Cystic fibrosis (CF), the most prevalent, fatal genetic disorder in the Caucasian population, is caused by mutations of CF transmembrane conductance regulator (CFTR). The mutations of this chloride channel alter the transport of chloride and associated liquid and thereby impair lung defenses. Patients typically succumb to chronic bacterial infections and respiratory failure. Restoration of the abnormal CFTR function to CF airway epithelium is considered the most direct way to treat the disease. In this report, we explore the potential of adult stem cells from bone marrow, referred to as mesenchymal or marrow stromal stem cells (MSCs), to provide a therapy for CF. We found that MSCs possess the capacity of differentiating into airway epithelia. MSCs from CF patients are amenable to CFTR gene correction, and expression of CFTR does not influence the pluripotency of MSCs. Moreover, the CFTR-corrected MSCs from CF patients are able to contribute to apical Cl ^- secretion in response to cAMP agonist stimulation, suggesting the possibility of developing cell-based therapy for CF. The ex vivo coculture system established in this report offers an invaluable approach for selection of stem-cell populations that may have greater potency in lung differentiation.

Chloride anion is essential for myeloperoxidase (MPO) to produce hypochlorous acid (HOCl) in polymorphonuclear 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 dose-dependent manner. By using specific inhibitors to selectively block NADPH oxidase, MPO, and cystic fibrosis transmembrane conductance regulator (CFTR) functions, neutrophil-mediated killing of PsA could be attributed to three distinct mechanisms: CFTR-dependent and oxidant-dependent; chloride-dependent but not CFTR- and oxidant-dependent; and independent of any of the tested factors. Therefore, chloride anion is involved in oxidant- and nonoxidant-mediated bacterial killing. We previously reported that neutrophils from CF patients are defective in chlorination of ingested bacteria [1], suggesting that the chloride channel defect might impair the MPO-hydrogen peroxide-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 one-third 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.

CFTR transports chloride anions necessary for HOCl production and optimal microbicidal activity in human neutrophil phagosomes. Chloride serves as a critical component of innate host defense against infection, providing the substrate for MPO-catalyzed production of HOCl in the phagosome of human neutrophils. Here, we used halide-specific fluorescent sensors covalently coupled to zymosan particles to investigate the kinetics of chloride and iodide transport in phagosomes of human neutrophils. Using the self-ratioable fluorescent probe specific for chloride anion, we measured chloride dynamics within phagosomes in response to extracellular chloride changes by quantitative fluorescence microscopy. Under the experimental conditions used, normal neutrophils showed rapid phagosomal chloride uptake with an initial influx rate of 0.31 ± 0.04 mM/s (n=5). GlyH-101, a CFTRinh, decreased the rate of uptake in a dose-dependent manner. Neutrophils isolated from CF patients showed a significantly slower rate of chloride uptake by phagosomes, having an initial influx rate of 0.043 ± 0.012 mM/s (n=5). Interestingly, the steady-state level of chloride in CF phagosomes was ∼26 mM, significantly lower than that of the control (∼68 mM). As CFTR transports chloride as well as other halides, we conjugated an iodide-sensitive probe as an independent approach to confirm the results. The dynamics of iodide uptake by neutrophil phagosomes were monitored by flow cytometry. CFTRinh172 blocked 40–50% of the overall iodide uptake by phagosomes in normal neutrophils. In a parallel manner, the level of iodide uptake by CF phagosomes was only 20–30% of that of the control. Taken together, these results implicate CFTR in transporting halides into the phagosomal lumen.

Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on the generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H₂O₂ produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that 95-99% of lysosomal-associated membrane protein 1 (LAMP-1)-positive mature phagosomes were CFTR positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed enhanced green fluorescent protein (EGFP) alone, EGFP-wt-CFTR and EGFP-DF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and colocalize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-DF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, the CFTR corrector compound VRT-325 facilitated the recruitment of DF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function.

Cystic fibrosis (CF) is a common and deadly inherited disease, caused by mutations in the CFTR gene that encodes a cAMP-activated chloride channel. One outstanding manifestation of the disease is the persistent bacterial infection and inflammation in the lung, which claims over 90% of CF mortality. It has been debated whether neutrophil-mediated phagocytic innate immunity has any intrinsic defect that contributes to the host lung defense failure. Here we compared phagosomal CFTR targeting, hypochlorous acid (HOCl) production, and microbial killing of the neutrophils from myeloid Cftr-inactivated (Myeloid-Cftr−/−) mice and the non-inactivated control (Cftr^fl10) mice. We found that the mutant CFTR that lacked Exon-10 failed to target to the neutrophil phagosomes. This dysfunction resulted in impaired intraphagosomal HOCl production and neutrophil microbial killing. In vivo lung infection with a lethal dose of Pseudomonas aeruginosa caused significantly higher mortality in the myeloid CF mice than in the controls. The myeloid-Cftr−/− lungs were deficient in bacterial clearance, and had sustained neutrophilic inflammation and stalled transition from early to late immunity. These manifestations recapitulated the symptoms of human CF lungs. The data altogether suggest that myeloid CFTR expression is critical to normal host lung defense. CFTR dysfunction in neutrophils compromises the phagocytic innate immunity, which may predispose CF lungs to infection.