Role of Mutant CFTR in Hypersusceptibility of Cystic Fibrosis Patients to Lung Infections

Abstract

Cystic fibrosis (CF) patients are hypersusceptible to chronic Pseudomonas aeruginosa lung infections. Cultured human airway epithelial cells expressing the ΔF508 allele of the cystic fibrosis transmembrane conductance regulator (CFTR) were defective in uptake of P. aeruginosa compared with cells expressing the wild-type allele. Pseudomonas aeruginosa lipopolysaccharide (LPS)-core oligosaccharide was identified as the bacterial ligand for epithelial cell ingestion; exogenous oligosaccharide inhibited bacterial ingestion in a neonatal mouse model, resulting in increased amounts of bacteria in the lungs. CFTR may contribute to a host-defense mechanism that is important for clearance of P. aeruginosa from the respiratory tract.Among the most serious manifestations of CF are chronic pulmonary infections with the bacterium P. aeruginosa. The basis for hypersusceptibility of CF patients to this bacterium is not well understood, and the role of mutant CFTR, if any, is not clear. Binding and internalization of respiratory pathogens by epithelial cells followed by desquamation could be an important mechanism for clearing bacteria from the lung. This mechanism has been shown to be important in protecting against bladder infections (1).To investigate whether the most common and severe CFTR mutation (ΔF508) affected uptake of P. aeruginosa, we performed bacterial invasion assays (2) with four cell lines: CFT1, an airway epithelial cell line derived from a CF patient homozygous for ΔF508 CFTR and that is transformed with human papilloma virus 18 E6/E7; CFT1-ΔF508, which expresses a third copy of ΔF508 CFTR introduced by a retrovirus; CFT1-LC3, which expresses a control gene (β-galactosidase) introduced by the same retrovirus; and CFT1-LCFSN, which expresses a functional wild-type human CFTR gene (3). We tested a standard laboratory strain of P. aeruginosa, designated PAOI, and two nonmucoid, LPSsmooth clinical isolates from CF patients (4). Compared with CFT1-LCFSN cells, the three lines expressing ΔF508 CFTR internalized significantly fewer bacterial cells (Fig. 1A). The ΔF508 mutation causes inefficient processing of CFTR, a defect that is partially corrected if the cells are grown at 26°C (5). When epithelial cells were cultured for 72 hours at 26°C there were no longer significant differences in uptake of the P. aeruginosa strains by the cells expressing wild-type or mutant CFTR (Fig. 1B). Because the overall uptake of bacteria at 26°C was low, we performed additional experiments with cells grown for 72 hours at 26°C in which the invasion assay was performed at 37°C for 3 hours, conditions under which surface expression of mutant ΔF508 CFTR is maintained (5). No significant difference in bacterial cell uptake was measured (Fig. 1C), and overall amounts of internalization approached those of the CFT1-LCFSN cells at 37°C. Returning cells expressing ΔF508 CFTR to 37°C for 24 hours after growth for 72 hours at 26°C removes CFTR from the cell surface (5); under these conditions internalization of the bacterial strains ...

Airway epithelial control of Pseudomonas aeruginosa infection in cystic fibrosis

“…In transgenic CFTR-knockout mice, NF-κB nuclear translocation occurs much later in the airway epithelial cells [28], probably contributing more to the harmful inflammation that ensues in this environment instead of the protective inflammation that occurs when functional CFTR is present. Overall, this scenario wherein WT-CFTR binding of P. aeruginosa leads to protection and failure of this interaction in CF leads to infection is supported by data obtained in numerous in vitro and, importantly, in υivo, studies demonstrating CFTR-dependent responses to P. aeruginosa in a variety of lung epithelial cell lines and in transgenic animals [8][9][10][11][12][27][28][29]31,36]. Thus, it seems that CFTR facilitates bacterial clearance and modulates innate immunity towards P. aeruginosa in lung epithelial cells by being the linchpin needed for coordinating multiple host responses involved in resisting infection and maintaining tissue homeostasis in the long run.…”

“…To wit, results showing that WT-CFTR is a receptor for P. aeruginosa that mediates effective innate immunity and that this receptor is not functional in CF [9,10] are the ones most consistent with clinical and experimental observations. One curious additional observation about this interaction has also been reported: the mucoid strains of P. aeruginosa responsible for chronic infection lose the bacterial ligand for CFTR [8,11,12], indicating that the P. aeruginosa-CFTR direct interaction is likely to be primarily of relevance to the early stages of infection, whereas in the later stages of infection other properties of the bacterium and the CF host conspire to allow for maintenance of the chronic infectious state.…”

“…P. aeruginosa recognition by WT-CFTR is due to the specific binding of amino acids 108-117 to the conserved bacterial outer core LPS [11,12,27]. CFTR binding to P. aeruginosa results in release of interleukin-1b from preformed stores in lung epithelial cells within 2-15 min [28], followed by rapid formation (5-15 min) of CFTR-containing lipid rafts in the plasma membrane, with P. aeruginosa cells attached to the CFTR.…”

“…To wit, results showing that WT-CFTR is a receptor for P. aeruginosa that mediates effective innate immunity and that this receptor is not functional in CF [9,10] are the ones most consistent with clinical and experimental observations. One curious additional observation about this interaction has also been reported: the mucoid strains of P. aeruginosa responsible for chronic infection lose the bacterial ligand for CFTR [8,11,12], indicating that the P. aeruginosa-CFTR direct interaction is likely to be primarily of relevance to the early stages of infection, whereas in the later stages of infection other properties of the bacterium and the CF host conspire to allow for maintenance of the chronic infectious state.However, there are clearly investigators who hypothesize that the major role of the airway epithelium in susceptibility to P. aeruginosa lung infection is through defects in ion transport, leading to dehydrated airway surface layer (ASL) and thickened mucus that cannot be cleared by the ciliary escalator. This is a necessary but not sufficient component of the pathogenesis of P. aeruginosa in CF that is well documented and supported by the work of Boucher and colleagues [13].…”

The Role of Sphingolipids and Ceramide in Pulmonary Inflammation in Cystic Fibrosis

“…demonstrated a defect of the innate immune function of epithelial cells in the respiratory tract of cystic fibrosis mice. They showed that the lipopolysaccharide (LPS)-molecule of P. aeruginosa binds to a short amino acid sequence in CFTR (AA 103-117) [13]. This interaction mediates internalisation of P. aeruginosa into lung epithelial cells.…”