Identification of molecular heterogeneity associated with cardiovascular disease, clinical evolution and therapeutic response in Systemic Autoimmune pathologies

Las enfermedades autoinmunes son enfermedades multifactoriales, resultado de la interacción entre factores genéticos, factores ambientales y alteraciones del propio sistema inmune. Entre ellas, el lupus eritematoso sistémico (LES) y la artritis reumatoide (AR) son enfermedades autoinmunes sistémicas que comparten una etiología autoinmune, pero difieren en los mecanismos inmunológicos, determinantes de sus distintas manifestaciones clínicas. Ambas enfermedades muestran un riesgo incrementado de enfermedad cardiovascular (ECV). La inflamación y los elementos autoinmunes presentes en estas patologías, tales como los autoanticuerpos, la activación endotelial, el estrés oxidativo y la función alterada de las células inmunes, parecen ser los principales factores desencadenantes de la ECV. No obstante, las características específicas que definen cada enfermedad pueden asimismo contribuir a determinar mecanismos únicos de ECV en cada condición autoinmune. Por tanto, la caracterización de la base molecular de las anomalías que conducen a la autoinmunidad y la inflamación son fundamentales para la comprensión de la patogénesis del LES y la AR. Estudios recientes en análisis genómicos y epigenéticos [metiloma y microRNAs (miRNAs)] han permitido identificar firmas específicas en dichas enfermedades autoinmunes y obtener información sobre los mecanismos subyacentes al desarrollo de comorbilidades asociadas. Además, se han caracterizado diversos miRNAs implicados en el control de proteínas asociadas al estatus proinflamatorio, el riesgo CV y la respuesta terapéutica en pacientes con LES y AR. Asimismo, numerosas evidencias derivadas de otros estudios han demostrado la implicación directa de las células del sistema inmune (linfocitos T y B, células dendríticas, monocitos y neutrófilos), los autoanticuerpos y el perfil inflamatorio específicos del LES y la AR en la patogenia, el desarrollo de ECV, la evolución clínica y la respuesta terapéutica en pacientes autoinmunes. Durante la evolución clínica de estas enfermedades, asociados a la ocurrencia de brotes, daño orgánico y eventos cardiovasculares, la identificación de dichos cambios moleculares podría asimismo ayudar a discernir los mecanismos implicados en estos procesos. Se ha demostrado también la eficacia del uso de terapias biológicas en el LES y la AR. No obstante, su efecto sobre la patología CV está aún poco definido. Estudios longitudinales de seguimiento clínico/molecular, y respuesta a nuevas terapias aportarían información valiosa para identificar dichos cambios, lo que podría favorecer la toma de decisiones clínicas. En base a estas premisas, el Objetivo Principal de esta tesis doctoral es caracterizar los mecanismos moleculares subyacentes al desarrollo de las EAS, sus comorbilidades y respuesta terapéutica, a fin de identificar las causas de su heterogeneidad clínica y favorecer el desarrollo de nuevos biomarcadores y dianas terapéuticas útiles para su seguimiento clínico y manejo terapéutico. Principales resultados obtenidos: 1. Hemos demostrado por primera vez el papel relevante de los anticuerpos anti-dsDNA como un responsable más que contribuye al aumento del riesgo CV en pacientes con LES. Mediante el uso de aproximaciones transcriptómicas y proteómicas, hemos caracterizado in vivo e in vitro los mecanismos moleculares que subyacen a los efectos de estos autoanticuerpos sobre la actividad específica de las células inmunitarias y vasculares, que conducen al aumento del riesgo CV presente en dichos pacientes. La positividad y persistencia de anticuerpos anti-dsDNA en pacientes con LES se asoció a disfunción endotelial, dislipidemia proaterogénica y aterosclerosis acelerada. Paralelamente, los anticuerpos anti-dsDNA se relacionaron con la activación aberrante de las células inmunitarias innatas, de modo que los monocitos de pacientes con LES positivos para la presencia de anticuerpos anti-dsDNA mostraron perfiles distintivos de expresión y actividad alterada de genes y proteínas, mientras que los neutrófilos fueron más propensos a sufrir NETosis en comparación con pacientes con LES negativos para anticuerpos anti-dsDNA. Pacientes con anticuerpos anti-dsDNA también mostraron niveles alterados de numerosos mediadores circulantes relacionados con la inflamación, la NETosis y el riesgo cardiovascular. In vitro, anticuerpos anti-dsDNA aislados a partir del suero de pacientes, promovieron NETosis en neutrófilos y apoptosis en monocitos, moduló la expresión de moléculas relacionadas con la inflamación y la trombosis e indujo activación endotelial. Dichos efectos fueron promovidos en parte mediante mecanismos dependientes de unión a receptores Fc. En suma, se ha demostrado que los anticuerpos anti-dsDNA aumentan el riesgo cardiovascular en pacientes con LES al alterar los procesos moleculares clave que impulsan una activación inmune y vascular distintiva y coordinada, constituyendo una herramienta clínica adicional para el manejo de esta comorbilidad. 