A functionally distinct neutrophil landscape in severe COVID-19 reveals opportunities for adjunctive therapies

  1. Rachita Panda
  2. Fernanda V.S. Castanheira
  3. Jared M. Schlechte
  4. Bas G.J. Surewaard
  5. Hanjoo Brian Shim
  6. Amanda Z. Zucoloto
  7. Zdenka Slavikova
  8. Bryan G. Yipp
  9. Paul Kubes
  10. Braedon McDonald

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Abstract

Acute respiratory distress syndrome (ARDS) is a life-threatening syndrome, constituted by respiratory failure and diffuse alveolar damage that results from dysregulated local and systemic immune activation, causing pulmonary vascular, parenchymal, and alveolar damage. SARS-CoV-2 infection has become the dominant cause of ARDS worldwide, and emerging evidence implicates neutrophils and their cytotoxic arsenal of effector functions as central drivers of immune-mediated lung injury in COVID-19 ARDS. However, key outstanding questions are whether COVID-19 drives a unique program of neutrophil activation or effector functions that contribute to the severe pathogenesis of this pandemic illness and whether this unique neutrophil response can be targeted to attenuate disease. Using a combination of high-dimensional single-cell analysis and ex vivo functional assays of neutrophils from patients with COVID-19 ARDS, compared with those with non-COVID ARDS (caused by bacterial pneumonia), we identified a functionally distinct landscape of neutrophil activation in COVID-19 ARDS that was intrinsically programmed during SARS-CoV-2 infection. Furthermore, neutrophils in COVID-19 ARDS were functionally primed to produce high amounts of neutrophil extracellular traps. Surprisingly, this unique pathological program of neutrophil priming escaped conventional therapy with dexamethasone, thereby revealing a promising target for adjunctive immunotherapy in severe COVID-19.

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  1. Version published to 10.1172/jci.insight.152291
  2. ScreenIT

    SciScore for 10.1101/2021.06.07.21258484: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Study design: Written informed consent was obtained from all study participants or most appropriate surrogate decision maker for patients who were unable to provide consent.
    IRB: This study was approved by the conjoint health research ethics board of the University of Calgary and Alberta Health Services (
    Sex as a biological variablenot detected.
    RandomizationTo visualize the neutrophil metacluster landscape, tSNE dimensionality reduction was performed on 2,500 randomly selected events from each sample using a perplexity of 70 for 5000 iterations.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Experimental Models: Organisms/Strains
    SentencesResources
    Between April 1 2020 and March 30 2021, consecutive patients were screened for the following inclusion criteria: adult patients (>18 years of age) with an index admission to one of four multi-system intensive care units in Calgary, AB, Canada, with a diagnosis of ARDS based on the Berlin Criteria (30), associated with a diagnosis of SARS-CoV-2 infection (based on clinical laboratory qPCR assay from nasopharyngeal swab or endotracheal tube aspirate) or bacterial pneumonia (based on standard clinical and microbiologic criteria).
    AB
    suggested: RRID:BDSC_203)
    Software and Algorithms
    SentencesResources
    For mass cytometry data analysis, FCS files were imported into Cytobank (Cytobank.org) for data visualization and manual gating.
    Cytobank
    suggested: (Cytobank, RRID:SCR_014043)
    Single-cell events were then exported from CytoBank as FCS files and loaded into the CATALYST package (32) in R (R Development Core Team)
    CATALYST
    suggested: (CATALYST, RRID:SCR_017127)
    R Development Core
    suggested: (R Project for Statistical Computing, RRID:SCR_001905)
    For neutrophil clustering and analysis, CD45+CD66b+ neutrophils were analyzed by FlowSOM (33) and a Consensus Clustering method (34) was employed on all neutrophil single-cell events within CATALYST based on the expression of neutrophil markers (see Fig 1C).
    FlowSOM
    suggested: (FlowSOM, RRID:SCR_016899)
    Data visualization was generated using the built-in functions of CATALYST and the ggplot2 package in R.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    Neutrophils were gated based on their forward and side scatter profiles, and bacterial phagocytosis was measured by as the percentage of neutrophils positive for GFP+ bacteria using FlowJo software (FloJo, BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Principal component analysis of neutrophil markers expression between patient groups was performed using the procomp function and VEGAN package in R, and plotted using ggplot2.
    VEGAN
    suggested: (vegan, RRID:SCR_011950)
    Statistical analysis and graphic generation were performed using Graphpad Prism (GraphPad Software) and R (R Development Core Team).
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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: 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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04402957Active, not recruitingLSALT Peptide vs. Placebo to Prevent ARDS and Acute Kidney I…

    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 10.1101/2021.06.07.21258484 on medRxiv