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Quantitative evaluation of citrullinated fibrinogen for detection of neutrophil extracellular traps

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Abstract

Activated neutrophils release neutrophil extracellular traps (NETs) composed of chromatin filaments containing bactericidal proteins and enzymes. This process, known as NETosis, is an innate host defense mechanism. However, NET accumulation can lead to uncontrolled inflammation and organ damage. Therefore, NET detection provides clinically important information for the assessment of inflammatory conditions. We investigated whether quantification of citrullinated fibrinogen (C-Fbg), which is catalyzed by peptidylarginine deiminase (PAD) released during NETosis, can be used to detect NETs. Human neutrophils were stimulated with fibrinogen using phorbol 12-myristate 13-acetate (PMA). The myeloperoxidase (MPO)-DNA complex and C-Fbg concentrations in the culture supernatants were quantified using an enzyme-linked immunosorbent assay. The protein levels of peptidylarginine deiminase 2 and 4 in culture supernatants and mRNA levels in PMA-stimulated neutrophils were also assessed. The levels of the MPO-DNA complex in the supernatants of PMA-stimulated neutrophils increased, indicating NETosis. C-Fbg level also increased, which was suppressed by both NETosis and PAD inhibitors. PAD2 was detected in the culture supernatant; however, PAD4, but not PAD2, mRNA levels increased in PMA-stimulated neutrophils. This study quantitatively demonstrates that fibrinogen is citrullinated by PAD derived from PMA-stimulated neutrophils upon NETosis. Although further studies are needed for clinical application, quantification of C-Fbg in blood may help detect the presence of NETs.

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Data Availability

Data will be made available by the corresponding author upon reasonable request.

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Acknowledgements

All figures used in this study were originally prepared by the authors and have not been published elsewhere.

Funding

This work was supported by Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant No. JP20K07822).

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Authors and Affiliations

  1. Department of Health and Medical Sciences, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan

    Tsubasa Sue, Tomoki Ichikawa, Shu Hattori, Hikaru Otani, Satoshi Fujimura & Yumiko Higuchi

  2. Department of Laboratory Medicine, Shinshu University Hospital, Matsumoto, Japan

    Tomoki Ichikawa & Satoshi Fujimura

  3. Department of General Pediatrics, Nagano Children’s Hospital, Azumino, Japan

    Tsukasa Higuchi

  4. Life Science Research Center, Nagano Children’s Hospital, Azumino, Japan

    Tsukasa Higuchi

  5. Department of Biomedical Laboratory Sciences, Shinshu University School of Medicine, Matsumoto, Japan

    Nobuo Okumura & Yumiko Higuchi

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  1. Tsubasa Sue
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Contributions

Conceptualization: TS, TH, NO, and YH. Writing—original draft: TS, TH, NO, and YH. Investigation: TS, TI, SH, HO, SF, and YH. Data curation—formal analysis: TS, TI, SH, HO, SF, and YH. All the authors have read and approved the final version of this manuscript.

Corresponding author

Correspondence to Yumiko Higuchi.

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Research involving human participants

This study was conducted in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki) and approved by the Ethical Review Board of Shinshu University School of Medicine (approval number 5342).

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Informed consent was obtained from all individual participants included in the study.

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Sue, T., Ichikawa, T., Hattori, S. et al. Quantitative evaluation of citrullinated fibrinogen for detection of neutrophil extracellular traps. Immunol Res (2023). https://doi.org/10.1007/s12026-023-09446-5

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