Non-diabetic Non-obese Diabetic Mice Research Articles

Inflammatory status of the pancreas in NOD mice that do not develop overt diabetes.

Type 1 diabetes (T1D) is an autoimmune disease in which immune cells target the pancreatic islets and destroy the β-cells, resulting in hyperglycemia and decreased plasmatic insulin levels. The non-obese diabetic (NOD) mouse is the most used animal model for studying diabetes because it spontaneously develops T1D and shares similarities with the human disease. A hallmark feature of this model is the appearance of insulitis, defined as an inflammatory cell infiltration of the pancreatic islets. However, a small percentage of NOD mice do not develop overt diabetes even after 28–35 weeks of age. Thus, we questioned the status of the pancreatic islets in these non-diabetic NOD mice, with particular focus on islet inflammation and plasmatic insulin levels, in comparison to pre-diabetic (11 weeks old) and new-onset diabetic mice. Diabetes progression was evaluated by assessing blood glucose and pancreas histology. The inflammatory score was determined on Hematoxylin–Eosin (HE)-stained sections of pancreas. Plasma insulin was detected by enzyme-linked immunosorbent assay (ELISA). The results showed that inflammation increased in an age-dependent manner in all mice, irrespective of their diabetic status. Mostly affected within the analyzed groups were the 28 weeks old non-diabetic NOD mice, in which insulin production was reduced and inversely correlated with the inflammatory status. We conclude that in NOD mice, pancreatic inflammation progresses independently of diabetes onset and clinical signs of disease. Most likely, the NOD females that do not develop overt diabetes preserve a small mass of functional β-cells, which is able to provide the physiological insulin levels and avoid diabetes onset.

Pancreatic beta cell autophagy is impaired in type 1 diabetes

Aims/hypothesisPancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development.MethodsPancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5).ResultsWe observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3–LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3–LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002).Conclusions/interpretationWe provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes.Graphical abstract

Dietary Early Glycation Products Protect Type 1 Diabetic Mice against Hyperglycemia through Altering Immune Homeostasis

Abstract Early glycation products (EGPs) are proteins modified with reducing sugar moieties. They are generated in the first step of Maillard reaction/glycation, and are precursors for the advanced glycation end-products (AGEs). This modification enhances the physiochemical properties of proteins (e.g. solubility), and has been proposed as a strategy to improve food protein qualities. However, the health effects of EGPs are rarely known. Our previous studies suggested that EGPs are anti-inflammatory. In contrast, AGEs are pro-inflammatory, and tightly associated with aging and hyperglycemia. It was hypothesized that EGPs were protective for type 1 diabetes (T1D) through modulating the immune homeostasis. Food-borne EGPs were produced from a whey protein isolate-glucose model system. Six-week-old nondiabetic non-obese diabetic (NOD) mice were dosed with water, non-reacted samples (NR) and EGPs up for 8 wks. Body weight and blood glucose levels (BGLs) were measured weekly; diabetic incidence (BGLs &amp;gt; 200 mg/dL) was recorded; glucose tolerance test and insulin tolerance test were conducted monthly; sera were separated; and spleen and thymus were harvested for flow cytometry analysis of the immune cell populations. The results showed that EGP-treated NOD mice exhibited significantly lower BGLs, delayed diabetes onset, less responsive to glucose challenge, and more sensitive to insulin challenge than water and NR-treated mice. Moreover, the diabetic NOD females displayed lower or even normal BGLs after EGP treatment. The flow cytometric data showed that EGP-treated mice had a significantly higher CD4/CD8 ratio than NR-treated mice. In conclusion, EGPs protected T1D mice against hyperglycemia through altering immune balance.

