Diabetes In Non-obese Diabetic Mice Research Articles

Nanoparticles Containing an Insulin-ChgA Hybrid Peptide Prevent Transfer of Type 1 Diabetes in NOD Mice by Expanding Foxp3+ Tregs

Abstract In Type 1 Diabetes (T1D) autoreactive T cells destroy insulin-producing β-cells in the pancreatic islets. A major focus of our lab has been to identify novel peptide ligands for diabetogenic CD4 T cells isolated from non-obese diabetic (NOD) mice. We have identified a novel post-translational modification whereby a peptide from chromogranin A is covalently bound to a fragment from proinsulin, resulting in formation of a hybrid insulin peptide (2.5HIP) that is a potent ligand for the CD4 T cell clone BDC-2.5. Using an MHC class II tetramer to track the frequency and phenotype of endogenous 2.5HIP-reactive T cells in NOD mice, we found that these cells play a critical role in the disease process. The purpose of this project was to determine if poly(lactide-co-glycolide) (PLG) nanoparticles (NPs) loaded with 2.5HIP could be used to prevent induction of diabetes by transfer of BDC-2.5 TCR transgenic T cells cells into NOD.scid mice. We have found that i.v. infusion of 2.5HIP-coupled NPs (2.5HIP-PLG) one day after T cell transfer can almost completely prevent diabetes. Treatment with 2.5HIP-PLG leads to reduced T cell trafficking to the pancreas early on, as well as in vivo expansion of long-lived Foxp3+ regulatory T cells (Tregs) that express inhibitory receptors, membrane bound TGFβ-1/LAP, and stability markers. We predict that stable long-lived Tregs are critical for suppressing pathogenic IFN-γ producing T cells and maintaining long-term tolerance in this system. The end goal of this study is to determine if NPs containing HIPs can be used to prevent and/or reverse spontaneous disease in NOD mice. Results from these studies could have significant implications for the development of an antigen-specific therapy for T1D patients.

Beta‐cell‐specific loss of the inhibitory G protein, Gαz, prevents development of Type 1 Diabetes in NOD mice.

The mechanisms that underlie the beta‐cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our lab has discovered a role for the unique inhibitory G‐protein, Gαz, in beta‐cell function, survival, and replication in both normal conditions and those of pathological stress. Gαz, when acting as a canonical inhibitory G‐protein, reduces production of cyclic adenosine monophosphate (cAMP) through inhibition of adenylate cyclase. cAMP acts as essential player in the amplification of glucose‐stimulated insulin secretion, while also mediating pathways that regulate beta‐cell death and replication. We've shown previously that removal of this inhibitory protein is sufficient to promote insulin secretion, beta‐cell growth and survival using chemically‐induced beta‐cell death and diabetes in C57BL/6J mice. Previous work in our lab has also shown that non‐obese diabetic (NOD) mice with global loss of Gαz are protected from developing T1D‐like hyperglycemia through a multi‐faceted maintenance of beta‐cell health. Whole body Gαznull animals exhibited a significant increase in beta‐cell function and replication and decreased beta‐cell death. Most interestingly, islets from Gαz‐null NOD animals exhibited a marked decrease in immune infiltration. In order to determine if Gαz in the beta‐cell was responsible for the protected phenotype, we generated a beta‐cell‐specific Gαz knockout by breeding Gz Flox/Flox mice with Rat Insulin Promoter (RIP) driven CreHerr mice, on the NOD background. After 10 generations of backcrossing, we began by determining if loss of beta cell Gαz was sufficient to protect NOD animals from developing T1D‐like hyperglycemia. Weekly random‐fed blood glucose measurements were taken beginning at weaning. In the initial cohort, we saw a 12% penetrance of hyperglycemia in male Cre‐positive NOD mice and a 23% penetrance of hyperglycemia in female Cre‐positive NOD mice by 30 weeks of age. In contrast, Gαzβ‐cell‐KO males and females were entirely protected from developing hyperglycemia during this period. Despite this stark contrast, fasting blood glucose was no different between genotypes, grouped by sex, at either 12 or 28 weeks of age. Glucose tolerance was also not different between Cre‐positive NOD controls and Gαzβ‐cell‐KO animals. Experiments are ongoing to further investigate other facets of beta‐cell health, including function, replication and survival. Additionally, it will be crucial to evaluate the presence and extent of insulitis in our Gαzβ‐cell‐KO animals. Thus far, our results show a critical role for beta‐cell Gαz in promoting T1D pathophysiology, although the precise mechanism remains to be determined.Support or Funding InformationNIH F31 DK109698 to RFThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Loss of the Inhibitory G‐Protein, Gαz, in the Cell Protects Against Spontaneous and Streptozotocin‐Induced Diabetes in NOD Mice

