Dendritic Cells Transduced to Express Interleukin 4 Reduce Diabetes Onset in Both Normoglycemic and Prediabetic Nonobese Diabetic Mice

Background

We and others have previously demonstrated that treatment with bone marrow derived DC genetically modified to express IL-4 reduce disease pathology in mouse models of collagen-induced arthritis and delayed-type hypersensitivity. Moreover, treatment of normoglycemic NOD mice with bone marrow derived DC, genetically modified to express interleukin 4 (IL-4), reduces the onset of hyperglycemia in a significant number of animals. However, the mechanism(s) through which DC expressing IL-4 function to prevent autoimmune diabetes and whether this treatment can reverse disease in pre-diabetic NOD mice are unknown.

Methodology/Principal Findings

DC were generated from the bone marrow of NOD mice and transduced with adenoviral vectors encoding soluble murine IL-4 (DC/sIL-4), a membrane-bound IL-4 construct, or empty vector control. Female NOD mice were segregated into normoglycemic (<150mg/dL) and prediabetic groups (between 150 and 250 mg/dL) on the basis of blood glucose measurements, and randomized for adoptive transfer of 10⁶ DC via a single i.v. injection. A single injection of DC/sIL-4, when administered to normoglycemic 12-week old NOD mice, significantly reduced the number of mice that developed diabetes. Furthermore, DC/sIL-4, but not control DC, decreased the number of mice progressing to diabetes when given to prediabetic NOD mice 12–16 weeks of age. DC/sIL-4 treatment also significantly reduced islet mononuclear infiltration and increased the expression of FoxP3 in the pancreatic lymph nodes of a subset of treated animals. Furthermore, DC/sIL-4 treatment altered the antigen-specific Th2:Th1 cytokine profiles as determined by ELISPOT of splenocytes in treated animals.

Conclusions

Adoptive transfer of DC transduced to express IL-4 into both normoglycemic and prediabetic NOD mice is an effective treatment for T1D.     

IL-2 Immunotherapy Reveals Potential for Innate Beta Cell Regeneration in the Non-Obese Diabetic Mouse Model of Autoimmune Diabetes

Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy.     

Differential impact of T cell repertoire diversity in diabetes-prone or -resistant IL-10 transgenic mice

Expression of IL-10 transgene (tg) in pancreatic beta cells failed to induce autoimmune insulitis and diabetes in (BALB/c x NOD)F1 mice. However, IL-10-expressing tg littermates from backcrosses (N2 and N3) with NOD mice became diabetic at 5 to 10 weeks of age in an MHC-dependent manner. In this study, we tested the possibility that enhancement in frequency of islet antigen (Ag)-specific T cells overrides the protective effects of a diabetes-resistant genetic background and promotes diabetes in IL-10 tg (BALB/c x NOD)F1 mice. For this test, we introduced the IL-10 transgene into tg BDC2.5 mice expressing the islet Ag-specific Vbeta4 T cell repertoire by breeding Ins-IL-10+/BALB/c mice with BDC2.5 mice. The progeny (Ins-IL-10+/BALB/c x BDC2.5+)F1 mice doubly tg for IL-10 and Vbeta4 (BDC2.5) T cell repertoire, developed diabetes at 10 to 18 weeks of age with a much more aggressive T cell infiltrate in the pancreatic islets than in single tg mice. Surprisingly, these diabetic mice were free from acute pancreatitis but had apoptotic beta cells in the islet infiltrate. Conversely, mice tg for Vbeta4 (BDC2.5) T cell repertoire but not IL-10 had no diabetes and no apoptotic beta cells in the islet infiltrate. Therefore, an increase in the frequency of islet-specific T cells apparently overcomes the protection from diabetes by a resistant genetic background. Interestingly, N2 backcross mice doubly tg for Vbeta4 (BDC2.5) T cell repertoire and IL-10, compared to N2 backcross mice tg for IL-10 only, eventually became diabetic but with a delayed onset and reduced incidence of disease. These findings demonstrate that, along with IL-10, an increase in frequency of islet antigen-specific T cells (a) overrides the protective effect of genetic resistance to autoimmune diabetes in F1 mice and (b) delays the onset of an otherwise accelerated diabetes in (Ins-IL-10+/NOD)N2 backcross mice.     

