Airway hyperresponsiveness reflects corticosteroid-sensitive mast cell involvement across asthma phenotypes.

BACKGROUND: Airway hyperresponsiveness is a hallmark of asthma across asthma phenotypes. Airway hyperresponsiveness to mannitol specifically relates to mast cell infiltration of the airways, suggesting inhaled corticosteroids to be effective in reducing the response to mannitol, despite low levels of type 2 inflammation. OBJECTIVE: We sought to investigate the relationship between airway hyperresponsiveness and infiltrating mast cells, and the response to inhaled corticosteroid treatment. METHODS: In 50 corticosteroid-free patients with airway hyperresponsiveness to mannitol, mucosal cryobiopsies were obtained before and after 6 weeks of daily treatment with 1600 µg of budesonide. Patients were stratified according to baseline fractional exhaled nitric oxide (Feno) with a cutoff of 25 parts per billion. RESULTS: Airway hyperresponsiveness was comparable at baseline and improved equally with treatment in both patients with Feno-high and Feno-low asthma: doubling dose, 3.98 (95% CI, 2.49-6.38; P < .001) and 3.85 (95% CI, 2.51-5.91; P < .001), respectively. However, phenotypes and distribution of mast cells differed between the 2 groups. In patients with Feno-high asthma, airway hyperresponsiveness correlated with the density of chymase-high mast cells infiltrating the epithelial layer (ρ, -0.42; P = .04), and in those with Feno-low asthma, it correlated with the density in the airway smooth muscle (ρ, -0.51; P = .02). The improvement in airway hyperresponsiveness after inhaled corticosteroid treatment correlated with a reduction in mast cells, as well as in airway thymic stromal lymphopoietin and IL-33. CONCLUSIONS: Airway hyperresponsiveness to mannitol is related to mast cell infiltration across asthma phenotypes, correlating with epithelial mast cells in patients with Feno-high asthma and with airway smooth muscle mast cells in patients with Feno-low asthma. Treatment with inhaled corticosteroids was effective in reducing airway hyperresponsiveness in both groups.

Impact of former smoking exposure on airway eosinophilic activation and autoimmunity in patients with severe asthma.

INTRODUCTION: Severe eosinophilic asthma is characterised by frequent exacerbations and a relative insensitivity to steroids. Experimentally, smoking may induce eosinophilic airway inflammation, but the impact in patients with severe asthma is not clear. OBJECTIVE: To investigate the association between smoking exposure in patients with severe asthma, and eosinophilic inflammation and activation, as well as airway autoimmunity and steroid responsiveness. METHODS: Patients with severe asthma according to European Respiratory Society/American Thoracic Society criteria were assessed with sputum samples, analysed by cell differential count, and for the presence of free eosinophil granules (FEGs), autoantibodies against eosinophil peroxidase (EPX) and macrophage receptor with collagenous structure (MARCO). A subgroup of patients with eosinophilic airway inflammation was re-assessed after a 2-week course of prednisolone. RESULTS: 132 severe asthmatics were included in the study. 39 (29.5%) patients had ≥10 pack-years of smoking history: 36 (27.3%) were former smokers and three (2.3%) current smokers; and 93 (70.5%) had <10 pack-years exposure. Eosinophilic airway inflammation was more prevalent among patients with ≥10 pack-years (66.7%), compared to patients with <10 pack-years (38.7%, p=0.03), as was the level of FEGs (p=0.001) and both anti-EPX and anti-MARCO (p<0.05 and p<0.0001, respectively). Omitting current smokers did not affect these associations. Furthermore, prednisolone reduced, but did not normalise, sputum eosinophils in patients with a ≥10 pack-year smoking history. CONCLUSION: In patients with severe asthma, a former smoking history is associated with eosinophilic airway inflammation and activation and relative insensitivity to steroids, as well as airway autoimmunity.

Phenotype and severity of asthma determines bronchial epithelial immune responses to a viral mimic.

BACKGROUND: Asthma is characterised by an aggravated immune response to respiratory viral infections. This phenomenon is a clinically well-recognised driver of acute exacerbations, but how different phenotypes of asthma respond immunologically to viruses is unclear. OBJECTIVES: To describe the association between different phenotypes and severity of asthma and bronchial epithelial immune responses to viral stimulation. METHODS: In the Immunoreact study, healthy subjects (n=10) and 50 patients with asthma were included; 30 (60%) were atopic, and 34 (68%) were eosinophilic; 14 (28%) had severe asthma. All participants underwent bronchoscopy with collection of bronchial brushings. Bronchial epithelial cells (BECs) were expanded and stimulated with the viral replication mimic poly (I:C) (Toll-like receptor (TLR)3 agonist) in vitro. The expression of TLR3-induced pro-inflammatory and antiviral responses of BECs were analysed using reverse transcriptase quantitative PCR and multiplex ELISA and compared across asthma phenotypes and severity of disease. RESULTS: Patients with atopic asthma had increased induction of interleukin (IL)-4, interferon (IFN)-ß, IL-6, tumour necrosis factor-α, and IL-1ß after poly (I:C) stimulation compared to non-atopic patients, whereas in patients with eosinophilic asthma only IL-6 and IL-8 induction was higher than in non-eosinophilic asthma. Patients with severe asthma displayed a decreased antiviral IFN-ß, and increased expression of IL-8, most pronounced in atopic and eosinophilic asthmatics. Furthermore, induction of IL-33 in response to poly (I:C) was increased in severe atopic and in severe eosinophilic asthma, but thymic stromal lymphopoietin only in severe eosinophilic asthma. CONCLUSIONS: The bronchial epithelial immune response to a viral mimic stimulation differs between asthma phenotypes and severities, which may be important to consider when targeting novel asthma treatments.

