RESUMO
RATIONALE: Recent data suggest that the localisation of airway epithelial cells in the distal lung in idiopathic pulmonary fibrosis (IPF) may drive pathology. We set out to discover whether chemokines expressed in these ectopic airway epithelial cells may contribute to the pathogenesis of IPF. METHODS: We analysed whole lung and single-cell transcriptomic data obtained from patients with IPF. In addition, we measured chemokine levels in blood, bronchoalveolar lavage (BAL) of IPF patients and air-liquid interface cultures. We employed ex vivo donor and IPF lung fibroblasts and an animal model of pulmonary fibrosis to test the effects of chemokine signalling on fibroblast function. RESULTS: By analysis of whole-lung transcriptomics, protein and BAL, we discovered that CXCL6 (a member of the interleukin-8 family) was increased in patients with IPF. Elevated CXCL6 levels in the BAL of two cohorts of patients with IPF were associated with poor survival (hazard ratio of death or progression 1.89, 95% CI 1.16-3.08; n=179, p=0.01). By immunostaining and single-cell RNA sequencing, CXCL6 was detected in secretory cells. Administration of mCXCL5 (LIX, murine CXCL6 homologue) to mice increased collagen synthesis with and without bleomycin. CXCL6 increased collagen I levels in donor and IPF fibroblasts 4.4-fold and 1.7-fold, respectively. Both silencing of and chemical inhibition of CXCR1/2 blocked the effects of CXCL6 on collagen, while overexpression of CXCR2 increased collagen I levels 4.5-fold in IPF fibroblasts. CONCLUSIONS: CXCL6 is expressed in ectopic airway epithelial cells. Elevated levels of CXCL6 are associated with IPF mortality. CXCL6-driven collagen synthesis represents a functional consequence of ectopic localisation of airway epithelial cells in IPF.
Assuntos
Fibrose Pulmonar Idiopática , Animais , Humanos , Camundongos , Bleomicina , Quimiocina CXCL6/metabolismo , Quimiocinas/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/genética , Pulmão/patologiaRESUMO
BACKGROUND: Pentraxin 3 (PTX3) influences innate immunity and inflammation, host defence, the complement cascade and angiogenesis. PTX3 expression in lung and blood of subjects with tobacco exposure, and its potential relationship with disease pattern and clinical outcome are poorly understood. METHODS: Using independent platforms and cohorts, we identified associations of PTX3 gene expression in lung tissue and plasma from current and former tobacco smokers (with and without chronic obstructive pulmonary disease, COPD) to disease phenotypes including quantitative CT determined emphysema, lung function, symptoms and survival. Two putative regulatory variants of the PTX3 gene were examined for association with COPD manifestations. The relationship between plasma PTX3 and hyaluronic acid levels was further examined. RESULTS: PTX3 gene expression in lung tissue was directly correlated with emphysema severity (p<0.0001). Circulating levels of PTX3 were inversely correlated with FEV1 (p=0.006), and positively associated with emphysema severity (p=0.004) and mortality (p=0.008). Two PTX3 gene regulatory variants were associated with a lower risk for emphysema and expiratory airflow obstruction, and plasma levels of PTX3 and hyaluronic acid were related. CONCLUSIONS: These data show strong and overlapping associations of lung and blood PTX3 levels, and PTX3 regulatory gene variants, with the severity of airflow obstruction, emphysema and mortality among smokers. These findings have potential implications regarding the pathogenesis of smoking-related lung diseases and warrant further exploration for the use of PTX3 as a predictive biomarker.
Assuntos
Proteína C-Reativa/metabolismo , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/mortalidade , Componente Amiloide P Sérico/metabolismo , Fumantes , Adulto , Idoso , Biomarcadores/metabolismo , Proteína C-Reativa/genética , Feminino , Expressão Gênica , Humanos , Ácido Hialurônico/metabolismo , Masculino , Pessoa de Meia-Idade , Fenótipo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Enfisema Pulmonar/fisiopatologia , Testes de Função Respiratória , Componente Amiloide P Sérico/genética , Taxa de Sobrevida , Tomografia Computadorizada por Raios XRESUMO
Rationale: Gene expression of BAL cells, which samples the cellular milieu within the lower respiratory tract, has not been well studied in severe asthma.Objectives: To identify new biomolecular mechanisms underlying severe asthma by an unbiased, detailed interrogation of global gene expression.Methods: BAL cell expression was profiled in 154 asthma and control subjects. Of these participants, 100 had accompanying airway epithelial cell gene expression. BAL cell expression profiles were related to participant (age, sex, race, and medication) and sample traits (cell proportions), and then severity-related gene expression determined by correlating transcripts and coexpression networks to lung function, emergency department visits or hospitalizations in the last year, medication use, and quality-of-life scores.Measurements and Main Results: Age, sex, race, cell proportions, and medications strongly influenced BAL cell gene expression, but leading severity-related genes could be determined by carefully identifying and accounting for these influences. A BAL cell expression network enriched for cAMP signaling components most differentiated subjects with severe asthma from other subjects. Subsequently, an in vitro cellular model showed this phenomenon was likely caused by a robust upregulation in cAMP-related expression in nonsevere and ß-agonist-naive subjects given a ß-agonist before cell collection. Interestingly, ELISAs performed on BAL lysates showed protein levels may partly disagree with expression changes.Conclusions: Gene expression in BAL cells is influenced by factors seldomly considered. Notably, ß-agonist exposure likely had a strong and immediate impact on cellular gene expression, which may not translate to important disease mechanisms or necessarily match protein levels. Leading severity-related genes were discovered in an unbiased, system-wide analysis, revealing new targets that map to asthma susceptibility loci.
