Telomere Dysfunction and Senescence-associated Pathways in Bronchiectasis

To the Editor:

Non–cystic fibrosis bronchiectasis, hereafter bronchiectasis, is a common inflammatory lung disease characterized by irreversible dilation of the bronchi (1). The prevalence of bronchiectasis and associated mortality is increasing in the United Kingdom, particularly in older patients (2). Chronic cycles of infection and inflammation, perpetuated by airway structural damage, are characteristic of bronchiectasis, but the underlying mechanisms are poorly understood (1).

An emerging concept is that aging-associated mechanisms, such as cellular senescence, contribute to the development of respiratory diseases, including chronic obstructive pulmonary disease (COPD) (3). Senescence is a state of irreversible cell cycle arrest, which contributes to organismal aging and the development of age-related diseases (4). Telomeres, sequences of DNA that protect the ends of linear chromosomes, are major drivers of senescence (5). Telomeres shorten with each cell division, and if a critical length is reached, a DNA damage response (DDR) is triggered, leading to cell cycle arrest and, potentially, senescence (5). Telomere-associated DDR foci (TAF) accumulate with age and under conditions of chronic inflammation (3, 6). Telomere dysfunction and senescence have been associated with COPD and are thought to contribute to impaired cellular regeneration and the proinflammatory phenotype characteristic of this disease (3). Whether senescence occurs in bronchiectasis is unknown. Therefore, we investigated whether telomere-induced senescence was associated with bronchiectasis.

Explant lung tissue from patients with bronchiectasis and research biopsies from healthy, aged-matched volunteers (control subjects) were retrieved from the Freeman Hospital, Newcastle upon Tyne, United Kingdom (Table 1). The work was performed under approval of the Newcastle 1 Research Ethics Committee (11/NE/0291), and all patients gave written informed consent. Samples from the central airways were formalin-fixed and paraffin-embedded, and telomere length and TAF frequencies were determined by telomere-specific quantitative fluorescence in situ hybridization combined with immunofluorescence, as previously described (3). Expression of the cell-cycle inhibitors p16 and p21 and the histone deacetylase sirtuin 1 (SIRT1) were assessed using immunohistochemistry, as in previous work (3). The following primary antibodies were used: anti–phospho-H2A.X (9718, 1:400; Cell Signaling, Hertfordshire, UK), anti-p16 (CINtec Histology Kit, 725-4713, as per the manufacturer’s instructions; Roche, Burgess Hill, West Sussex, UK), anti-p21 (ab7960, 1:100; Abcam, Cambridge, UK), and anti-SIRT1 (ab13749, 1:100; Abcam). Images were captured with a DFC360FX camera (Leica DM5500B microscope; Leica, Germany) or a DFC420 camera (Nikon eclipse E800 microscope; Nikon, Tokyo, Japan) and quantified using ImageJ (National Institutes of Health, Bethesda, MD). Data are presented as mean ± SD or median. Differences between groups were analyzed using unpaired t test or Mann-Whitney U test with GraphPad Prism version 6.0 (San Diego, CA).

Table 1.

Clinical Characteristics of Control Subjects and Patients with Bronchiectasis

	Control (n = 7)	Bronchiectasis (n = 8)
Age, yr	57 ± 6.76	54 ± 4.57
Females, n	4	1
Smoking history, pack-years	10 ± 15.56	12.25 ± 17.45
FEV1, % predicted	102.2 ± 15.51	24.5 ± 7.37*

Values are expressed as mean ± SD.

^*P < 0.001 as compared to control subjects.

Results

We found a significant decrease in median telomere intensity by quantitative fluorescence in situ hybridization (P = 0.0018), with an increased proportion of very short telomeres in patients with bronchiectasis (Figure 1A). Quantitative fluorescence in situ hybridization combined with immunofluorescence revealed a significant increase in the percentage of large airway epithelial cells containing TAF in patients with bronchiectasis (Figures 1A and 1B). Pathways downstream of the DDR, which are associated with senescence, were then investigated. p21, a cyclin-dependent kinase inhibitor, was increased in the large airway epithelium of patients with bronchiectasis (Figure 1C). Levels of SIRT1, a nicotinamide adenine dinucleotide–dependent deacetylase, whose expression is negatively associated with dysfunctional telomeres and senescence (3, 7), were decreased (Figure 1C). p16 was not significantly changed in patients with bronchiectasis (not shown).

Figure 1.

