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. 2014 Aug 18;111(35):12930–12935. doi: 10.1073/pnas.1406414111

Fig. 4.

Fig. 4.

P. aeruginosa-triggered ASL secretion requires expression of flagellin. (A) PAKΔfliC-laden agarose beads produced less ASL than PAK-laden agarose beads, and flagellin-laden (10 ng/mL) agarose beads (FLA) triggered a similar response as PAK-laden beads (n = 12 bacteria-free, n = 7 PAK, n = 10 PAKΔfliC, and n = 15 FLA; P < 0.05, ANOVA and Bonferroni's multiple comparison test). Columns labeled with different letters differ significantly. (B) Secretion assay data shows that the addition of flagellin (10 ng/mL) to the Krebs solution bathing the submucosal glands did not stimulate ASL secretion, whereas the addition of IL-1β (50 ng/mL) did (n = 10 for flagellin and n = 9 for IL-1β; P < 0.05, ANOVA and Bonferroni's multiple comparison test). Columns labeled with different letters differ significantly. (C) Airway surface epithelia (Upper) express the flagellin receptor TLR5, whereas submucosal glands do not (Lower). The lung is a positive control for TLR5, and + and − indicate RT positive and negative, respectively. (D) The Krebs solution bathing the submucosa of trachea preparations incubated with P. aeruginosa strain PAK on the mucosal side stimulated ASL secretion in a secretion assay, whereas PAKΔfliC and PBS did not (n = 9 for all groups; P < 0.05, ANOVA and Bonferroni's multiple comparison test). Columns labeled with different letters differ significantly. (E) Confocal immunofluorescence of IL-1β receptor IL-1R1 (Left), lysozyme (Middle), and both merged (Right) in submucosal glands. The images show that IL-1R1 is colocalized with lysozyme, a marker for serous cells. (F) Airway submucosal glands express IL-1R1. The heart is a positive control for IL-1R1, and + and − indicate RT positive and negative, respectively. (G and H) Bacteria-triggered ASL secretion is not limited to P. aeruginosa. Both S. aureus (G) (n = 8 bacteria-free, n = 10 S. aureus, and n = 12 S. aureus + CFTRinh172; P < 0.05, ANOVA and Bonferroni's multiple comparison test) and H. influenzae (H) stimulate ASL secretion that is inhibited by CFTRinh172 (n = 19 bacteria-free, n = 11 H. influenzae, and n = 10 H. influenzae + CFTRinh172; P < 0.05, ANOVA and Bonferroni's multiple comparison test). Columns labeled with different letters differ significantly.