Effect of insulin receptor expressing T cell infiltration in islets on beta cell functionality

Abstract T cells have a major role in the progression of type 1 diabetes (T1D) in human and the non-obese diabetic (NOD) mouse model. Activated T cells have increased insulin receptor (IR) expression on their surface. When T cells with high density of IR from NOD mice were transferred into young irradiated non-diabetic NOD mice, recipient mice developed insulitis and diabetes. Irradiated non-diabetic NOD mice that received T cells with low to negative density of IR had neither insulitis nor diabetes. In order to understand the effect of IR expressing T cells in T1D development, our lab has developed a novel transgenic mouse model, C57BL/6-CD3FLAGmIR/mfm, genetically modified to express FLAG tagged mouse insulin receptor (mIR) on T cells. It was observed that FLAG-mIR expressing T cells can chemotax to the islet leading to insulitis. However, C57BL/6-CD3FLAGmIR/mfm mice were not hyperglycemic at any age. Metabolic studies were performed to evaluate if FLAG-mIR expressing T cell infiltration in the transgenic mice has effect on beta cell functionality. Impaired in vitro islet insulin secretion on treatment with non-glucose beta cells secretagogue and abnormal glucose tolerance in the transgenic mice resemble the pathological changes during prediabetes in human and NOD mice. Islet and Metabolic changes were also found in immunocompromised RAG mice that received transgenic mice splenocytes confirming that the transgenic phenotype can be passaged to immunocompromised RAG mice in a short time frame. This study provides a new perspective to understand the pathology of T1D and design a novel therapeutic approach targeting IR overexpressing T cells.

Chemotaxis to islets based on cellular insulin receptor expression

Abstract In the non-obese diabetic (NOD) mouse model, T1D is an autoimmune disease characterized by insulitis and T cell-mediated destruction of pancreatic islet beta cells. Insulin receptor (IR) is a chemotactic receptor capable of driving T cell movement in response to insulin. In published work, purified T cells, obtained from diabetic mice, and sorted into IR positive and negative cells were passaged into irradiated nondiabetic NOD mice. Only recipients that received IR positive T cells had insulitis and diabetes. If the IR expressing cells are moving into the pancreas based on an insulin gradient, then artificially making T cells with high IR expression should allow for movement into the pancreas in mice that are not susceptible to T1D. A novel transgenic mouse model was made that has a FLAG tagged mouse insulin receptor behind a CD3 promoter for expression on T cells in a mouse not susceptible to T1D development. Transgenic BL/6-CD3FLAGmIR mice were successfully made, and show evidence for T cell trafficking into the pancreas. Although insulitis occurs, no diabetes has developed. Characterizing IR expression on T cells may be of key importance for understanding the pathogenesis of diabetes. If IR expression is established as a mechanism to move T cells into islets, then down regulation of IR expression specifically in T cells will provide a new therapeutic target to block cell movement into the pancreas, thus preventing T1D.

Intrinsic Gastrointestinal Macrophages: Their Phenotype and Role in Gastrointestinal Motility.

There is an increasing awareness of the role of macrophages in the regulation and maintenance of gastrointestinal function in health and disease. This work has proceeded in the context of an increased understanding of the complex phenotypic variation in macrophages throughout the body and has shown previously unidentified roles for macrophages in diseases such as gastroparesis, postoperative ileus, and inflammatory bowel disease. Opportunities for exploiting the phenotypic modulation of tissue resident macrophages have been identified as possible therapies for some of these diseases. In addition, macrophages are an established component of the innate immune system and can respond to variations and changes in the intestinal microbiome and potentially mediate part of the impact of the microbiota on intestinal health. We reviewed the latest work on novel concepts in defining macrophage phenotype, discuss possible mechanisms of action for tissue-resident macrophages in the gut, address the significance of microbiome effects on macrophage phenotype, and review the known and possible roles of macrophages in motility disorders of the gastrointestinal tract.

Host Factors Alter Effects of Angiopoietin-Like Protein 8 on Glucose Homeostasis in Diabetic Mice.

