Elsevier

Immunology Letters

Volume 160, Issue 2, August 2014, Pages 186-194
Immunology Letters

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

https://doi.org/10.1016/j.imlet.2014.03.003Get rights and content

Highlights

  • 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.

Abstract

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.

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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