A severe asthma phenotype of excessive airway Haemophilus influenzae relative abundance associated with sputum neutrophilia.

BACKGROUND: Severe asthma (SA) encompasses several clinical phenotypes with a heterogeneous airway microbiome. We determined the phenotypes associated with a low α-diversity microbiome. METHODS: Metagenomic sequencing was performed on sputum samples from SA participants. A threshold of 2 standard deviations below the mean of α-diversity of mild-moderate asthma and healthy control subjects was used to define those with an abnormal abundance threshold as relative dominant species (RDS). FINDINGS: Fifty-one out of 97 SA samples were classified as RDSs with Haemophilus influenzae RDS being most common (n = 16), followed by Actinobacillus unclassified (n = 10), Veillonella unclassified (n = 9), Haemophilus aegyptius (n = 9), Streptococcus pseudopneumoniae (n = 7), Propionibacterium acnes (n = 5), Moraxella catarrhalis (n = 5) and Tropheryma whipplei (n = 5). Haemophilus influenzae RDS had the highest duration of disease, more exacerbations in previous year and greatest number on daily oral corticosteroids. Hierarchical clustering of RDSs revealed a C2 cluster (n = 9) of highest relative abundance of exclusively Haemophilus influenzae RDSs with longer duration of disease and higher sputum neutrophil counts associated with enrichment pathways of MAPK, NF-κB, TNF, mTOR and necroptosis, compared to the only other cluster, C1, which consisted of 7 Haemophilus influenzae RDSs out of 42. Sputum transcriptomics of C2 cluster compared to C1 RDSs revealed higher expression of neutrophil extracellular trap pathway (NETosis), IL6-transignalling signature and neutrophil activation. CONCLUSION: We describe a Haemophilus influenzae cluster of the highest relative abundance associated with neutrophilic inflammation and NETosis indicating a host response to the bacteria. This phenotype of severe asthma may respond to specific antibiotics.

Species-level, metagenomic and proteomic analysis of microbe-immune interactions in severe asthma.

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.

Airway proteomics reveals broad residual anti-inflammatory effects of prednisolone in mepolizumab-treated asthma.

BACKGROUND: Mepolizumab is an anti-IL-5 mAb treatment for severe eosinophilic asthma that reduces asthma exacerbations. Residual airway inflammation with mepolizumab therapy may lead to persistent exacerbations. Oral corticosteroids remain the main treatment for these residual exacerbations. OBJECTIVE: Our study aimed to explore the corticosteroid responsiveness of airway inflammation after mepolizumab treatment to find potentially treatable inflammatory mechanisms beyond the IL-5 pathway. METHODS: The MAPLE trial was a multicenter, randomized, double-blind, placebo-controlled, crossover study of 2 weeks of high-dose oral prednisolone treatment at stable state in 27 patients treated with mepolizumab for severe eosinophilic asthma. We analyzed paired sputum (n = 16) and plasma (n = 25) samples from the MAPLE trial using high-throughput Olink proteomics. We analyzed additional sputum proteins using ELISA. RESULTS: In patients receiving mepolizumab, prednisolone significantly downregulated sputum proteins related to type 2 inflammation and chemotaxis including IL-4, IL-5, IL-13, CCL24, CCL26, EDN, CCL17, CCL22, OX40 receptor, FCER2, and the ST2 receptor. Prednisolone also downregulated cell adhesion molecules, prostaglandin synthases, mast cell tryptases, MMP1, MMP12, and neuroimmune mediators. Neutrophilic pathways were upregulated. Type 2 proteins were also downregulated in plasma, combined with IL-12, IFN-γ, and IP-10. IL-10 and amphiregulin were upregulated. CONCLUSIONS: At stable state, prednisolone has broad anti-inflammatory effects on top of mepolizumab. These effects are heterogeneous and may be clinically relevant in residual exacerbations.

Deep Learning-based Segmentation of Computed Tomography Scans Predicts Disease Progression and Mortality in Idiopathic Pulmonary Fibrosis.

