The clinical and molecular cardiometabolic fingerprint of an exploratory psoriatic arthritis cohort is associated with the disease activity and differentially modulated by methotrexate and apremilast.

OBJECTIVES: (1) To evaluate clinical and molecular cardiovascular disease (CVD) signs and their relationship with psoriatic arthritis (PsA) features and (2) to identify a clinical patient profile susceptible to benefit from methotrexate (MTX) and/or apremilast regarding CVD risk. METHODS: This cross-sectional study included 100 patients with PsA and 100 age-matched healthy donors. In addition, an exploratory cohort of 45 biologically naïve patients treated for 6 months with apremilast, MTX or combined therapy according to routine clinical practice was recruited. Extensive clinical and metabolic profiles were obtained. Ninety-nine surrogate CVD-related molecules were analysed in plasma and peripheral blood mononuclear cells (PBMCs). Hard cluster analysis was performed to identify the clinical and molecular phenotypes. Mechanistic studies were performed on adipocytes. RESULTS: Cardiometabolic comorbidities were associated with disease activity and long-term inflammatory status. Thirty-five CVD-related proteins were altered in the plasma and PBMCs of PsA patients and were associated with the key clinical features of the disease. Plasma levels of some of the CVD-related molecules might distinguish insulin-resistant patients (MMP-3, CD163, FABP-4), high disease activity (GAL-3 and FABP-4) and poor therapy outcomes (CD-163, LTBR and CNTN-1). Hard cluster analysis identified two phenotypes of patients according to the rates of cardiometabolic comorbidities with distinctive clinical and molecular responses to each treatment. CONCLUSIONS: (1) Novel CVD-related proteins associated with clinical features could be emerging therapeutic targets in the context of PsA and (2) the pleiotropic action of apremilast could make it an excellent choice for the management of PsA patients with high CVD risk, targeting metabolic alterations and CVD-related molecules.

Splicing machinery is impaired in rheumatoid arthritis, associated with disease activity and modulated by anti-TNF therapy.

OBJECTIVES: To characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response. METHODS: Leucocyte subtypes from 129 patients with RA and 29 healthy donors (HD) were purified, and 45 selected SM elements (SME) were evaluated by quantitative PCR-array based on microfluidic technology (Fluidigm). Modulation by anti-tumour necrosis factor (TNF) therapy and underlying regulatory mechanisms were assessed. RESULTS: An altered expression of several SME was found in RA leucocytes. Eight elements (SNRNP70, SNRNP200, U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1 and SRSF10) were equally altered in all leucocytes subtypes. Logistic regressions revealed that this signature might: discriminate RA and HD, and anti-citrullinated protein antibodies (ACPAs) positivity; classify high-disease activity (disease activity score-28 (DAS28) >5.1); recognise radiological involvement; and identify patients showing atheroma plaques. Furthermore, this signature was altered in RA synovial fluid and ankle joints of K/BxN-arthritic mice. An available RNA-seq data set enabled to validate data and identified distinctive splicing events and splicing variants among patients with RA expressing high and low SME levels. 3 and 6 months anti-TNF therapy reversed their expression in parallel to the reduction of the inflammatory profile. In vitro, ACPAs modulated SME, at least partially, by Fc Receptor (FcR)-dependent mechanisms. Key inflammatory cytokines further altered SME. Lastly, induced SNRNP70-overexpression and KHDRBS1-overexpression reversed inflammation in lymphocytes, NETosis in neutrophils and adhesion in RA monocytes and influenced activity of RA synovial fibroblasts. CONCLUSIONS: Overall, we have characterised for the first time a signature comprising eight dysregulated SME in RA leucocytes from both peripheral blood and synovial fluid, linked to disease pathophysiology, modulated by ACPAs and reversed by anti-TNF therapy.

Molecular Changes in the Adipose Tissue Induced by Rheumatoid Arthritis: Effects of Disease-Modifying Anti-Rheumatic Drugs.

