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.

List of scientific papers

I. Heyder T, Kohler M, Tarasova NK, Haag S, Rutishauser D, Rivera NV, Sandin C, Mia S, Malmstrom V, Wheelock AM, Wahlstrom J, Holmdahl R, Eklund A, Zubarev RA, Grunewald J, Ytterberg AJ. Approach for Identifying Human Leukocyte Antigen (HLA)-DR Bound Peptides from Scarce Clinical Samples. Mol Cell Proteomics. 2016: 15(9): 3017-3029.
https://doi.org/10.1074/mcp.M116.060764

II. Heyder T, Wiklundh E, Eklund A, Grunewald J, Zubarev RA, Lundstrom SL. Altered Fc-galactosylation status in IgG4 is a potential serum biomarker for chronic sarcoidosis. [Submitted]

III. Heyder T, Yang M, Kohler M, Merikallio H, Forsslund H, Li CX, Karimi R, Eklund A, Grunewald J, Kaarteenaho R, Sihlbom C, Skold CM, Wheelock AM. Genderenhanced alteration in airway epithelial proteome in COPD related to xenobiotic metabolism. [Submitted]

IV. Naz S, Kolmert J, Yang M, Reinke SN, Kamleh MA, Snowden S, Heyder T, Levanen B, Erle DJ, Skold CM, Wheelock AM, Wheelock CE. Metabolomics analysis identifies sex-associated metabotypes of oxidative stress and the autotaxin-lysoPA axis in COPD. [Accepted]
https://doi.org/10.1183/13993003.02322-2016