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. 2021 Mar 15;203(6):707-717.
doi: 10.1164/rccm.202004-1274OC.

Senescence of Alveolar Type 2 Cells Drives Progressive Pulmonary Fibrosis

Changfu Yao  1   2 , Xiangrong Guan  1 , Gianni Carraro  1 , Tanyalak Parimon  1 , Xue Liu  1 , Guanling Huang  1 , Apoorva Mulay  1 , Harmik J Soukiasian  3 , Gregory David  4 , Stephen S Weigt  5 , John A Belperio  5 , Peter Chen  1 , Dianhua Jiang  1 , Paul W Noble  1 , Barry R Stripp  1   2
Affiliations

Senescence of Alveolar Type 2 Cells Drives Progressive Pulmonary Fibrosis

Changfu Yao et al. Am J Respir Crit Care Med. .

Erratum in

Abstract

Rationale: Idiopathic pulmonary fibrosis (IPF) is an insidious and fatal interstitial lung disease associated with declining pulmonary function. Accelerated aging, loss of epithelial progenitor cell function and/or numbers, and cellular senescence are implicated in the pathogenies of IPF.Objectives: We sought to investigate the role of alveolar type 2 (AT2) cellular senescence in initiation and/or progression of pulmonary fibrosis and therapeutic potential of targeting senescence-related pathways and senescent cells.Methods: Epithelial cells of 9 control donor proximal and distal lung tissues and 11 IPF fibrotic lung tissues were profiled by single-cell RNA sequencing to assesses the contribution of epithelial cells to the senescent cell fraction for IPF. A novel mouse model of conditional AT2 cell senescence was generated to study the role of cellular senescence in pulmonary fibrosis.Measurements and Main Results: We show that AT2 cells isolated from IPF lung tissue exhibit characteristic transcriptomic features of cellular senescence. We used conditional loss of Sin3a in adult mouse AT2 cells to initiate a program of p53-dependent cellular senescence, AT2 cell depletion, and spontaneous, progressive pulmonary fibrosis. We establish that senescence rather than loss of AT2 cells promotes progressive fibrosis and show that either genetic or pharmacologic interventions targeting p53 activation or senescence block fibrogenesis.Conclusions: Senescence of AT2 cells is sufficient to drive progressive pulmonary fibrosis. Early attenuation of senescence-related pathways and elimination of senescent cells are promising therapeutic approaches to prevent pulmonary fibrosis.

Keywords: alveolar type 2 cells; cellular senescence; pulmonary fibrosis.

