Urinary metabotype of severe asthma evidences decreased carnitine metabolism independent of oral corticosteroid treatment in the U-BIOPRED study.

INTRODUCTION: Asthma is a heterogeneous disease with poorly defined phenotypes. Patients with severe asthma often receive multiple treatments including oral corticosteroids (OCS). Treatment may modify the observed metabotype, rendering it challenging to investigate underlying disease mechanisms. Here, we aimed to identify dysregulated metabolic processes in relation to asthma severity and medication. METHODS: Baseline urine was collected prospectively from healthy participants (n=100), patients with mild-to-moderate asthma (n=87) and patients with severe asthma (n=418) in the cross-sectional U-BIOPRED cohort; 12-18-month longitudinal samples were collected from patients with severe asthma (n=305). Metabolomics data were acquired using high-resolution mass spectrometry and analysed using univariate and multivariate methods. RESULTS: A total of 90 metabolites were identified, with 40 significantly altered (p<0.05, false discovery rate <0.05) in severe asthma and 23 by OCS use. Multivariate modelling showed that observed metabotypes in healthy participants and patients with mild-to-moderate asthma differed significantly from those in patients with severe asthma (p=2.6×10-20), OCS-treated asthmatic patients differed significantly from non-treated patients (p=9.5×10-4), and longitudinal metabotypes demonstrated temporal stability. Carnitine levels evidenced the strongest OCS-independent decrease in severe asthma. Reduced carnitine levels were associated with mitochondrial dysfunction via decreases in pathway enrichment scores of fatty acid metabolism and reduced expression of the carnitine transporter SLC22A5 in sputum and bronchial brushings. CONCLUSIONS: This is the first large-scale study to delineate disease- and OCS-associated metabolic differences in asthma. The widespread associations with different therapies upon the observed metabotypes demonstrate the need to evaluate potential modulating effects on a treatment- and metabolite-specific basis. Altered carnitine metabolism is a potentially actionable therapeutic target that is independent of OCS treatment, highlighting the role of mitochondrial dysfunction in severe asthma.

Combined Blockade of TNF-α and IL-17A Alleviates Progression of Collagen-Induced Arthritis without Causing Serious Infections in Mice.

The cytokines TNF-α and IL-17A are elevated in a variety of autoimmune diseases, including rheumatoid arthritis. Both cytokines are targets of several biologic drugs used in the clinic, but unfortunately many patients are refractory to these therapies. IL-17A and TNF-α are known to mediate signaling synergistically to drive expression of inflammatory genes. Hence, combined blockade of TNF-α and IL-17A represents an attractive treatment strategy in autoimmune settings where monotherapy is not fully effective. However, a major concern with this approach is the potential predisposition to opportunistic infections that might outweigh any clinical benefits. Accordingly, we examined the impact of individual versus combined neutralization of TNF-α and IL-17A in a mouse model of rheumatoid arthritis (collagen-induced arthritis) and the concomitant susceptibility to infections that are likely to manifest as side effects of blocking these cytokines (oral candidiasis or tuberculosis). Our findings indicate that combined neutralization of TNF-α and IL-17A was considerably more effective than monotherapy in improving collagen-induced arthritis disease even when administered at a minimally efficacious dose. Encouragingly, however, dual cytokine blockade did not cooperatively impair antimicrobial host defenses, as mice given combined IL-17A and TNF-α neutralization displayed infectious profiles and humoral responses comparable to mice given high doses of individual anti-TNF-α or anti-IL-17A mAbs. These data support the idea that combined neutralization of TNF-α and IL-17A for refractory autoimmunity is likely to be associated with acceptable and manageable risks of opportunistic infections associated with these cytokines.

I_MDS: an inflammatory bowel disease molecular activity score to classify patients with differing disease-driving pathways and therapeutic response to anti-TNF treatment.

