Telomere dysfunction causes alveolar stem cell failure

Abstract

Telomere syndromes have their most common manifestation in lung disease that is recognized as idiopathic pulmonary fibrosis and emphysema. In both conditions, there is loss of alveolar integrity, but the underlying mechanisms are not known. We tested the capacity of alveolar epithelial and stromal cells from mice with short telomeres to support alveolar organoid colony formation and found that type 2 alveolar epithelial cells (AEC2s), the stem cell-containing population, were limiting. When telomere dysfunction was induced in adult AEC2s by conditional deletion of the shelterin component telomeric repeat-binding factor 2, cells survived but remained dormant and showed all the hallmarks of cellular senescence. Telomere dysfunction in AEC2s triggered an immune response, and this was associated with AEC2-derived up-regulation of cytokine signaling pathways that are known to provoke inflammation in the lung. Mice uniformly died after challenge with bleomycin, underscoring an essential role for telomere function in AEC2s for alveolar repair. Our data show that alveoloar progenitor senescence is sufficient to recapitulate the regenerative defects, inflammatory responses, and susceptibility to injury that are characteristic of telomere-mediated lung disease. They suggest alveolar stem cell failure is a driver of telomere-mediated lung disease and that efforts to reverse it may be clinically beneficial.

Keywords: emphysema; idiopathic pulmonary fibrosis; senescence; telomerase.

Fig. 1.

Short telomeres in AEC2s, but not in stromal cells, limit alveolosphere formation. (A) Design of experiments to test the role of telomere length in alveolosphere formation. The H&E-stained image represents a single alveolosphere that was imaged 14 d after AEC2 were plated. (B) Colony-forming efficiency for Pdgfrα⁺ cells that were sorted from wild-type, mTR^−/− first-generation (mTR^−/−G1), and mTR^−/− fourth-generation (mTR^−/−G4) mice that were plated with Sftpc lineage-labeled wild-type AEC2s. Alveolosphere colonies were counted in triplicate on day 14 for each mouse. (C) AEC2s were sorted from wild-type, mTR^−/−G1, and mTR^−/−G4 mice, mixed with lineage-labeled Pdgfrα⁺ cells, and counted as in B. (D) 53BP1 foci were enumerated in AEC2s marked by ATP-binding cassette subfamily A member 3 (Abca3) by immunofluorescence (n = 6 mice per group). Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, Student's t test.

Fig. 2.

Telomere dysfunction in AEC2s provokes senescence. (A) Telomere FISH using a labeled peptide nucleic acid probe that contains the telomere sequence (TTAGGG)₄ and staining for 53BP1 shows telomere-induced DNA damage foci in sorted AEC2s 4 d after a single tamoxifen dose. (B) Representative images and quantification of 53BP1 foci. 53BP1 foci (green) were enumerated in AEC2s, identified by Abca3 staining, and in Club cells, identified by Club cell secretory protein (also known as CCSP/CC10). Immunofluorescence was performed on day 5 after tamoxifen (n = 3 mice per group). (C) mRNA levels of p53 targets in sorted AEC2s on days 7 and 21 after tamoxifen as measured by quantitative real-time PCR (qRT-PCR) and normalized to hypoxanthine phosphoribosyltransferase (Hprt) levels (n = 3 mice per group). (D) Lineage trace of AEC2s. Trf2^Fl/+;mTmG;Sftpc-CreER and Trf2^Fl/Fl;mTmG;Sftpc-CreER mice were given a single injection of tamoxifen (TAM), and lungs were harvested 7, 14, and 21 d later. The fraction of lineage-labeled GFP⁺ AEC2s, marked by Sftpc, is quantified (n = 3 mice per group). (E) The proliferating fraction of AEC2s following Trf2 deletion. Proliferation was measured after a 14-d EdU label, and proliferating AEC2s were identified by costaining for Sftpc and EdU (n = 5–7 mice per group). (F–H) Trf2^Fl/+;mTmG;Sftpc-CreER and Trf2^Fl/Fl;mTmG;Sftpc-CreER mice were given a single injection of tamoxifen, and lungs were harvested 7 d later. (F) Histogram of GFP⁺ lung cells 1 wk after lineage labeling was induced. (G) Low-power images of alveolospheres growing in the Matrigel/Transwell system. (Scale bars: 50 µm.) (H) Quantification of the colony-forming capacity of AEC2s after 7 and 14 d in culture (n = 3 or 4 mice per group). (I) Immunohistochemical staining of alveolospheres after 14 d in culture. Lineage-labeled cells were identified by GFP staining (green). AEC1s (purple), and AEC2s (red) were identified by podoplanin and prosurfactant protein C staining, respectively. (Scale bars, 50 µm.) (J) Quantification of the number of alveolospheres that contain podoplanin⁺ cells. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.

