Genetic studies linking LSP1 function in T cells to Inflammatory Bowel Disease

T 细胞中 LSP1 功能与炎症性肠病相关的遗传学研究

• 批准号: 10636526
• 负责人: BENJAMIN JOACHIM SCHMIEDEL
• 金额: $ 40.26万
• 依托单位: LA JOLLA INSTITUTE FOR IMMUNOLOGY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10636526
• 关键词: Address; Affect; Apoptosis; Arthritis; Autoimmune Diseases; Automobile Driving; Binding; Biological Assay; Biological Sciences; CD4 Positive T Lymphocytes; CRISPR interference; Cell physiology; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Colitis; Collaborations; Colonic inflammation; Crohn' s disease; DNA Sequence; Databases; Delayed Hypersensitivity; Disease; Disease Progression; Enhancers; F-actin-binding proteins; Gene Expression; Genes; Genetic; Genetic Polymorphism; Genetic study; Human; Immune; Immunology; Impairment; Infiltration; Inflammation; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Inherited; Knowledge; Leukocytes; Link; Luciferases; Mediating; Modeling; Molecular; Morbidity - disease rate; Mus; Outcome; Pathogenesis; Pathogenicity; Pathologic; Pathway interactions; Patients; Play; Production; Proliferating; Proteins; Quantitative Trait Loci; RNA Interference; Regulation; Reporter; Reporting; Rest; Risk; Role; Science; Signal Pathway; Small Interfering RNA; T cell therapy; T-Cell Activation; T-Lymphocyte; T-Lymphocyte Subsets; Testing; Translating; Ulcerative Colitis; Untranslated RNA; Variant; Work; causal variant; cell motility; cell type; chromatin immunoprecipitation; cytokine; cytotoxicity; disorder risk; epigenomics; genetic association; genetic variant; genome wide association study; improved; insight; loss of function; mortality; mouse model; murine colitis; new therapeutic target; overexpression; phase 3 study; prevent; programs; promoter; response; restraint; risk variant; therapeutic target; transcription factor; tumor

PROJECT SUMMARY/ABSTRACT Inflammatory bowel diseases (IBD) cause substantial mortality and morbidity. Current treatments that block pathological inflammatory responses have improved clinical outcomes in some patients but they are not highly effective in modifying disease progression or preventing relapses. Hence, there is a large unmet need to develop novel therapeutic targets. Genome-wide association studies (GWAS) offer an unbiased approach to identify therapeutic targets in relevant immune cell types such as CD4+ T cells that play key roles in IBD pathogenesis. Because T cells are quiescent in the absence of extrinsic stimulation, it is not possible to fully examine the effects of disease-risk variants on functionally relevant effector genes under resting conditions. To identify IBD-risk genes in activated CD4+ T cells, we performed the first large-scale single-cell eQTL study on activated CD4+ T cells. We found that reduced expression of Leukocyte-specific protein 1 (LSP1), specifically in activated CD4+ T-cell subsets such as TH1 and TH17 cells, was associated with the risk of IBD. In this R01 proposal, we will investigate how reduced levels of LSP1 influences the differentiation and function of CD4+ T cells to drive disease pathogenesis, and will test the hypothesis that LSP1 plays a key role in restraining the re-programming of CD4+ T cells into a more pathogenic cell state in IBD patients. In Aim 1, we will determine the functional IBD-risk variants that reduce LSP1 expression in CD4+ T cells. We will employ luciferase reporter assays to determine functional variants in the enhancers and promoter of LSP1, perform CRISPR-mediated editing of prioritized IBD-risk eQTLs to define causal variants, and perform CRISPRi and ChIP assays to determine functional enhancers, relevant up-stream regulators and whether the functional LSP1 eQTLs directly perturb the binding of key transcription factors that modulate LSP1 expression. In Aim 2, we will determine the role of LSP1 in reprogramming of CD4+ T cells into the pathogenic state observed in IBD. To determine whether LSP1 influences the differentiation and pathogenic function of CD4+ T cells from healthy and IBD donors, we will reduce and increase LSP1 levels and assess the effects on CD4+ T-cell activation, apoptosis, proliferation and proinflammatory cytokine production. In an adoptive T cell transfer model of colitis, we will compare the ability of Lsp1- sufficient (wild-type) and Lsp1-deficient CD4+ T cells in driving colonic inflammation and pathogenic TH1 and TH17 differentiation. We will determine whether reducing Lsp1 expression in CD4+ T cells enhances their pathogenicity in mouse models of colitis, thus implicating an important T cell-intrinsic role for LSP1 in IBD pathogenesis. Overall, this study, examining the function, expression, activation and regulation of LSP1 in CD4+ T cells, will provide important mechanistic insights into the genetic basis of risk for inflammatory bowel disease.

