Activation of Gpr109a, receptor for niacin and the commensal metabolite butyrate, suppresses colonic inflammation and carcinogenesis

Abstract

Commensal gut microflora and dietary fiber protect against colonic inflammation and colon cancer through unknown targets. Butyrate, a bacterial product from fermentation of dietary fiber in the colon, has been implicated in this process. GPR109A (encoded by Niacr1) is a receptor for butyrate in the colon. GPR109A is also a receptor for niacin, which is also produced by gut microbiota and suppresses intestinal inflammation. Here we showed that Gpr109a signaling promoted anti-inflammatory properties in colonic macrophages and dendritic cells and enabled them to induce differentiation of Treg cells and IL-10-producing T cells. Moreover, Gpr109a was essential for butyrate-mediated induction of IL-18 in colonic epithelium. Consequently, Niacr1(-/-) mice were susceptible to development of colonic inflammation and colon cancer. Niacin, a pharmacological Gpr109a agonist, suppressed colitis and colon cancer in a Gpr109a-dependent manner. Thus, Gpr10a has an essential role in mediating the beneficial effects of gut microbiota and dietary fiber in colon.

Figure 1. Pro-inflammatory phenotype of colonic CD4⁺T cells from Niacr1^−/− mice

(A) Foxp3 expression by CD4⁺ T cells from colonic lamina propria (LP) and spleens of WT and Niacr1^−/− mice. (B) Percent of Foxp3⁺ in CD4⁺ populations from colonic LP and spleens of WT and Niacr1^−/− mice (n=3). (C and E) IL-10 and IL-17 expression by (PMA+ionomycin)-stimulated colonic LP and splenic CD4⁺ T cells from WT and Niacr1^−/− mice. (D and F) Quantification of IL-10 and IL-17 producing CD4⁺ T cells shown in C and E respectively (n=3). The numbers in A, C and E represent percentage of positive cells in indicated area. Error bars represent standard deviation (SD) of mean. *p<0.002. A representative of 3 independent experiments is shown.

Figure 2. Butyrate and niacin induce anti-inflammatory properties in DCs and macrophages in a Gpr109a-dependent manner

(A) Foxp3 expression by OT-II CD4⁺ T cell differentiated in the presence of TGFβ1, IL-2 and cognate peptide with colonic LP CD11c⁺ (CD45⁺I-A^b+CD11c⁺) and CD11b⁺ (CD45⁺I-A^b+CD11b⁺) cells from WT or Niacr1^−/− mice. (B) OT-II CD4⁺ T cell differentiated as described in A were restimulated with anti-CD3 and anti-CD28 antibodies. One day later, IL-10 and IL-17 in culture supernatants were quantified by ELISA. (C) Expression of Il10, Il6 and Aldh1a1 by colonic LP DCs and macrophages isolated from WT and Niacr1^−/− mice were quantified by qPCR. (D) Sorted splenic DCs were cultured with butyrate or niacin. Two days later, cells were harvested, and expression of Il10 and Aldh1a1 was measured by qPCR. (E) Naïve OT-II CD4+ T cells were differentiated with butyrate- or niacin-treated splenic DCs from indicated mice as described in A. Shown is the FoxP3 expression by differentiated CD4⁺ T cells. (F) WT and Niacr1^−/− mice were treated with antibiotics in the presence or absence of niacin in drinking water. Four weeks later, colons were harvested and FoxP3 expression by colonic CD4⁺ T cell was analyzed. The numbers in A, E and F represent percentage of positive cells in indicated area. Error bars represent SD of mean (n=2–3). A representative of at least 2 independent experiments is shown.

Figure 3. Gpr109a is required for butyrate- and niacin-mediated IL-18 induction

(A) Induction of IL-18 in neonatal colon. Colons of 6-day-old WT or Niacr1^−/− mice were cultured in medium with or without butyrate or niacin for 24 h, and Il18 mRNA was quantified using qPCR (n=3). (B) Expression of Il18 mRNA and protein in colonic epithelial cells isolated from WT or Niacr1^−/− mice were quantified using qPCR and ELISA, respectively (n=3). (C) One day after oral administration of butyrate or niacin in vivo, expression of Il18 in colonic epithelium of WT or Niacr1^−/− mice was quantified (n=3). Error bars represent SD of mean. A representative of 3 independent experiments is shown.

