NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase–DNA and neutrophil elastase–DNA complexes
Introduction
Cystic fibrosis (CF) is an inherited disorder resulting from dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) anion channel [1]. Lung complications are responsible for the majority of morbidity and mortality in CF [2]. CF lungs are characterized by chronic bacterial infections, mucus overproduction and large numbers of infiltrating neutrophils [3]. Pseudomonas aeruginosa is the main bacterial pathogen infecting the CF respiratory tract [2].
Neutrophils are unable to eliminate bacteria from CF airways and are thought to contribute to lung damage instead [3]. Neutrophil azurophilic granule components, myeloperoxidase (MPO) and human neutrophil elastase (HNE) are found in high concentrations in CF sputum and bronchoalveolar lavage, and their levels correlate with lung disease severity [4], [5], [6], [7], [8], [9]. DNA can also be found in large quantities in CF airways, and DNAse treatment has been used to improve pulmonary function in CF patients [10], [11]. The mechanism for release of neutrophil-derived inflammatory mediators in CF is unknown.
Neutrophil extracellular traps (NETs) provide a potential mechanism for release of neutrophil-derived mediators in CF (DNA, MPO, HNE) [12]. NETs have been detected in CF sputum [13], [14]. P. aeruginosa likely triggers NETs in CF airways. P. aeruginosa has been shown to induce extracellular DNA release in human neutrophils in vitro [15], [16], [17]. However, most of these experiments used only laboratory standard strains of P. aeruginosa [15], [17], [18]. Even when CF clinical isolates were used, only extracellular DNA release was measured that alone does not equal measurement of NET formation [15].
Our aim was to quantify NET release induced by CF clinical isolates of P. aeruginosa and compare them to its standard strains. We modified a previously established method to measure MPO–DNA complexes [19], [20] and also established a new assay for measuring HNE–DNA complexes, both of which are only present in NETs. Our data show that all P. aeruginosa CF isolates tested triggered NET release in normal human neutrophils at levels comparable to the standard strains (PAO1, PA14). Our results confirm that PAO1 and PA14 serve as adequate models to study Pseudomonas-induced NET release and strengthen the view that P. aeruginosa can be the main NET-inducing factor in CF airways.
Section snippets
Human subject statement
Healthy human volunteers recruited under the guidelines of IRB-approved protocol (University of Georgia, UGA# 2012-10769-9) provided written informed consent for blood donation.
Human subjects also included CF patients enrolled in an observational study of CF lung disease severity, “Genetics of CF Lung Disease” (Seattle Children's Hospital institutional review board approved protocol 10,855 and Partners Healthcare Systems/Massachusetts General Hospital institutional review board approved
Results and discussion
To quantify NETs, we slightly modified a previously established ELISA assay that detects MPO–DNA complexes [19], [20]. We also established a new assay that detects HNE–DNA complexes in biological samples. Apoptotic neutrophils do not release protein–DNA complexes; only cells undergoing NET formation do this [19]. Therefore levels of MPO–DNA or HNE–DNA complexes determine the extent of NET formation. These ELISA methods use anti-MPO or anti-HNE capture antibodies and a horse radish
Conflicts of interest
The authors have no financial conflict of interest to declare.
Acknowledgments
We are thankful for the laboratory staff at the University of Georgia Health Center for performing phlebotomy on the recruited volunteers. This work was supported by the startup fund of B. Rada obtained from The Office of the Vice President for Research, UGA (grant no. 1026AR211001).
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