Tanja Arndt

Islet infiltration, cytokine expression and beta cell death in the NOD mouse, BB rat, Komeda rat, LEW.1AR1-iddm rat and humans with type 1 diabetes

Aims/hypothesisResearch on the pathogenesis of type 1 diabetes relies heavily on good animal models. The aim of this work was to study the translational value of animal models of type 1 diabetes to the human situation.MethodsWe compared the four major animal models of spontaneous type 1 diabetes, namely the NOD mouse, BioBreeding (BB) rat, Komeda rat and LEW.1AR1-iddm rat, by examining the immunohistochemistry and in situ RT-PCR of immune cell infiltrate and cytokine pattern in pancreatic islets, and by comparing findings with human data.ResultsAfter type 1 diabetes manifestation CD8+ T cells, CD68+ macrophages and CD4+ T cells were observed as the main immune cell types with declining frequency, in infiltrated islets of all diabetic pancreases. IL-1β and TNF-α were the main proinflammatory cytokines in the immune cell infiltrate in NOD mice, BB rats and LEW.1AR1-iddm rats, as well as in humans. The Komeda rat was the exception, with IFN-γ and TNF-α being the main cytokines. In addition, IL-17 and IL-6 and the anti-inflammatory cytokines IL-4, IL-10 and IL-13 were found in some infiltrating immune cells. Apoptotic as well as proliferating beta cells were observed in infiltrated islets. In healthy pancreases no proinflammatory cytokine expression was observed.Conclusions/interpretationWith the exception of the Komeda rat, the animal models mirror very well the situation in humans with type 1 diabetes. Thus animal models of type 1 diabetes can provide meaningful information on the disease processes in the pancreas of patients with type 1 diabetes.

Diabetes Prevention by Immunomodulatory FTY720 Treatment in the LEW.1AR1-iddm Rat Despite Immune Cell Activation

The prevention of diabetes by the immunomodulatory agent FTY720 (fingolimod) was studied in the LEW.1AR1-iddm (IDDM) rat, an animal model of human type 1 diabetes. Immune cell subtypes and cytokine profiles in pancreatic islets, secondary lymphoid tissue, and serum were analyzed for signs of immune cell activation. Animals were treated with FTY720 (1 mg/kg body weight) for 40 d starting on d 50 of life. Changes in gene and protein expression of cytokines, CD8 markers, monocyte chemoattractant protein-1, inducible NO synthase, and caspase 3 were evaluated. Treatment with FTY720 prevented diabetes manifestation and islet infiltration around d 60 of life, the usual time of spontaneous diabetes development. On d 120, 30 d after the end of FTY720 therapy, diabetes prevention persisted. However, six of 12 treated animals showed increased gene expression of IL-1beta, TNF-alpha, and CD8 markers in pancreas-draining lymph nodes, indicating immune cell activation. In parallel, serum concentrations of these proinflammatory cytokines were increased. These six animals also showed macrophage infiltration without proinflammatory cytokine expression in a small minority (2-3%) of islets. Interestingly, regulatory T lymphocytes were significantly increased in the efferent vessels of the pancreas-draining lymph nodes only in animals without signs of immune cell activation but not in the rats with immune cell activation. This provides an indication for a lack of protective capacity in the animals with activated immune cells. Thus, FTY720 treatment prevented the manifestation of diabetes by promoting the retention of activated immune cells in the lymph nodes, thereby avoiding islet infiltration and beta-cell destruction by proinflammatory cytokines.

Prevention of spontaneous immune-mediated diabetes development in the LEW.1AR1-iddm rat by selective CD8 + T cell transfer is associated with a cytokine shift in the pancreas-draining lymph nodes