Crohn's disease and ulcerative colitis are driven by both common and distinct underlying mechanisms of pathobiology. Both diseases, exhibit heterogeneity underscored by the variable clinical responses to therapeutic interventions. We aimed to identify disease-driving pathways and classify individuals into subpopulations that differ in their pathobiology and response to treatment. We applied hierarchical clustering of enrichment scores derived from gene set variation analysis of signatures representative of various immunological processes and activated cell types, to a colonic biopsy dataset that included healthy volunteers, Crohn's disease and ulcerative colitis patients. Patient stratification at baseline or after anti-TNF treatment in clinical responders and non-responders was queried. Signatures with significantly different enrichment scores were identified using a general linear model. Comparisons to healthy controls were made at baseline in all participants and then separately in responders and non-responders. Fifty-nine percent of the signatures were commonly enriched in both conditions at baseline, supporting the notion of a disease continuum within ulcerative colitis and Crohn's disease. Signatures included T cells, macrophages, neutrophil activation and poly:IC signatures, representing acute inflammation and a complex mix of potential disease-driving biology. Collectively, identification of significantly enriched signatures allowed establishment of an inflammatory bowel disease molecular activity score which uses biopsy transcriptomics as a surrogate marker to accurately track disease severity. This score separated diseased from healthy samples, enabled discrimination of clinical responders and non-responders at baseline with 100% specificity and 78.8% sensitivity, and was validated in an independent data set that showed comparable classification. Comparing responders and non-responders separately at baseline to controls, 43% and 70% of signatures were enriched, respectively, suggesting greater molecular dysregulation in TNF non-responders at baseline. This methodological approach could facilitate better targeted design of clinical studies to test therapeutics, concentrating on patient subsets sharing similar underlying pathobiology, therefore increasing the likelihood of clinical response.

Epithelial IL-6 trans-signaling defines a new asthma phenotype with increased airway inflammation.

BACKGROUND: Although several studies link high levels of IL-6 and soluble IL-6 receptor (sIL-6R) to asthma severity and decreased lung function, the role of IL-6 trans-signaling (IL-6TS) in asthmatic patients is unclear. OBJECTIVE: We sought to explore the association between epithelial IL-6TS pathway activation and molecular and clinical phenotypes in asthmatic patients. METHODS: An IL-6TS gene signature obtained from air-liquid interface cultures of human bronchial epithelial cells stimulated with IL-6 and sIL-6R was used to stratify lung epithelial transcriptomic data (Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes [U-BIOPRED] cohorts) by means of hierarchical clustering. IL-6TS-specific protein markers were used to stratify sputum biomarker data (Wessex cohort). Molecular phenotyping was based on transcriptional profiling of epithelial brushings, pathway analysis, and immunohistochemical analysis of bronchial biopsy specimens. RESULTS: Activation of IL-6TS in air-liquid interface cultures reduced epithelial integrity and induced a specific gene signature enriched in genes associated with airway remodeling. The IL-6TS signature identified a subset of patients with IL-6TS-high asthma with increased epithelial expression of IL-6TS-inducible genes in the absence of systemic inflammation. The IL-6TS-high subset had an overrepresentation of frequent exacerbators, blood eosinophilia, and submucosal infiltration of T cells and macrophages. In bronchial brushings Toll-like receptor pathway genes were upregulated, whereas expression of cell junction genes was reduced. Sputum sIL-6R and IL-6 levels correlated with sputum markers of remodeling and innate immune activation, in particular YKL-40, matrix metalloproteinase 3, macrophage inflammatory protein 1ß, IL-8, and IL-1ß. CONCLUSIONS: Local lung epithelial IL-6TS activation in the absence of type 2 airway inflammation defines a novel subset of asthmatic patients and might drive airway inflammation and epithelial dysfunction in these patients.

"T2-high" in severe asthma related to blood eosinophil, exhaled nitric oxide and serum periostin.

