BACKGROUND:Bronchodilator responsiveness (BDR) reflects airflow variability in asthma and is typically assessed after withholding therapy. However, its relevance under ongoing maintenance therapy remains unclear. OBJECTIVE:To investigate the frequency and clinical relevance of residual bronchodilator responsiveness (rBDR) in patients with asthma receiving maintenance therapy. METHODS:In 248 adults with moderate-to-severe asthma receiving inhaled corticosteroids, with or without long-acting bronchodilators, rBDR was assessed according to European Respiratory Society / American Thoracic Society 2005 and 2022 criteria. Maintenance therapy was continued, while short-acting bronchodilators were withheld for at least 12 hours before spirometry. Type 2 biomarkers, lung function including small airway function, patient-reported outcomes, and exacerbation rates were analyzed according to rBDR status. RESULTS:rBDR was present in approximately 23% of patients and was consistent across both European Respiratory Society / American Thoracic Society criteria. Maintenance therapy did not differ by rBDR status. Patients with rBDR had higher type 2 biomarkers, despite high-dose inhaled corticosteroid, including sputum eosinophils and fractional exhaled nitric oxide, more frequent fixed airflow obstruction, and greater small airway dysfunction. They also exhibited increased clinical disease burden, with poorer asthma control and frequent exacerbations, independent of underlying lung function and type 2 inflammation. CONCLUSIONS:rBDR is frequent and identifies patients with refractory type 2 inflammation and poor disease control. rBDR integrates inflammatory activity and functional impairment into a simple, widely available measure and may serve as a practical marker to identify residual disease burden and guide treatment optimization in routine clinical care.

Objectives:Asthma is a heterogeneous disease regarding its pathophysiology, clinical symptoms, and response to treatment. Eicosanoids are important inflammatory mediators, able to either promote or attenuate the underlying chronic airway inflammation. We compared eicosanoid expression patterns in the blood circulation and in stimulated blood leukocytes of asthma patients to identify differences in eicosanoid release which may be related to airway inflammation. Methods:Blood was collected from 198 adult asthmatic patients and 63 healthy controls, participating in the German Center for Lung Research (DZL) ALLIANCE cohort. Eicosanoid release from leukocytes was analyzed using heparinized whole blood after in vitro stimulation with zymosan. Additionally, circulating eicosanoids were measured directly from ethylenediaminetetraacetic acid (EDTA) plasma. Eicosanoids were extracted via solid phase extraction and quantified by high-performance-liquid-chromatography-tandem-mass-spectrometry (HPLC-MS2). Results:Eicosanoid levels were low in blood circulation with no significant differences between asthmatics and controls, except for leukotriene E4 (LTE4) which was slightly elevated in asthmatics. After in vitro stimulation we observed an inhibition of prostaglandin and thromboxane biosynthesis only in patients with severe asthma which was related to the regular use of systemic corticosteroids. In contrast, a significant increase was shown for formation of the 5-Lipoxygenase (5-LOX) product LTE4 in steroid-naïve asthmatics with moderate as well as severe disease severity but not in subjects with systemic steroid treatment. Furthermore 15-Hydorxyeicosatetraenoic acid (15-HETE) production was elevated in asthmatic patients with mild-to-moderate disease activity but dropped down in severe asthmatics. Conclusions:Profiling of eicosanoid production in stimulated whole blood samples showed a specific biosynthesis pattern of asthmatic patients, which is influenced by the use of systemic corticosteroids.

