Pos1414 type i interferons induce tie2-mediated endothelial cell dysfunction in systemic lupus erythematosus

Background Endothelial cell (EC) dysfunction is a hallmark of Systemic Lupus Erythematosus (SLE) and has been generally accepted to be one of the important factors contributing to the higher risk of thrombosis and atherosclerotic events observed in SLE patients. Although the presence of traditional factors (smoking, diabetes, increased age, obesity) and the presence of autoantibodies are associated with atherosclerosis and thrombotic events, they do not completely explain the higher risk of these events in SLE, suggesting the existence of other mechanism/factors. Tie2 is a tyrosine kinase receptor essential for vascular development and blood vessel remodeling through interaction with its ligands angiopoietin-1 (Ang-1) and Ang-2. In homeostatic conditions, both Ang-1 and Ang-2 activate Tie2 signaling and induce vascular stabilization in a Tie1-dependent manner. However, inflammatory processes induce Tie1 cleavage, leading to the inhibition of Ang-1-induced Tie2 activation, and to the increase of Ang-2 now acting as a Tie2 antagonist, culminating in vascular dysfunction and EC activation [1,2]. Importantly, this process has been implicated in both atherosclerosis and thrombosis. Objectives As type I Interferons (IFN-α and IFN-β) are key cytokines in the pathogenesis of SLE, the aim of this study is to determine whether these cytokines induce Tie2 signaling-mediated endothelial cell dysfunction. Methods Serum levels of Ang-1, Ang-2 and sTie1 in SLE patients (n=48) and healthy control (HC, n=29) were measured by ELISA. Human Umbilical Vein EC (HUVEC) were stimulated with SLE serum (20%), IFN-α and IFN-β (1000 IU) for 5, 15, 30 min and 1, 2, 4, 6, 8, 12, 24, 48 and 72 hours and mRNA and protein expression of Ang-1, Ang-2, Tie1 and Tie2 were determined by quantitative PCR (qPCR) and ELISA, respectively. The phosphorylation of Tie2 was determined by Western Blot and HUVEC viability by calcein assay. The angiogenic capacity was measured by tube formation assay. Silencing assays were performed with siRNAs addressed to IFNAR1 and Tie1 receptors. Results Type I IFNs, mainly IFN-β, significantly reduced TIE1 and TIE2 levels. IFN-β stimulation significantly increased the secretion of the Tie1 ectodomain (sTie1). Both IFNs significantly reduced protein secretion of Ang-1 after 24h of stimulation. Also, IFN-β induced Ang-2 secretion at early time points (<4h). Furthermore, IFN-α and IFN-β stimulation reduced Tie2 activation (Figure 1). Both type I IFNs significantly reduced the viability of HUVEC. SLE serum increased Ang-2 and sTie1 secretion levels in HUVEC at early time points (<1h). We found reduced levels of Ang-1 and elevated Ang-2 and sTie1 in SLE patients compared to HC. Also, IFN-β induced tube formation at short times (4h) and decreased at 24h. Remarkably, this effect was reversed by silencing the receptors Tie1 and IFNAR1. Figure 1. Ang-1, Ang-2, Tie1 and Tie2 mRNA expression and protein secretion in HUVEC stimulated with IFN-α or IFN-β (1000 IU/ml) for the indicated time points. Means and SEM are shown. * p<0.05, ** p<0.01 and **** p<0.0001. Also, representative immunoblot of Tie2 phosphorilation in HUVEC stimulated with IFN-α or IFN-β (1000 IU/ml) for 24 h is shown. Conclusion Our results demonstrate that type I IFNs play a relevant role in the stability of endothelial cells by inhibiting Tie2 signaling, suggesting that these processes may be implicated in the cardiovascular events observed in SLE patients. References [1]Kim, M. et al . Journal of Clinical Investigation 126, 3511–3525 (2016). [2]Saharinen, P., Eklund, L. & Alitalo, K. Nature Reviews Drug Discovery 16, 635–661 (2017). Acknowledgements: NIL. Disclosure of Interests None Declared.