D Shaw

S92 Matrix metalloproteinase-1 activation by mast cell tryptase causes airway remodelling and is associated with bronchial hyper-responsiveness in patients with asthma

Introduction Matrix Metalloproteinase-1 (MMP-1) is a collagenase, which is present, in its inactive form, in the airways, lung parenchyma and in broncho-alveolar lavage (BAL) fluid of patients with asthma. We hypothesised that MMP-1 could be activated during asthma exacerbations leading to extra-cellular matrix (ECM) processing which contributes to airway remodelling. Methods Patients with stable, BTS stage 2/3 asthma, and healthy controls underwent Juniper asthma questionnaire, spirometry, methacholine challenge and bronchoscopy. Bronchial washings were processed for MMP-1 protein and activation. A second cohort of 14 patients with mild and 16 with moderate asthma and 10 controls underwent rhinovirus inoculation and had BAL fluid collected 14 days before and 4 days after inoculation. MMP-1 activity was assessed by fluorescent activity assay. ECM was prepared from decellularised airway smooth muscle (ASM) cultures. Cell proliferation was measured by MTT reduction assay and cell counts. Mast cell supernatants were obtained from cultures of HMC-1 cells activated using Phorbol 12-myristate 13-acetate/Calcium ionophore. Results Pro-MMP-1 was expressed more strongly in bronchial washings in asthma than controls (P = 0.0003). After rhinovirus inoculation, asthma symptoms increased and lung function fell. BAL MMP-1 activity increased in asthma patients compared with controls (P = 0.047). MMP-1 protein and activity was positively associated with fall in FEV1 (R square = 0.3618) (P = 0.0039) post viral inoculation. Activated, but not control, mast cell supernatants increased both expression of pro- and active MMP-1 by ASM cell cultures. This was blocked almost completely by inhibitors of tryptase but not chymase or MMPs. Recombinant tryptase activated MMP-1 in vitro. ECM obtained from both control and asthma derived ASM cells treated with activated mast cell supernatants during matrix synthesis and ECM treated directly after decellularization with active MMP-1 (10 ng/ml) enhanced subsequent ASM growth by 1.5 fold (P < 0.05). Conclusions MMP-1 expression and activity in bronchial fluid is enhanced during asthma exacerbations and is associated with increased BHR. MMP-1 activation by mast cell tryptase processes ASM derived ECM to enhance ASM growth in-vitro. Our findings suggest that ASM/mast cell interactions during exacerbations may contribute to airway remodelling by generating a pro-proliferative matrix.

S86 Extracellular matrix deposited by asthmatic human airway smooth muscle cells enhances basal activation of tgfβ

TGFß is a widely distributed, pleiotropic cytokine implicated in tissue remodelling in many diseases including severe asthma. It is secreted in a latent form that requires activation for function. Mechanotransduction of intracellular forces through cell surface integrins can lead to TGFß activation. We have previously shown that human airway smooth muscle (HASM) cells can activate TGFβ via αvβ5 integrins. In the present study we have investigated the effect of extracellular matrix (ECM) on basal TGFβ activation in primary asthmatic and non-asthmatic HASM cells. We assessed basal TGFβ activation in non-asthmatic (n=9) and asthmatic (n=7) HASM cells. TGFβ activation was assessed using a TGFβ reporter cell assay and a phosphorylated Smad2 ELISA. Cell contractility was assessed using a collagen gel contraction assay and traction force microscopy. ECM was isolated from HASM cells and cross-over experiments performed where non-asthmatic cells were cultured on asthmatic ECM and vice-versa then TGFβ activity determined. Expression of ECM crosslinking enzymes was determined at the mRNA and protein levels. Finally, expression of LOXL2 was determined in an Aspergillus fumigatus model of asthma.Asthmatic HASM cells activated 3-fold higher levels of TGFβ basally than non-asthmatic cells (p<0.01). Both diseased and control HASM cells increased TGFβ activation in response to methacholine confirming our previous data (Tatler et al 2011). A collagen gel contraction assay demonstrated that asthmatic HASM cells were hypercontractile compared with non-asthmatic cells under basal conditions (p<0.05) and that contractility weakly correlated with amount of TGFβ activated (p<0.05). Importantly, culturing non-asthmatic HASM cells on asthmatic ECM led to increased TGFβ activation (p<0.05) and culturing asthmatic HASM cells on non-asthmatic ECM decreased TGFβ activation (p<0.05). mRNA Expression of the ECM crosslinking enzymes LOXL2 and LOXL3 was significantly increased in asthmatic HASMs (p<0.05). Finally, LOXL2 protein was increased in asthmatic HASMs cells, and increased in the airway smooth muscle layer of animals challenged repeatedly with Asp. f compared with control challenged animals. In conclusion HASM cells derived from asthmatic patients exhibit enhanced activation of TGFβ compared with non-asthmatic HASM cells. This may be driven by the diseased ECM since asthmatic HASMs cells exhibit aberrant expression of ECM crosslinking enzymes.

