Rapid endothelial infection, endothelialitis and vascular damage characterise SARS-CoV-2 infection in a human lung-on-chip model
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Abstract
Severe cases of COVID-19 present with hypercoagulopathies and systemic endothelialitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of immune cell mediated cytokine storms is unknown. This is in part because in vitro models are typically epithelial cell monocultures or fail to recapitulate vascular physiology. We use a vascularised lung-on-chip model where, consistent with monoculture reports, low numbers of SARS-CoV-2 virions are released apically from alveolar epithelial cells. However, rapid infection of the underlying endothelial layer leads to the generation of clusters of endothelial cells with low or no CD31 expression, a progressive loss of endothelial barrier integrity, and a pro-coagulatory microenvironment. These morphological changes do not occur if these cells are exposed to the virus apically. Viral RNA persists in individual cells, which generates a response that is skewed towards NF-KB mediated inflammation, is typified by IL-6 secretion even in the absence of immune cells, and is transient in epithelial cells but persistent in endothelial cells. Perfusion with Tocilizumab, an inhibitor of trans IL-6 signalling slows the loss of barrier integrity but does not prevent the formation of endothelial cell clusters with reduced CD31 expression. SARS-CoV-2 mediated endothelial cell damage occurs despite a lack of rapid viral replication, in a cell-type specific manner and independently of immune-cell mediated cytokine storms, whose effect would only exacerbate the damage.
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SciScore for 10.1101/2020.08.10.243220: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The secondary antibodies used were: Donkey anti-mouse Alexa Fluor 488 (A21206, Thermo Fisher), or Donkey anti-mouse Alexa Fluor 568 (A10037, Thermo Fisher), or Donkey anti-mouse Alexa Fluor 647 (A31573, Thermo Fisher), and were chosen to complement the fluorophores already assigned to RNAscope labelling. anti-mousesuggested: (Thermo Fisher Scientific Cat# A10037, RRID:AB_2534013)A21206suggested: (Molecular Probes Cat# A-21206, RRID:AB_2535792)A10037suggeste…SciScore for 10.1101/2020.08.10.243220: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The secondary antibodies used were: Donkey anti-mouse Alexa Fluor 488 (A21206, Thermo Fisher), or Donkey anti-mouse Alexa Fluor 568 (A10037, Thermo Fisher), or Donkey anti-mouse Alexa Fluor 647 (A31573, Thermo Fisher), and were chosen to complement the fluorophores already assigned to RNAscope labelling. anti-mousesuggested: (Thermo Fisher Scientific Cat# A10037, RRID:AB_2534013)A21206suggested: (Molecular Probes Cat# A-21206, RRID:AB_2535792)A10037suggested: (Thermo Fisher Scientific Cat# A10037, RRID:AB_2534013)A31573suggested: NoneExperimental Models: Cell Lines Sentences Resources Virus used for all experiments in this manuscript was at passage 3 in Vero E6 cells. Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resources CD14+ monocytes were isolated using positive selection (CD14 ultrapure isolation kit, Miltenyi Biosciences) and cultured in RPMI medium supplemented with 10% FBS and differentiated for 7 days with 20 ng/mL recombinant human Miltenyi Biosciencessuggested: NoneAmplicon specificity was confirmed by melting-curve analysis. Ampliconsuggested: (Amplicon, RRID:SCR_003294)Custom-written software in MATLAB was used to segment and identify the 3D volume, mean intensity, and number of RNA dots in each field of view using the nestedSortStruct algorithm for MATLAB written by the Hughey Lab (https://www.github.com/hugheylab/nestedSortStruct, GitHub). MATLABsuggested: (MATLAB, RRID:SCR_001622)Statistical analysis was performed using Origin 9.2 (OriginLabs) and P-values were calculated using a Kruskal-Wallis one-way ANOVA test, with the null hypothesis that the medians of each population were equal. Originsuggested: (Origin, RRID:SCR_014212)Results from OddPub: Thank you for sharing your data.
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: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- No funding statement was detected.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.08.10.243220: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Briefly, the chips were incubated with a blocking solution of 2% Bovine Serum Albumin (BSA) in PBS (‘blocking buffer’), followed by overnight incubation with the primary antibody: mouse anti-human CD45 (abcam NUMBER), or mouse anti-SARS-CoV-2 spike protein (Genetex, NUMBER ) at a concentration of 1:100 in the blocking buffer at 4℃. …anti-human CD45suggested: NoneSciScore for 10.1101/2020.08.10.243220: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Briefly, the chips were incubated with a blocking solution of 2% Bovine Serum Albumin (BSA) in PBS (‘blocking buffer’), followed by overnight incubation with the primary antibody: mouse anti-human CD45 (abcam NUMBER), or mouse anti-SARS-CoV-2 spike protein (Genetex, NUMBER ) at a concentration of 1:100 in the blocking buffer at 4℃. anti-human CD45suggested: Noneanti-SARS-CoV-2 spike proteinsuggested: NoneThe secondary antibodies used were: Donkey anti-mouse Alexa Fluor 488 (A21206, Thermo Fisher), or Donkey anti-mouse Alexa Fluor 568 (A10037, Thermo Fisher), or Donkey anti-mouse Alexa Fluor 647(A31573, Thermo Fisher), and were chosen to complement the fluorophores already assigned to RNAscope labelling. A21206suggested: (Molecular Probes Cat# A-21206, RRID:AB_2535792)A10037suggested: (Thermo Fisher Scientific Cat# A10037, RRID:AB_2534013)anti-mousesuggested: NoneA31573suggested: NoneExperimental Models: Cell Lines Sentences Resources Virus used for all experiments in this manuscript was at passage 3 in Vero E6 cells. Vero E6suggested: RRID:CVCL_XD71)Software and Algorithms Sentences Resources CD14+ monocytes were isolated using positive selection (CD14 ultrapure isolation kit, Miltenyi Biosciences) and cultured in RPMI medium supplemented with 10% FBS and differentiated for 7 days with 20 ng/ml recombinant human Macrophage-Colony Stimulating Factor protein (M-CSF) (Thermo Fisher Scientific), and 100U/L of penicillin-streptomycin solution (Thermo Fisher Scientific) to avoid bacterial contamination. Miltenyi Biosciencessuggested: NoneAmplicon specificity was confirmed by melting-curve analysis. Ampliconsuggested: (Amplicon, RRID:SCR_003294)Custom-written software in MATLAB was used to segment and identify the 3D volume, mean intensity, and number of RNA dots in each field of view using the nestedSortStruct algorithm for MATLAB written by the Hughey Lab (https://www.github.com/hugheylab/nestedSortStruct, GitHub). MATLABsuggested: (MATLAB, RRID:SCR_001622)Statistical analysis was performed using Origin 9.2 (OriginLabs) and p-values were calculated using a Kruskal-Wallis one-way ANOVA test, with the null hypothesis that the medians of each population were equal. Originsuggested: (Origin, RRID:SCR_014212)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 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: We did not find any issues relating to colormaps.
About SciScore
SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.
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