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The role of Gch1 and Nos2 in the macrophage response to BCG infection

ABSTRACT: Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of mycobacteria, presumed to be via nitric oxide (NO) mediated killing. However, NOS enzymes can also signal through NO-independent pathways, and production of NO by NOS requires the cofactor tetrahydrobiopterin (BH4). We compared Nos2-/- mice to mice with macrophage BH4 deficiency (Gch1fl/flTie2cre), due to a leukocyte-specific deletion of Gch1, to uncover the specific contribution of NO-independent NOS functions to anti-mycobacterial immunity. We used microarrays to detail the global programme of gene expression in uninfected and BCG infected macrophages that were either deficient in iNOS (Nos2-/- vs C57bl6/J) or BH4/Gch1 (GCHfl/flTie2cre vs GCHfl/fl)

ORGANISM(S): Mus musculus

PROVIDER: GSE107543 | GEO | 2018/11/30

REPOSITORIES: GEO

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Project description:The nitric oxide synthase (NOS) co-factor, tetrahydrobiopterin (BH4), is a redox-active molecule that regulates nitric oxide (NO) and reactive oxygen species (ROS) production by NOS, and is an example of redox-dependent signalling in the endothelium that underlies both the maintenance of vascular homeostasis and the development of cardiovascular disease (CVD). Loss of endothelial BH4 is observed in CVD states and results in decreased NO production and increased ROS generation by endothelial NO synthase (uncoupling). Genetic mouse models of augmented endothelial BH4 synthesis have shown proof of concept that endothelial BH4 can alter CVD pathogenesis. However, clinical trials of BH4 therapy in vascular disease have been limited by systemic oxidation and limited endothelial uptake of BH4, highlighting the need to explore the wider roles of BH4 in order to find novel therapeutic targets. In this study we aim to elucidate the effects of BH4 depletion on mitochondrial function and bioenergetics using targeted knockdown of the BH4 synthetic enzyme GTPCH in vitro. Knockdown of GTPCH (and, therefore, intracellular BH4 levels) by >90% lead to a striking induction of ROS generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH4 depleted GCH fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH4 in mitochondrial redox signalling. Furthermore, this BH4-dependent, mitochondrial-derived ROS oxidized mitochondrial BH4 and is seen alongside distinct changes in the thioredoxin and glutathione antioxidant pathways, revealed by mass spectrometry using an unbiased proteomic approach. These changes are accompanied by a modest increase in mitochondrial size, attenuated respiratory function, and marked changes in the cellular metabolic profile; including succinate accumulation. Taken together, these data reveal a novel NOS-independent role for BH4 in the regulation of mitochondrial redox signalling and metabolism.

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The role of Gch1 and Nos2 in the macrophage response to BCG infection

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