SARS‐CoV‐2 Spike Protein Enhances Toxicant‐Induced Blood‐Brain Barrier Dysfunction
Authors: Kelly Gillian C. , Watase Colby K. , Rooks Elizabeth A. , Ho Dao H. 
Published: May 2022

Exposure to toxicants such as benzo(a)pyrene (BaP), 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), or perfluorooctane sulfonate (PFOS), or viruses such as severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is associated with neuropathology and blood‐brain barrier (BBB) dysfunction. Recently, toxicant exposure has been reported to result in poorer health outcomes of (SARS‐CoV‐2) infection. Thus, we hypothesized that co‐exposure to toxicants and SARS‐CoV‐2 will cause greater BBB disruption than exposure to toxicants or SARS‐CoV‐2 alone.BBB permeability was assessed in vitro by measuring transendothelial electrical resistance (TEER; ohms cm2) of a monolayer of primary adult human brain microvascular endothelial cells (hBMECs). Cells were allowed to form a confluent monolayer on a 96‐well plate outfitted with circuit electrodes. Cells were treated with BaP (2, 10, and 50 uM), TCDD (1.5, 15, and 150 nM), PFOS (3, 30, and 300 uM), or recombinant SARS‐CoV‐2 spike protein (SP; 0.1, 1, and 10 nM). In experiments to test the synergistic effects of toxicants and SP, cells were treated with toxicants, then immediately were treated with SP (10 nM). Cell viability was assessed 24 hours after treatment using Cell Proliferation Kit I (Roche Diagnostics, Inc). Gene expression of tight junction protein 1 (TJP1) and vascular cell adhesion molecule‐1 (VCAM‐1) was measured by RT‐qPCR. One‐ and two‐way ANOVA followed by Holm‐Sidak post‐hoc comparisons were performed to detect differences in TEER (resistance, ohms cm2) and cell viability (absorbance, nm). T‐tests were performed to compare RT‐qPCR data. Statistical significance was reached at p < 0.05. Technical triplicates were averaged on 4 different days to obtain N = 4/group.Recombinant SARS‐CoV‐2 spike protein did not significantly affect the resistance or leakiness of the monolayer of hBMECs at any of the concentrations tested. However, when hBMECs were treated with SP (10 nM) in conjunction with BaP or PFOS, TEER was significantly reduced beyond treatment with the toxicants alone. When compared to BaP treatment alone, SP significantly lowered TEER in hBMECs treated with 10 uM or 50 uM of BaP at 9 to 24 hours after treatment (Holm‐Sidak, t’s > 2.507, p’s < 0.04), indicating a synergistic effect of SP and BaP in increasing leakiness of the hBMEC barrier. Similarly, SP caused a significant reduction in TEER of hBMECs treated with 3 uM PFOS starting at 15 hours post treatment when compared to PFOS treatment alone (Holm‐Sidak, t’s > 2.273, p’s < 0.04). TEER of TCDD‐treated hBMECs were not affected by SP treatment. At 24 hours after treatment, cell viability and expression of VCAM‐1 and TJP1 were not altered by treatment by SP alone, nor did SP affect cell viability or gene expression of toxicant‐treated hBMECs. These results suggest that disruption of the hBMEC monolayer was not due to cell death, or expression levels of VCAM‐1 and TJP1.To our knowledge, this is the first report of the synergistic effects of SARS‐CoV‐2 spike protein and environmental toxicants in an in vitro model of the BBB. These findings suggest that individuals who are exposed to environmental pollutants may suffer greater damage of their BBB if subsequently exposed to SARS‐CoV‐2 spike proteins or SARS‐CoV‐2.