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Cellular senescence contributes to the pathophysiology of chronic obstructive pulmonary disease (COPD) and cardiovascular disease. Using endothelial colony-forming-cells (ECFC), we have demonstrated accelerated senescence in smokers and patients with COPD compared with non-smokers. Subgroup analysis suggests that ECFC from patients with COPD on inhaled corticosteroids (ICS) (n=14; eight on ICS) exhibited significantly reduced senescence (Senescence-associated-beta galactosidase activity, p21CIP1), markers of DNA damage response (DDR) and IFN-γ-inducible-protein-10 compared with patients with COPD not on ICS. In vitro studies using human-umbilical-vein-endothelial-cells showed a protective effect of ICS on the DDR, senescence and apoptosis caused by oxidative stress, suggesting a protective molecular mechanism of action of corticosteroids on endothelium.

Rationale: Mast cells (MCs) play a role in inflammation and both innate and adaptive immunity, but their involvement in severe asthma (SA) remains undefined. Objectives: We investigated the phenotypic characteristics of the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes) asthma cohort by applying published MC activation signatures to the sputum cell transcriptome. Methods: Eighty-four participants with SA, 20 with mild/moderate asthma (MMA), and 16 healthy participants without asthma were studied. We calculated enrichment scores (ESs) for nine MC activation signatures by asthma severity, sputum granulocyte status, and three previously defined sputum molecular phenotypes or transcriptome-associated clusters (TACs) 1, 2, and 3 using gene set variation analysis. Measurements and Main Results: MC signatures except unstimulated, repeated FceR1-stimulated and IFN-g–stimulated signatures were enriched in SA. A FceR1-IgE–stimulated and a single-cell signature from asthmatic bronchial biopsies were highly enriched in eosinophilic asthma and in the TAC1 molecular phenotype. Subjects with a high ES for these signatures had elevated sputum amounts of similar genes and pathways. IL-33– and LPS-stimulated MC signatures had greater ES in neutrophilic and mixed granulocytic asthma and in the TAC2 molecular phenotype. These subjects exhibited neutrophil, NF-kB (nuclear factor-kB), and IL-1b/TNF-a (tumor necrosis factor-a) pathway activation. The IFN-g–stimulated signature had the greatest ES in TAC2 and TAC3 that was associated with responses to viral infection. Similar results were obtained in an independent ADEPT (Airway Disease Endotyping for Personalized Therapeutics) asthma cohort. Conclusions: Gene signatures of MC activation allow the detection of SA phenotypes and indicate that MCs can be induced to take on distinct transcriptional phenotypes associated with specific clinical phenotypes. IL-33–stimulated MC signature was associated with severe neutrophilic asthma, whereas IgE-activated MC was associated with an eosinophilic phenotype.

IntroductionCigarette smoking and oxidative stress are common risk factors for the multi-morbidities associated with chronic obstructive pulmonary disease (COPD). Elevated levels of advanced glycation endproducts (AGE) increase the risk of cardiovascular disease (CVD) comorbidity and mortality. The enzyme fructosamine-3-kinase (FN3K) reduces this risk by lowering AGE levels.MethodsThe distribution and expression of FN3K protein in lung tissues from stable COPD and control subjects, as well as an animal model of COPD, was assessed by immunohistochemistry. Serum FN3K protein and AGE levels were assessed by ELISA in patients with COPD exacerbations receiving metformin. Genetic variants within the FN3K and FN3K-RP genes were evaluated for associations with cardiorespiratory function in the Subpopulations and Intermediate Outcome Measures in COPD Study cohort.ResultsThis pilot study demonstrates that FN3K expression in the blood and human lung epithelium is distributed at either high or low levels irrespective of disease status. The percentage of lung epithelial cells expressing FN3K was higher in control smokers with normal lung function, but this induction was not observed in COPD patients nor in a smoking model of COPD. The top five nominal FN3K polymorphisms with possible association to decreased cardiorespiratory function (pDiscussionThe data highlight that low and high FN3K expressors exist within our study cohort and metformin induces FN3K levels, highlighting a potential mechanism to reduce the risk of CVD comorbidity and mortality.

