Autoimmune Diabetes In Non-obese Diabetic Mice Research Articles

Fucoidan alleviated autoimmune diabetes in NOD mice by regulating pancreatic autophagy through the AMPK/mTOR1/TFEB pathway.

The present study investigated the effect and its underlying mechanisms of fucoidan on Type 1 diabetes mellitus (T1DM) in non-obese diabetic (NOD) mice. Twenty 7-week-old NOD mice were used in this study, and randomly divided into two groups (10 mice in each group): the control group and the fucoidan treatment group (600 mg/kg. body weight). The weight gain, glucose tolerance, and fasting blood glucose level in NOD mice were detected to assess the development of diabetes. The intervention lasted for 5 weeks. The proportions of Th1/Th2 cells from spleen tissues were tested to determine the anti-inflammatory effect of fucoidan. Western blot was performed to investigate the expression levels of apoptotic markers and autophagic markers. Apoptotic cell staining was visualized through TdT-mediated dUTP nick-end labeling (TUNEL). The results suggested that fucoidan ameliorated T1DM, as evidenced by increased body weight and improved glycemic control of NOD mice. Fucoidan down-regulated the Th1/Th2 cells ratio and decreased Th1 type pro-inflammatory cytokines' level. Fucoidan enhanced the mitochondrial autophagy level of pancreatic cells and increased the expressions of Beclin-1 and LC3B II/LC3B I. The expression of p-AMPK was up-regulated and p-mTOR1 was inhibited, which promoted the nucleation of transcription factor EB (TFEB), leading to autophagy. Moreover, fucoidan induced apoptosis of pancreatic tissue cells. The levels of cleaved caspase-9, cleaved caspase-3, and Bax were up-regulated after fucoidan treatment. Fucoidan could maintain pancreatic homeostasis and restore immune disorder through enhancing autophagy via the AMPK/mTOR1/TFEB pathway in pancreatic cells.

Arachidonic acid alleviates autoimmune diabetes in NOD mice

BackgroundArachidonic acid (AA) is considered to link nutrient metabolism, to inflammation and immunity, suggesting it may have a role in autoimmune diseases. Our previous study suggests that DPP-4 inhibitors (DPP-4i) might regulate AA – relative signaling in type 1 diabetes. AimsTo examine the effect of AA on autoimmune diabetes and its cross-talk with DPP-4i in The Non-Obese Diabetic (NOD) mice. MethodsThe NOD mice were divided randomly and equally into three groups: AA group, AA plus DPP-4i group and control group. The incidence of diabetes, blood glucose, insulitis and cytokine profiles were monitored. At the end of the experiment, pancreatic tissues were stained by H&E. Serum cytokine profiles were examined using a Mesco Scale Discovery multiplexed-assay kit. ResultsEven though AA or AA plus DPP-4i treatment has no effect on incidence of diabetes and weight, AA treatment reduces blood glucose, preserves islet morphology and alleviates inflammatory cell infiltration into pancreatic islets in NOD mice, accompanying with increased serum levels of IL-10, IL-1 β, IL-6, IL-5, KC/GRO and TNF-α and decreased serum levels of IL-2. ConclusionWe observed that AA treatment alleviates autoimmune diabetes in NOD mice by reducing hyperglycemia, alleviating insulitis and improving cytokine profiles. DPP-4i might alleviate the effect of AA by cross-talk. We provide evidence of AA treatment to alleviate type 1 diabetes in NOD mice, which may provide a novel therapeutic option for type 1 diabetes.

High-fat diet prevents the development of autoimmune diabetes in NOD mice.

