Cellular and molecular delineation of pathologic fibroblasts in pulmonary fibrosis

肺纤维化中病理性成纤维细胞的细胞和分子描绘

• 批准号: 10300943
• 负责人: Tatsuya Tsukui
• 金额: $ 15.77万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-05 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10300943
• 关键词: Ablation; Adoptive Transfer; Alveolar; Apoptosis; Award; Binding; Biological Assay; Bleomycin; Candidate Disease Gene; Cell Count; Cell Cycle Kinetics; Cell physiology; Cells; ChIP-seq; Chromatin; Chronic; Cicatrix; Collagen; Collagen Gene; Computer Analysis; Data; Deposition; Deterioration; Development; Disease; Effector Cell; Epigenetic Process; Extracellular Matrix; Fibrillar Collagen; Fibroblasts; Fibrosis; Gases; Gene Expression; Gene-Modified; Genes; Genetic; Genetic Transcription; Genome; Genomic approach; Goals; Human; Impairment; In Situ Hybridization; Inflammation; Injury; Knowledge; Label; Lead; Light; Lung; Malignant neoplasm of lung; Malignant neoplasm of pancreas; Methodology; Modeling; Molecular; Morphology; Mouse Strains; Mus; Normal tissue morphology; Pathologic; Patients; Phase; Phenotype; Play; Population; Process; Proteins; Protocols documentation; Pulmonary Fibrosis; RNA; Regulator Genes; Reporter; Resolution; Respiratory physiology; Role; Site; Survival Rate; Therapeutic; Time; Transcriptional Regulation; Transposase; Work; XCL1 gene; conditional knockout; design; epigenetic regulation; experimental study; fibrogenesis; functional genomics; genetic approach; idiopathic pulmonary fibrosis; in vitro Assay; in vivo; indium-bleomycin; injured; innovation; knock-down; lung development; migration; mouse development; novel; overexpression; programs; single-cell RNA sequencing; skills; success; therapeutic development; therapeutic target; tool; training opportunity; transcription factor; transcriptome sequencing; triple helix; wound healing

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.

项目摘要 肺纤维化是一种慢性且顽固性的疾病，其存活率与胰腺或 肺癌。肺纤维化中呼吸功能的恶化是由进行性替代引起的 与原纤维胶原蛋白的正常组织一起交换与密集的纤维化疤痕。病理成纤维细胞 积聚在纤维发生部位，作为过度胶原蛋白沉积的效应细胞起作用。 缺乏缺乏 了解病理成纤维细胞的细胞谱系和分子细节。在我们以前的研究中，我们 对小鼠和人类的正常和纤维化肺进行了单细胞RNA序列，并具有专门的 识别所有产生胶原蛋白的细胞的方案。我们确定了几个本地化在不同的成纤维细胞子集 肺部的室。小鼠和人类的纤维化肺中出现的成纤维细胞子集之一 显示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CTHRC1的特定表达（胶原蛋白三螺旋重复1重复1）为特征，并本地化 特发性肺纤维化的成纤维细胞灶，表明它们在肺纤维化中的病理作用。我们 最近生成并验证了一种新型的小鼠菌株CTHRC1-CREER，这使我们能够特别 操纵肺纤维化中的病理成纤维细胞种群。此K99/R00建议的目标是 通过使用我们的 创新的鼠工具。 AIM 1（K99阶段）将通过磨料来揭示病理成纤维细胞的作用和命运 CTHRC1+细胞或谱系追踪CTHRC1-creer标记的细胞在博来霉素诱导的肺部过程中 纤维化。 AIM 2（K99阶段）将揭示病理成纤维细胞的转录和表观遗传景观 在多个时间点的多个时间点执行纯化的CTHRC1-Creer标记的细胞的RNA-Seq，ChIP-Seq和Atac-Seq 肺纤维化寻求主调节剂进行激活和失活。我们还将寻求 人肺纤维化中CTHRC1+细胞的转录调节。 AIM 3（R00阶段）将证明 通过使用气管内适应性转移的成纤维细胞的基因来调节病理成纤维细胞的作用 慢病毒基因修饰并通过有条件地敲除肺成纤维细胞中的候选基因 纤维化。这些使用鼠遗传工具对病理成纤维细胞高度特异的研究将揭示 肺纤维化中病理成纤维细胞的细胞功能和转录调节。该提议是 还旨在为候选人提供培训机会，以获得鼠遗传方法的技能 寻找治疗靶标和功能基因组学方法，以整合RNA-SEQ，CHIP-SEQ和ATAC- seq。该项目的成功将使候选人能够在肺部领域建立他的专业知识 纤维化并导致候选人在奖励期间过渡到科学独立性。