项目概要/摘要 炎症性肠病（IBD）导致大量死亡率和发病率。目前的治疗方法可以阻断 病理性炎症反应改善了一些患者的临床结果，但效果并不明显 有效改变疾病进展或预防复发。因此，存在大量未满足的开发需求 新的治疗靶点。全基因组关联研究（GWAS）提供了一种公正的方法来识别 相关免疫细胞类型的治疗靶点，例如在 IBD 发病机制中发挥关键作用的 CD4+ T 细胞。 由于 T 细胞在没有外部刺激的情况下处于静止状态，因此无法全面检查其影响 静息条件下功能相关效应基因的疾病风险变异。识别 IBD 风险 激活的 CD4+ T 细胞中的基因，我们对激活的 CD4+ T 细胞进行了首次大规模单细胞 eQTL 研究 细胞。我们发现白细胞特异性蛋白 1 (LSP1) 的表达减少，特别是在活化的 CD4+ 中 T 细胞亚群（例如 TH1 和 TH17 细胞）与 IBD 风险相关。在这个 R01 提案中，我们将 研究 LSP1 水平降低如何影响 CD4+ T 细胞的分化和功能以驱动疾病 发病机制，并将检验 LSP1 在抑制 CD4+ 重编程中发挥关键作用的假设 IBD 患者的 T 细胞进入更具致病性的细胞状态。 在目标 1 中，我们将确定降低 CD4+ T 细胞中 LSP1 表达的功能性 IBD 风险变异。我们将 采用荧光素酶报告基因检测来确定 LSP1 增强子和启动子的功能变异， 对优先 IBD 风险 eQTL 进行 CRISPR 介导的编辑，以定义因果变异，并执行 CRISPRi 和 ChIP 测定，以确定功能增强剂、相关上游调节剂以及功能是否 LSP1 eQTL 直接干扰调节 LSP1 表达的关键转录因子的结合。 在目标 2 中，我们将确定 LSP1 在将 CD4+ T 细胞重编程为观察到的致病状态中的作用 在炎症性肠病中。确定 LSP1 是否影响 CD4+ T 细胞的分化和致病功能 对于健康的 IBD 捐献者，我们将降低和增加 LSP1 水平并评估对 CD4+ T 细胞的影响 激活、细胞凋亡、增殖和促炎细胞因子的产生。在过继性 T 细胞转移模型中 对于结肠炎，我们将比较 Lsp1 充足（野生型）和 Lsp1 缺乏的 CD4+ T 细胞驱动的能力 结肠炎症和致病性 TH1 和 TH17 分化。我们将确定是否减少Lsp1 CD4+ T 细胞中的表达增强了其在结肠炎小鼠模型中的致病性，因此暗示了一个重要的 LSP1 在 IBD 发病机制中的 T 细胞内在作用。 总的来说，这项研究检查了 CD4+ T 细胞中 LSP1 的功能、表达、激活和调节，将 为炎症性肠病风险的遗传基础提供重要的机制见解。