SARS-CoV-2 infection of olfactory epithelial cells and neurons drives acute lung injury and lethal COVID-19 in mice

  1. Alan T. Tang
  2. David W. Buchholz
  3. Katherine M. Szigety
  4. Brian Imbhiaka
  5. Siqi Gao
  6. Maxwell Frankfurter
  7. Min Wang
  8. Jisheng Yang
  9. Peter Hewins
  10. Patricia Mericko-Ishizuka
  11. N Adrian Leu
  12. Stephanie Sterling
  13. Isaac A. Monreal
  14. Julie Sahler
  15. Avery August
  16. Xuming Zhu
  17. Kellie A. Jurado
  18. Mingang Xu
  19. Edward E. Morrisey
  20. Sarah E. Millar
  21. Hector C. Aguilar
  22. Mark L. Kahn

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Abstract

Lethal COVID-19 is associated with respiratory failure that is thought to be caused by acute respiratory distress syndrome (ARDS) secondary to pulmonary infection. To date, the cellular pathogenesis has been inferred from studies describing the expression of ACE2, a transmembrane protein required for SARS-CoV-2 infection, and detection of viral RNA or protein in infected humans, model animals, and cultured cells. To functionally test the cellular mechanisms of COVID-19, we generated hACE2 fl animals in which human ACE2 (hACE2) is expressed from the mouse Ace2 locus in a manner that permits cell-specific, Cre-mediated loss of function. hACE2 fl animals developed lethal weight loss and hypoxemia within 7 days of exposure to SARS-CoV-2 that was associated with pulmonary infiltrates, intravascular thrombosis and patchy viral infection of lung epithelial cells. Deletion of hACE2 in lung epithelial cells prevented viral infection of the lung, but not weight loss, hypoxemia or death. Inhalation of SARS-CoV-2 by hACE2 fl animals resulted in early infection of sustentacular cells with subsequent infection of neurons in the neighboring olfactory bulb and cerebral cortex— events that did not require lung epithelial cell infection. Pharmacologic ablation of the olfactory epithelium or Foxg1 Cre mediated deletion of hACE2 in olfactory epithelial cells and neurons prevented lethality and neuronal infection following SARS-CoV-2 infection. Conversely, transgenic expression of hACE2 specifically in olfactory epithelial cells and neurons in Foxg1 Cre ; LSL- hACE2 mice was sufficient to confer neuronal infection associated with respiratory failure and death. These studies establish mouse loss and gain of function genetic models with which to genetically dissect viral-host interactions and demonstrate that lethal disease due to respiratory failure may arise from extrapulmonary infection of the olfactory epithelium and brain. Future therapeutic efforts focused on preventing olfactory epithelial infection may be an effective means of protecting against severe COVID-19.

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    SciScore for 10.1101/2021.12.04.471245: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: The University of Pennsylvania Institutional Animal Care and Use Committee (IACUC) approved all animal protocols, and all procedures were performed in accordance with these protocols (#806811).
    Sex as a biological variableChimeras were mated to B6D2F1/J (Jackson Laboratory, 100006) females to generate germline F1 mice.
    RandomizationRandomization of animals was not performed, favoring use of littermates to control for the mixed strain background and given the complexity of the genetic intercrosses.
    BlindingStatistics: Animals were inoculated with SARS-CoV-2 in blinded fashion without knowledge of genotypes.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies used for immunoblotting: pan-ACE2 (1:1000; R&D Systems; AF933), hACE2 (1:1000; Atlas Antibodies; AMAB91259),
    hACE2
    suggested: None
    Sections were then incubated with anti-NFH (chicken 1:5000, Aves Labs, NFH) and anti-KRT8 (rabbit 1:500, Sigma-Aldrich, SAB4501654) overnight at 4°C, followed by incubation with secondary antibodies.
    NFH
    suggested: (LSBio (LifeSpan Cat# LS-C73369-500, RRID:AB_1601169)
    anti-KRT8
    suggested: None
    Antibody validation: ACE2 antibodies were validated for IHC and Western blotting using hACE2del/y tissues.
    ACE2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Properly targeted ES cell clones were confirmed using PCR screening of the 5’ and 3’ arms of homology and the entire targeted knock-in was PCR amplified for Sanger sequencing prior to microinjection into V6.5 ES cells.
    V6.5
    suggested: RRID:CVCL_C865)
    Infectious stocks were grown in Vero-E6 cells and stored at −80 °C.
    Vero-E6
    suggested: None
    Vero E6 cells were seeded in 12 well plates with complete DMEM and incubated at 37°C for 24 hours prior to infection.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Chimeras were mated to B6D2F1/J (Jackson Laboratory, 100006) females to generate germline F1 mice.
    B6D2F1/J
    suggested: RRID:IMSR_JAX:100006)
    Shh-Cre (stock 005622), Sox2-Cre (stock 008454), Sftpc-CreERT2 (stock 028054), R26-LSL-tdTomato (Ai14, stock 007914) animals were purchased from Jackson Laboratories on the congenic C57BL/6J strain28, 53, 54.
    Sox2-Cre
    suggested: None
    The Foxg1-IRES-Cre was a kind gift from Nada Jabado38.
    Foxg1-IRES-Cre
    suggested: None
    Experimental mice were maintained on a mixed C57BL/6J, 129S1/SvJ, and DBA/2J background and utilized between 2-4 months of age for infection studies.
    C57BL/6J
    suggested: None
    DBA/2J
    suggested: None
    Recombinant DNA
    SentencesResources
    qPCR was performed using the SARS-CoV-2 Research Use Only qPCR Probe Kit (IDT 10006713) with a standard curve using a positive control N-protein plasmid (IDT 10006625).
    N-protein
    suggested: None
    Software and Algorithms
    SentencesResources
    Graph generation and statistical analyses performed with GraphPad Prism 9.2.0.
    GraphPad Prism
    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: We detected the following sentences addressing limitations in the study:
    Interpretation of this study must consider several limitations. First, the mouse OE is more developed compared to human OE; thus, a mouse model may bias toward disease mechanisms involving the OE. In this regard, it is notable that we see complete lethality after exposure to virus in hACE2 mice while most humans experience mild disease. This outcome may also reflecs the administration of high viral titers that greatly exceed the inoculum received by most human patients. Second, since mice are obligate nose-breathers, our studies utilize intranasal SARS-CoV-2 administration. Unlike mice, humans are capable of mouth-breathing; thus, transmission may be less dependent on the OE. However, anosmia is pathognomonic for human disease and often precedes the onset of respiratory failure—a sequence consistent with that we observed in hACE2 mice. Overall, our mouse models likely reflect the most severe disease course experienced by human patients, e.g. those with the largest viral inocula and/or the greatest number of preexisting conditions that act as disease risk factors. Studies of human patients that more directly examine infection of the OE and brain early in disease are challenging but will be required to fully validate and extend these findings. In addition, the molecular mechanism by which SARS-CoV-2 infection of olfactory epithelial cells and neurons leads to acute lung injury in hACE2 mice is an important future direction with clear therapeutic implications. Finally, our studi...

    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 pages 46, 50, 61 and 38. 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.

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

    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 10.1101/2021.12.04.471245v1 on bioRxiv