NAD+ metabolism and its roles in cellular processes during ageing

Box 1 |. Generation of NAD+ via the NAM salvage pathway

The nicotinamide (NAM) salvage pathway generates nicotinamide adenine dinucleotide (NAD+) from the precursor NAM or the upstream NAD+ vitamin precursor nicotinamide riboside (NR) or nicotinamide mononucleotide (NMN) (see FIG. 1a), which can be found in a variety of daily foods such as milk, fruits, vegetables and meat178,199. As NAD+ is not cell permeable, it was thought that all the dietary precursors, including nicotinic acid, NAM, NR and tryptophan, are imported directly into the cells and made available for NAD+ biosynthesis, with the exception of NMN. In support of this, extracellular NMN must be converted to NR by the 5′-nucleotidase CD73 before being imported, thus creating a rate-limiting conversion back to NMN intracellularly by nicotinamide riboside kinase 1 (NRK1)245. A recent study revealed the presence of a specific NMN transporter, SLC12A8, that is highly expressed in the small intestine, suggesting that NMN represents a direct entry point into the NAD+ biosynthetic pathway246. Further studies are necessary to determine the physiological relevance of this transporter to disease, the kinetics and mechanisms of NMN uptake and those of other NAD+ precursors, and their selective expression and effects in each tissue and/or cell in mammals.

NAD+ recycling via the NAM salvage pathway is a fundamental step to restore NAD+ levels after irreversible degradation mediated by the different classes of NAD+-consuming enzymes, including glycohydrolases (CD38, CD157 and SARM1), protein deacylases (sirtuins) and poly(ADP-ribose) polymerases (PARPs). Although the activities and uses of NAD+ differ for each of these enzymes (see FIG. 2), all NAD+-consuming enzymes produce NAM as a by-product of NAD+ degradation; this is converted by the NAM salvage pathway enzyme nicotinamide phosphoribosyltransferase (NAMPT) to NMN. NMN can also be generated from NR by NRK1 and NRK2 (REFS5,245) and converted to NAD+ during the last step of the salvage pathway by the nicotinamide mononucleotide adenylyltransferases NMNAT1, NMNAT2 and NMNAT3 (REFS247,248). Three isoforms of NMNAT have different subcellular localization (NMNAT1, nucleus; NMNAT2, cytosolic face of the Golgi apparatus; NMNAT3, mitochondria) and were reported to regulate NAD+ levels in their respective cellular compartment, but also to influence other intracellular NAD+ stores4,249,250.

NAMPT is ubiquitously expressed and mostly upregulated in processes that depend on high NAD+ levels, such as immune cell activation251 and genotoxic stress252. NAMPT-mediated NAD+ biosynthesis influences cellular metabolism and responses to inflammatory, oxidative and genotoxic stress253,254 by modulating activities of sirtuins and PARP100,253,255. Moreover, NAMPT expression is regulated by the circadian clock machinery (CLOCK–BMAL1) in a feedback loop involving SIRT1, and this correlates with the circadian oscillation of NAD+ levels in vivo10,11. NAMPT exists in two forms in mammals: intracellular NAMPT (iNAMPT) and extracellular NAMPT (eNAMPT). To function as an NAD+ biosynthetic enzyme, NAMPT forms homodimers256. Many cell types produce eNAMPT130, and its secretion is actively regulated by SIRT1 or SIRT6-mediated deacetylation257 in adipose tissue and cancer cell lines258, respectively. More recently, it was shown that eNAMPT is carried in extracellular vesicles in the blood circulation of mice and humans (see FIG. 1a) and it is responsible for enhancing the intracellular NAD+ biosynthesis in primary hypothalamic neurons259. In addition to its role in regulating NAM salvage, eNAMPT was previously identified as a presumptive cytokine (also known as pre-B cell colony-enhancing factor (PBEF))260. The levels of eNAMPT monomer in the serum are increased in a number of immunological and metabolic disorders261,262. However, it is unclear whether the enzymatic activity of NAMPT is necessary for its cytokine-like function. Therefore, discriminating between monomer and dimer forms of eNAMPT could be a way to resolve the current controversy about enzymatic activity263 and function of eNAMPT. Collectively, these studies suggest that eNAMPT acts to maintain NAD+ in distant cells in an autocrine fashion and, remarkably, also acts as an endocrine signal to influence NAD+ levels and inflammation in distant tissues.

