Endotypes of Exacerbation in Bronchiectasis: An Observational Cohort Study.

Rationale: Bronchiectasis is characterized by acute exacerbations, but the biological mechanisms underlying these events are poorly characterized. Objectives: To investigate the inflammatory and microbial characteristics of exacerbations of bronchiectasis. Methods: A total of 120 patients with bronchiectasis were enrolled and presented with acute exacerbations within 12 months. Spontaneous sputum samples were obtained during a period of clinical stability and again at exacerbation before receipt of antibiotic treatment. A validated rapid PCR assay for bacteria and viruses was used to classify exacerbations as bacterial, viral, or both. Sputum inflammatory assessments included label-free liquid chromatography-tandem mass spectrometry and measurement of sputum cytokines and neutrophil elastase activity. 16 s rRNA sequencing was used to characterize the microbiome. Measurements and Main Results: Bronchiectasis exacerbations showed profound molecular heterogeneity. At least one bacterium was identified in 103 samples (86%), and a high bacterial load (total bacterial load > 107 copies/g) was observed in 81 patients (68%). Respiratory viruses were identified in 55 (46%) patients, with rhinovirus being the most common virus (31%). PCR testing was more sensitive than culture. No consistent change in the microbiome was observed at exacerbation. Exacerbations were associated with increased neutrophil elastase, proteinase-3, IL-1ß, and CXCL8. These markers were particularly associated with bacterial and bacterial plus viral exacerbations. Distinct inflammatory and microbiome profiles were seen between different exacerbation subtypes, including bacterial, viral, and eosinophilic events in both hypothesis-led and hypothesis-free analysis using integrated microbiome and proteomics, demonstrating four subtypes of exacerbation. Conclusions: Bronchiectasis exacerbations are heterogeneous events with contributions from bacteria, viruses, and inflammatory dysregulation.

Airway IL-1ß is related to disease severity and mucociliary function in bronchiectasis.

RATIONALE: The inflammasome is a key regulatory complex of the inflammatory response leading to interleukin-1ß (IL-1ß) release and activation. IL-1ß amplifies inflammatory responses and induces mucus secretion and hyperconcentration in other diseases. The role of IL-1ß in bronchiectasis has not been investigated. OBJECTIVES: To characterise the role of airway IL-1ß 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-1ß 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-1ß 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 interface culture were used to study the effect of IL-1ß on cilia function. RESULTS: Patients with high sputum IL-1ß 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-1ß. The active-dominant form of IL-1ß was associated with increased disease severity. High IL-1ß was related to higher relative abundance of Proteobacteria in the microbiome and increased mucus solid content and viscoelastic properties. Chronic IL-1ß 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-1ß, suggesting pulmonary inflammasome activation is linked with more severe disease, airway infection, mucus dehydration and epithelial dysfunction.

Inflammatory Molecular Endotypes in Bronchiectasis: A European Multicenter Cohort Study.

Rationale: Although inflammation and infection are key disease drivers in bronchiectasis, few studies have integrated host inflammatory and microbiome data to guide precision medicine. Objectives: To identify clusters among patients with bronchiectasis on the basis of inflammatory markers and to assess the association between inflammatory endotypes, microbiome characteristics, and exacerbation risk. Methods: Patients with stable bronchiectasis were enrolled at three European centers, and cluster analysis was used to stratify the patients according to the levels of 33 sputum and serum inflammatory markers. Clusters were compared in terms of microbiome composition (16S ribosomal RNA sequencing) and exacerbation risk over a 12-month follow-up. Measurements and Main Results: A total of 199 patients were enrolled (109 [54.8%] female; median age, 69 yr). Four clusters of patients were defined according to their inflammatory profiles: cluster 1, milder neutrophilic inflammation; cluster 2, mixed-neutrophilic and type 2; cluster 3, most severe neutrophilic; and cluster 4, mixed-epithelial and type 2. Lower microbiome diversity was associated with more severe inflammatory clusters (P < 0.001), and ß-diversity analysis demonstrated distinct microbiome profiles associated with each inflammatory cluster (P = 0.001). Proteobacteria and Pseudomonas at phylum and genus levels, respectively, were more enriched in clusters 2 and 3 than in clusters 1 and 4. Furthermore, patients in cluster 2 (rate ratio [RR], 1.49; 95% confidence interval [CI], 1.16-1.92) and cluster 3 (RR, 1.61; 95% CI, 1.12-2.32) were at higher risk of exacerbation over a 12-month follow-up compared with cluster 1, even after adjustment for prior exacerbation history. Conclusions: Bronchiectasis inflammatory endotypes are associated with distinct microbiome profiles and future exacerbation risk.

