Double deficiency in IL-17 and IFN-γ signalling significantly suppresses the development of diabetes in the NOD mouse

Aims/hypothesis

T helper type (Th) 17 cells have been shown to play important roles in mouse models of several autoimmune diseases that have been classified as Th1 diseases. In the NOD mouse, the relevance of Th1 and Th17 is controversial, because single-cytokine-deficient NOD mice develop diabetes similarly to wild-type NOD mice.

Methods

We studied the impact of IL-17/IFN-γ receptor double deficiency in NOD mice on the development of insulitis/diabetes compared with IL-17 single-deficient mice and wild-type mice by monitoring diabetes-related phenotypes. The lymphocyte phenotypes were determined by flow cytometric analysis.

Results

IL-17 single-deficient NOD mice showed delayed onset of diabetes and reduced severity of insulitis, but the cumulative incidence of longstanding diabetes in the IL-17-deficient mice was similar to that in wild-type mice. The IL-17/IFN-γ receptor double-deficient NOD mice showed an apparent decline in longstanding diabetes onset, but not in insulitis compared with that in the IL-17 single-deficient mice. We also found that double-deficient NOD mice had a severe lymphopenic phenotype and preferential increase in regulatory T cells among CD4⁺ T cells compared with the IL-17 single-deficient mice and wild-type NOD mice. An adoptive transfer study with CD4⁺CD25^? T cells from young non-diabetic IL-17 single-deficient NOD mice, but not those from older mice, showed significantly delayed disease onset in immune-deficient hosts compared with the corresponding wild-type mice.

Conclusions/interpretation

These results indicate that IL-17/Th17 participates in the development of insulitis and that both IL-17 and IFN-γ signalling may synergistically contribute to the development of diabetes in NOD mice.     

Astragalus polysaccharides: an effective treatment for diabetes prevention in NOD mice

BACKGROUND: Type 1 diabetes mellitus (DM) is a chronic autoimmune disease that is related to the disequilibrium state of Th1 and Th2 subgroups of helper T lymphocyte (Th) and their cytokines. Astragalus polysaccharides (APS) are bioactive components extracted from one of the traditional Chinese herbs, used to enhance the function of human immune system. OBJECTIVE: To investigate the effects of APS on preventing type 1 DM and Th1/Th2-subtype cytokines, we compared the results of administration of APS and normal saline (NS) on non-obese diabetic (NOD) mice. SUBJECTS AND METHODS: APS or NS was administered to 4-week-old mice at a dose of 2.0 g/kg per day for 10 weeks. At 40 weeks, blood glucose, serum C-peptide (C-P) and GAD antibody were measured; pancreas was examined histologically; the intra-islet lymphocyte infiltration and T lymphocyte subsets in the spleen were analysed; the gene expression of IL-1 beta, IL-2, IL-6, IL-12, TNF-alpha, INF-gamma, IL-4, IL-5, IL-10, TGF-beta, Bcl-2, SOD, Fas and iNOS were measured by RT-PCR. RESULTS: The results showed that APS-administered NOD mice had a lower incidence rate of type 1 DM, lower serum C-P level, better histologic findings of pancreatic islets, and a lower D4+/CD8+ ratio of T lymphocytes from the spleen and the infiltrated islets. RT-PCR analysis showed gene expression levels are lower in IL-1 beta, IL-2, IL-6, IL-12, TNF-alpha, INF-gamma, Fas, iNOS, and higher in IL-4, IL-5, IL-10, TGF-beta, Bcl-2, SOD in the pancreatic tissue from APS-administered NOD mice as compared to the NS group.CONCLUSIONS: These results demonstrated the effects of Astragalus polysaccharides on the prevention of type 1 DM in NOD mice by correcting the imbalance between the Th1/Th2 cytokines.     

