Smoking and human airway inflammatory cells : studies with focus on T cells in the development of COPD

Chronic obstructive pulmonary disease (COPD), the fourth leading cause of death worldwide, is characterized by persistent airflow limitation and a chronic inflammation of the lungs. One of the major risk factors is long-term exposure to cigarette smoking. The key inflammatory cells in the pathogenesis of COPD are macrophages, neutrophils and CD8+ T lymphocytes. The heterogeneity of COPD and the need for identifying patient subgroups is becoming increasingly recognized. Besides, the differences in the immunopathology of current- and exsmokers with COPD are uncertain. Characterizing the inflammatory mechanisms driving the disease in different subtypes of patients, including differences between men and women, will likely aid diagnostic procedures and tailoring of treatment strategies and caretaking. To investigate the airway and systemic inflammatory profile and the role for T cells in smoke-induced inflammation and COPD, bronchoalveolar lavage (BAL) fluid, blood and chest high resolution computed tomograpthy (HRCT) scans were collected from a genderand age-matched cohort of 40 never-smokers, 40 smokers with normal lung function and 38 COPD patients (27 current smokers and 11 ex-smokers). BAL characteristics, including the distribution of inflammatory cells, were assessed. T cells subsets in BAL and blood were phenotyped using flow cytometry, and the levels of cytokines, chemokines and growth factors in BAL fluid were assessed with a multi-plex bead-based assay. HRCT images of the lungs were analyzed for the investigation of morphological patterns and correlated with the cellular inflammatory patterns of the lungs. In addition to univariate analysis, multivariate data analysis with OPLS-modeling was used for discovering within- and between group variations, and potential biomarkers. The frequencies of several T cell subsets, including CD8+ T cells and NKT-like cells, both with cytotoxic capacities, and CD103+CD8+ T cells, were increased in BAL from smokers, regardless of airway obstruction. In COPD ex-smokers, the levels were similar to those of never-smokers. A more detailed phenotypic characterization of T cells revealed subsets specifically altered in smokers with normal lung function or COPD patients, including FOXP3+ regulatory T cells. A type 1 T cell-driven inflammation was indicated for female, but not male, COPD patients. Lung density, as measured by HRCT, was higher in smokers compared to both never-smokers and COPD patients and correlated with the cellular inflammation in the lungs. Taken together, these findings suggest that current smoking status has a larger impact on the distribution of lymphocytes in BAL than does airway obstruction. A detailed characterization of T cell subsets is important for finding disease-specific alterations. Gender-specific differences among smokers and COPD patients can be detected on a cellular level

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

Proteomic profiling of lung immune cells reveals dysregulation of phagocytotic pathways in female-dominated molecular COPD phenotype

Abstract Background Smoking is the main risk factor for chronic obstructive pulmonary disease (COPD). Women with COPD who smoke experienced a higher risk of hospitalization and worse decline of lung function. Yet the mechanisms of these gender-related differences in clinical presentations in COPD remain unknown. The aim of our study is to identify proteins and molecular pathways associated with COPD pathogenesis, with emphasis on elucidating molecular gender difference. Method We employed shotgun isobaric tags for relative and absolute quantitation (iTRAQ) proteome analyses of bronchoalveolar lavage (BAL) cells from smokers with normal lung function (n = 25) and early stage COPD patients (n = 18). Multivariate modeling, pathway enrichment analysis, and correlation with clinical characteristics were performed to identify specific proteins and pathways of interest. Results More pronounced alterations both at the protein- and pathway- levels were observed in female COPD patients, involving dysregulation of the FcγR-mediated phagocytosis-lysosomal axis and increase in oxidative stress. Alterations in pathways of the phagocytosis-lysosomal axis associated with a female-dominated COPD phenotype correlated well with specific clinical features: FcγR-mediated phagocytosis correlated with FEV1/FVC, the lysosomal pathway correlated with CT < −950 Hounsfield Units (HU), and regulation of actin cytoskeleton correlated with FEV1 and FEV1/FVC in female COPD patients. Alterations observed in the corresponding male cohort were minor. Conclusion The identified molecular pathways suggest dysregulation of several phagocytosis-related pathways in BAL cells in female COPD patients, with correlation to both the level of obstruction (FEV1/FVC) and disease severity (FEV1) as well as emphysema (CT < −950 HU) in women. Trial registration No.: NCT02627872, retrospectively registered on December 9, 2015

Additional file 2: of 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

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. Figure S2. Leukocyte transendothelial migration was significantly altered in joint smokers. ITGAM, P11215, Integrin alpha-M (CD11b); ITGB2, P05107, Integrin beta-2 (CD18); PECAM1, P16284, Platelet endothelial cell adhesion molecule; JAM-A, Q9Y624, Junctional adhesion molecule A; MLC-2, O14950, Myosin regulatory light chain 12B; CDC42, P60953, Cell division control protein 42 homolog; Actin, P60709, actin cytoplasmic 1; α-actin, P12814, O43707, α-actinin-1, α-actinin-4, respectively; NOX2, P04839, Cytochrome b-245 heavy chain; p40phox, Q15080, Neutrophil cytosol factor 4; RAC2, P15153, Ras-related C3 botulinum toxin substrate 2; RAP1A, P62834, Ras-related protein Rap-1A. (DOC 1606 kb

