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ETS domain-containing protein-1 (ELK1) is a transcriptional repressor important in regulating αvβ6 integrin expression. αvβ6 integrins activate the profibrotic cytokine Transforming Growth Factor β1 (TGFβ1) and are increased in the alveolar epithelium in idiopathic pulmonary fibrosis (IPF). IPF is a disease associated with aging and therefore we hypothesised that aged animals lacking Elk1 globally would develop spontaneous fibrosis in organs where avb6 mediated TGFb activation has been implicated. Here we identify that Elk1-knockout (Elk1(−/0)) mice aged to one year developed spontaneous fibrosis in the absence of injury in both the lung and the liver but not in the heart or kidneys. The lungs of Elk1(−/0) aged mice demonstrated increased collagen deposition, in particular collagen 3α1, located in small fibrotic foci and thickened alveolar walls. Despite the liver having relatively low global levels of ELK1 expression, Elk1(−/0) animals developed hepatosteatosis and fibrosis. The loss of Elk1 also had differential effects on Itgb1, Itgb5 and Itgb6 genes expression in the four organs potentially explaining the phenotypic differences in these organs. To understand the potential causes of reduced ELK1in human disease Finally we exposed human cells and murine lung slices to cigarette smoke extract which lead to reduced ELK1 expression which may explain the loss of ELK1 in human disease. These data support a fundament role for ELK1 in protecting against the development of progressive fibrosis via transcriptional regulation of beta integrin subunit genes, and demonstrate that loss of ELK1 can be caused by cigarette smoke.

Introduction: A hallmark of IPF is the excess accumulation of extracellular matrix (ECM) in the lungs. When ECM is degraded by metalloproteinases (MMPs), free-circulating protein fragments (neoepitopes) are generated. INMARK (NCT02788474) is an ongoing trial assessing the effects of nintedanib on changes in biomarkers of ECM turnover and on the association between changes in such biomarkers and disease progression in patients with IPF and limited FVC impairment (FVC ≥80% predicted). Aim: To summarise the baseline characteristics of patients participating in INMARK. Methods: Patients were randomised 1:2 to receive nintedanib 150 mg bid or placebo for 12 weeks followed by open-label nintedanib for 40 weeks. The primary endpoint is the rate of change in serum c-reactive protein degraded by MMP-1/8 (CRPM) from baseline to week 12. The proportion of patients with disease progression (defined as absolute decline in FVC ≥10% predicted or death) over 52 weeks is a key secondary endpoint. Results: Recruitment for the INMARK trial is complete. A total of 346 patients have been treated. At baseline, mean (SD) age was 70.3 (7.4) years; the majority of patients were white (61.8%), male (75.7%) and former or current smokers (72.8%). Mean (SD) FVC was 97.5 (13.5) % predicted and DLco was 63.8 (19.5) % predicted. Conclusion: The INMARK trial will provide insights into the association between changes in biomarkers of ECM turnover and disease progression, and whether treatment with nintedanib affects the rate of change in such biomarkers, in patients with IPF and limited FVC impairment. Results will be presented in 2019.

IntroductionA feature of the pathogenesis of idiopathic pulmonary fibrosis (IPF) is the excess accumulation of extracellular matrix (ECM) in the lungs. Cleavage of the ECM by metalloproteinases (MMPs) generates free-circulating protein fragments known as neoepitopes. The PROFILE study suggested that changes in ECM turnover proteins may be of value as markers of disease progression in patients with IPF. Nintedanib is an approved treatment for IPF that slows disease progression by reducing decline in forced vital capacity (FVC).Methods and analysisThe INMARK® trial is evaluating the effect of nintedanib on the rates of change of biomarkers of ECM turnover in patients with IPF, the value of changes in these biomarkers as predictors of disease progression and whether nintedanib affects the associations between changes in these biomarkers and disease progression. Following a screening period, 347 patients with IPF and FVC ≥80% predicted were randomised 1:2 to receive nintedanib 150 mg two times a day or placebo for 12 weeks, followed by an open-label period in which all patients will receive nintedanib for 40 weeks. The primary endpoint is the rate of change in C reactive protein degraded by MMP-1/8 from baseline to week 12.Ethics and disseminationThis trial is being conducted in compliance with the protocol, the ethical principles detailed in the Declaration of Helsinki and in accordance with the International Conference on Harmonisation Harmonised Tripartite Guideline for Good Clinical Practice. The results of the trial will be presented at national and international meetings and published in peer-reviewed journals.Trial registration numberNCT02788474.

Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide and the need for novel biomarkers and therapeutic strategies to improve diagnosis and surveillance is obvious. This study aims to identify β

Experimental models are critical for the understanding of lung health and disease and are indispensable for drug development. However, the pathogenetic and clinical relevance of the models is often unclear. Further, the use of animals in biomedical research is controversial from an ethical perspective.The objective of this task force was to issue a statement with research recommendations about lung disease models by facilitating in-depth discussions between respiratory scientists, and to provide an overview of the literature on the available models. Focus was put on their specific benefits and limitations. This will result in more efficient use of resources and greater reduction in the numbers of animals employed, thereby enhancing the ethical standards and translational capacity of experimental research.The task force statement addresses general issues of experimental research (ethics, species, sex, age,ex vivoandin vitromodels, gene editing). The statement also includes research recommendations on modelling asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, lung infections, acute lung injury and pulmonary hypertension.The task force stressed the importance of using multiple models to strengthen validity of results, the need to increase the availability of human tissues and the importance of standard operating procedures and data quality.

Idiopathic pulmonary fibrosis (IPF) is a progressive, irreversible, and typically fatal lung disease characterised by subpleural fibrosis, subepithelial fibroblast foci, and microscopic honeycombing. Although understanding of the pathogenic mechanisms continues to evolve, evidence indicates that distal airway and alveolar epithelial cells are central drivers of the disease. In this Viewpoint, we review the history of naming and classifications used to define the disease now referred to as IPF, in the context of understanding the clinical presentation, causes, and pathogenesis of the disease. We aim to generate discussion on whether, given the substantial progress made in understanding the clinical, genetic, cellular, and molecular mechanisms involved in the development of IPF, a change of name should be considered. To initiate this discussion, we offer new suggestions to update the name of this disease and new approaches to classify all forms of pulmonary fibrosis.

This document provides clinical recommendations for the diagnosis of idiopathic pulmonary fibrosis (IPF). It represents a collaborative effort between the American Thoracic Society, European Respiratory Society, Japanese Respiratory Society, and Latin American Thoracic Society.The evidence syntheses were discussed and recommendations formulated by a multidisciplinary committee of IPF experts. The evidence was appraised and recommendations were formulated, written, and graded using the Grading of Recommendations, Assessment, Development, and Evaluation approach.The guideline panel updated the diagnostic criteria for IPF. Previously defined patterns of usual interstitial pneumonia (UIP) were refined to patterns of UIP, probable UIP, indeterminate, and alternate diagnosis. For patients with newly detected interstitial lung disease (ILD) who have a high-resolution computed tomography scan pattern of probable UIP, indeterminate, or an alternative diagnosis, conditional recommendations were made for performing BAL and surgical lung biopsy; because of lack of evidence, no recommendation was made for or against performing transbronchial lung biopsy or lung cryobiopsy. In contrast, for patients with newly detected ILD who have a high-resolution computed tomography scan pattern of UIP, strong recommendations were made against performing surgical lung biopsy, transbronchial lung biopsy, and lung cryobiopsy, and a conditional recommendation was made against performing BAL. Additional recommendations included a conditional recommendation for multidisciplinary discussion and a strong recommendation against measurement of serum biomarkers for the sole purpose of distinguishing IPF from other ILDs.The guideline panel provided recommendations related to the diagnosis of IPF.

