Contribution of β-Cell- & Immune Cell-Derived Lipids to β-Cell Death and Diabetes

1型糖尿病（T1 D）是胰岛β细胞的自身免疫性破坏的结果， 这一过程的根本原因尚未完全了解。脂质的作用尚待确定 在这个过程中产生的β细胞和免疫细胞。胰岛β细胞和免疫细胞表达Ca 2 +- 独立的磷脂酶A2β（iPLA 2 β），在sn-2位水解膜磷脂， 产生脂质，促进炎症反应。我们假设iPLA 2 β衍生脂质（iDL） 提供关键信号，将免疫细胞、β细胞和ER应激与β细胞死亡联系起来， 自身免疫性糖尿病作为支持，我们发现（a）iPLA 2 β由促炎细胞因子诱导， 通过iPLA 2 β抑制、（B）β细胞和免疫抑制逆转苦参碱介导的ER应激和β细胞凋亡。 自身免疫性糖尿病自发模型（非肥胖糖尿病小鼠，NOD）中的细胞表达较高的iPLA 2 β 在糖尿病前期，（c）向NOD小鼠施用iPLA 2 β-选择性抑制剂（FKGK 18 保留β-细胞群并降低糖尿病发病率、胰岛炎和自身免疫， 具有FKGK 18的免疫缺陷小鼠通过T细胞减少糖尿病的过继转移，（e）FKGK 18的过表达， β细胞中的iPLA 2 β加速并增加NOD中糖尿病的发病率。（f）NOD中iPLA 2 β的减少 小鼠模拟FKGK 18给药的保护作用。（g）M1炎性巨噬细胞 iPLA 2 β缺乏时，极化降低;（h）iPLA 2 β与ER之间存在反馈调节 以及炎症相关转录因子与iPLA 2 β之间的关系。我们的假设将被测试， 整个动物（在NOD或免疫缺陷-NOD背景下具有改变的iPLA 2 β表达的小鼠模型）， 基于机制的体外和翻译（人T1 D和自身抗体阳性，但非糖尿病受试者） 方法通过以下目的：1.描述免疫细胞产生的iDL对 自身免疫性糖尿病2.描述β细胞产生的iDL对自身免疫性疾病的影响 糖尿病发展3.描述iDL诱导自身免疫性β细胞死亡的细胞机制 糖尿病4.评估iDL对人类T1 D发展的贡献。这些目标将包括 自身免疫性糖尿病发展过程中胰岛和免疫细胞脂质体的产生;过继转移 区分免疫细胞和β细胞产生的iDL对糖尿病发展的重要性的方案; 评估iDL对胰岛抗原性影响的共培养试验;蛋白质和信息分析，以解决 ER应激，iDL和炎症之间的关系; ChIP和CRISPR/Cas9分析以检查转录 iPLA 2 β的调节;和人类受试者，以评估iPLA 2 β作为T1 D易感性生物标志物的潜力。 我们的长期目标是阐明iPLA 2 β衍生的脂质信号有助于 T1 D的发病机制，因此，新的目标，可能是候选药物干预，以对付T1 D 可以开发。

Type 1 diabetes (T1D) is a consequence of autoimmune destruction of pancreatic islet β-cells and the underlying causes for this process are not completely understood. Yet to be defined are the roles of lipids generated in β-cells and immune cells in this process. Islet β-cells and immune cells express Ca2+- independent phospholipase A2β (iPLA2β), which hydrolyzes membrane phospholipids at the sn-2 position to generate lipids that can promote inflammatory responses. We hypothesize that iPLA2β-derived lipids (iDLs) provide critical signals linking immune cells, β-cells, and ER stress with β-cell death associated with autoimmune diabetes. In support, we find that (a) iPLA2β is induced by pro-inflammatory cytokines and cytokine-mediated ER stress and β-cell apoptosis are reversed by iPLA2β inhibition, (b) β-cells and immune cells in a spontaneous model of autoimmune diabetes (non-obese diabetic mice, NOD) express higher iPLA2β during the pre-diabetic phase, (c) administration of an iPLA2β-selective inhibitor (FKGK18) to NOD mice preserves β-cells mass and reduces diabetes incidence, insulitis, and autoimmunity, (d) pre-treatment of immunodeficient mice with FKGK18 reduces adoptive transfer of diabetes by T-cells, (e) overexpression of iPLA2β in β-cells accelerates and increases the incidence of diabetes in NOD, (f) reduction of iPLA2β in NOD mice mimics the protective effects seen with FKGK18 administration, (g) M1 inflammatory macrophage polarization is reduced with iPLA2β deficiency, and (h) there is feedback regulation between iPLA2β and ER stress, and between inflammation-related transcription factors and iPLA2β. Our hypothesis will be tested using whole animal (mouse models with altered iPLA2β expression on a NOD or immunodeficient-NOD background), mechanism-based in vitro, and translational (human T1D and autoantibody positive but not diabetic subjects) approaches through the following Aims: 1. Delineate the impact of iDLs generated by immune cells on autoimmune diabetes development. 2. Delineate the impact of iDLs generated by β-cells on autoimmune diabetes development. 3. Delineate the cellular mechanisms by which iDLs induce β-cell death in autoimmune diabetes. 4. Assess the contribution of iDLs to human T1D development. These Aims will encompass generation of islet and immune cell lipidome during autoimmune diabetes development; adoptive transfer protocols to distinguish importance of iDLs generated by immune cells and β-cells to diabetes development; co-culture assays to assess impact of iDLs on islet antigenicity; protein and message analyses to address link between ER stress, iDLs, and inflammation; ChIP and CRISPR/Cas9 analyses to examine transcriptional regulation of iPLA2β; and human subjects to asses the potential of iPLA2β as a biomarker of T1D susceptibility. Our long-term goal is to elucidate underlying mechanisms by which iPLA2β-derived lipid signals contribute to the pathogenesis of T1D, so that novel targets that might be candidates for drug intervention to counter T1D can be developed.