Assuntos
Asma/genética , Líquido da Lavagem Broncoalveolar/citologia , Expressão Gênica/genética , Agonistas Adrenérgicos beta/farmacologia , Adulto , Asma/metabolismo , Estudos de Casos e Controles , AMP Cíclico/metabolismo , Eosinófilos/metabolismo , Células Epiteliais/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Técnicas In Vitro , Linfócitos/metabolismo , Macrófagos Alveolares/metabolismo , Masculino , Neutrófilos/metabolismo , Análise de Sequência de RNA , Índice de Gravidade de Doença , Transdução de Sinais/genética , Células THP-1/metabolismoRESUMO
Idiopathic pulmonary fibrosis (IPF) is a disease characterized by the accumulation of apoptosis-resistant fibroblasts in the lung. We have previously shown that high expression of the transcription factor Twist1 may explain this prosurvival phenotype in vitro. However, this observation has never been tested in vivo. We found that loss of Twist1 in COL1A2+ cells led to increased fibrosis characterized by very significant accumulation of T cells and bone marrow-derived matrix-producing cells. We found that Twist1-null cells expressed high levels of the T cell chemoattractant CXCL12. In vitro, we found that the loss of Twist1 in IPF lung fibroblasts increased expression of CXCL12 downstream of increased expression of the noncanonical NF-κB transcription factor RelB. Finally, blockade of CXCL12 with AMD3100 attenuated the exaggerated fibrosis observed in Twist1-null mice. Transcriptomic analysis of 134 IPF patients revealed that low expression of Twist1 was characterized by enrichment of T cell pathways. In conclusion, loss of Twist1 in collagen-producing cells led to increased bleomycin-induced pulmonary fibrosis, which is mediated by increased expression of CXCL12. Twist1 expression is associated with dysregulation of T cells in IPF patients. Twist1 may shape the IPF phenotype and regulate inflammation in fibrotic lung injury.
Assuntos
Quimiocina CXCL12/metabolismo , Fibroblastos/fisiologia , Fibrose Pulmonar Idiopática/imunologia , Pulmão/patologia , Células-Tronco Mesenquimais/patologia , Linfócitos T/imunologia , Proteína 1 Relacionada a Twist/metabolismo , Idoso , Animais , Bleomicina , Células Cultivadas , Quimiocina CXCL12/genética , Colágeno Tipo I/metabolismo , Feminino , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Proteína 1 Relacionada a Twist/genética , Regulação para CimaRESUMO
Inadequate DNA repair is implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, the mechanisms that underlie inadequate DNA repair in COPD are poorly understood. We applied an integrative genomic approach to identify DNA repair genes and pathways associated with COPD severity.We measured the transcriptomic changes of 419 genes involved in DNA repair and DNA damage tolerance that occur with severe COPD in three independent cohorts (n=1129). Differentially expressed genes were confirmed with RNA sequencing and used for patient clustering. Clinical and genome-wide transcriptomic differences were assessed following cluster identification. We complemented this analysis by performing gene set enrichment analysis, Z-score and weighted gene correlation network analysis to identify transcriptomic patterns of DNA repair pathways associated with clinical measurements of COPD severity.We found 15 genes involved in DNA repair and DNA damage tolerance to be differentially expressed in severe COPD. K-means clustering of COPD cases based on this 15-gene signature identified three patient clusters with significant differences in clinical characteristics and global transcriptomic profiles. Increasing COPD severity was associated with downregulation of the nucleotide excision repair pathway.Systematic analysis of the lung tissue transcriptome of individuals with severe COPD identified DNA repair responses associated with disease severity that may underlie COPD pathogenesis.
