FIG. 1.

(A) EPEC supernatants induce IL-8 secretion from Caco-2 cells. Bacterial culture supernatant from overnight cultures of WT and mutant EPEC grown in LB broth were filter sterilized; 200 to 400 μl/well of supernatant was added to the growth medium of Caco-2 cell monolayers in 6- or 12-well plates for 3 h, cells were washed, and fresh medium was added. The cell culture supernatant was collected after 24 h, and the IL-8 concentration was quantified by ELISA. The bacteria were grown overnight in LB broth. IL-1β (10 ng/ml) was included as a positive control. The error bars indicate standard errors of the mean. *, P < 0.05 versus unstimulated. (B) Direct infection by EPEC bacteria triggers IL-8 secretion in a partially flagellin-dependent manner. WT EPEC, FliC-deficient EPEC (ΔfliC), complemented ΔfliC EPEC (Comp ΔfliC), T3SS-deficient EPEC (ΔescN), and a double mutant lacking FliC and T3SS (ΔfliC/ΔescN) were grown overnight in LB broth as described in Materials and Methods, and 10 μl of bacterial culture was used to infect Caco-2 cells in 2 ml of DMEM-F-12 in the absence of antibiotics and serum. After 3 h, the cells were washed twice with DMEM, and the medium was replaced with medium containing serum and antibiotics with gentamicin added to prevent the growth of extracellular bacteria. The cell culture supernatant was collected for IL-8 ELISA. The error bars indicate standard errors of the mean. *, P < 0.05 versus uninfected; **, P < 0.05 versus ΔfliC EPEC; ***, P < 0.05 versus WT EPEC; †, P < 0.05 versus ΔfliC EPEC.