P238 Investigating genome wide dna methylation in airway smooth muscle cells from asthmatic and non-asthmatic donors

Rationale Genetic mechanisms fail to fully explain asthma pathogenesis and environmental factors are considered to play an important role. Environmental factors may lead to permanent changes in epigenetic patterns and contribute to asthma. Epigenetics is the study of heritable changes in gene expression that are not due to changes in DNA sequence. DNA methylation is a reversible modification of DNA structure in which a methyl group is added to cytosine residues. Parental smoking affects the methylation of buccal cell DNA from children and children with early onset wheeze have an altered blood DNA methylation profile to healthy individuals. No studies have compared DNA methylation profiles in the disease relevant cell type of airway smooth muscle (ASM) cells. Methods DNA was isolated from ASM cells at passage 5 and bisulphite treated to convert epigenetic information into sequence-based information. Site specific, quantitative genome wide methylation was determined using the Illumina 450K Infinium Methylation BeadChip array. Hits were validated by Pyrosequencing. RNA was extracted simultaneously for mRNA expression analysis by real time PCR. Results There were no independent CpG sites associated with asthmatic status of ASM cells following multiple test correction. Without correction over 13000 CpG sites showed a significant difference in methylation (linear modelling, p value >0.05) between asthmatic and non-asthmatic cells, and a biologically relevant difference in methylation of greater that 10% (β value >0.1 ). 10 of these sites were selected as top hits. 7 sites positively validated by pyrosequencing. They were associated with 7 different genes; LGALS3BP, ATP11A, ZNF696, KLF6, TBX1, RUNX3, and SPINT2. Expression of these genes was measured in ASM cells isolated from asthmatic and non-asthmatic donors. LGALS3BP expression was undetectable while ATP11A and ZNF696 displayed no difference in expression between cells from asthmatic and non-asthmatic donors. KLF6 and SPINT2 showed a trend towards increased expression in cells from asthmatic donors while RUNX3 and TBX1 showed a trend towards decreased expression. Conclusions Differences in CpG methylation exist between ASM isolated from asthmatic and non-asthmatic donors. Future work will focus on identifying differentially methylated regions of DNA and further defining the association to gene and protein expression.

P236 The role of histone arginine methylation in gene expression of airway smooth muscle cells in asthma

