RESUMO
Introduction: Autopsies of COVID-19 patients demonstrate the presence of SARS-CoV-2 in the brain endothelium, cerebrospinal fluid, glial cells, and neuronal tissue;while emerging clinical data suggest that ~40% of COVID-19 patients develop neurological symptoms. We examined the effects of SARS-COV-2 RBD spike protein on blood brain barrier (BB) integrity, and on the expression of tight junction proteins (TJP) that maintain BBB permeability and function. Materials / Methods: Primary human BMVEC were treated with recombinant SARS-COV-2 Spike protein (BEI Resources Inc) for 24-48 hrs, followed by immunofluorescent staining to quantify ACE2 receptor expression. Pro-inflammatory cytokines were quantified in culture supernatants using BioLegend's LEGENDLplexTM bead-based immunoassay. Additionally, a well validated 2D in-vitro BBB model was used to examine the effects of SARS-COV-2 on BBB integrity as measured by transendothelial electrical resistance (TEER) across the membrane, and TJ protein gene expression levels were measured using real time quantitative PCR. Result(s): Data demonstrates that primary human BMVEC express the ACE2 receptor and treatment with SARS-COV-2 spike protein significant increases in ACE2 receptor expression. We observed a significant increase in the levels of the pro-inflammatory cytokines TNF-alpha (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-COV-2 spike protein compared to untreated controls. A 30% (p<0.05) decrease in TEER occurred in the BBB treated with SARS-COV-2 spike protein as compared to untreated controls, and SARS-COV-2 decreased TJP expression. Data demonstrates that SARS-COV-2 treatment decreased gene expression for the 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 untreated controls. Discussion(s): SARS COV-2 mediates its effects via the ACE2 receptor and therefore an increase in ACE2 expression on BMVEC suggests that neuroinvasion by SARS- COV2 is mediated via endothelial inflammation. Further, SARS-COV-2 induced levels of pro-inflammatory cytokines IL-6, TNF-alpha, IL-8, and IL-10 corroborates the induction of a neuroinflammatory response, confirming hypercytokinemia, which may underlie neuroinflammation in COVID-19 associated encephalopathy. Our data suggest that the significant decrease in TJP gene expression levels directly affect BBB integrity and function thus enabling neuro-invasion and potential subsequent COVID-19 associated neuropathology. Conclusion(s): BMVEC have a paracrine-autocrine role in maintaining CNS homeostasis and the SARS-COV2 associated endothelial cell dysfunction preludes the neuropathology observed in COVID-19 infected patients. Potentially, anti-cytokine based therapeutics may be effective in treating patients with COVID- 19 associated neurological disease. Acknowledgements: Authors gratefully acknowledge funding from SUNY Research Seed Grant Program 2019-20 -RFP #20-03-COVID that was crucial to obtain data for this pilot project. Learning Objectives: Examine the basic neuromodulatory mechanisms that underlie SARS-COV-2 mediated neuropathology. Keywords: SARS-COV2;transendothelial electrical resistance;Blood Brain Barrier;permeability, Tight junction Copyright © 2022
RESUMO
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.