Dietary αKG inhibits SARS CoV-2 infection and rescues inflamed lungs to restore normal O 2 saturation in animals

  1. Sakshi Agarwal
  2. Simrandeep Kaur
  3. Tejeswara Rao Asuru
  4. Garima Joshi
  5. Nishith M Shrimali
  6. Anamika Singh
  7. Oinam Ningthemmani Singh
  8. Puneet Srivastva
  9. Tripti Shrivastava
  10. Sudhanshu Vrati
  11. Milan Surjit
  12. Prasenjit Guchhait

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Abstract

Our recent works described the rescue effect of α-ketoglutarate (αKG, a metabolite of Krebs cycle) on thrombosis and inflammation in animals. αKG augments activity of prolyl hydroxylase 2 (PHD2), which in turn degrades proline residues of substrates like phosphorylated Akt (pAkt) and hypoxia inducible factor (HIF)α. Here we describe the inhibitory effect of octyl αKG on pAkt as well as on HIF1α/HIF2α, and in turn decreasing SARS CoV-2 replication in Vero E6 cells. αKG failed to inhibit the viral replication and Akt phosphorylation in PHD2-knockdown U937 cells transiently expressing ACE2. Contrastingly, triciribine (TCN, an Akt-inhibitor) inhibited viral replication alongside a downmodulation of pAkt in PHD2-KD cells. Dietary αKG significantly inhibited viral infection and rescued hamsters from thrombus formation and inflammation in lungs, the known causes of acute respiratory distress syndrome (ARDS) in COVID-19. αKG supplementation also reduced the apoptotic death of lung tissues in infected animals, alongside a downmodulation of pAkt and HIF2α. αKG supplementation neither affected IgG levels against SARS CoV-2 RBD protein nor altered the neutralization antibody response against SARS CoV-2. It did not interfere with the percentage of interferon-γ positive (IFNγ+) CD4+ and IFNγ+CD8+ T cells in infected animals. The extended work in balb/c mice transiently expressing ACE2 showed a similar effect of αKG in reducing accumulation of inflammatory immune cells and cytokines, including IL6, IL1β and TNFα, in lungs as well as in circulation of infected animals. Pro-thrombotic markers like platelet microparticles and platelet-leukocyte aggregates were reduced significantly in infected mice after αKG supplementation. Importantly, αKG supplementation restored the O 2 saturation (SpO 2 ) in circulation of SARS CoV-2 infected hamsters and mice, suggesting a potential therapeutic role of this metabolite in COVID-19 treatment.

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    SciScore for 10.1101/2022.04.02.486853: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics: Ethics approval was obtained from the Institutional Animal Ethics Committee (IAEC) (ref. no. RCB/ IAEC/2021/093), and Institutional Biosafety Committee (
    IACUC: Ethics: Ethics approval was obtained from the Institutional Animal Ethics Committee (IAEC) (ref. no. RCB/ IAEC/2021/093), and Institutional Biosafety Committee (
    Sex as a biological variableMale hamsters of 8 weeks old were infected with one-time SARS CoV-2 via nasal route inoculation using 1×105 plaque-forming units (PFU).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: The Vero E6 cell line was validated for free of mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    The lysate samples were further processed for SDS-PAGE followed by immunoblotting using primary antibodies against pAkt-Ser473, Akt, pAkt1-Thr308, Akt1, HIF1α, HIF2α, β-Actin (Cell Signalling, USA), as described in our work (15).
    pAkt-Ser473 , Akt , pAkt1-Thr308
    suggested: None
    Akt1
    suggested: None
    HIF1α
    suggested: (ABclonal Cat# A17906, RRID:AB_2861751)
    HIF2α
    suggested: None
    β-Actin
    suggested: None
    The plates were incubated at room temperature (RT) for 1 h and then washed three times with washing buffer (PBS + 0.1 % tween 20) and incubated with HRP conjugated antihamster IgG antibody at 1:10,000 dilution for another 1 h and washed four times with the washing buffer and incubated further with 100 μl of TMB substrate (Thermo Fisher Scientific), The reaction stopped with 1N H2SO4 solution.
    HRP conjugated antihamster IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    SARS CoV-2 infection in Vero and U937 cells: Monkey kidney epithelial cell line Vero E6 (0.1×106 cells/well) was infected with P-3 Wuhan SARS CoV-2 (world reference # USA-WA-1/2020, as mentioned in our earlier work (15) at 0.01 MOI for 1 hr in Dulbecco’s Modified Eagle Medium (DMEM) with 2% FBS, supplemented with 1% non-essential amino acids.
    Vero
    suggested: None
    Vero E6
    suggested: None
    Confocal microscopy: Vero E6, U937 cells or U937/PHD2-KD cells from above experiment were fixed in 4% paraformaldehyde for 20 min at room temperature.
    U937
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    Experiments using BALB/c mouse strain (RRID: IMSR_JAX_000651) and Syrian golden hamster (available form ICMR-National Institute of Nutrition, Hyderabad, India) were conducted within the guidelines of IAEC in the Biosafety level 3 (BSL3) facility of the institute.
    BALB/c
    detected: (IMSR Cat# JAX_000651, RRID:IMSR_JAX:000651)
    Recombinant DNA
    SentencesResources
    The lysate samples were further processed for SDS-PAGE followed by immunoblotting using primary antibodies against pAkt-Ser473, Akt, pAkt1-Thr308, Akt1, HIF1α, HIF2α, β-Actin (Cell Signalling, USA), as described in our work (15).
    pAkt1-Thr308
    suggested: None
    Software and Algorithms
    SentencesResources
    Imaging was performed using Z-stacks at 0.25 μm per slice by sequential scanning and Image J Fiji software was used to obtain maximum intensity projection images.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Cells were then washed and acquired on BD FACS Verse and were analyzed with FlowJo software (Tree Star, USA)
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Graph Pad Prism version 8.0 software was used for data analysis and P-values.
    Graph Pad 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: 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

    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 10.1101/2022.04.02.486853v1 on bioRxiv