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Review 1: "Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19"

This preprint uses single cell RNA-seq (scRNA-seq) to reconstruct nasopharyngeal tissue reorganization in COVID-19 patients. Reviewers deemed the manuscript's main claims well-substantiated, carefully qualified, and significantly novel.

Published onApr 09, 2021
Review 1: "Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19"
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Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID-19
Description

ABSTRACTInfection with SARS-CoV-2, the virus that causes COVID-19, can lead to severe lower respiratory illness including pneumonia and acute respiratory distress syndrome, which can result in profound morbidity and mortality. However, many infected individuals are either asymptomatic or have isolated upper respiratory symptoms, which suggests that the upper airways represent the initial site of viral infection, and that some individuals are able to largely constrain viral pathology to the nasal and oropharyngeal tissues. Which cell types in the human nasopharynx are the primary targets of SARS-CoV-2 infection, and how infection influences the cellular organization of the respiratory epithelium remains incompletely understood. Here, we present nasopharyngeal samples from a cohort of 35 individuals with COVID-19, representing a wide spectrum of disease states from ambulatory to critically ill, as well as 23 healthy and intubated patients without COVID-19. Using standard nasopharyngeal swabs, we collected viable cells and performed single-cell RNA-sequencing (scRNA-seq), simultaneously profiling both host and viral RNA. We find that following infection with SARS-CoV-2, the upper respiratory epithelium undergoes massive reorganization: secretory cells diversify and expand, and mature epithelial cells are preferentially lost. Further, we observe evidence for deuterosomal cell and immature ciliated cell expansion, potentially representing active repopulation of lost ciliated cells through coupled secretory cell differentiation. Epithelial cells from participants with mild/moderate COVID-19 show extensive induction of genes associated with anti-viral and type I interferon responses. In contrast, cells from participants with severe lower respiratory symptoms appear globally muted in their anti-viral capacity, despite substantially higher local inflammatory myeloid populations and equivalent nasal viral loads. This suggests an essential role for intrinsic, local epithelial immunity in curbing and constraining viral-induced pathology. Using a custom computational pipeline, we characterized cell-associated SARS-CoV-2 RNA and identified rare cells with RNA intermediates strongly suggestive of active replication. Both within and across individuals, we find remarkable diversity and heterogeneity among SARS-CoV-2 RNA+ host cells, including developing/immature and interferon-responsive ciliated cells, KRT13+ “hillock”-like cells, and unique subsets of secretory, goblet, and squamous cells. Finally, SARS-CoV-2 RNA+ cells, as compared to uninfected bystanders, are enriched for genes involved in susceptibility (e.g., CTSL, TMPRSS2) or response (e.g., MX1, IFITM3, EIF2AK2) to infection. Together, this work defines both protective and detrimental host responses to SARS-CoV-2, determines the direct viral targets of infection, and suggests that failed anti-viral epithelial immunity in the nasal mucosa may underlie the progression to severe COVID-19.

RR:C19 Evidence Scale rating by reviewer:

  • Reliable. The main study claims are generally justified by its methods and data. The results and conclusions are likely to be similar to the hypothetical ideal study. There are some minor caveats or limitations, but they would/do not change the major claims of the study. The study provides sufficient strength of evidence on its own that its main claims should be considered actionable, with some room for future revision.

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Review:

The main claims of the study by Ziegler et al - Impaired local intrinsic immunity to SARS-CoV-2 infection in severe COVID19 - are well-justified by the data and analytical methods used. There are some minor caveats or limitations, but they do not change the major claims of the study. The study’s main claims should be considered conclusive and actionable, with some room for future revision. My rapid evaluation is to accept with minor revisions.

In this work, the authors present a very nice comprehensive analysis by cutting edge single-cell transcriptomics (scRNAseq) of the human nasopharynx upon SARS-Cov-2 infection. Starting from a collection of swabs from patients at different levels of severity of COVID-19 they established protocols to retrieve and process the few cells to scRNAseq. They also provide a sophisticated pipeline for data analysis. The major findings are that: (i) SARS-CoV-2 infection cause depletion of ciliated cells, while there is an expansion of immature precursor ciliated cells and deuterosomal cells; (ii) replicating SARS-CoV-2 appears associated with specific cell types (ciliated, goblet, secretory and squamous); (iii) cells from milder diseases show induction of ISGs, either IFN-stimulated or from direct stimulation by the virus. Conversely, cells from severe cases often show a blunted antiviral response, which appears skewed towards an inflammatory response. (iv) infected cells compared to bystander non-infected cells are enriched of antiviral factors induced by the virus (PAMPS), also this phenotype negatively correlates with the severity of the disease.

The study is well-presented and took full advantage of scRNAseq to dissect the cellular response at the site of virus entry identifying specific infected cell populations and correlating a blunted IFN response to the severity of the disease. Based on a single technique they could establish cellular targets of infection as well as cellular response to the virus providing information on the first phases of the infection, valuable for the identification of biomarkers of progression as well as for therapy (i.e. interferons administered intranasally or modulation of cholesterol pathway).

Overall, the work is solid and informative, albeit limited to a single technique. Some of the more sophisticated analyses suffer from the lower numbers from sub-sampling of cells and their conclusions would need further validation. The very nature of the sample (i.e. swabs) has its own limitation limiting the analysis to surface cells, but the possibility of running this analysis on these cells is per se a great achievement. The RNA velocity method used to map the differentiation relationship between cell types is introduced only briefly and it is difficult to understand its basic principles by just reading the results (i.e. maybe a few lines of clarification would benefit the manuscript). Some closely related work is not discussed (i.e. Fiege et al PLOS Path 2021). The cholesterol involvement is discussed only in the Discussion, not in the Results. Given its growing importance, this session would benefit because it is a viable therapeutic option. Virologically, viral variants could modify tropism in this tissue and therefore show different characteristics, this could be discussed.

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