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BackgroundInterleukin (IL)-33 is an upstream regulator of type 2 (T2) eosinophilic inflammation and has been proposed as a key driver of some asthma phenotypes.ObjectiveTo derive gene signatures from in vitro studies of IL-33-stimulated cells and use these to determine IL-33-associated enrichment patterns in asthma.MethodsSignatures downstream of IL-33 stimulation were derived from our in vitro study of human mast cells and from public datasets of in vitro stimulated human basophils, type 2 innate lymphoid cells (ILC2), regulatory T cells (Treg) and endothelial cells. Gene Set Variation Analysis (GSVA) was used to probe U-BIOPRED and ADEPT sputum transcriptomics to determine enrichment scores (ES) for each signature according to asthma severity, sputum granulocyte status and previously defined molecular phenotypes.ResultsIL-33-activated gene signatures were cell-specific with little gene overlap. Individual signatures, however, were associated with similar signalling pathways (TNF, NF-κB, IL-17 and JAK/STAT signalling) and immune cell differentiation pathways (Th17, Th1 and Th2 differentiation). ES for IL-33-activated gene signatures were significantly enriched in asthmatic sputum, particularly in patients with neutrophilic and mixed granulocytic phenotypes. IL-33 mRNA expression was not elevated in asthma whereas the expression of mRNA for IL1RL1, the IL-33 receptor, was up-regulated in the sputum of severe eosinophilic asthma. The mRNA expression for IL1RAP, the IL1RL1 co-receptor, was greatest in severe neutrophilic and mixed granulocytic asthma.ConclusionsIL-33-activated gene signatures are elevated in neutrophilic and mixed granulocytic asthma corresponding with IL1RAP co-receptor expression. This suggests incorporating T2-low asthma in anti-IL-33 trials.

BackgroundChanges in microRNA (miRNA) expression can contribute to the pathogenesis of many diseases, including asthma. We aimed to identify miRNAs that are differentially expressed between asthma patients and healthy controls, and explore their association with clinical and inflammatory parameters of asthma.MethodsDifferentially expressed miRNAs were determined by small RNA sequencing on bronchial biopsies of 79 asthma patients and 82 healthy controls using linear regression models. Differentially expressed miRNAs were associated with clinical and inflammatory asthma features. Potential miRNA–mRNA interactions were analysed using mRNA data available from the same bronchial biopsies, and enrichment of pathways was identified with Enrichr and g:Profiler.ResultsIn total, 78 differentially expressed miRNAs were identified in bronchial biopsies of asthma patients compared with controls, of which 60 remained differentially expressed after controlling for smoking and inhaled corticosteroid treatment. We identified several asthma-associated miRNAs, including miR-125b-5p and miR-223-3p, based on a significant association with multiple clinical and inflammatory asthma features and their negative correlation with genes associated with the presence of asthma. The most enriched biological pathway(s) affected by miR-125b-5p and miR-223-3p were inflammatory response and cilium assembly/organisation. Of interest, we identified that lower expression of miR-26a-5p was linked to more severe eosinophilic inflammation as measured in blood, sputum as well as bronchial biopsies.ConclusionCollectively, we identified miR-125b-5p, miR-223-3p and miR-26a-5p as potential regulators that could contribute to the pathogenesis of asthma.

RATIONALE: Type 2 innate lymphoid cells (ILC2s) are significant sources of type 2 cytokines, which are implicated in the pathogenesis of asthma and asthma exacerbations. The role of ILC2s in virus-induced asthma exacerbations is not well characterized. OBJECTIVES: To characterize pulmonary ILC responses following experimental rhinovirus challenge in patients with moderate asthma and healthy subjects. METHODS: Patients with moderate asthma and healthy subjects were inoculated with rhinovirus-16 and underwent bronchoscopy at baseline and at Day 3, and Day 8 after inoculation. Pulmonary ILC1s and ILC2s were quantified in bronchoalveolar lavage using flow cytometry. The ratio of bronchoalveolar lavage ILC2:ILC1 was assessed to determine their relative contributions to the clinical and immune response to rhinovirus challenge. MEASUREMENTS AND MAIN RESULTS: At baseline, ILC2s were significantly higher in patients with asthma than in healthy subjects. At Day 8, ILC2s significantly increased from baseline in both groups, which was significantly higher in patients with asthma than in healthy subjects (all comparisons P

