BACKGROUND AND AIMS
Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory lung disease that affects nearly 10% of people over the age of 40 years.1 COPD is a disease of accelerated ageing,2 associated with accumulation of senescent cells in the lung.3,4 Senescent cells are in cell-cycle arrest and therefore do not proliferate. However, they remain metabolically active and secrete a milieu of proinflammatory mediators, known as the senescence-associated secretory phenotype,5 which is the same inflammatory profile seen in the lungs of COPD patients.6 These cells may, therefore, play a role in COPD pathogenesis through the loss of lung repair mechanisms and release of inflammatory mediators. Altered iron metabolism is important in the pathogenesis of COPD, with iron chelation therapy being shown to be protective at early time points in vivo.7Elevated levels of iron are found within bronchoalveolar lavage fluid of COPD patients,8 but the consequences of this are not fully elucidated. Recently, data have suggested elevated levels of intracellular iron are found in senescent cells, with this potentially driving cellular senescence.9 However, this has not been studied in the context of COPD. The aim of this study was to assess the relationship between excess intracellular iron and cellular senescence in COPD small airway epithelial cells (SAEC).
Total intracellular iron was detected in SAEC from non-smokers (NS) and COPD patients by graphite furnace atomic absorbance spectrometry. SAEC were treated with ammonia iron (Fe3+) citrate (FAC), the iron chelator deferoxamine, or doxorubicin to induce senescence. Iron and senescence markers were detected by Western blot and quantitative reverse transcription PCR in lung homogenate samples and SAEC.
Both intracellular and haem iron were significantly increased in COPD SAEC compared to NS. Treatment of NS and COPD SAEC with FAC caused a significant increase in total iron, with COPD SAEC taking up significantly more iron than NS cells. Chelation of iron significantly reduced intracellular iron levels in COPD SAEC, but not NS. Significant increases in the gene expression of senescence markers p21Cip1 and BCL-2 were detected in lung homogenate samples of COPD patients compared to NS, with significantly increased expression of the transferrin receptor and decreased expression of ferroportin also observed. Elevated protein levels of the transferrin heavy chain and transferrin receptor were detected in COPD SAEC compared to NS, and these correlated with changes in the expression of p21 and the anti-ageing molecules sirtuin-1 and -6. Induction of cellular senescence using doxorubicin led to elevated gene expression of both p21 and the transferrin receptor, suggesting a link between senescence and elevated iron uptake.
Elevated levels of intracellular iron were observed in COPD SAEC compared to NS. COPD SAEC had increased capacity to uptake extracellular iron, which could be chelated. COPD SAEC and lung homogenate samples had increased senescence markers, as well as altered iron metabolism proteins. Senescence induction led to increased expression of the iron import protein, the transferrin receptor. Overall, these data suggest both elevated intracellular iron levels and senescence in COPD SAEC. Further work is needed to elucidate how these two processes are linked.