Box 2 |. Metabolic catabolism of NAD+ via NNMT and NADK

Nicotinamide N-methyltransferase (NNMT) is an enzyme that uses S-methyladenosine (SAM) as a methyl donor to methylate the ring nitrogen of nicotinamide (NAM), converting it to N1-methylnicotinamide (MNAM) (see FIG. 1a). MNAM can be further oxidized via aldehyde oxidase to produce N1-methyl-2-pyridone-5-carboxamide and N1-methyl-4-pyridone-3-carboxamide, which along with MNAM are secreted in the urine. NNMT conversion of NAM to MNAM effectively diverts NAM from being recycled back to nicotinamide adenine dinucleotide (NAD+) by the NAM salvage pathway and affects global NAD+ levels. Thus, there has been growing interest in the role of NNMT as a key regulator of NAD+ levels during disease states, such as obesity, cancer and ageing264. For example, NNMT expression increases in visceral white adipose tissue and liver during obesity, and appears to have mostly negative consequences265–268. This includes the depletion of the methyl donor SAM and NAD+ in white adipose tissue and the liver in response to a high-fat diet. As a result, increased NNMT activity leads to reduced methylation of the promoters of genes involved in fibrosis and aberrant gene expression of metabolic and inflammatory genes266. Thus, NNMT appears to be a critical metabolic enzyme linking NAD+ metabolism to control of gene expression via its ability to regulate levels of bioactive molecules, such as consumption of NAD+ and SAM, to produce MNAM.

The role of NNMT in regulating NAD+ suggests that NNMT is important in ageing. In support of this, the Caenorhabditis elegans homologue of NNMT, ANMT-1, regulates the production of MNAM. In worms, MNAM serves as a substrate for the aldehyde oxidase GAD-3, which produces hydrogen peroxide, which, in turn, acts as a hormesis signal to promote longevity. Worms lacking anmt-1 no longer benefit from sir-2.1-dependent lifespan extension191. These data suggest that consumption of NAD+ via NNMT is beneficial for lifespan extension at least in C. elegans. However, it is unclear what role NNMT activation plays in mammalian ageing, although NNMT expression and activity do appear to increase in rodents as they age. For example, our group recently saw NNMT gene expression increase in the hepatocytes of ageing mice45, and a similar increase was observed in hepatocytes of older mice fed a high-protein diet269. Additionally, treating old mice with an NNMT inhibitor activated senescent muscle stem cells and promoted muscle regeneration during ageing270. Thus, in contrast to C. elegans, in mammals early evidence suggests that NNMT may promote ageing-related diseases. Thus, targeting NNMT may provide a viable therapeutic avenue to treat ageing-related diseases.

Another metabolic fate of NAD+ is direct phosphorylation by NAD+ kinase (NADK) to produce NADP(H), which has a key role as a major source of reducing power that regulates intracellular redox balance and anabolic processes, such as lipogenesis. A recent study using in vitro isotopic tracing label incorporation into NADP(H) showed that NADK accounts for 10% of NAD+ consumption5. However, the total NADP+ pool is 20 times less than the NAD+ pool. Furthermore, in conditions where NAD+ levels decline, such as in ageing, NADP+ levels also decline. Thus, NADP+ levels appear to be directly linked to NAD+ levels and rise and fall in tandem, making it unlikely that NADK is a major NAD+ sink. However, NADK has recently been shown to be a direct target of AKT kinase, phosphorylating the residues Ser44, Ser46 and Ser48 and leading to enhanced activation of NADK271. Thus, given that AKT signalling is abnormally increased during ageing272, aberrant activation of NADK may account for some of the decline in NAD+ levels during ageing. Therefore, further studies will be needed to determine the role of NADK in regulating NAD+ and NADP+ pools in ageing and ageing-related diseases.

Consumption by sirtuins.