Intravenous transfusion of BCR-activated B cells protects NOD mice from type 1 diabetes in an IL-10-dependent manner

Although B cells play a pathogenic role in the initiation of type 1 diabetes (T1D) in NOD mice, it is not known whether activated B cells can maintain tolerance and transfer protection from T1D. In this study, we demonstrate that i.v. transfusion of BCR-stimulated NOD spleen B cells into NOD mice starting at 5-6 wk of age both delays onset and reduces the incidence of T1D, whereas treatment initiated at 9 wk of age only delays onset of T1D. This BCR-activated B cell-induced protection from T1D requires IL-10 production by B cells, as transfusion of activated B cells from NOD.IL-10(-/-) mice does not confer protection from T1D. Consistent with this result, severe insulitis was observed in the islets of NOD recipients of transfused NOD.IL-10(-/-) BCR-stimulated B cells but not in the islets of NOD recipients of transfused BCR-stimulated NOD B cells. The therapeutic effect of transfused activated NOD B cells correlates closely with the observed decreased islet inflammation, reduced IFN-gamma production and increased production of IL-4 and IL-10 by splenocytes and CD4(+) T cells from NOD recipients of BCR-stimulated NOD B cells relative to splenocytes and CD4(+) T cells from PBS-treated control NOD mice. Our data demonstrate that transfused BCR-stimulated B cells can maintain long-term tolerance and protect NOD mice from T1D by an IL-10-dependent mechanism, and raise the possibility that i.v. transfusion of autologous IL-10-producing BCR-activated B cells may be used therapeutically to protect human subjects at risk for T1D.     

T cell response mediated by myeloid cell-derived IL-12 is responsible for Porphyromonas gingivalis-induced periodontitis in IL-10-deficient mice

Periodontal disease is a chronic inflammatory disease in the oral cavity, which culminates in alveolar bone loss. Porphyromonas gingivalis is a consensus periodontal pathogen that has been implicated in adult forms of periodontitis. We previously demonstrated that IL-10-deficient mice exhibit a hyperinflammatory phenotype and are highly susceptible to P. gingivalis-induced periodontitis, indicating an important anti-inflammatory effect of IL-10 in suppressing bone loss. In this study, we analyzed the pathway(s) by which IL-10 deficiency leads to severe P. gingivalis-induced periodontitis. Because Stat3 is essential in IL-10 signaling, immune cell-specific Stat3-deficient mice were subjected to P. gingivalis infection to identify the key IL-10-responsive cells in preventing periodontitis. Myeloid cell-specific Stat3-deficient mice exhibited increased periodontal bone loss (p < 0.001), whereas T cell- and B cell-specific Stat3 mice were resistant, suggesting that macrophages (MP) and/or polymorphonuclear leukocytes are the key target cells normally suppressed by IL-10. Myeloid cell-specific Stat3-deficient mice exhibited elevated gingival CD40L gene expression in vivo compared with wild-type controls (p < 0.01), and Stat3-deficient MPs exhibited vigorous P. gingivalis-stimulated IL-12 production in vitro and induced elevated Ag-specific T cell proliferation compared with wild-type MPs (p < 0.01). Of importance, both IL-12p40/IL-10 and T cell/IL-10 double-deficient mice were resistant to P. gingivalis-induced periodontitis, demonstrating roles for both IL-12p40 and T cells in pathogenesis in a hyperinflammatory model of disease. These data demonstrate that P. gingivalis-induced periodontitis in IL-10-deficient mice is dependent upon IL-12p40-mediated proinflammatory T cell responses.     

