TOLL-LIKE RECEPTOR-4 INNITIATES AN INFLAMMATORY IMMUNE RESPONE IN ACANTHAMOEBA KERATITIS

Purpose: The purpose of this study is to determine if Toll-like receptors (TLRs) on corneal epithelial cells are responsible for recognition of Acanthamoeba trophozoites. We hypothesize that TLR-4 recognizes the parasite and activates an initiate inflammatory responses. Methods: Human corneal epithelial (HCE), Chinese hamster corneal epithelial (HCORN) cells, or Human embryonic kidney (HEK 293) cells were cultured until confluent. The cells were then stimulated with either 1X 105/mL Acanthamoeba castellanii trophozoites, ultrapure lipopolysaccharides (LPS) at 10µg/mL as a positive control, or left un-stimulated for 24 hours. Once the incubation period was complete, the supernatant was removed and used for ELISA analysis for the production of IL-8 or CXCL2. RNA was collected from each experimental group and RT-PCR was performed using TLR-4 and chemokine primers. A TLR-4 neutralizing antibody was pre-incubated with the corneal cells 1 hr. prior to stimulation, the supernatant was collected for quantification of IL-8 production. Immunocytochemistry was used to visualize the TLR-4 protein on the cell membrane. The pathogenic Acanthamoeba castellanii and non-pathogenic Acanthamoeba neff strains were also compared utilizing this methodology. Results: Up-regulation of TLR-4 mRNA expression was seen in HCE cells and HCORN cells 24 hrs. after treatment. Increased production of cytokine/chemokine mRNA and IL-8 or CXCL2 protein was observed in the cells that were stimulated with Acanthamoeba castellanii trophozoites compared to un-stimulated control cells. This increase, was mitigated when a TLR-4 neutralizing antibody was added to the culture 1 hr. prior to treatment. We found that only the pathogenic Acanthamoeba castellanii strain up-regulates TLR-4 mRNA and chemokine production. Conclusions: These findings suggest that TLR-4 is responsible for the signaling cascade involved in the inflammatory response to Acanthamoeba infection. This cascade begins with the recognition of Acanthamoeba and leads to the production of cytokines and chemokines which attract neutrophils to the site of infection to combat the parasite.