IL-21 signaling is critical for the development of type I diabetes in the NOD mouse

IL-21 is a pleiotropic type I cytokine that shares the common cytokine receptor γ chain and plays important roles for normal Ig production, terminal B cell differentiation to plasma cells, and Th17 differentiation. IL-21 is elevated in several autoimmune diseases, and blocking its action has attenuated disease in MRL/lpr mice and in collagen-induced arthritis. The diabetes-associated Idd3 locus is at the Il2/Il21 locus, and elevated IL-21 was observed in the nonobese diabetic (NOD) mouse and suggested to contribute to diabetes by augmenting T cell homeostatic proliferation. To determine the role of IL-21 in diabetes, Il21r-knockout (KO) mice were backcrossed to NOD mice. These mice were devoid of lymphocytic infiltration into the pancreas, and only 1 of 20 animals had an elevated glucose compared with 60% of NOD mice on a wild-type (WT) background. Although TCR and Treg-related responses were normal, these mice had reduced Th17 cells and significantly higher levels of mRNAs encoding members of the Reg (regenerating) gene family whose transgenic expression protects against diabetes. Our studies establish a critical role for IL-21 in the development of type I diabetes in the NOD mouse, with obvious potential implications for type I diabetes in humans.     

Suppression of cyclophosphamide induced diabetes development and pancreatic Th1 reactivity in NOD mice treated with the interleukin (IL)-12 antagonist IL-12(p40)2

Summary The macrophage product interleukin (IL)-12 is known to drive Th1 reactions in physiological and pathological immune responses. Here we report that treatment with the homodimeric IL-12p40 subunit, an antagonist of the bioactive IL-12p35/p40 heterodimer, suppresses diabetes development in cyclophosphamide-injected NOD mice. Female mice of 70 days old received cyclophosphamide (250 mg/kg) to accelerate and synchronize diabetes development, and daily injections of 1 μg IL-12(p40)₂. While there was no delay of the first diabetes cases, the incidence of overt diabetes was significantly decreased in treated mice (46 vs 23 %, p < 0.05). Analysis of mRNA expression in the pancreas showed that administration of the IL-12 antagonist had dampened interferon-gamma gene expression, decreased the ratio of interferon-gamma/IL-10 mRNA levels and in parallel suppressed the expression of the inducible nitric oxide synthase. At the same time intra-islet infiltration was significantly decreased (p < 0.001). Interestingly, the administration of IL-12(p40)₂ also affected IL-12 gene expression, by downregulation of p35 mRNA. We conclude that IL-12 p40 homodimer suppresses diabetes development in the NOD mouse by dampening islet inflammation via selective downregulation of Th1 type responses. The naturally occurring IL-12 antagonist IL-12(p40)₂ represents a new and specific Th1 directed approach to prevent autoimmune diabetes. [Diabetologia (1997) 40: 641–646] Received: 7 January 1997 and in revised form: 10 March 1997     

The anti-inflammatory compound lisofylline prevents Type I diabetes in non-obese diabetic mice

AIMS/HYPOTHESIS: Pro-inflammatory cytokines are increased during the active stages of Type I (insulin-dependent) diabetes mellitus. The aim of this study was to investigate the applicability of using a new anti-inflammatory compound, Lisofylline, to prevent diabetes in non-obese diabetic (NOD) mice. Lisofylline has previously been shown to block Th1 cell differentiation and to reduce IL-1 beta-induced dysfunction in rat islets. METHODS: Lisofylline was added to isolated NOD islets in vitro, with or without IL-1 beta. Insulin secretion and DNA damage of the islets was assessed. Lisofylline was administered to female non-obese diabetic mice starting at 4, 7 and 17 weeks of age for 3 weeks. Cytokines and blood glucose concentrations were monitored. Histology and immunohistochemistry were carried out in pancreatic sections. Splenocytes isolated from donor mice were intravenously injected into immunodeficient NOD (NOD.scid) mice. RESULTS: In vitro, Lisofylline preserved beta-cell insulin secretion and inhibited DNA damage of islets in the presence of IL-1 beta. In vivo, Lisofylline suppressed IFN-gamma production, reduced the onset of insulitis and diabetes, and inhibited diabetes after transfer of splenocytes from Lisofylline-treated donors to NOD.scid recipients. However, cotransfer of splenocytes from both Lisofylline-treated and diabetic NOD donors did not suppress diabetes in recipient mice. CONCLUSION/INTERPRETATION: Lisofylline prevents the onset of autoimmune diabetes in NOD mice by a mechanism that does not seem to enhance the function of regulatory T cells, but could be associated with suppression of proinflammatory cytokines and reduction of cellular infiltration in islets. This study suggests that Lisofylline could have therapeutic benefits in preventing the onset of Type I diabetes.     

