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. 2012 Jun 15;302(12):G1405-15.
doi: 10.1152/ajpgi.00543.2011. Epub 2012 Apr 19.

Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells

Mary A Zimmerman  1 , Nagendra Singh, Pamela M Martin, Muthusamy Thangaraju, Vadivel Ganapathy, Jennifer L Waller, Huidong Shi, Keith D Robertson, David H Munn, Kebin Liu
Affiliations

Butyrate suppresses colonic inflammation through HDAC1-dependent Fas upregulation and Fas-mediated apoptosis of T cells

Mary A Zimmerman et al. Am J Physiol Gastrointest Liver Physiol. .

Abstract

Butyrate, an intestinal microbiota metabolite of dietary fiber, has been shown to exhibit protective effects toward inflammatory diseases such as ulcerative colitis (UC) and inflammation-mediated colorectal cancer. Recent studies have shown that chronic IFN-γ signaling plays an essential role in inflammation-mediated colorectal cancer development in vivo, whereas genome-wide association studies have linked human UC risk loci to IFNG, the gene that encodes IFN-γ. However, the molecular mechanisms underlying the butyrate-IFN-γ-colonic inflammation axis are not well defined. Here we showed that colonic mucosa from patients with UC exhibit increased signal transducer and activator of transcription 1 (STAT1) activation, and this STAT1 hyperactivation is correlated with increased T cell infiltration. Butyrate treatment-induced apoptosis of wild-type T cells but not Fas-deficient (Fas(lpr)) or FasL-deficient (Fas(gld)) T cells, revealing a potential role of Fas-mediated apoptosis of T cells as a mechanism of butyrate function. Histone deacetylase 1 (HDAC1) was found to bind to the Fas promoter in T cells, and butyrate inhibits HDAC1 activity to induce Fas promoter hyperacetylation and Fas upregulation in T cells. Knocking down gpr109a or slc5a8, the genes that encode for receptor and transporter of butyrate, respectively, resulted in altered expression of genes related to multiple inflammatory signaling pathways, including inducible nitric oxide synthase (iNOS), in mouse colonic epithelial cells in vivo. Butyrate effectively inhibited IFN-γ-induced STAT1 activation, resulting in inhibition of iNOS upregulation in human colon epithelial and carcinoma cells in vitro. Our data thus suggest that butyrate delivers a double-hit: induction of T cell apoptosis to eliminate the source of inflammation and suppression of IFN-γ-mediated inflammation in colonic epithelial cells, to suppress colonic inflammation.

