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RATIONALE: Allergic asthma is linked to impaired bronchial epithelial secretion of IFNs, which may be causally linked to the increased risk of viral exacerbations. We have previously shown that allergen immunotherapy (AIT) effectively reduces asthma exacerbations and prevents respiratory infections requiring antibiotics; however, whether AIT alters antiviral immunity is still unknown. OBJECTIVES: To investigate the effect of house dust mite sublingual AIT (HDM-SLIT) on bronchial epithelial antiviral and inflammatory responses in patients with allergic asthma. METHODS: In this double-blind, randomized controlled trial (VITAL [The Effect of Allergen Immunotherapy on Anti-viral Immunity in Patients with Allergic Asthma]), adult patients with HDM allergic asthma received HDM-SLIT 12-SQ or placebo for 24 weeks. Bronchoscopy was performed at baseline and at Week 24, which included sampling for human bronchial epithelial cells. Human bronchial epithelial cells were cultured at baseline and at Week 24 and stimulated with the viral mimic polyinosinic:polycytidylic acid (poly(I:C)). mRNA expression was quantified using qRT-PCR, and protein concentrations were measured using multiplex ELISA. MEASUREMENTS AND MAIN RESULTS: Thirty-nine patients were randomized to HDM-SLIT (n = 20) or placebo (n = 19). HDM-SLIT resulted in increased polyinosinic:polycytidylic acid–induced expression of IFN-β at both the gene (P = 0.009) and protein (P = 0.02) levels. IFN-λ gene expression was also increased (P = 0.03), whereas IL-33 tended to be decreased (P = 0.09). On the other hand, proinflammatory cytokines IL-6 (P = 0.009) and TNF-α (tumor necrosis factor-α) (P = 0.08) increased compared with baseline in the HDM-SLIT group. There were no significant changes in TSLP (thymic stromal lymphopoietin), IL-4, IL-13, and IL-10. CONCLUSIONS: HDM-SLIT improves bronchial epithelial antiviral resistance to viral infection. These results potentially explain the efficacy of HDM-SLIT in reducing exacerbations in allergic asthma. Clinical trial registered with www.clinicaltrials.gov (NCT 04100902).

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 2inflammation.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 mg of budesonide. Patients were stratified according to baseline fractional exhaled nitric oxide (FENO) with a cutoff of 25 partsper 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 cellsdiffered 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 (p, -0.42; P = .04), and in those with FENO-low asthma, it correlated with the density in the airway smooth muscle (p,-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.

Evaluation of perioperative hypersensitivity (POH) is challenging, and accurate screening tools are needed to optimize the diagnostic process. We aimed to assess and validate the diagnostic value of a published algorithm (using tryptase and clinical presentation) to identify appropriate individuals for further testing for IgE-mediated POH.We analysed the clinical presentation (tryptase elevation, cardiovascular, respiratory, skin involvement) of patients proceeding to testing for possible IgE-mediated POH at a single tertiary referral centre, relative to subsequent skin testing and specific IgE results. Clinical presentations by drug class were also determined.In 293 consecutive patients, the use of a published algorithm based on one or more of; (i) defined increase in serum tryptase, (ii) involvement of at least two-organ systems, or (iii) presentation with new urticaria and/or angioedema; was highly sensitive [98.8% (CI95: 95.7-99.9%)] but less specific [34.6% (CI95: 25.7-44.4%)] in identifying patients testing positive on skin testing and/or specific IgE. Presentation with cardiovascular symptoms was also sensitive [89.8%(CI95: 84.2-94.0%)], while the combination of respiratory symptoms and increased tryptase was most specific [85.9%(CI95:76.6-92.5%)]. Respiratory involvement was more common in neuromuscular blocking agent allergy, while urticaria/angioedema was more common in antibiotic allergy.The published algorithm (of tryptase rise, two-organ involvement or new urticaria/angioedema) is highly sensitive, and appropriate as a screening tool to identify patients suitable for testing for IgE-mediated POH.

Epithelial damage and increase of intraepithelial mast cells (MC) are characteristics of asthma. The role of MC mediator tryptase and the protease-activated receptor-2 (PAR2) on epithelial wound healing is not fully investigated. Stimulation of bronchial epithelial cells (BECs) with tryptase promoted gap closure, migration and cellular speed compared to controls. Stimulated BECs had higher expression of migration marker CD151 compared to controls. Proliferation marker KI67 was upregulated in tryptase-stimulated BECs compared to controls. Treatment with PAR2 antagonist I-191 reduced gap closure, migration and cell speed compared to BECs stimulated with tryptase. We found that tryptase enhances epithelial wound healing by increased migration and proliferation, which is in part regulated via PAR2. Our data suggest that tryptase might be beneficial in tissue repair under baseline conditions. However, in a pathological context such as asthma with increased numbers of activated MCs, it might lead to epithelial remodeling and loss of function.

