Immunopeptidomics and autoantigens of interstitial lung diseases

Abstract

We investigate Sarcoidosis and Idiopathic Pulmonary Fibrosis, IPF, to find markers for early fibrosis development. The aetiologies for both diseases are unknown and there is no specific treatment, moreover, there is a lack of diagnostic biomarkers for both diagnose and disease activity. Overall, the diseases have a considerable effect on patients’ physical health and quality of life. To identify patients at risk of rapid development of fibrosis, it is vital to improve patient care. We hypothesize that identification of specific antigens can help in the exposition of the pathogenesis of sarcoidosis as well as of IPF, and in the long perspective this could lead to identification of therapeutic targets.

In project I we investigated the presence of proteins in BAL and serum from sarcoidosis patients and controls to discover disease associated proteins. In total eight proteins had increased levels in sarcoidosis patients, two were the most prominent (Fibronectin 1, FN1 and C-C motif chemokine 2, CCL2) that displayed the greatest differences between cohorts. Furthermore, the protein cadherin 5 (CDH5) had a positive association to lymphocyte count in BAL, interesting since T-lymphocytes are the major cell type in sarcoidosis. Potentially this could provide a way of monitoring lymphocyte presence in the lung through blood sample.

In project II the large source of antigens from the Human Protein Atlas Project, a large library of protein fragments and antibodies that represent virtually all proteins in the body, was used to screen the immunoglobulin G specificity towards 3072 selected antigens in serum and BAL samples from patients with either of the sarcoidosis subcategories (Löfgren's syndrome (LS), non-Löfgren's sarcoidosis (nLS)), and asthma as well as healthy controls. A selected set of antigens went on to be analyzed in mSBA analysis. Sarcoidosis patients demonstrated an elevated reactivity frequencies toward Zinc finger protein 688 (ZNF688) and mitochondrial ribosomal protein L43 (MRPL43), particularly MRLP43 displayed a higher frequency in patients with non-Löfgren syndrome. Even though the protein fragment representing adenosine diphosphate-ribosylation factor GTPase activating protein 1 (ARFGAP1) showed high reactivity frequency in all sample groups, it was still significantly elevated in patients with sarcoidosis compared to the other cohorts.

In project III we used a mass spectrometry-based method to analyze and characterize the Fc regions of human IgGs in paired serum and BAL fluid. Antibodies were isolated using a fast and reliable approach via MelonGel extraction. The isolated IgGs were digested by trypsin and separated by nLC (nano liquid chromatography) as in conventional proteomics, but peptide fragmentation was performed by both high resolution HCD MS/MS (Higher-energy C-trap dissociation) and high-resolution ETD MS/MS (Electron-transfer dissociation).

We identified a candidate marker IgG4, which corresponded well to inflammatory activity in chronic lung diseases while also correlating between BAL and serum (R2=0.95), thus being readily available for sample collection. The IgG galactosylation marker could prove to be useful in clinical settings by monitoring chronic pulmonary inflammation status. Based on the results of project II we concluded that the Fc galactosylation status of IgG4 could potentially be used as a serum marker for severity in pulmonary inflammation.

In project IV project, we used the mSBA approach for both sarcoidosis and IPF samples in order to evaluate similarities and differences between fibrosis associated diseases. Autoantigens were found in a majority of patient samples, including healthy controls, although that cohort displayed a lower frequency of autoreactivity and also comparatively lower titers. Altogether, autoreactivity was higher in IPF and in nLS patients compared to all other groups; both groups included a higher fibrosis rate than the other diseases potentially linking a general presence of autoreactive antibodies to fibrosis development. Reactivity toward collagen 5A1 (COL5A1) was discovered with a statistical significant higher frequency in patients with IPF compared to all other groups apart from the nLS group. The protein epitope of COL5A1 is proposed as an autoimmune target and/or marker of fibrosis, and is of interest for further investigation.

In this thesis we have profiled the repertoire of proteins and autoantibodies in serum and BAL from patients with sarcoidosis, in addition we also investigated and compared the presence of autoreactive antibodies in patients with sarcoidosis and IPF, as well as control subjects and various comparable diseases. We have also proposed the ratio between agalactosylated and galactosylated forms of the Fc region in IgG4 as a marker for severe chronic lung disease. These discoveries open up for more studies to characterize, and test functionality, of these autoantibodies and proteins in the setting of sarcoidosis, IPF, or inflammatory and/or pulmonary diseases.