Distinct spatial arrangements of ACE2 and TMPRSS2 expression in Syrian hamster lung lobes dictates SARS-CoV-2 infection patterns

  1. Ilhan Tomris
  2. Kim M. Bouwman
  3. Youri Adolfs
  4. Danny Noack
  5. Roosmarijn van der Woude
  6. Gius Kerster
  7. Sander Herfst
  8. Rogier W. Sanders
  9. Marit J. van Gils
  10. Geert-Jan Boons
  11. Bart L. Haagmans
  12. R. Jeroen Pasterkamp
  13. Barry Rockx
  14. Robert P. de Vries

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Abstract

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRSS2) to facilitate viral-host membrane fusion. ACE2 and TMPRSS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq levels. However, transcriptomic data and actual protein validation convey conflicting information regarding the distribution of the biologically relevant protein receptor in whole tissues. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRSS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals was stained using antibodies against ACE2 and TMPRSS2, combined with SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize their expression and related infection patterns. The data demonstrate that infection is restricted to sites containing both ACE2 and TMPRSS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the bronchioles and alveoli. Conversely, infection completely overlaps where ACE2 and TMPRSS2 co-localize in the tertiary bronchi, bronchioles, and alveoli.

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  1. Version published to 10.1371/journal.ppat.1010340
  2. ScreenIT

    SciScore for 10.1101/2021.03.24.435771: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: ACE2 antibody abcam, ab27269 1:400 TMPRSS2 antibody Santa Cruz sc-515727 1:400 Anti-NP, sino-biological 40143-MM05 1:400 Anti-NP, sino-biological 40143-R001 1:400 Anti-NP, thermofisher MA1-7401 1:400 K8/K18, progen, 90001 1:400 Donkey-anti-rabbit750, abcam ab175731
    Antibodies: ACE2
    suggested: None
    TMPRSS2
    suggested: None
    Anti-NP
    suggested: (Bio-Rad Cat# MCA400, RRID:AB_2151884)
    With rigorous washing steps in between the proteins were detected with mouse anti-strep-tag-antibodies (IBA) and goat anti-mouse IgG antibodies (Life Biosciences). IDISCO: SARS-CoV-2 infected and non-infected Syrian hamster lungs were provided in 10% formalin, for longer storage hamster lungs were kept in PBS and 0.01% sodium azide.
    anti-mouse IgG
    suggested: None
    Software and Algorithms
    SentencesResources
    Obtained image stacks were analyzed with ImageJ v1.54f and Imaris 9.6.
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    For the 3D renders obtained imaging data was imported into Imaris, display adjustments were made for each channel.
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    The generated surfaces were subsequently used to mask the TMPRSS2 and ACE2 channels, after which statistical values were obtained and exported to GraphPad Prism 9.
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    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 6. 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

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  3. Version published to 10.1101/2021.03.24.435771 on bioRxiv