Fig. 3.

Telomere dysfunction in AEC2s recruits inflammation and impairs repair after injury. (A–I) Trf2^Fl/+;Sftpc-CreER and Trf2^Fl/Fl;Sftpc-CreER mice were treated with tamoxifen and examined 21 d later. (A) Total lung capacity (n = 6–8 mice per group). (B) High-power image of air space enlargement seen occasionally in Trf2^Fl/Fl;Sftpc-CreER mice. (Scale bars: 50 µm.) (C) Bronchoalveolar lavage cellularity. (D) Representative Cytospin images showing macrophages and lymphocytes (marked by arrowheads). (Insets) Differential counts. For C and D, n = 5 mice per group. For differential counts, 250 cells were counted per mouse. (E–H) Representative images from control (E) and Trf2-deleted lungs showing pigmented macrophages (F, arrow) and peribronchiolar (G), and perivascular (H) inflammation. (Scale bars: 100 µm in E and F; 25 µm in G and H.) br, bronchiole; bv, blood vessel. (I) Macrophage quantification per high-powered field (HPF) by Mac-3 immunohistochemistry (n = 4 or 5 mice per group; P value is one-sided). (J) Heat map of gene-expression microarray data from purified AEC2s from Trf2^Fl/+;Sftpc-CreER and Trf2^Fl/Fl;Sftpc-CreER mice (n = 3 mice per group) 7 d after tamoxifen. Red indicates up-regulated genes; blue indicates down-regulated genes. The fold-change based on color is shown in the key below. (K) Pathways identified by Ingenuity analysis of the up-regulated genes. P is calculated by Fisher’s exact test (right-tailed); R is the ratio of the number of genes in the indicated pathway divided by the total number of genes that make up that pathway. (L and M) Mice treated with tamoxifen 1 wk before bleomycin challenge, were weighed every other day (L), and their survival was monitored (M). The log-rank test was used in the Kaplan–Meier survival analysis. (N) EdU incorporation of AEC2s following bleomycin challenge. Mice were challenged with bleomycin and injected with EdU for 3 d before harvest on day 14 (n = 5 mice per group). Data are expressed as mean ± SEM. *P < 0.05. Unless otherwise noted, Student’s t test was used to calculate P values.

Fig. 4.

Telomere dysfunction in epithelial cells signals mesenchymal abnormalities via p53. (A) Pups showing cyanosis in mutants after birth (Right). (B) Newborn lungs showing a morphogenesis defect (Right). (C) E-cadherin staining identifies epithelial luminal cells; the remaining panels show DNA damage signaling marked by γH2AX, p53^Ser15, and p21 immunohistochemistry. (Scale bars: 50 µm.) (D) E-cadherin staining identifies luminal branches during lung morphogenesis. Images were used for quantification shown in E. (Scale bars: 100 microns.) (E) Lung branching was quantified by enumerating the number of E-cadherin⁺ lumina per lung area (n = 3–8 mice per group). (F) Number of apoptotic epithelial and mesenchymal (G) cells (n = 2–8 mice per group). (H and I) mRNA quantification of p53 target genes (n = 4 mice per group). (J) E-cadherin whole-mount staining typical of the embryo lungs analyzed (n = 4 per group). (Scale bar: 500 μm.) Unless otherwise noted, studies shown were performed on embryonic day 14.5 lungs. Data are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, Student’s t test.