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

项目摘要/摘要 炎症性肠病(IBD)导致相当大的死亡率和发病率。当前的治疗方法会阻止 病理性炎症反应改善了一些患者的临床结果，但不是很明显 在抑制疾病进展或防止复发方面有效。因此，有大量未得到满足的需求需要开发 新的治疗靶点。全基因组关联研究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，如在IBD发病机制中发挥关键作用的CD4T细胞。 因为T细胞在没有外在刺激的情况下是静止的，所以不可能完全检查其影响 在静息条件下功能相关效应基因上的疾病风险变异。识别IBD风险 基因，我们首次对活化的CD4T细胞进行了大规模的单细胞eQTL研究 细胞。我们发现，白细胞特异性蛋白1(LSP1)的表达减少，特别是在激活的CD4 T细胞亚群，如TH1和TH17细胞，与IBD的风险相关。在这份R01提案中，我们将 研究LSP1水平降低如何影响CD4T细胞的分化和驱动疾病的功能 发病机制，并将检验LSP1在抑制CD4重新编程方面发挥关键作用的假设 在IBD患者中，T细胞转化为更致病的细胞状态。 在目标1中，我们将确定降低CD4T细胞中LSP1表达的功能性IBD风险变体。我们会 使用荧光素酶报告分析来确定LSP1的增强子和启动子中的功能变体， 执行CRISPR介导的IBD风险eQTL的优先顺序编辑，以定义因果变量，并执行CRISPRI 和芯片检测以确定功能增强剂、相关的上游调节因子以及功能是否 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CD4T细胞重新编程到观察到的致病状态中的作用 在IBD。确定LSP1是否影响CD4T细胞的分化和致病功能 健康和IBD捐赠者，我们将降低和提高LSP1水平，并评估其对CD4T细胞的影响 活化、凋亡、增殖和促炎细胞因子的产生。在采用T细胞转移模型中 对于结肠炎，我们将比较Lsp1充足的(野生型)和Lsp1缺乏的CD4T细胞在驱动 结肠炎与致病性TH1、TH17分化。我们将确定是否降低LSP1 在小鼠结肠炎模型中，CD4T细胞的表达增强了它们的致病性，从而暗示了一种重要的 T细胞-LSP1在IBD发病中的内在作用。 总体而言，这项研究，检查了CD4T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洞察力。

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.