Background/Purpose: This study aimed to: 1- Evaluate the levels of NAD⁺ and gene transcripts involved in its metabolism in Rheumatoid Arthritis (RA) patients. 2- Analyse the effect of anti-TNF therapy in the NAD⁺ metabolism. 3- Explore mechanistically the anti-inflammatory effects of NAD⁺ boosters in RA leukocytes.

Methods: Plasma and PBMCs were purified from 100 RA patients and 50 healthy donors (HDs). NAD⁺ levels were tested by NAD/NADH-Glo Assay. Gene expression analysis of markers related to the synthesis (NAMPT, NMNATs, etc), transplasma membrane transport (Cx43), and consumption (PARPs, SIRTs, CD38, etc) of NAD⁺ were performed in PBMCs by RT-PCR. Public datasets from GEO repository were used as validation. A panel of 92 inflammatory mediators from Olink was also analyzed in the RA serum. In a second cohort of 50 RA patients treated with anti-TNF therapy, NAD⁺ levels were analyzed before and after 6 months. PBMCs from a third cohort of 10 active RA patients were treated ex vivo with NAD⁺ boosters [nicotinamide (NAM) and nicotinamide riboside (NR)] and inhibitors of NAMPT (FK866) and Cx43 (GAP19). Olink inflammatory panel was used to evaluate anti-inflammatory effects.

Results: NAD⁺ levels were significantly reduced in the plasma of RA patients while the expression of genes involved in the consumption (PARPs, SIRTs, CD38, CD157) and biosynthesis (NMNATs) of NAD⁺ were increased and reduced respectively in RA PBMCs. NAMPT and Cx43 were also upregulated in those patients. The results were validated in an independent dataset.

Unsupervised clustering analysis of the inflammatory proteome identified 3 clusters of patients: C1 showed the highest levels of inflammatory mediators and disease activity (DAS28) and the lowest NAD⁺ levels; C3 displayed reduced levels of inflammatory mediators and DAS28 and high NAD⁺ levels. C2 showed intermediate profiles.

Anti-TNF restored altered NAD⁺ levels in RA patients to those exhibited by HD. Moreover, changes in DAS28 and NAD⁺ were correlated. In vitro, NAD⁺ boosters promoted a significant increase in NAD⁺ levels of RA PBMCs. FK866 treatment highlighted a key role for NAMPT in the generation of NAD⁺ promoted by NAM. The inhibition of the extracellular transport of NAD⁺ with GAP19 further increased the intracellular NAD⁺ content in the presence of NAM and NR. Both boosters reduced the levels of numerous secreted inflammatory mediators in the supernatant, which was enhanced by effect of GAP19. In parallel, the gene expression of key inflammatory mediators was deeply reduced along with markers of oxidative stress and apoptosis.

Conclusion: 1- The NAD⁺ metabolism is altered in RA patients and directly linked to their inflammatory profile and disease activity. 2- Anti-TNF therapy restore NAD⁺ altered levels in line with the clinical response. 3- NR and NAM increase the NAD⁺ levels in PBMCs from active RA patients promoting an extensive anti-inflammatory effect which can be further enhanced through Cx43 inhibition.

NAD⁺ boosters might be considered a novel therapeutic approach to reduce inflammation along with the available therapies in RA.

Supported by ISCIII (PI21/005991 and RICOR-RD21/0002/0033) co-financed by FEDER; CTEICU, JA (P20_01367); RTI2018-100695-B-I00, P18-RT-4264, CVI276, PRE2019-087438.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/restoration-of-nad-levels-in-rheumatoid-arthritis-with-nad-boosters-as-a-novel-therapeutic-strategy-to-resolve-acute-inflammation/