BET proteins are associated with the induction of small airway fibrosis in COPD.

RationaleIn COPD, small airway fibrosis occurs due to increased extracellular matrix (ECM) deposition in and around the airway smooth muscle (ASM) layer. Studies of immune cells and peripheral lung tissue have shown that epigenetic changes occur in COPD but it is unknown whether airway mesenchymal cells are reprogrammed.ObjectivesDetermine if COPD ASM cells have a unique epigenetic response to profibrotic cytokine transforming growth factor β1 (TGF-β1).MethodsPrimary human ASM cells from COPD and non-COPD smoking patients were stimulated with TGF-β1. Gene array analysis performed to identify differences in ECM expression. Airway accumulation of collagen 15α1 and tenascin-C proteins was assessed. Aforementioned ASM cells were stimulated with TGF-β1 ± epigenetic inhibitors with qPCR quantification of COL15A1 and TNC. Global histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity were assessed. chromatin immunoprecipitation (ChIP)-qPCR for histone H3 and H4 acetylation at COL15A1 and TNC promoters was carried out. Effects of bromoterminal and extraterminal domain (BET) inhibitor JQ1(+) on expression and acetylation of ECM target genes were assessed.Measurements and main resultsCOPD ASM show significantly higher COL15A1 and TNC expression in vitro and the same trend for higher levels of collagen 15α1 and tenascin-c deposited in COPD airways in vivo. Epigenetic screening indicated differential response to HDAC inhibition. ChIP-qPCR revealed histone H4 acetylation at COL15A1 and TNC promoters in COPD ASM only. ChIP-qPCR found JQ1(+) pretreatment significantly abrogated TGF-β1 induced histone H4 acetylation at COL15A1 and TNC.ConclusionsBET protein binding to acetylated histones is important in TGF-β1 induced expression of COL15A1 and TNC and maintenance of TGF-β1 induced histone H4 acetylation in cell progeny

Aberrant DNA methylation and expression of SPDEF and FOXA2 in airway epithelium of patients with COPD

Background: Goblet cell metaplasia, a common feature of chronic obstructive pulmonary disease (COPD), is associated with mucus hypersecretion which contributes to the morbidity and mortality among patients. Transcription factors SAM-pointed domain-containing Ets-like factor (SPDEF) and forkhead box protein A2 (FOXA2) regulate goblet cell differentiation. This study aimed to (1) investigate DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation and (2) compare this in airway epithelial cells from patients with COPD and controls during mucociliary differentiation. Methods: To assess DNA methylation and expression of SPDEF and FOXA2 during goblet cell differentiation, primary airway epithelial cells, isolated from trachea (non-COPD controls) and bronchial tissue (patients with COPD), were differentiated by culture at the air-liquid interface (ALI) in the presence of cytokine interleukin (IL)-13 to promote goblet cell differentiation. Results: We found that SPDEF expression was induced during goblet cell differentiation, while FOXA2 expression was decreased. Importantly, CpG number 8 in the SPDEF promoter was hypermethylated upon differentiation, whereas DNA methylation of FOXA2 promoter was not changed. In the absence of IL-13, COPD-derived ALI-cultured cells displayed higher SPDEF expression than control-derived ALI cultures, whereas no difference was found for FOXA2 expression. This was accompanied with hypomethylation of CpG number 6 in the SPDEF promoter and also hypomethylation of CpG numbers 10 and 11 in the FOXA2 promoter. Conclusions: These findings suggest that aberrant DNA methylation of SPDEF and FOXA2 is one of the factors underlying mucus hypersecretion in COPD, opening new avenues for epigenetic-based inhibition of mucus hypersecretion

The Microbiome in Bronchial Biopsies from Smokers and Ex-Smokers with Stable COPD - A Metatranscriptomic Approach

