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International audience; Eosinophils play a homeostatic role in the body's immune responses. These cells are involved in combating some parasitic, bacterial, and viral infections and certain cancers and have pathologic roles in diseases including asthma, chronic rhinosinusitis with nasal polyps, eosinophilic gastrointestinal disorders, and hypereosinophilic syndromes. Treatment of eosinophilic diseases has traditionally been through nonspecific eosinophil attenuation by use of glucocorticoids. However, several novel biologic therapies targeting eosinophil maturation factors, such as interleukin (IL)-5 and the IL-5 receptor or IL-4/IL-13, have recently been approved for clinical use. Despite the success of biologic therapies, some patients with eosinophilic inflammatory disease may not achieve adequate symptom control, underlining the need to further investigate the contribution of patient characteristics, such as comorbidities and other processes, in driving ongoing disease activity. New research has shown that eosinophils are also involved in several homeostatic processes, including metabolism, tissue remodeling and development, neuronal regulation, epithelial and microbiome regulation, and immunoregulation, indicating that these cells may play a crucial role in metabolic regulation and organ function in healthy humans. Consequently, further investigation is needed into the homeostatic roles of eosinophils and eosinophil-mediated processes across different tissues and their varied microenvironments. Such work may provide important insights into the role of eosinophils not only under disease conditions but also in health. This narrative review synthesizes relevant publications retrieved from PubMed informed by author expertise to provide new insights into the diverse roles of eosinophils in health and disease, with particular emphasis on the implications for current and future development of eosinophil-targeted therapies.

Background Sputum cytology is currently the gold standard to evaluate cellular inflammation in the airways and phenotyping patients with airways diseases. Sputum eosinophil proportions have been used to guide treatment for moderate to severe asthma. Furthermore, raised sputum neutrophils are associated with poor disease control and impaired lung function in both asthma and COPD and small airways disease in cystic fibrosis. However, induced-sputum analysis is subjective and resource heavy, requiring dedicated specialist processing and assessment; this limits its utility in most clinical settings. Indirect blood eosinophil measures have been adopted in clinical care. However, there are currently no good peripheral blood biomarkers of airway neutrophils. A resource-light sputum processing approach could thus help integrate induced sputum more readily into routine clinical care. New mechanical disruption (MD) methods can rapidly obtain viable single cell suspensions from sputum samples. Aims The aim of this study was to compare MD sputum processing to traditional methods for cell viability, granulocyte proportions and sputum cytokine analysis. Methods Sputum plugs were split and processed using traditional methods and the MD method, and samples were then compared. Results The MD method produced a homogeneous cell suspension in 62 s; 70 min faster than the standard method used. No significant difference was seen between the cell viability (p = 0.09), or the concentration of eosinophils (p = 0.83), neutrophils (p = 0.99) or interleukin-8 (p = 0.86) using MD. Conclusion This cost-effective method of sputum processing could provide a more pragmatic, sustainable means of directly monitoring the airway milieu. Therefore, we recommend this method be taken forward for further investigation.

Background: IL-13 is considered an archetypal T2 cytokine central to the clinical disease expression of asthma. The IL-13 response genes, which are upregulated in central airway bronchial epithelial of asthma patients, can be normalized by high-dose inhaled steroid therapy in severe asthma. However, this is not the case within the peripheral airways. We have sought to further understand IL-13 in the peripheral airways in severe asthma through bronchoalveolar analysis. Methods: bronchoalveolar lavage samples were collected from 203 asthmatic and healthy volunteers, including 78 with severe asthma. Inflammatory mediators were measured using a multiple cytokine immunoassay platform. This analysis was replicated in a further 59 volunteers, in whom 16S rRNA analysis of BAL samples was undertaken by terminal restriction fragment length polymorphism. Results: severe asthma patients with high BAL IL-13, despite treatment with high-dose inhaled corticosteroids, had more severe lung function and significantly higher BAL neutrophil percentages, but not BAL eosinophils than those with normal BAL-13 concentrations. This finding was replicated in the second cohort, which further associated BAL IL-13 and neutrophilia with a greater abundance of potentially pathogenic bacteria in the peripheral airways. Conclusion: our findings demonstrate a steroid unresponsive source of IL-13 that is associated with BAL neutrophilia and bacterial dysbiosis in severe asthma. Our findings highlight the biological complexity of severe asthma and the importance of a greater understanding of the innate and adaptive immune responses in the peripheral airways in this disease.

Background Patients with severe asthma may have a greater risk of dying from COVID-19 disease. Angiotensin converting enzyme-2 (ACE2) and the enzyme proteases, transmembrane protease serine 2 (TMPRSS2) and FURIN, are needed for viral attachment and invasion into host cells. Methods We examined microarray mRNA expression of ACE2, TMPRSS2 and FURIN in sputum, bronchial brushing and bronchial biopsies of the European U-BIOPRED cohort. Clinical parameters and molecular phenotypes, including asthma severity, sputum inflammatory cells, lung functions, oral corticosteroid (OCS) use, and transcriptomic-associated clusters, were examined in relation to gene expression levels. Results ACE2 levels were significantly increased in sputum of severe asthma compared to mild-moderate asthma. In multivariate analyses, sputum ACE2 levels were positively associated with OCS use and male gender. Sputum FURIN levels were significantly related to neutrophils (%) and the presence of severe asthma. In bronchial brushing samples, TMPRSS2 levels were positively associated with male gender and body mass index, whereas FURIN levels with male gender and blood neutrophils. In bronchial biopsies, TMPRSS2 levels were positively related to blood neutrophils. The neutrophilic molecular phenotype characterised by high inflammasome activation expressed significantly higher FURIN levels in sputum than the eosinophilic Type 2-high or the pauci-granulocytic oxidative phosphorylation phenotypes. Conclusion Levels of ACE2 and FURIN may differ by clinical or molecular phenotypes of asthma. Sputum FURIN expression levels were strongly associated with neutrophilic inflammation and with inflammasome activation. This might indicate the potential for a greater morbidity and mortality outcome from SARS-CoV-2 infection in neutrophilic severe asthma.