Introduction: Allergen exposure worsens viral-triggered asthma exacerbations and could predispose the host to secondary bacterial infections. We have previously demonstrated that exposure to house dust mite (HDM) reduced TLR-3-induced IFN-β in human bronchial epithelial cells (HBECs) from healthy donors. We hypothesize that HDM sensitization in different ways may be involved in both viral and bacterial resistance of HBECs in asthma. In this study, the role of HDM sensitization and effects of HDM exposure on viral stimulus-challenged HBECs from asthmatic donors have been explored with regard to expression and release of molecules involved in anti-viral and anti-bacterial responses, respectively. Methods: HBECs from HDM-sensitized (HDM+) and unsensitized (HDM-) patients with asthma were used. HBECs were exposed to HDM or heat inactivated (hi)-HDM (20 μg/ml) for 24 h prior to stimulation with the viral infection mimic, Poly(I:C), for 3 or 24 h. Samples were analyzed with ELISA and RT-qPCR for β-defensin-2, IFN-β, TSLP, and neutrophil-recruiting mediators: IL-8 and TNF-⍺. NFκB signaling proteins p105, p65, and IκB-⍺ were analyzed by Western blot. Results: Poly(I:C)-induced IFN-β expression was reduced in HBECs from HDM + compared to HDM- patients (p = 0.05). In vitro exposure of HBECs to HDM furthermore reduced anti-microbial responses to Poly(I:C) including β-defensin-2, IL-8, and TNF-⍺, along with reduced NFκB activity. This was observed in HBECs from asthma patients sensitized to HDM, as well as in non-sensitized patients. By contrast, Poly (I:C)-induced release of TSLP, a driver of T2 inflammation, was not reduced with exposure to HDM. Conclusion: Using HBECs challenged with viral infection mimic, Poly(I:C), we demonstrated that allergic sensitization to HDM was associated with impaired anti-viral immunity and that HDM exposure reduced anti-viral and anti-bacterial defense molecules, but not TSLP, across non-allergic as well as allergic asthma. These data suggest a role of HDM in the pathogenesis of asthma exacerbations evoked by viral infections including sequential viral-bacterial and viral-viral infections.

BackgroundIn 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.Aims and ObjectivesTo compare quality and safety of mucosal cryobiopsies versus conventional forceps biopsies in patients with asthma.MethodsEndobronchial biopsies were obtained with forceps and cryoprobe from patients with asthma not currently treated with inhaled steroids and evaluated histologically.ResultsA 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 4 times larger compared with forceps. Stretches of intact epithelium were detected in in all cryobiopsies compared to 33% in forceps biopsies. Further, the length of intact epithelium was on average 4 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.ConclusionObtainment 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.

Background The mannitol test is an indirect bronchial challenge test widely used in diagnosing asthma. Response to the mannitol test correlates with the level of eosinophilic and mast cell airway inflammation, and a positive mannitol test is highly predictive of a response to anti-inflammatory treatment with inhaled corticosteroids. The response to mannitol is a physiological biomarker that may, therefore, be used to assess the response to other anti-inflammatory treatments and may be of particular interest in early phase studies that require surrogate markers to predict a clinical response. The main objectives of this review were to assess the practical aspects of using mannitol as an endpoint in clinical trials and provide the clinical researcher and respiratory physician with recommendations when designing early clinical trials. Methods The aim of this review was to summarise previous uses of the mannitol test as an outcome measure in clinical intervention studies. The PubMed database was searched using a combination of MeSH and keywords. Eligible studies included intervention or repeatability studies using the standard mannitol test, at multiple timepoints, reporting the use of PD15 as a measure, and published in English. Results Of the 193 papers identified, 12 studies met the inclusion criteria and data from these are discussed in detail. Data on the mode of action, correlation with airway inflammation, its diagnostic properties, and repeatability have been summarised, and suggestions for the reporting of test results provided. Worked examples of power calculations for dimensioning study populations are presented for different types of study designs. Finally, interpretation and reporting of the change in the response to the mannitol test are discussed. Conclusions The mechanistic and practical features of the mannitol test make it a useful marker of disease, not only in clinical diagnoses, but also as an outcome measure in intervention trials. Measuring airway hyperresponsiveness to mannitol provides a novel and reproducible test for assessing efficacy in intervention trials, and importantly, utilises a test that links directly to underlying drivers of disease.