Germ line deletion of the CD1 locus exacerbates diabetes in the NOD mouse

Quantitative and qualitative defects in CD1-restricted natural killer T cells have been reported in several autoimmune-prone strains of mice, including the nonobese diabetic (NOD) mouse. These defects are believed to be associated with the emergence of spontaneous autoimmunity. Here we demonstrate that both CD1d-null NOD and CD1d-null NOD/BDC2.5 T cell receptor transgenic mice have an accelerated onset and increased incidence of diabetes when compared with CD1d(+/-) and CD1d(+/+) littermates. The acceleration of disease did not seem to result from changes in the T helper (Th)1/Th2 balance because lymphocytes purified from lymphoid organs and pancreatic islets of wild-type and CD1d-null mice secreted equivalent amounts of IFN-gamma and IL-4 after stimulation. In contrast, the pancreata of CD1d-null mice harbored significantly higher numbers of activated memory T cells expressing the chemokine receptor CCR4. Notably, the presence of these T cells was associated with immunohistochemical evidence of increased destructive insulitis. Thus, CD1d-restricted T cells are critically important for regulation of the spontaneous disease process in NOD mice.     

Imbalance in Th cell polarization and its relevance in type 1 diabetes mellitus

Functional polarization of T helper (Th) subsets of lymphocytes has been implicated in promoting or conferring risk to Type 1 diabetes mellitus (T1DM) development in human and diabetic animal models. It is assumed that an immoderate preponderance of type 1 immunity establishes the prerequisite for this development. Over the past years, various immune-intervention strategies have been tested to protect diabetic animals from developing overt diabetes. These protocols implicate a protective mechanism that is attributed to a change in the set of autoreactive Th cells from their Th1 to the Th2 phenotype. The studies were aimed at improving the effectiveness of Th2 cells to secrete the principal cytokines, IL-4 and IL-10, in order to mediate protection from diabetes in NOD mice. In contrast, some immune-modulation protocols utilizing non-specific reagents report that diabetes protection is apparently attributed to preferential survival of both Th1 and Th2 cells, rather than via a shift from their Th1 to Th2 phenotypes. Even though we know that excessive immune responses against self antigens are also controlled and terminated by regulatory T cells, this article focuses on the polarization of Th effector cells and discusses the controversial findings regarding the Th1/Th2 hypothesis to draw a conclusion on its relevance in T1DM from the existing knowledge.     

An update on cytokines in the pathogenesis of insulin-dependent diabetes mellitus