Recovery of functional beta cell mass offers a biological cure for type 1 diabetes. However, beta cell mass is difficult to regain once lost since the proliferation rate of beta cells after youth is very low. Angiopoietin like-protein 8 (ANGPTL8), a peptide that has a role in the regulation of lipoprotein lipase activity, was reported to increase beta cell proliferation in mice in 2013. Subsequent studies of human ANGPTL8 for short term (3 to 8 days) in non-diabetic mice showed little or no increase in beta cell proliferation. Here, we examined the effect of ANGPTL8 on glucose homeostasis in models that have not been examined previously. We expressed mouse ANGPTL8 using adenovirus in 2 mouse models of diabetes (streptozotocin and Non-Obese Diabetic (NOD) mice) over 2 weeks. Also, we tested ANGPTL8 in NOD mice deficient in leukocyte 12-lipoxygenase (12LO), an enzyme that contributes to insulitis and loss of beta cell function in NOD, in an effort to determine whether 12LO deficiency alters the response to ANGPTL8. Adenovirus-mediated expression of ANGPTL8 lowered blood glucose levels in streptozotocin treated mice without an increase in beta cell proliferation or serum insulin concentration. While ANGPTL8 did not reverse hyperglycemia in overtly hyperglycemic NOD mice or alter glucose homeostasis of non-diabetic NOD mice, ANGPTL8 reduced blood glucose levels in 12LOKO NOD mice. However, the lower glucose levels in 12LOKO NOD were not associated with higher serum insulin levels or beta cell proliferation. In summary, while mouse ANGPTL8 does not increase beta cell proliferation in NOD mice or streptozotocin treated mice in agreement with studies in non-diabetic mice, it lowers blood glucose levels in multiple low-dose streptozotocin induced diabetes and 12LO deficiency indicating that host factors influence the impact of ANGPTL8 on glucose homeostasis.

Upregulation of Bpifb1 expression in the parotid glands of non-obese diabetic mice.

To define the increased mRNA expression of Bpifb1, a member of the bactericidal/permeability-increasing protein family, in parotid acinar cells from non-obese diabetic (NOD) mice, an animal model for Sjögren's syndrome. Parotid acinar cells were prepared from female NOD (NOD/ShiJcl) mice with or without diabetes, as well as from control (C57BL/6JJcl) mice. Total RNA and homogenate were prepared from the parotid acinar cells. Embryonic cDNA from a Mouse MTC(™) Panel I kit was used. The expression of Bpifb1 was determined by cDNA microarray analysis, RT-PCR, real-time PCR, northern blotting and in situ hybridization. The expression of Bpifb1 mRNA was high in parotid acinar cells from diabetic and non-diabetic NOD mice at 5-50 weeks of age. Acinar cells in the C57BL/6 mice had a low expression of Bpifb1 mRNA at an age >8 weeks, but had a relatively high expression in the foetus and infantile stages. Bpifb1 mRNA is upregulated in parotid acinar cells in NOD mice, but its expression is not related to the onset of diabetes. These findings suggest that high expression levels of Bpifb1 might predict disease traits before the onset of autoimmunity.

Deregulation of Protein Phosphatase 2A and Hyperphosphorylation of τ Protein Following Onset of Diabetes in NOD Mice

The histopathological hallmarks of Alzheimer disease (AD) include intraneuronal neurofibrillary tangles composed of abnormally hyperphosphorylated τ protein. Insulin dysfunction might influence AD pathology, as population-based and cohort studies have detected higher AD incidence rates in diabetic patients. But how diabetes affects τ pathology is not fully understood. In this study, we investigated the impact of insulin dysfunction on τ phosphorylation in a genetic model of spontaneous type 1 diabetes: the nonobese diabetic (NOD) mouse. Brains of young and adult female NOD mice were examined, but young NOD mice did not display τ hyperphosphorylation. τ phosphorylation at τ-1 and pS422 epitopes was slightly increased in nondiabetic adult NOD mice. At the onset of diabetes, τ was hyperphosphorylated at the τ-1, AT8, CP13, pS262, and pS422. A subpopulation of diabetic NOD mice became hypothermic, and τ hyperphosphorylation further extended to paired helical filament-1 and TG3 epitopes. Furthermore, elevated τ phosphorylation correlated with an inhibition of protein phosphatase 2A (PP2A) activity. Our data indicate that insulin dysfunction in NOD mice leads to AD-like τ hyperphosphorylation in the brain, with molecular mechanisms likely involving a deregulation of PP2A. This model may be a useful tool to address further mechanistic association between insulin dysfunction and AD pathology.