Since their discovery, the sirtuins have received much attention because they regulate key metabolic processes, stress responses and ageing biology7. The mammalian sirtuin family is composed of seven genes and proteins (SIRT1–SIRT7) with different subcellular localizations (nucleus for SIRT1 and SIRT6; nucleolus for SIRT7; mitochondria for SIRT3, SIRT4 and SIRT5; and cytosol for SIRT1, SIRT2 and SIRT5) (FIG. 1b), enzymatic activities and downstream targets. The sub-cellular localization of these NAD+-dependent enzymes emphasizes how local fluctuations of intracellular NAD+ pools, which are themselves regulated by sirtuins, may selectively impact organelle-specific sirtuin functions and cellular metabolism.

Sirtuins are continuously active in our cells. SIRT1 and SIRT2 seem to be responsible for about one third of the total NAD+ consumption under basal conditions5. Moreover, a rise of NAD+ levels is strongly correlated with sirtuin activation during fasting and caloric restriction8,9. It is also worth noting that sirtuin activity is coupled to the circadian clock, whereby SIRT1 and SIRT6 regulate the activity of the core clock transcription factors and downstream circadian transcriptome8. In addition, SIRT1 and a key enzyme of the NAD+ salvage pathway, nicotinamide phosphoribosyltransferase (NAMPT) (BOX 1), are key players in the circadian regulation of NAD+ levels, and NAMPT is regulated by the circadian clock, which provides a feedback loop, resulting in circadian oscillation of NAD+ levels10,11.

Early studies in yeast demonstrated roles of sirtuins in gene silencing, which in 2000 was attributed to their activity as NAD+-dependent deacetylases12. Sirtuin enzymatic activity was initially reported to encompass mainly removal of an acetyl group from lysine residues of target proteins in a two-step process: first NAD+ is cleaved to NAM and ADP-ribose, and second, the acetyl group on the target protein is transferred to ADP-ribose, allowing the formation of the intermediate peptidyl-ADP-ribose. Acetyl-ADP-ribose is subsequently released (FIG. 2a). Additionally, some members of the sirtuin family mediate non-acetyl lysine acylations (for example, succinylation, malonylation and fatty acid acylation)13–15, the cellular functions of which are so far poorly understood16. SIRT4 and SIRT6 also function as ADP-ribosyltransferases, indicating that sirtuins contribute to cell regulation also through mechanisms beyond modulation of protein acetylation, which require further investigation17,18.

 

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Fig. 2 |

Three main classes of NAD+-consuming enzymes.

a | Sirtuins remove acyl groups from lysine residues on target proteins using nicotinamide adenine dinucleotide (NAD+) as a co-substrate. NAD+ is cleaved, generating nicotinamide (NAM) and ADP-ribose, where ADP-ribose serves as an acyl group acceptor, generating acetyl-ADP-ribose (acetyl-ADPR). b | Poly(ADP-ribose) polymerases (PARP1–PARP3) use NAD+ as a co-substrate to mono(ADP-ribosyl)ate or poly(ADP-ribosyl)ate target proteins, generating NAM as a by-product. c | Reactions of NAD+ glycohydrolases and cyclic ADP-ribose (cADPR) synthases (CD38, CD157 and SARM1). The main catalytic activity of this group of proteins is the hydrolysis of NAD+ to NAM and ADP-ribose. To a lesser extent, CD38, CD157 and SARM1 have ADP-ribosyl cyclase activity, generating NAM and cADPR from NAD+. In acidic conditions, CD38 can also perform a base-exchange reaction, swapping the NAM of NAD(P)+ for nicotinic acid (NA), generating nicotinic acid adenine dinucleotide (phosphate) (NAAD(P)). CD38 and CD157 are reported to be able to use alternative substrates in their catalytic reactions. CD38 can degrade NMN to NAM and ribose monophosphate (RMP), while CD157 can degrade NR, generating NAM and ribose (R). NR, nicotinamide riboside.

