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.
