Background:NAD+ is an important cofactor and second messenger for multiple cellular processes that exhibits antioxidant, anti-apoptotic and anti-inflammatory properties. Pre-clinical studies in animal models of Rheumatoid Arthritis (RA) have demonstrated the therapeutic potential of NAD+ boosters in the control of the disease activity. However, to date no studies have been set up to evaluate the NAD+ metabolism and the therapeutic effects of NAD+ boosters in RA patients.Objectives:1- To study the NAD+ metabolism in RA patients and its association with key clinical features. 2- To evaluate the effect of anti-TNF therapy in the NAD+ metabolism.3- To analyze the beneficial effects of NAD+ boosters in leukocytes from active RA patients.Methods:Plasma and PBMCs were purified from 100 RA patients and 50 healthy donors (HDs). Moreover, an additional cohort of 50 RA patients treated with Anti-TNF therapy was analyzed before and after 6 months of treatment. NAD+ levels were determined by using the NAD/NADH-Glo Assay. NAD+-consuming genes expression were analyzed by RT-PCR. In parallel, PBMCs from eight HDs and eight active RA patients were treated ex vivo with 1 mM of NAD+ boosters including nicotinamide (NAM), nicotinamide riboside (NR), and nicotinamide mononucleotide (NMN). After 24 hours, intracellular reactive oxygen species (ROS) levels (DFCHDA) and the percentage of apoptotic PBMCs (annnexin V/PI) were assessed by flow cytometry. Lastly, a panel of key pro-inflammatory genes were evaluated by RT-PCR.Results:NAD+ and NADH levels were significantly reduced in plasma and PBMCs of RA patients compared with HDs and directly related to disease activity (DAS28, CDAI, SDAI). Accordingly, the expression levels of genes involved in the consumption of NAD+ such as SIRT-1, CD38 and PARP-1 were found up-regulated in PBMCs from RA patients. Anti-TNF therapy for 6 months restored the altered NAD+ levels towards those showed by HDs. Furthermore, the clinical response promoted by Anti-TNF therapy (changes in DAS28) correlated with changes in NAD+ levels. The in vitro treatments of PBMCs isolated from active RA patients with NAD+ boosters significantly increased the NAD+ levels and promoted a deep reduction of intracellular ROS levels, the percentage of apoptotic cells and the expression levels of key inflammatory mediators, such as IL-6, IL-8, IL-1ß, TNF-α, CCL2, IL-23, and STAT-3.Conclusion:1. NAD+ metabolism is altered and associated with the disease activity of RA patients, involving both, reduced NAD+ levels and increased expression of NAD+-consuming genes. 2. Anti-TNF therapy restored NAD+ levels, which were directly linked to the clinical effectiveness. 3. NAD+ boosters reduced the oxidative, apoptotic and inflammatory profiles of RA leukocytes through the parallel increase of intracellular NAD+ levels. Thus, NAD+ boosters might be considered novel therapeutic tools for RA patients.Acknowledgements:Supported by PI18/00837, RIER RD16/0012/0015, RTI2018-100695-B-I00, P18-RT-4264 and CVI276, co-funded with FEDER.Disclosure of Interests:None declared
BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy, tissue fibrosis and activation of the innate and adaptive immune system. Clinical features of the disease consist of skin thickening and internal organ involvement. Due to the heterogeneous nature of the disease, there is an unmet need of biomarkers for diagnosis, disease progression and response to treatment.ObjectivesThe aim of this study was to explore new serum proteomic fingerprints of clinically defined forms of SSc.MethodsHighly specific detection of nighty two proteins from a panel related to organ damage was performed, by using the breakthrough technology proximity extension immunoassay (PEA, Olink), in the serum of 72 patients with SSC and 18 age-matched healthy donors (HD). Main disease complications in the SSC cohort, including lung fibrosis, skin fibrosis, renal, vascular, and esophageal involvement were assessed, and prevalence of circulating autoantibodies was tested, along with standard demographic and inflammatory parameters. Unsupervised hierarchical clustering methodologies were applied to identify subgroups of patients based on their proteomic profiles. Gene ontology enrichment was used to interrogate the biological meaning of the distinctive molecular signatures identified.ResultsSixteen circulating proteins related to organ damage were coordinately altered in the serum of SSc patients in relation to HD. Unsupervised clustering analyses differentiated 3 patients clusters presenting different proteomic profiles. Clinically, patients belonging to cluster 1 were characterized by a significant prevalence of multiple organ involvement (84%) in relation to clusters 2 (52%) and 3 (43%), mostly encompassing lung and skin fibrosis and esophageal dysmotility. Immunologically, cluster 1 further displayed the highest percentage of positivity for anti-scl70 antibodies.Nineteen serum proteins, not previously reported in the serum of SSC patients (BANK1, BID, CALR, ERBB2IP, FGR, FOSB, FOXO1, INPPL1, MAEA, MAGED1, MAP4K5, NBN, NCF2, PRKAB1, RASSF2, RCOR1, SMAD1, STXBP3, VASH1) were found deregulated between clusters, with a significant increase in the levels of all of them in cluster 1 compared with clusters 2 and 3. These deregulated proteins were mostly involved in biological processes such as cell proliferation, apoptosis, cell adhesion, migration, and immune response. Among them, two were functionally linked with cutaneous diseases [Calreticulin (CALR) and B-cell scaffold protein with ankryn repeats (BANK1)], two with digestive disorders (Tyrosine-protein kinase Fgr (FGR) and syntaxin-binding protein 3 (STXBP3)] and three with lung disfunction [protein FosB (FOSB), mothers against decapentaplegic homolog 1 (SMAD1) and forkhead box protein O1 (FOXO1)]. Interestingly, levels of some overexpressed proteins in C1 [BH3-interacting domain death agonist (BID), phosphatidylinositol 3,4,5-triphosphate 5-phosphatase 2 (INPPL1), Erbin (ERBB2IP), BANK1 and FOSB] were further related to the positivity for anti-scl70, the specific SSC-autoantibody known to be mostly associated to a bad prognosis and multiple organ involvement in SSC patients.Conclusion:1) Stratification based on serum proteomic profile could be of use for a better clinical classification of SSc patients, adding new insights to the underlying pathophysiological mechanisms.2) Combination of disease classifying autoantibodies with principal pathophysiological processes and serum proteomic profiles can help to elucidate and strengthen the diagnosis as well as the prognosis in SSC.AcknowledgementsSupported by ISCIII (RICOR-RD21/0002/0033) co-financed with FEDER.Disclosure of InterestsNone declared.
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