miR-409-3p is reduced in plasma and islet immune infiltrates of NOD diabetic mice and is differentially expressed in people with type 1 diabetes.

AIMS/HYPOTHESIS: MicroRNAs (miRNAs) are a novel class of potential biomarkers emerging in many diseases, including type 1 diabetes. Here, we aim to analyse a panel of circulating miRNAs in non-obese diabetic (NOD) mice and individuals with type 1 diabetes. METHODS: We adopted standardised methodologies for extracting miRNAs from small sample volumes to evaluate a profiling panel of mature miRNAs in paired plasma and laser-captured microdissected immune-infiltrated islets of recently diabetic and normoglycaemic NOD mice. Moreover, we validated the findings during disease progression and remission after anti-CD3 therapy in NOD mice, as well as in individuals with type 1 diabetes. RESULTS: Plasma levels of five miRNAs were downregulated in diabetic vs normoglycaemic mice. Of those, miR-409-3p was also downregulated in situ in the immune islet infiltrates of diabetic mice, suggesting an association with disease pathogenesis. Target-prediction tools linked miR-409-3p to immune- and metabolism-related signalling molecules. In situ miR-409-3p expression correlated with insulitis severity, and CD8+ central memory T cells were found to be enriched in miR-409-3p. Plasma miR-409-3p levels gradually decreased during diabetes development and improved with disease remission after anti-CD3 antibody therapy. Finally, plasma miR-409-3p levels were lower in people recently diagnosed with type 1 diabetes compared with a non-diabetic control group, and levels were inversely correlated with HbA1c levels. CONCLUSIONS/INTERPRETATION: We propose that miR-409-3p may represent a new circulating biomarker of islet inflammation and type 1 diabetes severity.

MicroRNAs as Regulators of Insulin Signaling: Research Updates and Potential Therapeutic Perspectives in Type 2 Diabetes.

The insulin signaling pathway is composed of a large number of molecules that positively or negatively modulate insulin specific signal transduction following its binding to the cognate receptor. Given the importance of the final effects of insulin signal transduction, it is conceivable that many regulators are needed in order to tightly control the metabolic or proliferative functional outputs. MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively modulate gene expression through their specific binding within the 3'UTR sequence of messenger RNA (mRNA), thus causing mRNA decoy or translational inhibition. In the last decade, miRNAs have been addressed as pivotal cellular rheostats which control many fundamental signaling pathways, including insulin signal transduction. Several studies demonstrated that multiple alterations of miRNAs expression or function are relevant for the development of insulin resistance in type 2 diabetes (T2D); such alterations have been highlighted in multiple insulin target organs including liver, muscles, and adipose tissue. Indirectly, miRNAs have been identified as modulators of inflammation-derived insulin resistance, by controlling/tuning the activity of innate immune cells in insulin target tissues. Here, we review main findings on miRNA functions as modulators of insulin signaling in physiologic- or in T2D insulin resistance- status. Additionally, we report the latest hypotheses of prospective therapies involving miRNAs as potential targets for future drugs in T2D.

MicroRNA Expression Analysis of In Vitro Dedifferentiated Human Pancreatic Islet Cells Reveals the Activation of the Pluripotency-Related MicroRNA Cluster miR-302s.

ß-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 ß-cell lineage tracing experiments, demonstrated that dedifferentiated cells derive from ß-cells, thus representing a potential in vitro model of ß-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 ß-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 ß-cell mature phenotype.

Long-term outcomes with emicizumab in hemophilia A without inhibitors: results from the HAVEN 3 and 4 studies.

