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

基本信息

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
项目类别：
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

项目摘要

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）的特征是在过量酒精的情况下进行强烈的肝感染和损伤摄取。细胞因子和趋化因子上调会导致免疫细胞浸润，并在AH中驱动注射。肝正弦内皮细胞（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”的过程中吸引免疫电池。路径对AH肝RNA的分析分析表明，IL17在介导LSEC血管分泌信号传导中的潜在作用。 IL17是一种参与许多自身免疫性和炎症性疾病的细胞因子。我们的初步数据表明 IL17通过TNF协同刺激CXCL趋化因子的产生，但基本机制尚不清楚。 T细胞的IL17产生的调节也需要进一步研究。超级增强剂是DNA调节的与靶基因启动子复合以驱动基因表达的元素。我们以前的工作突出了超级增强子在AH炎症反应中的作用。在这里，我们假设LSEC增加了炎性趋化因子表达并增强免疫细胞传播到肝实质中为了响应T细胞IL17通过超级增强剂的激活上调。为了检验我们的假设，我们将采用完全细胞生物学和体内方法研究以下特定目的：目标1。LSEC增强 CXCL1依赖性嗜中性粒细胞跨性皮迁移响应IL17；目标2。治疗 T细胞中超级增强剂的靶向可下调IL17并改善AH中的注射。目标 1，我们将探讨IκB𝜁先前在IL17信号传导中实现的转录因子的作用，在介导 IL17和TNF信号通路之间的相互作用。我们还将评估IL17的转录组变化通过RNA测序，在LSEC上进行TNF刺激和IκB𝜁沉默。使用微流体设备模拟肝微血管圆，我们将直接观察IL17/TNF模拟和IκB𝜁抑制作用的影响在中性粒细胞趋化性上。在AIM 2中，我们将通过染色体构象来识别IL17超级增强剂捕获评估。我们将评估CRISPR介导的序列特异性抑制IL17的可行性超级增强剂。我们将使用依赖CRE的DCAS9-KRAB敲击蛋白小鼠，并使用AAV6病毒递送单一指南 RNA靶向体内IL17超级增强子。我们旨在实现T细胞特异性抑制IL17表达并评估其对鼠模型中AH炎症反应的影响。确实，体内CRISPR基因编辑有已经在临床上应用于治疗遗传疾病，强调了精确的翻译承诺基因组靶向疗法。更好地了解IL17介导的血管分泌信号传导过程和IL17 超增强剂调节可能揭示了用于治疗AH的新型治疗靶标。因此，我们的整体目标和方法与NIAAA的使命保持一致，以进一步理解和治疗酒精 - 相关的肝病。