Targeting NAD⁺ metabolism to modulate autoimmunity and inflammation

Abstract

NAD⁺ biology is involved in controlling redox balance, functioning as a coenzyme in numerous enzymatic reactions, is a cofactor for Sirtuin enzymes and a substrate for multiple regulatory enzyme reactions within and outside the cell. At the same time, NAD⁺ levels are diminished with aging and are consumed during the development of inflammatory and autoimmune diseases linked to aberrant immune activation. Direct NAD⁺ augmenation via the NAD⁺ salvage and Priess-Handler pathways are being investigated as putative therapeutic interventions to improve healthspan in inflammation-linked diseases. In this review, we survey NAD⁺ biology and its pivotal roles in the regulation of immunity and inflammation. Furthermore, we discuss emerging studies evaluating NAD⁺-boosting in murine models and in human diseases and highlight areas of research that remain unresolved in understanding the mechanisms of actions of these nutritional supplementation strategies.

Introduction

Nicotinamide adenine dinucleotide (NAD⁺) biology was initially understood in the context of its bidirectional flux with NADH or between NADP⁺ and NADPH in oxidation-reduction reactions, for energy transduction and redox homeostasis. The intracellular NAD⁺/NADH and NADP⁺/NADPH pools are highly compartmentalized and their disruption contribute to aging and disease (1, 2). Concurrently, non-redox effects of NAD⁺, where NAD⁺ transport, biosynthesis, and catabolism enable diverse roles in the regulation of energy transduction, in cell signaling, as a post-translational modifier, and as an epigenetic mediator have become apparent (3). At the organelle level, homeostatic control of mitochondrial integrity, function and metabolic signaling is governed, in part, by NAD⁺ biochemistry, and the activity of this organelle is tightly integrated with NAD⁺ levels (4, 5). In the immune system, these concepts are intergrated in that perturbed mitochondrial fidelity and function and abberations in NAD⁺ metabolism are linked components of autoimmune and inflammatory degenerative diseases and aging (6, 7). This pathophysiological connection between mitochondria and inflammation is partially underpinned where mitochondrial infidelity and dysfunction, operate as signaling platforms to initiate intracellular and extracellular immune activation signaling (8–10).

Therapeutic strategies including nutritional supplements and agents that modify NAD⁺ levels are being investigated as approaches to modulate degenerative diseases, including inflammatory and autoimmune diseases (11–14). In this review, we focus on highlighting NAD⁺ metabolism in the immune system, and uncover the emerging understanding of how NAD⁺-boosting functions as a potential therapeutic strategy targeting inflammatory and autoimmune-linked diseases.

Overview of Metabolic Demands in Immunity

In principle, the regulation of cellular metabolism is coordinated with energy demands and to fulfill biosynthetic, and redox control processes within cells. This concept was initially advanced by Otto Warburg, where he decribed how metabolic substrates orchestrated demands above and beyond bioenergetic needs (15). This effect in cancer cells implicated aerobic glycolysis as the preferential energy production source, enabling mitochondrial metabolism divertion to generate intermediates for biomass synthesis (e.g., nucleotides, amino acids, and lipids) to facilitate increased cell division (15). The spectrum of energy demand in immune cells show similar dynamism, as immune cells exhibit diverse functional fates spanning from quiescent to proliferative, and for undergoing differentiation and/or activation (8). Furthermore, cellular energetic requirements are increased to enable immune cell migration and phagocytosis (8). Some of these immune cell fates align with the metabolic mileau of cancer, where immune cells similarly display the Warburg metabolic profiles (8). At the same time, it should be noted that anaerobic glycolysis is not the only dominant fuel substrate in high-metabolic demand cells. Glutaminolysis and glutamine oxidation similarly function in this role, which in turn, diverts other substrates to biosynthetic functions to support cellular proliferation and function, whether in cancer or immune cell regulation (16, 17).