Restoring Ciliary Function: Gene Therapeutics for Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) is a genetic disease characterized by defects in motile cilia, which play an important role in several organ systems. Lung disease is a hallmark of PCD, given the essential role of cilia in airway surface defense. Diagnosis of PCD is complicated due to its reliance on complex tests that are not utilized by every clinic and also its phenotypic overlap with several other respiratory diseases. Nonetheless, PCD is increasingly being recognized as more common than once thought. The disease is genetically complex, with several genes reported to be associated with PCD. There is no cure for PCD, but gene therapy remains a promising therapeutic strategy. In this review, we provide an overview of the clinical symptoms, diagnosis, genetics, and current treatment regimens for PCD. We also describe PCD model systems and discuss the therapeutic potential of different gene therapeutics for targeting the intended cellular target, the ciliated cells of the airway.

Endotyping Chronic Obstructive Pulmonary Disease, Bronchiectasis, and the "Chronic Obstructive Pulmonary Disease-Bronchiectasis Association".

Rationale: Bronchiectasis and chronic obstructive pulmonary disease (COPD) are two disease entities with overlapped clinical features, and codiagnosis frequently occurs (termed the "COPD-bronchiectasis association"). Objectives: To investigate the sputum microbiome and proteome in patients with bronchiectasis, COPD, and the COPD-bronchiectasis association with the aim of identifying endotypes that may inform treatment. Methods: Sputum microbiome and protein profiling were carried out using 16S rRNA amplicon sequencing and a label-free proteomics workflow, respectively, in a cohort comprising patients with COPD (n = 43), bronchiectasis (n = 30), and the COPD-bronchiectasis association (n = 48). Results were validated in an independent cohort of 91 patients (n = 28-31 each group) using targeted measurements of inflammatory markers, mucins, and bacterial culture. Measurements and Main Results: Principal component analysis of sputum microbiome and protein profiles showed a partial separation between the COPD and the "COPD-bronchiectasis association" group. Further analyses revealed that patients with the "COPD-bronchiectasis association" had a higher abundance of proteobacteria, higher expression of mucin-5AC and proteins from the "neutrophil degranulation" pathway compared to those with COPD. In contrast, patients with COPD had an elevated expression of mucin-5B and several peptidase inhibitors, higher abundance of common commensal taxa, and a greater microbiome diversity. The profiles of "COPD-bronchiectasis association" and bronchiectasis groups were largely overlapping. Five endotypes were proposed with differential inflammatory, mucin, and microbiological features. The key features related to the "COPD-bronchiectasis association" were validated in an independent cohort. Conclusions: Neutrophilic inflammation, differential mucin expression, and Gram-negative infection are dominant traits in patients with the "COPD-bronchiectasis association."

Characterization of Eosinophilic Bronchiectasis: A European Multicohort Study.

Rationale: Bronchiectasis is classically considered a neutrophilic disorder, but eosinophilic subtypes have recently been described. Objectives: To use multiple datasets available through the European Multicentre Bronchiectasis Audit and Research Collaboration to characterize eosinophilic bronchiectasis as a clinical entity focusing on the impact of eosinophils on bronchiectasis exacerbations. Methods: Patients were included from five countries to examine the relationships between blood eosinophil counts and clinical phenotypes after excluding coexisting asthma. 16S rRNA sequencing was used to examine relationships between eosinophil counts and the sputum microbiome. A post hoc analysis of the PROMIS (Inhaled Promixin in the Treatment of Non-Cystic Fibrosis Bronchiectasis) phase 2 trial was used to examine the impact of blood eosinophil counts on exacerbations in patients with Pseudomonas aeruginosa infection. Measurements and Main Results: A relationship between sputum and blood eosinophil counts was demonstrated in two cohorts. In analysis of 1,007 patients from five countries, 22.6% of patients had blood eosinophil counts of ⩾300 cells/µl. Counts of <100 cells/µl were associated with higher bronchiectasis severity and increased mortality. There was no clear relationship with exacerbations. Blood eosinophil counts of ⩾300 cells/µl were associated with both Streptococcus- and Pseudomonas-dominated microbiome profiles. To investigate the relationship of eosinophil counts with exacerbations after controlling for the confounding effects of infection, 144 patients were studied in a clinical trial after treatment with antipseudomonal antibiotics. Compared with patients with blood eosinophil counts of <100 cells/µl (reference), elevated eosinophil counts of 100-299 cells/µl (hazard ratio, 2.38; 95% confidence interval, 1.33-4.25; P = 0.003) and ⩾300 cells/µl (hazard ratio, 3.99; 95% confidence interval, 2.20-7.85; P < 0.0001) were associated with shorter time to exacerbation. Conclusions: Eosinophilic bronchiectasis affects approximately 20% of patients. After accounting for infection status, raised blood eosinophil counts are associated with shortened time to exacerbation.

