Eicosanoid signaling as a therapeutic target in middle-aged mice with severe COVID-19

  1. Lok-Yin Roy Wong
  2. Jian Zheng
  3. Kevin Wilhelmsen
  4. Kun Li
  5. Miguel E. Ortiz
  6. Nicholas J. Schnicker
  7. Alejandro A. Pezzulo
  8. Peter J. Szachowicz
  9. Klaus Klumpp
  10. Fred Aswad
  11. Justin Rebo
  12. Shuh Narumiya
  13. Makoto Murakami
  14. David K. Meyerholz
  15. Kristen Fortney
  16. Paul B. McCray
  17. Stanley Perlman

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Abstract

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations 1 . Resolution of the COVID-19 pandemic has been advanced by the recent development of SARS-CoV-2 vaccines, but vaccine efficacy is partly compromised by the recent emergence of SARS-CoV-2 variants with enhanced transmissibility 2 . The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially in aged populations. Here, we describe the isolation of a new set of highly virulent mouse-adapted viruses and use them to test a novel therapeutic drug useful in infections of aged animals. Initially, we show that many of the mutations observed in SARS-CoV-2 during mouse adaptation (at positions 417, 484, 501 of the spike protein) also arise in humans in variants of concern (VOC) 2 . Their appearance during mouse adaptation indicates that immune pressure is not required for their selection. Similar to the human infection, aged mice infected with mouse-adapted SARS-CoV-2 develop more severe disease than young mice. In murine SARS, in which severity is also age-dependent, we showed that elevated levels of an eicosanoid, prostaglandin D2 (PGD 2 ) and of a phospholipase, PLA 2 G2D, contributed to poor outcomes in aged mice 3,4 . Using our virulent mouse-adapted SARS-CoV-2, we show that infection of middle-aged mice lacking expression of DP1, a PGD 2 receptor, or PLA 2 G2D are protected from severe disease. Further, treatment with a DP1 antagonist, asapiprant, protected aged mice from a lethal infection. DP1 antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, and demonstrates that the PLA 2 G2D-PGD 2 /DP1 pathway is a useful target for therapeutic interventions. (Words: 254)

