Cellular crosstalk regulating cholangiocyte proliferation following extrahepatic bile duct injury

细胞串扰调节肝外胆管损伤后胆管细胞增殖

基本信息

批准号：
10648254
负责人：
Nataliya Razumilava
金额：
$ 11.7万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
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：
2023-04-15 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10648254
关键词：
Acute Affect Agonist Alleles Archives Automobile Driving Biliary Binding Sites CXCL1 gene Cell Culture Techniques Cell Separation Cells Chemotactic Factors Cholangiocarcinoma Chromatin Complex Data Dependence Disease Epithelial Cell Proliferation Epithelium Erinaceidae Extrahepatic Bile Ducts FOSL1 gene Fibroblasts Flow Cytometry Functional disorder GLI gene Gene Expression Genes Genetic Genetic Transcription Health Hour Human Immunohistochemistry In Situ Hybridization In Vitro Inflammation Inflammation Mediators Inflammatory Injury Interleukin-1 beta Ligands Ligation Link LoxP-flanked allele Maps Mediating Mission Mitogen-Activated Protein Kinases Modeling Molecular Mus Neutrophil Infiltration Organoids Pathogenicity Patient-Focused Outcomes Patients Persons Prevention Primary Cell Cultures Process Proliferating Public Health Publishing Quality of life Quantitative Reverse Transcriptase PCR Regenerative Medicine Regulation Reporter Research Role Signal Induction Signal Pathway Signal Transduction Sorting Stromal Cells Testing Therapeutic Tissues Transcription Factor AP-1 Transcriptional Activation United States National Institutes of Health Wild Type Mouse bile duct biliary tract candidate identification chemokine cholangiocyte cytokine effective therapy improved improved outcome in vivo inhibitor injured morphogens mouse model neutrophil novel novel therapeutic intervention overexpression pharmacologic prevent receptor repaired response response to injury severe injury single-cell RNA sequencing smoothened signaling pathway tissue injury tissue repair tool transcription factor transcriptome sequencing treatment strategy

项目摘要

Cholangiopathies are incurable biliary tree disorders characterized by tissue injury with neutrophil recruitment, cholangiocyte hyperproliferation, and stromal cell expansion. Signals driving extrahepatic bile duct (EHBD) inflammation and proliferation are poorly understood. This application aims to answer fundamental questions about injury-driven processes promoting biliary proliferation. The rationale for these studies is that understanding molecular mechanisms of EHBD inflammatory and proliferative responses can inform therapeutic strategies to prevent and treat complications of cholangiopathies. My preliminary data shows a massive increase in cholangiocyte proliferation within 24 hours after acute injury. Further, increased cholangiocyte Hedgehog (HH) ligand targets stromal cell CXCL1, which recruits neutrophils to the injured tissue. My project focuses on uncovering the mechanisms of this complex crosstalk involving epithelial cholangiocyte – stromal fibroblast – neutrophil signaling to promote biliary proliferation and facilitate tissue repair. Biliary hyperproliferation is associated with HH-dependent expression of FOSL1, a transcription factor known to mediate proliferative responses in other tissues. IL1β is increased post EHBD injury in neutrophils and can regulate FOSL1. The overarching hypothesis for this proposal is that in response to EHBD injury, cholangiocytes secrete Indian HH which induces GLI1+ fibroblasts to produce CXCL1, leading to neutrophil recruitment and promotion of cholangiocyte proliferation through a IL1β/FOSL1-dependent mechanism. I will use state-of-the art in vivo genetic mouse and pharmacologic approaches combined with ex vivo human and mouse organoid and primary culture models to define the molecular mechanisms of cellular crosstalk regulating EHBD repair. Bile duct ligation (BDL) will be used as an acute biliary injury model. CUT&RUN chromatin mapping, cell isolation by flow cytometry, and RNA-seq approaches will be conducted within this proposal. Findings will be validated using archived biliary tissues from patients with/without cholangiopathy. Aim 1 will focus on GLI1+ stromal cells and regulation of Cxcl1 expression to determine if HH signaling induces fibroblasts to express Cxcl1 via direct transcriptional activation by GLI1. This aim will use reporter mice to isolate GLI1+ cells for downstream in vivo and ex vivo analyses. Pharmacological activators and inhibitors of HH signaling will be used to test effects of GLI1 modulation on gene expression. Aim 2 will focus on neutrophil-derived pro-proliferative signals to test whether neutrophils promote biliary proliferation via a IL1β/FOSL1-dependent mechanism and identify candidate neutrophil-derived factors. Aim 2 will use reporter mice to isolate neutrophils for downstream in vivo and ex vivo analyses. A genetic mouse model will test effects of Fosl1 deletion on biliary proliferation post EHBD injury. This proposal will allow me to gain proficiency in state-of-the-art tools and generate preliminary data for a successful R01 application. Ultimately, defining the cellular and molecular mechanisms regulating EHBD injury responses may inform novel therapeutic strategies to improve outcomes of patients with cholangiopathies.