Gαz is an inhibitory G protein that contributes to β‐cell death and diabetes pathogenesis. Loss of Gαz in the nonobese diabetic (NOD) mouse model of type I diabetes mellitus (T1DM) results in protection from hyperglycemia and significantly higher diabetes‐free survival compared to wild‐type mice despite a similar initial immune infiltration profile. Wild type islets from histologically stained pancreas sections revealed a higher insulitis profile and lower insulin positive area than their Gαz‐null counterparts at 17 weeks of age, and the protected Gαz null mice displayed significantly lower inflammation and immune infiltration. Loss of Gαz also increased the percentage of ki‐67‐positive β‐cells, a marker of proliferation, and lead to a decrease in β‐cell death as measured by percentage of terminal deoxynucleotidyltransferase‐mediated dUTP nick end labeling (TUNEL)‐positive cells. These data suggest that Gαz plays a central role in β‐cell survival and T1DM pathophysiology. Diabetes pathogenesis in the NOD model is initiated by immune infiltration into the pancreatic islet, followed by β‐cell death, which causes further recruitment of immune cells. To confirm the diabetes protection of Gαz‐null NOD mice is due specifically to loss of Gαz in the β‐cell, and not the infiltrating immune cells, a β‐cell‐specific Gαz knockout mouse was created in the NOD background. The specificity of the knockout was confirmed in the C57Bl/6J model by western blot analysis, revealing specific loss of Gαz from the pancreatic islets and wild‐type expression levels in other tissues. These mice were backcrossed for 10 generations into the NOD strain and were SNP genotyped to confirm that NOD loci were preserved. Preliminary data show a similar disease penetrance and diabetes‐free survival profile between whole‐body Gαz‐null and β‐cell‐specific Gαz null NOD mice. To increase the penetrance and temporal control of the diabetes phenotype, we tested different dosing regimens of the β‐cell toxin, streptozotocin (STZ), designed to be less potent than the standard 5‐day, multiple low‐dose protocol normally applied to ensure rapid and severe diabetes development. A pilot study revealed 3 consecutive low doses at 9–10 weeks of age produced moderate and reliable hyperglycemia within 2 weeks of STZ administration. Supporting the β‐cell‐centric role of Gαz in T1D pathophysiology, both the whole‐body Gαz‐null and β‐cell‐specific Gαz‐null NOD mice are protected from hyperglycemia after STZ administration as compared to the appropriate littermate controls. As the β‐cell‐specific NOD mice reach the study endpoint, pancreas samples will be collected and histologically stained. This data will allow us to confirm that it is solely β‐cell Gαz responsible for differences in insulitis, immune infiltration, proliferation, and insulin positive area seen between wild‐type and Gαz‐null NOD mice. These data would support Gαz as a strong target for therapeutics aimed to prevent or reverse insulitis and β‐cell death in early T1DM.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Carbamazepine, a beta-cell protecting drug, reduces type 1 diabetes incidence in NOD mice