AimsType 1 diabetes (T1D) has a strong genetic predisposition and requires an environmental trigger to initiate the beta‐cell autoimmune destruction. The rate of childhood obesity has risen in parallel to the proportion of T1D, suggesting high‐fat diet (HFD)/obesity as potential environmental triggers for autoimmune diabetes. To explore this, non‐obese diabetic (NOD) mice were subjected to HFD and monitored for the development of diabetes, insulitis and beta‐cell stress.Materials and MethodsFour‐week‐old female NOD mice were placed on HFD (HFD‐NOD) or standard chow‐diet. Blood glucose was monitored weekly up to 40 weeks of age, and glucose‐ and insulin‐tolerance tests performed at 4, 10 and 15 weeks. Pancreata and islets were analysed for insulin secretion, beta‐cell mass, inflammation, insulitis and endoplasmic reticulum stress markers. Immune cell levels were measured in islets and spleens. Stool microbiome was analysed at age 4, 8 and 25 weeks.ResultsAt early ages, HFD‐NOD mice showed a significant increase in body weight, glucose intolerance and insulin resistance; but paradoxically, they were protected from developing diabetes. This was accompanied by increased insulin secretion and beta‐cell mass, decreased insulitis, increased splenic T‐regulatory cells and altered stool microbiome.ConclusionsThis study shows that HFD protects NOD mice from autoimmune diabetes and preserves beta‐cell mass and function through alterations in gut microbiome, increased T‐regulatory cells and decreased insulitis. Further studies into the exact mechanism of HFD‐mediated prevention of diabetes in NOD mice could potentially lead to interventions to prevent or delay T1D development in humans.

Fatty acid binding protein 4 promotes autoimmune diabetes by recruitment and activation of pancreatic islet macrophages.

Both innate and adaptive immune cells are critical players in autoimmune destruction of insulin-producing β cells in type 1 diabetes. However, the early pathogenic events triggering the recruitment and activation of innate immune cells in islets remain obscure. Here we show that circulating fatty acid binding protein 4 (FABP4) level was significantly elevated in patients with type 1 diabetes and their first-degree relatives and positively correlated with the titers of several islet autoantibodies. In nonobese diabetic (NOD) mice, increased FABP4 expression in islet macrophages started from the neonatal period, well before the occurrence of overt diabetes. Furthermore, the spontaneous development of autoimmune diabetes in NOD mice was markedly reduced by pharmacological inhibition or genetic ablation of FABP4 or adoptive transfer of FABP4-deficient bone marrow cells. Mechanistically, FABP4 activated innate immune responses in islets by enhancing the infiltration and polarization of macrophages to proinflammatory M1 subtype, thus creating an inflammatory milieu required for activation of diabetogenic CD8+ T cells and shift of CD4+ helper T cells toward Th1 subtypes. These findings demonstrate FABP4 as a possible early mediator for β cell autoimmunity by facilitating crosstalk between innate and adaptive immune cells, suggesting that pharmacological inhibition of FABP4 may represent a promising therapeutic strategy for autoimmune diabetes.

Tolerance to Proinsulin-1 Reduces Autoimmune Diabetes in NOD Mice.

T-cell responses to insulin and its precursor proinsulin are central to islet autoimmunity in humans and non-obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes. Mice have two proinsulin genes proinsulin -1 and 2 that are differentially expressed, with predominant proinsulin-2 expression in the thymus and proinsulin-1 in islet beta-cells. In contrast to proinsulin-2, proinsulin-1 knockout NOD mice are protected from autoimmune diabetes. This indicates that proinsulin-1 epitopes in beta-cells maybe preferentially targeted by autoreactive T cells. To study the contribution of proinsulin-1 reactive T cells in autoimmune diabetes, we generated transgenic NOD mice with tetracycline-regulated expression of proinsulin-1 in antigen presenting cells (TIP-1 mice) with an aim to induce immune tolerance. TIP-1 mice displayed a significantly reduced incidence of spontaneous diabetes, which was associated with reduced severity of insulitis and insulin autoantibody development. Antigen experienced proinsulin specific T cells were significantly reduced in in TIP-1 mice indicating immune tolerance. Moreover, T cells from TIP-1 mice expressing proinsulin-1 transferred diabetes at a significantly reduced frequency. However, proinsulin-1 expression in APCs had minimal impact on the immune responses to the downstream antigen islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) and did not prevent diabetes in NOD 8.3 mice with a pre-existing repertoire of IGRP reactive T cells. Thus, boosting immune tolerance to proinsulin-1 partially prevents islet-autoimmunity. This study further extends the previously established role of proinsulin-1 epitopes in autoimmune diabetes in NOD mice.

