Intestinal mucosa responds to acute inflammation with increased cytokine production and mucosal barrier breakdown. This process may involve the TLR4 pathway. While intestinal mucosa is resistant to the LPS-induced signaling at baseline, it may become sensitive to LPS during acute inflammation. The mechanisms underlying this phenomenon are not fully understood. We hypothesized that treatment of intestinal epithelial cells with the pro-inflammatory cytokine TNFα would alter LPS mediated TLR4 pathway signaling.Methods: Caco2 cells, a human intestinal epithelial cell line unresponsive to LPS at baseline, were grown to confluence and treated with serum free media for 24h prior to treatment with TNFα (100ng/mL). TLR4 expression was analyzed by Western blot. In LPS response experiments, cells were pretreated for 24h with TNFα, then media was changed to include a second 24h treatment with TNFα alone, TNFα and LPS, or TNFα, LPS, and sMD-2 (0.1 nM), an important TLR4 cofactor. Supernatant IL8 content was measured by ELISA. Results: TLR4 expression was markedly increased after 24h and 48h of TNFα treatment. LPS treatment alone did not induce IL8. After TNFα pretreatment, addition of LPS resulted in increased IL8 production. Treatment with a combination of TNFα, LPS, and low dose sMD-2 resulted in a maximal IL8 production (Figure).Conclusion: In the present study, Caco2 cells stimulated with TNFα demonstrate increased TLR4 protein expression and increased LPS responsiveness, especially in the presence of sMD-2. These findings are important because they indicate that pro-inflammatory cytokines such as TNFα may increase enterocyte response to LPS during acute inflammation.


IL-8 after sequential treatment with TNFα, then as indicated (p<0.01 by ANOVA/Tukey test)