Pancreatic beta-cells are selectively destroyed by the host immune system in type 1 diabetes. Thus, drugs that preserve beta-cell mass and/or function have the potential to prevent or slow the progression of this disease. We recently reported that the use-dependent sodium channel blocker, carbamazepine, protects beta-cells from inflammatory cytokines in vitro. Here, we tested the effects of carbamazepine treatment in female non-obese diabetic (NOD) mice by supplementing LabDiet 5053 with 0.5% w/w carbamazepine to achieve serum carbamazepine levels of 14.98 ± 3.19 µM. Remarkably, diabetes incidence over 25 weeks, as determined by fasting blood glucose, was ~50% lower in carbamazepine treated animals. Partial protection from diabetes in carbamazepine-fed NOD mice was also associated with improved glucose tolerance at 6 weeks of age, prior to the onset of diabetes in our colony. Less insulitis was detected in carbamazepine treated NOD mice at 6 weeks of age, but we did not observe differences in CD4+ and CD8+ T cell composition in the pancreatic lymph node, as well as circulating markers of inflammation. Taken together, our results demonstrate that carbamazepine reduces the development of type 1 diabetes in NOD mice by maintaining functional beta-cell mass.

Two Novel MicroRNA Biomarkers Related to β-Cell Damage and Their Potential Values for Early Diagnosis of Type 1 Diabetes.

New strategies and biomarkers are needed in the early detection of β-cell damage in the progress of type 1 diabetes mellitus (T1DM). To explore whether serum microRNAs (miRNA) should be served as biomarkers for T1DM. The miRNA profile was established with miRNA microarray in discovery phase (six T1DM, six controls). A miRNA-based model for T1DM diagnosis was developed using logistic regression analysis in the training dataset (40 T1DM, 56 controls) and then validated with leave-one-out cross validation and another independent validation dataset (33 T1DM, 29 controls). Quantitative reverse transcription polymerase chain reaction was applied to confirm the differences of candidate miRNAs between T1DM and controls. Area under the receiver-operating characteristic (ROC) curve (AUC) was used to evaluate diagnostic accuracy. INS-1 cells, streptozotocin-treated mice (n = 4), and nonobese diabetic (NOD) mice (n = 12) were used to evaluate the association of miRNAs with β-cell damage. A miRNA -based model was established in the training dataset with high diagnostic accuracy for T1DM (AUC = 0.817) based on six candidate differential expressed miRNAs identified in discovery phase. The validation dataset showed the model's satisfactory diagnostic performance (AUC = 0.804). Secretions of miR-1225-5p and miR-320c were significantly increased in streptozotocin-treated mice and INS-1 cells. Noteworthy, the elevation of these two miRNAs was observed before glucose elevation in the progress of diabetes in NOD mice. Two miRNA biomarkers (miR-1225-5p and miR-320c) related to β-cell damage were identified in patients with recent-onset T1DM. The miRNA-based model established in this study exhibited a good performance in diagnosis of T1DM.

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.

Construction of a novel vaccine by conjugating a B-cell epitope of DPP4 to peptide IA2(5)-P2-1 to significantly control type 1 diabetes in NOD mice.

Type 1 diabetes is a chronic organ-specific autoimmune disease in which selective destruction of insulin-producing β cells leads to impaired glucose metabolism and its attendant complications. IA2(5)P2-1, a potent immunogenic carrier which designed by our laboratory, can induce high titer specific antibodies when carry a B cell epitope, such as B cell epitopes of DPP4, xanthine oxidase, and Urate transporter protein. In this report, we describe a novel multi-epitope vaccine composing a peptide of DPP4, an anti-diabetic B epitope of Insulinoma antigen-2(IA-2) and a Th2 epitope (P2:IPALDSLTPANED) of P277 peptide in human heat shock protein 60 (HSP60). Immunization with the multi-epitope vaccine in non-obese diabetic (NOD) mice successfully induced specific anti-DPP4 antibody, inhibited plasma DPP4 activity, and increased serum GLP-1 level. Moreover, this antibody titer was correlated with the dose of immunization (20μg, 100μg). Inoculation of this vaccine in NOD mice significantly control blood glucose level, improved glucose excursion and increased insulin level in vivo. Consistent with a lower diabetic and insulitis incidence, a induced splenic T cells proliferation and tolerance were observed. IFN-γ secretion reduced and IL-10 increased significantly in the D41-IA2(5)-P2-1 treated mice compared to P277 and control group due to the potential immunomodulatory effect of the epitope in the vaccine. Immunohistochemical analysis and cytometry showed a rebalance of Th1/Th2 in NOD mice. Our results demonstrate that this multi-epitope vaccine may serve as a promising therapeutic approach for type 1 diabetes.

