An essential role of Ffar2 (Gpr43) in dietary fibre-mediated promotion of healthy composition of gut microbiota and suppression of intestinal carcinogenesis

Abstract

Composition of the gut microbiota has profound effects on intestinal carcinogenesis. Diet and host genetics play critical roles in shaping the composition of gut microbiota. Whether diet and host genes interact with each other to bring specific changes in gut microbiota that affect intestinal carcinogenesis is unknown. Ability of dietary fibre to specifically increase beneficial gut microbiota at the expense of pathogenic bacteria in vivo via unknown mechanism is an important process that suppresses intestinal inflammation and carcinogenesis. Free fatty acid receptor 2 (FFAR2 or GPR43) is a receptor for short-chain fatty acids (acetate, propionate and butyrate), metabolites of dietary fibre fermentation by gut microbiota. Here, we show FFAR2 is down modulated in human colon cancers than matched adjacent healthy tissue. Consistent with this, Ffar2(-/-) mice are hypersusceptible to development of intestinal carcinogenesis. Dietary fibre suppressed colon carcinogenesis in an Ffar2-dependent manner. Ffar2 played an essential role in dietary fibre-mediated promotion of beneficial gut microbiota, Bifidobacterium species (spp) and suppression of Helicobacter hepaticus and Prevotellaceae. Moreover, numbers of Bifidobacterium is reduced, whereas those of Prevotellaceae are increased in human colon cancers than matched adjacent normal tissue. Administration of Bifidobacterium mitigated intestinal inflammation and carcinogenesis in Ffar2(-/-) mice. Taken together, these findings suggest that interplay between dietary fibre and Ffar2 play a key role in promoting healthy composition of gut microbiota that stimulates intestinal health.

Figure 1

Expression of FFAR2 is diminished in human colon cancers. (a) Expression of FFAR2 was assessed by quantitative polymerase chain reaction in 14 colon cancer and matched adjacent normal tissue. (b) Data from a was pooled and re-plotted. ***P<0.0001.

Figure 2

Ffar2 suppresses colon carcinogenesis. WT and Ffar2^−/− mice (littermates) were fed with conventional mouse chow. (a) Experimental model to induce inflammation-associated colon carcinogenesis. (b) Survival of WT and Ffar2^−/− mice subjected to AOM/DSS treatment as described in a (n=8 mice). (c) A representative photograph of H&E stained cross-section of colons of untreated (UT) or AOM/DSS treated WT and Ffar2^−/− mice. Original magnification 200 ×. (d) After the completion of first cycle of DSS, mice were gavaged with FITC-dextran and 6 h later FITC was quantified in serum (n=5 mice per genotype). (e) A representative photographs of luminal side of colons from WT and Ffar2^−/− animals on day 70 after AOM/DSS treatment. (f) Polyp burden in WT and Ffar2^−/− mice after AOM/DSS treatment (n=6 mice). A representative or pooled data from 2 experiments are shown. Error bars represent standard deviation. **P<0.002.

Figure 3

Ffar2 regulates tumorigenesis in Apc^Min/+ mice. (a) Ffar2 mRNA expression by colonic and small intestinal epithelium (n=3 mice). A representative of two experiments is shown. (b) Polyp burden in colon and small intestine of Apc^Min/+ (n=7) and Ffar2^−/− Apc^Min/+ mice (n=6). *P<0.01, **P<0.002.

Figure 4

An essential role of Ffar2 in dietary fibre-mediated promotion of Bifidobacterium and suppression of Prevotellaceae and H. hepaticus. (a) Fecal DNA from Ffar2^−/− mice and their WT littermates were evaluated for abundance of indicated gut microbiota. (b) Ffar2^−/− mice and their WT littermates maintained on conventional diet (Conv) were fed with FF or FP diets. The abundance of indicated bacterial groups in feces was quantified 1 day before (Conv) switching the diet or after 1 week on experimental diets (FF or FP). Shown is the relative abundance of indicated bacterial groups to that of total bacteria. *P<0.05, **P<0.01, ***P<0.0001. (n=4 mice). A representative of two experiments is shown.

Figure 5

An essential role of Ffar2 in dietary fibre-mediated suppression of colonic inflammation and carcinogenesis. WT and Ffar2^−/− mice (littermates) were fed with FF and FP diets and 1 month later were injected with AOM (i.p.). After 1 week, all the mice were given 1.5% DSS in drinking water for next 6 days. (a) Weight loss during and after DSS treatment. (b) A representative photograph of H&E stained cross-section of colons of mice after first cycle of DSS as treated in a. (c) Histopathological score (inflammation+epithelial damage) of colons from mice 4 days after completion of DSS treatment. (d) Polyp burden in colons of indicated mice at the end of experiment. A representative of two experiments is shown (n=4 mice). **P<0.01, ***P<0.0001.

Figure 6

Bifidobactreia suppress colonic inflammation and carcinogenesis in Ffar2^−/− mice. (a) Ffar2^−/− mice fed with FP diet and 1 month later were challenged with AOM/DSS and B. infantis as shown. B. infantis was gavaged everyday (2 × 10⁸ cfu/mouse) as indicated from day of AOM injection till the completion of DSS treatment. (b) Relative abundance of Bifidobacterium in mice gavaged with B. infantis or saline as in a. Shown is the weight loss (c) during and after DSS challenge. (d) A representative photograph of H&E stained cross-section of colons of mice 4 days after cessation of DSS treatment. (e) Histopathological score (inflammation+epithelial damage) of colons. (f) Polyp burden in colons of mice treated as described in a at experimental endpoint. A representative of two experiments is shown (n=4 mice). *P<0.05, **P<0.01, ***P<0.0001.

Figure 7

Decreased abundance of Bifidobacterium spp in human colon cancers. (a) Relative abundance of indicated bacterial groups in DNA extracted from colon cancers and matched adjacent normal tissue was measured by quantitative polymerase chain reaction. (b) Relative levels of Bifidobacterium in colon cancer and matched adjacent normal tissue. The horizontal line represents the median value. *P<0.05. UD, undetectable. Statistical significance was calculated using Mann–Whitney test with two-tailed analysis.