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Radiation therapy results in the recruitment of immune cells into tumors and healthy tissue. As a key player in this process, the irradiated vascular endothelium captures immune cells and actively promotes their passage to the injured tissue by transendothelial migration. However, the regulation of immune cell recruitment is not well understood in the general context and after radiotherapy. The interactions between endothelial cells and monocytes involve binding between monocyte receptors and glycosylations carried by endothelial membrane proteins. Endothelial high-mannose N-glycans are structures that could play an important role in inflammation in different pathological contexts such as atherosclerosis. Trimmed by class I alpha-mannosidases, these glycans are quite rare under normal conditions but particularly abundant after irradiation of endothelial cells. They are, at least in part, responsible for the interaction between monocytes and irradiated endothelial cells. The objective of this work was to better understand how high-mannose N-glycans are regulated in endothelial cells exposed to radiation.

To better understand the role of high-mannose N-glycans in endothelium-monocyte interactions, we used primary human endothelial cells (HUVECs) and a human monocyte cell line (THP-1). HUVECs were irradiated at 20 Gy with a LINAC. Two days after irradiation, two crucial steps in monocyte recruitment via endothelial cells, namely monocyte adhesion and transendothelial migration, were assessed in vitro by flow adhesion experiments videomicroscopy and transmigration experiments using real-time imaging. The role of the classe-1 alpha-mannosidase MAN1C1 was explored by performing the functional assays after inhibiting (by siRNA) or overexpressing (by lentiviral vectors) the gene encoding the mannosidase. The N-glycan content was identified and quantified by liquid chromatography-tandem mass spectrometry under the different experimental conditions.

We show that the expression of the endothelial MAN1C1 endogenous protein decreases after irradiation. Inhibition of MAN1C1 expression by siRNA gene silencing increases the abundance of high-mannose N-glycans, improves the adhesion rate of monocytes on endothelial cells in flow conditions and, in contrast, decreases radiation-induced transendothelial migration of monocytes. Consistently, overexpression of MAN1C1 in endothelial cells using lentiviral vectors decreases the abundance of high-mannose N-glycans and monocyte adhesion, and enhances transendothelial migration of monocytes.

Overall, this research supports the theory that radiation-induced downregulation of, at least, MAN1C1 is involved in monocyte adhesion to irradiated endothelial cells through the increased expression of high-mannose N-glycans. Hence, we propose a role for endothelial MAN1C1 in the recruitment of monocytes, particularly in the adhesion step to the endothelium.