Effects of Nitric Oxide onPseudomonas aeruginosaInfection of Epithelial Cells from a Human Respiratory Cell Line Derived from a Patient with Cystic Fibrosis

Abstract

Cystic fibrosis (CF) is characterized by airway inflammation and chronic bacterial lung infection, most commonly with Pseudomonas aeruginosa, an opportunistic human pathogen. Despite the persistent airway inflammation observed in patients with CF, although phagocyte inducible nitric oxide synthase (iNOS) production is upregulated, expression of iNOS in the respiratory epithelium is markedly reduced. Given the antimicrobial action of NO, this may contribute to the chronic airway infection of this disease. To define the role of epithelium-derived NO in airway defense against P. aeruginosa, we infected differentiated human bronchial epithelial cells derived from a patient with CF (CFBE41o-cells) with different strains of this pathogen at low multiplicities of infection. Using cells transfected with human iNOS cDNA, we studied the effect of NO on P. aeruginosa replication, adherence, and internalization. P. aeruginosa adherence to iNOSexpressing cells was reduced by 44 to 72% (P ‫؍‬ 0.02) compared with control values. Absolute P. aeruginosa uptake into these cells was reduced by 44%, but uptake expressed as a percentage of adherent bacteria did not differ from the control uptake. Survival of P. aeruginosa within iNOS-expressing cells was reduced at late times postinfection (P ‫؍‬ 0.034). NO production did not alter host cell viability. NO production reduced P. aeruginosa adherence to human bronchial epithelial cells and enhanced killing of internalized bacteria, suggesting that a lack of epithelial iNOS in patients with CF may contribute to P. aeruginosa infection and colonization.

Transcriptome Analysis of the ArgR Regulon inPseudomonas aeruginosa

“…It is intriguing that arginine serves as the substrate of nitric oxide synthetase for the synthesis of an important second messenger molecule, NO (21). Perhaps maintaining such a sophisticated arginine metabolic network in P. aeruginosa provides this opportunistic human pathogen an advantage in establishing infections, such as in cystic fibrosis patients (5).…”

Airway epithelial cell signaling in response to bacterial pathogens

“…84 NO has important local effects in vasodilatation as well as direct anti-bacterial activities. 85,86 Studies in iNOS deficient mice have shown an anti-inflammatory role for NO. The lack of iNOS induced a significant increase in leukocyte rolling and neutrophil recruitment to the lungs.…”

“…Bacterial killing activity could be restored in airway epithelial cells derived from a patient with cystic fibrosis, which have reduced expression of iNOS, by transfection with human iNOS cDNA. 85 Excessive NO production due to bacterial and cytokine induction of iNOS, however, is a major cause of lung injury. Nitrosylation by nitric oxide compounds such as peroxynitrite formed with reactive oxygen species in the proximity of activated neutrophils, can cause cell necrosis by disruption of mitochondrial metabolism and enhance apoptosis by inhibition of the anti-apoptotic transcription factor NF-kB.…”

Inflammatory Levels of Nitric Oxide Inhibit Airway Epithelial Cell Migration by Inhibition of the Kinase ERK1/2 and Activation of Hypoxia-inducible Factor-1α

“…Indeed, elevated concentrations of exhaled NO and increased epithelial expression of NOS2 are characteristic features of chronic inflammatory airway diseases such as asthma (1,2). The biological roles of NOS2 within the airway epithelium include contribution to innate host defense (3,4), regulation of epithelial ion transport (5), and maintenance of epithelial barrier integrity (1,6,7). In addition, our recent studies showed that physiological concentrations of NO can promote airway epithelial cell migration and repair in response to in vitro injury, which was associated with increased expression and activation of gelatinase B (matrix metalloproteinase-9; MMP-9) (8).…”