Background: Emicizumab, a bispecific monoclonal antibody, bridges activated factor (F) IX and FX, mimicking the function of missing or deficient activated FVIII in people with hemophilia A (HA). Objectives: To evaluate the long-term efficacy and safety of emicizumab prophylaxis in people with HA without FVIII inhibitors in the HAVEN 3 and 4 studies. Methods: HAVEN 3 and 4 were phase 3 open-label studies. Participants received emicizumab maintenance doses of 1.5 mg/kg every week or 3 mg/kg every 2 weeks (HAVEN 3), or 6 mg/kg every 4 weeks (HAVEN 4). Long-term efficacy and safety were assessed. Results: A total of 151 and 40 individuals without FVIII inhibitors received emicizumab in HAVEN 3 and 4, respectively. At the last patient, last visit dates (May 12, 2022 [HAVEN 3] and June 29, 2022 [HAVEN 4]), the median (range) duration of emicizumab exposure across the 2 studies was 248.1 (6.1-287.1) weeks. The mean (95% CI) annualized bleed rate for treated bleeds was 2.0 (0.23-7.15) for weeks 1 to 24, decreasing to 0.9 (0.01-5.28) by weeks 217 to 240. Overall, 188 (98.4%) participants experienced ≥1 adverse event (AE), with 185 treatment-related AEs in 71 (37.2%) participants. Forty-four (23.0%) participants reported a serious AE. Two thromboembolic events were reported, which were deemed unrelated to emicizumab by the investigator. No thrombotic microangiopathies were reported. Conclusion: With nearly 5 years of emicizumab exposure across the HAVEN 3 and 4 studies in people with HA without inhibitors, these data indicate continued bleed control with no new safety signals observed during long-term follow-up.

Emicizumab use in females with moderate or mild hemophilia A without factor VIII inhibitors who warrant prophylaxis.

Background: Hemophilia A (HA) is predominantly associated with males due to X-linked inheritance. Males and females with HA have shared unmet medical needs, highlighting the necessity for comprehensive care irrespective of sex. Objectives: This analysis investigated the efficacy and safety of emicizumab prophylaxis in 3 females with HA. Methods: HAVEN 6 (NCT04158648) is a phase III study of emicizumab in people with non-severe HA without factor (F)VIII inhibitors warranting prophylaxis per investigator assessment, and the study methodology has been reported previously. Female-specific endpoints included menstruation-related quality of life and menstruation heaviness. Results: HAVEN 6 enrolled 3 females aged ≥18 years and within reproductive age (n = 2 mild HA; n = 1 moderate HA; n = 2 receiving prior FVIII prophylaxis; n = 1 receiving prior episodic FVIII). Participants presented with diverse bleeding phenotypes at baseline: 2 had no bleeds in the 24 weeks prior to enrollment, while 1 had an annualized bleed rate for all bleeds of 208.6. On-study annualized bleed rates for all bleeds were 0, 2.8, and 11.6, respectively. The 2 evaluable participants indicated improved menstruation-related quality of life vs baseline. Two participants experienced 3 grade 1/2 treatment-related adverse events; no new safety signals were identified. All 3 participants preferred emicizumab over their previous treatment and reported a better score for treatment burden and preoccupation domains of the Comprehensive Assessment Tool of Challenges in Hemophilia questionnaire. Conclusion: Overall, results were consistent with those reported in the male population enrolled in the HAVEN 6 study, suggesting efficacy and a favorable safety profile for emicizumab in females with non-severe HA warranting prophylaxis.

A Plasma miR-193b-365 Signature Combined With Age and Glycemic Status Predicts Response to Lactococcus lactis-Based Antigen-Specific Immunotherapy in New-Onset Type 1 Diabetes.

