Recognition of Corynebacterium pseudodiphtheriticum by Toll-like receptors and up-regulation of antimicrobial peptides in human corneal epithelial cells.

Bacterial keratitis is a major cause of corneal ulcers in developing and industrialized nations. In this study, we examined the host innate immune responses to Corynebacterium pseudodiphtheriticum, often overlooked as commensal, in human corneal epithelial cells. The expressions of innate immune mediators were determined by quantitative PCR from corneal ulcers of patients and immortalized human corneal epithelial cells (HCEC). We have found an elevated expression of Toll like receptors (TLRs) along with IL-6 and IL-1ß from both ulcers and epithelial cells infected with C. pseudodiphtheriticum. Activation of NF-κB and MAPK signaling pathways were also observed in HCEC in response to C. pseudodiphtheriticum. In addition, we found a significant increase in the expression of antimicrobial peptides S100A8, S100A9 and human ß-defensin 1 from both corneal ulcers and HCEC.

Mutations in the Corneal Endothelial Dystrophy-Associated Gene SLC4A11 Render the Cells More Vulnerable to Oxidative Insults.

PURPOSE: To investigate the effect of mutations in SLC4A11 on cellular localization of the protein, mitochondrial function, and apoptosis due to oxidative stress. Mutations in SLC4A11 have been associated with 2 different forms of corneal endothelial dystrophy that lead to degeneration of the corneal endothelium, causing opacity of the cornea and gradual vision loss. METHODS: HEK 293 cells were transfected with wild-type SLC4A11 or mutants, Ser213Leu, Arg233Cys, Gly418Asp, and Thr584Lys, and exposed to oxidative stress. Cellular localization of the proteins was detected by confocal microscopy, whereas mitochondrial dysfunction, reactive oxygen species (ROS) generation, and apoptosis were analyzed by flow cytometry and a colorimetric assay. Expressions of antioxidant genes were quantitated by real-time polymerase chain reaction. RESULTS: Although wild-type SLC4A11 was localized on the cell membrane, mutant proteins were found diffused in the cytoplasm. Mutations in SLC4A11 caused an increase in generation of ROS and mitochondrial dysfunction due to oxidative stress. NRF2, HO-1, and NQO expression decreased significantly, and a higher rate of apoptosis was detected in cells with mutant proteins under oxidative stress. CONCLUSIONS: Our data suggest that mutations in SLC4A11 cause retention of the protein in the cytoplasm and generate increased reactive oxygen species. We found that cells containing mutant SLC4A11 are more vulnerable to oxidative and mitochondrial damage, less able to overcome oxidative stress through the expression of sufficient levels of antioxidant genes, and are more prone to apoptotic death.