Background
The number of habitual smokers is estimated to one billion worldwide [
1]. The direct effect of tobacco smoking was responsible for the death of 6 million people in 2015, with 27% of them resulting from COPD [
2]. Although one-third of long-term smokers develop COPD [
3], the molecular mechanisms by which smoking-induced inflammation triggers the development of disease remain unclear.
Proteomic profiling of blood, urine and lung tissue from smokers has improved our understanding of the molecular impact of smoking [
4‐
6]. Lung immune cells, especially alveolar macrophages, play a key role in the immune responses to smoking [
7]. However, quantitative proteomics analysis of resident immune cells in the lung, particularly with respect to the inflammatory disorder in the response to smoking, are scarce. Our previous work revealed specific alterations of the bronchoalveolar lavage (BAL) cell proteome in current-smoker COPD patients [
8]. However, in order to define molecular disease mechanisms occurring prior to disease manifestations, alterations of the BAL cell proteome in response to smoking needs to be defined.
The aim of this study was to investigate the impact of long-term habitual smoking on the BAL immune cell proteome. The proteomes of long-term smokers at elevated risk of developing COPD were contrasted to healthy never-smokers, with the specific emphasis on identifying alterations of molecular pathways occurring prior to the presentation of symptoms or decline of lung function associated with COPD diagnosis.
Discussion
Here we used high-resolution iTRAQ based proteomics to investigate the BAL cell proteome of smokers with normal lung function (Smokers) vs. healthy never-smokers (Never-smokers), with the aim to explore alterations in specific proteins and molecular pathways occurring in response to long-term cigarette smoking in otherwise healthy individuals. The results clearly show that smoking exerted a considerable impact on the BAL cell proteome, with the abundances of half of the detected proteins being altered in long-term smokers. Downstream pathway enrichment analyses revealed that the 506 significantly altered proteins were associated with 15 molecular pathways (q < 0.05). Two of these pathways, the phagosome and leukocyte transendothelial migration, were significantly correlated with the proportion of CD8+ T-cell in BAL, as well as with the level of airway obstruction (FEV1/FVC) in the Smoker group. Some of the identified molecular pathways may represent molecular events preceding the development of smoking-related disease in susceptible individuals, while others may represent protective mechanisms in individuals that prove to be resilient to disease development in spite of decades of smoking.
The increase of CD8
+ (cytotoxic) T-cells is one of the culprits of the inflammation and tissue destruction in the lung induced by long-term smoking in mice [
24]. The level of lung cytotoxicity of CD8
+ cells has been reported to increase with COPD severity [
25], indicating that these cells were contributing to the pathogenesis of COPD. Increased levels of CD8
+ T cells in lung parenchyma and pulmonary arteries have been reported as well [
26]. In these studies, we observed an increase in the proportion of CD8
+ T-cells in BAL cells of Smokers compared to Never-smokers, which is in line with our previous reports from the full Karolinska COSMIC cohort [
9]. CD69 is a leukocyte activation marker, involved in the pathogenesis of chronic inflammation and activation of leukocyte subpopulations [
27]. Recent studies have shown that CD69 is required for cigarette smoking-induced inflammation in the lung as well as the development of COPD [
28,
29]. The significant correlations of proteins in the phagosome- and LTM pathways with activated cytotoxic T-cells (CD69+) and the level of obstruction (FEV
1/FVC) in smokers, both hallmarks of COPD, suggest that dysregulation of these two pathways may play an important role in the molecular events preceding the development of COPD in susceptible smokers. The observations of further dysregulation in the phagosomal pathways in female smokers with mild to moderate COPD [
15] further supports this theory.