The Foundational Role of Metabolic Remodeling in Immune Cell Fate

The functional contribution of metabolic pathways in immune cell function are now recognized given that molecular alteration of metabolite availability remodels immune cell fates. Examples of this include restraint in polarization of CD4⁺ helper cells (T_H) to T_H17 cells by disruption of either de novo fatty acid synthesis or glucose uptake (18, 19). In both instances, T_H17 polarization becomes impeded due to metabolic and epigenetic regulatory control and manifests with reduced autoimmune disease susceptibility (18, 19). Concurrently, specific intrinsic metabolic pathways are remodeled directly during immune cell activation. For example, innate immune cell activation constipates specific catabolic transitions within the mitochondrial tricarboxylic acid cycle (TCA) resulting in accumulation of upstream substrates including citrate and succinate. These intermediates then orchestrate proinflammatory signaling via a myriad of signaling mechanisms (20). Additionally, lipopolysaccharide (LPS) upregulates the immune responsive gene 1 (irg1) encoded enzyme which decarboxylates the TCA cycle intermediate cis-aconitate to generate itaconate. Itaconate then directly orchestrates anti-inflammatory and anti-microbial effects (21–24). The role of itaconate as a signaling metabolite is similarly operational in T cells, albeit via indirect control. Here, myeloid cell derived itaconate is imported into CD8⁺ cytotoxic T lymphocytes (CTLs), resulting in blunting of nucleotide synthesis, with amelioration of proliferative and cytolytic activity of these tumor infiltrating cells (23). Together, these diverse contributions of metabolic pathways to immune cell regulation are termed immunometabolism and are recognized to play pivotal roles in overall immunoregulation (20, 25).

The Central Role of NAD⁺ in Cellular Metabolism and Homeostasis

Cellular compartment specific NAD⁺/NADH ratios broadly reflect redox balance supporting metabolism through innumerable metabolic oxidoreductive enzyme reactions within the cytoplasm and mitochondria. Here, NAD⁺ is recognized as an essential recycling co-enzyme to maintain metabolism and cellular homeostasis (3). To sustain its non-redox enzyme functions, intracellular NAD⁺ levels are controlled by the balance between NAD⁺ uptake, intracellular biosynthesis, enzymatic NAD⁺ consumption and the regulation of different intracellular NAD⁺ pools. The major intracellular NAD⁺ pools reside within mitochondria and the cytosol, which, together with the nucleus, are the major sites for non-redox functions (26). In mammalian cells NAD⁺ is synthesized by three main pathways: (i) de novo biosynthesis pathway, through the Kynurenine pathway, where tryptophan as the precursor, is catabolized to quinolinic acid, to generate NAD⁺; (ii) the Priess-Handler pathway utlizes nicotinic acid (NA) as its initial substrate; and (iii) the salvage pathway employs the NAD⁺ intermediate nicotinamide (NAM), or its nucleosides, nicotinamide riboside (NR) and nicotinic acid riboside (NAR) to restore NAD⁺ levels (3, 26). A recent study in mice additionally showed that the gut microbiome plays an important role in endogenous NAD⁺ biology, where circulating NAM and oral NR are converted to NA which, in turn, are reabsorbed into the circulation as a precursor for the Priess-Handler pathway (27). Furthermore, as biologic membranes are impermeable to pyridine nucleotides, NAD⁺ synthesis may be compartment specific. Consistent with this, there are distinct subcellular enzyme isoforms governing NAD⁺ biosynthetic pathways (26, 28), as epitomized by distinct nicotinamide mononucleotide adenyltransferes (NMNAT) nuclear, cytosolic and mitochondrial isoforms (29). A more recent discovery of a mitochondrial NAD⁺ transporter SLC25A51 on the inner mitochondrial membrane identifies yet another mechanism for targeted intracellular NAD⁺ transport (30–32).

