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2.
Allergy ; 74(6): 1102-1112, 2019 06.
Article in English | MEDLINE | ID: mdl-30667542

ABSTRACT

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.


Subject(s)
Airway Remodeling/genetics , Asthma/immunology , Eosinophils/immunology , Matrix Metalloproteinase 10/genetics , Matrix Metalloproteinase 10/metabolism , Proto-Oncogene Proteins c-met/genetics , Proto-Oncogene Proteins c-met/metabolism , Adult , Asthma/pathology , Biopsy , Bronchi/pathology , Cohort Studies , Eosinophilia/immunology , Extracellular Matrix/genetics , Female , Humans , Immunohistochemistry , Inflammation/genetics , Male , Middle Aged , Proto-Oncogene Protein c-ets-1/metabolism , SOX Transcription Factors/metabolism , Transcriptome
3.
Eur Respir J ; 53(1)2019 01.
Article in English | MEDLINE | ID: mdl-30578390

ABSTRACT

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.


Subject(s)
Asthma/blood , Cell Adhesion Molecules/blood , Eosinophilia/diagnosis , Sputum/chemistry , Adult , Biomarkers , Breath Tests , Case-Control Studies , Eosinophilia/blood , Eosinophils/cytology , Female , Humans , Immunoglobulin E/blood , Interleukins/analysis , Leukocyte Count , Male , Middle Aged , Nitric Oxide/analysis , Phenotype , Prospective Studies , Smoking/adverse effects
4.
Eur Respir J ; 51(5)2018 05.
Article in English | MEDLINE | ID: mdl-29650557

ABSTRACT

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.


Subject(s)
Asthma/metabolism , Ex-Smokers , Proteomics/methods , Smokers , Sputum/metabolism , Adult , Aged , Asthma/complications , Biomarkers/metabolism , Bronchi/pathology , Eosinophils/metabolism , Exhalation , Female , Gene Expression , Gene Expression Profiling , Humans , Leukocyte Count , Male , Middle Aged , Nitric Oxide/metabolism , Smoking/metabolism , Spirometry
5.
J Allergy Clin Immunol ; 141(4): 1280-1290, 2018 04.
Article in English | MEDLINE | ID: mdl-28756296

ABSTRACT

BACKGROUND: Adult-onset severe asthma is characterized by highly symptomatic disease despite high-intensity asthma treatments. Understanding of the underlying pathways of this heterogeneous disease is needed for the development of targeted treatments. Gene set variation analysis is a statistical technique used to identify gene profiles in heterogeneous samples. OBJECTIVE: We sought to identify gene profiles associated with adult-onset severe asthma. METHODS: This was a cross-sectional, observational study in which adult patients with adult-onset of asthma (defined as starting at age ≥18 years) as compared with childhood-onset severe asthma (<18 years) were selected from the U-BIOPRED cohort. Gene expression was assessed on the total RNA of induced sputum (n = 83), nasal brushings (n = 41), and endobronchial brushings (n = 65) and biopsies (n = 47) (Affymetrix HT HG-U133+ PM). Gene set variation analysis was used to identify differentially enriched predefined gene signatures of leukocyte lineage, inflammatory and induced lung injury pathways. RESULTS: Significant differentially enriched gene signatures in patients with adult-onset as compared with childhood-onset severe asthma were identified in nasal brushings (5 signatures), sputum (3 signatures), and endobronchial brushings (6 signatures). Signatures associated with eosinophilic airway inflammation, mast cells, and group 3 innate lymphoid cells were more enriched in adult-onset severe asthma, whereas signatures associated with induced lung injury were less enriched in adult-onset severe asthma. CONCLUSIONS: Adult-onset severe asthma is characterized by inflammatory pathways involving eosinophils, mast cells, and group 3 innate lymphoid cells. These pathways could represent useful targets for the treatment of adult-onset severe asthma.


