Neutrophil Extracellular Trap (NET)-Mediated Killing of Pseudomonas aeruginosa: Evidence of Acquired Resistance within the CF Airway, Independent of CFTR

Abstract

The inability of neutrophils to eradicate Pseudomonas aeruginosa within the cystic fibrosis (CF) airway eventually results in chronic infection by the bacteria in nearly 80 percent of patients. Phagocytic killing of P. aeruginosa by CF neutrophils is impaired due to decreased cystic fibrosis transmembrane conductance regulator (CFTR) function and virulence factors acquired by the bacteria. Recently, neutrophil extracellular traps (NETs), extracellular structures composed of neutrophil chromatin complexed with granule contents, were identified as an alternative mechanism of pathogen killing. The hypothesis that NET-mediated killing of P. aeruginosa is impaired in the context of the CF airway was tested. P. aeruginosa induced NET formation by neutrophils from healthy donors in a bacterial density dependent fashion. When maintained in suspension through continuous rotation, P. aeruginosa became physically associated with NETs. Under these conditions, NETs were the predominant mechanism of killing, across a wide range of bacterial densities. Peripheral blood neutrophils isolated from CF patients demonstrated no impairment in NET formation or function against P. aeruginosa. However, isogenic clinical isolates of P. aeruginosa obtained from CF patients early and later in the course of infection demonstrated an acquired capacity to withstand NET-mediated killing in 8 of 9 isolates tested. This resistance correlated with development of the mucoid phenotype, but was not a direct result of the excess alginate production that is characteristic of mucoidy. Together, these results demonstrate that neutrophils can kill P. aeruginosa via NETs, and in vitro this response is most effective under non-stationary conditions with a low ratio of bacteria to neutrophils. NET-mediated killing is independent of CFTR function or bacterial opsonization. Failure of this response in the context of the CF airway may occur, in part, due to an acquired resistance against NET-mediated killing by CF strains of P. aeruginosa.

Distinct Susceptibilities of Corneal Pseudomonas aeruginosa Clinical Isolates to Neutrophil Extracellular Trap-Mediated Immunity

“…Whether NETs trap and kill P. aeruginosa is controversial. We, along with others, reported that the nonmucoid cystic fibrosis-derived P. aeruginosa strains were sensitive to NET trapping while the mucoid strains were resistant (13)(14)(15). In contrast, Parker et al reported that P. aeruginosa was not sensitive to NET-mediated killing (16).…”

“…Based on the proteomic profile of NETs, the expectation was that NETs carry antimicrobial activity. The bactericidal properties of NETs have attracted scientific interest; however, the question of whether NETs are capable of trapping or killing P. aeruginosa remains contentious (14,16,28). To determine the functional significance of NETs, in vitro experiments addressing NET-mediated capture of P. aeruginosa and killing were carried out.…”

NETosis: how vital is it?

“…82 Whereas NETs are sufficient to kill Pseudomonas grown in suspension, during diseases such as cystic fibrosis, the bacteria changes to a mucoid type and can become NET resistant. 83 By contrast, other groups have not been able to demonstrate significant direct killing by NETs. In the case of S aureus, despite clear trapping by the NETs, DNase treatment liberated living bacteria that were capable of proliferation.…”

Evasion of Neutrophil Extracellular Traps by Respiratory Pathogens

“…The authors in this study showed that treatment with sialidase or use of P. aeruginosa strains lacking in sialic acids led to increased NET production compared with sialic acid-positive strains. Furthermore, paired P. aeruginosa strains isolated from patients with CF at early and late stages of disease showed that resistance to NET-mediated killing evolved over time, and correlated with the development of the mucoid phenotype characterized by excess alginate production (34). However, a mutant P. aeruginosa strain that overexpressed alginate did not show increased survival on incubation with PMA-treated neutrophils, suggesting that the acquired resistance of P. aeruginosa to NETmediated killing in the CF airway was due to an as-yet unknown mechanism (34).…”