Influence of Tyrphostin AG490 on the expression of diabetes-associated markers in human adipocytes

Tyrosine kinase inhibitors (TKi) hold promise as a treatment for a variety of disorders ranging from those in oncology to diseases thought as immune mediated. Tyrphostin AG490 is a potent Jak-Stat TKi shown effective in the prevention of allograft transplant rejection, experimental autoimmune disease, as well as the treatment of cancer. However, given its ability to modulate this important but pleiotropic intracellular pathway, we thought that it is important to examine its effects on glucose metabolism and expression of major transcription factors and adipokines associated with insulin insensitivity and diabetes. We investigated the metabolic effects of AG490 on glucose levels in vivo using an animal model of diabetes, nonobese diabetic (NOD) mice, and transcription factor expression through assessment of human adipocytes. AG490 treatment of young nondiabetic NOD mice significantly reduced blood glucose levels (p = 0.002). In vitro, treatment of adipocytes with rosiglitazone, an insulin sensitizer that binds to peroxisome proliferator-activated receptor (PPAR) receptors and increases the adipocyte response to insulin, significantly increased the expression of the antidiabetic adipokine adiponectin. Importantly, the combination of rosiglitazone plus Tyrphostin AG490 further increased this effect and was specifically associated with significant upregulation of C-enhanced binding protein (C/EBP) (p < 0.0001). In terms of the mechanism underlying this action, regulatory regions of the PPARγ, ADIPOQ, and C/EBP contain the Stat5 DNA-binding sequences and were demonstrated, by gel shift experiments in vitro. These data suggest that blocking Jak-Stat signaling with AG490 reduces blood glucose levels and modulates the expression of transcription factors previously associated with diabetes, thereby supporting its potential as a therapy for this disease.

An Immunohistochemical Study on the Distribution and Frequency of T Regulatory Cells in Pancreatic Islets of NOD Mice During Various Stages of Spontaneous and Cyclophosphamide-Accelerated Diabetes

To determine if there is an abrupt change in the frequency of intraislet T regulatory (Treg) cells in female nonobese diabetic (NOD) mice preceding and following spontaneous diabetes and during cyclophosphamide-accelerated disease. The frequency of intraislet CD4-positive Treg cells was analyzed between days 21 and 250, at onset, and at 1, 2, and 3 weeks of diabetes by dual-label immunohistochemistry. Tissues were also analyzed between days 0 and 14 after injection of cyclophosphamide or diluent. In the spontaneous group, intraislet Treg cells were first observed on day 30 in CD4 T cells and increased from day 45. There was no statistical difference in the frequency of Treg cells in nondiabetic NOD mice on days 45, 60, and 90. A sustained frequency at and after 1, 2, and 3 weeks of diabetes was also observed. In the cyclophosphamide group, there was a sharp decline in the frequency of Treg cells on day 4, which remained lower on day 7 but increased by days 11 and 14. During spontaneous diabetes and after onset, the frequency of intraislet CD4-positive Treg cells remains unchanged. They may possess diminished immunoregulatory function and thus unable to counteract the increasing tempo of immune-mediated beta-cell destruction.

Glucose Homeostasis in Pre-diabetic NOD and Lymphocyte-Deficient NOD/SCID Mice During Gestation

Unlike other strains, spontaneously type 1 non-obese diabetic (NOD) experience transient hyperinsulinemia after weaning. The same applies for NOD/SCID mice, which lack functional lymphocytes, and unlike NOD mice, do not develop insulitis and diabetes like NOD mice. Given that beta-cell stimulation is a natural feature of gestation, we hypothesized that glucose homeostasis is disturbed in gestate pre-diabetic NOD and non-diabetic NOD/SCID mice, which may accelerate the onset of diabetes and increase diabetes prevalence. During gestation and postpartum, mice were analyzed under basal feed conditions followed by glucose injection (1 g/kg, i.p.) after overnight fast, using glucose tolerance test (GTT). Glycemia, corticosteronemia, blood and pancreatic insulin, glucagon levels, islet size, and islet morphology were evaluated. Glycemia and mortality were assessed after successive gestations in NOD mice mated for the first time at 2 different ages. 1. Basal glucagonemia rose markedly in first-gestation fed NOD mice. 2. beta-cell hyperactivity was present earlier in first-gestation non-diabetic fasted NOD and NOD/SCID mice than in age-matched C57BL/6 mice, assessed by increased insulin/glucose ratio after GTT. 3. Overnight fasting increased corticosteronemia rapidly and sharply in pre-diabetic gestate NOD and NOD/SCID mice. 4. Islet size increased in non-diabetic gestate NOD mice compared with C57BL/6 mice. 5. Successive gestations accelerated diabetes onset, and contributed to increased mortality in NOD mice. First-gestation pre-diabetic NOD and non-diabetic NOD/SCID mice exhibited beta-cell hyperactivity and deregulation of glucagon and/or corticosterone secretion. This amplified normally occurring insulin resistance, further exhausted maternal beta-cells, and accelerated diabetes in NOD mice.

