The role of chloride anion and CFTR in killing ofPseudomonas aeruginosaby normal and CF neutrophils

Abstract

Chloride anion is essential for myeloperoxidase to produce hypochlorous acid (HOCl) in neutrophils (PMNs). To define whether chloride availability to PMNs affects their HOCl production and microbicidal capacity, we examined how extracellular chloride concentration affects killing of Pseudomonas aeruginosa (PsA) by normal neutrophils. PMN-mediated bacterial killing was strongly dependent on extracellular chloride concentration. Neutrophils in a chloride-deficient medium killed PsA poorly. However, as the chloride level was raised, the killing efficiency increased in a dosedependent fashion. By using specific inhibitors to selectively block NADPH-oxidase, MPO and CFTR functions, neutrophil-mediated killing of PsA could be attributed to three distinct mechanisms: 1) CFTR-dependent and oxidant-dependent, 2) chloride-dependent but not CFTR-and oxidantdependent, and 3) independent of any of the tested factors. Therefore, chloride anion is involved in both oxidant-and non-oxidant-mediated bacterial killing. We previously reported that neutrophils from cystic fibrosis (CF) patients are defective in chlorination of ingested bacteria, suggesting that the chloride channel defect might impair the MPO-H 2 O 2 -chloride microbicidal function. Here, we compared the competence of killing PsA by neutrophils from normal donors and CF patients. The data demonstrate that the killing rate by CF neutrophils was significantly lower than that by normal neutrophils. CF neutrophils in a chloride-deficient environment had only 1/3 of the bactericidal capacity of normal neutrophils in a physiological chloride environment. These results suggest that CFTR-dependent chloride anion transport contributes significantly to killing PsA by normal neutrophils and, when defective as in CF, may compromise the ability to clear PsA.

Burkholderia cenocepacia-induced delay of acidification and phagolysosomal fusion in cystic fibrosis transmembrane conductance regulator (CFTR)-defective macrophages

“…This is in agreement with a recent detailed analysis using fluorescence ratio imaging to measure the endosomal pH against an internal standard, which convincingly demonstrated that phagolysosomal acidification in macrophages is CFTR-independent (Haggie & Verkman, 2007). It has been reported that in human neutrophils CFTR channel dysfunction affects neutrophil chlorination of phagocytized bacteria (Painter et al, 2006(Painter et al, , 2008, raising the possibility that CFTR contributes to bacterial clearance rather than phagosomal acidification. Our experiments with B. cenocepacia-infected CFTR-defective macrophages showing an extended delay in the trafficking of BcCVs to lysosomes do indeed support a role for CFTR in the mechanism of clearance of the intracellular infection, as we have shown before that B. cenocepacia localized to the lysosome rapidly loses cell envelope integrity (Lamothe et al, 2007).…”

Cystic fibrosis neutrophils have normal intrinsic reactive oxygen species generation

“…Reduced expression of the patternrecognition receptor Toll-like receptor (TLR)2 has been reported on peripheral blood neutrophils from CF patients, and this was correlated with increased systemic tumour necrosis factor-a, but more dramatic effects on TLR expression are seen on transmigrated CF neutrophils [34], suggesting an important acquired effect secondary to factors within the lung microenvironment. Different groups have reported either normal [35] or somewhat reduced [36] ability of isolated peripheral blood CF neutrophils to kill Pseudomonas aeruginosa in vitro; importantly, HARTL et al [35] also found that CF neutrophils obtained from sputum or bronchoalveolar lavage had a dramatically attenuated bactericidal capacity with respect to comparable cells from healthy subjects or to the peripheral blood CF cells, strongly implicating an acquired rather than an intrinsic defect.…”

Low Sphingosine-1–Phosphate Impairs Lung Dendritic Cells in Cystic Fibrosis

“…The dysfunction or lack of CFTR expression in macrophages, neutrophils, and dendritic cells (DCs) results in an inflammatory phenotype (4,5,(8)(9)(10)(11). In addition, antigen presentation is affected in CF.…”

“…The dysregulated cytokine secretion of CF epithelial cells plays an important role in creating the inflammatory milieu (3). It has become increasingly clear that the cystic fibrosis transmembrane regulator (CFTR) also plays a role in lung immune cells, and that the dysfunction of the CFTR affects immune cell responses (4)(5)(6)(7)(8)(9)(10)(11). The dysfunction of pulmonary immune cells in CF could result from the lack of their own CFTR function, or may be induced by the altered milieu created by defective CFTR function in epithelial cells (5,8).…”