Long-term smoking alters abundance of over half of the proteome in bronchoalveolar lavage cell in smokers with normal spirometry, with effects on molecular pathways associated with COPD

Background Smoking represents a significant risk factor for many chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). Methods To identify dysregulation of specific proteins and pathways in bronchoalveolar lavage (BAL) cells associated with smoking, isobaric tags for relative and absolute quantitation (iTRAQ)-based shotgun proteomics analyses were performed on BAL cells from healthy never-smokers and smokers with normal lung function from the Karolinska COSMIC cohort. Multivariate statistical modeling, multivariate correlations with clinical data, and pathway enrichment analysis were performed. Results Smoking exerted a significant impact on the BAL cell proteome, with more than 500 proteins representing 15 molecular pathways altered due to smoking. The majority of these alterations occurred in a gender-independent manner. The phagosomal- and leukocyte trans endothelial migration (LTM) pathways significantly correlated with FEV1/FVC as well as the percentage of CD8+ T-cells and CD8+CD69+ T-cells in smokers. The correlations to clinical parameters in healthy never-smokers were minor. Conclusion The significant correlations of proteins in the phagosome- and LTM pathways with activated cytotoxic T-cells (CD69+) and the level of airway obstruction (FEV1/FVC) in smokers, both hallmarks of COPD, suggests that these two pathways may play a role in the molecular events preceding the development of COPD in susceptible smokers. Both pathways were found to be further dysregulated in COPD patients from the same cohort, thereby providing further support to this hypothesis. Given that not all smokers develop COPD in spite of decades of smoking, it is also plausible that some of the molecular pathways associated with response to smoking exert protective mechanisms to smoking-related pathologies in resilient individuals. Trial registration ClinicalTrials.gov identifier NCT02627872; Retrospectively registered on December 9, 2015. Electronic supplementary material The online version of this article (10.1186/s12931-017-0695-6) contains supplementary material, which is available to authorized users.


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][5][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.

Methods
The details of methods are provided in Supplemental materials.

Study subjects and design
This study was carried out on subjects from the Karolinska COSMIC cohort [8][9][10][11][12][13], a three-group crosssectional study including age-(45-65 years) and sexmatched groups of healthy never-smokers (hereafter referred to as Never-smokers), smokers with normal lung function (hereafter referred to as Smokers; >10 pack years; >10 cigarettes/day the past 6 months), and smokers with COPD (GOLD stage I-II/A-B, FEV 1 > 50% of predicted and FEV 1 /FVC < 0.7). Sixty-nine subjects were selected for proteome analyses (Fig. 1). The current segment included Never-smokers (n = 17) and Smokers (n = 25). Acute effects of smoking were excluded by requesting that participants to refrain from smoking at least 8 h prior to biospecimen collection, as confirmed by exhaled carbon monoxide monitoring [14]. Results from the Smokers compared to the COPD group are presented in the companion paper [15]. All subjects underwent extensive clinical examinations, including computed tomography [10,13] and spirometry (Table 1, Additional file 1: Table S1). BAL cells were collected during bronchoscopy as previously described [16]. Clinical characteristics are summarized in Table 1. The study was approved by the Stockholm regional ethical board (Case no. 2006/959-31/1), and written informed consent was obtained from all subjects. Fig. 1 Flow chart of the study design outlining groups of the current paper as well as the companion manuscript [15]. A total of 69 subjects from the Karolinska COSMIC cohort, age-and gender-matched, were selected for iTRAQ proteomic investigations, including 18 healthy Never-smokers, 25 Smokers with normal lung function, 18 current smokers with COPD, and 8 ex-smokers with COPD. The current analysis consists of 17 Never-smokers (one male Never-smoker did not pass QC and was excluded from further analysis), 25 Smokers with normal lung function. This report focuses on the alterations in proteomes and pathways related to smokingwhereas the pathways related to pathogenesis of COPD are reported in a companion paper [15]

Statistical analyses
Univariate statistical analyses were performed by Student's test followed by correction for multiple testing according to Storey (q) [17]. Multivariate statistical modeling was performed using SIMCA 14.1 (MKS Umetrics, Umeå, Sweden) using principal component analysis (PCA), orthogonal projection to latent structurediscriminant analysis (OPLS-DA) [18] and partial least square (PLS) regression.
In contrast to the more commonly used PCA modeling, OPLS analysis is a supervised method designed to separate structured noise unrelated (orthogonal, often intragroup variance) to the predictive variance of interest (e.g., COPD patients vs healthy subjects). The resulting "noise filter" increases the interpretability of the multivariate model, particularly in deriving the observed group separation back to the specific proteins driving the separation. For more information of how to interpret these models, or the related model statistics, please see [19].
Proteins with a value of the scaled loadings of the first predictive component (|p(corr) [1]|) greater than the critical value of the Pearson correlation coefficient (p < 0.05) was considered significant for OPLS-DA models. Model performance is reported as the goodness of fit (R 2 ), the  Table S2). Stratification by gender revealed a highly significant separation in females (b; p(CV-ANOVA) = 3.2 × 10 −10 ), fitted using 401 significant proteins (Additional file 1: Table S3), with a near perfect predictive power of 98% (R 2 = 0.98, Q 2 = 0.94). Also in males, the group separation was significant (c; p(CV-ANOVA) = 4.5 × 10 −6 ), fitted with 301 significant proteins (Additional file 1: Table S4) [20] indicating the significance of group separation, and 200 times permutation test to determine the level of model statistics that may occur by random for the specific data set [21]. Multivariate correlation analysis between the specific proteins from identified pathways with clinical data was performed using PLS, and inner relations with Pearson correlation coefficient of R 2 > 0.5 and p < 0.05 were considered significant.