Insulin Receptor Substrate-2 in β-Cells Decreases Diabetes in Nonobese Diabetic Mice

Insulin receptor substrate-2 (Irs2) integrates insulin-like signals with glucose and cAMP agonists to regulate beta-cell growth, function, and survival. This study investigated whether increased Irs2 concentration in beta-cells could reduce beta-cell destruction and the incidence of type 1 diabetes in nonobese diabetic (NOD) mice. NOD mice were intercrossed with C57BL/6 mice overexpressing Irs2 specifically in beta-cells to create NOD(Irs2) mice. After backcrossing NOD(Irs2) mice for 12 generations, glucose homeostasis and diabetes incidence were compared against NOD littermates. Compared with 12-wk-old NOD mice, the progression of severe insulitis was reduced and islet mass was increased in NOD(Irs2) mice. Moreover, the risk of diabetes decreased 50% in NOD(Irs2) mice until the experiment was terminated at 40 wk of age. Nondiabetic NOD(Irs2) mice displayed better glucose tolerance than nondiabetic NOD mice throughout the duration of the study and up to the age of 18 months. The effect of Irs2 to increase islet mass and improve glucose tolerance raised the possibility that NOD(Irs2) mice might have an increased capacity to respond to anti-CD3 antibody, which can induce remission of overt diabetes in some NOD mice. Anti-CD3 antibody injections restored glucose tolerance in newly diabetic NOD and NOD(Irs2) mice; however, anti-CD3-treated NOD(Irs2) mice were less likely than NOD mice to relapse during the experimental period because they displayed 10-fold greater beta-cell mass and mitogenesis. In conclusion, increased Irs2 attenuated the progression of beta-cell destruction, promoted beta-cell mitogenesis, and reduced diabetes incidence in NOD(Irs2) mice.

Cellular and humoral immune responses in the early stages of diabetic nephropathy in NOD mice

This study was designed to examine immunopathology of diabetic nephropathy in non-obese diabetic (NOD) mice and to investigate the involvement of cellular and humoral immunity at various time points after diabetes onset. We found that the glomeruli of diabetic NOD mice were infiltrated with T and B cells, as well as CD11c+ dendritic cells, which had close contact with CD4+ and CD8+ T cells in the infiltrates. We also found that IgG deposits in the glomeruli of diabetic NOD mice were accompanied by the presence of complement C3. Moreover, the serum from diabetic mice contained autoantibodies directed towards components of the glomeruli and these antibodies were not present in non-diabetic NOD mice. The immune changes in the kidney occurred together with increasing kidney weight and urinary albumin excretion along with duration of diabetes. We provide evidence that infiltrating lymphocytes and anti-kidney autoantibodies may be involved in diabetic nephropathy in autoimmune diabetes in the NOD mouse. Understanding the role that the immune system plays in the pathogenesis of diabetic nephropathy could lead to identification of new strategies and/or additional therapeutic targets for prevention and treatment of diabetic nephropathy.

