We have previously shown that selected commensal bacteria contribute to colorectal carcinogenesis through bystander effects. Specifically, Enterococcus faecalis, a human intestinal commensal, induces colitis and colon cancer in Il10−/- mice. In this study, we hypothesized that tissue macrophages triggered by E. faecalis were exclusively responsible for epithelial cell transformation. We evaluated the role of macrophages by depleting them from the colon in these mice and observing their loss on colorectal carcinogenesis.

To deplete colon macrophages in Il10−/ and control mice, we administered encapsulated clodronate liposomes (ECL) via weekly rectal enemas. Mice were colonized with E. faecalis strain OG1RFSS. Controls consisted of: 1) Il10−/- mice given ECL enemas but not colonized with enterococci; 2) Il10−/ mice colonized with E. faecalis but given sham liposome enemas containing no clodronate (SL); and 3) Il10+/+ mice colonized with E. faecalis and given ECL enemas. Mice were treated for 9 months and colon biopsies evaluated by immunohistochemical staining for numbers of macrophages; inflammation; cancer; presence of 4-hydroxy-2-nonenal protein adducts; and expression of cyclooxygenase (COX)-2, TNF-α, netrin-1, and WNT/β-catenin signaling.

Staining of colon biopsies using F4/80 (a murine macrophage marker) showed significant decreases in numbers of macrophages for mice administered ECL enemas compared to mice given SL enemas (P <0.001). As expected, no colitis or cancer occurred in E. faecalis-colonized Il10+/+ mice. For E. faecalis-colonized Il10−/ mice administered SL enemas, severe colitis and colon cancer (5 of 12 mice) developed. In contrast, for E. faecalis-colonized Il10−/ mice administered ECL enemas, no colitis (P <0.0001) or cancers (P = 0.037) developed. ECL enemas significantly decreased the expression of COX-2, TNF-α, and netrin-1, and presence of 4-hydroxy-2-nonenal protein adducts, suggesting that macrophage depletion by clodronate eliminated bystander effects. Finally, macrophage depletion also significantly decreased β-catenin signaling in colon biopsies.

These findings strongly suggest that the activation of colon macrophages by E. faecalis is necessary for inflammation and cancer development in the IL-10 knockout model. The reduction or elimination of expression of COX-2, TNF-α, netrin-1, and 4-hydroxy-2-nonenal protein adducts further supports a key role for these innate immune cells in generating bystander effects. Our results provide potential new strategies for preventing colorectal cancer.

Citation Format: Yonghong Yang, Xingmin Wang, Thomas Huycke, Danny Moore, Stanley Lightfoot, Mark Huycke, Department of Veterans Affairs Medical Center. Depletion of colon macrophages prevents colitis and colon cancer triggered by commensal bacteria. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2868. doi:10.1158/1538-7445.AM2013-2868