Microbiota Metabolite Butyrate Differentially Regulates Th1 and Th17 Cells' Differentiation and Function in Induction of Colitis

Abstract

Background: How the gut microbiota regulates intestinal homeostasis is not completely clear. Gut microbiota metabolite short-chain fatty acids (SCFAs) have been reported to regulate T-cell differentiation. However, the mechanisms underlying SCFA regulation of T-cell differentiation and function remain to be investigated.

Methods: CBir1, an immunodominant microbiota antigen, transgenic T cells were treated with butyrate under various T-cell polarization conditions to investigate butyrate regulation of T-cell differentiation and the mechanism involved. Transfer of butyrate-treated CBir T cells into Rag1-/- mice was performed to study the in vivo role of such T cells in inducing colitis.

Results: Although butyrate promoted Th1 cell development by promoting IFN-γ and T-bet expression, it inhibited Th17 cell development by suppressing IL-17, Rorα, and Rorγt expression. Interestingly, butyrate upregulated IL-10 production in T cells both under Th1 and Th17 cell conditions. Furthermore, butyrate induced T-cell B-lymphocyte-induced maturation protein 1 (Blimp1) expression, and deficiency of Blimp1 in T cells impaired the butyrate upregulation of IL-10 production, indicating that butyrate promotes T-cell IL-10 production at least partially through Blimp1. Rag1-/- mice transferred with butyrate-treated T cells demonstrated less severe colitis, compared with transfer of untreated T cells, and administration of anti-IL-10R antibody exacerbated colitis development in Rag-/- mice that had received butyrate-treated T cells. Mechanistically, the effects of butyrate on the development of Th1 cells was through inhibition of histone deacetylase but was independent of GPR43.

Conclusions: These data indicate that butyrate controls the capacity of T cells in the induction of colitis by differentially regulating Th1 and Th17 cell differentiation and promoting IL-10 production, providing insights into butyrate as a potential therapeutic for the treatment of inflammatory bowel disease.

Keywords: IL-10; Th1 cells; Th17 cells; butyrate; colitis.

FIGURE 1.

Butyrate differentially regulated the Th1 and Th17 cells’ differentiation. A and B, CBir1 Tg-naïve CD4⁺ T cells were cultured with irradiated APCs and CBir1 peptide in the presence or absence of butyrate (0.5 mM) under Th1 (IL-12) (A) and Th17 (TGF-β and IL-6) (B) conditions for 5 days. The expression of IL-10, IFN-γ, and IL-17 was examined by flow cytometry. C and D, The IFN-γ, IL-17, and IL-10 production in Th1 (C) and Th17 (D) cell culture supernatants was measured by ELISA. E, CBir1 Tg CD4⁺ T cells were injected IV into groups of Rag1^-/- mice, and the mice were fed with or without butyrate (300 mM) in drinking water. Lamina propria CD4⁺ T-cell cytokine production was determined by flow cytometry 10 days after T-cell transfer. Plot numbers represented the percentage of CD4⁺ T cells in the respective quadrants. Results were shown as mean ± SD. One representative of 3 experiments was performed. Student t test, *P < 0.05; ^**P < 0.01.

FIGURE 2.

Butyrate differentially regulated T-cell gene expression under Th1 and Th17 conditions. CBir1 Tg-naïve CD4⁺ T cells were cultured under Th1 and Th17 conditions with or without butyrate (0.5 mM) as indicated. A and B, The gene expression of T-bet under Th1 conditions (A) and Rorα, Rorγt, Runx1, and Batf under Th17 conditions (B) was examined by real-time PCR and normalized against gapdh. The results are shown as mean ± SD. One representative of 3 experiments was performed. Student t test, *P < 0.05; ^**P < 0.01; ^***P < 0.001.

FIGURE 3.

