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Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma.

ABSTRACT: Management of severe asthma remains a challenge despite treatment with glucocorticosteroid therapy. The majority of studies investigating disease mechanisms in treatment-resistant severe asthma have previously focused on the large central airways, with very few utilizing transcriptomic approaches. The small peripheral airways, which comprise the majority of the airway surface area, remain an unexplored area in severe asthma and were targeted for global epithelial gene expression profiling in this study. Differences between central and peripheral airways were evaluated using transcriptomic analysis (Affymetrix HG U133 plus 2.0 GeneChips) of epithelial brushings obtained from severe asthma patients (N = 17) and healthy volunteers (N = 23). Results were validated in an independent cohort (N = 10) by real-time quantitative PCR. The IL-13 disease signature that is associated with an asthmatic phenotype was upregulated in severe asthmatics compared to healthy controls but was predominantly evident within the peripheral airways, as were genes related to mast cell presence. The gene expression response associated with glucocorticosteroid therapy (i.e. FKBP5) was also upregulated in severe asthmatics compared to healthy controls but, in contrast, was more pronounced in central airways. Moreover, an altered epithelial repair response (e.g. FGFBP1) was evident across both airway sites reflecting a significant aspect of disease in severe asthma unadressed by current therapies. A transcriptomic approach to understand epithelial activation in severe asthma has thus highlighted the need for better-targeted therapy to the peripheral airways in severe asthma, where the IL-13 disease signature persists despite treatment with currently available therapy.

SUBMITTER: Singhania A

PROVIDER: S-EPMC5207492 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Changes in airway epithelial cell differentiation, driven in part by IL-13, are important in asthma. Micro-RNAs (miRNAs) regulate cell differentiation in many systems and could contribute to epithelial abnormalities in asthma.To determine whether airway epithelial miRNA expression is altered in asthma and identify IL-13-regulated miRNAs.We used miRNA microarrays to analyze bronchial epithelial brushings from 16 steroid-naive subjects with asthma before and after inhaled corticosteroids, 19 steroid-using subjects with asthma, and 12 healthy control subjects, and the effects of IL-13 and corticosteroids on cultured bronchial epithelial cells. We used quantitative polymerase chain reaction to confirm selected microarray results.Most (12 of 16) steroid-naive subjects with asthma had a markedly abnormal pattern of bronchial epithelial miRNA expression by microarray analysis. Compared with control subjects, 217 miRNAs were differentially expressed in steroid-naive subjects with asthma and 200 in steroid-using subjects with asthma (false discovery rate < 0.05). Treatment with inhaled corticosteroids had modest effects on miRNA expression in steroid-naive asthma, inducing a statistically significant (false discovery rate < 0.05) change for only nine miRNAs. qPCR analysis confirmed differential expression of 22 miRNAs that were highly differentially expressed by microarrays. IL-13 stimulation recapitulated changes in many differentially expressed miRNAs, including four members of the miR-34/449 family, and these changes in miR-34/449 family members were resistant to corticosteroids.Dramatic alterations of airway epithelial cell miRNA levels are a common feature of asthma. These alterations are only modestly corrected by inhaled corticosteroids. IL-13 effects may account for some of these alterations, including repression of miR-34/449 family members that have established roles in airway epithelial cell differentiation. Clinical trial registered with www.clinicaltrials.gov (NCT 00595153).

Project description:Previous metagenomic studies in asthma have been limited by inadequate sequencing depth for species-level bacterial identification and by heterogeneity in clinical phenotyping. We hypothesize that chronic bacterial airways infection is a key "treatable trait" whose prevalence, clinical phenotype and reliable biomarkers need definition. In this study, we have applied a method for Oxford Nanopore sequencing for the unbiased metagenomic characterization of severe asthma. We optimized methods to compare performance of Illumina MiSeq, Nanopore sequencing, and RT-qPCR on total sputum DNA extracts against culture/MALDI-TOF for analysis of induced sputum samples from highly phenotyped severe asthma during clinical stability. In participants with severe asthma (n = 23) H. influenzae was commonly cultured (n = 8) and identified as the dominant bacterial species by metagenomic sequencing using an optimized method for Illumina MiSeq and Oxford Nanopore. Alongside superior operational characteristics, Oxford Nanopore achieved near complete genome coverage of H. influenzae and demonstrated a high level of agreement with Illumina MiSeq data. Clinically significant infection was confirmed with validated H. influenzae plasmid-based quantitative PCR assay. H. influenzae positive patients were found to have sputum neutrophilia and lower FeNO. In conclusion, using an optimized method of direct sequencing of induced sputum samples, H. influenzae was identified as a clinically relevant pathogen in severe asthma and was identified reliably using metagenomic sequencing. Application of these protocols in ongoing analysis of large patient cohorts will allow full characterization of this clinical phenotype. IMPORTANCE The human airways were once thought sterile in health. Now metagenomic techniques suggest bacteria may be present, but their role in asthma is not understood. Traditional culture lacks sensitivity and current sequencing techniques are limited by operational problems and limited ability to identify pathogens at species level. We optimized a new sequencing technique-Oxford Nanopore technologies (ONT)-for use on human sputum samples and compared it with existing methods. We found ONT was effective for rapidly analyzing samples and could identify bacteria at the species level. We used this to show Haemophilus influenzae was a dominant bacterium in the airways in people with severe asthma. The presence of Haemophilus was associated with a "neutrophilic" form of asthma - a subgroup for which we currently lack specific treatments. Therefore, this technique could be used to target chronic antibiotic therapy and in research to characterize the full breadth of bacteria in the airways.

Project description:RationaleSevere asthma (SA) is a heterogeneous disease with multiple molecular mechanisms. Gene expression studies of bronchial epithelial cells in individuals with asthma have provided biological insight and underscored possible mechanistic differences between individuals.ObjectivesIdentify networks of genes reflective of underlying biological processes that define SA.MethodsAirway epithelial cell gene expression from 155 subjects with asthma and healthy control subjects in the Severe Asthma Research Program was analyzed by weighted gene coexpression network analysis to identify gene networks and profiles associated with SA and its specific characteristics (i.e., pulmonary function tests, quality of life scores, urgent healthcare use, and steroid use), which potentially identified underlying biological processes. A linear model analysis confirmed these findings while adjusting for potential confounders.Measurements and main resultsWeighted gene coexpression network analysis constructed 64 gene network modules, including modules corresponding to T1 and T2 inflammation, neuronal function, cilia, epithelial growth, and repair mechanisms. Although no network selectively identified SA, genes in modules linked to epithelial growth and repair and neuronal function were markedly decreased in SA. Several hub genes of the epithelial growth and repair module were found located at the 17q12-21 locus, near a well-known asthma susceptibility locus. T2 genes increased with severity in those treated with corticosteroids but were also elevated in untreated, mild-to-moderate disease compared with healthy control subjects. T1 inflammation, especially when associated with increased T2 gene expression, was elevated in a subgroup of younger patients with SA.ConclusionsIn this hypothesis-generating analysis, gene expression networks in relation to asthma severity provided potentially new insight into biological mechanisms associated with the development of SA and its phenotypes.

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Altered Epithelial Gene Expression in Peripheral Airways of Severe Asthma.

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