Involvement of Corneal Epithelial Cells in the TH17 Response in an In Vitro Bacterial Inflammation Model

Abstract

Purpose: : Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA) are very often the cause of bacterial keratitis, an inflammatory process that can lead to vision loss. We wanted to determine the degree of involvement of corneal epithelial cells in the T_H17 response in an in vitro bacterial inflammation model, verifying the expression of cytokines involved in this signalling pathway: IL-6, IL-17, and their specific receptors.

Methods: : Bacterial supernatants were prepared from SA and PA cultures. The HCE cell line (Human Corneal Epithelial, Araki-Sasaki et al, IOVS 1995) was stimulated with both SA and PA supernatants in different dilutions (1:100 and 1:50). Cell culture supernatants were harvested after different stimulation times (6, 24, and 72 h) and protein and RNA were isolated. Expression levels of protein and mRNA for IL-6, the soluble receptor for IL-6 (sIL-6R), and the soluble gp130 signalling subunit (sgp130), IL-17 and its main receptor (IL-17RA) were determined by ELISA, immunofluorescence, Western blotting, or real time RT-PCR (RT²-PCR) respectively.

Results: : HCE cells secreted IL-6 in a time-dependent manner. Expression of IL-6 was significantly increased in SA-stimulated cells, but not in PA-stimulated cells after incubation for 6 h. IL-6 mRNA expression was rapidly increased in stimulated cells and dropped to control mRNA levels after 72 h. HCE cells secreted sIL-6R and sgp130 in a time-dependent manner, without statistically significant differences. HCE cells secreted IL-17 in a time-dependent manner, reaching lower levels than IL-6. IL-17RA was present in HCE cell cytosol and nucleus, as determined by immunofluorescence. The quantification of IL-17RA by Western blotting showed only statistically significant differences in 1:50 dilution-stimulated cells.

Conclusions: : Corneal epithelial cells are able to react against bacterial inflammation by secreting IL-6, IL-6 soluble receptor, gp130 soluble mediator, IL-17 and its main receptor IL-17RA. All these molecules are implicated in the T_H17 differentiation pathway. This leads us to think that corneal epithelial cells may act as indirect participants in the T_H17 signalling pathway.

Keywords: cornea: epithelium • inflammation • cytokines/chemokines