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

1型糖尿病(T1D)是胰岛β细胞自身免疫破坏的结果， 这一过程的根本原因尚不完全清楚。尚未确定的是脂质的作用 在这一过程中产生于β-细胞和免疫细胞。胰岛β-细胞和免疫细胞表达钙离子 独立磷脂酶A2β(iPLA2β)，它能将sn-2位的膜磷脂水解成 产生能促进炎症反应的脂类。我们假设iPLA2β衍生的脂类(IDL) 提供关键信号，将免疫细胞、β细胞和内质网应激与β细胞死亡联系起来 自身免疫性糖尿病。在Support中，我们发现：(A)iPLA2β是由促炎细胞因子和 细胞因子介导的内质网应激和β细胞凋亡被iPLA2β抑制逆转，(B)β细胞和免疫 自发性自身免疫性糖尿病模型(非肥胖糖尿病小鼠，NOD)的细胞表达较高的iPLA2β 在糖尿病前期，(C)给NOD小鼠注射iPLA2β选择性抑制剂(FKGK18) 保留β细胞团并减少糖尿病发病率、胰岛素炎和自身免疫，(D) 带有FKGK18的免疫缺陷小鼠减少T细胞过继转移糖尿病，(E)过表达 IPLA2β在β细胞中的表达加速并增加NOD中糖尿病的发病率，(F)NOD中iPLA2β的减少 小鼠模拟给予FKGK18，(G)M1炎性巨噬细胞的保护作用 IPLA2β缺失导致极化降低；(H)iPLA2β与ER之间存在反馈调节 应激，以及炎症相关转录因子与iPLA2β之间的关系。我们的假设将通过以下方式进行验证 整个动物(在NOD或免疫缺陷NOD背景下iPLA2β表达改变的小鼠模型)， 基于机制的体外和翻译(人T1D和自身抗体阳性但不是糖尿病患者) 方法通过以下目标：1.描绘免疫细胞产生的IDL对 自身免疫性糖尿病的发展。2.描述β-细胞产生的IDL对自身免疫的影响 糖尿病的发展。3.阐明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T1D发育的贡献。这些目标将包括 自身免疫性糖尿病发生过程中胰岛和免疫细胞类脂体的生成；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；处理链接的蛋白质和消息分析 内质网应激、IDLS和炎症之间；CHIP和CRISPR/CAS9分析以检查转录 IPLA2β的调节；以及人类受试者评估iPLA2β作为T1D易感性生物标志物的潜力。 我们的长期目标是阐明iPLA2β衍生的脂质信号对 T1D的发病机制，使可能成为抗T1D药物干预的新靶点 可以被开发出来。

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.