Genetic characterization of bronchoalveolar lavage fluid cells from patients with IPF and sarcoidosis
ABSTRACT: 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. Overall design: The microarray experiments were performed using 21 samples: 9 IPF and 12 sarcoidosis.
INSTRUMENT(S): [HuGene-2_1-st] Affymetrix Human Gene 2.1 ST Array [probe set (exon) version]
Project description:Background: New biomarkers are urgently needed to facilitate diagnosis in Interstitial Lung Diseases (ILD), thus reducing the need for invasive procedures, and to enable tailoring and monitoring of medical treatment. Methods: In this study we investigated if patients with idiopathic pulmonary fibrosis (IPF; n = 21), non-IPF ILDs (n = 57) and other lung diseases (chronic obstructive pulmonary disease (COPD) n = 24, lung cancer (LC) n = 16) as well as healthy subjects (n = 20) show relevant differences in exhaled NO (FeNO; Niox MINO), or in eicosanoid (PGE2, 8-isoprostane; enzyme-linked immunosorbent assay (ELISA)) levels as measured in exhaled breath condensates (EBC) and bronchoalveolar lavage fluids (BALF). Results: There was no significant difference in FeNO values between IPF, non-IPF ILDs and healthy subjects, although some individual patients showed highly elevated FeNO. On the basis of the FeNO signal, it was neither possible to differentiate between the kind of disease nor to detect exacerbations. In addition, there was no correlation between FeNO values and lung function. The investigation of the eicosanoids in EBCs was challenging (PGE2) or unreliable (8-isoprostane), but worked out well in BALF. A significant increase of free 8-isoprostane was observed in BALF, but not in EBCs, of patients with IPF, hypersensitivity pneumonitis (HP) and sarcoidosis, possibly indicating severity of oxidative stress. Conclusions: FeNO-measurements are not of diagnostic benefit in different ILDs including IPF. The same holds true for PGE2 and 8-isoprostane in EBC by ELISA.
Project description:Background:The role of the lectin pathway of complement in the pathogenesis of interstitial lung diseases (ILDs) is largely unknown. Pattern recognition receptors (PRR) of the lectin pathway are involved in the clearance of apoptotic cells either via activation of the complement system or as direct opsonins. As recent findings suggest a role of apoptosis in the development of pulmonary fibrosis, the influence of plasma lectins has lately been considered in various ILDs, but data on local concentrations in the lungs are lacking. This study investigated the role of mannose-binding lectin (MBL), ficolin-2 and ficolin-3 in ILD patients with a focus on idiopathic pulmonary fibrosis (IPF) and sarcoidosis. Methods:A case control study was conducted involving 80 patients with different forms of ILD as well as 40 control patients undergoing routine flexible bronchoscopy with bronchoalveolar lavage (BAL). Plasma and BAL fluid (BALF) levels of MBL, ficolin-2 and ficolin-3 as well as complement split products C4d and C5a (only in BALF) were measured by enzyme-linked immunosorbent assays. Eight single-nucleotide polymorphisms (SNPs) of MBL and ficolin-2 were determined by genotyping and tested for their association with ILDs. Results:We included 35, 35, 10, and 40 patients with sarcoidosis, idiopathic pulmonary fibrosis (IPF), other ILD, and a control group, respectively. BALF but not plasma levels of the three PRR were significantly elevated in sarcoidosis patients compared to a control group without ILD (MBL: median 66.8 vs. 24.6 ng/ml, p = 0.02, ficolin-2: 140 vs. 58.8 ng/ml, p = 0.01, ficolin-3: 2523 vs. 1180 ng/ml, p = 0.02), whereas the frequency of the investigated SNPs was similar. In line, complement split products were markedly elevated in BALF of sarcoidosis patients (C4d, median 97.4 vs. 0 ng/ml, p = 0.10; C5a, 23.9 vs. 9.1 ng/ml, p = 0.01). There was a weak positive correlation of BALF ficolin-3 with serum neopterin, a marker of sarcoidosis activity. In IPF patients, we observed numerically higher MBL plasma and BALF levels (plasma, median 1511 vs. 879 ng/ml, p = 0.44; BALF, 37.5 vs. 24.6 ng/ml, p = 0.7) as well as lower ficolin-2 plasma levels (plasma 1111 vs. 1647 ng/ml, p = 0.11). Ficolin-2 plasma levels were inversely correlated with the forced vital capacity (r = 0.55, p = 0.1). Conclusion:This is the first study to simultaneously assess systemic and local lectin pathway protein levels in ILD patients. Our data suggest an involvement of PRR of the lectin pathway in the pathogenesis of sarcoidosis given the significantly higher BALF levels compared to a control group. Additional analyses in a larger patient cohort are required to confirm or refute a potential effect of local and/or systemic ficolin-2 levels in IPF patients.
