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

Abstract

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.

Keywords: Bronchoalveolar lavage; COPD; Inflammation; Proteomics; Smoking.

Conflict of interest statement

Ethics approval and consent to participate

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.

Consent for publication

Not applicable

Competing interests

The authors declare that they have no competing interests.

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Figures

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]

Fig. 2

a) Scores plots of OPLS-DA modeling of BAL cell proteome alterations between Smokers (circle) vs Never-smokers (open triangles) OPLS-DA models suggested a perfect separation and predictive power between Smokers and Never-smokers p(CV-ANOVA) = 6.2 × 10−19) using 506 significant proteins (|p(corr)| > 0.34, n = 42, Additional file 1: 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% (R2 = 0.98, Q2 = 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), with a predictive power of 87% (R2 = 0.95, Q2 = 0.87) . As displayed by the Venn diagram in panel d, a total of 199 proteins were altered in both female and male Smokers, while 202 proteins were altered only in female Smokers, and 102 proteins were altered only in male Smokers. An additional 75 proteins were found to be altered only in the joint gender model. The majority of the significantly altered proteins from the gender stratified models were altered also in the joint gender model; 95% in male and 86% female Smokers. Keys: t[1]: scores of OPLS-DA predictive component; to[1]: scores of the first orthogonal component from OPLS-DA model; p:cross-validated (CV)-ANOVA p-value for significance of group separation in the model

Fig. 3

The phagosomal pathway was enriched in Smokers vs Never-smokers in the joint gender model (p = 1.4 × 10−6, FDR = 2.7 × 10−5). Proteins with significantly increased levels are highlighted in red, and decreased levels in dark blue. (FcγR, P08637, Low affinity immunoglobulin γ Fc region receptor III-A; CR3, P11215/P05107, CD11b/CD18; αMβ2, P11215/P05107, CD11b/CD18; Collectins, Q8IWL1, Pulmonary surfactant-associated protein A2; SRA1, P21757, Macrophage scavenger receptor types I and II; MARCO, Q9UEW3, Macrophage receptor MARCO; TAP1, Q03518, Antigen peptide transporter 1; TAP2, Q03519, Antigen peptide transporter 2; F-actin, P60709, β-actin; Stx13, Q86Y82, Syntaxin-12; TfR, P02786, Transferrin receptor protein 1; LAMP, P13473, lysosome-associated membrane glycoprotein 2; Cathepsin S: P25774, Cathepsin S; Cathepsin L1: P07711, Cathepsin L1; vATPase: P38606, P21281, P61421, P21283, Q9Y5K8, P36543, O75348, Q9UI12, V-ATPase subunit A, B2, d1, C1, D, E1, G1, H, respectively; gp91, P04839, Cytochrome b-245 heavy chain; p40phox, Q15080, Neutrophil cytosol factor 4. Protein details are listed in Additional file 1: Table S5)

Fig. 4

Gender-specific alterations in glycolysis/gluconeogenesis in Smokers. a The increased expression levels (in red) of five proteins (HK2, FBP1, FBP2, ALDOA and ENO1) in the female Smoker. b The decreased levels (in blue) of four proteins (HK2, PGK1, PGH2 and LDHA) in male Smokers. The levels of four proteins (DLAT, PDHA1, DLD and PDHB) in pathway pyruvate metabolism were increased in both gender Smokers

Fig. 5

Pathways altered both due to smoking (left panels) and COPD (right panels) in females. The majority of expression levels in oxidative phosphorylation increased in the female Smoker compared to Never-smoker groups (a), and were further elevated in female current-smoker COPD compared with female Smoker groups (b), with six shared proteins (S). The levels of proteins in the citrate cycle were elevated in female Smokers compared to Never-smokers (c), and further increased in female smokers with COPD (d), with 4 proteins in common (S). The majority of protein levels in the lysosomal pathway were increased in female Smokers (e), but decreased in female smokers with COPD (f), with one common protein altered in the opposite direction (S). The bars display the scaled loadings, p(corr) of the predictive component from OPLS-DA models. Positive p(corr)[1] indicates increased protein levels. Full protein names are provided in Additional file 1: Table S3

Fig. 6

The proportion of CD8+ T cells in BAL increased and CD4+ T-cells decreased in Smokers compared to Never-smokers (a). Proteins from the leukocyte transendothelial migration (LTM) pathway and phagosome pathway correlated with proportion of CD8+ T-cells (b-c), as well as with the proportion of CD8+ CD69+ T-cells (e-f). Protein levels from the LTM pathway also correlated with the level of lung obstruction; FEV1/FVC (d). R2 and p-values refer to PLS inner relation

Fig. 7

Comparison of the proteomes investigated with the bottom-up iTRAQ peptidomics platform and the top-down 2D–DIGE intact proteomics platform. a) Venn diagram displaying the overlap of proteins identified with the two platforms indicates that the majority of proteins identified with iTRAQ (90%) were novel as compared to 2D–DIGE. The proteins that overlapped were generally altered in a similar fashion, thereby providing validation across platforms. b) Protein 60S acidic ribosomal protein P0 with decreased level in ribosomal pathway in Smokers (2-D DIGE: p = 5.1 × 10−8; iTRAQ: p = 0.009); c) Protein Cytochrome b-c1 complex subunit Rieske with elevated level in pathway oxidative phosphorylation in Smokers (2-D DIGE: p = 4.5 × 10−9; iTRAQ: p = 1.5 × 10−5); d) Protein Cathepsin D with increased level in lysosomal pathway in Smokers (2D DIGE: p = 4.0 × 10−17; iTRAQ: p = 4.3 × 10−19); e) ATP synthase subunit d in mitochondria with upregulated level in citrate cycle in Smokers (2-D DIGE: p = 4.0 × 10−5; iTRAQ: p = 6.3 × 10−5); f) Enzyme Aldehyde dehydrogenase in mitochondria with downregulated level in pathway valine, leucine and isoleucine degradation in Smokers (2-D DIGE: p = 8.4 × 10−8; iTRAQ: p = 0.02); g) Protein actin, cytoplasmic 1 with decreased level in pathway phagosome in smokers (2-D DIGE: p = 4.4 × 10−6; iTRAQ: p = 0.007)