Assuntos
Reparo do DNA/genética , Pulmão/patologia , Doença Pulmonar Obstrutiva Crônica/genética , Transcriptoma , Idoso , Dano ao DNA , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
RATIONALE: Severe asthma (SA) is a heterogeneous disease with multiple molecular mechanisms. Gene expression studies of bronchial epithelial cells in individuals with asthma have provided biological insight and underscored possible mechanistic differences between individuals. OBJECTIVES: Identify networks of genes reflective of underlying biological processes that define SA. METHODS: Airway epithelial cell gene expression from 155 subjects with asthma and healthy control subjects in the Severe Asthma Research Program was analyzed by weighted gene coexpression network analysis to identify gene networks and profiles associated with SA and its specific characteristics (i.e., pulmonary function tests, quality of life scores, urgent healthcare use, and steroid use), which potentially identified underlying biological processes. A linear model analysis confirmed these findings while adjusting for potential confounders. MEASUREMENTS AND MAIN RESULTS: Weighted gene coexpression network analysis constructed 64 gene network modules, including modules corresponding to T1 and T2 inflammation, neuronal function, cilia, epithelial growth, and repair mechanisms. Although no network selectively identified SA, genes in modules linked to epithelial growth and repair and neuronal function were markedly decreased in SA. Several hub genes of the epithelial growth and repair module were found located at the 17q12-21 locus, near a well-known asthma susceptibility locus. T2 genes increased with severity in those treated with corticosteroids but were also elevated in untreated, mild-to-moderate disease compared with healthy control subjects. T1 inflammation, especially when associated with increased T2 gene expression, was elevated in a subgroup of younger patients with SA. CONCLUSIONS: In this hypothesis-generating analysis, gene expression networks in relation to asthma severity provided potentially new insight into biological mechanisms associated with the development of SA and its phenotypes.
Assuntos
Asma/genética , Asma/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Adulto , Asma/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Índice de Gravidade de DoençaRESUMO
Small RNA sequencing can be used to gain an unprecedented amount of detail into the microRNA transcriptome. The relatively high cost and low throughput of sequencing bases technologies can potentially be offset by the use of multiplexing. However, multiplexing involves a trade-off between increased number of sequenced samples and reduced number of reads per sample (i.e., lower depth of coverage). To assess the effect of different sequencing depths owing to multiplexing on microRNA differential expression and detection, we sequenced the small RNA of lung tissue samples collected in a clinical setting by multiplexing one, three, six, nine, or 12 samples per lane using the Illumina HiSeq 2000. As expected, the numbers of reads obtained per sample decreased as the number of samples in a multiplex increased. Furthermore, after normalization, replicate samples included in distinct multiplexes were highly correlated (R > 0.97). When detecting differential microRNA expression between groups of samples, microRNAs with average expression >1 reads per million (RPM) had reproducible fold change estimates (signal to noise) independent of the degree of multiplexing. The number of microRNAs detected was strongly correlated with the log2 number of reads aligning to microRNA loci (R = 0.96). However, most additional microRNAs detected in samples with greater sequencing depth were in the range of expression which had lower fold change reproducibility. These findings elucidate the trade-off between increasing the number of samples in a multiplex with decreasing sequencing depth and will aid in the design of large-scale clinical studies exploring microRNA expression and its role in disease.
Assuntos
MicroRNAs/metabolismo , Perfilação da Expressão Gênica , Humanos , Pulmão/metabolismo , MicroRNAs/genética , Análise de Sequência de RNA , TranscriptomaRESUMO
RATIONALE: Relaxin is a hormone that has been considered as a potential therapy for patients with fibrotic diseases. OBJECTIVES: To gauge the potential efficacy of relaxin-based therapies in idiopathic pulmonary fibrosis (IPF), we studied gene expression for relaxin/insulin-like family peptide receptor 1 (RXFP1) in IPF lungs and controls. METHODS: We analyzed gene expression data obtained from the Lung Tissue Research Consortium and correlated RXFP1 gene expression data with cross-sectional clinical and demographic data. We also employed ex vivo donor and IPF lung fibroblasts to test RXFP1 expression in vitro. We tested CGEN25009, a relaxin-like peptide, in lung fibroblasts and in bleomycin injury. MEASUREMENTS AND MAIN RESULTS: We found that RXFP1 is significantly decreased in IPF. In patients with IPF, the magnitude of RXFP1 gene expression correlated directly with diffusing capacity of the lung for carbon monoxide (P < 0.0001). Significantly less RXFP1 was detected in vitro in IPF fibroblasts than in donor controls. Transforming growth factor-ß decreased RXFP1 in both donor and IPF lung fibroblasts. CGEN25009 was effective at decreasing bleomycin-induced, acid-soluble collagen deposition in vivo. The relaxin-like actions of CGEN25009 were abrogated by RXFP1 silencing in vitro, and, in comparison with donor lung fibroblasts, IPF lung fibroblasts exhibited decreased sensitivity to the relaxin-like effects of CGEN25009. CONCLUSIONS: IPF is characterized by the loss of RXFP1 expression. RXFP1 expression is directly associated with pulmonary function in patients with IPF. The relaxin-like effects of CGEN25009 in vitro are dependent on expression of RXFP1. Our data suggest that patients with IPF with the highest RXFP1 expression would be predicted to be most sensitive to relaxin-based therapies.