Immunomodulation combined with antigen therapy holds great promise to arrest autoimmune type 1 diabetes, but clinical translation is hampered by a lack of prognostic biomarkers. Low-dose anti-CD3 plus Lactococcus lactis bacteria secreting proinsulin and IL-10 reversed new-onset disease in nonobese diabetic (NOD) mice, yet some mice were resistant to the therapy. Using miRNA profiling, six miRNAs (i.e., miR-34a-5p, miR-125a-3p, miR-193b-3p, miR-328, miR-365-3p, and miR-671-3p) were identified as differentially expressed in plasma of responder versus nonresponder mice before study entry. After validation and stratification in an independent cohort, plasma miR-193b-3p and miR-365-3p, combined with age and glycemic status at study entry, had the best power to predict, with high sensitivity and specificity, poor response to the therapy. These miRNAs were highly abundant in pancreas-infiltrating neutrophils and basophils with a proinflammatory and activated phenotype. Here, a set of miRNAs and disease-associated parameters are presented as a predictive signature for the L. lactis-based immunotherapy outcome in new-onset type 1 diabetes, hence allowing targeted recruitment of trial participants and accelerated trial execution. ARTICLE HIGHLIGHTS: Low-dose anti-CD3 combined with oral gavage of genetically modified Lactococcus lactis bacteria secreting human proinsulin and IL-10 holds great promise to arrest autoimmune type 1 diabetes, but the absence of biomarkers predicting therapeutic success hampers clinical translation. A set of cell-free circulation miRNAs together with age and glycemia at baseline predicts a poor response after L. lactis-based immunotherapy in nonobese mice with new-onset diabetes. Pancreas-infiltrating neutrophils and basophils are identified as potential cellular sources of discovered miRNAs. The prognostic signature could guide targeted recruitment of patients with newly diagnosed type 1 diabetes in clinical trials with the L. lactis-based immunotherapy.

Emicizumab in people with moderate or mild haemophilia A (HAVEN 6): a multicentre, open-label, single-arm, phase 3 study.

BACKGROUND: Clinical trial data are scarce for the use of prophylaxis in people with non-severe haemophilia A. The HAVEN 6 study aims to assess safety and efficacy of emicizumab prophylaxis in people with non-severe haemophilia A without factor VIII (FVIII) inhibitors. METHODS: HAVEN 6 is a multicentre, open-label, single-arm, phase 3 study taking place in 22 specialty clinics and hospitals in Europe, North America, and South Africa. Eligible participants were people of all ages weighing at least 3 kg with a diagnosis of moderate (FVIII activity ≥1%-≤5%) or mild (FVIII >5%-<40%) haemophilia A without FVIII inhibitors requiring prophylaxis as assessed by the treating physician. Participants received subcutaneous emicizumab 3 mg/kg of bodyweight once weekly for 4 weeks, followed by the participant's choice of maintenance dose: 1·5 mg/kg once weekly, 3 mg/kg every 2 weeks, or 6 mg/kg every 4 weeks. Safety was the primary objective of the study. Safety endpoints included adverse events, serious adverse events, and adverse events of special interest including thromboembolic events and thrombotic microangiopathies. The primary efficacy endpoint was the annualised bleed rate for treated bleeds. Analyses were done for participants who received at least one dose of emicizumab. This study is registered with ClinicalTrials.gov, number NCT04158648, and is active but not recruiting. FINDINGS: Between Feb 10, 2020, and Aug 31, 2021, we assigned 73 people to treatment. 72 participants received at least one dose of emicizumab (51 moderate [71%]; 21 mild [29%]; 69 male [96%]; three female [4%]; and 61 White [85%]). Median age was 23·5 years (IQR 12·0-36·0); median follow-up was 55·6 weeks (IQR 52·3-61·6) weeks. At baseline, 24 participants (33%) had target joints and 37 (51%) were receiving FVIII prophylaxis. 60 participants (83%) had at least one adverse event; the most common adverse events were headache (in 12 participants [17%]), injection-site reaction (12 [17%]), and arthralgia (11 [15%]). 15 (21%) had at least one emicizumab-related adverse event; no adverse events led to treatment withdrawal, modification, or interruption. Eight participants (11%) reported ten serious adverse events in total, none emicizumab-related. There were no deaths or thrombotic microangiopathies. One participant had grade 1 thrombosed haemorrhoids (classified as a thromboembolic event), unrelated to emicizumab. The annualised bleed rate was 0·9 (95% CI 0·55-1·52) for treated bleeds. 48 participants (67%) had no treated bleeds. All-bleed annualised bleed rates were 10·1 (95% CI 6·93-14·76) from 24 weeks pre-study and 2·3 (1·67-3·12) on-study after a median follow-up of 55·6 weeks. INTERPRETATION: These data show efficacy and a favourable safety profile of emicizumab in people with non-severe haemophilia A without FVIII inhibitors who warrant prophylaxis, confirming emicizumab as a valuable treatment option in this population. FUNDING: F Hoffmann-La Roche.

