FIGURE 12.

Differential stimulation of TLR5 signaling by glycosylated and nonglycosylated forms of flagellin. A, HEK293 cells, stably expressing TLR5, were stimulated for 24 h in the absence (NT, nontreated) or presence of varying concentrations of fully glycosylated wild-type (WT) or nonglycosylated (Δ0111) forms of flagellin purified from the B. cenocepacia parental or ΔBCAL0111 strains, respectively. Conditioned medium was assayed for expression levels of IL-8. B, HEK293 cells, stably expressing TLR5, were transfected with a NFκB-regulated luciferase reporter gene and stimulated for 24 h as indicated above. Cell lysates were assayed for NFκB-regulated firefly luciferase activity, and fold induction levels of NFκB-regulated luciferase are expressed relative to nontreated (NT) cells. Data are representative of three independent experiments. Statistical analysis was performed by paired t test using two-tailed p values. Significant differences between samples from WT and Δ0111-treated cells are indicated by * (p < 0.05). HEK293 cells, stably expressing TLR5, were stimulated for indicated times with WT and Δ0111 flagellin (500 ng/ml). Cell lysates were immunoblotted for phosphorylated (p-) and total levels of p65 (C) and p38 (D), JNK and ERK MAPKs. β-Actin was used as a loading control.