NAD⁺ Biochemistry in Immune Cell Function

Immunomodulation linked to NAD⁺biosynthetic pathways

Given the metabolic ‘remodeling’ link to immune cell fate and function, NAD⁺ plays a central role in the regulation of immunity. At the level of NAD⁺ biosynthesis, the de novo NAD⁺ synthesis through the Kynurerine pathway, is operational in murine macrophages (33). This is evident in that genetic and pharmacologic disruption of de novo NAD⁺ biosynthesis impair macrophage phagocytosis and resolution of inflammation (33). Interestingly, these features recapitulate the aging macrophage phenotype and are mediated, in part, by disrupting macrophage mitochondrial bioenergetic capacity (33). The regulation is more complex though, as LPS triggered innate immunity upregulates proximal, but blunts distal enzymes, in the de novo NAD⁺ biosynthesis pathway. This results in accumulation of intermediates downstream of tryptophan, in parallel with reduced NAD⁺ and its precursors NA mononucleotide (NAMN) and NA adenine dinucleotide (NAAD) (33). Interestingly, constipation of this pathway with subsequent pro-inflammatory effects recapitulate concepts operational in the TCA cycle as described above. Furthermore, catabolism of tryptophan is not restricted to the Kynurerine pathway, as tryptophan is also metabolized to 5-hydroxytryptophan and indole derivatives by the liver and gut microbiome (34). Intermediates in these pathways confer context specific pro- and anti-inflammatory effects in multiple organs (34). Given the promiscuity of the catabolism of tryptophan, its use as a NAD⁺-boosting supplement is therefore limited and not extensively discussed further in this review. The most robust data on immunoregulation is evident following supplemention of intermediates that feed the NAD⁺ salvage and/or Priess-Handler pathways. These data are expanded upon later in the review, in the context of animal models and human diseases linked to inflammation and autoimmunity.

Immunomodulation linked to NAD⁺consumption pathways

The major NAD⁺ consumption pathways in immune cells include: the NAD⁺ glycohydrolase (NADase) - CD38; the sterile alpha and HEAT/Armadillo motif (SARM) - a Toll/IL-1 receptor adaptor family (TIR) member (SARM1); the poly(ADP-ribose) polymerase (PARP) family of enzymes, and sirtuin deacylase enzymes. Their immune roles are summarized below.

CD38 has immunomodulatory effects that are dependent or independent on its NADase activity. NADase independent effects include CD38 cyclase activity which modulates ADP ribose levels (35), and CD38 functions as a structural component of the B cell receptor complex (36). Additionally, CD38 usually exhibits a type II membrane orientation, with its catalytic site being extracellular, although an intracellular orientation and soluble intra- and extra-cellular forms are evident (37). In mice CD38 levels increase with aging and associate with impaired immunity of inflammaging (37). CD38 levels similarly rise with aggregate systemic lupus erythematosus (SLE) disease activity (38). Here, elevated CD8⁺ T cell’s CD38 levels blunt cellular cytotoxicity (39), and in parallel with CD38 expression, whole blood NAD⁺ levels diminish with increasing SLE disease activity (13). Interestingly, in young CD38 KO mice, there was no change in immune cell NAD⁺ levels and CD38 was not required for LPS-induced M1 macrophage polarization (37). In contrast, with aging, genotoxic stress, and senescence-associated secretory phenotype elevated macrophage CD38 levels promote the decline of NAD⁺ and nicotinamide mononucleotide (NMN) levels in a CD38-dependent manner, in parallel with increased M1 macrophage polarization (37, 40).

SARM1 also possesses NADase activity (41). Interestingly, SARM1 conveys anti-rather than pro-inflammatory regulation in contrast to other TIR family members, such as MyD88 and TRIF, by inhibiting toll-like receptor (TLR) signaling through interactions with TRIF and MyD88 (41). The molecular mechanism whereby SARM1 functions as an NADase has recently been uncovered, where dimerization of the SARM1 TIR domain possesses NADase activity (42). Despite this, the role of NAD⁺ consumption in SARM1 immunomodulatory effects remains unresolved.

