John Tedrow

miR-199a-5p Is upregulated during fibrogenic response to tissue injury and mediates TGFbeta-induced lung fibroblast activation by targeting caveolin-1.

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 TGFβ 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 TGFβ 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.

C-X-C Motif Chemokine 13 (CXCL13) Is a Prognostic Biomarker of Idiopathic Pulmonary Fibrosis

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.

An Airway Epithelial iNOS-DUOX2-Thyroid Peroxidase Metabolome Drives Th1/Th2 Nitrative Stress in Human Severe Asthma

Severe refractory asthma is associated with enhanced nitrative stress. To determine the mechanisms for high nitrative stress in human severe asthma (SA), 3-nitrotyrosine (3NT) was compared with Th1 and Th2 cytokine expression. In SA, high 3NT levels were associated with high interferon (IFN)-γ and low interleukin (IL)-13 expression, both of which have been reported to increase inducible nitric oxide synthase (iNOS) in human airway epithelial cells (HAECs). We found that IL-13 and IFN-γ synergistically enhanced iNOS, nitrite, and 3NT, corresponding with increased H2O2. Catalase inhibited whereas superoxide dismutase enhanced 3NT formation, supporting a critical role for H2O2, but not peroxynitrite, in 3NT generation. Dual oxidase-2 (DUOX2), central to H2O2 formation, was also synergistically induced by IL-13 and IFN-γ. The catalysis of nitrite and H2O2 to nitrogen dioxide radical (NO2•) requires an endogenous peroxidase in this epithelial cell system. Thyroid peroxidase (TPO) was identified by microarray analysis ex vivo as a gene distinguishing HAEC of SA from controls. IFN-γ induced TPO in HAEC and small interfering RNA knockdown decreased nitrated tyrosine residues. Ex vivo, DUOX2, TPO, and iNOS were higher in SA and correlated with 3NT. Thus, a novel iNOS–DUOX2–TPO–NO2• metabolome drives nitrative stress in HAEC and likely in SA.

Relationship of DNA Methylation and Gene Expression in Idiopathic Pulmonary Fibrosis

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.

A novel genomic signature with translational significance for human idiopathic pulmonary fibrosis.

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-β1-treated primary human lung fibroblasts and transforming growth factor-β1-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.

Cytokine-Like Factor 1 Gene Expression Is Enriched in Idiopathic Pulmonary Fibrosis and Drives the Accumulation of CD4+ T Cells in Murine Lungs: Evidence for an Antifibrotic Role in Bleomycin Injury

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.

Gene Expression Correlated with Severe Asthma Characteristics Reveals Heterogeneous Mechanisms of Severe Disease

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.

Suppression of NLRX1 in chronic obstructive pulmonary disease

Cigarette smoke (CS) and viruses promote the inflammation and remodeling associated with chronic obstructive pulmonary disease (COPD). The MAVS/RIG-I-like helicase (MAVS/RLH) pathway and inflammasome-dependent innate immune pathways are important mediators of these responses. At baseline, the MAVS/RLH pathway is suppressed, and this inhibition must be reversed to engender tissue effects; however, the mechanisms that mediate activation and repression of the pathway have not been defined. In addition, the regulation and contribution of MAVS/RLH signaling in CS-induced inflammation and remodeling responses and in the development of human COPD remain unaddressed. Here, we demonstrate that expression of NLRX1, which inhibits the MAVS/RLH pathway and regulates other innate immune responses, was markedly decreased in 3 independent cohorts of COPD patients. NLRX1 suppression correlated directly with disease severity and inversely with pulmonary function, quality of life, and prognosis. In murine models, CS inhibited NLRX1, and CS-induced inflammation, alveolar destruction, protease induction, structural cell apoptosis, and inflammasome activation were augmented in NLRX1-deficient animals. Conversely, MAVS deficiency abrogated this CS-induced inflammation and remodeling. Restoration of NLRX1 in CS-exposed animals ameliorated alveolar destruction. These data support a model in which CS-dependent NLRX1 inhibition facilitates MAVS/RHL activation and subsequent inflammation, remodeling, protease, cell death, and inflammasome responses.

Integrative phenotyping framework (iPF): integrative clustering of multiple omics data identifies novel lung disease subphenotypes

BackgroundThe 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.ResultsIn 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.ConclusionsIdentification 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.

Pneumocystis jirovecii colonization is associated with enhanced Th1 inflammatory gene expression in lungs of humans with chronic obstructive pulmonary disease

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.