Importance of immune-cell lipid signaling in events leading to type 1 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，并且该过程的根本原因尚未完全了解。我们的工作表明，巨噬细胞和CD 4 + T细胞提供的新型脂质信号传导影响T1 D发病率。特别是，相关脂质似乎是由免疫细胞中表达的Ca 2+非依赖性磷脂酶A2β（iPLA 2 β）产生的。iPLA 2 β在sn-2位水解膜磷脂以释放溶血磷脂和脂肪酸。当脂肪酸是花生四烯酸时，它可以代谢产生生物活性氧化脂质或类二十烷酸，其中许多是促炎性的。我们发现，（a）当给予自发性糖尿病易感的非肥胖糖尿病（NOD）小鼠时，iPLA 2 β选择性抑制剂保留了β细胞质量，降低了T1 D发病率和胰岛炎，（B）iPLA 2 β活性促进M1巨噬细胞促炎表型和CD 4 + T细胞产生TNFα，（c）巨噬细胞产生选定的促炎类花生酸（PGE 2、白三烯、12-HETE、DH 3）在NOD中增加，在具有减少的iPLA 2 β的巨噬细胞中减少（NOD.iPLA2β-/+），（d）在NOD.iPLA2β-/+中T1 D发病率降低，（e）NOD.iPLA2β-/-巨噬细胞或脾细胞的过继转移降低T1 D发病率并改善葡萄糖耐量。我们假设CD 4 +/CD 8 + T细胞和巨噬细胞产生的iPLA 2 β衍生脂质（iDL）在T1 D发展中起关键作用，并将在以下目标下解决这一问题：确定CD 4 +/CD 8 + T细胞iDL对T1 D的贡献。我们建议利用来自WT和iPLA 2 β缺陷型NOD的CD 4+和CD 8 + T细胞制备物来（a）确定糖尿病诱导对T细胞iDL的需求，（B）定量T细胞脂质产生并鉴定iDL，（c）评估这些选择的iDL对胰岛功能和存活的影响。2.确定巨噬细胞iDL对T1 D的贡献。我们建议（a）利用来自WT和iPLA 2 β缺陷型NOD的巨噬细胞制备物来确定巨噬细胞iDL对糖尿病诱导的时间需求，（B）评估具有巨噬细胞iPLA 2 β选择性缺陷的NOD中的糖尿病发展，（c）确定选择的巨噬细胞iDL对胰岛功能和存活的影响。3.评估CD 4 +/CD 8 + T细胞或巨噬细胞来源的iDL对胰岛iPLA 2 β的影响。初步结果表明，iPLA 2 β在应激β细胞中由NFκB诱导，并且已报道PGE 2诱导NFκB。我们将测试巨噬细胞和/或CD 4 +/CD 8 + T细胞iDL诱导β细胞iPLA 2 β的可能性，预期其将参与维持和放大免疫应答。我们将利用过继转移、系统水平脂质组学、条件性敲除、免疫印迹、信息、IF和选择性抑制脂质生成酶方案来解决这些目标。R21目标。短期内鉴定和评价对T1 D发展至关重要的CD 4 +/CD 8 + T细胞和巨噬细胞iDL。太过分了这可能会导致在免疫治疗的背景下，靶向和防止这些脂质的产生或修饰免疫细胞中负责脂质产生的酶的策略。

Type 1 diabetes (T1D) is a consequence of autoimmune destruction of pancreatic islet β-cells and the underlying causes for this process are incompletely understood. Our work suggests novel lipid signaling provided by macrophages and CD4+ T-cells impacts T1D incidence. In particular, the relevant lipids appear to be generated by the Ca2+-independent phospholipase A2β (iPLA2β), which is expressed in immune cells. iPLA2β hydrolyzes membrane phospholipids at the sn-2 position to release a lysophospholipid and a fatty acid. When the fatty acid is arachidonic acid, it can be metabolized to generate bioactive oxidized lipids, or eicosanoids, many of which are pro-inflammatory. We find that (a) an iPLA2β-selective inhibitor, when administered to spontaneous diabetes-prone non-obese diabetic (NOD) mice preserves β-cell mass, reduces T1D incidence and insulitis, (b) iPLA2β activity promotes M1 macrophage pro-inflammatory phenotype and TNFα production from CD4+ T-cells, (c) macrophage production of select pro-inflammatory eicosanoids (PGE2, leukotrienes, 12-HETE, DHETs) is increased in NOD and reduced in macrophages with reduced iPLA2β (NOD.iPLA2β-/+), (d) T1D incidence is reduced in NOD.iPLA2β-/+, (e) adoptive transfer of NOD.iPLA2β-/- macrophages or splenocytes decreases T1D incidence and improves glucose tolerance. We hypothesize that iPLA2β-derived lipids (iDLs) produced by CD4+/CD8+ T-cells and macrophages play critical roles in T1D development and will address this under the following Aims: 1. Determine the contribution of CD4+/CD8+ T-cell-iDLs to T1D. We propose to utilize CD4+ and CD8+ T-cell preparations from WT and iPLA2β-deficient NOD to (a) determine the requirement of T-cell iDLs for diabetes induction, (b) quantitate T-cell lipid production and identify the iDLs, (c) assess the impact of these select iDLs on islet function and survival. 2. Determine the contribution of macrophage-iDLs to T1D. We propose to (a) utilize macrophage preparations from WT and iPLA2β-deficient NOD to determine the temporal requirement of macrophage iDLs on diabetes induction, (b) assess diabetes development in NOD with selective deficiency in macrophage iPLA2β, (c) determine the impact of select macrophage iDLs on islet function and survival. 3. Assess the impact of CD4+/CD8+ T-cell-or macrophage-derived iDLs on islet iPLA2β. Preliminary results suggest that iPLA2β is induced in stressed β-cells by NFκB, and PGE2 has been reported to induce NFκB. We will test the possibility that macrophage and/or CD4+/CD8+ T-cell iDLs induce β-cell iPLA2β, which would be expected to participate in maintaining and amplifying immune responses. We will utilize adoptive transfer, systems level lipidomics, conditional knockouts, immunoblotting, message, IF, and select inhibition of lipid-generating enzymes protocols to address these Aims. R21 Goals. Short-term. Identify and evaluate CD4+/CD8+ T-cell and macrophage iDLs critical for T1D development. Overarching. This could lead to strategies to target and prevent generation of those lipids or modify the responsible lipid-generating enzyme in immune cells, in the context of immunotherapy.