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.

Impaired microRNA processing in neutrophils from rheumatoid arthritis patients confers their pathogenic profile. Modulation by biological therapies.

The aim of this study was to investigate the microRNA (miRNA) expression pattern in neutrophils from rheumatoid arthritis (RA) patients and its contribution to their pathogenic profile and to analyze the effect of specific autoantibodies or inflammatory components in the regulation of miRNA in RA neutrophils and its modulation by biological therapies. Neutrophils were isolated from paired peripheral blood (PB) and synovial fluid samples of 40 patients with RA and from PB of 40 healthy donors. A miRNA array was performed using nCounter technology. Neutrophils from healthy donors were treated in vitrowith antibodies to citrullinated protein antigens isolated from RA patients and tumor necrosis factor-a (TNF-a) or interleukin-6. A number of cytokines and chemokines were analyzed. In vitro treatments of RA-neutrophils with tocilizumab or infliximab were carried out. Transfections with pre-miRNA and DICER downregulation experiments were further performed. RA-neutrophils showed a global downregulation of miRNA and genes involved in their biogenesis, alongside with an upregulation of various potential mRNA targets related to migration and inflammation. Decreased levels of miRNA and DICER correlated with autoimmunity, inflammation and disease activity. Citrullinated protein antigens and TNF-a decreased the expression of numerous miRNA and their biogenesis-related genes, increasing their potential mRNA targets. Infliximab reversed those effects. Transfections with pre-miRNA-223, -126 and -148a specifically modulated genes regulating inflammation, survival and migration whereas DICER depletion influenced the inflammatory profile of neutrophils. Taken together RA-neutrophils exhibited a global low abundance of miRNA induced by autoantibodies and inflammatory markers, which potentially contributed to their pathogenic activation. miRNA biogenesis was significantly impaired in RAneutrophils and further associated with a greater downregulation of miRNA mainly related to migration and inflammation in synovial fluid neutrophils. Finally, anti-TNF-a and anti-interleukin-6 receptor treatments can modulate miRNA levels in the neutrophils, minimizing their inflammatory profile.