BACKGROUND:The airway microbiome in severe asthma has not been characterised at species-level by metagenomic sequencing, nor have the relationships between specific species and mucosal immune responses in 'type-2 low', neutrophilic asthma been defined. We performed an integrated species-level metagenomic data with inflammatory mediators to characterise prevalence of dominant potentially pathogenic organisms and host immune responses. METHODS:Sputum and nasal lavage samples were analysed using long-read metagenomic sequencing with Nanopore and qPCR in two cross-sectional adult severe asthma cohorts, Wessex (n = 66) and Oxford (n = 30). We integrated species-level data with clinical parameters and 39 selected airway proteins measured by immunoassay and O-link. RESULTS:The sputum microbiome in health and mild asthma displayed comparable microbial diversity. By contrast, 23% (19/81) of severe asthma microbiomes were dominated by a single respiratory pathogen, namely H. influenzae (n = 10), M. catarrhalis (n = 4), S. pneumoniae (n = 4) and P. aeruginosa (n = 1). Neutrophilic asthma was associated with H. influenzae, M. catarrhalis, S. pneumoniae and T. whipplei with elevated type-1 cytokines and proteases; eosinophilic asthma with higher M. catarrhalis, but lower H. influenzae, and S. pneumoniae abundance. H. influenzae load correlated with Eosinophil Cationic Protein, elastase and IL-10. R. mucilaginosa associated positively with IL-6 and negatively with FGF. Bayesian network analysis also revealed close and distinct relationships of H. influenzae and M. catarrhalis with type-1 airway inflammation. The microbiomes and cytokine milieu were distinct between upper and lower airways. CONCLUSIONS:This species-level integrated analysis reveals central, but distinct associations between potentially pathogenic bacteria and airways inflammation in severe asthma.

Background: Breathing pattern disorder (BPD) reflects altered biomechanical patterns of breathing that drive breathing difficulty and commonly accompanies difficult-to-treat asthma. Diagnosis of BPD has no gold standard, but Nijmegen Questionnaire (NQ) >23 is commonly used. Objectives: We sought to advance clinical characterization of BPD and better understand the clinical utility of NQ in difficult asthma in patients from the Wessex AsThma CoHort of difficult asthma (WATCH) study. Methods: Associations between demographic and clinical factors in difficult asthma and BPD, ascertained by clinical diagnosis (yes/no, n = 476), by NQ scores (<= 23: normal [no suggestion of BPD] and >23: abnormal [suggested BPD], n = 372), as well as the continuous raw NQ scores were assessed in univariate models to identify significant risk factors associated with the 3 BPD outcomes. For the clinician-diagnosed and NQ-based BPD, associations of continuous factors were assessed using the independent samples t test or the Mann-Whitney U test as appropriate for the data distribution or by the Spearman correlation test. Dichotomous associations were evaluated using chi(2) tests. Multivariable logistic (dichotomous outcomes) and linear regression models (continuous outcomes) were developed to identify predictive factors associated with clinician-diagnosed and NQ-based BPD, dichotomous and continuous. Patients with data on NQ scores were grouped into NQ quartiles (low, moderate, high, and very high). The patterns of association of the quartiles with 4 health-related questionnaire outcomes were assessed using linear regression analyses. Results: Multivariable regression identified that clinically diagnosed BPD was associated with female sex (odds ratio [OR]: 1.85; 95% confidence interval [CI]: 1.07, 3.20), comorbidities (rhinitis [OR: 2.46; 95% CI: 1.45, 4.17], gastroesophageal reflux disease [GORD] [OR: 2.77; 95% CI: 1.58, 4.84], inducible laryngeal obstruction [OR: 4.37; 95% CI: 2.01, 9.50], and any psychological comorbidity [OR: 1.86; 95% CI: 1.13, 3.07]), and health care usage (exacerbations [OR: 1.07; 95% CI: 1.003, 1.14] and previous intensive care unit (ICU) admissions [OR: 2.03; 95% CI: 1.18, 3.47]). Abnormal NQ-based BPD diagnosis was associated with history of eczema (OR: 1.83; 95% CI: 1.07, 3.14), GORD (OR: 1.94; 95% CI: 1.15, 3.27), or any psychological comorbidity (OR: 4.29; 95% CI: 2.64, 6.95) at multivariable regression. Differences between clinical and NQ-based BPD traits were also found with 42% discordance in BPD state between these definitions. Multivariable linear regression analysis with NQ as a continuous outcome showed positive association with worse asthma outcomes (admission to ICU, P = .037), different phenotypic traits (female sex, P = .001; ever smoker, P = .025), and greater multimorbidity (GORD, P = .002; sleep apnea, P = .04; and any psychological comorbidity, P < .0001). Conclusion: BPD is associated with worse health outcomes and negative health impacts in difficult asthma within a multimorbidity disease model. It therefore merits better recognition and prompt treatment. Clinical diagnosis and NQ offer different perspectives on BPD, so this goal may be best addressed by considering clinical features alongside the magnitude of NQ. (c) 2023 The Authors. Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology.