BACKGROUND: Sputum analysis in asthmatic patients is used to define airway inflammatory processes and might guide therapy. OBJECTIVE: We sought to determine differential gene and protein expression in sputum samples from patients with severe asthma (SA) compared with nonsmoking patients with mild/moderate asthma. METHODS: Induced sputum was obtained from nonsmoking patients with SA, smokers/ex-smokers with severe asthma, nonsmoking patients with mild/moderate asthma (MMAs), and healthy nonsmoking control subjects. Differential cell counts, microarray analysis of cell pellets, and SOMAscan analysis of sputum analytes were performed. CRID3 was used to inhibit the inflammasome in a mouse model of SA. RESULTS: Eosinophilic and mixed neutrophilic/eosinophilic inflammation were more prevalent in patients with SA compared with MMAs. Forty-two genes probes were upregulated (>2-fold) in nonsmoking patients with severe asthma compared with MMAs, including IL-1 receptor (IL-1R) family and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NRLP3) inflammasome members (false discovery rate

Severe asthma patients with a significant smoking history have airflow obstruction with reported neutrophilia. We hypothesise that multi-omic analysis will enable the definition of smoking and ex-smoking severe asthma molecular phenotypes.The U-BIOPRED cohort of severe asthma patients, containing current-smokers (CSA), ex-smokers (ESA), nonsmokers and healthy nonsmokers was examined. Blood and sputum cell counts, fractional exhaled nitric oxide and spirometry were obtained. Exploratory proteomic analysis of sputum supernatants and transcriptomic analysis of bronchial brushings, biopsies and sputum cells was performed.Colony-stimulating factor (CSF)2 protein levels were increased in CSA sputum supernatants, with azurocidin 1, neutrophil elastase and CXCL8 upregulated in ESA. Phagocytosis and innate immune pathways were associated with neutrophilic inflammation in ESA. Gene set variation analysis of bronchial epithelial cell transcriptome from CSA showed enrichment of xenobiotic metabolism, oxidative stress and endoplasmic reticulum stress compared to other groups. CXCL5 and matrix metallopeptidase 12 genes were upregulated in ESA and the epithelial protective genes, mucin 2 and cystatin SN, were downregulated.Despite little difference in clinical characteristics, CSA were distinguishable from ESA subjects at the sputum proteomic level, with CSA patients having increased CSF2 expression and ESA patients showing sustained loss of epithelial barrier processes.

Background: The Group 1 innate lymphoid cell (Gp 1 ILC), which includes ILC1 and natural killer (NK) cells, express T-bet and IFNγ, and are cytotoxic. Their role in asthma is unclear. Aim: Explore the enrichment of ILC1, ILC2, ILC3 and NK activation signatures in asthmatic transcriptomic samples. Methods: U-BIOPRED participants were classified as moderate to severe asthma eosinophilic, neutrophilic, mixed and paucigranulocytic, based on sputum cell counts, and healthy. We applied Gene Set Variation Analysis to sputum, nasal, bronchial brush and bronchial biopsies using published signatures [Bjorklund et al. Nat Immunol 2016,17:451-60]. Results: ILC1 but not ILC2 or ILC3 ES were higher in non-smoking severe sputum, particularly in neutrophilic and mixed patients. This phenotype has been associated with inflammasome, IFNγ and TNF activation [Kuo at al. Eur Respir J.2017, 49, in press]. In eosinophilic asthma, ILC1 ES was lower, ILC2 higher and ILC3 similar across groups (Fig1A). ILC1 and NK ES correlated with M1 macrophage signature in all compartments (Fig1B). ILC2 ES correlated with M2 macrophage signature, except in nasal brush. Conclusion: A neutrophilic severe asthma phenotype has been characterized by Group 1 ILC activation, associated with inflammasome, IFNγ, and TNF, which may reflect an antimicrobial response and contribute to chronic inflammation and remodelling in severe asthma.

Chronic cough is associated with airway inflammation and remodelling. Abnormal airway smooth muscle cell (ASMC) function may underlie mechanisms of chronic cough. Our objective was to examine the transcriptome and focused secretome of ASMCs from chronic cough patients and healthy non-cough volunteers. ASMC gene expression profiling was performed at baseline and/or after stimulation with polyinosinic:polycytidylic acid (poly(I:C)) to mimic viral infection. Supernatants were collected for multiplex analysis. Our results showed no significant differentially expressed genes (DEGs, false discovery rate (FDR) 1.5 fold-change, FDR