Disease severity, progression and response to therapy might be worse in obese rheumatoid arthritis (RA) patients, but paradoxically, obesity also might protect from radiographic joint damage. Thus, the intricate relationship between obesity and RA needs urgent clarification. The aim of this study was to assess the influence of obesity on the onset and development of RA and to determine whether arthritis could modify the adipose tissue biology and whether conventional Disease Modifying Anti-Rheumatic Drugs (cDMARDs) can modulate these alterations. Two strategies were followed: (1) clinical profiling of two cohorts of RA: non-obese and obese patients; and (2) mechanistic studies carried out in both a collagen-induced arthritis (CIA) in an obese mouse model and 3T3-L1 adipocytes treated with cDMARDs (leflunomide, methotrexate, and hydroxychloroquine). In our cohort of RA patients with low-moderate disease activity, the presence of obesity was not related to a higher activity of the disease; actually, disease activity score 28-erythrocyte sedimentation rate (DAS28-ESR) was reduced in the obese RA patients. However, the induction of arthritis promoted transcriptomic changes in the adipose tissue under obesity condition in the obese CIA model. Treatment with hydroxychloroquine reduced weight and insulin resistance, accompanied by beneficial metabolic effects in the adipose tissue. These molecular changes in adipose tissue were also observed after methotrexate administration. In sum, arthritis might affect directly the inflammatory burden and metabolic alterations associated with obesity in adipose tissue. Clinicians should be cautious measuring the activity of the disease in obesity and managing the best therapeutic options for the metabolic comorbidities of these patients, where the combination of hydroxychloroquine and methotrexate should be considered to improve adipose tissue dysfunction in obese RA.

Expression of DDX11 and DNM1L at the 12p11 Locus Modulates Systemic Lupus Erythematosus Susceptibility.

OBJECTIVES: This study employed genetic and functional analyses using OASIS meta-analysis of multiple existing GWAS and gene-expression datasets to identify novel SLE genes. METHODS: Four hundred and ten genes were mapped using SNIPPER to 30 SLE GWAS loci and investigated for expression in three SLE GEO-datasets and the Cordoba GSE50395-dataset. Blood eQTL for significant SNPs in SLE loci and STRING for functional pathways of differentially expressed genes were used. Confirmatory qPCR on SLE monocytes was performed. The entire 12p11 locus was investigated for genetic association using two additional GWAS. Expression of 150 genes at this locus was assessed. Based on this significance, qPCRs for DNM1L and KRAS were performed. RESULTS: Fifty genes were differentially expressed in at least two SLE GEO-datasets, with all probes directionally aligned. DDX11, an RNA helicase involved in genome stability, was downregulated in both GEO and Cordoba datasets. The most significant SNP, rs3741869 in OASIS locus 12p11.21, containing DDX11, was a cis-eQTL regulating DDX11 expression. DDX11 was found repressed. The entire 12p11 locus showed three association peaks. Gene expression in GEO datasets identified DNM1L and KRAS, besides DDX11. Confirmatory qPCR validated DNM1L as an SLE susceptibility gene. DDX11, DNM1L and KRAS interact with each other and multiple known SLE genes including STAT1/STAT4 and major components of IFN-dependent gene expression, and are responsible for signal transduction of cytokines, hormones, and growth-factors, deregulation of which is involved in SLE-development. CONCLUSION: A genomic convergence approach with OASIS analysis of multiple GWAS and expression datasets identified DDX11 and DNM1L as novel SLE-genes, the expression of which is altered in monocytes from SLE patients. This study lays the foundation for understanding the pathogenic involvement of DDX11 and DNM1L in SLE by identifying them using a systems-biology approach, while the 12p11 locus harboring these genes was previously missed by four independent GWAS.

Anti-dsDNA Antibodies Increase the Cardiovascular Risk in Systemic Lupus Erythematosus Promoting a Distinctive Immune and Vascular Activation.