PARP enzymes consume NAD⁺ where the PARP catalytic domains transfer ADP-ribose moieties from NAD⁺ to amino acid residues leading to protein mono- or poly-ADP-ribosylation (MARylation or PARylation). These modifications affect DNA repair, cell differentiation, gene transcription and signal transduction. There are 17 human PARPs, and different members catalyze ribosylation reactions in different cell types and within different subcellular compartments. The immunoregulatory effects of these isoforms have been reviewed (43), and only a few examples will be discussed here. LPS-mediated induction of macrophage activation upregulates PARP1, which then PARylates the NF-κB subunit p65/relA to transduce inflammation (44). This activation similarly increases macrophage NAD⁺ consumption (33). In contrast PARP9 and PARP14 confer counterregulatory effects on macrophage polarization towards the M2 fate (45). The genetic KO of PARP1 and PARP2 exhibit regulatory roles in CD4⁺ T cell differentiation, in B cell development and in antibody production (43, 46). Although the role of PARylation in lymphocyte biology has not been comprehensively defined (43, 46), immunomodulatory effects of PARPs appear to be independent of their enzymatic activities (43). This points to the complexity of this regulation implicates that PARP enzymes function via both NAD⁺ consumption dependent and independent pathways.

Finally, NAD⁺ functions as a cofactor for Sirtuin enzyme activation and its catabolism generates nicotinamide and 2’-O-acetyl-ADP-ribose. The 7 Sirtuin enzymes (SIRT1 through SIRT7) initiate numerous deacylase effects, including posttranslational modification such as deacetylation and desuccinylation (47, 48) on histones and non-histone proteins. SIRT1, SIRT2 and SIRT6 mediated deacetylation of the NF-κB p65 subunit blunts its transcriptional activity to limit macrophage inflammation (49). In parallel, LPS-mediated metabolic remodeling to favor glycolysis as a component of macrophage activation and IL-1β production, is associated with the downregulation of SIRT5 and increased succinylation of glycolytic enzymes (50). Furthermore, succinylation stabilizes the HIF-1α transcription factor which augments glycolysis, and SIRT5’s anti-inflammatory effects operate, in part, via desuccinylation of glycolytic intermediates to modify glycolysis and the nuclear regulation of HIF-1α (48, 50). The mitochondrial enriched SIRT3 similarly blunts the NLRP3 inflammasome via deacetylation and activation of the mitochondrial superoxide dismutase SOD2 (51). The Sirtuin enzymes are also operational in adaptive immune cells, where SIRT1 silences the transcription factor which upregulates IL-2 production, thereby decreasing T_H1 activation (52). In parallel, SIRT1-deficient T cells are more proliferative, produce more IL-2 and exhibit greater susceptibility to experimental autoimmune encephalomyelitis (EAE) (53). SIRT1 and SIRT3 additionally have immunomodulatory roles in regulatory CD8⁺ T cell and in B cell function (54). Finally, consistent with the role of NAD⁺ in Sirtuin activation, the genetic disruption of de novo NAD⁺ biosynthesis in mouse peritoneal macrophages directly blunts SIRT3 and SOD2 activity (33).

In summary, emerging evidence points to extensive contributions of NAD⁺ consumption pathways, and their subcellular compartmentalization in the modulation of innate and adaptive immunity. Together these highlight multiple levels of regulation and ultimately need to be understood in an integrated manner to appreciate the role of NAD⁺ biology in fine-tuning immunoregulation.

NAD⁺-Boosting: Approaches and Consequences

Dietary supplementation with intermediates including NAM, NMN and NR have all been shown to boost NAD⁺ levels in mice and humans (55–58). Furthermore, NAD⁺ has been administered via intraperitoneal administration (59). Finally, the pharmacologic inhibition of NAD⁺ consumption pathways may similarly confer therapeutic effects, which will also be described below. Prior to exploring these pharmacologic and dietary supplemental strategies, data in cell culture further implicate regulatory roles of NAD⁺ biology in immunity. Examples of this include regulation of the mitochondrial NAD⁺ transporter, SLC25A51 during CD4⁺ T cell activation (60). Furthermore, the genetic induction of nicotinamide phosphoribosyltransferase (NAMPT), a salvage pathway enzyme, in primary human and mouse monocytes cofers polarization towards inflammatory M1 macrophages and similarly NAMPT inhibition blunts this polarization (61). NAMPT inhibition similarly impairs NLRP3-dependent inflammation in primary human monocytes (62).

