Abstract P262: Spike Protein 1 Of Sars-cov-2 Increases Interferon Stimulated Genes And Induces An Immune/inflammatory Responses In Human Endothelial Cells.

Abstract

Introduction: Interferon (IFN) alpha (IFNα) and lambda3 (IFNL3) constitute the first line of immunity against SARS-CoV-2 infection by increasing interferon-stimulated genes (ISGs). IFNs influence the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. Here we hypothesized that in human microvascular endothelial cells (EC) IFNL3 and IFNα influence ACE2 and immune/inflammatory responses mediated by S1P. Methods: EC were stimulated with S1P of SARS-CoV-2 (1 μg/10^6 cells), IFNα (100 ng/mL) or IFNL3 (100 IU/mL). Because ACE2, metalloproteinase domain 17 (ADAM17) and type II transmembrane serine protease (TMPRSS2) are important for SARS-CoV-2 infection, cells were treated with inhibitors of ADAM17 (marimastat, 3.8nM and TAPI-1, 100nM), ACE2 (MLN4760, 440pM), and TMPRSS2 (camostat, 50μM). Expression of ISGs (ISG15, IFIT1, and MX1) was investigated by real-time PCR (5h) and protein expression by immunoblotting (24h). Results: EC stimulated with S1P increased expression of ISGs: ISG15 (2 fold), IFIT1 (6 fold), MX1 (6 fold) (n=12, p<0.05). EC exhibited higher responses to IFNα (ISG15: 16 fold, IFIT1: 21 fold, MX1: 31 fold) than to IFNL3 (ISG15: 1.7 fold, IFIT1: 1.9 fold, MX1: 1.7 fold) (p<0.05). S1P increased gene expression of IL-6 (1.3 fold), TNFα (6.2 fold) and IL-1β (3.3 fold), effects that were maximized 100% by IFNα. Only marimastat inhibited S1P effects. IL-6 was increased by IFNα (1230 pg/mL) and IFNL3 (1124 pg/mL) vs control (591pg/mL). IFNα increased expression of ACE2 (75 kDa) (63%), ADAM17 (36%), and TMPRSS2 (65%). This was associated with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). Nitric oxide production and eNOS phosphorylation (Ser1177) were reduced by IFNα and (40%) and IFNL3 (40%). Conclusions: In human microvascular endothelial cells, S1P, IFNα and IFNL3 induced an immune response characterized by increased expression of interferon-stimulated genes and IL-6 production, processes that involve ADAM17. Inflammation induced by S1P was amplified by IFNα. Our novel findings demonstrate that S1P induces an endothelial immune/inflammatory response that may be important in endotheliitis associated with COVID-19.