Endothelial cells elicit a pro-inflammatory response to SARS-CoV-2 without productive viral infection

  1. Lilian Schimmel
  2. Keng Yih Chew
  3. Claudia Stocks
  4. Teodor Yordanov
  5. Patricia Essebier
  6. Arutha Kulasinghe
  7. James Monkman
  8. Anna Flavia Ribeiro dos Santos Miggiolaro
  9. Caroline Cooper
  10. Lucia de Noronha
  11. Anne K. Lagendijk
  12. Kate Schroder
  13. Larisa I. Labzin
  14. Emma J. Gordon
  15. Kirsty R. Short

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Abstract

Objectives

Thrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious.

Methods

Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier to show that primary human endothelial cells express very low levels the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2.

Results

Accordingly, endothelial cells can only be infected when SARS-CoV-2 is present at very high concentrations. However, this is not a productive infection (i.e. no infectious virus is produced) and viral entry induces an inflammatory response. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. They do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines.

Conclusions

Taken together, these data suggest that in vivo , endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection is only likely to occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells are still likely to play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

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  1. Version published to 10.1101/2021.02.14.431177v2 on bioRxiv
  2. ScreenIT

    SciScore for 10.1101/2021.02.14.431177: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board StatementIRB: Autopsy and biopsy materials were obtained from the Pontificia Universidade Catolica do Parana PUCPR the National Commission for Research Ethics (CONEP) under ethics approval numbers 2020001792/30188020.7.1001.0020 and 2020001934/30822820.8.000.0020.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: The following antibodies were used: mouse anti-ACE2 (Santa Cruz, sc-390851, IF1:200) goat anti-ACE2 (R&D systems, AF933, WB 1:1000), mouse anti-dsRNA (Millipore, MABE1134, IF1:100)
    anti-ACE2
    suggested: (Santa Cruz Biotechnology Cat# sc-390851, RRID:AB_2861379)
    anti-dsRNA
    suggested: (Millipore Cat# MABE1134, RRID:AB_2819101)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: Human umbilical vein endothelial cells (HUVECs), and human microvascular endothelial cells from lungs (HMVEC-L) purchased from Lonza (Cat# CC-2935, and CC-2527 respectively) were cultured until passage 8 in EGM-Plus or EGM-2MV medium, supplemented with singlequots (Lonza Cat# CC-5035, CC-3102)
    HUVECs
    suggested: None
    Calu-3 cells purchased from ATCC (Cat# HTB-55) were maintained in MEM (Invitrogen), containing 10% (v/v) heat-inactivated foetal bovine serum (Cytiva), 100 U/ml penicillin and streptomycin (Life Technologies Australia), and grown in EGM-2MV for endothelial co-culture experiments.
    Calu-3
    suggested: ATCC Cat# HTB-55, RRID:CVCL_0609)
    Monocultures of HUVEC, HMVEC or Calu-3 cells were performed on 24 well cell culture inserts (Corning 6.5 mm Transwell, 0.4 µm polycarbonate membrane Cat#3413) coated with 5 µg/ml FN (Sigma).
    HUVEC
    suggested: None
    Virus was grown on Vero cells and titred [38].
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    Image analysis: Analysis of immunofluorescent images was performed using ImageJ version 2.0.0-rc-69/1.52n.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Imaris (Bitplane) version 8 was used to create the XZ projection in Figure 4B’’.
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    Statistical analysis: All statistical analysis was performed using Graphpad Prism version 9.0.
    Graphpad Prism
    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: We detected the following sentences addressing limitations in the study:
    The present study was subject to several limitations. Firstly, coagulation, thrombosis and induction of angiogenesis in the lungs of deceased COVID-19 patients has been described in addition to endothelial dysfunction [3]. This angiogenic response is thought to be predominantly mediated through intussusceptive angiogenesis, where a new blood vessel is formed by splitting of an existing vessel. This effect appears to be specific to COVID-19 patients, as lungs from decreased influenza patients do not display increased angiogenic features. Whether this is due to relative hypoxia in the lungs remains unclear, although elevated angiogenic growth factors such as VEGF-A and VEGF-C have been associated with COVID-19 [3, 39]. In this study we have not addressed the effect of SARS-CoV-2 exposure on the angiogenic capacity of endothelial cells in culture. Given the intimate association between inflammation, endothelial dysfunction and angiogenesis [40, 55], these are critical aspects of COVID-19 pathology that remain to be addressed in subsequent studies. An additional limitation of the present study is that, due to the reductionist nature of the in vitro systems used herein, we were unable to address the contribution of immune cells to endothelial cell dysfunction during SARS-CoV-2 infection. Multiple studies attribute the sustained inflammatory response directly to immune cells, wherein macrophage activation, monocyte NLRP3 inflammasome signalling, complement activation and extrusion ...

    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:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • 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.

    About SciScore

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  3. Version published to 10.1101/2021.02.14.431177v1 on bioRxiv