Both spontaneous Ins2+/− and streptozotocin‐induced type I diabetes cause bone loss in young mice

The adolescent skeleton undergoes accelerated growth determining overall bone density, length, and quality. Diseases such as type 1 diabetes (T1D), most often diagnosed in adolescents, can alter bone processes and promote bone loss. Studies examining type 1 diabetic (T1D) bone pathologies typically utilize adult mice and rely on pharmacologic models such as streptozotocin (STZ)‐induced diabetic rodents. To test the effect of T1D on adolescent bone growth/density we used a novel juvenile genetic model (Ins2^+/? mice) that spontaneously develop T1D at approximately 5 weeks of age and compared our findings with STZ‐induced T1D mice. Compared to controls, both Ins2^+/? and STZ‐induced T1D mice displayed blood glucose levels greater than 300 mg/dl and reduced body, fat and muscle mass as well as femur trabecular bone density. STZ mice exhibited greater bone loss compared to Ins2^+/? mice despite having lower blood glucose levels. Cortical bone was affected in STZ but not Ins2^+/? mice. Osteocalcin serum protein and bone RNA levels decreased in both models. Consistent with studies in adult mice, STZ adolescent mice displayed increased marrow adiposity, however this was not observed in the Ins2^+/? mice. Reduced femur length, decreased growth plate thickness and decreased collagen II expression in both model simplies impaired cartilage formation. In summary, both pharmacologic and spontaneous adolescent T1D mice demonstrated a bone synthesis and growth defect. STZ appears to cause a more severe phenotype. Thus, the Ins2^+/? mouse could serve as a useful model to study adolescent T1D bone loss with fewer complications. J. Cell. Physiol. 228: 689–695, 2013. © 2012 Wiley Periodicals, Inc.     

Molecular mechanisms for gender differences in susceptibility to T cell-mediated autoimmune diabetes in nonobese diabetic mice

Nonobese diabetic (NOD) mice spontaneously develop diabetes with a strong female prevalence; however, the mechanisms for this gender difference in susceptibility to T cell-mediated autoimmune diabetes are poorly understood. This investigation was initiated to find mechanisms by which sex hormones might affect the development of autoimmune diabetes in NOD mice. We examined the expression of IFN-gamma, a characteristic Th1 cytokine, and IL-4, a characteristic Th2 cytokine, in islet infiltrates of female and male NOD mice at various ages. We found that the most significant difference in cytokine production between sexes was during the early stages of insulitis at 4 wk of age. IFN-gamma was significantly higher in young females, whereas IL-4 was higher in young males. CD4(+) T cells isolated from lymph nodes of female mice and activated with anti-CD3 and anti-CD28 Abs produced more IFN-gamma, but less IL-4, as compared with males. Treatment of CD4(+) T cells with estrogen significantly increased, whereas testosterone treatment decreased the IL-12-induced production of IFN-gamma. We then examined whether the change in IL-12-induced IFN-gamma production by treatment with sex hormones was due to the regulation of STAT4 activation. We found that estrogen treatment increased the phosphorylation of STAT4 in IL-12-stimulated T cells. We conclude that the increased susceptibility of female NOD mice to the development of autoimmune diabetes could be due to the enhancement of the Th1 immune response through the increase of IL-12-induced STAT4 activation by estrogen.     

Diabetes acceleration or prevention by a coxsackievirus B4 infection: critical requirements for both interleukin-4 and gamma interferon