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    SciScore for 10.1101/2021.04.20.440676: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All animal studies were approved by the University of Iowa Animal Care and Use Committee and meet stipulations of the Guide for the Care and Use of Laboratory Animals.
    Euthanasia Agents: Histology and Immunohistochemistry: Mice were anesthetized by intraperitoneal injection of ketamine-xylazine and perfused transcardially with PBS.
    Sex as a biological variableTreatment with asapiprant: For asapiprant treatment, 7-8 month old, male and female C57BL/6 mice were infected with SARS2-N501YMA30 (5000 PFU).
    Randomizationnot detected.
    BlindingSerial in vivo passaging of virus in mice: Serial blind passage of rSARS2-N501YP0 through mouse lungs was performed in 8-to-10-week-old BALB/c mice.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    For SARS-CoV-2 antigen detection, slides were incubated with blocking reagent (10% normal goat serum x 30 minutes) followed by rabbit monoclonal antibody against SARS-CoV-2 N protein (1:20,000 dilution x 60 minutes, #40143-R019, Sino Biological US Inc., Wayne, PA, USA), then incubated with Rabbit Envision (Dako) and diaminobenzidine (Dako) as chromogen.
    SARS-CoV-2 N protein
    suggested: (ABclonal Cat# A20021, RRID:AB_2862924)
    Cells were then washed and blocked with 1 μg α-CD16/α-CD32 (2.4G2) antibody at 4 °C for 20 minutes and surface stained with the following antibodies at 4°C for 30 minutes: V450 α-mouse CD45 (clone 30-F11; Cat. No.: 560501) APC α-mouse CD220 (clone RA3-6B2, Cat. No.: 553092); APC/Cyanine 7 α-mouse CD3e (clone 145-2C11, Cat. No.: 100330); APC/Cyanine 7 α-mouse CD11c (clone HL3; Cat. No.: 561241); FITC α-mouse Ly6G (clone 1A8; Cat. No.: 127606); PE α-mouse CD11b (clone M1/70; Cat. No.: 101208); PE/Cyanine 7 α-mouse CD8 (clone 53-6.7; Cat. No.: 100722); PerCP/Cyanine 5.5 α-mouse CD4 (clone RM4.5; Cat. No.: 550954); FITC α-mouse Ly6G (1A8; Cat. No.: 127606); PerCP/Cyanine 5.5 α-mouse Ly6C (clone HK1.4; Cat. No.: 128012); CD 64 (clone X54-5/7.1; Cat. No.: 139304) Cells were washed, fixed and permeabilized with Cytofix/Cytoperm (BD Biosciences).
    α-CD16/α-CD32
    suggested: None
    CD45
    suggested: (BD Biosciences Cat# 560501, RRID:AB_1645275)
    CD220
    suggested: (Miltenyi Biotec Cat# 130-126-536, RRID:AB_2889509)
    CD3e
    suggested: (Thermo Fisher Scientific Cat# 88-7774-75, RRID:AB_476399)
    CD11c
    suggested: (BD Biosciences Cat# 561241, RRID:AB_10611727)
    Ly6G
    suggested: (BioLegend Cat# 127606, RRID:AB_1236494)
    CD11b
    suggested: (BioLegend Cat# 101208, RRID:AB_312791)
    CD8
    suggested: None
    CD4
    suggested: (BD Biosciences Cat# 550954, RRID:AB_393977)
    Ly6C
    suggested: None
    Cells were then labeled for cell-surface markers, fixed/permeabilized with Cytofix/Cytoperm Solution (BD Biosciences), and labeled with α-IFN-γ and α-TNF antibody.
    α-TNF
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    2 μg of N501Y-SARS-CoV-2 BAC were transfected into Vero E6 (ATCC) with Lipofectamine 3000 (Invitrogen) in a 6-well plate according to manufacturer’s protocol.
    Vero E6
    suggested: None
    Cultures were harvested when CPE was >50% by freezing at -80°C. rSARS2-N501YP0 was further passaged in Calu-3 cells in DMEM supplemented with 20% FBS.
    Calu-3
    suggested: None
    SARS2-N501YMA30 was further propagated in Calu-3 2B4 cells.
    Calu-3 2B4
    suggested: RRID:CVCL_YZ47)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice, cells and virus: 6-to-10-week- or 6-month-old BALB/c and C57BL/6 mice were obtained from Charles River Laboratories.
    BALB/c
    suggested: None
    C57BL/6
    suggested: None
    Recombinant DNA
    SentencesResources
    In brief, forward and reverse primers with overlapping SARS-CoV-2 spike sequence with the N501Y mutation followed by sequence complementary to the target plasmid (pEP-KanS) were synthesized (Invitrogen).
    pEP-KanS
    suggested: None
    Software and Algorithms
    SentencesResources
    Cells were then labeled for cell-surface markers, fixed/permeabilized with Cytofix/Cytoperm Solution (BD Biosciences), and labeled with α-IFN-γ and α-TNF antibody.
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    All flow cytometry data were acquired using a BD FACSVerse and analyzed with FlowJo software.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    In silico structural modelling and analysis: A homology model for mouse ACE2 (NP_001123985.1) was generated using YASARA and docked into the crystal structure of complexed human ACE2 and spike RBD (PDB: 6M0J).
    YASARA
    suggested: (YASARA, RRID:SCR_017591)
    Figures were generated in PyMOL.
    PyMOL
    suggested: (PyMOL, RRID:SCR_000305)
    Statistical analysis: Differences in mean values between groups were analysed by ANOVA and Student’s t-tests and differences in survival were analysed by log-rank (Mantel–Cox) tests using GraphPad Prism 8.
    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: 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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04705597RecruitingStudy to Evaluate the Safety, Tolerability, and Efficacy of …

    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

    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 10.1101/2021.04.20.440676v1 on bioRxiv