BackgroundAsthma 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.ObjectivesTo describe the association between different phenotypes and severity of asthma and bronchial epithelial immune responses to viral stimulation.MethodsIn 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.ResultsPatients with atopic asthma had increased induction of interleukin (IL)-4, interferon (IFN)-β, IL-6, tumour necrosis factor-α, and IL-1β 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-β, 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.ConclusionsThe 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.

Background Combining the antibiotic azithromycin and hydroxychloroquine induces airway immunomodulatory effects, with the latter also having in vitro antiviral properties. This may improve outcomes in patients hospitalised for COVID-19. Methods Placebo-controlled double-blind randomised multicentre trial. Patients ≥18 years, admitted to hospital for≤48 h (not intensive care) with a positive SARS-CoV-2 RT-PCR test, were recruited. The intervention was 500 mg daily azithromycin for 3 days followed by 250 mg daily azithromycin for 12 days combined with 200 mg twice daily hydroxychloroquine for all 15 days. The control group received placebo/placebo. The primary outcome was days alive and discharged from hospital within 14 days (DAOH14). Results After randomisation of 117 patients, at the first planned interim analysis, the data and safety monitoring board recommended stopping enrolment due to futility, based on pre-specified criteria. Consequently, the trial was terminated on February 1, 2021. A total of 61 patients received the combined intervention and 56 patients received placebo. In the intervention group, patients had a median of 9.0 DAOH14 (IQR, 3–11) versus. 9.0 DAOH14 (IQR, 7–10) in the placebo group (p=0.90). The primary safety outcome, death from all causes on day 30, occurred for 1 patient in the intervention group versus. 2 patients receiving placebo (p=0.52), and readmittance or death within 30 days occurred for 9 patients in the intervention group versus. 6 patients receiving placebo (p=0.57). Conclusions The combination of azithromycin and hydroxychloroquine did not improve survival or length of hospitalisation in patients with COVID-19., There are no beneficial or harmful effects from the combined intervention of hydroxychloroquine and azithromycin for hospitalised patients with confirmed coronavirus disease 2019 (COVID-19).

Rationale and objectives Thymic stromal lymphopoietin (TSLP), an epithelial upstream cytokine, initiates production of type-2 (T2) cytokines with eosinophilia and possibly airway hyperresponsiveness (AHR) in asthma. This study aimed to determine whether tezepelumab (a human monoclonal antibody targeting TSLP) decreases AHR and airway inflammation in patients with symptomatic asthma on maintenance treatment with inhaled corticosteroids. Methods and measurements In this double-blind, placebo-controlled randomised trial adult patients with asthma and AHR to mannitol received either 700 mg tezepelumab or placebo intravenously at 4-week intervals for 12 weeks. AHR to mannitol was assessed, and a bronchoscopy was performed at baseline and after 12 weeks. The primary outcome was the change in AHR from baseline to week-12 and secondary outcomes were changes in airway inflammation. Results Forty patients were randomised to receive either tezepelumab (n=20) or placebo (n=20). The mean change in PD15 with tezepelumab was 1.9 DD (95% CI 1.2 to 2.5) versus 1·0 (95% CI 0.3 to 1.6) with placebo; p=0.06. Nine (45%) tezepelumab and three (16%) placebo patients had a negative PD15 test at week-12, p=0.04. Airway tissue and BAL eosinophils decreased by 74% (95% CI −53 to −86) and 75% (95% CI −53 to −86) respectively with tezepelumab compared with an increase of 28% (95% CI −39 to 270) and a decrease of 7% (95% CI −49 to 72) respectively with placebo, p=0.004 and p=0.01. Conclusions Inhibiting TSLP-signalling with tezepelumab reduced the proportion of patients with AHR and decreased eosinophilic inflammation in BAL and airway tissue.

Monoclonal antibody therapies have significantly improved treatment outcomes for patients with severe asthma; however, a significant disease burden remains. Available biologic treatments, including anti-immunoglobulin (Ig)E, anti-interleukin (IL)-5, anti-IL-5Rα and anti-IL-4Rα, reduce exacerbation rates in study populations by approximately 50% only. Furthermore, there are currently no effective treatments for patients with severe, type 2-low asthma. Existing biologics target immunological pathways that are downstream in the type 2 inflammatory cascade, which may explain why exacerbations are only partly abrogated. For example, type 2 airway inflammation results from several inflammatory signals in addition to IL-5. Clinically, this can be observed in how fractional exhaled nitric oxide (FeNO), which is driven by IL-13, may remain unchanged during anti-IL-5 treatment despite reduction in eosinophils, and how eosinophils may remain unchanged during anti-IL-4Rα treatment despite reduction in FeNO. The broad inflammatory response involving cytokines including IL-4, IL-5 and IL-13 that ultimately results in the classic features of exacerbations (eosinophilic inflammation, mucus production and bronchospasm) is initiated by release of “alarmins” thymic stromal lymphopoietin (TSLP), IL-33 and IL-25 from the airway epithelium in response to triggers. The central, upstream role of these epithelial cytokines has identified them as strong potential therapeutic targets to prevent exacerbations and improve lung function in patients with type 2-high and type 2-low asthma. This article describes the effects of alarmins and discusses the potential role of anti-alarmins in the context of existing biologics. Clinical phenotypes of patients who may benefit from these treatments are also discussed, including how biomarkers may help identify potential responders., Blocking epithelial alarmins, upstream mediators triggered early in the asthma inflammatory response that orchestrate broad inflammatory effects, is a promising alternative approach to asthma treatment, which may be effective in a broad patient population https://bit.ly/2zqoXAw