Type-2 (T2) immune responses in airway epithelial cells (AECs) classifies mild-moderate asthma into a T2-high phenotype. We examined whether currently available clinical biomarkers can predict AEC-defined T2-high phenotype within the U-BIOPRED cohort.The transcriptomic profile of AECs obtained from brushings of 103 patients with asthma and 44 healthy controls was obtained and gene set variation analysis used to determine the relative expression score of T2 asthma using a signature from interleukin (IL)-13-exposed AECs.37% of asthmatics (45% nonsmoking severe asthma, n=49; 33% of smoking or ex-smoking severe asthma, n=18; and 28% mild-moderate asthma, n=36) were T2-high using AEC gene expression. They were more symptomatic with higher exhaled nitric oxide fraction (F eNO) and blood and sputum eosinophils, but not serum IgE or periostin. Sputum eosinophilia correlated best with the T2-high signature. F eNO (≥30 ppb) and blood eosinophils (≥300 cells·µL-1) gave a moderate prediction of T2-high asthma. Sputum IL-4, IL-5 and IL-13 protein levels did not correlate with gene expression.T2-high severe asthma can be predicted to some extent from raised levels of F eNO, blood and sputum eosinophil counts, but serum IgE or serum periostin were poor predictors. Better bedside biomarkers are needed to detect T2-high.

Contribution of airway eosinophils in airway wall remodeling in asthma: Role of MMP-10 and MET.

BACKGROUND: Eosinophils play an important role in the pathophysiology of asthma being implicated in airway epithelial damage and airway wall remodeling. We determined the genes associated with airway remodeling and eosinophilic inflammation in patients with asthma. METHODS: We analyzed the transcriptomic data from bronchial biopsies of 81 patients with moderate-to-severe asthma of the U-BIOPRED cohort. Expression profiling was performed using Affymetrix arrays on total RNA. Transcription binding site analysis used the PRIMA algorithm. Localization of proteins was by immunohistochemistry. RESULTS: Using stringent false discovery rate analysis, MMP-10 and MET were significantly overexpressed in biopsies with high mucosal eosinophils (HE) compared to low mucosal eosinophil (LE) numbers. Immunohistochemical analysis confirmed increased expression of MMP-10 and MET in bronchial epithelial cells and in subepithelial inflammatory and resident cells in asthmatic biopsies. Using less-stringent conditions (raw P-value < 0.05, log2 fold change > 0.5), we defined a 73-gene set characteristic of the HE compared to the LE group. Thirty-three of 73 genes drove the pathway annotation that included extracellular matrix (ECM) organization, mast cell activation, CC-chemokine receptor binding, circulating immunoglobulin complex, serine protease inhibitors, and microtubule bundle formation pathways. Genes including MET and MMP10 involved in ECM organization correlated positively with submucosal thickness. Transcription factor binding site analysis identified two transcription factors, ETS-1 and SOX family proteins, that showed positive correlation with MMP10 and MET expression. CONCLUSION: Pathways of airway remodeling and cellular inflammation are associated with submucosal eosinophilia. MET and MMP-10 likely play an important role in these processes.

Sputum transcriptomics reveal upregulation of IL-1 receptor family members in patients with severe asthma.

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 < 0.05). The inflammasome proteins nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain containing 1 (NLRP1), NLRP3, and nucleotide-binding oligomerization domain (NOD)-like receptor C4 (NLRC4) were associated with neutrophilic asthma and with sputum IL-1ß protein levels, whereas eosinophilic asthma was associated with an IL-13-induced TH2 signature and IL-1 receptor-like 1 (IL1RL1) mRNA expression. These differences were sputum specific because no activation of NLRP3 or enrichment of IL-1R family genes in bronchial brushings or biopsy specimens in patients with SA was observed. Expression of NLRP3 and of the IL-1R family genes was validated in the Airway Disease Endotyping for Personalized Therapeutics cohort. Inflammasome inhibition using CRID3 prevented airway hyperresponsiveness and airway inflammation (both neutrophilia and eosinophilia) in a mouse model of severe allergic asthma. CONCLUSION: IL1RL1 gene expression is associated with eosinophilic SA, whereas NLRP3 inflammasome expression is highest in patients with neutrophilic SA. TH2-driven eosinophilic inflammation and neutrophil-associated inflammasome activation might represent interacting pathways in patients with SA.

Sputum proteomics and airway cell transcripts of current and ex-smokers with severe asthma in U-BIOPRED: an exploratory analysis.