Assuntos
Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/terapia , Receptores Acoplados a Proteínas G/genética , Receptores de Peptídeos/genética , Relaxina/uso terapêutico , Estudos Transversais , Feminino , Expressão Gênica/genética , Humanos , Fibrose Pulmonar Idiopática/fisiopatologia , Immunoblotting , Pulmão/fisiopatologia , Masculino , Análise em Microsséries , Pessoa de Meia-IdadeRESUMO
RATIONALE: Despite shared environmental exposures, idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease are usually studied in isolation, and the presence of shared molecular mechanisms is unknown. OBJECTIVES: We applied an integrative genomic approach to identify convergent transcriptomic pathways in emphysema and IPF. METHODS: We defined the transcriptional repertoire of chronic obstructive pulmonary disease, IPF, or normal histology lungs using RNA-seq (n = 87). MEASUREMENTS AND MAIN RESULTS: Genes increased in both emphysema and IPF relative to control were enriched for the p53/hypoxia pathway, a finding confirmed in an independent cohort using both gene expression arrays and the nCounter Analysis System (n = 193). Immunohistochemistry confirmed overexpression of HIF1A, MDM2, and NFKBIB members of this pathway in tissues from patients with emphysema or IPF. Using reads aligned across splice junctions, we determined that alternative splicing of p53/hypoxia pathway-associated molecules NUMB and PDGFA occurred more frequently in IPF or emphysema compared with control and validated these findings by quantitative polymerase chain reaction and the nCounter Analysis System on an independent sample set (n = 193). Finally, by integrating parallel microRNA and mRNA-Seq data on the same samples, we identified MIR96 as a key novel regulatory hub in the p53/hypoxia gene-expression network and confirmed that modulation of MIR96 in vitro recapitulates the disease-associated gene-expression network. CONCLUSIONS: Our results suggest convergent transcriptional regulatory hubs in diseases as varied phenotypically as chronic obstructive pulmonary disease and IPF and suggest that these hubs may represent shared key responses of the lung to environmental stresses.
Assuntos
Redes Reguladoras de Genes/genética , Fibrose Pulmonar Idiopática/genética , Doença Pulmonar Obstrutiva Crônica/genética , Adulto , Enfisema/genética , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas I-kappa B/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismoRESUMO
As miRNAs are associated with normal cellular processes, deregulation of miRNAs is thought to play a causative role in many complex diseases. Nevertheless, the precise contribution of miRNAs in fibrotic lung diseases, especially the idiopathic form (IPF), remains poorly understood. Given the poor response rate of IPF patients to current therapy, new insights into the pathogenic mechanisms controlling lung fibroblasts activation, the key cell type driving the fibrogenic process, are essential to develop new therapeutic strategies for this devastating disease. To identify miRNAs with potential roles in lung fibrogenesis, we performed a genome-wide assessment of miRNA expression in lungs from two different mouse strains known for their distinct susceptibility to develop lung fibrosis after bleomycin exposure. This led to the identification of miR-199a-5p as the best miRNA candidate associated with bleomycin response. Importantly, miR-199a-5p pulmonary expression was also significantly increased in IPF patients (94 IPF versus 83 controls). In particular, levels of miR-199a-5p were selectively increased in myofibroblasts from injured mouse lungs and fibroblastic foci, a histologic feature associated with IPF. Therefore, miR-199a-5p profibrotic effects were further investigated in cultured lung fibroblasts: miR-199a-5p expression was induced upon TGFß exposure, and ectopic expression of miR-199a-5p was sufficient to promote the pathogenic activation of pulmonary fibroblasts including proliferation, migration, invasion, and differentiation into myofibroblasts. In addition, we demonstrated that miR-199a-5p is a key effector of TGFß signaling in lung fibroblasts by regulating CAV1, a critical mediator of pulmonary fibrosis. Remarkably, aberrant expression of miR-199a-5p was also found in unilateral ureteral obstruction mouse model of kidney fibrosis, as well as in both bile duct ligation and CCl4-induced mouse models of liver fibrosis, suggesting that dysregulation of miR-199a-5p represents a general mechanism contributing to the fibrotic process. MiR-199a-5p thus behaves as a major regulator of tissue fibrosis with therapeutic potency to treat fibroproliferative diseases.