BACKGROUND: Asthma is a heterogeneous disease in which there is a differential response to asthma treatments. This heterogeneity needs to be evaluated so that a personalised management approach can be provided.OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinico-physiological parameters and performed an -omics analysis of sputum.METHODS: Partition-around-medoid clustering was applied to a training set of 266 asthma participants from the European U-BIOPRED adult cohort using 8 pre-specified clinic-physiological variables. This was repeated in a separate validation set of 152 asthmatics. The clusters were compared based on sputum proteomic and transcriptomic data.RESULTS: Four reproducible and stable clusters of asthmatics were identified. The training set cluster T1 consists of well-controlled moderate-to-severe asthmatics, while cluster T2 is a group of late-onset severe asthmatics with history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of non-smokers. Cluster T4 is predominantly composed of obese female uncontrolled severe asthmatics with increased exacerbations, but with normal lung function. The validation set exhibited similar clusters, demonstrating reproducibility of the classification. There were significant differences in sputum proteomics and transcriptomics between the clusters. The severe asthma clusters, T2, T3 and T4, had higher sputum eosinophilia than T1 with no differences in sputum neutrophil counts, exhaled nitric oxide and serum IgE levels.CONCLUSION: Clustering based on clinico-physiological parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.CLINICAL IMPLICATIONS: The definition of four distinct clusters of asthma linked to different pathobiological pathways provides a better template for the phenotyping and personalised treatment of severe asthma, where high unmet needs remain.CAPSULE SUMMARY: Unsupervised clustering of asthma on clinical features alone has led to the definition of four phenotypes. Sputum 'omics' analysis has revealed different biological pathways pointing towards potential new treatments.

SummaryBackgroundThe presence of lipids in alveolar macrophages (AMs) may impair their phagocytic response, and determine airway inflammation and obstruction.ObjectiveTo determine the factors such as severity of asthma, chronic cough, airway inflammation and obesity that may influence the presence of lipids in lung macrophages.MethodsBronchoalveolar lavage fluid (BALF) was obtained from 38 asthmatics (21 severe and 17 mild/moderate), 16 subjects with chronic cough and 11 healthy control subjects. The presence of lipids in macrophages was detected using an Oil-red-O stain and an index of lipid-laden macrophages (LLMI) was obtained.ResultsLLMI scores were higher in healthy subjects (median 48 [IQR 10–61]) and the severe asthma group (37 [11.5–61]) compared to mild/moderate asthmatics (7 [0.5–37]; p

Background and Purpose Corticosteroid insensitivity is a major therapeutic problem for some inflammatory diseases including chronic obstructive pulmonary disease (COPD), and it is known to be induced by reduced histone deacetylase (HDAC)-2 activities via activation of the phosphoinositide 3-kinase (PI3K) pathway. The aim of this study is to evaluate effects of a novel macrolide/fluoroketolide, solithromycin (SOL, CEM-101), on corticosteroid sensitivity induced by oxidative stress.

Chronic obstructive pulmonary disease (COPD) has been recently characterized as a disease of accelerated lung aging. The prevalence of COPD is age-dependent suggesting an intimate relationship between the pathogenesis of COPD and aging. Lung function decline, the hallmark feature of COPD evolution, is more prominent with increasing age and this decline is greater in smoking individuals. One of the major goals of COPD pharmacotherapy is the development of drugs that would be able to result in a decrease of the decline in lung function over years. However, till nowadays smoking cessation is the only known intervention which is able to decelerate lung function decline. Several mechanisms of aging, including oxidative stress, inflammation and telomere shortening have been shown to be implicated in COPD. Furthermore, numerous anti-aging molecules, including sirtuins and Nrf-2 are reduced, and pathways such as mTOR and genes such as Klotho have also been shown to be abnormal in the lungs of COPD patients. The above mechanisms have been associated with the accelerated lung aging in COPD patients. Numerous therapeutic interventions have been studied in an attempt to reverse accelerated lung aging, and some of them have already been tested in clinical trials. The aim of the present review is to summarize the mechanisms associated with the accelerated lung aging in COPD and to provide information about the possible therapeutic implications targeting those mechanisms.

Rationale. COPD is characterised by chronic and progressive airway obstruction and associated with other conditions including obesity and diabetes. We determined the effects of high-fat feeding on airway inflammation and lung function in the chronic ozone mouse model. Methods. Male C57BL/6 mice were fed a low- or high-fat diet for 10 weeks. During the last 6 weeks mice were exposed to ozone (3ppm) twice a week for 3 hours per exposure. An IPGTT was performed one day before the final ozone exposure. One day after the final exposure AHR was determined and BAL, plasma and tissue samples collected. Results. High-fat feeding alone induced insulin resistance with a mild pulmonary inflammation consisting of elevated BAL cells (p Conclusion. Ozone-induced pulmonary inflammation, AHR and associated mitochondrial dysfunction are amplified during high-fat feeding. High-fat diets in man may enhance the effects of environmental pollution particularly in subjects with airways disease.

