The Extracellular NADome Modulates Immune Responses

Abstract

The term NADome refers to the intricate network of intracellular and extracellular enzymes that regulate the synthesis or degradation of nicotinamide adenine dinucleotide (NAD) and to the receptors that engage it. Traditionally, NAD was linked to intracellular energy production through shuffling electrons between oxidized and reduced forms. However, recent data indicate that NAD, along with its biosynthetic and degrading enzymes, has a life outside of cells, possibly linked to immuno-modulating non-enzymatic activities. Extracellular NAD can engage puriginergic receptors triggering an inflammatory response, similar - to a certain extent - to what described for adenosine triphosphate (ATP). Likewise, NAD biosynthetic and degrading enzymes have been amply reported in the extracellular space, where they possess both enzymatic and non-enzymatic functions. Modulation of these enzymes has been described in several acute and chronic conditions, including obesity, cancer, inflammatory bowel diseases and sepsis. In this review, the role of the extracellular NADome will be discussed, focusing on its proposed role in immunomodulation, together with the different strategies for its targeting and their potential therapeutic impact.

Keywords: DAMPs; NAD; immune cell regulation; immunometabolism; nucleotides; signaling; tumor microenvironment.

Figure 1

Schematic representation of the NADome. Schematic representation of the network of substrates/ligands, NAD-metabolizing cell surface and intracellular enzymes and their products in the extracellular and intracellular space. Biological functions regulated by NAD-related enzymes and products are listed. NAD, nicotinamide adenine dinucleotide; NADP, NAD phosphate; eNAD, extracellular NAD; Nam, nicotinamide; NR, nicotinamide riboside; Na, nicotinic acid; NAMPT, nicotinamide phosphoribosyltransferase; NAPRT, nicotinate phosphoribosyltransferase; NRK, nicotinamide riboside kinase; ARTs, mono adenosine diphosphate (ADP)-ribose transferases; PARPs, poly ADP-ribose polymerases; ADPR, ADP ribose; cADPR, cyclic ADP ribose; NAADP, nicotinic acid adenine dinucleotide phosphate; Ca²⁺, calcium; NMN, nicotinamide mononucleotide; ADO, adenosine; AMP, adenosine monophosphate; ENPP1; ectonucleotide pyrophosphatase/phosphodiesterases; TLR4, toll-like receptor 4.

Figure 2

eNAD and purinergic signaling. Pathological or physiological stimuli, including hypoxic and inflammatory conditions, metabolic and cellular stress, promote the release of NAD from the cell. NAD can then bind to P2X (isoforms P2X1, P2X4, P2X7) and P2Y (isoforms P2Y1, P2Y11), activating several intracellular signaling and modulating immune responses.

Figure 3

eNAD and enzymatic/functional extracellular machinery in regulating immune responses. Extracellular NAD can also be metabolized by a series of enzymes of the cell surface that are involved in scavenging of nucleotides. The end product of the reaction, adenosine, can then be internalized and reconverted to related nucleosides (e.g., ATP or NAD). In particular, CD38 hydrolyzes NAD to generate intermediates (cADPR and ADPR), potent intracellular Ca²⁺-mobilizing agents, through binding RyR or TRPM2 receptors. CD38 activation induces migration, proliferation, and modulation of immune responses, specifically T cell functions, as detailed in the text. In addition, CD38 activity releases nicotinamide (the main NAD precursor) that can be internalized into the cell and, together with a second precursor nicotinic acid, converted in NAD by NAMPT and NAPRT activities, respectively, increasing intracellular NAD levels and affecting sirtuins and PARPs (NAD-consuming enzymes) functions. NAMPT and NAPRT can be secreted/released in the extracellular space acting as cytokine-like proteins. Finally, NAD and ATP can be converted in adenosine. ATP is metabolized by CD39 to AMP that is further hydrolyzed by ecto-5’-nucleotidase/CD73 which promotes the formation of adenosine. Adenosine then activates adenosine receptors (purinergic receptor P1). The final outcome depends on the relative concentrations of substrates and products and on the expression of nucleotide-metabolizing ecto-enzymes and NAD-biosynthetic enzymes. NAD, nicotinamide adenine dinucleotide; NADP, NAD phosphate; NAMPT, nicotinamide phosphoribosyltransferase; NAPRT, nicotinate phosphoribosyltransferase; PARPs, poly ADP-ribose polymerases; ADPR, ADP ribose; cADPR, cyclic ADP ribose; NAADP, nicotinic acid adenine dinucleotide phosphate; ATP, adenosine triphosphate; AMP, adenosine monophosphate; P1, adenosine purinergic receptor; RYR, ryanodine receptors; TRPM2, transient receptor potential melastatin-related 2; i, intracellular; e, extracellular.

Figure 4

CD38, CD73 and NAMPT as markers and therapeutic targets in pathological conditions. CD38, CD73 and NAMPT expression levels increase in several pathological conditions. These molecules become markers of aggressive disease. In the lower part of the cartoon specific pharmacological inhibitors and/or blocking antibodies, currently in preclinical or clinical trials for each target are listed.