Type 1 diabetes acceleration in nonobese diabetic (NOD) mice through coxsackievirus B4 (CVB4) infection requires a preexisting critical mass of autoreactive T cells in pancreatic islets, and in the absence of this insulitic threshold, CVB4 infection leads to long-term disease protection. To understand this acceleration and protection process, we challenged 8- and 12-week-old NOD mice containing a disruption in interleukin-4 (IL-4) or gamma interferon (IFN-gamma) genes (NOD IL-4-/- and NOD IFN-gamma-/-, respectively) with a diabetogenic, pancreatropic Edwards strain of CVB4. The elimination of IL-4 did not alter the rate of insulitis or diabetes development in NOD mice, while the elimination of IFN-gamma delayed these events several weeks. CVB4 infection in 8-week-old mice only significantly accelerated the onset of diabetes in a subset of standard, but not IL-4- or IFN-gamma-deficient, NOD mice. Long-term diabetes protection was established in standard NOD mice as well as in the NOD IFN-gamma-/- mice that did not rapidly develop disease following CVB4 infection at 8 weeks of age. When mice were infected at 12 weeks of age, the onset of diabetes was accelerated in NOD IL-4-/- mice, while neither acceleration nor long-term protection was elicited in NOD IFN-gamma-/- mice. No differences were observed in the kinetics of CVB4 clearance in pancreases from NOD, NOD IL-4-/-, and NOD IFN-gamma-/- mice. Collectively, these results suggest that at the insulitis threshold at which CVB4 infection can first accelerate the onset of diabetes in NOD mice, IL-4 as well as IFN-gamma contributes to this pathogenic process. The protective mechanism against diabetes elicited in NOD mice infected with CVB4 prior to the development of a critical threshold level of insulitis requires neither IL-4 nor IFN-gamma.     

Prominent bone loss mediated by RANKL and IL-17 produced by CD4+ T cells in TallyHo/JngJ mice

Increasing evidence that decreased bone density and increased rates of bone fracture are associated with abnormal metabolic states such as hyperglycemia and insulin resistance indicates that diabetes is a risk factor for osteoporosis. In this study, we observed that TallyHo/JngJ (TH) mice, a polygenic model of type II diabetes, spontaneously developed bone deformities with osteoporotic features. Female and male TH mice significantly gained more body weight than control C57BL/6 mice upon aging. Interestingly, bone density was considerably decreased in male TH mice, which displayed hyperglycemia. The osteoblast-specific bone forming markers osteocalcin and osteoprotegerin were decreased in TH mice, whereas osteoclast-driven bone resorption markers such as IL-6 and RANKL were significantly elevated in the bone marrow and blood of TH mice. In addition, RANKL expression was prominently increased in CD4+ T cells of TH mice upon T cell receptor stimulation, which was in accordance with enhanced IL-17 production. IL-17 production in CD4+ T cells was directly promoted by treatment with leptin while IFN-γ production was not. Moreover, blockade of IFN-γ further increased RANKL expression and IL-17 production in TH-CD4+ T cells. In addition, the osteoporotic phenotype of TH mice was improved by treatment with alendronate. These results strongly indicate that increased leptin in TH mice may act in conjunction with IL-6 to preferentially stimulate IL-17 production in CD4+ T cells and induce RANKL-mediated osteoclastogenesis. Accordingly, we propose that TH mice could constitute a beneficial model for osteoporosis.     

Are the immune responses different in middle-aged and young mice following bone fracture, tissue trauma and hemorrhage?

Although immune responses following soft-tissue trauma-hemorrhage are markedly different in young (6-8 weeks) and aged (18-20 months) mice, it remains unknown if there are any differences in immune responses in middle-aged and young mice following bone fracture, soft-tissue trauma-hemorrhage (Fx-TH). To study this, young (6-8 weeks) and middle-aged (approximately 12 months) C3H/HeN male mice were subjected to sham operation or Fx-TH followed by resuscitation with Ringer's lactate. The mice were sacrificed 2 h thereafter and splenocytes, bone marrow cells (BM) and Kupffer cells (KC) were harvested, purified and stimulated with ConA (for splenocytes) or LPS (for BM and KC) in vitro. Splenocyte release of Th1 (IL-2 and IFN-gamma) cytokines was decreased and Th2 (IL-4 and IL-10) cytokine release was increased following Fx-TH in both young and middle-aged mice. However, the decrease in IL-2 and increase in IL-10 were significantly more in middle-aged mice compared to young mice (p < 0.05). Furthermore, splenocyte proliferation was decreased more in middle-aged mice compared to young mice following Fx-TH (p < 0.05). Additionally, TNF-alpha production was more in BM from middle-aged compared to BM from young mice after Fx-TH (p < 0.05). The production of IL-6 and IL-10 was also significantly higher in KC from middle-aged mice compared to young ones following Fx-TH. These results suggest that at middle age, the immune responses to Fx-TH are significantly different from those observed in young mice in different compartments of the body. Although the mechanism of the difference in various compartments in middle-aged vs. young mice following Fx-TH remains unknown, the decreased IL-2 production along with other altered T cell and macrophage functions may contribute to an increased susceptibility to sepsis in middle-aged vs. young individuals.     

