NAMPT-Mediated Salvage Synthesis of NAD+ Controls Morphofunctional Changes of Macrophages (original) (raw)
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Archives of Biochemistry and Biophysics, 1999
The parent pyridine nucleotide NAD is the end product of oxidative tryptophan catabolism via the kynurenine pathway. Indoleamine 2,3-dioxygenase, the rate-limiting enzyme for this pathway, is induced by the proinflammatory cytokine interferon-␥. The aim of this study was to investigate the effect of interferon-␥ treatment on intracellular NAD concentration in the murine macrophage cell line, RAW 264.7. A significant increase in intracellular NAD concentration was observed following 24 h exposure to interferon-␥. This cytokine-mediated increase in NAD concentration was markedly enhanced by the inhibition of poly(ADP-ribose) polymerase or nitric oxide synthase or following treatment with the synthetic glucocorticoid dexamethasone. NAD production was dependent on both the presence of tryptophan in the culture medium and on functional indoleamine 2,3-dioxygenase activity. In agreement with previous studies a marked increase in nitric oxide production was observed in these cells following activation with interferon-␥. These results provide evidence for the first time that de novo synthesis of NAD from tryptophan is increased concomitantly with free radical production in RAW 264.7 macrophages stimulated with interferon-␥. This increase in NAD biosynthesis may provide an improved supply of substrate to the nuclear repair enzyme poly(ADP-ribose) polymerase assisting in DNA repair and hence cell viability.
Macrophage de novo NAD+ synthesis specifies immune function in aging and inflammation
Nature Immunology, 2018
Recent advances highlight a pivotal role for cellular metabolism in programming immune responses. Here, we demonstrate that cell-autonomous generation of nicotinamide adenine dinucleotide (NAD +) via the kynurenine pathway (KP) regulates macrophage immune function in aging and inflammation. Isotope tracer studies revealed that macrophage NAD + derives substantially from KP metabolism of tryptophan. Genetic or pharmacological blockade of de novo Reprints and permissions information is available at www.nature.com/reprints.
Frontiers in Immunology, 2022
Nicotinamide adenine dinucleotide (NAD) metabolism plays an important role in the regulation of immune function. However, a complete picture of how NAD, its metabolites, precursors, and metabolizing enzymes work together in regulating immune function and inflammatory diseases is still not fully understood. Surprisingly, few studies have compared the effect of different forms of vitamin B3 on cellular functions. Therefore, we investigated the role of NAD boosting in the regulation of macrophage activation and function using different NAD precursors supplementation. We compared nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM) supplementation, with the recently described potent NAD precursor NRH. Our results show that only NRH supplementation strongly increased NAD + levels in both bone marrow-derived and THP-1 macrophages. Importantly, NRH supplementation activated a pro-inflammatory phenotype in resting macrophages, inducing gene expression of several cytokines, chemokines, and enzymes. NRH also potentiated the effect of lipopolysaccharide (LPS) on macrophage activation and cytokine gene expression, suggesting that potent NAD + precursors can promote inflammation in macrophages. The effect of NRH in NAD + boosting and gene expression was blocked by inhibitors of adenosine kinase, equilibrative nucleoside transporters (ENT), and IkB kinase (IKK). Interestingly, the IKK inhibitor, BMS-345541, blocked the mRNA expression of several enzymes and transporters involved in the NAD boosting effect of NRH, indicating that IKK is also a regulator of NAD metabolism. In conclusion, NAD precursors such as NRH may be important tools to understand the role of NAD and NADH metabolism in the inflammatory process of other immune cells, and to reprogram immune cells to a pro-inflammatory phenotype, such as the M2 to M1 switch in macrophage reprogramming, in the cancer microenvironment.
American Journal of Respiratory Cell and Molecular Biology, 2007
M-CSF induces PI 3-kinase activation, resulting in reactive oxygen species (ROS) production. Previously, we reported that ROS mediate macrophage colony-stimulating factor (M-CSF)-induced extracellular regulated kinase (Erk) activation and monocyte survival. In this work, we hypothesized that M-CSF-stimulated ROS products modulated Akt1 and p38 activation. Furthermore, we sought to clarify the source of these ROS and the role of ROS and Akt in monocyte/ macrophage survival. Macrophages from p47 phox-/mice, lacking a key component of the NADPH oxidase complex required for ROS generation, had reduced cell survival and Akt1 and p38 mitogenactivated protein kinase (MAPK) phosphorylation compared with wild-type macrophages in response to M-CSF stimulation, but had no difference in M-CSF-stimulated Erk. To understand how ROS affected monocyte survival and signaling, we observed that NAC and DPI decreased cell survival and Akt1 and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from mice expressing constitutively activated Akt1 (Myr-Akt1) or transfecting Myr-Akt1 constructs into human peripheral monocytes, we concluded that Akt is a positive regulator of monocyte survival. Moreover, the p38 MAPK inhibitor, SB203580, inhibited p38 activity and M-CSF-induced monocyte survival. These findings demonstrate that ROS generated from the NADPH oxidase complex contribute to monocyte/macrophage survival induced by M-CSF via regulation of Akt and p38 MAPK.