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.

Subcutaneous golimumab for children with active polyarticular-course juvenile idiopathic arthritis: results of a multicentre, double-blind, randomised-withdrawal trial.

OBJECTIVE: This report aims to determine the safety, pharmacokinetics (PK) and efficacy of subcutaneous golimumab in active polyarticular-course juvenile idiopathic arthritis (polyJIA). METHODS: In this three-part randomised double-blinded placebo-controlled withdrawal trial, all patients received open-label golimumab (30 mg/m2 of body surface area; maximum: 50 mg/dose) every 4 weeks together with weekly methotrexate during Part 1 (weeks 0-16). Patients with at least 30% improvement per American College of Rheumatology Criteria for JIA (JIA ACR30) in Part 1 entered the double-blinded Part 2 (weeks 16-48) after 1:1 randomisation to continue golimumab or start placebo. In Part 3, golimumab was continued or could be restarted as in Part 1. The primary outcome was JIA flares in Part 2; secondary outcomes included JIA ACR50/70/90 responses, clinical remission, PK and safety. RESULTS: Among 173 patients with polyJIA enrolled, 89.0% (154/173) had a JIA ACR30 response and 79.2%/65.9%/36.4% demonstrated JIA ACR50/70/90 responses in Part 1. At week 48, the primary endpoint was not met as treatment groups had comparable JIA flare rates (golimumab vs placebo: 32/78=41% vs 36/76=47%; p=0.41), and rates of clinical remission were comparable (golimumab vs placebo: 10/78=12.8% vs 9/76=11.8%). Adverse event and serious adverse event rates were similar in the treatment groups during Part 2. Injection site reactions occurred with <1% of all injections. PK analysis confirmed adequate golimumab dosing for polyJIA. CONCLUSION: Although the primary endpoint was not met, golimumab resulted in rapid, clinically meaningful, improvement in children with active polyJIA. Golimumab was well tolerated, and no unexpected safety events occurred. CLINICAL TRIAL REGISTRATION: NCT01230827; Results.

A phase 2a study of toreforant, a histamine H4 receptor antagonist, in eosinophilic asthma.

BACKGROUND: Data from preclinical and clinical studies support the evaluation of histamine 4 receptor antagonists in the treatment of asthma. Toreforant is a selective histamine 4 receptor antagonist that could be effective in patients with eosinophilic asthma. OBJECTIVE: To evaluate the efficacy and safety of toreforant in patients with eosinophilic, persistent asthma that was inadequately controlled despite current treatment. METHODS: In this phase 2a, multicenter, randomized, double-blinded, parallel-group, placebo-controlled, proof-of-concept study, 162 eligible patients were randomized (1:1) to placebo or 30 mg of toreforant once daily through week 24 and followed for 4 weeks. The primary end point was change from baseline in pre-bronchodilator percent-predicted forced expiratory volume in 1 second at week 16. Secondary end points included change from baseline at week 16 in postbronchodilator percent-predicted forced expiratory volume in 1 second, Asthma Control Questionnaire scores, weekly averages of Daytime and Nighttime Asthma Diary Symptom Scores, and weekly average of number of puffs in a day that rescue medication was used. RESULTS: There was no significant difference between groups in pre-bronchodilator percent-predicted forced expiratory volume in 1 second at week 16 (difference in least-square means -0.19%; 95% confidence interval -3.01 to 2.64; P = .90). Similarly, there were no significant differences between groups at week 16 in changes from baseline in the secondary end points (P ≥ .30). Toreforant was generally well tolerated. No deaths or serious adverse events were reported at any time point. CONCLUSION: Toreforant, at the dose tested, failed to provide therapeutic benefit in this population of patients with uncontrolled, eosinophilic, persistent asthma. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01823016.

A Transcriptome-driven Analysis of Epithelial Brushings and Bronchial Biopsies to Define Asthma Phenotypes in U-BIOPRED.

