MicroRNAs: markers of β-cell stress and autoimmunity

Purpose of review We discuss current knowledge about microRNAs (miRNAs) in type 1 diabetes (T1D), an autoimmune disease leading to severe loss of pancreatic β-cells. We describe: The role of cellular miRNAs in regulating immune functions and pathways impacting insulin secretion and β-cell survival; circulating miRNAs as disease biomarkers. Recent findings Studies examined miRNAs in experimental models and patients, including analysis of tissues from organ donors, peripheral blood cells, and circulating miRNAs in serum, plasma, and exosomes. Studies employed diverse designs and methodologies to detect miRNAs and measure their levels. Selected miRNAs have been linked to the regulation of key biological pathways and disease pathogenesis; several circulating miRNAs are associated with having T1D, islet autoimmunity, disease progression, and immune and metabolic functions, for example, C-peptide secretion, in multiple studies. Summary A growing literature reveals multiple roles of miRNAs in T1D, provide new clues into the regulation of disease mechanisms, and identify reproducible associations. Yet challenges remain, and the field will benefit from joint efforts to analyze results, compare methodologies, formally test the robustness of miRNA associations, and ultimately move towards validating robust miRNA biomarkers. Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved

Anthropometric characteristics of young Italian tennis players

Purpose. Aim of our study is to observe specific body differences induced by training in young agonist tennis players at pre-pubertal and pubertal age, using anthropometry. Method. We analyzed 101 tennis players (27 females and 74 males) coming from South Italy, aged 8-14, which played tennis from at least 1 year. Anthropometric measurements like wrist, mid-thigh, mid-arm circumferences and arms length were compared between dominant and not dominant side of the body. Results. The mean Z-score for females was 0.9627, corresponding to the 83.22th centile of Italian growth chart, the mean Z-score for males was 1.0157, corresponding to 84.51th centile of Italian growth chart. For weight the mean Z-score for females was 0.2394 (59.46th centile) and the mean Z-score for males was 0.4032 (65.66th centile). The mean Z-score for females Body Mass Index was -0.1451 (44.23th centile), instead the mean Z-score for males was -0.0768 (46.94th centile). The Wilcoxon test reported a statistically significant difference between dominant and not dominant wrist circumferences regardless of sex (p-value=1.87e-10). Conclusions. Our study revealed that playing tennis starting from childhood may be useful for a regular growth. Analysis of wrist circumference and height may confirm the osteogenic potential of the sport

MicroRNA expression analysis of in vitro dedifferentiated human pancreatic islet cells reveals the activation of the pluripotency-related microRNA cluster miR-302s

β-cell dedifferentiation has been recently suggested as an additional mechanism contributing to type-1 and to type-2 diabetes pathogenesis. Moreover, several studies demonstrated that in vitro culture of native human pancreatic islets derived from non-diabetic donors resulted in the generation of an undifferentiated cell population. Additional evidence from in vitro human β-cell lineage tracing experiments, demonstrated that dedifferentiated cells derive from β-cells, thus representing a potential in vitro model of β-cell dedifferentiation. Here, we report the microRNA expression profiles analysis of in vitro dedifferentiated islet cells in comparison to mature human native pancreatic islets. We identified 13 microRNAs upregulated and 110 downregulated in islet cells upon in vitro dedifferentiation. Interestingly, among upregulated microRNAs, we observed the activation of microRNA miR-302s cluster, previously defined as pluripotency-associated. Bioinformatic analysis indicated that miR-302s are predicted to target several genes involved in the control of β-cell/epithelial phenotype maintenance; accordingly, such genes were downregulated upon human islet in vitro dedifferentiation. Moreover, we uncovered that cell-cell contacts are needed to maintain low/null expression levels of miR-302. In conclusion, we showed that miR-302 microRNA cluster genes are involved in in vitro dedifferentiation of human pancreatic islet cells and inhibits the expression of multiple genes involved in the maintenance of β-cell mature phenotype

