H2 Inhibits the Formation of Neutrophil Extracellular Traps

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

doi:10.1016/j.jacbts.2021.11.005

H₂ Inhibits the Formation of Neutrophil Extracellular Traps

JACC Basic Transl Sci. 2022 Jan 12;7(2):146-161. doi: 10.1016/j.jacbts.2021.11.005. eCollection 2022 Feb.

Authors

Kohsuke Shirakawa^{1

2

3}, Eiji Kobayashi^{2

3

4}, Genki Ichihara³, Hiroki Kitakata³, Yoshinori Katsumata^{2

3}, Kazuhisa Sugai⁵, Yoji Hakamata⁵, Motoaki Sano^{2

3}

Affiliations

¹ Department of Cardiovascular Medicine, Graduate School of Medicine, Juntendo University, Tokyo, Japan.
² Center for Molecular Hydrogen Medicine, Keio University, Tokyo, Japan.
³ Department of Cardiology, School of Medicine, Keio University, Tokyo, Japan.
⁴ Department of Organ Fabrication, School of Medicine, Keio University, Tokyo, Japan.
⁵ Department of Basic Sciences, Faculty of Veterinary Sciences, School of Veterinary Nursing and Technology, Nippon Veterinary and Life Science University, Tokyo, Japan.

Abstract

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. CXCR4^high 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.

Keywords: BAL, bronchoalveolar lavage; CVD, cardiovascular disease; CitH3, citrullinated histone H3; H2; HOCl, hypochlorous acid; LPS, lipopolysaccharide; MI, myocardial infarction; MPO, myeloperoxidase; NAC, N-acetyl-L-cysteine; NET, neutrophil extracellular trap; PA, pulmonary artery; PADI4, peptidyl arginine deiminase 4; PMA, phorbol-12-myristate-13-acetate; ROS, reactive oxygen species; dsDNA, double-stranded DNA; neutrophil extracellular traps; phorbol-12-myristate-13-acetate.