Exopolysaccharide from Leuconostoc pseudomesenteroides XG5 delay the onset of autoimmune diabetes by modulating gut microbiota and its metabolites SCFAs in NOD mice

• XG5 EPS delay the onset of autoimmune diabetes in NOD mice. • XG5 EPS expanded the Foxp3 + Tregs frequency of the spleen in NOD mice. • XG5 EPS altered the gut microbiota composition. • XG5 EPS increased the short-chain fatty acids levels of colon contents. Exopolysaccharide produced by Leuconostoc pseudomesenteroides XG5 (XG5 EPS) is a potential prebiotic. The study objective is to assess the effect of XG5 EPS on autoimmune diabetes using non-obese diabetic (NOD) mouse strain. Our results indicated that XG5 EPS administration delayed the onset of autoimmune diabetes, reduced the level of diabetogenic cytokines IL-21 and promoted the frequency of Foxp3 + regulatory T cells in NOD mice. Moreover, XG5 EPS altered the gut microbiota composition, especially 50 mg·kg −1 XG5 EPS administration increased the ratio of Firmicutes to Bacteroidetes at the phylum level and the concentrations of total short-chain fatty acids (SCFAs) in NOD mice. Considering that the influence of viscosity on utilization of XG5 EPS, a dose dependent effect was not established. Taken together, oral administration of 50 mg·kg −1 XG5 EPS has a potential to delay the onset of autoimmune diabetes by modulating gut microbiota and its metabolites SCFAs in NOD mice.

Prevention of Autoimmune Diabetes in NOD Mice by Dimethyl Fumarate.

Oxidative stress plays critical roles in the pathogenesis of diabetes. This study tested the hypothesis that by protecting β-cells against oxidative stress and inflammation, an Nrf2 activator, dimethyl fumarate (DMF), may prevent or delay the onset of type 1 diabetes in non-obese diabetic (NOD) mice. Firstly, islet isolation was conducted to confirm the antioxidative effects of DMF oral administration on islet cells. Secondly, in a spontaneous diabetes model, DMF (25 mg/kg) was fed to mice once daily starting at the age of 8 weeks up to the age of 22 weeks. In a cyclophosphamide-induced accelerated diabetes model, DMF (25 mg/kg) was fed to mice twice daily for 2 weeks. In the islet isolation study, DMF administration improved the isolation yield, attenuated oxidative stress and enhanced GCLC and NQO1 expression in the islets. In the spontaneous model, DMF significantly reduced the onset of diabetes compared to the control group (25% vs. 54.2%). In the accelerated model, DMF reduced the onset of diabetes from 58.3% to 16.7%. The insulitis score in the islets of the DMF treatment group (1.6 ± 0.32) was significantly lower than in the control group (3.47 ± 0.21). The serum IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-9, IL-12p70, IFN-γ, TNF-α, MCP-1 and CXCL16 levels in the DMF-treated group were lower than in the control group. In conclusion, DMF may protect islet cells and reduce the incidence of autoimmune diabetes in NOD mice by attenuating insulitis and proinflammatory cytokine production.

