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

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 TGFβ signaling was highly upregulated in the cell cycle arrest subpopulation and relatively downregulated in transdifferentiating cells. In cultured AEC2s, TGFβ was necessary for cell cycle arrest but impeded transdifferentiation. We conclude that during regeneration after LPS-induced lung injury, TGFβ 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.

Keywords: Adult stem cells; Pulmonology.

Figure 1. scRNAseq reveals naive lung epithelial cell types.

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.

Figure 2. scRNAseq reveals proliferating, cell cycle arrest, and transdifferentiating AEC2-derived subpopulations.

(A–E) Expression levels (natural log of normalized counts) of proliferation (A), G₁ arrest (B), AEC1 (C), and AEC2 (D) markers in the Naive AEC1 and AEC2 and Injured AEC2-Derived cells. Blue circles (cluster 10), proliferating subpopulation; green circles (cluster 8), cell cycle arrest subpopulation; red circles (cluster 6), transdifferentiating subpopulation. (E) Expression levels of markers by the identified subpopulations. (A–E) n = 2 mice per group.

Figure 3. Immunofluorescence/in situ hybridization validates proliferating, cell cycle arrest, and transdifferentiating AEC2-derived subpopulations.

Immunofluorescence staining of lung sections for GFP (A–C), Ki67 (A), and T1α (C) and in situ hybridization for p15 (B). Insets and arrowheads indicate double-positive cells. Scale bars: 25 μm (A), 50 μm (B and C). n ≥ 4 mice per group.

Figure 4. Differential gene expression in regenerative subpopulations.

(A) Subpopulations of regenerating AEC2-derived cells identified as proliferating, cell cycle arrest, transdifferentiating, and other naive and injured cell types. (B) Heatmap of the top 30 most differentially expressed genes in the proliferating, cell cycle arrest, and transdifferentiating subpopulations compared with all the other Injured AEC2-Derived cells. TGF-β pathway genes are indicated in red font. Genes are ranked in order of Bonferroni-corrected P value. (C) Top 3 upstream regulators of gene expression by cells from all 3 regenerative subpopulations combined compared with the Other Injured AEC2-Derived cells, as determined by Ingenuity Pathway Analysis. n = 2 mice per group.

Figure 5. TGF-β pathway is activated in cell cycle arrest subpopulation.

(A and B) Expression of TGF-β signaling molecules in naive and regenerative subpopulations as determined by scRNAseq. Green circles (cluster 8), cell cycle arrest subpopulation. n = 2 mice per group. (C) Immunofluorescence for GFP and in situ hybridization for TGFβ2, and β₆ integrin on fixed lung sections. Insets indicate double-positive cells. Scale bar: 50 μm. n ≥ 4 mice per group.

Figure 6. TGF-β induces AEC2 cell cycle arrest in cultured AEC2s.

Primary rat AEC2s were cultured in the presence or absence of the TGFβRI inhibitor LY364947 (LY) or the TGF-β–neutralizing antibody 1D11 with or without EdU. (A, D, and F) Quantitative PCR was performed. Data are presented as fold change relative to day 0. (B and E) EdU incorporation is shown. (C) Western blotting of cell lysates for p-Smad3 and actin. Densitometry of p-Smad3 corrected for actin is shown. Experiments were performed at least 3 times, each with 2 technical replicates. Two-tailed t test or 1- or 2-way ANOVA with post hoc analysis for multiple comparisons was performed and corrected for repeated measures. Mean ± SEM is shown. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared with day 0 (A and D) or day 1 (C) or LY364947 vs. DMSO or 1D11 vs. IgG1 (E and F).

Figure 7. TGF-β inhibits AEC2-to-AEC1 transdifferentiation in cultured AEC2s.

(A–E) Primary rat AEC2s were cultured in the presence or absence of the TGFβRI inhibitor LY364947 (LY) or the TGF-β–neutralizing antibody 1D11 for the indicated time periods. (A, C, and D) Quantitative PCR (qPCR) for AEC1 markers. (B and E) Western blotting for AEC1 markers with densitometry. Vertical white lines indicate that the lanes were run on the same gel but were noncontiguous. Data are shown as fold change compared with day 0 (A–C) or fold change compared with IgG or DMSO controls (D and E). (F and G) AEC2s were isolated from Tgfbr2^fl/fl mice, transduced with AdGFP or AdCre, and cultured for 3 days. qPCR (F) and Western blotting with densitometry (G) shown as fold change in AdCre samples compared with AdGFP samples. Each experiment was performed at least 3 times, except the 1D11 experiments with 2 technical replicates each. Two-tailed t test or 1- or 2-way ANOVA with post hoc analysis for multiple comparisons was performed and corrected for repeated measures. Mean ± SEM is shown. *P < 0.05, **P < 0.01, ***P < 0.001, †P = 0.07 calculated for fold change relative to day 0 (A–C) or DMSO vs. LY or IgG1 vs. 1D11 (D and E) or AdGFP vs. AdCre (F and G).