Abstract B58: Enterococcus faecalis-infected macrophages produce tumor necrosis factor-α and induce netrin-1 expression in colonic epithelial cells

Enterococcus faecalis , a human intestinal commensal, causes inflammation and colorectal cancer in interleukin (IL)-10 knockout mice. Understanding mechanisms by which commensals contribute to colorectal carcinogenesis will help develop new prevention strategies. Our laboratory has reported that E. faecalis -infected macrophages generate diffusible clastogens that cause chromosomal instability through a bystander effect. The potential role of cytokines in this pathogenic mechanism remains unknown. Here we show that E. faecalis -infected macrophages produce tumor necrosis factor-α (TNFα) as part of the bystander effect, and that this cytokine leads to netrin-1 (Ntn1) expression in vitro and in vivo in IL-10 −/− mice. TNFα production by RAW264.7 murine macrophages substantially increased following E. faecalis infection at a multiplicity of infection of 1,000 colony forming units compared to untreated macrophages. Colon biopsies of IL-10 −/− mice colonized with E. faecalis for 9 months showed inflammation, dysplasia and cancer. Immunohistochemical staining of these biopsies showed increased numbers of macrophages in the lamina propria (69.6 ± 10.3 cells staining for F4/80 per 20X field for IL-10 −/− mice vs. 3.3 ± 3.6 cells for shams) and intense staining for TNFα (50.8 ± 19.1 cells per 20X field for IL-10 −/− mice vs. 1.2 ± 1.1 for shams). TNFα colocalized to macrophages suggesting E. faecalis activated these cells. To explore mechanisms by which TNFα contributed to E. faecalis -induced inflammation and cancer, we investigated Ntn1 expression in mouse primary colonic epithelial cells (YAMC) following exposure to TNFα, and to E. faecalis -infected macrophages using a dual chamber co-culture system. Ntn1 is an anti-apoptotic ligand that promotes colon tumors by dysregulating cell survival. Previously, using E. faecalis , we reported upregulation of Ntn1 in a short-term colonic ligation model. In this study, TNFα induced Ntn1 in YAMC cells in a dose-dependent manner. Similar results were found for cells exposed to E. faecalis -infected macrophages. Ntn1 expression, however, was not noted using heat-inactivated TNFα or anti-TNFα antibody, suggesting that these findings were specific for Ntn1 expression. Finally, colonic epithelial cells in IL-10 −/− mice colonized with E. faecalis were strongly stained for Ntn1 compared to sham colonized mice, supporting the in vitro findings. We next determined whether NFκB was involved in TNFα-mediated Ntn1 activation. The NFκB inhibitor Bay11–7085 significantly decreased TNFα-induced Ntn1 activation, suggesting that Ntn1 expression is NFκB dependent. Finally, we investigated TNFα receptor involvement in Ntn1 activation. Western blots showed no difference in Tnfr1 expression for YAMC cells treated with TNFα compared to untreated controls. Similarly, there was no change Tnfr1 expression by immunohistochemical staining for IL-10 −/− mice colonized with E. faecalis compared to sham, suggesting constitutive expression. However, Tnfr2 significantly increased for TNFα-treated YAMC cells and colonic epithelial cells in biopsies from E. faecalis colonized IL-10 −/− mice. Finally, for YAMC cells, TNFα-induced Ntn1 expression was substantially decreased following silencing of Tnfr2 by siRNA. These results indicate that Ntn1 induction by TNFα occurs via Tnfr2. In summary, TNFα from E. faecalis -infected macrophages induces Ntn1 expression in colonic epithelial cells and potentially contributes to colorectal cancer. These findings highlight a novel bystander mechanism that promotes colorectal cancer and suggest new targets for chemoprevention. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B58.