Project description:Although the pathogenesis of sarcoidosis is not fully understood, immunological characterization has elucidated highly polarized expression of the type 1 T helper cell response. Mucosal-associated invariant T (MAIT) cells are innate T cells that recognize bacterial riboflavin and rapidly produce cytokines such as interferon γ and tumor necrosis factor α. We prospectively evaluated the proportion of MAIT cells and the expression levels of cell surface markers in peripheral blood from 40 sarcoidosis patients and 28 healthy controls. MAIT cells in bronchoalveolar lavage fluid (BALF) were also examined in 12 sarcoidosis patients. In peripheral blood, the proportion of MAIT cells was lower (P = 0.0002), but the expression levels of CD69 and programmed death 1 on MAIT cells were higher in sarcoidosis patients than in healthy controls. Moreover, CD69 expression levels were significantly correlated with clinical biomarkers. Sarcoidosis patients with parenchymal infiltration in the lungs showed a significantly higher proportion and number of MAIT cells in BALF compared to patients without parenchymal infiltration. CD69 expression levels on MAIT cells in BALF were higher than levels in peripheral blood. The activation status of MAIT cells might reflect the disease activity of sarcoidosis. Therefore, it is a potential target for sarcoidosis treatment.
Project description:OBJECTIVES:Matrix metalloproteinase-8 (MMP-8) promotes lung fibrotic responses to bleomycin in mice. Although prior studies reported that MMP-8 levels are increased in plasma and bronchoalveolar lavage fluid (BALF) samples from IPF patients, neither the bioactive forms nor the cellular sources of MMP-8 in idiopathic pulmonary fibrosis (IPF) patients have been identified. It is not known whether MMP-8 expression is dys-regulated in IPF leukocytes or whether MMP-8 plasma levels correlate with IPF outcomes. Our goal was to address these knowledge gaps. METHODS:We measured MMP-8 levels and forms in blood and lung samples from IPF patients versus controls using ELISAs, western blotting, and qPCR, and assessed whether MMP-8 plasma levels in 73 IPF patients correlate with rate of lung function decline and mortality. We used immunostaining to localize MMP-8 expression in IPF lungs. We quantified MMP-8 levels and forms in blood leukocytes from IPF patients versus controls. RESULTS:IPF patients have increased BALF, whole lung, and plasma levels of soluble MMP-8 protein. Active MMP-8 is the main form elevated in IPF lungs. MMP-8 mRNA levels are increased in monocytes from IPF patients, but IPF patients and controls have similar levels of MMP-8 in PMNs. Surprisingly, macrophages and airway epithelial cells are the main cells expressing MMP-8 in IPF lungs. Plasma and BALF MMP-8 levels do not correlate with decline in lung function and/or mortality in IPF patients. CONCLUSION:Blood and lung MMP-8 levels are increased in IPF patients. Active MMP-8 is the main form elevated in IPF lungs. Surprisingly, blood monocytes, lung macrophages, and airway epithelial cells are the main cells in which MMP-8 is upregulated in IPF patients. Plasma and BALF MMP-8 levels are unlikely to serve as a prognostic biomarker for IPF patients. These results provide new information about the expression patterns of MMP-8 in IPF patients.
Project description:Pulmonary involvement occurs in up to 95% of sarcoidosis cases. In this pilot study, we examine lung compartment-specific protein expression to identify pathways linked to development and progression of pulmonary sarcoidosis. We characterized bronchoalveolar lavage (BAL) cells and fluid (BALF) proteins in recently diagnosed sarcoidosis cases. We identified 4,306 proteins in BAL cells, of which 272 proteins were differentially expressed in sarcoidosis compared to controls. These proteins map to novel pathways such as integrin-linked kinase and IL-8 signaling and previously implicated pathways in sarcoidosis, including phagosome maturation, clathrin-mediated endocytic signaling and redox balance. In the BALF, the differentially expressed proteins map to several pathways identified in the BAL cells. The differentially expressed BALF proteins also map to aryl hydrocarbon signaling, communication between innate and adaptive immune response, integrin, PTEN and phospholipase C signaling, serotonin and tryptophan metabolism, autophagy, and B cell receptor signaling. Additional pathways that were different between progressive and non-progressive sarcoidosis in the BALF included CD28 signaling and PFKFB4 signaling. Our studies demonstrate the power of contemporary proteomics to reveal novel mechanisms operational in sarcoidosis. Application of our workflows in well-phenotyped large cohorts maybe beneficial to identify biomarkers for diagnosis and prognosis and therapeutically tenable molecular mechanisms.