Background: Asthma treatment guidelines recommend increasing corticosteroid dose to control symptoms and reduce exacerbations. This approach is potentially flawed because symptomatic asthma can occur without corticosteroid responsive type-2 (T2)-driven eosinophilic inflammation, and inappropriately high-dose corticosteroid treatment might have little therapeutic benefit with increased risk of side-effects. We compared a biomarker strategy to adjust corticosteroid dose using a composite score of T2 biomarkers (fractional exhaled nitric oxide [FENO], blood eosinophils, and serum periostin) with a standardised symptom–risk-based algorithm (control). Methods: We did a single-blind, parallel group, randomised controlled trial in adults (18–80 years of age) with severe asthma (at treatment steps 4 and 5 of the Global Initiative for Asthma) and FENO of less than 45 parts per billion at12 specialist severe asthma centres across England, Scotland, and Northern Ireland. Patients were randomly assigned (4:1) to either the biomarker strategy group or the control group by an online electronic case-report form, in blocks of ten, stratified by asthma control and use of rescue systemic steroids in the previous year. Patients were masked to study group allocation throughout the entirety of the study. Patients attended clinic every 8 weeks, with treatment adjustment following automated treatment-group-specific algorithms: those in the biomarker strategy group received a default advisory to maintain treatment and those in the control group had their treatment adjusted according to the steps indicated by the trial algorithm. The primary outcome was the proportion of patients with corticosteroid dose reduction at week 48, in the intention-to-treat (ITT) population. Secondary outcomes were inhaled corticosteroid (ICS) dose at the end of the study; cumulative dose of ICS during the study; proportion of patients on maintenance oral corticosteroids (OCS) at study end; rate of protocol-defined severe exacerbations per patient year; time to first severe exacerbation; number of hospital admissions for asthma; changes in lung function, Asthma Control Questionnaire-7 score, Asthma Quality of Life Questionnaire score, and T2 biomarkers from baseline to week 48; and whether patients declined to progress to OCS. A secondary aim of our study was to establish the proportion of patients with severe asthma in whom T2 biomarkers remained low when corticosteroid therapy was decreased to a minimum ICS dose. This study is registered with ClinicalTrials.gov, NCT02717689 and has been completed. Findings: Patients were recruited from Jan 8, 2016, to July 12, 2018. Of 549 patients assessed, 301 patients were included in the ITT population and were randomly assigned to the biomarker strategy group (n=240) or to the control group(n=61). 28·4% of patients in the biomarker strategy group were on a lower corticosteroid dose at week 48 compared with 18·5% of patients in the control group (adjusted odds ratio [aOR] 1·71 [95% CI 0·80–3·63]; p=0·17). In the per-protocol (PP) population (n=121), a significantly greater proportion of patients were on a lower corticosteroid dose at week 48 in the biomarker strategy group (30·7% of patients) compared with the control group (5·0% of patients; aOR 11·48 [95% CI 1·35–97·83]; p=0·026). Patient choice to not follow treatment advice was the principle reason for loss to PP analysis. There was no difference in secondary outcomes between study groups and no loss of asthma control among patients in the biomarker strategy group who reduced their corticosteroid dose. Interpretation: Biomarker-based corticosteroid adjustment did not result in a greater proportion of patients reducing corticosteroid dose versus control. Understanding the reasons for patients not following treatment advice in both treatment strategies is an important area for future research. The prevalence of T2 biomarker-low severe asthma was low.

Background: Asthma is a complex disease with multiple phenotypes that may differ in disease pathobiology and treatment response. IL33 single nucleotide polymorphisms (SNPs) have been reproducibly associated with asthma. IL33 levels are elevated in sputum and bronchial biopsies of patients with asthma. The functional consequences of IL33 asthma SNPs remain unknown. Objective: This study sought to determine whether IL33 SNPs associate with asthma-related phenotypes and with IL33 expression in lung or bronchial epithelium. This study investigated the effect of increased IL33 expression on human bronchial epithelial cell (HBEC) function. Methods: Association between IL33 SNPs (Chr9: 5,815,786-6,657,983) and asthma phenotypes (Lifelines/DAG [Dutch Asthma GWAS]/GASP [Genetics of Asthma Severity & Phenotypes] cohorts) and between SNPs and expression (lung tissue, bronchial brushes, HBECs) was done using regression modeling. Lentiviral overexpression was used to study IL33 effects on HBECs. Results: We found that 161 SNPs spanning the IL33 region associated with 1 or more asthma phenotypes after correction for multiple testing. We report a main independent signal tagged by rs992969 associating with blood eosinophil levels, asthma, and eosinophilic asthma. A second, independent signal tagged by rs4008366 presented modest association with eosinophilic asthma. Neither signal associated with FEV 1, FEV 1/forced vital capacity, atopy, and age of asthma onset. The 2 IL33 signals are expression quantitative loci in bronchial brushes and cultured HBECs, but not in lung tissue. IL33 overexpression in vitro resulted in reduced viability and reactive oxygen species–capturing of HBECs, without influencing epithelial cell count, metabolic activity, or barrier function. Conclusions: We identify IL33 as an epithelial susceptibility gene for eosinophilia and asthma, provide mechanistic insight, and implicate targeting of the IL33 pathway specifically in eosinophilic asthma.