2. Hemos demostrado que la maquinaria de splicing está profundamente alterada en los leucocitos de pacientes AR y estrechamente relacionada con la fisiopatología de la enfermedad, lo cual no había sido descrito hasta la fecha. Identificamos ocho componentes (SNRNP70, SNRNP200 U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1, SRSF10) comúnmente alterados en neutrófilos, monocitos y linfocitos, cuyos niveles de expresión permitieron distinguir a pacientes con AR de donantes sanos e identificar a pacientes con alta actividad de la enfermedad, afectación articular y aterosclerosis temprana. Además, ampliamos estas observaciones examinando la relación entre los niveles alterados de la maquinaria de splicing y mediadores inflamatorios notablemente involucrados en el perfil clínico de estos pacientes. Estos resultados se validaron aún más en células mononucleares obtenidas del líquido sinovial de pacientes con AR, donde el daño inflamatorio es más pronunciado, y en las articulaciones de un modelo de ratón con AR, reforzando así la relevancia clínica de los datos obtenidos. Los estudios ex vivo e in vitro identificaron además los posibles mecanismos subyacentes a estos procesos. Por un lado, los anticuerpos ACPA y las citoquinas inflamatorias modularon la expresión de los componentes de la maquinaria de splicing. Po otro lado, la sobreexpresión inducida de SNRNP70 y KHDRBS1 revirtió la inflamación en los linfocitos, la NETosis en los neutrófilos y la adhesión en los monocitos de la AR, e indirectamente moduló la actividad de fibroblastos sinoviales (SF). Finalmente, la terapia anti-TNF a los tres y seis meses revirtió la expresión de algunos de los componentes de la maquinaria de splicing en paralelo a la reducción del perfil inflamatorio. En suma, hemos caracterizado por primera vez una firma que conforma 8 componentes de la maquinaria de splicing desregulados en leucocitos de AR tanto de sangre periférica como de líquido sinovial, vinculados a la fisiopatología de la enfermedad, modulados por ACPA y revertidos por la terapia anti-TNF. 3. Hemos caracterizado molecularmente pacientes AR que inician tratamiento con terapia anti-TNF, incluidos en un estudio multicéntrico, identificando subgrupos de pacientes cuyo perfil molecular circulante se asoció a la respuesta terapéutica tras 6 meses de tratamiento. En concreto, se encontraron biomoléculas alteradas inflamatorias, oxidativas y derivadas de NETosis en pacientes con AR en relación con donantes sanos, estrechamente interconectadas y asociadas con perfiles específicos de miRNA. Este perfil molecular alterado permitió la división no supervisada de tres grupos de pacientes con AR, que mostraron fenotipos clínicos distintivos, vinculados aún más a la eficacia de fármacos anti-TNF. Además, esta terapia revirtió dichas alteraciones moleculares en paralelo a la respuesta clínica. Mediante herramientas de aprendizaje automático (machine learning) se identificaron tanto firmas clínicas como moleculares como posibles predictores de la respuesta al tratamiento anti-TNF con alta precisión, la cual aumentó aún más cuando ambas características se integraron en un modelo mixto. Estos resultados se confirmaron en una cohorte independiente de validación. En su conjunto, estos datos sugieren que el análisis integrado de perfiles clínicos y moleculares utilizando herramientas computacionales avanzadas permite la identificación de firmas novedosas como posibles predictores de la respuesta terapéutica a la terapia anti-TNF. En conclusión, los resultados globales obtenidos en esta tesis doctoral han permitido identificar potenciales biomarcadores del estatus de la enfermedad, sus comorbilidades asociadas y la respuesta a determinadas terapias en pacientes con LES y AR, así como diversos mecanismos moleculares asociados a procesos patológicos clave en estas enfermedades. Dichos resultados podrían sentar las bases para la realización de futuros estudios, cuyo fin sea desarrollar una medicina personalizada dirigida a optimizar el cuidado de pacientes con enfermedades autoinmunes sistémicas.Autoimmune diseases are multifactorial diseases, resulting from the interaction between genetic factors, environmental factors, and alterations