Methylation of insulin DNA in response to proinflammatory cytokines during the progression of autoimmune diabetes in NOD mice

Abstract

Aims/hypothesis Type 1 diabetes is caused by the immunological destruction of pancreatic beta cells. Preclinical and clinical data indicate that there are changes in beta cell function at different stages of the disease, but the fate of beta cells has not been closely studied. We studied how immune factors affect the function and epigenetics of beta cells during disease progression and identified possible triggers of these changes. Methods We studied FACS sorted beta cells and infiltrating lymphocytes from NOD mouse and human islets. Gene expression was measured by qRT-PCR and methylation of the insulin genes was investigated by high-throughput and Sanger sequencing. To understand the role of DNA methyltransferases, Dnmt3a was knocked down with siRNA. The effects of cytokines on methylation and expression of the insulin gene were studied in humans and mice. Results During disease progression in NOD mice, there was an inverse relationship between the proportion of infiltrating lymphocytes and the beta cell mass. In beta cells, methylation marks in the Ins1 and Ins2 genes changed over time. Insulin gene expression appears to be most closely regulated by the methylation of Ins1 exon 2 and Ins2 exon 1. Cytokine transcription increased with age in NOD mice, and these cytokines could induce methylation marks in the insulin DNA by inducing methyltransferases. Similar changes were induced by cytokines in human beta cells in vitro. Conclusions/interpretation Epigenetic modification of DNA by methylation in response to immunological stressors may be a mechanism that affects insulin gene expression during the progression of type 1 diabetes.

Biomarkers of β-Cell Stress and Death in Type 1 Diabetes

“…Notably, Yang et al [82] demonstrated that islets from subjects with type 2 diabetes showed greater methylation at several CpG sites compared to islets from control subjects, suggesting that β-cells might exhibit increasing methylation of the INS gene with progression of disease states. In agreement with this observation, a recent report by the Herold group [87•] showed that the methylation status of the Ins1/Ins2 gene in the β-cell becomes more methylated with disease progression in the NOD mouse.…”

β Cells that Resist Immunological Attack Develop during Progression of Autoimmune Diabetes in NOD Mice

“…The relatively rapid appearance of the subpopulation most likely reflects epigenetic changes in these cells, but further studies will be needed to address this mechanism. Our previous studies showed that cytokines may cause changes in DNA methyltransferases and Ins1/2 DNA methylation (Rui et al, 2016), and we found increased expression of several inflammatory pathways in Btm cells by RNA-seq, including HMGB1(p = 2.11E00), Cxcl15(p = 1.91E00), and IL-6 (p = 2.18E00), among others. Epigenetic changes in other β cell genes are likely to occur, but further study will be needed to identify these mechanisms.…”

“…Dissociated islet cells were stained with FluoZin and tetramethylrhodamine ethyl ester perchlorate (TMRE) to identify viable β cells (Jayaraman, 2011; Rui et al, 2016) and with anti-CD45 monoclonal antibody (mAb) to identify islet infiltrates. Representative β cell profiles showing side scatter (SSC) and forward scatter (FSC) in mice of different ages are shown (Figure 1A).…”

Progress and challenges for treating Type 1 diabetes

“…In addition, in NOD mice, epigenetic changes are induced in β cells by immune mediators (88). The consequences of these and other alterations may include impaired synthetic function but studies with other mediators of cellular stress raise the possibility that cellular transdifferentiation may account for β cell failure (89, 90).…”