Spike protein 1 of SARS‐CoV‐2 induces an immune and inflammatory response and increases expression of ACE2 in human endothelial cells

Abstract

Introduction Interferon (IFN) alpha (IFNα) and lambda3 (IFNλ3) constitute the first line of immunity against viral infection by increasing expression of interferon-stimulated genes (ISGs). Both are produced in SARS-CoV-2 infection and have been considered therapeutically in COVID-19. In epithelial cells IFNs have been shown to influence the expression of angiotensin-converting enzyme 2 (ACE2), the receptor for S-protein (S1P) of SARS-CoV-2. ACE2 is part of the vascular renin angiotensin system and may be important in COVID-19-associated endotheliitis. Whether SP1 influences immune responses in endothelial cells unknown. We investigated effects of SP1 on ACE2 expression and immune responses in human endothelial cells. Methods Culturedhuman Microvascular, Lymphatic, Aortic and Pulmonary Endothelial Cells (MEC, LEC, AEC, and PEC respectively) were stimulated with SP1 of SARS-CoV-2 (1µg/106 cells), IFNα (100ng/mL) or IFNλ3 (100IU/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), cytokine by ELISA and protein expression by immunoblotting (24h). Results AEC, LEC, MEC, and PEC stimulated with SP1 exhibited increased expression of ISGs: ISG15 (1.9-2.3-fold), IFIT1 (2.0-7.9-fold), MX1 (3.1-5.8-fold), indicating activation of the IFN pathway. MEC and LEC exhibited higher responses to IFNα activation. MEC (ISG15: 16-fold, IFIT1: 21-fold, MX1: 31-fold) and LEC (ISG15: 48-fold, IFIT1: 105-fold, MX1: 134-fold). Only MEC exhibited ISG expression induced by IFNλ3 (ISG15: 1.7-fold, IFIT1: 1.9-fold, MX1: 1.7-fold) (P<0.05). IL-6 production was increased in MEC stimulated with IFNα (1230pg/mL) and IFNλ3 (1124pg/mL) vs control (591pg/mL). Marimastat, but not camostat or MLN4760 inhibited SP1 effects. IFNα induced IL-6 production in AEC and PEC (178pg/mL vs control 109pg/mL). No changes in IL-6 were observed in LEC. IFNα increased expression of the short form of ACE2 (75kDa) (63%), ADAM17 (36%), and TMPRSS2 (65%) in MECs. This was associated with increased activation of pro-inflammatory pathways with increased phosphorylation of Stat1 (134%), Stat2 (102%), ERK1/2 (42%). IFNλ3 also increased phosphorylation of Stat2 (60%) and ERK1/2 (75%). In MEC, phosphorylation of the eNOS activation motif Ser1177 was reduced by IFNLα and (40%) and IFNλ3 (40%). Conclusions In human endothelial cells from multiple vascular beds, SP1, IFNα and IFNλ3 induced an immune response characterised by increased ISG expression and IL-6 production, processes that involve ADAM17. These effects were associated with increased expression of the short form of ACE2 and activation of pro-inflammatory pathways. Our novel findings demonstrate that SP-1 induces an endothelial immune and inflammatory response that may be important in endotheliitis associated with COVID-19.