Mechanisms of epithelial repair and remodeling in pulmonary fibrosis

肺纤维化上皮修复与重塑机制

基本信息

批准号：
10431866
负责人：
Jonathan Andrew Kropski
金额：
$ 55.09万
依托单位：
VANDERBILT UNIVERSITY MEDICAL CENTER
依托单位国家：
美国
项目类别：
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/10431866
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 Population Predisposition Pulmonary Fibrosis Regulation Role Series Structure of parenchyma of lung Syndrome System Testing Tracheal Epithelium Transgenic Mice Transgenic Organisms Work airway epithelium alveolar epithelium cell type directed differentiation epithelial repair epithelium regeneration exome sequencing experimental study fibrotic lung in vivo induced pluripotent stem cell innovation loss of function mutant novel programs rare variant receptor repaired risk variant single-cell RNA sequencing stem cell model time use

项目摘要

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，代表慢性肺实质纤维化的终末期。远端肺上皮的功能失调修复已被视为 PF 的核心。发病机制，但损伤后控制上皮修复的机制仍不完全清楚。为了全面分析驱动 PF 的细胞类型和基因表达程序，我们进行了对 PF 和对照肺的外周组织进行单细胞 RNA 测序 (scRNA-seq)，并发现了显着的差异 PF 肺上皮细胞类型、状态和表达程序的变化，包括以前的未描述的 KRT5-/KRT17+“远端基底细胞”(DBC) 群体，可产生病理性细胞外基质。独立地，使用全外显子组测序在肺纤维化家族中进行基因发现（家族性间质性肺炎，FIP），我们发现了孤儿 G 蛋白偶联受体的罕见突变 (GPR87) 与疾病分离，表明 GPR87 是一种新的 FIP 风险基因。表明散发病例患者肺组织中 GPR87 基因表达显着增加 IPF，并专门定位于这些新描述的产生病理性 ECM 的小鼠 DBC，如在小鼠中。在人类中，Gpr87 在外周肺中的表达量较低；然而，在博来霉素损伤后，它定位于远端基底细胞，我们产生了表达 FIP- 的小鼠。使用 CRISPR-Cas9 基因编辑策略相关的 Gpr87 突变形式，发现携带与对照小鼠相比，单拷贝突变（Gpr87mut/wt）的肺纤维化增加单剂量博来霉素携带双等位基因突变（Gpr87mut/mut）的未受攻击的小鼠自发发育。体内气道上皮重塑和显着的非典型增生与这些发现一致。气液界面 (ALI) 和 3D 类器官系统中的 Gpr87mut/mut 小鼠气管上皮细胞 (MTEC) 总之，我们的初步数据表明 DBC 与 PF 发病机制有关。并表明 GPR87 调节这些细胞的命运和功能我们的假设是 GPR87。调节远端基底细胞的增殖和分化，这是有效修复肺泡所必需的我们的具体目标是：1) 确定 Gpr87- 的作用。表达远端基底细胞促进肺纤维化2)确定调节远端的机制。严重和慢性肺泡损伤中的细胞基础命运和功能 3) 研究 GPR87 依赖性。在下面提出的研究中，我们将使用创新的方法来调节基底细胞功能和分化。基于转基因小鼠、类器官和诱导多能干细胞（iPSC）的模型来研究 GPR87 促进纤维化易感性以及适应性与病理性肺的机制上皮修复。