Patients with severe uncontrolled asthma represent a distinct endotype with persistent airway inflammation and remodeling that is refractory to corticosteroid treatment. To determine T cell subsets and effector molecules that drive pathogenesis of severe asthma, we performed single-cell transcriptome analysis of >50,000 airway CD4+ T cells isolated from bronchoalveolar lavage (BAL) samples from 30 patients with mild and severe asthma. We observed striking heterogeneity in the nature of CD4+ T cells present in asthmatics' airways with tissue-resident memory (TRM) cells making a dominant contribution. Notably, in severe asthmatics a subset of CD4+ TRM cells (CD103-expressing) was significantly increased, comprising nearly 65% of all CD4+ T cells in the airways of male patients with severe asthma when compared to mild asthma (13%). This subset was enriched for transcripts linked to T cell receptor (TCR) activation (HLA-DRB1, HLA-DPA1, CD40LG) and cytotoxicity (GZMB, GZMH), and following stimulation expressed high levels of transcripts encoding for pro-inflammatory non-TH2 cytokines (CCL3, CCL4, CCL5, TNF, LIGHT) that could fuel persistent airway inflammation and remodeling. Our findings indicate the need to look beyond the traditional T2 model of severe asthma to better understand the heterogeneity of this disease. This work was supported by (i) NIH research grant R01HL114093 (PV) and equipment grants (S10RR027366 - BD FACSAria Fusion, and S10OD025052 - Illumina Novaseq 6000); the William K. Bowes Jr. Foundation (P.V.). The WATCH study is supported by the Southampton NIHR Biomedical Research Centre and the Southampton NIHR Clinical Research Facility which are funded by the NIHR and are a partnership between the University of Southampton and University Hospital Southampton NHS Foundation Trust.

Background: Despite most of the asthma population having mild disease, the mild asthma phenotype is poorly understood. Here, we aim to address this gap in knowledge by extensively characterising the mild asthma phenotype and comparing this with difficult-to-treat asthma.Methods: We assessed two real-world adult cohorts from the South of England using an identical methodology: the Wessex AsThma CoHort of difficult asthma (WATCH) (n=498) and a mild asthma cohort from the comparator arm of the Epigenetics Of Severe Asthma (EOSA) study (n=67). Data acquisition included detailed clinical, health and disease-related questionnaires, anthropometry, allergy and lung function testing, plus biological samples (blood and sputum) in a subset. Results: Mild asthma is predominantly early-onset and is associated with type-2 (T2) inflammation (atopy, raised fractional exhaled nitric oxide (FeNO), blood/sputum eosinophilia) plus preserved lung function. A high prevalence of comorbidities and multimorbidity was observed in mild asthma, particularly depression (58.2%) and anxiety (56.7%). In comparison to difficult asthma, mild disease showed similar female predominance (>60%), T2-high inflammation and atopy prevalence, but lower peripheral blood/airway neutrophil counts and preserved lung function. Mild asthma was also associated with a greater prevalence of current smokers (20.9%). A multi-component T2-high inflammatory measure was comparable between the cohorts; T2-high status 88.1% in mild asthma and 93.5% in difficult asthma. Conclusion: Phenotypic characterisation of mild asthma identified early-onset disease with high prevalence of current smokers, T2-high inflammation and significant multimorbidity burden. Early comprehensive assessment of mild asthma patients could help prevent potential later progression to more complex severe disease.