Rationale: Despite evidence demonstrating a prognostic role for computed tomography (CT) scans in idiopathic pulmonary fibrosis (IPF), image-based biomarkers are not routinely used in clinical practice or trials. Objectives: To develop automated imaging biomarkers using deep learning-based segmentation of CT scans. Methods: We developed segmentation processes for four anatomical biomarkers, which were applied to a unique cohort of treatment-naive patients with IPF enrolled in the PROFILE (Prospective Observation of Fibrosis in the Lung Clinical Endpoints) study and tested against a further United Kingdom cohort. The relationships among CT biomarkers, lung function, disease progression, and mortality were assessed. Measurements and Main Results: Data from 446 PROFILE patients were analyzed. Median follow-up duration was 39.1 months (interquartile range, 18.1-66.4 mo), with a cumulative incidence of death of 277 (62.1%) over 5 years. Segmentation was successful on 97.8% of all scans, across multiple imaging vendors, at slice thicknesses of 0.5-5 mm. Of four segmentations, lung volume showed the strongest correlation with FVC (r = 0.82; P < 0.001). Lung, vascular, and fibrosis volumes were consistently associated across cohorts with differential 5-year survival, which persisted after adjustment for baseline gender, age, and physiology score. Lower lung volume (hazard ratio [HR], 0.98 [95% confidence interval (CI), 0.96-0.99]; P = 0.001), increased vascular volume (HR, 1.30 [95% CI, 1.12-1.51]; P = 0.001), and increased fibrosis volume (HR, 1.17 [95% CI, 1.12-1.22]; P < 0.001) were associated with reduced 2-year progression-free survival in the pooled PROFILE cohort. Longitudinally, decreasing lung volume (HR, 3.41 [95% CI, 1.36-8.54]; P = 0.009) and increasing fibrosis volume (HR, 2.23 [95% CI, 1.22-4.08]; P = 0.009) were associated with differential survival. Conclusions: Automated models can rapidly segment IPF CT scans, providing prognostic near and long-term information, which could be used in routine clinical practice or as key trial endpoints.

Associations of Breathing Pattern Disorder and Nijmegen Score With Clinical Outcomes in Difficult-to-Treat 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 χ2 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.

Comprehensive Characterization of Difficult-to-Treat Asthma Reveals Near Absence of T2-Low Status.

BACKGROUND: Asthma is conventionally stratified as type 2 inflammation (T2)-high or T2-low disease. Identifying T2 status has therapeutic implications for patient management, but a real-world understanding of this T2 paradigm in difficult-to-treat and severe asthma remains limited. OBJECTIVES: To identify the prevalence of T2-high status in difficult-to-treat asthma patients using a multicomponent definition and compare clinical and pathophysiologic characteristics between patients classified as T2-high and T2-low. METHODS: We evaluated 388 biologic-naive patients from the Wessex Asthma Cohort of difficult asthma (WATCH) study in the United Kingdom. Type 2-high asthma was defined as 20 parts per billion or greater FeNO , 150 cells/µL or greater peripheral blood eosinophils, the need for maintenance oral corticosteroids, and/or clinically allergy-driven asthma. RESULTS: This multicomponent assessment identified T2-high asthma in 93% of patients (360 of 388). Body mass index, inhaled corticosteroid dose, asthma exacerbations, and common comorbidities did not differ by T2 status. Significantly worse airflow limitation was found in T2-high compared with T2-low patients (FEV1/FVC 65.9% vs 74.6%). Moreover, 75% of patients defined as having T2-low asthma had raised peripheral blood eosinophils within the preceding 10 years, which left only seven patients (1.8%) who had never had T2 signals. Incorporation of sputum eosinophilia 2% or greater into the multicomponent definition in a subset of 117 patients with induced sputum data similarly found that 96% (112 of 117) met criteria for T2-high asthma, 50% of whom (56 of 112) had sputum eosinophils 2% or greater. CONCLUSIONS: Almost all patients with difficult-to-treat asthma have T2-high disease; less than 2% of patients never display T2-defining criteria. This highlights a need to assess T2 status comprehensively in clinical practice before labeling a patient with difficult-to-treat asthma as T2-low.

Predicting the risk of chest radiograph abnormality 12-weeks post hospitalisation with SARS CoV-2 PCR confirmed COVID-19.

