SARS-CoV-2-induced brain endothelial barrier disruption and neuroinflammation are ameliorated by α5 integrin inhibitor ATN 161

Abstract

Introduction: The novel coronavirus SARS-CoV-2 infection can cause numerous symptoms, especially in patients with comorbidities such as chronic lung or cerebrovascular diseases. Viral spike protein, used to bind and infect host cells, encodes an arginine-glycine-aspartic acid (RGD) motif that it may use to bind integrin receptors vital for cerebrovascular integrity. Here we investigate the role of integrin α5β1 and its immunological consequences on brain vasculature during SARS-CoV-2 infection. Methods: Mouse brain microvascular-endothelial cells (bEnd.3) were treated with SARS-CoV-2 (Isolate USA- WA1/2020) or delta variant of SARS-CoV-2 spike protein for 24 h then later exposed to hypoxia for 6h (to model in-vivo pulmonary infection). Cells were pretreated with clinically-validated integrin α5β1 inhibitor, ATN-161 (10µM), 1h before SARS-CoV-2 and hypoxia challenge. In addition, BALB/c mice were inoculated intranasally with 2 × 104-PFU of the mouse-adapted (MA)-10 strain of SARS- CoV-2 and treated daily with saline or 1 mg/kg of ATN-161, retro-orbitally. The brains were collected 3-days post-infection and evaluated for changes in markers of inflammation and blood- brain barrier disruption by qPCR. Results: WA and delta SARS-CoV-2 variant spike protein inoculations induced integrin α5 and decreased claudin-5 expression in bEnd.3 cells in a dose-dependent manner. SARS-CoV-2 WA spike protein challenge at 0.5 µg for 24 h followed by hypoxia for 6h resulted in increased α5 and decreased claudin-5 expression in either hypoxia or SARS-CoV-2+hypoxia combination. ATN-161 pretreatment inhibited SARS-CoV-2+hypoxia-induced α5 upregulation and restored claudin-5 loss in bEnd.3 cells. ATN-161 also blocked SARS-CoV-2 and its variants spike protein by ELISA-based assays. qPCR results from the in vivo studies showed a significant increase in the pro-inflammatory response as measured by cytokine IL-6 expression and a decrease in barrier integrity as measured by tight junction claudin-5 expression in the brains of MA10-infected BALB/c mice as compared to mock controls. ATN-161 treatment decreased IL-6 expression and increased claudin 5 expression in the infected mice. Discussion: Our results suggest that targeting integrin α5β1 with ATN-161 offers a promising therapeutic strategy for attenuating SARS-CoV-2 viral load and mitigating the immunological impact on brain vasculature. This approach may be pivotal in reducing both acute and chronic neurological morbidities associated with COVID-19.