LOX-1 + immature neutrophils predict severe COVID-19 patients at risk of thrombotic complications

  1. Béhazine Combadiere
  2. Lucille Adam
  3. Paul Quentric
  4. Pierre Rosenbaum
  5. Karim Dorgham
  6. Olivia Bonduelle
  7. Christophe Parizot
  8. Delphine Sauce
  9. Julien Mayaux
  10. Charles-Edouard Luyt
  11. Alexandre Boissonnas
  12. Zahir Amoura
  13. Valérie Pourcher
  14. Makoto Miyara
  15. Guy Gorochov
  16. Amélie Guihot
  17. Christophe Combadière

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Abstract

Rational

Lymphopenia and neutrophil/lymphocyte ratio may have prognostic value in coronavirus disease 2019 (COVID-19) severity.

Objective

We sought to investigate the representation of neutrophil subsets in severe and critical COVID-19 patients based on Intensive Care Units (ICU) and non-ICU admission.

Methods

We developed a multi-parametric neutrophil profiling strategy based on known neutrophil markers to distinguish COVID-19 phenotypes in critical and severe patients.

Results

Our results showed that 80% of ICU patients develop strong myelemia with CD10 − CD64 + immature neutrophils. Cellular profiling revealed two distinct neutrophil subsets expressing either the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) or the Interleukin-3 receptor alpha (CD123), both significantly overrepresented in ICU patients compared to non-ICU patients. The proportion of LOX-1-expressing immature neutrophils positively correlated with clinical severity, with the cytokine storm (IL-1 β , IL-6, IL-8, TNF α ), and with intravascular coagulation. Importantly, high proportions of LOX-1 + -immature neutrophils are associated with high risks of severe thrombosis.

Conclusions

Together these data suggest that point of care enumeration of LOX-1-immature neutrophils might help distinguish patients at risk of thrombosis complication and most likely to benefit from intensified anticoagulant therapy.

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  1. ScreenIT

    SciScore for 10.1101/2020.09.15.293100: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Study approval: The study was conducted in accordance with the Declaration of Helsinki and the International Conference on Harmonization Good Clinical Practice guidelines and approved by the relevant regulatory and independent ethics committees.
    Consent: All patients gave oral informed consent.
    IACUC: The study was registered and approved by local ethical committee of Sorbonne-Université/assistance-publique hopitaux de Paris for standard hospitalized patients (N°2020-CER2020-21) and ICU patients (N° CER-2020-31).
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The antibody panel (Supplementary Table S2) included: CD15-BV786, CD14-BUV737, CD10-BUV395 (BD, Le Pont de Claix, France); CRTH2-FITC, CD123-PE, LOX-1-BV421, CD64-BV605 and PD-L1-BV711 (Biolegend, San Diego, USA).
    LOX-1-BV421
    suggested: None
    CD64-BV605
    suggested: None
    PD-L1-BV711
    suggested: None
    One hundred µl of whole blood were additionally stained for several patients with an FMO mix missing of antibodies targeting CD123, LOX-1, and PD-L1, in order to determine the threshold of expression of these markers.
    CD123
    suggested: None
    LOX-1
    suggested: None
    PD-L1
    suggested: None
    Software and Algorithms
    SentencesResources
    FlowJo software 10.0 was used for analysis of marker expression on neutrophils.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Data presentation and statistical analysis: Statistical analyses of the immunological data and graphic representations were performed with Prism 8.0 (GraphPad Software Inc.
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    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: We detected the following sentences addressing limitations in the study:
    The principal limitation of this study is the relatively low number of patients and needs further confirmation in a larger cohort. Our data indicated that patients’ severity could be predicted based on the proportion of immature CD10−CD64+ neutrophils using both unsupervised and expert-gating strategies. This neutrophil subset has been described as an immature subset (20) unlike the CD64+-activated neutrophils which still express the neutral endopeptidase (CD10) and the low-affinity immuno-globulin-Fc fragment III (CD16). Numerous studies have shown an association between circulating immature neutrophils and bacterial sepsis (21). Here, we provided evidence that this immature subset may serve as a severity biomarker in COVID-19. We recently reported that the proportion of CD123-expressing immature neutrophils correlated with bacterial sepsis severity (17). Here, we showed that the expression of CD123 on CD10−CD64+ neutrophils was related to a higher SOFA score among critically-ill COVID-19 patients. Indeed, both CD123 (the alpha chain of the Interleukin-3 receptor) and its cognate ligand, the IL-3 cytokine, were suggested to play an important role in sepsis. Recent studies demonstrated that a high IL-3 plasma levels were associated with lung inflammation, lung injury and high mortality rates in an animal model, but also in humans (Weber 2015, Tong 2020). In addition, these studies showed that IL3 neutralization and anti-CD123 treatment improved mice outcome by decreasing infl...

    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 10.1101/2020.09.15.293100 on bioRxiv