BACKGROUND: Routine follow-up of patients hospitalised with COVID-19 is recommended, however due to the ongoing high number of infections this is not without significant health resource and economic burden. In a previous study we investigated the prevalence of, and risk factors for, persistent chest radiograph (CXR) abnormalities post-hospitalisation with COVID-19 and identified a 5-point composite score that strongly predicted risk of persistent CXR abnormality at 12-weeks. Here we sought to validate and refine our findings in an independent cohort of patients. METHODOLOGY: A single-centre prospective study of consecutive patients attending a virtual post-hospitalisation COVID-19 clinic and CXR as part of their standard clinical care between 2nd March - 22nd June 2021. Inpatient and follow-up CXRs were scored by the assessing clinician for extent of pulmonary infiltrates (0-4 in each lung) with complete resolution defined as a follow-up score of zero. RESULTS: 182 consecutive patients were identified of which 31% had persistent CXR abnormality at 12-weeks. Patients with persistent CXR abnormality were significantly older (p < 0.001), had a longer hospital length of stay (p = 0.005), and had a higher incidence of both level 2 or 3 facility admission (level 2/3 care) (p = 0.003) and ever-smoking history (p = 0.038). Testing our composite score in the present cohort we found it predicted persistent CXR abnormality with reasonable accuracy (area under the receiver operator curve [AUROC 0.64]). Refining this score replacing obesity with Age ≥ 50 years, we identify the SHADE-750 score (1-point each for; Smoking history, Higher-level care (level 2/3 admission), Age ≥ 50 years, Duration of admission ≥ 15 days and Enzyme-lactate dehydrogenase (LDH ≥ 750U/L), that accurately predicted risk of persistent CXR abnormality, both in the present cohort (AUROC 0.73) and when retrospectively applied to our 1st cohort (AUROC 0.79). Applied to both cohorts combined (n = 213) it again performed strongly (AUROC 0.75) with all patients with a score of zero (n = 18) having complete CXR resolution at 12-weeks. CONCLUSIONS: In two independent cohorts of patients hospitalised with COVID-19, we identify a 5-point score which accurately predicts patients at risk of persistent CXR abnormality at 12-weeks. This tool could be used by clinicians to identify patients in which radiological follow-up may not be required.

Measurement of Exhaled Volatile Organic Compounds as a Biomarker for Personalised Medicine: Assessment of Short-Term Repeatability in Severe Asthma.

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.

Nontypeable Haemophilus influenzae infection of pulmonary macrophages drives neutrophilic inflammation in severe asthma.

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.

Vulnerability to acid reflux of the airway epithelium in severe asthma.

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.

The Clinical Implications of Aspergillus Fumigatus Sensitization in Difficult-To-Treat Asthma Patients.

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.

Real-world Omalizumab and Mepolizumab treated difficult asthma phenotypes and their clinical outcomes.

BACKGROUND: Omalizumab and Mepolizumab are biologic drugs with proven efficacy in clinical trials. However, a better understanding of their real-world effectiveness in severe asthma management is needed. OBJECTIVES: To better understand the real-world effectiveness of Omalizumab and Mepolizumab, elucidate the clinical phenotypes of patients treated with these drugs, identify baseline characteristics associated with biologic response and assess the spectrum of responses to these medications. METHODS: Using real-world clinical data, we retrospectively phenotyped biologic naïve patients from the Wessex AsThma CoHort of difficult asthma (N = 478) commenced on Omalizumab (N = 105) or Mepolizumab (N = 62) compared to severe asthma patients not receiving biologics (SNB, N = 178). We also assessed multiple clinical endpoints and identified features associated with response. RESULTS: Compared to SNB, Omalizumab patients were younger, diagnosed with asthma earlier, and more likely to have rhinitis. Conversely, compared to SNB, Mepolizumab patients were predominantly older males, diagnosed with asthma later, and more likely to have nasal polyposis but less dysfunctional breathing. Both treatments reduced exacerbations, Acute Healthcare Encounters [AHE] (emergency department or hospital admissions), maintenance oral corticosteroid dose, and improved Asthma Control Questionnaire 6 (ACQ6) scores. Omalizumab response was independently associated with more baseline exacerbations (p = .024) but fewer AHE (p = .050) and absence of anxiety (p = .008). Lower baseline ACQ6 was independently associated with Mepolizumab response (p = .007). A composite group of non-responders demonstrated significantly more psychopathologies and worse baseline subjective disease compared to responder groups. CONCLUSIONS AND CLINICAL RELEVANCE: In a difficult asthma cohort, Omalizumab and Mepolizumab were used in distinct clinical phenotypes but were both multidimensionally efficacious. Certain baseline clinical characteristics were associated with poorer biologic responses, such as psychological co-morbidity, which may assist clinicians in biologic selection. These characteristics also emphasize the need for comprehensive approaches to support these patients.