Background: The effect of inhaled corticosteroids (ICS) in patients with type 2 low (T2-low) asthma is unpredictable. Biomarkers are lacking to identify patients likely to benefit from ICS. We speculate that airway hyperresponsiveness (AHR) to mannitol, an indirect bronchial provocation test reflecting mast cell activation, predicts response to ICS in T2-low asthma. Aims and Objectives: To compare the response in AHR to mannitol in patients with T2-high and T2-low asthma after treatment with ICS. Methods: The RECONSTRUCT-study was a prospective intervention study with 50 steroid-free patients with asthma and AHR to mannitol. All patients were assessed before and after 6 weeks of daily treatment with 1600 µg budesonide. Patients were stratified into T2-high (FeNO≥25 ppb) and T2-low asthma (FeNO Results: In total, 46 patients, 25 with T2-high and 21 with T2-low asthma completed the study. AHR to mannitol was comparable at baseline: Gmean PD15: 126mg (95% CI:73-215) and 134mg (95% CI:77-232), P=0.87 and increased after treatment corresponding to a doubling dose of: 3.98 (95% CI:2.49-6.38, P Conclusion: Patients with T2-high and T2-low asthma and AHR to mannitol improve in AHR and ACQ after treatment with inhaled corticosteroids. Mannitol challenge may be a useful physiological marker to identify patients with T2-low asthma who benefit from inhaled corticosteroids.

Background: Mast cells play a key role in airway hyperresponsiveness (AHR) in asthma but infiltration of airway mast cells and the effect of inhaled corticosteroids (ICS) across Type 2 high (T2-high) and T2-low disease have only been poorly described. Aims and Objectives: To study the effect of inhaled corticosteroids (ICS) on airway mast cell density and phenotypes in patients with T2-high and T2-low asthma. Methods: The RECONSTRUCT-study was a prospective intervention study with 50 steroid-free patients with asthma stratified into T2-high (FeNO≥25 ppb) and T2-low (FeNO Results: MCT density was higher in T2-low asthma: Median: 0.20% (IQR: 0.18) than in T2-high asthma: 0.09% (IQR: 0.16), P=0.025. In comparison, MCTC density was higher in T2-high asthma: 0.39% (IQR: 0.30) than T2-low asthma: 0.17% (IQR: 0.09), p Conclusion: T2-low asthma is characterized by an MCT-predominant mast cell infiltration, whereas T2-high asthma is characterized by a higher density of MCTC. ICS decreases mast cell density in T2-high and T2-low asthma, suggesting infiltrating mast cells to be steroid-sensitive disease drivers in T2-low asthma.

Introduction: Allergen inhalation can induce cell death in the airways. Intracellular DAMPs and alarmins released from dead cells could be involved in allergic lung inflammation. Therefore, the molecular pathways implicated in house dust mite (HDM)-induced cell death in asthma need to be further explored. Methods: Human bronchial epithelial cells (HBECs) from asthmatics (N=5) were included for studies with HDM. 3 times/week during 3-weeks of intranasal (i.n.) HDM-challenge (25 µg) was used to induce lung inflammation in mice. Differential cell count, lactate dehydrogenase (LDH) activity and uric acid (UA) release were determined in bronchoalveolar lavage (BAL) fluid. H&E, IL-33 ELISAs were analyzed in lungs. RT-qPCR was used for analyzing gene expression. Results: I.n. administration of HDM in mice increased total cell, eosinophils, neutrophils and lymphocytes counts, as well as UA levels in BAL, and lung tissue IL-33 levels (p Conclusion: HDM challenge induces inflammation and cell necrosis in the airways, which is not related to bronchial epithelium damage. Treatment strategies targeting cell death could be beneficial in a way to dampen local tissue inflammation during asthma.