Current knowledge about the respiratory microbiome is mainly based on 16S ribosomal RNA gene sequencing. Newer sequencing approaches, such as metatranscriptomics, offer the technical ability to measure the viable microbiome response to environmental conditions such as smoking as well as to explore its functional role by investigating host-microbiome interactions. However, knowledge about its feasibility in respiratory microbiome research, especially in lung biopsies, is still very limited. RNA sequencing was performed in bronchial biopsies from clinically stable smokers (n=5) and ex-smokers (n=6) with COPD not using (inhaled) steroids. The Trinity assembler was used to assemble non-human reads in order to allow unbiased taxonomical and microbial transcriptional analyses. Subsequently, host-microbiome interactions were analyzed based on associations with host transcriptomic data. Ultra-low levels of microbial mass (0.009%) were identified in the RNA-seq data. Overall, no differences were identified in microbiome diversity or transcriptional profiles of microbial communities or individual microbes between COPD smokers and ex-smokers in the initial test dataset as well as a larger replication dataset. We identified an upregulated host gene set, related to the simultaneous presence of Bradyrhizobium, Roseomonas, Brevibacterium.spp., which were related to PERK-mediated unfolded protein response (UPR) and expression of the microRNA-155-5p. Our results show that metatranscriptomic profiling in bronchial biopsy samples from stable COPD patients yields ultra-low levels of microbial mass. Further, this study illustrates the potential of using transcriptional profiling of the host and microbiome to gain more insight into their interaction in the airways

The Pharmacogenomics of Inhaled Corticosteroids and Lung Function Decline in COPD

Inhaled corticosteroids (ICS) are widely prescribed for patients with chronic obstructive pulmonary disease (COPD), yet with variable outcomes and adverse reactions which may be genetically determined. The primary aim of the study was to identify the genetic determinants for FEV1 changes related to ICS therapy. In the Lung Health Study 2 (LHS-2), 1116 COPD patients were randomised to the ICS, triamcinolone acetonide (n=559), or placebo (n=557) with spirometry performed every 6 months for 3 years. We performed a pharmacogenomic genome-wide association study (GWAS) for the genotype-by-ICS treatment effect on 3 years of forced expiratory volume in 1 s (FEV1) changes (estimated as slope) in 802 genotyped LHS-2 participants. Replication was performed in 199 COPD patients randomised to the ICS, fluticasone or placebo. A total of five loci showed genotype-by-ICS interaction at p&lt;5×10-6; of these, SNP rs111720447 on chromosome 7 was replicated (discovery p=4.8×10-6, replication p=5.9×10-5) with the same direction of interaction effect. ENCODE data revealed that in glucocorticoid treated (dexamethasone) A549 alveolar cell line, glucocorticoid receptor binding sites were located near SNP rs111720447. In stratified analyses of LHS-2, genotype at SNP rs111720447 was significantly associated with rate of FEV1 decline in patients taking ICS (C allele beta=56.35 mL·year-1, 95% confidence interval (CI)=29.96, 82.76 mL·yr-1) and also in patients who were assigned to placebo, though the relationship was weaker and in the opposite direction than that in the ICS group (C allele beta=-27.57 mL·year-1, 95% CI=-53.27, -1.87 mL·yr-1). The study uncovered genetic factors associated with FEV1 changes related to ICS in COPD patients, which may provide new insight on the potential biology of steroid responsiveness in COPD.</p

High-Resolution Sonography: A New Technique to Detect Nerve Damage in Leprosy

Mycobacterium leprae, which causes leprosy, infects peripheral nerves resulting in functional impairment, ulcer formation and stigmatizing deformities. Early diagnosis of nerve involvement is important to avoid nerve related complications. We used non-invasive, high-resolution sonography (US) and color Doppler (CD) imaging to study the ulnar (UN), median (MN), lateral popliteal (LP) and posterior tibial (PT) nerves in 20 leprosy patients and compared 30 healthy Indian controls. The nerves were significantly thicker in the patients (p<0.0001 for each nerve). One of the key signs of leprosy is the presence of enlarged nerves. The kappa for clinical palpation and nerve enlargement by sonography was 0.30 for all examined nerves. Increased neural vascularity, the sign of inflammation was observed in 26% (39/152) of nerves by CD imaging. Increased CD was observed in multiple nerves in 3 of 4 patients with type 2 reaction. Significant correlation was observed between clinical parameters of grade of thickening, sensory loss and muscle weakness and US abnormalities of nerve echotexture, endoneural flow and cross-sectional area (p<0.001). We conclude that sonography is a better diagnostic tool to predict nerve damage as compared to clinical assessment. Nerve damage was sonographically more extensive and was observed in nerves considered clinically normal

Identifying a nasal gene expression signature associated with hyperinflation and treatment response in severe COPD

