Neuraminidase inhibitors rewire neutrophil function in vivo in murine sepsis and ex vivo in COVID-19

  1. Rodrigo de Oliveira Formiga
  2. Flávia C. Amaral
  3. Camila F. Souza
  4. Daniel A. G. B. Mendes
  5. Carlos W. S. Wanderley
  6. Cristina B. Lorenzini
  7. Adara A. Santos
  8. Juliana Antônia
  9. Lucas F. Faria
  10. Caio C. Natale
  11. Nicholas M. Paula
  12. Priscila C. S. Silva
  13. Fernanda R. Fonseca
  14. Luan Aires
  15. Nicoli Heck
  16. Márick R. Starick
  17. Celso M. Queiroz-Junior
  18. Felipe R. S. Santos
  19. Filipe R. O. de Souza
  20. Vivian V. Costa
  21. Shana P. C. Barroso
  22. Alexandre Morrot
  23. Johan Van Weyenbergh
  24. Regina Sordi
  25. Frederico Alisson-Silva
  26. Fernando Q. Cunha
  27. Edroaldo L. Rocha
  28. Sylvie Chollet-Martin
  29. Maria Margarita Hurtado-Nedelec
  30. Clémence Martin
  31. Pierre-Régis Burgel
  32. Daniel S. Mansur
  33. Rosemeri Maurici
  34. Matthew S. Macauley
  35. André Báfica
  36. Véronique Witko-Sarsat
  37. Fernando Spiller

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Abstract

Neutrophil overstimulation plays a crucial role in tissue damage during severe infections. Neuraminidase (NEU)-mediated cleavage of surface sialic acid has been demonstrated to regulate leukocyte responses. Here, we report that antiviral NEU inhibitors constrain host NEU activity, surface sialic acid release, ROS production, and NETs released by microbial-activated human neutrophils. In vivo , treatment with Oseltamivir results in infection control and host survival in peritonitis and pneumonia models of sepsis. Single-cell RNA sequencing re-analysis of publicly data sets of respiratory tract samples from critical COVID-19 patients revealed an overexpression of NEU1 in infiltrated neutrophils. Moreover, Oseltamivir or Zanamivir treatment of whole blood cells from severe COVID-19 patients reduces host NEU-mediated shedding of cell surface sialic acid and neutrophil overactivation. These findings suggest that neuraminidase inhibitors can serve as host-directed interventions to dampen neutrophil dysfunction in severe infections.

At a Glance

In a severe systemic inflammatory response, such as sepsis and COVID-19, neutrophils play a central role in organ damage. Thus, finding new ways to inhibit the exacerbated response of these cells is greatly needed. Here, we demonstrate that in vitro treatment of whole blood with the viral neuraminidase inhibitors Oseltamivir or Zanamivir, inhibits the activity of human neuraminidases as well as the exacerbated response of neutrophils. In experimental models of severe sepsis, oseltamivir decreased neutrophil activation and increased the survival rate of mice. Moreover, Oseltamivir or Zanamivir ex vivo treatment of whole blood cells from severe COVID-19 patients rewire neutrophil function.

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    SciScore for 10.1101/2020.11.12.379115: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementConsent: All participants gave their written informed consent for blood collection after being informed on procedures.
    IRB: The research protocol followed the World Medical Association Declaration of Helsinki and was approved by the Institutional Review Board of the Federal University of Santa Catarina (CAAE #82815718.2.0000.0121).
    IACUC: Protocols were approved by the Animal Use Ethics Committee of UFSC (CEUA #8278290818).
    RandomizationA group of E. coli-septic mice was randomly pretreated (2 hr before infection) and post treated by per oral (PO, 12/12 hr) via with saline or Oseltamivir phosphate (10 mg/kg, Eurofarma, Brazil) for 4 days to survival analysis.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The mix of antibodies against CD66b (G10F5; BioLegend, San Diego, CA, USA), CD62L (DREG-56; BioLegend), CD16 (3G8; BioLegend), isotypes or Maackia amurensis Lectin II biotinylated (MAL-II, Vector Labs, San Diego, CA, USA) coupled to Streptavidin (Biolegend), plus human BD Fc BlockTM (BD PharmingenTM) and Fixable Viability Stain (FVS, BD HorizonTM, San Jose, CA, USA) were added to leukocytes for 30 min at 4 °C.
    CD66b
    suggested: None
    CD62L
    suggested: None
    CD16
    suggested: None
    Lectin II biotinylated ( MAL-II , Vector Labs , San Diego , CA
    suggested: None
    Briefly, an anti-MPO antibody bound to a 96-well flat-bottom plate captured the enzyme MPO (5 μg/ml; Abcam), and the amount of DNA bound to the enzyme was quantified using the Quant-iT™ PicoGreen® kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
    anti-MPO
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    C57BL/6 (Jackson Laboratory, Bar Harbor, ME, USA) mice (8–10 weeks old) and Swiss mice (10–12 weeks old) were housed in cages at 21 ± 2°C with free access to water and food at the Animal Facility of the Department of Microbiology, Immunology, and Parasitology and Department of Pharmacology from UFSC, respectively.
    C57BL/6
    suggested: None
    Swiss
    suggested: None
    Software and Algorithms
    SentencesResources
    Cells were washed, resuspended in FACS buffer, acquired in a FACSVerse cytometer and analyzed using FlowJo software (FlowJo LLC).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    The lane intensities were quantified using the Image J software.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Data was analyzed using GraphPad Prism version 8.00 for Mac (GraphPad Software, USA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    Results from OddPub: Thank you for sharing your data.

    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 10.1101/2020.11.12.379115 on bioRxiv