Fucoidan prevent murine autoimmune diabetes via suppression TLR4-signaling pathways, regulation DC/Treg induced immune tolerance and improving gut microecology

BackgroundThis study was to investigate the effect and its possible mechanism of fucoidan on the development of spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice.Methods7-week-old NOD mice were randomly divided into three groups: control group, low-dose (300 mg/kg) and high-dose (600 mg/kg) fucoidan-treatment groups. After 5 weeks of treatment, 10 mice per group were randomly selected to be sacrificed after feces collection. The remaining 12 mice per group were fed until 26 weeks of age to assess the incidence of diabetes.ResultsTreatment with fucoidan increased serum insulin level, delayed the onset and decreased the development of diabetes in NOD mice. Fucoidan reduced the levels of strong Th1 proinflammatory cytokines, but induced Th2-bias ed. cytokine response. And dentridic cells (DCs) in fucoidan treatment group were characterized as low expression of MHC class II and CD86 molecules. TLR4 expressions and the downstream molecules in pancreas were down-regulated in fucoidan-treated groups. There were significant differences in the composition of gut flora between NOD control group and fucoidan group. Lactobacillus and Akkermansia were significantly enriched in fucoidan group.ConclusionsFucoidan could prevent the development of autoimmune diabetes in NOD mice via regulating DC/Treg induced immune tolerance, improving gut microecology, down-regulating TLR4 signaling pathway, and maintaining pancreatic internal environment.

Effect of Arginase-1 Inhibition on the Incidence of Autoimmune Diabetes in NOD Mice

Metabolism of the amino acid L-arginine is implicated in many physiological and pathophysiological processes including autoimmune conditions such as type 1 diabetes (T1D). Alternate arginine metabolism through the citrulline-nitric oxide (NO) or the ornithine pathways can lead to proinflammatory or immune regulatory effects, respectively. In this report, we blocked the arginine-ornithine metabolic pathway by inhibiting the enzyme arginase-1 with Nω-hydroxy-nor-arginine (nor-NOHA) to make arginine more available to the alternate citrulline pathway for augmented NO production and increased incidence of autoimmune T1D in female non-obese diabetic (NOD) mice. Unexpectedly, mice receiving nor-NOHA did not develop diabetes although increased NO production is proinflammatory and expected to increase diabetes incidence. These results warrant further studies of the mechanism of action of nor-NOHA, and highlight its potential as a therapeutic agent for the treatment or prevention of T1D.

β Cells that Resist Immunological Attack Develop during Progression of Autoimmune Diabetes in NOD Mice

Type 1 diabetes (T1D) is a chronic autoimmune disease that involves immune-mediated destruction of β cells. How β cells respond to immune attack is unknown. We identified a population of β cells during the progression of T1D in non-obese diabetic (NOD) mice that survives immune attack. This population develops from normal β cells confronted with islet infiltrates. Pathways involving cell movement, growth and proliferation, immune responses, and cell death and survival are activated in these cells. There is reduced expression of β cell identity genes and diabetes antigens and increased immune inhibitory markers and stemness genes. This new subpopulation is resistant to killing when diabetes is precipitated with cyclophosphamide. Human β cells show similar changes when cultured with immune cells. These changes may account for the chronicity of the disease and the long-term survival of β cells in some patients.

Vaccination with a co-expression DNA plasmid containing GAD65 fragment gene and IL-10 gene induces regulatory CD4(+) T cells that prevent experimental autoimmune diabetes.