Intestinal Delivery of Proinsulin and IL-10 via Lactococcus lactis Combined With Low-Dose Anti-CD3 Restores Tolerance Outside the Window of Acute Type 1 Diabetes Diagnosis.

A combination treatment (CT) of proinsulin and IL-10 orally delivered via genetically modified Lactococcus lactis bacteria combined with low-dose anti-CD3 (aCD3) therapy successfully restores glucose homeostasis in newly diagnosed non-obese diabetic (NOD) mice. Tolerance is accompanied by the accumulation of Foxp3+ regulatory T cells (Tregs) in the pancreas. To test the potential of this therapy outside the window of acute diabetes diagnosis, we substituted autoimmune diabetic mice, with disease duration varying between 4 and 53 days, with syngeneic islets at the time of therapy initiation. Untreated islet recipients consistently showed disease recurrence after 8.2 ± 0.7 days, while 32% of aCD3-treated and 48% of CT-treated mice remained normoglycemic until 6 weeks after therapy initiation (P < 0.001 vs. untreated controls for both treatments, P < 0.05 CT vs. aCD3 therapy). However, mice that were diabetic for more than 2 weeks before treatment initiation were less efficient at maintaining normoglycemia than those treated within 2 weeks of diabetes diagnosis, particularly in the aCD3-treated group. The complete elimination of endogenous beta cell mass with alloxan at the time of diabetes diagnosis pointed toward the significance of continuous feeding of the islet antigen proinsulin at the time of aCD3 therapy for treatment success. The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (ovalbumin) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Sustained tolerance was accompanied with a reduction of IGRP+CD8+ autoreactive T cells and an increase in insulin-reactive (InsB12-20 or InsB13-2) Foxp3+CD4+ Tregs, with a specific accumulation of Foxp3+ Tregs around the insulin-containing islet grafts after CT with proinsulin. The combination of proinsulin and IL-10 via oral Lactococcus lactis with low-dose aCD3 therapy can restore tolerance to beta cells in autoimmune diabetic mice, also when therapy is started outside the window of acute diabetes diagnosis, providing persistence of insulin-containing islets or prolonged beta cell function.

MicroRNAs: markers of ß-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 ß-cells. We describe: the role of cellular miRNAs in regulating immune functions and pathways impacting insulin secretion and ß-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.

Serum Levels of miR-148a and miR-21-5p Are Increased in Type 1 Diabetic Patients and Correlated with Markers of Bone Strength and Metabolism.

Type 1 diabetes (T1D) is characterized by bone loss and altered bone remodeling, resulting into reduction of bone mineral density (BMD) and increased risk of fractures. Identification of specific biomarkers and/or causative factors of diabetic bone fragility is of fundamental importance for an early detection of such alterations and to envisage appropriate therapeutic interventions. MicroRNAs (miRNAs) are small non-coding RNAs which negatively regulate genes expression. Of note, miRNAs can be secreted in biological fluids through their association with different cellular components and, in such context, they may represent both candidate biomarkers and/or mediators of bone metabolism alterations. Here, we aimed at identifying miRNAs differentially expressed in serum of T1D patients and potentially involved in bone loss in type 1 diabetes. We selected six miRNAs previously associated with T1D and bone metabolism: miR-21; miR-24; miR-27a; miR-148a; miR-214; and miR-375. Selected miRNAs were analyzed in sera of 15 T1D patients (age: 33.57 ± 8.17; BMI: 21.4 ± 1.65) and 14 non-diabetic subjects (age: 31.7 ± 8.2; BMI: 24.6 ± 4.34). Calcium, osteocalcin, parathormone (PTH), bone ALkaline Phoshatase (bALP), and Vitamin D (VitD) as well as main parameters of bone health were measured in each patient. We observed an increased expression of miR-148a (p = 0.012) and miR-21-5p (p = 0.034) in sera of T1D patients vs non-diabetic subjects. The correlation analysis between miRNAs expression and the main parameters of bone metabolism, showed a correlation between miR-148a and Bone Mineral Density (BMD) total body (TB) values (p = 0.042) and PTH circulating levels (p = 0.033) and the association of miR-21-5p to Bone Mineral Content-Femur (BMC-FEM). Finally, miR-148a and miR-21-5p target genes prediction analysis revealed several factors involved in bone development and remodeling, such as MAFB, WNT1, TGFB2, STAT3, or PDCD4, and the co-modulation of common pathways involved in bone homeostasis thus potentially assigning a role to both miR-148a and miR-21-5p in bone metabolism alterations. In conclusion, these results lead us to hypothesize a potential role for miR-148a and miR-21-5p in bone remodeling, thus representing potential biomarkers of bone fragility in T1D.

