Toll-like receptor 3 signaling attenuated colitis-associated cancer development in mice

Abstract

Inflammatory bowel disease is associated with a high risk of colitis-associated cancer (CAC). We evaluated the role of TLR3 in CAC using a murine model. Wild-type (WT) and TLR3-knockout (TLR3^-/-) mice received azoxymethane (AOM) 12.5 mg/kg intraperitoneally on day zero, followed by three cycles of 2% dextran sulfate sodium (DSS) for five days and free water for two weeks. We evaluated clinical indices, such as weight change, colon length, histological severity of colitis, and tumor number. We performed immunofluorescence assays for phospho-IκB kinase and β-catenin in colon tissues. To elucidate the antitumorigenic mechanism of TLR3 signaling, we injected poly(I: C) or phosphate-buffered saline intraperitoneally into an AOM/DSS-induced tumorigenesis model in WT mice. We also evaluate the direct antitumor effect of TLR signaling in AOM-treated WT and TLR3^-/- mice without DSS. TLR3 deficiency increased tumor burden and colitis severity in the colon tissue than in the WT mice. β-catenin immunoreactivity was higher in TLR3^-/- mice, while phospho-IκB kinase expression was similar. TLR3 activation by poly(I: C) did not reduce tumor burden in WT mice, but long-term AOM administration without DSS significantly increased tumor burden in TLR3^-/- mice. TLR3 signaling attenuates CAC development, suggesting it may be a target for preventing CAC in inflammatory bowel disease.

Fig. 1

Clinical and histological changes in WT and TLR3^−/− mice after AOM/DSS treatment. (A) Body weight change of AOM/DSS-treated WT and TLR3^−/− mice. TLR3^−/− mice showed marked weight loss during the DSS cycle compared with WT. (B, C) Gross evaluation of tumor burden. The number of tumors was significantly higher in AOM/DSS-treated TLR3^−/− mice than in WT mice. (D, E) Histological score of colon tissue. The histopathologic score of TLR3^−/− mice was higher in all three sections of colon tissue than in WT mice. (F) Quantification of inflammatory cytokines from mouse colon lamina propria mononuclear cells (LPMC) by PCR. The level of TNF-α and IFN-γ were significantly elevated in TLR3^−/− mice compared with that in WT mice. The symbols: ***p < 0.001, **p < 0.01 and *p < 0.05.

Fig. 2

Histological images from non-tumor site and tumor site in AOM/DSS-treated WT and TLR3^−/− mice. (A) H&E and immunofluorescence assays of AOM/DSS-treated WT and TLR3^−/− mice. Overall inflammation on both non-tumor and tumor sites was more severe in TLR3^−/− mice compared with that in the WT mice. (B) Fluorescence intensity of IKK, NF-κB and β-catenin. The expression of β-catenin was significantly more amplified in tumor site of TLR3^−/− mice than in the WT mice. However, there was no significant difference of the expression IKK in tumor site between WT and TLR3^−/− mice. The symbol: *p < 0.05.

Fig. 3

Clinical and histological changes in AOM/DSS-treated WT mice with or without Poly(I: C) injection. (A) There was no significant difference of body weight change between Poly(I: C) injected and control groups. (B, C) Gross evaluation of tumor burden did not show significant difference between the two groups. (D, E) There was no significant difference in histologic score between the two groups.

Fig. 4

Clinical and histological changes in WT and TLR3^−/− mice after AOM treatment. (A) TLR3^−/− mice showed significantly decreased weight gain over 12 weeks compared with that of WT mice. (B, C) Gross evaluation of tumor burden. The number of tumors showed no significant difference between WT and TLR3^−/− mice. (D, E) There was no significant difference of histological score from all three sections of colon tissue in WT and TLR3^−/− mice. The symbol: *p < 0.05.

Fig. 5

Histological images from non-tumor site and tumor site in AOM-treated WT and TLR3^−/− mice. (A) H&E and immunofluorescence assays of AOM-treated WT and TLR3^−/− mice. Overall inflammation on both non-tumor and tumor sites showed no significant difference in WT and TLR3^−/− mice. (B) Fluorescence intensity of IKK, NF-κB and β-catenin. The expression of β-catenin was significantly more amplified in tumor site of TLR3^−/− mice than WT mice. However, there was no significant difference of the expression IKK in tumor site between WT and TLR3^−/− mice. The symbols: **p < 0.01 and *p < 0.05.