Figure 3

hBD-3 release induced byL. pneumophilain alveolar epithelial cells is controlled by TLR2, TLR5 and TLR9. (A) SAEC were incubated with the TLR2 ligand Mapl 2 [1 μg/ml], purified flagellin to activte TLR5 [1 μg/ml] and ODN as TLR9 ligand [1 μM]. After 24 h hBD-3 release was measure by ELISA. RNAi experiments in A549 cells to inhibit expression of endogenous TLR2, TLR5 or TLR9 respectively were performed. Cells were incubated for 72 h with TLR2- and for 48 h with TLR5- and TLR9-specific siRNA as well as a non-silencing control siRNA (C-siRNA). Infection with L. pneumophila strain 130b (L. p.), incubation with TNFα [100 ng/ml] or (B) the TLR2 ligand Malp 2 [1 μg/ml] and heat inactivated L. pneumophila, Corby, CorbyΔflaA or (C, D) incubated with flagellin [1 μg/ml] and (E) the TLR9 ligand ODN [1 μM] and DNA [5 μg/ml] from Legionella. After 24 h hBD-3 release was measure by ELISA. ELISA data presented are means ± SEM of four separate experiments. *, p < 0.05 compared with uninfected control cells; #, p < 0.05 control siRNA versus target siRNA.