Figure 4. Increased susceptibility of Niacr1^−/− mice to colonic inflammation

(A) Experimental paradigm for induction of colonic inflammation and inflammation-associated colon cancer in mice; azoxymethane (AOM) by intraperitoneal injection; DSS in drinking water. At days 20 and 70, colons of mice were analyzed for inflammation and cancer, respectively. (B–D), Change in body weight, diarrhea and rectal bleeding of WT and Niacr1^−/− mice subjected to AOM+DSS (n≥4). On 20^th day of AOM+DSS treatment, colons were harvested and analyzed for histopathology. (E) Representative images of H&E-stained colonic sections from untreated or (AOM+DSS)-treated WT and Niacr1^−/− mice (original magnification, 200×). (F) Claudin-3 staining of colonic sections from untreated (UT) or (AOM+DSS)-treated (20^th day) WT and Niacr1^−/− mice (original magnification, 200×). (G) IL-10, IL-17 and IL-18 levels in colons of WT and Niacr1^−/− mice before and after AOM+DSS treatment (n=5). Error bars represent standard deviation of mean. Values are mean ± SD or representative of at least 2 independent experiments.

Figure 5. Increased susceptibility of Niacr1^−/− mice to colon cancer

(A) Representative photographs of dissected colons from WT and Niacr1^−/− mice on day 70 after animals were treated with AOM+DSS as described in Figure 4A. (B and C) Number and size distribution of colonic polyps induced by AOM+DSS treatment in WT and Niacr1^−/− mice (n=6). (C) Representative photographs of dissected colons of 3-month-old Apc^Min/+ and Niacr1^−/−Apc^Min/+ mice. (D) Number of polyps in colon and small intestine in Apc^Min/+ and Niacr1^−/−Apc^Min/+ mice (n=4). Values are mean ± SD or representative of 2 independent experiments.

Figure 6. Role of Gpr109a expressed in hematopoietic and non-hematopoietic cells in regulation of colitis and colitis-associated colon cancer

Reciprocal bone marrow chimeras of WT and Niacr1^−/− mice were subjected to (AOM+DSS)-induced model of colitis-associated colon cancer. (A) Weight loss and diarrhea after the first cycle of DSS (n≥4). *; significant difference between WT → Niacr1^−/− and Niacr1^−/− → Niacr1^−/− group for weight loss (p<0.05) and diarrhea (p<0.02). **; Significant difference between WT → WT and Niacr1^−/− → WT group for weight loss and diarrhea (p<0.02). (B) Representative photographs of dissected colons from bone marrow chimeric mice. (C) Tumor burden in bone marrow chimeras subjected to AOM+DSS induced colon cancer (n=4). Error bars represent SD of mean. A representative of 2 independent experiments is shown.

Figure 7. Niacin suppresses colonic inflammation and carcinogenesis in absence of gut microbiota and dietary fibers via Gpr109a

(A) Experimental paradigm for antibiotic treatment (gentamicin sulfate, ciprofloxacin, streptomycin and bacitracin in drinking water) and niacin administration to mice in the (AOM+DSS)-induced colon cancer model. (B) Weight loss in WT and Niacr1^−/− mice treated with antibiotics in the presence or absence of antibiotics and subjected to AOM+DSS as in A. (C) Tumor burden in WT and Niacr1^−/− mice under various treatment conditions. Error bars represent standard deviation of mean (n≥4). (D) Representative photographs of dissected colons of WT and Niacr1^−/− mice subjected to various treatments as described in A. (E) Niacr1^−/− mice were treated as in Figure 4A. Some mice also received rIL-10 or rIL-18 intraperitoneally (50 ng/mouse/injection) starting day 2 and every alternate day till day 60. Shown are the weight loss and diarrhea during first cycle of DSS. (F) Tumor burden on day 70 in Niacr1^−/− mice treated as described in (E) (n=4). (G) Two-month-old, Apc^Min/+ and Niacr1^−/−Apc^Min/+ mice were fed with dietary fiber containing normal chow (FC) or fiber-free (FF) chow. Some mice in FF diet group also received niacin in drinking water ad libitum. Five weeks later, mice were sacrificed and colonic polyps were counted (n=2–5). * p<0.007 (H) A representative photographs of dissected colon from mice subjected to experimental protocol described in G. Values represent mean ± SD or representative of at least 2 independent experiments.