Subject(s)
Asthma/genetics , Transcriptome/immunology , Adult , Age of Onset , Asthma/immunology , Cross-Sectional Studies , Female , Gene Expression Profiling , Genetic Markers , Humans , Male , Middle Aged , Oligonucleotide Array Sequence Analysis , Phenotype , Severity of Illness Index
6.
Biosci Rep ; 37(6)2017 Dec 22.
Article in English | MEDLINE | ID: mdl-28842514

ABSTRACT

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) <0.05) between chronic cough and healthy non-cough ASMCs at baseline. Poly(I:C) stimulation resulted in 212 DEGs (>1.5 fold-change, FDR <0.05) in ASMCs from chronic cough patients compared with 1674 DEGs in healthy non-cough volunteers. The top up-regulated genes included chemokine (C-X-C motif) ligand (CXCL) 11 (CXCL11), CXCL10, chemokine (C-C motif) ligand (CCL) 5 (CCL5) and interferon-induced protein 44 like (IFI44L) corresponding with inflammation and innate immune response pathways. ASMCs from cough subjects had enhanced activation of viral response pathways in response to poly(I:C) compared with healthy non-cough subjects, reduced activation of pathways involved in chronic inflammation and equivalent activation of neuroregulatory genes. The poly(I:C)-induced release of inflammatory mediators, including CXCL8, interleukin (IL)-6 and CXCL1, from ASMCs from cough patients was significantly impaired compared with healthy non-cough subjects. Addition of fluticasone propionate (FP) to poly(I:C)-treated ASMCs resulted in greater gene expression changes in healthy non-cough ASMCs. FP had a differential effect on poly(I:C)-induced mediator release between chronic cough and healthy non-cough volunteers. In conclusion, altered innate immune and inflammatory gene profiles within ASMCs, rather than infiltrating cells or nerves, may drive the cough response following respiratory viral infection.


Subject(s)
Airway Remodeling/genetics , Cough/immunology , Gene Expression Profiling/methods , Immunity, Innate/genetics , Myocytes, Smooth Muscle/immunology , Airway Remodeling/immunology , Anti-Inflammatory Agents/pharmacology , Antigens/genetics , Antigens/metabolism , Bronchi/cytology , Bronchi/immunology , Bronchi/metabolism , Bronchoscopy , Chronic Disease , Cytokines/genetics , Cytokines/metabolism , Cytoskeletal Proteins/genetics , Cytoskeletal Proteins/metabolism , Female , Fluticasone/pharmacology , Humans , Immunity, Innate/immunology , Inflammation/genetics , Inflammation/metabolism , Male , Middle Aged , Myocytes, Smooth Muscle/metabolism , Pilot Projects , Poly C/pharmacology
7.
Eur Respir J ; 49(2)2017 02.
Article in English | MEDLINE | ID: mdl-28179442

ABSTRACT

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.


Subject(s)
Asthma/genetics , Eosinophils/immunology , Sputum , Th2 Cells/immunology , Transcriptome , Adrenal Cortex Hormones/therapeutic use , Adult , Aged , Area Under Curve , Asthma/immunology , Biomarkers , C-Reactive Protein/analysis , Case-Control Studies , Female , Forced Expiratory Volume , Humans , Interleukin-13/immunology , Leukocyte Count , Male , Middle Aged , Nitric Oxide/blood , Phenotype , United Kingdom
8.
Am J Respir Crit Care Med ; 195(4): 443-455, 2017 Feb 15.
Article in English | MEDLINE | ID: mdl-27580351

ABSTRACT

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.


Subject(s)
Adrenal Cortex Hormones/immunology , Asthma/genetics , Bronchi/pathology , Adrenal Cortex Hormones/pharmacology , Adrenal Cortex Hormones/therapeutic use , Adult , Asthma/drug therapy , Asthma/immunology , Asthma/pathology , Biomarkers/analysis , Biopsy , Breath Tests , Bronchoscopy/instrumentation , Bronchoscopy/methods , Cohort Studies , Drug Resistance/genetics , Drug Resistance/immunology , Eosinophils/cytology , Eosinophils/immunology , Female , Gene Expression Profiling/instrumentation , Gene Expression Profiling/methods , Humans , Inflammation , Leukocyte Count , Male , Middle Aged , Phenotype , Severity of Illness Index , Sputum/cytology , Sputum/immunology , Th2 Cells/cytology , Th2 Cells/immunology
9.
Cell Immunol ; 284(1-2): 119-28, 2013.
Article in English | MEDLINE | ID: mdl-23973875

ABSTRACT

Plasmacytoid dendritic cells [pDC], also known as type I interferon [IFN] producing cells, play a significant role in the pathogenesis of systemic lupus erythematosus [SLE]. The current study was undertaken to identify novel SLE autoantibody specificities associated with interferon-inducing activity in human pDCs. We found that immune complex mixtures from some Interferon signature negative [IFN-] and all interferon signature positive [IFN+] SLE patients could trigger type I IFN production by pDCs. IgGs from IFN- and IFN+ SLE patients were subsequently screened via a high throughput protein microarray to identify novel auto-antibody specifities that mediate type I IFN production by pDCs. This approach identified five novel autoantibodies that may contribute to type I IFN production by pDCs via a nucleic acid dependent mechanism. The newly identified autoantibody specificities function in a myriad of cell processess and, to date, have not been implicated in SLE pathogenesis.


