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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 6

Expression of altered KEGG pathways in human IPF and control single cells.

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Expression of altered KEGG pathways in human IPF and control single cell...
(A) The heatmap shows the top 25 pathways and differentially expressed genes identified using a 1-tailed Welch’s t test of gene expression between the control AT2 cells (C1) and IPF cell clusters (C2, C3, and C4) using the following criteria: P < 0.01, expressed (TPM ≥1) in at least 80% of cell type with induced gene expression. KEGG pathways enriched or suppressed in IPF epithelium were determined by the following criteria: (a) at least 5 genes in the pathway were expressed (TPM ≥1), (b) at least 30% of expressed genes were differentially expressed, and (c) the ratio between the number of C1 differentially expressed genes and the number of IPF differentially expressed genes in the pathway was ≥1.5 or ≤0.67. 4). Pathways were ranked based on the ratios. The expression of a pathway in a cell was measured by the average expression (TPM + 1, log2 transformed) of differentially expressed genes associated in the pathway. Pathways were clustered using hierarchical clustering analysis with Spearman’s correlation–based distance measure and complete linkage. Cancer- or disease-related pathways were excluded. (B) Representative TGF-β signaling pathway genes (BMP1, BMPR1B, INHBA, INHBB, TGFBR1, TGFB1, TGFB2, and SMAD3) in control (n = 215), IPF (n = 316), and relatively normal IPF cells that clustered with control AT2 cells (n = 9). Data are presented as dot plot with mean ± SEM. P values were determined by Student’s t test. **P < 0.05.
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