Immune Effects of NAD⁺ Modulation in Animal Models

NAD⁺-boosting was initially shown to modulate immunoregulation following NMN supplementation in aging mice. Here, 12-months of NMN suppressed gene signatures of age-associated adipose tissue inflammation and altered the leucocyte population by reducing neutrophils and increasing lymphocyte levels (63). Three months of NR supplementation similarly reduced neuroinflammation in a genetic Alzheimer’s disease (AD) model (64). In a subsequent study in transgenic β-amyloid induced AD, NR supplementation from 7 to 9 months of age broadly reduced transcript levels of inflammatory mediators in the hippocampus, reduced brain and plasma cytokines levels and diminished astrocytes and microglia activation, in part, by blunting the NLRP3 inflammasome (65). In parallel, NR diminished neuronal DNA damage, and the concomitant DNA damage sensing inflammatory pathway mediated by intracellular cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) (65). NR and NMN supplementation prior to and during cisplatin and kidney ischemia-reperfusion injury, blunted renal damage via an NAD⁺-dependent reduction in mitochondrial RNA release (66). This, in turn ameliorated activation of the cytosolic pattern recognition receptor retinoic acid-inducible gene I (RIG-I), to attenuate renal leukocyte infiltration (66).

The most comprehensive immunological work on NAD⁺-boosting is in the murine model of myelin oligodendrocyte glycoprotein (MOG) induced EAE. Here, NAM supplementation at the time of MOG inoculation or starting 10-days post-innoculation reduced axonal demyelination and CD4⁺ T cell infiltration into the spinal cord (67). Similarly, intraperitoneal NAD⁺ preceding, in conjunction with, or delayed administration after MOG inoculation, attenuated EAE by: (i) increasing levels of the anti-inflammatory cytokines IL-10 in T_H1 cells and TGF-β1 in T_H17 cells (59); (ii) activating AMPK and SIRT1 mediated blunting of T_H1 and T_H17 cytokine production (68); (iii) reducing myeloid cell NF-κB signaling (69); and (iv) via activation of autophagy in parallel with NLRP3 inflammasome attenuation (70). The role of NAD⁺ consumptive enzymes has also begun to be investigated in EAE, where CD38 levels are induced in multiple sclerosis (71, 72), and CD38 KO attenuates EAE (72).

On a note of caution, the indiscriminate augmentation of NAD⁺ biosynthesis to blunt autoimmunity may be too simplistic a concept. This circumspection is based on contradictory data, as evident by the blunting of EAE via pharmacologic inhibition of NAMPT by FK866 (73). This is due to diminution of T-cell viability resulting in blunted T-cell mediated autoimmunity (73). Interestingly, FK866-mediated NAD⁺ depletion is evident in activated but not resting T lymphocytes, highlighting differences in NAD⁺ biosynthetic and consumption pathway functional roles aligned with cell fate-dependent cellular bioenergetic demands. In a similar vain, pharmacologic inhibition of the kynurenine pathway with the QPRT inhibitor, 3-nitropyriine-2-thione or the NMNAT inhibitor, tannic acid similarly blunted murine EAE (72). Here, the kynurenine pathway was shown to be induced by EAE in myeloid derived microglial cells rather than in astrocytes. The complexity of this biology was further realized, where primary astrocytes in response to EAE modeling by LPS and IFNγ supplementation, showed activation of the salvage pathway, rather than the kynurenine pathway, and FK866 similarly blunted astrocytic inflammation (72). Interestingly, astocytes show elevated expression of the gene encoding CD38 (71, 74) and induction of CD38 by EAE exacerbated NAD⁺ depletion. However, in contrast to blocking the salvage pathway, the inhibition or genetic depletion of CD38 in this study blunted EAE. The mechanisms underlying the proinflammatory effects of CD38 include signaling through cyclic adenosine diphosphate ribose (cADPR) and ADPR, which are derived during NAD⁺ hydrolysis. These signaling intermediates, promoted chemokine signaling to augment astrocytic chemoattraction of inflammatory monocytes (72). Together these data implicate that the modulation of NAD⁺ levels in lymphoid, myeloid and non-professional immune cells (astrocytes) contribute to the susceptibility of experimental autoimmune encephalomyelitis. At the same time, divergent data with NAD⁺ supplementation, CD38 modulation and inhibition of NAD⁺ biosynthesis implicate that: (i) spatial, temporal and/or absolute levels of NAD⁺ may have opposing effects; (ii) NAD⁺ hydrolysis intermediates have independent pro-inflammatory signaling; and/or that (iii) pharmacologic inhibition may have broader and yet undetermined off-target effects on the overall inflammatory signatures in in vivo models.