MicroRNA miR-124a, a negative regulator of insulin secretion, is hyperexpressed in human pancreatic islets of type 2 diabetic patients

MicroRNAs are a class of negative regulators of gene expression, which have been demonstrated to be involved in the development of endocrine pancreas and in the regulation of insulin secretion. Type 2 Diabetes (T2D) is a metabolic disease characterized by insulin-resistance in target tissues and by the functional alteration of pancreatic insulin-secreting beta-cells. Recently, we characterized the expression levels of microRNAs miR-124a and miR-375, both involved in the control of beta cell function, in human pancreatic islets obtained from T2D and from age-matched non-diabetic organ donors. We observed the hyperexpression of miR-124a in human pancreatic islets obtained from T2D patients vs non-diabetic subjects, while miR-375 did not result differentially expressed. Moreover, we demonstrated that miR-124a overexpression in MIN6-pseudoislets reduced glucose-stimulated insulin secretion. Among predicted miR-124a target genes we focused on Foxa2 and Mtpn, which are both involved in the regulation of insulin secretion and of glucose sensing. Indeed, using luciferase assay, we validated miR-124a targeting Foxa2 and Mtpn 3’UTR sequences. Accordingly, upon miR-124a inhibition in MIN6 pseudoislets, we detected the upregulation of Foxa2 and Mtpn and of other selected miR-124a predicted target genes such as Akt3, Flot2, Sirt1, and NeuroD1, indicating a possible role for such a microRNA in the control of several beta-cell functions. In conclusion, we uncovered a major hyperexpression of miR-124a in T2D islets, whose silencing resulted in increased expression of target genes of major importance for beta cell function and whose overexpression impaired glucose stimulated insulin secretion, leading to the hypothesis that an altered miR-124a expression may contribute to beta cell dysfunction in type 2 diabete

Regulatory T-cells from pancreatic lymphnodes of patients with type-1 diabetes express increased levels of microRNA miR-125a-5p that limits CCR2 expression

Autoimmune type 1 diabetes (T1D) is thought to be caused by a defective immune regulation with regulatory T (Treg) cells playing a fundamental role in this process. Tolerance mechanisms depend on tunable responses that are sensitive to minor perturbations in the expression of molecules that can be carried out by multiple epigenetic mechanisms, including regulation by microRNAs. In this study, microRNA expression profile was investigated in Treg cells isolated from peripheral blood (PB) and from pancreatic draining lymph nodes (PLN) of T1D patients and non-diabetic subjects. Among 72 microRNAs analyzed, miR-125a-5p resulted specifically hyper-expressed in Treg cells purified from PLN of T1D patients. TNFR2 and CCR2 were identified as miR-125a-5p target genes. Elevated miR-125a-5p was detected in Treg cells isolated from PLN but not from PB of donors with T1D and was associated with reduced CCR2 expression. A specific beta-cell expression of the CCR2-ligand (CCL2) was observed in the pancreata of cadaveric donors, suggesting that beta-cells are prone to attract CCR2+ Treg cells. These novel data propose a mechanism, occurring in PLNs of T1D patients, involving increased expression of miR-125a-5p on Treg cells which results into reduced expression of CCR2, thus limiting their migration and eventual function in the pancreas

Circulating microRNA as biomarkers of autoimmune type 1 diabetes – Assessing the relevance of a target organ specific microRNA signature in type 1 diabetes

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

MicroRNAs: Novel Players in the Dialogue between Pancreatic Islets and Immune System in Autoimmune Diabetes

