Female Non-obese Diabetic Mice Research Articles

Exploiting T cell co-inhibition to delay autoimmune disease recurrence

Abstract Type 1 diabetes (T1D) results from T cell-mediated destruction of insulin-producing pancreatic β cells. Individuals with long-term disease are at risk of developing life-threatening complications. β cell replacement is a therapy for T1D but is limited by recurrent autoreactive T cell targeted β cell death. Thus, β cells better equipped to inhibit local T cell responses may survive longer in autoimmune recipients. Programmed-death 1 (PD-1) signaling through its ligand PD-L1 inhibits T cells, and may serve as a prominent defense in T1D. Using flow cytometric analysis, in the absence of T cells in NOD.RAG−/− mice we do not detect β cell PD-L1 expression. However, with T cells, we observed an increased proportion of β cells expressing PD-L1 in female non-obese diabetic (NOD) mice which had not developed diabetes. In addition, the majority of remaining live β cells at diabetes onset in NOD mice continue to express high levels of PD-L1. These three situations suggest that islet β cells may increase PD-L1 expression as a last line of defense to limit infiltrating T cell mediated destruction. To manipulate β cell PD-L1 expression prior to transplantation, we screened a panel of diabetes-related cytokines and found that IFN-γ enhances β cell PD-L1 expression. Unfortunately, islet transplant survival was not prolonged, which we hypothesized was due to enhanced MHC class I expression, facilitating CD8+ T cell-mediated killing. We therefore de-coupled PD-L1 from enhanced MHC I expression. Using this approach, enforced β cell PD-L1 expression delays disease recurrence. These data support our hypothesis that β cells expressing T cell co-inhibitory molecules, like PD-L1, can locally inhibit autoreactive T cells which may prevent transplant destruction.

IDO1-targeting intervention by proteasome inhibition in autoimmune diabetes

Abstract Indoleamine 2,3-dioxygenase 1 (IDO1) is a metabolic enzyme involved in the catabolism of tryptophan. Its metabolic activity has become pivotal in the regulation of the immune responses. The functional expression of IDO1 enzyme confers a tolerogenic phenotype to different subsets of dendritic cells (DCs). Our previous data suggested a defective IDO1 expression in a prediabetic phase of nonobese diabetic (NOD) mice, a prototypic model of human type 1 diabetes. Based on the recent finding that IDO1 may be subjected to regulatory proteolysis, which is triggered by IL-6 and mediated by the immunoproteasome, this pathway was explored in splenic plasmacytoid dendritic cells (pDCs) of NOD mice. The in vitro conditioning of murine NOD pDCs with the proteasome inhibitor Bortezomib, increased the functional expression of IDO1 enzyme, conferring those cells an immunoregulatory phenotype that is strongly inhibited for presenting in vivo the diabetogenic antigen IGRP, an effect accompanied by increased IGRP-specific regulatory T cells in pancreatic lymph nodes. The inhibition of IDO1 by 1-methyl tryptophan (MT) completely abolished the immunoregulatory effect of Bortezomib in NOD pDCs, indicating the involvement of the enzyme. Moreover, the pharmacologic treatment of NOD female mice with Bortezomib during prediabetic phase (8–10 weeks old) prevented the onset of hyperglycemia, reducing the diabetes incidence. Overall, our data suggest that the control of proteasomal degradation in pDCs may represent an innovative strategy for inducing/potentiating IDO1-mediated immunoregulation in autoimmune diabetes.

Protective role of adenovirus vector-mediated interleukin-10 gene therapy on endogenous islet β-cells in recent-onset type 1 diabetes in NOD mice.