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Figures

Fig. 1.
Fig. 1.
Signal transducer and activator of transcription 1 (STAT1) hyperactivation is linked to T cell accumulation in the colonic mucosa of human ulcerative colitis (UC) specimens. Immunohistochemical staining of CD3+ T cells (A), and cytoplasmic (cSTAT1) and nuclear (pSTAT1) protein (B) in normal colon tissues and colonic tissue specimens of human patients with UC. CD3 and STAT1 immunoreactivity is shown as the brown color, whereas cells that are unreactive are indicated by the blue counterstain. Shown are representative images. a and c: Normal colon tissues from patients with colon cancer b and d: colon tissues from human patients with UC. 1: Image of entire tissue microarray (TMA) tissue disc; 2: high magnification of 1. C: quantification of the number of CD3+ T cells and STAT1-positive cells in the colon tissues. The number of CD3+ T cells in each specimen printed on the TMA was counted, and cSTAT1 and nSTAT1 were scored. Left: difference in the number of T cells between normal (n = 7) and UC (n = 6) colon tissues. Middle: difference in the percentage of cSTAT1-positive colonic epithelial cells between normal and UC colon tissues. Right: difference in the percent of nSTAT1-positive colonic epithelial cells between normal and UC colon tissues.
Fig. 2.
Fig. 2.
Butyrate inhibits CD4+ T cell activation. A: T cell activation kinetics. Spleen cells were cultured in anti-CD3/CD28 mAb-coated plates and analyzed by staining with CD4- and CD25-specific mAbs, or CD4- and CD69-specific mAbs, respectively, at the indicated time points. Percentages of CD25- and CD69-positive cells in the CD4+ T cell population and mean fluorescence intensity (MFI) of CD25 and CD69 were quantified. B: inhibition of T cell activation by butyrate. Spleen cells were cultured as described above in the presence of butyrate for 24 h and analyzed for CD25- and CD69-positive cells in the CD4+ T cell population, as well as the MFI of CD25 and CD69 of the CD4+ T cell population.
Fig. 3.
Fig. 3.
Butyrate induces T cell apoptosis. Purified CD4+ (A) and CD8+ (B) T cells were cultured in anti-CD3/CD28 mAb-coated plates for 24 h. Butyrate was then added to the culture for another 24 h. Cellular proliferation/survival was analyzed using MTT assays. Cellular proliferation in the absence of butyrate was set as 100%. C: spleen cells were cultured in the absence (control) and presence (+Anti-CD3/CD28) of anti-CD3/CD28 mAbs for 24 h. Various concentrations of butyrate were then added to the cultures for another 24 h. Cells were collected and stained with Annexin V and CD4 mAb and analyzed by flow cytometry for apoptosis. Left: apoptosis profiles of CD4+ T cells are shown. Right: apoptotic cell death of CD4+ T cells was quantified by the formula: % Annexin V cells = % Annexin V-positive cells in the presence of butyrate − % Annexin V-positive cells in the absence of butyrate. Shown are representative results from 1 of 3 experiments. D: CD8+ T cells apoptosis in response to butyrate was analyzed as in C.
Fig. 4.
Fig. 4.
Butyrate inhibits Fas promoter-bound histone deacetylase 1 (HDAC1) to induce Fas promoter hyperacetylation and Fas upregulation in T cells. A: induction of T cell apoptosis by butyrate is Fas dependent. Spleen cells from Fasgld and Faslpr were cultured in anti-CD3/CD28 mAb-coated plates with butyrate for 24 h. Cells were then collected and analyzed for apoptosis of CD4+ (A) and CD8+ (B) T cells as in Fig. 2. B: Fas expression level is upregulated by butyrate treatment. Spleen cells were cultured in anti-CD3/CD28-coated plates in the absence or presence of various concentrations of butyrate for 24 h. Cells were then stained with CD4- and Fas-specific mAbs (top, left) or CD8- and Fas-specific mAbs (top, right) and analyzed by flow cytometry. The Fas protein level is represented by the mean fluorescent intensity. The butyrate concentrations are indicated at the upper left corner of each plot, and the Fas MFI is indicated at the upper right corner of each plot. Shown is 1 representative result of 3 independent experiments. Bottom: Fas MFI in CD4+ and CD8+ T cells after butyrate treatment was quantified and presented. Data are means ± SD. C: HDAC1 association with the Fas promoter and butyrate-induced Fas promoter hyperacetylation. Left: mouse Fas promoter structure showing the chromatin immunoprecipitation (ChIP) PCR amplified region. Right: ChIP analysis of HDAC1, HDAC2, and acetyl-H3K9 association with the mouse Fas promoter. The HDAC1-, HDAC2-, and acetyl-H3K9-bound chromatin fragments were immunoprecipitated with the indicated antibodies and analyzed by PCR using Fas promoter sequence-specific primers. D: inhibition of HDAC1 activity by butyrate. Recombinant human HDAC1 was assayed for its activity using a commercially available kit in the absence or presence of butyrate. WT, wild-type.
Fig. 5.
Fig. 5.
Expression of the butyrate transporter and receptor in T cells and its relevance to butyrate-induced cell death. A: slc5a8 and gpr109a promoter structure. The 5′ regulatory region (−500 to +1,000 relative to the transcription initiation site) of the slc5a8 (left) and gpr109a (right) were analyzed with the program, Methprimer, for CpG island identification. The bisulfite sequencing PCR primer regions are indicated. B: methylation status of the slc5a8 (top) and gpr109a (bottom) promoter regions. ●: methylated CpGs; ○: unmethylated CpGs. C: slc5a8 and gpr109a expression in mouse T cells. CD4+ T cells were purified from WT mouse spleen and analyzed for slc5a8 and gpr109a expression levels by RT-PCR. D: sensitivity of T cells from slc5a8−/− and gpr109a−/− mice to butyrate. Spleen cells were cultured in anti-CD3/CD28-coated plates overnight, followed by incubation with butyrate at the indicated concentrations for 24 h. Cells were then stained with CD4-specific mAb and Annexin V (left), or CD8-specific mAb and Annexin V (right), respectively, and analyzed by flow cytometry as above. Data are means ± SD.
Fig. 6.
Fig. 6.
slc5a8- and gpr109a-mediated expression of genes in the cytokine and TGF-β signaling pathways in mouse colon tissues in vivo. Total RNA was isolated from fresh colons of WT, gpr109a−/−, and slc5a8−/− mice and used for RT-PCR analysis of the indicated genes. KO, knockout; iNOS, inducible nitric oxide synthase; SOCS, suppressor of cytokine signaling.
Fig. 7.
Fig. 7.
Butyrate inhibits IFN-γ-induced STAT1 activation and iNOS induction in human colonic epithelial cells in vitro. A: IFN-γR level on CCD-841 cells. CCD-841 cells were stained with IFN-γR-specific mAb and analyzed by flow cytometry. Shaded area: IgG isotype control, solid line: IFN-γR-specific staining. B: butyrate inhibits IFN-γ signaling. CCD-841 cells were cultured in the presence of butyrate at the indicated concentrations for 2 h. IFN-γ (100 U/ml) was then added and cultured for 24 h. The cells were analyzed by Western blotting with pSTAT1- and β-actin-specific mAbs, sequentially. C: colon carcinoma cell line T84 cells were cultured in the absence or presence of IFN-γ (100 U/ml) and LPS (10 μg/ml) or IFN-γ, LPS and butyrate (3 mM) for ∼20 h, and analyzed for iNOS transcript level by RT-PCR.
Fig. 8.
Fig. 8.
Model of butyrate function. Butyrate delivers a double-hit to suppress colonic inflammation: first, butyrate inhibits STAT1 hyperactivation in colonic epithelial cells to inhibit IFN-γ-mediated chronic inflammation; second, butyrate inhibits Fas promoter-bound HDAC1 activity to induce Fas promoter hyperacetylation and Fas upregulation, resulting in enhanced apoptosis of T cells, which leads to decreased accumulation of T cells in the inflamed colonic mucosa and consequently elimination of the source of inflammation. IBD, inflammatory bowel disease; CRC, colorectal cancer.
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