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.