Correlation studies between cytokines expressed in islets and autoimmune diabetes development in NOD mice and BB rats have demonstrated that β-cell destructive insulitis is associated with increased expression of proinflammatory cytokines (IL-1, TNFα, and IFNα) and type 1 cytokines (IFNγ, TNFβ, IL-2 and IL-12), whereas non-destructive (benign) insulitis is associated with increased expression of type 2 cytokines (IL-4 and IL-10) and the type 3 cytokine (TGFβ). Cytokines (IL-1, TNFα, TNFβ and IFNγ) may be directly cytotoxic to β-cells by inducing nitric oxide and oxygen free radicals in the β-cells. In addition, cytokines may sensitize β-cells to T-cell-mediated cytotoxicity in vivo by upregulating MHC class I expression on the β-cells (an action of IFNγ), and inducing Fas (CD95) expression on β-cells (actions of IL-1, and possibly TNFα and IFNγ). Transgenic expression of cytokines in β-cells of non-diabetes-prone mice and NOD mice has suggested pathogenic roles for IFNα, IFNγ, IL-2 and IL-10 in insulin-dependent diabetes mellitus (IDDM) development, and protective roles for IL-4, IL-6 and TNFα. Systemic administrations of a wide variety of cytokines can prevent IDDM development in NOD mice and/or BB rats; however, a given cytokine may retard or accelerate IDDM development, depending on the dose and frequency of administration, and the age and the diabetes-prone animal model studied (NOD mouse or BB rat). Islet-reactive CD4⁺ T-cell lines and clones that adoptively transfer IDDM into young NOD mice have a Th1 phenotype (IFNγ-producing), but other islet-specific Th1 clones that produce TGFβ can adoptively transfer protection against IDDM in NOD mice. NOD mice with targeted deletions of IL-12 and IFNγ genes still develop IDDM, albeit delayed and slightly less often. In contrast, post-natal deletions of IL-12 and IFNγ, also IL-1, TNFα, IL-2, and IL-6—by systemic administrations of neutralizing antibodies, soluble receptors and receptor antagonists, and receptor-targeted cytotoxic drugs—significantly decrease IDDM incidence in NOD mice and/or BB rats. These cytokine deletion studies have provided the best evidence for pathologic roles for proinflammatory cytokines (IL-1, TNFα, and IL-6) and type 1 cytokines (IFNγ, IL-2 and IL-12) in IDDM development. © 1998 John Wiley & Sons, Ltd.     

The novel inosine analogue, INO-2002, protects against diabetes development in multiple low-dose streptozotocin and non-obese diabetic mouse models of type I diabetes

Endogenous purines including inosine have been shown to exert immunomodulatory and anti-inflammatory effects in a variety of disease models. The dosage of inosine required for protection is very high because of the rapid metabolism of inosine in vivo. The aim of this study was to determine whether a metabolic-resistant purine analogue, INO-2002, exerts anti-inflammatory effects in two animal models of type I diabetes. Type I diabetes was induced chemically with streptozotocin or genetically using the non-obese diabetic (NOD) female mouse model. Mice were treated with INO-2002 or inosine as required at 30, 100, or 200 mg/kg per day, while blood glucose and diabetes incidence were monitored. The effect of INO-2002 on the pancreatic cytokine profile was also determined. INO-2002 reduced both the hyperglycaemia and incidence of diabetes in both streptozotocin-induced and spontaneous diabetes in NOD mice. INO-2002 proved to be more effective in protecting against diabetes than the naturally occurring purine, inosine, when administered at the same dose. INO-2002 treatment decreased pancreatic levels of interleukin (IL)-12 and tumour necrosis factor-alpha, while increasing levels of IL-4 and IL-10. INO-2002 also reduced pancreatic levels of the chemokine MIP-1 alpha. The inosine analogue, INO-2002, was protected more effectively than the naturally occurring purine, inosine, against development of diabetes in two separate animal models. INO-2002 exerts protective effects by changing the pancreatic cytokine expression from a destructive Th1 to a protective Th2 profile. The use of analogues of inosine such as INO-2002 should be considered as a potential preventative therapy in individuals susceptible to developing type I diabetes.     

Altered chemokine levels in individuals at risk of Type 1 diabetes mellitus.