> Ignorance, allied with power, is the most ferocious enemy justice can have. James Baldwin On the eve of President Trump’s inauguration in 2017, we published an editorial setting out the threat that climate change poses to lung health. We urged the incoming President to face up to his responsibilities and take steps to address this growing existential threat.1 We drew a parallel with the actions of two conservative leaders in the 1980s, Ronald Reagan and Margaret Thatcher who, faced with unequivocal scientific evidence, led the decisive steps necessary to avert the destruction of the ozone layer.2 3 Rather than being contrary to his stated goals, acting on climate change, we wrote ‘is one way in which President Trump can make good on his promises to improve the wellbeing of Americans, increase America’s energy independence, and act with fiscal prudence. Investing in green infrastructure creates jobs. It reduces healthcare costs, as 1/4 - 1/3 of the costs of decarbonising come straight back as health economic gains’.4 Sadly, 2 years on our worst fears have been realised. The USA is experiencing an unprecedented wave of natural disasters attributable to climate change, including widespread wildfires, destructive storms and temperature swings. However, rather than providing global leadership, the Trump administration has not merely abdicated responsibility but is actively sabotaging efforts to mitigate climate change by announcing plans to withdraw from the Paris Climate accords.5 Clear evidence in the most recent Intergovernmental Panel on Climate Change report sets …

Purpose: Asthma is a disease of increasing worldwide importance that calls for new investigative methods. Ex vivo lung tissue is being increasingly used to study functional respiratory parameters independent of confounding systemic considerations but also to reduce animal numbers and associated research costs. In this work a straightforward laboratory method is advanced to probe dynamic changes in gas inhalation patterns by utilizing an ex vivo small animal ovalbumin (OVA) model of human asthma. Methods: Hyperpolarized (hp) 129Xe was actively inhaled by the excised lungs exposed to a constant pressure differential that mimicked negative pleural cavity pressure. The method enabled hp 129Xe MRI of airway responsiveness to intravenous methacholine (MCh) and airway challenge reversal through salbutamol. Results: Significant differences were demonstrated between control and OVA challenged animals on global lung hp 129Xe gas inhalation with p < 0.05 at MCh dosages above 460 μg. Spatial mapping of the regional hp gas distribution revealed an approximately 3 fold increase in heterogeneity for the asthma model organs. Conclusion: The experimental results from this proof of concept work suggest that the ex vivo hp noble gas imaging arrangement and the applied image analysis methodology may be useful as an adjunct to current diagnostic techniques.

Idiopathic pulmonary fibrosis (IPF) and fibrotic nonspecific interstitial pneumonitis are progressive interstitial lung diseases (ILDs) with limited treatment options and poor survival. However, the rate of disease progression is variable, implying there may be different endotypes of disease. We hypothesised that immunophenotyping biopsies from ILD patients might reveal distinct endotypes of progressive fibrotic disease, which may facilitate stratification when undertaking clinical trials of novel therapies for IPF.43 paraffin-embedded, formalin-fixed lung tissue sections were immunostained for five molecules implicated in the pathogenesis of the fibrosis: α-smooth muscle actin (αSMA), αvβ6 integrin, pro-surfactant protein C (SP-C), hepatocyte growth factor (HGF) and tenascin-C (TenC). Levels of immunostaining and numbers of fibroblastic foci were quantified using operator-dependent and -independent methods. The relationship of all these markers to overall survival was analysed.Staining revealed high levels of αSMA, αvβ6 integrin, pro-SP-C, HGF and TenC, and fibroblastic foci. Immunostaining varied across samples for all molecules but only the extent of αvβ6 integrin immunostaining was associated with increased mortality. There was no association with the other markers measured.Our data suggest high levels of αvβ6 integrin may identify a specific endotype of progressive fibrotic lung disease.

Influenza infection exacerbates chronic pulmonary diseases, including idiopathic pulmonary fibrosis. A central pathway in the pathogenesis of idiopathic pulmonary fibrosis is epithelial injury leading to activation of transforming growth factor β (TGFβ). The mechanism and functional consequences of influenza-induced activation of epithelial TGFβ are unclear. Influenza stimulates toll-like receptor 3 (TLR3), which can increase RhoA activity, a key event prior to activation of TGFβ by the αvβ6 integrin. We hypothesized that influenza would stimulate TLR3 leading to activation of latent TGFβ via αvβ6 integrin in epithelial cells. Using H1152 (IC50 6.1 μm) to inhibit Rho kinase and 6.3G9 to inhibit αvβ6 integrins, we demonstrate their involvement in influenza (A/PR/8/34 H1N1) and poly(I:C)-induced TGFβ activation. We confirm the involvement of TLR3 in this process using chloroquine (IC50 11.9 μm) and a dominant negative TLR3 construct (pZERO-hTLR3). Examination of lungs from influenza-infected mice revealed augmented levels of collagen deposition, phosphorylated Smad2/3, αvβ6 integrin, and apoptotic cells. Finally, we demonstrate that αvβ6 integrin-mediated TGFβ activity following influenza infection promotes epithelial cell death in vitro and enhanced collagen deposition in vivo and that this response is diminished in Smad3 knock-out mice. These data show that H1N1 and poly(I:C) can induce αvβ6 integrin-dependent TGFβ activity in epithelial cells via stimulation of TLR3 and suggest a novel mechanism by which influenza infection may promote collagen deposition in fibrotic lung disease.

Rationale: Interstitial lung diseases (ILDs) are associated with oxidative stress. Plasma biomarkers that are directly linked to oxidative stress responses in this disease have not been identified. Stable oxidation products of tyrosine residues in proteins may reflect the oxidative microenvironment in the lung or a systemic inflammatory state.

Rationale Idiopathic pulmonary fibrosis is a chronic fibroproliferative disease with a median survival of approximately 3 years. αvβ6 integrins are upregulated in lung fibrosis and are associated with increased activation of the profibrotic cytokine TGF-β. The transcription factor Elk1 can repress gene expression of β6. We therefore hypothesise that animals lacking functional Elk1 (Elk1-/0) will develop age related pulmonary fibrosis. Methods Elk1 knock-out (Elk1-/0) and wild-type (Elk1+/0) mice were allowed to age for 365 days. At 365 days old mice were sacrificed and their lungs harvested for evaluation of collagen gene expression, lung hydroxyproline concentration and histological assessment. Results Lungs were extracted and lung wet weights were measured in Elk1-/0 mice and wild-type (Elk1+/0) controls and no significant difference between the two genotypes was shown (175.7 mg, 161.8 mg respectively n=6–8). However, assessment of total lung hydroxyproline established that there was significantly more hydroxyproline in Elk1-/0 mice compared with Elk1+/0 controls (852.3, 758.4 µg/lung set, respectively, n=5–8, p=0.0346). Assessment of Masson’s trichrome stained Elk1-/0 lung tissue sections found a small number of fibrotic lesions were present. Furthermore, there was a trend towards increased alveolar wall median thickness in Elk1-/0 mice compared with Elk1+/0 animals (5.94 vs 5.56 µm respectively). In a small number of 12 week old mice we identified a trend towards increased α-smooth muscle actin (αSMA) mRNA expression in the lungs of Elk1-/0 mice compared with Elk1+/0 controls (9.40, 2.24 median relative expression, respectively n=3). We therefore performed immunohistochemical staining for αSMA in the lungs of mice aged to 1 year and demonstrated visible increases in expression of αSMA in the alveolar epithelium of Elk1-/0 mice but not in Elk1+/0 controls. Conclusion These data suggest that Elk1 gene deletion result in age-related early fibrotic changes associated with the development of pulmonary fibrosis.

