Periodic Reporting for period 1 - EXAUTOIMMUNE (Extracellular vesicles’ transcriptomics: unravelling the role of short non-coding RNAs in autoimmunity)
Periodo di rendicontazione: 2020-10-01 al 2022-09-30
It is estimated that over 36 million Europeans suffer from some autoimmune disease (AID) and this represents the second cause of illness and the third cause of social security disability, after heart disease and cancer. In fact, according to the European League against Rheumatism (EULAR) one third of people of all ages are affected at some point during their lifetime. AID are a significant clinical problem because of their chronic nature and are thought to be associated with a failure of regulatory mechanisms. However, most of the current therapies target the terminal phase of inflammation and do not address the fundamental events responsible for the initiation and progression of the autoimmune process. Tackling these diseases at their source will require an understanding of how the abnormal immune reactions arise, how they are sustained, and the intrinsic mechanisms used to suppress these responses in healthy individuals. In this sense, research efforts should focus on better understanding of the molecular and cellular basis of AID for the development of curative and preventive strategies. EXAUTOIMMUNE project uses Sjögren’s syndrome (SS) as a model disease to create innovating strategies for addressing the fundamental processes involved in the initiation of the autoimmune responses.
SS is a rather common systemic autoimmune disease, as common as Rheumatoid Arthritis. Salivary gland epithelial cells (SGEC) in SS lesions are active participants in the induction and perpetuation of the inflammatory process, from early onset to chronic state of the disease. Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, are secreted by almost all cell types and mediate intercellular communication acting as nanoshuttles and transfering information among cells. EVs contain various classes of short non-coding RNAs of approximately 20-35 nucleotides in length. They can act as regulators of gene function, and are emerging as pivotal regulators of immune responses.
The OVERALL OBJECTIVE of the EXAUTOIMMUNE project is to define the immunomodulatory function of EVs and their RNA-cargo in SS. We hypothesize that SGEC through the secretion of RNA-containing EVs, modulate the regulatory mechanisms in favor of autoimmunity.
SS is a rather common systemic autoimmune disease, as common as Rheumatoid Arthritis. Salivary gland epithelial cells (SGEC) in SS lesions are active participants in the induction and perpetuation of the inflammatory process, from early onset to chronic state of the disease. Extracellular vesicles (EVs), including exosomes, microvesicles and apoptotic bodies, are secreted by almost all cell types and mediate intercellular communication acting as nanoshuttles and transfering information among cells. EVs contain various classes of short non-coding RNAs of approximately 20-35 nucleotides in length. They can act as regulators of gene function, and are emerging as pivotal regulators of immune responses.
The OVERALL OBJECTIVE of the EXAUTOIMMUNE project is to define the immunomodulatory function of EVs and their RNA-cargo in SS. We hypothesize that SGEC through the secretion of RNA-containing EVs, modulate the regulatory mechanisms in favor of autoimmunity.
In order to demonstrate whether SGEC-derived EVs from SS and control groups have immunomodulatory functions, first, we performed a comprehensive analysis of the different fractions, combining biology and nanoscience tools. SGEC culture media from the SS and Ct donors were purified and enriched using Silicon Carbide resin-based columns. EV morphology was analyzed by Transmission Electron Microscopy. The size and concentration of EVs were assessed by nanotracking analysis through the instrument Nanosight and the levels of specific exosomal protein markers were tested by immunoblotting. Collectivelly, our results indicate that the vast majority of SGEC-derived EVs fall within the exosome fraction (average size 70 nm) and, thus, they will be called exosomes for the rest of the project. The exosome phenotype was supported by the cup-shape morphology revealed by electron microscopy and the presence of an exosome-specific membrane protein. To answer the key question of the project: If exosomes have immunoregulatory roles, we performed next generation RNA sequencing combined with bioinformatics analysis. The resulting data show that exosomes from SGEC display altered noncoding RNA cargo in Sjogren’s syndrome. The various molecules with differential expression showed enrichment in pathways associated with TNF signaling, T cell receptor, Th1 and Th2 cell differentiation signaling. These data define for the first time the core transcriptomic differences of SGEC-secreted exosomes between SS and Ct, and reveal new targetable molecules and pathways towards treatment of autoimmune reactivity in Sjogren’s syndrome.
