SARS-CoV-2 spike protein induces brain pericyte immunoreactivity in absence of productive viral infection

Abstract

COVID-19 is a respiratory disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via still elusive mechanisms. SARS-CoV-2 infects host cells by binding to angiotensin-converting enzyme 2 (ACE2), a transmembrane receptor that recognizes the viral spike (S) protein. Brain pericytes were recently shown to express ACE2 at the neurovascular interface, outlining their possible implication in microvasculature injury in COVID-19. Yet, pericyte responses to SARS-CoV-2 is still to be fully elucidated. Using cell-based assays, we report that ACE2 expression in human brain vascular pericytes is highly dynamic and is increased upon S protein stimulation. Pericytes exposed to S protein underwent profound phenotypic changes translated by increased expression of contractile and myofibrogenic proteins, namely α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). These changes were associated to an altered intracellular calcium (Ca²⁺) dynamic. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signalling pathway, which was potentiated by hypoxia, a condition associated to vascular comorbidities, which exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely interleukin-8 (IL-8), IL-18, macrophage migration inhibitory factor (MIF), and stromal cell-derived factor-1 (SDF-1). Finally, we found that S protein could reach the mouse brain via the intranasal route and that reactive ACE2-expressing pericytes are recruited to the damaged tissue undergoing fibrotic scarring in a mouse model of cerebral multifocal micro-occlusions, a main reported vascular-mediated neurological condition associated to COVID-19. Our data demonstrate that the released S protein is sufficient to mediate pericyte immunoreactivity, which may contribute to microvasculature injury in absence of a productive viral infection. Our study provides a better understanding for the possible mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.