In Vivo Quantification of SARS-CoV-2 Induced Endothelial-Leukocyte Interactions

Objective: The hallmark of severe SARS-CoV-2 infection is endothelial (EC) dysfunction, resulting in microvascular thrombosis. Venous thrombosis initiates via leukocyte adhesion to endothelial cellular adhesion molecules (CAMs). EC-leukocyte interactions in SARs-CoV-2 (CoV2) related thrombosis, such as microCLOTs subtype of ARDS, remains understudied.

Methods:
Publicly available single cell RNA sequencing from human, post-mortem, CoV2 pneumonia affected lungs were analyzed with CellChat.  Murine venous endothelial cells (VECs) were infected with murine coronavirus (MHVA59) as in vitro CoV2 model for ELISA and cellular adhesion assay.  Intravital cremasteric microscopy performed on MHVA59 infected (8x10⁵ PFU intranasal inoculation) 12-week old C57bl6 mice versus sham controls. Three days post infection, cremasteric venules were exposed microsurgically. Rat anti-mouse GP1B DyLight488 conjugated antibody and rat anti-mouse Ly6G AlexaFluor 647 conjugated antibody were administered via an internal jugular cannula. Thrombi were induced using laser ablation.



Results: Pathway analysis of human lung endothelial venous, arterial, and capillary ECs but not lymphatic ECs, predicted increased leukocyte-EC interactions in fatal CoV2 infection (Figure 1A). In vitro models demonstrate MHVA59 infection significantly increased CAM protein levels, p < 0.01 (Figure 1B). Ex vivo leukocyte adhesion assay demonstrates significant increase in leukocyte adhesion to MHVA59-infected VECs compared to uninfected controls (Figure 1C). Intravital microscopic endothelial-leukocyte imaging demonstrated significant increases in leukocyte adhesion time in MHVA59-infected mice compared to healthy controls (Figure 1D).

Conclusions: The proadhesive propensity of ECs in SARS-CoV-2 infection may act as the inciting event behind the microthrombotic phenotype by promoting endothelial-leukocyte interactions. Using a new model combining CoV2 in vivo murine model with microvascular injury under intravital imaging, endothelial-leukocyte interaction can be quantified in induced microthrombi and strategies to mitigate microthrombosis can be assessed.