SARS-CoV-2 spike protein promotes IL-6 trans-signaling by activation of angiotensin II receptor signaling in epithelial cells

Abstract

Cytokine storm is suggested as one of the major pathological characteristics of SARS-CoV-2 infection, although the mechanism for initiation of a hyper-inflammatory response, and multi-organ damage from viral infection is poorly understood. In this virus-cell interaction study, we observed that SARS-CoV-2 infection or viral spike protein expression alone inhibited angiotensin converting enzyme-2 (ACE2) receptor protein expression. The spike protein promoted an angiotensin II type 1 receptor (AT1) mediated signaling cascade, induced the transcriptional regulatory molecules NF-κB and AP-1/c-Fos via MAPK activation, and increased IL-6 release. SARS-CoV-2 infected patient sera contained elevated levels of IL-6 and soluble IL-6R. Up-regulated AT1 receptor signaling also influenced the release of extracellular soluble IL-6R by the induction of the ADAM-17 protease. Use of the AT1 receptor antagonist, Candesartan cilexetil, resulted in down-regulation of IL-6/soluble IL-6R release in spike expressing cells. Phosphorylation of STAT3 at the Tyr705 residue plays an important role as a transcriptional inducer for SOCS3 and MCP-1 expression. Further study indicated that inhibition of STAT3 Tyr705 phosphorylation in SARS-CoV-2 infected and viral spike protein expressing epithelial cells did not induce SOCS3 and MCP-1 expression. Introduction of culture supernatant from SARS-CoV-2 spike expressing cells on a model human liver endothelial cell line (TMNK-1), where transmembrane IL-6R is poorly expressed, resulted in the induction of STAT3 Tyr705 phosphorylation as well as MCP-1 expression. In conclusion, our results indicated that the presence of SARS-CoV-2 spike protein in epithelial cells promotes IL-6 trans-signaling by activation of the AT1 axis to initiate coordination of a hyper-inflammatory response. The mechanisms of SARS-CoV-2 induced excessive inflammatory response associated with disease severity needs to be critically studied for therapeutic intervention. Cytokine storm is typical of macrophage activation; which leads to tissue damage, lung injury, and acute respiratory distress syndrome. Lung epithelial cells; a primary host for SARS coronavirus infection, can generate a cytokine response leading to an expanded pathology. Our present study was designed to understand SARS-CoV-2 and human epithelial cell interactions, by delineating their contribution to inflammatory cytokine systems with special emphasis on ectopic expression of the viral spike protein. We observed that SARS-CoV-2 infection or spike protein expression in human epithelial cells inhibits ACE2 expression leading to the induction of AT1 signaling, and promotion of IL-6/soluble IL-6R release. IL-6 is one of the major mediators of the hyper-inflammatory response, and can exert signaling through two main pathways referred to as classical or trans- signaling to coordinate prominent pro-inflammatory properties. Our experimental observations suggest the potential for IL-6 trans-signaling during SARS-CoV-2 infection of epithelial cells. These observations will aid in the development of new therapeutic modalities for combating COVID-19 associated disease severity. Aggressive tissue damage via increased IL-6 secretion and other cytokines leading to a hyper-inflammatory response are in line with COVID-19 disease severity. We propose that SARS-CoV-2 infection in pulmonary epithelial cells induces IL-6 trans-signaling for secretion of chemokines; like MCP-1, from pulmonary vascular endothelial cells to attract monocytes/macrophages and create a hyper-inflammatory state leading to enhanced disease severity and acute respiratory distress syndrome.