Background Mepolizumab (100 mg delivered s.c. every 4 weeks) is indicated for add-on maintenance treatment for patients with severe eosinophilic asthma. Mepolizumab has been shown to reduce exacerbations and the requirement for daily oral corticosteroids, and improve asthma control and symptoms. However, data on the durability of the response to mepolizumab during dosing periods are limited. The aim of this study was to investigate the efficacy profile in patients with severe eosinophilic asthma over the 4-weekly dosing period for various fixed mepolizumab doses. Methods This was a post hoc analysis of data from the phase IIb/III DREAM study. Patients ≥12 years of age with severe eosinophilic asthma were randomised (1:1:1:1) to receive intravenous mepolizumab 75 mg (equivalent to 100 mg s.c.), 250 mg, 750 mg or placebo, plus standard of care, every 4 weeks for 52 weeks. The number of exacerbations and eDiary data (peak expiratory flow, rescue medication use and symptom scores) from two periods in each 4-weekly dosing interval (days 1–14 and 15–28) over the 52-week treatment period were analysed. Findings eDiary data and the proportion of patients experiencing ≥1 exacerbation were similar during the first and second 2 weeks of a dosing period across all mepolizumab doses. Interpretation These results demonstrate that the response to mepolizumab is sustained over the 4-weekly dosing period with no differences across a 10-fold dose range and supports the use of the current mepolizumab dosing regimen in patients with severe eosinophilic asthma., Post hoc analysis of data from DREAM demonstrated a sustained response to mepolizumab across the 4-weekly dosing period, suggesting therapeutic benefit is maintained between each mepolizumab dose following long-term treatment https://bit.ly/3843lH6

Background: Transcriptomic changes in patients who respond clinically to biological therapies may identify responses in other tissues or diseases. Objective: We sought to determine whether a disease signature identified in atopic dermatitis (AD) is seen in adults with severe asthma and whether a transcriptomic signature for patients with AD who respond clinically to anti–IL-22 (fezakinumab [FZ]) is enriched in severe asthma. Methods: An AD disease signature was obtained from analysis of differentially expressed genes between AD lesional and nonlesional skin biopsies. Differentially expressed genes from lesional skin from therapeutic superresponders before and after 12 weeks of FZ treatment defined the FZ-response signature. Gene set variation analysis was used to produce enrichment scores of AD and FZ-response signatures in the Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes asthma cohort. Results: The AD disease signature (112 upregulated genes) encompassing inflammatory, T-cell, T H2, and T H17/T H22 pathways was enriched in the blood and sputum of patients with asthma with increasing severity. Patients with asthma with sputum neutrophilia and mixed granulocyte phenotypes were the most enriched (P H22/IL-22 pathways. Conclusions: The FZ-response signature in AD identifies severe neutrophilic asthmatic patients as potential responders to FZ therapy. This approach will help identify patients for future asthma clinical trials of drugs used successfully in other chronic diseases.

IntroductionAsthma 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.MethodsBaseline 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.ResultsA total of 90 metabolites were identified, with 40 significantly altered (p−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.ConclusionsThis 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.

Background: fungal sensitivity has been associated with severe asthma outcomes. However, the clinical implication of Aspergillus fumigatus sensitization in difficult-to-treat (or difficult) asthma is unclear.Objectives: to characterize the clinical implications of A fumigatus sensitization in a large difficult asthma cohort.Methods: participants who underwent both skin prick and specific IgE testing to A fumigatus (n = 318) from the longitudinal real-life Wessex AsThma CoHort of difficult asthma, United Kingdom, were characterized by A fumigatus sensitization (either positive skin prick test result or specific IgE) and allergic bronchopulmonary aspergillosis status using clinical/pathophysiological disease measures.Results: a fumigatus sensitization was found in 23.9% (76 of 318) of patients with difficult asthma. Compared with A fumigatus nonsensitized subjects, those with sensitization were significantly more often male (50% vs 31%), older (58 years) with longer asthma duration (33 years), higher maintenance oral corticosteroid (39.7%) and asthma biologic use (27.6%), raised current/maximum log10 total IgE+1 (2.43/2.72 IU/L), worse prebronchodilator airflow obstruction (FEV1 62.2% predicted, FEV1/forced vital capacity 61.2%, forced expiratory flow between 25% and 75% exhalation 30.9% predicted), and frequent radiological bronchiectasis (40%), but had less psychophysiologic comorbidities. Allergic bronchopulmonary aspergillosis diagnosis was associated with higher treatment needs and stronger eosinophilic signals. Factors independently associated with A fumigatus sensitization in difficult asthma included maintenance oral corticosteroid use (odds ratio [OR], 3.34) and maximum log10 total IgE+1 (OR, 4.30), whereas for allergic bronchopulmonary aspergillosis included maintenance oral corticosteroid use (OR, 6.98), maximum log10 total IgE+1 (OR, 4.65), and radiological bronchiectasis (OR, 4.08).Conclusions: a fumigatus sensitization in difficult asthma identifies a more severe form of airways disease associated with greater morbidity, treatment need, and airways dysfunction/damage, but fewer psychophysiologic comorbidities. Screening of A fumigatus status should be an early element in the comprehensive assessment of patients with difficult asthma.