The non-obese diabetic (NOD) mouse is a commonly used animal model for studying type 1 diabetes (T1D). The aims of our study were to explore the diabetes-preventive effect in NOD mice and the potential mechanisms of an optimized co-expression DNA vaccine containing GAD65 fragment gene with the IL-10 gene (SGAD65190-315 /IL-10). Female NOD mice at the age of 3-4weeks old were randomly divided into two groups and received intra-muscular injection of either blank pBudCE4.l vector (n = 34) or pBudCE4.l carrying the SGAD65190-315 /IL-10 (n = 32). The incidence of diabetes was monitored up to 30weeks of age. The severity of insulitis, apoptosis rate of β cells and relevant mechanisms were examined. Administration with SGAD65190-315 /IL-10 blocked the onset of autoimmune diabetes in NOD mice, significantly suppressed islet inflammation, inhibited the apoptosis of islet β cells, induced immune tolerance to autoantigen GAD65 and proinsulin and shifted the Th1/Th2 balance towards Th2. More importantly, the frequencies of CD4(+) CD25(+) Foxp3(+) regulatory T cells (Tregs) in the spleen and pancreatic lymph nodes in vaccine-immunized mice were significantly increased, and these Tregs were GAD65-reactive. In addition, Treg depletion by anti-CD25 mAb administration abolished the protective effects of SGAD65190-315 /IL-10 on diabetes and insulitis. Moreover, depletion of CD4(+) CD25(+) T cells using magnetic-activated cell sorting impaired the protective effect of SGAD65190-315 /IL-10 vaccination on adoptive transfer of diabetes. Our data suggested that SGAD65190-315 /IL-10 DNA vaccine had protective effects on T1D by upregulating autoantigen-reactive Tregs. Our findings may provide a novel preventive therapy for T1D. Copyright © 2016 John Wiley & Sons, Ltd.

Inhibition of Autoimmune Diabetes in NOD Mice by miRNA Therapy.

Autoimmune destruction of the pancreatic islets in Type 1 diabetes is mediated by both increased proinflammatory (Teff) and decreased regulatory (Treg) T lymphocytes resulting in a significant decrease in the Treg:Teff ratio. The non-obese diabetic (NOD) mouse is an excellent in vivo model for testing potential therapeutics for attenuating the decrease in the Treg:Teff ratio and inhibiting disease pathogenesis. Here we show for the first time that a bioreactor manufactured therapeutic consisting of a complex of miRNA species (denoted as TA1) can effectively reset the NOD immune system from a proinflammatory to a tolerogenic state thus preventing or delaying autoimmune diabetes. Treatment of NOD mice with TA1 resulted in a systemic broad-spectrum upregulation of tolerogenic T cell subsets with a parallel downregulation of Teff subsets yielding a dramatic increase in the Treg:Teff ratio. Moreover, the murine-derived TA1 was highly effective in the inhibition of allorecognition of HLA-disparate human PBMC. TA1 demonstrated dose-responsiveness and exhibited equivalent or better inhibition of allorecognition driven proliferation than etanercept (a soluble TNF receptor). These findings demonstrate that miRNA-based therapeutics can effectively attenuate or arrest autoimmune disease processes and may be of significant utility in a broad range of autoimmune diseases including Type 1 diabetes.

TAK1 inhibition prevents the development of autoimmune diabetes in NOD mice

Transforming growth factor-β activated kinase-1 (TAK1, Map3k7), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, is essential in innate and adaptive immune responses. We postulated that blockade of TAK1 would affect autoimmune diabetes in non-obese diabetic (NOD) mice. Administration of 5Z-7-oxozeaenol (OZ), a TAK1 inhibitor, decreased the incidence and delayed the onset of autoimmune diabetes in both spontaneous and accelerated (cyclophosphamide-induced) experimental NOD mice. OZ also reduced insulitis, preserved islet function, increased the expression of α1- antitrypsin (AAT), and severely inhibited NF-κB and JNK/AP-1 signaling pathways in immune organs and pancreatic tissues. Importantly, TAK1 inhibition by OZ elicited a Th1 to Th2 cytokine shift, and increased TGF-β1 production in cultured T lymphocytes supernatants. Systemic TAK1 inhibition induced immature DCs with lower expressions of MHC-II and CD86, attenuated DC-mediated T cell proliferation in allogeneic MLR, and production of cytokine IL-12p70 in DCs suspensions. The results indicate that TAK1 inhibition with OZ was associated with a lower frequency of autoimmune diabetes in NOD mice. The net effect of TAK1 inhibition in NOD mice therefore appears to be protective rather than disease-enhancing. Strategies targeting TAK1 specifically in NOD mice might prove useful for the treatment of autoimmune diabetes in general.

