E. Coli-Epithelial Contact Is Key to Host Response Figure 6. Structural components of the TTSS affect the IEC innate immune response. HEp-2 cells were either co-cultured with E69, E69 DescN, E69 DespA (MOI = 60), or stimulated with IL-1b (10 ng/ml) as a positive control. At 4 or 16 h post-infection co-culture supernatants were harvested and IL-8 (a) and CCL20 (b) were evaluated by ELISA. Data shown is mean (+SD, */+P#0.02) induction of three independent experiments. doi:10.1371/journal.pone.0027030.g006 immune responses in our model system, IL-8 mRNA expression of HEp-2 cells was determined 4 h post inoculation. At 4 h the E69 WT induced a significant up-regulation of IL-8 mRNA expression (Figure 8 & Figure S1), suggesting an overall balance towards the host pro-inflammatory response. On exposure to the flagella mutant (DfliC) and the rough/smooth LPS mutants significant IL- 8 mRNA expression was observed however, there was no significant difference in expression compared to the WT. There was no induction of IL-8 mRNA expression with the flagella enhanced strain (fliC+) and the TTSS mutant DespA, thus showing Figure 7. EPEC effector proteins can either induce or modulate IEC innate immune responses. HEp-2 cells were either co-cultured with E69, E69 DespC, E69 DespF, E69 DlifA (MOI = 60), or stimulated with IL-1b (10 ng/ml) as a positive control. At 4 or 16 h post-infection co-culture supernatants were harvested and IL-8 (a) and CCL20 (b) were evaluated by ELISA. Data shown is mean (+SD, */+P#0.05) induction of three independent experiments. doi:10.1371/journal.pone.0027030.g007 PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e27030