Roles Played by Toll-like Receptor-9 in Corneal Endothelial Cells after Herpes Simplex Virus Type 1 Infection

PURPOSE. To determine the roles played by toll-like receptor 9 (TLR9) in cultured human corneal endothelial (HCEn) cells after herpes simplex virus type 1 (HSV-1) infection and to characterize the TLR9-mediated antiviral responses. METHOD. Immortalized HCEn cells were examined for TLR expression. The upregulation of inflammatory cytokines after HSV-1 infection was determined by real-time RT-PCR or protein array analyses. The TLR9-mediated HSV-1 replication was determined by real-time PCR and plaque assay. To determine whether there was an activation of the signal transduction pathway, HCEn cells that were transfected with pathway-focused transcription factor reporters were examined for promoter activity. RESULTS. TLR9 was abundantly expressed intracellularly in HCEn cells. The CpG oligonucleotide, a TLR9 ligand, stimulated the NF-kappa B activity in HCEn cells. HSV-1 infection also stimulated NF-kappa B and induced NF-kappa B-related inflammatory cytokines, including RANTES, IP-10, MCP-2, MIF, MCP-4, MDC, MIP-3 alpha, IL-5, TARC, MCP-1, and IL-6. The induction of these cytokines was significantly reduced by blocking the activity of TLR9. In addition, viral replication in HCEn cells was significantly reduced by the inhibition of TLR9, but was preserved by a concomitant activation of the NF-kappa B cascade. Of the different HSV-1-induced inflammatory cascade-related transcription factors, TLR9 was found to activate NF-kappa B, cyclic AMP response element (CRE), and the CCAAT-enhancer-binding proteins (C/EBP) the most. CONCLUSIONS. Corneal endothelial cells transcriptionally initiate inflammatory programs in response to HSV-1 infection related to NF-kappa B, CRE, and C/EBP and express arrays of inflammatory cytokine induction by TLR9. On the other hand, HSV-1 exploits TLR9-mediated NF-kappa B activation for its own replication. (Invest Ophthalmol Vis Sci. 2011; 52: 6729-6736) DOI: 10.1167/iovs.11-7805