FIG. 3.

P. aeruginosa activates p38 MAPK through TLR5 but not through TLR2. (A) TLR5-dependent activation of p38 MAPK by PAO1 in 293 cells. 293 cells were seeded in 24-well plates and transfected with 0.5 μg DNA of an empty vector (Mock) or murine TLR5. Twenty-four hours later, cells were incubated with heat-killed PAO1 at an MOI of 100. Phosphorylated and total p38 MAPK were analyzed by Western blotting. (B) Effect of DNhTLR5 and DNhTLR2 on PAO1- and LTA-stimulated NF-κB activation. HAECs were seeded in 24-well plates and transfected with empty vector (1 μg), DNhTLR5 (1 μg), or DNhTLR2 (1 μg) in combination with plasmids containing the hBD2 promoter-driven luciferase gene (25 ng) and the Renilla luciferase gene (1 ng). Twenty-four hours later, cells were stimulated with medium, heat-killed PAO1 (MOI of 100), or LTA (10 μg/ml) for 18 h. Induction of luciferase activity was measured with the dual-luciferase assay. (C) Effect of DNhTLR5 and DNhTLR2 on PAO1-stimulated p38 MAPK activation in HAECs. HAECs were seeded in 24-well plates and transfected with empty vector (1 μg), DNhTLR5 (1 μg), or DNhTLR2 (1 μg). Twenty-four hours after transfection, cells were incubated with heat-killed PAO1 at an MOI of 100 for 30 min. Phosphorylated and total p38 MAPK were analyzed by Western blotting. (D) Effect of DNhTLR5 and DNhTLR2 on the activation of p38 MAPK by P. aeruginosa flagellin protein. HAECs were seeded in 24-well plates and transfected with empty vector (1 μg), DNhTLR5 (1 μg), or DNhTLR2 (1 μg). Twenty-four hours after transfection, cells were incubated with 1 μg/ml flagellin protein purified from PAK (lanes F) or mock flagellin protein prepared from PAK/fliC (lanes F/fliC) for 30 min. Phosphorylated and total p38 MAPK were analyzed by Western blotting. All images are representative of at least two independent experiments. One asterisk, P < 0.01 for a comparison with the control.