Footprint of pancreas infiltrating and circulating immune cells throughout type 1 diabetes development.

Introduction: Type 1 diabetes (T1D) is defined by immune cell infiltration of the pancreas, in particular the islets of Langerhans, referred to as insulitis, which is especially prominent during the early disease stages in association with decreased beta cell mass. An in-depth understanding of the dynamics and phenotype of the immune cells infiltrating the pancreas and the accompanying changes in their profiles in peripheral blood during T1D development is critical to generate novel preventive and therapeutic approaches, as well as to find biomarkers for the disease process. Methods: Using multi-parameter flow cytometry, we explored the dynamic changes of immune cells infiltrating the pancreas and the pancreatic draining lymph nodes (PLN), compared to those in peripheral blood in female and male non-obese diabetic (NOD) mice during T1D progression. Results: The early stages of T1D development were characterized by an influx of innate dendritic cells and neutrophils in the pancreas. While dendritic cells seemed to move in and out (to the PLN), neutrophils accumulated during the pre-symptomatic phase and reached a maximum at 8 weeks of age, after which their numbers declined. During disease progression, CD4+ and CD8+ T cells appeared to continuously migrate from the PLN to the pancreas, which coincided with an increase in beta cell autoimmunity and insulitis severity, and a decline in insulin content. At 12 weeks of age, CD4+ and especially CD8+ T cells in the pancreas showed a dramatic shift from naïve to effector memory phenotype, in contrast to the PLN, where most of these cells remained naïve. A large proportion of pancreas infiltrating CD4+ T cells were naïve, indicating that antigenic stimulation was not necessary to traffic and invade the pancreas. Interestingly, a pre-effector-like T cell dominated the peripheral blood. These cells were intermediates between naïve and effector memory cells as identified by single cell RNA sequencing and might be a potential novel therapeutic target. Conclusion: These time- and tissue-dependent changes in the dynamics and functional states of CD4+ and CD8+ T cells are essential steps in our understanding of the disease process in NOD mice and need to be considered for the interpretation and design of disease-modifying therapies.

Preventing type 1 diabetes in late-stage pre-diabetic NOD mice with insulin: A central role for alum as adjuvant.

Background: Restoration of immune tolerance to disease-relevant antigens is an appealing approach to prevent or arrest an organ-specific autoimmune disease like type 1 diabetes (T1D). Numerous studies have identified insulin as a key antigen of interest to use in such strategies, but to date, the success of these interventions in humans has been inconsistent. The efficacy of antigen-specific immunotherapy may be enhanced by optimising the dose, timing, and route of administration, and perhaps by the inclusion of adjuvants like alum. The aim of our study was to evaluate the effect of an insulin peptide vaccine formulated with alum to prevent T1D development in female non-obese diabetic (NOD) mice when administered during late-stage pre-diabetes. Methods: Starting at 10 weeks of age, female NOD mice received four weekly subcutaneous injections of an insulin B:8-24 (InsB:8-24) peptide with (Ins+alum) or without Imject® alum (Ins) as adjuvant. Diabetes incidence was assessed for up to 30 weeks of age. Insulin autoantibodies and C-peptide concentrations were measured in plasma and flow cytometric analysis was performed on pancreatic-draining lymph nodes (PLN) and pancreas using an InsB:12-20-reactive tetramer. Results: InsB:8-24 peptide formulated in alum reduced diabetes incidence (39%), compared to mice receiving the InsB:8-24 peptide without alum (71%, P < 0.05), mice receiving alum alone (76%, P < 0.01), or mice left untreated (70%, P < 0.01). This was accompanied by reduced insulitis severity, and preservation of C-peptide. Ins+alum was associated with reduced frequencies of pathogenic effector memory CD4+ and CD8+ T cells in the pancreas and increased frequencies of insulin-reactive FoxP3+ Tregs in the PLN. Of interest, insulin-reactive Tregs were enriched amongst populations of Tregs expressing markers indicative of stable FoxP3 expression and enhanced suppressive function. Conclusion: An InsB:8-24 peptide vaccine prevented the onset of T1D in late-stage pre-diabetic NOD mice, but only when formulated in alum. These findings support the use of alum as adjuvant to optimise the efficacy of antigen-specific immunotherapy in future trials.

High Serum Vitamin D Concentrations, Induced via Diet, Trigger Immune and Intestinal Microbiota Alterations Leading to Type 1 Diabetes Protection in NOD Mice.

The hormonally-active form of vitamin D, 1,25-dihydroxyvitamin D3, can modulate both innate and adaptive immunity, through binding to the nuclear vitamin D receptor expressed in most immune cells. A high dose of regular vitamin D protected non-obese diabetic (NOD) mice against type 1 diabetes (T1D), when initiated at birth and given lifelong. However, considerable controversy exists on the level of circulating vitamin D (25-hydroxyvitamin D3, 25(OH)D3) needed to modulate the immune system in autoimmune-prone subjects and protect against T1D onset. Here, we evaluated the impact of two doses of dietary vitamin D supplementation (400 and 800 IU/day), given to female NOD mice from 3 until 25 weeks of age, on disease development, peripheral and gut immune system, gut epithelial barrier function, and gut bacterial taxonomy. Whereas serum 25(OH)D3 concentrations were 2.6- (400 IU/day) and 3.9-fold (800 IU/day) higher with dietary vitamin D supplementation compared to normal chow (NC), only the 800 IU/day vitamin D-supplemented diet delayed and reduced T1D incidence compared to NC. Flow cytometry analyses revealed an increased frequency of FoxP3+ Treg cells in the spleen of mice receiving the 800 IU/day vitamin D-supplemented diet. This vitamin D-induced increase in FoxP3+ Treg cells, also expressing the ecto-5'-nucleotidase CD73, only persisted in the spleen of mice at 25 weeks of age. At this time point, the frequency of IL-10-secreting CD4+ T cells was also increased in all studied immune organs. High-dose vitamin D supplementation was unable to correct gut leakiness nor did it significantly modify the increased gut microbial diversity and richness over time observed in NOD mice receiving NC. Intriguingly, the rise in alpha-diversity during maturation occurred especially in mice not progressing to hyperglycaemia. Principal coordinates analysis identified that both diet and disease status significantly influenced the inter-individual microbiota variation at the genus level. The abundance of the genera Ruminoclostridium_9 and Marvinbryantia gradually increased or decreased, respectively in faecal samples of mice on the 800 IU/day vitamin D-supplemented diet compared to mice on the 400 IU/day vitamin D-supplemented diet or NC, irrespective of disease outcome. In summary, dietary vitamin D reduced T1D incidence in female NOD mice at a dose of 800, but not of 400, IU/day, and was accompanied by an expansion of Treg cells in various lymphoid organs and an altered intestinal microbiota signature.