Assuntos
Caveolina 1 , Fibrose Pulmonar Idiopática , Pulmão , MicroRNAs , Fator de Crescimento Transformador beta , Animais , Bleomicina/toxicidade , Caveolina 1/genética , Caveolina 1/metabolismo , Diferenciação Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/fisiopatologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica , Fator de Crescimento Transformador beta/administração & dosagem , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
The bleomycin-induced rodent lung fibrosis model is commonly used to study mechanisms of lung fibrosis and to test potential therapeutic interventions, despite the well recognized dissimilarities to human idiopathic pulmonary fibrosis (IPF). Therefore, in this study, we sought to identify genomic commonalities between the gene expression profiles from 100 IPF lungs and 108 control lungs that were obtained from the Lung Tissue Research Consortium, and rat lungs harvested at Days 3, 7, 14, 21, 28, 42, and 56 after bleomycin instillation. Surprisingly, the highest gene expression similarity between bleomycin-treated rat and IPF lungs was observed at Day 7. At this point of maximal rat-human commonality, we identified a novel set of 12 disease-relevant translational gene markers (C6, CTHRC1, CTSE, FHL2, GAL, GREM1, LCN2, MMP7, NELL1, PCSK1, PLA2G2A, and SLC2A5) that was able to separate almost all patients with IPF from control subjects in our cohort and in two additional IPF/control cohorts (GSE10667 and GSE24206). Furthermore, in combination with diffusing capacity of carbon monoxide measurements, four members of the translational gene marker set contributed to stratify patients with IPF according to disease severity. Significantly, pirfenidone attenuated the expression change of one (CTHRC1) translational gene marker in the bleomycin-induced lung fibrosis model, in transforming growth factor-ß1-treated primary human lung fibroblasts and transforming growth factor-ß1-treated human epithelial A549 cells. Our results suggest that a strategy focused on rodent model-human disease commonalities may identify genes that could be used to predict the pharmacological impact of therapeutic interventions, and thus facilitate the development of novel treatments for this devastating lung disease.
Assuntos
Células Epiteliais/patologia , Fibroblastos/metabolismo , Fibrose Pulmonar Idiopática/genética , Pulmão/patologia , Transdução de Sinais/genética , Animais , Bleomicina/metabolismo , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Expressão Gênica/fisiologia , Genômica , Humanos , Pulmão/metabolismo , Biossíntese de Proteínas , Ratos Sprague-DawleyRESUMO
BACKGROUND: The increased multi-omics information on carefully phenotyped patients in studies of complex diseases requires novel methods for data integration. Unlike continuous intensity measurements from most omics data sets, phenome data contain clinical variables that are binary, ordinal and categorical. RESULTS: In this paper we introduce an integrative phenotyping framework (iPF) for disease subtype discovery. A feature topology plot was developed for effective dimension reduction and visualization of multi-omics data. The approach is free of model assumption and robust to data noises or missingness. We developed a workflow to integrate homogeneous patient clustering from different omics data in an agglomerative manner and then visualized heterogeneous clustering of pairwise omics sources. We applied the framework to two batches of lung samples obtained from patients diagnosed with chronic obstructive lung disease (COPD) or interstitial lung disease (ILD) with well-characterized clinical (phenomic) data, mRNA and microRNA expression profiles. Application of iPF to the first training batch identified clusters of patients consisting of homogenous disease phenotypes as well as clusters with intermediate disease characteristics. Analysis of the second batch revealed a similar data structure, confirming the presence of intermediate clusters. Genes in the intermediate clusters were enriched with inflammatory and immune functional annotations, suggesting that they represent mechanistically distinct disease subphenotypes that may response to immunomodulatory therapies. The iPF software package and all source codes are publicly available. CONCLUSIONS: Identification of subclusters with distinct clinical and biomolecular characteristics suggests that integration of phenomic and other omics information could lead to identification of novel mechanism-based disease sub-phenotypes.
Assuntos
Biologia Computacional/métodos , Fenótipo , Algoritmos , Análise por Conglomerados , Simulação por Computador , Conjuntos de Dados como Assunto , Análise Discriminante , Genômica/métodos , Humanos , Pneumopatias/etiologia , Pneumopatias/metabolismo , Anotação de Sequência Molecular , Fluxo de TrabalhoRESUMO
RATIONALE: C-X-C motif chemokine 13 (CXCL13) mediates B-cell trafficking and is increased, proportionately to disease activity, in many antibody-mediated syndromes. Dysregulated B cells have recently been implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis. OBJECTIVES: To determine if CXCL13 is associated with IPF progression. METHODS: CXCL13 was measured in lungs by DNA microarray and immunohistochemistry, and in plasma by ELISA. MEASUREMENTS AND MAIN RESULTS: CXCL13 mRNA was threefold and eightfold greater in IPF lungs (n = 92) compared with chronic obstructive pulmonary disease (COPD) (n = 191) and normal (n = 108) specimens, respectively (P < 0.0001). IPF lungs also showed increased CXCL13 staining. Plasma CXCL13 concentrations (pg/ml) were greater in 95 patients with IPF (94 ± 8) than in 128 subjects with COPD (53 ± 9) and 57 normal subjects (35 ± 3) (P < 0.0001). Circulating CXCL13 levels were highest in patients with IPF with pulmonary artery hypertension (P = 0.01) or acute exacerbations (P = 0.002). Six-month survival of patients with IPF in the highest quartile of plasma CXCL13 was 65 ± 10% versus 93 ± 10% in the others (hazard ratio, 5.5; 95% confidence interval, 1.8-16.9; P = 0.0008). CXCL13 increases by more than 50% in IPF serial assays, irrespective of initial values, also presaged respiratory failure (hazard ratio, 7.2; 95% confidence interval, 1.3-40.0; P = 0.008). In contrast, CXCL13 clinical associations in subjects with COPD were limited to modest correlations with FEV1 (P = 0.05) and progression of radiographic emphysema (P = 0.05). CONCLUSIONS: CXCL13 is increased and is a prognostic biomarker in patients with IPF, and more so than in patients with COPD. This contrast indicates CXCL13 overexpressions are intrinsic to IPF, rather than an epiphenomenon of lung injury. The present data implicate CXCL13 and B cells in IPF pathogenesis, and support considerations for trials of specific B-cell-targeted therapies in patients with this intractable disease.