Patients with severe asthma may have a greater risk of dying from COVID-19 disease. Angiotensin converting enzyme-2 (ACE2) and the enzyme proteases, transmembrane protease serine 2 (TMPRSS2) and FURIN, are needed for viral attachment and invasion into host cells.We examined microarray mRNA expression of ACE2, TMPRSS2 and FURIN in sputum, bronchial brushing and bronchial biopsies of the European U-BIOPRED cohort. Clinical parameters and molecular phenotypes, including asthma severity, sputum inflammatory cells, lung functions, oral corticosteroid (OCS) use, and transcriptomic-associated clusters, were examined in relation to gene expression levels.ACE2 levels were significantly increased in sputum of severe asthma compared to mild-moderate asthma. In multivariate analyses, sputum ACE2 levels were positively associated with OCS use and male gender. Sputum FURIN levels were significantly related to neutrophils (%) and the presence of severe asthma. In bronchial brushing samples, TMPRSS2 levels were positively associated with male gender and body mass index, whereas FURIN levels with male gender and blood neutrophils. In bronchial biopsies, TMPRSS2 levels were positively related to blood neutrophils. The neutrophilic molecular phenotype characterised by high inflammasome activation expressed significantly higher FURIN levels in sputum than the eosinophilic Type 2-high or the pauci-granulocytic oxidative phosphorylation phenotypes.Levels of ACE2 and FURIN may differ by clinical or molecular phenotypes of asthma. Sputum FURIN expression levels were strongly associated with neutrophilic inflammation and with inflammasome activation. This might indicate the potential for a greater morbidity and mortality outcome from SARS-CoV-2 infection in neutrophilic severe asthma.

Background: understanding why patients with severe asthma do not follow healthcare provider (HCP) advice to adjust treatment is critical to achieving personalised disease management.Methods: we reviewed patient choice to follow HCP advice to adjust asthma treatment in a randomised, controlled, single-blind (study participant), multi-centre, parallel group 48-week clinical study comparing biomarker directed treatment adjustment to standard care in severe asthma.Results: of 1572 treatment advisories (301 participants), instructions were followed in 1377 cases (87.6%). Patients were more likely to follow advice to remain on treatment (96.7%) than to either reduce (70.3%) or increase (67.1%) their treatment, with 64% of patients following all treatment advice. Multivariate analysis associated belonging to an ethnic minority group (OR: 3.10; 95% CI: 1.68, 5.73) and prior study medication changes (≥2 OR: 2.77, 95% CI: 1.51, 5.10) with failure to follow treatment advice. In contrast, emergency room attendance in the prior year (OR: 0.54, 95% CI: 0.32, 0.92) was associated with following treatment advice. The largest effect was seen with transition onto or off oral corticosteroids (OR: 29.28; 95% CI: 16.07, 53.36) when compared to those requested to maintain treatment. Centre was also an important determinant regarding the likelihood of patients to follow treatment advice.Conclusions: belonging to an ethnic minority group and multiple prior treatment adjustments were associated with not following HCP treatment advice. Patients also responded differently to HCP advice across UK specialist centres. These findings have implications for generalisability for models of care in severe asthma and require further focussed studies.

Background: Interleukin 5, an archetypal type 2 cytokine, is fundamental to eosinophilic airways disease, considered to signal the bone marrow to promote eosinophil progenitor maturation and increase levels of eosinophils in the circulation. Aim: To investigate the relationship between airway IL-5 concentrations and inflammatory phenotypes of severe asthma. Methods: Healthy volunteers and biologic naive severe asthma patients were clinically characterised and had blood and induced sputum samples collected. Sputum was analysed for differential cell count and supernatant (PBS processed) proteins, measured by single plex ELISA. Eosinophilic asthma was defined as sputum eosinophils >2%. Between group comparisons were assessed by Kruskal Wallis and relationships between variables by spearman rank correlations. Results: 17 healthy controls, 53 non-eosinophilic and 26 eosinophilic patients with severe asthma participated in the study. In severe asthma, there were modest correlations between sputum IL-5 and sputum eosinophils (r = 0.519, p Discussion: Despite high levels of steroid treatment, there is persistent IL-5 generation within the airways of severe asthma.

Asthma is a heterogeneous disease with multiple clinical presentations (phenotypes). Neutrophilic asthma is characterised by increased sputum neutrophils and generally has a poor response to corticosteroids and limited other therapeutic options. Neutrophilia originates from different factors, including the defective resolution of inflammation or bacterial infections. An association between airway bacterial imbalance (disturbance) and the neutrophilic phenotype has been reported, suggesting that airway microbiota composition is involved in neutrophilic asthma. Rather than being a separate entity, neutrophilic asthma may be in part, an alliance between innate immunity and microbiota composition that prompts protective mechanisms against invading pathogens.

Background: blood eosinophil measurement is essential for the phenotypic characterization of patients with difficult asthma and in determining eligibility for anti-IL-5/IL-5Rα biological therapies. However, assessing such measures over limited time spans may not reveal the true underlying eosinophilic phenotype, as treatment, including daily oral corticosteroid therapy, suppresses eosinophilic inflammation and asthma is intrinsically variable.Methods: we interrogated the electronic healthcare records of patients in the Wessex AsThma CoHort of difficult asthma (WATCH) study (UK). In 501 patients being evaluated in this tertiary care centre for difficult to control asthma, all requested full blood count test results in a 10-year retrospective period from the index WATCH assessment were investigated (n = 11,176).Results: in 235 biological therapy-naïve participants who had 10 or more measures in this time period, 40.3% were eosinophilic (blood eosinophils ≥300 cells/µl) at WATCH enrolment whilst an additional 43.1%, though not eosinophilic at enrolment, demonstrated eosinophilia at least once in the preceding decade. Persistent eosinophilia was associated with worse post-bronchodilator airway obstruction and higher Fractional exhaled Nitric Oxide (FeNO). In contrast, the 16.6% of patients who never demonstrated eosinophilia at this blood eosinophil threshold showed preserved lung function and lower markers of Type 2 inflammation.Conclusions: this highlights the central role that type 2 inflammation, as indicated by blood eosinophilia, has in difficult asthma and suggests that longitudinal electronic healthcare record analysis can be an important tool in clinical asthma phenotyping, providing insight that may help understand disease progression and better guide more specific treatment approaches.

