Neutrophil extracellular traps are a recently described mechanism by which neutrophils kill a variety of pathogens. However, significant variability appears to exist in the relative susceptibility to NET-mediated killing by clinically important microbes, and the relative biologic roles of NET versus phagocytic killing have yet to be clearly elucidated in health and disease (i.e., in specific clinical infections or within specific anatomic compartments). Herein, we demonstrate that P. aeruginosa induces formation of NETs and is susceptible to NET-mediated killing ex vivo under specific conditions. Under conditions typically used to study NETs, where bacteria and neutrophils were layered together on a motionless surface, we found that phagocytic killing of P. aeruginosa was the predominant response. In contrast, under conditions in which the bacteria and neutrophils are maintained in suspension with constant motion and mild shear forces, nearly all killing of P. aeruginosa was NET-mediated. To our knowledge, these experimental test conditions have not previously been examined. Under these conditions, NET-mediated killing was efficient even with a very low ratio of P. aeruginosa to neutrophils and was not dependent on opsonization of the bacteria. This method was optimized to test the parameters of NET-mediated killing of P. aeruginosa; optimal NET-mediated killing of other pathogens may require different experimental conditions.
In the context of CF lung disease, we explored the possibility that CF neutrophils might fail to produce effective NETs, prompted by observations that a number of neutrophil responses are abnormal in the setting of non-functional CFTR 
. Specifically, previous reports have indicated that CFTR deficiency results in reduced phagolysosomal function in neutrophils and macrophages, and impaired phagocytic killing of P. aeruginosa 
. In contrast, our results indicate that CF neutrophils produce functional NETs comparable to those of neutrophils with functional CFTR. Thus, NET-mediated killing is a CFTR-independent arm of the innate immune response that may assume greater importance in CF patients. Despite many reports identifying impaired response by CF neutrophils, these cells clearly have substantial antimicrobial capabilities, as bacterial infections outside the airway are not a feature of the disease. Our findings suggest that NET-mediated killing is fully functional in CF patients, and thus may account for the general absence of invasive infection despite a massive bacterial burden within the CF airway.
Our data suggest a role for NET-mediated clearance of initial P. aeruginosa
infection in healthy individuals, and possibly in the early stages of CF lung disease. Episodic exposure of humans to P. aeruginosa
is likely a common occurrence. Even within the CF airway, P. aeruginosa
is often cleared effectively, and progression to chronic infection may not occur for years 
. One possibility is that resident macrophages within the lung could be responsible for this initial clearance. However, another possibility is that NET-mediated killing by neutrophils may account for early clearance. Conditions that favor NET-mediated killing are likely present within both CF and normal airways. The lung is in constant motion, and environmental exposures typically involve small inocula of bacteria with limited direct contact with neutrophils, in the presence of low plasma concentrations and without effective opsonization 
. Consistent with this premise, our results indicate that P. aeruginosa
strains isolated from CF airways early in the course of infection are effectively killed by NETs.
Strong evidence indicates that neutrophil defenses ultimately fail as CF lung disease progresses and P. aeruginosa
infection becomes persistent. For CF patients, inhaled DNase therapy improves lung function and reduces infectious exacerbations 
. Since DNase disrupts killing by NETs 
, these results support the conclusion the NET-mediated killing is not effective within the airway of CF patients who have established infection. Given the evidence suggesting that NETs do not facilitate bacterial clearance later in the course of CF, NET formation may actually be detrimental by promoting hyperviscosity of airway secretions, release of neutrophil proteases, and development of P. aeruginosa
One factor which could contribute to ineffective NET-mediated killing within the CF airway is acquired P. aeruginosa
resistance to this arm of innate host defense. The hypermutability of P. aeruginosa
within the CF airway is well-described and it is not surprising that in this intense inflammatory environment, mutants with increased resistance to NETs would emerge 
. We tested the capacity of CF strains of P. aeruginosa
to acquire resistance to NET-mediated killing. Using paired isogenic clinical isolates of P. aeruginosa
, we showed that decreasing susceptibility to NET-mediated killing evolves over time in the CF airway. The development of mucoidy (i.e.
increased alginate production) is an acquired P. aeruginosa
virulence factor that is closely associated with acceleration of CF lung disease 
. Among the nine pairs of isolates tested, conversion to a mucoid phenotype coincided with a decline in susceptibility to NETs, raising the possibility that increased alginate production decreases interactions with NETs, or otherwise interferes with killing by NET-associated granule proteins. However, two independently derived mucA
mutants of PAO1 failed to display the NET resistance seen for late CF airway isolates, suggesting that increased exopolysaccharide production alone does not explain this phenotype.
Presumably, increased expression of other, as yet unidentified, determinants may act either independently or concurrently with increased alginate production to mediate NET resistance. Several pathogens possess specific mechanisms that disrupt NET-mediated killing, including Streptococcus pneumoniae
and GAS, which produce nucleases that degrade NETs 
. In addition, capsule formation, in concert with D-alanylation of lipoteichoic acids, enhances resistance of S. pneumoniae
to NET-mediated killing 
. GAS strains expressing the M1 protein resist NET-mediated killing by virtue of their resistance to the human cathelicidin peptide LL-37, an important antimicrobial component of NETs 
. P. aeruginosa,
with its extremely large genome, is capable of tremendous versatility and environmental adaptability 
. It encodes deoxyribonucleases, and the possibility exists that these, or related enzymes, could act on NETs 
, as well as yet undescribed mechanisms of disrupting NET-mediated killing. Although not tested here, extrinsic features of the CF airway could also contribute to ineffective NET function in the setting of chronic P. aeruginosa
infection. The CF airway is a complex environment characterized by altered airway mucus, high levels of proteases, large amounts of neutrophil-derived DNA and F-actin, and abundant P. aeruginosa 
. One or more of these features may disrupt NET killing by preventing NET formation, disassembling or altering the antibacterial components of NETs, preventing physical interaction between P. aeruginosa
and NETs, or competing for binding sites on NETs.
We postulate that NETs contribute to early clearance of P. aeruginosa
from the CF airway, but that, later in the disease, features of the CF airway or an adaptation of the organism render NETs ineffective, and possibly detrimental. If in fact NET-mediated killing is effective in the initial contact between P. aeruginosa
and neutrophils suspended within the CF airway secretions, this has important implications both for development of new therapies and for early CF airway disease. This notion may also help guide the use of inhaled DNase in other lung conditions for which benefit might be assumed based on results in CF patients. For example, disruption of effective NET killing could explain the increased rates of infection reported with DNase use in non-CF bronchiectasis 
. Understanding the role of NETs in controlling P. aeruginosa
at different stages of airway infection is particularly important given ongoing clinical trials evaluating the use of this agent in very young children 
, and in devising strategies to prevent initial infection by enhancing host defenses.