Assuntos
Quimiocina CXCL13/análise , Fibrose Pulmonar Idiopática/sangue , Fibrose Pulmonar Idiopática/diagnóstico , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Estudos de Casos e Controles , Quimiocina CXCL13/sangue , Quimiocina CXCL13/genética , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/mortalidade , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Valor Preditivo dos Testes , Prognóstico , Doença Pulmonar Obstrutiva Crônica/sangue , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Fatores de Risco , Sensibilidade e Especificidade , Índice de Gravidade de DoençaRESUMO
RATIONALE: Although asthma is recognized as a heterogeneous disease associated with clinical phenotypes, the molecular basis of these phenotypes remains poorly understood. Although genomic studies have successfully broadened our understanding in diseases such as cancer, they have not been widely used in asthma studies. OBJECTIVES: To link gene expression patterns to clinical asthma phenotypes. METHODS: We used a microarray platform to analyze bronchial airway epithelial cell gene expression in relation to the asthma biomarker fractional exhaled nitric oxide (FeNO) in 155 subjects with asthma and healthy control subjects from the Severe Asthma Research Program (SARP). MEASUREMENTS AND MAIN RESULTS: We first identified a diverse set of 549 genes whose expression correlated with FeNO. We used k-means to cluster the patient samples according to the expression of these genes, identifying five asthma clusters/phenotypes with distinct clinical, physiological, cellular, and gene transcription characteristics-termed "subject clusters" (SCs). To then investigate differences in gene expression between SCs, a total of 1,384 genes were identified that highly differentiated the SCs at an unadjusted P value < 10(-6). Hierarchical clustering of these 1,384 genes identified nine gene clusters or "biclusters," whose coexpression suggested biological characteristics unique to each SC. Although genes related to type 2 inflammation were present, novel pathways, including those related to neuronal function, WNT pathways, and actin cytoskeleton, were noted. CONCLUSIONS: These findings show that bronchial epithelial cell gene expression, as related to the asthma biomarker FeNO, can identify distinct asthma phenotypes, while also suggesting the presence of underlying novel gene pathways relevant to these phenotypes.
Assuntos
Asma/genética , Expressão Gênica/genética , Óxido Nítrico/metabolismo , Adulto , Asma/metabolismo , Biomarcadores , Brônquios/citologia , Brônquios/metabolismo , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Família Multigênica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
RATIONALE: Idiopathic pulmonary fibrosis (IPF) is an untreatable and often fatal lung disease that is increasing in prevalence and is caused by complex interactions between genetic and environmental factors. Epigenetic mechanisms control gene expression and are likely to regulate the IPF transcriptome. OBJECTIVES: To identify methylation marks that modify gene expression in IPF lung. METHODS: We assessed DNA methylation (comprehensive high-throughput arrays for relative methylation arrays [CHARM]) and gene expression (Agilent gene expression arrays) in 94 patients with IPF and 67 control subjects, and performed integrative genomic analyses to define methylation-gene expression relationships in IPF lung. We validated methylation changes by a targeted analysis (Epityper), and performed functional validation of one of the genes identified by our analysis. MEASUREMENTS AND MAIN RESULTS: We identified 2,130 differentially methylated regions (DMRs; <5% false discovery rate), of which 738 are associated with significant changes in gene expression and enriched for expected inverse relationship between methylation and expression (P < 2.2 × 10(-16)). We validated 13/15 DMRs by targeted analysis of methylation. Methylation-expression quantitative trait loci (methyl-eQTL) identified methylation marks that control cis and trans gene expression, with an enrichment for cis relationships (P < 2.2 × 10(-16)). We found five trans methyl-eQTLs where a methylation change at a single DMR is associated with transcriptional changes in a substantial number of genes; four of these DMRs are near transcription factors (castor zinc finger 1 [CASZ1], FOXC1, MXD4, and ZDHHC4). We studied the in vitro effects of change in CASZ1 expression and validated its role in regulation of target genes in the methyl-eQTL. CONCLUSIONS: These results suggest that DNA methylation may be involved in the pathogenesis of IPF.
