Immune Response to Insulin and Changes in the Gut Immune System in Children with or at Risk for Type 1 Diabetes

Type 1 diabetes (T1D), an insulin-dependent diabetes, is considered to be an autoimmune disease. The cause of T1D is the destruction of insulin-producing cells in the pancreatic islets and thus lifelong insulin treatment is necessary. The pancreatic islets are infiltrated with the cells of the immune system and the destruction is considered to be T-cell-mediated. The autoimmune nature of T1D is characterized by the presence of autoreactive T-cells and autoantibodies against cell molecules in the circulation of the patients. Autoantibodies to islet cells and to islet cell derived autoantigens, such as insulin, are used together with genetic screening in the prediction of the disease. Insulin is the only -cell-specific autoantigen associated with T1D but the insulin autoantibodies (IAAs) are difficult to measure with proper sensitivity. T-cell assays for detection of autoreactive Tcells, such as insulin-specific T-cells, have also proven to be difficult to perform. The genetic risk of T1D is associated with the human leukocyte antigen (HLA) class II gene region as is many other autoimmune diseases. However, genetic risk is only part of the risk of developing T1D: environmental factors also play an important role. The most studied environmental risk factors of T1D are enteroviruses and cow’s milk. Both of these factors affect the immune system through the gut and there is increasing evidence suggesting that the gut immune system plays a role in the development of T1D. One hypothesis is that the insulin-specific immune response develops against bovine insulin in cow’s milk during early infancy and later spreads to include human insulin, developing into a condition with autoaggressive characteristics. The aims of this study were to determine whether the separation of immunoglobulin (Ig)G from plasma would improve the sensitivity of the IAA assay and how insulin treatment affects the cellular immune response to insulin in newly diagnosed patients. Furthermore, the effect of insulin concentration in mother’s breast milk on the development of antibodies to dietary insulin in the child was also examined. Small intestinal biopsies were also obtained from children with T1D to characterize any immunological changes associated with T1D in the gut. The isolation of the IgG fraction from the plasma of T1D patients negative for plasma IAAs led to detectable IAA levels that exceeded those in the control children. Thus the isolation of IgG and dissociation of immune complexes may improve the sensitivity of the IAA assay. The effect of insulin treatment on insulin-specific T-cells was studied by culturing peripheral blood mononuclear cells in vitro with both human and bovine insulin. The insulin stimulation in vitro induced higher expression of regulatory T-cell markers at the messenger ribonucleic acid (mRNA) level in those patients treated with insulin than in patients examined before initiating insulin treatment. The insulin-induced expression of the regulatory T-cell marker Foxp3 was also demonstrated at the protein level. This finding suggests that insulin treatment in patients with T1D stimulates regulatory T-cells in vivo and this may partly explain the difficulties in measuring autoantigen-specific T-cell responses in recently diagnosed patients. The stimulation of regulatory T-cells by insulin treatment may also explain the remission period often seen after initiating insulin treatment. In the third study we showed that the insulin concentration in breast milk is increased in mothers affected with T1D. The insulin concentration in mother’s breast milk also correlated inversely with the levels of bovine insulin-specific antibodies in those children who were exposed to cow’s milk proteins in their diet. This finding suggests that human insulin in breast milk induces tolerance to dietary bovine insulin. However, in infants who later developed T1D-associated autoantibodies, the insulin concentration in their mother’s breast milk was increased. This finding may indicate that in those children prone to -cell autoimmunity, breast milk insulin does not promote tolerance to insulin but may in contrast enhance insulin-specific immunity. In the small intestinal biopsies the presence of several immunological markers, including markers for effector and regulatory T-cells, were quantified with the reverse transcriptase-polymerase chain reaction (RT-PCR). From these markers the expression of the interleukin (IL)-18 cytokine was significantly increased in the gut in patients with T1D compared with children with celiac disease or control children. The expression of regulatory T-cell markers in the intestine was not increased in children with T1D despite the presence of low-grade inflammation. The increased IL-18 expression lends further support for the hypothesis that the gut immune system is involved in the pathogenesis of T1D, whereas no activation of regulatory T-cells is seen in the intestinal immune activation related to T1D.