The SARS-CoV-2 spike protein alters barrier function in 2D static and 3D microfluidic in vitro models of the human blood–brain barrier

Abstract

As researchers across the globe have focused their attention on understanding SARS-CoV-2, the picture that is emerging is that of a virus that has serious effects on the vasculature in multiple organ systems including the cerebral vasculature. Observed effects on the central nervous system includes neurological symptoms (headache, nausea, dizziness), fatal microclot formation and in rare cases encephalitis. However, our understanding of how the virus causes these mild to severe neurological symptoms and how the cerebral vasculature is impacted remains unclear. Thus, the results presented in this report explored whether deleterious outcomes from the SARS-COV-2 viral spike protein on primary human brain microvascular endothelial cells (hBMVECs) could be observed. First, using postmortem brain tissue, we show that the angiotensin converting enzyme 2 or ACE2 (a known binding target for the SARS-CoV-2 spike protein), is expressed throughout various caliber vessels in the frontal cortex. Additionally, ACE2 was also detectable in primary human brain microvascular endothelial (hBMVEC) maintained under cell culture conditions. Analysis for cell viability revealed that neither the S1, S2 or a truncated form of the S1 containing only the RBD had minimal effects on hBMVEC viability within a 48hr exposure window. However, when the viral spike proteins were introduced into model systems that recapitulate the essential features of the Blood-Brain Barrier (BBB), breach to the barrier was evident in various degrees depending on the spike protein subunit tested. Key to our findings is the demonstration that S1 promotes loss of barrier integrity in an advanced 3D microfluid model of the human BBB, a platform that most closely resembles the human physiological conditions at this CNS interface. Subsequent analysis also showed the ability for SARS-CoV-2 spike proteins to trigger a pro-inflammatory response on brain endothelial cells that may contribute to an altered state of BBB function. Together, these results are the first to show the direct impact that the SARS-CoV-2 spike protein could have on brain endothelial cells; thereby offering a plausible explanation for the neurological consequences seen in COVID-19 patients.

Neuropathobiology of COVID-19: The Role for Glia

“…Meanwhile, autoimmunity may be another underlying mechanism for both the neurological (such as encephalitis and encephalopathy; Costello and Dalakas, 2020;Ellul et al, 2020) and psychiatric (such as psychosis and delirium; Hosseini et al, 2020;Rogers et al, 2020;Steardo et al, 2020b) manifestations in COVID-19. Autoimmune responses can be induced by viral pathogens, possibly through "molecular mimicry" (Cappello et al, 2020;Lucchese and Floel, 2020) and breakdown of physiological barriers (Alam et al, 2020;Buzhdygan et al, 2020), in which unleashed autoimmune cells cross-react to exposed autoantigens, hence causing autoimmune pathology. As an immune privileged site, nervous tissue is most vulnerable for autoimmune attack, which instigates various neurological and psychiatric diseases, such as the demyelinating multiple sclerosis and Guillain-Barré syndrome (Giovannoni and Hartung, 1996), as well as the recently depicted autoimmune encephalitis (Dalmau et al, 2007;Granerod et al, 2010;Crisp et al, 2016) and autoimmune psychosis (Kayser et al, 2013;Al-Diwani et al, 2019;Pollak et al, 2020).…”

Neurological consequences of COVID-19: what have we learned and where do we go from here?

“…Neurotropism is commonly observed in coronaviruses, with neuro-invasive properties well documented in SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, and porcine hemagglutinating encephalomyelitis coronavirus (PHE) [11,13,27,28]. The SARS-CoV-2 spike protein also alters barrier function in human models of the blood-brain barrier, providing an additional mechanism of potential CNS entry [29]. Given the genetic similarity and conserved viral structure with SARS-CoV, it appears likely that SARS-CoV-2 may also exhibit neurotropic properties [30,31].…”

SARS-CoV-2 Infection Reaches the Human Nervous System: How?

“…Under physiologic conditions, the BBB is relatively impermeable, though recently the list of biomolecules capable of modulating the permeability, integrity, and tightness of the BBB was extended by inclusion of the SARS-CoV-2 spike protein [151]. It was shown here that introduction of the viral spike proteins into the model systems recapitulating the essential features of the BBB resulted in a breach of the barrier.…”

“…It was shown here that introduction of the viral spike proteins into the model systems recapitulating the essential features of the BBB resulted in a breach of the barrier. Furthermore, SARS-CoV-2 spike protein was shown to increase the MMP3, CCL5, CXCL10, ICAM-1, and VCAM-1 (which are cell adhesion molecules, CAMs) gene expression levels, alter mRNA levels of interleukins IL-1β and IL-6, and trigger a pro-inflammatory response on brain endothelial cells that may further contribute to an altered state of BBB function [151]. These observations were used to support a hypothesis that SARS-CoV-2 is potentially a neuroinvasive virus since it can turn on the machinery to enable the migration of infected immune cells into the brain parenchyma [151].…”

“…Furthermore, SARS-CoV-2 spike protein was shown to increase the MMP3, CCL5, CXCL10, ICAM-1, and VCAM-1 (which are cell adhesion molecules, CAMs) gene expression levels, alter mRNA levels of interleukins IL-1β and IL-6, and trigger a pro-inflammatory response on brain endothelial cells that may further contribute to an altered state of BBB function [151]. These observations were used to support a hypothesis that SARS-CoV-2 is potentially a neuroinvasive virus since it can turn on the machinery to enable the migration of infected immune cells into the brain parenchyma [151]. In blood vessels, the increase of VCAM1 and ICAM1 in response to the pro-inflammatory cytokines plays a crucial role in the adhesion of leukocytes, including macrophages and neutrophils, with the end result being disruption of the BBB and inflammation of the brain [97].…”