MicroRNA-26a Promotes Regulatory T cells and Suppresses Autoimmune Diabetes in Mice.

Type-1 diabetes (TID) is an autoimmune disease in which the body's own immune cells attack islet β cells, the cells in the pancreas that produce and release the hormone insulin. Mir-26a has been reported to play functions in cellular differentiation, cell growth, cell apoptosis, and metastasis. However, the role of microRNA-26a (Mir-26a) in autoimmune TID has never been investigated. In our current study, we found that pre-Mir-26a (LV-26a)-treated mice had significantly longer normoglycemic time and lower frequency of autoreactive IFN-γ-producing CD4(+) cells compared with an empty lentiviral vector (LV-Con)-treated non-obese diabetic (NOD) mice. Mir-26a suppresses autoreactive T cells and expands Tregs in vivo and in vitro. Furthermore, in our adoptive transfer study, the groups receiving whole splenocytes and CD25-depleted splenocytes from LV-Con-treated diabetic NOD mice develop diabetes at 3 to 4weeks of age. In comparison, mice injected with undepleted splenocytes obtained from LV-26a-treated reversal NOD mice develop diabetes after 6-8weeks. And depletion of CD25(+) cells in the splenocytes of reversed mice abrogates the delay in diabetes onset. In conclusion, Mir-26a suppresses autoimmune diabetes in NOD mice in part through promoted regulatory T cells (Tregs) expression.

HCELL Expression on Murine MSC Licenses Pancreatotropism and Confers Durable Reversal of Autoimmune Diabetes in NOD Mice.

Type 1 diabetes (T1D) is an immune-mediated disease resulting in destruction of insulin-producing pancreatic beta cells. Mesenchymal stem cells (MSCs) possess potent immunomodulatory properties, garnering increasing attention as cellular therapy for T1D and other immunologic diseases. However, MSCs generally lack homing molecules, hindering their colonization at inflammatory sites following intravenous (IV) administration. Here, we analyzed whether enforced E-selectin ligand expression on murine MSCs could impact their effect in reversing hyperglycemia in nonobese diabetic (NOD) mice. Although murine MSCs natively do not express the E-selectin-binding determinant sialyl Lewis(x) (sLe(x) ), we found that fucosyltransferase-mediated α(1,3)-exofucosylation of murine MSCs resulted in sLe(x) display uniquely on cell surface CD44 thereby creating hematopoietic cell E-/L-selectin ligand (HCELL), the E-selectin-binding glycoform of CD44. Following IV infusion into diabetic NOD mice, allogeneic HCELL(+) MSCs showed threefold greater peri-islet infiltrates compared to buffer-treated (i.e., HCELL(-) ) MSCs, with distribution in proximity to E-selectin-expressing microvessels. Exofucosylation had no effect on MSC immunosuppressive capacity in in vitro assays; however, although engraftment was temporary for both HCELL(+) and HCELL(-) MSCs, administration of HCELL(+) MSCs resulted in durable reversal of hyperglycemia, whereas only transient reversal was observed following administration of HCELL(-) MSCs. Notably, exofucosylation of MSCs generated from CD44(-/-) mice induced prominent membrane expression of sLe(x) , but IV administration of these MSCs into hyperglycemic NOD mice showed no enhanced pancreatotropism or reversal of hyperglycemia. These findings provide evidence that glycan engineering to enforce HCELL expression boosts trafficking of infused MSCs to pancreatic islets of NOD mice and substantially improves their efficacy in reversing autoimmune diabetes. Stem Cells 2013;33:1523-1531.