Airway gene expression identifies subtypes of type 2 inflammation in severe asthma.

BACKGROUND: Type 2 inflammation is characterized by enhanced activity of interleukin (IL)-4, -5 and -13, and treatments targeting these pathways are available for treatment of severe asthma. At present, the pattern of pathway activity and the implications overlapping of pathway activity are unknown. OBJECTIVE: We hypothesized that clustering of airway mRNA expression would identify distinct molecular subtypes of severe asthma and thereby uncover the prevalence and overlap of pathway activity. METHODS: Sputum mRNA expression of genes related to expression of IL-5(CLC, CPA3 and DNASE1L3), IL-13(IL13Ra1, TNFSF14 and SERPINB2), T1/Th17 activity(IL1B, ALPL and CXCR2) and in vitro response to corticosteroids (FKBP512) and mepolizumab (ARAP3) was analysed in patients (n = 109) with severe asthma and healthy controls (n = 22). A cluster analysis of gene expression was performed. The response to a short course of OCS was assessed in a subset of patients (n = 29). RESULTS: Five molecular clusters were identified. Three had abundant T2 gene expression of which two (n = 39 and n = 9) were characterized by abundant expression of both IL-13- and IL-5-related genes. The last (n = 6) had only abundant IL-5-related gene expression. These T2-high molecular clusters could not be distinguished using T2 biomarkers. T2- and Th1/Th17-related mRNA expression were co-expressed across all clusters. OCS significantly reduced T2 gene expression (CLC, IL13Ra1, SERPINB2 and ARAP3) and significantly increase expression of Th1/Th17-related genes (ALPL and CXCR2). CONCLUSIONS AND CLINICAL RELEVANCE: Clustering of airway mRNA expression identified five molecular clusters of severe asthma of which three were considered T2 high. Co-expression of IL-5- and IL-13-related genes at moderate levels was present in almost half of patients, while marked elevated expression of both was rare. In contrast to IL-5, clusters with isolated IL-13- and Th1/Th17-related gene expression were not identified.

Automated cell differential count in sputum is feasible and comparable to manual cell count in identifying eosinophilia.

INTRODUCTION: Cell differential count (CDC) of induced sputum is considered the gold standard for inflammatory phenotyping of asthma but is not implemented in routine care due to its heavy time- and staff demands. Digital Cell Morphology is a technique where digital images of cells are captured and presented preclassified as white blood cells (neutrophils, eosinophils, lymphocytes, macrophages, and unidentified) and nonwhite blood cells for review. With this study, we wanted to assess the accuracy of an automated CDC in identifying the key inflammatory cells in induced sputum. METHODS: Sputum from 50 patients with asthma was collected and processed using the standard processing protocol with one drop 20% albumin added to hinder cell smudging. Each slide was counted automatically using the CellaVision DM96 and manually by an experienced lab technician. Sputum was classified as eosinophilic or neutrophilic using 3% and 61% cutoffs, respectively. RESULTS: We found a good agreement using intraclass correlation for all target cells, despite significant differences in the cell count rate. The automated CDC had a sensitivity of 65%, a specificity of 93%, and a kappa-coefficient of 0.61 for identification of sputum eosinophilia. In contrast, the automated CDC had a sensitivity of 29%, a specificity of 100%, and a kappa-coefficient of 0.23 for identification of sputum neutrophilia. CONCLUSION: Automated- and manual cell counts of sputum agree with regards to the key inflammatory cells. The automated cell count had a modest sensitivity but a high specificity for the identification of both neutrophil and eosinophil asthma.

Missing sputum samples are common in asthma intervention studies and successful collection at follow-up is related to improvement in clinical outcomes.

Several factors significantly impact ability to produce a sputum sample after an anti-inflammatory intervention and these authors argue that the widely used complete-case analysis is inappropriate for paired sputum-based outcome measures https://bit.ly/3qN2pk5.

Mucosal cryobiopsies: a new method for studying airway pathology in asthma.

