SARS-CoV-2 causes severe alveolar inflammation and barrier dysfunction

Abstract

Infections with SARS-CoV-2 lead to mild to severe coronavirus disease-19 (COVID-19) with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses.

To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human alveolus-on-a-chip model composed of epithelial, endothelial, and mononuclear cells.

Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including the interferon expression. By contrast, the adjacent endothelial layer was no infected and did neither show productive virus replication or interferon release. With prolonged infection, both cell types are damaged, and the barrier function is deteriorated, allowing the viral particles to overbear.

In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokine release, which results in the damage of the alveolar barrier function and viral dissemination.

SciScore for 10.1101/2020.08.31.276725: (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
) IgG monoclonal, primary antibodies and AlexaFluor® goat anti-mouse IgG polyclonal antibodies (Dianova; # 115-545-146).	anti-mouse IgG suggested: (Jackson ImmunoResearch Labs Cat# 115-545-146, RRID:AB_2307324)
Rabbit anti-E-cadherin IgG monoclonal (CellSignaling; 3195S) or rabbit anti-VE-cadherin polyclonal, primary antibodies (CellSignaling; 2158S) and Cy5 goat anti-mouse IgG polyclonal antibodies (Dianova; #111-175-144) were used to detect cell borders of …

SciScore for 10.1101/2020.08.31.276725: (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
) IgG monoclonal, primary antibodies and AlexaFluor® goat anti-mouse IgG polyclonal antibodies (Dianova; # 115-545-146).	anti-mouse IgG suggested: (Jackson ImmunoResearch Labs Cat# 115-545-146, RRID:AB_2307324)
Rabbit anti-E-cadherin IgG monoclonal (CellSignaling; 3195S) or rabbit anti-VE-cadherin polyclonal, primary antibodies (CellSignaling; 2158S) and Cy5 goat anti-mouse IgG polyclonal antibodies (Dianova; #111-175-144) were used to detect cell borders of Calu-3 or HUVEC cells on the membrane of the alveolus-on-a-chip model, respectively.	anti-E-cadherin IgG suggested: None anti-VE-cadherin suggested: (Cell Signaling Technology Cat# 2158, RRID:AB_2077970)
For the detection of SARS-CoV-2 spike protein a rabbit polyclonal anti-SARS-CoV-2 spike S2 antibody (Sino Biological #40590-T62) was used.	anti-SARS-CoV-2 spike S2 suggested: (Thermo Fisher Scientific Cat# PA5-112048, RRID:AB_2866784)
Experimental Models: Cell Lines
Sentences	Resources
For the cultivation of the human alveolus-on-a-chip model we used Calu-3 cells and macrophages at the epithelial side, and HUVECs at the endothelial side.	HUVECs suggested: None
For infection of Vero-76 or Calu-3 cells, cells were washed with PBS and either left uninfected (mock) or infected with SARS-CoV-2 with a multiplicity of infection (MOI) of 1 for 120 min in medium (EMEM with HEPES modification and 5 mM L-Glutamine for Vero-76 cells and RPMI 1640 for Calu-3 cells) supplemented with 10 % FCS.	Vero-76 suggested: None Calu-3 suggested: None
Rabbit anti-E-cadherin IgG monoclonal (CellSignaling; 3195S) or rabbit anti-VE-cadherin polyclonal, primary antibodies (CellSignaling; 2158S) and Cy5 goat anti-mouse IgG polyclonal antibodies (Dianova; #111-175-144) were used to detect cell borders of Calu-3 or HUVEC cells on the membrane of the alveolus-on-a-chip model, respectively.	HUVEC suggested: KCB Cat# KCB 200648YJ, RRID:CVCL_2959)
Software and Algorithms
Sentences	Resources
The cDNA preparation was performed using the SuperScript IV (Thermofisher), followed by a multiplex PCR to generate overlapping 400 nt amplicons using version 3 of the primer set (https://github.com/artic-network/artic-ncov2019/tree/master/primer_schemes/nCoV-2019/V3).	Thermofisher suggested: (ThermoFisher; SL 8; Centrifuge, RRID:SCR_020809)
Infection by SARS-CoV-2 was visualized using mouse anti-SARS-CoV-2 spike (GeneTex; #GTX632604	GeneTex suggested: (GeneTex, RRID:SCR_000069)
Images were acquired using an Axio Observer.Z1 microscope (Zeiss) with Plan Apochromat 20x/0.8 objective (Zeiss), ApoTome.2 (Zeiss) and Axiocam 503 mono (Zeiss) and the software Zen 2.6 (blue edition; Zeiss).	Zen suggested: None
Fiji V 1.52b (ImageJ) was used for further image processing, including Z-stack merging with maximum intensity projection and gamma correction.	Fiji suggested: (Fiji, RRID:SCR_002285) ImageJ suggested: (ImageJ, RRID:SCR_003070)
Statistical analysis: Statistical analyses were performed using Prism 8 (GraphPad Software).	GraphPad suggested: (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 did not find any issues relating to the usage of bar graphs.

Results from JetFighter: We did not find any issues relating to colormaps.

Results from rtransparent:

No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
No protocol registration statement was detected.

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Abstract

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