Additional file 1: of 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

Table S1. Clinical characteristics of subjects, stratified by gender. Table S2. Proteins significantly altered between Smoker vs Never-smoker groups. Table S3. Proteins significantly altered between female Smoker vs Never-smoker groups. Table S4. Proteins significantly altered between male Smoker vs Never-smoker groups. Table S5. Significantly enriched pathways and associated proteins when comparing Smoker and Never-smoker groups. Table S6. Significantly enriched pathways following stratification by gender when comparing Smoker and Never-smoker groups. (XLSX 1321 kb

Additional file 1: of Proteomic profiling of lung immune cells reveals dysregulation of phagocytotic pathways in female-dominated molecular COPD phenotype

Supplementary Methods. Figure S1. Analysis of Share and Unique Structure (SUS) between OPLS-DA models of female Smoker vs COPD (x-axis) and Never-smoker vs ex-smoker with COPD (exCOPD) (y-axis). Figure S2. Multivariate sensitivity analysis of the impact of menopausal status on proteomic profiling in female COPD patients. Figure S3. The percentage of CT attenuation values <−950 HU in the Smoker and COPD groups, stratified by gender. (DOC 1849 kb

Additional file 2: of Proteomic profiling of lung immune cells reveals dysregulation of phagocytotic pathways in female-dominated molecular COPD phenotype

Table S1. Heterogeneity indeces (I2) for proteins significantly altered between Smoker vs COPD groups joint gender as well as gender stratified models. Table S2. Protein identities and model statistics of proteins of interest from OPLS-DA models comparing Smoker and COPD groups for joint gender as well as gender stratified models. Table S3. Uniprot accessions, gene names, protein names, as well as and direction of alteration for proteins involved in pathways significantly altered due to COPD. Table S4. Pathways significantly enriched in female vs male COPD patients. Table S5. Names and MS/MS data of proteins used in statistical analyses. Table S6. Significantly altered proteins i Smoker vs COPD groups, stratified by gender. (XLSX 338 kb

Gender differences in the T-cell profiles of the airways in COPD patients associated with clinical phenotypes

Abstract T lymphocytes are believed to play an important role in the pathogenesis of chronic obstructive pulmonary disease (COPD). How T cells are recruited to the lungs and contribute to the inflammatory process is largely unknown. COPD is a heterogeneous disease, and discriminating disease phenotypes based on distinct molecular and cellular pathways may provide new approaches for individualized diagnosis and therapies. Bronchoalveolar lavage (BAL) and blood samples were obtained from 40 never-smokers, 40 smokers with normal lung function, and 38 COPD patients. T-cell chemokine receptor expression was analyzed with flow cytometry, and soluble BAL cytokines and chemokines were measured using a cytokine multiplex assay. Correlations with gender and clinical characteristics including lung imaging were investigated using multivariate modeling. Th1/Tc1- and Th2/Tc2-associated soluble analytes and T-cell chemokine receptors were analyzed as cumulative Th1/Tc1 and Th2/Tc2 immune responses. A higher expression of chemokine receptor CCR5 on CD8⁺ T cells in BAL and higher percentage of CXCR3⁺CD8⁺ T cells in blood was found in female smokers with COPD compared to those without COPD. CCR5 expression on CD4⁺ and CD8⁺ T cells was lower in BAL from male smokers with COPD compared to those without COPD. Among female smokers with COPD, Th1/Tc1 immune response was linked to BAL macrophage numbers and goblet cell density, and Th2/Tc2 response was associated with the measures of emphysema on high-resolution computed tomography. The highly gender-dependent T-cell profile in COPD indicates different links between cellular events and clinical manifestations in females compared to males. Our findings may reveal mechanisms of importance for the difference in clinical course in female COPD patients compared to males

Mucins 3A and 3B are expressed in the epithelium of human large airway

Abstract Aberrant mucus secretion is a hallmark of chronic obstructive pulmonary disease (COPD). Expression of the membrane-tethered mucins 3A and 3B (MUC3A, MUC3B) in human lung is largely unknown. In this observational cross-sectional study, we recruited subjects 45–65 years old from the general population of Stockholm, Sweden, during the years 2007–2011. Bronchial mucosal biopsies, bronchial brushings, and bronchoalveolar lavage fluid (BALF) were retrieved from COPD patients (n = 38), healthy never-smokers (n = 40), and smokers with normal lung function (n = 40). Protein expression of MUC3A and MUC3B in bronchial mucosal biopsies was assessed by immunohistochemical staining. In a subgroup of subjects (n = 28), MUC3A and MUC3B mRNAs were quantified in bronchial brushings using microarray. Non-parametric tests were used to perform correlation and group comparison analyses. A value of p %lt; 0.05 was considered statistically significant. MUC3A and MUC3B immunohistochemical expression was localized to ciliated cells. MUC3B was also expressed in basal cells. MUC3A and MUC3B immunohistochemical expression was equal in all study groups but subjects with emphysema had higher MUC3A expression, compared to those without emphysema. Smokers had higher mRNA levels of MUC3A and MUC3B than non-smokers. MUC3A and MUC3B mRNA were higher in male subjects and correlated negatively with expiratory air flows. MUC3B mRNA correlated positively with total cell concentration and macrophage percentage, and negatively with CD4/CD8 T cell ratio in BALF. We concluded that MUC3A and MUC3B in large airways may be a marker of disease or may play a role in the pathophysiology of airway obstruction