Introduction Idiopathic pulmonary fibrosis (IPF) is an incurable and progressive fibrotic condition of the lung with a poor prognosis. Activation of the profibrotic cytokine transforming growth factor-β (TGFβ) is central to the pathogenesis of IPF. Mechanical signals are emerging as key mediators of TGFβ activation, and cyclical stretch is a ubiquitous stimulus in the lungs. Stretch of fibrotic lung induces TGFβ activation, and alveolar epithelial Gαq/11 signalling is important in stretch-mediated TGFβ activation. However, the role of Gαq/11 signalling in stretch-mediated TGFβ activation by lung fibroblasts is unclear. Methods Murine embryonic fibroblasts (MEFs) and human lung fibroblasts (HLFs) were subject to cyclical stretch using the Flexcell system, and stretch regimens designed to mimic tidal breathing in the relevant organism (15% elongation unless otherwise stated, frequency: 1 Hz in MEFs, 0.3 Hz in HLFs). Phosphorylated Smad2 (pSmad2), total Smad2, and alpha smooth muscle actin (αSMA) protein expression were assessed by western blot. TGFβ activation was determined using the ratio of pSmad2 to total Smad2 on densitometry. Comparisons of stretch-mediated TGFβ activation were made between wild-type (WT), Gα12/13-, and Gαq/11-deficient MEFs, and between HLFs from patients with and without IPF. Results Cyclical stretch induced a significant increase in pSmad2 expression after 48 hours in both MEFs and HLFs (p Conclusion Cyclical stretch is a physiologically relevant stimulus that drives Gαq/11-mediated TGFβ activation in lung fibroblasts. IPF fibroblasts have enhanced stretch-mediated TGFβ activation, indicating that dysregulation of breathing-related stretch signalling is a potential mechanism of IPF progression. A greater understanding of these pathways may identify targets for new therapies that could halt the progression of IPF.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with high mortality, uncertain cause, and few treatment options. Studies have identified a significant genetic risk associated with the development of IPF; however, mechanisms by which genetic risk factors promote IPF remain unclear. We aimed to identify genetic variants associated with IPF susceptibility and provide mechanistic insight using gene and protein expression analyses.Methods: We used a two-stage approach: a genome-wide association study in patients with IPF of European ancestry recruited from nine different centres in the UK and controls selected from UK Biobank (stage 1) matched for age, sex, and smoking status; and a follow-up of associated genetic variants in independent datasets of patients with IPF and controls from two independent US samples from the Chicago consortium and the Colorado consortium (stage 2). We investigated the effect of novel signals on gene expression in large transcriptomic and genomic data resources, and examined expression using lung tissue samples from patients with IPF and controls.Findings: 602 patients with IPF and 3366 controls were selected for stage 1. For stage 2, 2158 patients with IPF and 5195 controls were selected. We identified a novel genome-wide significant signal of association with IPF susceptibility near A-kinase anchoring protein 13 (AKAP13; rs62025270, odds ratio [OR] 1·27 [95% CI 1·18–1·37], p=1·32 × 10−9) and confirmed previously reported signals, including in mucin 5B (MUC5B; rs35705950, OR 2·89 [2·56–3·26], p=1·12 × 10−66) and desmoplakin (DSP; rs2076295, OR 1·44 [1·35–1·54], p=7·81 × 10−28). For rs62025270, the allele A associated with increased susceptibility to IPF was also associated with increased expression of AKAP13 mRNA in lung tissue from patients who had lung resection procedures (n=1111). We showed that AKAP13 is expressed in the alveolar epithelium and lymphoid follicles from patients with IPF, and AKAP13 mRNA expression was 1·42-times higher in lung tissue from patients with IPF (n=46) than that in lung tissue from controls (n=51).Interpretation: AKAP13 is a Rho guanine nucleotide exchange factor regulating activation of RhoA, which is known to be involved in profibrotic signalling pathways. The identification of AKAP13 as a susceptibility gene for IPF increases the prospect of successfully targeting RhoA pathway inhibitors in patients with IPF.Funding: UK Medical Research Council, National Heart, Lung, and Blood Institute of the US National Institutes of Health, Agencia Canaria de Investigación, Innovación y Sociedad de la Información, Spain, UK National Institute for Health Research, and the British Lung Foundation.

The potent and pluripotent cytokine TGFβ has important roles in normal homeostasis and disease pathogenesis. Once released from cells, TGFβ exists in both latent and functionally active forms. Large amounts of latent TGFβ are secreted from cells and sequestered in extracellular matrix, only a small proportion of which is activated at any given time. Accurate assessment of TGFβ activity levels is an important measurement in biological research and requires methods distinct from measuring total levels of TGFβ expression as small changes in TGFβ activity levels could be masked by the large amounts of latent TGFβ available to be measured. In this chapter, we describe detailed experimental methods for assessing levels of active TGFβ in cells and tissues. This chapter includes methods for the assessment of TGFβ activity in cells in vitro, in ex vivo precision cut tissue, and in vivo.

Numerous compounds have shown efficacy in limiting development of pulmonary fibrosis using animal models, yet few of these compounds have replicated these beneficial effects in clinical trials. Given the challenges associated with performing clinical trials in patients with idiopathic pulmonary fibrosis (IPF), it is imperative that preclinical data packages be robust in their analyses and interpretations to have the best chance of selecting promising drug candidates to advance to clinical trials. The American Thoracic Society has convened a group of experts in lung fibrosis to discuss and formalize recommendations for preclinical assessment of antifibrotic compounds. The panel considered three major themes (choice of animal, practical considerations of fibrosis modeling, and fibrotic endpoints for evaluation). Recognizing the need for practical considerations, we have taken a pragmatic approach. The consensus view is that use of the murine intratracheal bleomycin model in animals of both genders, using hydroxyproline measurements for collagen accumulation along with histologic assessments, is the best-characterized animal model available for preclinical testing. Testing of antifibrotic compounds in this model is recommended to occur after the acute inflammatory phase has subsided (generally after Day 7). Robust analyses may also include confirmatory studies in human IPF specimens and validation of results in a second system using in vivo or in vitro approaches. The panel also strongly encourages the publication of negative results to inform the lung fibrosis community. These recommendations are for preclinical therapeutic evaluation only and are not intended to dissuade development of emerging technologies to better understand IPF pathogenesis.

This article reviews the British Thoracic Society Winter Meeting 2013, the annual scientific meeting attended by over 2000 delegates and representing the depth and breadth of UK respiratory medicine. This year's meeting from 4 to 6 December in London featured cutting-edge research alongside keynote symposia from international experts in respiratory science, epidemiology and clinical trials. This article reviews the key symposia and selected abstract sessions from the 2013 meeting.

IntroductionThe Its Not JUST Idiopathic pulmonary fibrosis Study (INJUSTIS) is a multicentre, prospective, observational cohort study. The aims of this study are to identify genetic, serum and other biomarkers that may identify specific molecular mechanisms, reflecting disease endotypes that are shared among patients with progressive pulmonary fibrosis regardless of aetiology. Furthermore, it is anticipated that these biomarkers will help predict fibrotic activity that may identify patterns of disease behaviour with greater accuracy than current clinical phenotyping.Methods and analysis200 participants with the multidisciplinary team confirmed fibrotic lung disease (50 each of rheumatoid-interstitial lung disease (ILD), asbestosis, chronic hypersensitivity pneumonitis and unclassifiable ILD) and 50 idiopathic pulmonary fibrosis participants, recruited as positive controls, will be followed up for 2 years. Participants will have blood samples, lung function tests, quality of life questionnaires and a subgroup will be offered bronchoscopy. Participants will also be given the option of undertaking blinded home handheld spirometry for the first 3 months of the study. The primary end point will be identification of a biomarker that predicts disease progression, defined as 10% relative change in forced vital capacity (FVC) or death at 12 months.Ethics and disseminationThe trial has received ethical approval from the National Research Ethics Committee Nottingham (18/EM/0139). All participants must provide written informed consent. The trial will be overseen by the INJUSTIS steering group that will include a patient representative, and an independent chairperson. The results from this study will be submitted for publication in peer-reviewed journals and disseminated at regional and national conferences.Trial registration numberNCT03670576.

