Airway Epithelial Cell-pathogen Interaction

The respiratory tract epithelium is a critical interface that protects the host from infection by pathogens. Airway epithelial cells not only function as a mechanical barrier to prevent the lung from the entrance of bacteria, but also at cellular and molecular level the cells have evolved a variety of complex mechanisms against microbes. Pseudomonas aeruginosa and Klebsiella pneumoniae both are intractable opportunistic pathogens, which frequently cause severe infectious diseases in patients with impaired host defense or damaged epithelium. In the past decade, it is becoming increasingly clear that airway epithelial cells play an important role in defense against opportunistic pathogens. This study aims at probing into whether the phenomena of the internalization of Pseudomonas aeruginosa by airway epithelial cells is a mechanism by which the epithelium clear invasive pathogens, and whether cytoplasm pattern-recognition receptors Nods are involved in this process. Also we investigate whether human neutrophil a-defensin (HNP) could potentiate airway epithelial defense against Klebsiella pneumoniae by enhancing the adherence to and the entrance into airway epithelial cells of the bacteria.In the study of the internalization of Pseudomonas aeruginosa by airway epithelial cells, viable bacteria were incubated with primary human epithelial cells or A549 cells for 2 hours and then cells were washed with PBS and cultured for another 2 hours in the presence of the gentamicin tokill any extracelluar bacteria. Cells were lysed at 4 hours or 24 hours respectively by Triton X-100 to release intracellular viable bacteria. Colony counting method was preformed to quantify bacteria. The results indicated that there was a continuous decrease of viable bacteria in the airway epithelial cells, which suggested the internalized bacteria were cleared by epithelial cells. The expression level of IL-8, an inflammatory response marker, in airway epithelial cells incubated with viable bacteria was determined by ELISA and the results indicate that Pseudomonas aeruginosa could strongly induce the secretion of IL-8 from epithelial cells.The role of Nods in the inflammation response induced by Pseudomonas aeruginosa and in the clearance of Pseudomonas aeruginosa by airway epithelial cells was investigated. A549 cells treated with or without digitonin, a modest detergent, were stimulated with sonicated bacterial extract from Pseudomonas aeruginosa. The expression level of IL-8 was detected using ELISA. The result showed that the sonicated bacterial extract only caused a modest, less than 1 fold, increase in IL-8 secretion by digitonin-untreated A549 cells, whereas in the digitonin-permeabilized A549 cells, sonicated bacterial extract strongly induced the expression of IL-8, a 3 fold increase over the control. These data suggested that there are some intracellular pattern recognition receptors in the airway epithelial cells that respond to the inflammation induced by sonicated bacterial extract. The intracellular pattern recognition receptors Nodi and Nod2 were detected in human lung epithelial cells by RT-PCR. It was proved that Nodi and Nod2 were expressed in both primary cultured human tracheobronchial epithelial cells and A549 cells. Two expression plasmids encoding dominant negative mutant Nodi and dominant negative mutant Nod2 were constructed respectively and weretransfected into A549 cells. The number of viable internalized Pseudomonas aeruginosa in A549 cells transfected with plasmid expressing mutant Nod2 was higher than un-transfected cells or MOCK transfectant. In contrast, transfection of mutant Nodi plasmid had no effect on the number of viable internalized Pseudomonas aeruginosa in A549 cells.To validate our hypothesis that a-defensin can stimulate the adherence of some pathogens to lung epithelial cells and further enhance the entrance of bacteria into epithelial cells, A549 cells were incubated with viable klebsiella pneumoniae in the presence or absent of HNP for 4 hours. The number of bacteria adherent to cells was quantified by colony counting method. Meanwhile, the gentamicin protection assay was performed to investigate the effect of HNP on the internalization of klebsiella pneumoniae by A549 cells. HNP, at the concentration of 20 u g/ml, caused a 12-fold increase in the number of bacteria adherent to A549 cells and in the presence of HNP, much more bacteria were internalized by A549 cells.It can be concluded that bacteria internalization by human airway epithelial cells is an important host defense mechanism against pathogen. The intracellular pattern recognition receptor Nod2 may participate in the process of the clearance of pathogens by airway epithelial cells. a-Defensins can enhance the adherence of some pathogen to airway epithelial cells epithelial cells and may contribute to the clearance of pathogens in the lung...