Exploiting iPLA2β-modified macrophages as immunotherapy for T1D

利用 iPLA2β 修饰巨噬细胞作为 T1D 的免疫疗法

基本信息

批准号：
10620299
负责人：
SASANKA RAMANADHAM
金额：
$ 18万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-10 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10620299
关键词：
Acids Address Adoptive Transfer Anti-Inflammatory Agents Antigen-Presenting Cells Arachidonic Acids Autoimmune Diseases B-Lymphocytes Beta Cell Biological Assay Blood Glucose Bone Marrow CRISPR/Cas technology Cell Death Cells Child Clinical Clustered Regularly Interspaced Short Palindromic Repeats Development Diabetes Mellitus Diabetes autoantibodies Diagnosis Disease Eicosanoids Environment Enzymes Epoxide hydrolase Event Exhibits Exploratory/Developmental Grant Family Fatty Acids Gene Modified Generations Genetic Glucose tolerance test Human Immune Immunotherapeutic agent Immunotherapy Incidence Infiltration Inflammation Inflammatory Insulin Insulin-Dependent Diabetes Mellitus Investigation Knock-in Knowledge Leukocytes Lipids Lysophospholipids Macrophage Mass Spectrum Analysis Membrane Mission Modification Mus Non obese Pancreas Pathway interactions Phase Phenotype Phospholipase Phospholipase A2 Phospholipids Plasma Play Positioning Attribute Prediabetes syndrome Process Production Protocols documentation Reporting Role Scheme Signal Transduction Source Stains Stimulus Structure of beta Cell of islet T-Lymphocyte Testing autoimmune pathogenesis diabetes mellitus therapy effective therapy eicosanoid metabolism genetic selection glucose tolerance high reward high risk insulin dependent diabetes mellitus onset insulin secretion insulitis islet knockout gene lipidome member monocyte non-diabetic novel oxidized lipid prevent recruit

项目摘要

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.

项目摘要 1型糖尿病（T1D）是一种自身免疫性疾病，其特征是破坏胰腺B细胞，但是，尚未完全了解导致B细胞破坏的机制。我们确定了一个关键 T1D发作中巨噬细胞（Mφ）产生的促炎性脂质的作用。这样的脂质是由 Ca2+非依赖性磷脂酶A2β（IPLA2B）的激活。作为PLA2家族的成员IPLA2B 水解在SN-2位置的膜磷脂，以释放脂肪酸，例如花生四烯酸（AA），可以将其代谢为生物活性氧化物脂质（eicosanoids）。这些脂质中的几种是深刻的促炎，我们发现它们在糖尿病前期显着升高，表明它们对T1D发作的关键贡献。 Mφ是最早浸润胰岛的之一，可以将其诱导到促炎（M1）或抗炎（M2）表型。在T1D中，MφM1占主导地位。我们发现抑制作用或IPLA2B的遗传全球降低可降低促炎IPLA2B衍生的脂质（PIDLS）的生产。在糖尿病阶段，不散发MφM1，并降低非OBase的胰岛炎和T1D发病率自发糖尿病（点头）小鼠。重要的是，患有T1D的非糖尿病儿童有高风险，展示类似的等离子体脂质签名。这些发现提出了通过减少mφ-ipla2b，pidls的可能性可以缓解MφM1的生产和诱导，这将有益于预防或延迟T1D 发作。在PIDL中是DHETE，通过CYP450/可溶性环氧水解酶在IPLA2B下游产生（SEH）。据报道，炎症缓解了SEH抑制作用，为某些IDL生成目标提供了计数器T1D。我们假设减少Mφ-PIDL将不利于MφM1和计数器T1D发展。 CRISPR-CAS9领域的最新进展已制备了细胞特异性并选择的遗传修饰基因。我们的发现提出了一部小说，尚未考虑场景，其中使用Mφ减少IPLA2B CRISPR-CAS9可以发展为免疫疗法，以对抗人类的T1D。我们建议解决这个问题在AIM 1下。评估Mφ-IDLS对T1D发展的影响。我们将确定mφ-idls的影响在T1D发作和使用NOD小鼠的进展中，在Mφ中有条件修饰IPLA2B。目标2。建立遗传改性的MF作为抵抗T1D发育的潜在免疫疗法。发展crispr- CAS9协议以生成和评估来自点头骨髓单核细胞的Mφ的功能 IPLA2B或SEH1表达降低，并确定它们对T1D事件的影响。意义。我们的建议解决了两个新颖的概念：（1）Mφ产生的PIDL是t1d中B细胞死亡的重要因素（2）在Mφ（一个易于访问的池）中操纵脂质生成酶可以对抗T1D。我们感到利用这些将允许产生新型免疫治疗途径，以防止或延迟T1D诊断临床环境。此前提符合R21机制的高风险/高奖励任务，很容易我们小组可测试，并预计将在T1D治疗领域取得重大进展。