SARS‐CoV‐2 Spike RBD‐Induced Inflammatory Response by CD14+ Monocytes Causes Endothelial Barrier Dysfunction

Abstract

IntroductionEmerging research and clinical evidence suggest that severe COVID‐19 is a microvascular disease, where SARS‐CoV‐2 infection triggers a rapid inflammatory vascular response that leads to endothelial dysfunction and thrombosis. Underlying medical conditions such as obesity, type 2 diabetes and hypertension, have been associated with increased risk for severe illness. SARS‐CoV‐2 enters cells by surface spike protein binding to host cell receptor angiotensin‐converting enzyme 2 (ACE2). Isolated S protein of SARS‐CoV‐1 has been shown to induce an innate immune response. Here we investigate the cytokine response of monocytes with and without comorbidities (obesity and hypertension) exposed to SARS‐CoV‐2 spike protein, and the effects of this response on endothelial barrier function.MethodsHuman CD14+ monocytes (Lonza, Precision for Medicine) from two Caucasian males in their 50s, one with obesity and hypertension (HTN) and one without, were stimulated with LPS (100 ng/mL [positive control]), recombinant spike receptor‐binding domain (RBD, 10 ug/mL) or serum‐free media (negative control) for 48 hours. Monocyte conditioned media (MCM) was collected and cytokines (IL‐6, IL‐8, IL‐10, IL‐12p70, IL‐1beta, IFN‐gamma, TNF‐alpha, IL‐4) were analyzed by multiplex ELISA. Human lung microvascular endothelial cells (HLMVECs) from a healthy donor were exposed to MCM from healthy or HTN subjects. Endothelial barrier function was measured by electric cell‐substrate impedance sensing (ECIS). Briefly, HLMVECs were seeded on ECIS chambers to proliferate until impedance plateaued. Half of the media was then replaced with MCM. Differences in impedance drops were compared among various conditions. Western blotting was used to investigate Spike RBD internalization.ResultsSpike RBD and resulted in increased IL‐6, IL‐8, IL‐12p70, and TNF‐alpha compared to negative control (Fig1A). Monocytes from HTN subject showed increased secretion of IL‐6, IL‐8 and IL‐12p70 compared to those from the healthy subject (Fig 1B). HLMVECs showed moderate long‐term barrier disruption, when treated with Spike RBD alone and Spike RBD with MCM from healthy subjects and no difference in transient barrier disruption or recovery. HLMVECs exposed to MCM from HTN subjects showed increased long‐term barrier dysfunction, especially with Spike RBD present, and recovery of barrier function was also hampered (Fig 2). Spike RBD was observed to be internalized by both monocytes and HLMVECs.DiscussionSpike RBD alone induces inflammatory cytokine secretion by CD14+ monocytes and is further increased in monocytes from the obese/HTN subject. Exposure to Spike RBD and MCM from both healthy and HTN subjects caused significant long‐term endothelial barrier dysfunction in HLMVECs with increased dysfunction seen for the obese/HTN MCM. These results are consistent with clinical experience with the development of acute respiratory distress syndrome, increased disease severity, and worse outcome in COVID‐19 patients with comorbidities. Further work is being done to characterize the cytokine profile and mechanisms of barrier dysfunction.