Cellular crosstalk regulating cholangiocyte proliferation following extrahepatic bile duct injury

胆管病是一种无法治愈的胆道疾病，其特征是组织损伤伴中性粒细胞聚集， 胆管细胞过度增殖，间质细胞扩张。信号驱动肝外胆管(EHBD) 人们对炎症和增殖知之甚少。此应用程序旨在回答基本问题 关于损伤驱动的促进胆管增殖的过程。这些研究的基本原理是，理解 EHBD炎症和增殖反应的分子机制可以为治疗策略提供参考 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) Ligand靶向基质细胞CXCL1，后者将中性粒细胞招募到损伤组织中。我的项目重点是 揭示这种复杂的串扰涉及上皮胆管细胞-间质成纤维细胞- 中性粒细胞信号转导促进胆管增殖和促进组织修复。胆管过度增殖是 与依赖于HH的FOSL1表达有关，FOSL1是一种已知的介导增殖的转录因子 在其他组织中的反应。中性粒细胞IL-1FOSL1在高血压病损伤后升高，对β有调节作用。这个 这一理论的主要假设是，作为对EHBD损伤的反应，胆管细胞分泌印度 HH诱导GLI1+成纤维细胞产生CXCL1，导致中性粒细胞募集和促进 通过IL 1β/FOSL1依赖的机制促进胆管细胞增殖。我将使用最先进的技术 体内遗传小鼠和药理学方法结合体外人和小鼠有机化合物和 初步培养模型，以确定细胞串扰调节EHBD修复的分子机制。胆汁 胆管结扎术(BDL)将被用作急性胆道损伤模型。切割并运行染色质图谱，细胞分离 流式细胞术和RNA-SEQ方法将在该提案范围内进行。调查结果将通过以下方式进行验证 有/无胆管病变患者的存档胆管组织。目标1将专注于GLI1+基质细胞和 调节CXCL1的表达以确定HH信号是否直接诱导成纤维细胞表达CXCL1 GLI1转录激活。这一目标将使用报告小鼠分离GLI1+细胞，用于体内下游 和体外分析。HH信号的药理激活剂和抑制剂将用于测试 Gli1对基因表达的调控。目标2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IL-1β/FOSL1依赖机制促进胆管增殖并确定候选细胞 中性粒细胞衍生因子。AIM 2将使用报告小鼠分离中性粒细胞，用于体内和体外下游 分析。遗传小鼠模型将测试Fosl1缺失对EHBD损伤后胆管增殖的影响。这 计划书将使我能够熟练掌握最先进的工具，并为成功的 R01应用程序。最终，确定调控EHBD损伤反应的细胞和分子机制 可能会提供新的治疗策略，以改善胆管病患者的预后。

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.