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Bronchoalveolar lavage fluid IFN-?+ Th17 cells and regulatory T cells in pulmonary sarcoidosis.

ABSTRACT: In sarcoidosis, increased Th17 cell fractions have been reported in bronchoalveolar lavage fluid, and elevated numbers of Th17 cells producing IFN- ? have been observed in peripheral blood. The balance between Th1, Th17, and FoxP3(+) CD4(+) T cell subsets in sarcoidosis remains unclear. Bronchoalveolar lavage fluid cells, from 30 patients with sarcoidosis, 18 patients with other diffuse parenchymal lung diseases, and 15 healthy controls, were investigated with flow cytometry for intracellular expression of FoxP3. In a subset of the patients, expression of the cytokines IL17A and IFN- ? was investigated. The fractions of FoxP3(+) CD4(+) T cells and Th17 cells were both lower in sarcoidosis compared to controls (P = 0.017 and P = 0.011, resp.). The proportion of Th17 cells positive for IFN- ? was greater in sarcoidosis than controls (median 72.4% versus 31%, P = 0.0005) and increased with radiologic stage (N = 23, rho = 0.45, and P = 0.03). IFN- ? (+) Th17 cells were highly correlated with Th1 cells (N = 23, rho = 0.64, and P = 0.001), and the ratio of IFN- ? (+) Th17/FoxP3(+) CD4(+) T cells was prominently increased in sarcoidosis. IFN- ? (+) Th17 cells may represent a pathogenic subset of Th17 cells, yet their expression of IFN- ? could be a consequence of a Th1-polarized cytokine milieu. Our results indicate a possible immune cell imbalance in sarcoidosis.

SUBMITTER: Tondell A

PROVIDER: S-EPMC4027000 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Bronchoalveolar lavage fluid IFN-γ+ Th17 cells and regulatory T cells in pulmonary sarcoidosis.

Tøndell Anders A Moen Torolf T Børset Magne M Salvesen Øyvind Ø Rø Anne Dorthea AD Sue-Chu Malcolm M

Mediators of inflammation 20140505

In sarcoidosis, increased Th17 cell fractions have been reported in bronchoalveolar lavage fluid, and elevated numbers of Th17 cells producing IFN- γ have been observed in peripheral blood. The balance between Th1, Th17, and FoxP3(+) CD4(+) T cell subsets in sarcoidosis remains unclear. Bronchoalveolar lavage fluid cells, from 30 patients with sarcoidosis, 18 patients with other diffuse parenchymal lung diseases, and 15 healthy controls, were investigated with flow cytometry for intracellular ex ...[more]

PMID: 24882950

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Project description:BackgroundSarcoidosis is a multisystem immuno-inflammatory disorder of unknown etiology that most commonly involves the lungs. We hypothesized that an unbiased approach to identify pathways activated in bronchoalveolar lavage (BAL) cells can shed light on the pathogenesis of this complex disease.MethodsWe recruited 15 patients with various stages of sarcoidosis and 12 healthy controls. All subjects underwent bronchoscopy with lavage. For each subject, total RNA was extracted from BAL cells and hybridized to an Affymetrix U133A microarray. Rigorous statistical methods were applied to identify differential gene expression between subjects with sarcoidosis vs.ControlsTo better elucidate pathways differentially activated between these groups, we integrated network and gene set enrichment analyses of BAL cell transcriptional profiles.ResultsSarcoidosis patients were either non-smokers or former smokers, all had lung involvement and only two were on systemic prednisone. Healthy controls were all non-smokers. Comparison of BAL cell gene expression between sarcoidosis and healthy subjects revealed over 1500 differentially expressed genes. Several previously described immune mediators, such as interferon gamma, were upregulated in the sarcoidosis subjects. Using an integrative computational approach we constructed a modular network of over 80 gene sets that were highly enriched in patients with sarcoidosis. Many of these pathways mapped to inflammatory and immune-related processes including adaptive immunity, T-cell signaling, graft vs. host disease, interleukin 12, 23 and 17 signaling. Additionally, we uncovered a close association between the proteasome machinery and adaptive immunity, highlighting a potentially important and targetable relationship in the pathobiology of sarcoidosis.ConclusionsBAL cells in sarcoidosis are characterized by enrichment of distinct transcriptional programs involved in immunity and proteasomal processes. Our findings add to the growing evidence implicating alveolar resident immune effector cells in the pathogenesis of sarcoidosis and identify specific pathways whose activation may modulate disease progression.

