Single-Cell Deconvolution of Fibroblast Heterogeneity in Mouse Pulmonary Fibrosis

Abstract

SUMMARYFibroblast heterogeneity has long been recognized in mouse and human lungs, homeostasis, and disease states. However, there is no common consensus on fibroblast subtypes, lineages, biological properties, signaling, and plasticity, which severely hampers our understanding of the mechanisms of fibrosis. To comprehensively classify fibro-blast populations in the lung using an unbiased approach, single-cell RNA sequencing was performed with mesenchymal preparations from either uninjured or bleomycin-treated mouse lungs. Single-cell transcriptome analyses classified and defined six mesenchymal cell types in normal lung and seven in fibrotic lung. Furthermore, delineation of their differentiation trajectory was achieved by a machine learning method. This collection of single-cell transcriptomes and the distinct classification of fibroblast subsets provide a new resource for understanding the fibroblast landscape and the roles of fibroblasts in fibrotic diseases.

Single Cell RNA-seq reveals ectopic and aberrant lung resident cell populations in Idiopathic Pulmonary Fibrosis

“…To 110 characterize myofibroblasts and fibroblasts in the IPF lung, we first focused on cell populations characterized by PDGFRB expression, and then removed cells which were characterized as smooth muscle cells (DES, ACTG2 and PLN) or pericytes (RGS5, COX4I2). This strategy allowed us to identify two distinct stromal populations: fibroblasts were characterized by expression of CD34, FBN1, FBLN2 and VIT, whereas myofibroblasts consistently express MYLK, NEBL, MYO10, 115 MYO1D, RYR2 and ITGA8, as described in the murine lung (24,25) (Fig. 4A).…”

“…Among epithelial cells, we identify a novel population of IPF enriched aberrant basaloid cells that 25 co-express basal epithelial markers, mesenchymal markers, senescence markers, developmental transcription factors and are located at the edge of myofibroblast foci in the IPF lung. Among vascular endothelial cells we identify an expanded cell population in IPF transcriptomically identical to vascular endothelial cells normally restricted to the bronchial circulation.…”

Origin and functional heterogeneity of fibroblasts

“…Building on scRNA sequencing in murine lung development, myofibroblasts ( Acta2 (αSMA), Myh11 (encoding smooth muscle myosin heavy chain 11), and Tagln (encoding a smooth muscle cytoskeletal protein named Transgelin)‐expressing fibroblast) expand in fibrotic lung in an inverse relationship with the transcript levels of Col1a1 and Acta2 , suggesting further heterogeneity may exists within this subtype . Fibrotic lung also displays a unique subpopulation of fibroblasts, albeit in low frequency when compared to healthy lung, characterized by high levels of transcripts encoding Pdgfrβ but which are not derived from pericytes . Matrix producing fibroblasts were further subclassified as Col13α1 + and Col14α1 + subpopulations, respectively, with distinct enrichment of transcripts discriminating the two subpopulations (eg, Cxcl14 and Itga8 in Col13α1 + fibroblasts, and Pi16 and Mmp3 in Col14α1 + fibroblasts).…”

Single-cell RNA-seq reveals spatially restricted multicellular fibrotic niches during lung fibrosis

“…of resident fibroblasts within spatially-restricted profibrotic niches. We queried both our asbestos and published bleomycin (Xie et al, 2018) alveolar macrophage single-cell RNA-seq datasets for the expression of ligands that have partnering receptors on fibroblasts and could be involved in their proliferation. Using curated database of the ligand-receptor pairs (Ramilowski et al, 2015) we identified that macrophages from AM3 cluster in both asbestos-and bleomycin-treated mice expressed Pdgfa (Figure 6A, B).…”