of the immune system itself. Among them, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are systemic autoimmune diseases that share an autoimmune etiology but differ in the immunological mechanisms that determine their different clinical manifestations. Both diseases show an increased risk of cardiovascular disease (CVD). Inflammation and autoimmune elements present in these pathologies, such as autoantibodies, endothelial activation, oxidative stress and altered immune cell function, seem to be the main triggers of CVD. However, the specific characteristics that define each disease may also contribute to determining unique CVD mechanisms in each autoimmune condition. Therefore, characterization of the molecular basis of the abnormalities that lead to autoimmunity and inflammation is essential for understanding the pathogenesis of SLE and RA. Recent studies in genomic and epigenetic analyzes [methylome and microRNAs (microRNAs)] have made it possible to identify specific signatures in these autoimmune diseases and obtain information on the mechanisms underlying the development of associated comorbidities. In addition, various miRNAs involved in the control of proteins associated with proinflammatory status, CV risk, and therapeutic response in patients with SLE and RA have been characterized. Likewise, numerous pieces of evidence derived from other studies have shown the direct involvement of cells of the immune system (T and B lymphocytes, dendritic cells, monocytes, and neutrophils), autoantibodies and the specific inflammatory profile of SLE and RA in the pathogenesis, CVD development, clinical course and therapeutic response in autoimmune patients. During the clinical evolution of these diseases, associated with the occurrence of outbreaks, organ damage and cardiovascular events, the identification of these molecular changes could also help to discern the mechanisms involved in these processes. The efficacy of the use of biological therapies in SLE and RA has also been demonstrated. However, its effect on CV pathology is still poorly defined. Longitudinal studies of clinical/molecular follow-up and response to new therapies would provide valuable information to identify these changes, which could favour clinical decision-making. Based on these premises, the Main Objective of this doctoral thesis is to characterize the molecular mechanisms underlying the development of SAD, their comorbidities and therapeutic response, to identify the causes of their clinical heterogeneity and favour the development of new biomarkers and targets. therapeutic useful for clinical follow-up and therapeutic management. Main results obtained: 1. We have shown for the first time the relevant role of anti-dsDNA antibodies as one more factor that contributes to the increased CV risk in patients with SLE. Using transcriptomic and proteomic approaches, we have characterized in vivo and in vitro the molecular mechanisms underlying the effects of these autoantibodies on the specific activity of immune and vascular cells, leading to the increased CV risk present in these patients. The positivity and persistence of anti-dsDNA antibodies in patients with SLE was associated with endothelial dysfunction, proatherogenic dyslipidemia, and accelerated atherosclerosis. In parallel, anti-dsDNA antibodies were associated with aberrant activation of innate immune cells, such that monocytes from SLE patients positive for the presence of anti-dsDNA antibodies showed distinctive profiles of altered gene and protein expression and activity. while neutrophils were more prone to NETosis compared to anti-dsDNA antibody negative SLE patients. Patients with antidsDNA antibodies also showed altered levels of numerous circulating mediators related to inflammation, NETosis, and cardiovascular risk. In vitro, anti-dsDNA antibodies isolated from patient serum promoted NETosis in neutrophils and apoptosis in monocytes, modulated the expression of molecules related to inflammation and thrombosis, and induced endothelial activation. These effects were promoted in part by mechanisms dependent on binding to Fc receptors. In sum, anti-dsDNA antibodies have been shown to increase cardiovascular risk in patients with SLE by altering the key molecular processes that drive distinctive and coordinated immune and vascular activation, constituting an additional clinical tool for the management of this comorbidity. 