Background: In vivo studies of airway pathology in obstructive lung disease are limited by poor quality of specimens obtained with forceps. Obtainment of cryobiopsies has increased diagnostic yield in cancer and interstitial lung disease but has not been used in patients with asthma. In a recent pilot study, we found mucosal cryobiopsies to be larger and more intact than conventional forceps biopsies. The aim of the present study was to compare quality and safety of mucosal cryobiopsies versus conventional forceps biopsies in patients with asthma. Methods: Endobronchial biopsies were obtained with forceps and cryoprobe from patients with asthma not currently treated with inhaled steroids and evaluated histologically. Results: A total of 240 cryobiopsies and 288 forceps biopsies were obtained from 48 patients. Bleeding from the biopsy site was common but self-limiting. No major complications related to the procedure were seen. Cryobiopsy cross areas were four times larger compared with forceps. Stretches of intact epithelium were detected in all cryobiopsies compared to 33% in forceps biopsies. Further, the length of intact epithelium was on average four times longer in the cryobiopsies. Importantly, there was a good preservation of both antigens and mRNA in the cryobiopsies ensuring a suitability and robustness for immunohistochemistry and in situ hybridisation. Conclusion: Obtainment of mucosal cryobiopsies in patients with asthma is safe and yields biopsies that are significantly larger and morphologically better preserved compared with traditional forceps biopsies. The cryotechnique thus seems to be a promising tool for future in vivo studies of airway pathology.

The Prevalence of Subtypes of Type 2 Inflammation in an Unselected Population of Patients with Severe Asthma.

BACKGROUND: With the introduction of different targeted therapies for type 2 (T2)-high asthma, there is an urgent need for markers to guide the choice of treatment. T2-high asthma includes different clinical phenotypes of asthma, but the prevalence and impact of activation of different T2 inflammatory pathways is unknown. OBJECTIVE: To describe the level of coexpression of clinically available T2 inflammatory markers in patients with severe asthma, and the relationship with clinical characteristics and comorbidities. METHODS: Patients with severe asthma according to European Respiratory Society/American Thoracic Society guidelines were examined prospectively including sputum induction and grouped according to T2 biomarkers: blood eosinophilia (≥0.3 × 109/L), total IgE (≥150 U/mL), and fractional exhaled nitric oxide (≥25 parts per billion). RESULTS: We found 116 (70%) of the 166 patients to have at least 1 T2 biomarker elevated: 39% had 2 or more elevated biomarkers, whereas 31% had only 1 biomarker elevated. Concomitant airway and systemic eosinophilia was present in 28% of all patients, corresponding to half (53%) of the patients with either. Expression patterns of the T2 biomarkers were associated with differences in allergic sensitization and the coexistence of nasal polyposis. CONCLUSIONS: Most patients with severe asthma showed at least 1 T2 inflammatory trait. Coexpression of T2 biomarkers was highly heterogeneous, and different expression patterns were associated with distinct clinical characteristics.

Clinical characteristics of the BREATHE cohort - a real-life study on patients with asthma and COPD.

Background: The BREATHE study is a cross-sectional study of real-life patients with asthma and/or COPD in Denmark and Sweden aiming to increase the knowledge across severities and combinations of obstructive airway disease. Design: Patients with suspicion of asthma and/or COPD and healthy controls were invited to participate in the study and had a standard evaluation performed consisting of questionnaires, physical examination, FeNO and lung function, mannitol provocation test, allergy test, and collection of sputum and blood samples. A subgroup of patients and healthy controls had a bronchoscopy performed with a collection of airway samples. Results: The study population consisted of 1403 patients with obstructive airway disease (859 with asthma, 271 with COPD, 126 with concurrent asthma and COPD, 147 with other), and 89 healthy controls (smokers and non-smokers). Of patients with asthma, 54% had moderate-to-severe disease and 46% had mild disease. In patients with COPD, 82% had groups A and B, whereas 18% had groups C and D classified disease. Patients with asthma more frequently had childhood asthma, atopic dermatitis, and allergic rhinitis, compared to patients with COPD, asthma + COPD and Other, whereas FeNO levels were higher in patients with asthma and asthma + COPD compared to COPD and Other (18 ppb and 16 ppb vs 12.5 ppb and 14 ppb, p < 0.001). Patients with asthma, asthma + COPD and Other had higher sputum eosinophilia (1.5%, 1.5%, 1.2% vs 0.75%, respectively, p < 0.001) but lower sputum neutrophilia (39.3, 43.5%, 40.8% vs 66.8%, p < 0.001) compared to patients with COPD. Conclusions: The BREATHE study provides a unique database and biobank with clinical information and samples from 1403 real-life patients with asthma, COPD, and overlap representing different severities of the diseases. This research platform is highly relevant for disease phenotype- and biomarker studies aiming to describe a broad spectrum of obstructive airway diseases.