Preclinical Characterization of Pharmacologic NAD+ Boosting as a Promising Therapeutic Approach in Rheumatoid Arthritis.

OBJECTIVE: We analyzed NAD+ metabolism in patients with rheumatoid arthritis (RA), its association with disease activity and clinical outcomes of RA, and the therapeutic potential of pharmacologic NAD+ boosting. METHODS: Our study included 253 participants. In the first cohort, comprising 153 RA patients and 56 healthy donors, we assessed NAD+ levels and NAD+ -related gene pathways. We analyzed 92 inflammatory molecules by proximity extension assay. In the second cohort, comprising 44 RA patients starting anti-tumor necrosis factor (anti-TNF) drugs, we evaluated changes in NAD+ levels and their association with clinical response after 3 months. Mechanistic studies were performed ex vivo on peripheral blood mononuclear cells (PBMCs) from patients with RA to test the beneficial effects of NAD+ boosters, such as nicotinamide and nicotinamide riboside. RESULTS: Reduced NAD+ levels were found in RA samples, in line with altered activity and expression of genes involved in NAD+ consumption (sirtuins, poly[ADP-ribose] polymerase, CD38), transport (connexin 43), and biosynthesis (NAMPT, NMNATs). Unsupervised clustering analysis identified a group of RA patients with the highest inflammatory profile, the lowest NAD+ levels, and the highest disease activity (as shown by the Disease Activity Score in 28 joints). NAD+ levels were modulated by anti-TNF therapy in parallel with the clinical response. In vitro studies using PBMCs from RA patients showed that nicotinamide riboside and nicotinamide increased NAD+ levels via NAMPT and NMNAT and reduced their prooxidative, proapoptotic, and proinflammatory status. CONCLUSION: RA patients display altered NAD+ metabolism, directly linked to their inflammatory and disease activity status, which was reverted by anti-TNF therapy. The preclinical beneficial effects of NAD+ boosters, as shown in leukocytes from RA patients, along with their proven clinical safety, might pave the way for the development of clinical trials using these compounds.

Achilles enthesitis on physical examination leads to worse outcomes after 2 years of follow up in patients with ankylosing spondylitis from REGISPONSER-AS registry.

BACKGROUND: Enthesitis represents one of the most important peripheral musculoskeletal manifestations in patients with axial spondyloarthritis (axSpA). However, studies specifically evaluating Achilles tendon enthesitis and its impact over time are scarce. The objectives of this study were to evaluate the impact of Achilles' tendon enthesitis found at baseline during physical examination on the outcome measures after 2 years of follow-up in patients with ankylosing spondylitis (AS). METHODS: This was an observational and prospective study conducted during 2 years of follow-up in the REGISPONSER-AS registry. Linear regression models adjusted for age, body mass index (BMI), and anti-TNF intake were conducted to evaluate the association between the presence of Achilles enthesitis at baseline and the patient-reported outcome (PRO) scores at baseline. The impact of this feature on PROs over 2 years of follow-up was evaluated using mixed models for repeated measures adjusted for age, BMI, and anti-TNF intake. RESULTS: Among the 749 patients included, 46 patients (6.1%) showed Achilles' tendon enthesitis during physical examination at the baseline study visit. Patients with Achilles enthesitis had an increase in the global VAS score, BASDAI, mBASDAI, ASDAS-CRP, and BASFI scores in comparison with patients without this feature. In addition, the mean global VAS, BASDAI, and ASDAS-CRP scores were significantly higher among patients with Achilles enthesitis over the 2 years of follow-up after adjusting for age, BMI, and current anti-TNF intake. The percentage of patients achieving ASDAS low disease activity (ASDAS < 2.1) after 2 years of follow-up was 15.9% and 31.5% for patients with and without Achilles enthesitis, respectively (p = 0.030). CONCLUSIONS: In patients with AS, the presence of Achilles' tendon enthesitis was associated with worse scores on the outcome measures after 2 years of follow-up, leading to a lower probability of achieving low disease activity.

Subclinical Atherosclerosis Measure by Carotid Ultrasound and Inflammatory Activity in Patients with Rheumatoid Arthritis and Spondylarthritis.