Idiopathic pulmonary fibrosis is a progressive and fatal interstitial lung disease. We aimed to determine if patient response to a palliative assessment survey could predict disease progression or death.We undertook a cross-sectional study in a UK clinical cohort of incident cases. Rasch-based methodology provided a disease distress value from an abridged 11 item model of the original 45 item survey. Distress values were compared with measures of lung function. Disease progression or mortality alone was predicted at twelve months from survey completion, with risk of death assessed at three, six and twelve months.Disease distress values were negatively correlated with lung function (r=-0.275 percent predicted DLCO). Expected survey scores computed from distress values could distinguish disease progression, 8.8 (p=0.004), and people who died, 10.2 (p=0.002), from those who did not progress, 6.9. Actual survey scores predicted disease progression and mortality with an area under the curve of 0.60 and 0.64, respectively. Each point increment in actual score increased risk of twelve-month mortality by 10%, almost 43% of people scoring above 18 did not survive beyond 105 days.We define a short questionnaire that can score disease distress and predict prognosis, assisting clinical decision making in progressive fibrosis.

Transforming growth factor b activation by the avb6 integrin is central to the pathogenesis of idiopathic pulmonary fibrosis. Expression of the avb6 integrin is increased in fibrotic lung tissue and is a promising therapeutic target for treatment of the disease. Currently, measurement of avb6 integrin levels in the lung requires immunohistochemical analysis of biopsy samples. This procedure is clinically impractical for many patients with pulmonary fibrosis, and a noninvasive strategy for measuring avb6 integrin levels in the lungs is urgently required to facilitate monitoring of disease progression and therapeutic responses. Methods: Using a murine model of bleomycin-induced lung injury, we assessed the binding of intravenously administered 111In-labeled avb6-specific (diethylenetriamine pentaacetate-tetra [DTPA]-A20FMDV2) or control (DTPAA20FMDVran) peptide by nanoSPECT/CT imaging. Development of fibrosis was assessed by lung hydroxyproline content, and avb6 protein and itgb6 messenger RNA were measured in the lungs. Results: Maximal binding of 111In-labeled A20FMDV2 peptide to avb6 integrins was detected in the lungs 1 h after intravenous administration. No significant binding was detected in mice injected with control peptide. Integrin binding was increased in the lungs of bleomycin-, compared with saline-, exposed mice and was attenuated by pretreatment with avb6-blocking antibodies. Levels of 111 In-labeled A20FMDV2 peptide correlated positively with hydroxyproline, avb6 protein, and itgb6 messenger RNA levels. Conclusion: We have developed a highly sensitive, quantifiable, and noninvasive technique for measuring avb6 integrin levels within the lung. Measurement of avb6 integrins by SPECT/CT scanning has the potential for use in stratifying therapy for patients with pulmonary fibrosis.

Heterotrimeric guanine nucleotide–binding protein (G protein) signaling is a ubiquitous signaling system that links hundreds of G protein–coupled receptors (GPCRs) with four G protein signaling pathways. Two of these pathways, one mediated by Gq and G11 and the other by G12 and G13, are implicated in the force-dependent activation of transforming growth factor–β (TGFβ) in lung epithelial cells. Reduced TGFβ activation in alveolar cells leads to emphysema, whereas enhanced TGFβ activation promotes acute lung injury, and idiopathic pulmonary fibrosis, therefore precise control of alveolar TGFβ activation is essential for alveolar homeostasis. Here, we investigated whether the Gq/G11 or G12/G13 pathways in epithelial cells are required to generate TGFβ and suppress alveolar inflammation. Mice deficient in both Gαq and Gα11 developed inflammation primarily due to alternatively activated (M2-polarized) macrophages, enhanced production of matrix metalloprotease 12 (MMP12), and age-related alveolar airspace enlargement consistent with emphysema. We found that mice with impaired Gq/G11 signaling had reduced stretch-mediated generation of TGFβ by epithelial cells and elevated macrophage MMP12 synthesis, but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the pleiotropic cytokine interleukin-33 (IL-33), was increased in these alveolar epithelial cells resulting in the M2-type polarization of alveolar macrophages independently of the effect on TGFβ. Our results suggest that alveolar Gq/G11 signaling maintains alveolar homeostasis and is likely to independently upregulate mechanotransduced epithelial TGFβ activation and downregulate epithelial IL-33 synthesis. Together, these findings suggest that disruption of Gq/G11 signaling promotes inflammatory emphysema, but protects against mechanostransduced lung injury.

Fibrosis is the formation of scar tissue due to injury or long-term inflammation and is a leading cause of morbidity and mortality in disorders that include IPF. The αvβ6 integrin has been identified as playing a key role in the activation of TGFβ that is hypothesized to be pivotal in …

When we accepted the prestigious role of Joint Editors-in-Chief of Thorax 1 year ago we were fully aware of the enormous responsibility that came with the role as well as the challenges that we would face as the editorial team. We were wholly cognisant that we were representing more than just a journal, but indeed we were representing an important national body, the British Thoracic Society (BTS). Our key aims were to focus on originality, excellence and rigour in respiratory, sleep and critical care medicine to ensure that Thorax delivers interesting, educational and impactful content to all its readers, most importantly BTS members. We were tasked with building up still further the publication metrics. In the last year, our top read articles have included the pulmonary nodule guidelines,1 read over 9000 times and cited 17 times, original research looking at the use of venous blood gas analysis in COPD,2 read over 6700 times, clinical trial data from the Lebrikizumab trials,3 read over 5000 times and cited 16 times, basic science studies investigating the role of Toll-like receptor 7 in regulating …