Rationale:Asthma remission is a state of low to no disease activity. To date, little is known about predictors and the achievability of long-term asthma remission. Objective:To identify clinical predictors and trends of long-term remission in a cohort of adults with mild to severe asthma. Methods:This study included 203 adults with mild to severe asthma from the All Age Asthma Cohort, followed over 6 years. Participants attended 5 visits, during which type 2 inflammation markers (blood and sputum eosinophils, fractional exhaled nitric oxide), lung function measurements (oscillometry, spirometry), atopy and systemic comorbidities were assessed. Clinical remission was defined by an Asthma Control Test score of ≥20 plus the absence of both severe exacerbations and systemic corticosteroid use in the past 12 months, and normal or stable lung function. Long-term remission was defined as remission lasting at least 3 consecutive years, while short-term remission lasted 1 or 2 consecutive years. Results:The frequencies of long-term, short-term, and no remission were 27%, 34%, and 39%, respectively. 16% of all patients with severe asthma achieved long-term remission, compared to 65% of those with mild-to-moderate disease. Over one-third of all patients never achieved remission and had persistent T2 markers despite high-dose ICS. Predictors of no asthma remission included number of persistent T2-markers (OR:0.26, CI: 0.11, 0.61), frequency dependence of resistance (FDR, R5-R20Hz; OR:0.36, CI: 0.15, 0.82), FEV1/FVC (OR:0.16, CI: 0.06, 0.37), GERD (OR:0.23, CI: 0.1, 0.5), CVD (OR:0.44, CI: 0.22, 0.87), dyslipidemia (OR:0.38, CI: 0.13, 1.05), whereas sensitization to house dust mite was associated with a higher remission rate (OR:2.06, CI: 1.03, 4.17). During long-term follow-up, significant adjusted predictors of no remission were sputum eosinophils, small airway dysfunction, and airflow obstruction. Conclusion:This study highlights a substantial unmet need in achieving long-term remission, particularly in patients with persistent type 2 inflammation and impaired lung function, prompting re-evaluation of targeting T2 inflammation earlier to prevent lung function impairment.

Long-term exposure to noxious gases and particulate matter from cigarette smoke (CS) or air pollution is a major cause of chronic obstructive pulmonary disease (COPD). However, only a portion of smokers develop the disease. Besides, COPD progression trajectories vary among patients. The molecular mechanisms underlying susceptibility to COPD development and progression remain largely undefined. To study the mechanisms of resistance against CS, an in vitro model of a CS-resistant cell line was recently established by our laboratory. Transcriptomic profiles between resistant and control cells were analysed and compared. Human lung samples were obtained to verify the findings from our in vitro model. Following our previous work, further studies on protein homeostasis were conducted using inhibitors of protein synthesis and degradation. Resistant phenotype was conferred by an enhanced heme oxygenase-1 (HO-1)-mediated antioxidant defence. Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) was identified as a novel repressor of HO-1. Moreover, adeno-associated virus (AAV)-mediated CEACAM6 overexpression sensitised precision-cut lung slices (PCLS) from healthy donor lungs to CS-induced damage. Strikingly, HO-1 was regulated at post-transcriptional level in the resistant cells, accompanied by distinct patterns of HO-1 protein synthesis and degradation. Our research takes a significant step toward unravelling the complex mechanisms of CS-induced chronic lung diseases. The discovery of CEACAM6, and new insights into the post-transcriptional regulation of HO-1 protein in CS-resistant cells may lay the groundwork for further therapeutic strategies tailored to susceptible individuals.

Abstract Background Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory condition. Given patients with COPD continue to experience exacerbations despite the availability of effective therapies, anti-inflammatory treatments targeting novel pathways are needed. Kinases, notably the phosphoinositide 3-kinases (PI3K), are thought to be involved in chronic airway inflammation, with this pathway proposed as a critical regulator of inflammation and oxidative stress response in COPD. CHF6523 is an inhaled PI3Kδ inhibitor that has shown positive preclinical results. This manuscript reports the results of a study of CHF6523 in patients with stable COPD (chronic bronchitis phenotype), and who had evidence of type-2 inflammation. Methods This randomised, double-blind, placebo-controlled, two-way crossover study comprised two 28-day treatment periods separated by a 28-day washout. Patients (N = 44) inhaled CHF6523 in one period, and placebo in the other, both twice daily. The primary objective was to assess the safety and tolerability of CHF6523; the secondary objective was to assess CHF6523 pharmacokinetics. Exploratory endpoints included target engagement (the relative reduction in phosphatidylinositol (3,4,5)-trisphosphate [PIP3]), pharmacodynamic evaluations such as airflow obstruction, and hyperinflation, and to identify biomarker(s) of drug response using proteomics and transcriptomics. Results CHF6523 plasma pharmacokinetics were characterised by an early maximum concentration (Cmax), reached 15 and 10 min after dosing on Days 1 and 28, respectively, followed by a rapid decline. Systemic exposure on Day 28 showed limited accumulation, with ratios < 1.6 for Cmax and area under the curve from 0 to 12 h post-dose, and with steady state achieved on Day 20. Target engagement was confirmed by a significant 29.7% reduction from baseline in induced sputum PIP3 (29.5% reduction vs. placebo; adjusted ratio 0.705 [0.580, 0.856]; p = 0.001), but this did not translate into an anti-inflammatory pharmacodynamic effect, as assessed through measures including biomarkers and multi-omics. Additionally, although CHF6523 was generally well-tolerated, 95.2% of patients reported cough as an adverse event, most mild to moderate and resolving within one-hour post-dose. Conclusions These data, together with those from other PI3K inhibitors, suggest that PI3Kδ is not a suitable pathway for the management of COPD, as the achieved target engagement did not translate into any pharmacodynamic anti-inflammatory effect. Trial registration ClinicalTrials.gov (NCT04032535); posted 23rd July 2019.