Rationale: Activation of multiple cytokine receptors is reported to play an important role in asthma via the Janus kinases (JAK) pathway. IL-13 is known to activate JAKs. We investigated whether this pathway was activated in the sputum and blood of patients from the U-BIOPRED cohort. Methods: Sputum transcriptomics data from 120 subjects with severe asthma (SA) who were smokers (SAsm) or non-smokers (SAn), mild moderate asthmatics (MMA) or healthy controls (HC). Blood transcriptomic data from 312 subjects was also obtained. We used gene set variation analysis (GSVA) to study the KEGG JAK-STAT signalling pathway. IL-13 expression in sputum was detected by SomaScan. Results: The JAK-STAT signalling pathway was enriched in the sputum of asthmatic subjects compared to HC although this was only significant in the SAn group (p=0.01). Subgrouping asthmatics according to their sputum granulocyte status indicated that patients with neutrophilic and mixed granulocytic asthma had the highest JAK-STAT enrichment compared to HC, eosinophilic asthma and paucigranulocytic asthma (all FDR Conclusions: High activation of JAK-STAT signalling in neutrophilic and mixed granulocytic asthma suggests that these subjects may be suitable for inhaled anti-JAK therapy.

BACKGROUND AND PURPOSE Patients with chronic obstructive pulmonary disease (COPD) show a poor response to corticosteroids, which has been linked to oxidative stress. Here we show that the long-acting β2-agonist formoterol (FM) reversed corticosteroid insensitivity under oxidative stress via inhibition of phosphoinositide-3-kinase (PI3K) signalling.

Background: Although previous studies have implicated tissue CD4 + T cells in the development and maintenance of the inflammatory response in asthmatic patients, little is known about the role of CD8 + T cells. There is now accumulating evidence that microRNAs and other noncoding RNAs are important regulators of T-cell function. Objectives: We sought to use transcriptomics to determine the activation state of circulating CD4 + and CD8 + T cells in patients with nonsevere and severe asthma. Methods: mRNA and noncoding RNA expression in circulating T cells was measured by means of microarray, quantitative real-time PCR, or both. Results: Comparison of mRNA expression showed widespread changes in the circulating CD8 + but not CD4 + T cells from patients with severe asthma. No changes were observed in the CD4 + and CD8 + T cells in patients with nonsevere asthma versus those in healthy control subjects. Bioinformatics analysis showed that the changes in CD8 + T-cell mRNA expression were associated with multiple pathways involved in T-cell activation. As with mRNAs, we also observed widespread changes in expression of noncoding RNA species, including natural antisense, pseudogenes, intronic long noncoding RNAs (lncRNAs), and intergenic lncRNAs in CD8 + T cells from patients with severe asthma. Measurement of the microRNA expression profile showed selective downregulation of miR-28-5p in CD8 + T cells and reduction of miR-146a and miR-146b in both CD4 + and CD8 + T cells. Conclusions: Severe asthma is associated with the activation of circulating CD8 + T cells but not CD4 + T cells. This response is correlated with the downregulation of miR-146a/b and miR-28-5p, as well as changes in the expression of multiple species of lncRNA that might regulate CD8 + T-cell function. © 2011 American Academy of Allergy, Asthma & Immunology.

Asthma is a heterogeneous inflammatory disease caused by association of genetic and environmental factors and its incidence has significantly increased over the latest years. The clinical manifestations of asthma are the result of airway hyper-reactivity to a variety of triggers such as aeroallergens, viral and bacterial components. Toll-like receptors (TLRs) are pathogen associated molecular pattern receptors, which are also expressed in the lung tissue as well as in several cells of the innate and adaptive immune system. Ligation of TLRs results in alterations in the expression of several inflammatory and anti-inflammatory mediators, which are known to be involved in the pathogenesis of asthma. The endosomal TLRs have been shown to be associated with the induction of asthmatic inflammation (TLR3), and with disease exacerbations (TLR7, TLR8 and TLR9). Targeting these receptors seems to be an effective choice for suppressing airway inflammation, eosinophilia and airway hyperresponsiveness in asthmatic patients. In this review we provide information regarding endosomal TLRs and their role in the pathogenesis of asthma as well as their potential use as targets for the development of novel treatments for the therapy of asthma.