Project description:BACKGROUND: Idiopathic pulmonary fibrosis (IPF) and pulmonary sarcoidosis are typical interstitial lung diseases with unknown etiology that cause lethal lung damages. There are notable differences between these two pulmonary disorders, although they do share some similarities. Gene expression profiles have been reported independently, but differences on the transcriptional level between these two entities have not been investigated. METHODS/RESULTS: All expression data of lung tissue samples for IPF and sarcoidosis were from published datasets in the Gene Expression Omnibus (GEO) repository. After cross platform normalization, the merged sample data were grouped together and were subjected to statistical analysis for finding discriminate genes. Gene enrichments with their corresponding functions were analyzed by the online analysis engine "Database for Annotation, Visualization and Integrated Discovery" (DAVID) 6.7, and genes interactions and functional networks were further analyzed by STRING 9.0 and Cytoscape 3.0.0 Beta1. One hundred and thirty signature genes could potentially differentiate one disease state from another. Compared with normal lung tissue, tissue affected by IPF and sarcoidosis displayed similar signatures that concentrated on proliferation and differentiation. Distinctly expressed genes that could distinguish IPF from sarcoidosis are more enriched in processes of cilium biogenesis or degradation and regulating T cell activations. Key discriminative network modules involve aspects of bone morphogenetic protein receptor two (BMPR2) related and v-myb myeloblastosis viral oncogene (MYB) related proliferation. CONCLUSIONS: This study is the first attempt to examine the transcriptional regulation of IPF and sarcoidosis across different studies based on different working platforms. Groups of significant genes were found to clearly distinguish one condition from the other. While IPF and sarcoidosis share notable similarities in cell proliferation, differentiation and migration, remarkable differences between the diseases were found at the transcription level, suggesting that the two diseases are regulated by overlapping yet distinctive transcriptional networks.
Project description:BACKGROUND:Patients with idiopathic pulmonary fibrosis (IPF) often experience acute exacerbation (AE) after an episode of common cold. AIMS:To establish a mouse model of virus infection-induced AE-IPF and investigate the mechanism underlying the AE-IPF. METHODS:Herpes simplex virus 1 (HSV1) was inoculated intranasally to wild-type (WT) and IL-17A gene knockout (IL-17A-/- ) mice 21 days after intratracheal administration of bleomycin (BLM). RESULTS:HSV1 infection caused acute exacerbation in mice with BLM-induced fibrosis. Compared with the BLM+Saline mice, the mice with BLM+HSV1 showed significantly higher acute lung injury (ALI) score (P < 0.0001), lower survival rate (100% vs 21.4%, P < 0.0001), poorer lung function and higher inflammatory response representing by increased total inflammatory cells in bronchoalveolar lavage fluid (BALF) (P = 0.0323), increased proportion of Th17 cells in peripheral blood (P = 0.0004) and higher inflammatory factors in BALF. In addition, HSV1 infection increased the expression of endoplasmic reticulum stress (ERS)-related proteins in mice with BLM-induced fibrosis. The inhibition of ERS by tauroursodeoxycholic acid (TUDCA, an ERS inhibitor) significantly reduced the IL-17A levels in BALF (P = 0.0140) and TH17 cells in the peripheral blood (P = 0.0084) of mice with BLM+HSV1, suggesting that suppression of ERS may reduce TH17 response in mice with AE-IPF. Compared with WT mice with BLM+HSV1, IL-17A-/- mice with BLM+HSV1 had lower ALI score (P = 0.0119), higher survival rate (78.6% vs 21.4%, P = 0.004), improved lung function, and milder inflammatory response. CONCLUSIONS:HSV1 infection in addition to BLM-induced IPF can successfully establish AE-IPF in mice. IL-17A and ERS promote lung inflammation in AE-IPF development.
