Mucin 4 Protects Female Mice from Coronavirus Pathogenesis

  1. Jessica A. Plante
  2. Kenneth S. Plante
  3. Lisa E. Gralinski
  4. Anne Beall
  5. Martin T. Ferris
  6. Daniel Bottomly
  7. Richard Green
  8. Shannon K. McWeeney
  9. Mark T. Heise
  10. Ralph S. Baric
  11. Vineet D. Menachery

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Abstract

Using incipient lines of the Collaborative Cross (CC), a murine genetic reference population, we previously identified a quantitative trait loci (QTL) associated with low SARS-CoV titer. In this study, we integrated sequence information and RNA expression of genes within the QTL to identify mucin 4 ( Muc4 ) as a high priority candidate for controlling SARS-CoV titer in the lung. To test this hypothesis, we infected Muc4 -/- mice and found that female, but not male, Muc4 -/- mice developed more weight loss and disease following infection with SARS-CoV. Female Muc4 -/- mice also had more difficulty breathing despite reduced lung pathology; however, no change in viral titers was observed. Comparing across viral families, studies with chikungunya virus, a mosquito-borne arthralgic virus, suggests that Muc4’s impact on viral pathogenesis may be widespread. Although not confirming the original titer QTL, our data identifies a role for Muc4 in the SARS-CoV disease and viral pathogenesis.

Importance

Given the recent emergence of SARS-CoV-2, this work suggest that Muc4 expression plays a protective role in female mice not conserved in male mice following SARS-CoV infection. With the SARS-CoV-2 outbreak continuing, treatments that modulate or enhance Muc4 activity may provide an avenue for treatment and improved outcomes. In addition, the work highlights the importance of studying host factors including host genetics and biological sex as key parameters influencing infection and disease outcomes.

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  1. ScreenIT

    SciScore for 10.1101/2020.02.19.957118: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    BlindingAll scoring was conducted in a blinded fashion on a severity scale of 0 (none) to 3 (severe).
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Rabbit anti-SARS-CoV nucleocapsid polyclonal antibody (PA1-41098, Thermo Scientific, Rockford, IL) diluted 1:2,000 in blocking buffer was allowed to bind overnight at 4°C.
    anti-SARS-CoV
    suggested: None
    Slides were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (ab97051, Abcam, Cambridge, MA) diluted 1:1,000 in blocking buffer for one hour in a humidity box, then washed three times in TBST.
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero E6 cell monolayers in 6-well plates were infected with 200μl of diluted lung homogenate (10−1-10−6) for one hour at 37°C with 5% CO2.
    Vero E6
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Infection with SARS-CoV or Chikungunya Virus: The generation of C57BL/6NTac Muc4tm1Unc mice, kindly provided by Dr. Scott Randell of the University of North Carolina at Chapel Hill and hereafter referred to as Muc4-/- mice, was described by Rowson-Hodel et al (Rowson-Hodel et al., 2018).
    C57BL/6NTac Muc4tm1Unc
    suggested: None
    Muc4-/-
    suggested: None
    The C57BL/6NTac genetic background of these mice was confirmed in our hands via utilization of the MiniMUGA genotyping array (Neogen Inc, Lincoln, NE), a new genotyping array which includes diagnostic markers for the substrain origin of many inbred mouse strains.
    C57BL/6NTac
    suggested: RRID:IMSR_TAC:b6)
    Software and Algorithms
    SentencesResources
    (GraphPad Software, La Jolla, CA)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Buxco data was transformed as previously described (Menachery et al., 2015a) Daily results for all titer-related data types (log10-transformed viral load and immunohistochemistry) and cytokine levels were analyzed by two-way ANOVA using RStudio version 1.1.383 with R version 3.2.0 (R Core Team,
    RStudio
    suggested: (RStudio, RRID:SCR_000432)
    When three-way ANOVA results indicated significant differences on the basis of sex (weight loss, histopathology, and footpad swelling), the strain-based differences between infected animals of a single sex were analyzed using multiple t-tests with the Benjamini and Hochberg correction for false discoveries in Prism.
    Prism
    suggested: (PRISM, RRID:SCR_005375)

    Results from OddPub: Thank you for sharing your data.

    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: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • 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 10.1101/2020.02.19.957118v1 on bioRxiv