An Immune Cell Atlas Reveals Dynamic COVID-19 Specific Neutrophil Programming Amenable to Dexamethasone Therapy

  1. Sarthak Sinha
  2. Nicole L. Rosin
  3. Rohit Arora
  4. Elodie Labit
  5. Arzina Jaffer
  6. Leslie Cao
  7. Raquel Farias
  8. Angela P. Nguyen
  9. Luiz G. N. de Almeida
  10. Antoine Dufour
  11. Amy Bromley
  12. Braedon McDonald
  13. Mark Gillrie
  14. Marvin J. Fritzler
  15. Bryan Yipp
  16. Jeff Biernaskie

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Abstract

SARS-CoV-2 is a novel coronavirus that causes acute respiratory distress syndrome (ARDS), death and long-term sequelae. Innate immune cells are critical for host defense but are also the primary drivers of ARDS. The relationships between innate cellular responses in ARDS resulting from COVID-19 compared to other causes of ARDS, such as bacterial sepsis is unclear. Moreover, the beneficial effects of dexamethasone therapy during severe COVID-19 remain speculative, but understanding the mechanistic effects could improve evidence-based therapeutic interventions. To interrogate these relationships, we developed an scRNA-Seq and plasma proteomics atlas ( biernaskielab.ca/COVID_neutrophil ). We discovered that compared to bacterial ARDS, COVID-19 was associated with distinct neutrophil polarization characterized by either interferon (IFN) or prostaglandin (PG) active states. Neutrophils from bacterial ARDS had higher expression of antibacterial molecules such as PLAC8 and CD83. Dexamethasone therapy in COVID patients rapidly altered the IFN active state, downregulated interferon responsive genes, and activated IL1R2 +ve neutrophils. Dexamethasone also induced the emergence of immature neutrophils expressing immunosuppressive molecules ARG1 and ANXA1, which were not present in healthy controls. Moreover, dexamethasone remodeled global cellular interactions by changing neutrophils from information receivers into information providers. Importantly, male patients had higher proportions of IFN active neutrophils, a greater degree of steroid-induced immature neutrophil expansion, and increased mortality benefit compared to females in the dexamethasone era. Indeed, the highest proportion of IFN active neutrophils was associated with mortality. These results define neutrophil states unique to COVID-19 when contextualized to other life-threatening infections, thereby enhancing the relevance of our findings at the bedside. Furthermore, the molecular benefits of dexamethasone therapy are also defined, and the identified pathways and plasma proteins can now be targeted to develop improved therapeutics.

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    SciScore for 10.1101/2021.04.18.440366: (What is this?)

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

    Table 1: Rigor

    EthicsConsent: Participants were required to have a definitive diagnosis and appropriate consent and samples collected within 72hrs of admission to the ICU in order to be included.
    IRB: This study was approved by the Conjoint Health Research Ethics Board (CHREB) at the University of Calgary (Ethics ID: REB20-0481) and is consistent with and the Declaration of Helsinki.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Experimental Models: Cell Lines
    SentencesResources
    IL-28a, IL-33, IP-10, LIF, MCP-1, MCP-2, MCP-3, MCP-4, MDC, MIP-1α, MIP-1β, MIP-1d, PDGF-AA, PDGF-AB/BB, RANTES, SDF-1 a+b, sCD40L, SCF,TARC, TGFa, TNFa, TNFb, TPO, TRAIL, TSLP, VEGF) and a 14 MilliPLEX soluble cytokine (sCD30, sEGFR, sgp130, sIL-1RI, sIL-1RII, sIL-2Ra, sIL-4R, sIL-6R, sRAGE, sTNF RI, sTNF RII, sVEGF R1, sVEGF R2, sVEGF R3) arrays (Millipore Sigma, Oakville, ON, Canada) on a Luminex Model 200 Luminometer (Luminex Corporation, Austin, TX)
    MCP-1
    suggested: None
    Software and Algorithms
    SentencesResources
    Cell density was quantified with a hemacytometer, cell viability was assessed with Trypan Blue staining (T8154; Sigma Aldrich), and 7500 live lymphocytes were transferred to a sterile 1.5 mL microcentrifuge tube.
    T8154; Sigma Aldrich
    suggested: None
    Sequencing reads were aligned using CellRanger 3.1.0 pipeline33 to the standard pre-built GRCh38 reference genome.
    CellRanger
    suggested: (SCIGA, RRID:SCR_021002)
    Boxplots comparing cell type composition were generated using the ggplot2 package.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    Differential cell-cell interaction networks were reconstructed using the Connectome R toolkit v0.2.240 and CellChat v1.0.0 41.
    Connectome R toolkit
    suggested: None
    Differential edge list was passed through CircosDiff (a wrapper around the R package ‘circlize’) and CellChat’s netVisual_chord_gene to filter receptor-ligand edges and generate Circos plots.
    Circos
    suggested: (Circos, RRID:SCR_011798)
    Briefly, neutrophils were subsetted from scVelo-realigned Seurat object and processed using default and recommended parameters specified in SCENIC’s vignette (https://github.com/aertslab/SCENIC) using the hg19 RcisTarget reference.
    https://github.com/aertslab/SCENIC
    suggested: (SCENIC, RRID:SCR_017247)
    Rds’) was profiled using g:Profiler’s functional enrichment analysis and genes intersecting with the Interferon pathway were plotted using iRegulon (Cytoscape plugin)45. COVID Neutrophil Atlas: To enable intuitive exploration of single-cell datasets, a web portal (http://biernaskielab.ca/covid_neutrophil or http://biernaskielab.com/covid_neutrophil) was built using RShiny v1.1.0, shinyLP v.1.1.2, and shinythemes v.
    Cytoscape
    suggested: (Cytoscape, RRID:SCR_003032)

    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.

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  2. Version published to 10.1101/2021.04.18.440366 on bioRxiv