Acute exacerbation of idiopathic pulmonary fibrosis has been defined as an acute, clinically significant, respiratory deterioration of unidentifiable cause. The objective of this international working group report on acute exacerbation of idiopathic pulmonary fibrosis was to provide a comprehensive update on the topic. A literature review was conducted to identify all relevant English text publications and abstracts. Evidence-based updates on the epidemiology, etiology, risk factors, prognosis, and management of acute exacerbations of idiopathic pulmonary fibrosis are provided. Finally, to better reflect the current state of knowledge and improve the feasibility of future research into its etiology and treatment, the working group proposes a new conceptual framework for acute respiratory deterioration in idiopathic pulmonary fibrosis and a revised definition and diagnostic criteria for acute exacerbation of idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a devastating, progressive disease with poor survival rates and limited treatment options. Upregulation of αvβ6 integrins within the alveolar epithelial cells is a characteristic feature of IPF and correlates with poor patient survival. The pro-fibrotic cytokine TGFβ1 can upregulate αvβ6 integrin expression but the molecular mechanisms driving this effect have not previously been elucidated. We confirm that stimulation with exogenous TGFβ1 increases expression of the integrin β6 subunit gene (ITGB6) and αvβ6 integrin cell surface expression in a time- and concentration-dependent manner. TGFβ1-induced ITGB6 expression occurs via transcriptional activation of the ITGB6 gene, but does not result from effects on ITGB6 mRNA stability. Basal expression of ITGB6 in, and αvβ6 integrins on, lung epithelial cells occurs via homeostatic αvβ6-mediated TGFβ1 activation in the absence of exogenous stimulation, and can be amplified by TGFβ1 activation. Fundamentally, we show for the first time that TGFβ1-induced ITGB6 expression occurs via canonical Smad signalling since dominant negative constructs directed against Smad3 and 4 inhibit ITGB6 transcriptional activity. Furthermore, disruption of a Smad binding site at -798 in the ITGB6 promoter abolishes TGFβ1-induced ITGB6 transcriptional activity. Using chromatin immunoprecipitation we demonstrate that TGFβ1 stimulation of lung epithelial cells results in direct binding of Smad3, and Smad4, to the ITGB6 gene promoter within this region. Finally, using an adenoviral TGFβ1 over-expression model of pulmonary fibrosis we demonstrate that Smad3 is crucial for TGFβ1-induced αvβ6 integrin expression within the alveolar epithelium in vivo. Together, these data confirm that a homeostatic, autocrine loop of αvβ6 integrin activated TGFβ1-induced ITGB6 gene expression regulates epithelial basal αvβ6 integrin expression, and demonstrates that this occurs via Smad-dependent transcriptional regulation at a single Smad binding site in the promoter of the β6 subunit gene. Active TGFβ1 amplifies this pathway both in vitro and in vivo, which may promote fibrosis.

Trans-epithelial migration (TEpM) of leukocytes during inflammation requires engagement with receptors expressed on the basolateral surface of the epithelium. One such receptor is Coxsackie and Adenovirus Receptor (CAR) that binds to Junctional Adhesion Molecule-like (JAM-L) expressed on leukocytes. Here we provide the first evidence that efficient TEpM of monocyte-derived THP-1 cells requires and is controlled by phosphorylation of CAR. We show that TNFα acts in a paracrine manner on epithelial cells via a TNFR1-PI3K-PKĆ pathway leading to CAR phosphorylation and subsequent transmigration across cell junctions. Moreover, we show that CAR is hyper-phosphorylated in vivo in acute and chronic lung inflammation models and this response is required to facilitate immune cell recruitment. This represents a novel mechanism of feedback between leukocytes and epithelial cells during TEpM and may be important in controlling responses to pro-inflammatory cytokines in pathological settings.

Introduction and objectives: Idiopathic Pulmonary Fibrosis (IFP) is a fatal interstitial lung disease with unknown aetiology. Lung myofibroblasts (activated fibrobalsts) are the major effector cells in the pathogenesis of IPF. Transforming growth factor-β (TGF-β1) is a potent activator of fibroblasts. Lack of effective treatment options necessitates novel therapeutic approaches. Epigenetic drugs, by inhibiting chromatin modifying enzymes involved in gene expression control, represent promising agents capable of modulating the cellular phenotype. We previously demonstrated that the cyclooxygenase-2 (COX-2) gene is epigenetically silenced in lung fibroblasts from IPF patients (F-IPF)1 and epigenetic inhibitors and restore COX-2 expression. However, whether epigenetic inhibitors can alter fibroblast phenotype remains unknown. This study aimed to investigate the effect of four different epigenetic enzyme inhibitors on fibroblast phenotype change in IPF. Methods: F-IPF and fibroblasts from non-fibrotic lung (F-NL) treated with TGF-β1 were cultured to test the effects of the epigenetic inhibitors BIX01294 (BIX, G9a histone methyltransferase inhibitor), 3-deazaneplanocin A (DZNep, EZH2 histone methyltransferase inhibitor), SAHA (histone deacetylases inhibitor) and Decitabine (DAC, DNA demethylating agent), in comparison with the COX-2 products prostaglandin E2 (PGE2). The expression of COX-2 and myofibroblast markers collagen 1 (COL1) and α-smooth muscle actin (α-SMA) was assessed. The COX-2 DNA promoter methylation level was analysed by bisulfite sequencing. Results: TGF-β1 induced a myofibroblast phenotype in F-NL characterised by COL1 and α-SMA upregulation and COX-2 downregulation, similar to F-IPF. PGE2 and SAHA were able to maintain/restore COX-2 expression in TGF-β1-induced myofibroblasts and F-IPF. DAC demonstrated similar effect in TGF-β1 treated F-NL only. SAHA also reduced COL1 and α-SMA expression. But DZNep and BIX showed no effect. No differences in the COX-2 promoter methylation was detected between F-NL and F-IPF. Conclusions: Among the epigenetic inhibitors tested, SAHA shows a promising antifibrotic effect by inhibiting fibroblast activation and the underlying molecular mechanisms are currently under investigation.

Background: Pulmonary fibrosis encompasses a group of lung-scarring disorders that occur owing to known or unknown insults and accounts for significant morbidity and mortality. Despite intense investigation spanning decades, much remains to be learned about the natural history, pathophysiology, and biologic mechanisms of disease. Purpose: To identify the most pressing research needs in the lung fibrosis community and to provide a roadmap of priorities to investigators, funding agencies, patient advocacy groups, and other interested stakeholders. Methods: An ad hoc international working group of the American Thoracic Society with experience in clinical, translational, and bench-based research in fibrotic lung diseases was convened. The group used an iterative consensus process to identify successes and challenges in pulmonary fibrosis research. Measurements and Main Results: The group identified five main priority areas in which substantial resources should be invested to advance our understanding and to develop novel therapies for patients with pulmonary fibrosis. These priorities include develop newer models of human lung fibrosis, engage current and new stakeholders to provide sustained funding for the initiatives, create a global infrastructure for storing patient-derived materials, establish collaborative preclinical and clinical research networks in fibrotic lung disease, and create a global lung fibrosis initiative that unites these multifaceted efforts into a single virtual umbrella structure. Conclusions: Despite recent advances in the treatment of some forms of lung fibrosis, many gaps in knowledge about natural history, pathophysiology, and treatment remain. Investment in the research priorities enumerated above will help address these shortcomings and enhance patient care worldwide. &nbsp

This review describes the challenges created by the existence of multiple molecular pathways leading to fibrosis and proposes that attention be focused on targeting the fibroblasts and myofibroblasts which themselves produce multiple cytokines and growth factors as well as the extracellular matrix, which is the hallmark of fibrotic lung disease.The last 20 years have seen remarkable progress in our understanding of the molecular pathogenesis of pulmonary fibrosis leading to multiple programmes in drug discovery, and there are currently 15 actively recruiting trials registered on http://www.clinicaltrials.gov. Unfortunately, at this time only one new drug, pirfenidone, has progressed to approval for use in patients. Part of the frustration is that drugs that are effective in targeting inflammatory pathways have been ineffective in lung fibrosis. This may result from the inability to treat patients early in the disease process but it is also likely that pathways independent of inflammation can drive fibrosis.We further propose that this approach should inhibit fibrosis independent of cell type or the signalling cascade that is activating these cells. We are hopeful that the next 20 years will see many more therapeutic options for patients suffering with fibrotic lung disease.