The aim of the present study was to provide an animal experimental basis for the protective effect of the adenoviral vector-mediated interleukin-10 (Ad-mIL-10) gene on islet β-cells during the early stages of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. A total of 24 female NOD mice at the onset of diabetes were allocated at random into three groups (n=8 per group): Group 1, intraperitoneally injected with 0.1 ml Ad-mIL-10; group 2, intraperitoneally injected with 0.1 ml adenovirus vector; and group 3, was a diabetic control. In addition to groups 1, 2 and 3, 8 age- and gender-matched NOD mice were intraperitoneally injected with 0.1 ml PBS and assigned to group 4 as a normal control. All mice were examined weekly for body weight, urine glucose and blood glucose values prior to onset of diabetes, and at 1, 2 and 3 weeks after that, and all mice were sacrificed 3 weeks after injection. Serum levels of interleukin (IL)-10, interferon (IFN)-γ, IL-4, insulin and C-peptide were evaluated, and in addition the degree of insulitis and the local expression of IL-10 gene in the pancreas were detected. The apoptosis rate of pancreatic β-cells was determined using a TUNEL assay. Compared with groups 2 and 3, IL-10 levels in the serum and pancreas were elevated in group 1. Serum IFN-γ levels were decreased while serum IL-4 levels and IFN-γ/IL-4 ratio were significantly increased in group 1 (P<0.01). C-peptide and insulin levels were higher in group 1 compared with groups 2 and 3, (P<0.01). Furthermore, compared with groups 2 and 3, the degree of insulitis, islet β-cell apoptosis rate and blood glucose values did not change significantly (P>0.05). The administration of the Ad-mIL-10 gene induced limited immune regulatory and protective effects on islet β-cell function in NOD mice with early T1D, while no significant reduction in insulitis, islet β-cell apoptosis rate and blood glucose was observed.

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.

Regulatory T and B lymphocytes in a spontaneous autoimmune polyneuropathy.

B7-2(-/-) non-obese diabetic (NOD) mice develop a spontaneous autoimmune polyneuropathy (SAP) that mimics the progressive form of chronic inflammatory demyelinating polyradiculoneuropathy (CIDP). In this study, we focused on the role of regulatory T cells (Tregs ) and regulatory B cells (Bregs ) in SAP. We found that deletion of B7-2 in female NOD mice led to a lower frequency and number of Tregs and Bregs in spleens and lymph nodes. Tregs but not Bregs suppressed antigen-stimulated splenocyte proliferation, whereas Bregs inhibited the T helper type 1 (Th1) cytokine response. Both Tregs and Bregs induced an increase in CD4(+) interleukin (IL)-10(+) cells, although less effectively in the absence of B7-2. Adoptive transfer studies revealed that Tregs , but not Bregs , suppressed SAP, while Bregs attenuated disease severity when given prior to symptom onset. B cell deficiency in B cell-deficient (muMT)/B7-2(-/-) NOD mice prevented the development of SAP, which would indicate that the pathogenic role of B cells predominates over its regulatory role in this model. We conclude that Bregs and Tregs control the immunopathogenesis and progression of SAP in a non-redundant fashion, and that therapies aimed at expansion of Bregs and Tregs may be an effective approach in autoimmune neuropathies.

The IL-1β Receptor Antagonist SER140 Postpones the Onset of Diabetes in Female Nonobese Diabetic Mice.

The cytokine interleukin-1β (IL-1β) is known to stimulate proinflammatory immune responses and impair β-cell function and viability, all critical events in the pathogenesis of type 1 diabetes (T1D). Here we evaluate the effect of SER140, a small peptide IL-1β receptor antagonist, on diabetes progression and cellular pancreatic changes in female nonobese diabetic (NOD) mice. Eight weeks of treatment with SER140 reduced the incidence of diabetes by more than 50% compared with vehicle, decreased blood glucose, and increased plasma insulin. Additionally, SER140 changed the endocrine and immune cells dynamics in the NOD mouse pancreas. Together, the data suggest that SER140 treatment postpones the onset of diabetes in female NOD mice by interfering with IL-1β activated pathways.