We previously described the identification of the 3'end of an unknown gene CDK7 using differential display which appeared to be up-regulated in diabetic kidneys [R.A. Page, C.A. Morris, J.D. Williams, C.J. von Ruhland, A.N. Malik, Isolation of diabetes-associated kidney genes using differential display, Biochem. Biophys. Res. Commun. 232 (1997) 49-53]. Here we show that CDK7 is a putative thiol related gene which is regulated by glucose in human and rat renal cells. CDK7 mRNA increased by >threefold in cultured human mesangial cells grown in high glucose for 4 days. In the kidneys of the GK rat, a model of type II diabetes, CDK7 showed a steady age-related increase in mRNA, increasing to >sixfold in 40 week GK rats compared to normoglycemic age-matched Wistar rat kidneys, this increase correlates with progressive hyperglycemia. CDK7 mRNA is widely expressed, showing particularly high levels of expression in rat and human liver, and encodes a putative 338 amino acids highly conserved peptide with several conserved domains, including a cys-pro-arg-cys domain conserved in 15 diverse species which is similar to the catalytic centre of thioredoxin, suggesting a role in oxidative stress.

We hypothesized that Gene Set Variation Analysis (GSVA) of gene expression in bronchial brushings can effectively classify airway Th2-high status in U-BIOPRED asthmatics. Airway Th2-status was defined as IL-13-induced transcriptional activity, evaluated as enrichment of IL-13 in-vitro stimulation (IVS) or expression of POSTN and SERPINB2 in human bronchial epithelial cell transcriptomic profiles from U-BIOPRED asthma patients. Based on IL-13 IVS GSVA enrichment scores above the 95 th percentile for healthy subjects, 53% of severe asthma (SA) non-smokers (n=49), 67% of SA smokers (n=18) and 36% of non-severe asthmatics (n=36) were classified as Th2-high. Eosinophilic inflammation marker values are shown in Figure 1. Using the mean normalised expression of POSTN and SERPINB2 as an alternative Th2 stratification factor, 35% of SA non-smokers, 22% of SA smokers, and 14% of non-severe asthmatics were Th2-high, mostly subsets of the IL-13 IVS-defined Th2-high asthma groups and consistent with the modest correlation (r=0.39) between the IL-13 IVS enrichment scores and POSTN/SERPINB2 scores. IL-13 IVS and POSTN/SERPINB2 based definitions of Th2 activity showed associations with eosinophilic and atopy markers, with good agreement for Th2-high vs. -low calls, although the latter approach may be too conservative. IL-13 IVS enrichment identified a broader set of subjects with airway Th2 activity when compared to POSTN and SERPINB2. Funded by IMI.

Rationale: Airway inflammation of asthma involves diverse cell types. We hypothesize that gene expression profile of bronchial biopsy in asthma is associated with specific inflammatory cell infiltration. Methods: Bronchial biopsies were obtained from moderate/severe asthma cohort and gene expression analyzed. Submucosal inflammatory cell counts of eosinophil (EOS), neutrophil (NEU) and CD4+ T cell (CD4) were studied by immunohistochemistry. Each cell type was clustered by partition around medoids and support vector machine (Y Lee et al.Bioinformatics2003 19:1132-39) was used to address classification. Results: 81 biopsy samples were available for analysis. 4 cluster medoids of IHC counts were noted for EOS (0, 2.2, 5.7 and 13.6/mm2), NEU (4.6, 12.1, 20.0 and 50.8/mm2) and CD4 (4.4, 12.2, 35.2 and 111.9/mm2). IHC counts of each higher two medoids (high group) were significantly higher than control. MMP10 (log2FC:1.31, adjusted-p=0.035) and MET (Met proto-oncogene) (log2FC:0.42, adjusted-p=0.035) were differentially expressed in the EOS high group (cutoff 4.0/mm2). The most differentially expressed genes in the CD4 high (DOCK10, adjusted-p=0.175) group did not reach statistical significance. ROC curve showed MMP10 and MET as classifiers of high bronchial EOS (AUC: 0.853, 95%CI: 0.758-0.937, p=3.97e-08), providing the overall best accuracy at 82.7%. Conclusion:MMP10 and MET are novel gene markers of bronchial eosinophilic asthma.

Asthma and COPD are major causes of mortality and morbidity worldwide. Whilst the aetiology underlying both diseases are different, chronic inflammation and structural changes to the airways, termed remodelling, (including mucous hypersecretion, fibrosis and airway wall thickening) is observed in both diseases. We have developed a novel 3D cell culture model (bronchospheres) of the airways using p63+/Krt6+ primary human basal epithelial cells. Bronchospheres capture more of the relevant complexity of the airways when compared to 2D cultures such as air liquid interface (ALI) cultures. Normal Human Epithelial (NHBEC), Asthmatic Human Epithelial (AHBEC) and COPD Human Epithelial (CHBEC) cells were obtained from Lonza and seeded onto matrigel at Passage 2. Basal cells were cultured for 14-18 days being fed on day 2 and day 8. Basal cell to bronchosphere transformation was visualised using light and video microscopy. NHBEC, AHBEC and CHBEC formed bronchospheres developing a fully functional pseudostratified epithelial structure with an inner lumen, mucus secreting goblet cells and beating cilia. There are differences between the rate at, which basal cells from different disease states form bronchospheres (NHBEC= 18 days, AHBEC= 20 days and CHBEC = 24 days) and the average size that they reach (Figure 1). The physiology of bronchospheres make them ideal intermediates between 2D and in vivo models.