Intestinal type 1 regulatory T cells migrate to periphery to suppress diabetogenic T cells and prevent diabetes development

Growing insight into the pathogenesis of autoimmune diseases and numerous studies in preclinical models highlights the potential of regulatory T cells to restore tolerance. By using non-obese diabetic (NOD) BDC2.5 TCR-transgenic (Tg), and IL-10 and Foxp3 double-reporter mice, we demonstrate that alteration of gut microbiota during cohousing experiments or treatment with anti-CD3 mAb significantly increase intestinal IL-10-producing type 1 regulatory T (Tr1) cells and decrease diabetes incidence. These intestinal antigen-specific Tr1 cells have the ability to migrate to the periphery via a variety of chemokine receptors such as CCR4, CCR5, and CCR7 and to suppress proliferation of Th1 cells in the pancreas. The ability of Tr1 cells to cure diabetes in NOD mice required IL-10 signaling, as Tr1 cells could not suppress CD4+ T cells with a dominant-negative IL-10R. Taken together, our data show a key role of intestinal Tr1 cells in the control of effector T cells and development of diabetes. Therefore, modulating gut-associated lymphoid tissue to boost Tr1 cells may be important in type 1 diabetes management.

Anti-TCRβ mAb in Combination With Neurogenin3 Gene Therapy Reverses Established Overt Type 1 Diabetes in Female NOD Mice.

Insulin-producing β cells in patients with type 1 diabetes (T1D) are destroyed by T lymphocytes. We investigated whether targeting the T-cell receptor (TCR) with a monoclonal antibody (mAb) abrogates T-cell response against residual and newly formed islets in overtly diabetic nonobese diabetic (NOD) mice. NOD mice with blood glucose levels of 250 to 350 mg/dL or 350 to 450 mg/dL were considered as new-onset or established overt diabetes, respectively. These diabetic NOD mice were transiently treated with an anti-TCR β chain (TCRβ) mAb, H57-597, for 5 days. Two weeks later, some NOD mice with established overt diabetes further received hepatic gene therapy using the islet-lineage determining gene Neurogenin3 (Ngn3), in combination with the islet growth factor gene betacellulin (Btc). We found that anti-TCRβ mAb (50 µg/d) reversed >80% new-onset diabetes in NOD mice for >14 weeks by reducing the number of effector T cells in the pancreas. However, anti-TCRβ mAb therapy alone reversed only ∼20% established overt diabetes in these mice. Among those overtly diabetic NOD mice whose diabetes was resistant to anti-TCRβ mAb treatment, ∼60% no longer had diabetes when they also received Ngn3-Btc hepatic gene transfer 2 weeks after initial anti-TCRβ mAb treatment. This combination of Ngn3-Btc gene therapy and anti-TCRβ mAb treatment induced the sustained formation of periportal insulin-producing cells in the liver of overtly diabetic mice. Therefore, directly targeting TCRβ with a mAb potently reverses new-onset T1D in NOD mice and protects residual and newly formed gene therapy-induced hepatic neo-islets from T-cell‒mediated destruction in mice with established overt diabetes.

AAV-mediated pancreatic overexpression of Igf1 counteracts progression to autoimmune diabetes in mice