Assuntos
Metilação de DNA/genética , Epigênese Genética/fisiologia , Fibrose Pulmonar Idiopática/genética , Locos de Características Quantitativas/genética , Transcriptoma/genética , Corticosteroides/uso terapêutico , Estudos de Casos e Controles , Feminino , Expressão Gênica , Marcadores Genéticos , Humanos , Imunossupressores/uso terapêutico , Masculino , Pessoa de Meia-Idade , Fumar/epidemiologiaRESUMO
Chronic obstructive pulmonary disease (COPD) is a complex disease, the pathogenesis of which remains incompletely understood. Colonization with Pneumocystis jirovecii may play a role in COPD pathogenesis; however, the mechanisms by which such colonization contributes to COPD are unknown. The objective of this study was to determine lung gene expression profiles associated with Pneumocystis colonization in patients with COPD to identify potential key pathways involved in disease pathogenesis. Using COPD lung tissue samples made available through the Lung Tissue Research Consortium (LTRC), Pneumocystis colonization status was determined by nested PCR. Microarray gene expression profiles were performed for each sample and the profiles of colonized and non-colonized samples compared. Overall, 18 participants (8.5%) were Pneumocystis-colonized. Pneumocystis colonization was associated with fold increase in expression of four closely related genes: INF-γ and the three chemokine ligands CXCL9, CXCL10, and CXCL11. These ligands are chemoattractants for the common cognate receptor CXCR3, which is predominantly expressed on activated Th1 T-lymphocytes. Although these ligand-receptor pairs have previously been implicated in COPD pathogenesis, few initiators of ligand expression and subsequent lymphocyte trafficking have been identified: our findings implicate Pneumocystis as a potential trigger. The finding of upregulation of these inflammatory genes in the setting of Pneumocystis colonization sheds light on infectious-immune relationships in COPD.
Assuntos
Quimiocinas CXC/genética , Pulmão/imunologia , Pneumocystis carinii/crescimento & desenvolvimento , Pneumonia por Pneumocystis/microbiologia , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/microbiologia , Células Th1/imunologia , Adulto , Idoso , Quimiocinas CXC/imunologia , Feminino , Humanos , Pulmão/microbiologia , Masculino , Pessoa de Meia-Idade , Pneumonia por Pneumocystis/genética , Pneumonia por Pneumocystis/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Regulação para CimaRESUMO
Idiopathic pulmonary fibrosis (IPF) is a progressive and typically fatal lung disease. To gain insight into the pathogenesis of IPF, we reanalyzed our previously published gene expression data profiling IPF lungs. Cytokine receptor-like factor 1 (CRLF1) was among the most highly up-regulated genes in IPF lungs, compared with normal controls. The protein product (CLF-1) and its partner, cardiotrophin-like cytokine (CLC), function as members of the interleukin 6 (IL-6) family of cytokines. Because of earlier work implicating IL-6 family members in IPF pathogenesis, we tested whether CLF-1 expression contributes to inflammation in experimental pulmonary fibrosis. In IPF, we detected CLF-1 expression in both type II alveolar epithelial cells and macrophages. We found that the receptor for CLF-1/CLC signaling, ciliary neurotrophic factor receptor (CNTFR), was expressed only in type II alveolar epithelial cells. Administration of CLF-1/CLC to both uninjured and bleomycin-injured mice led to the pulmonary accumulation of CD4(+) T cells. We also found that CLF-1/CLC administration increased inflammation but decreased pulmonary fibrosis. CLF-1/CLC leads to significantly enriched expression of T-cell-derived chemokines and cytokines, including the antifibrotic cytokine interferon-γ. We propose that, in IPF, CLF-1 is a selective stimulus of type II alveolar epithelial cells and may potentially drive an antifibrotic response by augmenting both T-helper-1-driven and T-regulatory-cell-driven inflammatory responses in the lung.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Fibrose Pulmonar Idiopática/metabolismo , Receptores de Citocinas/biossíntese , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Lesão Pulmonar Aguda/prevenção & controle , Animais , Bleomicina , Subunidade alfa do Receptor do Fator Neutrófico Ciliar/metabolismo , Colágeno/metabolismo , Interações Medicamentosas , Células Epiteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Humanos , Fibrose Pulmonar Idiopática/genética , Fibrose Pulmonar Idiopática/imunologia , Macrófagos Alveolares/metabolismo , Masculino , Camundongos , Alvéolos Pulmonares/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de Citocinas/genética , Receptores de Citocinas/imunologia , Receptores de Citocinas/uso terapêutico , Proteínas Recombinantes/uso terapêutico , Proteínas Recombinantes/toxicidade , Regulação para Cima/fisiologiaRESUMO
