High mobility group box 1, ATP , lipid mediators, and tissue factor are elevated in COVID ‐19 patients: HMGB1 as a biomarker of worst prognosis

  1. Amanda Roberta Revoredo Vicentino
  2. Vanderlei da Silva Fraga‐Junior
  3. Matheus Palazzo
  4. Natalia Recardo Amorim Tasmo
  5. Danielle A. S. Rodrigues
  6. Shana Priscila Coutinho Barroso
  7. Sâmila Natiane Ferreira
  8. Anna Cristina Neves‐Borges
  9. Diego Allonso
  10. Marcelo Rosado Fantappié
  11. Julio Scharfstein
  12. Ana Carolina Oliveira
  13. Rosane Vianna‐Jorge
  14. André Macedo Vale
  15. Robson Coutinho‐Silva
  16. Luiz Eduardo Baggio Savio
  17. Claudio Canetti
  18. Claudia Farias Benjamim

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Abstract

The severe acute respiratory syndrome coronavirus 2, the agent of the ongoing coronavirus disease 2019 (COVID‐19) pandemic, has spread worldwide since it was first identified in November 2019 in Wuhan, China. Since then, progress in pathogenesis linked severity of this systemic disease to the hyperactivation of network of cytokine‐driven pro‐inflammatory cascades. Here, we aimed to identify molecular biomarkers of disease severity by measuring the serum levels of inflammatory mediators in a Brazilian cohort of patients with COVID‐19 and healthy controls (HCs). Critically ill patients in the intensive care unit were defined as such by dependence on oxygen supplementation (93% intubated and 7% face mask), and computed tomography profiles showing ground‐glass opacity pneumonia associated to and high levels of D‐dimer. Our panel of mediators included HMGB1, ATP, tissue factor, PGE 2 , LTB 4 , and cys‐LTs. Follow‐up studies showed increased serum levels of every inflammatory mediator in patients with COVID‐19 as compared to HCs. Originally acting as a transcription factor, HMGB1 acquires pro‐inflammatory functions following secretion by activated leukocytes or necrotic tissues. Serum levels of HMGB1 were positively correlated with cys‐LTs, D‐dimer, aspartate aminotransferase, and alanine aminotransferase. Notably, the levels of the classical alarmin HMGB1 were higher in deceased patients, allowing their discrimination from patients that had been discharged at the early pulmonary and hyperinflammatory phase of COVID‐19. In particular, we verified that HMGB1 levels above 125.4 ng/ml is the cutoff that distinguishes patients that are at higher risk of death. In conclusion, we propose the use of serum levels of HMGB1 as a biomarker of severe prognosis of COVID‐19.

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  1. Version published to 10.1111/cts.13475
  2. ScreenIT

    SciScore for 10.1101/2022.05.26.22275611: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: Ethics statement: The present study was performed in accordance with regulations guidelines and approved by institutional ethical review boards from Clementino Fraga Filho University Hospital and Marcílio Dias Naval Hospital Ethics Committees (protocol numbers 4.551.702, protocol ID 361-20 and 32382820.3.0000.5256 respectively), with written informed consents obtained from all participants or their legal representatives
    Consent: Ethics statement: The present study was performed in accordance with regulations guidelines and approved by institutional ethical review boards from Clementino Fraga Filho University Hospital and Marcílio Dias Naval Hospital Ethics Committees (protocol numbers 4.551.702, protocol ID 361-20 and 32382820.3.0000.5256 respectively), with written informed consents obtained from all participants or their legal representatives
    Sex as a biological variablenot detected.
    RandomizationWe received samples from 73 patients randomly chosen from the HMMD repository of COVID-19 severe cases (322 total cases), with at least two samples collected in different hospitalization days.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Briefly, the wells of a 96-well microtiter plate (Greiner Bio-One, Austria) were coated overnight at 4 °C with anti-HMGB1 mouse monoclonal antibody (No H9537, Sigma-Aldrich, San Luis, MO) in PBS buffer (8.06 mM sodium phosphate, 1.94 mM potassium phosphate, 2.7 mM KCl, and 137 mM NaCl) at pH 7.4.
    anti-HMGB1
    suggested: None
    Subsequently, the wells were incubated with rabbit-produced anti-rHMGB1 polyclonal antibody diluted in PBS buffer for 1 h at 37 °C, and then incubated for 1 h at the same temperature with anti-IgG rabbit antibody conjugated to horseradish peroxidase (No W4011, Promega, Madison, WI).
    anti-rHMGB1
    suggested: None
    anti-IgG
    suggested: (Promega Cat# W4011, RRID:AB_430833)
    Measurement of serum anti-SARS-CoV-2 antibodies: For quantitative analysis of anti-SARS-CoV-2 spike protein IgM and IgG antibodies, we performed the S-UFRJ test, as described previously (47).
    quantitative analysis of anti-SARS-CoV-2 spike protein IgM
    suggested: None
    IgG antibodies
    suggested: None
    Then, the plate was washed with 150 μL of PBS (5x) and 50 μL of 1:10000 goat anti-human IgM and IgG (Fc)-horseradish peroxidase antibody (Sourthen Biotech, Birmingham, AL) were added, and the plate was incubated for 1.5 h at RT.
    1:10000 goat anti-human IgM
    suggested: None
    IgG ( Fc)-horseradish peroxidase antibody ( Sourthen Biotech , Birmingham , AL )
    suggested: None
    Software and Algorithms
    SentencesResources
    The bands corresponding to both proteins were quantified using Image J software (NIH, Bethesda, MD) and the ratio between tissue factor and transferrin was calculated.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    The optical density (OD) was read at 450 nm with 655 nm background compensation in a microplate reader (Bio-Rad Laboratories, Inc, CA).
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)

    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

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  3. Version published to 10.1101/2022.05.26.22275611v1 on medRxiv