MicroRNA expression profiles of human iPSCs differentiation into insulin-producing cells.

AIMS: MicroRNAs are a class of small noncoding RNAs, which control gene expression by inhibition of mRNA translation. MicroRNAs are involved in the control of biological processes including cell differentiation. Here, we aim at characterizing microRNA expression profiles during differentiation of human induced pluripotent stem cells (hiPSCs) into insulin-producing cells. METHODS: We differentiated hiPSCs toward endocrine pancreatic lineage following a 18-day protocol. We analyzed genes and microRNA expression levels using RT real-time PCR and TaqMan microRNA arrays followed by bioinformatic functional analysis. RESULTS: MicroRNA expression profiles analysis of undifferentiated hiPSCs during pancreatic differentiation revealed that 347/768 microRNAs were expressed at least in one time point of all samples. We observed 18 microRNAs differentially expressed: 11 were upregulated (miR-9-5p, miR-9-3p, miR-10a, miR-99a-3p, miR-124a, miR-135a, miR-138, miR-149, miR-211, miR-342-3p and miR-375) and 7 downregulated (miR-31, miR-127, miR-143, miR-302c-3p, miR-373, miR-518b and miR-520c-3p) during differentiation into insulin-producing cells. Selected microRNAs were further evaluated during differentiation of Sendai-virus-reprogrammed hiPSCs using an improved endocrine pancreatic beta cell derivation protocol and, moreover, in differentiated NKX6.1+ sorted cells. Following Targetscan7.0 analysis of target genes of differentially expressed microRNAs and gene ontology classification, we found that such target genes belong to categories of major significance in pancreas organogenesis and development or exocytosis. CONCLUSIONS: We detected a specific hiPSCs microRNAs signature during differentiation into insulin-producing cells and demonstrated that differentially expressed microRNAs target several genes involved in pancreas organogenesis.

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.

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.

MicroRNA-124a is hyperexpressed in type 2 diabetic human pancreatic islets and negatively regulates insulin secretion.

AIMS: MicroRNAs are a class of negative regulators of gene expression, which have been shown to be involved in the development of endocrine pancreas and in the regulation of insulin secretion. Since type 2 diabetes (T2D) is characterized by beta cell dysfunction, we aimed at evaluating expression levels of 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. METHODS: We analyzed miR-124a and miR-375 expression by real-time qRT-PCR in human pancreatic islets and evaluated the potential role of miR-124a by overexpressing or silencing such miRNA in MIN6 pseudoislets. RESULTS: We identified a major miR-124a hyperexpression in T2D human pancreatic islets with no differential expression of miR-375. Of note, miR-124a overexpression in MIN6 pseudoislets resulted in an impaired glucose-induced insulin secretion. In addition, miR-124a silencing in MIN6 pseudoislets resulted in increased expression of predicted target genes (Mtpn, Foxa2, Flot2, Akt3, Sirt1 and NeuroD1) involved in beta cell function. For Mtpn and Foxa2, we further demonstrated the actual binding of miR-124a to their 3UTR sequences by luciferase assay. CONCLUSIONS: 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.