Caffeine is a commonly used food additive found naturally in many products. In addition to potently stimulating the central nervous system caffeine is able to affect various systems within the body including the cardiovascular and respiratory systems. Importantly, caffeine is used clinically to treat apnoea and bronchopulmonary dysplasia in premature babies. Recently, caffeine has been shown to exhibit antifibrotic effects in the liver in part through reducing collagen expression and deposition, and reducing expression of the profibrotic cytokine TGFβ. The potential antifibrotic effects of caffeine in the lung have not previously been investigated. Using a combined in vitro and ex vivo approach we have demonstrated that caffeine can act as an antifibrotic agent in the lung by acting on two distinct cell types, namely epithelial cells and fibroblasts. Caffeine inhibited TGFβ activation by lung epithelial cells in a concentration-dependent manner but had no effect on TGFβ activation in fibroblasts. Importantly, however, caffeine abrogated profibrotic responses to TGFβ in lung fibroblasts. It inhibited basal expression of the α-smooth muscle actin gene and reduced TGFβ-induced increases in profibrotic genes. Finally, caffeine reduced established bleomycin-induced fibrosis after 5 days treatment in an ex vivo precision-cut lung slice model. Together, these findings suggest that there is merit in further investigating the potential use of caffeine, or its analogues, as antifibrotic agents in the lung.

This is a story from recent history that we believe incoming President Trump urgently needs to hear. In 1985, a huge and growing hole in the planet's ozone layer was identified. Ozone in the stratosphere blocks some of the sun's ultraviolet radiation from reaching the Earth's surface, thus protecting its biosphere (which includes humans) from DNA damage that would otherwise occur. The hole was caused by the action of chlorofluorocarbons (CFCs) used in refrigeration and as aerosol propellants.1 Margaret Thatcher is admired by many on both sides of the Atlantic, including President Trump2 as a strong politician, a person with clear beliefs on which she acted; the ‘Iron Lady’ as Donald Trump has described her on Twitter. A scientist by background, Thatcher appreciated the magnitude of the threat immediately, throwing her weight behind international efforts to address this. Working with Ronald Reagan, within 2 years the 1987 Montreal Protocol was signed to phase out the production and use of CFCs. Despite this, because CFCs persist in the atmosphere for more than a century, the ozone layer will not recover completely until 2060. A failure of leadership at that time would have been catastrophic. CFCs are also powerful greenhouse gases and without the Montreal agreement the world would already …

Severe asthma is associated with airway remodeling, characterized by structural changes including increased smooth muscle mass and matrix deposition in the airway, leading to deteriorating lung function. TGF-β is a pleiotropic cytokine leading to increased synthesis of matrix molecules by human airway smooth muscle (HASM) cells and is implicated in asthmatic airway remodeling. TGF-β is synthesized as a latent complex, sequestered in the extracellular matrix, and requires activation for functionality. Activation of latent TGF-β is the rate-limiting step in its bioavailability. This study investigated the effect of the contraction agonists LPA and methacholine on TGF-β activation by HASM cells and its role in the development of asthmatic airway remodeling. The data presented show that LPA and methacholine induced TGF-β activation by HASM cells via the integrin αvβ5. Our findings highlight the importance of the β5 cytoplasmic domain because a polymorphism in the β5 subunit rendered the integrin unable to activate TGF-β. To our knowledge, this is the first description of a biologically relevant integrin that is unable to activate TGF-β. These data demonstrate that murine airway smooth muscle cells express αvβ5 integrins and activate TGF-β. Finally, these data show that inhibition, or genetic loss, of αvβ5 reduces allergen-induced increases in airway smooth muscle thickness in two models of asthma. These data highlight a mechanism of TGF-β activation in asthma and support the hypothesis that bronchoconstriction promotes airway remodeling via integrin mediated TGF-β activation.

IPF (idiopathic pulmonary fibrosis) is a chronic progressive disease of unknown aetiology without effective treatment. IPF is characterized by excessive collagen deposition within the lung. Recent evidence suggests that the lung epithelium plays a key role in driving the fibrotic response. The current paradigm suggests that, after epithelial injury, there is impaired epithelial proliferation and enhanced epithelial apoptosis. This in turn promotes lung fibrosis through impaired basement membrane repair and increased epithelial-mesenchymal transition. Furthermore, fibroblasts are recruited to the wounded area and adopt a myofibroblast phenotype, with the up-regulation of matrix-synthesizing genes and down-regulation of matrix-degradation genes. There is compelling evidence that the cytokine TGFbeta (transforming growth factor beta) plays a central role in this process. In normal lung, TGFbeta is maintained in an inactive state that is tightly regulated temporally and spatially. One of the major TGFbeta-activation pathways involves integrins, and the role of the (alpha)vbeta6 integrin has been particularly well described in the pathogenesis of IPF. Owing to the pleiotropic nature of TGFbeta, strategies that inhibit activation of TGFbeta in a cell- or disease-specific manner are attractive for the treatment of chronic fibrotic lung conditions. Therefore the molecular pathways that lead to integrin-mediated TGFbeta activation must be precisely defined to identify and fully exploit novel therapeutic targets that might ultimately improve the prognosis for patients with IPF.

Activation of latent transforming growth factor β (TGF-β) by αvβ6 integrin is critical in the pathogenesis of lung injury and fibrosis. We have previously demonstrated that the stimulation of protease activated receptor 1 promotes αvβ6 integrin-mediated TGF-β activation via RhoA, which is known to modulate cell contraction. However, whether other G protein-coupled receptors can also induce αvβ6 integrin-mediated TGF-β activation is unknown; in addition, the αvβ6 integrin signaling pathway has not yet been fully characterized. In this study, we show that lysophosphatidic acid (LPA) induces αvβ6-mediated TGF-β activation in human epithelial cells via both RhoA and Rho kinase. Furthermore, we demonstrate that LPA-induced αvβ6 integrin-mediated TGF-β activity is mediated via the LPA2 receptor, which signals via Gα q . Finally, we show that the expression levels of both the LPA2 receptor and αvβ6 integrin are up-regulated and are spatially and temporally associated following bleomycin-induced lung injury. Furthermore, both the LPA2 receptor and αvβ6 integrin are up-regulated in the overlying epithelial areas of fibrosis in patients with usual interstitial pneumonia. These studies demonstrate that LPA induces αvβ6 integrin-mediated TGF-β activation in epithelial cells via LPA2, Gα q , RhoA, and Rho kinase, and that this pathway might be clinically relevant to the development of lung injury and fibrosis.

Transforming growth factor beta (TGFbeta) is a key remodelling factor in asthma. It is produced as a latent complex and the main limiting step in TGFbeta bioavailability is its activation. Mast cell tryptase has been shown to stimulate the release of functionally active TGFbeta from human airway smooth muscle (ASM) cells [P. Berger, P.O. Girodet, H. Begueret, O. Ousova, D.W. Perng, R. Marthan, A.F. Walls, J.M. Tunon de Lara, Tryptase-stimulated human airway smooth muscle cells induce cytokine synthesis and mast cell chemotaxis, FASEB J. 17 (2003) 2139-2141]. The aim of this study was to determine if tryptase could cause TGFbeta activation as well as expression in ASM cells via its receptor, proteinase-activated receptor 2 (PAR2). Tryptase caused TGFbeta activation without affecting levels of total TGFbeta. This effect was inhibited by the selective tryptase inhibitor FUT175 and leupeptin but not mimicked by the PAR2 activating peptide SLIGKV-NH(2). Furthermore, the ASM cells used in the study did not express PAR2. The results indicate that tryptase activates TGFbeta via a PAR2-independent proteolytic mechanism in human ASM cells and may help understanding the role of tryptase in asthma.