Background: Asthma is a chronic respiratory disease with significant heterogeneity in its clinical presentation and pathobiology. There is need for improved understanding of respiratory lipid metabolism in asthma patients and its relation to observable clinical features.Objective: We performed a comprehensive, prospective, cross-sectional analysis of the lipid composition of induced sputum supernatant obtained from asthma patients with a range of disease severities, as well as from healthy controls.Methods: Induced sputum supernatant was collected from 211 adults with asthma and 41 healthy individuals enrolled onto the U-BIOPRED (Unbiased Biomarkers for the Prediction of Respiratory Disease Outcomes) study. Sputum lipidomes were characterized by semiquantitative shotgun mass spectrometry and clustered using topologic data analysis to identify lipid phenotypes.Results: Shotgun lipidomics of induced sputum supernatant revealed a spectrum of 9 molecular phenotypes, highlighting not just significant differences between the sputum lipidomes of asthma patients and healthy controls, but also within the asthma patient population. Matching clinical, pathobiologic, proteomic, and transcriptomic data helped inform the underlying disease processes. Sputum lipid phenotypes with higher levels of nonendogenous, cell-derived lipids were associated with significantly worse asthma severity, worse lung function, and elevated granulocyte counts.Conclusion: We propose a novel mechanism of increased lipid loading in the epithelial lining fluid of asthma patients resulting from the secretion of extracellular vesicles by granulocytic inflammatory cells, which could reduce the ability of pulmonary surfactant to lower surface tension in asthmatic small airways, as well as compromise its role as an immune regulator. (J Allergy Clin Immunol 2023;152:117-25.)