Bioscience reports, 2016
Metabolism and immune responses have been shown to be closely linked and as our understanding increases, so do the intricacies of the level of linkage. NAD(+) has previously been shown to regulate TNF- α synthesis and TNF-α has been shown to regulate NAD(+) homeostasis providing a link between a pro-inflammatory response and redox status. Here we have used THP-1 differentiation into pro- (M1-like) and anti- (M2-like) inflammatory macrophage subset models to investigate this link further. Pro- and anti-inflammatory macrophages showed different resting NAD(+) levels and expression levels of NAD(+) homeostasis enzymes. Challenge with Bacterial lipopolysaccharide, a pro-inflammatory stimulus for macrophages, caused a large, biphasic and transient increase in NAD(+) levels in pro- but not anti-inflammatory macrophages that were correlated with TNF-α release and inhibition of certain NAD(+) synthesis pathways blocked TNF-α release. Lipopolysaccharide stimulation also caused changes in mRN...
Built for the kill. Studies on the neutrophil NADPH oxidase
2004
Inn this study, the translocation of the NADPH oxidase components p67 phox and Rac22 was studied during phagocytosis in living cells. For this purpose, GFP-tagged versionss of these proteins were expressed in the myeloid cell-line PLB-985. First, thee correct localization of p67GFP and GFP-Rac2 was shown during phagocytosis off serum-treated zymosan (STZ) by wild-type PLB-985 cells and PLB-985 X-CGD cells,, which lack expression of flavocytochrome b$ 5 &. Subsequently, these constructss were used for fluorescence recovery after photobleaching (FRAP) studiess to elucidate the turnover of these proteins on the phagosomal membrane. Thee turnover of p67GFP and GFP-Rac2 proved to be very high, indicating a continuouss exchange of flavocytochrome 6 5 5 8-bound p67GFP and GFP-Rac2 for cytosolic,, free p67GFP and GFP-Rac2. Furthermore, the importance of an intact actinn cytoskeleton for correct localization of these proteins was investigated by disruptingg the actin cytoskeleton with cytochalasin B. However, cytochalasin B treatmentt of PLB-985 cells did not alter the localization of p67GFP and GFP-Rac2 oncee phagocytosis was initiated. In addition, the continuous exchange of flavocytochromeflavocytochrome è 55 8-bound p67GFP and GFP-Rac2 for cytosolic p67GFP GFP-Rac22 was still intact in cytochalasin B-treated cells, indicating that translocationn of these proteins does not depend on a rearrangement of the actin cytoskeleton. .
Proceedings of the National Academy of Sciences, 2010
The phagosomal lumen in macrophages is the site of numerous interacting chemistries that mediate microbial killing, macromolecular degradation, and antigen processing. Using a non-hypothesisbased screen to explore the interconnectivity of phagosomal functions, we found that NADPH oxidase (NOX2) negatively regulates levels of proteolysis within the maturing phagosome of macrophages. Unlike the NOX2 mechanism of proteolytic control reported in dendritic cells, this phenomenon in macrophages is independent of changes to lumenal pH and is also independent of hydrolase delivery to the phagosome. We found that NOX2 mediates the inhibition of phagosomal proteolysis in macrophages through reversible oxidative inactivation of local cysteine cathepsins. We also show that NOX2 activity significantly compromises the phagosome's ability to reduce disulfides. These findings indicate that NOX2 oxidatively inactivates cysteine cathepsins through sustained ablation of the reductive capacity of the phagosomal lumen. This constitutes a unique mechanism of spatiotemporal control of phagosomal chemistries through the modulation of the local redox environment. In addition, this work further implicates the microbicidal effector NOX2 as a global modulator of phagosomal physiologies, particularly of those pertinent to antigen processing. phagocytosis | cathepsin | disulfide reduction | antigen processing | lysosome
PLoS ONE, 2008
Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased proinflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFa levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders. Citation: Busso N, Karababa M, Nobile M, Rolaz A, Gool FV, et al. (2008) Pharmacological Inhibition of Nicotinamide Phosphoribosyltransferase/Visfatin Enzymatic Activity Identifies a New Inflammatory Pathway Linked to NAD. PLoS ONE 3(5): e2267.
The FASEB Journal, 2007
Neutrophils generate microbicidal oxidants through activation of a multicomponent enzyme called NADPH oxidase. During activation, the cytosolic NADPH oxidase components (p47 phox , p67 phox , p40 phox , and Rac2) translocate to the membranes, where they associate with flavocytochrome b 558 , which is composed of gp91 phox /NOX2 and p22 phox , to form the active system. During neutrophil stimulation, p47 phox , p67 phox , p40 phox , and p22 phox are phosphorylated; however, the phosphorylation of gp91 phox /NOX2 and its potential role have not been defined. In this study, we show that gp91 phox is phosphorylated in stimulated neutrophils. The gp91 phox phosphoprotein is absent in neutrophils from chronic granulomatous disease patients deficient in gp91 phox , which confirms that this phosphoprotein is gp91 phox. The protein kinase C inhibitor GF109203X inhibited phorbol 12-myristate 13-acetate-induced phosphorylation of gp91 phox , and protein kinase C (PKC) phosphorylated the recombinant gp91 phoxcytosolic carboxy-terminal flavoprotein domain. Twodimensional tryptic peptide mapping analysis showed that PKC phosphorylated the gp91 phox-cytosolic tail on the same peptides that were phosphorylated on gp91 phox in intact cells. In addition, PKC phosphorylation increased diaphorase activity of the gp91 phox flavoprotein cytosolic domain and its binding to Rac2, p67 phox , and p47 phox. These results demonstrate that gp91 phox is phosphorylated in human neutrophils by PKC to enhance its catalytic activity and assembly of the complex. Phosphorylation of gp91 phox /NOX2 is a novel mechanism of NADPH oxidase regulation.