Papel de CD69 en el balance TH17 / Treg mediado por miRNAs

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 28-01-2016Esta tesis tiene embargado el acceso al texto completo hasta el 28-01-2018El antígeno de activación leucocitario CD69 es un modulador negativo de la respuesta inmune inflamatoria, que inhibe la diferenciación y la función de las células TH17. Los ratones deficientes en CD69 muestran una inflamación exacerbada en los modelos animales de artritis inducida por colágeno, asma alérgico, dermatitis por contacto, miocarditis autoinmune y colitis. Dado que el equilibrio entre las células TH17 y Tregs es crítico para la regulación de la respuesta inmune, uno de los objetivos de esta tesis fue investigar el papel de CD69 en la diferenciación de las células Tregs naturales e inducibles en el modelo murino de CD69, así como en su capacidad para inducir tolerancia. De esta forma se ha observado que la expresión de CD69 es crítica tanto para la generación de las células nTreg en el timo como para la función supresora de las mismas, a través de la inducción de la secreción de IL-10. Por otro lado, los microRNAs (miRNAs) desempeñan un papel crítico en la regulación del sistema inmune, controlando la diferenciación y funciones inmunológicas de muchos tipos celulares. La expresión o función de los miRNAs se encuentra alterada en muchas enfermedades inflamatorias y cardiovasculares, por lo que constituyen una herramienta muy útil como biomarcadores y/o dianas terapéuticas. Numerosos trabajos relacionan las células TH17 y Tregs con enfermedades cardiovasculares, sin embargo, el papel de los miRNAs presentes en estas células está prácticamente inexplorado en estas patologías. Por ello, otro de los objetivos de esta tesis fue investigar la regulación de la expresión de miRNAs y sus dianas predictivas mediada por CD69 en los linfocitos CD4+ naive, TH17 y Treg, así como su papel en el modelo animal de miocarditis experimental autoinmune (EAM) y en pacientes con miocarditis y cardiomiopatías. Así se ha comprobado que CD69 regula diferencialmente la expresión de miR-721 en las células CD4+ naive, TH17 y Tregs. En el modelo de EAM, se ha encontrado que la expresión de miR-721 en el suero y en el miocardio de los ratones se correlaciona con la severidad de la enfermedad, sugiriendo que CD69 podría controlar la respuesta pro-inflamatoria TH17 en la EAM mediante la regulación de miR-721. En línea con estas observaciones, también se ha descrito que los porcentajes de linfocitos T CD4+, TH17 y Treg en sangre están alterados en los pacientes con miocarditis y cardiomiopatías dilatadas y que los niveles de expresión de miR-721 en el plasma de los pacientes están muy aumentados respecto a los niveles normales de los individuos sanos. Todos estos datos corroboran el papel de CD69 como modulador negativo de la respuesta inmune inflamatoria y señalan un nuevo rol de CD69 en el control de la respuesta pro-inflamatoria TH17 en la miocarditis mediante la regulación del microRNA miR-721.The leukocyte activation antigen CD69 is a negative modulator of the inflammatory immune response that inhibits the differentiation and function of TH17 cells. CD69 deficient mice show an exacerbated inflammation in animal models of collagen-induced arthritis, allergic asthma, contact dermatitis, autoimmune myocarditis, and colitis. Since the balance between Tregs and TH17 is critical for the regulation of the immune response, one of the objectives of this thesis was to investigate the role of CD69 in the differentiation of natural and inducible Tregs in the murine model of CD69, as well as its capacity to induce tolerance. Thus it has been observed that expression of CD69 is critical to both the generation of nTreg cells in the thymus and their suppressor capacity, through the induction of IL-10 secretion. On the other hand, microRNAs (miRNAs) play a critical role in regulating the immune system, controlling differentiation and immune functions of many cell types. The expression or function of miRNAs are altered in many inflammatory and cardiovascular diseases, which are a very useful tool as biomarkers and/or therapeutic targets. Numerous articles relate TH17 and Tregs with cardiovascular diseases, however, the role of miRNAs present in these cells is largely overlooked in these pathologies. Therefore, another objective of this thesis was to investigate the regulation of the expression of miRNAs and their predictive targets mediated CD69 in CD4+ naive, TH17 and Treg cells and their role in the animal model of experimental autoimmune myocarditis (EAM) and in patients with myocarditis and cardiomyopathies. Thus it has been found that CD69 differentially regulates expression of miR-721 in CD4+ naive, TH17 and Tregs. In the model of EAM, it has been found that the expression of miR-721 in serum and myocardium of mice correlates with disease severity, suggesting that CD69 could control TH17 pro-inflammatory response in the EAM by the regulation of miR-721. In line with these observations, it has also been described that the percentages of CD4+, TH17 and Treg lymphocytes in blood are altered in patients with myocarditis and dilated cardiomyopathies and expression levels of miR-721 in the plasma of patients are greatly increased over normal levels of healthy individuals. All these data support the role of CD69 as a negative modulator of the immune response and point at a new role of CD69 in the control of pro-inflammatory TH17 response in myocarditis by regulating the microRNA miR-721

