Exploiting iPLA2β-modified macrophages as immunotherapy for T1D

项目总结 1型糖尿病(T1D)是一种以胰腺b细胞破坏为特征的自身免疫性疾病， 然而，导致b细胞破坏的机制并不完全清楚。我们确定了一名危急的 巨噬细胞产生的促炎脂质(M-Φ)在T1D发病中的作用这种脂质是由以下物质产生的 激活非钙依赖性磷脂酶A2β(IPLA2b)。作为PLA2家族的成员，iPLA2b 水解膜上sn-2位的磷脂以释放脂肪酸，如花生四烯酸(AA)， 它可以被代谢成生物活性的氧化类脂(二十烷基类)。其中几种脂质对人体健康有很大影响 促炎作用，我们发现它们在糖尿病前期显著升高，这表明他们 对T1D发病的关键贡献。MΦ是最早渗透到胰岛的细胞之一，它们可以被诱导成 促炎(M1)或抗炎(M2)表型。在T1D中，MΦM1占优势。我们发现抑制作用 或遗传的iPLA2b的全局减少减少了MΦ产生的促炎性iPLA2b衍生的脂类(PiDls) 在糖尿病前期，不利于MΦM1，并减少非肥胖者的胰岛素炎和T1D发生率 自发性糖尿病易感(NOD)小鼠。重要的是，非糖尿病儿童患T1D的风险很高， 表现出相似的血脂特征。这些发现增加了一种可能性，即通过减少MΦ-iPLA2b，piDLs 可以减轻MΦM1的产生和诱导，并且这将有利于预防或延迟T1D 开始了。其中包括通过细胞色素P450/可溶性环氧化物水解酶在iPLA2b下游产生的DHETE (Seh)。据报道，sEH抑制可以缓解炎症，为IDL的产生提供了一个选择性的靶点 计数器T1D。我们假设，减少MΦ-piDL将不利于MΦM1，并阻碍T1D的发展。 CRISPR-Cas9领域的最新进展促进了细胞特异性和选择性的基因修饰 基因。我们的发现提出了一种新的、尚未考虑的情景，即在MΦ中使用 CRISPR-Cas9可以开发成一种免疫疗法来对抗人类的T1D。我们建议解决这个问题 在目标1下，评估MΦ-IDL对T1D发育的影响。我们将确定MΦ-IDL的影响 M-Φ中iPLA2b条件修饰NOD小鼠T1D的发生和发展目标2.建立 转基因MF作为潜在的免疫疗法来对抗T1D的发展。开发CRISPR-- CAS9方案生成和评估NOD骨髓单核细胞来源的MΦ的功能 减少iPLA2b或sEH1的表达，并确定它们对T1D发病的影响。意义重大。我们的建议 提出了两个新的概念：(1)由MΦ产生的piDls是T1D和T1D中b细胞死亡的重要贡献者 (2)在MΦ(一个容易接近的池)中操纵脂质生成酶可以对抗T1D。我们觉得 利用这些将允许产生新的免疫治疗途径来预防或推迟T1D的诊断 临床环境。这一前提符合R21机制的高风险/高回报使命，很容易 可由我们小组测试，并有望在T1D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.