BACKGROUND:Dipeptidyl peptidase-1 (DPP1) inhibitors prevent the activation of neutrophil serine proteases and reduce exacerbations in people with bronchiectasis. We previously identified a novel effect of DPP1 inhibitors in reducing the neutrophil pseudoenzyme azurocidin-1 (AZU1). The aim of this study was to investigate the role of AZU1 in the pathophysiology of bronchiectasis. METHODS:Sputum AZU1 concentrations were analysed in multiple cohorts. These consisted of two observational cohorts of patients with bronchiectasis (EMBARC BRIDGE cohort 1 and cohort 2) and a cohort of patients with chronic obstructive pulmonary disease (COPD; TARDIS COPD cohort) to correlate AZU1 with disease severity and exacerbations. A rhinovirus challenge study was used to investigate AZU1 concentrations during experimental exacerbation in COPD, people who smoke, and controls. A post-hoc analysis of the phase 2 WILLOW trial of brensocatib versus placebo was used to assess the effect of DPP1 inhibition on airway AZU1. FINDINGS:Higher AZU1 sputum concentration was associated with increased bronchiectasis disease severity index (p<0·0001), decreased percentage predicted forced expiratory volume in 1 second (r=-0·4662, p<0·001), and increased exacerbation frequency (p<0·0019; EMBARC cohort 1, n=197). AZU1 was associated with radiological severity (Reiff score), symptoms (quality of life bronchiectasis respiratory symptom score), and bacterial infection (sputum microbiology and 16S microbiome alpha diversity; highest levels of AZU1 were found in airway samples with Pseudomonas aeruginosa; p<0·0001; EMBARC cohort 2, n=144). Bronchiectasis patients with bacterial and viral exacerbations had increased concentrations of AZU1 (p=0·0003; n=96). These findings were extended to COPD, in which AZU1 was related to COPD severity (COPD cohort, n=101), and in patients with COPD challenged with rhinovirus A16, AZU1 was increased at day 9 post-challenge (p<0·001; n=9). In-vitro AZU1 impaired ciliary function and epithelial integrity, suggesting a mechanism by which AZU1 drives disease pathogenesis. In a post-hoc analysis of the WILLOW trial, AZU1 was the most downregulated protein with brensocatib treatment (brensocatib 10 mg, n=71; brensocatib 25 mg, n=73; and placebo, n=71). Over 24 weeks, AZU1 was significantly reduced by DPP1 inhibition (p<0·0001). INTERPRETATION:AZU1 was identified as a novel marker of disease severity in bronchiectasis, associated with bacterial infection and exacerbation, and targeted by DPP1 inhibition. FUNDING:EMBARC3 and Insmed.

