The specific p38 mitogen-activated protein kinase pathway inhibitor FR167653 keeps insulitis benign in nonobese diabetic mice

The p38 mitogen-activated protein kinase (MAPK) pathway is important in Th1 immunity, macrophage activation, and apoptosis. Since they may be associated with beta-cell destruction during the development of type 1 diabetes, we investigated the role of the p38 MAPK pathway in female nonobese diabetic (NOD) mice. Phosphorylated p38 MAPK was observed immunohistochemically in CD4+ cells that had infiltrated into the islets and part of beta-cells, increasing in proportion to the severity of insulitis. Continuous oral administration of 0.08% FR167653, a specific p38 MAPK pathway inhibitor, significantly reduced the ex vivo production of interferon-gamma by splenic Th1 cells without affecting interleukin-4 production by Th2 cells. FR167653 administration from 4-30 weeks of age prevented NOD mice from developing diabetes without affecting the severity of insulitis. Treatment with FR167653 after insulitis had developed (i.e. from 10-30 weeks of age) also prevented diabetes, further suggesting that treatment with the p38 MAPK pathway inhibitor keeps insulitis benign in NOD mice, partly by inhibiting Th1 immunity. These findings suggest that p38 MAPK is a key mediator that switches insulitis from benign to destructive in the development of type 1 diabetes.     

TSG-6 protein expression in the pancreatic islets of NOD mice

The histologic hallmark of the development of type 1 diabetes (T1D) is insulitis, characterized by leukocytic infiltration of the pancreatic islets. The molecules controlling the early influx of leukocytes into the islets are poorly understood. Tumor necrosis factor α (TNFα)-stimulated gene 6 (TSG-6) is involved in inflammation, extracellular matrix formation, cell migration, and development. In the present study, we examined the expression and cellular localization of TSG-6 protein in islets of female non-obese diabetic (NOD) mice using frozen section immunofluorescence staining. Pancreata from nondiabetic (8 and 25 weeks old), prediabetic (230–280 mg/dl blood glucose) and diabetic (>300 mg/dl blood glucose) NOD mice were stained for TSG-6, insulin, CD3, CD11c, Mac3 and CD31. TSG-6 protein was detected in 67% of islets of prediabetic mice, 27% of islets of 25-week old nondiabetic mice, and less than 7% of islets of diabetic mice and 8-week old nondiabetic mice. Lastly, islet-derived TSG-6 protein was localized to the infiltrating CD3 and CD11c positive leukocytes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Combined insulin B:9-23 self-peptide and polyinosinic-polycytidylic acid accelerate insulitis but inhibit development of diabetes by increasing the proportion of CD4+Foxp3+ regulatory T cells in the islets in non-obese diabetic mice

Insulin peptide B:9-23 is a major autoantigen in type 1 diabetes. Combined treatment with B:9-23 peptide and polyinosinic-polycytidylic acid (poly I:C), but neither alone, induce insulitis in normal BALB/c mice. In contrast, the combined treatment accelerated insulitis, but prevented diabetes in NOD mice. Our immunofluorescence study with anti-CD4/anti-Foxp3 revealed that the proportion of Foxp3 positive CD4⁺CD25⁺ regulatory T cells (Tregs) was elevated in the islets of NOD mice treated with B:9-23 peptide and poly I:C, as compared to non-treated mice. Depletion of Tregs by anti-CD25 antibody hastened spontaneous development of diabetes in non-treated NOD mice, and abolished the protective effect of the combined treatment and conversely accelerated the onset of diabetes in the treated mice. These results indicate that poly I:C combined with B:9-23 peptide promotes infiltration of both pathogenic T cells and predominantly Tregs into the islets, thereby inhibiting progression from insulitis to overt diabetes in NOD mice.     

Presence of residual beta cells and co-existing islet autoimmunity in the NOD mouse during longstanding diabetes: a combined histochemical and immunohistochemical study

During type 1 diabetes, most beta cells die by immune processes. However, the precise fate and characteristics of beta cells and islet autoimmunity after onset are unclear. Here, the extent of beta cell survival was determined in the non-obese diabetic (NOD) mouse during increasing duration of disease and correlated with insulitis. Pancreata from female NOD mice at diagnosis and at 1, 2, 3 and 4 weeks thereafter were analysed immunohistochemically for insulin, glucagon and somatostatin cells and glucose transporter-2 (glut2) and correlated with the degree of insulitis and islet immune cell phenotypes. Insulitis, although variable, persisted after diabetes and declined with increasing duration of disease. During this period, beta cells also declined sharply whereas glucagon and somatostatin cells increased, with occasional islet cells co-expressing insulin and glucagon. Glut2 was absent in insulin-containing cells from 1 week onwards. CD4 and CD8 T cells and macrophages persisted until 4 weeks, in islets with residual beta cells or extensive insulitis. We conclude that after diabetes onset, some beta cells survive for extended periods, with continuing autoimmunity and expansion of glucagon and somatostatin cells. The absence of glut2 in several insulin-positive cells suggests that some beta cells may be unresponsive to glucose.