ARTICLE ABSTRACTMacrophage-induced bystander effects have been implicated as an important mediator of chromosomal instability and colon cancer triggered by Enterococcus faecalis, a human intestinal commensal bacteria. There is little understanding about how inflammatory cytokines mediate bystander effects, but questions in this area are important because of the pivotal contributions made by inflammatory processes to cancer initiation and progression. Here, we report that the central proinflammatory cytokine TNF-α acts as a diffusible mediator of the bystander effects induced by macrophages, an effect caused by a proliferation of macrophages that trigger epithelial cell production of Netrin-1, a neuronal guidance molecule. TNF-α-mediated bystander assays used a murine coculture system of primary colonic epithelial cells and E. faecalis-infected macrophages (in vitro), with an interleukin 10 (IL-10)-deficient mouse model of colon cancer that involves long-term colonization with E. faecalis (in vivo). In cell cocultures, we observed increased expression of the TNF-α receptor Tnfrsf1b and Netrin-1. These effects were blocked by anti-TNF-α antibody or by pretreatment with an inhibitor of NF-κB signaling. RNAi-mediated attenuation of Tnfrsf1b decreased TNF-α-induced netrin-1 production and augmented epithelial cell apoptosis in culture. Extending these observations, colon biopsies from E. faecalis-colonized IL-10−/− mice exhibited crypt hyperplasia and increased staining for macrophages, TNF-α, netrin-1, NF-κB, Tnfrsf1b, and the proliferation marker proliferating cell nuclear antigen while also displaying a reduction in epithelial cell apoptosis. Together, our results define a pathway for macrophage-induced bystander effects in which TNF-α triggers TNFRSF1b receptor signaling leading to increased production of Netrin-1, crypt hyperplasia, and decreased epithelial cell apoptosis. In elucidating an important commensal-associated proinflammatory mechanism in the intestinal microenvironment, our work highlights the role of Netrin-1 and a specific TNF-α receptor as candidate targets to prevent or treat colorectal cancer. Cancer Res; 72(20); 5219–29. ©2012 AACR.