Human nasal and lung tissues infected ex vivo with SARS-CoV-2 provide insights into differential tissue-specific and virus-specific innate immune responses in the upper and lower respiratory tract

  1. Or Alfi
  2. Arkadi Yakirevitch
  3. Ori Wald
  4. Ori Wandel
  5. Uzi Izhar
  6. Esther Oiknine-Djian
  7. Yuval Nevo
  8. Sharona Elgavish
  9. Elad Dagan
  10. Ory Madgar
  11. Gilad Feinmesser
  12. Eli Pikarsky
  13. Michal Bronstein
  14. Olesya Vorontsov
  15. Wayne Jonas
  16. John Ives
  17. Joan Walter
  18. Zichria Zakay-Rones
  19. Menachem Oberbaum
  20. Amos Panet
  21. Dana G. Wolf

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Abstract

The nasal-mucosa constitutes the primary entry site for respiratory viruses including SARS-CoV-2. While the imbalanced innate immune response of end-stage COVID-19 has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo , coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local-mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with rapid increase in tissue-associated viral sub-genomic mRNA, and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon stimulated genes, cytokines and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tract, that are distinct to SARS-CoV-2. The studies shed light on the role of the nasal-mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19.

IMPORTANCE

In order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal-mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues, infected in parallel with SARS-CoV-2 and influenza virus, we have revealed distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal-mucosal infection model can be employed to assess the impact of viral evolutionary changes, and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

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    SciScore for 10.1101/2021.03.08.434404: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: The studies were approved by the Hadassah Medical Center and the Sheba Medical Center Institutional Review Boards.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following primary antibodies were used: Ep-CAM (Mouse monoclonal,
    Ep-CAM
    suggested: None
    The following secondary antibodies were used: Donkey anti-Mouse IgG pre-adsorbed, Alexa Fluor® 568 (1:250
    anti-Mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells and viruses: Simian kidney Vero E6 (ATCC CRL-1586) and Madin-Darby Canine Kidney (MDCK, ATCC® CCL-34™) cells were maintained in Eagle’s Minimum Essential Medium (EMEM; Biological Industries, Beit Haemek, Israel), supplemented with 10% fetal bovine serum, 2 mM L-Glutamine, 10 IU/ml Penicillin, and 10 μg/ml streptomycin (Biological Industries, Beit Haemek, Israel).
    E6
    suggested: None
    SARS-CoV-2 clinical isolate (SARS-CoV-2 isolate Israel-Jerusalem-854/2020) was isolated on Vero E6 cells from a positive nasopharyngeal swab sample, obtained at the Hadassah Hospital Clinical Virology Laboratory.
    Vero E6
    suggested: RRID:CVCL_XD71)
    The virus titers of cleared infected cells- and tissue supernatants were determined by a standard TCID50 assay on Vero E6 cells (SARS-CoV-2) or MDCK cells (influenza virus).
    MDCK
    suggested: CLS Cat# 602280/p823_MDCK_(NBL-2, RRID:CVCL_0422)
    Software and Algorithms
    SentencesResources
    Whole-mount tissues were visualized using a Nikon A1R confocal microscope and were analyzed using NIS Elements software (Nikon)
    NIS Elements
    suggested: (NIS-Elements, RRID:SCR_014329)
    Normalization and differential expression were done with the DESeq2 package (version 1.22.2).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Pathway and molecular function and disease enrichment analysis of the significantly differentially expressed genes was carried out using the Ingenuity Pathway Analysis (IPA®)
    Ingenuity Pathway Analysis
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
    Dot plots of selected IPA® canonical pathways (based on IPA® values for BH P-values and number of genes) were generated using ggplot2 R graphical package (Wickham H (2016). ggplot2: Elegant Graphics for Data Analysis.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    ISBN 978-3-319-24277-4, https://ggplot2.tidyverse.org.) Statistical analysis: All data, presented as means ± standard errors of the mean (SEM), were analyzed using unpaired, two-tailed Student’s t test in GraphPad Prism 8 software (GraphPad Software Inc.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Data availability: The transcriptomic data described in this publication have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO series accession number GSE163959.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Our study has several limitations. The cellular heterogeneity within the tissues limits the resolution of isolated molecular pathways. Additionally, native respiratory tissues in organ culture are relatively short-lived (∼a week) compared to primary human airway epithelial cells and to the self-renewable stem cell-derived organoid cultures, which have proven useful for the studies of SARS-CoV-2 infection and cellular response (9–11, 43, 44). Nonetheless, our studies recapitulate viral infection and host response within the authentic multicellular and morphologically-intact tissue microenvironment - containing tissue epithelial, vascular endothelial, stromal and immune cells, and the specific extracellular matrix. It is notable that the comparative data between the human nasal and lung tissues were not obtained from the same individuals. Yet, we believe that the findings, based on extensive analyses of independent tissues from different individuals, faithfully support the generalizability of the observed tissue-specific patterns. Notwithstanding, the comparison of SARS-CoV-2 and Influenza infections was done in parallel within the same donor tissue. Furthermore, these studies capture the inherent person-to-person variability of innate immune responses, thereby paving the way to future studies of personal host features which determine the innate responses to viral infection along the respiratory tract. In summary, we have demonstrated the active replication of SARS-CoV-2 in nat...

    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 45. 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 10.1101/2021.03.08.434404 on bioRxiv