BACKGROUND:Bronchiectasis and diabetes commonly coexist and are associated with immune dysfunction and increased susceptibility to infection. Although diabetes is associated with worse prognosis in cystic fibrosis-related bronchiectasis, data are scarce for its impact on non-cystic fibrosis bronchiectasis. This study aimed to characterise the impact of diabetes on clinical outcomes and microbial and inflammatory profiles in patients with bronchiectasis. METHODS:This analysis comprised data from the European Bronchiectasis Registry (EMBARC), Respiratory Research Network of India (EMBARC-India), Chinese Bronchiectasis Registry (BE-China), and Australian Bronchiectasis Registry (ABR); 30 263 patients with CT-confirmed bronchiectasis in 33 countries were included in the analysis: 16 963 from EMBARC (Jan 12, 2015, to April 12, 2022), 2361 from EMBARC-India plus additional Asian countries (June 1, 2015, to Sept 1, 2017), 10 324 from BE-China (Jan 10, 2020, to March 31, 2024), and 615 from the ABR (March 7, 2016, to Sept 11, 2018). Clinical data were compared between patients with and without diabetes. Long-term outcome data were available in EMBARC and EMBARC-India. Microbiome and inflammatory profiles were characterised in a sub-cohort of EMBARC patients by sputum 16S rRNA sequencing (n=433) and serum Olink (n=479). FINDINGS:2487 (8·2%) of 30 263 patients with bronchiectasis had diabetes. Patients with diabetes had a higher prevalence of comorbidities than those without diabetes, including cardiovascular disorders (53·5% vs 21·8%, p<0·0001), asthma (27·5% vs 21·0%, p<0·0001), and chronic obstructive pulmonary disease (34·3% vs 19·0%, p<0·0001). Patients with diabetes had more severe disease than those without diabetes, with higher Bronchiectasis Severity Index scores (8 [IQR 5-12] vs 7 [4-10], p<0·0001) and UK Medical Research Council (MRC) dyspnoea scores (p<0·0001) and more hospital admissions in the previous year (p<0·0001). After adjustment for confounders, outcomes were significantly worse in patients with diabetes than in those without diabetes, including more frequent exacerbations (incidence rate ratio [IRR] 1·18 [95% CI 1·09-1·28], p<0·0001), hospital admissions (IRR 1·57 [1·40-1·76], p<0·0001), and higher 5-year mortality (hazard ratio 1·80 [1·53-2·12], p<0·0001). The sputum microbiome was significantly altered in patients with diabetes compared to those without diabetes, with increased isolation of Enterobacteriaceae (p<0·0001), Moraxella catarrhalis (p=0·0035), and Haemophilus influenzae (p=0·046). In serum, Gal-4 and GDF-15, established biomarkers of disease severity and cardiovascular risk in diabetes, were significantly increased in patients with diabetes (Gal-4, p<0·0001; GDF-15, p=0·0019). INTERPRETATION:Patients with diabetes and bronchiectasis are a high-risk population with more severe disease, worse outcomes, increased comorbidities, and increased risk of infections compared with patients without diabetes. These findings support inclusion of diabetes as a risk factor in individualised risk assessments for bronchiectasis. FUNDING:European Respiratory Society, Armata, AstraZeneca, Boehringer Ingelheim, Chiesi, CSL Behring, GSK, Grifols, Insmed, Janssen, Lifearc, Roche, Verona Pharma, Zambon, National Natural Science Foundation of China, Innovation Program of the Shanghai Municipal Education Commission, Program of the Shanghai Municipal Science and Technology Commission, Program of the Shanghai Shenkang Development Center, EU/European Federation of Pharmaceutical Industries and Associations, Innovative Medicines Initiative, and Inhaled Antibiotics in Bronchiectasis and Cystic Fibrosis Consortium.

BACKGROUND:There is substantial overlap between features of COPD, asthma, bronchiectasis, and cystic fibrosis (CF). Each is characterized by inflammation and mucociliary dysfunction. RESEARCH QUESTION:Is there a relationship between inflammation and mucociliary clearance in chronic respiratory conditions, and can biology, rather than disease labels, stratify patients into therapeutically relevant subtypes? STUDY DESIGN AND METHODS:Patients were categorized according to primary disease and clinical characteristics recorded. Spontaneous sputum was collected, and inflammatory markers (neutrophil elastase and 18 cytokines), sputum properties (DNA content, mucins, rheology, dry weight), and microbiome (long-read 16S sequencing) were measured. K-means clustering was performed and parameters compared between and within disease groups. Control participants were individuals who had formerly smoked but were without respiratory disease. RESULTS:The study included patients with asthma (n = 76), COPD (n = 91), bronchiectasis (n = 54), CF (n = 24), and control participants (n = 26). Nine cytokines (interferon-γ, IL-4, IL-5, eotaxin, eotaxin-3, thymus and activation regulated chemokine, granulocyte colony-stimulating factor, fractalkine, IL-22), neutrophil elastase, dry weight, mucins, and sputum rheology parameters were significantly different between disease groups and control participants (P < .05). K-means clustering identified 2 clusters defined by neutrophilic or T helper 2 (Th2) inflammation. The Th2 cluster was associated with lower sputum dry weight and DNA content and higher mucin-5B. Rheological parameters G', G'', and G∗ were significantly higher in the Th2 group, whereas the tangent of the loss angle δ was higher in the neutrophilic group, indicating a higher viscous to elastic ratio (P < .05 all comparisons). The neutrophilic cluster was associated with decreased alpha diversity (P = .04) and increased presence of Proteobacteria in their sputum microbiome compared with the Th2 cluster (P = .01). More neutrophilic inflammation was present in CF and bronchiectasis (42% of patients with COPD and 46% of patients with asthma were neutrophilic vs 78% of bronchiectasis and 87% of CF (P < .0001). Both clusters were present in all disease groups. INTERPRETATION:Our results indicate that airway diseases have heterogeneous mucus properties. Patients were shown to cluster according to inflammatory endotype rather than disease label. Assessment based on disease labels may be aided by endotyping using inflammatory and mucociliary clearance biomarkers.