To the Editor, Severe asthma is a heterogeneous disease comprising numerous endophenotypes.1 In recent years, there has been increased focus on eosinophils and type 2 (T2) inflammatory pathways as treatable traits in severe asthma, as these can be effectively targeted by an emerging portfolio of biologic therapies.2 Traditionally, the presence of increased eosinophils in induced sputum was used to define eosinophilic asthma. Sputum induction and analysis, however, require specialist expertise, preventing its use in routine clinical care. Consequently, the classification of severe eosinophilic asthma by blood eosinophil status has become commonplace in clinical practice. There are, however, limitations in defining patients as eosinophilic or not, as severe asthma patients have high oral or inhaled corticosteroid burden which may suppress blood eosinophils and mask the underlying eosinophilic nature of the disease. Particularly if this trait is defined by limited timespan assessment in this variable disease.1, 3, 4 Consistent with this, we recently reported that repeated blood eosinophil counts (BECs) over time can help unmask the underlying T2 asthma status not revealed by a single snapshot measure.3 This longitudinal approach demonstrated a higher burden of T2 asthma status than previously reported. Thus, rigorous approaches are needed to correctly define non-eosinophilic and eosinophilic forms of severe asthma to accurately guide clinical management. Following our longitudinal analysis of blood eosinophilia in severe asthma, the International Severe Asthma Registry (ISAR) consortium recently presented a novel multidimensional algorithm to determine the probability of an eosinophilic phenotype within a severe asthma population.5 This algorithm combined BECs with relevant clinical indices (on anti- interleukin-5 [IL-5] or anti-IL-5Rα therapy, on maintenance oral corticosteroids [OCS], history of nasal polyps [NP], fraction of exhaled nitric oxide [FeNO] and adult-onset asthma). The resulting probability of eosinophilic disease ranged from Grade 0 (unlikely) to Grade 3 (most likely). There were similarities between findings reported for this ISAR algorithm and our recent assessment of eosinophilic T2 difficult-to-treat/severe asthma status using longitudinal measures of BECs in the Wessex AsThma CoHort of difficult asthma (WATCH) study.3 The ISAR eosinophil gradient algorithm report identified two points that merit further understanding and which could be addressed by our WATCH cohort data: firstly, how longitudinal BEC assessment might consolidate these algorithm-derived phenotypes; secondly, how the identification of eosinophilic asthma via the ISAR algorithm aligns with the traditional method of sputum analysis. Wessex AsThma CoHort of difficult asthma is a real-world study of patients with difficult-to-treat/severe asthma attending a tertiary centre (Southampton, United Kingdom). The study had national research ethics committee (REC) approval (reference 14/WM/1226), and all patients provided written informed consent. The WATCH study protocol and methodology has been described previously.6 The WATCH dataset includes current and historic BEC data plus induced sputum data in a subset of patients with severe asthma. Sputum was induced in poorly controlled patients who were at least 4 weeks clear of an exacerbation. We applied the ISAR algorithm to the WATCH population to explore its generalizability, using both a single BEC taken at enrolment and, where available, observations of BECs up to 15 years prior. Additionally, we correlated induced sputum granulocyte phenotypes with the ISAR-defined phenotype in a subset of WATCH patients. Replication of the ISAR algorithm in 471 patients from the WATCH cohort confirmed the predominance of an eosinophilic status regardless of the BEC observation timeframe applied (Table 1). The weakest alignment in frequency of Grade 3 status between WATCH and ISAR was found when a single snapshot BEC measure was used: though 70% of patients considered at least "likely eosinophilic" (Grade 2), only 45% of the population being considered "most likely eosinophilic" (Grade 3). Progressively greater alignment in the proportions of ISAR Grades was found by increasing the retrospective reach of BEC observation in the WATCH cohort (Table 1), which increased the proportion of patients classified as Grade 3 and decreased the proportion of patients classified as Grade 0. Highest BEC Cells/µl WATCH (latest result test only) WATCH (highest result in last 5 years) WATCH (highest result in last 10 years) WATCH (highest result ever) Grade 3 Most likely Grade 3 Most likely Grade 3 Most likely Grade 3 Most likely Grade 2 Likely Grade 1 Least likely Grade 1 Least likely Grade 0 Unlikely (non) As in ISAR, no differences were observed between WATCH patients classified as Grade 2 or higher compared to patients classified as Grade 0 with regard to BMI (29.8 vs. 29.7 p = .625), presence of rhinitis (67.1% vs. 63.2% p = .915), asthma control (ACQ6 score 2.5 vs. 2.1 p = .354), or number of annual exacerbations (3.0 vs. 2.5 p = .452). In addition, as seen in ISAR, Grade 2 or higher patients were older (age at enrolment 55.0 vs. 40.0 p = .002), had a higher FeNO (20.3 vs. 11.0 p = .004) and had a later disease onset (age at asthma diagnosis 21.5 vs. 6.5 p=<.0001). Grade 0 patients were less likely than Grade 2 or higher to be male (9.5% vs. 37.3%, p = .019) and had better preserved lung function (median post-BD FEV1% predicted 89.9% vs. 74.6% p = .05, median post-BD FEV1:FVC ratio 80.0 vs. 67.0 p = .02). Unlike in ISAR, no difference was found in the prevalence of eczema ever (25.5% vs. 30.0% p = .853), atopy (67.6% vs. 68.4% p = .862) or median total IgE (89.7 vs. 50.9 p = .1). Similar ISAR eosinophil phenotype distributions were observed in the subset (n = 130) of WATCH patients with paired sputum analysis: 107 (82.3%) patients were classified as Grade 3 (Table 2). Only 1 patient (0.8%) was classed as Grade 0; this patient presented with a pauci granular sputum phenotype (Table 2). When sputum eosinophilia (≥2% eosinophils) alone is used to define eosinophilic asthma, less than half of patients are found to be eosinophilic (36% patients with ≥2% sputum eosinophils and 12% with mixed granular sputum [≥2% eosinophils, ≥61% sputum neutrophils]). The pauci granular population had fewer patients reported to have CT scan evidence of bronchiectasis than those with sputum granular disease; however, no significant difference in the presence of bronchiectasis was identified between sputum neutrophilic and sputum eosinophilic patients. 