The proteasome, the main enzymatic complex for degradation of intracellular proteins, is emerging as a potential biomarker for chronic inflammatory diseases. Its activity is impaired by cigarette smoke and in lung tissue of COPD patients. Population-based studies focusing on proteasome profiling from peripheral blood mononuclear cells (PBMC) for stratifying COPD severity and progression are missing. We profiled proteasome activity and expression of COSYCONET patients and validate proteasome function as a biomarker for COPD severity and endotypes. PBMCs were obtained from 378 COSYCONET patients. Activity-based probes were used to quantify total proteasome activity and the six distinct standard (β1, β2, β5) and immunoproteasome (LMP2, MECL1, LMP7) activities. Relative RNA expression of multiple proteasome subunits (PSMA3, PSMB[5-10], PSMC3, PSMD11, PSME1) was determined by RTqPCR. Linear regression was used to analyze the association of COPD stages and BODE index with proteasome function. The activity ratio of the catalytic subunits LMP7 and β5 significantly decreased with increasing BODE index among females (%difference -48.5% (95%CI -70.4; -10.1) for index 1-2 and -46.5% (-70.0; -4.8) for index ≥3 compared to index 0) but not among males. These changes in activity levels were not accompanied by significantly altered expression levels of the subunits PSMB5 (encoding β5) and PSMB8 (encoding LMP7). Non-transcriptional mechanisms are associated with the downregulation of proteasome activity in COPD. Further proteasome function analyses of healthy subjects of the KORA cohort are in preparation to provide insight in proteasome activity and expression patterns among COPD patients compared to lung healthy controls.

Rationale: Tobacco smoking and air pollution are primary causes of chronic obstructive pulmonary disease (COPD). However, only a minority of smokers develop COPD. The mechanisms underlying the defense against nitrosative/oxidative stress in nonsusceptible smokers to COPD remain largely unresolved. Objectives: To investigate the defense mechanisms against nitrosative/oxidative stress that possibly prevent COPD development or progression. Methods: Four cohorts were investigated: 1) sputum samples (healthy, n = 4; COPD, n = 37), 2) lung tissue samples (healthy, n = 13; smokers without COPD, n = 10; smoker1COPD, n = 17), 3) pulmonary lobectomy tissue samples (no/mild emphysema, n = 6), and 4) blood samples (healthy, n = 6; COPD, n = 18). We screened 3-nitrotyrosine (3-NT) levels, as indication of nitrosative/oxidative stress, in human samples. We established a novel in vitro model of a cigarette smoke extract (CSE)-resistant cell line and studied 3-NT formation, antioxidant capacity, and transcriptomic profiles. Results were validated in lung tissue, isolated primary cells, and an ex vivo model using adeno-associated virus-mediated gene transduction and human precision-cut lung slices. Measurements and Main Results: 3-NT levels correlate with COPD severity of patients. In CSE-resistant cells, nitrosative/oxidative stress upon CSE treatment was attenuated, paralleled by profound upregulation of heme oxygenase-1 (HO-1). We identified carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) as a negative regulator of HO-1-mediated nitrosative/oxidative stress defense in human alveolar type 2 epithelial cells (hAEC2s). Consistently, inhibition of HO-1 activity in hAEC2s increased the susceptibility toward CSE-induced damage. Epithelium-specific CEACAM6 overexpression increased nitrosative/oxidative stress and cell death in human precision-cut lung slices on CSE treatment. Conclusions: CEACAM6 expression determines the hAEC2 sensitivity to nitrosative/oxidative stress triggering emphysema development/progression in susceptible smokers.

