Adaptive epigenetic mechanisms of beta and immune cells in autoimmune diabetes

项目摘要 1型糖尿病(T1D)是一种慢性自身免疫性疾病，导致产生胰岛素的β细胞被破坏 在临床表现之前和之后的一段时间内。然而，并不是所有的β细胞都被杀死了，因为 对长期患有T1D的人的UP研究发现，即使在T1D发生多年后仍有剩余的胰岛素产生 疾病的发作。这项工作的前提是，基于这些和其他观察，存在自适应的 β细胞对免疫攻击的反应，可能会阻止它们的破坏。我们之前发现， 一些β细胞经历了“去分化”，β细胞转录因子表达水平降低， 免疫原性，并受到保护而不被杀害。该提案的总体目标是确定适应性变化 在β细胞中，并使用这些信息来提高它们在免疫攻击背景下的存活率。我们在那里发现了 在人和小鼠β中，表观基因组修饰物DNMT3a和TET2的表达增加 细胞在体外，在炎性细胞因子暴露期间，或在体内，在自身免疫期间。我们发现增加了 自身免疫性胰腺炎患者胰岛β细胞中TET2的表达及T1D的表达 然而，从我们对NOD小鼠糖尿病进展过程中β细胞的分析来看，那些抵抗自身免疫的小鼠 杀伤可降低TET2的表达。TET2诱导CpG位点甲基化，这是 将压抑标记转化为允许的表观遗传标记的第一步。我们创造了TET2/-NOD小鼠和领养小鼠 移植研究、骨髓移植和炎性细胞因子与糖尿病的直接培养 免疫细胞研究表明，TET2基因的缺失可以阻止对β细胞的自身免疫杀伤。我们的数据表明， TET2基因缺陷的β细胞不仅能抵抗免疫杀伤，还能调节自身免疫反应。我们 假设TET2可以修饰β细胞并影响其对自身免疫杀伤的敏感性，并计划对此进行测试 小鼠模型系统和人类细胞中的假说。我们将在单细胞的基础上分析转录组 和来自WT和TET2-/-小鼠的β细胞的表观基因组。我们将鉴定DNA结合序列 TET2在β细胞中的表达。为了明确TET2在β细胞中的作用并确定其与时间的关系 TET2表达与杀伤易感性的关系我们将建立组织特异性缺失TET2的小鼠，并诱导 在T1D的整个发育过程中，有时会发生缺失。我们将分析TET2中免疫细胞的差异 足够的和-/-老鼠。在第二个目标中，我们将分析TET2的表达及其相关基因的表达和 NPOD、自身免疫性胰腺炎患者和对照组样本中的表观遗传学特征 研究对象。最后，我们将评估TET2在人类胚胎干细胞来源的β细胞中的表达 在体外和移植到小鼠体内后没有TET2的表达。这些研究将确定 TET2可以控制β细胞对免疫攻击的反应并可能识别一条预防免疫攻击的途径 它们的破坏适用于临床环境。

Project Summary Type 1 diabetes (T1D) is a chronic autoimmune disease that lead to the destruction of insulin producing β cells over a period of years before clinical presentation and afterwards. However, not all β cells are killed since follow- up studies of individuals with long standing T1D have identified residual insulin production even years after the onset of disease. The premise of this work, based on these and other observations, is that there are adaptive responses of the β cells to the immunologic attack that may prevent their destruction. We previously found that some β cells undergo “dedifferentiation”, express lower levels of β cell transcription factors, have reduced immunogenicity, and are protected from killing. The overall goal of this proposal is to identify adaptive changes in β cells and use this information to enhance their survival in the setting of immune attack. We found that there was increased expression of modifiers of the epigenome such as DNMT3a and Tet2 in human and murine β cells in vitro, during exposure to inflammatory cytokines, or in vivo during autoimmunity. We found increased expression of TET2 in β cells in islets from humans with autoimmune pancreatitis and T1D in nPOD samples. However, from our analysis of β cells during diabetes progression in NOD mice, those that resist autoimmune killing have decreased expression of Tet2. Tet2 induces hydroxymethylation of methylated CpG site which is the first step in converting a repressive to permissive epigenetic mark. We created Tet2-/- NOD mice and in adoptive transfer studies, bone marrow transplants and direct cultures with inflammatory cytokines and diabetogenic immune cells showed that deletion of Tet2 prevents autoimmune killing of β cells. Our data indicates that the Tet2-deficient β cells are not only resistant to immune killing but also modify the autoimmune responses. We hypothesize that Tet2 can modify β cells and affect their susceptibility to autoimmune killing and plan to test this hypothesis in murine model systems and human cells. We will analyze on a single cell basis the transcriptome and epigenome (by ATACseq) of β cells from WT and Tet2-/- mice. We will identify the DNA binding sequences of Tet2 in β cells. To specifically identify the role of Tet2 in β cells and determine the relationship between timing of Tet2 expression and susceptibility to killing we will create mice with tissue specific deletion of Tet2 and induce the deletion at times throughout the development of T1D. We will analyze the differences in immune cells in Tet2 sufficient and -/- mice. In the 2nd aim we will analyze TET2 expression and associated gene expression and epigenetic signatures in human samples from nPOD, patients with autoimmune pancreatitis, and control subjects. Finally, we will assess the role of TET2 expression in human embryonic stem cell-derived- β cells with and without TET2 expression in vitro and after transplantation into mice. These studies will determine the mechanisms whereby TET2 can control β cell responses to immune attack and may identify a pathway to prevent their destruction that is applicable to clinical settings.