RATIONALE: Asthma is a heterogeneous disease driven by diverse immunologic and inflammatory mechanisms. OBJECTIVES: Using transcriptomic profiling of airway tissues, we sought to define the molecular phenotypes of severe asthma. METHODS: The transcriptome derived from bronchial biopsies and epithelial brushings of 107 subjects with moderate to severe asthma were annotated by gene set variation analysis using 42 gene signatures relevant to asthma, inflammation, and immune function. Topological data analysis of clinical and histologic data was performed to derive clusters, and the nearest shrunken centroid algorithm was used for signature refinement. MEASUREMENTS AND MAIN RESULTS: Nine gene set variation analysis signatures expressed in bronchial biopsies and airway epithelial brushings distinguished two distinct asthma subtypes associated with high expression of T-helper cell type 2 cytokines and lack of corticosteroid response (group 1 and group 3). Group 1 had the highest submucosal eosinophils, as well as high fractional exhaled nitric oxide levels, exacerbation rates, and oral corticosteroid use, whereas group 3 patients showed the highest levels of sputum eosinophils and had a high body mass index. In contrast, group 2 and group 4 patients had an 86% and 64% probability, respectively, of having noneosinophilic inflammation. Using machine learning tools, we describe an inference scheme using the currently available inflammatory biomarkers sputum eosinophilia and fractional exhaled nitric oxide levels, along with oral corticosteroid use, that could predict the subtypes of gene expression within bronchial biopsies and epithelial cells with good sensitivity and specificity. CONCLUSIONS: This analysis demonstrates the usefulness of a transcriptomics-driven approach to phenotyping that segments patients who may benefit the most from specific agents that target T-helper cell type 2-mediated inflammation and/or corticosteroid insensitivity.

Identification of airway mucosal type 2 inflammation by using clinical biomarkers in asthmatic patients.

BACKGROUND: The Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study profiled patients with mild, moderate, and severe asthma and nonatopic healthy control subjects. OBJECTIVE: We explored this data set to define type 2 inflammation based on airway mucosal IL-13-driven gene expression and how this related to clinically accessible biomarkers. METHODS: IL-13-driven gene expression was evaluated in several human cell lines. We then defined type 2 status in 25 healthy subjects, 28 patients with mild asthma, 29 patients with moderate asthma, and 26 patients with severe asthma based on airway mucosal expression of (1) CCL26 (the most differentially expressed gene), (2) periostin, or (3) a multigene IL-13 in vitro signature (IVS). Clinically accessible biomarkers included fraction of exhaled nitric oxide (Feno) values, blood eosinophil (bEOS) counts, serum CCL26 expression, and serum CCL17 expression. RESULTS: Expression of airway mucosal CCL26, periostin, and IL-13-IVS all facilitated segregation of subjects into type 2-high and type 2-low asthmatic groups, but in the ADEPT study population CCL26 expression was optimal. All subjects with high airway mucosal CCL26 expression and moderate-to-severe asthma had Feno values (≥35 ppb) and/or high bEOS counts (≥300 cells/mm3) compared with a minority (36%) of subjects with low airway mucosal CCL26 expression. A combination of Feno values, bEOS counts, and serum CCL17 and CCL26 expression had 100% positive predictive value and 87% negative predictive value for airway mucosal CCL26-high status. Clinical variables did not differ between subjects with type 2-high and type 2-low status. Eosinophilic inflammation was associated with but not limited to airway mucosal type 2 gene expression. CONCLUSION: A panel of clinical biomarkers accurately classified type 2 status based on airway mucosal CCL26, periostin, or IL-13-IVS gene expression. Use of Feno values, bEOS counts, and serum marker levels (eg, CCL26 and CCL17) in combination might allow patient selection for novel type 2 therapeutics.

U-BIOPRED clinical adult asthma clusters linked to a subset of sputum omics.