TSG-6 protein expression in the pancreatic islets of NOD mice

The histologic hallmark of the development of type 1 diabetes (T1D) is insulitis, characterized by leukocytic infiltration of the pancreatic islets. The molecules controlling the early influx of leukocytes into the islets are poorly understood. Tumor necrosis factor alpha (TNFalpha)-stimulated gene 6 (TSG-6) is involved in inflammation, extracellular matrix formation, cell migration, and development. In the present study, we examined the expression and cellular localization of TSG-6 protein in islets of female non-obese diabetic (NOD) mice using frozen section immunofluorescence staining. Pancreata from nondiabetic (8 and 25 weeks old), prediabetic (230-280 mg/dl blood glucose) and diabetic (>300 mg/dl blood glucose) NOD mice were stained for TSG-6, insulin, CD3, CD11c, Mac3 and CD31. TSG-6 protein was detected in 67% of islets of prediabetic mice, 27% of islets of 25-week old nondiabetic mice, and less than 7% of islets of diabetic mice and 8-week old nondiabetic mice. Lastly, islet-derived TSG-6 protein was localized to the infiltrating CD3 and CD11c positive leukocytes.

Gluten‐free but also gluten‐enriched (gluten+) diet prevent diabetes in NOD mice; the gluten enigma in type 1 diabetes

Environmental factors such as nutrition or exposure to infections play a substantial role in the pathogenesis of type 1 diabetes (T1D). We have previously shown that gluten-free, non-purified diet largely prevented diabetes in non-obese diabetic (NOD) mice. In this study we tested hypothesis that early introduction of gluten-enriched (gluten+) diet may increase diabetes incidence in NOD mice. Standard, gluten-free, gluten+ modified Altromin diets and hydrolysed-casein-based Pregestimil diet were fed to NOD females and diabetes incidence was followed for 310 days. Insulitis score and numbers of gut mucosal lymphocytes were determined in non-diabetic animals. A significantly lower diabetes incidence (p < 0.0001) was observed in NOD mice fed gluten-free diet (5.9%, n = 34) and Pregestimil diet (10%, n = 30) compared to mice on the standard Altromin diet (60.6%, n = 33). Surprisingly, gluten+ diet also prevented diabetes incidence, even at the level found with the gluten-free diet (p < 0.0001, 5.9%, n = 34). The minority of mice, which developed diabetes on all the three diabetes-protective (gluten+, gluten-free, Pregestimil) diets, did that slightly later compared to those on the standard diet. Lower insulitis score compared to control mice was found in non-diabetic NOD mice on the gluten-free, and to a lesser extent also gluten+ and Pregestimil diets. No substantial differences in the number of CD3(+), TCR-gammadelta(+), and IgA(+) cells in the small intestine were documented. Gluten+ diet prevents diabetes in NOD mice at the level found with the non-purified gluten-free diet. Possible mechanisms of the enigmatic, dual effect of dietary gluten on the development of T1D are discussed.

Diabetes in non‐obese diabetic mice is not associated with quantitative changes in CD4+ CD25+ Foxp3+ regulatory T cells

The role of regulatory T cells (Tregs) in maintaining self tolerance has been intensively researched and there is a growing consensus that a decline in Treg function is an important step towards the development of autoimmune diseases, including diabetes. Although we show here that CD25+ cells delay diabetes onset in non-obese diabetic (NOD) mice, we found, in contrast to previous reports, neither an age-related decline nor a decline following onset of diabetes in the frequency of CD4+ CD25+ Foxp3+ regulatory T cells. Furthermore, we demonstrate that CD4+ CD25+ cells from both the spleen and pancreatic draining lymph nodes of diabetic and non-diabetic NOD mice are able to suppress the proliferation of CD4+ CD25- cells to a similar extent in vitro. We also found that pretreatment of NOD mice with anti-CD25 antibody allowed T cells with a known reactivity to islet antigen to proliferate more in the pancreatic draining lymph nodes of NOD mice, regardless of age. In addition, we demonstrated that onset of diabetes in NOD.scid mice is faster when recipients are co-administered splenocytes from diabetic NOD donors and anti-CD25. Finally, we found that although diabetic CD4+ CD25+ T cells are not as suppressive in cotransfers with effectors into NOD.scid recipients, this may not indicate a decline in Treg function in diabetic mice because over 10% of CD4+ CD25+ T cells are non-Foxp3 and the phenotype of the CD25- contaminating population significantly differs in non-diabetic and diabetic mice. This work questions whether onset of diabetes in NOD mice is associated with a decline in Treg function.

