Identificación de la heterogeneidad molecular asociada a la enfermedad cardiovascular, la evolución clínica y la respuesta terapéutica en patologías Autoinmunes Sistémicas

Abstract

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 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.