Identification of antigens in BAL fluid in Sarcoidosis

AbstractActivated T lymphocytes of the Th-1 type are typically accumulated in the lungs of patients with sarcoidosis, and there they may recognize specific sarcoidosis antigens, presented by antigen presenting cells such as dendritic cells and/or alveolar macrophages. In line with this, previous studies have indicated alveolar macrophages to contain proteins that could induce granuloma formation. Also, signs of a specific immune response in the lungs of HLA-DRB1*0301 positive sarcoidosis patients suggest the presentation of specific antigens by HLA class IImolecules on BAL cells. A number of peptides have therefore been eluted from HLA-DR molecules of BAL cells of HLA-DRB1*0301 positive sarcoidosis patients and these peptides are now used in stimulation tests to analyse their capacity to elicit T cell responses, using an elispot assay. Interestingly, all peptides identified so far were derived from self proteins, indicating autoimmune reactions in the lungs of these patients. These studies may lead to the identification of (a) sarcoidosis specific antigen(s)

Increased pulmonary Wnt (wingless/integrated)-signaling in patients with sarcoidosis

SummaryBackgroundSarcoidosis is an inflammatory multisystemic granulomatous disease of unknown aetiology commonly affecting the lungs, and pulmonary fibrosis often develops in chronic sarcoidosis. It has been suggested that Wnt (Wingless/integrated)-signaling has a role in inflammatory and fibrotic processes in the lungs, but its role in sarcoidosis has not been investigated. We hypothesised that Wnts secreted from T cells or other inflammatory cells have a role in the pathogenesis of sarcoidosis.MethodsBrush biopsies and bronchoalveolar lavage (BAL) were obtained through bronchoscopy from healthy controls (n = 18) and patients with sarcoidosis (n = 48). Semi-quantitative RT-PCR, electrophoretic mobility shift assay (EMSA) and immunocytochemistry were performed to analyse Wnt expression and activation of the Wnt-signal transducer β-catenin.ResultsAltered expression of Wnt5A, Wnt7A and Wnt7B mRNA in BAL cells was observed, as well as an increased activation of β-catenin, measured by EMSA and confirmed with immunocytochemistry, in resident lung cells from patients with sarcoidosis. More pronounced changes in Wnt expression were seen with advancing disease stage. Thus, by three independent methods, we have found evidence of increased pulmonary Wnt-activation in sarcoidosis.ConclusionsIn the lungs of patients with sarcoidosis there is a previously unappreciated increased Wnt-signal activation that could contribute to the inflammatory processes

Sex differences in the genetics of sarcoidosis across European and African ancestry populations

BackgroundSex differences in the susceptibility of sarcoidosis are unknown. The study aims to identify sex-dependent genetic variations in two clinical sarcoidosis phenotypes: Löfgren’s syndrome (LS) and non-Löfgren’s syndrome (non-LS).MethodsA meta-analysis of genome-wide association studies was conducted on Europeans and African Americans, totaling 10,103 individuals from three population-based cohorts, Sweden (n = 3,843), Germany (n = 3,342), and the United States (n = 2,918), followed by an SNP lookup in the UK Biobank (UKB, n = 387,945). A genome-wide association study based on Immunochip data consisting of 141,000 single nucleotide polymorphisms (SNPs) was conducted in the sex groups. The association test was based on logistic regression using the additive model in LS and non-LS sex groups independently. Additionally, gene-based analysis, gene expression, expression quantitative trait loci (eQTL) mapping, and pathway analysis were performed to discover functionally relevant mechanisms related to sarcoidosis and biological sex.ResultsWe identified sex-dependent genetic variations in LS and non-LS sex groups. Genetic findings in LS sex groups were explicitly located in the extended Major Histocompatibility Complex (xMHC). In non-LS, genetic differences in the sex groups were primarily located in the MHC class II subregion and ANXA11. Gene-based analysis and eQTL enrichment revealed distinct sex-specific gene expression patterns in various tissues and immune cell types. In LS sex groups, a pathway map related to antigen presentation machinery by IFN-gamma. In non-LS, pathway maps related to immune response lectin-induced complement pathway in males and related to maturation and migration of dendritic cells in skin sensitization in females were identified.ConclusionOur findings provide new evidence for a sex bias underlying sarcoidosis genetic architecture, particularly in clinical phenotypes LS and non-LS. Biological sex likely plays a role in disease mechanisms in sarcoidosis

CD4\u3csup\u3e+\u3c/sup\u3e T cells in the lungs of acute sarcoidosis patients recognize an Aspergillus nidulans epitope

