Soluble Signal Inhibitory Receptor on Leukocytes-1 Is Released from Activated Neutrophils by Proteinase 3 Cleavage

  1. Helen J. von Richthofen
  2. Geertje H.A. Westerlaken
  3. Doron Gollnast
  4. Sjanna Besteman
  5. Eveline M. Delemarre
  6. Karlijn Rodenburg
  7. Petra Moerer
  8. Daphne A.C. Stapels
  9. Anand K. Andiappan
  10. Olaf Rötzschke
  11. Stefan Nierkens
  12. Helen L. Leavis
  13. Louis J. Bont
  14. Suzan H.M. Rooijakkers
  15. Linde Meyaard

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Abstract

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is an immune inhibitory receptor expressed on human granulocytes and monocytes that dampens antimicrobial functions. We previously showed that sputum neutrophils from infants with severe respiratory syncytial virus (RSV) bronchiolitis have decreased SIRL-1 surface expression compared with blood neutrophils and that SIRL-1 surface expression is rapidly lost from in vitro activated neutrophils. This led us to hypothesize that activated neutrophils lose SIRL-1 by ectodomain shedding. Here, we developed an ELISA and measured the concentration of soluble SIRL-1 (sSIRL-1) in patients with RSV bronchiolitis and hospitalized patients with COVID-19, which are both characterized by neutrophilic inflammation. In line with our hypothesis, sSIRL-1 concentration was increased in sputum compared with plasma of patients with RSV bronchiolitis and in serum of hospitalized patients with COVID-19 compared with control serum. In addition, we show that in vitro activated neutrophils release sSIRL-1 by proteolytic cleavage and that this diminishes the ability to inhibit neutrophilic reactive oxygen species production via SIRL-1. Finally, we found that SIRL-1 shedding is prevented by proteinase 3 inhibition and by extracellular adherence protein from Staphylococcus aureus. Notably, we recently showed that SIRL-1 is activated by PSMα3 from S. aureus, suggesting that S. aureus may counteract SIRL-1 shedding to benefit from preserved inhibitory function of SIRL-1. In conclusion, we report that SIRL-1 is released from activated neutrophils by proteinase 3 cleavage and that endogenous sSIRL-1 protein is present in vivo.

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  1. Version published to 10.4049/jimmunol.2200169
  2. ScreenIT

    SciScore for 10.1101/2022.03.03.482795: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: All samples were collected in accordance with the Institutional Review Board of the University Medical Center (UMC) Utrecht or the National University of Singapore.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To determine PR3 cleavage, 20 μg/mL sSIRL-1ecto or VSTM1-v2 were incubated with 2 μg/mL PR3 in PBS with 0.5M NaCl and incubated 3h at 25°C, followed by SDS-PAGE and Western blot analysis as described above, except that rabbit-anti-mouse IgG-HRP (DAKO; 1:10.000 diluted in TBS-T with 1% BSA) was used as secondary antibody.
    rabbit-anti-mouse IgG-HRP
    suggested: None
    DAKO
    suggested: None
    Recombinant DNA
    SentencesResources
    The gBlocks were inserted into a pcDNA 3.1+ Zeocine plasmid using Gibson assembly master mix (New England Biolabs).
    pcDNA 3.1+
    suggested: None
    Software and Algorithms
    SentencesResources
    Proteins were separated by SDS-PAGE on Any kD Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad Laboratories) and either stained directly with coomassie blue (Merck) or transferred onto 0.45 μm PVDF membranes (Merck) for western blot analysis.
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
    , cathepsin G (Biocentrum), or proteinase 3 (Elastin Products Company) (all 1 μM), followed by flow cytometry analysis.
    Biocentrum
    suggested: None
    Cells were analyzed using the BD FACS Canto II and FlowJo software (Treestar, Ashland, OR).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Statistical analysis: Statistical analyses were performed using GrapPad Prism software (version 8.3.0).
    GrapPad Prism
    suggested: None

    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: 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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  3. Version published to 10.1101/2022.03.03.482795v1 on bioRxiv