Norio Abiru

Prime role for an insulin epitope in the development of type 1 diabetes in NOD mice

A fundamental question about the pathogenesis of spontaneous autoimmune diabetes is whether there are primary autoantigens. For type 1 diabetes it is clear that multiple islet molecules are the target of autoimmunity in man and animal models. It is not clear whether any of the target molecules are essential for the destruction of islet beta cells. Here we show that the proinsulin/insulin molecules have a sequence that is a primary target of the autoimmunity that causes diabetes of the non-obese diabetic (NOD) mouse. We created insulin 1 and insulin 2 gene knockouts combined with a mutated proinsulin transgene (in which residue 16 on the B chain was changed to alanine) in NOD mice. This mutation abrogated the T-cell stimulation of a series of the major insulin autoreactive NOD T-cell clones. Female mice with only the altered insulin did not develop insulin autoantibodies, insulitis or autoimmune diabetes, in contrast with mice containing at least one copy of the native insulin gene. We suggest that proinsulin is a primary autoantigen of the NOD mouse, and speculate that organ-restricted autoimmune disorders with marked major histocompatibility complex (MHC) restriction of disease are likely to have specific primary autoantigens.

Evidence for a primary islet autoantigen (preproinsulin 1) for insulitis and diabetes in the nonobese diabetic mouse

It has been reported that an insulin 2 gene knockout, when bred onto nonobese diabetic (NOD) mice, accelerates diabetes. We produced insulin 1 gene knockout congenic NOD mice. In contrast to insulin 2, diabetes and insulitis were markedly reduced in insulin 1 knockout mice, with decreased and delayed diabetes in heterozygous females and no insulitis and diabetes in most homozygous female mice. Lack of insulitis was found for insulin 1 female homozygous knockout mice at 8, 12, and 37 weeks of age. Despite a lack of insulitis, insulin 1 homozygous knockout mice spontaneously expressed insulin autoantibodies. Administration of insulin peptide B:9-23 of both insulin 1 and 2 to NOD mice induced insulin autoantibodies. Insulin 1 is not the only lymphocytic target of NOD mice. Insulin 1 homozygous knockout islets, when transplanted into recently diabetic wild-type NOD mice, became infiltrated with lymphocytes and only transiently reversed diabetes. These observations indicate that loss of either insulin gene can influence progression to diabetes of NOD mice and suggest that the preproinsulin 1 gene is crucial for the spontaneous development of NOD insulitis and diabetes.

Induction and acceleration of insulitis/diabetes in mice with a viral mimic (polyinosinic-polycytidylic acid) and an insulin self-peptide

Polyinosinic-polycytidylic acid (PolyIC), a “mimic” of double-stranded viral RNA, can induce diabetes when administered to rats with RT1u, and immunization of normal H-2d mice (e.g., BALB/c) with insulin B:9–23 peptide (but not H-2b) results in the rapid induction of insulin autoantibodies. Because a mouse model of PolyIC/antigen-induced diabetes is lacking, we sought to produce insulitis and diabetes with either PolyIC and/or B:9–23 peptide immunization. Simultaneous administration of PolyIC and B:9–23 peptide to BALB/c mice (but with neither alone) induced insulitis. CD4 T lymphocytes predominated within islets, and the mice did not progress to hyperglycemia. Islets with transgene-induced expression of the costimulatory B7–1 molecule have enhanced diabetes susceptibility. Diabetes was frequently induced in B7–1 transgenic mice with H-2d in contrast to H-2b mice after PolyIC administration. Disease induction was accelerated by adding B:9–23 immunization to PolyIC. These studies demonstrate that “normal” mice have autoreactive T lymphocytes able to rapidly target islets and insulin given appropriate MHC alleles and that a peripherally administered insulin peptide (an altered peptide ligand of which is in clinical trials) can enhance specific anti-islet autoimmunity. These first PolyIC/insulin-induced murine models should provide an important tool to study the pathogenesis of type 1 diabetes with experimental autoimmune diabetes.

Evidence That a Peptide Spanning the B-C Junction of Proinsulin Is an Early Autoantigen Epitope in the Pathogenesis of Type 1 Diabetes

The expression of pro(insulin) in the thymus may lead to the negative selection of pro(insulin) autoreactive T cells and peripheral tolerance to this autoantigen in type 1 diabetes (T1D). We investigated whether proinsulin is expressed in the thymus of young nonobese diabetic (NOD) mice, whether T cells from naive NOD female mice at weaning are reactive to mouse proinsulin, and the role of proinsulin as a pathogenic autoantigen in T1D. Proinsulin II mRNA transcripts were detected in the thymus of 2-wk-old NOD mice at similar levels to other control strains. Despite this expression, proinsulin autoreactive T cells were detected in the periphery of 2- to 3-wk-old naive NOD mice. Peripheral T cells reactive to the insulin, glutamic acid decarboxylase 65 (GAD65), GAD67, and islet cell Ag p69 autoantigens were also detected in these mice, indicating that NOD mice are not tolerant to any of these islet autoantigens at this young age. T cell reactivities to proinsulin and islet cell Ag p69 exceeded those to GAD67, and T cell reactivity to proinsulin in the spleen and pancreatic lymph nodes was directed mainly against a p24–33 epitope that spans the B chain/C peptide junction. Intraperitoneal immunization with proinsulin perinatally beginning at 18 days of age delayed the onset and reduced the incidence of T1D. However, s.c. immunization with proinsulin initiated at 5 wk of age accelerated diabetes in female NOD mice. Our findings support the notion that proinsulin p24–33 may be a primary autoantigen epitope in the pathogenesis of T1D in NOD mice.