Hyperinflation contributes to dyspnea intensity in COPD. Little is known about the molecular mechanisms underlying hyperinflation and how inhaled corticosteroids (ICS) affect this important aspect of COPD pathophysiology. To investigate the effect of ICS/long-acting β2-agonist (LABA) treatment on both lung function measures of hyperinflation, and the nasal epithelial gene-expression profile in severe COPD. 117 patients were screened and 60 COPD patients entered a 1-month run-in period on low-dose ICS/LABA budesonide/formoterol (BUD/F) 200/6 one inhalation b.i.d. Patients were then randomly assigned to 3-month treatment with either a high dose BDP/F 100/6 two inhalations b.i.d. (n = 31) or BUD/F 200/6 two inhalations b.i.d. (n = 29). Lung function measurements and nasal epithelial gene-expression were assessed before and after 3-month treatment and validated in independent datasets. After 3-month ICS/LABA treatment, residual volume (RV)/total lung capacity (TLC)% predicted was reduced compared to baseline (p < 0.05). We identified a nasal gene-expression signature at screening that associated with higher RV/TLC% predicted values. This signature, decreased by ICS/LABA treatment was enriched for genes associated with increased p53 mediated apoptosis was replicated in bronchial biopsies of COPD patients. Finally, this signature was increased in COPD patients compared to controls in nasal, bronchial and small airways brushings. Short-term ICS/LABA treatment improves RV/TLC% predicted in severe COPD. Furthermore, it decreases the expression of genes involved in the signal transduction by the p53 class mediator, which is a replicable COPD gene expression signature in the upper and lower airways.Trial registration: ClinicalTrials.gov registration number NCT01351792 (registration date May 11, 2011), ClinicalTrials.gov registration number NCT00848406 (registration date February 20, 2009), ClinicalTrials.gov registration number NCT00158847 (registration date September 12, 2005)

miR449 Protects Airway Regeneration by Controlling AURKA/HDAC6-Mediated Ciliary Disassembly

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449−/− mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449−/− mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449−/− mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449−/− cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis

Taming trilogues: the EU's law-making process in a comparative perspective.

Trilogues have become the modus operandi of EU decision-making. They are an informal but institutionalised mechanism providing for in camera discussions of legislative texts between the three main EU decision-making institutions, with a view to securing legislative compromises. Trilogues present risks to an organ of parliamentary representation through their potential to depoliticise conflict and by reducing the accountability and transparency of the decision-making process. We examine how the European Parliament (EP) has responded to trilogues and what this response tells us about the development of the EP as an institutionalised organ of representative democracy. We compare these with arrangements for bicameral conflict resolution in the United States, where similar issues are presented by informal mechanisms of decision-making. We assess the institutionalisation of trilogues from a democratic perspective, highlighting achievements and future challenges, and the value of these findings for the ongoing reflection on the EP as a normal parliament and the role of informal institutions in EU law-making

miR449 Protects Airway Regeneration by Controlling AURKA/HDAC6-Mediated Ciliary Disassembly

Airway mucociliary regeneration and function are key players for airway defense and are impaired in chronic obstructive pulmonary disease (COPD). Using transcriptome analysis in COPD-derived bronchial biopsies, we observed a positive correlation between cilia-related genes and microRNA-449 (miR449). In vitro, miR449 was strongly increased during airway epithelial mucociliary differentiation. In vivo, miR449 was upregulated during recovery from chemical or infective insults. miR0449-/- mice (both alleles are deleted) showed impaired ciliated epithelial regeneration after naphthalene and Haemophilus influenzae exposure, accompanied by more intense inflammation and emphysematous manifestations of COPD. The latter occurred spontaneously in aged miR449-/- mice. We identified Aurora kinase A and its effector target HDAC6 as key mediators in miR449-regulated ciliary homeostasis and epithelial regeneration. Aurora kinase A is downregulated upon miR449 overexpression in vitro and upregulated in miR449-/- mouse lungs. Accordingly, imaging studies showed profoundly altered cilia length and morphology accompanied by reduced mucociliary clearance. Pharmacological inhibition of HDAC6 rescued cilia length and coverage in miR449-/- cells, consistent with its tubulin-deacetylating function. Altogether, our study establishes a link between miR449, ciliary dysfunction, and COPD pathogenesis