or activation between no stimulated and TCR bead-stimulated conditions and between butyrate or apicidin concentrations versus no butyrate/apicidin conditions in the presence or absence (C only) of TCR beads. *P < 0.05, **P < 0.01, ^#P ≤ 0.07.

Fig. 6.

GPR43 agonist phenocopies butyrate’s ability to decrease LP CD4 T cell proliferation. CFSE-labelled purified LP CD4 T cells (N = 4) were cultured with TCR beads, with or without butyrate (0.5 mM) or GPR agonists (1‒100 μM; N = 3 for 1 μM and 100 μM) for four days and percentages of LP CD4 T cells that proliferated (CFSE^dim) determined using multi-color flow cytometry. Values are shown as mean ± SEM of net proliferation (TCR-stimulated minus unstimulated). Statistical analysis: Paired t tests were conducted to determine differences in proliferation between butyrate or GPR agonist concentrations versus no butyrate or GPR agonist conditions. *P < 0.05, **P < 0.01.