ObjectiveType 1 diabetes is characterized by autoimmune destruction of β-cells leading to severe insulin deficiency. Although many improvements have been made in recent years, exogenous insulin therapy is still imperfect; new therapeutic approaches, focusing on preserving/expanding β-cell mass and/or blocking the autoimmune process that destroys islets, should be developed. The main objective of this work was to test in non-obese diabetic (NOD) mice, which spontaneously develop autoimmune diabetes, the effects of local expression of Insulin-like growth factor 1 (IGF1), a potent mitogenic and pro-survival factor for β-cells with immunomodulatory properties. MethodsTransgenic NOD mice overexpressing IGF1 specifically in β-cells (NOD-IGF1) were generated and phenotyped. In addition, miRT-containing, IGF1-encoding adeno-associated viruses (AAV) of serotype 8 (AAV8-IGF1-dmiRT) were produced and administered to 4- or 11-week-old non-transgenic NOD females through intraductal delivery. Several histological, immunological, and metabolic parameters were measured to monitor disease over a period of 28–30 weeks. ResultsIn transgenic mice, local IGF1 expression led to long-term suppression of diabetes onset and robust protection of β-cell mass from the autoimmune insult. AAV-mediated pancreatic-specific overexpression of IGF1 in adult animals also dramatically reduced diabetes incidence, both when vectors were delivered before pathology onset or once insulitis was established. Transgenic NOD-IGF1 and AAV8-IGF1-dmiRT-treated NOD animals had much less islet infiltration than controls, preserved β-cell mass, and normal insulinemia. Transgenic and AAV-treated islets showed less expression of antigen-presenting molecules, inflammatory cytokines, and chemokines important for tissue-specific homing of effector T cells, suggesting IGF1 modulated islet autoimmunity in NOD mice. ConclusionsLocal expression of Igf1 by AAV-mediated gene transfer counteracts progression to diabetes in NOD mice. This study suggests a therapeutic strategy for autoimmune diabetes in humans.

Restoration of Immune Tolerance in Type 1 Diabetes by Modulating Interleukin-2 Receptor Signaling

Abstract Type 1 diabetes (T1D) is an organ-specific autoimmune disease, characterized by the destruction of insulin-producing beta cells in pancreatic islets. A variety of lymphocytes, including T cells and natural killer (NK) cells infiltrate into the islets. Their roles in T1D are rather different--effector T cells and NK cells accelerate, while regulatory T cells, dampen the development of T1D. Signaling through interleukin-2 (IL-2) receptor beta (IL-2Rβ, also known as CD122) is critical for the differentiation and function of both T cells and NK cells. However, it remains poorly understood how these cells respond to IL-2Rβ signaling during the development of T1D. Here we show that in non-obese diabetic (NOD) mice, the most commonly used animal model for human T1D, administration of a non-immunogenic monoclonal antibody (mAb) selectively ablates pathogenic cells, including memory CD4+ T cells, CD8+ T cells and NK cells. In contrast, CD4+ Foxp3+ regulatory T (Treg) cells are only mildly affected. This leads to significantly elevated ratios between Treg cells and pathogenic cells. This reset balance preferentially occurs in pancreatic islets, without systemic immunosuppression. Importantly, IL-2Rβ blockade suppresses both insulitis and diabetes in NOD mice. Moreover, IL-2Rβ blockade significantly suppresses interferon gamma (IFN-γ) production in T cells and NK cells, but promotes interleukin-17 (IL-17) production in CD4+ T cells. Therefore, the modulation of IL-2Rβ signaling represents a novel and efficient approach to restore immune tolerance in T1D.

Targeting ABL-IRE1α Signaling Spares ER-Stressed Pancreatic β Cells to Reverse Autoimmune Diabetes

In cells experiencing unrelieved endoplasmic reticulum (ER) stress, the ER transmembrane kinase/endoribonuclease (RNase)-IRE1α-endonucleolytically degrades ER-localized mRNAs to promote apoptosis. Here we find that the ABL family of tyrosine kinases rheostatically enhances IRE1α's enzymatic activities, thereby potentiating ER stress-induced apoptosis. During ER stress, cytosolic ABL kinases localize to the ER membrane, where they bind, scaffold, and hyperactivate IRE1α's RNase. Imatinib-an anti-cancer tyrosine kinase inhibitor-antagonizes the ABL-IRE1α interaction, blunts IRE1α RNase hyperactivity, reduces pancreatic β cell apoptosis, and reverses type 1 diabetes (T1D) in the non-obese diabetic (NOD) mouse model. A mono-selective kinase inhibitor that allosterically attenuates IRE1α's RNase-KIRA8-also efficaciously reverses established diabetes in NOD mice by sparing β cells and preserving their physiological function. Our data support a model wherein ER-stressed β cells contribute to their own demise during T1D pathogenesis and implicate the ABL-IRE1α axis as a drug target for the treatment of an autoimmune disease.

