Introduction

The COVID-19 pandemic has dominated the headlines of every major news outlet for the majority of 2020. The disease COVID-19 is caused by SARS-coronavirus 2 (SARS-CoV-2), which belongs to the coronavirus group of viruses. SARS-CoV, which caused the 2002 outbreak, has previously been described to infect human cells by its viral spike (S) protein interacting with the human angiotensin-converting enzyme 2 (ACE2) receptor (Li et al., 2003). This entry is assisted by the virus utilising the human entry-associated protease TMPRSS2, although other proteins are thought to be capable of substituting TMPRSS2 (Glowacka et al., 2011; Matsuyama et al., 2010; Shulla et al., 2011). ACE2 and TMPRSS2 are therefore referred to as entry factors. More recently, SARS-CoV-2 has been shown to be highly likely to use these same cellular entry proteins (Hoffmann et al., 2020). Which cells in the human body co-express these entry factors? SARS-CoV of the 2002 outbreak was shown to infect ciliated cells of the nasal and bronchiolar epithelium and cause ciliated cell death in vitro (Sims et al., 2005). The bronchiolar proximal airway epithelium consists of various luminal cell types including ciliated, goblet, club, neuroendocrine, ionocyte and tuft cells with basal cells sheltering between the luminal cell types. Transcriptomic analysis of datasets available from the Human Cell Atlas consortium have suggested that goblet, club and ciliated cells of the proximal airways are likely targets of SARS-CoV-2 as a result of their co-expression of the entry factors (Sungnak et al., 2020). These studies necessitated in vitro experimentation on human bronchiolar epithelium to determine which cell type in the airway is hijacked by SARS-CoV-2 for entry and replication. Ravindra et al. have performed this in vitro experimentation and determined that ciliated cells are the cell type that SARS-CoV-2 primarily targets.

 

Key findings

Ciliated cells are the point of entry into the bronchiolar epithelium
The authors established air-liquid interface (ALI) cultures using human bronchiolar epithelial cells (HBECs). ALI culture mimics the natural condition of the human bronchiolar epithelium, i.e. the apical side being exposed to air. They then infected the ALI cultures with SARS-CoV-2 from the apical side, as infection would occur in the human body, and sequenced samples at days 1, 2 and 3 post-infection (Figure A). This allowed assessment of which cell was primarily targeted and also if more than one cell type could become infected. Clustering of single cell transcriptomes revealed that the SARS-CoV2 genome was detectable at the greatest extent in the ciliated cell cluster. 83% of infected cells at 1 day post-infection were ciliated cells. Ciliated cells were also reported to have an increase in apoptosis-associated genes. These findings mirror the cellular targets of SARS-CoV previously reported.

 

 

SARS-CoV-2 can replicate in ciliated, basal, club and basal-club intermediate cells
The authors revealed that from 1 – 3 days post-infection ciliated, basal, club and basal-club cells all had increasing viral reads in the sequencing data. Contrastingly, although there was some increase in viral reads, it was determined to be to a much lesser extent in the neuroendocrine, ionocyte, tuft and goblet cell populations.

 

Induction of interferon stimulated genes in infected and bystander cells
Next, the authors segmented the cells in the “infected” condition into truly infected cells and bystander cells (which did not have sufficient SARS-CoV-2 reads). Assessing interferon (IFN) signatures revealed upregulation of type I and type III interferons in the truly infected cell populations. Although infected, IFN signatures were not observed in the neuroendocrine, ionocyte, tuft or goblet populations. Importantly, they reported that interferon stimulated genes are upregulated in both truly infected and bystander populations.

 

 

Why I chose this preprint

It is critical to understand the cellular targets of SARS-CoV-2. In this study the authors have carefully considered their experimental plan, allowing them to differentiate how uninfected “bystander” cells in the cultures subjected to infection respond compared to the infected cells. They report that ciliated cells are the main cellular target of SARS-CoV-2 and that the virus can also effectively replicate in basal, club and basal-club like cells, too. Finally, they report that infected cells cause upregulation of interferon stimulated genes in themselves and in bystander, uninfected cells sharing the same culture condition.