Paricalcitol modulates ACE2 shedding and renal ADAM17 in NOD mice beyond proteinuria

Circulating and renal activity of angiotensin-converting enzyme 2 (ACE2) is increased in non-obese diabetic (NOD) mice. Because paricalcitol has been reported to protect against diabetic nephropathy, we investigated the role of paricalcitol in modulating ACE2 in these mice. In addition, renal ADAM17, a metalloprotease implied in ACE2 shedding, was assessed. NOD female and non-diabetic control mice were studied for 21 days after diabetes onset and divided into various treatment groups. Diabetic animals received either vehicle; 0.4 or 0.8 μg/kg paricalcitol, aliskiren, or a combination of paricalcitol and aliskiren. We then studied the effect of paricalcitol on ACE2 expression in proximal tubular epithelial cells. Paricalcitol alone or in combination with aliskiren resulted in significantly reduced circulating ACE2 activity in NOD mice but there were no changes in urinary albumin excretion. Serum renin activity was significantly decreased in mice that received aliskiren but no effect was found when paricalcitol was used alone. Renal content of ADAM17 was significantly decreased in animals that received a high dose of paricalcitol. Renal and circulating oxidative stress (quantified by plasma H2O2 levels and immunolocalization of nitrotyrosine) were reduced in high-dose paricalcitol-treated mice compared with non-treated diabetic mice. In culture, paricalcitol incubation resulted in a significant increase in ACE2 expression compared with nontreated cells. In NOD mice with type 1 diabetes, paricalcitol modulates ACE2 activity, ADAM17, and oxidative stress renal content independently from the glycemic profile and urinary albumin excretion. In tubular cells, paricalcitol may modulate ACE2 by blocking its shedding. In the early stage of diabetic nephropathy, paricalcitol treatment counterbalances the effect of diabetes on circulating ACE2 activity. Our results suggest that additional use of paricalcitol may be beneficial in treating patients with diabetes under standard therapeutic strategies.

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.

Estrogen Therapy Delays Autoimmune Diabetes and Promotes the Protective Efficiency of Natural Killer T-Cell Activation in Female Nonobese Diabetic Mice

Therapeutic strategies focused on restoring immune tolerance remain the main avenue to prevent type 1 diabetes (T1D). Because estrogens potentiate FoxP3+ regulatory T cells (Treg) and invariant natural killer T (iNKT) cells, two regulatory lymphocyte populations that are functionally deficient in nonobese diabetic (NOD) mice, we investigated whether estradiol (E2) therapy influences the course of T1D in this model. To this end, female NOD mice were sc implanted with E2- or placebo-delivering pellets to explore the course of spontaneous and cyclophosphamide-induced diabetes. Treg-depleted and iNKT-cell-deficient (Jα18(-/-)) NOD mice were used to assess the respective involvement of these lymphocyte populations in E2 effects. Early E2 administration (from 4 wk of age) was found to preserve NOD mice from both spontaneous and cyclophosphamide-induced diabetes, and a complete protection was also observed throughout treatment when E2 treatment was initiated after the onset of insulitis (from 12 wk of age). This delayed E2 treatment remained fully effective in Treg-depleted mice but failed to entirely protect Jα18(-/-) mice. Accordingly, E2 administration was shown to restore the cytokine production of iNKT cells in response to in vivo challenge with the cognate ligand α-galactosylceramide. Finally, transient E2 administration potentiated the previously described protective action of α-galactosylceramide treatment in NOD females. This study provides original evidence that E2 therapy strongly protects NOD mice from T1D and reveals the estrogen/iNKT cell axis as a new effective target to counteract diabetes onset at the stage of insulitis. Estrogen-based therapy should thus be considered for T1D prevention.

