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

项目概要/摘要 炎症性肠病（IBD）引起大量死亡率和发病率。目前的治疗方法， 病理性炎症反应改善了一些患者的临床结果，但它们并不高 有效地改变疾病进展或预防复发。因此，有一个巨大的未满足的需求， 新的治疗靶点。全基因组关联研究（GWAS）提供了一种无偏见的方法来识别 在IBD发病机制中起关键作用的相关免疫细胞类型如CD 4 + T细胞中的治疗靶点。 由于T细胞在没有外界刺激的情况下是静止的，因此不可能完全检查其作用。 疾病风险变异的功能相关的效应基因在休息条件下。识别IBD风险 我们首次对活化的CD 4 + T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（LSP 1）的表达减少，特别是在活化的CD 4+细胞中， T细胞亚群如TH 1和TH 17细胞与IBD的风险相关。在R 01提案中，我们将 研究LSP 1水平的降低如何影响CD 4 + T细胞的分化和功能，以驱动疾病 发病机制，并将测试LSP 1在抑制CD 4+细胞重编程中起关键作用的假设。 在IBD患者中，T细胞转化为更具致病性的细胞状态。 在目标1中，我们将确定降低CD 4 + T细胞中LSP 1表达的功能性IBD风险变体。我们将 采用荧光素酶报告基因测定来确定LSP 1的增强子和启动子中的功能变体， 对优先的IBD风险eQTL进行CRISPR介导的编辑以定义因果变异，并进行CRISPRi 和ChIP测定，以确定功能增强子、相关上游调节子以及功能增强子和相关上游调节子是否具有功能性。 LSP 1 eQTL直接干扰调节LSP 1表达的关键转录因子的结合。 在目标2中，我们将确定LSP 1在CD 4 + T细胞重编程为所观察到的致病状态中的作用 在IBD。为了确定LSP 1是否影响CD 4 + T细胞的分化和致病功能， 在健康和IBD供体中，我们将降低和增加LSP 1水平，并评估对CD 4 + T细胞的影响。 活化、凋亡、增殖和促炎细胞因子产生。在过继性T细胞转移模型中， 我们将比较Lsp 1充足（野生型）和Lsp 1缺陷型CD 4 + T细胞在驱动结肠炎中的能力。 结肠炎症和致病性TH 1和TH 17分化。我们将确定是否减少Lsp 1 在小鼠结肠炎模型中，CD 4 + T细胞的表达增强了它们的致病性，因此暗示了一个重要的 LSP 1在IBD发病机制中的T细胞内在作用 总之，本研究通过检测LSP 1在CD 4 + T细胞中的功能、表达、活化和调节， 为炎症性肠病风险的遗传基础提供了重要的机制性见解。

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.