Introduction and objectives Asthma is estimated to affect at least 300 million people globally. About 25% of the patients do not respond to therapy; therefore we need to develop novel treatments. ASM cells have a crucial role in asthma, contributing to airway remodelling, inflammation and airflow obstruction. We have previously shown that epigenetic histone modifications, particularly histone lysine acetylation and methylation regulate the secretion of inflammatory mediators from ASM cells. Here we tested the hypothesis that histone arginine changes are also involved. Protein arginine N-methyltransferases (PRMTs) are the enzymes which catalyse histone arginine methylation (HRme, the addition of a methyl group to arginine residues on the N-terminal tails of histones), and inhibiting them represents a strategy to reduce the secretion of inflammatory mediators from ASM cells. Methods Studies were performed in cultured human ASM cells from asthmatic and non-asthmatic donors at passage 6. PRMT expression in human ASM cells was investigated by qPCR. Protein levels of four PRMTs in human ASM cells were investigated by western blotting. As PMRT1 has previously been suggested to play a role in mouse asthma models, we studied the association of PRMT1 with eotaxin, IL-6, IP-10 and CXCL8 promoters in healthy ASM cells, under basal conditions and following stimulation with TNF-α (1ng/ml), by chromatin immunoprecipitation (ChIP). IgG was used as a negative control, while acetylated histone H4 (AcH4) was used as a positive control. Results We found that ASM cells express the PRMT1, PRMT2, PRMT3, CARM1, PRMT5, PRMT6, PRMT7 and FBX011 mRNA and PRMT1, CARM1, PRMT5, and PRMT6 protein. The analysis showed no difference in the levels of expression between cells isolated from asthmatic and non-asthmatic donors. Under basal conditions, PRMT1 was associated with all of the promoters and association increased following 1 hour stimulation with TNF-α. Conclusions ASM cells express a number of PRMTs at mRNA and protein levels. PRMT1 associates with a number of chemokine and cytokine promoters after TNF-α stimulation. PRMTs may have an important role in regulating chemokine production from ASM cells in asthma, and are a promising target for future investigations in asthma.

P44 Chronic obstructive pulmonary disease exacerbation and respiratory acidosis: patient outcomes at 6 months

Introduction Recognition of hypercapnic respiratory failure is a vital part of the assessment and management of the patient with an acute exacerbation of chronic obstructive pulmonary disease (COPD). Several studies have demonstrated that respiratory acidosis in the context of an acute exacerbation is associated with worse inpatient outcomes. Our study compares the outcomes of patients admitted with an acute exacerbation, between those with respiratory acidosis and those who had a normal pH and PaCO2 on arterial blood gas (ABG) analysis. Methods Patients requiring hospital treatment for an acute exacerbation of COPD had an ABG taken on admission. Patients were subsequently assessed for the following outcomes: inpatient mortality, outpatient mortality up to six months after discharge and hospital re-admission rates in the six months post discharge. Chi-squared test was applied to assess the relationship between respiratory acidosis and our outcomes. Results 234 patients had an admission ABG and were subsequently followed up to the point of death or six months post discharge. Patients with a PaCO2 of >6 Kpa were 2.33 times (95% CI 1.11 to 4.96) more likely to die in hospital as compared to those patients with a normal value. Patients with a lower arterial pH (<7.35) were 2.32 times (95% CI 1.07 to 4.96) more likely to die in hospital as compared to those with a pH of >7.35. The increased risk in mortality was only seen for in-hospital mortality and there was no association with death in the 6 months following discharge, hospital re-admission or re-admission for a respiratory problem. Conclusion This data supports previous studies that suggest hypercapnia and respiratory acidosis are associated with increased inpatient mortality, therefore further demonstrating the usefulness of pH and PaCO2 as prognostic markers for inpatient outcomes. However our study does suggest that patients with respiratory acidosis on admission, who survive until discharge from hospital, do not have an increased risk of six month mortality or readmission compared to those with a normal admission ABG.

P219 Potential impact of air pollution coverage in the media on respiratory disease admissions