Background: Asthma is a heterogeneous disease characterized by distinct phenotypes with associated microbial dysbiosis. Objectives: Our aim was to identify severe asthma phenotypes based on sputum microbiome profiles and assess their stability after 12 to 18 months. A further aim was to evaluate clusters’ robustness after inclusion of an independent cohort of patients with mild-to-moderate asthma. Methods: In this longitudinal multicenter cohort study, sputum samples were collected for microbiome profiling from a subset of the Unbiased Biomarkers in Prediction of Respiratory Disease Outcomes adult patient cohort at baseline and after 12 to 18 months of follow-up. Unsupervised hierarchical clustering was performed by using the Bray-Curtis β-diversity measure of microbial profiles. For internal validation, partitioning around medoids, consensus cluster distribution, bootstrapping, and topological data analysis were applied. Follow-up samples were studied to evaluate within-patient clustering stability in patients with severe asthma. Cluster robustness was evaluated by using an independent cohort of patients with mild-to-moderate asthma. Results: Data were available for 100 subjects with severe asthma (median age 55 years; 42% males). Two microbiome-driven clusters were identified; they were characterized by differences in asthma onset, smoking status, residential locations, percentage of blood and/or sputum neutrophils and macrophages, lung spirometry results, and concurrent asthma medications (all P values < .05). The cluster 2 patients displayed a commensal-deficient bacterial profile that was associated with worse asthma outcomes than those of the cluster 1 patients. Longitudinal clusters revealed high relative stability after 12 to 18 months in those with severe asthma. Further inclusion of an independent cohort of 24 patients with mild-to-moderate asthma was consistent with the clustering assignments. Conclusion: Unbiased microbiome-driven clustering revealed 2 distinct robust phenotypes of severe asthma that exhibited relative overtime stability. This suggests that the sputum microbiome may serve as a biomarker for better characterizing asthma phenotypes.

Background: Airway remodelling, which may include goblet cell hyperplasia / hypertrophy, changes in epithelial integrity, accumulation of extracellular matrix components, smooth muscle hypertrophy and thickening of the lamina reticularis, is a feature of severe asthma and contributes to the clinical phenotype. Objective: Within the U-BIOPRED severe asthma study, we have assessed histological elements of airway remodelling and their relationship to computed tomography (CT) measures of proximal airway dimensions. Methods: Bronchial biopsies were collected from two severe asthma groups, one non-smoker (SAns, n=28) and one current/ex-smoker (SAs/ex, n=13), and a mild-moderate asthma group (MMA, n=28) classified and treated according to GINA guidelines, plus a healthy control group (HC, n=33). A Movat’s pentachrome technique was used to identify mucin, elastin and total collagen in these biopsies. The number of goblet cells (mucin+) were counted as a percentage of the total number of epithelial cells and the percentage mucin epithelial area measured. The percentage area of elastic fibres and total collagen within the submucosa were also measured, and the morphology of the elastic fibres classified. Participants in the asthma groups also had a CT scan to assess large airway morphometry.Results: The submucosal tissue elastin percentage was higher in both severe asthma groups (16.1% SAns, 18.9% SAs/ex) compared to the HC (9.7%) but did not differ between asthma groups. There was a positive relationship between elastin and airway wall area measured by CT (n= 18-20, rho=0.544, p=0.024), which also related to an increase in elastic fibres with a thickened lamellar morphological appearance. Mucin epithelial area and total collagen were not different between the four groups. Due to small numbers of suitable CT scans it was not feasible to compare airway morphometry between the asthma groups. Conclusion: These findings identify a link between extent of elastin deposition and airway wall thickening in severe asthma.

Introduction Efficacy of mepolizumab, an anti-interleukin-5 monoclonal antibody, was demonstrated in randomised controlled trials; data on its real-world impact in routine clinical practice are starting to emerge. We assessed the effectiveness and safety of mepolizumab prescribed for patients in the real world. Methods REALITI-A is a global, prospective, observational cohort study, collecting data from routine healthcare visits from patients with asthma. Patients newly prescribed mepolizumab for severe asthma with 12 months of relevant medical history pre-mepolizumab (collected retrospectively) were enrolled. An initial analysis of data from early initiators who had completed 1 year of follow-up (as of February 28, 2019) was conducted. The primary objective was to compare the rate of clinically significant exacerbations (requiring oral corticosteroids (OCS) and/or hospitalisation and/or emergency department visit) before and after mepolizumab; exacerbations requiring hospitalisation and/or emergency department visit and change in maintenance OCS use were secondary objectives. Treatment-related adverse events were reported. Results Overall, 368 mepolizumab-treated patients were included. Rates of clinically significant exacerbations were reduced by 69% from 4.63 per person per year pre-treatment to 1.43 per person per year during follow-up (p, Mepolizumab has demonstrated efficacy in patients with severe eosinophilic asthma in the controlled environment of clinical trials. These initial data from the prospective REALITI-A study show that similar results are obtained in a real-world setting. https://bit.ly/3hINnFO

