Impact of human airway epithelial cellular composition on SARS-CoV-2 infection biology

  1. Ying Wang
  2. Melissa Thaler
  3. Dennis K. Ninaber
  4. Anne M. van der Does
  5. Natacha S. Ogando
  6. Hendrik Beckert
  7. Christian Taube
  8. Clarisse Salgado-Benvindo
  9. Eric J. Snijder
  10. Peter J. Bredenbeek
  11. Pieter S. Hiemstra
  12. Martijn J. van Hemert

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Abstract

Infection biology and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), are incompletely understood. Here, we assessed the impact of airway epithelial cellular composition on infection in air-liquid interface (ALI) cultures of differentiated primary human tracheal (PTEC) and bronchial epithelial cells (PBEC). We first compared SARS-CoV-2 infection kinetics, related antiviral and inflammatory responses, and viral entry factors in PTEC and PBEC. Next, the contribution of differentiation time was investigated by differentiating ALI-PTEC/PBEC for 3-5 weeks and comparing dynamics of viral replication/spread, cellular composition and epithelial responses. We observed a gradual increase in viral load with prolonged culture duration. Ciliated and goblet cells were predominantly infected in both PTEC and PBEC. Immunofluorescence analysis and RT-qPCR showed that compared to other cell types mainly ciliated and goblet cell numbers were affected by increased culture duration. An increased proportion of these two target cell types was associated with increased viral load. Furthermore, modulation of cellular composition using IL-13 and the Notch signaling inhibitor DAPT, underlined the importance of both ciliated and goblet cells for infection. DAPT treatment resulted in a lower viral load and a relative increase in ciliated cells at the expense of goblet cells, compared to IL-13 treated cultures in which both cell types were present and viral load was higher.

In conclusion, our results identify cellular composition as a contributing factor to airway epithelial susceptibility to SARS-CoV-2.

IMPORTANCE

In this study, we determined an effect of culture duration and airway cellular composition of ALI-PBEC and ALI-PTEC cultures on SARS-CoV-2 infection. We found that SARS-CoV-2 infection was increased with prolonged cell culture time and the total percentage and proportion of ciliated and goblet cells played an important role in infection level, suggesting that airway epithelial differentiation/maturation levels may in part determine susceptibility of SARS-CoV-2 infection.

The development of effective therapies either targeting virus replication or pathogenesis against SARS-CoV-2 requires robust cell culture-based infection models to test small molecules and biologicals. Therefore, it is important to identify factors that are essential for reliably modeling SARS-CoV-2-airway epithelial cell interactions. This study sheds light on virus-airway epithelial cell interactions and adds to the complexity of SARS-CoV-2 cell tropism in the airways. In addition, the effect of IL-13 on viral infection hints at a causal connection between SARS-CoV-2 infection and (allergic) asthma.

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    SciScore for 10.1101/2021.07.21.453304: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Use of such donor tissue for research was approved by the ethical committee of the Medical faculty of the University Duisburg-Essen (ID: 19-8717-BO).
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Membranes were excised from the insert and cut into 4 pieces that were incubated overnight at 4°C with specific antibodies at the following dilutions: rabbit anti-SARS-CoV-2 N antibody (JUC3,1:500, (46)), mouse anti-MUC5AC antibody (1:200; Thermo Fisher Scientific), mouse anti-acetylated α-tubulin (1/100; Sigma Aldrich) or goat anti-FOXJ1 antibody (1:200; R&D, Minneapolis, MN, USA).
    anti-MUC5AC
    suggested: None
    anti-FOXJ1
    suggested: None
    After washing, membranes were incubated with corresponding secondary antibodies: donkey anti-rabbit, donkey anti-mouse or donkey anti-goat Alexa-fluor antibodies (all diluted 1:200, Thermo Fisher Scientific) and 4’,6-diamidino-2-phenylindole (DAPI, 1:200, Sigma-Aldrich) in the dark for 30 min at room temperature.
    anti-rabbit
    suggested: (Thermo Fisher Scientific Cat# 18-8816-33, RRID:AB_469529)
    anti-mouse
    suggested: None
    anti-goat
    suggested: (Thermo Fisher Scientific Cat# 18-8814-33, RRID:AB_469527)
    Experimental Models: Cell Lines
    SentencesResources
    Vero E6 cells (master stock MM-3 from dept of Medical Microbiology collection, characterized by full genome sequencing) were maintained in Dulbecco’s modified Eagle’s medium with 4.5 g/l glucose with L-Glutamin (DMEM; Lonza), supplemented with 8% fetal calf serum (FBS; CapriCorn Scientific) and 100 U/ml of penicillin/streptomycin (Sigma-Aldrich).
    Vero E6
    suggested: RRID:CVCL_XD71)

    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:
    However, there are also some limitations in this study, like the sources of cells. PBEC were derived from tumor-free resected bronchial tissue from (ex-)smoking non-COPD patients with lung cancer, and PTEC were from lung donors without lung disease, which may have affected the comparison between PBEC and PTEC. In summary, we show that culture time of airway epithelial cells at the air-liquid interface is a determinant of their susceptibility to SARS-CoV-2 infection, which can only be partly explained by differentiation status based on the amount of ciliated and goblet cells. Differences in expression of entry-associated factors, like ACE2 or TMPRSS2 do not explain the increased susceptibility of airway epithelial cell cultures upon prolonged culture. Our observation that IL-13 treatment causes a limited increase in SARS-CoV-2 infection may be relevant for understanding the impact of type 2 allergic inflammation on SARS-CoV-2 susceptibility. Decreased infection following prolonged inhibition of Notch signaling by DAPT highlights the importance of the presence of both ciliated and goblet cells and warrants further investigation. Ultimately, cellular maturation/differentiation seems intertwined with virus load and spread of the infection over the culture. The proposed cell culture set-up provides a robust tool to test antiviral compounds and acquire additional insight into infection biology of SARS-CoV-2.

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 34. 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:
    • 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.

    Results from scite Reference Check: We found no unreliable references.

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  2. Version published to 10.1101/2021.07.21.453304 on bioRxiv