RATIONALE AND OBJECTIVE:Plasma extracellular vesicles (EVs) represent a vital source of molecular information about health and disease states. Due to their heterogenous cellular sources, EVs and their cargo may predict specific pathomechanisms behind disease phenotypes. Here we aimed to utilize EV microRNA (miRNA) signatures to gain new insights into underlying molecular mechanisms of obesity-associated low type-2 asthma. METHODS:Obese low type-2 asthma (OA) and non-obese low type-2 asthma (NOA) patients were selected from an asthma cohort conjointly with healthy controls. Plasma EVs were isolated and characterised by nanoparticle tracking analysis. EV-associated small RNAs were extracted, sequenced and bioinformatically analysed. RESULTS:Based on EV miRNA expression profiles, a clear distinction between the three study groups could be established using a principal component analysis. Integrative pathway analysis of potential target genes of the differentially expressed miRNAs revealed inflammatory cytokines (e.g., interleukin-6, transforming growth factor-beta, interferons) and metabolic factors (e.g., insulin, leptin) signalling pathways to be specifically associated with OA. The miR-17-92 and miR-106a-363 clusters were significantly enriched only in OA. These miRNA clusters exhibited discrete bivariate correlations with several key laboratory (e.g., C-reactive protein) and lung function parameters. Plasma EV miRNA signatures mirrored blood-derived CD4+ T-cell transcriptome data, but achieved an even higher sensitivity in identifying specifically affected biological pathways. CONCLUSION:The identified plasma EV miRNA signatures and particularly the miR-17-92 and -106a-363 clusters were capable to disentangle specific mechanisms of the obesity-associated low type-2 asthma phenotype, which may serve as basis for stratified treatment development.

Plasma extracellular vesicles (EVs) and their cargo represent a vital source of molecular information about health and disease states and may predict specific pathomechanisms behind disease phenotypes. Here we aimed to utilize EV microRNA (miRNA) signatures to gain new insights into underlying molecular mechanisms of obesity-associated low type-2 asthma. Ten obese (OA) and ten non-obese low type-2 asthma (NOA) patients were selected from an asthma cohort alongside with ten healthy controls. Plasma EVs were isolated and characterised by nanoparticle tracking analysis, EV-associated small RNAs were extracted, sequenced and bioinformatically analysed. Based on EV miRNA expression profiles a clear distinction could be established between the three study groups by a principal component analysis. An integrative pathway analysis of potential target genes of the differentially present miRNAs revealed inflammatory cytokine (e.g., interleukin-6, transforming growth factor-beta, interferons) and metabolic factors (e.g., insulin, leptin) signalling pathways to be specifically associated with OA. The miR-17~92 and miR-106a~363 clusters were significantly enriched only in OA. These miRNA clusters exhibited discrete bivariate correlations with several key laboratory and lung function parameters. Further, plasma EVs miRNA signatures mirrored blood-derived CD4+ T-cells transcriptome data with even higher sensitivity in identifying specifically affected biological pathways. Thus, the identified plasma EV miRNA signatures and particularly the mentioned miRNA clusters were specifically linked to pathomechanims of obesity-associated low type-2 asthma.

