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.
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.
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-? (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.
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.