FIG. 6.

Campylobacter-induced IL-8 secretion depends on Campylobacter-secreted CDT and the TLR signaling adaptor MyD88 in polarized intestinal epithelial cells. (A) Polarized T84 cells were pretreated with MyD88 inhibitory peptide (100 μM) (bars III, V, and VII) or its control peptide (bars II, IV, and VI) for 24 h or with chloroquine (5 μM) (bars VIII, IX, and XIII) for 30 min before being incubated with the conditioned supernatant (ConSup) from the coculture of T84 cells with wt C. jejuni 81-176 (bars II, III, and VIII), its cdtB mutant (bars IV, V, and IX), or a C. jejuni 81-176 DNA extract (25 μg/ml) (bars VI and VII) at 37°C for 24 h. MyD88 inhibitory peptide, its control peptide, or chloroquine was also included during the 24-h incubation. Polarized T84 cells incubated with TNF-α only (bar XII) or TNF-α plus chloroquine (bar XIII) were used as controls for the effect of chloroquine on IL-8 secretion. Polarized T84 cells incubated with the medium alone for 24 h (bars I) or with live C. jejuni 81-176 for 4 h (bars X), after which the conditioned supernatants were collected, or 24 h (bars XI) were used as controls. In the 24-h incubation with live bacteria, unbound bacteria were removed at 6 h. No significant difference was detected between the transepithelial resistance before incubation and that after incubation. The apical and basolateral media were collected individually, and the IL-8 concentrations were determined by ELISA. The data are means for three independent experiments; error bars, standard deviations. (B) Polarized T84 cells were pretreated with chloroquine (5 μM) for 30 min before incubation with C. jejuni 81-176 (MOI, ∼10) at 37°C for 4 h. The abilities of C. jejuni 81-176 to adhere to, invade, and transcytose across chloroquine-treated T84 cells were analyzed as described in the legend to Fig. 3. Averages for three independent experiments with triplicate samples are shown; error bars, standard deviations.