Mechanisms of epithelial repair and remodeling in pulmonary fibrosis

摘要 肺纤维化（pulmonary fibrosis，PF）是慢性肺实质病变终末期的临床综合征 肺部疾病远端肺上皮的功能性修复被假设为PF的核心 发病机制，但上皮修复损伤后的机制仍然不完全理解。 为了全面分析细胞类型和驱动PF的基因表达程序，我们进行了 来自PF和对照肺的外周组织的单细胞RNA测序（scRNA-seq），并鉴定出显著的 PF肺上皮细胞类型、状态和表达程序的变化，包括先前的 未描述的KRT 5-/KRT 17+“远端基底细胞”（DBC）群体，其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），我们发现罕见的突变，在孤儿G蛋白偶联受体 （GPR 87）与疾病分离，暗示GPR 87是新的FIP风险基因。我们的初步数据 表明在来自散发病例患者肺组织中GPR 87基因表达显著增加 的IPF，并专门定位于这些新描述的病理ECM产生的DBC。在老鼠身上， 在人类中，Gpr 87在外周肺中的表达较低;然而， 在博来霉素损伤后，其定位于远端基底细胞。我们制造了表达FIP的小鼠， 使用CRISPR-Cas9基因编辑策略研究了Gpr 87相关突变形式，发现携带Gpr 87的小鼠 与对照小鼠相比，单拷贝突变（Gpr 87 mut/wt）的肺纤维化增加， 单剂量博莱霉素携带双等位基因突变（Gpr 87 mut/mut）的未激发小鼠自发发生 气道上皮重塑和明显的不典型增生。与这些发现一致， 气液界面（ALI）和3D类器官系统中的Gpr 87 mut/mut小鼠气管上皮细胞（MTEC） 导致异常的上皮分化。总之，我们的初步数据暗示DBCs在PF发病机制 并表明GPR 87调节这些细胞的命运和功能。我们的假设是GPR 87 调节远端基底细胞的增殖和分化，这是肺泡上皮细胞有效修复所必需的。 严重或重复性损伤后的上皮细胞。我们的具体目标是：1）确定GPR 87的作用- 表达远端基底细胞促进肺纤维化。2)确定远端调节机制 严重和慢性肺泡损伤中的基底细胞命运和功能。3)调查GPR 87依赖性 调节基底细胞功能和分化。在下面的研究中，我们将使用创新的 转基因小鼠，类器官和诱导性多能干细胞（iPSC）为基础的模型，以研究 GPR 87促进纤维化易感性和适应性与病理性肺的机制 上皮修复

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.