L-Arginine and tetrahydrobiopterin supported nitric oxide production is crucial for the microbicidal activity of neutrophils

Abstract

Recent report from this lab has shown role of Rac2 in the translocation of inducible nitric oxide synthase (iNOS) to the phagosomal compartment of polymorphonuclear leukocytes (PMNs) following phagocytosis of beads. This study was undertaken to further assess the status and role of tetrahydrobiopterin (BH₄), a redox-sensitive cofactor, L-arginine, and the substrate of nitric oxide synthase (NOS) in sustained nitric oxide (˙NO) production in killing of phagocytosed microbes (Escherichia coli) by human PMNs. Time-dependent study revealed consistent NO and reactive oxygen species (ROS) production in the PMNs following phagocytosis of beads. In addition, levels of L-arginine and BH₄ were maintained or increased simultaneously to support the enzymatic activity of NOS in the bead activated PMNs. Moreover, translocation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) subunits along with iNOS was reconfirmed in the isolated phagosomes. We demonstrate that increase in the level of NO was supported by L-arginine and BH₄ to kill E. coli, by using PMNs from NOS2^−/− mice, human PMNs treated with biopterin inhibitor, N-acetyl serotonin (NAS), or by suspending human PMNs in L-arginine deficient medium. Altogether, this study demonstrates that following phagocytosis, sustained. NO production in the PMNs was well-maintained by redox sensitive cofactor, BH₄ and substrate, and L-arginine to enable microbial killing. Further results suggest NO production in the human PMNs, along with ROS and myeloperoxidase (MPO) is important to execute antimicrobial activity.

Keywords:

• iNOS
• l-arginine
• microbial killing
• NADPH oxidase
• neutrophils
• nitric oxide
• phagosomes
• tetrahydrobiopterin

Acknowledgements

Award of research fellowships to SN and SS from University of Grant commission and DA from Council of Scientific and Industrial Research, India is acknowledged. Mr. A L. Vishwakarma and Mrs. M Chaturvedi for the excellent technical assistance during the flow cytometry experiments and Ms. K Singh, Mr. Dhananjay and Mrs. R Ray Sarkar for confocal imaging. S.N. performed most of the experiments, and was involved in writing the article. S.S and D.A performed HPLC and phagosomes experiments, respectively. T.C provided the buffy coat samples. K.J provided the knock out animals. S.K involved in writing the manuscript and gave critical inputs during the experiments. M.D the corresponding author guided designed and conceptualised the work and was involved in writing and finalising the article.

Disclosure statement

There are no conflicting financial interests.

Additional information

Funding

This study was supported by financial assistance provided to Dr. Madhu Dikshit from the JC Bose National fellowship from DST-SERB (SB/S2/JCB-017/2015).Award of research fellowships to SN and SS from University of Grant commission and DA from Council of Scientific and Industrial Research, India is acknowledged. CDRI Communication No.9800.