MicroRNAs are small noncoding RNA molecules that regulate gene expression in all cell types. Therefore, these tiny noncoding RNA molecules are involved in a wide range of biological processes, exerting functional effects at cellular, tissue, and organ level. In pancreatic islets of Langerhans, including beta-cells, microRNAs are involved in cell differentiation as well as in insulin secretion, while in immune cells they have been shown to play pivotal roles in development, activation, and response to antigens. Indeed, it is not surprising that microRNA alterations can lead to the development of several diseases, including type 1 diabetes (T1D). Type 1 diabetes is the result of a selective autoimmune destruction of insulin-producing beta-cells, characterized by islet inflammation (insulitis), which leads to chronic hyperglycemia. Given the growing importance of microRNA in the pathophysiology of T1D, the aim of this review is to summarize the most recent data on the potential involvement of microRNAs in autoimmune diabetes. Specifically, we will focus on three different aspects: (i) microRNAs as regulators of immune homeostasis in autoimmune diabetes; (ii) microRNA expression in pancreatic islet inflammation; (iii) microRNAs as players in the dialogue between the immune system and pancreatic endocrine cells

Circulating microRNAs and diabetes mellitus: a novel tool for disease prediction, diagnosis, and staging?

Diabetes is a complex, multifactorial group of metabolic diseases characterized by chronic hyperglycaemia due to pancreatic beta-cell dysfunction and/or loss. It is characterized by an asymptomatic and highly variable prodromic phase, which renders diabetes mellitus difficult to be predicted with sufficient accuracy. Despite several efforts in the identification and standardization of newly trustable. Biomarkers able to predict and follow-up diabetes and to specifically subtype its different forms, few of them have proven of clinical utility. Recently, a new class of endogenous non-coding small RNAs, namely microRNAs, have been indicated as putative biomarkers, being released by cells and tissues and found in a cell-free circulating form in many biological fluids, including serum and/or plasma. MicroRNAs have been initially identified as promising biomarkers in cancer, and nowadays their application has been extended to other diseases, including diabetes. Although an increasing number of studies focused on the evaluation of circulating microRNAs in diabetes, few reproducibly identified microRNAs as biomarkers for disease prediction or follow-up. Technological problems as well as the need to obtain highly standardized operating procedures and methods are still an issue in such research field. In this review, we comprehensively resume the main and most recent findings on circulating microRNAs, and their possible use as biomarkers to predict and follow-up diabetes and its complications, as well as the methodological challenges to standardize accurate operating procedures for their analysis

microRNA miR-124a, a negative regulator of insulin secretion, is hyperexpressed in human pancreatic islets of type 2 diabetic patients: DOI: 10.14800/rd.593

microRNAs are a class of negative regulators of gene expression, which have been demonstrated to be involved in the development of endocrine pancreas and in the regulation of insulin secretion. Type 2 Diabetes (T2D) is a metabolic disease characterized by insulin-resistance in target tissues and by the functional alteration of pancreatic insulin-secreting beta-cells. Recently, we characterized the expression levels of microRNAs miR-124a and miR-375, both involved in the control of beta cell function, in human pancreatic islets obtained from T2D and from age-matched non-diabetic organ donors. We observed the hyperexpression of miR-124a in human pancreatic islets obtained from T2D patients vs non-diabetic subjects, while miR-375 did not result differentially expressed. Moreover, we demonstrated that miR-124a overexpression in MIN6-pseudoislets reduced glucose-stimulated insulin secretion. Among predicted miR-124a target genes we focused on Foxa2 and Mtpn, which are both involved in the regulation of insulin secretion and of glucose sensing. Indeed, using luciferase assay, we validated miR-124a targeting Foxa2 and Mtpn 3’UTR sequences. Accordingly, upon miR-124a inhibition in MIN6 pseudoislets, we detected the upregulation of Foxa2 and Mtpn and of other selected miR-124a predicted target genes such as Akt3, Flot2, Sirt1, and NeuroD1, indicating a possible role for such a microRNA in the control of several beta-cell functions. In conclusion, we uncovered a major hyperexpression of miR-124a in T2D islets, whose silencing resulted in increased expression of target genes of major importance for beta cell function and whose overexpression impaired glucose stimulated insulin secretion, leading to the hypothesis that an altered miR-124a expression may contribute to beta cell dysfunction in type 2 diabetes