Background: Airway eosinophilic inflammation is usually linked to recurrent exacerbations in COPD. However, little is known about its role in SAD in patients with mild or at risk for COPD. We aimed to describe the association between airway eosinophilic inflammation and SAD in these patients. Methods: We induced sputum in 45 patients with mild stable COPD (post FEV1/FVC<70% + FEV1≥70%pred) and in 24 smokers at risk (post FEV1/FVC≥70%, ≥10py and CAT ≥10 or long-acting bronchodilator) from CAPTO-COPD. Lung function included spirometry, body plethysmography and SF6 multiple breath washout (MBW). Patients were stratified based on sputum eosinophil count in eosinophil-high (≥2%) and eosinophil-low. Results: 19% of patients (8 COPD, 5 at risk) were eosinophil-high (mean eosinophil count: 5.6± 6.0%). Markers of SAD indicated increased acinar (Sacin 0.66±0.51 vs 0.38±0.27), global (LCI 10.6±1.2 vs 9.5±1.5) ventilation inhomogeneity and air trapping (RV/TLC 48±7 vs 41±6%) in eosinophil-high patients. Age (64±8 vs 65±8y), smoking (43±16 vs 45±25 py), airway neutrophilia (72±11 vs 70±17%) and airflow obstruction (FEV1/FVC 58±9 vs 62±8%, FEV1 75±12 vs 83±13%pred) did not differ between both groups. Significant correlation between airway eosinophils and SAD (r=0.48 to 0.30, all p<0.05) was found while no correlation between eosinophils and airflow obstruction was be established. Conclusion: In a subgroup of smokers at risk or patients with mild stable COPD, eosinophilic airway inflammation was associated with more severe SAD. Interventional trials are needed to demonstrate the benefit of early targeting airway eosinophilia on disease progression.

BACKGROUND:Comprehensive studies investigated the role of T-cells in asthma which led to personalised treatment options targeting severe eosinophilic asthma. However, little is known about the contribution of B-cells to this chronic inflammatory disease. In this study we investigated the contribution of various B-cell populations to specific clinical features in asthma. METHODS:In the All Age Asthma Cohort (ALLIANCE), a subgroup of 154 adult asthma patients and 28 healthy controls were included for B-cell characterisation by flow cytometry. Questionnaires, lung function measurements, blood differential counts and allergy testing of participants were analysed together with comprehensive data on B-cells using association studies and multivariate linear models. RESULTS:Patients with severe asthma showed decreased immature B-cell populations while memory B-cells were significantly increased compared with both mild-moderate asthma patients and healthy controls. Furthermore, increased frequencies of IgA+ memory B-cells were associated with impaired lung function and specifically with parameters indicative for augmented resistance in the peripheral airways. Accordingly, asthma patients with small airway dysfunction (SAD) defined by impulse oscillometry showed increased frequencies of IgA+ memory B-cells, particularly in patients with mild-moderate asthma. Additionally, IgA+ memory B-cells significantly correlated with clinical features of SAD such as exacerbations. CONCLUSIONS:With this study we demonstrate for the first time a significant association of increased IgA+ memory B-cells with asthma and SAD, pointing towards future options for B-cell-directed strategies in preventing and treating asthma.

Mustafa Abdo,1 Frauke Pedersen,1,2 Frederik Trinkmann,3,4 Felix JF Herth,3 Klaus F Rabe,1 Anne-Marie Kirsten,2 Henrik Watz2 1LungenClinic Grosshansdorf, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Grosshansdorf, Germany; 2Pulmonary Research Institute at LungenClinic Grosshansdorf, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Grosshansdorf, Germany; 3Pneumology and Critical Care Medicine, Thoraxklinik at University Hospital Heidelberg, Translational Lung Research Center Heidelberg (TLRC), Member of German Center for Lung Research (DZL), Heidelberg, Germany; 4Department of Biomedical Informatics (DBMI) at the Center for Preventive Medicine and Digital Health Baden-Württemberg (CPD-BW), University Medical Center Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, GermanyCorrespondence: Mustafa Abdo, LungenClinic Grosshansdorf, Airway Research Center North (ARCN), German Center for Lung Research (DZL), Wöhrendamm 80, Grosshansdorf, 22927, Germany, Email m.abdo@lungenclinic.de

Hintergrund Die eosinophile Atemwegsentzündung ist bei COPD mit der Exazerbationsfrequenz verbunden. Unklar ist derzeit jedoch, inwieweit eine eosinophile Atemwegsentzündung für die Dysfunktion der kleinen Atemwege (small airway disease, SAD) bei leichter COPD und symptomatischen (ex)-Rauchern von Bedeutung ist.