Objective: Systemic lupus erythematosus (SLE) is associated to boosted atherosclerosis development and a higher cardiovascular disease risk. This study aimed to delineate the role of anti-double stranded DNA (anti-dsDNA) antibodies on the molecular profile and the activity of immune and vascular cells, as well as on their enhanced cardiovascular risk. Approach and Results: Eighty SLE patients were included. Extensive clinical/analytical evaluation was performed, including cardiovascular disease parameters (endothelial function, proatherogenic dyslipidemia, and carotid intima-media thickness). Gene and protein expression profiles were evaluated in monocytes from patients diagnosed positive or negative for anti-dsDNA antibodies by using NanoString and cytokine arrays, respectively. NETosis and circulating inflammatory profile was assessed in both neutrophils and plasma. Positivity and persistence of anti-dsDNA antibodies in SLE patients were associated to endothelial dysfunction, proatherogenic dyslipidemia, and accelerated atherosclerosis. In parallel, anti-dsDNA antibodies were linked to the aberrant activation of innate immune cells, so that anti-dsDNA(+) SLE monocytes showed distinctive gene and protein expression/activity profiles, and neutrophils were more prone to suffer NETosis in comparison with anti-dsDNA(−) patients. Anti-dsDNA(+) patients further displayed altered levels of numerous circulating mediators related to inflammation, NETosis, and cardiovascular risk. In vitro, Ig-dsDNA promoted NETosis on neutrophils, apoptosis on monocytes, modulated the expression of inflammation and thrombosis-related molecules, and induced endothelial activation, at least partially, by FcR (Fc receptor)-binding mechanisms. Conclusions: Anti-dsDNA antibodies increase the cardiovascular risk of SLE patients by altering key molecular processes that drive a distinctive and coordinated immune and vascular activation, representing a potential tool in the management of this comorbidity.

Therapeutic Potential and Immunomodulatory Role of Coenzyme Q10 and Its Analogues in Systemic Autoimmune Diseases.

Coenzyme Q10 (CoQ10) is a mitochondrial electron carrier and a powerful lipophilic antioxidant located in membranes and plasma lipoproteins. CoQ10 is endogenously synthesized and obtained from the diet, which has raised interest in its therapeutic potential against pathologies related to mitochondrial dysfunction and enhanced oxidative stress. Novel formulations of solubilized CoQ10 and the stabilization of reduced CoQ10 (ubiquinol) have improved its bioavailability and efficacy. Synthetic analogues with increased solubility, such as idebenone, or accumulated selectively in mitochondria, such as MitoQ, have also demonstrated promising properties. CoQ10 has shown beneficial effects in autoimmune diseases. Leukocytes from antiphospholipid syndrome (APS) patients exhibit an oxidative perturbation closely related to the prothrombotic status. In vivo ubiquinol supplementation in APS modulated the overexpression of inflammatory and thrombotic risk-markers. Mitochondrial abnormalities also contribute to immune dysregulation and organ damage in systemic lupus erythematosus (SLE). Idebenone and MitoQ improved clinical and immunological features of lupus-like disease in mice. Clinical trials and experimental models have further demonstrated a therapeutic role for CoQ10 in Rheumatoid Arthritis, multiple sclerosis and type 1 diabetes. This review summarizes the effects of CoQ10 and its analogs in modulating processes involved in autoimmune disorders, highlighting the potential of these therapeutic approaches for patients with immune-mediated diseases.

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.

Characterization of Antiphospholipid Syndrome Atherothrombotic Risk by Unsupervised Integrated Transcriptomic Analyses.

OBJECTIVE: Our aim was to characterize distinctive clinical antiphospholipid syndrome phenotypes and identify novel microRNA (miRNA)-mRNA-intracellular signaling regulatory networks in monocytes linked to cardiovascular disease. Approach and Results: Microarray analysis in antiphospholipid syndrome monocytes revealed 547 differentially expressed genes, mainly involved in inflammatory, cardiovascular, and reproductive disorders. Besides, this approach identified several genes related to inflammatory, renal, and dermatologic diseases. Functional analyses further demonstrated phosphorylation of intracellular kinases related to thrombosis and immune-mediated chronic inflammation. miRNA profiling showed altered expression of 22 miRNAs, enriched in pathways related to immune functions, cardiovascular disease, and autoimmune-associated pathologies. Unbiased integrated mRNA-miRNA analysis identified a signature of 9 miRNAs as potential modulators of 17 interconnected genes related to cardiovascular disease. The altered expression of that miRNA-mRNA signature was proven to be stable along time and distinctive of nonautoimmune thrombotic patients. Transfection studies and luciferase assays established the relationship between specific miRNAs and their identified target genes and proteins, along with their involvement in the regulation of monocytes procoagulant activity and cell adhesion. Correlation analyses showed relationship among altered miRNAs and their interconnected genes with aPL (antiphospholipid antibodies)-titers, along with microvascular endothelial dysfunction. In vitro studies demonstrated modulation in healthy monocytes by IgG-aPLs of several genes/miRNAs, which further intermediated downstream effects on endothelial function. The identified transcriptomic signature allowed the unsupervised division of three clusters of patients with antiphospholipid syndrome showing distinctive clinical profiles, mainly associated with their prothrombotic risk (thrombosis, autoantibody profile, cardiovascular risk factors, and atherosclerosis). CONCLUSIONS: Extensive molecular profiling of monocytes in patients with primary antiphospholipid syndrome might help to identify distinctive clinical phenotypes, thus enabling new patients' tailored treatments.