46% of patients classified as Grade 3 did not have evidence of sputum eosinophilia. Number (%) Eosinophilic (Eos ≥2%, Neut <61%) Mixed granular (Eos ≥2%, Neut ≥61%) Neutrophilic (Eos <2%, Neut ≥61%) Pauci-granular (Eos <2%, Neut <61%) Grade 3, most likely Grade 2 Likely Grade 1 Least likely Grade 0 Unlikely (non) We validated the ISAR eosinophil gradient algorithm in the WATCH cohort and demonstrated that extended longitudinal BEC monitoring within that framework increases the probability of identifying Grade 3 patients. It is notable that in both ISAR and WATCH datasets, a very high percentage of subjects were Grade 3 "most likely eosinophilic" using this algorithm. We previously showed remarkably similar overwhelming prevalence of underlying eosinophilic status in WATCH using an alternative perspective of longitudinal BEC monitoring. While there is potential selection bias with that latter approach (given increased propensity to perform blood counts when patients are exacerbating), the ISAR algorithm corroborates that observation by including clinical characteristics, which is particularly relevant when longitudinal BEC data are limited. When present, however, the inclusion of multiple historic BECs, mitigates against granulocyte count instability and treatment effects, in the detection of an underlying eosinophilic phenotype. Our WATCH data thus support Heaney et al's findings and those of others,7 that severe asthma is mostly an eosinophilic disease.5 It is worth noting that while the terms eosinophilic and T2 asthma are often applied interchangeably, the ISAR algorithm does not incorporate measures of atopic predisposition such as total IgE or specific allergen sensitization. Since allergy is a T2-associated process that omission might raise concerns that the ISAR algorithm could miss a proportion of T2 patients. However, the ISAR algorithm still finds overwhelming prevalence of eosinophilic status. As no differences were identified in eczema, atopy or total IgE between patients considered Grade 2 and above vs. those considered Grade 0 these definitions cannot be considered interchangeable using the ISAR algorithm in the WATCH cohort. Our sputum findings emphasize the value of longitudinal repeat measures in truly understanding the underlying phenotypic propensity and are consistent with a recent publication that also highlighted that single sputum measures underestimate the likelihood of being classified as eosinophilic.4 Variable granulocyte measures are also associated with poorer disease outcome,3, 8 highlighting the prognostic and diagnostic advantages of longitudinal repeat measurements. In addition, we are yet to fully understand the longitudinal stability of granulocyte phenotypes, the frequency of phenotype switching and how such events influence factors like disease severity and remission. We are also blinded to the influence granulocytes have on each other within this notoriously variable disease. Importantly, the presence of an eosinophilic phenotype does not exclude the concomitant existence of additional biology, as 25% of those identified as eosinophilic by sputum measures in WATCH also had neutrophilic airways disease. Such additional biology is not apparent with the use of BEC for phenotypic classification. There thus remains an unmet need for peripheral blood biomarkers that reflect airway biology additional to that linked to eosinophilic inflammation. One such measure is Chitinase 3 Like 1 (CHI3L1/YKL-40). Liu and colleagues identified higher measures of the T1 biomarker CHI3L1/YKL-40 in serum of patients with normal sputum eosinophil levels,9 and CHI3L1/YKL-40 was also identified in our previous severe asthma cohort as a sub-phenotype in severe asthma.1 Thus, monitoring a broad spectrum of airway inflammatory markers in asthma should be an important future consideration in addition to the use of classifications like the ISAR algorithm. In conclusion, though the term "non-eosinophilic asthma" is used to describe severe asthma patients without current evidence of raised eosinophils while on high dose steroids. It is imprecise: many of these patients will have an underlying eosinophilic phenotype. Nevertheless, though, they may underestimate the presence of an eosinophilic phenotype. It is important to reiterate that contemporaneous blood and sputum measurements are proven theragnostic biomarkers, predicting response to anti IL-5 and steroid treatment for reducing asthma exacerbations. The authors would like to thank the patients who participated in the study. They also wish to acknowledge the support of the Southampton Biomedical Research Centre funded by the National Institute for Health Research UK and the WATCH study investigators. The WATCH study has received funding support from Novartis and the Asthma Allergy Inflammation Research (AAIR) Charity. Dr. Hitasha Rupani reports Speaker and consultancy fees from AstraZeneca, GlaxoSmithKline, Teva, Novartis and Chiesi. Professor Peter Howarth reports employment by GSK outside of the submitted work. Clair Barber, Adnan Azim, Colin Newell, Aref Kyyaly, Hans Michael Haitchi and Ramesh Kurukulaaratchy declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. CB, AA, CN, AK, HR, HMH, PH, RJK all contributed to study development, conduct and governance. AA performed the statistical analysis. CB wrote the first draft of the manuscript AA, CN, AK, HR, HMH, PH, RJK all contributed to manuscript development. RJK acts as guarantor for the manuscript. All authors have read and approved the manuscript before submission.

