Cystic fibrosis (CF) lung disease features persistent neutrophil accumulation to the airways from the time of infancy. CF children are frequently exposed to Pseudomonas aeruginosa, and by adulthood, 80% of CF patients are chronically infected. The formation of biofilms is a particularly important phenotypic characteristic of P. aeruginosa that allows for bacterial survival despite aggressive antibiotic therapy and an exuberant immune response. Here, we show that the presence of neutrophils enhances initial P. aeruginosa biofilm development over a period of 72 h through the formation of polymers comprised of actin and DNA. F-actin was found to be a site of attachment for P. aeruginosa. These actin and DNA polymers are present in CF sputum, and disruption of the polymers dispersed the associated P. aeruginosa cells and reduced biofilm development. These findings demonstrate a potential maladaptation of the primary innate response. When the host fails to eradicate the infection, cellular components from necrotic neutrophils can serve as a biological matrix to facilitate P. aeruginosa biofilm formation.

The bone marrow is the primary site for neutrophil production and release into the circulation. Because the CXC chemokine receptor-4/stromal derived factor-1 (CXCR4/ SDF-1) axis plays a central role in the interactions of hematopoietic stem cells, lymphocytes, and developing neutrophils in the marrow, we investigated whether reciprocal CXCR4-dependent mechanisms might be involved in neutrophil release and subsequent return to the marrow following circulation. Neutralizing antibody to CXCR4 reduced marrow retention of infused neutrophils (45.7% ؎ 0.5% to 6.9% ؎ 0.5%) and was found to mobilize neutrophils from marrow (34.4% ؎ 4.4%). Neutrophil CXCR4 expression and SDF-1-induced calcium flux decreased with maturation and activation of the cells, corresponding to the decreased marrow homing associated with these characteristics in vivo. Infusion of the inflammatory mediator and CXCR2 ligand KC led to mobilization of neutrophils from marrow by itself and was augmented 3-fold by low doses of CXCR4-blocking antibody that otherwise had no mobilizing effect. Examination of KC and SDF-1 calcium signaling demonstrated that the effect of KC may, in part, be due to heterologous desensitization to SDF-1. These results suggest that the CXCR4/SDF-1 axis is critical in circulating neutrophil homeostasis and that it may participate in the rapid release of neutrophils from the marrow during inflammation through a novel interaction with inflammatory CXC chemokines. (Blood. 2004;104: 565-571)

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.

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