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.

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.

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.

Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples.

Immune-mediated diseases strongly associating with human leukocyte antigen (HLA) alleles are likely linked to specific antigens. These antigens are presented to T cells in the form of peptides bound to HLA molecules on antigen presenting cells, e.g. dendritic cells, macrophages or B cells. The identification of HLA-DR-bound peptides presents a valuable tool to investigate the human immunopeptidome. The lung is likely a key player in the activation of potentially auto-aggressive T cells prior to entering target tissues and inducing autoimmune disease. This makes the lung of exceptional interest and presents an ideal paradigm to study the human immunopeptidome and to identify antigenic peptides.Our previous investigation of HLA-DR peptide presentation in the lung required high numbers of cells (800 × 10(6) bronchoalveolar lavage (BAL) cells). Because BAL from healthy nonsmokers typically contains 10-15 × 10(6) cells, there is a need for a highly sensitive approach to study immunopeptides in the lungs of individual patients and controls.In this work, we analyzed the HLA-DR immunopeptidome in the lung by an optimized methodology to identify HLA-DR-bound peptides from low cell numbers. We used an Epstein-Barr Virus (EBV) immortalized B cell line and bronchoalveolar lavage (BAL) cells obtained from patients with sarcoidosis, an inflammatory T cell driven disease mainly occurring in the lung. Specifically, membrane complexes were isolated prior to immunoprecipitation, eluted peptides were identified by nanoLC-MS/MS and processed using the in-house developed ClusterMHCII software. With the optimized procedure we were able to identify peptides from 10 × 10(6) cells, which on average correspond to 10.9 peptides/million cells in EBV-B cells and 9.4 peptides/million cells in BAL cells. This work presents an optimized approach designed to identify HLA-DR-bound peptides from low numbers of cells, enabling the investigation of the BAL immunopeptidome from individual patients and healthy controls in order to identify disease-associated peptides.

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.

Altered Fc galactosylation in IgG4 is a potential serum marker for chronic lung disease.

Characterising chronic lung diseases is challenging. New, less invasive diagnostics are needed to decipher disease pathologies and subphenotypes. Fc galactosylation is known to affect IgG function, and is altered in autoimmune disorders and under other pathological conditions. We tested how well Fc glycans in IgG from bronchoalveolar lavage fluid (BALF) and serum correlated, and if the Fc glycan profile could reveal pulmonary inflammation. A shotgun proteomics approach was used to profile Fc glycans in serum and BALF of controls (n=12) and sarcoidosis phenotypes (Löfgren's syndrome (LS), n=11; and non-LS, n=12). Results were further validated in severe asthma (SA) (n=20) and published rheumatoid arthritis (RA) patient data (n=13) including clinical information. Intra-individually, Fc-galactosylation status of IgG1 (R2=0.87) and IgG4 (R2=0.95) correlated well between matrixes. Following GlycoAge-index correction, the ratio between agalactosylated and digalactosylated Fc glycans of IgG4 could distinguish sarcoidosis and SA from healthy and RA subjects with a mean±se area under the curve (AUC) of 78±6%. The AUC increased to 83±6% using the more chronic lung disease types (non-LS and SA) and most strikingly, to 87±6% for the SA subgroup. The results indicate that the Fc galactosylation status of IgG4 is a potential blood test marker for chronic lung inflammation.