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Single-cell RNA sequencing identifies TGF-β as a key regenerative cue following LPS-induced lung injury
Kent A. Riemondy, … , Jay R. Hesselberth, Rachel L. Zemans
Kent A. Riemondy, … , Jay R. Hesselberth, Rachel L. Zemans
Published March 26, 2019
Citation Information: JCI Insight. 2019;4(8):e123637. https://doi.org/10.1172/jci.insight.123637.
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Research Article Pulmonology

Single-cell RNA sequencing identifies TGF-β as a key regenerative cue following LPS-induced lung injury

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Abstract

Many lung diseases result from a failure of efficient regeneration of damaged alveolar epithelial cells (AECs) after lung injury. During regeneration, AEC2s proliferate to replace lost cells, after which proliferation halts and some AEC2s transdifferentiate into AEC1s to restore normal alveolar structure and function. Although the mechanisms underlying AEC2 proliferation have been studied, the mechanisms responsible for halting proliferation and inducing transdifferentiation are poorly understood. To identify candidate signaling pathways responsible for halting proliferation and inducing transdifferentiation, we performed single-cell RNA sequencing on AEC2s during regeneration in a murine model of lung injury induced by intratracheal LPS. Unsupervised clustering revealed distinct subpopulations of regenerating AEC2s: proliferating, cell cycle arrest, and transdifferentiating. Gene expression analysis of these transitional subpopulations revealed that TGF-β signaling was highly upregulated in the cell cycle arrest subpopulation and relatively downregulated in transdifferentiating cells. In cultured AEC2s, TGF-β was necessary for cell cycle arrest but impeded transdifferentiation. We conclude that during regeneration after LPS-induced lung injury, TGF-β is a critical signal halting AEC2 proliferation but must be inactivated to allow transdifferentiation. This study provides insight into the molecular mechanisms regulating alveolar regeneration and the pathogenesis of diseases resulting from a failure of regeneration.

Authors

Kent A. Riemondy, Nicole L. Jansing, Peng Jiang, Elizabeth F. Redente, Austin E. Gillen, Rui Fu, Alyssa J. Miller, Jason R. Spence, Anthony N. Gerber, Jay R. Hesselberth, Rachel L. Zemans

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Figure 1

scRNAseq reveals naive lung epithelial cell types.

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scRNAseq reveals naive lung epithelial cell types.
SftpcCreERT2;mTmG mic...
SftpcCreERT2;mTmG mice were treated with or without LPS. At day 7, non-AEC2 epithelial cells and AEC2s from naive mice and AEC2-derived cells from LPS-treated mice were subjected to scRNAseq. (A) tSNE plot of all cells sequenced. (B) Locations within the tSNE plot of Naive Non-AEC2 Epithelial, Naive AEC2, and Injured AEC2-Derived cells. (C) Unsupervised clustering with a resolution of 0.6. (D and E) Gene expression of canonical AEC1 (D) and AEC2 (E) markers (natural log of normalized counts). (F) Based on gene expression patterns, specific epithelial and nonepithelial cell types were identified. (G) Expression levels of canonical cell markers by the identified cell types. (H) Heatmap of the 20 most differentially expressed genes in naive AEC2 and AEC1s compared with other naive epithelial cell types, ranked in order of Bonferroni-corrected P value. Green circles (cluster 14), AEC1s; red circles (cluster 5), AEC2s; dark orange circles (cluster 13), Scgb1a1+Sftpc+ cells; light orange circles (cluster 9), club cells; yellow circles (cluster 15), ciliated cells; blue circles (cluster 7), basal cells; pink circles (cluster 11), hematopoietic cells; purple circles (cluster 12), endothelial cells/fibroblasts. n = 2 mice per group.

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