IL17 dependent angiocrine signaling drives inflammation in alcohol associated hepatitis

IL17 依赖性血管分泌信号传导驱动酒精相关性肝炎的炎症

• 批准号: 10837927
• 负责人: Mengfei Liu
• 金额: $ 19.06万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10837927
• 关键词: Affect; Alcoholic Hepatitis; Alcoholic Liver Diseases; Autoimmune; Biological; Biological Assay; Blood Vessels; CXCL1 gene; Cells; Chemotaxis; Chromatin Conformation Capture and Sequencing; Circulation; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; DNA; Data; Development; Discontinuous Capillary; Disease; Dominant-Negative Mutation; Endothelial Cells; Endothelium; Enhancers; Family; Gene Expression; Genes; Genome; Guide RNA; Heavy Drinking; Hereditary Disease; IL17 gene; Immune; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Interleukin Suppression; Knock-in; Knock-in Mouse; Knockout Mice; Knowledge; Ligands; Liver; Liver parenchyma; Mediating; Microfluidic Microchips; Mission; Modeling; Molecular Target; Mus; National Institute on Alcohol Abuse and Alcoholism; Neutrophil Infiltration; Nuclear; Paracrine Communication; Pathogenesis; Pathway Analysis; Pathway interactions; Patients; Phosphorylation; Process; Production; Protein Isoforms; Regulation; Regulator Genes; Regulatory Element; Role; Signal Pathway; Signal Transduction; Small Interfering RNA; Source; System; T-Lymphocyte; T-Lymphocyte Subsets; TNF gene; Testing; Threonine; Tissues; Transcriptional Regulation; Transgenic Mice; Up-Regulation; Variant; Viral; Work; alcohol abuse therapy; chemokine; chromatin immunoprecipitation; chromosome conformation capture; cytokine; experimental study; gene network; immune activation; immune cell infiltrate; in vivo; inhibitor; liver inflammation; liver injury; member; migration; mouse model; neutrophil; new therapeutic target; novel; novel therapeutics; paracrine; pharmacologic; promoter; response; synergism; targeted treatment; therapeutic target; transcription factor; transcriptome sequencing; transcriptomics

Project Abstract Alcoholic hepatitis (AH) is characterized by intense liver inflammation and injury in the setting of excess alcohol ingestion. Cytokine and chemokine upregulation leads to immune cell infiltration and drives inflammation in AH. Liver sinusoidal endothelial cells (LSEC) are an important source of chemokine expression in the liver and participate in paracrine signaling to attract immune cells in a process termed “angiocrine signaling”. Pathway analysis of AH liver RNA-sequencing suggests a potential role of IL17 in mediating LSEC angiocrine signaling. IL17 is a cytokine involved in many autoimmune and inflammatory disorders. Our preliminary data shows that IL17 synergically stimulate CXCL chemokine production with TNF, but the underlying mechanism is not clear. The regulation of IL17 production from T cells also requires further study. Super enhancers are DNA regulatory elements that complex with target gene promoters to drive gene expression. Our previous work has highlighted the role of super enhancers in AH inflammation response. Here, we hypothesize that LSECs increase inflammatory chemokine expression and enhance immune cell transmigration into the liver parenchyma in response to T cell IL17 upregulation by super enhancer activation. To test our hypothesis, we will employ complementary cell biologic and in vivo approaches to study the following specific aims: Aim 1. LSECs enhance CXCL1-dependent neutrophil transendothelial migration in response to IL17; Aim 2. Therapeutic targeting of a super enhancer in T cells downregulates IL17 and ameliorates inflammation in AH. In Aim 1, we will explore the role of IκB𝜁, a transcription factor previously implicated in IL17 signaling, in mediating the interaction between IL17 and TNF signaling pathways. We will also assess the transcriptomic changes of IL17 and TNF stimulation and IκB𝜁 silencing on LSECs by RNA-sequencing. Using microfluidic devices to simulate liver microvascular circulation, we will directly observe the effects of IL17/TNF stimulation and IκB𝜁 inhibition on neutrophil chemotaxis. In Aim 2, we will identify the IL17 super enhancer by chromosome conformation capture assays. We will assess the feasibility of CRISPR-mediated sequence-specific suppression of the IL17 super enhancer. We will use a Cre dependent dCas9-KRAB knockin mice with AAV6 viral delivery of single guide RNA to target the IL17 super enhancer in vivo. We aim to achieve T cell-specific suppression of IL17 expression and assess its effect on AH inflammatory response in a murine model. Indeed, in vivo CRISPR gene-editing has already been applied clinically to treat hereditary disorders, highlighting the translational promise of precision genome-targeting therapies. Better understanding of the IL17 mediated angiocrine signaling process and IL17 super enhancer regulation may reveal novel therapeutic targets for treatment of AH. Therefore, our overall aims and approaches are aligned with the mission of NIAAA to further understanding and treatment of alcohol- associated liver diseases.

项目摘要 酒精性肝炎（AH）的特点是严重的肝脏炎症和损伤的设置过量酒精 摄入细胞因子和趋化因子上调导致免疫细胞浸润并驱动AH中的炎症。 肝窦内皮细胞（LSEC）是肝脏中趋化因子表达的重要来源， 参与旁分泌信号传导以在称为“血管分泌信号传导”的过程中吸引免疫细胞。途径 AH肝RNA测序的分析表明IL 17在介导LSEC血管分泌信号传导中的潜在作用。 IL 17是参与许多自身免疫性和炎性病症的细胞因子。我们的初步数据显示， IL 17与TNF α协同刺激CXCL趋化因子的产生，但其潜在机制尚不清楚。 从T细胞产生IL 17的调节也需要进一步研究。超级增强子是DNA调控 与靶基因启动子复合以驱动基因表达的元件。我们之前的工作 超级增强子在AH炎症反应中的作用。在这里，我们假设LSEC增加 炎性趋化因子表达和增强免疫细胞向肝实质的迁移 响应于通过超级增强子激活的T细胞IL 17上调。为了验证我们的假设，我们将使用 补充细胞生物学和体内方法来研究以下具体目标：目的1. LSEC增强 CXCL 1依赖性中性粒细胞跨内皮迁移响应IL 17;目的2.治疗 靶向T细胞中的超级增强子下调IL 17并改善AH中的炎症。在Aim中 1，我们将探讨IκBβ，一种先前参与IL 17信号转导的转录因子，在介导IL 17信号转导中的作用。 IL 17和TNF α信号通路之间的相互作用。我们还将评估IL 17的转录组学变化， 和TNF β刺激和IκBβ沉默。利用微流体装置模拟 直接观察IL 17/TNF β刺激和IκB抑制对肝脏微血管循环的影响 对中性粒细胞趋化性的影响在目标2中，我们将通过染色体构象鉴定IL 17超级增强子 捕获测定。我们将评估CRISPR介导的IL 17序列特异性抑制的可行性。 超级增强剂我们将使用Cre依赖性dCas 9-KRAB敲入小鼠，其中AAV 6病毒递送单向导RNA。 RNA在体内靶向IL 17超级增强子。我们的目标是实现T细胞特异性抑制IL 17表达 并评估其对小鼠模型中AH炎症反应的作用。事实上，体内CRISPR基因编辑 已经在临床上应用于治疗遗传性疾病，突出了精确的转化承诺 基因组靶向治疗更好地理解IL 17介导的血管分泌信号传导过程和IL 17 超级增强子调控可能揭示治疗AH的新的治疗靶点。因此，我们的总体目标 和方法与NIAAA的使命一致，以进一步了解和治疗酒精- 相关的肝脏疾病。