SARS-CoV-2 Exploits Sexually Dimorphic and Adaptive IFN and TNFa Signaling to Gain Entry into Alveolar Epithelium

  1. Yan Wang
  2. Sreeharsha Gurrapu
  3. Hong Chen
  4. Sara Laudato
  5. Emily Caggiano
  6. Yan Jiang
  7. Hsiang-Hsi Ling
  8. Filippo G. Giancotti

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Abstract

Infection of the alveolar epithelium constitutes a bottleneck in the progression of COVID-19 to SARS presumably due to the paucity of viral entry receptors in alveolar epithelial type 1 and 2 cells. We have found that the male alveolar epithelial cells express twice as many ACE2 and TMPRSS2 entry receptors as the female ones. Intriguingly, IFN and TNF-α signaling are preferentially active in male alveolar cells and induce binding of the cognate transcription factors to the promoters and lung-active enhancers of ACE2 and TMPRSS2 . Cotreatment with IFN-I and III dramatically increases expression of the receptors and viral entry in alveolar epithelial cells. TNFα and IFN-II, typically overproduced during the cytokine storm, similarly collaborate to induce these events. Whereas JAK inhibitors suppress viral entry induced by IFN-I/III, simultaneous inhibition of IKK/NF- κ B is necessary to block viral entry induced by TNFα and IFN-II. In addition to explaining the increased incidence of SARS in males, these findings indicate that SARS-Cov-2 hijacks epithelial immune signaling to promote infection of the alveolar epithelium and suggest that JAK inhibitors, singly and in combination with NF-KB inhibitors, may exhibit efficacy in preventing or treating COVID-19 SARS.

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  1. ScreenIT

    SciScore for 10.1101/2021.07.23.453505: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variableStudy Design: We have used public scRNAseq datasets to compare the level of expression of SARS-CoV-2 entry receptors in male and female lung alveolar epithelial cells.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Whole cell extracts were incubated with either STAT1, STAT2, or control rabbit IgG antibody and protein G beads (Invitrogen, 10003D) overnight at 4 °C while rotating.
    STAT2
    suggested: None
    Afterward, membranes were probed with antibodies against STAT1, STAT2, IRF9, and rabbit IgG.
    STAT1
    suggested: None
    IRF9
    suggested: None
    The cells were then incubated with primary antibodies overnight, followed by incubation with the fluorochrome-conjugated secondary anti-mouse or anti-rabbit IgG (H+L) for 1 hour at 37 °C.
    anti-mouse
    suggested: None
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Calu-3 cells were incubated with Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin.
    Calu-3
    suggested: None
    Co-immunoprecipitation (co-IP): Calu3 cells were incubated with either IFN-α and IFN-β, or IFN-λ for 3 hours.
    Calu3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    Lipofectamine 3000 was used to co-transfect transfer plasmids and packaging vectors in 293T cells.
    293T
    suggested: CCLV Cat# CCLV-RIE 1018, RRID:CVCL_0063)
    Software and Algorithms
    SentencesResources
    Single cell RNA sequencing datasets: Three publicly available scRNA-seq datasets were obtained as follows: 1) processed data including count and metadata tables of healthy lung tissue was downloaded from Figshare (https://doi.org/10.6084/m9.figshare.11981034.v1); 2) h5 files of normal lungs were extracted from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) under accession number GSE122960; and 3) processed data including count and metadata tables of human lung tissue was acquired from GSE130148.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    Accession numbers for all ENCODE datasets used can be found in Encode Data Sets Table (Supplementary Table 3).
    ENCODE
    suggested: (Encode, RRID:SCR_015482)
    Predicted enhancer regions of TMPRSS2 were identified using the GeneHancer tool within Track Data Hubs of UCSC genome browser (85, 86).
    GeneHancer
    suggested: None
    UCSC genome browser
    suggested: (UCSC Genome Browser, RRID:SCR_005780)
    Gene set enrichment analysis: Gene set enrichment analyses (GSEA) were performed according to the instructions.
    Gene set enrichment analyses
    suggested: None
    Gene sets of Hallmark Collection, Canonical Pathway (including KEGG Pathway, Biocarta Pathway, Reactome Pathway and PID Pathway), and GO Biological Process were used.
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    GO Biological
    suggested: None
    Statistics: Statistical analysis used R and GraphPad Prism 8 software.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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:
    • 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

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  2. Version published to 10.1101/2021.07.23.453505v1 on bioRxiv