Rationale: Granulocytic inflammation in sputum may be linked to diverse mechanisms. Clustering of the sputum transcriptome may help define granulocyte-specific disease drivers. Methods: In the UBIOPRED cohort, s putum eosinophils (EOS) ≥1.5% was used to define an EOS or ≤1.5% non-EOS phenotype. Differentially-expressed genes (DEGs) were obtained that differentiated between EOS, non-EOS and healthy volunteers (HV). Gene signatures were refined using nearest shrunken centroids algorithm (Robert et al. PNAS 2002, 99; 6567). Results: Clustering of 478 DEGs yielded clusters defined by 3 disease drivers, namely EOS- (n=31) and neutrophil- (NEU, n=31) enriched as well as a non-EOS non-NEU enriched (n=56) cluster (Fig 1). In the EOS-enriched cluster, signature refinement revealed 28 DEGs encoding multiple cytokine receptors and proteases including IL1RL1, TSLPR, CCR3, VSTM1, IL3RA, PRSS33 and CLC . For the NEU-enriched cluster, 27 DEGs mainly related to TNF and interferon regulation were found: TNFIP3, TNFSF10, CASP4, GBP1P1, CLEC4D, IFIT2, IFIH1, IFIT3 and IFI16 . Conclusion: Clustering on sputum cell genes reveal diverse mechanisms associated with sputa from asthma patients, with TNF- and IFN-associated genes in neutrophil-rich sputa.

Cell proliferation and intracellular oxidative stress are enhanced in asthma. Mitochondria are associated with these processes. Regulation of mitochondrial function therefore provides a novel target in understanding disease mechanisms. We investigated mitochondrial function through changes in activity, membrane potential, reactive oxygen species production and pro-inflammatory cytokine release in airway smooth muscle cells (ASMCs) isolated from healthy control, non-severe and severe asthmatic patients, at baseline and following treatment with hyperproliferative stimuli (TGFβ and FBS) and oxidative stress (H 2 O 2 ) over 4, 24 and 48 hours. Mitochondrial function was altered in response to TGFβ+FBS and to H 2 O 2 , with an accumulation of mitochondrial reactive oxygen species (Fig 1), disruption to the mitochondrial membrane potential (ΔΨm, significantly decreased at 4 hours after TGFβ+FBS in all patient types, p These data suggest that an intrinsic ASMC mitochondrial dysfunction per se is not driving the asthmatic phenotype but is a secondary response to the inflammatory/proliferative environment in the lung.

Background: Severe asthma is characterised by poor therapeutic response to corticosteroids and a diverse airway inflammation profile. We have shown that inflammasome-associated genes are highly differentially expressed in sputum in the U-BIOPRED adult severe asthma (SA) cohort. Aims & Objectives: To investigate mediator profiles in sputum supernatants from subjects in U-BIOPRED. Methods: 1129 analytes were measured in sputum supernatants (Somalogic SOMAscan TM V3), with 111 samples passing quality control (with squamous cell content Results: 54 analytes were differentially expressed between SA non-smokers (n=43) and healthy subjects (n=18) (FDR 2-fold /healthy cohort). Pregnancy-associated plasma protein A (PAPP-A), serum amyloid P (SAP) and C-reactive protein (CRP) were highly up-regulated in both smoking and non-smoking SA (14.7-, 6.27- and 3.21-fold). PAPP-A (r=087;p -10 ), SAP (r=0.6;p -10 ) and periostin (r=0.55;p -10 ) correlated with sputum eosinophils, while b-endorphin (r=0.67;p -10 ), azurocidin (r=0.63;p -10 ), IL-5 (r=0.6;p -10 ) and IL-8 (r=0.51;p -8 ) with sputum neutrophils. These data are consistent with transciptomic data showing up-regulation of IL-1 receptor family gene transcripts, which are upstream of IL-6/acute phase response signalling, in SA. A similar profile of mediators has been seen in the Airways Disease Endotyping for Personalised Therapeutics (ADEPT) study. Conclusion: Severe asthma is associated with acute phase and eosinophil- and neutrophil- associated analytes in sputum. Inflammasome activation appears strongest in eosinophilic asthma.

