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: 10570615
• 负责人: Mengfei Liu
• 金额: $ 19.06万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2023-02-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10570615
• 关键词: Affect; Alcoholic Hepatitis; Alcoholic Liver Diseases; Alcohols; Autoimmune; Biological; Biological Assay; Blood Circulation; CXCL1 gene; Cells; Chemotaxis; Chromatin Conformation Capture and Sequencing; 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; Hepatitis; Hereditary Disease; Immune; Infiltration; Inflammation; Inflammatory; Inflammatory Response; Interleukin Suppression; Interleukin-17; Knock-in; Knock-in Mouse; Knockout Mice; Knowledge; Lead; Ligands; Liver; Liver diseases; 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; Pharmacology; 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; factor A; gene network; in vivo; inhibitor; liver inflammation; liver injury; member; migration; mouse model; neutrophil; new therapeutic target; novel; novel therapeutics; paracrine; 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) 的特点是过量饮酒导致严重的肝脏炎症和损伤 摄入。细胞因子和趋化因子上调导致免疫细胞浸润并引发 AH 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吸引免疫细胞，这一过程称为“血管分泌信号传导”。途径 AH 肝脏 RNA 测序分析表明 IL17 在介导 LSEC 血管分泌信号传导中具有潜在作用。 IL17 是一种参与许多自身免疫和炎症性疾病的细胞因子。我们的初步数据表明 IL17与TNFα协同刺激CXCL趋化因子的产生，但其潜在机制尚不清楚。 T 细胞产生 IL17 的调节也需要进一步研究。超级增强子是DNA调节剂 与靶基因启动子复合以驱动基因表达的元件。我们之前的工作已经强调 超级增强剂在 AH 炎症反应中的作用。在这里，我们假设 LSEC 增加 炎症趋化因子表达并增强免疫细胞迁移至肝实质 通过超级增强子激活来响应 T 细胞 IL17 上调。为了检验我们的假设，我们将采用 互补细胞生物学和体内方法来研究以下具体目标： 目标 1. LSEC 增强 IL17 响应的 CXCL1 依赖性中性粒细胞跨内皮迁移；目标 2. 治疗 T 细胞中的超级增强子的靶向可下调 IL17 并改善 AH 中的炎症。瞄准 1，我们将探讨 IκB𝜁（一种先前参与 IL17 信号转导的转录因子）在介导 IL17 和 TNFα 信号通路之间的相互作用。我们还将评估 IL17 的转录组变化 以及通过 RNA 测序对 LSEC 进行 TNFα 刺激和 IκB𝜁 沉默。使用微流控装置进行模拟 肝脏微血管循环，我们将直接观察IL17/TNFα刺激和IκB𝜁抑制的效果 关于中性粒细胞的趋化性。在目标2中，我们将通过染色体构象鉴定IL17超级增强子 捕获分析。我们将评估 CRISPR 介导的 IL17 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 dCas9-KRAB 敲入小鼠，并通过单向导 AAV6 病毒递送 体内靶向 IL17 超级增强子的 RNA。我们的目标是实现 T 细胞特异性抑制 IL17 表达 并评估其对小鼠模型中 AH 炎症反应的影响。事实上，体内 CRISPR 基因编辑已经 已应用于临床治疗遗传性疾病，凸显了精准的转化前景 基因组靶向疗法。更好地了解 IL17 介导的血管分泌信号传导过程和 IL17 超级增强子调节可能揭示治疗 AH 的新治疗靶点。因此，我们的总体目标 和方法与 NIAAA 进一步了解和治疗酒精的使命相一致 相关肝脏疾病。