Mechanisms of epithelial repair and remodeling in pulmonary fibrosis

肺纤维化上皮修复与重塑机制

• 批准号: 10215620
• 负责人: Jonathan Andrew Kropski
• 金额: $ 55.21万
• 依托单位: VANDERBILT UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10215620
• 关键词: 3-Dimensional; Acute; Air; Alveolar; Atypical hyperplasia; Automobile Driving; Basal Cell; Bleomycin; CRISPR/Cas technology; Cell Differentiation process; Cell physiology; Cells; Chronic; Clinical; Data; Disease; Distal; Dose; Epithelial; Epithelial Cells; Exhibits; Extracellular Matrix; Family; Family Study; Fibrosis; G-Protein-Coupled Receptors; Gene Expression; Genes; Genetic; Genomic approach; Human; Human Cell Line; Influenza; Injury; Interstitial Pneumonia; Liquid substance; Lung; Lung diseases; Modeling; Mus; Mutation; Organoids; Orphan; Pathogenesis; Pathologic; Pathway interactions; Patients; Peripheral; Play; Pluripotent Stem Cells; Population; Predisposition; Pulmonary Fibrosis; Regulation; Role; Series; Structure of parenchyma of lung; Syndrome; System; Testing; Tissues; Tracheal Epithelium; Transgenic Mice; Transgenic Organisms; Work; airway epithelium; alveolar epithelium; cell type; epithelial repair; epithelium regeneration; exome sequencing; experimental study; in vivo; innovation; loss of function; mutant; novel; programs; rare variant; receptor; repaired; risk variant; single-cell RNA sequencing; stem cell model; time use

Abstract Pulmonary fibrosis (PF) is a clinical syndrome that represents the end-stage of chronic parenchymal lung diseases. Dysfunctional repair of the distal lung epithelium has been hypothesized as central to PF pathogenesis, but the mechanisms governing epithelial repair following injury remain incompletely understood. In order to comprehensively profile the cell types and gene expression programs driving PF, we performed single-cell RNA-sequencing (scRNA-seq) of peripheral tissue from PF and control lungs and identified dramatic changes in cell types, states, and expression programs in PF lung epithelium including a previously undescribed KRT5-/KRT17+ “distal basal cell” (DBC) population that produces pathologic extracellular matrix. Independently, using whole-exome sequencing for genetic discovery in families with pulmonary fibrosis (Familial Interstitial Pneumonia, FIP), we identified rare mutations in an orphan G-protein coupled receptor (GPR87) that segregate with disease, implicating GPR87 as a novel FIP risk gene. Our preliminary data indicate that GPR87 gene expression is dramatically increased in lung tissue from patients with sporadic cases of IPF, and localizes specifically to these newly described pathologic ECM-producing DBCs. In mice, as in humans, Gpr87 expression was low in the peripheral lung; however, expression increases substantially after following bleomycin injury, where it localized to distal basal cells. We generated mice expressing an FIP- associated mutant form of Gpr87 using a CRISPR-Cas9 gene editing strategy and found that mice carrying a single-copy of the mutation (Gpr87mut/wt) had increased lung fibrosis compared to control mice following a single-dose bleomycin. Unchallenged mice carrying biallelic mutations (Gpr87mut/mut) develop spontaneous airway epithelial remodeling and striking atypical hyperplasia in vivo. Consistent with these findings, culture of Gpr87mut/mut mouse tracheal epithelial cells (MTECs) in air-liquid interface (ALI) and 3D organoid systems resulted in aberrant epithelial differentiation. Together, our preliminary data implicate DBCs in PF pathogenesis and suggest that GPR87 regulates the fate and function of these cells. Our hypothesis is that GPR87 regulates proliferation and differentiation of distal basal cells, which are required for efficient repair of alveolar epithelium after severe or repetitive injury. Our specific aims are: 1) Determine the role of Gpr87- expressing distal basal cells in promoting lung fibrosis. 2) Identify mechanisms regulating distal basal cell fate and function in severe and chronic alveolar injury. 3) Investigate GPR87-dependent regulation of basal cell function and differentiation. In studies proposed below, we will use innovative transgenic mouse, organoid and inducible pluripotent stem cell (iPSC)-based models to investigate the mechanisms through which GPR87 contributes to fibrotic susceptibility and adaptive versus pathologic lung epithelial repair.

抽象的 肺纤维化（PF）是一种临床综合征，代表慢性副群的终阶段 肺部疾病。远端肺上皮的功能失调已被认为是PF的中心 发病机理，但是损伤后上皮修复的机制尚未完全理解。 为了全面介绍驱动PF的细胞类型和基因表达程序，我们进行了 PF和对照肺的外周组织的单细胞RNA序列（SCRNA-SEQ），并鉴定出戏剧性的 PF肺上皮中细胞类型，状态和表达程序的变化，包括先前 未描述的KRT5-/KRT17+“远端基细胞”（DBC）群体产生病理性细胞外基质。 独立地，使用全外活体测序进行肺纤维化家族的遗传发现 （家族性肺炎，FIP），我们在孤儿G蛋白偶联受体中鉴定了稀有突变 （GPR87）与疾病分离，隐式GPR87是一种新型的FIP风险基因。我们的初步数据 表明患有零星病例的患者的肺组织中GPR87基因表达显着增加 IPF的of，并专门定位于这些新描述的病理性ECM产生的DBC。在老鼠中，如 人类，GPR87的表达在周围肺中很低。但是，表达在 博来霉素损伤之后，它本地化为远端碱性细胞。我们产生了表达FIP-的小鼠 使用CRISPR-CAS9基因编辑策略的GPR87相关突变形式，发现携带A的小鼠 与对照小鼠相比 单剂量博来霉素。携带双重突变（GPR87MUT/MUT）的无挑战小鼠发展 气道上皮重塑和体内醒目的非典型增生。与这些发现一致 GPR87MUT/MUT小鼠气管上皮细胞（MTEC）在空气界面（ALI）和3D器官系统中 导致异常上皮分化。总之，我们的初步数据暗示了DBC在PF发病机理中 并建议GPR87调节这些细胞的命运和功能。我们的假设是GPR87 调节远端基线细胞的增殖和分化，这是有效修复肺泡所必需的 严重或重复损伤后上皮。我们的具体目的是：1）确定GPR87-的作用 在促进肺纤维化中表达圆盘山层细胞。 2）确定调节盘式的机制 严重和慢性肺泡损伤中的基本细胞命运和功能。 3）研究GPR87依赖性 调节基本细胞功能和分化。在下面提出的研究中，我们将使用创新 转基因小鼠，器官和诱导多能干细胞（IPSC）的模型，以研究该模型 GPR87有助于纤维化易感性和自适应与病理肺的机制 上皮修复。