Background: Allergic sensitization is associated with severe asthma, but assessment of sensitization is not recommended by most guidelines. Objective: We hypothesized that patterns of IgE responses to multiple allergenic proteins differ between sensitized participants with mild/moderate and severe asthma. Methods: IgE to 112 allergenic molecules (components, c-sIgE) was measured using multiplex array among 509 adults and 140 school-age and 131 preschool children with asthma/wheeze from the Unbiased BIOmarkers for the PREDiction of respiratory diseases outcomes cohort, of whom 595 had severe disease. We applied clustering methods to identify co-occurrence patterns of components (component clusters) and patterns of sensitization among participants (sensitization clusters). Network analysis techniques explored the connectivity structure of c-sIgE, and differential network analysis looked for differences in c-sIgE interactions between severe and mild/moderate asthma. Results: Four sensitization clusters were identified, but with no difference between disease severity groups. Similarly, component clusters were not associated with asthma severity. None of the c-sIgE were identified as associates of severe asthma. The key difference between school children and adults with mild/moderate compared with those with severe asthma was in the network of connections between c-sIgE. Participants with severe asthma had higher connectivity among components, but these connections were weaker. The mild/moderate network had fewer connections, but the connections were stronger. Connectivity between components with no structural homology tended to co-occur among participants with severe asthma. Results were independent from the different sample sizes of mild/moderate and severe groups. Conclusions: The patterns of interactions between IgE to multiple allergenic proteins are predictors of asthma severity among school children and adults with allergic asthma.

Introduction: Maintenance OCS use in severe asthma (SA) is associated with significant side-effects and has the potential to mask the systemic type-2 inflammatory biomarker signal required for initiation of biologic therapies. Method: WSAC is a large published cohort of 342 well characterised SA patients fulfilling the ATS/ERS 2014 definition of SA undertaken prior to the widespread use of biologic treatments. We compared the clinical, physiological & biological characteristics of patients requiring & not requiring maintenance OCS. Results: Discussion: Maintenance OCS use is associated with a significantly greater burden of comorbidities, poorer quality of life & disease control with more frequent exacerbations & ITU admissions. Despite this no significant differences were identified in lung function, atopic status, inflammatory cell counts or the other extensive biological measures assessed in the cohort. 44% of patients on maintenance OCS showed no biomarker signal of T2 inflammation. 77% of those on maintenance OCS with a sputum eos ≥2% had a blood eos count ≥0.3x109/L whilst only 60% had a FeNO ≥25ppb (43% FeNO ≥50ppb) suggesting blood eos remains a useful biomarker of T2 inflammation.

Introduction: Increasing corticosteroids (CS) to control asthma symptoms and exacerbations has potential for inappropriately high and difficult to down-titrate CS doses. Aims and Objectives: We tested whether a T2 biomarker-based strategy (BS) to guide CS reduction (factoring fractional exhaled nitric oxide (FeNO), blood eosinophils, serum periostin) vs. a symptom-based control would see fewer exacerbations and better asthma control and lung function. Methods: In this randomised (4:1/BS:control), single-blind parallel group trial, patients (pts) with severe asthma and FeNO Results: In the intention-to-treat population (240 BS vs. 61 control pts), the proportion of BS pts on lower CS dose at Wk48 was 28.4% vs. 18.5% in the controls (adjusted OR: 1.71; 95%CI 0.80, 3.63; p=0.165). In the per protocol (PP) population (n=121), significantly more pts had lower CS doses at Wk48 in the BS vs. the control group (30.7% vs. 5.0% (OR: 11.48; 95%CI 1.35, 97.83; p=0.026). Failure to follow treatment advice was the main reason for ITT and PP population differences. There was no difference in secondary outcomes between study arms. Conclusion: Biomarker based CS adjustment did not result in a greater proportion of pts reducing CS vs. control, probably because many pts did not follow treatment advice. The biomarker strategy seemed beneficial in pts where symptoms and T2 biomarker profile were discordant.

Background: In asthmatic patients, airway eosinophilia identifies a phenotype of patients that suffer from recurrent exacerbations and respond to biologic therapies. Direct measurement, through sputum induction, is impractical as part of routine clinical care so blood eosinophil counts are commonly used as a surrogate marker. We sought to assess the degree of concordance between these measures in our cohort of severe asthma patients. Methods: WATCH patients with severe asthma had blood tests, FeNO measurement and sputum induction performed on the same day. Spearman’s rank correlation coefficients and classification tables were calculated between sputum eosinophil, blood eosinophil, serum IgE and FeNO measures. Prediction of sputum eosinophilia (>3%) was assessed using receiver operating curves. Results: 203 patients, treated with high dose inhaled corticosteroids, were included in the analysis. Using commonly used cut-offs for blood eosinophilia (>300 cells/μL) and sputum eosinophilia (>3%), measurements were concordant in 138 patient (73.0%). 22 patients (11.6%) demonstrated blood eosinophilia alone and 29 patients (15.3%) sputum eosinophilia alone. Conclusions: We find that in a proportion of patients with severe asthma have sputum eosinophilia not recognised by blood. A better understanding of the nature of these airway eosinophils and the basis for the lack of apparent systemic IL5 signalling would help understand if these are a potentially biologically overlooked population or represent a different disease endotype.

Introduction: Depression is a well-recognised comorbidity in SA associated with poorer disease control & quality of life (QOL) but it remains unclear whether this is the consequence of altered pathobiology. Method: We compared the clinical, physiological & biological characteristics of patients with & without depression (defined by HADS depression score ≥8) within WSAC, a large published cohort of well characterised patients fulfilling the ATS/ERS 2014 definition of SA. Results: Discussion: SA patients with depression report poorer disease control & QOL associated with a greater burden of co-morbidities including obesity, rhinosinusitis & reflux. However, no statistically significant differences were identified in their other clinical or physiological characteristics. Inflammatory phenotypes did not differ between the groups but those with depression were found to have significantly higher serum hs-CRP & sputum IL-6 levels (not seen when subdivided for BMI). There is a well-recognised association between inflammation & depression with elevated serum IL-6 levels previously reported. Our findings raise the possibility that depression influences the pathobiology of asthma & thus reflects a targetable trait.