The measurement of exhaled volatile organic compounds (VOCs) in exhaled breath (breathomics) represents an exciting biomarker matrix for airways disease, with early research indicating a sensitivity to airway inflammation. One of the key aspects to analytical validity for any clinical biomarker is an understanding of the short-term repeatability of measures. We collected exhaled breath samples on 5 consecutive days in 14 subjects with severe asthma who had undergone extensive clinical characterisation. Principal component analysis on VOC abundance across all breath samples revealed no variance due to the day of sampling. Samples from the same patients clustered together and there was some separation according to T2 inflammatory markers. The intra-subject and between-subject variability of each VOC was calculated across the 70 samples and identified 30.35% of VOCs to be erratic: variable between subjects but also variable in the same subject. Exclusion of these erratic VOCs from machine learning approaches revealed no apparent loss of structure to the underlying data or loss of relationship with salient clinical characteristics. Moreover, cluster evaluation by the silhouette coefficient indicates more distinct clustering. We are able to describe the short-term repeatability of breath samples in a severe asthma population and corroborate its sensitivity to airway inflammation. We also describe a novel variance-based feature selection tool that, when applied to larger clinical studies, could improve machine learning model predictions.

Introduction: Corticosteroid-resistant severe asthma is associated with significant morbidity and lacks targeted therapies. Emerging evidence implicates chronic airways infection, particularly with Haemophilus influenzae, and innate immune dysregulation during stable disease as drivers of this phenotype. Previous asthma metagenomic studies lack adequate phenotyping and sequencing depth for bacterial species-level identification. We hypothesise chronic airways infection is a ‘treatable-trait’ but its prevalence, clinical phenotype and biomarkers need definition. Methods: We performed Oxford Nanopore sequencing, and RT-qPCR of total DNA extracts on induced sputum samples (n=52) from the Wessex Severe Asthma Cohort and identified sputum cytokines associated with airways infection. Results: A dominant pathogenic airway organism (H.influenzae, Streptococcus pneumoniae or Moraxella catarrhalis, Figure 1) was identified in 21%of patients with severe asthma by metagenomic sequencing and PCR. Infection was associated with sputum neutrophilia and elevated sputum pro-inflammatory cytokines (including IL-1, IL-6, IL-8, TNF; p<0.01, unpaired t-test, Benjamini-Hochberg correction). Conclusions: Airways infection in asthma is reliably identified using molecular microbiological methods and associated with neutrophilic inflammation. Ongoing analyses will fully characterise this phenotype including mucosal immune responses within the airway.

BACKGROUND:Nontypeable Haemophilus influenzae (NTHi) is a respiratory tract pathobiont that chronically colonizes the airways of asthma patients and is associated with severe, neutrophilic disease phenotypes. The mechanism of NTHi airway persistence is not well understood, but accumulating evidence suggests NTHi can persist within host airway immune cells such as macrophages. We hypothesized that NTHi infection of pulmonary macrophages drives neutrophilic inflammation in severe asthma.METHODS:Bronchoalveolar lavage (BAL) samples from 25 severe asthma patients were assessed by fluorescence in situ hybridisation to quantify NTHi presence. Weighted gene correlation network analysis (WGCNA) was performed on RNASeq data from NTHi-infected monocyte-derived macrophages to identify transcriptomic networks associated with NTHi infection.RESULTS:NTHi was detected in 56% of BAL samples (NTHi+) and was associated with longer asthma duration (34 vs 22.5 years, p = .0436) and higher sputum neutrophil proportion (67% vs 25%, p = .0462). WGCNA identified a transcriptomic network of immune-related macrophage genes significantly associated with NTHi infection, including upregulation of T17 inflammatory mediators and neutrophil chemoattractants IL1B, IL8, IL23 and CCL20 (all p < .05). Macrophage network genes SGPP2 (p = .0221), IL1B (p = .0014) and GBP1 (p = .0477) were more highly expressed in NTHi+ BAL and moderately correlated with asthma duration (IL1B; rho = 0.41, p = .041) and lower prebronchodilator FEV1/FVC% (GBP1; rho = -0.43, p = .046 and IL1B; rho = -0.42, p = .055).CONCLUSIONS:NTHi persistence with pulmonary macrophages may contribute to chronic airway inflammation and T17 responses in severe asthma, which can lead to decreased lung function and reduced steroid responsiveness. Identifying therapeutic strategies to reduce the burden of NTHi in asthma could improve patient outcomes.