From the beginning of EXAUTOIMMUNE the data that have generated have been disseminated internally and externally with various ways. Internally, the results have been progressively presented during the Institute’s seminars series twice a year for the last two years, and externally different datasets have been presented in two international scientific meetings as oral and poster presentations.
At the level of exploitation, the project has provided us with a handful of testable signaling pathways and RNA molecules which are readily applicable ex vivo and in vivo in an animal setting. Testing this outcome on a Sjogren’s mouse model through clinical phenotyping, will enable successful transition to molecule/pathway targeting and development of an effective therapeutic approach. With this exploitation strategy we will increase the impact of the outcome to the in vivo setting and improve translation of the findings from preclinical level into the clinical setting of Sjogren’s.
From the beginning of EXAUTOIMMUNE the data that have generated have been disseminated internally and externally with various ways. Internally, the results have been progressively presented during the Institute’s seminars series twice a year for the last two years, and externally different datasets have been presented in two international scientific meetings as oral and poster presentations.
At the level of exploitation, the project has provided us with a handful of testable signaling pathways and RNA molecules which are readily applicable ex vivo and in vivo in an animal setting. Testing this outcome on a Sjogren’s mouse model through clinical phenotyping, will enable successful transition to molecule/pathway targeting and development of an effective therapeutic approach. With this exploitation strategy we will increase the impact of the outcome to the in vivo setting and improve translation of the findings from preclinical level into the clinical setting of Sjogren’s.
In the recent years new biologic therapies targeting the molecules involved in the pathogenesis of chronic inflammatory disease have been developed. However, at the current stage, the majority of autoimmune diseases can be treated symptomatically but they cannot be cured because of insufficient information on causes and early pathogenic events. Although biologic regimes have been tested in Sjogren’s, currently, no biologic treatment has been approved in Europe and early phase clinical trials have shown controversial results. Our project reveals new factors and signals that may initiate the abnormal immune responses, resulting in break of immune tolerance. The newly generated results of EXAUTOIMMUNE identify new players implicated in the initial causes of immune deregulation in Sjogren’s. In summary, our study identified new potential targets aiming at reinstallation of immune tolerance and prevention of Sjogren’s. Understanding the role of exosomal RNAs in Sjogren’s represents a step forward the state-of-the art while opening the arena for new therapeutic approaches.
The EXAUTOIMMUNE project has helped the fellow acquire the competences and skills necessary to challenge the state of the art and apply innovative ideas to the current knowledge of autoimmune diseases. In particular, the emerging applications of next generation RNAseq and its exciting future in biomarker discovery and Precision Medicine make this project highly impactful for the fellow’s career while allowing for a more holistic understanding of the autoimmune diseases in society.
The fellow’s goal for the near future is to define molecular targets for pathogenesis and treatment of autoimmune diseases that can be tested in predictive models. The results obtained from these mouse models will lead to novel treatment approaches for the human disease while at the same time increasing the output of research and innovation (R&I) and resulting to more knowledge and ideas converted into therapies in Europe.The dissemination activities of the project result in broader and better communication of R&I results to society, demonstrating that science is not (only) for scientists, but aims at helping European citizens in diagnosis, treatment and cure of common diseases. The researcher’ s permanent return to Europe strengthens European human capital base in R&I and leads to enhanced and stronger scientific networks that produce long-term synergies and define EU and ERA as world leaders in the battle against autoimmune diseases.
The EXAUTOIMMUNE project has helped the fellow acquire the competences and skills necessary to challenge the state of the art and apply innovative ideas to the current knowledge of autoimmune diseases. In particular, the emerging applications of next generation RNAseq and its exciting future in biomarker discovery and Precision Medicine make this project highly impactful for the fellow’s career while allowing for a more holistic understanding of the autoimmune diseases in society.
The fellow’s goal for the near future is to define molecular targets for pathogenesis and treatment of autoimmune diseases that can be tested in predictive models. The results obtained from these mouse models will lead to novel treatment approaches for the human disease while at the same time increasing the output of research and innovation (R&I) and resulting to more knowledge and ideas converted into therapies in Europe.The dissemination activities of the project result in broader and better communication of R&I results to society, demonstrating that science is not (only) for scientists, but aims at helping European citizens in diagnosis, treatment and cure of common diseases. The researcher’ s permanent return to Europe strengthens European human capital base in R&I and leads to enhanced and stronger scientific networks that produce long-term synergies and define EU and ERA as world leaders in the battle against autoimmune diseases.