Background In all chronic airway diseases, the dynamics of airway function are influenced by underlying airway inflammation and bronchial hyperresponsiveness along with limitations in reversibility owing to airway and lung remodeling as well as mucous plugging. The relative contribution of each component translates into specific clinical patterns of symptoms, quality of life, exacerbation risk, and treatment success. Objective We aimed to evaluate whether subgrouping of patients with obstructive airway diseases according to patterns of fluctuation in lung function allows identification of specific phenotypes with distinct clinical characteristics. Methods We applied the novel method of fluctuation-based clustering (FBC) to twice-daily FEV1 measurements recorded over a 1-year period in a mixed group of 134 adults with mild-to-moderate asthma, severe asthma, or chronic obstructive pulmonary disease from the European BIOAIR cohort. Results Independently of clinical diagnosis, FBC divided patients into 4 fluctuation-based clusters with progressively increasing alterations in lung function that corresponded to patterns of increasing clinical severity, risk of exacerbation, and lower quality of life. Clusters of patients with airway disease with significantly elevated levels of biomarkers relating to remodeling (osteonectin) and cellular senescence (plasminogen activator inhibitor-1), accompanied by a loss of airway reversibility, pulmonary hyperinflation, and loss of diffusion capacity, were identified. The 4 clusters generated were stable over time and revealed no differences in levels of markers of type 2 inflammation (blood eosinophils and periostin). Conclusion FBC-based phenotyping provides another level of information that is complementary to clinical diagnosis and unrelated to eosinophilic inflammation, which could identify patients who may benefit from specific treatment strategies or closer monitoring.

SummaryBackgroundPatients with severe asthma may have a greater risk of dying from COVID-19 disease caused by SARS-CoV-2 virus. Angiotensin converting enzyme 2 (ACE2) receptor and enzyme proteases, transmembrane protease, serine 2 (TMPRSS2) and furin are needed for the attachment and invasion of the virus into host cells. We determined whether their expression in the airways of severe asthma patients is increased.MethodWe examined the microarray mRNA expression of ACE2, TMPRSS2 and furin in the sputum, bronchial brush and bronchial biopsies of participants in the European U-BIOPRED cohort.ResultsACE2 and furin sputum gene expression was significantly increased in severe non-smoking asthma compared to mild-moderate asthma and healthy volunteers. By contrast, TMPRSS2 expression in bronchial biopsy and bronchial brushings was increased in severe smoking and ex-smoking asthmatics, and so was furin expression in bronchial brushings. Several clinical parameters including male gender, oral steroid use and nasal polyps were positively associated with ACE2, TMPRSS2 and furin expression levels. There was a higher expression of ACE2 and furin in the sputum neutrophilic molecular phenotype with inflammasome activation compared to the eosinophilic Type2-high or paucigranulocytic phenotypes. The enrichment score of the IL-13-Type2 gene signature was positively correlated with ACE2, TMPRSS2 and furin levels.ConclusionThese key determinants of virus entry into the lungs may contribute to the poorer outcomes from COVID-19 disease in patients with severe asthma.“take home” messageIn severe asthma, gene expression of ACE, TMPRSS2 and furin are elevated compared to mild-moderate asthma and healthy volunteers, particularly in neutrophilic asthma. This might explain the increased risk of death in severe asthma afflicted with COVID19.

To-date, there has not been a clear signal suggesting that asthma or treatment with inhaled steroids are a risk factor for severe COVID-19 disease. We have therefore explored ACE2 receptor mRNA expression, and co-factors for Sars-CoV-2 infectivity (TMPRSS2 and furin) in bronchial brushes and biopsies from people with asthma and healthy controls, and looked for relationships between asthma severity, Th2- and IL-17 dependent gene signatures, and clinical demographics (age, sex). We have looked at a cohort of 356 research participants from previously described studies. The only significant association was a positive correlation between ACE2 and IL-17-dependent gene expression, and an inverse correlation between ACE2 and Th2-cytokine-dependent gene expression. These data suggest that differences in ACE2, TMPRSS2 and furin epithelial and airway gene expression are unlikely to confer enhanced COVID-19 pneumonia risk in patients with asthma across all treatment intensities and severity., Expression of mRNA for ACE2, the Sars-CoV-2 receptor, is similar in the lower airways of healthy controls and people with mild-severe asthma. Altered ACE2 expression is unlikely to confer enhanced COVID-19 pneumonia risk in asthma.

International audience; Background: Asthma is a diverse condition that differs with age and sex. However, it remains unclear how sex, age of asthma-onset, and/or their interaction, influence clinical expression of more problematic adult “difficult” asthma.Objectives: To better understand the clinical features of difficult asthma within a real-world clinical setting using novel phenotypic classification, stratifying subjects by sex and age of asthma-onset.Methods: Participants in a longitudinal difficult asthma clinical cohort study (Wessex AsThma CoHort of difficult asthma; WATCH), United Kingdom, (n=501) were stratified into 4 difficult asthma phenotypes based on sex and age of asthma-onset (early