Introduction Air pollution can exacerbate respiratory disease. However, when air quality warnings are provided by authoritative bodies (e.g. MET office) media coverage may be disproportionate. We explored whether there is an association between respiratory admissions and media pollution coverage via non-linear predictive models, to potentially predict respiratory admissions. Methods The association was examined as follows: Baseline regression models were generated to predict daily respiratory admission episodes from 1st January 2009–9th April 2014. Predictors consisted of daily logs for PM10 particulate matter, PM2.5, Nitric oxide, Nitrogen dioxide, Ozone, Black carbon, Mean Temperature, Precipitation (obtained from National Oceanic and Atmospheric Administration data) and the DAQI Air Pollution Index. Models were optimised via cross-validation using daily respiratory admission levels sourced from Nottingham University Hospitals Trust data (ICD10 codes J39 – J9999). Time series of levels of media coverage were generated by applying kernel density estimation at a range of bandwidths (using linear and exponential kernels at bandwidths of 1, 10, 25, 50 and 100 days) to daily counts of online news articles featuring pollution and air quality issues over the period 01.01.2013 – 9.04. 2014. Predictive model accuracies were compared following integration of these time series of media coverage levels as an additional predictor. Results Of the predictive models tested, random forests parameterized provided optimal results for air-quality predictors. When predicting daily respiratory admissions, the model’s accuracy was 19.90% better than simply predicting mean daily admissions, with an average root mean square error (RMSE) of 7.5031. However, on introduction of the media-coverage variable, RMSE was reduced to 7.3210, representing a 21.85% improvement over mean prediction. While this reflected a slight improvement in admissions forecasting, a corrected t-test suggested these differences were not statistically significant, with a p-value of 0.0633. Conclusion Initial results indicate that consideration of media coverage may offer minor improvements in predicting respiratory admissions, but this effect was not statistically significant. While such a relationship requires further investigation, models informed by media coverage cannot currently be considered to be accurate enough for use in a practical setting. Better media data collection may improve prediction accuracy.

P157 Individual patterns of inhaler use and health outcomes in adolescents with asthma

Background Electronic monitoring devices with an audio-visual reminder function can significantly improve asthma inhaler adherence and control in children.1 However, the relationship between attitudes, patterns of medication use and clinical outcomes are unknown. Aim To examine individual patterns of inhaled corticosteroid use, and their relationships with clinical outcomes and qualitative feedback in adolescents with asthma. Methods An exploratory study based on previous qualitative research investigating the attitudes of adolescents with asthma towards inhaler monitoring and data sharing. Patients from a specialist severe asthma clinic had their preventer inhaler use electronically monitored for one-month with a SmartTrack (Nexus6, Auckland, NZ) device. Adherence data was obtained and participants completed a questionnaire and interview at the beginning, middle and end of the trial on their attitudes. Ten months later, participants’ case notes were examined for information related to their health before and after the study. Results Spirometric data was captured on 4/7 participants and is presented alongside adherence data in Table 1. Daily adherence ranged from 67%–93% with the largest FEV1 change (+0.95) observed in P1 who had an average daily adherence of 73%, and the smallest FEV1 change (-0.18) observed in P3 who had an average daily adherence of 93%. All changes occurred without intensification of treatment. This fits with the previous qualitative findings that participants were enthusiastic about the reminders the SmartTrack device provided and felt more conscious of adhering to their treatment plan when they knew someone would be monitoring it. Participants spoke positively for utilising the data to demonstrate their adherence to their parents or doctor.Abstract P157 Table 1 Forced Expiratory Volume (FEV1) Spirometry values for four of the seven participants pre and post study Pre Study Study Period Post-Study Participant FEV1 Value (L) Days Before Study Start Date Daily Adherence average (%) mean AM Adherence average (%) mean PM Adherence average (%) mean FEV1 Value (L) Change in FEV1 after study participation Days After Study End Date P1 2.15 28 73 71 76 3.1 +0.95 372 P2 4.76 0 93 97 90 5.43 +0.67 156 P3 2.86 23 93 94 92 2.68 -0.18 69 P4 2.36 118 67 66 68 2.61 +0.25 71 Conclusions Through examining inhaled corticosteroid use, attitudes and clinical outcomes we gain an understanding of each patient’s condition including their habits with their inhaler (e.g. better adherence in the evenings), their attitudes to their asthma treatment and the potential effects of inhaler use on their health. Doing so helps us to identify patients who could benefit from intervention, to improve their inhaler taking behaviour and potentially improve their asthma control. Reference 1 Chan A, Stewart AW, Harrison J, et al. The effect of an electronic monitoring device (…). Respir Med. 2015;3:210–19