Löfgren’s syndrome (LS) is an acute form of sarcoidosis characterized by a genetic association with HLA-DRB1*03 (HLA-DR3) and an accumulation of CD4+ T cells of unknown specificity in the bronchoalveolar lavage (BAL). Here, we screened related LS-specific TCRs for antigen specificity and identified a peptide derived from NAD-dependent histone deacetylase hst4 (NDPD) of Aspergillus nidulans that stimulated these CD4+ T cells in an HLA-DR3–restricted manner. Using ELISPOT analysis, a greater number of IFN-γ– and IL-2–secreting T cells in the BAL of DR3+ LS subjects compared with DR3+ control subjects was observed in response to the NDPD peptide. Finally, increased IgG antibody responses to A. nidulans NDPD were detected in the serum of DR3+ LS subjects. Thus, our findings identify a ligand for CD4+ T cells derived from the lungs of LS patients and suggest a role of A. nidulans in the etiology of LS

SNP Variants in Major Histocompatibility Complex Are Associate with Sarcoidosis Susceptibility - A Joint Analysis in Four European Populations

Sarcoidosis is a multiorgan inflammatory disorder with heritability estimates up to 66%. Previous studies have shown the major histocompatibility complex (MHC) region to be associated with sarcoidosis, suggesting a functional role for antigen-presenting molecules and immune mediators in the disease pathogenesis. To detect variants predisposing to sarcoidosis and to identify genetic differences between patient subgroups, we studied four genes in the MHC Class III region (LTA, TNF, AGER, BTNL2) and HLA-DRA with tag-SNPs and their relation to HLA-DRB1 alleles. We present results from a joint analysis of four study populations (Finnish, Swedish, Dutch, and Czech). Patients with sarcoidosis (n = 805) were further subdivided based on the disease activity and the presence of Lofgren's syndrome. In a joint analysis, seven SNPs were associated with non-Lofgren sarcoidosis (NL; the strongest association with rs3177928, P = 1.79E-07, OR = 1.9) and eight with Lofgren's syndrome [ Lofgren syndrome (LS); the strongest association with rs3129843, P = 3.44E-12, OR = 3.4] when compared with healthy controls (n = 870). Five SNPs were associated with sarcoidosis disease course (the strongest association with rs3177928, P = 0.003, OR = 1.9). The high linkage disequilibrium (LD) between SNPs and an HLA-DRB1 challenged the result interpretation. When the SNPs and HLA-DRB1 alleles were analyzed together, independent association was observed for four SNPs in the HLA-DRA/BTNL2 region: rs3135365 (NL; P = 0.015), rs3177928 (NL; P <0.001), rs6937545 (LS; P = 0.012), and rs5007259 (disease activity; P = 0.002). These SNPs act as expression quantitative trait loci (eQTL) for HLA-DRB1 and/or HLA-DRB5. In conclusion, we found novel SNPs in BTNL2 and HLA-DRA regions associating with sarcoidosis. Our finding further establishes that polymorphisms in the HLA-DRA and BTNL2 have a role in sarcoidosis susceptibility. This multi-population study demonstrates that at least a part of these associations are HLA-DRB1 independent (e.g., not due to LD) and shared across ancestral origins. The variants that were independent of HLA-DRB1 associations acted as eQTL for HLA-DRB1 and/or -DRB5, suggesting a role in regulating gene expression.Peer reviewe

Pulmonary sarcoidosis is associated with exosomal vitamin D-binding protein and inflammatory molecules

BACKGROUND: Sarcoidosis is an inflammatory granulomatous disorder characterized by accumulation of TH1-type CD4+T cells and immune effector cells within affected organs, most frequently the lungs. Exosomes are extracellular vesicles conveying intercellular communication with possible diagnostic and therapeutic applications.OBJECTIVES: Weaimed to provide an understanding of the proinflammatory role of bronchoalveolar lavage fluid (BALF) exosomes in patients with sarcoidosis and to find candidates for disease biomarkers.METHODS: Weperformed a mass spectrometric proteomics characterization of BALF exosomes from 15 patients with sarcoidosis and 5 healthy control subjects and verified the most interesting results with flow cytometry, ELISA, and Western blot analyses in an additional 39 patients and 22 control subjects.RESULTS: Morethan 690 proteins were identified in the BALF exosomes, several of which displayed significant upregulation in patients, including inflammation-associated proteins, such as leukotriene A4 hydrolase. Most of the complement-activating factors were upregulated, whereas the complement regulator CD55 was seen less in patients comparedwith healthy control subjects. In addition, for the first time, we detected vitamin D-binding protein in BALF exosomes, which was more abundant in patients. To evaluate exosome-associated vitamin D-binding protein as a biomarker for sarcoidosis, we investigated plasma exosomes from 23 patients and 11 healthy control subjects and found significantlyhigher expression in patients.CONCLUSION: Together,these data contribute to understanding the role of exosomes in lung disease and provide suggestions for highly warranted sarcoidosis biomarkers. Furthermore, the validation of an exosome-associated biomarker in the blood of patients provides novel, and less invasive, opportunities for disease diagnosis.</h4

