We sought to understand the mechanisms by which short telomeres might contribute to lung disease in humans by studying telomerase-null mice. We identified telomere length and telomerase deficiency as a susceptibility factor in emphysema. Late-generation telomerase-null mice developed emphysematous air space enlargement after a chronic CS exposure as evidenced by morphometric differences that affected lung function. Telomere length is heterogeneous and heritable across populations, and short telomeres accumulate with age (7
). Our data indicate that short telomere length is a genetic determinant of emphysema in mice, and may contribute to the susceptibility to CS-induced lung disease with age in humans.
Several pieces of evidence in our study point to epithelial injury being a primary mechanism of the telomere-associated CS-induced susceptibility. We show that the telomere-associated emphysema susceptibility does not depend on a telomere defect in bone marrow–derived cells. In adoptive transfer, short telomeres caused emphysema susceptibility independent of inflammatory cell genotype indicating that although the inflammatory response after CS is striking, the recruitment of macrophages per se
is not sufficient to induce emphysema in our model. In addition, we show that in epithelial cells, DNA damage caused by CS is additive to telomere dysfunction with short telomere mice carrying the greatest burden. Because of the slow turnover rate of lung epithelium, we developed an assay to track epithelial-specific proliferation in vivo
and show that it is dynamic during and after CS exposure. In parallel to the dynamics of epithelial proliferation, p21 levels are significantly down-regulated during the recovery phase (increased proliferation), and in short telomere mice there is a failure of this down-regulation. In situ
studies have also found p21 up-regulation in human lungs with emphysema (21
). These data suggest that p21 is a candidate effector of the senescence-like phenotype we observe and the associated emphysema susceptibility. Choudhury and coworkers (45
) have shown that p21 loss rescues telomere degenerative defects in the bone marrow. Future studies in compound p21 knockout mice with short telomeres can definitively establish whether rescue of the telomere-mediated emphysema susceptibility by p21 deletion is feasible and are ongoing. p21 is a known tumor suppressor in lung cancer and p21−/−
mice are prone to develop sponaneous lung adenocarcinoma with age (46). These data together highlight the role of p21 as a regulator of epithelial cell cycle.
How might epithelial proliferative defects in short telomere mice lead to alveolar breakdown? Our data suggest at least two possibilities: First, it is possible that the defective type 2 AEC proliferation directly leads to regenerative failure and remodeling of the lung. We identified proliferative defects up to 14 days after CS exposure, so in the setting of repetitive injury cycles as occurs with CS, it may be that defects become eventually irreversible and directly lead to regional alveolar loss. Chronic low-grade apoptosis caused by the combined effect of CS-induced and telomere damage may similarly contribute to alveolar loss. Another possibility is that the combined DNA damage induces a senescence phenotype and this indirectly contributes to alveolar destruction. Senescence is a complex process associated with gene expression changes and a cytokine and protease secretory phenotype (47
). A recent study showed that senescent alveolar cells are associated with a higher proinflammatory cytokine profile in vitro
); however, the relevance of this phenotype in vivo
has not been examined. Although our data do not entirely exclude other lung parenchymal cells or extrapulmonary factors, such as nutritional deficiency, as contributing to the telomere-mediated emphysema, our studies support a model where epithelial injury is the primary determinant of the emphysema susceptibility.
We did not identify de novo
pulmonary fibrosis or emphysema in the mice with short telomeres in the age groups we examined. Our findings are in contrast to a prior study that identified de novo
airspace changes in late-generation mTR−/−
mice, a difference that may reflect differences in phenotype severity caused by telomere length heterogeneity, or other strain-specific factors (49
). The absence of a phenotype in our mice within the relatively short mouse lifespan is consistent with the finding that in individuals who inherit telomerase mutations, disease onset is rare before the age of 40 (18
). Emphysema onset is also age-dependent and is rare before the sixth decade (3
). Thus, although short telomere length alone does not cause disease phenotypes, the combined telomere and CS-induced damage together overcomes a threshold and manifests as emphysema. The clinical observations, along with findings in our model, indicate that even though the short telomere defect alone is not sufficient to induce lung disease in mice, it serves as a first genetic hit in a likely multistep process that is cumulative with age, and accelerated with CS.
Previous work has established a causal role for short telomeres in IPF, and our studies here extend the role of short telomeres in age-related lung disease susceptibility to include emphysema. Emphysema and pulmonary fibrosis have traditionally been considered distinct clinical entities; however, in recent years it has become clear that as many as 20% of patients with emphysema have concurrent interstitial lung disease (2
). Here we show that within a single family, the pulmonary manifestations of telomerase insufficiency are heterogeneous and can include emphysema, IPF, and the combined syndrome. In a cohort of telomerase mutation carriers, 5% of cases were reported to have a history of spontaneous pneumothrax or had the diagnosis of chronic obstructive pulmonary disease (39
). It may therefore be that emphysema alone or combined with pulmonary fibrosis are rare manifestations of inherited telomerase mutations. Identifying the factors that determine whether the first presentation in telomerase mutation carriers is primarily emphysema, IPF, or both is important to examine in larger studies. Given the early onset of disease in the patients we describe, consideration of telomerase genetic testing may be indicated in young-onset patients with emphysema who have a personal or family history suspicious for telomere-mediated disease (17
In summary, we report that short telomere length is a susceptibility factor for CS-induced emphysema in mice. Our data indicate that short telomeres may contribute to the differential susceptibility to CS across populations, and with aging.