The phagosome is formed by the fusion of cell membrane around large particles or pathogens [
30], followed by invagination (phagocytosis). Nascent phagosomes interact with recycling endosomes, early endosomes and lysosomes, thereby acquiring digestive capacity and becoming phagolysosomes [
31], where pathogens are killed and subsequently degraded into fragments. The levels of three phagocytotic proteins were altered in Smokers compared to Never-smokers: Syntaxin 12 and transferrin receptor 1, co-localize in the recycling endosome, with the function of facilitating the fusion of the early phagosome with early endosome [
32]. LAMP2 is required for fusion of late phagosomes with lysosomes [
33]. The dysregulation of these three proteins may indicated a disturbance in phagosome maturation due to smoking. Acidification is essential for the phagosomes to mature [
34]. The observed increase in V-ATPases, which pump protons across the phagosomal membranes to maintain homeostasis of acidification, may indicate a dysregulation also in the local microenvironment of the phagosome. The decrease in F-actin and several receptors mediated in phagocytosis, including FcγR, SPA2, SRA1 and MARCO, may also contribute to defective phagocytosis in BAL immune cells due to smoking. Reduced protein levels of phagocytosis have been observed in female smokers with COPD [
35]. Further dysregulation of the phagocytotic pathway in female smoking COPD patients compared to the Smokers studied here is reported in the companion paper [
15]. As such, the level of dysregulation of the phagocytotic pathway in smokers may be of use as prognostic indicators of disease development.
The cell count of macrophages increased significantly in BAL fluid in Smokers. Alveolar macrophages predominantly originate from circulating blood monocytes [
36], and are recruited to the lung by a mechanism involving leukocyte transendothelial migration (LTM), an inflammatory response to tissue damage. Recruitment of leukocytes from the blood to the site of injury includes several steps, including adhesion of leukocytes to the blood vessel wall, traversing the endothelial basement membrane, and migrating through the interstitial tissue [
37]. Alterations of the LTM pathway were observed in the Smoker group, with a number of proteins being both up- and down-regulated.
Platelet/endothelial cell adhesion molecule 1 (PECAM-1, CD31), is expressed on platelets and leukocytes and concentrated at the borders of endothelial cells. PECAM-1 regulates the start of diapedesis and is required for transendothelial migration [
38,
39]. The level of PECAM-1 decreased in BAL cells in Smokers, suggesting that LTM decreased in Smokers. PECAM-1 has previously been reported to be decreased in the serum of smoker compared with nonsmokers [
40]. Myosin light-chain kinase (MLCK) and actin involve the actin-myosin fiber contraction, which helps endothelial cells separate [
37]. The levels of these two molecules were reduced in BAL cells of smokers, further supporting that LTM may be reduced due to smoking.
Macrophage-1 antigen (Mac-1, CD11b, ITGAM) is expressed on the surface of many leukocytes including monocytes, neutrophils and macrophages. CD11b plays a key role in the regulation of leukocyte adhesion and migration in the inflammatory response [
41], and in the recruitment of alveolar macrophage during e.g., pneumococcal infection [
42]. Integrin beta-2 (CD18, ITGB2) pairs with CD11b to form the CR3 heterodimer of integrin. The higher expression levels of CD11b and CD18 indicated that, while the recruitment of leukocytes to the lung is impaired, leukocyte adhesion in the lungs increased due to smoking.
Four of the pathways (lysosome, citrate cycle, oxidative phosphorylation and phagosome) altered due to smoking were also found to be significantly altered in female smokers with COPD compared to smokers with normal spirometry, [
8,
15]. The levels of the majority of proteins increased in the citrate cycle and oxidative phosphorylation pathways due to smoking were further increased in female smokers with COPD. The decreased levels of proteins in the lysosomal pathway observed in the female Smoker vs. Never-smoker groups were further decreased in female smoking COPD patients. As such, alteration of these pathways in smokers, with further enhanced dysregulation in smokers with mild to moderate COPD suggests that dysregulation of these pathways in healthy smokers may represent early signs of disease development in susceptible individuals. Even though the risk of developing COPD has been shown to increase linearly with the number of years smoked [
43], it is curious that many smokers may never develop COPD in spite of decades of smoking. It is therefore plausible that some of the molecular pathways altered in long-term smokers represent protective mechanisms in resilient individuals, rather than disease mechanisms. These associations will be investigated following the completion of the on-going 10-year clinical follow-up of the Karolinska COSMIC cohort.
Acknowledgements
We would like to thank research nurses Heléne Blomqvist, Margitha Dahl, and Gunnel de Forest as well as biomedical technician Benita Engvall for excellent assistance in collection and work-up of BAL samples. We would also like to extend our appreciation to the volunteer subjects that participated in the Karolinska COSMIC study.