Difficult asthma describes asthma in which comorbidities, inadequate treatment, suboptimal inhaler technique and/or poor adherence impede good asthma control. The association of anxiety and depression with difficult asthma outcomes (exacerbations, hospital admissions, asthma control, etc.) is unclear. This study assessed the clinical associations of anxiety and depression with difficult asthma outcomes in patients with a specialist diagnosis of difficult asthma. Using real-world data, we retrospectively phenotyped patients from the Wessex Asthma Cohort of Difficult Asthma (N = 441) using clinical diagnoses of anxiety and depression against those without anxiety or depression (controls). Additionally, we stratified patients by severity of psychological distress using the Hospital Anxiety and Depression Scale (HADS). We found that depression and/or anxiety were reported in 43.1% of subjects and were associated with worse disease-related questionnaire scores. Each psychological comorbidity group showed differential associations with difficult asthma outcomes. Anxiety alone (7.9%) was associated with dysfunctional breathing and more hospitalisations [anxiety, median (IQR): 0 (2) vs. controls: 0 (0)], while depression alone (11.6%) was associated with obesity and obstructive sleep apnoea. The dual anxiety and depression group (23.6%) displayed multimorbidity, worse asthma outcomes, female predominance and earlier asthma onset. Worse HADS-A scores in patients with anxiety were associated with worse subjective outcomes (questionnaire scores), while worse HADS-D scores in patients with depression were associated with worse objective (ICU admissions and maintenance oral corticosteroid requirements) and subjective outcomes. In conclusion, anxiety and depression are common in difficult asthma but exert differential detrimental effects. Difficult asthma patients with dual anxiety and depression experience worse asthma outcomes alongside worse measures of psychological distress. There is a severity-gradient association of HADS scores with worse difficult asthma outcomes. Collectively, our findings highlight the need for holistic, multidisciplinary approaches that promote early identification and management of anxiety and depression in difficult asthma patients.

BACKGROUND:Severe asthma is associated with multiple comorbidities, including gastro-oesophageal reflux disease (GORD), which can contribute to exacerbation frequency and poor quality of life. Since epithelial dysfunction is an important feature in asthma, we hypothesised that in severe asthma the bronchial epithelium is more susceptible to the effects of acid reflux.METHODS:We developed an in vitro model of GORD using differentiated bronchial epithelial cells (BECs) from normal or severe asthmatic donors exposed to a combination of pepsin, acid pH and bile acids using a multiple challenge protocol (MCP-PAB). In addition, we analysed bronchial biopsies and undertook RNA sequencing of bronchial brushings from controls and severe asthmatics without or with GORD.RESULTS:Exposure of BECs to the MCP-PAB caused structural disruption, increased permeability, interleukin (IL)-33 expression, inflammatory mediator release and changes in gene expression for multiple biological processes. Cultures from severe asthmatics were significantly more affected than those from healthy donors. Analysis of bronchial biopsies confirmed increased IL-33 expression in severe asthmatics with GORD. RNA sequencing of bronchial brushings from this group identified 15 of the top 37 dysregulated genes found in MCP-PAB treated BECs, including genes involved in oxidative stress responses.CONCLUSIONS AND CLINICAL IMPLICATION:By affecting epithelial permeability, GORD may increase exposure of the airway submucosa to allergens and pathogens, resulting in increased risk of inflammation and exacerbations. These results suggest the need for research into alternative therapeutic management of GORD in severe asthma.

