BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF), a molecular subclass of the common human commensal, B. fragilis, has been associated with inflammatory bowel disease. ETBF colitis is characterized by the activation of Stat3 and a Th17 immune response in the colonic mucosa. This study was designed to investigate the time course and cellular distribution of Stat3 activation in ETBF-colonized mice. METHODS: C57BL/6 wild-type, C57BL/6, or Rag-1 mice were inoculated with saline, nontoxigenic B. fragilis or ETBF. Histologic diagnosis and mucosal Stat activation (immunohistochemistry, Western blot, and/or electrophorectic mobility shift assay) were evaluated over time (6-24 h, 1-7 d, and 1-18 mo after inoculation). Mucosal permeability was evaluated at 16 hours, 1 day, and 3 days. Mucosal immune responses were evaluated at 1 week, and 12 and 18 months. RESULTS: ETBF induced rapid-onset colitis that persisted for up to 1 year. Stat3 activation (pStat3) was noted in the mucosal immune cells within 16 hours, with colonic epithelial cell activation evident at 24 hours after inoculation. ETBF-induced increased mucosal permeability was first observed at 24 hours after inoculation, after which the initial immune cell pStat3 activation was noted. Immune cell pStat3 was present in the absence of epithelial pStat3 (C57BL/6). Epithelial pStat3 was present in the absence of T and B cells (Rag-1 mice). pStat3 persisted in the epithelial and immune cells for 1 year, characterized by isolated pStat3-positive cell clusters, with varying intensity distributed through the proximal and distal colon. Similarly, mucosal Th17 immune responses persisted for up to 1 year. Loss of fecal ETBF colonization was associated with the loss of mucosal pStat3 and Th17 immune responses. CONCLUSIONS: ETBF rapidly induces immune cell pStat3, which is independent of epithelial pStat3. This occurs before ETBF-induced mucosal permeability, suggesting that ETBF, likely through B. fragilis toxin and its action on the colonic epithelial cell, triggers mucosal immune cell Stat3 activation. Peak mucosal Stat3 activation (immune and epithelial cells) occurs subsequently when other colonic bacteria may contribute to the ETBF-initiated immune response due to barrier dysfunction. ETBF induces long-lived, focal colonic Stat3 activation and Th17 immune responses dependent on the ongoing ETBF colonization. Further study is needed to evaluate the early mucosal signaling events, resulting in epithelial Stat3 activation and the sequelae of long-term colonic Stat3 activation.

Individuals with sporadic colorectal cancer (CRC) frequently harbor abnormalities in the composition of the gut microbiome; however, the microbiota associated with precancerous lesions in hereditary CRC remains largely unknown. We studied colonic mucosa of patients with familial adenomatous polyposis (FAP), who develop benign precursor lesions (polyps) early in life. We identified patchy bacterial biofilms composed predominately of Escherichia coli and Bacteroides fragilis Genes for colibactin (clbB) and Bacteroides fragilis toxin (bft), encoding secreted oncotoxins, were highly enriched in FAP patients colonic mucosa compared to healthy individuals. Tumor-prone mice cocolonized with E. coli (expressing colibactin), and enterotoxigenic B. fragilis showed increased interleukin-17 in the colon and DNA damage in colonic epithelium with faster tumor onset and greater mortality, compared to mice with either bacterial strain alone. These data suggest an unexpected link between early neoplasia of the colon and tumorigenic bacteria.

IL17-producing Th17 cells, generated through a STAT3-dependent mechanism, have been shown to promote carcinogenesis in many systems, including microbe-driven colon cancer. Additional sources of IL17, such as γδ T cells, become available under inflammatory conditions, but their contributions to cancer development are unclear. In this study, we modeled Th17-driven colon tumorigenesis by colonizing Min(Ap) (c+/-) mice with the human gut bacterium, enterotoxigenic Bacteroides fragilis (ETBF), to investigate the link between inflammation and colorectal cancer. We found that ablating Th17 cells by knocking out Stat3 in CD4(+) T cells delayed tumorigenesis, but failed to suppress the eventual formation of colonic tumors. However, IL17 blockade significantly attenuated tumor formation, indicating a critical requirement for IL17 in tumorigenesis, but from a source other than Th17 cells. Notably, genetic ablation of γδ T cells in ETBF-colonized Th17-deficient Min mice prevented the late emergence of colonic tumors. Taken together, these findings support a redundant role for adaptive Th17 cell- and innate γδT17 cell-derived IL17 in bacteria-induced colon carcinogenesis, stressing the importance of therapeutically targeting the cytokine itself rather than its cellular sources. Cancer Res; 76(8); 2115-24. ©2016 AACR.

Environmental factors clearly affect colorectal cancer (CRC) incidence, but the mechanisms through which these factors function are unknown. One prime candidate is an altered colonic microbiota. Here we show that the mucosal microbiota organization is a critical factor associated with a subset of CRC. We identified invasive polymicrobial bacterial biofilms (bacterial aggregates), structures previously associated with nonmalignant intestinal pathology, nearly universally (89%) on right-sided tumors (13 of 15 CRCs, 4 of 4 adenomas) but on only 12% of left-sided tumors (2 of 15 CRCs, 0 of 2 adenomas). Surprisingly, patients with biofilm-positive tumors, whether cancers or adenomas, all had biofilms on their tumor-free mucosa far distant from their tumors. Bacterial biofilms were associated with diminished colonic epithelial cell E-cadherin and enhanced epithelial cell IL-6 and Stat3 activation, as well as increased crypt epithelial cell proliferation in normal colon mucosa. High-throughput sequencing revealed no consistent bacterial genus associated with tumors, regardless of biofilm status. However, principal coordinates analysis revealed that biofilm communities on paired normal mucosa, distant from the tumor itself, cluster with tumor microbiomes as opposed to biofilm-negative normal mucosa bacterial communities also from the tumor host. Colon mucosal biofilm detection may predict increased risk for development of sporadic CRC.

Pro-carcinogenic bacteria have the potential to initiate and/or promote colon cancer, in part via immune mechanisms that are incompletely understood. Using Apc^Min mice colonized with the human pathobiont enterotoxigenic Bacteroides fragilis (ETBF) as a model of microbe-induced colon tumorigenesis, we show that the Bacteroides fragilis toxin (BFT) triggers a pro-carcinogenic, multi-step inflammatory cascade requiring IL-17R, NF-κB, and Stat3 signaling in colonic epithelial cells (CECs). Although necessary, Stat3 activation in CECs is not sufficient to trigger ETBF colon tumorigenesis. Notably, IL-17-dependent NF-κB activation in CECs induces a proximal to distal mucosal gradient of C-X-C chemokines, including CXCL1, that mediates the recruitment of CXCR2-expressing polymorphonuclear immature myeloid cells with parallel onset of ETBF-mediated distal colon tumorigenesis. Thus, BFT induces a pro-carcinogenic signaling relay from the CEC to a mucosal Th17 response that results in selective NF-κB activation in distal colon CECs, which collectively triggers myeloid-cell-dependent distal colon tumorigenesis.