Aims/hypothesisThe LEW.1AR1-iddm rat is an animal model of spontaneous type 1 diabetes mellitus. This study analysed how adoptive transfer of selective T cell subpopulations affects the incidence of diabetes.MethodsCD4+ or CD8+ T cells were isolated from diabetic LEW.1AR1-iddm rats or diabetes-resistant LEW.1AR1 rats. Cells were selectively transferred into athymic LEW.1AR1-Whnrnu or prediabetic LEW.1AR1-iddm rats. The animals were monitored for blood glucose, islet infiltration and immune cell composition of pancreas-draining lymph nodes.ResultsAfter adoptive transfer of CD4+ T cells from diabetic LEW.1AR1-iddm rats into athymic LEW.1AR1-Whnrnu rats, 50% of the recipients developed diabetes. Transfer of CD8+ T cells failed to induce diabetes. Only 10% of the athymic recipients became diabetic after co-transfer of CD4+ and CD8+ T cells. Adoptive transfer of CD8+ T cells from LEW.1AR1 or diabetic LEW.1AR1-iddm rats into prediabetic LEW.1AR1-iddm rats significantly reduced the incidence of diabetes. In protected normoglycaemic animals regulatory CD8+/CD25+ and CD4+/CD25+ T cell subpopulations that were also FOXP3-positive accumulated in the pancreas-draining lymph nodes. In this lymphatic organ, gene expression of anti-inflammatory cytokines was significantly higher than in diabetic rats.Conclusions/interpretationOur results show that adoptive transfer of CD4+ but not CD8+ T cells from diabetic LEW.1AR1-iddm rats induced diabetes development. Importantly, CD8+ T cells from diabetic LEW.1AR1-iddm rats and diabetes-resistant LEW.1AR1 rats provided protection against beta cell destruction. The accumulation of regulatory T cells in the pancreas-draining lymph nodes from protected rats indicates that transferred CD8+ T cells may have beneficial effects in the control of beta cell autoimmunity.

A Variable CD3+ T-Cell Frequency in Peripheral Blood Lymphocytes Associated with Type 1 Diabetes Mellitus Development in the LEW.1AR1-iddm Rat

Purpose The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes mellitus (T1DM), which arose through a spontaneous mutation within the MHC-congenic inbred strain LEW.1AR1 (RT1r2). In contrast to the diabetes-resistant LEW.1AR1 background strain in LEW.1AR1-iddm rats a highly variable T-cell frequency could be observed in peripheral blood lymphocytes (PBLs). Methods In this study we therefore characterised the T-cell repertoire within the PBLs of the two strains by flow cytometry analysis and identified the CD3+ T-cell phenotype and its possible linkage to diabetes susceptibility. To map loci conferring susceptibility to variable CD3+ T-cell frequency, backcross strains (N2) were generated with the genetically divergent BN and PAR rats for microsatellite analysis. Results The LEW.1AR1-iddm rat strain was characterised by a higher variability of CD3+ T-cells in PBLs along with a slightly decreased mean value compared to the LEW.1AR1 background strain. The reason for this reduction was a decrease in the CD4+ T-cell count while the CD8+ T-cell proportion remained unchanged. However, both T-cell subpopulations showed a high variability. This resulted in a lower CD4+/CD8+ T-cell ratio than in LEW.1AR1 rats. Like LEW.1AR1-iddm rats all animals of the backcross populations, N2 BN and N2 PAR rats, also showed large variations of the CD3+ T-cell frequency. The phenotype of variable CD3+ T-cell frequency mapped to the telomeric region of chromosome 1 (RNO1), which is identical with the already known Iddm8 diabetes susceptibility region. The data indicate that a variable CD3+ T-cell frequency in PBLs is genetically linked to diabetes susceptibility in the LEW.1AR1-iddm rat. Conclusion The T-cell variability in PBLs could be related to the previously reported imbalance between regulatory and effector T-cell populations which results in beta-cell autoimmunity. Since similar T-cell phenotypes have also been described in human T1DM the identification of the functional role of the observed variable CD3+ T-cell frequency may help to understand the mechanisms of autoimmunity in T1DM.

TNF-α Antibody Therapy in Combination With the T-Cell–Specific Antibody Anti-TCR Reverses the Diabetic Metabolic State in the LEW.1AR1-iddm Rat

Anti–tumor necrosis factor-α (TNF-α) therapy (5 mg/kg body weight), alone or combined with the T-cell–specific antibody anti–T-cell receptor (TCR) (0.5 mg/kg body weight), was performed over 5 days immediately after disease manifestation to reverse the diabetic metabolic state in the LEW.1AR1-iddm rat, an animal model of human type 1 diabetes. Only combination therapy starting at blood glucose concentrations below 15 mmol/L restored normoglycemia and normalized C-peptide. Increased β-cell proliferation and reduced apoptosis led to a restoration of β-cell mass along with an immune cell infiltration–free pancreas 60 days after the end of therapy. This combination of two antibodies, anti-TCR/CD3, as a cornerstone compound in anti–T-cell therapy, and anti–TNF-α, as the most prominent and effective therapeutic antibody in suppressing TNF-α action in many autoimmune diseases, was able to reverse the diabetic metabolic state. With increasing blood glucose concentrations during the disease progression, however, the proapoptotic pressure on the residual β-cell mass increased, ultimately reaching a point where the reservoir of the surviving β-cells was insufficient to allow a restoration of normal β-cell mass through regeneration. The present results may open a therapeutic window for reversal of diabetic hyperglycemia in patients, worthwhile of being tested in clinical trials.