Although measuring NAD+ levels and sirtuin activity in subcellular compartments has been hampered by technological challenges, owing to recent technical advancements (Supplementary Box 4) we now have some insights into distinct cellular roles of these enzymes. Specifically, recent evidence has shown that nuclear SIRT1, SIRT6 and SIRT7 are critical regulators of DNA repair and genome stability and that mitochondrial SIRT3, SIRT4 and SIRT5 and nuclear SIRT1 regulate mitochondrial homeostasis and metabolism7. In apparent contrast with its role in promoting mitochondrial biogenesis through peroxisome proliferator-activated receptor-γ co-activator 1α deacetylation19–21, SIRT1 has also been implicated in turnover of defective mitochondria by mitophagy22,23. However, in both cases SIRT1 seems to be a key factor in mitochondrial quality maintenance9. Overall, sirtuins have emerged as key actors in the efforts to understand and characterize how NAD+ levels affect cellular homeostasis in a wide variety of cellular processes that impact ageing (Supplementary Box 2), and boosting their activity is a key focus of therapies aimed at counteracting ageing.

Consumption by PARPs.

The human PARP protein family is composed of 17 proteins characterized by poly(ADP-ribosyl) polymerase or mono(ADP-ribosyl) polymerase activity24. Briefly, the PARP-mediated cleavage of NAD+ produces NAM and ADP-ribose as by-products, in which ADP-ribose is added as single or covalently linked polymers to PARP itself and other acceptor proteins, in a process called ‘poly(ADP-ribosyl)ation’ (PARylation) (FIG. 2b). Among all the PARPs, only PARP1, PARP2 and PARP3 localize to the nucleus (FIG. 1b) in response to early DNA damage and have a key role in DNA damage repair25–27 (Supplementary Box 2).

PARP1 is the best-characterized member of the group, and it alone is responsible for about 90% of total PARP activity at least in response to DNA damage28,29. On activation, PARP1 PARylates itself along with histones and other proteins that act as a scaffold to recruit and activate other DNA repair enzymes and proteins to the lesion site to initiate DNA repair30. Owing to high PARP1 activity, DNA damage is associated with massive NAD+ consumption. PARP1, in its role as an NAD+ responsive signalling molecule, has been widely associated with the ageing process. However, PARP1 is one of the major NAD+ consumers not only in cells with acute DNA damage but also under normal and other pathophysiological conditions5, supporting a key role of PARP1 in regulating NAD+ homeostasis. For example, in mice fed a high-fat diet, those that were treated with PARP inhibitors or lacking PARP1 and PARP2 had increased NAD+ levels, increased SIRT1 activity and improved mitochondrial function, and were protected from insulin resistance31,32. The strong correlation among PARP1 activation, decline in NAD+ levels and inhibition of SIRT1 activity is observed in patients with xeroderma pigmentosum group A, as well as progeroid diseases, ataxia telangiectasia and Cockayne syndrome. Notably, treatment of mice with Cockayne syndrome with PARP1 inhibitors or NAD+ supplements promoted lifespan extension and ameliorated the severe phenotypes caused by PARP1 hyperactivation, providing strong evidence that the negative consequences downstream of PARP1 activation are mediated by dysregulation of NAD+ homeostasis in response to extensive DNA damage and genotoxic stress33. Of note, the ability of PARP1 to antagonize SIRT1 activity is most likely because these enzymes are localized to the same cellular compartment — in this case, the nucleus — and compete for the same NAD+ pool. However, since PARP1 has lower Km and higher Vmax with regard to NAD+ than SIRT1, PARP1 likely outcompetes SIRT1 for NAD+ as a result of its greater binding affinity and faster kinetics34.

PARP2 is structurally related to PARP1. They share a similar catalytic domain required for several cellular processes, including DNA repair and transcriptional regulation, and account for about 10% of PARP activity35. PARP2 activity may also influence NAD+ bioavailability25,26. Like Parp1-knockout mice, Parp2-knockout mice show enhanced SIRT1 activity and improved metabolic function, and are protected from high-fat diet-induced obesity36. PARP3 is important in DNA repair as well27, suggesting a large overlap and potential redundancy in PARP1, PARP2 and PARP3. The functions of the other PARPs (PARP4–PARP17) in NAD+ homeostasis and global metabolism in cells or organs have not been fully determined, but they are thought to be less important in modulating intracellular NAD+ levels. Overall, targeting PARPs, and in particular PARP1, is a promising therapeutic strategy in the ageing field. However, more studies are needed to fully understand the contribution of PARPs to the age-related decline in NAD+ levels.

Consumption by CD38 and CD157.