Sodium meta-arsenite prevents the development of autoimmune diabetes in NOD mice

Sodium meta-arsenite (SA) is an orally available arsenic compound. We investigated the effects of SA on the development of autoimmune type 1 diabetes. Female non-obese diabetic (NOD) mice were orally intubated with SA (5mg/kg/day) from 8weeks of age for 8weeks. The cumulative incidence of diabetes was monitored until 30weeks of age, islet histology was examined, and lymphocytes including T cells, B cells, CD4+ IFN-γ+ cells, CD8+ IFN-γ+ cells, CD4+ IL-4+ cells, and regulatory T cells were analyzed. We also investigated the diabetogenic ability of splenocytes using an adoptive transfer model and the effect of SA on the proliferation, activation, and expression of glucose transporter 1 (Glut1) in splenocytes treated with SA in vitro and splenocytes isolated from SA-treated mice. SA treatment decreased the incidence of diabetes and delayed disease onset. SA treatment reduced the infiltration of immunocytes in islets, and splenocytes from SA-treated mice showed a reduced ability to transfer diabetes. The number of total splenocytes and T cells and both the number and the proportion of CD4+ IFN-γ+ and CD8+ IFN-γ+ T cells in the spleen were significantly reduced in SA-treated NOD mice compared with controls. The number, but not the proportion, of regulatory T cells was decreased in SA-treated NOD mice. Treatment with SA either in vitro or in vivo inhibited proliferation of splenocytes. In addition, the expression of Glut1 and phosphorylated ERK1/2 was decreased by SA treatment. These results suggest that SA reduces proliferation and activation of T cells, thus preventing autoimmune diabetes in NOD mice.

Premature CD4+ T Cell Aging and Its Contribution to Lymphopenia-Induced Proliferation of Memory Cells in Autoimmune-Prone Non-Obese Diabetic Mice

Lymphopenia-induced proliferation (LIP), a mechanism to maintain a constant number of T cells in circulation, occurs in both normal aging and autoimmune disease. The incidence of most autoimmune diseases increases with age, and premature CD4+ T cell aging has been reported in several autoimmune diseases. In this study, we tested the hypothesis that premature CD4+ T cell aging can cause autoimmune disease by examining whether premature CD4+ T cell aging exists and causes LIP in our mouse model. Non-obese diabetic (NOD) mice were used because, in addition to Treg defects, the LIP of T cells has been shown to plays a causative role in the development of insulin-dependent diabetes mellitus (IDDM) in these mice. We found that with advancing age, NOD mice exhibited an accelerated decrease in the number of CD4+ T cells due to the loss of naïve cells. This was accompanied by an increase in the percentage of memory cells, leading to a reduced naïve/memory ratio. In addition, both the percentage of CD28+ cells in CD4+ T cells and IL-2 production decreased, while the percentage of FAS+CD44+ increased, suggesting that NOD mice exhibit premature CD4+ T cell aging. This process preferentially contributed to LIP of memory cells. Therefore, our results suggest that premature CD4+ T cell aging underlies the development of IDDM in NOD mice. Given that CD28 and IL-2 play important roles in Treg function, the relationships between premature CD4+ T cell aging and lymphopenia as well as Treg defects in autoimmune-prone NOD mice are proposed.

Pharmacological inhibition of PI3Kδ opposes the progression of autoimmune diabetes in non-obese diabetic mice. (P5149)

Abstract B-lymphocytes have been implicated in many autoimmune diseases, and non-obese diabetic (NOD) mice that lack B cells do not develop spontaneous autoimmune diabetes. The catalytic activity of PI3Kδ is important for B cell migration, activation, proliferation, and antigen presentation. We tested whether a PI3Kδ inhibitor could delay the onset or reduce the incidence of autoimmune diabetes in NOD mice. We found that preventative treatment of pre-diabetic NOD mice with GS-548202 (IC87114), a highly selective small molecule inhibitor of PI3Kδ, reduced the infiltration of inflammatory cells into the pancreatic islets and, accordingly, delayed and reduced the loss of glucose homeostasis. Diabetes developed in about 75% of vehicle-treated NOD mice by 18 weeks of age but in only 33% of GS-548202-treated animals at this same time point. Moreover in a therapeutic treatment mode, GS-548202 treatment conferred prolonged protection from progression to overt diabetes in a number of animals. Within 28 days, all of the NOD mice that received only the vehicle developed overt diabetes, with blood glucose levels rising substantially above 300 mg/dl. In contrast, 4 of the 7 GS-548202-treated NOD mice maintained their blood glucose levels below 300 mg/dl for at least 60 days after initiating treatment when they first exhibited blood glucose levels of 200-300mg/dl. These findings suggest that PI3Kδ inhibitors could be useful for managing autoimmune diabetes.