Effects of Biological Therapies on Molecular Features of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is an autoimmune and chronic inflammatory disease primarily affecting the joints, and closely related to specific autoantibodies that mostly target modified self-epitopes. Relevant findings in the field of RA pathogenesis have been described. In particular, new insights come from studies on synovial fibroblasts and cells belonging to the innate and adaptive immune system, which documented the aberrant production of inflammatory mediators, oxidative stress and NETosis, along with relevant alterations of the genome and on the regulatory epigenetic mechanisms. In recent years, the advances in the understanding of RA pathogenesis by identifying key cells and cytokines allowed the development of new targeted disease-modifying antirheumatic drugs (DMARDs). These drugs considerably improved treatment outcomes for the majority of patients. Moreover, numerous studies demonstrated that the pharmacological therapy with biologic DMARDs (bDMARDs) promotes, in parallel to their clinical efficacy, significant improvement in all these altered molecular mechanisms. Thus, continuous updating of the knowledge of molecular processes associated with the pathogenesis of RA, and on the specific effects of bDMARDs in the correction of their dysregulation, are essential in the early and correct approach to the treatment of this complex autoimmune disorder. The present review details basic mechanisms related to the physiopathology of RA, along with the core mechanisms of response to bDMARDs.

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.

Subclinical cardiovascular risk signs in adults with juvenile idiopathic arthritis in sustained remission.

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) is one of the most common chronic diseases of childhood that often persists into adulthood and can result in significant long-term morbidity. As a long lasting chronic inflammatory disease, concern has been raised regarding the risk of premature development of cardiovascular disease (CVD) in JIA. This study aims to determine whether adults with JIA in clinical remission display clinical and subclinical signs of CVD risk: inflammatory mediators, adipokines, endothelial dysfunction and oxidative stress markers. METHODS: This is a cross-sectional study including 25 patients diagnosed with JIA according to the International League of Associations for Rheumatology criteria (ILAR 2001) and 25 age- and sex-matched controls. Remission was determined by JADAS10 < 1 and according to Wallace criteria. The presence of traditional CVD risk factors was analyzed. An extensive clinical analysis including body mass index (BMI), lipid profile, homeostatic model assessment - insulin resistance (HOMA-IR) and arterial blood pressure was performed. Intima media thickness of the common carotid artery (CIMT) was measured as a marker of subclinical atherosclerosis. Several proinflammatory cytokines, molecules involved in the endothelial dysfunction, oxidative stress and adipokines were quantified on serum by ELISA and on peripheral blood mononuclear cells (PBMCs) by RT-PCR. In vitro studies were carried out in healthy PBMCs, adipocytes and endothelial cells which were treated with serum from JIA patients under sustained remission. RESULTS: Mean duration of the disease was 13.47 ± 5.47 years. Mean age was 25.11 ± 7.21. Time in remission was 3.52 ± 3.33 years. Patients were in remission with no treatment (40%) and with treatments (60%). CVD risk factors and CIMT were similar in JIA patients and controls. However, cholesterol levels were significantly elevated in JIA patients. Levels of adipocytokines, oxidative stress and endothelial activation markers were elevated in serum and PBMCs from JIA patients. Serum of those JIA patients induced the activation of adipocytes, endothelial cells and healthy PBMCs. CONCLUSIONS: JIA adult patients in remission have subclinical signs of inflammation and CVD risk, showed by an increase in the levels of inflammatory cytokines, endothelial activation and oxidative stress markers and adipokines, molecules closely involved in the alteration of the vascular system.

Role of microRNAs in the Development of Cardiovascular Disease in Systemic Autoimmune Disorders.