Clinical evaluation of type 2 disease status in a real-world population of difficult to manage asthma using historic electronic healthcare records of blood eosinophil counts.

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.

Peripheral airways type 2 inflammation, neutrophilia and microbial dysbiosis in severe asthma.

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.

A novel ACE2 isoform is expressed in human respiratory epithelia and is upregulated in response to interferons and RNA respiratory virus infection.

Angiotensin-converting enzyme 2 (ACE2) is the main entry point in airway epithelial cells for SARS-CoV-2. ACE2 binding to the SARS-CoV-2 protein spike triggers viral fusion with the cell plasma membrane, resulting in viral RNA genome delivery into the host. Despite ACE2's critical role in SARS-CoV-2 infection, full understanding of ACE2 expression, including in response to viral infection, remains unclear. ACE2 was thought to encode five transcripts and one protein of 805 amino acids. In the present study, we identify a novel short isoform of ACE2 expressed in the airway epithelium, the main site of SARS-CoV-2 infection. Short ACE2 is substantially upregulated in response to interferon stimulation and rhinovirus infection, but not SARS-CoV-2 infection. This short isoform lacks SARS-CoV-2 spike high-affinity binding sites and, altogether, our data are consistent with a model where short ACE2 is unlikely to directly contribute to host susceptibility to SARS-CoV-2 infection.

Patient perceived barriers to exercise and their clinical associations in difficult asthma.

BACKGROUND: Exercise is recommended in guidelines for asthma management and has beneficial effects on symptom control, inflammation and lung function in patients with sub-optimally controlled asthma. Despite this, physical activity levels in patients with difficult asthma are often impaired. Understanding the barriers to exercise in people with difficult asthma is crucial for increasing their activity, and in implementing successful, disease modifying, and holistic approaches to improve their health. METHODS: 62 Patients within the WATCH Difficult Asthma Cohort (Southampton, UK) completed an Exercise Therapy Burden Questionnaire (ETBQ). The results were analyzed with contemporaneous asthma-related data to determine relationships between perceived exercise barriers and asthma and comorbidity characteristics. RESULTS: Patients were reflective of a difficult asthma cohort, 66% were female, and 63% were atopic. They had a high BMI (median [inter-quartile range]) of 29.3 [25.5-36.2], age of 53.5 [38.75, 65.25], impaired spirometry with FEV1 73% predicted [59.5, 86.6%] and FEV/FVC ratio of 72 [56.5, 78.0] and poor symptom control, as defined by an Asthma Control Questionnaire (ACQ6) result of 2.4 [1.28, 3.2]. A high perceived barriers to exercise score was significantly correlated with increased asthma symptoms (r = 0.452, p < 0.0001), anxiety (r = 0.375, p = 0.005) and depression (r = 0.363, p = 0.008), poor quality of life (r = 0.345, p = 0.015) and number of rescue oral steroid courses in the past 12 months (r = 0.257, p = 0.048). Lung function, blood eosinophil count, FeNO, Njimegen and SNOT22 scores, BMI and hospitalisations in the previous year were not related to exercise perceptions. CONCLUSION: In difficult asthma, perceived barriers to exercise are related to symptom burden and psychological morbidity. Therefore, exercise interventions combined with psychological input such as CBT to restructure thought processes around these perceived barriers may be useful in facilitating adoption of exercise.