Role of microARNs and inflammation in the pathogenesis of autoimmune diseases

Las enfermedades autoinmunes sistémicas se desarrollan como consecuencia de un conjunto de interacciones entre factores genéticos predisponentes, la perturbación del sistema inmune y diversos factores ambientales. El Síndrome Antifosfolípido (SAF) se caracteriza por la presencia de trombosis y complicaciones obstétricas, con especial implicación del sistema hemostático y mecanismos inflamatorios asociados. Por otro lado, la Artritis Reumatoide (AR), también asociada a un proceso inflamatorio crónico, se caracteriza por el deterioro progresivo de las articulaciones periféricas. Además, aunque el daño principal de la AR es la sinovitis articular, presenta también alteraciones metabólicas a nivel sistémico, tales como la resistencia a la insulina. De hecho, la enfermedad cardiovascular (ECV) es la principal causa de mortalidad en pacientes SAF y AR. La inflamación y los elementos autoinmunes presentes en estas patologías parecen ser los principales factores desencadenantes de la ECV. Estudios recientes muestran que los microARNs (miRNAs) juegan un papel relevante y novedoso en la regulación epigenética de la expresión de genes y proteínas claves en la patogénesis de estas enfermedades. Por ello, la identificación y caracterización de los miRNAs asociados a estos desordenes autoinmunes podría favorecer la identificación de nuevos biomarcadores que permitan la tipificación clínica de la enfermedad y faciliten su manejo terapéutico. El objetivo principal de esta tesis es investigar el papel de la inflamación y los miRNAs en la patogénesis del SAF y la AR, mediante la integración de aproximaciones inmunológicas, celulares, moleculares y epigenéticas. Principales resultados obtenidos: 1. Hemos identificado una firma molecular de miRNAs circulantes como potenciales biomarcadores de enfermedad en el SAF. Esta firma se asoció a la ocurrencia de perdidas fetales, aterosclerosis y trombosis y correlaciono con parámetros de inflamación, trombosis y autoinmunidad. Además, dicha firma permaneció estable en el tiempo, hecho demostrado por análisis realizados en un intervalo de tiempo de 3 meses. Asimismo, se demostró la especificidad de la firma de miRNAs en pacientes SAF, mediante el análisis paralelo de dos cohortes de pacientes con trombosis pero sin enfermedad autoinmune y pacientes con Lupus Eritematoso Sistémico sin anticuerpos antifosfolípido, los cuales presentaban perfiles diferenciales. De modo complementario, el análisis mediante clústeres permitió establecer grupos de pacientes con diferente riesgo trombótico y expresión de miRNAs. Estudios mecanísticos in vitro revelaron que los anticuerpos antifosfolípidos promovían la desregulación del perfil de miRNAs analizado y sus potenciales proteínas diana en monocitos y células endoteliales. En suma, se ha identificado una firma de miRNAs diferencialmente expresados en pacientes con SAF como potenciales biomarcadores de la enfermedad que permiten tipificar el estado aterotrombótico de estos pacientes, constituyendo una posible herramienta en la práctica clínica habitual. 2. El estudio del perfil de expresión de miRNAs en neutrófilos de pacientes con AR demostró una reducción global de su expresión, asociado al descenso de genes involucrados en su biogénesis (DICER), así como una sobre-expresión de sus potenciales genes diana involucrados en inflamación y migración celular. Además, dichos niveles reducidos de miRNAs y DICER correlacionaron con mediadores de autoinmunidad, inflamación y el índice de actividad de la enfermedad. Los estudios in vitro revelaron que los anticuerpos antipéptidos cíclico citrulinados (ACPAs) y el factor de necrosis tumoral alfa (TNFa) promovían una reducción tanto de la expresión de estos miRNAs como de los genes involucrados en su biogénesis, en paralelo al incremento de sus potenciales genes diana. El tratamiento concomitante con fármacos inhibidores de TNF-a revirtió dichos efectos. Transfecciones con agonistas de varios de estos miRNAs mostraron también la especificidad de regulación de sus genes diana asociados con inflamación, supervivencia y migración celular. Adicionalmente, el silenciamiento del gen clave en el proceso de biogénesis de miRNAs, DICER, influencio de manera significativa el perfil inflamatorio de los neutrófilos. En suma, los neutrófilos en el contexto de AR exhiben una reducida expresión de miRNAs, promovida por los principales inductores de la activación patogénica de este tipo celular: autoanticuerpos y mediadores inflamatorios. Asimismo, la maquinaria de biogénesis de miRNAs se encuentra significativamente alterada en los neutrófilos de estos pacientes y fuertemente asociada con una desregulación de los miRNAs relacionados con la migración e inflamación en neutrófilos sinoviales. Finalmente, terapias dirigidas contra TNF-a podrían modular estos niveles de expresión de miRNAs, minimizando de este modo el perfil inflamatorio. 3. El estudio de la actividad inflamatoria en pacientes con AR y su implicación en la alteración del metabolismo glucídico y lipídico demostró, en primer lugar, que los pacientes con AR mostraron una fuerte asociación entre el grado de inflamación sistémica y el desarrollo de resistencia a insulina. Estos resultados fueron corroborados mediante el desarrollo de un modelo murino de artritis inducida por colágeno que resulto en un estado de inflamación global caracterizado por un metabolismo defectuoso de la glucosa y de los lípidos en diferentes tejidos, siendo el tejido adiposo el principal tejido afectado y el más susceptible a las alteraciones inducidas por la AR. Estos resultados fueron confirmados asimismo mediante estudios in vitro, tras tratamiento de adipocitos con el suero de pacientes con AR. En su conjunto, los datos obtenidos en este estudio han mostrado como las alteraciones metabólicas observadas en pacientes AR dependen del grado de inflamación e identifica al tejido adiposo como el principal tejido diana para el desarrollo de resistencia a insulina. Por tanto, estrategias terapéuticas dirigidas a controlar el estatus inflamatorio podrían permitir normalizar o prevenir las alteraciones metabólicas asociadas con la AR. En suma, los resultados globales obtenidos en esta tesis doctoral han permitido identificar potenciales biomarcadores del estatus de la enfermedad y sus comorbilidades asociadas en pacientes con Síndrome Antifosfolípido y Artritis Reumatoide, así como diversos mecanismos moleculares asociados a procesos patológicos clave en estas enfermedades, tales como la