AIMS: The hypothesis was tested in an exploratory study that individuals at high risk of developing Type 1 diabetes mellitus have altered systemic levels of cytokines and chemokines. SUBJECTS AND METHODS: Forty-two non-diabetic first-degree relatives of patients with Type 1 diabetes mellitus were recruited. Of these, 18 had multiple islet autoantibodies (islet cell antibody, glutamic acid decarboxylase antibody, IA-2 antibody). Follow-up for 9-11 years confirmed high vs. moderate diabetes risk in islet autoantibody-positive vs. -negative relatives. Cytokines and chemokines were determined by enzyme-linked immunosorbent assay (ELISA). RESULTS: Serum concentrations of classic Th1-associated cytokines (IFN-gamma, IL-12, IL-18) or Th2/Treg-associated cytokines (IL-5, IL-10, IL-13) did not significantly differ in high vs. moderate diabetes risk group. However, of six chemokines analysed, levels of CCL3 and CCL4 were increased (P = 0.0442 and P = 0.0334) while CCL2 was decreased (P = 0.0318) in the multiple islet autoantibody-positive group. No significant differences were seen for CCL5, CCL11, CXCL10. There was a significant correlation between the two closely related chemokines CCL3 and CCL4 in individuals at risk (r = 0.84, P = 0.00005), but not in the autoantibody-negative group. CONCLUSION: Relatives at high risk of developing Type 1 diabetes mellitus have abnormal cellular immune regulation at the level of systemic chemokines. The up-regulation of CCL3 and CCL4 vs. down-regulation of CCL2 suggests opposed functions of these chemokines in the disease process. These findings need to be confirmed by independent studies.     

Human chorionic gonadotropin is an immune modulator and can prevent autoimmune diabetes in NOD mice

Aims/hypothesis Expression of T helper (Th)1 cytokine mRNA in pregnant women is known to be inversely correlated with serum human chorionic gonadotropin (hCG). Type 1 diabetes is a Th1-mediated autoimmune disease, in which intervention at an early stage of the autoimmune process can prevent disease progression. We hypothesised that immune modulation by treating young NOD mice with hCG may prevent diabetes. Methods Female NOD mice were treated with hCG or recombinant hCG from 3 to 15 weeks of age and the incidence of diabetes and development of insulitis was determined. CD4⁺ and CD8⁺ T cell populations, T cell proliferation, cytokine production and CD4⁺CD25⁺ regulatory T cells were examined and adoptive transfer experiments were performed. Results Both purified and recombinant hCG prevented development of diabetes in NOD mice. hCG decreased the proportion and number of CD4⁺ and CD8⁺ T cells and inhibited T cell proliferative responses against beta cell antigens. hCG treatment suppressed IFN-γ production, but increased IL-10 and TGF-β production in splenocytes stimulated with anti-CD3 antibody. hCG treatment also suppressed TNF-α production in splenocytes stimulated with lipopolysaccharide. Furthermore, hCG treatment increased the CD4⁺CD25⁺/CD4⁺ T cell ratio in spleen and pancreatic lymph nodes. Depletion of CD4⁺CD25⁺ T cells from splenocytes of hCG-treated NOD mice abolished their preventive effect on diabetes transfer. Conclusions/interpretation We conclude that hCG has an immunomodulatory effect by downregulating effector cells, including Th1 cells, CD8⁺ T cells and macrophages, and increasing the CD4⁺CD25⁺/CD4⁺ T cell ratio, thus preventing autoimmune diabetes in NOD mice. L.-Y. Khil, H.-S Jun and H. Kwon contributed equally to this work. Ji-Won Yoon sadly passed away on 6 April 2006.     

Oral insulin for diabetes prevention in NOD mice: potentiation by enhancing Th2 cytokine expression in the gut through bacterial adjuvant