胆管病是无法治愈的胆道树疾病，其特征是嗜中性粒细胞招募的组织损伤，胆管细胞高增殖和基质细胞膨胀。信号驱动ePARHEPATITITITIC胆管（EHBD）炎症和增殖知之甚少。该应用程序旨在回答基本问题关于促进胆道增殖的伤害驱动的过程。这些研究的理由是理解 EHBD炎症和增生反应的分子机制可以为治疗策略提供依据预防和治疗胆管病的并发症。我的初步数据显示急性损伤后24小时内胆管细胞增殖。此外，增加胆管细胞刺猬（HH）配体靶向基质细胞CXCL1，该细胞CXCL1将中性粒细胞募集到受伤的组织中。我的项目专注于揭示了涉及上皮胆管细胞 - 基质成纤维细胞 - 中性粒细胞信号传导以促进胆道增殖并促进组织修复。胆道高增殖是与FOSL1的HH依赖性表达相关，FOSL1是培养基增殖已知的转录因子其他组织中的反应。 IL1β在中性粒细胞中EHBD损伤后增加，可以调节FOSL1。这该提议的总体假设是，为了应对EHBD伤害，胆管细胞秘密印度 HH诱导Gli1+成纤维细胞产生CXCL1，从而导致中性粒细胞募集和促进通过IL1β/FOSL1依赖机制的胆管细胞增殖。我将使用最先进的艺术体内遗传小鼠和药物方法结合了体内人体和小鼠器官以及定义调节EHBD修复的细胞串扰的分子机制的主要培养模型。胆汁管道连接（BDL）将用作急性胆道损伤模型。切割和运行染色质映射，通过在此提案中将进行流式细胞仪和RNA-seq方法。调查结果将使用来自/没有胆管疾病的患者的存档胆道组织。 AIM 1将专注于Gli1+基质细胞和 CXCL1表达的调节以确定HH信号是否诱导成纤维细胞通过直接表达CXCL1 GLI1的转录激活。该目标将使用报告基因小鼠隔离gli1+细胞以进行体内下游并进行体内分析。 HH信号传导的药理激活剂和抑制剂将用于测试 GLI1调节基因表达。 AIM 2将专注于中性粒细胞衍生的促销信号进行测试中性粒细胞是否通过IL1β/FOSL1依赖机制促进胆道增殖并确定候选者中性粒细胞的因素。 AIM 2将使用记者小鼠分离中性粒细胞，用于体内下游和体内分析。遗传小鼠模型将测试FOSL1缺失对EHBD损伤后胆道增殖的影响。这提案将使我能够熟练精通最先进的工具，并生成初步数据以成功 R01应用程序。最终，定义调节EHBD损伤反应的细胞和分子机制可以为新的治疗策略提供信息，以改善胆管疾病患者的预后。