Neuromodulatory effects of SARS-CoV-2 on the blood-brain barrier

ABSTRACT

Background: Autopsies of the COVID-19 patients, show presence of SARS-CoV-2 in the brain endothelium, cerebrospinal fluid, glial cells, and neuronal tissue and emerging clinical data from the current pandemic suggests that ∼40% of the patients with COVID-19 developed neurological symptoms. We examined the effect of SARS-COV2 RBD spike protein and heat inactivated SARS-COV2 on Blood barrier barrier (BBB) integrity using a well validated 2D in-vitro Blood brain barrier model, and on the expression levels of tight junction proteins (TJP) that are key to BBB permeability and function. Methods: Our experimental paradigm included treating primary human BMVEC (Cat# ACBRI-376) with recombinant SARS-COV2 Spike protein ( BEI Resources Inc) for 24-48 hrs, followed by examining ACE2 receptor expression by immunofluorescent staining, quantification of levels of pro-inflammatory cytokines in culture supertanants using BioLegend's LEGENDLplex™ bead-based immunoassay. Additionally, we examined the effects of SARS-COV2 on BBB integrity using a well validated 2D in-vitro BBB model and modulation of TJ protein gene expression levels using real time quantitative PCR. Results: Our data shows that primary human BMVEC expressed the ACE2 receptor and that treatment with SARS-COV2 spike protein resulted in a significant increase in ACE 2 receptor expression by BMVEC. We observed a significant increase in the levels of pro-inflammatory cytokines such as TNF-α (p<0.01), IL-6 (p<0.0001), IL-10 (p<0.05), IL-23 (p<0.05) and IL-33 (p<0.01) in BMVEC treated with SARS-COV2 spike protein compared to the untreated controls. BBB integrity which was measured using the transendothelial electrical resistance (TEER) across membrane showed an ∼ 30% (p<0.05) decrease in TEER in BBB treated with SARS-COV2 spike protein as compared to the untreated control, and the functional translational of this effects was evident by the SARS-COV2 induced decrease in TJP expression. Our data showed that SARS-CoV-2 treatment resulted in a decrease in the gene expression of TJPs-ZO-1 (52%;p<0.05), ZO-2 (92%;p<0.001), Claudin-5 (97%;p<0.001) and JAM-2 (45%;p<0.05) as compared to the untreated controls. Conclusion: BMVEC have a paracrine-autocrine role in maintaining CNS homeostasis and that the SARS-COV2 associated endothelial cell dysfunction preludes the neuropathology associated with SARS-COV2 that is observed in COVID-19 infected patients. Potentially, anti-cytokine based therapeutics may be effective in treating patients with COVID-19 associated neurological disease.