Project description:Background: Sarcoidosis goes into remission in two-thirds of patients with sarcoidosis, but about 20 % of patients develop pulmonary fibrosis. The mechanisms of pulmonary fibrosis in sarcoidosis and differences in pathogenesis between clinical stages are still unclear. Objectives: The aim of this study was investigating proteins associated with clinical stages by comparing bronchoalveolar lavage fluid (BALF) protein between stage I and stage IV using proteome analysis. Methods: Proteomic differences in BALF were compared between stage I and stage IV by examining BALF from 8 stage I patients and 5 stage IV patients by two-dimensional gel electrophoresis and mass spectrometry. Results: In individual comparisons of BALF samples, the levels of apolipoprotein (Apo) A-I fragment, fibrinogen ? chain, calcyphosine, complement C3, and surfactant protein A were significantly higher in stage I than in stage IV. In contrast, none of the proteins examined significantly higher in stage IV than in stage I. To confirm the results of Apo A-I in the BALF proteome, we performed enzyme-linked immunosorbent assay (ELISA) in a larger group. The concentration of BALF Apo A-I was significantly higher in stage I patients than in stage IV patients (0.70 [0.13-0.89] vs. 0.15 [0.08-0.21] ng/?g protein, p=0.003). Conclusion: The involvement of BALF Apo A-I in sarcoidosis may differ between stage I and stage IV. (Sarcoidosis Vasc Diffuse Lung Dis 2018; 35: 5-15).
Project description:An increase in the number of mononuclear phagocytes in lung biopsies from patients with idiopathic pulmonary fibrosis (IPF) worsens prognosis. Chemokines that recruit mononuclear phagocytes, such as CC chemokine ligand 2 (CCL2), are elevated in bronchoalveolar lavage (BAL) fluid (BALF) from patients with IPF. However, little attention is given to the role of the mononuclear phagocyte survival and recruitment factor, macrophage colony-stimulating factor (M-CSF), in pulmonary fibrosis.To investigate the role of mononuclear phagocytes and M-CSF in pulmonary fibrosis.Wild-type, M-CSF-/-, or CCL2-/- mice received intraperitoneal bleomycin. Lung inflammation and fibrosis were measured by immunohistochemistry, ELISA, collagen assay, BAL differentials, real-time polymerase chain reaction, and Western blot analysis. Human and mouse macrophages were stimulated with M-CSF for CCL2 expression. BALF from patients with IPF was examined for M-CSF and CCL2.M-CSF-/- and CCL2-/- mice had less lung fibrosis, mononuclear phagocyte recruitment, collagen deposition, and connective tissue growth factor (CTGF) expression after bleomycin administration than wild-type littermates. Human and mouse macrophages stimulated with M-CSF had increased CCL2 production, and intratracheal administration of M-CSF in mice induced CCL2 production in BALF. Finally, BALF from patients with IPF contained significantly more M-CSF and CCL2 than BALF from normal volunteers. Elevated levels of M-CSF were associated with elevated CCL2 in BALF and the diagnosis of IPF.These data suggest that M-CSF contributes to the pathogenesis of pulmonary fibrosis in mice and in patients with IPF through the involvement of mononuclear phagocytes and CCL2 production.
Project description:BACKGROUND:The endogenous secretory receptor for advanced glycation end products (esRAGE) is a soluble isoform produced by alternative splicing of the RAGE gene. The isoform has anti-inflammatory properties due to its inhibition of the RAGE/ligand interaction and is reduced in the lung tissue of patients with idiopathic pulmonary fibrosis (IPF). This study aimed to investigate the association of esRAGE serum and bronchoalveolar lavage fluid (BALF) levels with progression of IPF. METHODS:This study included 79 IPF patients and 90 healthy controls. IPF and control serum esRAGE levels were compared, and the correlation between serum and BALF esRAGE levels was analyzed in 57 IPF patient samples. We also investigated the relationship of esRAGE serum and BALF levels with prognoses and lung function parameters in patients with IPF. RESULTS:Serum esRAGE levels in IPF patients were significantly lower than those in healthy controls (162.0?±?102.4?ng/ml and 200.7?±?107.3?ng/ml, p?=?0.009), although the baseline characteristics of age and smoking history were not matched. Serum levels of esRAGE were correlated with BALF esRAGE levels (rs?=?0.317). The BALF esRAGE levels were also correlated with diffusion capacity for carbon monoxide (rs?=?0.406). A Kaplan-Meier curve analysis and univariate/multivariate Cox hazard proportion analysis revealed that lower levels of esRAGE in blood and BALF were significantly associated with poorer prognoses in patients with IPF. CONCLUSIONS:Decreased esRAGE levels in BALF and blood were associated with poor prognoses in patients with IPF. These results suggest that esRAGE could be related to the pathophysiology of IPF and serum esRAGE could be a potential prognostic marker of IPF.