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 personalized management approach can be provided. OBJECTIVES: We stratified patients with moderate-to-severe asthma based on clinicophysiologic parameters and performed an omics analysis of sputum. METHODS: Partition-around-medoids clustering was applied to a training set of 266 asthmatic participants from the European Unbiased Biomarkers for the Prediction of Respiratory Diseases Outcomes (U-BIOPRED) adult cohort using 8 prespecified clinic-physiologic variables. This was repeated in a separate validation set of 152 asthmatic patients. The clusters were compared based on sputum proteomics and transcriptomics data. RESULTS: Four reproducible and stable clusters of asthmatic patients were identified. The training set cluster T1 consists of patients with well-controlled moderate-to-severe asthma, whereas cluster T2 is a group of patients with late-onset severe asthma with a history of smoking and chronic airflow obstruction. Cluster T3 is similar to cluster T2 in terms of chronic airflow obstruction but is composed of nonsmokers. Cluster T4 is predominantly composed of obese female patients with uncontrolled severe asthma 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 cluster T1, with no differences in sputum neutrophil counts and exhaled nitric oxide and serum IgE levels. CONCLUSION: Clustering based on clinicophysiologic parameters yielded 4 stable and reproducible clusters that associate with different pathobiological pathways.

T-helper cell type 2 (Th2) and non-Th2 molecular phenotypes of asthma using sputum transcriptomics in U-BIOPRED.

Asthma is characterised by heterogeneous clinical phenotypes. Our objective was to determine molecular phenotypes of asthma by analysing sputum cell transcriptomics from 104 moderate-to-severe asthmatic subjects and 16 nonasthmatic subjects.After filtering on the differentially expressed genes between eosinophil- and noneosinophil-associated sputum inflammation, we used unbiased hierarchical clustering on 508 differentially expressed genes and gene set variation analysis of specific gene sets.We defined three transcriptome-associated clusters (TACs): TAC1 (characterised by immune receptors IL33R, CCR3 and TSLPR), TAC2 (characterised by interferon-, tumour necrosis factor-α- and inflammasome-associated genes) and TAC3 (characterised by genes of metabolic pathways, ubiquitination and mitochondrial function). TAC1 showed the highest enrichment of gene signatures for interleukin-13/T-helper cell type 2 (Th2) and innate lymphoid cell type 2. TAC1 had the highest sputum eosinophilia and exhaled nitric oxide fraction, and was restricted to severe asthma with oral corticosteroid dependency, frequent exacerbations and severe airflow obstruction. TAC2 showed the highest sputum neutrophilia, serum C-reactive protein levels and prevalence of eczema. TAC3 had normal to moderately high sputum eosinophils and better preserved forced expiratory volume in 1 s. Gene-protein coexpression networks from TAC1 and TAC2 extended this molecular classification.We defined one Th2-high eosinophilic phenotype TAC1, and two non-Th2 phenotypes TAC2 and TAC3, characterised by inflammasome-associated and metabolic/mitochondrial pathways, respectively.

Transcriptomic gene signatures associated with persistent airflow limitation in patients with severe asthma.

A proportion of severe asthma patients suffers from persistent airflow limitation (PAL), often associated with more symptoms and exacerbations. Little is known about the underlying mechanisms. Here, our aim was to discover unexplored potential mechanisms using Gene Set Variation Analysis (GSVA), a sensitive technique that can detect underlying pathways in heterogeneous samples.Severe asthma patients from the U-BIOPRED cohort with PAL (post-bronchodilator forced expiratory volume in 1 s/forced vital capacity ratio below the lower limit of normal) were compared with those without PAL. Gene expression was assessed on the total RNA of sputum cells, nasal brushings, and endobronchial brushings and biopsies. GSVA was applied to identify differentially enriched predefined gene signatures based on all available gene expression publications and data on airways disease.Differentially enriched gene signatures were identified in nasal brushings (n=1), sputum (n=9), bronchial brushings (n=1) and bronchial biopsies (n=4) that were associated with response to inhaled steroids, eosinophils, interleukin-13, interferon-α, specific CD4+ T-cells and airway remodelling.PAL in severe asthma has distinguishable underlying gene networks that are associated with treatment, inflammatory pathways and airway remodelling. These findings point towards targets for the therapy of PAL in severe asthma.

Severe asthma exists despite suppressed tissue inflammation: findings of the U-BIOPRED study.

