Involvement Of Toll-like Receptors And Nuclear Factor-κB In Innate Immune Response Of Human Corneal Epithelial Cells To HSV-1 Infection

Herpetic epithelial and stromal keratitis is a sight-threatening ocular infection. Herpes simplex virus type 1 (HSV-1) has been identified to be the major pathogen of this disease. The corneal epithelial cells constitute the first line of defense against microbial infection and, therefore, must possess the ability to sense the presence of viruses. In reponse to HSV-1 infection, the epithelial cells may produce proinflammatory cytokines and interferons (IFNs) to enhance antiviral activity of residential cells of the cornea. However, the mechanism by which initiation of the inflammatory response to HSV-1 infection still remains to be elucidated. Recently, the identification of Toll-like Receptors (TLRs) leads to a brand new field where this receptor family is found to initiate the specific innate immune response to microbial infections by recognizing the pathogen-associated molecular patterns (PAMPs), and plays a crucial role in engaging differential signaling pathways, and as well as linking innate and acquired immunity closely.In current study, we used a telomerase-immortalized HCE cell line, HUCL, and primary human corneal epithelial cells (HCECs) as an in vitro model and infected the cells with HSV-1 (KOS strain). We performed a pilot study to screen the expression pattern of TLRs in HUCL cells by using reverse transcription polymerase chain reaction (RT-PCR) analysis. The data showed for the first time that most of members (TLR1, 2, 3, 4, 5, 6, 8, and 9) of TLR family were predominantly expressed in these cells, whereas no transcript of TLR7 and TLR10 was observed in these cells. Then, we investigated whether the expression of TLRs was influenced after HSV-1 challenge to HUCL cells. Surprisingly, the data reveals that TLR7 was dramatically induced by HSV-1 infection in epithelial cells and this, to date, is the first report of induced-TLR7 expression in non-myeloid cells. In addition, the induction of TLR7 could not be blocked, or even decreased with treatment of a TLR7-specific small interfering RNA (siRNA), suggesting HSV-1 infection was a strong stimulus to TLR7 induction. While TLR3 which predominantly expresses in non-infected HUCL cells was downregulated coincident with the induction of TLR7 by viral infection. Although the exact mechanism by which these TLRs were affected by HSV-1 infection is not clear yet, it was still suggested that HSV-1 infection indeed anddifferentially affects the expression of TLRs in HCECs, therefore mediates profound inflammatory response to viral infection.To study the role of epithelia in specific innate response to HSV-1 infection of the cornea, we determined the levels of the major components of three different signaling transduction pathways by Western blotting assay. We observed that HSV-1 infection activated nuclear factor-kappaB (NF-kB), mitogen-activated protein kinase (MAP kinase) and phosphoinositide-3-OH kinase (PI3 Kinase) signaling pathways in HCECs and resulted in a two-phase activation. Concomitant with the first peak of these signaling pathways activation, transcriptional expression of IL-6, IL-8, TNF-a and IL-lp were rapidly induced in HSV-1 infected cells. Subsequent secretion of IL-6, IL-8, and TNF-a was also observed in these cells. The induced expression and secretion of these cytokines and chemokines were dependent of NF-kB signaling pathway verified by treatments with NF-kB specific inhibitors.Our data also showed that HSV-1 infection induced sequential expression of interferons, IFN-p1 followed by IFN-a. More interestingly, IFN-y was found to be induced at late phase of HSV-1 infection in HUCL cells and this is the first time that IFN-y was reported to express in epithelial cells. The induction of TLR7 by HSV-1 infection was in coincidence with the upregulation of IFN-a. With the treatment of specific inhibitors, our data indicated that HSV-1 induced expression of TLR7 was in a NF-kB dependent and MAPK pathway independent manner. Finally, while accumulation of IFN-a/p in culture media was not detectable by ELISA analysis, functional blocking of their receptor resulted in reduced replication of viruses in HCECs, suggesting a type IIFN mediated autocrine/paracrine signaling in infected epithelial cells.Thus, in response to HSV-1 infection, corneal epithelial cells may produce early response proinflammatory cytokines leading to infiltration and also release of IFNs to enhance the antiviral activity in the cornea through either sequential activation or inactivation of TLRs. This study provides us a new insight of innate immune response of epithelia cells to viral infection.