A defect in mitochondrial activity can contributes to disease. We found that monolayers of T84 epithelial cells exposed to dinitrophenol (DNP; uncouples oxidative phosphorylation) and non-pathogenic E. coli display decreased barrier function. Here the impact of DNP on macrophages and the effect of TNF , DNP and E. coli on epithelial permeability were assessed. DNP treatment of THP-1 macrophages resulted in reduced ATP, and although hypo-responsive to LPS, these macrophages produced IL-1, IL-6 and TNF. Given the role of TNF in inflammatory bowel disease (IBD), and the association between increased permeability and IBD, TNF (10 ng/ml) was added to the DNP (0.1 mM)+E. coli (10⁶ cfu) and this resulted in a significantly greater lincrease in epithelial permeability than that elicited by DNP+E. coli. This increased permeability was not due to epithelial death and the enhanced E. coli translocation was reduced by pharmacological inhibitors of NF signaling (PTDC, NEMO-binding peptide and BAY 11-7082, and the proteosome inhibitor, MG132). In contrast, the drop in TER was unaffected by the inhibitors of NF. As an integrative model system, our findings support the induction of a positive feedback loop that can severely impair epithelial barrier function and contribute to existing inflammation or trigger relapses in IBD. Thus, metabolically-stressed epithelia display increased permeability in the presence of viable non-pathogenic E. coli that is exaggerated by TNF released by immune cells, such as macrophages, that retain this ability even if they also metabolically stressed.