Figure 6. Pathogenic but not non-pathogenic isolates of Acanthamoeba upregulate TLR4 and CXCL2 mRNA, and induce CXCL2 secretion in Chinese hamster corneal epithelial (HCORN) cells.
HCORN cells were stimulated with or without pathogenic (Clinical) isolates, A. castellanii (1×105 cells/ml) and A. culbertsoni (1×105 cells/ml), and non-pathogenic (Soil) isolates, A. castellanii Neff (1×105 cells/ml) and A. astronyxis (1×105 cells/ml), and LPS (10 μg/ml) for 24 hours incubation. Cells cultured without the trophozoites served as a control. Cells and supernatants were collected. Total RNA was isolated from cells and RT-PCR was performed to examine the mRNA expression of TLR2, TLR4, and CXCL2. Quantification was calculated by densitometry of bands relative to GAPDH. Densitometry of mRNA bands were quantified by three independent scanned presented as mean±SEM (6A – 6C). Supernatants from the harvested cells were subjected to CXCL2 specific ELISA (6D). The data are mean±SEM of three independent experiments. Asterisk indicates P value < 0.05 by unpaired Student’s t-test.