ECV. Dichos resultados podrían sentar las bases para la realización de estudios futuros, cuyo fin sea desarrollar una medicina personalizada dirigida a optimizar el cuidado de los pacientes con enfermedades autoinmunes sistémicas.Systemic autoimmune diseases (including antiphospholipid syndrome and rheumatoid arthritis) are the result of a complex interaction between predisposing genetic factors, disturbance of the immune system, and several environmental factors. Antiphospholipid Syndrome (APS) is characterized by the presence of thrombosis and obstetric complications, with special involvement of the hemostatic system and associated inflammatory mechanisms. On the other hand, Rheumatoid Arthritis (RA), also associated with a chronic inflammatory process, is characterized by the progressive deterioration of the peripheral joints. Furthermore, although the main focus of RA is joint synovitis, it also presents extra-articular manifestations such as metabolic disorders and cardiovascular disease. In fact, cardiovascular disease (CVD) is the main cause of mortality in both, APS and RA patients. Inflammation and the autoimmune elements seem to be the main triggers for CVD in these pathologies. Recent studies showed that microRNAs (miRNAs) are involved in crucial cellular processes and their dysregulation has been described in many cell types and fluids in a broad range of diseases. Therefore, the characterization of the miRNAs associated with the pathogenesis of systemic autoimmune disorders, such as APS and RA, could identify new biomarkers that might facilitate its diagnosis, outcome and therapeutic management. The main objective of this thesis is to investigate the role of inflammation and miRNAs in the pathogenesis of APS and RA, through the integration of immunological, cellular, molecular and epigenetic approaches. Main results obtained: 1. We have identified a molecular signature of circulating miRNAs as potential disease biomarkers in APS. This signature was associated with the occurrence of fetal loss, atherosclerosis, and thrombosis and correlated with parameters of inflammation, thrombosis, and autoimmunity. Furthermore, this signature remained stable over time, a fact demonstrated by analysis carried out in a time interval of 3 months. Likewise, the specificity of the signature of miRNAs in APS patients was demonstrated, through the parallel analysis of two cohorts of patients with thrombosis but without autoimmune disease and patients with Systemic Lupus Erythematosus without antiphospholipid antibodies, which displayed differential profiles. In addition, hard clustering analysis established groups of patients with different thrombotic risk and expression of miRNAs. In vitro mechanistic studies revealed that antiphospholipid antibodies promoted the dysregulation of the analyzed miRNA profile and its potential target proteins in monocytes and endotelial cells. In sum, differentially expressed circulating miRNAs in APS patients, modulated at least partially by antiphospholipid antibodies of IgG isotype, might have the potential to serve as novel biomarkers of disease features and to typify patients’ atherothrombotic status, thus constituting a useful tool in the management of the disease. 2. The study of the expression profile of miRNAs in neutrophils of RA patients showed an overall reduction in their expression, associated with the decrease in genes involved in their biogenesis (DICER), as well as an over-expression of their potential target genes involved in inflammation and cell migration. Furthermore, these reduced levels of miRNAs and DICER correlated with mediators of autoimmunity, inflammation and disease activity. In vitro studies revealed that anti-citrullinated protein antibodies (ACPAs) and tumor necrosis factor alpha (TNF-a) promoted a reduction in both the expression of these miRNAs and the genes involved in their biogenesis, in parallel with the increase in their potential target genes. Concomitant treatment with TNFinhibitors could reverse these effects. Transfections with agonists of several of these miRNAs also showed the regulatory specificity of their target genes associated with inflammation, survival, and cell migration. Additionally, the silencing of the key gene in the miRNA biogenesis process, DICER, significantly influenced the inflammatory profile of neutrophils. In sum, neutrophils in the context of RA exhibit reduced expression of miRNAs, promoted by the main inducers of pathogenic activation of this cell type: autoantibodies and inflammatory mediators. Likewise, the biogenesis machinery of miRNAs is significantly altered in the neutrophils of these patients and strongly associated with a dysregulation of miRNAs related to migration and inflammation in synovial neutrophils. Finally, therapies directed against TNF-a could modulate these expression levels of miRNAs, thus minimizing the inflammatory profile. 3. The study of the inflammatory activity in patients with RA and its implication in the alteration of the glucose and lipid metabolism showed, first of all, that there was a strong association between the degree of systemic inflammation and the development of insulin resistance. These results were corroborated through the development of a murine model of collagen-induced arthritis that resulted in a state of global inflammation characterized by a defective metabolism of glucose and lipids in different tissues, being adipose tissue the main affected tissue and the more susceptible to the alterations induced by RA. These results were also confirmed by in vitro studies, after treatment of adipocytes with the serum of RA patients. Taken together, the data obtained in this study indicates how the metabolic alterations observed in RA patients depend on the degree of inflammation and identify adipose tissue as the main target tissue for the development of insulin resistance. Therefore, therapeutic strategies focused on tightly controlling the inflammatory status could reduce or prevent the metabolic disorders associated with RA. In sum, the global results obtained in this doctoral thesis allowed the identification of potential biomarkers of the status of the disease and its associated comorbidities in patients with Antiphospholipid Syndrome and Rheumatoid Arthritis, as well as various molecular mechanisms related to key pathological processes in these diseases, such as CVD. These results could pave the way for future studies, based on the development of personalized medicine aimed at optimizing the care of patients with systemic autoimmune diseases

