Cell-type apoptosis in lung during SARS-CoV-2 infection

  1. Yakun Liu
  2. Tania M. Garron
  3. Qing Chang
  4. Zhengchen Su
  5. Changcheng Zhou
  6. Eric C. Gong
  7. Junying Zheng
  8. Yw Yin
  9. Thomas Ksiazek
  10. Trevor Brasel
  11. Yang Jin
  12. Paul Boor
  13. Jason E. Comer
  14. Bin Gong

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Abstract

The SARS-CoV-2 pandemic has inspired renewed interest in understanding the fundamental pathology of acute respiratory distress syndrome (ARDS) following infection because fatal COVID-19 cases are commonly linked to respiratory failure due to ARDS. The pathologic alteration known as diffuse alveolar damage in endothelial and epithelial cells is a critical feature of acute lung injury in ARDS. However, the pathogenesis of ARDS following SRAS-CoV-2 infection remains largely unknown.

In the present study, we examined apoptosis in post-mortem lung sections from COVID-19 patients and lung tissues from a non-human primate model of SARS-CoV-2 infection, in a cell-type manner, including type 1 and 2 alveolar cells and vascular endothelial cells (ECs), macrophages, and T cells. Multiple-target immunofluorescence (IF) assays and western blotting suggest both intrinsic and extrinsic apoptotic pathways are activated during SARS-CoV-2 infection. Furthermore, we observed that SARS-CoV-2 fails to induce apoptosis in human bronchial epithelial cells (i.e., BEAS2B cells) and primary human umbilical vein endothelial cells (HUVECs), which are refractory to SARS-CoV-2 infection. However, infection of co-cultured Vero cells and HUVECs or Vero cells and BEAS2B cells with SARS-CoV-2 induced apoptosis in both Vero cells and HUVECs/BEAS2B cells, but did not alter the permissiveness of HUVECs or BEAS2B cells to the virus. Post-exposure treatment of the co-culture of Vero cells and HUVECs with an EPAC1-specific activator ameliorated apoptosis in HUVECs. These findings may help to delineate a novel insight into the pathogenesis of ARDS following SARS-CoV-2 infection.

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  1. ScreenIT

    SciScore for 10.1101/2020.12.23.424254: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All animal experiments were performed according to protocols approved by the UTMB Institutional Animal Care and Use Committee (IACUC 2005057).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableTwo male and one female rhesus macaques (age:46-48 months, weight: 3-5 kg, Envigo, Alice, TX) and two female cynomolgus macaques (age: 84-112 months, 3-5 kg, Envigo) were used in a COVID-19 model development study.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    IF-TUNEL dual staining: For IF-TUNEL dual staining (55), after IF using CD31, caveolin-1, SPC, CD68, CD3, cytochrome C, FasL, or SARS-CoV-2 antibody, the TUNEL assay was done following the protocol above.
    CD31
    suggested: None
    caveolin-1
    suggested: None
    CD68
    suggested: None
    CD3
    suggested: None
    SARS-CoV-2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Co-culture of Vero cells and HUVECs or Vero cells and BEAS2B cells: For co-culturing of Vero cells and HUVECs or Vero cells and BEAS2B cells, Vero cells were seeded in the inserts of 24-well plates (0.4 µm polyester membrane, Costar, Thermo Fisher Scientific) while HUVECs or BEAS2B cells were grown in wells without inserts.
    HUVECs
    suggested: None
    After 48 hours, the insert with Vero cells were transferred to the well of HUVECs or BEAS2B cells.
    BEAS2B
    suggested: BCRJ Cat# 0395, RRID:CVCL_0168)
    The co-culture was kept in co-culture media (HUVECs and Vero cells co-culture media is ½ HUVEC media and ½ Vero cell media; Vero cells and BEAS2B cells co-culture media is Vero cell media) (51).
    Vero
    suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
    Software and Algorithms
    SentencesResources
    The extent of TUNEL signal-positive cells is normalized to total nuclear content (DAPI staining) in each filed using NIH ImageJ (54).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    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:
    There were major limitations in our present study. First, NHP data are limited due to the small number of animals and the fact that lung tissue samples were not collected until 21 days post challenge. This significantly restricts our profiling of the course of apoptosis in various cell types following SARS-CoV-2 infection. Further work is needed to characterize the apoptotic events during an active infection. Second, many cells undergo apoptosis as a host defense to restrict virus amplification (65). However, dysregulation of apoptosis is associated with pathological resolution following viral infections. Based on the current body of data, the present study fails to determine any potential regulatory roles of apoptosis during the pathogenesis of SARS-CoV-2 infection. Third, a dysregulated innate immune response and endothelial dysfunction have been proposed to play a role in the pathogenesis of SARS-CoV-19-associated MIS-C (20, 21). The presence of disease-associated autoantibodies against ECs was reported in a COVID-19 patient with MIS-C (95). Investigation of endothelial programed cell death in multiple organs will help define the pathological mechanism of MIS-C. Lastly, cell-type apoptosis contributes to the resolution of ARDS and vasculopathy in the lung (23, 24). Long-term deleterious or beneficial consequences of endothelial apoptosis triggered by SARS-CoV-2 infection, which may promote or prevent vasculopathy progression, will be another important research area in the ...

    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 20. 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.
    • 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

    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.

  2. Version published to 10.1101/2020.12.23.424254v1 on bioRxiv