Resistance to Pseudomonas aeruginosa chronic lung infection requires cystic fibrosis transmembrane conductance regulator-modulated interleukin-1 (IL-1) release and signaling through the IL-1 receptor

Abstract

Innate immunity is critical for clearing Pseudomonas aeruginosa from the lungs. In response to P. aeruginosa infection, a central transcriptional regulator of innate immunity-NF-kappaB-is translocated within 15 min to the nuclei of respiratory epithelial cells expressing wild-type (WT) cystic fibrosis (CF) transmembrane conductance regulator (CFTR). P. aeruginosa clearance from lungs is impaired in CF, and rapid NF-kappaB nuclear translocation is defective in cells with mutant or missing CFTR. We used WT and mutant P. aeruginosa and strains of transgenic mice lacking molecules involved in innate immunity to identify additional mediators required for P. aeruginosa-induced rapid NF-kappaB nuclear translocation in lung epithelia. We found neither Toll-like receptor 2 (TLR2) nor TLR4 nor TLR5 were required for this response. However, both MyD88-deficient mice and interleukin-1 receptor (IL-1R)-deficient mice failed to rapidly translocate NF-kappaB to the nuclei of respiratory epithelial cells in response to P. aeruginosa. Cultured human bronchial epithelial cells rapidly released IL-1beta in response to P. aeruginosa; this process was maximized by expression of WT-CFTR and dramatically muted in cells with DeltaF508-CFTR. The IL-1R antagonist blocked P. aeruginosa-induced NF-kappaB nuclear translocation. Oral inoculation via drinking water of IL-1R knockout mice resulted in higher rates of lung colonization and elevated P. aeruginosa-specific antibody titers in a manner analogous to that of CFTR-deficient mice. Overall, rapid IL-1 release and signaling through IL-1R represent key steps in the innate immune response to P. aeruginosa infection, and this process is deficient in cells lacking functional CFTR.

FIG. 1.

NF-κB nuclear translocation in sections of small airways from either WT or MyD88 knockout C57BL/6 mice determined at the indicated times after intratracheal infection with P. aeruginosa strain PAO1. Sections were stained for NF-κB (red), CFTR (green), and nuclear DNA (blue). First row: yellow/orange color represents overlap between cytoplasmic CFTR and cytoplasmic NF-κB. Second row: cytoplasmic NF-κB (red) and nuclear DNA (blue) only. Third and fourth rows: colocalization of translocated nuclear NF-κB (red) and nuclear DNA (blue), represented as pseudocolored magenta at two different magnifications. Magnifications are indicated on the right-hand side of each row.

FIG. 2.

NF-κB nuclear translocation in lung sections from the indicated strains of mice in response to intratracheal infection with P. aeruginosa strain PAO1 or strain PAO1 fliC::gent (ΔfliC) where indicated. Samples were taken 15 min postinfection for the WT mice infected with either PAO1 or the ΔfliC strain (second two columns) and for the TLR2 knockout (KO) and TLR4 dominant-negative (DN) mouse strains (C3H/HeJ) infected with P. aeruginosa PAO1. Maximal NF-κB nuclear translocation was seen at 75 min postinfection for the TNF-R1 knockout mouse strain infected with PAO1. Sections were stained for NF-κB (red), CFTR (green), and nuclear DNA (blue). First row: yellow/orange color represents overlap between cytoplasmic CFTR and cytoplasmic NF-κB. Second row: cytoplasmic NF-κB (red) and nuclear DNA (blue) only. Third and fourth rows: colocalization of translocated nuclear NF-κB (red) and nuclear DNA (blue), represented as pseudocolored magenta at two different magnifications. Magnifications are indicated on the right-hand side of each row.

FIG. 3.

NF-κB nuclear translocation in small airway sections of WT or IL-1R knockout (KO) mice at the indicated times in response to intratracheal infection with P. aeruginosa strain PAO1. Sections were stained for NF-κB (red), CFTR (green), and nuclear DNA (blue). First row: yellow/orange color represents overlap between cytoplasmic CFTR and cytoplasmic NF-κB. Second row: cytoplasmic NF-κB (red) and nuclear DNA (blue) only. Third and fourth rows: colocalization of translocated nuclear NF-κB (red) and nuclear DNA (blue), represented as pseudocolored magenta at two different magnifications. Magnifications are indicated on the right-hand side of each row.

FIG. 4.

IL-1β release in supernatants of WT-CFTR and ΔF508-CFTR human bronchial epithelial cells in response to P. aeruginosa strain PAO1. Panels A and B show results from two different experiments conducted under identical conditions but yielding somewhat different kinetics of IL-1β release. Asterisks indicate a significant difference (P < 0.05, t tests) in the levels of IL-1 released at the same time point when comparing WT-CFTR and ΔF508-CFTR cells.

FIG. 5.

Inhibition of NF-κB nuclear translocation by addition of IL-1ra to cultures of P. aeruginosa-infected human bronchial epithelial cells that express WT-CFTR. The left upper panel shows colocalization of red-stained NF-κB and blue-stained DNA in nuclei (pseudocolored magenta) 15 min postinfection with P. aeruginosa strain PAO1. In the presence of IL-1ra, NF-κB is present in the cytoplasm (middle upper panel, red) but does not translocate to the nucleus (right upper panel). Magnifications are ×400. The lower graph indicates the quantitative analysis of NF-κB nuclear translocation in isogenic cell lines with WT- or ΔF508-CFTR 15 min following P. aeruginosa infection. IL-1ra was added at the indicated amount of protein to triplicate assay wells (200-μl volume each). Specificity of binding of the nuclear NF-κB was assessed by inhibition with either a WT NF-κB probe or a mutant NF-κB probe added to the nuclear extract. P values represent both overall analysis of variance and pair-wise comparisons using Tukey's multiple comparisons test.

FIG. 6.

Oropharyngeal colonization of WT and IL-1R knockout mice after exposure to P. aeruginosa strain N6 in sterile drinking water. Mice (six per group) were administered nonabsorbable antibiotics (100 μg gentamicin/ml and 100 μg ceftazidime/ml) along with orally absorbable nitrofurantoin (100 μg/ml) in sterile drinking water prior to week 20 and after week 24 in order to distinguish between oropharyngeal colonization and lung infection. The probability of colonization for the IL-1R knockout mice throughout the study period was 93% whereas that for the WT mice was 17% (P < 0.001, generalized estimating equations).