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 more prevalent in males during childhood but becomes predominantly female in adulthood. Cohort studies and registries of adult severe asthma demonstrate a higher prevalence and severity of disease in females. Aim: To describe the differences in severe asthma patients in terms of biological characteristics across sex. Methods: Clinical characterisation and sputum induction was perfomed as part of the WATCH cohort study in biologic naïve patients severe asthma patients. Sputum was analysed for differential cell count and supernatant proteins (PBS processed), measured by singleplex ELISA with between group differences assessed by Mann Whitney U tests. Analysis was replicated in a second severe asthma cohort (WSAC cohort), who had undergone a similar characterisation process. Results: The analysis included 152 and 99 patients from WATCH and WSAC cohorts respectively. Both cohorts were predominantly female (57.9% and 60.6% respectively) with no difference between males and females in maintenance OCS use or exacerbation frequency in the last 12 months. Compared to females, male patients in the WATCH cohort had later onset disease and a higher frequency of nasal polyps whilst WSAC patients had higher serum total IgE and sputum eosinophil counts. In both cohorts, male patients had higher blood eosinophil counts, FeNO and sputum ECP. Additional differentiators were sputum EDN (measured in WATCH study) and sputum IL-5 (measured WSAC cohort). Conclusion: Sex differences in asthma and severe asthma are well described with various putative mechanisms proposed. We demonstrate that signals of type 2 inflammation in blood and sputum are more frequently seen in males than females, despite a comparable level of corticosteroid therapy.

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 naïve 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<0.001) and blood eosinophils (r=0.673, p<0.001). Those with severe eosinophilic asthma had significantly increased sputum eosinophils (8.2% [5.8,18.7]) and sputum IL-5 (11.6 ng/ml [2.1,18.9]), compared to those with non-eosinophilic asthma (both p<0.001). Though sputum eosinophil percentages in non-eosinophilic severe asthma (0.2% [0.0,1.0]) did not differ from in health (0.4% [0.0,0.8]), sputum IL-5 (0.2 ng/ml [0.1,0.9] was higher in this non-eosinophilic phenotype compared to that in healthy volunteers (0.0 ng/ml [0.0,0.0], p=0.002). Discussion: Despite high levels of steroid treatment, there is persistent IL-5 generation within the airways of severe asthma.

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: 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 dinical/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 log io 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 log lo total IgE + 1 (OR, 4.30), whereas for allergic bronchopulmonary aspergillosis included maintenance oral corticosteroid use (OR, 6.98), maximum log lo 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. (C) 2021 American Academy of Allergy, Asthma & Immunology

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 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.

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 <18 years or adult >= 18years) and characterized in relation to clinical and pathophysiological features. RESULTS: The cohort had more female participants (65%) but had similar proportions of participants with early- or adult-onset disease. Early-onset female disease was commonest (35%), highly atopic, with good spirometry and strong associations with some physical comorbidities but highest psychophysiologic comorbidities. Adult-onset females also had considerable psychophysiologic comorbidities and highest obesity, and were least atopic. Amongst male subjects, proportionately more had adult-onset disease. Early-onset male disease was rarest (14%) but associated with worst lung function, high smoking, atopy, and fungal sensitization. Despite shortest disease duration, adult-onset males had highest use of maintenance oral corticosteroid, poor lung function, and highest fractional exhaled nitrogen oxide in spite of highest smoking prevalence. CONCLUSIONS: This study shows that sex, age of asthma onset, and their interactions influence different clinical manifestations of difficult asthma and identifies a greater risk for lung function loss and oral corticosteroid dependence associated with smoking in adult-onset male subjects. Crown Copyright (C) 2020 Published by Elsevier Inc. on behalf of the American Academy of Allergy, Asthma & Immunology

Introduction: Identifying persistent T2 inflammation is vitally important in the management of SA, particularly in determining suitability for biologic therapies. Method: The Wessex Severe Asthma Cohort (WSAC) is a large published cohort of well characterised SA patients fulfilling the ATS/ERS 2014 definition of SA. We compared the clinical, physiological & biological characteristics of patients dependent upon the strength of their T2 signal. Results: Discussion: A strong T2 signal is associated with increased sinonasal comorbidity & lung function impairment alongside increased biological markers of the T2 inflammatory pathway. Patients with SA with an intermediate T2 signal suffer a similar burden of disease with high levels of co-morbidity, poor disease control, reduced quality of life & frequent severe exacerbations despite similar levels of ICS and maintenance OCS use. These patients currently fall outside of the treatment recommendations for anti-IL5 biologic therapies in the UK and thus reflect a significant unmet need.