The role of LTB4 in neutrophil-mediated inflammation in airway epithelium infected with pseudomonas
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Abstract
Inflammation plays a protective function in the immune system but is also a contributor of many diseases. Airway inflammation can be the result of excessive infiltration of leukocytes, specifically neutrophils, leading to tissue damage or death. A proposed mechanism of the lipoxygenase pathway by Tamang et al. in 2012 showed Hepoxilin A3, a neutrophil chemoattractant, can trigger the transmigration of neutrophils across airway epithelium. Activated neutrophils further produce another chemoattractant called leukotriene B4 (LTB4) to amplify the neutrophil migration to the airspace in the lungs. We investigated the role of LTB4 in neutrophil-mediated inflammation in airway epithelial infected with Pseudomonas by using a mouse airway transitional epithelial model cocultured with mouse neutrophils. We first established and assessed the in vitro coculture model using immunostaining and confocal microscope imaging to characterize the differentiation of the epithelium and RT-qPCR to investigate the gene expression of critical enzymes in the lipoxygenase pathway. Then we performed neutrophil transmigration assays in vitro using wild type C57BL/6 or alox5-/- as bone marrow polymorphonuclear cells (PMNs) cocultured with transitional airway epithelial cells. Myeloperoxidase (MPO) ELISA was performed to measure the amount of transmigrated neutrophils. Our results suggested that LTB4 had the potential of being a critical amplifier with higher migration percentages seen in C57 PMNs compared to alox5-/- PMNs. This was further tested in a mouse pneumonia model with wild type C57 and alox5-/- mice which showed a statistically non-significant difference in neutrophils transmigration to the air space in the lung based on FACS analysis, MPO and elastase 2 (ELA2) ELISA’s, and lactate dehydrogenase (LDH) and LTB4 assays. In vitro results suggest that LTB4 has the potential in being targeted as a critical mediator and amplifier in neutrophil-mediated inflammation in airway epithelial infected with Pseudomonas, but further research is needed to investigate its role in vivo.
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Attribution 4.0 International