Subject(s)
Antigen-Antibody Complex/immunology , Autoantibodies/immunology , Dendritic Cells/immunology , Interferon-alpha/biosynthesis , Interferon-alpha/immunology , Lupus Erythematosus, Systemic/immunology , Autoantibodies/blood , Cell Line , Female , Humans , Leukocytes, Mononuclear/immunology , Lupus Erythematosus, Systemic/blood , Protein Array Analysis
10.
Am J Respir Crit Care Med ; 185(1): 67-76, 2012 Jan 01.
Article in English | MEDLINE | ID: mdl-22016448

ABSTRACT

RATIONALE: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease of unknown etiology with a variable and unpredictable course. OBJECTIVES: The aim of this study was to identify and validate plasma proteins that are predictive of outcome in IPF. METHODS: Plasma samples were available for 241 patients with IPF (140 derivation and 101 validation). In the derivation cohort, concentrations of 92 proteins were analyzed using a multiplex bead-based immunoassay and concentrations of matrix metalloproteinase (MMP)-7, MMP-1, and surfactant protein D were assessed by ELISA. In the validation cohort concentrations of intercellular adhesion molecule (ICAM)-1, IL-8, and vascular cell adhesion molecule (VCAM)-1 were assessed by bead-based multiplex assay, and S100A12 and MMP-7 by ELISA. Associations of biomarkers with mortality, transplant-free survival, and disease progression were tested in the derivation and validation cohorts using nonparametric methods of survival analysis and the Cox proportional hazards model, and an integrated risk prediction score was derived and tested. MEASUREMENTS AND MAIN RESULTS: High concentrations of MMP-7, ICAM-1, IL-8, VCAM-1, and S100A12 predicted poor overall survival, poor transplant-free survival, and poor progression-free survival in the derivation cohort. In the independent validation cohort high concentrations of all five were predictive of poor transplant-free survival; MMP-7, ICAM-1, and IL-8 of overall survival; and ICAM-1 of poor progression-free survival. The personal clinical and molecular mortality prediction index derived in the derivation cohort was highly predictive of mortality in the validation cohort. CONCLUSIONS: Our results suggest that plasma proteins should be evaluated as a tool for prognosis determination in prioritization of patients for lung transplantation and stratification in drug studies.


Subject(s)
Cell Adhesion Molecules/blood , Idiopathic Pulmonary Fibrosis/blood , Idiopathic Pulmonary Fibrosis/mortality , Interleukin-8/blood , Matrix Metalloproteinases/blood , S100 Proteins/blood , Aged , Biomarkers/blood , Cohort Studies , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Intercellular Adhesion Molecule-1/blood , Male , Matrix Metalloproteinase 1/blood , Matrix Metalloproteinase 7/blood , Predictive Value of Tests , Proportional Hazards Models , S100A12 Protein , Survival Analysis , Vascular Cell Adhesion Molecule-1/blood
11.
12.
Bioorg Med Chem Lett ; 21(9): 2626-30, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21315584

ABSTRACT

We describe the systematic optimization, focused on the improvement of CV-TI, of a series of CCR2 antagonists. This work resulted in the identification of 10 (((1S,3R)-1-isopropyl-3-((3S,4S)-3-methoxy-tetrahydro-2H-pyran-4-ylamino)cyclopentyl)(4-(5-(trifluoromethyl)pyridazin-3-yl)piperazin-1-yl)methanone) which possessed a low projected human dose 35-45mg BID and a CV-TI=3800-fold.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Models, Molecular , Piperazines/chemistry , Piperazines/pharmacology , Pyridazines/chemistry , Pyridazines/pharmacology , Receptors, CCR2/agonists , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacokinetics , Biological Assay , Humans , Inhibitory Concentration 50 , Microsomes/drug effects , Microsomes/metabolism , Molecular Structure , Piperazines/pharmacokinetics , Protein Binding/drug effects , Pyridazines/pharmacokinetics , Receptors, CCR2/blood , Structure-Activity Relationship
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