Toll-like receptor 2 signaling protects mice from tumor development in a mouse model of colitis-induced cancer

Abstract

Inflammatory bowel disease (IBD) is a disorder of chronic inflammation with increased susceptibility to colorectal cancer. The etiology of IBD is unclear but thought to result from a dysregulated adaptive and innate immune response to microbial products in a genetically susceptible host. Toll-like receptor (TLR) signaling induced by intestinal commensal bacteria plays a crucial role in maintaining intestinal homeostasis, innate immunity and the enhancement of intestinal epithelial cell (IEC) integrity. However, the role of TLR2 in the development of colorectal cancer has not been studied. We utilized the AOM-DSS model for colitis-associated colorectal cancer (CAC) in wild type (WT) and TLR2(-/-) mice. Colons harvested from WT and TLR2(-/-) mice were used for histopathology, immunohistochemistry, immunofluorescence and cytokine analysis. Mice deficient in TLR2 developed significantly more and larger colorectal tumors than their WT controls. We provide evidence that colonic epithelium of TLR2(-/-) mice have altered immune responses and dysregulated proliferation under steady-state conditions and during colitis, which lead to inflammatory growth signals and predisposition to accelerated neoplastic growth. At the earliest time-points assessed, TLR2(-/-) colons exhibited a significant increase in aberrant crypt foci (ACF), resulting in tumors that developed earlier and grew larger. In addition, the intestinal microenvironment revealed significantly higher levels of IL-6 and IL-17A concomitant with increased phospho-STAT3 within ACF. These observations indicate that in colitis, TLR2 plays a protective role against the development of CAC.

Figure 1. TLR2-deficiency leads to increased development of colitis-associated colon cancer.

(A) Schematic overview of the CAC model. After initial AOM injection (12.5 mg/kg), DSS was given in the drinking water (boxed areas) followed by regular drinking water. Mice were sacrificed on days 14 or 61 post AOM injection (Day 61: n = 19 WT, n = 21 TLR2^−/− mice). (B) Percent weight change during AOM-DSS treatment. (C) Mouse mortality during AOM-DSS treatments. (D) Number of colorectal tumors per mouse induced by AOM-DSS treatment at day 61. (E) Number of tumors per mouse located in proximal or distal colons in WT or TLR2^−/− mice. (F) Size distribution of colorectal tumors formed in WT or TLR2^−/− mice. (G) Tumor burden in AOM-DSS treated WT or TLR2^−/− mice. All tests were performed using 95% confidence intervals. Data are expressed as means ± SEM. * = p<0.05, ** = p<0.01, *** = p<0.001.

Figure 2. TLR2-deficient colons have more advanced dysplasia and ß-catenin expression compared to WT colons.

Histopathology of colons at day 61 of AOM-DSS treatment. (A) Hematoxylin and eosin (H&E) (left) or BrdU stained (right) serial sections of WT or TLR2^−/− mice are shown. Original magnification 20x (upper panels) and 100x (lower panels) are shown. (B) Immunofluorescent staining for ß-catenin. Original magnification 100x (left panel) or 400x (right panel).

Figure 3. TLR2-deficiency leads to early formation of aberrant crypt foci.

Scoring of inflammation, necrosis and ACF at day 14 of AOM-DSS treatment (n = 5 WT and n = 5 TLR2^−/−). (A) Inflammatory scores of colons. (B) Extent of colonic necrosis. (C) Number of ACF per mouse located in proximal or distal colons. (D) H&E stains of serial sections of colons. Original magnification 40x (upper panel) or 100x (lower panel). (E) Immunohistochemical stains for BrdU. Original magnification 100x. (F) Immunofluorescent TUNEL staining. Original magnification 100x. All tests were performed using 95% confidence intervals. Data are expressed as means ± SEM. * = p<0.05, ** = p<0.01, *** = p<0.001.

Figure 4. Increased cell proliferation and reduced apoptosis in TLR2-deficient colons during early CAC development.

