Neutrophilic granule protein promotes lipopolysaccharide-induced iNOS/NO expression via JAK2/STAT1 signaling pathway and augments bacteria clearance of macrophages

Neutrophilic granule protein (NGP) was identified as a granular protein in neutrophils, but its function in macrophages has not been fully understood. In our prior investigation, NGP was observed to be significantly upregulated in macrophages following stimulation with lipopolysaccharide (LPS) or Escherichia coli (E.coli). Furthermore, NGP demonstrated an anti-inflammatory effect by inhibiting the NF-κB signaling pathway through its extracellular distribution. Nitric oxide (NO), as a crucial factor in the immune regulation of macrophages mainly controlled by inducible nitric oxide synthase (iNOS), plays an indispensable role in the eradication of bacteria. However, the regulatory effects of NGP on NO remains unknown. In this study, we observed an upregulation and central role of Nos2, the gene encoding iNOS, in macrophages with high NGP expression induced by LPS. Overexpression of NGP significantly enhanced NO production in LPS-induced macrophages, while NGP knockout attenuated NO production. Furthermore, exogenous complementation experiments confirmed that NGP primarily exerted its up-regulatory effect on iNOS/NO through intracellular distribution. Additionally, RNA sequencing (RNA-seq) and immunoprecipitation-mass spectrometry (IP-MS) analyses revealed that NGP predominantly regulated NO levels via the JAK2/STAT1 pathway. Finally, our in vitro and in vivo experiments provided evidence supporting the involvement of NGP in bacterial clearance through modulation of NO. The evidence suggests that NGP plays a crucial role in regulating NO production and promoting bacterial clearance in macrophages, making it a promising target for the prevention and treatment of infectious diseases.