Fibroblast heterogeneity in pulmonary fibrosis

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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.