Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication

  1. Jennifer S. Chen
  2. Mia Madel Alfajaro
  3. Jin Wei
  4. Ryan D. Chow
  5. Renata B. Filler
  6. Stephanie C. Eisenbarth
  7. Craig B. Wilen

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Abstract

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE 2 , one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE 2 receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE 2 signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE 2 signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on ACE2 expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients.

Importance

Public health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NSAIDs function by inhibiting the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). These enzymes are critical for the generation of prostaglandins, lipid molecules with diverse roles in maintaining homeostasis as well as regulating the inflammatory response. While COX-1/COX-2 signaling pathways have been shown to affect the replication of many viruses, their effect on SARS-CoV-2 infection remains unknown. We found that SARS-CoV-2 infection induced COX-2 expression in both human cell culture systems and mouse models. However, inhibition of COX-2 activity with NSAIDs did not affect SARS-CoV-2 entry or replication. Our findings suggest that COX-2 signaling may instead regulate the lung inflammation observed in COVID-19 patients, which is an important area for future studies.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.24.312769: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All work with SARS-CoV-2 or icSARS-CoV-2-mNG was performed in a biosafety level 3 facility with approval from the office of Environmental Health and Safety and the Institutional Animal Care and Use Committee at Yale University.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableOnly male mice were used due to availability.
    Cell Line AuthenticationContamination: All cell lines tested negative for Mycoplasma spp.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    The P1 stock was then used to inoculate Vero-E6 cells, and after three days, the supernatant was harvested and clarified by centrifugation (450 × g for 5 min), filtered through a 0.45-micron filter, and stored in aliquots at −80°C.
    Vero-E6
    suggested: None
    From GSE147507 (14), we re-analyzed the raw count data from Calu-3 and A549-ACE2 cells, comparing SARS-CoV-2 infection to matched mock controls.
    Calu-3
    suggested: None
    A549-ACE2
    suggested: None
    1 Spike Glycoprotein Gene, NR-52310, was produced under HHSN272201400008C and obtained through BEI Resources, NIAID, NIH. 293T cells were transfected with the pCAGGS vector expressing the SARS-CoV-2 spike glycoprotein and then incubated with replication-deficient VSV expressing Renilla luciferase for 1 hour at 37°C (27).
    293T
    suggested: None
    icSARS-CoV-2-mNG assay: 6.5 × 103 Calu-3 or 2.5 × 103 Huh7.5 cells were plated in 20 μl phenol red-free media containing 50 μM ibuprofen, 50 μM ibuprofen, or an equivalent amount of DMSO in each well of a black-walled, clear-bottom 384-well plate.
    Huh7.5
    suggested: RRID:CVCL_7927)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: C57BL/6J and K18-hACE2 [B6.Cg-Tg(K18-ACE2)2Prlmn/J (17)] were purchased from Jackson Laboratory.
    K18-hACE2 [B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: None
    K18-hACE2 mice were anesthetized using 30% vol/vol isoflurane diluted in propylene glycol (30% isoflurane) and administered 1.2 × 106 PFU of SARS-CoV-2 intranasally.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    C57BL/6J mice were anesthetized using 30% isoflurane and administered 30 mg/kg ibuprofen, 1 mg/kg meloxicam, or an equivalent amount of DMSO intraperitoneally in a volume of 10 ml/kg daily for 4 days.
    C57BL/6J
    suggested: None
    Software and Algorithms
    SentencesResources
    We performed differential expression analysis using the Wald test from DESeq2 (45), using a Benjamini-Hochberg adjusted p < 0.05 as the cutoff for statistical significance.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    For visualization of PTGS2 expression, the DESeq2-normalized counts were exported and plotted in GraphPad Prism.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Total cell numbers were quantified by Gen5 software for brightfield images.
    Gen5
    suggested: (Gen5, RRID:SCR_017317)

    Results from OddPub: Thank you for sharing your code.

    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.

    About SciScore

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  2. Version published to 10.1101/2020.09.24.312769v1 on bioRxiv