Summary Oral administration of insulin suppresses the development of diabetes in nonobese diabetic (NOD) mice and deviates the cytokine balance in the islets of Langerhans from a Th1 to a Th2 type cytokine pattern. However, the effect of oral insulin is limited and disease suppression is limited to a narrow dose range. Therefore we tried to improve the outcome of suboptimal insulin dosing by bacterial adjuvant. Mice treated with a suboptimal dose of oral insulin showed no change in diabetes incidence although a shift from Th1 towards Th2 cytokine expression occurred in inflamed islets. Significant suppression of diabetes development was only seen in NOD mice receiving both, insulin and the bacterial preparation OM-89 as adjuvant. OM-89 is a protein extract of Escherichia coli, with nonspecific immunostimulatory properties. Potentiation of the effect of oral insulin by the adjuvant was associated with upregulation of interleukin (IL)-4 Th2 cells in infiltrated islets and sustained local IL-2 gene expression. RT PCR analyses of cytokine expression in the gut showed a clear deviation to Th2 type reactivity and downregulation of inducible nitric oxide (NO) synthase (iNOS) expression by the bacterial adjuvant but not by oral insulin alone. Since macrophages are the primary target cells of adjuvant action we tested its effect on mouse macrophages in vitro. Treatment with OM-89 induced transient release of tumour necrosis factor alpha and nitrite but rendered macrophages refractory to restimulation by the potent macrophage activator lipopolysaccharide. In conclusion, the protective effect of oral insulin can be potentiated by pretreatment with the bacterial adjuvant OM-89. This effect correlates with enhanced Th2 cytokine and decreased iNOS gene expression in the gut, probably due to the downregulation of proinflammatory mediators by exposure to the adjuvant. [Diabetologia (1997) 40: 902–909] Received: 28 January 1997 and in revised form 16 April 1997     

Melatonin prolongs islet graft survival in diabetic NOD mice

Abstract:  Islet transplantation has been established as a potential therapy for type 1 diabetes. However, inflammation, allorejection, and on-going autoimmune damage contribute to early graft loss and failure of islet transplantation. Melatonin is the major secretory product of the pineal gland during the dark period of each day and displays multifunctional properties including the regulation of circadian and seasonal rhythms, antioxidation reactions and immune modulation. Based on the immunosuppressive properties of melatonin, we investigated whether melatonin treatment prolonged the survival of islet grafts in non-obese diabetic (NOD) mice. The mean islet graft survival time was 7.33 ± 1.51 and 7.75 ± 2.66 days in untreated controls and in the solvent-treated animals, respectively. Strikingly, the mean survival time of islet grafts in recipients treated with melatonin (200 mg/kg/bw) was 17 ± 7.76 days. Moreover, melatonin treatment reduced the proliferation of splenocytes in NOD mice. Using a T1 and T2 double transgenic mouse model, we found that T helper 1 (Th1) cells in mice treated with melatonin were significantly decreased. The reduction of Th1 cells and T cell proliferation may result from an increase in the immunosuppressive cytokine IL-10. Our results indicate that melatonin treatment suppresses autoimmune recurrence by inhibiting the proliferation of Th1 cells in NOD mice and thus prolongs the survival of syngeneic islet grafts.     

Low TCR signal strength induces combined expansion of Th2 and regulatory T cell populations that protect mice from the development of type 1 diabetes

Aims/hypothesis

Weak stimulation of CD4⁺ T cells induces expansion of CD4⁺ forkhead box P3⁺ regulatory T cells (Tregs) and can also promote T helper (Th) 2 responses, which have demonstrable beneficial effects on autoimmune diabetes. This study explored the feasibility of combined Treg/Th2 expansion for immunotherapy of type 1 diabetes in NOD mice.

Methods

We compared Treg and Th responses to dendritic cells (DC) presenting scaled antigen doses to islet-specific NOD CD4⁺ T cells. Flow cytometric and Luminex analyses were performed to determine the phenotype and cytokine profile of expanded T cells. The ability of expanded T cells to prevent type 1 diabetes was tested in an adoptive transfer model.

