Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

Eileen Mcneill,Mark J Crabtree,Natasha Sahgal,Jyoti Patel,Surawee Chuaiphichai,Asif J Iqbal,Ashley B Hale,David R Greaves,Keith M Channon

doi:10.1016/j.freeradbiomed.2014.10.575

Abstract

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1fl/flTie2cre). Macrophages from Gch1fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1fl/flTie2cre macrophages. These findings identify BH4-dependent iNOS regulation and NO generation as specific requirements for NRF2-dependent responses in macrophage inflammatory activation.

Highlights

Macrophages are pivotal cells of the innate immune system, and are a source of both pro- and anti-inflammatory cytokines, capable of phagocytosing apoptic cells and have a potent antimicrobial arsenal [1]
Deletion of Gch1 resulted in a near complete loss of cellular BH4 in bone marrowderived macrophages from Gch1fl/flTie2cre mice compared to wildtype controls (Po0.01, Fig. 1D)
We have created a novel mouse model of Gch1 deletion in order to test the specific requirements for BH4 in macrophage redox biology

Summary

Introduction

Macrophages are pivotal cells of the innate immune system, and are a source of both pro- and anti-inflammatory cytokines, capable of phagocytosing apoptic cells and have a potent antimicrobial arsenal [1]. Archetypal proinflammatory macrophages are activated by Abbreviations: As3MT, arsenic(III)-methyltransferase; BH4, tetrahydrobiopterin; [Fe(DETC)2], colloid iron (II) diethyldithiocarbamate; GTPCH, GTP cyclohydrolase 1; 2-HE, 2-hydroxyethidium; iNOS, inducible nitric oxide synthase; ROS, reactive oxygen species. Interferon-γ and microbial products such as LPS, leading to production of proinflammatory cytokines and high levels of nitric oxide (NO), due to the induction of nitric oxide synthase. Generation of NO by inducible nitric oxide synthase (iNOS, encoded by NOS2) is implicated in critical functions such as microbial killing and immune regulation [2]. Inflammatory cells including macrophages generate high levels of reactive oxygen specifies (ROS), that play key roles in host defense and immunity.

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Conclusion