P186 Mast cell mediators stimulate human airway smooth muscle growth, a feature of airway remodelling in asthma via matrix metalloproteinase (MMP-1) activity

Introduction and Objective : Increased airway smooth muscle (ASM) mass and infiltration by mast cells are key features of airway remodelling in asthma. We tested a hypothesis to investigate the relationship between ASM growth, mast cell mediators and the matrix metalloproteinase MMP-1 activity. Methods : Primary ASM cultures were derived from a healthy subject. ASM cells were cultured for up to 2 days firstly in the presence and then in absence of serum and treated with conditioned media either collected from activated mast cells cultures or inactive/ unstimulated mast cells cultures. Mast cells were grown in suspension and activated using phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (A23187) to release serine proteases such as histamine, beta-tryptase and chymase etc. We also performed western blots to determine MMP-1 activity in the supernatants of ASM cultures. Results ASM cells treated with stimulated mast cells conditioned media showed increased cell proliferation by almost 2 folds after 48 hours of incubation under serum free conditions confirmed by cell counting and MTT assay in comparison with untreated airway smooth muscle cells or ASM cells treated with inactive/ unstimulated mast cells culture media. Furthermore our experiments showed that matrix metalloproteinase (MMP -1) levels and activity was significantly increased in ASM cultures treated with activated mast cells as compared to other two control conditions as mentioned earlier. Conclusion These findings clearly indicate role of mast cell proteases in ASM proliferation and therefore airway remodeling in asthma, a mechanism that perhaps is modulated by MMP-1 activity. We further suggest that the pathway will prove susceptible to pharmacological intervention for treatment of chronic asthma.

P161 A randomised controlled trial of single combination budesonide/formoterol inhaler as maintenance and reliever therapy in asthma patients at risk of severe exacerbations

Introduction The Single budesonide/formoterol inhaler Maintenance And Reliever Therapy (SMART) regimen reduces severe asthma exacerbations, but it is uncertain whether it increases the risk of adverse effects due to high beta-agonist and corticosteroid doses with both short-term and cumulative exposure. Objectives Our hypothesis was that treatment with the SMART regimen would reduce the risk of beta-agonist overuse but that when such episodes occurred, patients were less likely to seek medical review and, that any reduction in severe exacerbations would be at the cost of a higher systemic corticosteroid burden. Methods This was a 24-week, open-label, parallel group trial in 303 asthma patients with a recent exacerbation, conducted at four primary healthcare practices and one secondary care hospital. Participants were randomised to 200/6µg budesonide/formoterol metered dose inhaler according to the SMART regimen (two actuations twice daily maintenance with one extra actuation as-needed) or a fixed-dose regimen (two actuations twice daily maintenance) with one to two actuations of 100µg salbutamol as-needed (‘Standard’), with electronic monitoring of actual inhaler use. The primary outcome was the proportion of participants with at least one high beta-agonist use episode (>12 budesonide/formoterol actuations/day in SMART or >16 salbutamol actuations/day in Standard). Results There was no significant difference between groups in the proportion of participants with at least one high use episode: SMART 84/151 (56%) vs Standard 68/152 (45%) participants; relative risk (95% CI) 1.24 (0.99–1.56), p = 0.058. There were fewer days of high use [mean (SD) 5.1 days (14.3) vs 8.9 days (20.9), relative rate (RR) (95% CI) 0.58 (0.39–0.88), p = 0.01] and days of high use without medical review [8.5 days (17.8) vs 18.3 days (24.8) per high use patient, RR 0.49 (0.31–0.75), p = 0.001] in the SMART group. The SMART regimen resulted in higher average daily inhaled corticosteroid exposure, but reduced oral corticosteroid exposure, with no difference in composite systemic corticosteroid exposure [ratio of means (95% CI) 1.03 (0.86–1.22), p = 0.76]. SMART participants had fewer severe asthma exacerbations [35 vs 66, RR 0.54 (0.36–0.82), p = 0.004]. Conclusions The SMART regimen has a favourable risk/benefit profile in patients at risk of severe asthma exacerbations.