CD38 and CD157 are multifunctional ectoenzymes with both glycohydrolase and ADP-ribosyl cyclase activities. The glycohydrolysis of NAD+ is the primary catalytic reaction that cleaves the glycosidic bond within NAD+ to generate NAM and ADP-ribose, whereas the ADP-ribosyl cyclase activity generates cyclic ADP-ribose (FIG. 2c). CD38 also performs a base-exchange reaction, by swapping the NAM of NAD(P)+ for NA in acidic conditions and generating nicotinic acid adenine dinucleotide (phosphate) (NAAD(P))37 (FIG. 2c). Of note, cyclic ADP-ribose, NAAD(P) and ADP-ribose are all key Ca2+-mobilizing second messengers, illustrating the pivotal role of CD38 in activating Ca2+ signalling and modulating essential cell processes, such as immune cell activation, survival and metabolism38–40. Importantly, other than NAD+ and NADP+, NMN is emerging as an alternative substrate of CD38 (REFS41,42), whereas CD157 consumes NR as an alternative substrate43,44 (FIG. 2c). Thus, targeting CD38 and CD157 with small-molecule inhibitors may make these commonly used NAD+ precursor metabolites more efficient in restoring NAD+ levels in ageing individuals. Very little is known about the purpose of CD157 enzymatic functions in cellular biology or ageing. However, recent evidence shows that, like CD38, CD157 is upregulated in ageing tissues45 and may have a role in ageing-related diseases, such as rheumatoid arthritis and cancer46.

Although CD38 and CD157 are genetically homologous and are members of the same enzymatic family, their structure, localization and role in diseases differ. CD38 is a transmembrane protein with a type ii orientation and/or a type III orientation identified in the late 1970s as a T cell activation marker but now known to be ubiquitously expressed, particularly during inflammation47,48. CD157 is a glycophosphatidylinositol-anchored protein, which was first identified in the myeloid compartment of the haematopoietic system49. However, CD157 is also expressed by other cells, including B cell progenitors, Paneth cells and endothelial cells in the gut, pancreas and kidney50.

Besides their enzymatic function, CD38 and CD157 act as cell receptors. For example, CD38 is an adhesion receptor interacting with CD31 to mediate immune cell trafficking and extravasation through the endothelium51,52. The CD38–CD31 axis seems to promote a proliferative response in chronic lymphocytic leukaemia lymphocytes, suggesting a detrimental role of CD38 in blood cancers53,54 (see Supplementary Box 3). However, very little is known about the functional consequences of CD38–CD31 interaction. Additionally, CD38 is thought to mediate immunity through antimicrobial functions, given that a major phenotype of Cd38-knockout mice is their inability to mount immune responses to bacteria55. It is still unknown whether the CD38 enzymatic function is necessary for its antibacterial functions and to what extent these functions depend on NAD+. However, it is plausible that the role of CD38 in antimicrobial resistance is related to sequestering NAD+ or NAD+-related metabolites from bacteria that require NAD+ for survival and growth56,57.

The role of CD157 as a receptor is still not fully explored. Several lines of evidence suggest that CD157 activation by specific monoclonal antibodies promotes trafficking of neutrophils and monocytes58. Moreover, CD157 interacts with integrins to form a receptor recognized by scrapie-responsive gene 1 protein (SCRG1), promoting self-renewal, migration and osteogenic differentiation of human mesenchymal stem cells43. However, whether these receptor functions are relevant to NAD+ metabolism remain unclear.

Consumption by SARM1.

SARM1 was only very recently assigned to the NAD+ glycohydrolase and cyclase family along with CD38 and CD157. The enzymatic activity of SARM1 (FIG. 2c) relies on the Toll/interleukin receptor (TIR) domain that was previously not known to possess catalytic activity and generally to be involved in protein–protein interaction59. Whether SARM1 regulates NAD+ under physiological conditions and to what extent is still unknown. However, SARM1-mediated NAD+ degradation plays a key role in axonal degeneration after axonal injury60 (see the section Neurodegeneration). SARM1 is primarily expressed in neurons and promotes neuronal morphogenesis and inflammation61,62; however, SARM1 is also expressed by immune cells such as macrophages and T lymphocytes, and regulates their functions63–65. SARM1 was originally discovered as a negative regulator of the innate immune response via direct interaction with TRIF (TIR domain-containing adapter protein inducing interferon-β), downstream of Toll-like receptor signalling63. However, the immunoregulatory role of SARM1 remains largely uncharacterized. Although SARM1 was originally reported to be required for chemokine CCL5 production in macrophages66, recent evidence revealed that SARM1 is not expressed in macrophages and that the observed chemokine phenotype was due to a background effect of the Sarm1-knockout mouse strain67. Despite its controversial role in immune cells, SARM1 plays an undisputed key role in axonal degeneration and is emerging as a therapeutic target to prevent or ameliorate neurodegenerative diseases and traumatic brain injuries (as discussed in the section Neurodegeneration).