Maintenance of immune tolerance by Foxp3+ regulatory T cells requires CD69 expression

Although FoxP3+ regulatory T cells are key players in the maintenance of immune tolerance and autoimmunity, the lack of specific markers constitute an obstacle to their use for immunotherapy protocols. In this study, we have investigated the role of the C-type lectin receptor CD69 in the suppressor function of Tregs and maintenance of immune tolerance towards harmless inhaled antigens. We identified a novel FoxP3+CD69+ Treg subset capable to maintain immune tolerance and protect to developing inflammation. Although CD69+ and CD69−FoxP3+ Tregs exist in homeostasis, only CD69-expressing Tregs express high levels of CTLA-4, ICOS, CD38 and GITR suppression-associated markers, secrete high amounts of TGFβ and have potent suppressor activity. This activity is regulated by STAT5 and ERK signaling pathways and is impaired by antibody-mediated down-regulation of CD69 expression. Moreover, immunotherapy with FoxP3+CD69+ Tregs restores the homeostasis in Cd69−/− mice, that fail to induce tolerance, and is also highly proficient in the prevention of inflammation. The identification of the FoxP3+CD69+ Treg subset paves the way toward the development of new therapeutic strategies to control immune homeostasis and autoimmunityThis work was supported by funding from the Spanish Ministry of Economy and Competitiveness: SAF2011-27330 to P.M., SAF2010-15106 to M.L.T and SAF2011-25834 to F.S-M.; grant INDISNET (S2010/BMD-2332) from Comunidad de Madrid and RETICS Enfermedades Cardiovasculares (RD12/0042/0056) from Instituto de Salud Carlos III to P.M and F. S-M; and ERC-2011-AdG294340-GENTRIS to F.S-M. J.R.C. was supported by a CNIC post-doctoral fellowship, R. S-D is funded with a pre-doctoral fellowship from Comunidad de Madrid and E.R.B. and A.M-M. were supported by a FPI pre-doctoral fellowship from the Spanish Ministry of Economy and Competitiveness. The CNIC is supported by the Spanish Ministry of Economy and Competitiveness and the Pro CNIC Foundatio

Immunosuppression-independent role of regulatory T cells against hypertension-driven renal dysfunctions