Hydrogen sulfide (H₂S) is a gas produced by respiratory cells including smooth muscle cells and may play a role as a cellular gasotransmitter. We evaluated whether H₂S levels in serum or sputum could represent a new biomarker of COPD in a cross-sectional study.H₂S levels in sputum and serum samples were measured using a sulfide-sensitive electrode in 64 patients with stable COPD (S-COPD), 29 COPD subjects during acute exacerbation (AE-COPD), 14 healthy smokers and 21 healthy non-smokers.Sputum H₂S levels in AE-COPD subjects were higher than those in S-COPD, healthy smoking and non-smoking subjects (p0.001), but serum H₂S levels in AE-COPD were lower than those in S-COPD (p0.001). Thus, the sputum-to-serum ratio of H₂S (H₂S ratio) in AE-COPD subjects were higher than those in stable COPD, healthy smoking and non-smoking subjects (p0.001). In 14 COPD subjects whose H₂S ratios were measured during and after an exacerbation, the mean ratio was increased during exacerbation (p0.05). H₂S ratio was positively correlated with St. George's Respiratory Questionnaire score, sputum neutrophils and IL-6 and IL-8 levels in sputum and serum (p0.01) but inversely correlated with sputum macrophages (%), FEV₁%predicted and FEV₁/FVC (p0.01). The cut-off level of H₂S ratio to indicate an exacerbation was ≥0.44 (sensitivity of 93.1% and specificity of 84.5%).The ratio of sputum-to-serum levels of H₂S may provide a useful marker of COPD indicative of obstructive neutrophilic inflammation and of potential ongoing exacerbation.

Inflammatory lung diseases are highly complex in respect of pathogenesis and relationships between inflammation, clinical disease and response to treatment. Sophisticated large-scale analytical methods to quantify gene expression (transcriptomics), proteins (proteomics), lipids (lipidomics) and metabolites (metabolomics) in the lungs, blood and urine are now available to identify biomarkers that define disease in terms of combined clinical, physiological and patho-biological abnormalities. The aspiration is that these approaches will improve diagnosis, i.e. define pathological phenotypes, and facilitate the monitoring of disease and therapy, and also, unravel underlying molecular pathways. Biomarker studies can either select predefined biomarker(s) measured by specific methods or apply an "unbiased" approach involving detection platforms that are indiscriminate in focus. This article reviews the technologies presently available to study biomarkers of lung disease within the 'omics field. The contributions of the individual 'omics analytical platforms to the field of respiratory diseases are summarised, with the goal of providing background on their respective abilities to contribute to systems medicine-based studies of lung disease.

Macrolides are reported to reduce exacerbation of chronic inflammatory respiratory disease, such as chronic obstructive pulmonary disease (COPD), and also show anti-inflammatory effects in vitro and in vivo. However the anti-inflammatory efficacies of current macrolides are relatively weak. Here we found that a novel macrolide/fluoroketolide solithromycin (CEM-101) showed superior anti-inflammatory effects to macrolides in current clinical use. The effects of solithromycin (SOL) on lipopolysaccharide-induced TNFα (tumor necrosis factor α) and/or CXCL8 (C-X-C motif chemokine ligand 8; interleukin-8) release, phorbol 12-myristate 13-acetate-induced MMP9 (matrix metalloproteinase 9) activity and NF-κB (nuclear factor-κB) activity under conditions of oxidative stress have been evaluated and compared with the effects of erythromycin, clarithromycin, azithromycin, and telithromycin in macrophage-like PMA-differentiated U937 cells and peripheral blood mononuclear cells (PBMC) obtained from COPD patients. We also examined effect of SOL on cigarette smoke-induced airway inflammation in mice. SOL exerted superior inhibitory effects on TNFα/CXCL8 production and MMP9 activity in monocytic U937 cells. In addition, SOL suppressed TNFα release and MMP9 activity in PBMC from COPD patients at 10 µM, which is 10 times more potent than the other macrolides tested. Activated NF-κB by oxidative stress was completely reversed by SOL. SOL also inhibited cigarette smoke-induced neutrophilia and pro-MMP9 production in vivo, although erythromycin did not inhibit them. Thus, SOL showed better anti-inflammatory profiles compared with macrolides currently used in the clinic and may be a promising anti-inflammatory and antimicrobial macrolide for the treatment of COPD in future.

