Inhibiting SARS-CoV-2 infection in vitro by suppressing its receptor, angiotensin-converting enzyme 2, via aryl-hydrocarbon receptor signal

  1. Keiji Tanimoto
  2. Kiichi Hirota
  3. Takahiro Fukazawa
  4. Yoshiyuki Matsuo
  5. Toshihito Nomura
  6. Nazmul Tanuza
  7. Nobuyuki Hirohashi
  8. Hidemasa Bono
  9. Takemasa Sakaguchi

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Abstract

Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1 . Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells.

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  1. Version published to 10.1038/s41598-021-96109-w
  2. ScreenIT

    SciScore for 10.1101/2021.03.04.433658: (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.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-ACE2 (GTX101395, GeneTex), anti-SARS-CoV/SARS-CoV-2 nucleocapsid (GTX632269, GeneTex), or anti-β-actin (A5441, Sigma-Aldrich) was used as the primary antibody, diluted 1:1000, 1:1000, or 1:5000, respectively.
    Anti-ACE2
    suggested: (GeneTex Cat# GTX101395, RRID:AB_1240451)
    anti-SARS-CoV/SARS-CoV-2 nucleocapsid ( GTX632269
    suggested: None
    anti-β-actin
    suggested: (Sigma-Aldrich Cat# A5441, RRID:AB_476744)
    A 1:2000 dilution of anti-mouse or anti-rabbit IgG horseradish peroxidase conjugate (#7076, #7074, Cell Signaling TECHNOLOGY) was used as a secondary antibody.
    anti-mouse
    suggested: (Cell Signaling Technology Cat# 7076, RRID:AB_330924)
    anti-rabbit IgG
    suggested: (Assay Biotech Cat# B7076, RRID:AB_10684258)
    Experimental Models: Cell Lines
    SentencesResources
    ) into HepG2 cells (1 x 106/10 cm diameter dish) for 12 hours, and then the cells were incubated with DMSO or FICZ for 24 hours.
    HepG2
    suggested: None
    Virus infection analysis: To observe the efficiency of infection of mammalian cells by SARS-CoV-2, SARS-CoV-2/JP/Hiroshima-46059T/2020 and TMPRSS2 transfected Vero E6 cells were employed (Matsuyama et al., 2020).
    Vero E6
    suggested: None
    Vero E6/TMPRSS2 cells in a 24 well plate were treated with FICZ or OMP for 24 hours before virus infections.
    Vero E6/TMPRSS2
    suggested: None
    Software and Algorithms
    SentencesResources
    The ikra pipeline automated the RNA-seq data-analysis process, including the quality control of reads [sra-tools v.2.10.7, Trim Galore v.0.6.3 (Krueger et al., 2020) using Cutadapt v.
    Trim Galore
    suggested: (Trim Galore, RRID:SCR_011847)
    1.9.1 (Martin, 2011)], and transcript quantification (Salmon v.0.14.0, using reference transcript sets in GENCODE release 31 for humans), and tximport v.1.6.0.
    Salmon
    suggested: (Salmon, RRID:SCR_017036)
    GENCODE
    suggested: (GENCODE, RRID:SCR_014966)
    Quantified transcript reads were filtered at a threshold of 0.5 counts per million (CPM) in at least one sample and transformed as log2(CPM + c) with EdgeR (3.28.0), with a pseudocount c value of 4.
    EdgeR
    suggested: (edgeR, RRID:SCR_012802)
    Gene set enrichment analysis was performed in iDEP with the fold-change values returned by DESeq2 (1.26.0).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    False positive rates of q < 0.05 were considered enriched and investigated further with Metascape (Zhou et al., 2019).
    Metascape
    suggested: (Metascape, RRID:SCR_016620)
    Statistical Analysis: Statistical tests were based on the ANOVA test, Student’s t test, Dunnett’s test, or the Gemes-Howell test in SPSS Statistics version 17.0 (IBM).
    SPSS
    suggested: (SPSS, RRID:SCR_002865)

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    One limitation of this strategy might be that these drugs do not target the SARS-CoV-2 virus itself but just modify cellular susceptibility to it. Combination therapies of AHR agonists with anti-virus drugs, such as favipiravir or remdesivir, are therefore possible strategies for clinical application. In addition to modifying cellular susceptibility to SARS-CoV-2, treatment with AHR agonists might stimulate immune response in the treated cells without virus infection. An RNA-seq dataset composed of SARS-CoV-2 infection experiments suggested that genes regulated by CSE, FICZ, and OMP overlapped with genes modified by SARS-CoV-2 infection, suggesting that stimulation of the immune system is involved (Figure 2h and k, Supplementary Figures 2d and h). These observations might signify a potentially useful clinical application, although further investigation will be necessary to elucidate the details. In conclusion, we here demonstrated that treatment with CSE or AHR agonists decreased expression of ACE2 in mammalian cells, resulting in suppression of SARS-CoV-2 infection (Figure 6). Application of these compounds in clinical research and in clinical practice might be warranted.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    About SciScore

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  3. Version published to 10.1101/2021.03.04.433658v1 on bioRxiv