Alterations in miRNA expression may play a role in the pathogenesis of asthma. Our aim was to identify miRNAs that are differentially expressed in bronchial biopsies of asthmatics compared to healthy controls and to explore their association with clinical asthma parameters. Small RNA sequencing data from bronchial biopsies of 79 asthmatics and 82 healthy controls was used to determine differential miRNA expression. The analysis was repeated in 30 non-smoking, ICS negative asthmatics and 42 non-smoking healthy controls. The differentially expressed miRNAs were associated with clinical parameters, including FEV1% predicted, neutrophil and eosinophil counts in blood, sputum and bronchial biopsies. In total 78 miRNAs were differentially expressed in bronchial biopsies from asthmatics compared to healthy controls. Of those 78 miRNAs, 62 were also differentially expressed in non-smoking, ICS negative asthmatics compared to non-smoking healthy controls. The most significant miRNAs were miR-16-5p, miR-451a, miR-125a/b and the let-7 family. Of the 62 asthma-associated miRNAs, 25 miRNAs correlated with one or more clinical asthma parameters. Lower levels of let-7a and let-7c were significantly associated with higher levels of blood and tissue eosinophils within asthma, while lower levels of miR-125a/b were associated with lower FEV1% predicted and higher blood eosinophils and neutrophils within asthma. In conclusion, various miRNAs including miR-125a/b and the let-7 family are differentially expressed in bronchial biopsies from asthmatics compared to healthy controls and are associate with multiple clinical asthma features. Based on the literature and our data, we suggest that those miRNAs might serve as potential asthma biomarker.

Approximately 40% of asthmatics experience remission of asthma symptoms. A better understanding of biological pathways leading to asthma remission may provide insight into new therapeutic targets for asthma. As an important mechanism of gene regulation, investigation of DNA methylation provides a promising approach. Our objective was to identify differences in epigenome wide DNA methylation levels in bronchial biopsies between subjects with asthma remission and subjects with persistent asthma or healthy controls.We analysed differential DNA methylation in bronchial biopsies from 26 subjects with persistent asthma, 39 remission subjects and 70 healthy controls, using the limma package. The comb-p tool was used to identify differentially methylated regions. DNA methylation of CpG-sites was associated to expression of nearby genes from the same biopsies to understand function.Four CpG-sites and 42 regions were differentially methylated between persistent asthma and remission. DNA methylation at two sites was correlated incis with gene expression at ACKR2 and DGKQ. Between remission subjects and healthy controls 1163 CpG-sites and 328 regions were differentially methylated. DNA methylation was associated with expression of a set of genes expressed in ciliated epithelium.CpGs differentially methylated between remission and persistent asthma identify genetic loci associated with resolution of inflammation and airway responsiveness. Despite the absence of symptoms, remission subjects have a DNA methylation profile that is distinct from that of healthy controls, partly due to changes in cellular composition, with a higher gene expression signal related to ciliated epithelium in remission versus healthy controls.

Human lungs enable efficient gas exchange and form an interface with the environment, which depends on mucosal immunity for protection against infectious agents. Tightly controlled interactions between structural and immune cells are required to maintain lung homeostasis. Here, we use single-cell transcriptomics to chart the cellular landscape of upper and lower airways and lung parenchyma in healthy lungs, and lower airways in asthmatic lungs. We report location-dependent airway epithelial cell states and a novel subset of tissue-resident memory T cells. In the lower airways of patients with asthma, mucous cell hyperplasia is shown to stem from a novel mucous ciliated cell state, as well as goblet cell hyperplasia. We report the presence of pathogenic effector type 2 helper T cells (TH2) in asthmatic lungs and find evidence for type 2 cytokines in maintaining the altered epithelial cell states. Unbiased analysis of cell-cell interactions identifies a shift from airway structural cell communication in healthy lungs to a TH2-dominated interactome in asthmatic lungs.

Background: Many children with asthma exhibit uncontrolled symptoms despite inhaled corticosteroid therapy. The molecular pathways linked to this phenotype are unknown. Aim: To investigate gene expression in paediatric severe and moderate/mild asthma blood samples. Methods: Gene expression analysis was applied on severe and mild asthma samples from pre- and school-age children (SApre,N=62; MApre,N=42; SAsc,N=75 and MAsc,N=37). Gene network analysis (WGCNA) was applied separately on the pre- and school-age data. Obtained gene modules were compared with the Jaccard Index. Results: No genes weresignificantly differentially-expressed in SA versus MA asthma in the two age groups (FDR Conclusion: WGCNA implicates mitochondrial oxidative stress in early onset severe asthma that needs further investigation.