Project description:Background: Sarcoidosis and idiopathic pulmonary fibrosis (IPF) are two most frequent forms of interstitial lung diseases (ILDs). Cellular and biochemical composition of bronchoalveolar lavage fluid (BALf) was shown to reflect the proliferative and fibrotic changes in the local environment in the lung. However, the usefulness of BALf cellular profile evaluation in the diagnosis of ILDs is limited. The aim of the study was a multivariate, molecular, comparative analysis of BALf cells from IPF and sarcoidosis patients. Methods: Transcriptomic measurements were carried out using Affymetrix Human Gene 2.1 ST ArrayStrip in 21 samples: 9 IPF and 12 sarcoidosis. The mRNA expression for the most significantly differentiating genes was evaluated by real time PCR in 32 samples (11 IPF and 21 sarcoidosis). Results: The number of genes differentially expressed between IPF and sarcoidosis groups were 4832 (13359 probesets). Our cluster analysis indicated that sarcoidosis BALf cells are characterized by increased mRNA expression of genes associated with ribosome biogenesis, transcription machinery. Clusters formed by genes with changed mRNA expression in IPF samples were implicated in the processes of cell adhesion and migration, metalloproteinase expression and negative regulation of cell proliferation. PCR verification showed higher expression of ANK3 in the sarcoidosis compared to the IPF group, and higher expression of MMP7 and PPBP in patients with IPF. The GO analysis indicated that predominant biological processes associated with the differential mRNA gene expression of BALf cells were upregulation of neutrophils in IPF and lymphocytes in sarcoidosis. Conclusions: Analysis of BALf from sarcoidosis and IPF showed highly different mRNA profile of cells present in bronchoalveolar compartment and provided new data on the pathobiology of these ILDs. The changes of most important biological processes observed at the molecular level in BALf cells were associated with ribosome biogenesis and proteasome apparatus in sarcoidosis and neutrophilic dysfunction in IPF.

Project description:BackgroundSarcoidosis is a multisystem granulomatous disease of unknown origin with a variable and often unpredictable course and pattern of organ involvement. In this study we sought to identify specific bronchoalveolar lavage (BAL) cell gene expression patterns indicative of distinct disease phenotypic traits.MethodsRNA sequencing by Ion Torrent Proton was performed on BAL cells obtained from 215 well-characterised patients with pulmonary sarcoidosis enrolled in the multicentre Genomic Research in Alpha-1 Antitrypsin Deficiency and Sarcoidosis (GRADS) study. Weighted gene co-expression network analysis and nonparametric statistics were used to analyse genome-wide BAL transcriptome. Validation of results was performed using a microarray expression dataset of an independent sarcoidosis cohort (Freiburg, Germany; n=50).ResultsOur supervised analysis found associations between distinct transcriptional programmes and major pulmonary phenotypic manifestations of sarcoidosis including T-helper type 1 (Th1) and Th17 pathways associated with hilar lymphadenopathy, transforming growth factor-β1 (TGFB1) and mechanistic target of rapamycin (MTOR) signalling with parenchymal involvement, and interleukin (IL)-7 and IL-2 with airway involvement. Our unsupervised analysis revealed gene modules that uncovered four potential sarcoidosis endotypes including hilar lymphadenopathy with increased acute T-cell immune response; extraocular organ involvement with PI3K activation pathways; chronic and multiorgan disease with increased immune response pathways; and multiorgan involvement, with increased IL-1 and IL-18 immune and inflammatory responses. We validated the occurrence of these endotypes using gene expression, pulmonary function tests and cell differentials from Freiburg.ConclusionTaken together, our results identify BAL gene expression programmes that characterise major pulmonary sarcoidosis phenotypes and suggest the presence of distinct disease molecular endotypes.

