Epigenetic modulation of β-cells by interferon-α via PNPT11-miR-26a-TET2 triggers autoimmune diabetes [RNA-seq]

Type 1 diabetes (T1D) is caused by autoimmune destruction of pancreatic β cells. Mounting evidence supports a central role for β-cell alterations in triggering the activation of self-reactive T-cells in T1D. However, the early deleterious events that occur in β cells, underpinning islet autoimmunity are not known. We hypothesized that epigenetic modifications induced in β cells by inflammatory mediators play a key role in initiating the autoimmune response. We analyzed DNA methylation (DNAm) patterns and gene expression in human islets exposed to IFNα, a cytokine associated with T1D development. We found that IFNα triggers DNA demethylation and increases expression of genes controlling inflammatory and immune pathways. We then demonstrated that DNA demethylation was caused by up-regulation of the exoribonuclease, PNPase Old-35 (PNPT1), which caused degradation of miR-26a. This in turn promoted the up-regulation of ten-eleven translocation TET2 enzyme and increased 5-hydoxymethylcytosine levels in human islets and pancreatic β-cells. Moreover, we showed that specific IFNα expression in the β cells of IFNα-INS1CreERT2 transgenic mice, led to development of T1D that was preceded by increased islet DNA hydroxymethylation through a PNPT1/TET2-dependent mechanism. Our results suggest a new mechanism through which IFNα regulates DNAm in β cells, leading to changes in expression of genes in inflammatory and immune pathways that can initiate islet autoimmunity in T1D. We exposed human pancreatic islets from three donors to 2000 IU IFNα and assessed gene expression by RNAseq. The cDNA library was prepared using Illumina TruSeq RNA Sample Prep Kits. Next generation sequencing was performed on Illumina HiSeq2000 using the Single-Read Cluster Generation kit v2 and SBS Sequencing kit v3. Image analysis and base calling were conducted using the SDS 2.5/RTA1.5 software.

1型糖尿病（Type 1 diabetes, T1D）是由胰腺β细胞自身免疫破坏所引发的疾病。越来越多的研究证据表明，β细胞异常在触发T1D中自身反应性T细胞活化的过程中发挥核心作用。然而，支撑胰岛自身免疫发生的β细胞早期有害事件仍未明确。本研究推测，炎症介质诱导β细胞产生的表观遗传修饰，在启动自身免疫应答的过程中扮演关键角色。我们对暴露于干扰素α（IFNα，一种与T1D发病相关的细胞因子）的人胰岛样本开展了DNA甲基化（DNA methylation, DNAm）谱与基因表达分析。研究发现，IFNα可诱导DNA去甲基化，并上调调控炎症与免疫通路的基因表达。后续研究证实，DNA去甲基化源于外核糖核酸酶PNPase Old-35（PNPT1）的表达上调，该酶可介导miR-26a的降解。这进而促进了十-十一易位酶TET2（ten-eleven translocation TET2）的表达上调，并提升了人胰岛与胰腺β细胞中的5-羟甲基胞嘧啶（5-hydoxymethylcytosine）水平。此外，本研究证实，在IFNα-INS1CreERT2转基因小鼠的β细胞中特异性表达IFNα，可诱导T1D发病，且胰岛DNA羟甲基化水平升高先于T1D发生，该过程依赖PNPT1/TET2介导的机制。本研究结果揭示了IFNα调控β细胞DNAm的全新机制，该机制可通过改变炎症与免疫通路基因的表达，进而启动T1D中的胰岛自身免疫反应。我们将3名供者来源的人胰腺胰岛暴露于2000国际单位的IFNα中，并通过RNA测序（RNAseq）评估基因表达情况。cDNA文库构建采用Illumina TruSeq RNA样本制备试剂盒完成。后续在Illumina HiSeq2000测序平台上，使用Single-Read Cluster Generation kit v2与SBS Sequencing kit v3开展二代测序。图像分析与碱基识别工作通过SDS 2.5/RTA1.5软件完成。