Beyond the main groups of NAD+-

Beyond the main groups of NAD+-consuming enzymes discussed so far, NAD+ is widely used as a cofactor or substrate for biochemical reactions, with more than 300 enzymes1 relying on NAD+ for their activity. Accordingly, NAD+ is a mediator of key cellular functions and adaptation to metabolic needs. Some of these critical cellular processes include metabolic pathways, redox homeostasis, maintenance and repair of DNA to safeguard genomic stability, epigenetic regulation and chromatin remodelling, and autophagy. Collectively, these functions are important for maintaining systemic health and homeostasis. However, during ageing, declining NAD+ levels can impinge on these processes and exacerbate ageing-related diseases (FIG. 3; Supplementary Box 2).

 

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Fig. 3 |

NAD+ metabolism in ageing.

A | Decreased nicotinamide adenine dinucleotide (NAD+) levels have been implicated in various processes associated with ageing (see also Supplementary Box 2). Aa | Ageing is associated with aberrant proinflammatory immune cell activation or ‘inflammageing’, leading to sustained low-grade inflammation. This is caused in part by the accumulation of senescent cells, which via a senescence-associated secretory phenotype (SASP) promote the phenotypic polarization of macrophages towards a proinflammatory M1 state, thereby driving inflammation. There is evidence that in response to the SASP in these macrophages expression of the NAD+-consuming enzymes CD38 and poly(ADP-ribose) polymerases (PARPs) increases, leading to NAD+ level decline, and that these mechanisms importantly contribute to the decrease of NAD+ levels in ageing. In addition, it has been shown that in aged T cells mitochondrial function declines, and this leads to increased secretion of proinflammatory cytokines that promote the state of inflammation and also induce senescence. Ab | Axonal degeneration, which is a precursor to many age-related neuronal disorders, is characterized by rapid NAD+ depletion. During normal physiological conditions, NAD+ biosynthetic enzymes, nicotinamide mononucleotide adenylyltransferases (NMNATs), are protective against axonal degeneration, and their expression supports maintenance of axons and prevents neurodegeneration. In particular, NMNAT2 is an important survival factor in axons, to which it needs to be constantly delivered from the soma — where it is synthesized — to account for its rapid turnover, and these transport processes are disturbed during axonal degeneration. Moreover, the NAD+-consuming enzyme SARM1 is activated by axonal injury and mediates axonal degeneration by promoting NAD+ degradation. Ac | Autophagy is a key cellular catabolic process that allows cells to adapt to variable nutrient availability and serves in cellular quality control, allowing removal of defective organelles and proteins. Autophagy is regulated downstream of NAD+ levels via sirtuins (mostly SIRT1). Decline of NAD+ levels reduces overall autophagic flux as well as selective removal of mitochondria via mitophagy, suggesting that defective autophagy can be a consequence of NAD+ depletion during ageing, contributing to cell dysfunction. B | Because NAD+ is a cofactor for various enzymes, loss of NAD+ impacts a plethora of cellular processes. For example, NAD+ is required for the activity of epigenetic regulators such as SIRT1, and decline in its level causes changes in histone modifications, thereby affecting chromatin organization and function in gene expression. There is evidence that the ageing-associated loss of NAD+ is related to increased expression of PARPs, which can be caused by increased levels of DNA damage and the need for DNA repair during ageing (panel Ba). NAD+ also affects transcriptional activity of the core clock components CLOCK and BMAL, thereby regulating circadian expression of important metabolic genes as well as nicotinamide phosphoribosyltransferase (NAMPT), which in turn is required for circadian oscillation in NAD+ levels (panel Bb). Decreased NAD+ levels also interfere with the activity of PARPs and sirtuins in DNA repair, leading to genomic instability: a hallmark of ageing and cancer (panel Bc). ADPR, ADP-ribose; ATG, autophagy-related protein; FOXO, forkhead box protein O; ROS, reactive oxygen species.

