H2 Inhibits the Formation of Neutrophil Extracellular Traps

Kohsuke Shirakawa, Eiji Kobayashi, Genki Ichihara, Hiroki Kitakata, Yoshinori Katsumata, Kazuhisa Sugai, Yoji Hakamata, Motoaki Sano

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)


Neutrophil extracellular traps (NETs) contribute to inflammatory pathogenesis in numerous conditions, including infectious and cardiovascular diseases, and have attracted attention as potential therapeutic targets. H2 acts as an antioxidant and has been clinically and experimentally proven to ameliorate inflammation. This study was performed to investigate whether H2 could inhibit NET formation and excessive neutrophil activation. Neutrophils isolated from the blood of healthy volunteers were stimulated with phorbol-12-myristate-13-acetate (PMA) or the calcium ionophore A23187 in H2-exposed or control media. Compared with control neutrophils, PMA- or A23187-stimulated human neutrophils exposed to H2 exhibited reduced neutrophil aggregation, citrullination of histones, membrane disruption by chromatin complexes, and release of NET components. CXCR4high neutrophils are highly prone to NETs, and H2 suppressed Ser-139 phosphorylation in H2AX, a marker of DNA damage, thereby suppressing the induction of CXCR4 expression. H2 suppressed both myeloperoxidase chlorination activity and production of reactive oxygen species to the same degree as N-acetylcysteine and ascorbic acid, while showing a more potent ability to inhibit NET formation than these antioxidants do in PMA-stimulated neutrophils. Although A23187 formed NETs in a reactive oxygen species–independent manner, H2 inhibited A23187-induced NET formation, probably via direct inhibition of peptidyl arginine deiminase 4-mediated histone citrullination. Inhalation of H2 inhibited the formation and release of NET components in the blood and bronchoalveolar lavage fluid in animal models of lipopolysaccharide-induced sepsis (mice and aged mini pigs). Thus, H2 therapy can be a novel therapeutic strategy for NETs associated with excessive neutrophil activation.

Original languageEnglish
Pages (from-to)146-161
Number of pages16
JournalJACC: Basic to Translational Science
Issue number2
Publication statusPublished - 2022 Feb


  • H
  • neutrophil extracellular traps
  • phorbol-12-myristate-13-acetate

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine


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