A novel Dock8 gene mutation confers diabetogenic susceptibility in the LEW.1AR1/Ztm-iddm rat, an animal model of human type 1 diabetes.

Aims/hypothesisThe LEW.1AR1-iddm rat, an animal model of human type 1 diabetes, arose through a spontaneous mutation within the inbred strain LEW.1AR1. A susceptibility locus (Iddm8) on rat chromosome 1 (RNO1) has been identified previously, which is accompanied by autoimmune diabetes and the additional phenotype of a variable CD3+ T cell frequency.MethodsIn the present study we characterised the Iddm8 region on RNO1 in backcross strains using the genetically divergent Brown Norway (BN) and Paris (PAR) rats. Candidate genes of the Iddm8 region were sequenced for mutation analysis.ResultsThe Iddm8 region could be subdivided by single nucleotide polymorphism (SNP) analyses. In the first region, a mutation in exon 44 of the Dock8 gene was identified resulting in an amino acid exchange in the protein from glutamine to glutamate. This exchange is unique for the LEW.1AR1-iddm rat. In the second region, a SNP was detected in exon 11 of the Vwa2 gene with an exchange from arginine to tryptophan. This SNP is also present in other rat strains.Conclusions/interpretationThe Dock8 mutation gave rise to a new type 1 diabetes rat model with very close similarity to type 1 diabetes in humans, providing a deepened insight into the impact of genes involved in diabetes development.

Variable immune cell frequencies in peripheral blood of LEW.1AR1‐iddm rats over time compared to other congenic LEW strains

The LEW.1AR1‐iddm rat is an animal model of human type 1 diabetes (T1D), which arose through a spontaneous mutation within the major histocompatibility complex (MHC)‐congenic background strain LEW.1AR1. The LEW.1AR1‐iddm rat is characterized by two phenotypes: diabetes development with a diabetes incidence of 60% and a variable T cell frequency in peripheral blood. In this study the immune cell repertoire of LEW.1AR1‐iddm rats was analysed over time from days 30 to 90 of life and compared to the background strain LEW.1AR1 and the LEW rat strain as well as the LEW.1WR1 rat strain. The LEW.1AR1‐iddm rats are characterized by a high variability of CD3+, CD4+ and CD8+ T cell frequencies in peripheral blood over time, and the frequency is unique for each animal. The variability within the frequencies resulted in changes of the CD4+ : CD8+ T cell ratio. The other three rat strains studied were characterized by a stable but nevertheless strain‐specific T cell frequency resulting in a specific CD4+ : CD8+ T cell ratio. The frequency of natural killer (NK) cells and B cells in LEW.1AR1‐iddm rats was increased, with a higher variability compared to the other strains. Only monocytes showed no differences in frequency and variability between all strains studied. These variabilities of immune cell frequencies in the LEW.1AR1‐iddm rats might lead to imbalances between autoreactive and regulatory T cells in peripheral blood as a prerequisite for diabetes development.

Changes in immune cell frequencies in primary and secondary lymphatic organs of LEW.1AR1-iddm rats, a model of human type 1 diabetes compared to other MHC congenic LEW inbred strains

The LEW.1AR1-iddm rat is an animal model of human type 1 diabetes, which arose through a spontaneous mutation in the Dock8 gene within the MHC congenic background strain LEW.1AR1. This mutation not only mediates diabetes development but also leads to a variable T cell frequency in peripheral blood. In this study, the immune cell frequencies of primary and secondary lymphatic organs of LEW.1AR1-iddm rats were analysed at days 40 and 60 and compared to other MHC congenic LEW rat strains. In LEW.1AR1-iddm rats, the secondary lymphatic organs such as lymph nodes and spleen showed a reduced, around 15% in comparison to all other strains, but very variable T cell frequency, mirroring the fluctuating T cell content in blood. On the other hand, the frequency of B cells was increased by 10% in the lymph nodes and by 5% in the spleen. Thus, the decreasing number of T cells in blood could not be caused by an increase of T cells in secondary lymphatic organs. The frequency of single- or double-positive T cells in the thymus was unaffected. The T cell frequencies in the other analysed strains were more stable and mostly higher in all secondary lymphatic organs. Obviously, the Dock8 mutation leads to variabilities of T cell frequencies in blood as well as in secondary lymphatic organs. In conclusion, the Dock8 mutation was responsible for changed immune cell frequencies in different compartments and together with the RT1B/Du haplotype causing immune imbalances and development of autoimmune diabetes.