Table 1 |

Innate immunity.

Chronic low-grade inflammation, characterized by aberrant activation of the innate immune system, enhanced expression of proinflammatory cytokines, such as tumour necrosis factor (TNF), IL-6 and IL-1β, and activation of immune complexes, such as the NLRP3 inflammasome, is now being recognized as a key driver of ageing-related and metabolic diseases. Additionally, it is now emerging that altered macrophage activation and phenotypic polarization is a key source of this inflammation. For example, in obese tissues such as the visceral fat, peripheral monocytes are recruited by stressed adipocytes, leading to a progressive infiltration of proinflammatory M1-like macrophages and the displacement of the resident, anti-inflammatory M2 macrophages96. This switch in macrophage polarization states in the visceral fat is accompanied by enhanced expression of proinflammatory cytokines, insulin resistance and reduced rates of lipolysis. Elie Metchnikoff, the discoverer of the macrophage, was the first to observe the increased abundance of macrophages in ageing tissues more than 100 years ago. Although this observation was initially disregarded, there is now growing evidence that ageing not only leads to increased macrophage abundance but is also accompanied by altered macrophage polarization state and function, which is emerging as a key driver of inflammageing98,99.

Some of the earliest work suggesting that NAD+ affects macrophage function showed that inhibition of NAMPT and subsequent depletion of NAD+ pools in macrophages decreased the secretion of proinflammatory cytokines, such as TNF, and caused morphological changes, such as reduced spreading in these cells100,101. Recent studies have shown that NAD+ is a critical regulator of macrophage function and that macrophage activation is associated with the upregulation of NAD+ biosynthetic or degradative pathways, depending on the acquired fate. For example, our laboratory recently showed that proinflammatory (M1) macrophage polarization is associated with enhanced expression of CD38, leading to increased NAD+ consumption45. Conversely, anti-inflammatory (M2) macrophage polarization was associated with an increase in NAD+ levels that depended on NAMPT45. Blocking the NAM salvage pathway (BOX 1) in both M1 macrophages and M2 macrophages significantly reduced gene expression of select genes associated with the M1 and M2 phenotypes. This effect could be rescued with supplementation with the NAD+ precursors NMN and NR, which bypass and rescue NAMPT inhibition45. M2 macrophages required significantly more NR/NMN to rescue macrophage activation than M1 macrophages, suggesting that NAD+ is a critical metabolite for general macrophage activation, and its metabolism is differentially regulated to control distinct biological processes and functions in M1 and M2 macrophages.

Consistent with our results, a recent study found that M1 macrophage polarization is associated with enhanced degradation of NAD+ and that inhibiting NAMPT blocks glycolytic shifts in M1 macrophages, limiting proinflammatory responses in vitro and reducing systemic inflammation in vivo in response to sepsis102. This study and another recent report6,102 suggest that this NAD+ turnover, particularly during the first few hours of macrophage M1 polarization, depends on reactive oxygen species-induced DNA damage and activation of PARP1. However, recently published results from our laboratory detected no evidence of DNA damage or PARP1 activation during M1 macropahge polarization45. By contrast, our results suggest that CD38 is the primary NAD+-consuming enzyme in M1 macrophages. These results are consistent with recent work showing that M1 macrophages are protected from reactive oxygen species-induced DNA damage as a result of increased transcription of genes involved in antioxidant defence, such as SOD2 (REFS102–104). Thus, these contradictory observations highlight a need to better delineate the major consumers of NAD+ during proinflammatory and anti-inflammatory macrophage polarization to determine whether the observed decline in NAD+ levels depends on context and time and to determine the molecular mechanisms through which NAD+ levels influence proinflammatory and anti-inflammatory gene expression and macrophage functions.