OBJECTIVE: To investigate the collapsibility of the lung and individual lobes in patients with COPD during inspiration/expiration and assess the association of whole lung and lobar volume changes with pulmonary function tests (PFTs) and disease severity. METHODS: PFT measures used were RV/TLC%, FEV1% predicted, FVC, FEV1/FVC%, DLco% predicted and GOLD category. A total of 360 paired inspiratory and expiratory CT examinations acquired in 180 subjects were analysed. Automated computerised algorithms were used to compute individual lobe and total lung volumes. Lung volume collapsibility was assessed quantitatively using the simple difference between CT computed inspiration (I) and expiration (E) volumes (I-E), and a relative measure of volume changes, (I-E)/I. RESULTS: Mean absolute collapsibility (I-E) decreased in all lung lobes with increasing disease severity defined by GOLD classification. Relative collapsibility (I-E)/I showed a similar trend. Upper lobes had lower volume collapsibility across all GOLD categories and lower lobes collectively had the largest volume collapsibility. Whole lung and left lower lobe collapsibility measures tended to have the highest correlations with PFT measures. Collapsibility of lung lobes and whole lung was also negatively correlated with the degree of air trapping between expiration and inspiration, as measured by mean lung density. All measured associations were statistically significant (P < 0.01). CONCLUSION: Severity of COPD appears associated with increased collapsibility in the upper lobes, but change (decline) in collapsibility is faster in the lower lobes. KEY POINTS: ⢠Inspiratory and expiratory computed tomography allows assessment of lung collapsibility ⢠Lobe volume collapsibility is significantly correlated with measures of lung function. ⢠As COPD severity increases, collapsibility of individual lung lobes decreases. ⢠Upper lobes exhibit more severe disease, while lower lobes decline faster.
Assuntos
Pulmão/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Doença Pulmonar Obstrutiva Crônica/diagnóstico , Idoso , Algoritmos , Feminino , Humanos , Pulmão/diagnóstico por imagem , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Interpretação de Imagem Radiográfica Assistida por Computador , Testes de Função Respiratória , Tomografia Computadorizada por Raios X/métodosRESUMO
OBJECTIVES: To determine the optimal threshold by quantitatively assessing the extent of emphysema at the level of the entire lung and at the level of individual lobes using a large, diverse dataset of computed tomography (CT) examinations. METHODS: This study comprises 573 chest CT examinations acquired from subjects with different levels of airway obstruction (222 none, 83 mild, 141 moderate, 63 severe and 64 very severe). The extent of emphysema was quantified using the percentage of the low attenuation area (LAA%) divided by the total lung or lobe volume(s). The correlations between the extent of emphysema, and pulmonary functions and the five-category classification were assessed using Pearson and Spearman's correlation coefficients, respectively. When quantifying emphysema using a density mask, a wide range of thresholds from -850 to -1,000 HU were used. RESULTS: The highest correlations of LAA% with the five-category classification and PFT measures ranged from -925 to -965 HU for each individual lobe and the entire lung. However, the differences between the highest correlations and those obtained at -950 HU are relatively small. CONCLUSION: Although there are variations in the optimal cut-off thresholds for individual lobes, the single threshold of -950 HU is still an acceptable threshold for density-based emphysema quantification.
Assuntos
Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Enfisema Pulmonar/diagnóstico por imagem , Enfisema Pulmonar/epidemiologia , Tomografia Computadorizada por Raios X/métodos , Causalidade , Comorbidade , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pennsylvania/epidemiologia , Prevalência , Reprodutibilidade dos Testes , Medição de Risco , Sensibilidade e EspecificidadeRESUMO
Chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD) are clinically and molecularly heterogeneous diseases. We utilized clustering and integrative network analyses to elucidate roles for microRNAs (miRNAs) and miRNA isoforms (isomiRs) in COPD and ILD pathogenesis. Short RNA sequencing was performed on 351 lung tissue samples of COPD (n = 145), ILD (n = 144) and controls (n = 64). Five distinct subclusters of samples were identified including 1 COPD-predominant cluster and 2 ILD-predominant clusters which associated with different clinical measurements of disease severity. Utilizing 262 samples with gene expression and SNP microarrays, we built disease-specific genetic and expression networks to predict key miRNA regulators of gene expression. Members of miR-449/34 family, known to promote airway differentiation by repressing the Notch pathway, were among the top connected miRNAs in both COPD and ILD networks. Genes associated with miR-449/34 members in the disease networks were enriched among genes that increase in expression with airway differentiation at an air-liquid interface. A highly expressed isomiR containing a novel seed sequence was identified at the miR-34c-5p locus. 47% of the anticorrelated predicted targets for this isomiR were distinct from the canonical seed sequence for miR-34c-5p. Overexpression of the canonical miR-34c-5p and the miR-34c-5p isomiR with an alternative seed sequence down-regulated NOTCH1 and NOTCH4. However, only overexpression of the isomiR down-regulated genes involved in Ras signaling such as CRKL and GRB2. Overall, these findings elucidate molecular heterogeneity inherent across COPD and ILD patients and further suggest roles for miR-34c in regulating disease-associated gene-expression.