At the same time, NAD⁺-boosting augments CD8⁺ CTLs function, which as effector cells, confer tumor cytotoxic effects and kill virally infected cells. Tumor infiltrating lymphocytes (TILs), functionally distinct CTLs, possess high metabolic demands to promote tumor lysis. In this context, CD8⁺ T cell exhaustion is inextricable linked to metabolic ‘failure’ which span from mitochondrial depolarization, reduced mitochondrial DNA (mtDNA) copy number to excess mitochondrial ROS production (75). Similar features are evident in hepatitis B virus targeting CTLs, and here, it was found that their incapacity to augment oxidative phosphorylation diminished their capacity to generate cytotoxic cytokines (76). As may be expected, in these cells, increasing NAD⁺ may augment cytotoxic function. This has been shown in TILs, where NR supplementation improved mitochondrial fitness, reduced mitochondrial ROS, and enhanced cytotoxic activity in vitro, and intratumoral NR administration diminished tumor growth in vivo (75). The role of NAD⁺ biology in TILs is further evident in that NAMPT expression is reduced in TILs, and NAD⁺ supplementation enhanced anti-tumor efficacy of chimeric antigen receptor (CAR) modified T cells (CAR-T) (77). Furthermore, daily NAM supplementation for 3 days enhanced CAR-T tumor-killing efficacy and boosted T cell-based immunotherapeutic efficacy in solid tumor models (77). Similarly, elevated CD38 levels impaired TIL immune responsiveness in metastatic cancer models (78), and CD38 depletion partially diminished NAD⁺ degradation and restores CD8⁺ T cell function. However, targeted disruption of CD38 perse was not sufficient to increase antitumor T cell therapy efficacy (79). Data on NAD⁺ manipulation for antiviral effects is less explored. Although, using murine hepatitis virus as a coronavirus model, the robust interferon response depressed cellular NAD⁺ in a manner that was opposed by supporting NAD⁺ status (80). More recently, in two murine models of SARS-CoV2-induced pneumonia, NAD⁺ and NMN supplementation for 3 days following inoculation reduced lung inflammatory infiltrates and improved survival, although the effects of NAD⁺-boosting on CTLs was not explored (81). The major roles of NAD⁺-boosting in the modulation of inflammation and autoimmunity in murine models is shown in Figure 1.

FIGURE 1.

Effects of NAD⁺-boosting in murine models of inflammaging, autoimmunity, cancer, kidney disease and viral infection. Strategies to boost NAD⁺ are shown on the left, although this review was restricted to the direct targeting of NAD⁺ metabolism and supplementation. The potential immunometabolic regulatory mechanisms are depicted in the box above the mouse image. To the right the different models are shown including how NAD⁺-boosting modulates immune responses.