Anti-serum with anti-autoantibody activity decreases autoantibody-positive B lymphocytes and type 1 diabetes of female NOD mice

Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterized by an autoimmune-mediated loss of insulin secreting β-cells. Each B lymphocyte clone that escapes immune tolerance produces a specific antibody. No specific treatment against autoantibodies is available for autoimmune diseases. We have developed a strategy to produce an antiserum against autoantibodies for the treatment of T1DM. Non-obese diabetic (NOD) but not Balb/c mouse serum contains autoantibodies. Antisera were produced by immunizing Balb/c mice with affinity-purified IgG from NOD or BALB/c mice along with the immune adjuvant (hereafter, NIgG or BIgG, respectively). A bolus administration of NIgG significantly reduced serum autoantibodies, autoantibody-positive B lymphocytes in the spleens of NOD mice, mortality and morbidity of diabetes, blood glucose and islet immune infiltration, whereas it increased islet mass in NOD mice for at least 26 weeks. NIgG antiserum treatment has no significant effect on CD3+, CD4+ or CD8+ T cells and B220+ or CD19+ B cells. BIgG also imparted a moderate therapeutic effect, although it was considerably lower than that of NIgG. NIgG did not cross-react with allogeneic serum. NIgG showed no effect on Balb/c mice. The results show the feasibility of producing antiserum against autoantibodies to prevent and treat autoimmune-induced T1DM with a single bolus administration.

Beneficial Effect of Naringin on Glucose Regulation in Type 1 Diabetic Mice

The present study investigated the effect of naringin on glucose metabolism in non-obese diabetic (NOD) mice, a good animal model for studying human autoimmune type 1 diabetes or insulin dependent diabetes. Female NOD mice were divided into control and naringin (0.02%, w/w) and fed experimental diet for 11 weeks. There was no significant difference in food intake between groups. However, naringin inhibited weight loss and hyperglycemia and increased survival rate. In addition, naringin improved glucose tolerance, increased plasma insulin and C-peptide levels and decreased plasma glucagon level. Similar to plasma insulin and glucagon levels, pancreatic insulin protein expression was increased by naringin, whereas pancreatic glucagon protein expression was decreased. Moreover, naringin decreased activity of gluconeogenic enzymes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, in liver, along with increased hepatic glucokinase activity and glycogen content. These results suggest that naringin may maintain plasma insulin and glucagon by protecting pancreatic α-cells and β-cells, thereby reducing gluconeogenesis and increasing glucose utilization in liver and ultimately preventing diabetes onset in type 1 diabetic NOD mice. Therefore, naringin may be useful phytochemical compound for improving and preventing type 1 diabetes.

Interleukin-10 gene transfer into insulin-producing β cells protects against diabetes in non-obese diabetic mice.

Type 1 diabetes is an autoimmune disorder, which occurs due to β cell damage. Interleukin (IL)-10, a pleotropic cytokine, has been reported to have anti‑inflammatory, immunosuppressive and immunostimulatory properties. Administration of IL‑10 is known to prevent autoimmune diabetes in non‑obese diabetic (NOD) mice. However, the mechanism of IL‑10‑induced protection in NOD mice requires further investigation. The aim of the present study was to evaluate the protective effect of transgenic IL‑10 expression in pancreatic β cells against autoimmune damage in NOD mice and to elucidate its mechanism of action. Female NOD mice (9 weeks old) were intraperitoneally injected with an adenovirus carrying either IL‑10 (Adv‑IL‑10) or green fluorescent protein (Adv‑GFP). Blood glucose was monitored weekly and the expression of IL‑10 was evaluated using reverse transcription quantitative polymerase chain reaction. IL‑10 and interferon (IFN)‑γ expression levels in serum and splenocytes were analyzed. CD4+CD25+FoxP3+ T regulatory (Treg) cells were determined by flow cytometry. Apoptosis of pancreatic β cells was determined using a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick‑end labeling assay and expression levels of Fas and caspase‑3 were estimated by immunohistochemistry analysis. The results revealed that mice treated with IL‑10 showed less severe insulitis and a lower incidence of diabetes compared with the saline control and Adv‑GFP groups. In addition, compared with the control group, IFN‑γ levels were decreased in sera and splenocytes, while IL‑10 expression was increased in sera only. The number of CD4+CD25+FoxP3+ Treg cells was increased in IL‑10‑injected mice. Furthermore, the expression levels of Fas and caspase‑3 were decreased in IL‑10‑injected mice compared with that of GFP‑injected and control mice, which was concomitant with a reduction in β cell apoptosis. In conclusion, the present study demonstrated that IL‑10 gene transfer reduced the expression of the inflammatory cytokines, attenuated pancreatic insulitis and inhibited β cell apoptosis. This therefore indicated that IL-10 reduced the incidence of diabetes in female NOD mice.