Pathway analysis
Pathway enrichment analysis was performed based on proteins found to be significantly altered in OPLS-DA models, as defined by OPLS-DA p(corr) above, using KOBAS 2.0 [22], with pathway enrichment analysis performed based on the KEGG pathway database [23].

Results
Clinical characteristics FEV 1 and percentage of neutrophils were lower (p < 0.05), while the proportion of macrophages was higher (p < 0.01) in Smokers compared to Never-smokers. No other differences between Smokers and Never-smokers, or between genders, were observed (Additional file 1: Table S1).

Proteome alteration due to smoking
Univariate analysis comparing Never-smoker vs.

Alteration of pathway
Pathway analysis based on the 506 proteins found significant in the OPLS-DA model between Smokers and Never-smokers resulted in 15 significantly enriched pathways (pFDR < 0.05; Table 2, Additional file 1: Table S5). The majority of proteins in metabolic pathways such as oxidative phosphorylation, and citrate cycle were up-regulated, while ribosomal and antigen presentation were downregulated in Smokers compared to Never-smokers. Proteins involved in the phagosomal (Fig. 3), LTM (Additional file 2: Figure S2) and lysosomal pathways were both up-and down-regulated (Table 2, Additional file 1: Table S5).
Gender-stratified pathway analysis showed that the majority of pathways mentioned above was altered in both genders in a similar fashion ( Table 2, Additional file 1:  Table S6), except the ribosomal-and glycolysis/gluconeogenesis pathways. In glycolysis/gluconeogenesis, five proteins were up-regulated in female Smokers (Fig. 4a), while four proteins were down-regulated in males (Fig. 4b).
Four pathways altered in Smokers were found to be altered in female Smokers with COPD: citrate cycle, oxidative phosphorylation, lysosome and phagosome [15]. The majority of protein levels in oxidative phosphorylation and citrate cycle increased in female Smokers (Fig. 5a, c) were further increased in COPD patients (Fig. 5b, d). For the lysosomal pathway, the  Table S5) majority of proteins increased in female Smokers, with the opposite trend in female smoking COPD patients, as indicated by the one shared protein (Fig. 5f ).

Proteomes identified by the iTRAQ vs. 2D-DIGE platforms
In complementary proteome investigations using 2D-DIGE on the same biospecimens from the same cohort, 149 proteins were identified [8]. Comparison of the proteomes investigated with the bottom-up iTRAQ peptidomics platform and the top-down 2D-DIGE intact proteomics platform indicates that the majority of proteins identified with iTRAQ (90%) were novel as compared to the previously published 2D-DIGE analyses Fig. 7a.
From the DIGE perspective, the majority of the proteins identified in the DIGE workflow were also identified using iTRAQ. However, it should be noted that there is a bias towards overlap since only proteins of interest are identified in the DIGE workflow. The proteins that overlapped were generally altered in a similar fashion, thereby providing validation across platforms. Some examples are given in Fig. 7b-g, showing the levels of six proteins from six pathways significantly altered in both platforms.

Discussion
Here we used high-resolution iTRAQ based proteomics to investigate the BAL cell proteome of smokers with normal lung function (Smokers) vs. healthy neversmokers (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 (FEV 1 /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 smokinginduced 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  Table S3 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 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.

Conclusion
Our high-resolution iTRAQ proteomics investigations of BAL cells from smokers with normal lung function and healthy never-smokers revealed that long-term smoking exerted a considerable impact on the BAL cell proteome, with almost half of the detected proteins representing 15 different molecular pathways being significantly altered. In contrast to the significant gender differences in proteome alterations observed between smokers with and without COPD diagnosis, the gender differences observed between Smokers and Never-smokers were minor. Proteins in the phagosome-and LTM pathways significantly correlated with the proportion of CD8 + T-cells, its subtype CD8 + CD69 + cells as well as the level of airway obstruction (FEV 1 /FVC) in the Smoker group, stressing the importance these pathways may play both in the protection against as well as the early pathophysiology of smokinginduced inflammatory lung disorders such as COPD.

Additional files
Additional file 1: Table S1. Clinical characteristics of subjects, stratified by gender. Additional file 2: Figure S1. The OPLS-DA modeling parameters for joint gender, female and male Smoker vs. Never-smoker. Permutation test was performed 200 times for each model.