Immune Effects of NAD⁺-Modulation in Human Health and Disease

The role of NR on the human immunity is evident where NR depresses inflammatory cytokines in older men (12), reduces the inflammasome (82) and blunts monocytic type I interferon signaling (13), diminishes cytokine production in PBMCs isolated from heart failure subjects (83), reduces systemic- and neuro-inflammation in Parkinson disease (11) and blunts T_H1 and T_H17 cell activation from healthy and psoriasis subjects (14). Although as in murine studies, contradictory data is also evident, which will be expanded upon below. A summary of these effects of NAD⁺ supplementation in human diseases and links to specific immune pathways is highlighted in Figure 2.

FIGURE 2.

The diseases explored using NAD⁺-boosting is shown in the outer wheel of the circle. The inner sections highlight effects of this supplementation on mitochondrial function and distinct immune pathways. The text delineated by the arrows emanating from NAD⁺ boosters show the approaches explored to uncover the effects of this metabolic intervention on immune function.

Mechanisms underpinning these effects have begun to be explored in primary human immune cells. In monocytes and macrophages, NR deacetylates and activates SIRT3, which in turn reduces mitochondrial ROS levels and NLRP3 activation (82). A subsequent study in murine macrophages showed that SIRT3 diminished mtDNA extrusion and blunted intracellular immunogenic triggers thereby dampening NLRP3 inflammasome assembly and activation (51). In healthy volunteers and in subjects with SLE, ex vivo NR similarly blunted monocytic type I interferon (13). This effect functioned via inosine signaling and blunting of autophagy, which in turn attenuated type 1 IFN signaling (13). Furthermmore, oral NR supplementation for 5 days to 12 weeks blunted the NLRP3 inflammasome, in patients with heart failure. This was due to enhancement of mitochondrial function and integrity with amelioration of immunogenic mtDNA extrusion (83, 84). An interesting hypothesis from these studies is that NAD⁺-boosting, by augmenting mitochondrial fidelity (85), may play a broader role in alleviating immune activation in response to aging, and in degenerative diseases (86). In the adaptive immune system, ex vivo NR, via arginine and fumarate biosynthesis activated anti-oxidant transcriptional regulation via NRF2 to blunt inflammation (14). Furthermore, in a phase 3 placebo-controlled double blinded study, oral nicotinamide supplementation for 12 months significantly reduced non-melanoma skin cancer recurrence in high-risk individuals (87). Although, effects on TIL function was not explored.

Finally, an interesting concept being explored is that activation of indoleamine 2,3-deoxygenase 1 (IDO1), an enzyme required for the degradation of tryptophan to kynurenine, within the tumor environment, leads to impaired effector T cell activity (88). The consequence of this, putatively via kynurenine metabolites, orchestrate immunosuppression and tumor immune evasion (89). The role of NAD⁺ biology in this context does not appear to have been well characterized and early IDO1 inhibitor therapy have not been shown to augment tumor immunotherapy (90). Nevertheless, the further evaluation of IDO1 in NAD⁺ biosynthesis and tumor immunomodulation warrant exploration on the role of NAD⁺ metabolism in immunoregulation.

Combining the findings of changes in both NAD⁺ production and consumption pathways in immunity implies that the balance of these processes may be crucial for immune responses and the progression of inflammatory diseases. Indeed, data shows that in SLE, whole blood NAD⁺ levels are elevated at lower disease activity and progressively fall with increasing disease activity (13). Furthermore, brain RNA-seq from patients with multiple sclerosis show that the NAD⁺ consuming enzyme encoding genes CD38 and PARP14, are significantly induced in demyelinating lesions (71), implicating increased NAD⁺ consumption in the disease pathophysiology. Subsequently, pharmacologic inhibition of CD38 in LPS and IFNγ stimulated primary human astrocytes, phenocopied the murine studies and showed that the inhibition of NAD⁺ hydrolysis in these cells, decreased chemokine signaling (72), supporting that astrocytic NAD⁺ biology may play an important role in human neuroinflammation. Furthermore, in a proof of concept study in two patients with life threatening SLE, Daratumumab, which targets CD38, reduced inflammatory signatures and the composite SLEDAI score, supporting that reduced NAD⁺ consumption may have systemic anti-inflammatory effects (91).