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

Funder: Europeo de Desarrollo Regional de la Unión EuropeaFunder: Rheumatic Diseases NetworkOBJECTIVE: 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

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

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

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Todo química

Mención especial del Concurso de Carteles "La Química de la vida cotidiana", celebrado con motivo del Año Internacional de la Química 2011

Nonalcoholic fatty liver disease in inflammatory arthritis: Relationship with cardiovascular risk.

Liver disease is one of the most important causes of morbidity and mortality worldwide whose prevalence is dramatically increasing. The first sign of hepatic damage is inflammation which could be accompanied by the accumulation of fat called non-alcoholic fatty liver disease (NAFLD), causing damage in the hepatocytes. This stage can progress to fibrosis where the accumulation of fibrotic tissue replaces healthy tissue reducing liver function. The next stage is cirrhosis, a late phase of fibrosis where a high percentage of liver tissue has been replaced by fibrotic tissue and liver functionality is substantially impaired. There is a close interplay of cardiovascular disease (CVD) and hepatic alterations, where different mechanisms mediating this relation between the liver and systemic vasculature have been described. In chronic inflammatory diseases such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA), in which the CVD risk is high, hepatic alterations seem to be more prevalent compared to the general population and other rheumatic disorders. The pathogenic mechanisms involved in the development of this comorbidity are still unraveled, although chronic inflammation, autoimmunity, treatments, and metabolic deregulation seem to have an important role. In this review, we will discuss the involvement of liver disease in the cardiovascular risk associated with inflammatory arthritis, the pathogenic mechanisms, and the recognized factors involved. Likewise, monitoring of the liver disease risk in routine clinical practice through both, classical and novel techniques and indexes will be exposed. Finally, we will examine the latest controversies that have been raised about the effects of the current therapies used to control the inflammation in RA and PsA, in the liver damage of those patients, such as methotrexate, leflunomide or biologics