BACKGROUND:The microbiome is associated with exacerbation risk, quality of life and mortality in COPD. Inhaled corticosteroid (ICS) treatment has been reported to alter the microbiome through modulating host defence. How ICS alters the microbiome and whether effects are equal between different ICS preparations is debated. The aim of the MUSIC trial was to investigate whether commonly used ICS therapies have different effects on the airway microbiome in COPD. METHODS:This was a multicentre randomised controlled trial. After a 4-week washout period during which they withdrew from ICS, patients with COPD (forced expiratory volume in 1 s <50% predicted at baseline and/or a history of two or more exacerbations per year) were randomised to one of four treatments (budesonide/formoterol 400/12 µg (BF400), fluticasone/salmeterol 500/50 µg (FS500), fluticasone/salmeterol 250/50 µg (FS250) or aclidinium/formoterol 340/12 µg, twice daily). Patients were followed-up for 3 months with monthly induced sputum, oropharyngeal and nasopharyngeal swabs for bacterial load and 16S rRNA sequencing to characterise the microbiome. Inflammatory markers were measured in sputum and blood. The primary outcome was bacterial load in oropharyngeal swabs comparing BF400 versus FS500, with sputum bacterial load the key secondary end-point. RESULTS:122 participants started the washout period. ICS withdrawal was poorly tolerated; 61 participants withdrew before randomisation with 45 experiencing an exacerbation. 61 patients were randomised. No statistically significant differences were observed for the primary comparison of BF400 versus FS500 in oropharyngeal bacterial load. There was, however, a significant increase in sputum bacterial load with FS500 compared to BF400 by month 3. This difference was not seen with FS250. No significant differences in microbiome α-diversity were observed over time. Adverse events were similar between the groups. CONCLUSION:FS500 increased sputum but not upper airway bacterial loads. ICS withdrawal was poorly tolerated in severe COPD.

Rationale The inflammasome is a key regulatory complex of the inflammatory response leading to interleukin-1[3 [3 (IL-1[3) [3 ) release and activation. IL-1[3 [3 amplifies inflammatory responses and induces mucus secretion and hyperconcentration in other diseases. The role of IL-1[3 [3 in bronchiectasis has not been investigated. Objectives To characterise the role of airway IL-1[3 [3 in bronchiectasis, including the association with mucus properties, ciliary function, airway inflammation, microbiome and disease severity. Methods Stable bronchiectasis patients were enrolled in an international cohort study (n=269). IL-1[3 [3 was measured in sputum supernatant. A validation cohort also had sputum rheology and hydration measured (n=53). For analysis, patients were stratified according to the median value of IL-1[3 [3 in the population (high versus low) to compare disease severity, airway infection, microbiome (16S rRNA sequencing), inflammation and caspase-1 activity. Primary human nasal epithelial cells grown in air-liquid - liquid interface culture were used to study the effect of IL-1[3 [3 on cilia function. Results Patients with high sputum IL-1[3 [3 had more severe disease, increased caspase-1 activity and an increased T-helper type 1, T-helper type 2 and neutrophil inflammatory response compared with patients with low IL-1[3. [3 . The active-dominant form of IL-1[3 [3 was associated with increased disease severity. High IL-1[3 [3 was related to higher relative abundance of Proteobacteria in the microbiome and increased mucus solid content and viscoelastic properties. Chronic IL-1[3 [3 treatment reduced the functionality of cilia and tight junctions of epithelial cells in vitro. Conclusions A subset of stable bronchiectasis patients show increased airway IL-1[3, [3 , suggesting pulmonary inflammasome activation is linked with more severe disease, airway infection, mucus dehydration and epithelial dysfunction.