Neutrophil extracellular traps, disease severity, and antibiotic response in bronchiectasis: an international, observational, multicohort study.

BACKGROUND: Bronchiectasis is predominantly a neutrophilic inflammatory disease. There are no established therapies that directly target neutrophilic inflammation because little is understood of the underlying mechanisms leading to severe disease. Neutrophil extracellular trap (NET) formation is a method of host defence that has been implicated in multiple inflammatory diseases. We aimed to investigate the role of NETs in disease severity and treatment response in bronchiectasis. METHODS: In this observational study, we did a series of UK and international studies to investigate the role of NETs in disease severity and treatment response in bronchiectasis. First, we used liquid chromatography-tandem mass spectrometry to identify proteomic biomarkers associated with disease severity, defined using the bronchiectasis severity index, in patients with bronchiectasis (n=40) in Dundee, UK. Second, we validated these biomarkers in two cohorts of patients with bronchiectasis, the first comprising 175 patients from the TAYBRIDGE study in the UK and the second comprising 275 patients from the BRIDGE cohort study from centres in Italy, Spain, and UK, using an immunoassay to measure NETs. Third, we investigated whether pathogenic bacteria had a role in NET concentrations in patients with severe bronchiectasis. In a separate study, we enrolled patients with acute exacerbations of bronchiectasis (n=20) in Dundee, treated with intravenous antibiotics for 14 days and proteomics were used to identify proteins associated with treatment response. Findings from this cohort were validated in an independent cohort of patients who were admitted to the same hospital (n=20). Fourth, to assess the potential use of macrolides to reduce NETs in patients with bronchiectasis, we examined two studies of long-term macrolide treatment, one in patients with bronchiectasis (n=52 from the UK) in which patients were given 250 mg of azithromycin three times a week for a year, and a post-hoc analysis of the Australian AMAZES trial in patients with asthma (n=47) who were given 500 mg of azithromycin 3 times per week for a year. FINDINGS: Sputum proteomics identified that NET-associated proteins were the most abundant and were the proteins most strongly associated with disease severity. This finding was validated in two observational cohorts, in which sputum NETs were associated with bronchiectasis severity index, quality of life, future risk of hospital admission, and mortality. In a subgroup of 20 patients with acute exacerbations, clinical response to intravenous antibiotic treatment was associated with successfully reducing NETs in sputum. Patients with Pseudomonas aeruginosa infection had a lessened proteomic and clinical response to intravenous antibiotic treatment compared with those without Pseudomonas infections, but responded to macrolide therapy. Treatment with low dose azithromycin was associated with a significant reduction in NETs in sputum over 12 months in both bronchiectasis and asthma. INTERPRETATION: We identified NETs as a key marker of disease severity and treatment response in bronchiectasis. These data support the concept of targeting neutrophilic inflammation with existing and novel therapies. FUNDING: Scottish Government, British Lung Foundation, and European Multicentre Bronchiectasis Audit and Research Collaboration (EMBARC).

A point-of-care neutrophil elastase activity assay identifies bronchiectasis severity, airway infection and risk of exacerbation.

INTRODUCTION: Neutrophil elastase activity in sputum can identify patients at high risk of airway infection and exacerbations in bronchiectasis. Application of this biomarker in clinical practice is limited, because no point-of-care test is available. We tested whether a novel semi-quantitative lateral flow device (neutrophil elastase airway test stick - NEATstik®) can stratify bronchiectasis patients according to severity, airway infection and exacerbation risk. METHODS: Sputum samples from 124 patients with stable bronchiectasis enrolled in the UK and Spain were tested using the NEATstik®, which scores neutrophil elastase concentration from 0 (<8 µg·mL-1 elastase activity) to 10 (maximum detectable neutrophil elastase activity). High neutrophil elastase activity was regarded as a NEATstik® grade >6. Severity of disease, airway infection from sputum culture and exacerbations over the 12 months were recorded. An independent validation was conducted in 50 patients from Milan, Italy. MEASUREMENTS AND MAIN RESULTS: Patients had a median age of 69 years and forced expiratory volume in 1 s (FEV1) 69%. High neutrophil elastase activity was associated with worse bronchiectasis severity using the bronchiectasis severity index (p=0.0007) and FEV1 (p=0.02). A high NEATstik® grade was associated with a significant increase in exacerbation frequency, incident rate ratio 2.75 (95% CI 1.63-4.64, p<0.001). The median time to next exacerbation for patients with a NEATstik® grade >6 was 103 days compared to 278 days. The hazard ratio was 2.59 (95% CI 1.71-3.94, p<0.001). Results were confirmed in the independent validation cohort. CONCLUSIONS: A novel lateral flow device provides assessment of neutrophil elastase activity from sputum in minutes and identifies patients at increasing risk of airway infection and future exacerbations.