B7-H4.Ig inhibits the development of Type 1 diabetes by regulating Th17 cells in NOD mice

Type 1 diabetes (T1D) is an autoimmune disease characterized by immunological destruction of insulin-producing pancreatic β-cells and subsequent hyperglycemia. The non-obese diabetic (NOD) mouse strain spontaneously develops a disease similar to human T1D and is commonly used as an animal model for studying this disease. We have previously shown that the administration of B7-H4-immunoglobulin fusion protein (B7-H4.Ig), a newly identified T-cell co-inhibitory signaling molecule, blocks the onset of diabetes in NOD mice. However, the mechanism(s) by which B7-H4 protects NOD mice from T1D is not fully understood. IL-17 is a pro-inflammatory cytokine, produced by Th17 cells, that activates T cells and other immune cells to produce a variety of cytokines and chemokines. Increasing evidence has shown that therapeutic agents targeting the IL-17 molecule or directly inhibiting IL-17-producing cells regulate autoimmune diabetes in NOD mice, suggesting that IL-17 is involved in the pathogenesis of this disease. In this study, we investigate whether B7-H4.Ig treatment inhibits the generation of Th17 cells which subsequently decreases IL-17 production and prevents the onset of T1D in NOD mice. Pre-diabetic female NOD mice were injected intraperitoneally with control mouse IgG or B7-H4.Ig starting at 4weeks of age for 12weeks. Our data showed that the frequency of Th17 cells in B7-H4.Ig-treated mice was significantly decreased. In addition, our data showed that B7-H4.Ig-treated mice had decreased levels of pro-inflammatory cytokines and Th17-associated cytokines, and an increased level of the potent Th17 inhibitor IFN-γ. To further investigate the effect of B7-H4.Ig on differentiation of Th17 cells, we co-cultured splenocytes with Th17-polarizing cytokines in the absence or presence of B7-H4.Ig. Our results indicated that splenocytes, under the Th17 driving conditions in the presence of B7-H4.Ig, had significantly decreased the numbers of Th17 cells compared to cells co-cultured in the absence of B7-H4.Ig. Together, this study suggests that blocking the generation of Th17 cells with the administration of B7-H4.Ig effectively inhibits the development of T1D in NOD mice.

The nonconventional MHC class II molecule DM governs diabetes susceptibility in NOD mice.

The spontaneous destruction of insulin producing pancreatic beta cells in non-obese diabetic (NOD) mice provides a valuable model of type 1 diabetes. As in humans, disease susceptibility is controlled by the classical MHC class II genes that guide CD4+ T cell responses to self and foreign antigens. It has long been suspected that the dedicated class II chaperone designated HLA-DM in humans or H-2M in mice also makes an important contribution, but due to tight linkage within the MHC, a possible role played by DM peptide editing has not been previously tested by conventional genetic approaches. Here we exploited newly established germ-line competent NOD ES cells to engineer a loss of function allele. DM deficient NOD mice display defective class II peptide occupancy and surface expression, and are completely protected against type 1 diabetes. Interestingly the mutation results in increased proportional representation of CD4+Foxp3+ regulatory T cells and the absence of pathogenic CD4+ T effectors. Overall, this striking phenotype establishes that DM-mediated peptide selection plays an essential role in the development of autoimmune diabetes in NOD mice.