Dendritic cell–expanded, islet-specific CD4+ CD25+ CD62L+ regulatory T cells restore normoglycemia in diabetic NOD mice

Most treatments that prevent autoimmune diabetes in nonobese diabetic (NOD) mice require intervention at early pathogenic stages, when insulitis is first developing. We tested whether dendritic cell (DC)–expanded, islet antigen–specific CD4+ CD25+ suppressor T cells could treat diabetes at later stages of disease, when most of the insulin-producing islet β cells had been destroyed by infiltrating lymphocytes. CD4+ CD25+ CD62L+ regulatory T cells (T reg cells) from BDC2.5 T cell receptor transgenic mice were expanded with antigen-pulsed DCs and IL-2, and were then injected into NOD mice. A single dose of as few as 5 × 104 of these islet-specific T reg cells blocked diabetes development in prediabetic 13-wk-old NOD mice. The T reg cells also induced long-lasting reversal of hyperglycemia in 50% of mice in which overt diabetes had developed. Successfully treated diabetic mice had similar responses to glucose challenge compared with nondiabetic NOD mice. The successfully treated mice retained diabetogenic T cells, but also had substantially increased Foxp3+ cells in draining pancreatic lymph nodes. However, these Foxp3+ cells were derived from the recipient mice and not the injected T reg cells, suggesting a role for endogenous T reg cells in maintaining tolerance after treatment. Therefore, inoculation of DC-expanded, antigen-specific suppressor T cells has considerable efficacy in ameliorating ongoing diabetes in NOD mice.

The Pathogenesis of Staphylococcus aureus Infection in the Diabetic NOD Mouse

Although Staphylococcus aureus is a major pathogen implicated in diabetic foot infections, little is known about the pathogenesis of this disease. A model of S. aureus infection in the hindpaw of nonobese diabetic (NOD) mice was developed. The experimental infection was exacerbated in diabetic mice (blood glucose levels > or =19 mmol/l) compared with nondiabetic mice, and the diabetic animals were unable to clear the infection over a 10-day period. Insulin-mediated control of glycemia in diabetic mice resulted in enhanced clearance of S. aureus from the infected tissue. Diabetic mice showed reduced tissue inflammation in response to bacterial inoculation compared with nondiabetic NOD animals, and this was consistent with the novel finding of significantly decreased tissue levels of the chemokines KC and MIP-2 in diabetic mice. Blood from nondiabetic and diabetic NOD mice killed S. aureus in vitro, whereas the bacteria multiplied in blood from diabetic mice with severe hyperglycemia. The impaired killing of S. aureus by diabetic mice was correlated with a diminished leukocytic respiratory burst in response to S. aureus in blood from diabetic animals. This animal model of hindpaw infection may be useful for the analysis of host defects in innate immunity that contribute to recalcitrant diabetic foot infections.

Bone Formation Is Impaired in a Model of Type 1 Diabetes

The effects of type 1 diabetes on de novo bone formation during tibial distraction osteogenesis (DO) and on intact trabecular and cortical bone were studied using nonobese diabetic (NOD) mice and comparably aged nondiabetic NOD mice. Diabetic mice received treatment with insulin, vehicle, or no treatment during a 14-day DO procedure. Distracted tibiae were analyzed radiographically, histologically, and by microcomputed tomography (microCT). Contralateral tibiae were analyzed using microCT. Serum levels of insulin, osteocalcin, and cross-linked C-telopeptide of type I collagen were measured. Total new bone in the DO gap was reduced histologically (P < or = 0.001) and radiographically (P < or = 0.05) in diabetic mice compared with nondiabetic mice but preserved by insulin treatment. Serum osteocalcin concentrations were also reduced in diabetic mice (P < or = 0.001) and normalized with insulin treatment. Evaluation of the contralateral tibiae by microCT and mechanical testing demonstrated reductions in trabecular bone volume and thickness, cortical thickness, cortical strength, and an increase in endosteal perimeter in diabetic animals, which were prevented by insulin treatment. These studies demonstrate that bone formation during DO is impaired in a model of type 1 diabetes and preserved by systemic insulin administration.