T6 Sputum proteomics identifies mechanisms of disease severity and treatment response in bronchiectasis

Introduction
Neutrophil extracellular traps (NETs) are a form of antimicrobial defence which have been implicated in multiple inflammatory diseases. This study investigated the role of NETs in bronchiectasis, a neutrophilic disease which lacks therapies that directly target neutrophilic inflammation, through a series of UK and international studies.

Methods
LC/MS proteomics was used to compare protein profiles in sputum between severe and mild bronchiectasis (20 vs 20 patients). Microbiome changes, using 16S rRNA sequencing, clinical characteristics and NET levels using a validated histone-elastase immunoassay were analysed. Results were validated in an independent European cohort. Proteomics was used to identify proteins associated with treatment response of acute exacerbations of bronchiectasis in 20 patients treated with intravenous antibiotics for 14 days. Two studies of long-term macrolide treatment, one in bronchiectasis and a post-hoc analysis of the AMAZES trial in asthma, investigated the effect of macrolide treatment on NETs.

Results
96 proteins were differentially expressed in sputum between severe and mild bronchiectasis, with known NET proteins being the most abundant and discriminating. The relationship between NETs and associated proteins were validated in two independent cohorts, a UK cohort (n=175) and a European cohort (n=275). Sputum NETs were associated with BSI (p < 0.0001), a history of severe exacerbations (p=0.0089), quality of life (p < 0.0001), time to first exacerbation (p < 0.0001) and mortality (p=0.009). High NET levels were associated with reduced microbial alpha-diversity, measured by Shannon-Weiner, and microbial dysbiosis (p < 0.0001, PERMANOVA) and elevated IL-8, IL-1beta, TNF-alpha, interferon-gamma and GM-CSF in sputum. Antibiotic treatment (n=20) significantly altered the expression of 39 sputum proteins, with the ‘neutrophil degranulation’ pathway being most strongly implicated in response. Patients with P. aeruginosa infection had a blunted proteomic and clinical response to treatment. Treatment with azithromycin was associated with a significant reduction in sputum NETs over 12 months in both bronchiectasis (n=52, p=0.016) and asthma (n=47, p < 0.0001).

Conclusion
NET-associated proteins are elevated in bronchiectasis sputum and are associated with disease severity, bacterial infection and mortality. Treatment response is linked to successfully reducing NET levels with intravenous antibiotic or macrolide therapies suggesting that NETs may be an important therapeutic target in bronchiectasis.