Extracellular vesicles from adipose-derived stem cells activate a pro-inflammatory phenothype in t cell from type і and type 2 diabetic patients

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BACKGROUND AND AIMS The concept of immunemediated inflammatory disease is established for type 1 diabetes, but recent evidence indicates type 2 diabetes as an auto-inflammatory disease, with signs of insulitis as well. Adipose tissue is the largest endocrine organ with immune function and adipocyte dysfunction correlates with the development of insulin-resistance type 2 diabetes and with impaired micro- and macro-vascular function. On the other hand, studies in animal models of type 1 diabetes reveal that adipocyte dysfunction and high levels of inflammatory cytokines can directly play a role in the onset and progression of the type 1 diabetes. Angiogenesis and cell therapy studies indicate that adipose-derived stem cells (ASCs) hold promise amongst stem cells of mesenchymal lineage, acting through paracrine mechanisms possibly involving the release of extracellular vesicles (EVs), a recognised integral component of the cellular network. Paralleling previous in vitro studies using EVs derived from bone-marrow mesenchymal stem cells (MSCs) indicated the promotion of an anti-inflammatory T cell response. We evaluated whether EVs derived from ASCs may effect inflammatory response in type 1 and type 2 diabetes, acting on T cell. MATERIALS AND METHODS EVs were purified from heterologous human subcutaneous adipose mesenchymal stem cells (ASCs) obtained from healthy donors by differential centrifugation. Protein array and gene array analysis showed high expression of some pro-inflammatory factors such as IFN-γ, IL-1, IL-17 and IL-6 and microRNA miR-126 in EVs. PBMCs were obtained from 6 patients with recent onset type 1 diabetes and 6 patients with long standing type 2 diabetes on metformin. Cultures were established with PBMCs and ASC-EVs for 48 hours in type 1 and type 2 diabetic patients. Responses to GAD65 stimulation were assessed by IFN-y ELISPOT analysis in type 1 patients. Levels of cytokines were measured in the supernatant by ELISA and by intracellular flow cytometry analyses. T helper 17 (Th17) analysis was performed by flow cytometry analyses. RESULTS: ASC- MVs were internalised by PBMCs, as assessed by confocal microscopy and flow cytometry analyses. ASC-MVs increased IFN-y spots in GAD65-stimulated PBMCs obtained from type 1 diabetes. Moreover, ASC-MVs increased levels of IFN-γ, IL-6, IL-10, TNF-α, IL-1-β in PBMCs obtained from type 1 and type 2 diabetes, and decreased transforming growth factor-β (TGF-β) levels. Furthermore, ASC- MVs increased the number of Th17 cells and the levels of IL-17. CONCLUSIONS ASCEVs appear to lack the antigen and non-antigen specific anti-inflammatory effects of MSCs, and induce a pro-inflammatory phenotype in T cells. In the context of type 1 diabetes, ASC-EVs may contribute to increased levels of cytokines thus inducing ß cell death, inhibit insulin production and possibly contributing to the loss of self-tolerance. In type 2 diabetes may contribute lipotoxicity and exacerbate inflammation, also in the context of pancreatic islets.