The Protective Role of Bacteroides fragilis in a Murine Model of Colitis-Associated Colorectal Cancer

Abstract

Many patients with chronic inflammation of the gut, such as that observed in inflammatory bowel disease (IBD), develop colorectal cancer (CRC). Recent studies have reported that the development of IBD and CRC partly results from an imbalanced composition of intestinal microbiota and that intestinal inflammation in these diseases can be modulated by the microbiota. The human commensal Bacteroides fragilis is best exemplified playing a protective role against the development of experimental colitis in several animal disease models. In this study, we found that gut inflammation caused by dextran sulfate sodium (DSS) treatment was inhibited by B. fragilis colonization in mice. Further, we reveal a protective role of B. fragilis treatment against colon tumorigenesis using an azoxymethane (AOM)/DSS-induced model of colitis-associated colon cancer in mice and demonstrate that the decreased tumorigenesis by B. fragilis administration is accompanied by inhibited expression of C-C chemokine receptor 5 (CCR5) in the gut. We show direct evidence that the inhibition of tumor formation provided by B. fragilis in colitis-associated CRC animals was dependent on the production of polysaccharide A (PSA) from B. fragilis and that Toll-like receptor 2 (TLR2) signaling was responsible for the protective function of B. fragilisIMPORTANCE The incidence of colorectal cancer (CRC) is rapidly growing worldwide, and there is therefore a greater emphasis on studies of the treatment or prevention of CRC pathogenesis. Recent studies suggested that consideration of the microbiota is unavoidable to understand inflammation and tumorigenesis in the gastrointestinal tract. We demonstrate, using a mouse model of colitis-associated CRC, that human commensal B. fragilis protects against colon tumorigenesis. The protective role against tumor formation provided by B. fragilis is associated with inhibition of expression of the chemokine receptor CCR5 in the colon. The molecular mechanism for protection against CRC provided by B. fragilis is dependent on polysaccharide A production and is mediated by TLR2 signaling. Our results suggest that the commensal microorganism B. fragilis can be used to prevent inflammation-associated CRC development and may provide an effective therapeutic strategy for CRC.

Keywords: Bacteroides fragilis; Toll-like receptor 2; colitis-associated colorectal cancer; inflammation; polysaccharide A.

FIG 1

B. fragilis colonization protects mice against dextran sulfate sodium (DSS)-induced colitis. C57BL/6J (B6) mice were treated orally with either PBS or B. fragilis three times a week starting a week prior to DSS treatment and continuing until the end of the experiment (5 mice per group). DSS (2.5%) was administered via the drinking water for 6 days followed by administration of regular drinking water. Mice were sacrificed on day 8. (A) Percent weight change during DSS-induced colitis. (B) Inflammation score of colon sections of the indicated groups. (C) Expression levels of IL-1β, IL-6, TNF-α, IL-10, CCL3, and CCR5 from colon homogenates of control or B. fragilis-colonized mice on day 8 of DSS treatment. Relative values were normalized to that of β-actin. Data are representative of results from at least two experiments. Statistical significance was calculated using unpaired Student's t tests and nonparametric Mann-Whitney tests. *, P < 0.05; **, P < 0.005 (in all panels).

FIG 2

Colonization with B. fragilis protects mice against development of colon cancer. (A) Schematic overview of the azoxymethane (AOM)/dextran sulfate sodium (DSS)-induced colorectal cancer model. C57BL/6J (B6) mice were treated orally with PBS (control), B. fragilis, or B. fragilis ΔPSA three times a week starting a week prior to AOM injection until the end of the experiment (8 mice per group). After an initial AOM intraperitoneal injection (10 mg/kg), 3% DSS was administered via the drinking water for 6 days followed by administration of regular drinking water. The mice underwent a second DSS treatment cycle with 3% DSS-treated water on day 25 for 6 days and a third cycle with 1.5% DSS-treated water on day 55 for 4 to 6 days. The mice were sacrificed on day 81 post-AOM injection. (B) Percent weight change in the indicated groups during DSS treatment. (C) Number of tumors in the distal colon (left) and the sum of tumor size (right) in the indicated groups on day 81 of AOM-DSS treatment. (D) Scores of hyperplasia (left) and dysplasia (right) from longitudinal sections of distal colon of the indicated groups on day 81 of AOM-DSS treatment. Data are representative of results from at least two experiments. Statistical significance was calculated using unpaired Student's t tests and nonparametric Mann-Whitney tests. *, P < 0.05 (in all panels).

FIG 3

B. fragilis colonization regulates expression of proinflammatory cytokines and gene signatures associated with inflammation and tumor pathogenesis during colon cancer development in mice. (A) Expression levels of the indicated genes from colon homogenates of control, B. fragilis-colonized, or B. fragilis ΔPSA-colonized mice on day 81 of AOM-DSS treatment. Relative values were normalized to that of β-actin. (B) Immunofluorescent staining of CCR5 in colonic tissues of untreated control and B. fragilis-colonized mice on day 81 of AOM-DSS treatment. Data are representative of results from at least two experiments. Statistical significance was calculated using unpaired Student's t tests. *, P < 0.05 (in all panels).

FIG 4

B. fragilis utilizes TLR2 signaling for its protective role against the development of colitis-associated cancer in mice. C57BL/6J (B6) or Tlr2^−/− mice were treated orally with either PBS or B. fragilis three times a week starting a week prior to AOM injection until the end of the experiment (8 mice per group). After an initial AOM intraperitoneal injection (10 mg/kg), 2.5% DSS was administered via the drinking water for 6 days followed by administration of regular drinking water. The mice underwent a second DSS treatment cycle with 2.5% DSS-treated water on day 25 for 6 days and a third cycle with 1.5% DSS-treated water on day 55 for 4 to 6 days. The mice were sacrificed on day 81 post-AOM injection. (A) Percent weight change in the indicated groups during DSS treatment. WT, wild type. (B) Number of tumors in the total (left), proximal (middle), or distal (right) colon of the indicated groups on day 81 of AOM-DSS treatment. (C) Scores of hyperplasia (left) and dysplasia (right) from longitudinal sections of colon of the indicated groups on day 81 of AOM-DSS treatment. Data are representative of results from at least two experiments. Statistical significance was calculated using unpaired Student's t tests and nonparametric Mann-Whitney tests. *, P < 0.05; **, P < 0.005; ****, P < 0.0001 (in all panels).