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Figures

Figure 1.
Figure 1.
Accumulation of senescent alveolar type 2 (AT2) cells in idiopathic pulmonary fibrosis (IPF) explant tissue. (A) Uniform manifold approximation and projection (UMAP) visualization of cell type clustering (AT1, AT2, club [SCGB3A2+, SCGB1A1+], SCGB3A2+ club-like [SCGB3A2+, SCGB1A1−], ciliated, preciliated [MCIDAS+], MUC5AC high goblet [MUC5AC+, MUC5B+], MUC5B high goblet [MUC5AC−, MUC5B+], KRT15+ basal [KRT5 high, KRT17+, KRT15 high], KRT17+ basal [KRT5 low, KRT17+, KRT15 low], and ionocyte) and cell origin of single-cell RNA sequencing data on isolated epithelial cells from donor lung tissues and fibrotic region of lung tissue from patients with IPF (9 donor patient samples and 11 IPF patient samples). (B) Heat-map visualization of cellular senescence score based on core senescence genes of all epithelial cell types. Color scale represents log-transformed ratio of cellular senescence score. (C) Violin plot visualization of cellular senescence score based on core senescence genes of the AT2 cell subset from human epithelial cell single-cell RNA sequencing. The dashed line indicates the median value of each group. (D) Heat map of the top 20 differential expression genes of the AT2 cell subset comparing IPF with donor and top canonical pathways of genes upregulated in IPF AT2 cells identified by Ingenuity Pathway Analysis; senescence pathway and senescence-related pathways are highlighted. In total, 506 significantly upregulated genes and 1,209 significantly downregulated genes comparing IPF AT2 cells with donor AT2 cells. IPA = Ingenuity Pathway Analysis; PNEC = pulmonary neuroendocrine cell.
Figure 2.
Figure 2.
Loss of Sin3a in adult mouse alveolar type 2 (AT2) cells leads to cellular senescence. (A) Schematic outline of experiment design. (B) Western blot detection of Sin3a knockout efficiency in isolated AT2 cells 2 weeks after tamoxifen treatment. (C) Time course of immunofluorescence staining for lineage reporter-GFP (green fluorescent protein) and pro-Sftpc in control and Sin3a-LOF (Sin3a loss of function) lung. Scale bars, 20 μm. (D) Representative immunofluorescence staining of membrane-bound GFP and Imaris surface rendering for surface area quantification. Scale bars, 20 μm. (E) AT2 cell surface area quantification. (F) Uniform manifold approximation and projection (UMAP) visualization of AT2 cell subset, origin, and clustering from mouse epithelial cell single-cell RNA sequencing. (G) Ingenuity Pathway Analysis canonical pathway analysis of genes upregulated in AT2 cells of Sin3a-LOF compared with control; senescence pathway and senescence-related pathways are highlighted. (H) Violin plot visualization of cellular senescence score based on core senescence genes comparing Sin3a-LOF AT2 cells with control AT2 cells. The dashed line indicates the median value of each group. (I) Representative immunofluorescence staining of lineage reporter gene (GFP) and pro-Sftpc, comparing lung tissue of control with Sin3a-LOF at different times after tamoxifen exposure. Scale bars, 20 μm. (J) Quantification of percentage of either lineage reporter GFP+ cells, pro-Sftpc+ cells, or the ratio of GFP+/pro-Sftpc+ cells as a function of total cells within a section of lung lobe of Sin3a-LOF mice over the time course. Control samples are SftpcCreER and RosamTmG mice 6 weeks after tamoxifen treatment. P values were calculated by nonparametric Mann-Whitney test. **P < 0.01 and ****P < 0.0001. CON = control; IPA = Ingenuity Pathway Analysis.
Figure 3.
Figure 3.
Loss of Sin3a in alveolar type 2 cells leads to progressive lung fibrosis. (A) Schematic outline of experiment design. (B) Body weight change for tamoxifen-exposed Sin3a-LOF (Sin3a loss of function) and control mice. (C) Survival curve for tamoxifen-exposed Sin3a-LOF and control mice. The P value was calculated by log-rank (Mantel-Cox) survival analysis. (D) Time course of Masson’s trichrome staining of lung tissue from tamoxifen-exposed Sin3a-LOF and control mice. Squares indicate zones of magnified images. Scale bars: top, 1 mm; bottom, 50 μm. (E) Representative immunofluorescence staining of Sin3a-LOF lung tissue 6 weeks after tamoxifen exposure for lineage reporter-GFP (green fluorescent protein), pro-Sftpc, and αSMA. The dashed line represents the boundary between fibrotic and nonfibrotic tissue. E′ and E″ are magnified images of representative regions from either nonfibrotic or fibrotic tissue. Scale bars: E, 200 μm; E′ and E″, 20 μm. (F) Western blot detection of αSMA abundance within lung tissue of Sin3a-LOF mice. (G) Hydroxyproline content of right lung between Sin3a-LOF and control mice 6 weeks after tamoxifen exposure (n = 10 for each group). The P value was calculated by two-tailed Student’s t test. ****P < 0.0001.