Introduction: No definite cure is available yet for asthma. A subset of asthmatics goes into spontaneous remission and becomes symptom free. Comparison of gene expression in patients with persistent asthma and asthma remission may provide better understanding of the biological mechanisms leading to the remission of asthma and the identification of novel therapeutic targets. Aim: To find differential gene expression in bronchial biopsies from patients with persistent asthma and subjects with well-documented asthma remission. Methods: We performed RNA sequencing in RNA isolated from bronchial biopsies from 25 patients with persistent asthma and 38 subjects with asthma remission, who did not use inhaled corticosteroids. Asthma remission was defined as no symptoms in the last 3 years and no medication in the past 3 months. Differences in raw read counts between persistent asthma and asthma remission were analysed using DESeq2 adjusting for age, gender and smoking status as possible confounders. The FDR was controlled at 10%. Results: We found that the expression of keratin 13 ( KRT13 ) was lower in subjects with complete asthma remission compared to persistent asthmatics (log2FC = 5.4, q-value = 0.078). KRT13 encodes a subunit of keratin intermediate filaments characteristic of squamous epithelium and (bronchial) basal cells. Conclusion: This is the first study on gene expression in bronchial biopsies from asthma remission subjects. We demonstrate that the expression of KRT13 is lower in bronchial biopsies from subjects in remission compared to persistent asthmatics, suggesting less squamous metaplasia and/or basal cell hyperplasia in the airways. Funded by GlaxoSmithKline

Rationale: Severe asthma (SA)cohorts now exist worldwide. Comparing them will enable validation of initial findings from bio-clinical phenotyping of the cohorts. Aims: The aim of the study was to compare SA from Brazil (ProAR cohort)and from Europe(U-BIOPRED cohort) Methods: The U-BIOPRED adult SA cohort was collected from 11 European countries.ProAR cohort is comprised of adults with previously untreated SA followed up for several years in one reference centre in Brazil.Data collection was harmonized when possible. In ProAR there was a subgroup of subjects with a typical history of asthma but no spirometric reversibility whereas in U-BIOPRED, those with no reversibility were required to fulfil other criteria eg.hyperresponsiveness to methacholine. Results: Similarities and differences between U-BIOPRED and ProAR cohorts of SA. Figure 1 Summary: These two cohorts, which markedly differ in their environment and ethnicity, have striking similarities on their mean age and FEV1.Age of onset was higher in U-BIOPRED and the proportion of females was greater in ProAR. The use of oral corticosteroids daily was frequent in U-BIOPRED, and still they had more exacerbations and higher ACQ scores. Regular use of oral corticosteroids was not observed in ProAR, after several years of tapering down. Comparisons between these two cohorts, however diverse they are, will provide the basis of global, external validation of SA phenotypes.

Background: We have previously reported altered gene expression in adults with asthma compared to healthy controls from the U-BIOPRED study (Bigler, 2016). Altered transcripts may define dysregulated biological pathways and identify novel therapeutic targets. We hypothesised that similar dysregulation would be seen in children with asthma. Aim: To compare blood transcriptomic profiles of children and adults with asthma. Methods: Affymetrix blood transcriptomic profiles of severe asthmatic adult non-smokers, n=152, were compared to mild moderate asthmatics, n=50 (Shaw, 2015; Fleming, 2015). Profiles of school-aged children with severe asthma, n=75, were compared to mild moderate asthmatics, n=37, and in the preschool age group severe wheeze, n=62, was compared to mild moderate wheeze, n=42. Differentially expressed genes (DEG) were identified as probe sets with maximum median group intensity >log2 5, with a significant (raw P≤0.01) change ≥ 20%. Overlapping genes were determined and pathway analysis performed. Results: We found 1887 DEG comparing severe and mild moderate asthmatic adults. Only 28 DEG were found between the severe wheeze and mild pre-school age children, with a larger signature (569 DEG) in the school aged children. 480 genes were specific to school-aged children and 1801 specific to adults, with 89 DEG in common between the adults and school-aged children. Conclusions: Preschool age children were poorly defined in terms of blood transcriptomics by the clinical definitions used. While the school-aged children showed some DEG overlap with the adults, they were distinct in many DEG and pathways indicating that childhood and adult asthma may be mechanistically different.