Anti-peptide autoantibodies and fatal anaphylaxis in NOD mice in response to insulin self-peptides B:9-23 and B:13-23

There is evidence that amino acids 9-23 of the insulin B chain are a major target of anti-islet autoimmunity in type 1 diabetes. Administration of this peptide to NOD mice prevents diabetes, and phase I trials of an altered peptide ligand of B:9-23 are underway in humans. We were interested in long-term subcutaneous therapeutic administration of B:9-23 without adjuvant. To our initial surprise, the peptide consistently induced fatal anaphylaxis in NOD mice after 6 weeks of administration. Anaphylaxis could be blocked by a combination of antihistamine and platelet-activating factor antagonist (but neither alone) or by a combination of anti-IgG receptor and anti-IgE antibodies. High titers of anti-B:9-23 antibodies were induced within 3-4 weeks of immunization with the peptide. Peptide B:13-23 also induced anaphylaxis and was more potent than peptide B:9-23. Antibodies induced by peptide B:9-23 and peptide B:13-23 did not cross-react with each other. Thus, the insulin peptides B:9-23 and B:13-23, even when administered subcutaneously in the absence of adjuvant, can induce a dramatic humoral response leading to fatal anaphylaxis in NOD mice.

Association Between Liver Fibrosis and Insulin Sensitivity in Chronic Hepatitis C Patients

BACKGROUND:Several clinical studies have suggested a possible link between chronic hepatitis caused by hepatitis C virus (HCV) and the development of diabetes mellitus. We investigated the association between liver fibrosis and glucose intolerance in HCV-infected patients by measuring insulin sensitivity and β-cell function.METHOD:A total of 83 chronic HCV-infected patients were recruited into this study. We evaluated insulin sensitivity and β-cell function of all patients in a fasting state (homeostasis model assessment of insulin resistance [HOMA-R] and homeostasis model assessment of β-cell function [HOMA-β]) and after an oral load of 75 g glucose (whole-body insulin sensitivity index [WBISI] and Δ-insulin/Δ-glucose 30).RESULTS:In a multivariate analysis, severe fibrosis was the only independent factor associated with insulin resistance. There were significant differences in both HOMA-R (P = 0.0063) and WBISI (P = 0.0159) between patients with mild fibrosis (N = 34) and those with severe fibrosis (N = 49). Although HOMA-β was increased significantly in the subjects with severe fibrosis compared with those with mild fibrosis (P = 0.0169), Δ-insulin/Δ-glucose 30 showed no significant difference in stage of liver fibrosis, suggesting an uncertain association between liver fibrosis and β-cell function.CONCLUSION:Our findings suggest that the development of liver fibrosis is associated with insulin resistance in HCV-infected patients.

T helper type 17 immune response plays an indispensable role for development of iodine-induced autoimmune thyroiditis in nonobese diabetic-H2h4 mice.

T helper type 1(Th1)/Th2 paradigm has been expanded by discovery of a novel effector T cell (T(eff)) subset, Th17 cells, which produce a proinflammatory cytokine IL-17. Th17 cells have recently been shown to play a major role in numerous autoimmune diseases that had previously been thought to be Th1-dominant diseases. We here studied the significance of Th17 cells in iodine-induced autoimmune thyroiditis in nonobese diabetic-H2(h4) mice, a mouse model of Hashimotos thyroiditis in humans, which spontaneously develop antithyroglobulin autoantibodies and intrathyroidal lymphocyte infiltration when supplied with iodine in the drinking water. We observed increased numbers of Th1 and Th17 cells in spleen and accumulation of both types of T(eff) in the thyroid glands of iodine-fed wild-type mice, indicating that Th17 cells as well as Th1 cells constitute thyroid lesions. Furthermore, the incidence and severity of intrathyroidal lymphocyte infiltration, and the titers of antithyroglobulin autoantibodies were markedly reduced in iodine-treated IL-17(-/-) mice as compared with wild-type mice. Of interest, IL-17(+/-) mice showed an intermediate phenotype. Therefore, the present study, together with a previous report demonstrating the importance of Th1, not Th2, immune response for developing thyroiditis using mice deficient for interferon-gamma or IL-4, clearly indicates that both Th1 and Th17 cells are critical T(eff) subsets for the pathogenesis of spontaneous autoimmune thyroiditis in nonobese diabetic-H2(h4) mice.

