Inhibitors of nitric oxide synthase can reduce extracellular traps from neutrophils in asthmatic children in vitro.

Abstract

This study aimed to explore the link between neutrophil extracellular traps (NETs) and childhood asthma, to investigate the ability of nitric oxide (NO) to induce NETs in asthmatic children and find inhibitors to reduce NETs in the NO synthesis pathway. A total of 49 children with mild persistent asthma were included in the study and 20 healthy children's blood samples were collected as healthy controls. Children with asthma were divided into symptomatic and asymptomatic groups according to the presence or absence of symptoms on the day of blood collection. Neutrophils in peripheral blood were isolated and plasma was preserved. NO donor (sodium nitroferricyanide(III) dehydrate [SNP]) could provide NO and proved by a fluorescent probe. A PicoGreen Kit was used to detect the NETs quantificationally. Fluorescence microscopy prepared to observe the main structures of NETs. We measured NETs components (extracellular free double-stranded DNA [dsDNA]) in healthy, symptomatic and asymptomatic groups' plasma samples, and we compared the ability of SNP with phosphate-buffered saline, lipopolysaccharides (LPS), and phorbol 12-myristate 13-acetate (PMA) to induce NETs. NO synthase (NOS) inhibitors were added to see the impact on NETs formation. Plasma was obtained from all blood samples of 69 children. The neutrophils of 40 asthmatic and 20 healthy children were successfully obtained, the recovery rate was over 95%, and the cell activity was over 80%. There was higher extracellular free dsDNA in the plasma of symptomatic group (n = 27) than asymptomatic group (n = 22) and healthy group (n = 20; P < .05). Studies on neutrophils from 40 children with asthma found that NO can be produced by adding SNP, PMA, and LPS. SNP could induce NETs with dose- and time-dependent. PMA (160 nM) had the strongest ability to induce NETs, LPS (200 ng/mL) followed, SNP (200 µM) was the weakest (P < .05), and the amount of NETs in the asthma group was significantly higher than that in the healthy group (P < .05). NOS inhibitors had the same blocking capacity for PMA- and LPS-induced NETs (P > .05), while NG-nitro- l-arginine methyl ester (500 µM) had the strongest inhibitory effect on SNP induction with time-dependent (P < .05). Inducible NOS was found in the NETs structure. Children with asthma had higher levels of NETs in peripheral blood, especially when they had asthma symptoms. We verified the ability of NO to induce NETs, and found neutrophils from asthmatic children can produce more NETs in vitro. NOS inhibitors blocked this process may provide new therapeutic targets for childhood asthma.