[The role of monocytes in Hantaan virus-induced vascular endothelial injury using a co-culture model].

Objective To investigate the role and regulatory mechanism of monocytes in the dysfunction of vascular endothelial cell induced by Hantaan virus(HTNV) infection. Methods The in vitro co-culture model based on Transwell chambers was established [Human umbilical vein endothelial cells (HUVECs) were seeded in the middle layer, Human embryonic kidney cell 293 (HEK293) cells in the lower layer, and peripheral blood mononuclear cells(PBMCs) or monocyte-depleted PBMCs in the upper layer]. The monocytes in PBMCs were removed by the adherent method and the efficiency of removal was verified by flow cytometry (FCM). The expression of HTNV nucleocapsid protein (NP) in tissue cells was detected by Western blot to evaluate the effects of monocytes on viral replication. The levels of cytokine secretion and the states of T cell activation in the co-culture model were detected by Bio Plex suspension chip system and FCM, respectively. Finally, HUVEC permeability was assessed by measuring transepithelial electrical resistance (TER) following monocyte depletion or the addition of an anti-tumor necrosis factor α (TNF-α) neutralizing antibody. This was designed to determine whether monocytes exacerbate endothelial injury after HTNV infection by disrupting immune microenvironment homeostasis. Results Depletion of monocytes significantly increased HTNV NP expression in both HEK293 cells and HUVECs. Concurrently, levels of pro-inflammatory cytokines, including TNF-α, interleukin 1β(IL-1β), and IL-6, in the upper chamber culture medium were significantly reduced. Furthermore, the activation of cytotoxic T lymphocytes (CTL), type 1 helper T(Th1), Th2, and Th17 cells in the PBMCs was inhibited. The decline in the transepithelial electrical resistance of HUVEC was attenuated following monocyte removal, and this monocyte-mediated increase in endothelial permeability was reversed by the anti-TNF-α neutralizing antibody. Conclusion In this study, the methodology of the Transwell experiment is improved, allowing a better simulation of the in vivo process of HTNV infection. The findings reveal a dual regulatory role of monocytes in HTNV-induced vascular endothelial cell injury. Monocytes could not only inhibit HTNV replication but also promote T cell activation and increase vascular endothelial permeability through the secretion of pro-inflammatory cytokines such as TNF-α. Intervention targeting TNF-α significantly alleviate endothelial cell injury caused by HTNV infection.