 

Questions for the authors

1. SARS-CoV-2 course of infection lasts longer than 72 hours in patients. Can the authors demonstrate what happens to the cells in the ALI culture conditions after longer culture time post-infection? (A) Does the epithelial barrier become compromised? (B) Do the cells simply die by 3 days post-infection? (C) Is there a proliferation response in an attempt to repair the effects of the viral insult?
2. Figures 1D, E and F all show an increase in infected cells in all populations, although it is lesser in some than others. It is known that neuroendocrine, ionocyte and tuft cells are much rarer than ciliated, club and basal cells in the bronchiolar epithelium. Is the conclusion that these cell types are more resistant to SARS-CoV-2 entry and replication taking into account their lower cell number in the culture?
3. The expression of ACE2 is low, evidence which is corroborated by other reports in the literature. Do the authors think that there is potentially another receptor which could be a likely candidate allowing SARS-CoV-2 entry into human bronchiolar epithelial cells?
4. Do the authors observe any evidence of shifting cellular identity in the bystander cell populations (perhaps to compensate for loss of differentiated luminal cells by apoptosis)?
5. Do the authors plan to perform similar analysis on nasal epithelium?

 

 

References

Glowacka, I., Bertram, S., Muller, M. A., Allen, P., Soilleux, E., Pfefferle, S., … Pohlmann, S. (2011). Evidence that TMPRSS2 Activates the Severe Acute Respiratory Syndrome Coronavirus Spike Protein for Membrane Fusion and Reduces Viral Control by the Humoral Immune Response. Journal of Virology, 85(9), 4122–4134. https://doi.org/10.1128/jvi.02232-10

Hoffmann, M., Kleine-Weber, H., Schroeder, S., Krüger, N., Herrler, T., Erichsen, S., … Pöhlmann, S. (2020). SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, 181(2), 271-280.e8. https://doi.org/10.1016/j.cell.2020.02.052

Li, W., Moore, M. J., Vasllieva, N., Sui, J., Wong, S. K., Berne, M. A., … Farzan, M. (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature, 426(6965), 450–454. https://doi.org/10.1038/nature02145

Matsuyama, S., Nagata, N., Shirato, K., Kawase, M., Takeda, M., & Taguchi, F. (2010). Efficient Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by the Transmembrane Protease TMPRSS2. Journal of Virology, 84(24), 12658–12664. https://doi.org/10.1128/jvi.01542-10

Shulla, A., Heald-Sargent, T., Subramanya, G., Zhao, J., Perlman, S., & Gallagher, T. (2011). A Transmembrane Serine Protease Is Linked to the Severe Acute Respiratory Syndrome Coronavirus Receptor and Activates Virus Entry. Journal of Virology, 85(2), 873–882. https://doi.org/10.1128/jvi.02062-10

Sims, A. C., Baric, R. S., Yount, B., Burkett, S. E., Collins, P. L., & Pickles, R. J. (2005). Severe Acute Respiratory Syndrome Coronavirus Infection of Human Ciliated Airway Epithelia: Role of Ciliated Cells in Viral Spread in the Conducting Airways of the Lungs. Journal of Virology, 79(24), 15511–15524. https://doi.org/10.1128/jvi.79.24.15511-15524.2005

Sungnak, W., Huang, N., Bécavin, C., Berg, M., Queen, R., Litvinukova, M., … Barnes, J. L. (2020). SARS-CoV-2 entry factors are highly expressed in nasal epithelial cells together with innate immune genes. Nature Medicine, 26(5), 681–687. https://doi.org/10.1038/s41591-020-0868-6

Tags: ciliated cell, covid-19, epithelium, infection, lung, sars-cov-2, tropism

doi: https://doi.org/10.1242/prelights.20637

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