Currently available glucocorticoids are relatively short acting and may be less effective in patients with chronic obstructive pulmonary disease (COPD) where high levels of oxidative stress are seen. Here we show that a novel glucocorticoid, fluticasone furoate (FF), has a longer duration of action in several cell systems compared with fluticasone propionate (FP) and budesonide, and unlike FP, FF is resistant to oxidative stress. FF had similar or slightly higher potency to FP and was 2-9 fold more potent than budesonide, when assessed at 4h, in inhibiting inflammatory cytokine production in epithelial cell lines (BEAS2B, A549), primary bronchial epithelial cells and a monocytic cell line (U937). The potency of FF was sustained beyond 16 h with or without washout compared with FP or budesonide, such that it showed a greater duration of action in this range of cellular assays. The activated YFP-conjugated glucocorticoid receptor was detectable in nuclei of FF treated BEAS2B cells for at least for 30 h, and FF had a longer duration of action than FP in inhibiting activation of transcription factors such as NF-κB and AP-1. In addition, FF showed superior effects to FP in peripheral blood mononuclear cells from patients with COPD and also in U937 cells or primary bronchial epithelial cells under conditions of oxidative stress. The longer duration of action and oxidative stress insensitivity of FF compared with FP has potential clinical implications for the control of inflammation in respiratory diseases, such as COPD.

3527 Y. Kobayashi, C. Rossios, P. J. Barnes, K. Ito Airway Disease Section, NHLI, Imperial College London, UK Rationale: We have recently demonstrated that the reduction of histone deacetylase (HDAC)2 activity via PI3K activation causes corticosteroid (CS) insensitivity in COPD (ATS 2007). Here we show that CEM-101, a novel macrolide/fluoroketolide, which has potent activities against many bacteria causing pneumonia (starting Phase 2), reverses oxidative stress-dependent CS insensitivity via inhibition of PI3K signaling. Methods: CS sensitivity was determined by calculation of IC50 value of dexamethasone on TNFα-induced IL8 production in U937 monocytic cell line. HDAC activity and phosphorylation level of Akt as a marker of PI3K activation were measured by fluorescence based activity assay and western blotting, respectively. HDAC2 mRNA expression were also determined in A549 type II alveolar epithelial cell line. Cells were exposed to oxidative stress such as hydrogen peroxide (H2O2) or cigarette smoke extract (CSE) to induce steroid insensitivity. Results: Oxidative stress decreased CS sensitivity with concomitant down-regulation of total HDAC activity/HDAC2 expression and increased Akt phosphorylation. Treatment with CEM-101 (10 μM) restored CS sensitivity in H2O2 exposed cells. In addition, CEM-101 (10 100 μM) restored HDAC activity and HDAC2 expression reduced under oxidative stress and also inhibited Akt phosphorylation, which effects were more potent than those of any macrolides currently used clinically. Conclusions: CEM-101 restores CS sensitivity in oxidative stress-dependent CS insensitive model via enhancement of HDAC activity/expression due to PI3K signaling inhibition. This novel fluoroketolide, CEM-101, has potential for a novel treatment of COPD or severe asthma, which are steroid insensitive.

Inappropriate elevation of matrix metalloproteinase-9 (MMP9) is reported to be involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). The object of this study was to identify the molecular mechanism underlying this increase of MMP9 expression, and here we show that oxidative stress-dependent reduction of a protein deacetylase, SIRT1, known as a putative antiaging enzyme, causes elevation of MMP9 expression. A sirtuin inhibitor, splitomycin, and SIRT1 knockdown by RNA interference led an increase in MMP9 expression in human monocytic U937 cells and in primary sputum macrophages, which was detected by RT-PCR, Western blot, activity assay, and zymography. In fact, the SIRT1 level was significantly decreased in peripheral lungs of patients with COPD, and this increase was inversely correlated with MMP9 expression and MMP9 promoter activation detected by a chromatin immunoprecipitation assay. H(2)O(2) reduced SIRT1 expression and activity in U937 cells; furthermore, cigarette smoke exposure also caused reduction of SIRT1 expression in lung tissue of A/J mice, with concomitant elevation of MMP9. Intranasal treatment of a selective and novel SIRT1 small molecule activator, SRT2172, blocked the increase of MMP9 expression in the lung as well as pulmonary neutrophilia and the reduction in exercise tolerance. Thus, SIRT1 is a negative regulator of MMP9 expression, and SIRT1 activation is implicated as a novel therapeutic approach to treating chronic inflammatory diseases, in which MMP9 is abundant.