INTRODUCTION
Type 1 diabetes is caused by autoimmune destruction of Langerhans islets' beta-cells, resulting in an absolute lack of one’s own insulin. Hence, individuals with type 1 diabetes require a life long intensive exogenous insulin therapy with glucose monitoring to prevent the development of diabetes-related complications, which can dramatically shorten their lifespan. The cause of type 1 diabetes is still unknown due to a lack of biomarkers and difficulties in gaining the insight into beta-cells functioning during the developmental stages of the disease. With their molecular cargo extracellular vesicles can modulate a target cell response and may affect the pathogenesis of the diseases. The extracellular vesicles containing miRNAs are often studied as disease biomarkers, but rarely as mediators of development of the disease. The role of extracellular vesicles derived miRNAs in type 1 diabetes is currently not well established. Our work aimed to evaluate the extracellular vesicles as biomarkers in type 1 diabetes and evaluate their role in the modulation of the immune system in the development of the disease.
METHODS
Transmission electron microscopy and vesicle labelling with specific antibodies was used to characterize the extracellular vesicles originating from insulin-producing pancreatic beta cells in blood plasma. In the next step, differentially expressed extracellular vesicles’ miRNAs were evaluated using the next-generation sequencing approach in ten individuals with type 1 diabetes at the disease onset, ten individuals with ten years duration of the disease, and ten healthy controls. To assess the extracellular vesicle miRNAs in intensive beta-cell destruction, Langerhans islet transplantation individuals’ chronological blood plasma samples were sequenced and analysed. Eight differentially expressed miRNAs in type 1 diabetes and Langerhans islets destruction were selected for the in vitro immunomodulation testing with the synthetic vesicle delivery system on the whole blood samples. Flow cytometry was applied to evaluate the immune system activation with the detection of CD107a degranulation and CD69 early activation markers on CD4+ and CD8+ T-cells and CD56+ NK cells. The vesicle miRNA accumulation in the cells of the immune system was evaluated with microscopy and flow cytometry after the whole blood cells exposure to synthetic vesicles and fluorescent labeled miRNA. The endosomal TLR7/8 inhibition in the transfection experiments was performed to evaluate the role of these receptors in the recognition of extracellular vesicle delivered miRNA in the immune system activation. Additionally, miRNAs regulate telomere lengths in the cells of a human organism and these are biomarkers of oxidative stress and organsim aging. Telomere lengths were assessed in the individuals with type 1 diabetes according to their glycemic control.
RESULTS
Transmission electron microscopy imaging confirmed Langerhans islets beta-cell-released extracellular vesicles in the blood plasma and potential beta-cells’ communication with other tissues. Based on the next-generation sequencing differential expression analysis results, eight significantly differentially expressed miRNA in type 1 diabetes and Langerhans transplantation individuals were selected (hsa-miR-122-5p, hsa-miR-192-5p, hsa-miR-193b-5p, hsa-miR-185-5p, hsa-miR-195-3p, hsa-miR-455-5p, hsa-miR-375-3p and hsa-miR-129-5p). The selected differentially expressed miRNA delivered with synthetic vesicles to the whole blood samples from participants with type 1 diabetes and healthy controls showed the immunomodulation activity with the overexpression of an early activation marker CD69 and a cytotoxicity marker CD107a on CD4+ and CD8+ T-cells and CD 56+ NK cells. The phagocytic cells accumulated vesicles with miRNAs in the endolysosomal pathway, where TLR7 and TLR8 were expressed. The potential involvement of the endolysosomal TLR7/8 in the activation of the immune system was shown with the endosomal TLR7/8 inhibitor chloroquine, which effectively inhibited a vesicle delivered miRNAs activation. The sequencing results also revealed differentially expressed miRNAs, whose role was reported in the telomere length regulation. The telomere length erosion in the individuals with type 1 diabetes with poor glyceamic control was accelerated compared to the good glycemic controlled individuals.
SUMMARY
Our results illustrate miRNAs derived from human blood plasma extracellular vesicles as modulators of the immune system in type 1 diabetes autoimmunity, providing a potentially new insight into the pathogenesis of the disease, and novel molecular targets for intervention and type 1 diabetes prevention.
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