2. We have shown that the splicing machinery is profoundly altered in the leukocytes of RA patients and is closely related to the pathophysiology of the disease, which had not been described to date. We identified eight components (SNRNP70, SNRNP200 U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1, SRSF10) commonly altered in neutrophils, monocytes, and lymphocytes, whose expression levels made it possible to distinguish RA patients from healthy donors and to identify patients with high activity of the disease, joint involvement and early atherosclerosis. Furthermore, we extend these observations by examining the relationship between altered levels of the splicing machinery and inflammatory mediators notably involved in the clinical profile of these patients. These results were further validated in mononuclear cells obtained from the synovial fluid of RA patients, where inflammatory damage is more pronounced, and in the joints of an RA mouse model, thus reinforcing the clinical relevance of the data obtained. Ex vivo and in vitro studies further identified possible mechanisms underlying these processes. On the one hand, ACPA antibodies and inflammatory cytokines modulated the expression of components of the splicing machinery. On the other hand, induced overexpression of SNRNP70 and KHDRBS1 reversed inflammation in lymphocytes, NETosis in neutrophils, and adhesion in RA monocytes, and indirectly modulated synovial fibroblast (SF) activity. Finally, anti-TNF therapy at three and six months reversed the expression of some of the components of the splicing machinery in parallel with the reduction of the inflammatory profile. In sum, we have characterized for the first time a signature that makes up 8 components of the deregulated splicing machinery in RA leukocytes from both peripheral blood and synovial fluid, linked to the pathophysiology of the disease, modulated by ACPA and reversed by anti-inflammatory therapy. -TNF. 3. We have molecularly characterized RA patients who start treatment with anti-TNF therapy, included in a multicenter study, identifying subgroups of patients whose circulating molecular profile was associated with therapeutic response after 6 months of treatment. Specifically, altered inflammatory, oxidative, and NETosis-derived biomolecules in RA patients were found relative to healthy donors, closely interconnected, and associated with specific miRNA profiles. This altered molecular profile allowed the unsupervised division of three groups of RA patients, which showed distinctive clinical phenotypes, further linked to the efficacy of anti- TNF drugs. In addition, this therapy reversed these molecular alterations in parallel to the clinical response. Using machine learning tools, both clinical and molecular signatures were identified as possible predictors of response to anti-TNF treatment with high precision, which increased even more when both characteristics were integrated into a mixed model. These results were confirmed in an independent validation cohort. Taken together, these data suggest that integrated analysis of clinical and molecular profiles using advanced computational tools allows the identification of novel signatures as potential predictors of therapeutic response to anti-TNF therapy. In conclusion, the global results obtained in this doctoral thesis have made it possible to identify potential biomarkers of the status of the disease, its associated comorbidities and the response to certain therapies in patients with SLE and RA, as well as various molecular mechanisms associated with key pathological processes in these diseases. These results could lay the groundwork for future studies, aimed at developing personalized medicine aimed at optimizing the care of patients with systemic autoimmune diseases.Autoimmune diseases are multifactorial diseases, resulting from the interaction between genetic factors, environmental factors, and alterations of the immune system itself. Among them, systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are systemic autoimmune diseases that share an autoimmune etiology but differ in the immunological mechanisms that determine their different clinical manifestations. Both diseases show an increased risk of cardiovascular disease (CVD). Inflammation and autoimmune elements present in these pathologies, such as autoantibodies, endothelial activation, oxidative stress and altered immune cell function, seem to be the main triggers of CVD. However, the specific characteristics that define each disease may also contribute to determining unique CVD mechanisms in each autoimmune condition. Therefore, characterization of the molecular basis of the abnormalities that lead to autoimmunity and inflammation is essential for understanding the pathogenesis of SLE and RA. Recent studies in genomic and epigenetic analyzes [methylome and microRNAs (microRNAs)] have made it possible to identify