Cellular and molecular delineation of pathologic fibroblasts in pulmonary fibrosis

项目总结 肺纤维化是一种慢性难治性疾病，5年存活率与胰腺或 肺癌。肺纤维化患者的呼吸功能恶化是由渐进性置换引起的 从正常组织的气体交换到致密的纤维性瘢痕伴纤维状胶原。病理性成纤维细胞 在纤维化部位堆积，作为过度胶原沉积的效应细胞。 靶向病理性成纤维细胞的治疗策略的开发因缺乏 对病理性成纤维细胞的细胞谱系和分子细节的理解。在我们之前的研究中，我们 对小鼠和人的正常肺和纤维化肺进行了单细胞RNA测序 识别所有胶原蛋白产生细胞的协议。我们确定了几个定位于不同组织中的成纤维细胞亚群 肺的隔膜。其中一个成纤维细胞亚群出现在小鼠和人的纤维化肺中，并 表现出最高水平的胶原基因表达和增强的迁移能力。这些成纤维细胞是 以Ctrc1(胶原三螺旋重复序列包含1)的特异性表达为特征，并定位于 特发性肺纤维化的成纤维细胞灶，提示其在肺纤维化中的病理作用。我们 最近产生并验证了一种新的小鼠品系Ctrc1-Creer，它允许我们特异性地 操纵肺纤维化中的病理性成纤维细胞群。这份K99/R00提案的目标是 应用OUR技术阐明病理性成纤维细胞在肺纤维化中的作用及转录调控 创新的老鼠工具。目标1(K99期)将通过消融揭示病理性成纤维细胞的作用和命运 博莱霉素性肺损伤过程中Ctrc1+细胞或Ctrc1-Creer标记细胞系的变化 纤维化症。目的2(K99期)将揭示病理性成纤维细胞的转录和表观遗传学图景 在多个时间点对纯化的Ctrc1-Creer标记细胞进行RNA-SEQ、ChIP-SEQ和ATAC-SEQ 肺纤维化寻求主控调节剂进行激活和失活。我们还将寻求 CTHRC1+细胞在人肺纤维化中的转录调控。目标3(R00阶段)将演示 气管内过继转移成纤维细胞对病理性成纤维细胞调控基因的作用 慢病毒基因修饰和条件性敲除肺成纤维细胞候选基因 纤维化症。这些使用对病理性成纤维细胞高度特异的小鼠遗传工具的研究将揭示 肺纤维化中病理性成纤维细胞的细胞功能和转录调控。这项建议是 还旨在为候选人提供培训机会，以获得小鼠遗传方法的技能集 寻找整合RNA-SEQ、CHIP-SEQ和ATAC的治疗靶点和功能基因组学方法。 序列号。该项目的成功将使候选人能够确立他在肺病领域的专业知识 并导致候选人在获奖过程中过渡到科学独立。

PROJECT SUMMARY Pulmonary fibrosis is a chronic and intractable disease with a 5-year survival rate comparable to pancreatic or lung cancers. Deterioration of respiratory function in pulmonary fibrosis is caused by progressive replacement of normal tissue for gas exchange to dense fibrotic scar with fibrillar collagens. Pathologic fibroblasts accumulate at the sites of fibrogenesis and work as effector cells for excessive collagen deposition. Development of therapeutic strategies for targeting pathologic fibroblasts is hindered by the lack of understanding to cellular lineage and molecular detail of pathologic fibroblasts. In our previous study, we performed single-cell RNA-sequencing of normal and fibrotic lungs of mouse and human with a specialized protocol to identify all collagen-producing cells. We identified several fibroblast subsets that localize in different compartments of the lung. One of the fibroblast subsets emerge in fibrotic lungs of both mouse and human and show the highest levels of collagen gene expression and enhanced migratory capacity. These fibroblasts are characterized by specific expression of Cthrc1 (collagen triple helix repeat containing 1) and localized within fibroblastic foci of idiopathic pulmonary fibrosis, suggesting their pathologic role in pulmonary fibrosis. We recently generated and validated a novel mouse strain, Cthrc1-CreER, which allows us to specifically manipulate the pathologic fibroblast population in pulmonary fibrosis. The goal of this K99/R00 proposal is to elucidate the role and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis by using our innovative murine tools. Aim 1 (K99 phase) will reveal the role and fate of pathologic fibroblasts by ablating Cthrc1+ cells or lineage-tracing Cthrc1-CreER-labeled cells over the course of bleomycin-induced pulmonary fibrosis. Aim 2 (K99 phase) will reveal the transcriptional and epigenetic landscape of pathologic fibroblasts by performing RNA-seq, ChIP-seq, and ATAC-seq of purified Cthrc1-CreER-labeled cells at multiple time points of pulmonary fibrosis to seek master regulators for activation and deactivation. We will also seek the transcriptional regulations of CTHRC1+ cells in human pulmonary fibrosis. Aim 3 (R00 phase) will demonstrate the role of genes regulating pathologic fibroblasts by using intratracheal adoptive transfer of fibroblasts with lentiviral gene modifications and by conditionally knocking out candidate genes in fibroblasts in pulmonary fibrosis. These studies using the murine genetic tool highly specific for pathologic fibroblasts will shed light on cellular function and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis. This proposal is also designed to provide the candidate with training opportunity to obtain skill sets for murine genetic approach in search of therapeutic targets and functional genomics approach integrating RNA-seq, ChIP-seq, and ATAC- seq. The success of this project will enable the candidate to establish his expertise in the field of pulmonary fibrosis and lead to the candidate’s transition to scientific independence over the course of award period.