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Background: The nicotinamide adenine dinucleotide (NAD + ) pathway plays a key role in essential biological processes and is closely linked to the main pathophysiological mechanisms of Chronic Inflammatory Rheumatic Diseases (CIRDs). These disorders are characterized by features of accelerated aging, including inflammation, oxidative stress, and immune response. Despite this strong biological relevance, the role of NAD + metabolism in CIRDs remains poorly characterized and warrants further investigation.
NAD + biosynthesis depends on multiple forms of vitamin B3, known as NAD + boosters, which have demonstrated therapeutic potential in several pathological conditions. However, their efficacy and impact in CIRDs have not yet been fully evaluated.
Objectives: This study aims to:
Characterize the NAD + metabolome in CIRDs by analyzing the levels of key pathway components and their association with clinical traits.
Evaluate the impact of 6-month anti-TNF therapy on NAD + metabolome alterations in these patients.
Test the preclinical potential of a broad group of NAD + boosters to restore NAD + pathway alterations and promote anti-inflammatory effects.
Assess the molecular effects of three months of oral nicotinamide (NAM) supplementation in patients with rheumatoid arthritis.
Methods: Whole blood samples and associated clinical data were collected from a total of 310 individuals, including 70 healthy donors (HDs), 80 patients with rheumatoid arthritis (RA), 80 with psoriatic arthritis (PsA), and 80 with spondyloarthritis (SpA). A panel of key metabolites from the NAD + pathway, including NAD + , NADH, NAM, NMN, NR, NADP and Tryp, was quantified using two-dimensional Nuclear Magnetic Resonance (DOSY-NMR) technology (Biosfer).
A longitudinal analysis was performed in 90 patients with CIRDs, 30 from each disease group, to assess therapy-induced changes in the NAD + metabolome before and after 6 months of anti-TNF treatment.
Peripheral blood mononuclear cells (PBMCs) isolated from 45 patients with CIRD (15 from each disease group) were treated ex-vivo with 1 nM of established and emerging NAD + boosters, including NAM, NR, NMN, NRT, NRH, and NAR, for 24 hours. Intracellular NAD + and NADH levels were measured using the NAD/NADH-Glo Assay Kit (Promega). In parallel, secreted inflammatory mediators were assessed using the Olink Target 96 Inflammation Panel (Cobiomic).
Finally, an interventional pilot study was performed in which ten patients with RA with moderate disease activity, receiving stable methotrexate therapy for at least three months, were administered oral nicotinamide (NAM) supplementation (500 mg twice daily) for three months. Peripheral blood samples were collected before and after supplementation to evaluate oxidative status and cellular metabolism using flow cytometry–based assays and functional metabolic profiling, including Seahorse extracellular flux analysis.
Results: RA, SpA, and PsA patients exhibited a significant reduction in the NAD + /NADH ratio, along with increased levels of NAM and NMN compared to HDs. Additionally, NAD + levels were reduced in RA, whereas NADH levels were elevated in SpA and PsA. Notably, SpA patients showed a decrease in NADP levels and an increase in Trp levels. These findings underscore disease-specific disruptions in NAD + metabolism, which may contribute to chronic inflammation and immune dysfunction.
The disease-specific alterations in the NAD + metabolome were directly associated with distinct clinical features across CIRDs. Anti-TNF therapy for 6 months induced disease-specific changes in NAD + metabolites, with distinct patterns observed across different conditions. Overall, the treatment appeared to restore NAD + metabolite levels closer to those observed in HDs, suggesting a potential role in rebalancing disrupted metabolic pathways.
All NAD + boosters significantly increased intracellular NAD + levels in PBMCs from all three diseases, although the response depended on the specific booster and disease type. Notably, NRH demonstrated the most potent effect in raising NAD + levels. The boosters also showed anti-inflammatory potential by reducing the secretion of key inflammatory mediators, with effects varying according to the booster and pathology, further highlighting their therapeutic potential in modulating disease-related inflammation.
Among the diseases analyzed, RA was selected to assess the in vivo effects of NAD + boosting through nicotinamide supplementation. At baseline, RA patients exhibited increased oxidative stress and enhanced basal mitochondrial respiration compared with healthy controls. Both parameters were significantly reduced after three months of treatment, supporting a beneficial physiological modulation of mitochondrial metabolism and redox homeostasis.
Conclusions: The NAD + metabolome is profoundly altered in CIRDs, characterized by disease-specific disruption patterns and associated with key clinical features.
Anti-TNF therapy partially restored NAD + metabolite levels toward those observed in HDs, suggesting a role for metabolic rebalancing in the therapeutic response.
Preclinical testing of NAD + boosters demonstrated their ability to effectively enhance intracellular NAD + levels and reduce inflammatory mediators, with both shared and disease-specific effects.
Oral nicotinamide supplementation restores mitochondrial and redox balance in rheumatoid arthritis, supporting NAD + boosting as a promising therapeutic approach.
Overall, dysregulation of NAD + metabolism emerges as a key feature of chronic inflammatory rheumatic diseases with clear therapeutic potential.
Supported by: CPS: MICIU (RYC2021-033828-I; PID2022-141500OA-I00); FAR2023-24; JA (PIP-0149-2024); AEI (DIN2022-012766). CLP: ISCIII (PI21/0591, PI24/00959, CD21/00187 y RICOR-21/0002/0033). JMV: RTI2018-100695-B-I00, PID2021-126280OB-I00, P18-RT-4264, 1263735-R, BIO-276 and PRE2019-08743. AEC: ISCIII (ICI23/00100). CLM, ECE y NBP: ISCIII(PI22/00539 y PMP21/00119); JA (I-0243-2022); MICIU (PID2023-152503OB). Cofinanciados por la UE.
REFERENCES: NIL.
Acknowledgments: NIL.
Disclosure of Interests: None declared.