specific signatures in these autoimmune diseases and obtain information on the mechanisms underlying the development of associated comorbidities. In addition, various miRNAs involved in the control of proteins associated with proinflammatory status, CV risk, and therapeutic response in patients with SLE and RA have been characterized. Likewise, numerous pieces of evidence derived from other studies have shown the direct involvement of cells of the immune system (T and B lymphocytes, dendritic cells, monocytes, and neutrophils), autoantibodies and the specific inflammatory profile of SLE and RA in the pathogenesis, CVD development, clinical course and therapeutic response in autoimmune patients. During the clinical evolution of these diseases, associated with the occurrence of outbreaks, organ damage and cardiovascular events, the identification of these molecular changes could also help to discern the mechanisms involved in these processes. The efficacy of the use of biological therapies in SLE and RA has also been demonstrated. However, its effect on CV pathology is still poorly defined. Longitudinal studies of clinical/molecular follow-up and response to new therapies would provide valuable information to identify these changes, which could favour clinical decision-making. Based on these premises, the Main Objective of this doctoral thesis is to characterize the molecular mechanisms underlying the development of SAD, their comorbidities and therapeutic response, to identify the causes of their clinical heterogeneity and favour the development of new biomarkers and targets. therapeutic useful for clinical follow-up and therapeutic management. Main results obtained: 1. We have shown for the first time the relevant role of anti-dsDNA antibodies as one more factor that contributes to the increased CV risk in patients with SLE. Using transcriptomic and proteomic approaches, we have characterized in vivo and in vitro the molecular mechanisms underlying the effects of these autoantibodies on the specific activity of immune and vascular cells, leading to the increased CV risk present in these patients. The positivity and persistence of anti-dsDNA antibodies in patients with SLE was associated with endothelial dysfunction, proatherogenic dyslipidemia, and accelerated atherosclerosis. In parallel, anti-dsDNA antibodies were associated with aberrant activation of innate immune cells, such that monocytes from SLE patients positive for the presence of anti-dsDNA antibodies showed distinctive profiles of altered gene and protein expression and activity. while neutrophils were more prone to NETosis compared to anti-dsDNA antibody negative SLE patients. Patients with antidsDNA antibodies also showed altered levels of numerous circulating mediators related to inflammation, NETosis, and cardiovascular risk. In vitro, anti-dsDNA antibodies isolated from patient serum promoted NETosis in neutrophils and apoptosis in monocytes, modulated the expression of molecules related to inflammation and thrombosis, and induced endothelial activation. These effects were promoted in part by mechanisms dependent on binding to Fc receptors. In sum, anti-dsDNA antibodies have been shown to increase cardiovascular risk in patients

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

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

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

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

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

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

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

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.This study was supported by grants from the Instituto de Salud Carlos III (ref. PI18/00837), cofinanciado por el Fondo Europeo de Desarrollo Regional de la Unión Europea ‘Una manera de hacer Europa’, Spain, the Spanish Inflammatory and Rheumatic Diseases Network (RIER), Instituto de Salud Carlos III (RD16/0012/0015) and the Servicio Andaluz de Salud (PI-0285-2017). C.L-P was supported by a contract from the Spanish Junta de Andalucía (‘Nicolas Monardes’ Program).Ye

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.This study was supported by grants from the Instituto de Salud Carlos III (PI18/00837), cofinanciado por el Fondo Europeo de Desarrollo Regional de la Unión Europea Una manera de hacer Europa, Spain, the Spanish Inflammatory and Rheumatic Diseases Network (RIER), Instituto de Salud Carlos III (RD16/0012/0015) and the Andalusian Regional Health System (ref. PI-0285-2017). CL-P was supported by a contract from the Spanish Junta de Andalucía (Nicolas Monardes program).Ye