Telomere length is a determinant of emphysema susceptibility

Abstract

Rationale: Germline mutations in the enzyme telomerase cause telomere shortening, and have their most common clinical manifestation in age-related lung disease that manifests as idiopathic pulmonary fibrosis. Short telomeres are also a unique heritable trait that is acquired with age.

Objectives: We sought to understand the mechanisms by which telomerase deficiency contributes to lung disease.

Methods: We studied telomerase null mice with short telomeres.

Measurements and main results: Although they have no baseline histologic defects, when mice with short telomeres are exposed to chronic cigarette smoke, in contrast with controls, they develop emphysematous air space enlargement. The emphysema susceptibility did not depend on circulating cell genotype, because mice with short telomeres developed emphysema even when transplanted with wild-type bone marrow. In lung epithelium, cigarette smoke exposure caused additive DNA damage to telomere dysfunction, which limited their proliferative recovery, and coincided with a failure to down-regulate p21, a mediator of cellular senescence, and we show here, a determinant of alveolar epithelial cell cycle progression. We also report early onset of emphysema, in addition to pulmonary fibrosis, in a family with a germline deletion in the Box H domain of the RNA component of telomerase.

Conclusions: Our data indicate that short telomeres lower the threshold of cigarette smoke-induced damage, and implicate telomere length as a genetic susceptibility factor in emphysema, potentially contributing to its age-related onset in humans.

Figure 1.

C57BL/6J mice with short telomeres develop emphysematous changes after cigarette smoke (CS) exposure. (A) Representative images show air space enlargement in G4 mice compared with controls after 6 months exposure (hematoxylin and eosin, original magnification ×100). RA = room air. The emphysematous changes shown in the lower right panel represent regional areas of emphysema that were seen in short telomeres CS-exposed mice. (B) Mean linear intercept (MLI) quantification in a blinded analysis from 12–15 mice per group. Mice were 9–11 months of age, and were sex-matched across groups. (C) Table shows mean values of body weight and pulmonary function studies. RV/TLC = ratio of the residual volume relative to the total lung capacity (n = 7–9 mice per group). V₁₀ = percentage of TLC at 10 cm H₂O. Specific compliance = compliance corrected for TLC. Error bars = SEM. * P < 0.05; ** P < 0.01. P values in C refer to comparisons with mean in respective RA-exposed control group. WT = wild-type.

Figure 2.

The telomere-mediated emphysema susceptibility is independent of circulating cells. (A) Representative images of alveolar macrophages detected by Mac-3 immunohistochemistry indicated by arrows. (B) Quantitation of alveolar macrophages in room air (RA) and cigarette smoke (CS) exposed groups after 6 months exposure. The mean number of macrophages was calculated based on counts per high power field (HPF) (n = 8–9 mice per group, eight images per lung). WT = wild-type. (C) WT mice transplanted with WT bone marrow (hematoxylin and eosin, original magnification 100×). (D) Representative image of air space enlargement in a G4 mouse that received a bone marrow transplant from a WT donor. (E) Mean linear intercept (MLI) after 6 months CS exposure (n = 3–5 mice per group). (F) Macrophage quantitation of transplanted mice shown in E. For E and F, mice were 11–14 months at the time of analysis. Error bars = SEM. * P < 0.05; ** P < 0.01.

Figure 3.

Cigarette smoke (CS) causes additive DNA damage to telomere dysfunction. (A) Representative images of terminal bronchioles identifies damage foci detected by 53BP1 nuclear foci (green) in Clara cells identified by cytoplasmic CC10 (red). (B) Percentage of cells containing DNA damage foci after 6 months CS exposure (n = 7–10 mice per group). WT = wild-type. (C) Merged triple color immunofluorescence images of telomere signals (red) in type 2 alveolar cells marked by SPC staining (green) and nuclei (blue). The number of nuclear signals per DAPI area is fewer in G4 cells. (D) The number of telomere signals relative to DAPI area is shown in the bar graph (n = 12–15 mice per group, 20–30 nuclei were imaged and quantified). Mice were exposed to CS for 6 months. AEC = alveolar epithelial cells; DAPI = 4′,6-diamidino-2-phenylindole; error bars = SEM. * P < 0.05; *** P < 0.001.

