NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase–DNA and neutrophil elastase–DNA complexes

• Limited DNAseI digestion of NETs allows better reproducibility.
• A modified MPO–DNA ELISA and a new HNE–DNA ELISA was used to quantify NETs.
• Cystic fibrosis isolates of Pseudomonas aeruginosa trigger respiratory burst in human neutrophils.
• Early P. aeruginosa cystic fibrosis isolates induce significantly more robust NET release than late isolates.

Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO–DNA complexes and established a new HNE–DNA ELISA. We show that these methods reliably quantify MPO–DNA and HNE–DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired “early” and “late” bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways.