SARS-CoV-2 spike protein induces brain pericyte immunoreactivity in absence of productive viral infection
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Abstract
COVID-19 is a respiratory disease caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). COVID-19 pathogenesis causes vascular-mediated neurological disorders via still elusive mechanisms. SARS-CoV-2 infects host cells by binding to angiotensin-converting enzyme 2 (ACE2), a transmembrane receptor that recognizes the viral spike (S) protein. Brain pericytes were recently shown to express ACE2 at the neurovascular interface, outlining their possible implication in microvasculature injury in COVID-19. Yet, pericyte responses to SARS-CoV-2 is still to be fully elucidated. Using cell-based assays, we report that ACE2 expression in human brain vascular pericytes is highly dynamic and is increased upon S protein stimulation. Pericytes exposed to S protein underwent profound phenotypic changes translated by increased expression of contractile and myofibrogenic proteins, namely α-smooth muscle actin (α-SMA), fibronectin, collagen I, and neurogenic locus notch homolog protein-3 (NOTCH3). These changes were associated to an altered intracellular calcium (Ca 2+ ) dynamic. Furthermore, S protein induced lipid peroxidation, oxidative and nitrosative stress in pericytes as well as triggered an immune reaction translated by activation of nuclear factor-kappa-B (NF-κB) signalling pathway, which was potentiated by hypoxia, a condition associated to vascular comorbidities, which exacerbate COVID-19 pathogenesis. S protein exposure combined to hypoxia enhanced the production of pro-inflammatory cytokines involved in immune cell activation and trafficking, namely interleukin-8 (IL-8), IL-18, macrophage migration inhibitory factor (MIF), and stromal cell-derived factor-1 (SDF-1). Finally, we found that S protein could reach the mouse brain via the intranasal route and that reactive ACE2-expressing pericytes are recruited to the damaged tissue undergoing fibrotic scarring in a mouse model of cerebral multifocal micro-occlusions, a main reported vascular-mediated neurological condition associated to COVID-19. Our data demonstrate that the released S protein is sufficient to mediate pericyte immunoreactivity, which may contribute to microvasculature injury in absence of a productive viral infection. Our study provides a better understanding for the possible mechanisms underlying cerebrovascular disorders in COVID-19, paving the way to develop new therapeutic interventions.
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SciScore for 10.1101/2021.04.30.442194: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Induction of cerebral micro-occlusions in mice: Animal experiments were performed according to the Canadian Council on Animal Care guidelines, as administered by the Université Laval Animal Welfare Committee. Sex as a biological variable Three months old C57BL6/j male mice were subjected to micro-occlusions to generate sporadic cerebral micro-infarcts via the unilateral injection of microspheres into the common carotid artery (CCA), as previously described [20]. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources The following primary antibodies were used; mouse anti-α-SMA Cy3-conjugated (Sigma-Aldrich; C6198; 1/250) anti-…SciScore for 10.1101/2021.04.30.442194: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IACUC: Induction of cerebral micro-occlusions in mice: Animal experiments were performed according to the Canadian Council on Animal Care guidelines, as administered by the Université Laval Animal Welfare Committee. Sex as a biological variable Three months old C57BL6/j male mice were subjected to micro-occlusions to generate sporadic cerebral micro-infarcts via the unilateral injection of microspheres into the common carotid artery (CCA), as previously described [20]. Randomization not detected. Blinding not detected. Power Analysis not detected. Table 2: Resources
Antibodies Sentences Resources The following primary antibodies were used; mouse anti-α-SMA Cy3-conjugated (Sigma-Aldrich; C6198; 1/250) anti-α-SMAsuggested: NoneAfterwards, cells were rinsed in 0.1M PBS, followed by a 2-hour incubation with the appropriate secondary antibodies; Cy3 AffiniPure goat anti-mouse IgG (H+L) (Jackson Immunoreasearch), or Cy5 AffiniPure goat anti-rabbit IgG (H+L) (Jackson Immunoreasearch) anti-mouse IgGsuggested: Noneanti-rabbit IgGsuggested: NoneThe following primary antibodies were used; goat anti-ACE2 (R&D systems; AF3437; 1/200) anti-ACE2suggested: (Millipore Cat# MAB5676, RRID:AB_2223314)The next day, brain sections were washed with KPBS and then incubated for 2 hours at room temperature in the dark with either a secondary antibody donkey anti-goat Alexa Fluor 647® (Jackson Immunoresearch) or donkey anti-rabbit Alexa Fluor 488® (Jackson Immunoresearch) diluted 1/10000 in KPBS. anti-goatsuggested: Noneanti-rabbitsuggested: NoneExperimental Models: Organisms/Strains Sentences Resources Three months old C57BL6/j male mice were subjected to micro-occlusions to generate sporadic cerebral micro-infarcts via the unilateral injection of microspheres into the common carotid artery (CCA), as previously described [20]. C57BL6/jsuggested: NoneSoftware and Algorithms Sentences Resources Digitized blots were analyzed with ImageJ software, corrected for protein loading by means of β-actin, and expressed as relative values comparing different groups. ImageJsuggested: (ImageJ, RRID:SCR_003070), rabbit anti-fibronectin (Abcam; ab18723; 1/250), rabbit anti-NOTCH3 (Abcam; ab23426; 1/250), rabbit anti-collagen I (Abcam; ab34710; 1/200), and mouse anti-ACE2 (Cell Signaling Technology; 15983; 1/200). Cell Signaling Technologysuggested: (Cell Signaling Technology, RRID:SCR_004431)Images were acquired every 10 seconds for 20 minutes and the recorded videos were analyzed using a custom-written script [25] in MATLAB (MathWorks). MATLABsuggested: (MATLAB, RRID:SCR_001622)All statistical analyses were performed using GraphPad Prism Version 8 for Mac (GraphPad Software, CA, USA). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: Please consider improving the rainbow (“jet”) colormap(s) used on page 42. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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