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Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis
Yan Xu, … , Barry R. Stripp, Jeffrey A. Whitsett
Yan Xu, … , Barry R. Stripp, Jeffrey A. Whitsett
Published December 8, 2016
Citation Information: JCI Insight. 2016;1(20):e90558. https://doi.org/10.1172/jci.insight.90558.
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Research Article Inflammation

Single-cell RNA sequencing identifies diverse roles of epithelial cells in idiopathic pulmonary fibrosis

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Abstract

Idiopathic pulmonary fibrosis (IPF) is a lethal interstitial lung disease characterized by airway remodeling, inflammation, alveolar destruction, and fibrosis. We utilized single-cell RNA sequencing (scRNA-seq) to identify epithelial cell types and associated biological processes involved in the pathogenesis of IPF. Transcriptomic analysis of normal human lung epithelial cells defined gene expression patterns associated with highly differentiated alveolar type 2 (AT2) cells, indicated by enrichment of RNAs critical for surfactant homeostasis. In contrast, scRNA-seq of IPF cells identified 3 distinct subsets of epithelial cell types with characteristics of conducting airway basal and goblet cells and an additional atypical transitional cell that contributes to pathological processes in IPF. Individual IPF cells frequently coexpressed alveolar type 1 (AT1), AT2, and conducting airway selective markers, demonstrating “indeterminate” states of differentiation not seen in normal lung development. Pathway analysis predicted aberrant activation of canonical signaling via TGF-β, HIPPO/YAP, P53, WNT, and AKT/PI3K. Immunofluorescence confocal microscopy identified the disruption of alveolar structure and loss of the normal proximal-peripheral differentiation of pulmonary epithelial cells. scRNA-seq analyses identified loss of normal epithelial cell identities and unique contributions of epithelial cells to the pathogenesis of IPF. The present study provides a rich data source to further explore lung health and disease.

Authors

Yan Xu, Takako Mizuno, Anusha Sridharan, Yina Du, Minzhe Guo, Jie Tang, Kathryn A. Wikenheiser-Brokamp, Anne-Karina T. Perl, Vincent A. Funari, Jason J. Gokey, Barry R. Stripp, Jeffrey A. Whitsett

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Figure 4

Single-cell RNA analysis identifies altered epithelial gene expression and epithelial cell types in IPF.

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Single-cell RNA analysis identifies altered epithelial gene expression a...
(A) Single cells from human IPF (n = 6) and donor (n = 3) distal lung (CD326+) were prepared using the Fluidigm C1 system. RNA was prepared and analyzed from a total of 325 single cells from IPF and 215 cells from donor lungs. Shown are lung epithelial cell markers: EPCAM and CDH1; alveolar type 1 cell markers: AGER and HOPX; alveolar type 2 cell markers: SFTPC, SLC34A2, and ABCA3; proximal lung epithelial cell markers: SOX2, PAX9, TP63, KRT5, KRT14, MUC5B, and SCGB1A1. Expression values were measured in TPM and square root (sqrt) normalized. Cells are shown in solid colors if the expressions of the markers were greater than 1 (TPM). (B) MUC5B, PAX9, and SOX2 were selectively expressed in subsets of IPF cells (MUC5B: n = 24, PAX9: n = 65; SOX2: n = 24) but not present in C1 control cells. Representative genes clustering with MUC5B, PAX9, and SOX2 in IPF cells are shown in the heatmaps. Equal numbers of control cells were randomly selected. IPF cells expressed a diversity of conducting airway epithelial markers not present in control cells, the latter expressing RNAs characteristic of AT2 cells. (C) Only 9 of 325 IPF cells clustered with control cells, the heatmap indicating “AT2”-like expression patterns; however, these 9 normal IPF cells also coexpressed some of the of IPF-associated disease markers. Expression data (TPM) were log10 transformed.
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