Between two lungs : proteomic and metabolomic approaches in inflammatory lung diseases

This doctoral thesis presents results on proteomic and metabolomic profiling of two distinct inflammatory lung diseases: sarcoidosis and chronic obstructive pulmonary disease (COPD). Chronic inflammatory lung diseases are debilitating conditions representing a major health problem throughout the world. However, up to this date disease pathobiology is not fully understood and clinical practise is lacking early diagnostic tools. The overall aim of the work has been the investigation of disease specific biomarkers as well as underlying molecular pathways. In addition, the characterization of clinical sub-phenotypes will provide deeper insights into the molecular mechanisms involved in disease pathophysiology. Moreover, for the purpose of profiling the immunopeptidome in sarcoidosis, an approach was developed to identify immunopeptides presented on antigen presenting cells with high sensitivity. The first part of this thesis focuses on sarcoidosis, an inflammatory T cell driven disease that mainly manifests in the lungs. It is hypothesised that specific antigenic substances trigger onset of sarcoidosis. Because lung samples from healthy non-smokers typically contains 10– 15×106 cells, there was a need for a sensitive approach to identify immunopeptides. Project I describes the optimization of an approach designed specifically to investigate the immunopeptidome on human leukocyte allele antigens (HLA)-DR from scarce clinical material. Epstein-Barr Virus immortalized B cells and antigen presenting cells obtained from bronchoalveolar lavage of sarcoidosis patients were utilised for investigating the HLA-DR immunopeptidome. The approach presents a valuable tool to profile and compare peptide repertoires in health and disease in order to reveal disease specific antigenic peptide(s) in sarcoidosis. Project II is based on the hypothesis that sarcoidosis patients potentially exhibit an expansion of certain antibody clones, supporting the concept of induced activation against a disease specific antigen. Specific features of polyclonal antibodies could be utilized for disease characterization via blood testing. The IgG Fc-galactosylation status in matched bronchoalveolar lavage fluid and serum samples was very well correlating. In particular, the ratio between the main agalactosylated (FA2) and main di-galactosylated Fc-glycans (FA2G2) of IgG 4 could successfully distinguish both sarcoidosis phenotypes and controls. The second part of this thesis concerns investigations of early stage COPD with a particular focus on identifying molecular gender differences related to distinct disease characteristics and clinical phenotypes between women and men. Disease related alterations in the airway epithelial proteome were characterised using two complementary proteomic platforms (project III); two-dimensional difference gel electrophoresis (2D-DIGE) and tandem mass tag-based shotgun proteomics (TMT-MS). Additionally, serum metabolites from the same study cohort were analysed with non-targeted liquid chromatography-high resolution mass spectrometry (project IV). Both molecular investigations revealed specific alterations between early stage COPD patients and smokers with normal lung function mainly driven by the female population in the cohort. Gender-enhanced alterations in xenobiotic metabolism were revealed in the airway epithelium as well as changes in oxidative phosphorylation and protein processing in the endoplasmic reticulum. Non-targeted metabolomics and downstream-targeted metabolomic validations confirmed enhanced metabolic dysregulation in women with COPD, which was linked to oxidative stress. Together, this suggests metabolic activation and detoxification in the airway epithelium, as well as dysregulation in the delicate balance between oxidative stress and the anti-oxidative defence system, influence homeostasis and tissue damage in early stage COPD. This work provides an optimized methodology that enables the investigation of the human immunopeptidome to reveal disease specific immunogenic peptides. Furthermore, proteomic and metabolomics approaches strive to add novel insights into biomarker discovery and disease mechanisms of inflammatory pulmonary diseases. The presented findings have the potential to provide clinical biomarkers and pharmaceutical targets to be used for early diagnosis and individualised treatment