Results

In vitro studies revealed a hierarchical, selective expansion of Treg and T effector (Teff) populations at different antigen doses. Thus, a single low dose produced a mixture of Tregs Th2 and type 1 regulatory (Tr1) cells, which prevented diabetes in NOD-SCID mice and increased the ratio of Treg/Teff cells infiltrating the pancreatic islets. Subcutaneous injection of DC, previously shown to prevent diabetes in NOD mice, induced expansion of the same mixture of Tregs Tr1 and Th2 cells. Low-dose expansion of Treg required MHC–T cell receptor interaction and was partly dependent on T cell derived TGF-β and IL-2. Autocrine IFN-γ was required for the promotion of diabetogenic Th1 cells at high antigen doses.

Conclusions/interpretation

Weak stimulation of CD4⁺ T cells with DC and low-dose antigen expands a combination of antigen-specific Tregs Th2 and Tr1 cells that prevent autoimmunity, without the need to target or purify specific Treg populations.     

Cytokine balance and lipid antigen presentation in the NOD mouse pancreas during development of insulitis

The role of cytokine balance and lipid antigen presentation in the development of diabetes was studied using immunohistochemistry of cytokines in the pancreas of non-obese diabetic mice (NOD) and BALB/c mice at various ages. In both the NOD and BALB/c mice, interleukin 10 (IL-10) was expressed in the islets. IL-10 was also present in the epithelial cells of the exocrine tissue in both strains. In the NOD mice, IL-10 disappeared from both the islets and the exocrine tissue at 16 weeks of age. At this age, IL-10 was still present in the islets and exocrine tissue of the BALB/c pancreata. IL-10 was not present in the pancreata of diabetic NOD mice. IL-6 first appeared in the pancreas at 10 weeks of age and disappeared at the age of 16 weeks in both NOD and BALB/c mice. It was present in the endothelial cells. Neither the pancreata of normal BALB/c mice nor NOD mice at 2-16 weeks of age contained tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-gamma), IL-4, or IL-12. At 8 weeks of age, a few IL-2+ cells were found in the pancreas of one of three NOD mice. CD1d was already present in both strains at 2 weeks of age but disappeared from the NOD mice at 16 weeks of age. CD1d localized to walls of tubular structures probably representing collecting tubules. These results suggest that in the NOD mice the disappearance of the T(H0), T(H1), and T(H2) responses inhibiting IL-10 from the islets at the age of 16 weeks may trigger the final stage of the immune response leading to overt diabetes. The simultaneous disappearance of CD1d suggests that activation of immune responses against lipid antigens does not play a role in this stage of the disease.     

High T-helper-1 cytokines but low T-helper-3 cytokines, inflammatory cytokines and chemokines in children with high risk of developing type 1 diabetes

BACKGROUND: Type 1 diabetes (T1D) is suggested to be of T-helper (Th)1-like origin. However, recent reports indicate a diminished interferon (IFN)-gamma secretion at the onset of the disease. We hypothesize that there is a discrepancy in subsets of Th-cells between children with a high risk of developing T1D, children newly diagnosed with T1D and healthy children. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from children at high risk for T1D (islet cells antibodies [ICA] >/= 20 IJDF-U), those newly diagnosed and healthy children carrying the HLA-risk gene DQB1*0302 or DQB1*0201 and DQA1*0501. Th1- (IFN-gamma, tumour necrosis factor [TNF]-beta, interleukin [IL]-2), Th2- (IL-4,-5,-13), Th3- (transforming growth factor [TGF-beta], IL-10) and inflammatory associated cytokines (TNF-alpha, IL-1alpha,-6) and chemokines (monocyte chemoattractant protein [MCP]-1,-2,-3, Monokine unregulated by IFN-gamma [MIG], Regulated on Activation, Normal T-cell Expressed and Secreted [RANTES], IL-7,-8,-15) were detected in cell-culture supernatants of PBMC, stimulated with glutamic acid decarboxylase 65 (GAD(65)) and phytohaemagglutinin (PHA), by protein micro array and enzyme linked immunospot (ELISPOT) technique. RESULTS: The Th1 cytokines IFN-gamma and TNF-beta, secreted both spontaneously and by GAD(65)- and mitogen stimulation, were seen to a higher extent in high-risk children than in children newly diagnosed with T1D. In contrast, TNF-alpha and IL-6, classified as inflammatory cytokines, the chemokines RANTES, MCP-1 and IL-7 as well as the Th3 cytokines TGF-beta and IL-10 were elevated in T1D children compared to high-risk children. CONCLUSION: High Th-1 cytokines were observed in children with high risk of developing TID, whereas in children newly diagnosed with T1D Th3 cytokines, inflammatory cytokines and chemokines were increased. Thus, an inverse relation between Th1-like cells and markers of inflammation was shown between children with high risk and those newly diagnosed with T1D.     