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.

Alterations in oviductal cilia morphology and reduced expression of axonemal dynein in diabetic NOD mice.

In the present study, we examined the morphology of cilia and expression of the dynein intermediate chain 2 (DNAI2) in the oviduct of non-obese diabetic (NOD) mice. Results obtained with immunohistochemistry showed that DNAI2 expression was reduced in oviducts of diabetic NOD (dNOD) mice, as compared to that observed in the normoglycemic NOD (cNOD) group, especially in the acyclic dNOD mice. Oviductal cilia of dNOD mice appeared to be reduced in number. Results obtained with Western blot analysis revealed that the expression of DNAI2 protein was significantly less in oviducts of dNOD mice as compared to that of cNOD mice corroborating the results obtained with immunohistochemistry. Electron microscopic examination and quantitative imaging of thin sections of Epon-embedded oviducts of both dNOD and cNOD mice confirmed the reduction of the number of cilia in the oviduct of the dNOD group which also displayed aberrant axonemal ultrastructure, including disorganization of the axoneme and alteration of microtubule doublets into singlets as well as disruption of the plasma membrane in many cilia. Taken together, the present findings suggest that structural alterations of oviductal cilia in female diabetic NOD mice might be detrimental to the normal function of these particular cell structures in gamete transport.

An insulin-IAPP hybrid peptide is an endogenous antigen for CD4 T cells in the non-obese diabetic mouse

BDC-6.9, a diabetogenic CD4 T cell clone isolated from a non-obese diabetic (NOD) mouse, responds to pancreatic islet cells from NOD but not BALB/c mice. We recently reported that a hybrid insulin peptide (HIP), 6.9HIP, formed by linkage of an insulin C-peptide fragment and a fragment of islet amyloid polypeptide (IAPP), is the antigen for BDC-6.9. We report here that the core 12-mer peptide from 6.9HIP, centered on the hybrid peptide junction, is also highly antigenic for BDC-6.9. In agreement with the observation that BALB/c islet cells fail to stimulate the T cell clone, a single amino acid difference in the BALB/c IAPP sequence renders the BALB/c version of the HIP only weakly antigenic. Mutant peptide analysis indicates that each parent molecule—insulin C-peptide and IAPP—donates residues critical for antigenicity. Through mass spectrometric analysis, we determine the distribution of naturally occurring 6.9HIP across chromatographic fractions of proteins from pancreatic beta cells. This distribution closely matches the profile of the T cell response to the fractions, confirming that 6.9HIP is the endogenous islet antigen for the clone. Using a new MHC II tetramer reagent, 6.9HIP-tet, we show that T cells specific for the 6.9HIP peptide are prevalent in the pancreas of diabetic NOD mice. Further study of HIPs and HIP-reactive T cells could yield valuable insight into key factors driving progression to diabetes and thereby inform efforts to prevent or reverse this disease.

Congenic mapping identifies a novel Idd9 subregion regulating type 1 diabetes in NOD mice.

Type 1 diabetes (T1D) results from complex interactions between genetic and environmental factors. The nonobese diabetic (NOD) mouse develops spontaneous T1D and has been used extensively to study the genetic control of this disease. T1D is suppressed in NOD mice congenic for the C57BL/10 (B10)-derived Idd9 resistance region on chromosome 4. Previous studies conducted by other investigators have identified four subregions (Idd9.1, Idd9.2, Idd9.3, and Idd9.4) where B10-derived genes suppress T1D development in NOD mice. We independently generated and characterized six congenic strains containing B10-derived intervals that partially overlap with the Idd9.1 and Idd9.4 regions. T1D incidence studies have revealed a new B10-derived resistance region proximal to Idd9.1. Our results also indicated that a B10-derived gene(s) within the Idd9.4 region suppressed the diabetogenic activity of CD4 T cells and promoted CD103 expression on regulatory T cells indicative of an activated phenotype. In addition, we suggest the presence of a B10-derived susceptibility gene(s) in the Idd9.1/Idd9.4 region. These results provide additional information to improve our understanding of the complex genetic control by the Idd9 region.