OBJECTIVE: To compare the effect of inflammation on subclinical atherosclerosis using carotid ultrasound in patients with rheumatoid arthritis (RA) and spondyloarthritis (SpA). METHODS: Cross-sectional study including 347 participants (148 RA, 159 SpA, and 40 controls). We measured the carotid intima media thickness (cIMT) and detection of atheromatous plaques using carotid ultrasound. We recorded disease activity (DAS28-CRP/ASDAS-CRP) and traditional cardiovascular risk factors. We performed descriptive, bivariate, and linear multivariate analyses (dependent variable: cIMT) to evaluate the influence of diagnosis on cIMT in all patients. Two additional multivariate analyses were performed by stratifying patients according to their inflammatory activity. RESULTS: cIMT correlated with the mean CRP during the previous 5 years in RA, but not with CRP at the cut-off date. We did not find such differences in patients with SpA. The first multivariate model revealed that increased cIMT was more common in patients with RA than in those with SpA (ß coefficient, 0.045; 95% confidence interval (95% CI), 0.0002-0.09; p = 0.048) after adjusting for age, sex, disease course, and differential cardiovascular risk factors (arterial hypertension, smoking, statins, and corticosteroids). The second model revealed no differences in cIMT between the 2 groups of patients classified as remission-low activity (ß coefficient, 0.020; 95% CI, -0.03 to 0.080; p = 0.500). However, when only patients with moderate-high disease activity were analysed, the cIMT was 0.112 mm greater in those with RA (95% CI, 0.013-0.212; p = 0.026) than in those with SpA after adjusting for the same variables. CONCLUSIONS: Subclinical atherosclerosis measured by carotid ultrasound in patients with RA and SpA is comparable when the disease is well controlled. However, when patients have moderate-high disease activity, cIMT is greater in patients with RA than in those with SpA after adjusting for age, sex, disease course, and cardiovascular risk factors. Our results point to greater involvement of disease activity in subclinical atherosclerosis in patients with RA than in those with SpA.

Integrative Clinical, Molecular, and Computational Analysis Identify Novel Biomarkers and Differential Profiles of Anti-TNF Response in Rheumatoid Arthritis.

Background: This prospective multicenter study developed an integrative clinical and molecular longitudinal study in Rheumatoid Arthritis (RA) patients to explore changes in serologic parameters following anti-TNF therapy (TNF inhibitors, TNFi) and built on machine-learning algorithms aimed at the prediction of TNFi response, based on clinical and molecular profiles of RA patients. Methods: A total of 104 RA patients from two independent cohorts undergoing TNFi and 29 healthy donors (HD) were enrolled for the discovery and validation of prediction biomarkers. Serum samples were obtained at baseline and 6 months after treatment, and therapeutic efficacy was evaluated. Serum inflammatory profile, oxidative stress markers and NETosis-derived bioproducts were quantified and miRNomes were recognized by next-generation sequencing. Then, clinical and molecular changes induced by TNFi were delineated. Clinical and molecular signatures predictors of clinical response were assessed with supervised machine learning methods, using regularized logistic regressions. Results: Altered inflammatory, oxidative and NETosis-derived biomolecules were found in RA patients vs. HD, closely interconnected and associated with specific miRNA profiles. This altered molecular profile allowed the unsupervised division of three clusters of RA patients, showing distinctive clinical phenotypes, further linked to the TNFi effectiveness. Moreover, TNFi treatment reversed the molecular alterations in parallel to the clinical outcome. Machine-learning algorithms in the discovery cohort identified both, clinical and molecular signatures as potential predictors of response to TNFi treatment with high accuracy, which was further increased when both features were integrated in a mixed model (AUC: 0.91). These results were confirmed in the validation cohort. Conclusions: Our overall data suggest that: 1. RA patients undergoing anti-TNF-therapy conform distinctive clusters based on altered molecular profiles, which are directly linked to their clinical status at baseline. 2. Clinical effectiveness of anti-TNF therapy was divergent among these molecular clusters and associated with a specific modulation of the inflammatory response, the reestablishment of the altered oxidative status, the reduction of NETosis, and the reversion of related altered miRNAs. 3. The integrative analysis of the clinical and molecular profiles using machine learning allows the identification of novel signatures as potential predictors of therapeutic response to TNFi therapy.