Background: The Na+/K+ ATPase may play a role in bronchoconstriction in asthma: Through active building of ion gradients across cell membranes, the Na+/K+ ATPase contributes to regulation of intracellular Ca2+ in airway smooth muscle via the Na+/Ca2+ exchanger. Corticosteroids increase Na+/K+ ATPase content in striated muscle but the effect has not been investigated in human airways. Aims and Objectives: To investigate the effect of inhaled corticosteroids (ICS) on the airway Na+/K+ ATPase content in patients with type 2 high (T2-high) and T2-low asthma. Methods: The RECONSTRUCT-study was a prospective intervention study with 50 steroid-free patients with asthma and 10 healthy controls. Airway Na+/K+ ATPase content was determined by the vanadate-facilitated [H3]-ouabain method in mucosal cryobiopsies before and after 6 weeks of daily treatment with 1600 µg budesonide. Patients were stratified into T2-high (FeNO≥25 ppb) and T2-low asthma (FeNO Results: Airway Na+/K+ ATPase content was comparable in healthy controls: Mean: 148ρmol/g (95% CI: 120-177), patients with T2-high asthma: 177ρmol/g (95% CI: 157-198) and patients with T2-low asthma: 189ρmol/g (95% CI: 156-222), P=0.20. After treatment with ICS, Na+/K+ ATPase content increased by 14% in patients with T2-high asthma to: 205ρmol/g (95% CI: 178-228), P=0.017, but not in patients with T2-low asthma: 201ρmol/g (95% CI: 174-228), P=0.54. Conclusion: Treatment with ICS increases airway Na+/K+ ATPase content in patients with T2-high asthma but not in patients with T2-low asthma. This effect could be protective against bronchoconstriction, but needs to be verified in further studies.

Type 2 low (T2-low) asthma is reported to respond less to anti-inflammatory treatment compared with Type 2 high (T2-high) asthma. Airway hyperresponsiveness (AHR) to mannitol, a marker of airway mast cell activation, may be indicative of response to treatment in patients with T2-low disease. We investigated whether AHR to mannitol improves in patients with T2-low asthma after specialist management.Patients with asthma or suspected asthma, referred to our specialist outpatient clinic, were enrolled consecutively and assessed with FeNO, asthma control, blood eosinophils, mannitol and methacholine tests and induced sputum. T2-low asthma was defined in patients with FeNO25ppbThirty-two patients (Females: 56%, age: 22 years (15-59)) were followed. Fourteen (44%) with T2-high and 18 (56%) with T2-low asthma. Baseline AHR to mannitol was comparable:Patients with asthma and AHR to mannitol improve similarly in responsiveness to mannitol after 12 months of specialist management regardless of Type 2 inflammatory biomarker levels. Mechanisms driving AHR in T2-low asthma need to be further elucidated.

Background Patients with asthma are heterogeneous in clinical presentation and in response to treatment. Despite this, tools to guide treatment are limited and include mainly measures of eosinophilic inflammation and symptoms. Airway hyperresponsiveness (AHR) to mannitol is present in patients across inflammatory phenotypes and improve with inhaled corticosteroids. Objective To investigate whether measuring AHR to mannitol in addition to eosinophilic inflammation and symptoms adds information to the phenotypic characterization of patients with asthma. Methods A total of 317 patients with asthma from 6 different cohorts were included in the analysis. All patients had measures of AHR to mannitol, blood eosinophils, and Asthma Control Questionnaire 5 available. A cluster analysis using Ward minimum variance method was performed. The distribution of fraction of exhaled nitric oxide, immunoglobulin E, lung function, induced sputum inflammatory cell count, age of onset, and severity of disease was compared between clusters. Results Four clusters were identified. Three of the clusters had proportionate levels of AHR, eosinophilic inflammation, and symptoms, but 1 cluster presented with low levels of eosinophilic inflammation and a significant symptom burden. Half of the subjects in this cluster presented with AHR to inhaled mannitol. Lung function, fraction of exhaled nitric oxide, body mass index, and immunoglobulin E were normal. Conclusions Information on AHR to mannitol in addition to blood eosinophils and symptoms identifies a subgroup of asthma patients with symptomatic, noneosinophilic disease. Airway hyperresponsiveness to mannitol may provide a treatable trait in a subgroup of patients with noneosinophilic asthma.

NORDSTAR is a longitudinal dataset for the study of asthma comprising 3.3 million individuals and over 50 million person-years in 4 Nordic countries. The data include diagnoses, medication dispensi ...