Assessment of proliferation by BrdU staining and apoptosis by TUNEL staining in proximal and distal colons from mice either treated for 14 days with the AOM-DSS regimen (Day 14) or untreated mice (Day 0) (n = 5), BrdU⁺ (A) and TUNEL⁺ (B) cells were counted in intact and well-oriented crypts. BrdU⁺ cells were quantified from at least 20 crypts per region from 4 different slides per animal. (C) Day 14 representative immunohistochemical stains for BrdU in colonic sections. Original magnification 100x. (D) Day 14 representative immunofluorescent stains for TUNEL stains in colonic sections. Original magnification 100x. All tests were performed using 95% confidence intervals. Data are expressed as means ± SEM. * = p<0.05, ** = p<0.01, *** = p<0.001.

Figure 5. TLR2^−/− mice have increased IL-6 and STAT3 activation during early intestinal tumorigenesis.

Panels A–C: IL-6 production in WT and TLR2^−/− mice at day 14 of AOM-DSS treatment was measured by ELISA. (A) Serum levels of IL-6 (n = 8 WT, n = 9 TLR2^−/−). (B) IL-6 concentration in colon homogenates was normalized to concentration of protein in the tissues (n = 3 WT, n = 5 TLR2^−/−). (C) IL-6 secretion from isolated colonic lamina propria cells treated with LPS (1 µg/ml) for 6 h (n = 3–5). (D) Immunofluorescent stains of phospho-Stat3 in colonic tissues of WT and TLR2^−/− mice at baseline. Original magnification 100x. (E) Immunofluorescent staining of phospho-Stat3 in colonic tissues of WT and TLR2^−/− mice treated for 14 days with the AOM-DSS regimen. Top panel original magnification 40x, bottom panel original magnification 400x. (F) Quantification of phospho-Stat3 intensity measured in ACF from greater than five focus fields in at least four slides per animal (n = 5 WT and n = 5 TLR2^−/−). All tests were performed using 95% confidence intervals. Data are expressed as means ± SEM. * = p<0.05, ** = p<0.01, *** = p<0.001.

Figure 6. TLR2-deficient mice develop increased T_H17 responses during early CAC development.

(A–D, F–H) Cytokines concentrations in colon homogenates in WT and TLR2^−/− mice at day 14 of AOM-DSS treatment were measured by ELISA. (A) IFN-g (n = 5–8). (B) IL-10 (n = 4). (C) IL-4 (n = 4–5). (D) IL-17A (n = 6). (E) IL-17A secretion from isolated chronic lamina propria cells treated with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) for 72 h (n = 4). (F) TGF-ß (n = 4–10). (G) IL-23p19 (Day 0: n = 8–10; Day 14: n = 4 = 10). (H) TNFa (n = 4–10). All tests were performed using 95% confidence intervals. Data are expressed as means ± SEM. * = p<0.05, ** = p<0.01, *** = p<0.001.

Figure 7. Colonic tissue from TLR2^−/− mice recruit inflammatory cells that have reduced NO production and fail to mount an adequate defense against tumor growth in early CAC.

(A) Chemokines concentrations quantified by ELISA in colon homogenates in WT and TLR2^−/− mice at day 14 of AOM-DSS treatment were measured by ELISA. KC (n = 6 WT, n = 9 TLR2−/−), MIP-2 (n = 6 WT, n = 9 TLR2−/−), MCP-1 (n = 6 WT, n = 4 TLR2−/−), RANTES (n = 4 WT, n = 10 TLR2−/−). (B) Immunofluorescent staining for nitrotyrosine in proximal (top panels) and distal (bottom panels) colon section of WT (left panels) and TLR2^−/− (right panels) mice treated for 14 days with the AOM-DSS regimen. Original magnification 100x. (C) Quantification of nitrotyrosine positive cells normalized by area (mm²) from greater than five focus fields in at least four slides per animal (n = 5 WT and n = 5 TLR2^−/−).