Role of the cystic fibrosis transmembrane conductance regulator in innate immunity to Pseudomonas aeruginosa infections

Abstract

Chronic Pseudomonas aeruginosa infection occurs in 75-90% of patients with cystic fibrosis (CF). It is the foremost factor in pulmonary function decline and early mortality. A connection has been made between mutant or missing CF transmembrane conductance regulator (CFTR) in lung epithelial cell membranes and a failure in innate immunity leading to initiation of P. aeruginosa infection. Epithelial cells use CFTR as a receptor for internalization of P. aeruginosa via endocytosis and subsequent removal of bacteria from the airway. In the absence of functional CFTR, this interaction does not occur, allowing for increased bacterial loads in the lungs. Binding occurs between the outer core of the bacterial lipopolysaccharide and amino acids 108-117 in the first predicted extracellular domain of CFTR. In experimentally infected mice, inhibiting CFTR-mediated endocytosis of P. aeruginosa by inclusion in the bacterial inoculum of either free bacterial lipopolysaccharide or CFTR peptide 108-117 resulted in increased bacterial counts in the lungs. CFTR is also a receptor on gastrointestinal epithelial cells for Salmonella enterica serovar Typhi, the etiologic agent of typhoid fever. There was a significant decrease in translocation of this organism to the gastrointestinal submucosa in transgenic mice that are heterozygous carriers of a mutant DeltaF508 CFTR allele, suggesting heterozygous CFTR carriers may have increased resistance to typhoid fever. The identification of CFTR as a receptor for bacterial pathogens could underlie the biology of CF lung disease and be the basis for the heterozygote advantage for carriers of mutant alleles of CFTR.

Figure 1

Inverse relationship between isolation of mucoid P. aeruginosa (but not Staphylococcus aureus or Haemophilus influenzae) and decline in percentage of predicted FEV₁, as compiled from the CF Foundation Patient Registry database for 1998.

Figure 2

Molecular consequences of CFTR mutations. [Reproduced with permission from ref. (Copyright 1995, Lap Chee Tsui)].

Figure 3

Invasion of transformed airway epithelial cell lines by three strains of P. aeruginosa (two clinical isolates from patients with CF, 324 and 149, and a laboratory strain, PAO1). Cells were grown for 72 h at the temperature indicated in each figure (“Cells”). Bacteria were allowed to invade the epithelial cells for 3 or 4 h at the temperature indicated by “Invasion” on the figures. Bars indicate the means of the determinations, and error bars indicate the standard deviation of the mean. (A) Cells grown at 37°C, a temperature that inhibits membrane expression of ΔF508 CFTR (44); the invasion assay was carried out for 4 h at 37°C. Ingestion of P. aeruginosa by cells expressing wild-type CFTR was significantly higher than in cells lacking wild-type CFTR (P < 0.01, ANOVA). (B) Cells grown at 26°C and invasion assessed at 26°C. (C) Cells grown at 26°C and invasion assessed at 37°C. In both assays where cells were grown at 26°C to promote surface expression of ΔF508 CFTR (44), there were no significant differences (P > 0.2, ANOVA) in bacterial invasion among the cell lines for any P. aeruginosa strain tested.

Figure 4

Effect of adding inhibitors of the CFTR–P. aeruginosa interaction on the course of infection in neonatal mouse lungs. (A) Addition of LPS core oligosaccharides to the P. aeruginosa challenge inoculum reduces cellular uptake and increases bacterial loads in the lung. Closed circles, no inhibitor; open squares, LPS core oligosaccharide, 10 μg/ml; closed squares, LPS incomplete core oligosaccharide control, 10 μg/ml. Each symbol indicates the median number of bacterial colony-forming units for 8–10 lungs obtained from each group, and the bars indicate the upper and lower quartiles. Differences among groups were analyzed by nonparametric statistics [P < 0.0001; Kruskal–Wallis nonparametric ANOVA; P < 0.001; Dunn procedure for individual pairwise differences between the groups at 1 and 24 h; also at 1 h, the group receiving the incomplete core oligosaccharide had a reduced level (P = 0.05; Dunn procedure) of intracellular bacteria compared with the group receiving nothing along with the inoculum]. At 48 h, the group treated initially with complete-core inhibitor had significantly more bacteria in the lungs (P = 0.003; Kruskal–Wallis; P < 0.05; Dunn procedure for all pairwise comparisons). (B) Effect of addition of synthetic peptides to the bacterial inoculum on P. aeruginosa infection in neonatal mice. (Upper) Amount of P. aeruginosa internalized by lung cells 24 h after infection. (Lower) Total amount of P. aeruginosa found in lungs 24 h after infection. Box plots indicate–from bottom to top–the 10th, 25th, 50th (median), 75th, and 90th percentiles. Circles above or below the 90th or 10th percentile indicate individual points outside this range. There were 12–14 total lung samples used in each group. For both groups of comparisons (A and B), the overall differences were significant at P < 0.001 (Kruskal–Wallis nonparametric ANOVA test), and the difference between the group receiving the first-domain peptide and the other three groups was significant at P < 0.001 (Dunn procedure for pairwise comparisons).

Figure 5

Increased expression of CFTR in the bronchial epithelial cells of mice infected with P. aeruginosa. At 1 h after instillation of P. aeruginosa into the lungs of neonatal BALB/c mice, the tissue was removed, fixed, and stained with monoclonal antibody CF3 specific to the first extracellular domain of CFTR (amino acids 103–117; ref. 61). Tissue sections stained with an irrelevant control monoclonal antibody had no visible fluorescence (not shown).

Figure 6

Translocation of S. enterica serovar Typhi across the GI epithelium of transgenic ΔF508 CF mice. (A) Decreased translocation of serovar Typhi strain Ty2 from the lumen of the GI tract of mice with the indicated genotype for murine Cftr. P < 0.001; ANOVA and Fisher probable least square differences for all three pair-wise comparisons. (B) Inhibition of translocation of serovar Typhi strain Ty2 from the GI lumen of BALB/c mice infected with this bacterium plus a synthetic peptide corresponding to the indicated amino acids in the first predicted extracellular domain of CFTR. P < 0.001; ANOVA and Fisher probable least square differences for pair-wise comparisons between amino acid 108–117 inhibitor and the other two groups. Bars indicate mean of the log₁₀ colony-forming units of serovar Typhi translocated; numbers indicate the antilog of the mean; error bars indicate the SEM.