The impact of diagnostic work-up in asthma management on medication redemption and probably also drug adherence is largely unknown, but we hypothesized that a confirmed diagnosis of asthma in a hospital-based out-patient clinic increases the willingness to subsequent medication redemption in a real life setting.In a retrospective register-based study, 300 medical records of patients referred with possible asthma during one year were examined, of whom 171 had asthma (57%). One-year data on dispensed medicine was collected using the Danish Registry of Medicinal Product Statistics. Patients who had a positive asthma (e.g. bronchial challenge) were classified as verified asthma, whereas unverified asthma refers to doctor's diagnosis of asthma with negative or no diagnostic tests performed.111 (65%) had a verified diagnosis and patients with verified asthma were more frequently prescribed new therapy compared to those with unverified asthma (88.9% vs. 65.0%, respectively, p0.001). No difference was found in first time redemption of prescriptions (72% vs. 64%, respectively, p = 0.3), whereas the second (52% vs. 27%, p = 0.001) and third or more asthma redeemed prescriptions (37% vs. 17%, p = 0.006) showed increased redemption of prescription and probably adherence in the verified compared with the unverified patients with asthma. Furthermore, the use of inhaled corticosteroid (ICS) was calculated as Percent Days Covered (PDC), which was higher in the verified group compared with the non-verified asthma group (88% vs. 30%, p = 0.004).Objective verification of a diagnosis of asthma using asthma tests was associated with an improved redemption of prescription.

SummaryBackground The major trigger of asthma exacerbations is infection with a respiratory virus, most commonly rhinovirus. Type 2 inflammation is known to be associated with an increased risk of exacerbations in general. Whether type 2 inflammation at baseline increases the risk of future virus-induced exacerbations is unknown. Objective To assess whether type 2 inflammation is associated with an increased risk of virus-induced exacerbations of asthma. Methods Stable asthmatics had spirometry, skin prick test, measurement of FeNO and sputum induced for differential cell counts. Patients were followed up for 18 months, during which they were assessed at the research unit when they had symptoms of an exacerbation. Nasal swabs collected at these assessments underwent viral detection by PCR. Results A total of 81 asthma patients were recruited, of which 22 (27%) experienced an exacerbation during the follow-up period. Of these, 15 (68%) had a respiratory virus detected at exacerbation. Sputum eosinophils >1% at baseline increased the risk of having a subsequent virus-induced exacerbation (HR 7.6 95% CI: 1.6-35.2, P=.010) as did having FeNO >25 ppb (HR 3.4 95% CI: 1.1-10.4, P=.033). Conclusion and Clinical Relevance Established type 2 inflammation during stable disease is a risk factor for virus-induced exacerbations in a real-life setting. Measures of type 2 inflammation, such as sputum eosinophils and FeNO, could be included in the risk assessment of patients with asthma in future studies.

Dysfunctional breathing (DB) is a respiratory disorder, which involves a pattern of breathing too deeply, too superficially and/or too rapidly. In asthma patients, DB may lead to an overestimation of the severity of asthma symptoms, and hence potentially to overtreatment. However, it is not known to which degree DB may affect estimates of asthma control, in a specialist clinical setting.The MAPOut-study examined all patients referred consecutively over a 12-months period for specialist assessment of asthma at the Respiratory Outpatient Clinic at Bispebjerg Hospital in Copenhagen. All patients were examined with the Nijmegen questionnaire with a DB defined as a score ≥23 and the ACQ questionnaire. Linear regression analysis of predictors of ACQ score was performed. Asthma was defined as asthma symptoms and a positive asthma test.Of the 256 patients referred to the lung clinic, data on both the Nijmegen questionnaire and ACQ score was obtained in 127 patients, who were included in the present analysis. Median (range) age: 30 (15-63) years, and 76 (59.8%) were females. DB was found in 31 (24.4%). Asthmatic patients with co-existing DB had a poorer asthma control compared to asthmatics without DB (Median (range) ACQ score: 2.40 (0.20-4.60) vs 1.20 (0.00-4.40); p 0.001.). A regression analysis showed that the effect of DB on asthma control was independent of airway hyperresponsiveness or airway inflammation in patients with DB.Dysfunctional breathing is common among asthma patients in a specialist setting, and results in a clinically significant underestimation of asthma control, which may potentially lead to overtreatment.