Large airway epithelial cells from patients with bronchiectasis have increased telomere dysfunction, p21, and decreased sirtuin 1 (SIRT1) expression. Explant lung tissue sections from patients with bronchiectasis and biopsies from healthy, aged-matched control subjects were analyzed for expression of γH2A.X and telomere-associated foci (TAF) by immunofluorescence in situ hybridization (FISH), using a telomere-specific Cy-3–labeled peptide nucleic acid probe (Panagene, Daejeon, Korea). (A) Histograms show frequencies of telomere intensity for control subjects and patients with bronchiectasis, determined using quantitative FISH. Dot plots represent the percentage of cells containing TAF for each individual subject, generated by quantifying Z-stacks of at least 100 cells per subject. (B) Representative images of quantitative FISH combined with immunofluorescence staining for γH2A.X (green) and telomeres (red) in large airway epithelial cells from patients with bronchiectasis and control subjects captured using ×100 oil objective. Arrows point to TAF, depicted by associated histograms and shown at higher magnification on the right (images from single Z-plane). (C) Immunohistochemistry was performed against p21 and SIRT1. Dot plots represent average percentage positivity of p21 expression and SIRT1 expression for each subject, generated by quantifying 10 images randomly taken using ×40 objective. Horizontal lines represent group median. Representative images of p21 and SIRT1 staining by immunohistochemistry. Black arrows point to positive cells. Statistics: Mann-Whitney U test *P < 0.05, **P < 0.01, as compared with control subjects. A.U. = arbitrary units.

Discussion

We report the novel finding that telomere shortening and telomeric DNA damage, along with increased p21 and decreased SIRT1, are features of the bronchiectatic large airway epithelium, which may be indicative of cellular senescence. Telomere dysfunction plays a causal role in senescence, and telomere regions are particularly sensitive to oxidative damage and less efficiently repaired compared with the bulk of the genome (8). We have previously shown that telomere-associated DNA damage increases in the lungs with age and in patients with COPD, irrespective of telomere length, by oxidation-induced damage within telomere repeats (3). Although we observed more short telomeres in patients with bronchiectasis, we also noted that relatively long telomeres colocalized with γH2A.X, suggesting that telomere dysfunction could be driven independent of telomere length.

DDR signaling may engage the p53–p21 or p16–pRb pathways, depending on stimuli and cell type (9). We failed to detect significant increases in p16 expression. Nevertheless, we found increases in TAF and p21 with decreased SIRT1, which are well-established features of senescence (3, 9). It has been shown that telomere-dependent senescence can occur through the activation of the p53–p21 pathway (9). Therefore, it could be that telomere dysfunction drives p21, but not p16, in the lungs of patients with bronchiectasis. SIRT1 plays roles in a number of processes, including aging, inflammation, and cell-cycle regulation. SIRT1 has been shown to interact directly with telomeres and attenuate age-dependent telomere dysfunction in vivo by increasing homologous recombination events (7). It is possible that decreased SIRT1 expression may contribute to bronchiectasis-associated telomere dysfunction.

In summary, this is the first report of telomere dysfunction and activation of senescence-associated pathways in the airways of patients with bronchiectasis. These preliminary data prompt the need for further research into the role of senescence in bronchiectasis with larger, more varied samples than used here. Because of the limited availability of bronchiectasis tissue, we were only able to acquire data from patients undergoing transplantation for end-stage disease compared with healthy control subjects. This is a major limitation of this work, as our data are only representative of a very unique patient population: predominantly males with severe bronchiectasis. It is therefore possible that senescence is not causally implicated in, but is a direct result of, the disease processes. Whether senescence is associated with milder bronchiectasis in patients that do not require transplantation will need to be determined. Investigating whether female patients with bronchiectasis show similar levels of senescence-associated pathway activation is also of interest. We focused on the large airway epithelium. However, it is possible that telomere dysfunction and senescence occur at other sites in the bronchiectatic lung, which should be explored further. Given that bronchiectasis is characterized by chronic inflammation (1) and the established links between telomere dysfunction and proinflammatory processes (6), our data may be of interest to the understanding of bronchiectasis pathogenesis and to development of therapeutics. Clinically, there is great interest in selectively clearing senescent cells or targeting the senescence-associated secretory phenotype (10). Antisenescence therapies are yet to be tested in the context of bronchiectasis but could allow dampening of inflammatory processes and slower progression of bronchiectasis.