DNA sensing via STING regulates autoimmunity and tumor immunity. (IRM5P.648)

Abstract The Stimulator of Interferon Genes (STING) is activated by cytosolic DNA sensors such as cyclic GMP-AMP Synthase (cGAS) to induce interferon type I (IFN-I). Dendritic cells (DCs) that engulf dying tumor cells incite anti-tumor T cell immunity by sensing tumor cell DNA to activate STING/IFN-I signaling. Paradoxically, we find that DNA sensing to activate STING/IFN-I signaling induces DCs to express indoleamine 2, 3 dioxygenase (IDO), which activates regulatory T cells (Tregs). Thus treatment with DNA nanoparticles (DNPs) or cyclic dinucleotides (CDNs) to activate STING attenuated Experimental Autoimmune Encephalomyelitis (EAE), arthritis, and therapeutic responses were dependent on STING/IFN-I signaling to induce IDO. DNP and CDN treatments were also effective in slowing Type I Diabetes (T1D) progression in susceptible female Non-Obese Diabetic (NOD) mice. STING ablation also abolished IDO-dependent regulatory responses to apoptotic cells, suggesting that DNA from dying cells is sensed to activate the STING/IFN-I pathway and induce IDO to suppress autoimmunity. Moreover, cGAS and STING ablation abolished the ability of Lewis Lung carcinoma (LLC) tumor cells to induce local IDO, which suppresses anti-tumor immunity. These findings support the hypothesis that DNA from dying cells is sensed to activate STING/IFN-I in regulatory DCs that suppress T cell immunity and autoimmunity at sites of chronic inflammation associated with autoimmunity and tumor growth.

Maternal obesity alters gut microbial ecology in offspring of NOD mice

BackgroundGut microbiota (GM) are increasingly recognized as a significant factor in the pathogenesis of T1D. High Bacteroidetes/Firmicutes (B/F) is associated with the development of T1D. Previously we reported that maternal obesity (MO) increases the incidence of T1D in the offspring of nonobese diabetic (NOD) mice, and the current study investigated the possible link of GM to the exaggerated insulitis in offspring due to MO. Methods: 4‐wk‐old female NOD mice were fed either a control or a high fat diet for 8 weeks before mating. Mice were maintained in their respective diets during pregnancy and lactation. All offspring were fed the control diet to 16‐wk‐old.ResultsPyrosequencing examination revealed that GM of offspring born to obese mothers was distinct from those of control mothers. At phylum level, MO increased Bacteroidetes, Defferibacteres and Proteobacteria, while decreased Firmicutes in offspring GM, resulting in increased B/F ratio in GM of obese offspring. Family level analysis indicated that GM of offspring from obese mothers had increased levels of Deferribacteraceae, Bacteroidaceae, Desulfovibrionaceae, Helicobacteraceaev, and Porphyromonadaceae. Reciprocally, the abundance of Rikenellaceae and Lachnospiraceae‐Ruminococcaceae were decreased in offspring from obese mothers. The analysis at the genus level revealed that Bacteriodes, Barnesiella, Desulfovibrio and Mucispirillum were more abundance, while Alistipes, Clostridium XIVa, Dorea and Osillibacter were less abundance in offspring of obese mothers.ConclusionMO resulted in a dysbiosis in offspring, suggesting a GM‐mediated mechanism on diabetogenesis by MO (NIH R15HD073864).