Butyrate induced IL-10 production by T cells through Blimp1. A, CBir1 Tg CD4⁺ T cells were cultured with Cbir1 peptide and irradiated APCs in the presence or absence of butyrate (0.5 mM). The gene expression of Blimp-1 and IL-10 was determinated by real-time PCR. B, Blimp1 expression in CD4⁺ T cells, cultured under Th1 and Th17 conditions, was examined by real-time PCR. C and D, CD4^CreBlimp1^+/fl (WT) and CD4^CreBlimp1^fl/fl (CKO) CD4⁺ T cells were cultured with irradiated APCs and anti-CD3 (5 μg/mL) in the presence or absence of butyrate (0.5 mM) for 5 days. The expression of IL-10 and IFN-γ in Th1 cells (C) and IL-10 and IL-17 in Th17 cells (D) was determined by flow cytometry. E, The production of IL-10 in Th1 culture supernatants and Th17 culture supernatants was measured by ELISA. Results were shown as mean ± SD. One representative of 3 experiments was performed. Student t test, *P < 0.05; ^**P < 0.01; ^***P < 0.001.

FIGURE 4.

Butyrate inhibited pathogenicity of CBir1 Tg T cells cultured under Th1 conditions. CBir1 Tg T cells cultured under Th1 conditions in the presence or absence of butyrate were injected IV into groups of Rag1^-/- mice. Mice were killed 4–6 weeks after T-cell transfer. A and B, Assessment of the severity of intestinal inflammation. Colonic histopathology was conducted using hematoxylin and eosin staining (A), and histological scores were examined and compared between the 2 groups by Mann-Whitney U test, *P < 0.05. B and C, LP CD4⁺ T-cell cytokine production was determined by flow cytometry. D, Colonic tissues were cultured ex vivo for 24 hours, and the relative cytokine production was measured by ELISA. Plot numbers represent the percentage of CD4⁺ T cells in the respective quadrants. Results are shown as mean ± SD. One representative of 3 experiments was performed. Student t test, *P < 0.05; ^**P < 0.01.

FIGURE 5.

Butyrate inhibited pathogenicity of CBir1 Tg T cells cultured under Th17 conditions. CBir1 Tg T cells cultured under Th17 conditions in the presence or absence of butyrate were injected IV into Rag1^-/- mice. Mice were killed, and the severity of intestinal inflammation was assessed 4–6 weeks after T-cell transfer. A and B, Colonic histopathology was conducted using hematoxylin and eosin staining (A), and colonic histological scores were examined and compared between the 2 groups by Mann-Whitney U test, *P < 0.05. B and C, LP CD4⁺ T-cell cytokine production was determined by flow cytometry. D, Colonic tissues were cultured for 24 hours, and cytokine production was measured by ELISA. Results are shown as mean ± SD. One representative of 3 experiments was performed. Student t test, *P < 0.05; ^**P < 0.01.

FIGURE 6.

Administration of anti-IL-10R antibody worsened the colitis induced by T cells treated with butyrate. Control or butyrate-treated T cells, cultured under Th1 conditions, were injected IV into groups of Rag1^-/- mice, respectively. A group of mice that received butyrate-treated T cells under Th1 conditions were administered with anti-IL-10R antibody intraperitoneally twice a week. The other 2 groups of mice were injected with IgG. Mice were killed 4–6 weeks after T-cell transfer. Colitis severity and histopathological scores are shown. Mann-Whitney U test, *P < 0.05; ^**P < 0.01. C, LP CD4⁺ T-cell cytokine expression was determined by flow cytometry. D, Colonic tissues were cultured ex vivo for 24 hours, and the cytokine production was measured by ELISA. Plot numbers represent the percentage of CD4⁺ T cells in the respective quadrants. Results are shown as mean ± SD. One representative of 3 experiments was performed. One-way ANOVA test, *P < 0.05; ^**P < 0.01.

FIGURE 7.

The effect of butyrate on Th1, but not Th17, differentiation is through HDAC inhibitory activity, independent of GPR43. GPR43^-/- and WT CBir1 Tg-naïve CD4⁺ T cells were cultured under Th1 and Th17 conditions for 5 days in the presence or absence of butyrate (0.5 mM). A and B, The expression of IFN-γ, IL-17, and IL-10 on Th1 cells (A) and Th17 cells (B) was examined by flow cytometry. Plot numbers represent the percentages of IFN-γ⁺, IL-17⁺, and IL-10⁺ T cells in the respective quadrants. C and D, CBir1 Tg T cells were treated with butyrate in addition to HDAC inhibitor TSA (30 nM) under Th1 conditions (C) and Th17 conditions (D), respectively, for 5 days. Results are shown as mean ± SD. One representative of 2 experiments was performed. Student t test, 1-way ANOVA test, *P < 0.05; ^**P < 0.01.