Introduction: Innate lymphoid cells (ILC) are a group of immune cells that mirror Th-cell functions but lack specific antigen receptors. Type 2 ILCs are increased in atopic asthma compared to healthy control subjects, but data on non-atopic and non-eosinophilic asthma are lacking. Aim: To report the distribution of ILC subpopulations in blood according to inflammatory phenotypes of asthma Methods: Peripheral blood mononuclear cells were isolated from n=22 non-smoking stable asthmatics treated with ICS. Data on atopy, lung function, blood differential count, FeNO and induced sputum were obtained. Viable circulating ILC1 (lin-CRTH2-CD127+), ILC2 (lin-CRTH2+CD127+) and ILC3 (lin-CRTH2-CD127+CD117+CD56+/-) were expressed as fractions of total blood lymphocytes. Patients were stratified based on blood eosinophils (≥0.3), sputum eosinophils (≥3%), sputum neutrophils (>61%) and atopy (positive skin prick test). Results: ILCs constituted a minor fraction of the total lymphocyte pool in peripheral blood: ILC1 0,024% (0.002-0.94%), ILC2 0.009% (0.001-0.079%), ILC3 0.033% (0.002-0.125%). No significant differences were found in ILC1 or ILC2 fractions when comparing eosinophilic and non-eosinophilic asthma whereas ILC3 was significantly higher in non-eosinophilia (Mann-Whitley P=0.04). When comparing atopic and non-atopic individuals no significant differences were found in any ILC populations, the same was true stratifying on sputum neutrophils or eosinophils. ILC3 fractions significantly correlated with the fractions of ILC2 (rho (S)=0.67, p=0.001), no correlation between remaining ILC subsets. Conclusion: Elevated blood ILC3 fractions may be associated with a non-eosinophilic asthma phenotype

Background: The mechanisms driving inflammation in severe asthma are not fully understood. Experimental studies have suggested type 2 innate lymphoid cells (ILC2) as a potential driver of severe steroid-refractory eosinophilic asthma by release of type 2 cytokines through a non-Th2-mediated innate immune response. Aim: We aimed to examine the association between the fraction of ILC2s in peripheral blood and asthma severity. Methods: A total of n=18 patients with mild-moderate and n=21 patients with severe asthma (according to ERS/ATS guidelines) were recruited and grouped based on asthma severity. Data on atopy, lung function, ICS dose, blood and sputum differential counts and FeNO was obtained. Circulating ILC2s (Lin−CD45+CD127+CRTH2+) were quantified using fluorescence-activated cell sorting. Results: Sex (50.0% vs. 42.9% female) and age (mean±SD 45.5±15.2 vs. 51.6±13.7 years)) did not differ between groups. Median daily dose of budesonide was 800 ug (min 400-max 1200) vs. 1600 ug (min 1600-max 3200). A significantly higher fraction of circulating ILC2 cells was detected in the blood of patients with severe asthma compared to mild-moderate asthma (Mann-Whitney U test; p = 0.0004). In a multiple linear regression model adjusting for asthma severity, sex, age, blood and sputum eosinophilia, FeNO, smoking and atopy, the difference in ILC2 fraction was 44% attributable to asthma severity (p=0.007); no other variables were significant. Conclusions: In this stable asthma cohort, the fraction of ILC2 cells in peripheral blood was strongly associated with asthma severity, independently of other inflammatory markers. Further analyses to identify the pathways involved are ongoing.

Introduction: Rhinovirus (RV) infections are commonly followed by secondary bacterial lung infections contributing to the severity of asthma exacerbations. We have shown that bronchial epithelial cells (BECs) have impaired interferon signaling in response to house dust mite (HDM) after viral-stimulus. However, little is known how interactions between allergen exposure and viral infection affect the epithelial antibacterial defense. Here we hypothesized that HDM reduces viral-stimulus induced production of anti-microbial s-defensin together with TNF-?, CCL5 and IL-8 involved in bacterial clearance. Methods: BECs from asthmatic donors were pre-exposed with HDM or heat-inactivated (hi)-HDM (20µg/ml) for 24 h then stimulated with the viral mimic dsRNA (10µg/mL) for 24h. Samples were analyzed with ELISA and RT-qPCR for CCL5, IL-8 and TNF-? and RT-qPCR for s-defensin. NF?B signaling proteins p105, p65 and I?B-? were analyzed for western blot. Results: dsRNA stimulation alone increased s-defensin, CCL5, IL-8 and TNF-? gene expression at 24 h compared to control (P>0.001), which were completely impaired in cells receiving HDM pre-exposure followed by dsRNA stimulation (P>0.05). This was accompanied by reduction in NF?B activity. Hi-HDM followed by dsRNA stimulation did not impair gene and protein expression of s-defensin, CCL5, IL-8 and TNF-? or NF?B activity. Conclusion: We propose that heat-sensitive components in HDM might reduce the ability of bronchial epithelial cells to produce factors involved in anti-bacterial defense. Future studies are now warranted to investigate if HDM are increasing the risk of getting a secondary bacterial infection.

