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管系统疾病，其特征是组织损伤和中性粒细胞募集，胆管细胞过度增殖和基质细胞扩张的信号驱动肝外胆管（EHBD）。人们对炎症和增殖知之甚少，该应用旨在回答基本问题。这些研究的基本原理是了解损伤驱动的过程促进胆道增殖。 EHBD 炎症和增殖反应的分子机制可以为治疗策略提供信息预防和治疗胆管病的并发症我的初步数据显示，胆管病的并发症大幅增加。急性损伤后24小时内胆管细胞增殖进一步增加，Hedgehog胆管细胞(HH)增加。配体靶向基质细胞 CXCL1，它将中性粒细胞募集到受伤的组织中。揭示这种涉及上皮胆管细胞 - 基质成纤维细胞 - 的复杂串扰机制中性粒细胞信号传导促进胆道增殖并促进组织修复。与 FOSL1 的 HH 依赖性表达相关，FOSL1 是一种已知介导增殖的转录因子 EHBD 损伤后中性粒细胞中 IL1β 的反应增加，并且可以调节 FOSL1。该提议的总体假设是，为了应对 EHBD 损伤，胆管细胞会分泌印度 HH 诱导 GLI1+ 成纤维细胞产生 CXCL1，导致中性粒细胞募集和促进我将通过 IL1β/FOSL1 依赖性机制来研究胆管细胞增殖。体内遗传小鼠和药理学方法与离体人类和小鼠类器官相结合原代培养模型定义细胞串扰调节 EHBD 修复的分子机制。胆管结扎 (BDL) 将用作急性胆道损伤模型，通过 CUT&RUN 染色质定位、细胞分离。流式细胞术和 RNA-seq 方法将在本提案中进行，结果将通过使用进行验证。目标 1 将重点关注 GLI1+ 基质细胞和来自患有/不患有胆管病的患者的存档胆管组织。调节 Cxcl1 表达以确定 HH 信号传导是否通过直接诱导成纤维细胞表达 Cxcl1 GLI1 的转录激活该目标将使用报告小鼠分离 GLI1+ 细胞用于体内下游。 HH 信号传导的药理学激活剂和抑制剂将用于测试效果。 GLI1 对基因表达的调节将重点测试中性粒细胞衍生的促增殖信号。中性粒细胞是否通过 IL1β/FOSL1 依赖性机制促进胆道增殖并确定候选者 Aim 2 将使用报告小鼠分离中性粒细胞以用于体内和离体下游。遗传小鼠模型将测试 Fosl1 缺失对 EHBD 损伤后胆道增殖的影响。提案将使我能够熟练掌握最先进的工具，并为成功的项目生成初步数据 R01 应用最终定义了调节 EHBD 损伤反应的细胞和分子机制。可能会为改善胆管病患者的预后提供新的治疗策略。