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BackgroundCystic fibrosis (CF) lung disease is defined by large numbers of neutrophils and associated damaging products in the airway. Delayed neutrophil apoptosis is described in CF although it is unclear whether this is a primary neutrophil defect or a response to chronic inflammation. Increased levels of neutrophil extracellular traps (NETs) have been measured in CF and we aimed to investigate the causal relationship between these phenomena and their potential to serve as a driver of inflammation. We hypothesised that the delay in apoptosis in CF is a primary defect and preferentially allows CF neutrophils to form NETs, contributing to inflammation.MethodsBlood neutrophils were isolated from patients with CF, CF pigs and appropriate controls. Neutrophils were also obtained from patients with CF before and after commencing ivacaftor. Apoptosis was assessed by morphology and flow cytometry. NET formation was determined by fluorescent microscopy and DNA release assays. NET interaction with macrophages was examined by measuring cytokine generation with ELISA and qRT-PCR.ResultsCF neutrophils live longer due to decreased apoptosis. This was observed in both cystic fibrosis transmembrane conductance regulator (CFTR) null piglets and patients with CF, and furthermore was reversed by ivacaftor (CFTR potentiator) in patients with gating (G551D) mutations. CF neutrophils formed more NETs and this was reversed by cyclin-dependent kinase inhibitor exposure. NETs provided a proinflammatory stimulus to macrophages, which was enhanced in CF.ConclusionsCF neutrophils have a prosurvival phenotype that is associated with an absence of CFTR function and allows increased NET production, which can in turn induce inflammation. Augmenting neutrophil apoptosis in CF may allow more appropriate neutrophil disposal, decreasing NET formation and thus inflammation.

Introduction Bronchiectasis is associated with a destructive cycle of bacterial infection, airway inflammation and airway structural damage. Ficolins are a family of recently described serum pattern recognition molecules capable of binding to micro-organisms and activating complement through MBL associated serine protease-2 (MASP-2). Their role in chronic lung disease has not previously been investigated. Methods Serum levels of Ficolin-2 and MASP-2 were determined in 470 patients with idiopathic non-cystic fibrosis bronchiectasis and 400 matched control subjects by ELISA. Single nucleotide polymorphisms were determined using TaqMan PCR based genotyping. Bacterial binding was determined using ELISA and flow cytometry. Neutrophils from healthy donors were isolated by percoll gradient centrifugation and used in phagocytosis assays with FITC-labelled Pseudomonas aeruginosa strain PA01. Results Genotyping success was >95% and all SNP9s were in Hardy–Weinburg equilibrium (p>0.05). Bronchiectasis was associated with a homozygous mutation in the promoter of ficolin-2 (rs3124952) (28.5% vs 19.5%, p=0.002) and a homozygous mutation in exon-8 of the ficolin-2 gene causing impaired binding (rs17549193) (12% vs 8.4%, p=0.04). Low serum levels ( P aeruginosa , including clinical isolates and mucoid strains, activating the lectin pathway of complement and promoting C3 and C4 deposition. Ficolin-2 also bound to Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus . Opsonisation with ficolin-2 promoted phagocytosis of P aeruginosa (PA01) by human neutrophils in a MASP-2 but not c1q dependent manner (p P aeruginosa colonisation (OR=2.8, p=0.0001) were independently associated with ficolin-2 insufficiency. These patients also had more frequent outpatient exacerbations (mean 3.2/yr vs 2.4/yr, p=0.01) and unscheduled hospital admissions for exacerbations (OR=2.3; p Conclusion Single nucleotide polymorphisms in the ficolin-2 gene affecting serum levels and carbohydrate binding are associated with non-CF bronchiectasis and increase susceptibility to colonisation with P aeruginosa.