Fibroblast heterogeneity in pulmonary fibrosis

摘要 肺纤维化，其特征在于细胞外基质（ECM）蛋白的积累， 功能障碍是残疾和死亡的主要原因。尽管已经做了大量的研究来确定 多种潜在的细胞谱系，可以产生负责ECM生产的成纤维细胞，它具有 人们普遍认为这些细胞是一种相对同质的称为肌成纤维细胞的细胞群， 其特征在于平滑肌肌动蛋白（aSMA）的高表达。然而，有关的信息有限， 这些细胞在体内的分子特征限制了我们对这些细胞的基本生物学基础的理解。 纤维化，阻碍了有效疗法的发展。为了解决这一重要差距，我们使用了 胶原蛋白产生细胞的单细胞RNA测序（scRNAseq），以鉴定多种不同的细胞类型， 在正常和纤维化鼠和人肺中产生胶原蛋白。使用原位邻位连接 通过PLISH技术，我们鉴定了具有不同分子特征的胶原蛋白产生细胞亚群， 集中在传导气道壁（支气管周围），支气管血管束周围 （外膜）和弥漫性分布在气体交换区（肺泡）。博莱霉素治疗后， 出现了一个独特的新亚群，表达高水平的胶原蛋白和其他ECM蛋白， 以表达含胶原蛋白三螺旋重复序列的蛋白1（cthrc 1）为标志。解离的scRNAseq 来自正常和纤维化人肺的细胞也鉴定出CTHRC 1表达标记的细胞群， 表达最高水平的胶原蛋白和其他ECM蛋白，并且只在来自 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 使用PLISH在单剂量和重复剂量博来霉素模型中cthrc 1+细胞的时间分布和谱系， 我们正在开发过继转移和新型ERcre系，以追踪来自支气管周围、外膜 和肺泡成纤维细胞。接下来，我们将评估cthrc 1+细胞的功能作用，使用过继转移到 正常或博来霉素治疗的小鼠，在体外研究与病理性 成纤维细胞，并通过与我们已经产生的新的cthrc 1-ERcre系杂交来删除这些细胞， 在Rosa位点表达lox-stop-lox dta的小鼠。最后，我们将检查每个功能的重要性 我们在正常肺的基线和模型中鉴定的主要胶原蛋白产生细胞群 使用类似的消融策略或通过删除先前显示的基因， 导致组织纤维化从这些研究中，我们希望获得对这些角色的新见解， 独特的成纤维细胞亚群在肺内环境稳定和疾病中起作用。

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.