Figure 4.

Short telomeres limit epithelial recovery after cigarette smoke (CS). (A) Type 2 alveolar epithelial proliferation was measured at three time points: at baseline, immediately after 14 days CS exposure, and 14 days after CS ended. For each time point, a mini-osmotic pump delivered EdU subcutaneously for 14 days, and proliferation in type 2 alveolar cells was identified by costaining with cytoplasmic SPC (n = 5 mice per group, 6–8 high power fields per lung). AEC = alveolar epithelial cells. (B) Proliferation of terminal bronchiolar Clara cells was measured similar to A, but with EdU colocalization with CC10 (n = 5 mice per group, 5–10 terminal bronchioles per lung). WT = wild-type. (C–F) Relative expression levels of cyclin-dependent kinase inhibitors from three groups of mice are shown from a long-term experiment: room air (RA)–exposed mice represent baseline time point, mice exposed to 6 months of CS, and mice that were exposed to 6 months of CS that were then allowed to recover in RA for 7 days (n = 5–8 mice for each data point, 9–11 mo old). (G–J) Relative expression levels of cyclin-dependent kinase inhibitors from three groups of mice are shown from a short-term experiment: RA-exposed mice represent baseline time point, mice exposed to 14 days CS exposure, and mice that were exposed to 14 days CS that were then allowed to recover in RA for 14 days (n = 5 mice for each data point, 6 mo old). Expression in whole lung lysates was measured by real-time polymerase chain reaction and normalized to Hprt and β₂m expression. Wild-type RA control group transcript levels were assigned a value of 1 for relative comparison. Error bars = SEM. *P < 0.05; **P < 0.01.

Figure 5.

p21 is a determinant of basal pulmonary epithelial proliferation. (A) Representative immunofluorescence showing EdU staining (red) in type 2 alveolar epithelial cells (AECs) marked by cytoplasmic SPC (green) in wild-type (WT) mice and in (B) p21^−/− mice. (C) The percentage of proliferating AECs in p21^−/− mice was higher as shown in the bar graph. (D and E) Representative images showing EdU staining (red) in terminal bronchiole Clara cells (green) in WT and p21^−/− lungs, respectively. (F) Increased proliferative fraction of EdU-CC10–positive cells in bar graph. (G and H) Intestinal tract epithelium shows EdU incorporation (red) in WT and p21^−/− mice, respectively. (I) Bar graph shows quantitation after twice-daily injections for 2 days. n = 5 mice per group, age 3–4 months, 10 HPF were analyzed per mouse. Error bars = SEM. * P < 0.05.

Figure 6.

Early onset emphysema as a manifestation of inherited telomerase mutation. (A) Pedigree with features of telomere syndrome including idiopathic pulmonary fibrosis (IPF), bone marrow failure, and premature hair graying shows autosomal-dominant inheritance. The proband is indicated by an arrow and the shaded squares and circles indicate mutation carrier. I1 is a probable carrier, and II3 is an obligate carrier. The summary of the clinical history is listed below with CS referring to the cigarette smoke exposure history. (B and C) Apical and mid-lung computed tomography (CT) images from the proband show evidence of emphysema with apical bullae and hyperinflation. (D and E) CT images of II3 show subcutaneous emphysema caused by a spontaneous pneumothorax, apical bullae, and mid-lung ground glass infiltrates with septal thickening consistent with a combined emphysema and interstitial lung disease phenotype. (F) Chromatogram showing heterozygous deletion in hTR, the RNA component of telomerase. (G) Secondary structure of hTR shows site of the three nucleotide deletion within the essential Box H domain in red. (H) Quantification of hTR levels in cells from unaffected relatives (n = 3), affected mutation carriers (n = 3), and DKC1 mutation carriers (n = 2). Data were generated by quantitative real-time polymerase chain reaction and hTR levels were normalized to ARF3 levels. ** P < 0.01. (I) Lymphocyte telomere length in proband and mutation carriers shows significant shortening relative to age-matched controls. Identifiers refer to pedigree in A.