Mechanisms of epithelial repair and remodeling in pulmonary fibrosis

摘要 摘要肺纤维化是一种代表慢性肺实质终末期的临床综合征。 肺部疾病。远端肺上皮功能障碍的修复被认为是肺纤维化的中枢。 其发病机制尚不清楚，但损伤后上皮修复的机制仍不完全清楚。 为了全面了解驱动PF的细胞类型和基因表达程序，我们进行了 肺纤维化和对照肺外周组织的单细胞RNA测序(scRNA-seq) 肺小管上皮细胞类型、状态和表达程序的变化，包括先前的 未描述的Krt5-/Krt17“远端基底细胞”(DBC)群体，产生病理性细胞外基质。 在肺纤维化家系中独立使用全外显子组测序进行基因发现 (家族性间质性肺炎，FIP)，我们发现了一种孤儿G蛋白偶联受体的罕见突变 (GPR87)与疾病分离，暗示GPR87是一个新的FIP风险基因。我们的初步数据 提示散发性肺组织中GPR87基因表达显著增加 并特异性地定位于这些新描述的产生ECM的病理性DBCs。在老鼠身上，如在 在人类，GPR87在外周肺中的表达很低；然而，在 博莱霉素损伤后，定位于远端基底细胞。我们产生了表达FIP的小鼠- 使用CRISPR-Cas9基因编辑策略的GPR87相关突变形式，并发现携带有 与对照组小鼠相比，单拷贝突变(Gpr87mut/wt)增加了 单剂博莱霉素。携带双等位基因突变(Gpr87mut/mut)的未受攻击的小鼠自发发展 体内呼吸道上皮重塑和显著的不典型增生。与这些发现相一致的是，文化 Gpr87mut/mut小鼠气管上皮细胞(MTECs)在气液界面(ALI)和三维有机物系统中的表达 导致异常的上皮分化。总之，我们的初步数据表明DBCS与PF的发病机制有关 并提示GPR87调控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 严重或反复损伤后的上皮细胞。我们的具体目标是：1)确定GPR87的作用- 远端基底细胞在促进肺纤维化中的表达2)确定调节远端的机制 重度和慢性肺泡损伤时基底细胞的命运和功能。3)调查依赖GPR87的情况 基本细胞功能和分化的调节。在下面提出的研究中，我们将使用创新 转基因小鼠、类器官和可诱导多能干细胞(IPSC)模型的研究 GPR87促进纤维化易感性和适应性肺病理改变的机制 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.