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<p>Presepsin (P-SEP), a soluble subtype of CD14, is widely recognized as a diagnostic biomarker for sepsis. However, the molecular mechanisms underlying its production remain poorly understood. In this study, we investigated whether M1 macrophages (M1 MΦs) contribute to P-SEP generation by phagocytosing neutrophil extracellular traps (NETs) and degrading NET-derived CD14 through intracellular proteinase 3 (PR3). M1 MΦs were differentiated from peripheral blood mononuclear cells obtained from healthy donors, and NETs were induced in neutrophils stimulated with <i>Escherichia coli</i> or phorbol 12-myristate 13-acetate PMA. Co-cultures of M1 MΦs and NETs were analyzed by immunofluorescence staining, flow cytometry, ELISA, and Western blotting. The effects of PR3 inhibition (phenylmethylsulfonyl fluoride or elafin) and phagocytosis inhibition (cytochalasin D or wortmannin) on P-SEP production were also evaluated. Recombinant CD14 (rCD14) was used in an in vitro cleavage assay to confirm PR3-dependent CD14 fragmentation. M1 MΦs actively phagocytosed citrullinated histone H3–positive NETs and produced intracellular P-SEP. P-SEP levels strongly correlated with the percentage of NETs (<i>r</i> = 0.907). Inhibition of PR3 activity or phagocytosis significantly reduced P-SEP production. Furthermore, incubation of rCD14 with PR3 produced P-SEP, and this effect was blocked by PR3 inhibitors. These findings demonstrate that PR3 contributes to P-SEP production through CD14 cleavage following NET phagocytosis by M1 MΦs. This study provides new mechanistic insight into the P-SEP biogenesis and advances the understanding of its role as a biomarker in sepsis.</p>

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Proteinase 3 is involved in presepsin production through neutrophil extracellular trap phagocytosis by macrophages

  • 期刊论文

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  • 发表日期：

    2025年12月19日

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  • Akishige Ikegame,
  • Akihiro Kondo,
  • Tatsuya Morinishi,
  • Yusuke Yamaguchi,
  • Yui Okutani,
  • Chinatsu Maegawa,
  • Satoshi Tada

摘要

Presepsin (P-SEP), a soluble subtype of CD14, is widely recognized as a diagnostic biomarker for sepsis. However, the molecular mechanisms underlying its production remain poorly understood. In this study, we investigated whether M1 macrophages (M1 MΦs) contribute to P-SEP generation by phagocytosing neutrophil extracellular traps (NETs) and degrading NET-derived CD14 through intracellular proteinase 3 (PR3). M1 MΦs were differentiated from peripheral blood mononuclear cells obtained from healthy donors, and NETs were induced in neutrophils stimulated with Escherichia coli or phorbol 12-myristate 13-acetate PMA. Co-cultures of M1 MΦs and NETs were analyzed by immunofluorescence staining, flow cytometry, ELISA, and Western blotting. The effects of PR3 inhibition (phenylmethylsulfonyl fluoride or elafin) and phagocytosis inhibition (cytochalasin D or wortmannin) on P-SEP production were also evaluated. Recombinant CD14 (rCD14) was used in an in vitro cleavage assay to confirm PR3-dependent CD14 fragmentation. M1 MΦs actively phagocytosed citrullinated histone H3–positive NETs and produced intracellular P-SEP. P-SEP levels strongly correlated with the percentage of NETs (r = 0.907). Inhibition of PR3 activity or phagocytosis significantly reduced P-SEP production. Furthermore, incubation of rCD14 with PR3 produced P-SEP, and this effect was blocked by PR3 inhibitors. These findings demonstrate that PR3 contributes to P-SEP production through CD14 cleavage following NET phagocytosis by M1 MΦs. This study provides new mechanistic insight into the P-SEP biogenesis and advances the understanding of its role as a biomarker in sepsis.