Proteins of the CLCA gene family have been proposed to mediate calcium-activated chloride currents. In this study, we used detailed bioinformatics analysis and found that no transmembrane domains are predicted in hCLCA1 or mCLCA3 (Gob-5). Further analysis suggested that they are globular proteins containing domains that are likely to be involved in protein-protein interactions. In support of the bioinformatics analysis, biochemical studies showed that hCLCA1 and mCLCA3, when expressed in HEK293 cells, could be removed from the cell surface and could be detected in the extracellular medium, even after short incubation times. The accumulation in the medium was shown to be brefeldin A-sensitive, demonstrating that hCLCA1 is constitutively secreted. The N-terminal cleavage products of hCLCA1 and mCLCA3 could be detected in bronchoalveolar lavage fluid taken from asthmatic subjects and ovalbumin-challenged mice, demonstrating release from cells in a physiological setting. We conclude that hCLCA1 and mCLCA3 are non-integral membrane proteins and therefore cannot be chloride channels in their own right.

Chronic inflammation in the lung has long been linked to the pathogenesis of asthma. Central to this airway inflammation is a T-cell response to allergens, with Th2 cytokines driving the differentiation, survival and function of the major inflammatory cells involved in the allergic cascade. PI3Kδ (phosphoinositide 3-kinase δ) is a lipid kinase, expressed predominantly in leucocytes, where it plays a critical role in immune receptor signalling. A selective PI3Kδ inhibitor is predicted to block T-cell activation in the lung, reducing the production of pro-inflammatory Th2 cytokines. PI3Kδ is also involved in B-cell and mast cell activation. Therefore the inhibition of PI3Kδ should dampen down the inflammatory cascade involved in the asthmatic response through a wide breadth of pharmacology. Current anti-inflammatory therapies, which are based on corticosteroids, are effective in controlling inflammation in mild asthmatics, but moderate/severe asthmatic patients remain poorly controlled, experiencing recurrent exacerbations. Corticosteroids have no effect on mast cell degranulation and do not act directly on B-cells, so, overall, a PI3Kδ inhibitor has the potential to deliver improvements in onset of action, efficacy and reduced exacerbations in moderate/severe asthmatics. Additionally, PI3Kδ inhibition is expected to block effects of Th17 cells, which are increasingly implicated in steroid-insensitive asthma.

Glucocorticoids are the mainstay of asthma therapy; however, a proportion of patients with asthma has a severe form of the disease that fails to respond to therapy. Understanding the molecular mechanisms behind glucocorticoid-insensitive asthma is therefore of clinical importance. Evidence in glucocorticoid-unresponsive Henrietta Lack (HeLa) cells indicated that cofilin-1 could act as an inhibitor of glucocorticoid function.To determine whether cofilin-1 expression is abnormally expressed in cells from patients with severe glucocorticoid-insensitive asthma and examine the effect of cofilin-1 overexpression on glucocorticoid function.Peripheral blood CD4(+) T cells were purified from 16 subjects with severe glucocorticoid-insensitive asthma and 16 subjects with mild glucocorticoid-sensitive asthma, and cofilin-1 expression was determined by quantitative real-time RT-PCR and Western blotting. The effect of dexamethasone on cofilin-1 expression was determined in Jurkat T cells, and the effect of cofilin-1 overexpression on anti-CD3/CD28-stimulated IL-2 release was measured.Peripheral blood CD4(+) T cells from subjects with severe glucocorticoid-insensitive asthma are less responsive to dexamethasone than cells from subjects with mild glucocorticoid-sensitive asthma. Cells from these patients express significantly (P.05) higher levels of cofilin-1 than cells from subjects with mild asthma. Dexamethasone did not affect cofilin-1 expression in Jurkat T cells. Functionally, dexamethasone suppression of anti-CD3/CD28-stimulated IL-2 was attenuated in Jurkat cells overexpressing cofilin-1.These results suggest that increased cofilin-1 expression may be important in the regulation of glucocorticoid sensitivity in peripheral blood lymphocytes of patients with severe treatment-insensitive asthma.Understanding the mechanisms of enhanced cofilin-1 expression may lead to the development of new therapies for severe treatment-insensitive asthma.