BACKGROUND:Little is known about the relationship between airway inflammatory phenotypes and some important asthma features such as small airway dysfunction (SAD). OBJECTIVE:To describe the longitudinal impact of airway inflammatory phenotypes on SAD and asthma outcomes. METHODS:We measured eosinophil and neutrophil counts in induced sputum at baseline and 1 year later to stratify 197 adult patients with asthma into 4 inflammatory phenotypes. We conducted a comprehensive assessment of lung function using spirometry, body plethysmography, impulse oscillometry, and inert gas single and multiple breath washouts. We compared lung function, asthma severity, exacerbation frequency, and symptom control between the phenotypes. We studied the longitudinal impact of persistent sputum inflammatory phenotypes and the change of sputum cell counts on lung function. RESULTS:Patients were stratified into eosinophilic (23%, n = 45), neutrophilic (33%, n = 62), mixed granulocytic (22%, n = 43), and paucigranulocytic (24%, n = 47) phenotypes. Patients with eosinophilic and mixed granulocytic asthma had higher rates of airflow obstruction and severe exacerbation as well as poorer symptom control than patients with paucigranulocytic asthma. All SAD measures were worse in patients with eosinophilic and mixed asthma than in those with paucigranulocytic asthma (all P values <.05). Eosinophilic asthma also indicated worse distal airflow obstruction, increased ventilation inhomogeneity (all P values <.05), and higher tendency for severe exacerbation (P = .07) than neutrophilic asthma. Longitudinally, persistent mixed granulocytic asthma was associated with the worst follow-up measures of SAD compared with persistent neutrophilic, persistent paucigranulocytic, or nonpersistent asthma phenotypes. In patients with stable forced expiratory volume in 1 second (FEV1), the mean increase in small airway resistance (R5-20) was greater in patients with persistent mixed granulocytic asthma (+103%) than in patients with persistent neutrophilic (+26%), P = .040, or persistent paucigranulocytic asthma (-41%), P = .028. Multivariate models adjusted for confounders and treatment with inhaled or oral corticosteroids or antieosinophilic biologics indicated that the change of sputum eosinophil rather than neutrophil counts is an independent predictor for the longitudinal change in FEV1, forced expiratory flow at 25% to 75% of forced vital capacity, specific effective airway resistance, residual lung volume, and lung clearance index. CONCLUSIONS:In asthma, airway eosinophilic inflammation is the main driver of lung function impairment and poor disease outcomes, which might also be aggravated by the coexistence of airway neutrophilia to confer a severe mixed granulocytic asthma phenotype. Persistent airway eosinophilia might be associated with dynamic SAD even in patients with stable FEV1.

Asthma is a widespread and chronic multifactorial disease, characterized by impaired lung function with airway hyperreactivity, obstruction and inflammation of the central and peripheral airways. An improved knowledge of disease phenotypes and endotypes paved the way for personalized therapeutic approaches, especially directed against T helper 2 cytokines. While asthma research mainly focused on T cells, very little is known about the influence of B cells. To better understand their role, we analysed various B cell subsets in regards to specific clinical traits in asthma. Circulating B cells of 154 adult asthma patients and 28 healthy controls of the All Age Asthma Cohort (ALLIANCE) were characterized by flow cytometry. Comprehensive clinical phenotyping by blood and sputum inflammation, lung function measurements, allergy testing, and questionnaires was analysed together with extensive data on B cells by association tests and multivariate linear models. Frequencies of immature B cells were decreased in severe asthma, while frequencies of memory B cells were elevated. In addition, increased relative numbers of immunoglobulin A positive (IgA+) memory B cells were associated with decreased lung function, especially with measures reflecting increased narrowing of the small airways. Thus, patients with asthma and small airway dysfunction (SAD), determined by impulse oscillometry parameter R5-R20, had elevated numbers of IgA+ memory B cells, which also correlated with severe exacerbations and impaired asthma control. We show for the first time a connection between IgA+ memory B cells, asthma and SAD, pointing towards a potential diagnostic option for IgA+ B cells as biomarker for SAD in asthma.