Background Toll-like receptors play an important role in the human immune system. This study was conducted to investigate the expression profiles and function of Toll-like receptor （TLR）1-9 in human corneal epithelium. Methods The expression of TLR1-9 mRNA in 20 human donor corneal epithelia samples abraded during photorefractive keratotomy （PRK） and cultivated telomerase-immortalized human corneal epithelial cells （THCEs） was examined by semi-quantitative reverse-transcriptase polymerase chain reaction （RT-PCR） analysis. Human peripheral blood mononuclear cells （PBMCs） were used as positive controls. The expression of the TLR2 and TLR4 proteins was detected by Western analysis. ELISA was used to detect IL-8 secretion from THCEs challenged with ligands for TLR3 and TLR4 with and without antibody blockade. Results The expression of TLR1-9 at the mRNA level was detected in the epithelia of 20 patients and in THCE. Significant differences among individuals were observed. One patient was found to lack of the expression of TLR3, 4, 6 and 8, whereas another did not express TLRS. The expression of TLR2 and TLR4 protein was detected in human corneal epithelial cells. As THCE cells express TLR1-9, cells were challenged with lipopolysaccharides （LPS） and poly I：C to determine whether TLR4 and TLR3 were functional. The results showed that secretion of IL-8 by cells stimulated with LPS and Poly I：C was 7 to 10 fold greater than secretion by unchallenged cells. Blocking TLR4 with an anti-TLR4 antibody significantly inhibited the LPS-induced IL-8 production by THCE （P〈0.05）. Conclusion Human corneal epithelial cells express multiple TLRs and are able to recognize LPS and poly I：C. Different expression profiles among individuals suggest that differences in the susceptibilities and sensitivities to bacterial and viral infection in human populations relate to different patterns of TLR expression.
The cornea is a highly specialized and unique organ in the human body. Its main function is to project light from the external environment onto the retina, and it has a specific transparency to perform its function properly. The transparency and integrity of the cornea is of vital importance. The corneal wound, especially laceration deep to Bowman＇s membrane and stroma, which will inevitably cause scar formation, may cause the degeneration or even loss of sight.
Background Cornea epithelial cells play eady and crucial roles in the initiation of ocular surface responses to pathogens. Participation of toll-like receptor （TLR） 2 and TLR4, which are major forms of fungi receptors, may be involved in Aspergillus fumigatus induced immune responses. The objective of the present study was to examine whether inactive Aspergillus fumigatus conidia induce NF-KB activation and production of proinflammaory cytokines, and whether the expression of TLR2 and TLR4 were amplified by conidia in cultured immortalized human corneal epithelial cells （THCEs）. This may contribute to our knowledge of the mechanism by which the host cornea can successfully defend against invasive fungi. Methods Aspergillus fumigatus conidia were used to challenge THCE cells. THCE cells were harvested after 0.5, 1, 2 or 4 hours incubation. Real-time quantitative PCR was performed to determine the expression of TLR2, TLR4, TNF-a and IL-8. Western blotting was performed to determine the expression of NF-KB. Enzyme-linked immunosorbent assay （ELISA） was performed to determine the expression of TNF-a and IL-8. And the release of TNF-a and IL-8 in the cell supematant were also assessed by ELISA with or without pretreatment with TLR2 and TLR4 neutralizing antibodies. Results Aspergillus fumigatus conidia elicited the expression of TLR2, TLR4, TNF-a and IL-8 mRNA in THCEs. Exposure of THCE cells to Aspergillus fumigatus conidia resulted in NF-KB activation, which increased at 30 minutes （increased from 11.35±2.74 in the controls to 19.12±3.48, p〈0.05） and thereafter increased steadily up to 4 hours after challenge （P 〈0.01）. Concomitant with NF-KB activation, secretion of TNF-α and IL-8 in conidia-challenged cells was increased in a time-dependent manner. Incubation of THCE cells with TLR2 antibody or TLR4 antibody before conidia challenge resulted in inhibition of conidia-induced TNF-α and IL-8 secretion （P 〈0.05）, TLR2 antibody and TLR4 antibody together significantly increased inhibit