Maternal obesity alters gut microbial ecology in offspring of NOD mice

BackgroundGut microbiota (GM) are increasingly recognized as a significant factor in the pathogenesis of T1D. High Bacteroidetes/Firmicutes (B/F) is associated with the development of T1D. Previously we reported that maternal obesity (MO) increases the incidence of T1D in the offspring of nonobese diabetic (NOD) mice, and the current study investigated the possible link of GM to the exaggerated insulitis in offspring due to MO.Methods4‐wk‐old female NOD mice were fed either a control or a high fat diet for 8 weeks before mating. Mice were maintained in their respective diets during pregnancy and lactation. All offspring were fed the control diet to 16‐wk‐old.ResultsPyrosequencing examination revealed that GM of offspring born to obese mothers was distinct from those of control mothers. At phylum level, MO increased Bacteroidetes, Defferibacteres and Proteobacteria, while decreased Firmicutes in offspring GM, resulting in increased B/F ratio in GM of obese offspring. Family level analysis indicated that GM of offspring from obese mothers had increased levels of Deferribacteraceae, Bacteroidaceae, Desulfovibrionaceae, Helicobacteraceaev, and Porphyromonadaceae. Reciprocally, the abundance of Rikenellaceae and Lachnospiraceae‐Ruminococcaceae were decreased in offspring from obese mothers. The analysis at the genus level revealed that Bacteriodes, Barnesiella, Desulfovibrio and Mucispirillum were more abundance, while Alistipes, Clostridium XIVa, Dorea and Osillibacter were less abundance in offspring of obese mothers.ConclusionMO resulted in a dysbiosis in offspring, suggesting a GM‐mediated mechanism on diabetogenesis by MO (NIH R15HD073864).

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.

Taurine Supplementation Enhances Insulin Secretion Without Altering Islet Morphology in Non-obese Diabetic Mice.

Taurine (TAU) is a sulfated amino acid that improves pancreatic islet function and regulates β-cell mass in pre- and diabetic states. We herein analyzed glucose homeostasis and islet morphofunction in non-obese diabetic (NOD) mice supplemented with 2 % TAU in their drinking water from birth until 90-days of age. TAU-supplemented female NOD mice (TAU group) showed a better glucose tolerance without modification in insulinemia, when compared to non-supplemented NOD mice (CTL). Glucose-induced insulin secretion was higher in islets isolated from female and male TAU groups. In addition, a better insulin release was observed at 30 mM K+ in islets from female TAU mice. These effects were accompanied by a higher total intracellular Ca2+ concentration in islets from female and male TAU mice. TAU-treated mice did not show any alteration in β-cell and islet areas, compared with CTL mice. Islets from TAU female mice presented a higher ratio of phosphorylated Akt and ERK (extracellular signal-regulated kinase) related to Akt and ERK protein content, respectively, in comparison with CTL islets. Additional experiments using isolated islets from Swiss mice showed that 3 mM TAU prevented the reduction in insulin secretion induced by 12 h incubation with IL1-β or IL1-β + IFN-γ. In conclusion, TAU supplementation improved NOD islet function without altering endocrine pancreatic morphometry, an effect that may be associated with a protective TAU effect upon cytokine-induced islet dysfunction, together with an improved protein expression of Akt and ERK.

Dietary polyherbal supplementation decreases CD3+ cell infiltration into pancreatic islets and prevents hyperglycemia in nonobese diabetic mice