The U-BIOPRED study is a multicentre European study aimed at a better understanding of severe asthma. It included three steroid-treated adult asthma groups (severe nonsmokers (SAn group), severe current/ex-smokers (SAs/ex group) and those with mild-moderate disease (MMA group)) and healthy controls (HC group). The aim of this cross-sectional, bronchoscopy substudy was to compare bronchial immunopathology between these groups.In 158 participants, bronchial biopsies and bronchial epithelial brushings were collected for immunopathologic and transcriptomic analysis. Immunohistochemical analysis of glycol methacrylate resin-embedded biopsies showed there were more mast cells in submucosa of the HC group (33.6 mm-2) compared with both severe asthma groups (SAn: 17.4 mm-2, p<0.001; SAs/ex: 22.2 mm-2, p=0.01) and with the MMA group (21.2 mm-2, p=0.01). The number of CD4+ lymphocytes was decreased in the SAs/ex group (4.7 mm-2) compared with the SAn (11.6 mm-2, p=0.002), MMA (10.1 mm-2, p=0.008) and HC (10.6 mm-2, p<0.001) groups. No other differences were observed.Affymetrix microarray analysis identified seven probe sets in the bronchial brushing samples that had a positive relationship with submucosal eosinophils. These mapped to COX-2 (cyclo-oxygenase-2), ADAM-7 (disintegrin and metalloproteinase domain-containing protein 7), SLCO1A2 (solute carrier organic anion transporter family member 1A2), TMEFF2 (transmembrane protein with epidermal growth factor like and two follistatin like domains 2) and TRPM-1 (transient receptor potential cation channel subfamily M member 1); the remaining two are unnamed.We conclude that in nonsmoking and smoking patients on currently recommended therapy, severe asthma exists despite suppressed tissue inflammation within the proximal airway wall.

Validated and longitudinally stable asthma phenotypes based on cluster analysis of the ADEPT study.

BACKGROUND: Asthma is a disease of varying severity and differing disease mechanisms. To date, studies aimed at stratifying asthma into clinically useful phenotypes have produced a number of phenotypes that have yet to be assessed for stability and to be validated in independent cohorts. The aim of this study was to define and validate, for the first time ever, clinically driven asthma phenotypes using two independent, severe asthma cohorts: ADEPT and U-BIOPRED. METHODS: Fuzzy partition-around-medoid clustering was performed on pre-specified data from the ADEPT participants (n = 156) and independently on data from a subset of U-BIOPRED asthma participants (n = 82) for whom the same variables were available. Models for cluster classification probabilities were derived and applied to the 12-month longitudinal ADEPT data and to a larger subset of the U-BIOPRED asthma dataset (n = 397). High and low type-2 inflammation phenotypes were defined as high or low Th2 activity, indicated by endobronchial biopsies gene expression changes downstream of IL-4 or IL-13. RESULTS: Four phenotypes were identified in the ADEPT (training) cohort, with distinct clinical and biomarker profiles. Phenotype 1 was "mild, good lung function, early onset", with a low-inflammatory, predominantly Type-2, phenotype. Phenotype 2 had a "moderate, hyper-responsive, eosinophilic" phenotype, with moderate asthma control, mild airflow obstruction and predominant Type-2 inflammation. Phenotype 3 had a "mixed severity, predominantly fixed obstructive, non-eosinophilic and neutrophilic" phenotype, with moderate asthma control and low Type-2 inflammation. Phenotype 4 had a "severe uncontrolled, severe reversible obstruction, mixed granulocytic" phenotype, with moderate Type-2 inflammation. These phenotypes had good longitudinal stability in the ADEPT cohort. They were reproduced and demonstrated high classification probability in two subsets of the U-BIOPRED asthma cohort. CONCLUSIONS: Focusing on the biology of the four clinical independently-validated easy-to-assess ADEPT asthma phenotypes will help understanding the unmet need and will aid in developing tailored therapies. TRIAL REGISTRATION: NCT01274507 (ADEPT), registered October 28, 2010 and NCT01982162 (U-BIOPRED), registered October 30, 2013.