Mucosal administration of CD3-specific monoclonal antibody inhibits diabetes in NOD mice and in a preclinical mouse model transgenic for the CD3 epsilon chain

CD3-specific monoclonal antibody (mAb) treats autoimmune disease in animal models and has shown promise in clinical trials of type 1 diabetes. Whereas intravenous administration of CD3-specific mAb acts primarily by transient depletion of activated effector T cells, oral CD3-specific mAb acts primarily by the induction Tregs. We investigated whether oral CD3-specific mAb inhibits disease in non obese diabetic (NOD) mice that spontaneously develop autoimmune diabetes, closely resembling human type 1 diabetes. We found that oral CD3-specific mAb treatment delayed onset and reduced incidence of diabetes in NOD mice, inducing changes in both effector and regulatory T cell compartments. The therapeutic effect was associated with decreased T cell proliferation, decreased IFNγ and IL-17 production, and increased TGF-β and IL-10 production in vitro. In vivo transfer experiments demonstrated that oral CD3-specific mAb decreased diabetogenicity of effector T cells and increased the function of regulatory T cells. Oral OKT3, a monoclonal antibody specific for human CD3 had equivalent effects in transgenic NOD mice expressing the human CD3 epsilon chain which serves as a preclinical model for testing human CD3-specific mAb. These results suggest that oral CD3-specific mAb has the potential for treating autoimmune diabetes in humans.

Perturbation of the molecular clockwork in the SCN of non-obese diabetic mice prior to diabetes onset

ABSTRACTCircadian disruption is associated with the development of diabetes. Non-obese diabetic (NOD) mice show abnormal diurnal profiles in energy balance and locomotor activity suggesting circadian misalignment. Therefore, we analyzed cFos and mPER1 as markers for rhythmic neuronal activity within the suprachiasmatic nucleus (SCN) of wildtype (WT) and non-diabetic (nNOD) as well as acutely diabetic NOD (dNOD) mice. cFos levels show a day/night difference in both WT and nNOD but not in dNOD. mPER1 levels did not show a day/night difference in both nNOD and dNOD. This suggests that disruption of SCN rhythmicity in NOD mice precedes the actual onset of diabetes.

Detection of vasostatin-1-specific CD8(+) T cells in non-obese diabetic mice that contribute to diabetes pathogenesis.

Chromogranin A (ChgA) is an antigenic target of pathogenic CD4(+) T cells in a non-obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Vasostatin-1 is a naturally processed fragment of ChgA. We have now identified a novel H2-K(d) -restricted epitope of vasostatin-1, ChgA 36-44, which elicits CD8(+) T cell responses in NOD mice. By using ChgA 36-44/K(d) tetramers we have determined the frequency of vasostatin-1-specific CD8(+) T cells in pancreatic islets and draining lymph nodes of NOD mice. We also demonstrate that vasostatin-1-specific CD4(+) and CD8(+) T cells constitute a significant fraction of islet-infiltrating T cells in diabetic NOD mice. Adoptive transfer of T cells from ChgA 36-44 peptide-primed NOD mice into NOD/severe combined immunodeficiency (SCID) mice led to T1D development. These findings indicate that vasostatin-1-specific CD8(+) T cells contribute to the pathogenesis of type 1 diabetes in NOD mice.

An intravital microscopy model to study early pancreatic inflammation in type 1 diabetes in NOD mice.

Intravital microscopy (IVM) of the pancreas has been proven to be an invaluable tool in pancreatitis, transplantation and ischemia/reperfusion research. Also in type 1 diabetes (T1D) pancreatic IVM offers unique advantages for the elucidation of the disease process. Female non-obese diabetic (NOD) mice develop T1D spontaneously by 40weeks of age. Our goal was to establish an IVM-based method to study early pancreatic inflammation in NOD mice, which can be used to screen novel medications to prevent or delay T1D in future studies. This included evaluation of leukocyte-endothelial interactions as well as disturbances of capillary perfusion in the pancreatic microcirculation.