Mass Cytometry Identifies Distinct Lung CD4+ T Cell Patterns in Löfgren’s Syndrome and Non-Löfgren’s Syndrome Sarcoidosis

Sarcoidosis is a granulomatous disorder of unknown etiology, characterized by accumulation of activated CD4+ T cells in the lungs. Disease phenotypes Löfgren’s syndrome (LS) and “non-LS” differ in terms of clinical manifestations, genetic background, HLA association, and prognosis, but the underlying inflammatory mechanisms largely remain unknown. Bronchoalveolar lavage fluid cells from four HLA-DRB1*03+ LS and four HLA-DRB1*03− non-LS patients were analyzed by mass cytometry, using a panel of 33 unique markers. Differentially regulated CD4+ T cell populations were identified using the Citrus algorithm, and t-stochastic neighborhood embedding was applied for dimensionality reduction and single-cell data visualization. We identified 19 individual CD4+ T cell clusters differing significantly in abundance between LS and non-LS patients. Seven clusters more frequent in LS patients were characterized by significantly higher expression of regulatory receptors CTLA-4, PD-1, and ICOS, along with low expression of adhesion marker CD44. In contrast, 12 clusters primarily found in non-LS displayed elevated expression of activation and effector markers HLA-DR, CD127, CD39, as well as CD44. Hierarchical clustering further indicated functional heterogeneity and diverse origins of T cell receptor Vα2.3/Vβ22-restricted cells in LS. Finally, a near-complete overlap of CD8 and Ki-67 expression suggested larger influence of CD8+ T cell activity on sarcoid inflammation than previously appreciated. In this study, we provide detailed characterization of pulmonary T cells and immunological parameters that define separate disease pathways in LS and non-LS. With direct association to clinical parameters, such as granuloma persistence, resolution, or chronic inflammation, these results provide a valuable foundation for further exploration and potential clinical application

Assessing Recent Smoking Status by Measuring Exhaled Carbon Monoxide Levels

The main expectations of applying proteomics technologies to clinical questions are the discovery of disease related biomarkers. Despite technological advancement to increase proteome coverage and depth to meet these expectations the number of generated biomarkers for clinical use is small. One of the reasons is that found potential biomarkers often are false discoveries. Small sample sizes, in combination with patient sample heterogeneity increase the risk of false discoveries. To be able to extract relevant biological information from such data, high demands are put on the experimental design and the use of sensitive and quantitatively accurate technologies. The overall aim of this thesis was to apply quantitative proteomics methods for biomarker discovery in clinical samples. A method for reducing bias by controlling for individual variation in smoking habits is described in paper I. The aim of the method was objective assessment of recent smoking in clinical studies on inflammatory responses. In paper II, the proteome of alveolar macrophages obtained from smoking subjects with and without the inflammatory lung disease chronic obstructive pulmonary disease (COPD) were quantified by two-dimensional gel-electrophoresis (2-DE). A gender focused analysis showed protein level differences within the female group, with down-regulation of lysosomal pathway and up-regulation of oxidative pathway in COPD patients. Paper III, a mass spectrometry based proteomics analysis of tumour samples, contributes to the molecular understanding of vulvar squamous cell carcinoma (VSCC) and we identified a high risk patient subgroup of HPV-negative tumours based on the expression of four proteins, further suggesting that this subgroup is characterized by an altered ubiquitin-proteasome signalling pathway. Paper III describes a data analysis workflow for the extraction of biological information from quantitative mass spectrometry based proteomics data. High patient-to-patient tumour proteome variability was addressed by using pathway profiling on individual tumour data, followed by comparison of pathway association ranks in a multivariate analysis. We show that pathway data on individual tumour level can detect subpopulations of patients and identify pathways of specific importance in pre-defined clinical groups by the use of multivariate statistics. In paper IV, the potentials and limits of quantitative mass spectrometry on clinical samples was evaluated by defining the quantitative accuracy of isobaric labels and label-free quantification. Quantification by isobaric labels in combination with pI pre-fractionation showed a lower limit of quantification (LOQ) than a label-free analysis without pI pre-fractionation, and 6-plex TMT were more sensitive than 8-plex iTRAQ. Precursor mixing measured by isolation interference (MS1 interference) is more linked to the quantitative accuracy of isobaric labels than reporter ion interference (MS2 interference). Based on that we could define recommendations for how much isolation interference that can be accepted; in our data <30% isolation interference had little effect the quantitative accuracy. In conclusion, getting biological knowledge from proteomics studies requires a careful study design, control of possible confounding factors and the use of clinical data to identify disease subtypes. Further, to be able to draw conclusions from the data, the analysis requires accurate quantitative data and robust statistical tools to detect significant protein alterations. Methods around these issues are developed and discussed in this thesis