Emerging Concepts That Require Further Investigation

Although the current evidence supports important roles of NAD⁺-boosting in immunoregulation, many questions remain unanswered. Some of the major ones are highlighted here: (i) Are different supplements metabolized differently conferring distinct effects and/or tissue/cell targeted effects? (ii) What role do additional NAD⁺ metabolic intermediates such as nicotinic acid riboside play in immunity? (iii) How does intracellular and extracellular NAD⁺ intermediates and NAD⁺ compartmentalization effect immune cell modulation? (iv) Given that subcellular compartements have distinct NAD⁺ levels, and NAD⁺ consumption enzymes have variable K_m values (92), a question arises as to which pathways, and in which compartments, NAD⁺-boosting may be operational? (v) Similarly, how does NAD⁺-boosting modulate distinct subcellular NAD⁺/NADH and NADP⁺/NADPH pools, and can we explore these effects by exploiting the emerging biosensors (93, 94) to characterize this dynamic biology? (vi) How does the interplay between NAD⁺ biosynthetic and consumptive pathways collectively regulate immune function? (vii) Given the emerging role of metabolic intermediates in signaling including succinate, itaconate, ketones inosine and arginine for example, begs the question whether other metabolites play regulatory roles in response to NAD⁺ modulation? (viii) In the same vain, could NAD⁺ biosynthesis intermediates such as kynurenic acid or quinolinic acid also be exploited to modulate immunoregulation? Finally, (ix) Does NAD⁺-boosting display immunomodulatory effects in the presence of canonical immunomodulatory therapies in inflammatory or autoimmune diseases? These questions require additional consideration for the future investigation. Furthermore, as the use of NAD⁺-boosting supplement is increasing in the population, longitudinal studies will need to be undertaken to evaluate whether these interventions have beneficial, neutral, or even possibly adverse longer-term effects on overall health.

Conclusions

The data reviewed here shows that innumerable components of NAD⁺ metabolic pathway orchestrate immune cell function. This complexity is further evident in the myriad of NAD⁺ biosynthesis and consumption pathways, including intracellular and extracellular components and functions. This is evident where depletion of NAD⁺ can prevent immune cell proliferation and differentiation, reduce viability, and blunt distinct immune cell processes such as phagocytosis, in parallel with NAD⁺-boosting blunting inflammation whilst at the same time increasing CTL activity. The importance of NAD⁺ hydrolysis and/or consumption in immune cell signaling is also emerging via generation of metabolic intermediates and through post-translational modifications. Meanwhile, mechanisms underlying beneficial effects of NAD⁺-boosting are being uncovered. These include the modulation of mitochondrial function and fidelity, augmentation of overall bioenergetic capacity, orchestration of immune signaling by distinct metabolic intermediates, epigenetic regulation, and/or posttranslational modifications in response to NAD⁺ consumption. Together these findings underscore the importance in our further understanding of NAD⁺ biology. An imperative of major importance, given that NAD⁺-boosting is an emerging field, in the immunomodulation of inflammatory, degenerative and oncogenic diseases.

Abbreviations in this article:

AD

Alzheimer’s disease

CD38

NAD⁺ glycohydrolase (NADase)

cGAS

cyclic GMP-AMP synthase

CTLs

cytotoxic T lymphocytes

EAE

experimental autoimmune encephalomyelitis

IDO1

indoleamine 2,3-deoxygenase 1

mtDNA

mitochondrial DNA

NA

nicotinic acid

NAM

nicotinamide

NAAD

NA adenine dinucleotide

NAMN

NA mononucleotide

NAD⁺

Nicotinamide adenine dinucleotide

NAMPT

nicotinamide phosphoribosyltransferase

NAR

nicotinic acid riboside

NMN

nicotinamide mononucleotide

NR

nicotinamide riboside

PARP

poly(ADP-ribose) polymerase

STING

stimulator of interferon genes

SARM

sterile alpha and HEAT/Armadillo motif

TCA

tricarboxylic acid cycle

TLR

tumor infiltrating lymphocytes

TLR

toll-like receptors