Objectives There is limited information about the prevalence of rhinosinusitis in the UK community. This study aims to identify its prevalence and investigate any association with demographic variables. The secondary aims are to determine the degree of impairment, impact on quality of life and any costs incurred by patients. Design We used a modified version (MSNOT-20) of a quality of life instrument; the sinonasal outcome test -20 (SNOT-20), in a small and successful pilot project. It was then used in a community-based survey and a second phase 6-months later to test repeatability. Nasal examination and comparison of its quality of life section with other health-related quality of life tools occurred in the second phase. Setting and Participants The questionnaire was administered by post to 2,000 Farnborough (UK) residents, selected through stratified randomisation. The relation of an abnormal MSNOT-20 score with hay fever, asthma, smoking, food allergy, work productivity and social limitation were also analysed. Main Outcome and Results The response rate was 79.8%; over thirty per cent of the community suffer from upper respiratory tract symptoms with impact on multiple domains of quality of life including emotional, financial costs and loss of days at work. The MSNOT-20 provided a more sensitive assessment of health related quality of life than the Short Form-36 questionnaire. Conclusion Rhinosinusitis is prevalent in the Farnborough community and associated with significant morbidity and impairment on quality of life. The MSNOT-20 is a useful disease-specific quality of life tool in rhinosinusitis. This article is protected by copyright. All rights reserved.

Rationale: Genetic polymorphisms in the asthma susceptibility gene, urokinase plasminogen activator receptor (uPAR/PLAUR) have been associated with lung function decline and uPAR blood levels in asthma subjects. Preliminary studies have identified uPAR elevation in asthma; however, a definitive study regarding which clinical features of asthma uPAR may be driving is currently lacking.Objectives: We aimed to comprehensively determine the uPAR expression profile in asthma and control subjects utilizing bronchial biopsies and serum, and to relate uPAR expression to asthma clinical features.Methods: uPAR levels were determined in control (n = 9) and asthmatic (n = 27) bronchial biopsies using immunohistochemistry, with a semi-quantitative score defining intensity in multiple cell types. Soluble-cleaved (sc) uPAR levels were determined in serum through ELISA in UK (cases n = 129; controls n = 39) and Dutch (cases n = 514; controls n = 96) cohorts.Measurements and main results: In bronchial tissue, uPAR was elevated in inflammatory cells in the lamina propria (P = 0.0019), bronchial epithelial (P = 0.0002) and airway smooth muscle cells (P = 0.0352) of patients with asthma, with uPAR levels correlated between the cell types. No correlation with disease severity or asthma clinical features was identified. scuPAR serum levels were elevated in patients with asthma (1.5-fold; P = 0.0008), and we identified an association between high uPAR serum levels and severe, nonatopic disease.Conclusions: This study provides novel data that elevated airway and blood uPAR is a feature of asthma and that blood uPAR is particularly related to severe, nonatopic asthma. The findings warrant further investigation and may provide a therapeutic opportunity for this refractory population.

Introduction: Asthmatics have been reported to have airway microbial dysbiosis [Chung JACI 2017]. We aimed to detect severe asthma phenotypes using induced sputum microbiome profiles and to assess cluster-wise stability after 12-18 months of prospective follow-up. Methods: Induced sputum samples were collected from a subset of the U-BIOPRED adult severe asthmatics. 16s rRNA sequencing and metagenomics were used to characterize the sputum microbiome. Unsupervised hierarchical clustering was performed by Bray-Curtis β-diversity measure of 16s microbiome data. The clustering was validated using partition around medoids, topological data analysis, consensus cluster distribution and bootstrapping. Sputum samples collected after 12-18 months were used to assess patient cluster migration. Results: Analysis of sputum samples of 100 severe asthmatics (median age: 55 years, 42% male) revealed two microbiome-driven clusters. The clusters were significantly different in: age of asthma onset, residential location, smoking status, percentage of sputum neutrophils, and spirometry (p-values Conclusion: Induced sputum microbiome identified two distinct severe asthma phenotypes. The relative stability of microbiome-driven phenotypic clusters indicates its suitability for diagnostic and therapeutic precision medicine approaches.

Background: Blood eosinophil counts ≥0.3x109/L are used to define Type-2, eosinophilic asthma. However, differential responses to T2 biologics of patients with eosinophilic asthma suggests that thi ...

Background: Common cold (rhinovirus, RV) infections are the most frequent trigger of asthma exacerbations. Asthma is a common chronic inflammatory disease of the airways. Asthmatics show interferon-deficient responses to RV; however, the mechanisms underlying increased RV-driven symptoms leading to exacerbations are inadequately understood. Aim: To determine the role of RNA processing in antiviral immunity in asthma. Methods: We applied Frac-seq (subcellular fractionation and RNA-seq) in primary bronchial epithelial cells (BECs) from asthmatics and healthy controls. Frac-seq determines which cytoplasmic mRNAs bind to polyribosomes, the cellular translational machinery. We employed RNA immunoprecipitation (RIP) and siRNA knock-down experiments in bronchial epithelial cells (BEAS-2B) infected with RV1C. Results: Frac-seq data showed decreased translation of UPF1 (upstream frameshift 1) in asthma BECs. UPF1 translational levels negatively correlated with reversibility (r=-0.7235 P=0.0067). UPF1 is the main mediator of nonsense mediated decay (NMD), an RNA surveillance pathway that degrades potentially deleterious mRNAs (e.g. mRNAs with premature termination codons) and postulated to regulate 10-30% of all mRNAs. Emetine (a translation inhibitor that leads to upregulation of NMD targets) resulted in increased RV RNA levels. RIP showed that UPF1 directly binds RV RNA. Depletion of UPF1 in bronchial epithelial cells resulted in deficient RV-induced interferon (beta and lambda) and genome-wide changes in antiviral responses. Conclusions: Our data suggest that decreased UPF1 levels in asthma BECs contribute to impaired antiviral immunity and RV-induced exacerbations in asthma.