Type 1 diabetes mellitus results from autoimmune-mediated destruction of pancreatic islet β-cells, a process associated with inflammatory signals. We hypothesized that dietary supplementation with botanicals known to contain anti-inflammatory properties would prevent losses in functional β-cell mass in nonobese diabetic (NOD) mice, a rodent model of autoimmune-mediated islet inflammation that spontaneously develops diabetes. Female NOD mice, a model of spontaneous autoimmune diabetes, were fed a diet supplemented with herbal extracts (1.916 g total botanical extracts per 1 kg of diet) over a 12-week period. The mice consumed isocaloric matched diets without (controls) and with polyherbal supplementation (PHS) ad libitum starting at a prediabetic stage (age 6 weeks) for 12 weeks. Control mice developed hyperglycemia (>180 mg/dL) within 16 weeks (n = 9). By contrast, mice receiving the PHS diet did not develop hyperglycemia by 18 weeks (n = 8). Insulin-positive cell mass within pancreatic islets was 31.9% greater in PHS mice relative to controls. We also detected a 26% decrease in CD3+ lymphocytic infiltration in PHS mice relative to mice consuming a control diet. In vitro assays revealed reduced β-cell expression of the chemokines CCL2 and CXCL10 after overnight PHS addition to the culture media. We conclude that dietary PHS delays initiation of autoimmune-mediated β-cell destruction and subsequent onset of diabetes mellitus by diminishing islet inflammatory responses.

Toll-like receptor 3 is critical for coxsackievirus B4-induced type 1 diabetes in female NOD mice.

Group B coxsackieviruses (CVBs) are involved in triggering some cases of type 1 diabetes mellitus (T1DM). However, the molecular mechanism(s) responsible for this remain elusive. Toll-like receptor 3 (TLR3), a receptor that recognizes viral double-stranded RNA, is hypothesized to play a role in virus-induced T1DM, although this hypothesis is yet to be substantiated. The objective of this study was to directly investigate the role of TLR3 in CVB-triggered T1DM in nonobese diabetic (NOD) mice, a mouse model of human T1DM that is widely used to study both spontaneous autoimmune and viral-induced T1DM. As such, we infected female wild-type (TLR3(+/+)) and TLR3 knockout (TLR3(-/-)) NOD mice with CVB4 and compared the incidence of diabetes in CVB4-infected mice with that of uninfected counterparts. We also evaluated the islets of uninfected and CVB4-infected wild-type and TLR3 knockout NOD mice by immunohistochemistry and insulitis scoring. TLR3 knockout mice were markedly protected from CVB4-induced diabetes compared with CVB4-infected wild-type mice. CVB4-induced T-lymphocyte-mediated insulitis was also significantly less severe in TLR3 knockout mice compared with wild-type mice. No differences in insulitis were observed between uninfected animals, either wild-type or TLR3 knockout mice. These data demonstrate for the first time that TLR3 is 1) critical for CVB4-induced T1DM, and 2) modulates CVB4-induced insulitis in genetically prone NOD mice.

The H1-receptor antagonist cetirizine ameliorates high-fat diet-induced glucose intolerance in male C57BL/6 mice, but not diabetes outcome in female non-obese diabetic (NOD) mice

Background. It has been proposed that the histamine 1-receptor (H1-receptor) not only promotes allergic reactions, but also modulates innate immunity and autoimmune reactions. In line with this, we have recently reported that the H1-receptor antagonist cetirizine partially counteracts cytokine-induced beta-cell signaling and destruction. Therefore, the aim of this study was to determine whether cetirizine affects diabetes in NOD mice, a model for human type 1 diabetes, and glucose intolerance in high-fat diet C57BL/6 mice, a model for human glucose intolerance.Methods. Female NOD mice were treated with cetirizine in the drinking water (25 mg/kg body weight) from 9 until 30 weeks of age during which precipitation of diabetes was followed. Male C57BL/6 mice were given a high-fat diet from 5 weeks of age. When the mice were 12 weeks of age cetirizine was given for 2 weeks in the drinking water. The effects of cetirizine were analyzed by blood glucose determinations, glucose tolerance tests, and insulin sensitivity tests.Results. Cetirizine did not affect diabetes development in NOD mice. On the other hand, cetirizine treatment for 1 week protected against high-fat diet-induced hyperglycemia. The glucose tolerance after 2 weeks of cetirizine treatment was improved in high-fat diet mice. We observed no effect of cetirizine on the insulin sensitivity of high-fat diet mice.Conclusion. Our results suggest a protective effect of cetirizine against high-fat diet-induced beta-cell dysfunction, but not against autoimmune beta-cell destruction.