Hypertension-associated cardiorenal diseases represent one of the heaviest burdens for current health systems. In addition to hemodynamic damage, recent results have revealed that hematopoietic cells contribute to the development of these diseases by generating proinflammatory and profibrotic environments in the heart and kidney. However, the cell subtypes involved remain poorly characterized. Here we report that CD39+ regulatory T (TREG) cells utilize an immunosuppression-independent mechanism to counteract renal and possibly cardiac damage during angiotensin II (AngII)-dependent hypertension. This mechanism relies on the direct apoptosis of tissue-resident neutrophils by the ecto-ATP diphosphohydrolase activity of CD39. In agreement with this, experimental and genetic alterations in TREG/TH cell ratios have a direct impact on tissue-resident neutrophil numbers, cardiomyocyte hypertrophy, cardiorenal fibrosis, and, to a lesser extent, arterial pressure elevation during AngII-driven hypertension. These results indicate that TREG cells constitute a first protective barrier against hypertension-driven tissue fibrosis and, in addition, suggest new therapeutic avenues to prevent hypertension-linked cardiorenal diseases.This work has been supported by grants from the Castilla-León Autonomous Government (CSI101U13), the Spanish Ministry of Economy and Competitiveness (SAF2012-31371, RD12/0036/0002), Worldwide Cancer Research, the Solórzano Foundation, and the Ramón Areces Foundation to X.R.B. P.M. is funded by the Spanish Ministry of Economy and Competitiveness (SAF2011-27330). S.F., M.M.-M., J.R.-V., and A.M.-M. were supported by the Spanish Ministry of Economy and Competitiveness through BES-2010-031386, CSIC JAE-Doc, Juan de la Cierva, and BES-2009-016103 contracts, respectively. Spanish government-sponsored funding to X.R.B. is partially supported by the European Regional Development Fund.Peer Reviewe

A Novel Circulating MicroRNA for the Detection of Acute Myocarditis.

The diagnosis of acute myocarditis typically requires either endomyocardial biopsy (which is invasive) or cardiovascular magnetic resonance imaging (which is not universally available). Additional approaches to diagnosis are desirable. We sought to identify a novel microRNA for the diagnosis of acute myocarditis. To identify a microRNA specific for myocarditis, we performed microRNA microarray analyses and quantitative polymerase-chain-reaction (qPCR) assays in sorted CD4+ T cells and type 17 helper T (Th17) cells after inducing experimental autoimmune myocarditis or myocardial infarction in mice. We also performed qPCR in samples from coxsackievirus-induced myocarditis in mice. We then identified the human homologue for this microRNA and compared its expression in plasma obtained from patients with acute myocarditis with the expression in various controls. We confirmed that Th17 cells, which are characterized by the production of interleukin-17, are a characteristic feature of myocardial injury in the acute phase of myocarditis. The microRNA mmu-miR-721 was synthesized by Th17 cells and was present in the plasma of mice with acute autoimmune or viral myocarditis but not in those with acute myocardial infarction. The human homologue, designated hsa-miR-Chr8:96, was identified in four independent cohorts of patients with myocarditis. The area under the receiver-operating-characteristic curve for this novel microRNA for distinguishing patients with acute myocarditis from those with myocardial infarction was 0.927 (95% confidence interval, 0.879 to 0.975). The microRNA retained its diagnostic value in models after adjustment for age, sex, ejection fraction, and serum troponin level. After identifying a novel microRNA in mice and humans with myocarditis, we found that the human homologue (hsa-miR-Chr8:96) could be used to distinguish patients with myocarditis from those with myocardial infarction. (Funded by the Spanish Ministry of Science and Innovation and others.).Supported by a grant (PI19/00545, to Dr. Martín) from the Ministry of Science and Innovation through the Carlos III Institute of Health–Fondo de Investigación Sanitaria; by a grant from the Biomedical Research Networking Center on Cardiovascular Diseases (to Drs. Martín, Sánchez-Madrid, and Ibáñez); by grants (S2017/BMD-3671-INFLAMUNE-CM, to Drs. Martín and Sánchez-Madrid; and S2017/BMD-3867-RENIM-CM, to Dr. Ibáñez) from Comunidad de Madrid; by a grant (20152330 31, to Drs. Martín, Sánchez-Madrid, and Alfonso) from Fundació La Marató de TV3; by grants (ERC-2011-AdG 294340-GENTRIS, to Dr. Sánchez-Madrid; and ERC-2018-CoG 819775-MATRIX, to Dr. Ibáñez) from the European Research Council; by grants (SAF2017-82886R, to Dr. Sánchez-Madrid; RETOS2019-107332RB-I00, to Dr. Ibáñez; and SAF2017-90604-REDT-NurCaMeIn and RTI2018-095928-BI00, to Dr. Ricote) from the Ministry of Science and Innovation; by Fondo Europeo de Desarrollo Regional (FEDER); and by a 2016 Leonardo Grant for Researchers and Cultural Creators from the BBVA Foundation to Dr. Martín. The National Center for Cardiovascular Research (CNIC) is supported by the Carlos III Institute of Health, the Ministry of Science and Innovation, the Pro CNIC Foundation, and by a Severo Ochoa Center of Excellence grant (SEV-2015-0505). Mr. Blanco-Domínguez is supported by a grant (FPU16/02780) from the Formación de Profesorado Universitario program of the Spanish Ministry of Education, Culture, and Sports. Ms. Linillos-Pradillo is supported by a fellowship (PEJD-2016/BMD-2789) from Fondo de Garantía de Empleo Juvenil de Comunidad de Madrid. Dr. Relaño is supported by a grant (BES-2015-072625) from Contratos Predoctorales Severo Ochoa para la Formación de Doctores of the Ministry of Economy and Competitiveness. Dr. Alonso-Herranz is supported by a fellowship from La Caixa–CNIC. Dr. Caforio is supported by Budget Integrato per la Ricerca dei Dipartimenti BIRD-2019 from Università di Padova. Dr. Das is supported by grants (UG3 TR002878 and R35 HL150807) from the National Institutes of Health and the American Heart Association through its Strategically Focused Research Networks.S

