High oxidative stress adversely affects NFκB mediated induction of inducible nitric oxide synthase in human neutrophils: Implications in chronic myeloid leukemia

• Diamide or PMA induced enhanced oxidative stress attenuate basal as well as LPS/cytokines induced iNOS/NO generation.
• High ROS in PMNs of CML subjects linked with reduced iNOS/NO, and were unresponsive to LPS/cytokines mediated induction.
• S-Glutathionylation of NFκB under excessive ROS limits its binding to iNOS promoter.
• DTT reverses NFκB S-glutathionylation, improves DNA binding activity and enhanced iNOS expression/NO generation.
• Enhanced NOX2, mitochondrial ROS and reduced GSH/Grxs regulate NFκB S-glutathionylation in CML.

Increasing evidence support bimodal action of nitric oxide (NO) both as a promoter and as an impeder of oxygen free radicals in neutrophils (PMNs), however impact of high oxidative stress on NO generation is less explored. In the present study, we comprehensively investigated the effect of high oxidative stress on inducible nitric oxide synthase (iNOS) expression and NO generation in human PMNs. Our findings suggest that PMA or diamide induced oxidative stress in PMNs from healthy volunteers, and high endogenous ROS in PMNs of chronic myeloid leukemia (CML) patients attenuate basal as well as LPS/cytokines induced NO generation and iNOS expression in human PMNs. Mechanistically, we found that under high oxidative stress condition, S-glutathionylation of NFκB (p50 and p65 subunits) severely limits iNOS expression due to its reduced binding to iNOS promoter, which was reversed in presence of DTT. Furthermore, by using pharmacological inhibitors, scavengers and molecular approaches, we identified that enhanced ROS generation via NOX2 and mitochondria, reduced Grx1/2 expression and GSH level associated with NFκB S-glutathionylation in PMNs from CML patients. Altogether data obtained suggest that oxidative status act as an important regulator of NO generation/iNOS expression, and under enhanced oxidative stress condition, NOX2-mtROS-NFκB S-glutathionylation is a feed forward loop, which attenuate NO generation and iNOS expression in human PMNs.