Figure 4.
Figure 4.
p53 pathway activation in Sin3a-LOF (Sin3a loss of function) alveolar type 2 (AT2) cells. (A) Violin plot visualization of p53 activation score calculated using p53 Kyoto Encyclopedia of Genes and Genomes pathway genes of the mouse single-cell RNA sequencing (scRNA-Seq) AT2 cell subset. The dashed line indicates the median value of each group. (B) Violin plot visualization of p53 activation score of the human scRNA-Seq AT2 cell subset. The dashed line indicates the median expression of each group. (C) Representative immunofluorescence staining of lineage reporter-GFP (green fluorescent protein) and Cdkn1a/p21, comparing control lung tissue with Sin3a-LOF 6 weeks after tamoxifen exposure. Scale bars, 10 μm. (D) Violin plot representation showing relative expression of Cdkn1a/p21 in mouse scRNA-Seq AT2 subset. The dashed line indicates the median expression of each group. (E) Schematic outline of experiment design for p53-LOF studies. (F) Survival curve for Sin3a-LOF and Sin3a/p53-LOF groups 6 weeks after tamoxifen treatment. The P value was calculated by log-rank (Mantel-Cox) survival analysis. (G) Hematoxylin and eosin and Masson’s trichrome staining of Sin3a-LOF and Sin3a/p53-LOF lung tissues 6 weeks after tamoxifen treatment. Squares indicate zones of magnified images. (H) Hydroxyproline content of right caudal lobe in Sin3a-LOF and Sin3a/p53-LOF mice 6 weeks after tamoxifen treatment (n = 10 for each group). P values were calculated by two-tailed Student’s t test. ****P < 0.0001. H&E = hematoxylin and eosin; IPF = idiopathic pulmonary fibrosis; ns = not significant.
Figure 5.
Figure 5.
Senescence rather than loss of alveolar type 2 (AT2) cells results in progressive lung fibrosis. (A) Schematic outline of experiment design for AT2 cell ablation. (B) Quantitative PCR of control and diphtheria toxin A (DTA) mouse lung tissues for relative changing expression of Sftpc, indicating AT2 cell ablation efficiency (n > 3). (C) Masson’s trichrome staining of Sin3a-LOF (Sin3a loss of function) mice after repeated tamoxifen treatment. Squares indicate zones of magnified images. Scale bars: top, 1 mm; bottom, 50 μm. (D) Masson’s trichrome staining of DTA mice after repeated tamoxifen treatment. Squares indicate zones of magnified images. Scale bars: top, 1 mm; bottom, 50 μm. (E) Hydroxyproline content in the right caudal lobe of Sin3a-LOF mice after 4 and 6 weeks repeated exposure of tamoxifen and control groups. (F) Hydroxyproline content in the right caudal lobe of DTA mice after 4, 6, and 8 weeks repeated exposure of tamoxifen and control groups. (G) Schematic outline of experiment design for senolytic drug treatment. (H) Masson’s trichrome staining of Sin3a-LOF mice 6 weeks after tamoxifen treatment comparing 5 mg/kg dasatinib and 50 mg/kg quercetin (D+Q) cocktail treatment and vehicle treatment groups. Squares indicate zones of magnified images. Scale bars: top, 1 mm; bottom, 50 μm. (I) Quantitative PCR of isolated AT2 cells from control, vehicle-treated, and D+Q-treated Sin3a-LOF mouse lung tissues for relative changing expression of senescence markers indicating senolytic drug treatment efficiency (n = 4). (J) Hydroxyproline content in right caudal lobe of Sin3a-LOF mice treated with either D+Q or vehicle 6 weeks after tamoxifen treatment (n > 10 for each group). P values were calculated by nonparametric Mann-Whitney test (comparison of two groups) or Kruskal-Wallis test (comparison of more than two groups). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. ns = not significant.
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References

    1. King TE, Jr, Pardo A, Selman M. Idiopathic pulmonary fibrosis. Lancet. 2011;378:1949–1961. - PubMed
    1. Schafer MJ, White TA, Iijima K, Haak AJ, Ligresti G, Atkinson EJ, et al. Cellular senescence mediates fibrotic pulmonary disease. Nat Commun. 2017;8:14532. - PMC - PubMed
    1. Lehmann M, Korfei M, Mutze K, Klee S, Skronska-Wasek W, Alsafadi HN, et al. Senolytic drugs target alveolar epithelial cell function and attenuate experimental lung fibrosis ex vivo. Eur Respir J. 2017;50:1602367. - PMC - PubMed
    1. Alder JK, Barkauskas CE, Limjunyawong N, Stanley SE, Kembou F, Tuder RM, et al. Telomere dysfunction causes alveolar stem cell failure. Proc Natl Acad Sci USA. 2015;112:5099–5104. - PMC - PubMed
    1. Minagawa S, Araya J, Numata T, Nojiri S, Hara H, Yumino Y, et al. Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-β-induced senescence of human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol. 2011;300:L391–L401. - PMC - PubMed

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