Metabolomics analysis identifies sex-associated metabotypes of oxidative stress and the autotaxin-lysoPA axis in COPD.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease and a leading cause of mortality and morbidity worldwide. The aim of this study was to investigate the sex dependency of circulating metabolic profiles in COPD.Serum from healthy never-smokers (healthy), smokers with normal lung function (smokers), and smokers with COPD (COPD; Global Initiative for Chronic Obstructive Lung Disease stages I-II/A-B) from the Karolinska COSMIC cohort (n=116) was analysed using our nontargeted liquid chromatography-high resolution mass spectrometry metabolomics platform.Pathway analyses revealed that several altered metabolites are involved in oxidative stress. Supervised multivariate modelling showed significant classification of smokers from COPD (p=2.8×10-7). Sex stratification indicated that the separation was driven by females (p=2.4×10-7) relative to males (p=4.0×10-4). Significantly altered metabolites were confirmed quantitatively using targeted metabolomics. Multivariate modelling of targeted metabolomics data confirmed enhanced metabolic dysregulation in females with COPD (p=3.0×10-3) relative to males (p=0.10). The autotaxin products lysoPA (16:0) and lysoPA (18:2) correlated with lung function (forced expiratory volume in 1 s) in males with COPD (r=0.86; p<0.0001), but not females (r=0.44; p=0.15), potentially related to observed dysregulation of the miR-29 family in the lung.These findings highlight the role of oxidative stress in COPD, and suggest that sex-enhanced dysregulation in oxidative stress, and potentially the autotaxin-lysoPA axis, are associated with disease mechanisms and/or prevalence

SpotLight Proteomics—A IgG-Enrichment Phenotype Profiling Approach with Clinical Implications

Sarcoidosis is a systemic interstitial lung disease of unknown aetiology. Less invasive diagnostics are needed to decipher disease pathology and to distinguish sub-phenotypes. Here we test if SpotLight proteomics, which combines de novo MS/MS sequencing of enriched IgG and co-extracted proteins with subsequent label-free quantification of new and known peptides, can differentiate controls and sarcoidosis phenotypes (Löfgrens and non-Löfgrens syndrome, LS and nonLS). Intra-individually matched IgG enriched from serum and bronchial lavage fluid (BALF) from controls (n = 12), LS (n = 11) and nonLS (n = 12) were investigated. High-resolution mass-spectrometry SpotLight proteomics and uni- and multivariate-statistical analyses were used for data processing. Major differences were particularly observed in control-BALF versus sarcoidosis-BALF. However, interestingly, information obtained from BALF profiles was still present (but less prominent) in matched serum profiles. By using information from orthogonal partial least squares discriminant analysis (OPLS-DA) differentiating 1) sarcoidosis-BALF and control-BALF and 2) LS-BALF vs. nonLS-BALF, control-serum and sarcoidosis-serum (p = 0.0007) as well as LS-serum and nonLS-serum (p = 0.006) could be distinguished. Noteworthy, many factors prominent in identifying controls and patients were those associated with Fc-regulation, but also features from the IgG-Fab region and novel peptide variants. Differences between phenotypes were mostly IgG-specificity related. The results support the analytical utility of SpotLight proteomics which prospectively have potential to differentiate closely related phenotypes from a simple blood test

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

Metabolomics analysis identifies sex-associated metabotypes of oxidative stress and the autotaxin-lysoPA axis in&nbsp;COPD.

Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease and a leading cause of mortality and morbidity worldwide. The aim of this study was to investigate the sex dependency of circulating metabolic profiles in COPD.Serum from healthy never-smokers (healthy), smokers with normal lung function (smokers), and smokers with COPD (COPD; Global Initiative for Chronic Obstructive Lung Disease stages I-II/A-B) from the Karolinska COSMIC cohort (n=116) was analysed using our nontargeted liquid chromatography-high resolution mass spectrometry metabolomics platform.Pathway analyses revealed that several altered metabolites are involved in oxidative stress. Supervised multivariate modelling showed significant classification of smokers from COPD (p=2.8×10-7). Sex stratification indicated that the separation was driven by females (p=2.4×10-7) relative to males (p=4.0×10-4). Significantly altered metabolites were confirmed quantitatively using targeted metabolomics. Multivariate modelling of targeted metabolomics data confirmed enhanced metabolic dysregulation in females with COPD (p=3.0×10-3) relative to males (p=0.10). The autotaxin products lysoPA (16:0) and lysoPA (18:2) correlated with lung function (forced expiratory volume in 1 s) in males with COPD (r=0.86; p&lt;0.0001), but not females (r=0.44; p=0.15), potentially related to observed dysregulation of the miR-29 family in the lung.These findings highlight the role of oxidative stress in COPD, and suggest that sex-enhanced dysregulation in oxidative stress, and potentially the autotaxin-lysoPA axis, are associated with disease mechanisms and/or prevalence

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