(A) TLR2 and MyD88 dominant-negative (DN) mutants, but not TLR4 DN mutant, inhibit NTHi-induced up-regulation of HBD-2 expression. (B) The MyD88 downstream signaling proteins, IRAK1 and TRAF6 are also involved in this up-regulation. (C) The kinetics of P38 phosphorylation: phosphorylation of p38 MAPK started at 15 minutes post-treatment, peaked at 45 minutes post-treatment and then disappeared after 60 minutes. (D) Inhibition of p38 MAPK by SB203580, a specific inhibitor of p38, abolishes HBD-2 up-regulation by NTHi. Cells were pretreated with 5 μM of SB203580, PD98059, and U0126 for 1 hour and then treated with NTHi WCL (5 μg/ml) for 4 hours. Only SB203580 exhibited inhibitory activity towards NTHi WCL-specific HBD-2 expression. The ERK inhibitors, PD98059 and U0126 showed little or no effect. In experiments A, B and D, values are given as mean ± standard deviation. N = 3. *: p < 0.05.