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– 15×10 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 IgG4 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. POPULAR SCIENCE SUMMARY Between Two Lungs presents a doctoral thesis studying two inflammatory lung diseases, sarcoidosis and COPD. Both diseases are chronic, however they show highly distinct clinical characteristics and therefore were studied separately. What brings the investigation in this thesis together are methodologies to identify and characterize proteins and metabolites and their alterations due to disease. The causes of both diseases are not completely understood, but genetic predisposition, gender and environmental factors present main risk factors in both cases. Symptoms for patients can be severe and life threatening in both instances. Therefore, markers that can be used for early diagnosis, as well as improved therapeutic strategies are in high demand to improve disease outcome and the life quality of patients. Disease symptoms in sarcoidosis and COPD are distinct and their prevalence throughout the world also differs. Sarcoidosis is a rather rare disease, with the highest incidences in Scandinavian countries with approximately 1,200 new cases in Sweden every year. It is a disease which presents with acute inflammation and granuloma formation in the affected organs, most commonly the lungs. Breathing complications constitute a major issue for these patients and occur due to tissue scaring, which decreases the elasticity of the lung. Certain features of sarcoidosis resemble autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis. Autoimmunity is triggered by molecules (known as antigens) which cause the immune system to react against its own tissue, and it has been suggested that sarcoidosis is induced by exposure peptides that resembles the body’s own proteins (auto antigens). In this thesis, a sensitive method was developed to identify such antigenic molecules in the lungs of individuals, both healthy controls as well as patients. This provides a platform for individual antigen profiling and comparison of peptide repertoires in heath and disease, and may reveal disease specific antigens and potential disease-specific triggers. This methodology can also be applied to other disease with unknown antigenic triggers and therefore presents a valuable tool in the hunt for specific antigens in complex, multifactorial diseases. Further, within the scope of biomarker discovery for the diagnosis of sarcoidosis, we studied the antibody repertoire in blood and lung fluid. A marker that could be measured by a simple blood test that reflects the inflammatory status in the lungs, would be of great value in clinical practice. Emphasis was put on attached sugar moieties on antibodies (glycosylation), a specific modification that is known to change with inflammation. A ratio between two different glycosylation stages was identified as a potential marker for successfully distinguishing sarcoidosis patients from healthy controls, while simultaneously reflecting the degree of inflammation in the lung. COPD, the second disease studied in this thesis, is characterized by lung inflammation and the narrowing of the airways, with obstructions resulting in difficulties in complete exhalation and therefore small amounts of air remaining in the lungs. COPD is the most common lung disease in the world and smoking presents the main risk factor for disease development. Focus of this part of the thesis was the discovery of disease-specific biomarkers for early diagnosis and the identification of biological mechanisms that could be relevant for COPD development. In general, women are more susceptible to developing the disease and also present with different clinical features compared to men. Therefore, we specifically focused on gender differences while comparing healthy controls with COPD patients. In order to study molecular changes attributed to disease, we characterized proteins in the airway epithelium, which presents the top cell layer in the airways and the first line of defence against any inhaled particles such as tobacco smoke. Additionally, we analysed metabolites in blood, which reflect all chemical processes that occur throughout the body. Distinct alterations in protein and metabolite profiles were observed in both female and male patients compared to healthy smokers. Discovered proteins and metabolites could therefore have potential as diagnostic marker for COPD. We were also able to link the observed changes to processes related to oxidative stress in women. Oxidative stress essentially indicates an imbalance between the production of reactive toxic compounds (free radicals) and the ability of the body to counteract or detoxify their harmful effects. Disturbance of this balance may result in tissue damage. This dysregulation could play a key role in the destruction of lung tissue in COPD. Our data indicates that men and women react differently to tobacco smoke, and are not equal in their ability to tolerate inhaled toxicants. This might also explain why males and females show different symptoms upon clinical investigation. This thesis presents different methods to study sarcoidosis and COPD, striving to identify disease-specific markers and reveal mechanisms that are of importance in the course of disease. We were able to provide tools developed to study specific antigens in inflammatory diseases, identify potential biomarkers and increase our knowledge of disease mechanisms in sarcoidosis and COPD. This might eventually contribute to improved diagnostic procedures, the development of new drugs, and eventually an improved quality of life for patients.