A Crucial Role of Flagellin in the Induction of Airway Mucus Production by Pseudomonas aeruginosa

Fatima Ben Mohamed,Reuben Ramphal,Mathieu Medina,Viviane Balloy,Michel Chignard,Lhousseine Touqui,Ignacio Garcia-Verdugo,Bernhard Ryffel

doi:10.1371/journal.pone.0039888

Fatima Ben Mohamed, Reuben Ramphal + Show 6 more

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https://doi.org/10.1371/journal.pone.0039888

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Journal: PLoS ONE	Publication Date: Jul 2, 2012
Citations: 71	License type: CC BY 4.0

Affiliation: University of Florida, Institut Pasteur, Inserm

Abstract

Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis.

Highlights

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human respiratory pathogen involved in a number of nosocomial infections [1,2]
These findings suggest that flagellin plays a critical role in stimulation of airway mucus secretion in P. aeruginosa-infected mice
These findings suggest that flagellin: (i) is involved in both MUC5AC and MUC2 expressions in NCI-H292 cells; and (ii) is released by bacteria at sufficient amounts to induce these expressions

Summary

Introduction

The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human respiratory pathogen involved in a number of nosocomial infections [1,2]. To protect its mucosal surfaces from infections by pathogens, the host uses sophisticated recognition systems including Toll-like receptors (TLRs) expressed by mucosal epithelial cells and macrophages, which sense conserved pathogen-associated molecular patterns (PAMPs) [3,4,5]. P. aeruginosa expresses numerous PAMPs [6] among which LPS and flagellin play a key role in host response to this bacterium, through interactions with TLR4 and TLR5, respectively [7]. The nucleotidebinding and oligomerization domain (NOD)-like receptor (NLR) family have been identified as intracellular pattern recognition molecules for various microbial pathogens, including P. aeruginosa [10]. Human NLR family is composed of numerous pattern recognition molecules including NOD1, NOD2, Ipaf and Naip reviewed in [10]. It is known that NOD1 and NOD2 interact with bacterial cell wall components, whereas Ipaf and

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