FIG. 1.

P. aeruginosa strongly stimulates NF-κB activation through TLR receptors in 293 cells. A screening analysis of TLRs and dual combinations of TLRs that respond to P. aeruginosa was performed with 293 cells. 293 cells were grown in 96-well plates and transiently transfected with 25 ng murine TLR1 to TLR9 DNA or dual combinations of TLRs together with 25 ng plasmid encoding firefly luciferase driven by the NF-κB promoter of human β-defensin 2 and 1 ng Renilla luciferase. Empty pIRES vector was used to ensure that equal quantities of DNA were used. Cells were challenged 24 h after transfection with PAO1 or were not challenged (medium) for 18 h, and then dual-luciferase assays were performed to measure the induction of the luciferase activity. (A) PAO1 activated NF-κB activation via TLR5 in a dose-dependent manner in 293 cells. (B) TLR5 mediated PAO1 (10 CFU/cell)-stimulated luciferase activity in CHO cells. (C) Synergetic activation of NF-κB through the TLR1/TLR2 combination in response to PAO1 in 293 cells. (D) TLR2/TLR6 combination mediated NF-κB activation by PAO1 in 293 cells. The data are means ± standard errors for at least two independent experiments. Samples were compared to a control. One asterisk indicates that the P value is <0.05; two asterisks indicate that the P value is <0.001.