HLA-DR immunopeptidomics and quantitative lipid mediator profiling for studying lung involvement in immune-mediated diseases

In this thesis, we developed mass spectrometry-based methodologies for HLA- DR immunopeptidomics and lipid mediator profiling for the study of rheumatoid arthritis (RA) and long-term outcomes of preterm birth respectively.

RA is an autoimmune disease affecting the joints. Smoking and a group of alleles of the HLA-DRB1 gene, termed shared epitope (SE) are major risk factors for developing RA. HLA-DR is a protein involved in antigen presentation, and its polymorphism affects peptide binding preference. Evidence suggests that the autoimmunity of RA might be triggered in the lung. To our knowledge, there have been no previous non-targeted studies on the HLA-DR antigen landscape in the RA lung to date.

In Paper I, we developed a method for HLA-DR immunopeptidomics analysis of lung immune cells sampled by bronchoalveolar lavage (BAL). We applied this method on treatment-naïve RA patients (n=9) and healthy controls (n=10), as well as RA patients following 6 months of treatment (n=5). As the cohort represented a high genetic variance of HLA-DR types, we deconvoluted peptides to predicted binding HLA-DRB1 alleles. Interestingly, some alleles were predicted to bind more peptides. These included all present SE alleles (*01:01, *04:01, *04:04, *04:05, *10:01) and only one non-SE allele (*04:07). Together with published transcriptomics data, this suggests allele-specific expression profiles. Furthermore, the peptides predicted to bind to SE alleles contained native versions of known citrullinated RA autoantigens, including a-enolase and fibrinogen B. This suggests that future immunogenicity studies on the identified SE-binding peptides might lead to the discovery of novel autoantigens in RA.

In the following studies we investigated lipid mediators, many of which have known biological activities in the context of lung diseases. In Paper II, we developed a method to profile octadecanoids in a stereospecific manner, which allows for inference of their biosynthetic route and study of potential stereospecific biological activities. Using chiral super-critical fluid chromatography coupled to tandem mass spectrometry (SFC-MS/MS), we enabled stereo-specific profiling of 103 octadecanoids in less than 13 min. Additionally, we developed a more sensitive achiral method using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), which includes octadecanoids produced exclusively by autooxidation. Both methods were validated according to EMA guidelines in solvent and surrogate matrix for linearity, lower limit of quantification, accuracy, precision, recovery, and matrix effects.

Next, we addressed the limitations of established validation protocols for LC- MS/MS of endogenous compounds, as they often rely on replicate analysis from one pooled sample material. Thereby, they do not account for the inter-individual matrix diversity encountered in cohort studies. In Paper III, we developed a workflow which enables the validation of phenotyping robustness using cohort measurements. The statistical approaches are reflecting those typically applied in downstream cohort studies, such as association and quantile analysis. Therefore, this workflow allows for praxis-oriented assessment of method performance. Applications are shown on a subset of sphingolipids, such as identification of coelutions, evaluation of sample preparation, and selection of the most suitable internal standard per analyte.

Lastly, we applied the developed methods to investigate long-term consequences of sustained oxygen support after preterm birth, termed bronchopulmonary dysplasia (BPD). We hypothesized that young adults with a history of BPD might have persisting alterations in their lipid mediator profile of the lung, which might help understand why impaired lung function is frequently observed in these individuals throughout life. In Paper IV, we quantified oxylipins and sphingolipid levels in bronchoalveolar lavage fluid (BALF) of young adults with a history of BPD, as well as three age- and sex-matched control groups. These comprised preterm born individuals without a history of BPD, healthy at-term born, and individuals with mild asthma. Following analysis in three LC-MS/MS methods, a total of 22 oxylipins and 44 sphingolipids were quantified passing quality control criteria. Following noise removal using lasso and ridge regression, groups were compared using multivariate orthogonal projection to latent structures discriminate analysis (OPLS-DA) modeling. These analyses suggest that female groups were primarily differing in their oxylipin profile, while male groups in their sphingolipid profile. The BALF of the female BPD group contained higher levels of 12-HHTrE compared to the other groups, and 9,10-EpOME was increased in both female BPD and asthma group compared to the healthy group. Our previous studies showed higher levels of these analytes in BALF of females with smoking-associated chronic obstructive pulmonary disease (COPD). The male BPD and asthma group showed increased levels of Cer(d18:/16:0), Cer(d18:1/18:0), and DhCer(18:0/16:0). Increased levels of the former two are known predictors of adverse cardiovascular outcomes.

Taken together, this PhD thesis provides methodologies to investigate the HLA- DR immunopeptidome and lipid mediator profiles. By applying these methodologies in RA and BPD respectively, we could report novel findings which might aid the understanding the immunological mechanisms driving these diseases, and associated molecular phenotypes in the lung.

List of scientific papers

I. Benedikt Zöhrer, Ákos Végvári, Martina Bonatti, Iryna Kolosenko, Nicole Wagner, Antonio Checa, Vijay Joshua, C. Magnus Sköld, Karin Lundberg, Lars Klareskog, Vivianne Malmström, Åsa M. Wheelock. Characterization of the HLA-DR immunopeptidome of bronchoalveolar lavage cells in patients with newly diagnosed rheumatoid arthritis and healthy current-smoker controls. [Manuscript]

II. Alessandro Quaranta, Benedikt Zöhrer, Johanna Revol-Cavalier, Kurt Benkestock, Laurence Balas, Camille Oger, Gregory S. Keyes, Åsa M. Wheelock, Thierry Durand, Jean-Marie Galano, Christopher E. Ramsden, Mats Hamberg, Craig E. Wheelock. Development of a Chiral Supercritical Fluid Chromatography-Tandem Mass Spectrometry and Reversed-Phase Liquid Chromatography-Tandem Mass Spectrometry Platform for the Quantitative Metabolic Profiling of Octadecanoid Oxylipins. Analytical Chemistry. 2022, 94 (42): 14618-14626. https://doi.org/10.1021/acs.analchem.2c02601

III. Benedikt Zöhrer, Cristina Gómez, Joaquim Jaumot, Helena Idborg, Signe S. Torekov, Asa M. Wheelock, Craig E. Wheelock, Antonio Checa. Cohort-based strategies as an in-house tool to evaluate and improve phenotyping robustness of LC-MS/MS lipidomics platforms. Analytical and Bioanalytical Chemistry. 2024, 416 (25): 5485-5496. https://doi.org/10.1007/s00216-024-05404-8

IV. Benedikt Zöhrer*, Nicole Wagner*, Antonio Checa, Alessandro Quaranta, Martina Bonatti, Iryna Kolosenko, Petra Um-Bergström, Anders Lindén, Eva Berggren-Broström, C. Magnus Sköld, Craig E. Wheelock, Åsa M. Wheelock. *Shared first authorship. Lipid mediator profiles in bronchoalveolar lavage fluid of young adults with a history of bronchopulmonary dysplasia are distinct from those of healthy controls and subjects with mild asthma. [Manuscript]