Introduction: Bronchiectasis (BE) and chronic obstructive pulmonary disease (COPD) share similar clinical characteristics and are frequently co-diagnosed (BE-COPD). BE-COPD is associated with worse outcomes but the mechanisms of this are unknown. Aim: To investigate differences in the lung microbiome in BE and BE-COPD overlap. Methods: Stable BE and BE-COPD patients were enrolled from a single UK centre. BE-COPD was defined using the objective ROSE criteria. The sputum microbiome was evaluated using 16S rRNA sequencing. Alpha-diversity was analysed using Shannon Wiener (SWDI), Chao1 (CI) and Simpson Index (SI). Results were validated in the EMBARC-BRIDGE cohort from UK, Italy and Spain. Results: 281 patients were enrolled (BE n=176, BE-COPD n=105), 52.3% female, age 68 (±12.6). Proteobacteria were the most abundant phyla, and Haemophilus and Streptococcus the most abundant genera. Alpha diversity was significantly lower in BE-COPD group (SWDI p=0.02, CI p=0.04, SI p=0.03). No difference in beta-diversity was seen between the groups (PERMANOVA, p=0.33). Random Forest analysis identified reduced commensal taxa in the BE-COPD group, including lower Neisseria, Prevotella, Campylobacter and Fusobacterium. 208 patients were enrolled in the EMBARC BRIDGE cohort (BE=147, BE-COPD=61). Alpha diversity was reduced in the BE-COPD group (SWDI p=0.06, CI p=0.02, SI p=0.03) and there were significant changes in beta-diversity (PERMANOVA, p=0.02). Similarly, Random Forest detected depletion of Neisseria, Prevotella, Campylobacter in the BE-COPD arm. Conclusion: We demonstrate in two large cohorts that BE-COPD is associated with reduced microbial diversity and depletion of anti-inflammatory commensal taxa.

Introduction: Inflammation is believed to be central to the pathophysiology of bronchiectasis. This study aimed to perform inflammatory endotyping in bronchiectasis and examine the validity of identified inflammatory clusters by comparing the microbiome profiles and exacerbation risk among endotypes of bronchiectasis. Methods: Patients with stable bronchiectasis were enrolled at three European centres. K-means cluster analysis was used to stratify the patients according to the levels of 33 sputum and serum inflammatory markers. Sputum microbiome composition was determined through 16S rRNA amplicon sequencing. Endotypes were compared for their risk of exacerbations over 12 months follow-up. Results: 199 patients were enrolled, and using cluster analysis, four endotypes were defined according to their inflammatory profiles: cluster 1 (milder neutrophilic inflammation), cluster 2 (mixed-neutrophilic and type 2 inflammation), cluster 3 (most severe neutrophilic), and cluster 4 (mixed-epithelial and type 2). In the sputum microbiome, Proteobacteria and Pseudomonas at phylum and genus levels, respectively, were more dominant in clusters 2 and 3 than in clusters 1 and 4. Although the four clusters were indistinguishable by clinical characteristics at baseline, patients in clusters 2 (rate ratio [RR] 1.53, 95% CI 1.19–1.97) and 3 (RR 1.46, 95% CI 1.02–2.09) were at higher risk of exacerbation and severe exacerbation over 12 months follow-up compared to cluster 1. Conclusion: Bronchiectasis inflammatory endotypes are associated with distinct microbiome profiles and future exacerbation risk.

Introduction: Adverse effects of inhaled corticosteroids (ICS), such as pneumonia, are believed to be due to effects on airway bacterial load and the microbiome. Different ICS molecules may have distinct effects in COPD. Objectives: To investigate whether commonly used ICS have different effects on the COPD airway microbiome. Methods: Multicentre randomized controlled trial. After a 4-week washout patients with COPD (FEV1 <50% predicted or ≥2 exacerbations per year) were randomized to one of 4 treatments: budesonide/formoterol 400/12 (BF), fluticasone/salmeterol 500 (FS500), fluticasone/salmeterol 250 (FS250) or aclidinium/formoterol (AF). Patients were treated for 3 months with monthly induced sputum, oropharyngeal (OP) and nasopharyngeal (NP) swabs for bacterial load and 16S sequencing. Inflammatory markers were measured in sputum. The primary outcome was bacterial load in OP swabs between BF and FS500, and the key secondary outcome was sputum bacterial load. Results: Following washout of 122 patients, 61 were randomized. After 3 months treatment there was no significant difference in OP bacterial load between FS500 and BF (difference 0.26 logunits, 95%CI 0.65-1.18, p=0.56), but there was a significant increase in bacterial load in sputum at 3 months comparing FS500 and BF (difference 0.87 logunits, 95%CI 0.29-1.44, p=0.0037). BF treatment did not increase bacterial load compared to AF. There was no significant effect of different ICS on Shannon diversity index or inflammatory biomarkers. Conclusion: Patients randomized to fluticasone/salmeterol at licensed doses had an increased bacterial load in sputum over time compared to budesonide/formoterol.