In ageing, declining NAD+ levels are associated with increased accumulation of proinflammatory M1-like resident macrophages in the liver and fat, which are characterized by increased expression of CD38 and heightened NADase activity45,105. By in vitro and in vivo methods, it was found that these CD38-overexpressing M1-like macrophages are activated directly by inflammatory cytokines secreted by senescent cells (discussed in more detail later)45,105,106. In addition, ageing macrophages are characterized by impaired de novo synthesis of NAD+, which in itself may impact macrophage function during ageing6.

With regard to inflammageing, the studies described above suggest that proinflammatory M1-like macrophages (in addition to senescent cells; see later) may be a major source of proinflammatory cytokines in ageing tissues. Ageing is associated with increased activation of the NLRP3 inflammasome45,107, which is primarily expressed by myeloid immune cells107,108, and a subsequent increase in the expression of IL-1β, which is a major cytokine implicated in promoting ageing-related disease107. Enhanced expression of proinflammatory cytokines may drive a vicious cycle of inflammageing, leading to greater inflammation, enhanced tissue and DNA damage, further activation of major consumers of NAD+, such as CD38 and PARPs, and accelerated physiological age-related decline.

Hence, targeting macrophage immunometabolism pathways, in particular those that regulate NAD+ biosynthetic or degradative pathways, could be explored as a therapeutic strategy to activate or suppress macrophage functions and regulate macrophage polarization states. This is certainly pertinent to regulating inflammageing, but could also serve to alleviate diseases driven by chronic inflammation, such as neurodegenerative diseases and autoinflammatory diseases, and also as a therapeutic strategy in cancer, where macrophage polarization can be either tumour promoting (M2) or tumour inhibitory (M1)109.

Adaptive immunity.

Like for the innate immune system, ageing is characterized by a reduced ability to build an effective adaptive immune response due to altered functions of adaptive immune cells, known as immunosenescence.

Ageing leads to an imbalance or skewing of immune cell populations, including decreased levels of naive T and B cells, loss of T cell antigen receptor diversity and an increase in levels of virtual memory T cells. The regulatory role of NAD+ and NAD+-consuming enzymes in T cell biology has been demonstrated; however, their contribution to the ageing of the adaptive immunity is largely uncharacterized. On the one hand, extracellular NAD+ has been proposed as a danger signal110 causing cell death in specific T cell subpopulations, such as regulatory T cells111. On the other hand, NAD+ seems to exhibit immunomodulatory properties, such as influencing T cell polarization112,113. However, whether NAD+ promotes a specific T cell phenotype and whether the manipulation of NAD+ metabolism with NAD+ precursors could result in similar immunomodulatory properties is still unknown.

An established hallmark of adaptive immune ageing is the expansion of the memory population of highly cytotoxic CD8+CD28− T cells114, characterized by high secretion of effector molecules such as granzyme B115. This cell population is characterized by decreased SIRT1 and FOXO1 levels, resulting in an enhanced glycolytic capacity and granzyme B production116. These studies highlight the potential of metabolic reprogramming via manipulation of NAD+-associated pathways in age-related adaptive immune dysfunction116. The upregulation of the NAD+–SIRT1–FOXO1 axis via CD38 inhibition increases effector functions in T helper 1 and 17 hybrid cells117, and future studies using CD38 inhibitors or NAD+ precursors may potentially show the therapeutic potential of targeting NAD+ in the ageing adaptive immune system.

Another immunological feature of adaptive immune ageing is the increase in the number of exhausted T cells, characterized by the expression of inhibitory receptor molecules (for example, PD1 and TIM3), decreased proliferative capacity and decreased effector functions118,119. PD1 is a component of the immune checkpoint, blockade of which is commonly used as an anticancer strategy120,121, but also has been proposed to restore the effector function of aged T cells122. With respect to NAD+ metabolism, a recent study showed that CD38 overexpression was associated with a dysfunctional and exhausted CD8 T population in PD1 blockade-resistant cancers123,124, highlighting the potential of expanding studies on CD38 inhibition to age-related exhausted T cells. However, although extremely intriguing, this hypothesis is yet to be explored in depth, and more studies will be needed to determine the preclinical efficacy of this approach.

Thus, overall, more work needs to be done to determine whether manipulating NAD+ levels is effective in reversing ageing-related immunological dysfunction in the adaptive immune system and, equally important, whether such manipulations are safe.

Table 2 |