Figures
- FIGURE 1
Telomere and DNA damage in fibrotic lung tissue measured by combined FISH and immunofluorescence staining techniques. a) Haematoxylin and eosin staining representing a typical idiopathic pulmonary fibrosis lung biopsy. b–d) Representative fluorescent stained examples of boxed areas in image (a), containing pro-surfactant protein C-positive alveolar type 2 cells (green) in the alveolus (A), CC10-positive club cells (light blue) in the bronchiolus (B) and α-smooth muscle actin-positive myofibroblasts (purple), in the fibroblast focus (FF). e–g) FISH-stained telomere signals (red dots) and h–j) immunofluorescence-stained γH2AX signals (yellow dots) in magnified boxed areas of images (b–d). k–m) Overlay pictures of telomere, γH2AX and DAPI stainings. All fluorescent pictures were captured and Z-stacked using a LSM700 laser scanning confocal microscope. FISH: fluorescence in situ hybridisation; DAPI: 4′,6-diamidino-2-phenylindole; γH2AX: phosphorylated histone protein from the H2A family.
- FIGURE 2
Cell-specific quantification of telomere and DNA damage signals in control and fibrotic lungs. Bar charts of telomere length (measured by FISH) and DNA damage signals (measured by immunofluorescence) in alveolar type 2 (AT2) cells, club cells and myofibroblasts in lungs of a and e) eight controls, b and f) six patients with pulmonary fibrosis and a telomerase reverse transcriptase (TERT-PF) mutation, c and g) 10 idiopathic pulmonary fibrosis (IPF) and d and h) five fibrotic hypersensitivity pneumonitis (fHP) patients. Controls contained long telomeres and low DNA damage signals in AT2 cells and club cells. In TERT-PF and IPF lungs, telomere length in AT2 cells was shorter than in club cells and myofibroblasts (p<0.0001) and DNA damage signals in AT2 cells were significantly higher than in club cells and myofibroblasts (p<0.0001). In fHP lungs, DNA damage signals in club cells were highly elevated compared to AT2 cells and myofibroblasts (p<0.0001), while telomere length in club cells was comparable with AT2 cells and myofibroblasts. Boxes represent medians and whiskers extend up to values within the third quartile. FISH: fluorescence in situ hybridisation; γH2AX: phosphorylated histone protein from the H2A family; Ctrl: control; NA: not applicable. ****: p<0.0001, calculated by Kruskal–Wallis multiple comparison tests.
- FIGURE 3
Observed (obs) and expected (exp) DNA damage signals in alveolar type 2 (AT2) cells, club cells and myofibroblasts of idiopathic pulmonary fibrosis (IPF) and fibrotic hypersensitivity pneumonitis (fHP) lungs. a) Inverse spearman correlation (r=−0.689, p=0.003) of median telomere length and median γH2AX signals in AT2 cells, club cells and myofibroblasts per biopsy specimen in six TERT-PF lungs resulting in a linear regression line with equation y=−4.23x + 5727. This equation was used to calculate expected DNA damage values from observed telomere length signals in b) IPF AT2 cells, c) IPF club cells, d) IPF myofibroblasts, e) fHP AT2 cells, f) fHP club cells, and g) fHP myofibroblasts. In IPF and fHP, club cells and myofibroblasts showed lower expected than observed DNA damage signals, while in AT2 cells no significant difference was found between expected and observed DNA damage signals. Statistical differences were computed by Mann–Whitney tests. γH2AX: phosphorylated histone protein from the H2A family; TERT-PF: patients with pulmonary fibrsosis and a telomerase reverse transcriptase (TERT) mutation. *: p<0.05; **: p<0.01.
- FIGURE 4
Experimental induction of telomere shortening and DNA damage signals by inhibition of telomerase by BIBR1532. Bar charts of telomere length (measured by FISH) and DNA damage signals (measured by immunofluorescence) in cytospins of a and e) A549, b and f) H460, c and g) 16HBE and d and h) MRC5 cell lines. Induced telomere shortening with 25 μM BIBR1532 was only observed in A549 (p=0.004) and H460 cells (p=0.013) compared to untreated cells, while in all cell lines increased DNA damage was found. Telomeres in MRC5 cells treated with 25 μM BIBR1532 were significantly longer than untreated cells (p=0.0001). Boxes represent medians and whiskers extend up to values within the third quartile. FISH: fluorescence in situ hybridisation; γH2AX: phosphorylated histone protein from the H2A family; BIBR1532: 2-[(E)-3-naphthalen-2-yl-but-2-enoylamino]-benzoic acid. *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001, calculated by Kruskal–Wallis multiple comparison tests.
- FIGURE 5
Graphical illustration summarising the main message of this study. The extent to which key cells in progressive fibrosing lung disease are affected by telomere shortening and DNA damage is so far unknown. This study revealed that in patients with idiopathic pulmonary fibrosis (IPF), telomere shortening and accumulation of DNA damage is primarily affecting alveolar type 2 (AT2) cells, further supporting the importance of AT2 cells in this disease, while in fibrotic hypersensitivity pneumonitis (fHP) the particularly high telomere-independent DNA damage signals in club cells underscores its bronchiolocentric pathogenesis.
Tables
- TABLE 1
Group comparison of telomere length and DNA damage in lung tissue
Controls TERT-PF IPF fHP Average TL (T/S ratio) of whole biopsy by MMqPCR (IQR) 0.932# (0.909–0.947) 0.772¶ (0.734–0.803) 0.862 (0.810–0.959) 0.849 (0.778–0.884) AT2 cell γH2AX signal (IQR) 1005 (246–3354) 4253+ (3019–6390) 3923 (2005–5364) 3661 (2028–4749) FISH TL (IQR) 1764# (999–2484) 464¶ (226–703) 729§ (342–1101) 1049 (679–1605) Club cell γH2AX signal (IQR) 1303 (248–3782) 1879 (902–4624) 1745 (487–3407) 6769+ (4025–8827) FISH TL (IQR) 1650# (769–2600) 813¶ (467–1147) 1405 (821–1949) 1155 (847–1864) Myofibroblast γH2AX signal (IQR) NA 752 (0–1637) 388 (9.2–2691) 2080 (681–7381) FISH TL (IQR) NA 924¶ (646–1345) 1518 (1139–2105) 1513 (1055–2069) Numbers describe median telomere length or DNA damage signal. TERT-PF: patients with pulmonary fibrosis and a telomerase reverse transcriptase mutation; IPF: idiopathic pulmonary fibrosis; fHP: fibrotic hypersensitivity pneumonitis; AT2: alveolar type 2; TL: telomere length; NA: not applicable. #: telomeres were significantly longer than in the other study groups (p<0.05); ¶: telomeres were significantly shorter than in the other study groups (p<0.05); +: DNA damage signal was significantly higher than in the other study groups (p<0.05); §: telomeres in AT2 cells of IPF lungs were significantly shorter than in AT2 cells of fHP lungs (p<0.0001).
Supplementary Material
Please note: supplementary material is not edited by the Editorial Office, and is uploaded as it has been supplied by the author.
Supplementary material Supplementary_material
