Fibroblast heterogeneity in pulmonary fibrosis

摘要 肺纤维化，以细胞外基质(ECM)蛋白积聚为特征，损害正常 功能障碍是致残和死亡的主要原因。尽管已经做了大量的研究来确定 多个潜在的细胞谱系可以产生负责细胞外基质产生的成纤维细胞，它有 人们普遍认为这些细胞是相对均一的称为肌成纤维细胞的细胞群， 以高表达平滑肌肌动蛋白(ASMA)为特征。然而，有限的信息关于 体内这些细胞的分子特征限制了我们对其基本生物学基础的了解 纤维化，阻碍了有效治疗方法的发展。为了解决这一重要差距，我们使用了 胶原产生细胞的单细胞RNA测序(ScRNAseq)以识别多种不同的细胞类型 在正常和纤维化的小鼠和人的肺中产生胶原。使用近距离原位结扎术 杂交(PLISH)我们鉴定了具有不同分子特征的胶原生成细胞亚群 集中在传导气道壁(支气管周围)、周围的支气管血管束内 (外膜)，弥漫分布于气体交换区(肺泡)。用博莱霉素治疗后， 出现了独特的新亚群，表达高水平的胶原蛋白和其他ECM蛋白，并且是唯一的 以含有蛋白1的胶原三螺旋重复序列(Ctrc1)表达为特征。解离的scRNAseq 来自正常和纤维化人肺的细胞也鉴定出了一组CTHRC1-表达的细胞 表达最高水平的胶原蛋白和其他ECM蛋白，只有在 肺纤维化患者。在这里提出的研究中，我们将首先确定地理和 应用PLISH，单次和多次给药博莱霉素模型中ctrc1细胞的时间分布和谱系 我们正在开发的用于追踪来自支气管周围、外膜的细胞的过继转移和新型ERcre系 和肺泡成纤维细胞。接下来，我们将通过过继转导的方法评估ctrc1细胞的功能。 正常或博莱霉素处理的小鼠，一系列与病理相关的行为的体外研究 成纤维细胞，并通过与我们产生的新的ctrc1-ERcre系杂交来删除这些细胞 在ROSA基因座表达lox-Stop-lox DTA的小鼠。最后，我们将检查每个组件的功能意义 在基线和模型中我们在正常肺中鉴定出的主要胶原生成细胞群 使用类似的消融策略或通过删除先前显示的基因来治疗肺泡和呼吸道纤维化 会导致组织纤维化。从这些研究中，我们希望对每一个角色都有新的见解 独特的成纤维细胞亚群在肺内稳态和疾病中发挥作用。

ABSTRACT Pulmonary fibrosis, characterized by accumulation of extracellular matrix (ECM) proteins that impair normal function, is a major cause of disability and death. Although considerable research has been done to identify multiple potential lineages of cells that can give rise to the fibroblasts responsible for ECM production, it has been widely assumed that these cells are a relatively homogeneous population of cells called myofibroblasts, characterized by high expression of a smooth muscle actin (aSMA). However, limited information about the molecular characteristics of these cells in vivo has limited our understanding of the basic biology underlying fibrosis and hampered the development of effective therapies. To address this important gap, we have used single cell RNA sequencing (scRNAseq) of collagen producing cells to identify multiple distinct cell types that produce collagen in the normal and fibrotic murine and human lung. Using proximity ligation in situ hybridization (PLISH) we identified subsets of collagen-producing cells with distinct molecular signatures that were concentrated within the walls of conducting airways (peribronchial), surrounding bronchovascular bundles (adventitial) and diffusely distributed in gas exchanging regions (alveolar). After treatment with bleomycin, a distinct new subset emerged that expressed high levels of collagens and other ECM proteins and was uniquely marked by expression of collagen triple helix repeat containing protein 1 (cthrc1). scRNAseq of dissociated cells from normal and fibrotic human lungs also identified a population of cells marked by CTHRC1-expression that expressed the highest levels of collagens and other ECM proteins and was only seen in lungs from patients with pulmonary fibrosis. In the studies proposed here, we will first determine the geographic and temporal distribution and lineage of cthrc1+ cells in single and repeated dose bleomycin models using PLISH, adoptive transfer and novel ERcre lines we are developing to track cells derived from peribronchial, adventitial and alveolar fibroblasts. Next, we will evaluate the functional roles of cthrc1+ cells using adoptive transfer into normal or bleomycin-treated mice, in vitro studies of an array of behaviors associated with pathologic fibroblasts, and through deletion of these cells by crossing a novel cthrc1-ERcre line we have generated to mice expressing lox-stop-lox dta in the Rosa locus. Finally, we will examine the functional significance of each of the major collagen-producing cell populations we have identified in normal lungs at baseline and in models of alveolar and airway fibrosis, using a similar ablation strategy or through deletion of genes previously shown to contribute to tissue fibrosis. From these studies we hope to gain novel insights into the roles each of these unique fibroblast subsets plays in lung homeostasis and disease.