isolation-vitro-culture-bone-marrow-derived-macrophages-for-study-no

Isolation and <em>In vitro</em> Culture of Bone Marrow-Derived Macrophages for the Study of NO-Redox Biology

Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and ...

University of Oxford

The redox co-factor tetrahydrobiopterin (BH) regulates nitric oxide (NO) and reactive oxygen species (ROS) production by endothelial NOS (eNOS) and is an important redox-dependent signalling molecule in the endothelium. Loss of endothelial BH is observed in cardiovascular disease (CVD) states and results in decreased NO and increased superoxide (O) generation via eNOS uncoupling. Genetic mouse models of augmented endothelial BH synthesis have shown proof of concept that endothelial BH can alter CVD pathogenesis. However, clinical trials of BH therapy in vascular disease have been limited by systemic oxidation, highlighting the need to explore the wider roles of BH to find novel therapeutic targets. In this study, we aimed to elucidate the effects of BH deficiency on mitochondrial function and bioenergetics using targeted knockdown of the BH synthetic enzyme, GTP Cyclohydrolase I (GTPCH). Knockdown of GTPCH by >90% led to marked loss of cellular BH and a striking induction of O generation in the mitochondria of murine endothelial cells. This effect was likewise observed in BH-depleted fibroblasts devoid of NOS, indicating a novel NOS-independent role for BH in mitochondrial redox signalling. Moreover, this BH-dependent, mitochondria-derived ROS further oxidised mitochondrial BH, concomitant with changes in the thioredoxin and glutathione antioxidant pathways. These changes were accompanied by a modest increase in mitochondrial size, mildly attenuated basal respiratory function, and marked changes in the mitochondrial proteome and cellular metabolome, including the accumulation of the TCA intermediate succinate. Taken together, these data reveal a novel NOS-independent role for BH in the regulation of mitochondrial redox signalling and bioenergetic metabolism.

A novel role for endothelial tetrahydrobiopterin in mitochondrial redox balance.

Mitochondrial creatine kinase (MtCK) couples ATP production via oxidative phosphorylation to phosphocreatine in the cytosol, which acts as a mobile energy store available for regeneration of ATP at times of high demand. We hypothesized that elevating MtCK would be beneficial in ischaemia-reperfusion (I/R) injury.

Over-expression of mitochondrial creatine kinase in the murine heart improves functional recovery and protects against injury following ischaemia-reperfusion.

Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics "biotin-switch" approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis.

Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation.

Herein, we maximize the labeling efficiency of cardiac progenitor cells (CPCs) using perfluorocarbon nanoparticles (PFCE-NP) and F MRI detectability, determine the temporal dynamics of single-cell label uptake, quantify the temporal viability/fluorescence persistence of labeled CPCs in vitro, and implement in vivo, murine cardiac CPC MRI/tracking that could be translatable to humans. FuGENE-mediated CPC PFCE-NP uptake is confirmed with flow cytometry/confocal microscopy. Epifluorescence imaging assessed temporal viability/fluorescence (up to 7 days [D]). Nonlocalized murine F MRS and cardiac MRI studied label localization in terminal/longitudinal tracking studies at 9.4 T (D1-D8). A 4-8 fold F concentration increase is evidenced in CPCs for FuGENE vs. directly labeled cells. Cardiac F signals post-CPC injections diminished in vivo to ~31% of their values on D1 by D7/D8. Histology confirmed CPC retention, dispersion, and macrophage-induced infiltration. Intra-cardiac injections of PFCE-NP-labeled CPCs with FuGENE can be visualized/tracked in vivo for the first time with F MRI.

Improved cellular uptake of perfluorocarbon nanoparticles for in vivo murine cardiac F MRS/MRI and temporal tracking of progenitor cells.

Classical activation of macrophages (M(LPS+IFNγ)) elicits the expression of inducible nitric oxide synthase (iNOS), generating large amounts of NO and inhibiting mitochondrial respiration. Upregulation of glycolysis and a disrupted tricarboxylic acid (TCA) cycle underpin this switch to a pro-inflammatory phenotype. We show that the NOS cofactor tetrahydrobiopterin (BH) modulates IL-1β production and key aspects of metabolic remodeling in activated murine macrophages via NO production. Using two complementary genetic models, we reveal that NO modulates levels of the essential TCA cycle metabolites citrate and succinate, as well as the inflammatory mediator itaconate. Furthermore, NO regulates macrophage respiratory function via changes in the abundance of critical N-module subunits in Complex I. However, NO-deficient cells can still upregulate glycolysis despite changes in the abundance of glycolytic intermediates and proteins involved in glucose metabolism. Our findings reveal a fundamental role for iNOS-derived NO in regulating metabolic remodeling and cytokine production in the pro-inflammatory macrophage.

Nitric Oxide Modulates Metabolic Remodeling in Inflammatory Macrophages through TCA Cycle Regulation and Itaconate Accumulation.

Macrophages are mononuclear phagocytes derived from haematopoietic progenitors that are widely distributed throughout the body. These cells participate in both innate and adaptive immune responses and lie central to the processes of inflammation, development, and homeostasis. Macrophage physiology varies depending on the environment in which they reside and they exhibit rapid functional adaption in response to external stimuli. To study macrophages in vitro, cells are typically cultured ex vivo from the peritoneum or alveoli, or differentiated from myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). BMDMs represent an efficient and cost-effective means of studying macrophage biology. However, the inherent sensitivity of macrophages to biochemical stimuli (such as cytokines, metabolic intermediates, and RNS/ROS) makes it imperative to control experimental conditions rigorously. Therefore, the aim of this study was to establish an optimised and standardised method for the isolation and culture of BMDMs. We used classically activated macrophages isolated from WT and nitric oxide (NO)-deficient mice to develop a standardised culture method, whereby the constituents of the culture media are defined. We then methodically compared our standardised protocol to the most commonly used method of BMDM culture to establish an optimal protocol for the study of nitric oxide (NO)-redox biology and immunometabolism in vitro.

Article	Year
A novel role for endothelial tetrahydrobiopterin in mitochondrial redox balance. Free radical biology & medicine\| PubMed ID: 28104455	2017
Over-expression of mitochondrial creatine kinase in the murine heart improves functional recovery and protects against injury following ischaemia-reperfusion. Cardiovascular research\| PubMed ID: 29509881	2018
Tetrahydrobiopterin modulates ubiquitin conjugation to UBC13/UBE2N and proteasome activity by S-nitrosation. Scientific reports\| PubMed ID: 30254268	2018
Improved cellular uptake of perfluorocarbon nanoparticles for in vivo murine cardiac F MRS/MRI and temporal tracking of progenitor cells. Nanomedicine : nanotechnology, biology, and medicine\| PubMed ID: 30448526	2019
Nitric Oxide Modulates Metabolic Remodeling in Inflammatory Macrophages through TCA Cycle Regulation and Itaconate Accumulation. Cell reports\| PubMed ID: 31269442	2019
Isolation and culture of murine bone marrow-derived macrophages for nitric oxide and redox biology. Nitric oxide : biology and chemistry\| PubMed ID: 32339668	2020

Andrew Shaw

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