Immunosuppression-Independent Role of Regulatory T Cells against Hypertension-Driven Renal Dysfunctions

Hypertension-associated cardiorenal diseases represent one of the heaviest burdens for current health systems. In addition to hemodynamic damage, recent results have revealed that hematopoietic cells contribute to the development of these diseases by generating proinflammatory and profibrotic environments in the heart and kidney. However, the cell subtypes involved remain poorly characterized. Here we report that CD39+ regulatory T (TREG) cells utilize an immunosuppression-independent mechanism to counteract renal and possibly cardiac damage during angiotensin II (AngII)-dependent hypertension. This mechanism relies on the direct apoptosis of tissue-resident neutrophils by the ecto-ATP diphosphohydrolase activity of CD39. In agreement with this, experimental and genetic alterations in TREG/TH cell ratios have a direct impact on tissue-resident neutrophil numbers, cardiomyocyte hypertrophy, cardiorenal fibrosis, and, to a lesser extent, arterial pressure elevation during AngII-driven hypertension. These results indicate that TREG cells constitute a first protective barrier against hypertension-driven tissue fibrosis and, in addition, suggest new therapeutic avenues to prevent hypertension-linked cardiorenal diseases.Fil: Fabbiano, Salvatore. Consejo Superior de Investigaciones Científicas; España. Universidad de Salamanca; España. Instituto de Biología Molecular y Celular del Cáncer de Salamanca; España; EspañaFil: Menacho Márquez, Mauricio Ariel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Consejo Superior de Investigaciones Científicas; España. Universidad de Salamanca; EspañaFil: Robles Valero, Javier. Consejo Superior de Investigaciones Científicas; España. Universidad de Salamanca; EspañaFil: Pericacho, Miguel. Universidad de Salamanca; EspañaFil: Matesanz Marín, Adela. Centro Nacional de Investigaciones Cardiovasculares; EspañaFil: García Macías, Carmen. Consejo Superior de Investigaciones Científicas; España. Universidad de Salamanca; EspañaFil: Sevilla, María A.. Universidad de Salamanca; EspañaFil: Montero, M. J.. Universidad de Salamanca; EspañaFil: Alarcón, Balbino. Consejo Superior de Investigaciones Científicas; EspañaFil: López Novoa, José M.. Universidad de Salamanca; EspañaFil: Martín, Pilar. Consejo Superior de Investigaciones Científicas; EspañaFil: Bustelo, Xosé R.. Consejo Superior de Investigaciones Científicas; España. Universidad de Salamanca; Españ