Pseudomonas Aeruginosa And Its Small Molecular Weight Secretory Factors Induce Human Airway Inflammation

The gram-negative bacterium P. aeruginosa is one of the most common pathogens in airway infectious diseases and is most frequently reported association with hospital infection. Moreover, it is commonly associated with the chronic infection and excessive inflammation that are the proximate causes of lung destruction and, ultimately, the death of the patients. Pseudomonas infections are characterized by a marked influx of polymorphonuclear cells (PMNs, neutrophils) and excessive inflammatory response. Interleukin-8 (IL-8) is the major PMNs chemoattractant responsible for PMNs influx. P. aeruginosa secretes several virulence factors that may contribute to this pathophysiologic effects, includes the small molecular weight secretary factor pyocyanin. However, the molecular mechanisms by which these factors exert their effects are poorly understood. In this study, we examined the ability of P. aeruginosa and pyocyanin to induce airway inflammation and the signaling pathway of pyocyanin-dependent IL-8 release.Three lines of airway epithelial cells, including primary culture of human bronchial epithelial (hTBE) cells, the type II human alveolar cell line A549 and the lung adenocarcinoma cell line SPC-A-1 were used in this experimental system. First, conditioned media from the culture of P. aeruginos remarkably increased IL-8 release in all the three lines of cells. Pyocyanin can stimulate IL-8 release of A549 and SPC-A-1 cells, while there have no effects on hTBE cells. Ultrafiltrate of the culture supernatant via Centricon filters with molecular weight cutoff of 3 kD or 1 kD after chloroform exractions to remove most phenazines, the <3 kD fraction also stimulated A549 cells IL-8 release, suggesting that there would be some unknown small molecular weight secretory factors of P. aeruginos that could sitmulate airway epithelial IL-8 expression.Next, we examined the mechanism of pyocyanin-dependent IL-8 release. Exposure of A549 cells to pyocyanin resulted in NF-kB and MAPK signaling pathway activation. Degradation of IθB-α. was found shortly after A549 cells were stimulated with pyocyanin. Western blot analysis also demonstrated that pyocyanin caused phosphorylation of MAPKs including EKR1/2, p38 and JNK in A549 cells. Pretreatment of A549 cells with specific inhibitors of MEK (U0126), the kinase that activates ERK 1/2, and of p38 (SB203580), both diminished the pyocyanin-induced IL-8 production. Pretreatment of A549 cells with inhibitor of protein tyrosine kinases (genistein) also inhibited the pyocyanin-dependent increase in IL-8 release.Finally, we investigated the role of Toll-like receptor (TLR)-mediated signaling in pyocyanin-dependent IL-8 expression. We constructed the expression plasmid of mutant TLR2 (TLR2 DN) and of mutant MyD88 (MyD88 DN), which is the key molecule in TLRs signaling pathway, and transfected A549 cells and then exposured to pyocyanin. The results indicated that both TLR2 DN and MyD88 DN can significantly inhibit P. aeruginos supernants-induced expression of IL-8 in A549 cells, and have no effects on pyocyanin-dependent IL-8 induction.