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Quantitative parameters of lymphocyte nuclear morphology in bronchoalveolar lavage fluid as novel biomarkers for sarcoidosis.

ABSTRACT:

Background

Bronchoalveolar lavage (BAL) is one of the fundamental examinations for the differential diagnosis of interstitial lung diseases (ILDs), and lymphocytosis strongly indicates alternative diagnoses rather than idiopathic pulmonary fibrosis. However, the BALF lymphocytosis is observed in several ILDs. We considered that quantitative evaluation of the BALF lymphocyte nuclear morphology would be useful in the differential diagnosis of ILDs with increased BALF lymphocyte fraction.

Results

One hundred and twenty-one patients with ILDs having increased BALF lymphocyte fraction were recruited (68 in the development cohort and 53 in the validation cohort). In the development cohort, BALF lymphocyte nuclei in sarcoidosis patients showed significantly smaller areas, shorter perimeters, lower radius ratios, and increased roundness than those of other ILD patients (p < 0.001 for each). Next, the fractions of lymphocytes with small areas, short perimeters, low radius ratios, and increased roundness, which were determined based on receiver operating characteristic (ROC) analyses-based thresholds, were demonstrated to be higher in sarcoidosis patients than in the other ILD patients (p < 0.001 for each). Furthermore, when we combined size-representing parameters with shape-representing parameters, the fraction of lymphocytes with small and round nuclei showed approximately 0.90 of area under the ROC curve in discriminating sarcoidosis both in the development cohort and the validation cohort.

Conclusion

This study is the first to demonstrate the usefulness of quantitative parameters of BALF lymphocyte nuclear morphology as novel biomarkers for sarcoidosis.

SUBMITTER: Horimasu Y

PROVIDER: S-EPMC8254956 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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 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: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.