FIG. 3.

(A) TLR2 and TLR1 recognize BspA. HEK293 cells were transiently transfected with plasmids expressing the indicated human TLRs or an empty vector (EVC). Cells were incubated for 6 h with zymosan (10 μg/ml), BspA (5 μg/ml), highly purified E. coli LPS (Ec LPS) (1 μg/ml), or no agonist. NF-κB activity in supernatants was quantified by the luciferase reporter assay. (B) NF-κB is activated by BspA through TLR2 and TLR1 but not through TLR2 and TLR6. Cells were transiently transfected with the TLR2 and TLR1 plasmids or with the TLR2 and TLR6 plasmids and incubated for 6 h with the indicated reagents. The data are the averages ± standard deviations for triplicate wells, normalized to the unstimulated cells for each TLR combination. Statistically significant cellular activation (P < 0.05) compared with the corresponding unstimulated control (A) or a statistically significant difference between TLR2/TLR1 and TLR2/TLR6 (B) as calculated by Student's t test is indicated by an asterisk. Pam3, Pam₃Cys. (C) Anti-human TLR2 antibody (Ab) inhibited BspA-mediated activation of NF-κB activity in HEK293 cells stably expressing human TLR2 and human TLR1. HEK293 (hTLR2/hTLR1) stable cells were transiently transfected with reporter plasmids, the firefly luciferase gene conjugated with NF-κB promoters (NF-κB-luc), and Renilla luciferase genes (R-luc). Cells were preincubated with anti-TLR2 MAb (TL2.1; 5 mg/ml) or an Ig isotype-matched control (IgG2a; 5 mg/ml) for 30 min before stimulation with Pam₃Cys (1 μg/ml) or BspA (5 μg/ml). After 6 h of stimulation, supernatants were collected, and the NF-κB activity in supernatants was quantified by a luciferase reporter assay. The data are the averages ± standard deviations for triplicate determinations. Statistically significant inhibition (P < 0.05) as calculated by Student's t test compared to the isotype control treatment is indicated by an asterisk. (D) HEK293 or HEK293 (hTLR2/hTLR1) cells were stimulated with BspA protein (5 μg/ml) for 16 h, and the amount of IL-8 released in the medium was determined by an ELISA. The data are the means ± standard deviations for triplicate determinations. Statistical significance (P < 0.05) as calculated by Student's t test is indicated by an asterisk.