Project description:IntroductionEquine asthma (EA) is a common disease of adult horses with chronic respiratory pathology and common neutrophilic airway inflammation. It presents with hyperreactivity to hay dust components such as molds, and underlying dysregulated T cell responses have been suggested. Thus far, T cells have been analysed in EA with conflicting results and the antigen reactivity of T cells has not been demonstrated. Serological and epidemiological data point to the relevance of Aspergillus fumigatus as an antigen source in EA. Here, we aimed to identify and characterise Aspergillus antigen-reactive T cells in EA.MethodsCryopreserved bronchoalveolar lavage cells (BALC) and peripheral blood mononuclear cells (PBMC) from healthy horses (HE, n=9) and those with mild-moderate (MEA, n=3) or severe asthma (SEA, n=8) were stimulated in vitro with the recombinant A. fumigatus antigens Asp f 1, or Asp f 7 combined with Asp f 8, to assess antigen reactivity, and with phorbol-12-myristat-13-acetate and ionomycin (P/i) to assess overall T cell reactivity. Stimulated cells were analysed by flow cytometry for CD4, CD8, IL-17, IL-4, and IFN-γ. Cytokine expression in all lymphocytes, and in CD4+ or CD8+ T cells, was quantified and compared between the groups. In BAL fluid (BALF), soluble cytokines and chemokines were quantified by bead-based assays.ResultsAntigen restimulation of BALC with Asp f 1 or Asp f 7/8 provoked higher frequencies of IL-17+ lymphocytes, CD4+IL-17+ Th17 cells, and CD4+IL-4+ Th2 cells in SEA than in HE, whereas MEA and HE were similar. Antigen stimulation of PBMC did not result in group differences. P/i stimulation of BALC resulted in increased IL-17+ lymphocyte and CD4+IL-17+ Th17 cell frequencies in MEA compared with HE but the limited number of horses with MEA must be considered. P/i-stimulated PBMC from MEA or SEA contained more IL-17+ lymphocytes compared with HE. Cytokines were hardly detected in BALF and similar between the groups but CCL2 and CCL5 concentrations were increased in BALF from SEA or MEA, respectively, compared with HE.ConclusionHorses with SEA have increased Aspergillus antigen-reactive Th17 cells in their airways, emphasising local T cell responses to this mold, which were quantified in EA for the first time here.

Project description:BackgroundSarcoidosis is an inflammatory disease that affects multiple organs. Cell analysis from bronchoalveolar lavage fluid (BALF) is a valuable tool in the diagnostic workup and differential diagnosis of sarcoidosis. Besides the expansion of lymphocyte expression-specific receptor segments (Vα2.3 and Vβ22) in some patients with certain HLA types, the relation between sarcoidosis susceptibility and BAL cell populations' quantitative levels is not well-understood.MethodsQuantitative levels defined by cell concentrations of BAL cells and CD4+/CD8+ ratio were evaluated together with genetic variants associated with sarcoidosis in 692 patients with extensive clinical data. Genetic variants associated with clinical phenotypes, Löfgren's syndrome (LS) and non-Löfgren's syndrome (non-LS), were examined separately. An association test via linear regression using an additive model adjusted for sex, age, and correlated cell type was applied. To infer the biological function of genetic associations, enrichment analysis of expression quantitative trait (eQTLs) across publicly available eQTL databases was conducted.ResultsMultiple genetic variants associated with sarcoidosis were significantly associated with quantitative levels of BAL cells. Specifically, LS genetic variants, mainly from the HLA locus, were associated with quantitative levels of BAL macrophages, lymphocytes, CD3+ cells, CD4+ cells, CD8+ cells, CD4+/CD8+ ratio, neutrophils, basophils, and eosinophils. Non-LS genetic variants were associated with quantitative levels of BAL macrophages, CD8+ cells, basophils, and eosinophils. eQTL enrichment revealed an influence of sarcoidosis-associated SNPs and regulation of gene expression in the lung, blood, and immune cells.ConclusionGenetic variants associated with sarcoidosis are likely to modulate quantitative levels of BAL cell types and may regulate gene expression in immune cell populations. Thus, the role of sarcoidosis-associated gene-variants may be to influence cellular phenotypes underlying the disease immunopathology.

Dataset Information

Bronchoalveolar lavage fluid IFN-?+ Th17 cells and regulatory T cells in pulmonary sarcoidosis.

Publications

Bronchoalveolar lavage fluid IFN-γ+ Th17 cells and regulatory T cells in pulmonary sarcoidosis.

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