Telomere shortening and DNA damage in culprit cells of different types of progressive fibrosing interstitial lung disease

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.

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.

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.