IL—4对NOD小鼠1型糖尿病免疫干预及其机制的探讨

目的：探讨IL－4对NOD鼠1型糖尿病发病率，胰岛炎的影响及其机制。方法：采用人类1型糖尿病动物模型NO（nonobese diabetic)鼠，IL－4处理后检测血糖，尿糖及糠愕病发病率，HE染色观察胰岛炎程度，流式细胞仪测定周围及中枢淋巴细胞亚群的变化，结果IL－4处理组糖尿病发病率为12．50％，明显低于对照组（62．50％）（P＜0．05），且胰岛炎的严重程度亦低于对照组；处理组周围单个核细胞（CD 4/CD 8及IL－2R＋细胞百分比增高，胸腺CD＋4，CD＋8细胞百分比增高而D＋4 CD＋8细胞百分比下降（P＜0．01）。结论 IL－4有降低NOD鼠1型糖尿病发病率及胰岛炎严重程度的作用，这种作用可能与IL－4影响了淋巴细胞亚群的分布从而纠正了免疫调节失衡有关。     

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

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

Combination therapy with low dose sirolimus and tacrolimus is synergistic in preventing spontaneous and recurrent autoimmune diabetes in non-obese diabetic mice

Aims/hypothesis: Sirolimus and tacrolimus are immunosuppressive drugs that prevent rejection of pancreatic islet allografts transplanted into patients with Type I (insulin-dependent) diabetes mellitus. This study aimed to determine whether sirolimus and tacrolimus can prevent autoimmune beta-cell destruction, and if so, what the mechanisms of action are. Methods: Sirolimus and tacrolimus were given separately and together to female non-obese diabetic (NOD) mice from age 12 to 35 weeks. Diabetes incidence was determined and pancreatic insulitis and insulin content were measured. Sirolimus and tacrolimus were also given separately and together to diabetic NOD mice from the time of syngeneic islet transplantation until the reappearance of hyperglycaemia. Islet grafts were examined by RT-PCR assay for expression of interferon (IFN)-γ, interleukin (IL)-2, IL-4, IL-10 and transforming growth factor (TGF)-β1. Results: Low doses of sirolimus (0.1 mg/kg) and tacrolimus (0.1 mg/kg) were synergistic in reducing insulitis, preserving pancreatic insulin content and preventing diabetes in female NOD mice (8 % diabetes incidence at 35 weeks vs 66 % in vehicle-treated mice). Also, the combination of sirolimus and tacrolimus prolonged syngeneic islet graft survival (median 34 days vs 13 days for vehicle-treated mice). Islet grafts from sirolimus plus tacrolimus-treated mice expressed significantly decreased mRNA contents of Th1-type cytokines (IFN-γ and IL-2) and the highest ratio of TGF-β1/IFN-γ mRNA. Conclusion/interpretation: These findings suggest that combination therapy with sirolimus and tacrolimus prevent autoimmune beta-cell destruction by upregulating expression of the immunoregulatory cytokine, TGF-β1 and reducing Th1 cytokines (IFN-γ and IL-2) expressed in the islets. Low-dose sirolimus and tacrolimus combination therapy could warrant consideration for prevention or early treatment of human Type I diabetes. [Diabetologia (2002) 45: 224–230] Received: 20 August 2001 and in revised form: 29 October 2001