Background: Increased mast cell (MC) densities in the airway epithelium is a hallmark of asthma. However, little is known regarding the role and effect of MC mediators on epithelium damaged by respiratory viruses. Aim: To investigate the density of MCs in biopsies in patients with different asthma phenotypes and the effect of MC mediator tryptase on wound-healing in bronchial epithelial cells. Methods: Intraepithelial MCs (IEMCs) were stained for tryptase (MCT) and tryptase/chymase (MCTC) in bronchial biopsies. In a BEAS-2B cell line, the effect of tryptase and the TLR3 agonist polyI:C on wound healing was studied using live cell imaging (Phase Holographic Imaging). Results: MCT, but not MCTC, were increased in the epithelium in ICS-naive mild asthma and ICS-responsive asymptomatic moderate and severe asthma when compared to ICS-unresponsive symptomatic moderate and severe asthmatics (p=0.03). Within the asthma groups, patients with atopy had the highest numbers of IEMCs compared to non-atopic asthmatics and controls (p=0.002). Stimulation of BEAS-2B with polyI:C resulted in impaired wound gap closure compared to untreated cells (p=0.05). However, MC tryptase enhanced cell proliferation and wound healing responses when compared to cells stimulated with polyI:C (p Conclusion: We showed that asymptomatic patients with different severities of asthma had a higher density of IEMCs regardless of treatment with ICS. Further, the MC mediator tryptase promoted epithelial proliferation and wound healing responses. Hence, IEMCs may have beneficial roles and MC localization may be an important determinant in asthma pathology.

Rationale: Deficient production of anti-viral interferon (IFN)-s may contribute to Rhinovirus (RV)-induced asthma exacerbations. However, the involved mechanisms are unclear. We have previously shown that IFN-s deficiency at exacerbation promotes necroptosis, a regulated cell death mechanism associated with inflammation (Cerps et al., 2018). Here we hypothesized that house dust mite (HDM) will skew bronchial epithelial cells (BECs) to go into necroptosis instead of apoptosis during viral stimuli. Methods: Primary BECs from asthmatic donors were pre-exposed with HDM (20µg/mL) for 24 hours then stimulated with the viral mimic dsRNA (10µg/mL) for 24 hours. Samples were analyzed with RT-qPCR for IFN-s and western blot for the apoptotic marker cleaved caspase-3 and the necroptosis marker phosphorylated MLKL. Results: dsRNA stimulation alone increased IFN-s gene expression and were followed by increased protein levels of the apoptotic marker cleaved caspsase-3 and necroptosis marker phosphorylated MLKL compared to control. Pre-exposure with HDM decreased dsRNA induced IFN-s gene expression and protein expression of cleaved caspase-3. However, dsRNA induced protein expression of phosphorylated MLKL was not affected by pre-exposure with HDM. Conclusion: HDM exposure prior to dsRNA stimulation reduced the expression of the apoptotic marker caspase-3 but not the necroptosis marker phosphorylated MLKL in BECs obtained from asthmatic donors. Our data suggest that HDM skews towards potential pathogenic epithelial cell death (necroptosis) during viral infection which could further aggravate inflammation at asthma exacerbation.

Oxidative stress is linked to the inflammation observed in asthma and COPD and might be involved in exacerbations. Both COPD and asthma exacerbations often involve rhinovirus (RV) infection. Impaired anti-viral signaling and attenuated viral clearance in the bronchial epithelium has been hypothesized for these diseases. However, it is not known whether oxidative stress affects the anti-viral signaling capacity. Bronchial epithelial cells from asthma and COPD donors were infected with RV or treated with the oxidative stressor H2O2 followed by exposure to the synthetic RV replication intermediate poly(I:C). Gene and protein levels of antioxidants and anti-viral responses were measured 3h and 24h post poly(I:C) exposure. RV infection and poly(I:C) stimulation induced protein levels of the antioxidants SOD1 and SOD2. poly(I:C) was used to ascertain a constant infection-like burden in the interaction experiments. Upon pre-treatment with increasing doses of H2O2 the antioxidant response to dsRNA was dose-dependently decreased. This data suggests exaggerated H2O2-induced oxidative stress. Further, poly(I:C)-induced IFNs gene expression was reduced after pre-treatment with H2O2. This was associated with a reduced expression of the pattern recognition receptors RIG-I, MDA5 and TLR3 both on gene and protein level. Knockdown of TLR3 but not RIG-I/MDA5 abrogated impairment of poly(I:C)-induced IFNs gene expression by H2O2. Oxidative stress impairs anti-viral signaling in asthma and COPD bronchial epithelium, likely by reducing RV-responsiveness of TLR3. These findings shed light on molecular mechanisms potentially causing reduced IFNs at exacerbation.