PTPN2 Regulates the Interferon Signaling and Endoplasmic Reticulum Stress Response in Pancreatic ß-Cells in Autoimmune Diabetes.

Type 1 diabetes (T1D) results from autoimmune destruction of ß-cells in the pancreas. Protein tyrosine phosphatases (PTPs) are candidate genes for T1D and play a key role in autoimmune disease development and ß-cell dysfunction. Here, we assessed the global protein and individual PTP profiles in the pancreas from nonobese mice with early-onset diabetes (NOD) mice treated with an anti-CD3 monoclonal antibody and interleukin-1 receptor antagonist. The treatment reversed hyperglycemia, and we observed enhanced expression of PTPN2, a PTP family member and T1D candidate gene, and endoplasmic reticulum (ER) chaperones in the pancreatic islets. To address the functional role of PTPN2 in ß-cells, we generated PTPN2-deficient human stem cell-derived ß-like and EndoC-ßH1 cells. Mechanistically, we demonstrated that PTPN2 inactivation in ß-cells exacerbates type I and type II interferon signaling networks and the potential progression toward autoimmunity. Moreover, we established the capacity of PTPN2 to positively modulate the Ca2+-dependent unfolded protein response and ER stress outcome in ß-cells. Adenovirus-induced overexpression of PTPN2 partially protected from ER stress-induced ß-cell death. Our results postulate PTPN2 as a key protective factor in ß-cells during inflammation and ER stress in autoimmune diabetes.

One hundred years of insulin therapy.

At the time of its first clinical application 100 years ago, insulin was presented as the cure for people with diabetes mellitus. That transpired to be an overstatement, yet insulin has proven to be the lifesaver for people with type 1 diabetes mellitus and an essential therapy for many with type 2 diabetes mellitus or other forms of diabetes mellitus. Since its discovery, insulin (a molecule of only 51 amino acids) has been the subject of pharmaceutical research and development that has paved the way for other protein-based therapies. From purified animal-extracted insulin and human insulin produced by genetically modified organisms to a spectrum of insulin analogues, pharmaceutical laboratories have strived to tailor the preparations to the needs of patients. Nonetheless, overall glycaemic control often remains poor as exogenous insulin is still not able to mimic the physiological insulin profile. Circumventing subcutaneous administration and the design of analogues with profiles that mimic that of physiological insulin are ongoing areas of research. Novel concepts, such as once-weekly insulins or glucose-dependent and oral insulins, are on the horizon but their real-world effectiveness still needs to be proven. Until a true cure for type 1 diabetes mellitus is found and the therapeutic arsenal for other forms of diabetes mellitus is expanded, insulin will remain central in the treatment of many people living with diabetes mellitus.

100 YEARS OF INSULIN: Arresting or curing type 1 diabetes: an elusive goal, but closing the gap.

Type 1 diabetes is one of the most common chronic diseases in children and adolescents, but remains unpreventable and incurable. The discovery of insulin, already 100 years ago, embodied a lifesaver for people with type 1 diabetes as it allowed the replacement of all functions of the beta cell. Nevertheless, despite all technological advances, the majority of type 1 diabetic patients fail to reach the recommended target HbA1c levels. The disease-associated complications remain the true burden of affected individuals and necessitate the search for disease prevention and reversal. The recognition that type 1 diabetes is a heterogeneous disease with an etiology in which both the innate and adaptive immune system as well as the insulin-producing beta cells intimately interact, has fostered the idea that treatment to specific molecular or cellular characteristics of the patient groups will be needed. Moreover, robust and reliable biomarkers to detect type 1 diabetes in the early (pre-symptomatic) phases are wanted to preserve functional beta cell mass. The pitfalls of past therapeutics along with the perspectives of current therapies can open up the path for future research.

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.

Vitamin D's Effect on Immune Function.

Ever since its discovery by Windhaus, the importance of the active metabolite of vitamin D (1,25-dihydroxyvitamin D3; 1,25-(OH)2D3) has been ever expanding. In this review, the attention is shifted towards the importance of the extra-skeletal effects of vitamin D, with special emphasis on the immune system. The first hint of the significant role of vitamin D on the immune system was made by the discovery of the presence of the vitamin D receptor on almost all cells of the immune system. In vitro, the overwhelming effect of supra-physiological doses of vitamin D on the individual components of the immune system is very clear. Despite these promising pre-clinical results, the translation of the in vitro observations to solid clinical effects has mostly failed. Nevertheless, the evidence of a link between vitamin D deficiency and adverse outcomes is overwhelming and clearly points towards avoidance of vitamin D deficiency especially in early life.