Dysregulated Neutrophil Phenotype and Function in Hospitalised Non-ICU COVID-19 Pneumonia

  1. Kylie B. R. Belchamber
  2. Onn S. Thein
  3. Jon Hazeldine
  4. Frances S. Grudzinska
  5. Aduragbemi A. Faniyi
  6. Michael J. Hughes
  7. Alice E. Jasper
  8. Kay Por Yip
  9. Louise E. Crowley
  10. Sebastian T. Lugg
  11. Elizabeth Sapey
  12. Dhruv Parekh
  13. David R. Thickett
  14. Aaron Scott

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Abstract

Rationale: Infection with the SARS-CoV2 virus is associated with elevated neutrophil counts. Evidence of neutrophil dysfunction in COVID-19 is based on transcriptomics or single functional assays. Cell functions are interwoven pathways, and understanding the effect across the spectrum of neutrophil function may identify therapeutic targets. Objectives: Examine neutrophil phenotype and function in 41 hospitalised, non-ICU COVID-19 patients versus 23 age-matched controls (AMC) and 26 community acquired pneumonia patients (CAP). Methods: Isolated neutrophils underwent ex vivo analyses for migration, bacterial phagocytosis, ROS generation, NETosis and receptor expression. Circulating DNAse 1 activity, levels of cfDNA, MPO, VEGF, IL-6 and sTNFRI were measured and correlated to clinical outcome. Serial sampling on day three to five post hospitalization were also measured. The effect of ex vivo PI3K inhibition was measured in a further cohort of 18 COVID-19 patients. Results: Compared to AMC and CAP, COVID-19 neutrophils demonstrated elevated transmigration (p = 0.0397) and NETosis (p = 0.0332), and impaired phagocytosis (p = 0.0036) associated with impaired ROS generation (p < 0.0001). The percentage of CD54+ neutrophils (p < 0.001) was significantly increased, while surface expression of CD11b (p = 0.0014) and PD-L1 (p = 0.006) were significantly decreased in COVID-19. COVID-19 and CAP patients showed increased systemic markers of NETosis including increased cfDNA (p = 0.0396) and impaired DNAse activity (p < 0.0001). The ex vivo inhibition of PI3K γ and δ reduced NET release by COVID-19 neutrophils (p = 0.0129). Conclusions: COVID-19 is associated with neutrophil dysfunction across all main effector functions, with altered phenotype, elevated migration and NETosis, and impaired antimicrobial responses. These changes highlight that targeting neutrophil function may help modulate COVID-19 severity.

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  1. Version published to 10.3390/cells11182901
  2. ScreenIT

    SciScore for 10.1101/2021.06.08.21258535: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: COVID-19 patients were recruited January to March 2021 from the Queen Elizabeth Hospital Birmingham, in accordance with ethics REC ref: 19/WA/0299 and 20/WA/0092 approved by the West Midlands – Solihull research ethics committee.
    Consent: Written informed consent was obtained where possible; patients unable to consent due to lack of capacity were consented by proxy; designated consultee via telephone or professional consultee.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power AnalysisA power calculation performed on isolated neutrophil NETosis data (80%, alpha 0.05) suggested 18 participants were required in each group (see supplement for details).

    Table 2: Resources

    No key resources detected.

    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: We detected the following sentences addressing limitations in the study:
    Limitations: This study was limited due to the safety measures required. All experiments were carried out within a BSL2 hood and methods chosen based on tolerance to inactivation/fixation with 4% PFA. Our patients did not include an ICU group, however, mild-moderate disease forms a larger proportion of overall COVID-19 patients, and we believe it is this point in the patient pathway which holds most potential for successful intervention. Conclusion: Our study shows that moderate COVID-19 is associated with alterations in neutrophil phenotype, increased migratory capacity and NETosis, and impaired antimicrobial function which contributes to the severity of COVID-19. Elevated NETosis in the lung is associated with disease severity, and elevated systemic NET production is likely to contribute to inflammation, which may drive ARDS associated damage, and thrombosis. Targeting neutrophils and their downstream effectors may be beneficial in the treatment of COVID-19.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04343898Active, not recruitingStudy of the Treatment and Outcomes in Critically Ill Patien…

    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.

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  3. Version published to 10.1101/2021.06.08.21258535v1 on medRxiv