THE ROLE OF ENDOTHELIAL MERTK DURING THE INFLAMMATORY RESPONSE IN LUNGS

As a key homeostasis regulator in mammals, the MERTK receptor tyrosine kinase is crucial for efferocytosis, a process that requires remodeling of the cell membrane and adjacent actin cytoskeleton. Membrane and cytoskeletal reorganization also occur in endothelial cells during inflammation, particularly during neutrophil transendothelial migration (TEM) and during changes in permeability. However, MERTK’s function in endothelial cells remains unclear. The goal of this dissertation is to evaluate the contribution of endothelial MERTK to neutrophil TEM and endothelial barrier function during inflammation. In vitro experiments using primary human pulmonary microvascular endothelial cells found that neutrophil TEM across the endothelial monolayers was enhanced when MERTK expression in endothelial cells was reduced by siRNA knockdown. Examination of endothelial barrier function revealed increased passage of dextran across the MERTK-depleted monolayers, suggesting that MERTK helps maintain endothelial barrier function. Pharmacological inhibition of MERTK also enhanced dextran leakage across endothelial monolayers, suggesting that the kinase function of MERTK is involved in regulating endothelial barrier function. In biochemical assays examining the signaling pathways through which MERTK regulates TEM and endothelial permeability, MERTK knockdown led to decreased Rac1 activity in endothelial cells both at basal level and in an in vitro inflammation model induced by TNFα. As higher Rac1 activity correlates with superior barrier function, its decrease in the absence of MERTK provides one pathway by which MERTK regulates endothelial permeability. In contrast, the baseline activity of another Rho family GTPase and important regulator of TEM, RhoG, was not affected by MERTK depletion in endothelial cells. To study MERTK’s function in vivo, inflammation in the lungs of Mertk-/- mice was examined during acute pneumonia. In response to Pseudomonas aeruginosa, more neutrophils were recruited to the lungs of Mertk-/- than wildtype mice. Vascular leakage of an intravenously injected Evans blue dye into the lung tissue was also greater in Mertk-/- mice during pneumonia compared to wildtype mice, suggesting elevated pulmonary vascular permeability in Mertk-/- mice. To analyze endothelial MERTK’s involvement in these processes, we generated inducible endothelial cell-specific (iEC) Mertk-/- mice. When similarly challenged with P. aeruginosa, iEC Mertk-/- mice demonstrated no difference in neutrophil TEM into the inflamed lungs or in vascular permeability compared to control mice. Taken together, these results suggest that deletion of MERTK in human pulmonary microvascular endothelial cells in vitro and in all cells in vivo aggravates the inflammatory response. However, selective MERTK deletion in endothelial cells in vivo failed to replicate this response. We attempted to explain the differences of results obtained from our in vitro and in vivo systems based on the current knowledge of MERTK and TEM. Future work of this dissertation will involve efforts to investigate the mechanisms that might have caused the discrepancies between the in vitro and in vivo results.Doctor of Philosoph