Hypoxia Activates Nuclear Translocation of NF-kB and Increases Expression of Its Receptor CFTR Along With Binding and Internalization of P. aeruginosa in Intact Corneal Tissues

Abstract

Abstract: : Purpose: P. aeruginosa utilizes the cystic fibrosis transmembrane conductance regulator (CFTR) as a receptor for corneal cell uptake, a process that allows these organisms to be protected from host defense mechanisms when they gain access to sub-surface epithelial cells following corneal injury. Additionally, inflammation is a major cause of tissue injury, involving NF-kB regulated inflammatory cytokines. P. aeruginosa is also a common cause of contact lens keratitis, but how this situation promotes infection in the absence of overt corneal injury is unclear. We hypothesized that hypoxia might affect CFTR expression and also perturb the expression of NF-kB leading to increased microbial adherence and corneal cell uptake and dysregulation of the inflammatory response on the corneal surface which could exacerbate damage from inflammation. Methods: Organ cultures of rabbit eyes, human corneal rims or cultures of human corneal cells were incubated for 24-72 hours in 15% O₂ :80%N₂:5%CO₂ or atmospheric O₂ plus 5% CO₂. A clinical isolate of P. aeruginosa was applied to the eye tissue or cells for 30 min. Corneas were excised from tissues and sectioned for staining while cell cultures were fixed and stained. CFTR expression and nuclear translocation of NF-kB were evaluated by confocal microscopy after reactions with specific antibodies and nuclear stains. Adherence and internalization were evaluated by measuring direct binding after 30 min. and internalization after 3 hr using gentamicin-exclusion assays. Results: There was increased expression of CFTR and translocation of NF-kB in intact tissues of human and rabbit corneal epithelial cells incubated in 15% O₂ compared to cells kept at atmospheric O₂. There was also a 3.5-fold increase in P. aeruginosaadherence and over 2-fold increase in internalization on otherwise undamaged corneal surfaces. Conclusion: Hypoxia disrupts the corneal epithelium, increasing adherence and ingestion of P. aeruginosain conjunction with increased CFTR expression. Hypoxia also causes nuclear translocation of NF-kB, which likely exacerbates inflammatory damage and potentially also compromises the innate immune response to P. aeruginosa.

Keywords: 428 hypoxia • 449 keratitis • 367 contact lens