L-4F treatment reduces adiposity, increases adiponectin levels, and improves insulin sensitivity in obese mice

We hypothesized that the apolipoprotein mimetic peptide L-4F, which induces arterial anti-oxidative enzymes and is vasoprotective in a rat model of diabetes, would ameliorate insulin resistance and diabetes in obese mice. L-4F (2 mg/kg/d) administered to ob/ob mice for 6 weeks limited weight gain without altering food intake, decreased visceral (P < 0.02) and subcutaneous (P < 0.045) fat content, decreased plasma IL-1beta and IL-6 levels (P < 0.05) and increased insulin sensitivity, resulting in decreased glucose (P < 0.001) and insulin (P < 0.036) levels. In addition, L-4F treatment increased aortic and bone marrow heme oxygenase (HO) activity and decreased aortic and bone marrow superoxide production (P < 0.001). L-4F treatment increased serum adiponectin levels (P < 0.037) and decreased adipogenesis in mouse bone marrow (P < 0.039) and in cultures of human bone marrow-derived mesenchymal stem cells (P < 0.022). This was manifested by reduced adiposity, improved insulin sensitivity, improved glucose tolerance, increased plasma adiponectin levels, and reduced IL-1beta and IL-6 levels in obese mice. This study highlights the existence of a temporal relationship between HO-1 and adiponectin that is positively affected by L-4F in the ob/ob mouse model of diabetes, resulting in the amelioration of the deleterious effects of diabetes.     

Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (10(4), 10(6), 10(8), and 10(9) infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (10(9) IU), or saline. Transduction with rAAV-IL-10 at 10(9) IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 10(8) IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.     

Prevention or Early Cure of Type 1 Diabetes by Intranasal Administration of Gliadin in NOD Mice

Induction of long-term tolerance to β-cell autoantigens has been investigated both in animal models and in human type 1 diabetes (T1D) in order to prevent the disease. As regards external compounds, the dietary plant protein fraction has been associated with high penetrance of the disease, whereas gluten-free diets prevent T1D in animal models. Herewith we investigated whether intranasal (i.n.) administration of gliadin or gluten may arrest the diabetogenic process. I.n. administration of gliadin to 4-week-old NOD mice significantly reduced the diabetes incidence. Similarly, the insulitis was lowered. Intranasal gliadin also rescued a fraction of prediabetic 13-week-old NOD mice from progressing to clinical onset of diabetes compared to OVA-treated controls. Vaccination with i.n. gliadin led to an induction of CD4⁺Foxp3⁺ T cells and even more significant induction of γδ T cells in mucosal, but not in non-mucosal lymphoid compartments. This prevention strategy was characterized by an increased proportion of IL-10 and a decreased proportion of IL-2, IL-4 and IFN-γ-positive CD4⁺Foxp3⁺ T cells, and IFN-γ-positive γδ T cells, preferentially in mucosal lymphoid organs. In conclusion, i.n. vaccination with gliadin, an environmental antigen with possible etiological influence in T1D, may represent a novel, safer strategy for prevention or even early cure of T1D.     

