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Pseudomonas aeruginosa Flagella Activate Airway Epithelial Cells through asialoGM1 and Toll-Like Receptor 2 as well as Toll-Like Receptor 5

Departments of Pediatrics and Pharmacology, College of Physicians and Surgeons, Columbia University, New York, New York

Address correspondence to: Alice Prince, Departments of Pediatrics and Pharmacology, College of Physicians and Surgeons, Columbia University, BB 416–650 W. 168th Street, New York, NY 10032. E-mail: asp7{at}columbia.edu

The distribution of specific toll-like receptors and components of the signaling pathways activated by Pseudomonas aeruginosa flagella were studied in airway epithelial cells. Initially flagella bound to the apical surface of polarized epithelial cells, where they prominently colocalized with asialoGM1. By 4 h of exposure to flagella, toll-like receptor (TLR)5 expression was induced, mobilized to the apical surface of the cells, and colocalized with superficial flagella. Interleukin-8 expression in airway cells was activated by flagella through induction of Ca²⁺ fluxes, Src, Ras, and extracellular signal–regulated kinase 1/2 mitogen-activated protein kinase and nuclear factor-B activation, a pathway previously associated with asialoGM1-mediated stimuli. There was evidence for participation of asialoGM1 and TLR2 as well as TLR5 in the response to flagella, and increased asialoGM1 correlated directly with increased signaling. TLR2 DN or TLR5 DN mutations inhibited interleukin-8 induction by 78% and 35%, respectively (P < 0.001 for each). The participation of TLR2 as well as TLR5 was confirmed in Chinese hamster ovary cells transfected with either human TLR2 or TLR5 in which flagella activated a nuclear factor-B–luciferase reporter to the same extent. Flagella signaling in airway cells can be initiated by interactions with asialoGM1 and TLR2 as well as by activation of TLR5. The availability of exposed receptors on the apical surface of polarized airway epithelial cells is a major factor in the activation of signaling pathways by flagella.

Abbreviations: gangliotetraosylceramide (Galß1,2GalNAcß1,4Galß1,4Glcß1,1Cer), asialoGM1 • 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid, BAPTA • Chinese hamster ovary, CHO • Dulbecco's modified Eagle's media, DMEM • enzyme-linked immunosorbent assay, ELISA • extracellular signal–regulated kinases(s), ERK(s) • fetal calf serum, FCS • interleukin, IL • interleukin-1 receptor–activated kinase, IRAK • lipopolysaccharide, LPS • mitogen-activated protein kinases, MAPKs • mass spectroscopy, MS • normal human nasal polyp, NHNP • phosphate-buffered saline, PBS • toll-like receptor, TLR