Exploiting iPLA2β-modified macrophages as immunotherapy for T1D

项目摘要 1型糖尿病（T1 D）是一种自身免疫性疾病，其特征在于胰腺b细胞的破坏， 然而，导致b细胞破坏的机制尚未完全了解。我们发现了一个 巨噬细胞（MΦ）产生的促炎脂质在T1 D发作中的作用。这些脂质是由 Ca 2+非依赖性磷脂酶A2β（iPLA 2b）的激活。作为PLA 2家族的一员，iPLA 2b 在sn-2位水解膜磷脂以释放脂肪酸，例如花生四烯酸（AA）， 其可代谢成生物活性氧化脂质（类二十烷酸）。这些脂质中有几种是深刻的 我们发现它们在糖尿病前期显著升高，这表明它们在糖尿病早期可能是促炎性的。 T1 D发病的关键贡献。MΦ是最先浸润胰岛的细胞之一，它们可以被诱导成一个细胞， 促炎（M1）或抗炎（M2）表型。T1 D以MΦM1为主。我们发现抑制 或iPLA 2b的遗传性整体减少减少了MΦ产生的促炎性iPLA 2b衍生的脂质（piDL） 在糖尿病前期，不利于MΦM1，并降低非肥胖者的胰岛炎和T1 D发病率 自发性糖尿病易感（NOD）小鼠。重要的是，非糖尿病儿童患T1 D的风险很高， 显示出相似的血脂特征。这些发现提出了通过减少MΦ-iPLA 2b， 可以减轻MΦM1的产生和诱导，这将有利于预防或延迟T1 D 发病在piDL中有DHETE，其通过CYP 450/可溶性环氧化物水解酶在iPLA 2b下游产生 （sEH）.据报道，sEH抑制可缓解炎症，提供了一个选择的iDL生成靶点， 计数器T1 D。我们假设减少MΦ-piDLs将不利于MΦM1并对抗T1 D发展。 CRISPR-Cas9领域的最新进展促进了细胞特异性和选择性遗传修饰 基因.我们的研究结果提出了一种新的，尚未考虑的方案，其中使用 CRISPR-Cas9可以被开发成一种免疫疗法来对抗人类的T1 D。我们建议解决这个问题 在目标1下。评估MΦ-iDL对T1 D发展的影响。我们将确定MΦ-iDL的影响 使用具有MΦ中iPLA 2b的条件性修饰的NOD小鼠对T1 D发作和进展的影响。目标二。建立 转基因MF作为对抗T1 D发展的潜在免疫疗法。开发CRISPR- Cas9方案产生和评估来源于NOD骨髓单核细胞的MΦ的功能性， 降低iPLA 2b或sEH 1表达，并确定其对T1 D发病率的影响。意义我们的建议 提出了两个新概念：（1）MΦ产生的piDL是T1 D中b细胞死亡的重要贡献者， (2)操纵MΦ中的脂质生成酶（一个容易获得的库）可以对抗T1 D。我们觉得 利用这些将允许产生新的免疫途径来预防或延迟T1 D诊断， 临床设置。这一前提符合R21机制的高风险/高回报使命， 我们的团队可以测试，并有望在T1 D治疗领域取得重大进展。

PROJECT SUMMARY Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of pancreatic b-cells, however, the mechanisms leading to b-cells destruction are not completely understood. We identified a critical role for proinflammatory lipids produced by macrophages (MΦ) in T1D onset. Such lipids were generated by activation of the Ca2+-independent phospholipase A2β (iPLA2b). As a member of the PLA2 family, iPLA2b hydrolyzes membrane phospholipids at the sn-2 position to release a fatty acid, such as arachidonic acid (AA), which can be metabolized to bioactive oxidized lipids (eicosanoids). Several of these lipids are profoundly proinflammatory and we find that they are dramatically elevated during the pre-diabetic phase, suggesting their critical contribution to T1D onset. The MΦ are among the first to infiltrate islets and they can be induced to a proinflammatory (M1) or anti-inflammatory (M2) phenotype. In T1D, MΦM1 predominate. We find that inhibition or genetic global reduction of iPLA2b decreases proinflammatory iPLA2b-derived lipids (piDLs) production by MΦ in the pre-diabetic phase, disfavors MΦM1, and reduces insulitis and T1D incidence in the non-obese spontaneous diabetes-prone (NOD) mice. Importantly, non-diabetic children at high risk for developing T1D, exhibit a similar plasma lipid signature. These findings raise the possibility that by reducing MΦ-iPLA2b, piDLs production and induction of MΦM1 can be mitigated and that this will be beneficial in preventing or delaying T1D onset. Among the piDLs are DHETEs, generated downstream of iPLA2b via CYP450/soluble epoxide hydrolase (sEH). Inflammation relief has been reported with sEH inhibition, offering a select iDL-generating target to counter T1D. We hypothesize that reducing MΦ-piDLs will disfavor MΦM1 and counter T1D development. Recent advances in the CRISPR-Cas9 field have facilitated cell-specific and select genetic modification of genes. Our findings present a novel, and not yet considered scenario, where reducing iPLA2b in MΦ using CRISPR-Cas9 can be developed into an immunotherapy to counter T1D in humans. We propose to address this under Aim 1. Assess the impact of MΦ-iDLs on T1D development. We will determine the impact of MΦ-iDLs on T1D onset and progression using NOD mice with conditional modification of iPLA2b in MΦ. Aim 2. Establish genetically-modified MF as potential immunotherapy to counter T1D development. Develop CRISPR- Cas9 protocols to generate and assess functionality of MΦ derived from NOD bone marrow monocytes with reduced iPLA2b or sEH1 expression and determine their impact on T1D incidence. Significance. Our proposal addresses two novel concepts: (1) piDLs produced by MΦ are important contributors to b-cell death in T1D and (2) manipulating lipid-generating enzymes in MΦ (a readily accessible pool) can counter T1D. We feel that exploiting these will allow generation of novel immunotherapeutic avenues to prevent or delay T1D diagnosis in the clinical setting. This premise is in line with the high risk/high reward mission of the R21 mechanism, readily testable by our group, and expected to yield significant advances in the field of T1D therapy.