Preventive effect of Ninjin-to (Ren-Shen-Tang), a Kampo (Japanese traditional) formulation, on spontaneous autoimmune diabetes in non-obese diabetic (NOD) mice

We previously found that ingestion of an extract of Ninjin-to (NJT; Ren-Shen-Tang) suppressed the development of autoimmune diabetes in C57BL/KsJ mice induced by multiple low doses of streptozotocin. To verify this effects on spontaneous autoimmune diabetes, the effects of NJT on NOD mice were investigated in the present study. NJT, provided in drinking water (0.25%, 450 mg/kg/day) from 6 weeks of age, significantly prevented the incidence of spontaneous diabetes in female NOD mice at 30 weeks of age (2/10) compared with that of the controls (7/10), with no effects on body growth or food intake. Even in non-diabetic mice, the blood glucose levels of the NOD controls gradually increased with age, while such increase in NJT-treated mice was significantly suppressed by preventing any deficiency of glucose tolerance. NJT also significantly suppressed the progression of insulitis, which causes insulin deficiency and diabetes. It is well known that NOD mice develop insulitis and diabetes because of their Th1-dominant autoimmune response. IFN-gamma production from splenic T lymphocytes stimulated with anti-CD3 monoclonal antibodies was increased, whereas IL-4 production was decreased in NOD controls compared to age- and sex-matched normal ICR mice. NJT-treatment reduced these deviations of cytokine production in NOD mice. These data all suggest that NJT can prevent spontaneous insulitis and diabetes by the modification of deviated cytokine production in NOD mice.     

Regulation of type 1 diabetes by a self-MHC class II peptide: role of transforming growth factor beta (TGF-beta).

The present study was undertaken to analyze the regulatory T cells generated in response to class I derived self-I-A beta(g7) (54-76) peptide. It was observed T cells from young unprimed type 1 diabetes (T1D) prone NOD mice did not respond to self-I-A beta(g7) (54-76) peptide although T cells from primed young NOD mice showed a strong response. T cells from young unprimed BALB/c mice responded to self-I-A beta(d) (62-78) peptide. However, a breakdown of tolerance to these peptides was observed with age in both the strains. Culture supernatant from I-A beta(g7) (54-76) peptide-primed cells secreted large amounts of TGF-beta and inhibited T cell responses in allogeneic-MLR. Further, I-A beta(g7) (54-76) peptide specific T cell lines from young (I-A.Y) and diabetic (I-A.D) NOD mice were established. I-A.Y secreted IL-4, TGF-beta and IL-10 while I-A.D T cell line secreted IL-10 and IFN-gamma. We found that I-A.D T cell line induced diabetes when transferred in NOD/SCID mice but I-A.Y T cell line did not induce disease. These results show that immunization of NOD mice with I-A beta(g7) (54-76) peptide at a younger age induces a regulatory T cell response suggesting that correcting the defects in immunoregulatory mechanisms using self-MHC peptides may be one of the approaches to prevent autoimmune diseases like T1D.     

Analysis of candidate susceptibility genes in canine diabetes

Canine diabetes is a complex genetic disease of unknown aetiology. It affects 0.005-1.5% of the canine population and shows a clear breed predisposition with the Samoyed being at high risk and the Boxer being at low risk of developing the disease. Canine diabetes is considered to be a disease homologue for human type 1 diabetes (T1D). It results in insulin deficiency as a consequence of autoimmune destruction of islet beta-cells in the pancreas and is believed to be mediated by Th1 cytokines (IFNgamma, TNFalpha, and IL-2). A number of genes have been associated with type 1 diabetes in humans, including the human leukocyte antigen region, the insulin variable number tandem repeat, PTPN22, CTLA4, IL-4, and IL-13. As yet, these genes have not been evaluated in canine diabetes. In this study, 483 cases of canine diabetes and 869 controls of known breed were analyzed for association with IFNgamma, IGF2, IL-10, IL-12beta, IL-6, insulin, PTPN22, RANTES, IL-4, IL-1alpha and TNFalpha. Minor allele frequencies were determined for these genes in each breed. These data were used for comparative analyses in a case-control study, and clear associations with diabetes were identified in some breeds with certain alleles of candidate genes. Some associations were with increased susceptibility to the disease (IFNgamma, IL-10, IL-12beta, IL-6, insulin, PTPN22, IL-4, and TNFalpha), whereas others were protective (IL-4, PTPN22, IL-6, insulin, IGF2, TNFalpha). This study demonstrates that a number of the candidate genes previously associated with human T1D also appear to be associated with canine diabetes and identifies an IL-10 haplotype which is associated with diabetes in the Cavalier King Charles Spaniel. This suggests that canine diabetes is an excellent comparative and spontaneously occurring disease model of human T1D.     

Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting allograft rejection and autoimmune diseases

Dendritic cells (DCs) not only induce but also modulate T cell activation. 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces DCs with a tolerogenic phenotype, characterized by decreased expression of CD40, CD80, and CD86 costimulatory molecules, low IL-12 and enhanced IL-10 secretion. We have found that a short treatment with 1,25(OH)(2)D(3) induces tolerance to fully mismatched mouse islet allografts that is stable to challenge with donor-type spleen cells and allows acceptance of donor-type vascularized heart grafts. This effect is enhanced by co-administration of mycophenolate mofetil (MMF), a selective inhibitor of T and B cell proliferation that has also effects similar to 1,25(OH)(2)D(3) on DCs. Graft acceptance is associated with an increased percentage of CD4(+)CD25(+) regulatory cells in the spleen and in the draining lymph node that can protect 100% of syngeneic recipients from islet allograft rejection. CD4(+)CD25(+) cells, able to inhibit the T cell response to a pancreatic autoantigen and to significantly delay disease transfer by pathogenic CD4(+)CD25(-) cells, are also induced by treatment of adult nonobese diabetic (NOD) mice with 1,25-dihydroxy-16,23Z-diene-26,27-hexafluoro-19-nor vitamin D(3) (BXL-698). This treatment arrests progression of insulitis and Th1 cell infiltration, and inhibits diabetes development at non-hypercalcemic doses. The enhancement of CD4(+)CD25(+) regulatory T cells, able to mediate transplantation tolerance and to arrest type 1 diabetes development by a short oral treatment with VDR ligands, suggests possible clinical applications of this approach.     

Accelerated Type 1 Diabetes Induction in Mice by Adoptive Transfer of Diabetogenic CD4+ T Cells

The nonobese diabetic (NOD) mouse spontaneously develops autoimmune diabetes after 12 weeks of age and is the most extensively studied animal model of human Type 1 diabetes (T1D). Cell transfer studies in irradiated recipient mice have established that T cells are pivotal in T1D pathogenesis in this model. We describe herein a simple method to rapidly induce T1D by adoptive transfer of purified, primary CD4+ T cells from pre-diabetic NOD mice transgenic for the islet-specific T cell receptor (TCR) BDC2.5 into NOD.SCID recipient mice. The major advantages of this technique are that isolation and adoptive transfer of diabetogenic T cells can be completed within the same day, irradiation of the recipients is not required, and a high incidence of T1D is elicited within 2 weeks after T cell transfer. Thus, studies of pathogenesis and therapeutic interventions in T1D can proceed at a faster rate than with methods that rely on heterogenous T cell populations or clones derived from diabetic NOD mice.     

Possible involvement of corticosterone in bone loss of genetically diabetic db/db mice.

The etiology of bone loss in non-insulin dependent diabetes mellitus is still unknown. We compared serum biochemical parameters and bone parameters of genetically diabetic db/db mice with those of their control non-diabetic +/+ mice. We found that serum corticosterone levels of the db/db mice were significantly elevated after 5 weeks while bone mineral density of femur metaphysis significantly decreased in the db/db mice after 12 weeks of age compared with age matched +/+ mice. To explore the causal relationship between the serum corticosterone levels and the bone loss, metyrapone (100 mg/kg, p.o., twice a day), a glucocorticoid synthesis inhibitor, was administered to these mice for 4 weeks after the age of 8 weeks. The compound significantly decreased serum corticosterone levels in both strains. Metyrapone prevented bone loss by increasing the bone mineral content of the metaphysis in the db/db mice. In addition, the treatment slightly improved the ratio of ash weight to dry weight in the db/db mice. These results suggest that increased serum corticosterone levels are concerned with the etiology of bone loss in non-insulin dependent diabetic db/db mice.