Chronic intestinal inflammation culminates in cancer and a link to TLR4 has been suggested by our observation that TLR4 deficiency prevents colitis-associated neoplasia. In the current study, we address the effect of the aberrant activation of epithelial TLR4 on induction of colitis and colitis-associated tumor development. We take a translational approach to address the consequences of increased TLR signaling in the intestinal mucosa. Mice transgenic for a constitutively-active TLR4 under the intestine-specific villin promoter (villin-TLR4 mice) were treated with DSS for acute colitis and azoxymethane-dextran sulfate sodium. TLR4 expression was analyzed by immunohistochemistry in colonic tissue from patients with ulcerative colitis and ulcerative colitis associated cancer. The effect of an antagonist TLR4 Ab was tested in prevention of colitis-associated neoplasia in the AOM-DSS model. Villin-TLR4 mice were highly susceptible to both acute colitis and colitis-associated neoplasia. Villin-TLR4 mice had increased epithelial expression of COX-2 and mucosal PGE2 production at baseline. Increased severity of colitis in villin-TLR4 mice was characterized by enhanced expression of inflammatory mediators and increased neutrophilic infiltration. In human UC samples, TLR4 expression was upregulated in almost all CAC and progressively increases with grade of dysplasia. As a proof of principle, a TLR4/MD-2 antagonist antibody inhibited colitis-associated neoplasia in the mouse model. Our results show that regulation of TLR's can affect the outcome of both acute colitis and its consequences—cancer. Targeting TLR4 and other TLR's may ultimately play a role in prevention or treatment of colitis-associated cancer.

Colonic bacteria have been implicated in the development of colon cancer. We have previously demonstrated that toll-like receptor 4 (TLR4), the receptor for bacterial lipopolysaccharide (LPS), is over-expressed in humans with colitis-associated cancer. Genetic epidemiologic data support a role for TLR4 in sporadic colorectal cancer (CRC) as well, with over-expression favoring more aggressive disease. The goal of our study was to determine whether TLR4 played a role as a tumor promoter in sporadic colon cancer. Using immunofluorescence directed to TLR4, we found that a third of sporadic human colorectal cancers over-express this marker. To mechanistically investigate this observation, we used a mouse model that over-expresses TLR4 in the intestinal epithelium (villin-TLR4 mice). We found that these transgenic mice had increased epithelial proliferation as measured by BrdU labeling, longer colonic crypts and an expansion of Lgr5+ crypt cells at baseline. In addition, villin-TLR4 mice developed spontaneous duodenal dysplasia with age, a feature that is not seen in any wild-type (WT) mice. To model human sporadic CRC, we administered the genotoxic agent azoxymethane (AOM) to villin-TLR4 and WT mice. We found that villin-TLR4 mice showed an increased number of colonic tumors compared to WT mice as well as increased β-catenin activation in non-dysplastic areas. Biochemical studies in colonic epithelial cell lines revealed that TLR4 activates β-catenin in a PI3K-dependent manner, increasing phosphorylation of β-cateninSer552, a phenomenon associated with activation of the canonical Wnt pathway. Our results suggest that TLR4 can trigger a neoplastic program through activation of the Wnt/β-catenin pathway. Our studies highlight a previously unexplored link between innate immune signaling and activation of oncogenic pathways, which may be targeted to prevent or treat CRC.

TRIF signaling triggers the amplification of macrophage bactericidal activity sufficient to eliminate invading intestinal pathogens through the sequential induction of IFN-β and IFN-γ from macrophages and NK cells, respectively.

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