An editorial board delivering basic science, translational science and clinical trials across paediatrics and adults We are incredibly proud and honoured to accept the position of Editors-in-Chief of Thorax. Thorax is the premier respiratory journal in the UK, and the official journal of the British Thoracic Society (BTS). It currently has an impact factor of 8.29, making it the third highest ranked respiratory journal in the world. We believe that Thorax has for many years reflected the strength of the British respiratory community publishing wide-ranging, high-calibre research across the translational spectrum. This view is supported by metrics showing that the UK has the highest H factor within Europe and the second highest H factor in the world after the USA. Furthermore, the UK is the third most cited country in the world, after the USA and China. The strong association with the BTS has been a major strength of the Journal because it represents the clinicians, clinical academics and scientists within respiratory medicine in the UK. We have ‘grown up’ with Thorax and the BTS and have seen how the Journal has grown from strength to strength under the strong, astute editorial guidance …

Idiopathic pulmonary fibrosis (IPF) is a progressive fibrotic lung disease with high mortality. Active TGFβ1 is considered central to the pathogenesis of IPF. A major mechanism of TGFβ1 activation in the lung involves the epithelially restricted αvβ6 integrin. Expression of the αvβ6 integrin is dramatically increased in IPF. How αvβ6 integrin expression is regulated in the pulmonary epithelium is unknown. Here we identify a region in the β6 subunit gene (ITGB6) promoter acting to markedly repress basal gene transcription, which responds to both the Ets domain-containing protein Elk1 (Elk1) and the glucocorticoid receptor (GR). Both Elk1 and GR can regulate αvβ6 integrin expression in vitro. We demonstrate Elk1 binding to the ITGB6 promoter basally and that manipulation of Elk1 or Elk1 binding alters ITGB6 promoter activity, gene transcription, and αvβ6 integrin expression. Crucially, we find that loss of Elk1 causes enhanced Itgb6 expression and exaggerated lung fibrosis in an in vivo model of fibrosis, whereas the GR agonist dexamethasone inhibits Itgb6 expression. Moreover, Elk1 dysregulation is present in epithelium from patients with IPF. These data reveal a novel role for Elk1 regulating ITGB6 expression and highlight how dysregulation of Elk1 can contribute to human disease.

Summary Background Idiopathic pulmonary fibrosis, a progressive and inevitably fatal disorder, has a highly variable clinical course. Biomarkers that reflect disease activity are urgently needed to inform patient management and for use as biomarkers of therapeutic response (theragnostic biomarkers) in clinical trials. We aimed to determine whether dynamic change in markers of extracellular matrix (ECM) turnover predicts progression of idiopathic pulmonary fibrosis as determined by change in forced vital capacity and death. Methods In this ongoing prospective, multicentre, observational cohort study (PROFILE), participants with idiopathic pulmonary fibrosis or idiopathic non-specific interstitial pneumonia diagnosed within the preceding 6 months were recruited from two coordinating centres (Nottingham, UK, and, Royal Brompton Hospital, London, UK). Serum samples were prospectively collected at baseline, 1 month, 3 months, and 6 months and were analysed for a panel of novel matrix metalloprotease (MMP)-degraded ECM proteins, by ELISA-based, neoepitope assay. 11 neoepitopes were tested in a discovery cohort of 55 patients to identify biomarkers of sufficient rigour for more detailed analyses. Eight were then further assessed in a validation cohort of 134 patients with 50 age-matched and sex-matched controls. Changes in biomarker concentrations were related to subsequent progression of idiopathic pulmonary fibrosis (defined as death or decline in forced vital capacity >10% at 12 months after study enrolment) using a repeated measures model. The PROFILE study is registered on ClinicalTrials.gov, numbers NCT01134822 and NCT01110694. Findings Of 214 eligible participants recruited between Sept 1, 2010, and March 31, 2012, 189 had a confirmed diagnosis of idiopathic pulmonary fibrosis and were included in subsequent analyses. In the discovery cohort, mean concentrations of seven neoepitopes (BGM, p=0·009; C1M, p=0·009; C3M, p=0·046; C6M, p=0·032; CRPM, p=0·008; ELM2, p=0·02; and VICM, p=0·0007) differed significantly between healthy controls and participants with idiopathic pulmonary fibrosis. Baseline concentrations of six neoepitopes (C1M, p=0·012; C3A, p=0·012; C3M, p=0·0005; C6M, p=0·0003; CRPM, p=0·021; and VICM, p=0·046) were significantly higher in patients with progressive idiopathic pulmonary fibrosis (n=32) than in those with stable disease (n=23). In the validation cohort, mean concentrations of C1M (p=0·001), C3M (p=0·044), C6M (p=0·003), and CRPM (p=0·024) at baseline were higher in patients with idiopathic pulmonary fibrosis than in healthy controls. When assessed longitudinally, concentrations of six neoepitopes (BGM, C1M, C3A, C3M, C6M, and CRPM) were significantly higher in patients with progressive idiopathic pulmonary fibrosis (n=71) than in patients with stable idiopathic pulmonary fibrosis (n=60) by 6 months. Baseline concentrations of two neoepitopes were associated with increased mortality (C1M: HR 1·62 [95% CI 1·14–2·31], p=0·0069; C3A: 1·91 [1·06–3·46], p=0·032). The rate of change between baseline and 3 months of six neoepitopes (BGM: HR 1·084 [95% CI 1·03–1·14], p=0·0019; C1M: 1·01 [1·003–1·017], p=0·0039; C3M: 1·106 [1·045–1·170], p=0·0005; C5M: 1·003 [1·001–1·005], p=0·0011; C6M: 1·042 [1·007–1·078], p=0·017; and CRPM: 1·38 [1·16–1·63], p=0·0002) was strongly predictive of overall survival, and the increased risk was proportional to the magnitude of change in neoepitope concentrations. The strongest association with 3-month rate of biomarker change was recorded for CRPM; greater than 0 ng/mL per month conferred a HR of 2·16 (95% CI 1·15–4·07), whereas a rate greater than 1 ng/mL per month resulted in an HR 4·08 (2·14–7·8), and a rate greater than 1·7 ng/mL per month was associated with an HR 6·61 (2·74–15·94). Interpretation Concentrations of protein fragments generated by MMP activity are increased in the serum of individuals with idiopathic pulmonary fibrosis compared with healthy controls. Increased neoepitope concentrations were associated with disease progression, and the rate of this increase predicted survival. Serial measurements of neoepitopes have potential to be used as theragnostic biomarkers in clinical trials and to guide management of idiopathic pulmonary fibrosis. Funding GlaxoSmithKline R&D and the Medical Research Council.

Purpose Asthma is a disease of increasing worldwide importance that calls for new investigative methods. Ex vivo lung tissue is being increasingly used to study functional respiratory parameters independent of confounding systemic considerations but also to reduce animal numbers and associated research costs. In this work, a straightforward laboratory method is advanced to probe dynamic changes in gas inhalation patterns by using an ex vivo small animal ovalbumin (OVA) model of human asthma. Methods Hyperpolarized (hp) 129Xe was actively inhaled by the excised lungs exposed to a constant pressure differential that mimicked negative pleural cavity pressure. The method enabled hp 129Xe MRI of airway responsiveness to intravenous methacholine (MCh) and airway challenge reversal through salbutamol. Results Significant differences were demonstrated between control and OVA challenged animals on global lung hp 129Xe gas inhalation with P

Activation of latent TGF-beta by the alpha(v)beta6 integrin is a critical step in the development of acute lung injury. However, the mechanism by which alpha(v)beta6-mediated TGF-beta activation is regulated has not been identified. We show that thrombin, and other agonists of protease-activated receptor 1 (PAR1), activate TGF-beta in an alpha(v)beta6 integrin-specific manner. This effect is PAR1 specific and is mediated by RhoA and Rho kinase. Intratracheal instillation of the PAR1-specific peptide TFLLRN increases lung edema during high-tidal-volume ventilation, and this effect is completely inhibited by a blocking antibody against the alpha(v)beta6 integrin. Instillation of TFLLRN during high-tidal-volume ventilation is associated with increased pulmonary TGF-beta activation; however, this is not observed in Itgb6-/- mice. Furthermore, Itgb6-/- mice are also protected from ventilator-induced lung edema. We also demonstrate that pulmonary edema and TGF-beta activity are similarly reduced in Par1-/- mice following bleomycin-induced lung injury. These results suggest that PAR1-mediated enhancement of alpha(v)beta6-dependent TGF-beta activation could be one mechanism by which activation of the coagulation cascade contributes to the development of acute lung injury, and they identify PAR1 and the alpha(v)beta6 integrin as potential therapeutic targets in this condition.