Rheumatoid Arthritis (RA), Systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS) are the systemic autoimmune diseases (SADs) most associated with an increased risk of developing cardiovascular (CV) events. Cardiovascular disease (CVD) in SADs results from a complex interaction between traditional CV-risk factors, immune deregulation and disease activity. Oxidative stress, dyslipidemia, endothelial dysfunction, inflammatory/prothrombotic mediators (cytokines/chemokines, adipokines, proteases, adhesion-receptors, NETosis-derived-products, and intracellular-signaling molecules) have been implicated in these vascular pathologies. Genetic and genomic analyses further allowed the identification of signatures explaining the pro-atherothrombotic profiles in RA, SLE and APS. However, gene modulation has left significant gaps in our understanding of CV co-morbidities in SADs. MicroRNAs (miRNAs) are emerging as key post-transcriptional regulators of a suite of signaling pathways and pathophysiological effects. Abnormalities in high number of miRNA and their associated functions have been described in several SADs, suggesting their involvement in the development of atherosclerosis and thrombosis in the setting of RA, SLE and APS. This review focusses on recent insights into the potential role of miRNAs both, as clinical biomarkers of atherosclerosis and thrombosis in SADs, and as therapeutic targets in the regulation of the most influential processes that govern those disorders, highlighting the potential diagnostic and therapeutic properties of miRNAs in the management of CVD.

Complement component 3 as biomarker of disease activity and cardiometabolic risk factor in rheumatoid arthritis and spondyloarthritis.

OBJECTIVE: To analyze the relationship between complement component 3 (C3) and the prevalence of cardiometabolic risk factors and disease activity in the rheumatic diseases having the highest rates of cardiovascular morbidity and mortality: rheumatoid arthritis (RA), psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA). METHODS: This is a cross-sectional study including 200 RA, 80 PsA, 150 axSpA patients and 100 healthy donors. The prevalence of cardiometabolic risk factors [obesity, insulin resistance, type 2 diabetes mellitus, hyperlipidemia, apolipoprotein B/apolipoprotein A (apoB/apoA) and atherogenic risks and hypertension] was analyzed. Serum complement C3 levels, inflammatory markers and disease activity were evaluated. Cluster analysis was performed to identify different phenotypes. Receiver operating characteristic (ROC) curve analysis to assess the accuracy of complement C3 as biomarker of insulin resistance and disease activity was carried out. RESULTS: Levels of complement C3, significantly elevated in RA, axSpA and PsA patients, were associated with the prevalence of cardiometabolic risk factors. Hard clustering analysis identified two distinctive phenotypes of patients depending on the complement C3 levels and insulin sensitivity state. Patients from cluster 1, characterized by high levels of complement C3 displayed increased prevalence of cardiometabolic risk factors and high disease activity. ROC curve analysis showed that non-obesity related complement C3 levels allowed to identify insulin resistant patients. CONCLUSIONS: Complement C3 is associated with the concomitant increased prevalence of cardiometabolic risk factors in rheumatoid arthritis and spondyloarthritis. Thus, complement C3 should be considered a useful marker of insulin resistance and disease activity in these rheumatic disorders.

Early restoration of immune and vascular phenotypes in systemic lupus erythematosus and rheumatoid arthritis patients after B cell depletion.

This translational multi-centre study explored early changes in serologic variables following B lymphocyte depletion by rituximab (RTX) treatment in systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) patients and investigated in vitro effects on the activity of other immune cells and the vascular endothelium. Eighty-five SLE patients, seventy-five RA patients and ninety healthy donors were enrolled. Two additional cohorts of selected SLE and RA patients were treated with RTX for 3 months. Changes in circulating levels of inflammatory mediators, oxidative stress markers and NETosis-derived bioproducts were evaluated. Serum miRNomes were identified by next-generation sequencing, and RTX-induced changes were delineated. Mechanistic in vitro studies were performed to assess activity profiles. Altered inflammatory, oxidative and NETosis-derived biomolecules were found in SLE and RA patients, closely interconnected and associated to specific miRNA profiles. RTX treatment reduced SLE and RA patients' disease activity, linked to a prominent alteration in those biomolecules and the reversal of altered regulating miRNAs. In vitro studies showed inhibition of NETosis and decline of pro-inflammatory profiles of leucocytes and human umbilical vein endothelial cells (HUVECs) after B cell depletion. This study provides evidence supporting an early RTX-induced re-setting of the pro-inflammatory status in SLE and RA, involving a re-establishment of the homeostatic equilibrium in immune system and the vascular wall.