Genetic association between interleukin-10 gene promoter region polymorphisms and type 1 diabetes age-at-onset

This study investigated whether interleukin-10 (IL-10) gene promoter region polymorphisms are associated with susceptibility to or clinical presentation of type 1 diabetes. The frequency of -1082G/A, -819C/T, and -592C/A polymorphisms was analyzed in 128 Japanese patients with type 1 diabetes and in 107 healthy control subjects in a case-controlled study. The allelic and haplotypic frequencies of the IL-10 gene promoter region polymorphisms were similar in patients with type 1 diabetes and in control subjects. However, the -819T and -592A allele were associated with adult-onset (>18 years) of the disease (p = 0.037). Furthermore, the frequency of ATA haplotype was increased in adult-onset patients than that in early-onset patients (< or =18 years; p = 0.037). Among the genotypes comprising ATA haplotype, the frequency of ATA/ATA was significantly higher in adult-onset patients than in early-onset patients (p = 0.004). These results suggest that the IL-10 gene promoter polymorphisms are associated with the age-at-onset in Japanese patients with type 1 diabetes.

Association between anti-ZnT8 autoantibody specificities and SLC30A8 Arg325Trp variant in Japanese patients with type 1 diabetes.

Aims/hypothesisWe analysed the association between humoral autoreactivity to zinc transporter-8 (ZnT8) and the SLC30A8 rs13266634 polymorphism (Arg325Trp), which is located at the most distal loop in the ZnT8 protein.MethodsAutoantibodies to ZnT8 were determined by RIA in 270 patients with type 1 diabetes using ZnT8 carboxy-terminal constructs (amino acids 268–369) carrying 325Trp(CW) and 325Arg(CR) and a hybrid construct (CW-CR). Forty-four ZnT8 autoantibody-positive sera with genomic DNA were used to examine the association between reactivity to ZnT8 constructs and the rs13266634 genotype.ResultsSeventy-five patients reacted to the CW-CR hybrid construct, whereas 37 and 36 patients reacted to the CW and CR constructs, respectively. All sera positive for either CW or CR autoantibodies were positive for CW-CR autoantibodies. Among 19 patients with a 325Arg(CC) genotype, 5% had CW-specific autoantibodies, 42% had CR-specific autoantibodies and 32% had dual reactivity. Conversely, 73% of 15 patients with the 325Trp(TT) genotype had CW-specific autoantibodies, no patients had CR-specific autoantibodies and 13% had dual reactivity. Nine of the ten patients (90%) with the CT genotype reacted with either CR or CW constructs. The titre of CR autoantibodies in patients carrying the C allele was significantly higher than that in TT homozygotes (p < 0.0001). In contrast, the titre of CW autoantibodies in patients carrying a T allele was significantly higher than that in CC homozygotes (p < 0.005). No evidence of an association between rs13266634 and type 1 diabetes was observed.Conclusions/interpretationThese results indicate that variant residue at amino acid 325 is a key determinant of humoral autoreactivity to ZnT8 and that the SLC30A8 genotype is an important determinant of autoantibody specificity.

Autoantibodies to Insulin, Insulinoma-Associated Antigen-2, and Zinc Transporter 8 Improve the Prediction of Early Insulin Requirement in Adult-Onset Autoimmune Diabetes

OBJECTIVE The aim of this study was to identify the predictive marker for early insulin requirement in adult-onset autoimmune diabetes in the Japanese populations. DESIGN/PATIENTS We analyzed insulin autoantibodies (IAA), insulinoma-associated antigen-2 (IA-2) autoantibodies (IA-2icA), and zinc transporter 8 (ZnT8) autoantibodies (ZnT8A) by radioimmunoassay in 47 Japanese patients with adult-onset autoimmune diabetes who were identified by native GAD autoantibody (nGADA) screening of approximately 3000 non-insulin-requiring diabetes patients and 302 nGADA-negative type 2 diabetes patients. Furthermore, GAD65 autoantibody-specific epitopes were also analyzed using GAD65/GAD67 chimeric constructs. RESULTS The prevalence of IAA, IA-2icA, and ZnT8A in nGADA-positive patients was 26, 15, and 19%, respectively, which was significantly higher than that in nGADA-negative type 2 diabetes (2, 2, and 2%; P < 0.0001). Among nGADA-positive patients, 38% had one or more of IAA, IA-2icA, or ZnT8A, and 15% had two or more of these autoantibodies, compared with none of the nGADA-negative patients (P < 0.0001). Thirty-six percent of nGADA-positive patients subsequently required insulin therapy; and high nGADA titer (log-rank P = 0.003), middle epitope recognition of GAD65A (P = 0.002), and the presence of one or more of IAA, IA-2icA, or ZnT8A (P = 0.002) at diagnosis marked the risk for early requirement of insulin therapy. Multivariate logistic regression analysis showed the multiple islet autoantibodies to be independently associated with the risk for insulin requirement (odds ratio = 13.77; 95% confidence interval, 2.77-68.45; P = 0.001). CONCLUSIONS These results indicate that the determination of IAA, IA-2icA, and ZnT8A improves the prediction of a future insulin insufficiency in adult-onset autoimmune diabetes, which appears to be superior to GADA titer and GAD65A-specific epitopes.