Idiopathic pulmonary fibrosis (IPF) is a devastating condition with a poor prognosis and few treatment options. However, recent research into this condition has led to considerable insights into the pathophysiology of the disease, resulting in the identification of potential biomarkers to aid diagnosis and stratification of patients and the development of novel therapies. In this review we will discuss the recent developments in this field and review how this knowledge has been translated into clinical trials and a paradigm shift in our approach to patients with IPF.

Pirfenidone works. There have been four randomised placebo-control trials of pirfenidone for the treatment of idiopathic pulmonary fibrosis (IPF); a phase II and phase III study in Japan, and two international multicentre phase III studies.1–3 In all four studies, patients treated with pirfenidone had slower rates of decline in lung volume (vital capacity (VC) in the Japanese studies and forced vital capacity (FVC) in the international studies) than placebo, and in three studies, the results were statistically significant. National Institute of Clinical Excellence (NICE) agrees that pirfenidone has a ‘modest but measurable effect on slowing the decline in lung function’.4 Therefore, whether patients receive pirfenidone depends on whether pirfenidone works enough to justify its cost. IPF is a chronic progressive disease of unknown aetiology, it is fatal with a median survival of approximately 3 years,5 or less than one electoral cycle. No therapy has been proven to improve survival. Despite the absence of good evidence, various combinations of N-acetylcysteine (NAC), prednisolone and azathioprine have been used for many years to treat patients with IPF. In the early 1990s, a small study suggested that prednisolone and azathioprine might be beneficial,6 and the initial British Thoracic Society (BTS) guidelines for the treatment of cryptogenic fibrosing alveolitis (as IPF was then known) recommended this therapy.7 In 2005, the Idiopathic Pulmonary Fibrosis International Group Exploring N-Acetylcysteine I Annual (IFIGENIA) study demonstrated that NAC might be beneficial in IPF.8 However, this study was criticised at the time for including azathioprine and prednisolone in both the placebo and treatment arms. The inclusion of this faux placebo lead some, possibly correctly, to suggest that NAC was inhibiting the adverse effects of prednisolone and azathioprine, rather than possessing any direct antifibrotic effect. The 2008 BTS IPF guidelines9 implicitly recommended treatment with triple …

Levels of prostaglandin E(2) (PGE(2)), a potent inhibitor of fibroblast function, are decreased in the lungs of patients with pulmonary fibrosis, which has been shown to be because of limited expression of cyclooxygenase-2 (COX-2). To further investigate the relative importance of COX-2 and PGE(2) in the development of fibrosis we have used a selective COX-2 inhibitor and COX-2-deficient ((-/-) and (+/-)) mice in studies of bleomycin-induced lung fibrosis. We demonstrate in wild-type mice that bleomycin-induced lung PGE(2) production is predominantly COX-2 mediated. Furthermore, COX-2(+/-) mice show limited induction of PGE(2) and an enhanced fibrotic response with increased lung collagen content compared with wild-type mice after bleomycin injury (P0.001). In contrast, COX-2(-/-) mice show increased levels of lung PGE(2), compared with wild-type mice after injury (P0.05), because of compensatory up-regulation of COX-1, which appears to be associated with macrophage/monocytes but not fibroblasts derived from these mice. COX-2(-/-) mice show an enhanced and persistent inflammatory response to bleomycin, however the fibrotic response to injury was unaltered compared with wild-type animals. These data provide further direct evidence for the importance of up-regulating COX-2 and PGE(2) expression in protecting against the development of fibrosis after lung injury.

Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator prostaglandin E2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF). This study explored the role of G9a- and enhancer of zeste homolog 2 (EZH2)-mediated methylation of histone H3 lysine 9 (H3K9me3) and histone H3 lysine 27 (H3K27me3) in COX-2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and re-ChIP assays demonstrated marked increases in H3K9me3, H3K27me3, and DNA methylation, together with their respective modifying enzymes G9a, EZH2, and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and methyl CpG binding protein 2 (MeCP2), at the COX-2 promoter in lung fibroblasts from patients with IPF (F-IPFs) compared with fibroblasts from nonfibrotic lungs. HP1, EZH2, and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPFs. G9a and EZH2 inhibitors and small interfering RNAs and the Dnmt1 inhibitor markedly reduced H3K9me3 (49−79%), H3K27me3 (44−81%), and DNA methylation (61−97%) at the COX-2 promoter. These reductions were correlated with increased histone H3 and H4 acetylation, resulting in COX-2 mRNA and protein reexpression in F-IPFs. Our results support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing, and interdependent crosstalk between histone hypermethylation and DNA methylation in COX-2 epigenetic silencing in IPF.—Coward, W. R., Feghali-Bostwick, C. A., Jenkins, G., Knox, A. J., Pang, L. A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis.

Prostaglandin E(2) (PGE(2)) inhibits fibroblast proliferation and collagen production. Its synthesis by fibroblasts is induced by profibrotic mediators including transforming growth factor (TGF)-beta(1). However, in patients with pulmonary fibrosis, PGE(2) levels are decreased. In this study we examined the effect of TGF-beta(1) on PGE(2) synthesis, proliferation, collagen production, and cyclooxygenase (COX) mRNA levels in fibroblasts derived from fibrotic and nonfibrotic human lung. In addition, we examined the effect of bleomycin-induced pulmonary fibrosis in COX-2-deficient mice. We demonstrate that basal and TGF-beta(1)-induced PGE(2) synthesis is limited in fibroblasts from fibrotic lung. Functionally, this correlates with a loss of the anti-proliferative response to TGF-beta(1). This failure to induce PGE(2) synthesis is because of an inability to up-regulate COX-2 mRNA levels in these fibroblasts. Furthermore, mice deficient in COX-2 exhibit an enhanced response to bleomycin. We conclude that a decreased capacity to up-regulate COX-2 expression and COX-2-derived PGE(2) synthesis in the presence of increasing levels of profibrotic mediators such as TGF-beta(1) may lead to unopposed fibroblast proliferation and collagen synthesis and contribute to the pathogenesis of pulmonary fibrosis.

BACKGROUND: Laboratory studies suggest that the clotting cascade is activated in fibrotic lungs. Since humans vary in their tendency to clot due to a variety of inherited or acquired defects, we investigated whether a prothrombotic state increases the chance of developing idiopathic pulmonary fibrosis (IPF) and/or worsens the prognosis of IPF. \ud \ud METHODS: We recruited 211 incident cases of IPF and 256 age-and sex-matched general population controls and collected data on medical history, medication, smoking habit, blood samples as well as lung function and high-resolution CT scans done as part of routine clinical care. A prothrombotic state was defined as the presence of at least one inherited or acquired clotting defect or marker of fibrinolytic dysfunction. We used logistic regression to quantify the association between a prothrombotic state and IPF adjusted for age, sex, smoking habit and highly sensitive C reactive protein. Cox regression was used to determine the influence of a prothrombotic state on survival. \ud \ud RESULTS: Cases were more than four times more likely than controls to have a prothrombotic state (OR 4.78, 95% CI 2.93 to 7.80; p