Type 1 diabetes (T1D) results from a T cell mediated destruction due to a breach in immune tolerance towards insulin producing β-cells, leading to an absolute insulin deficiency. To date, the pathogenesis of the disease is not fully understood and exogenous insulin still represents the gold standard as therapy. It is believed that in some genetically predisposed individuals environmental factors can lead to a breakdown in immunological tolerance. T regulatory cells (Tregs), a specialized subpopulation of T cell, are the major components of peripheral tolerance mechanisms due to their ability to restrain autoreactive effector T cells. Interestingly, microRNAs, small non-coding RNAs that negatively regulate gene expression, are emerging as crucial modulators of immune cell functions and their deregulation has been associated with autoimmune disease like T1D, thus representing major players in the regulation of immune homeostasis. Against this background, characterizing microRNA expression in Tregs deriving from tissues close to target organ in T1D patients will be crucial to gain a deeper insight into immune deregulation. Interestingly, microRNAs have also been detected extracellularly in biological fluids, thus representing new potential disease biomarkers. The need of new biomarkers in the field is underlined by the complexity and heterogeneity of T1D with the clinical presentation being preceded by a long asymptomatic phase in which most β-cells are destroyed. Moreover, immunomodulatory trials in T1D, although having failed their primary endpoints, have identified through post-hoc analysis subgroups of patients for whom interventions were beneficial. Therefore, the identification of new biomarkers would help to specifically select patients that would benefit from specific immunotherapies. Thus, the main purpose of this work was to gain insight into the microRNA regulation occurring in Tregs residing in the pancreatic draining lymph nodes (PLN) of T1D patients and to establish circulating microRNAs as new biomarkers in diabetes autoimmune prone mice in order to monitor the lymphocyte mediated inflammatory state in the pancreas and to predict therapeutic responses in an Ag-specific combination therapy. Tregs circulating in the peripheral blood are likely to differ from those residing in the LN draining the organ targeted by an autoimmune response, indeed a previous finding showed that only Tregs deriving from PLN of T1D patients have an impaired regulatory activity in vitro. Therefore, in the first project (Chapter 4) we investigated the microRNA expression profile in Tregs isolated from blood and PLN of patients with T1D and non-diabetic subjects. Among the 72 microRNAs detected in purified cells, miR-125a was highly expressed only in Treg cells purified from PLN of patients with T1D. Bioinformatic analysis of the target genes revealed CCR2 as a possible gene modulated by miR-125a. Accordingly, elevated miR-125a levels were detected in Tregs isolated from the PLN but not from the peripheral blood of donors with T1D and it was associated with a reduced CCR2 expression. A specific β-cell expression of the CCR2 ligand CCL2 (MCP1) was also observed in the pancreata of multi-organ donors, suggesting that β-cells are prone to attract CCR2+ Tregs. These data showed a reduced expression of CCR2 on Tregs deriving from PLN of T1D patients; this Treg status may limit their migration and eventual function in the pancreas. In the second project (Chapter 5) we aimed to investigate whether circulating microRNA alterations could reflect the pathologic process within the specific diseased tissue. Indeed, we identified miR-409-3p, a miRNA that negatively regulates the expression of IFNγ, as downregulated in both plasma and pancreatic lymphocytic infiltrates of recent-onset diabetic vs. normoglycemic NOD mice. Furthermore, we showed that circulating miR-409-3p plasma levels negatively correlated with in situ IFNγ expression in islet-infiltrating cells both in basal condition and following a therapeutic intervention with anti-CD3 treatment, thus highlighting the potential role of miR-409-3p as a biomarker of islet inflammation. Finally, we confirmed miR-409-3p downregulation in plasma samples derived from newly-diagnosed T1D patients vs. age-matched controls, indicating a possible application of circulating miR-409-3p as biomarker in order to monitor IFNγ mediated islet inflammation. The host lab previously showed that a combination therapy consisting of a 5 day course of anti-CD3 antibodies at disease onset along with a 6 weeks oral administration of live genetically modified Lactococcus lactis (L. lactis) producing human proinsulin and IL-10, restored durable normoglycemia in approximately 60% of NOD mice. Even though L. lactis therapy was successful in the reversal of autoimmune diabetes in around 60% of NOD mice, 40% of them were nonresponsive to therapy. The route to bring this successful antigen-based therapy from preclinical models to clinic will depend on implementation of biomarkers and profound understanding of diverse mechanisms underlining therapeutic success. Therefore, the aim of the third project (Chapter 6) was to establish whether circulating microRNAs could be used as predictor biomarkers for therapeutic response. Indeed, we found a circulating microRNA signature consisting of 6 microRNAs that were specifically upregulated at disease onset in non-responders mice. Moreover, the combination of 2 microRNAs were able to distinguish with good specificity and sensitivity. responder and non-responders at diabetes onset, thus emerging as a valuable tool to tailor this intervention therapy towards a more effective clinical study. Moreover, bioinformatics target analysis of the circulating microRNAs differentially expressed between responders and nonresponders in the plasma after treatment course, highlighted pathways related to the metabolic status of T cells, suggesting the induction of a T cell exhausted phenotype in responder mice. To conclude, this PhD project demonstrated the importance of assessing microRNA regulation in Tregs deriving from tissue sites close to the organ targeted by the autoimmune attack, as it differs from circulating Tregs. Moreover, plasma circulating microRNAs appear to be promising novel biomarkers in order to: i) monitor IFNγ mediated islet inflammation ii) predict therapeutic response and understand the tolerance induction mechanisms induced by L. lactis-combined therapy. Molecular understanding of Tregs function in the site of the autoimmune attack together with the discovery of new circulating biomarkers will surely help to monitor T1D progression and enhance the effective translation of this Ag-specific L.lactis intervention therapy in future clinical trials.
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|Titolo:||Circulating microRNA as biomarkers of autoimmune type 1 diabetes – Assessing the relevance of a target organ specific microRNA signature in type 1 diabetes|
|Appare nelle tipologie:||8.1 Tesi Dottorato|