Molecular Characterization of Monocyte Subsets Reveals Specific and Distinctive Molecular Signatures Associated With Cardiovascular Disease in Rheumatoid Arthritis.

Objectives: This study, developed within the Innovative Medicines Initiative Joint Undertaking project PRECISESADS framework, aimed at functionally characterize the monocyte subsets in RA patients, and analyze their involvement in the increased CV risk associated with RA. Methods: The frequencies of monocyte subpopulations in the peripheral blood of 140 RA patients and 145 healthy donors (HDs) included in the PRECISESADS study were determined by flow cytometry. A second cohort of 50 RA patients and 30 HDs was included, of which CD14+ and CD16+ monocyte subpopulations were isolated using immuno-magnetic selection. Their transcriptomic profiles (mRNA and microRNA), proinflammatory patterns and activated pathways were evaluated and related to clinical features and CV risk. Mechanistic in vitro analyses were further performed. Results: CD14++CD16+ intermediate monocytes were extended in both cohorts of RA patients. Their increased frequency was associated with the positivity for autoantibodies, disease duration, inflammation, endothelial dysfunction and the presence of atheroma plaques, as well as with the CV risk score. CD14+ and CD16+ monocyte subsets showed distinctive and specific mRNA and microRNA profiles, along with specific intracellular signaling activation, indicating different functionalities. Moreover, that specific molecular profiles were interrelated and associated to atherosclerosis development and increased CV risk in RA patients. In vitro, RA serum promoted differentiation of CD14+CD16- to CD14++CD16+ monocytes. Co-culture with RA-isolated monocyte subsets induced differential activation of endothelial cells. Conclusions: Our overall data suggest that the generation of inflammatory monocytes is associated to the autoimmune/inflammatory response that mediates RA. These monocyte subsets, -which display specific and distinctive molecular signatures- might promote endothelial dysfunction and in turn, the progression of atherosclerosis through a finely regulated process driving CVD development in RA.

New Biomarkers for Atherothrombosis in Antiphospholipid Syndrome: Genomics and Epigenetics Approaches.

Antiphospholipid Syndrome (APS) is an autoimmune disorder, characterized by pregnancy morbidity and/or a hyper coagulable state involving the venous or the arterial vasculature and associated with antiphospholipid antibodies (aPL), including anti-cardiolipin antibodies (aCL), anti-beta2-glycoprotein I (anti-ß2GPI), and Lupus anticoagulant (LA). In recent years there have been many advances in the understanding of the molecular basis of vascular involvement in APS. APS is of multifactorial origin and develops in genetically predisposed individuals. The susceptibility is determined by major histocompatibility complex (MHC). Different HLA-DR and HLA-DQ alleles have been reported in association with APS. Moreover, MHC II alleles may determine the autoantibody profile and, as such, the clinical phenotype of this disease. Besides, polymorphisms in genes related to the vascular system are considered relevant factors predisposing to clinical manifestations. Antiphospholipid antibodies (aPL) induce genomic and epigenetic alterations that support a pro- thrombotic state. Thus, a specific gene profile has been identified in monocytes from APS patients -related to aPL titres in vivo and promoted in vitro by aPL- explaining their cardiovascular involvement. Regarding epigenetic approaches, we previously recognized two miRNAs (miR-19b/miR-20a) as potential modulators of tissue factor, the main receptor involved in thrombosis development in APS. aPLs can further promote changes in the expression of miRNA biogenesis proteins in leukocytes of APS patients, which are translated into an altered miRNA profile and, consequently, in the altered expression of their protein targets related to thrombosis and atherosclerosis. MicroRNAs are further released into the circulation, acting as intercellular communicators. Accordingly, a specific signature of circulating miRNAs has been recently identified in APS patients as potential biomarkers of clinical features. Genomics and epigenetic biomarkers might also serve as indices for disease progression, clinical pharmacology, or safety, so that they might be used to individually predict disease outcome and guide therapeutic decisions. In that way, in the setting of a clinical trial, novel and specific microRNA-mRNA regulatory networks in APS, modified by effect of Ubiquinol treatment, have been identified. In this review, current and previous studies analyzing genomic/epigenetic changes related to the clinical profile of APS patients, and their modulation by effect of specific therapies, are discussed.