Abstract
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. This study evaluated the contribution of endothelial MERTK to neutrophil TEM and endothelial barrier function. 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. MERTK knockdown also altered adherens junction structure, decreased junction protein levels, and reduced basal Rac1 activity in endothelial cells, providing potential mechanisms of how MERTK regulates endothelial barrier function. To study MERTK’s function in vivo, inflammation in the lungs of global Mertk-/- mice was examined during acute pneumonia. In response to P. aeruginosa, more neutrophils were recruited to the lungs of Mertk-/- than wildtype mice. Vascular leakage of Evans blue dye into the lung tissue was also greater 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. 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.
Highlights
Expressed in many different tissues, the Mer receptor tyrosine kinase (MERTK) plays important roles during developmental, physiological, and pathological processes [1,2,3,4,5,6]
MERTK-depleted endothelial cells (ECs) allowed more neutrophil transendothelial migration (TEM) in the transwell assay, as demonstrated by increased fluorescence intensity (Fig 2E). These results suggest that the expression of MERTK in ECs lessens neutrophil TEM in vitro
We report that in vitro, MERTK contributes to endothelial barrier function in unstimulated ECs and after TNFα, modulating both neutrophil TEM and endothelial permeability
Summary
Expressed in many different tissues, the Mer receptor tyrosine kinase (MERTK) plays important roles during developmental, physiological, and pathological processes [1,2,3,4,5,6]. All three TAM kinases are expressed in many adult tissues such as the nervous system, reproductive organs, the lungs, hematopoietic cells, the vasculature, and more [5,6,7] This widespread expression pattern correlates with their involvement in various key cellular processes, including growth and differentiation, adhesion, migration, and immune regulation [6,7,8,9]. Among these, their importance in maintaining tissue homeostasis has been underscored by studies with single, double, and triple TAM kinase knockout mice [10,11,12]. Inability to clear apoptotic cells due to the lack of MERTK can lead to retinitis pigmentosa [9]
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