Hara Yuko, Shiraishi Atsushi, Kobayashi Takeshi, Kadota Yuko, Shirakata Yuji, Hashimoto Koji, Ohashi Yuichi
Department of Ophthalmology, Ehime University School of Medicine, Shitsukawa, Japan.
Mol Vis. 2009 May 8;15:937-48.
The toll-like receptor 3 (TLR3) recognizes viral double-stranded RNA and its synthetic analog polyriboinosinic-polyribocytidylic acid (poly(I:C)), and the activation of TLR3 is known to induce the production of type I interferon (IFN) and inflammatory cytokines/chemokines. The purpose of this study was to determine the role played by innate responses to a herpes simplex virus 1 (HSV-1) infection of the corneal epithelial cells. In addition, we determined the effects of immunosuppressive drugs on the innate responses.
Cultured human corneal epithelial cells (HCECs) were exposed to poly(I:C), and the expressions of the mRNAs of the cytokines/chemokines macrophage-inflammatory protein 1 alpha (MIP1-alpha), macrophage-inflammatory protein 1 beta (MIP1-beta), interleukin-6 (IL-6), interleukin-8 (IL-8), regulated on activation, normal T cell expressed and secreted (RANTES), Interferon-beta (IFN-beta), and TLR3 were determined using real-time reverse transcription-polymerase chain reaction (RT-PCR). The effects of dexamethasone (DEX, 10(-6) or 10(-5) M) and cyclosporine A (CsA, 10(-6) or 10(-5) M) on the expression of these cytokines and TLR3 were also determined using real-time RT-PCR. Levels of MIP1-alpha, MIP1-beta, IL-6, IL-8, RANTES, and IFN-beta were measured using the enzyme-linked immunosorbent assay (ELISA). The activation of nuclear factor kappa B (NFkappaB) and interferon regulatory factor 3 (IRF3) in HCECs was assessed by immunohistochemical staining. The effects of DEX and CsA on HCECs exposed to HSV-1 (McKrae strain) were also examined.
The expressions of MIP1-alpha, MIP1-beta, IL-6, IL-8, RANTES, IFN-beta, and TLR3 were up-regulated in HCECs exposed to poly(I:C). The poly(I:C)-induced expressions of IL-6 and IL-8 were down-regulated by both DEX and CsA, while the expressions of IFN-beta and TLR3 were suppressed by DEX alone. Similarly, the poly(I:C)-induced activation of NFkappaB was decreased by both DEX and CsA, and the activation of IRF3 was reduced by DEX alone. When HCECs were inoculated with HSV-1, DEX led to a decrease in the expression of IL6, IFN-beta, and TLR3, and an extension of plaque formation.
These results indicate that DEX may increase the susceptibility of HCECs to viral infections by altering the TLR3 signaling pathways.
Toll样受体3（TLR3）可识别病毒双链RNA及其合成类似物聚肌苷酸-聚胞苷酸（聚（I:C）），已知TLR3的激活可诱导I型干扰素（IFN）以及炎性细胞因子/趋化因子的产生。本研究的目的是确定角膜上皮细胞对单纯疱疹病毒1（HSV-1）感染的固有免疫反应所起的作用。此外，我们还确定了免疫抑制药物对固有免疫反应的影响。
将培养的人角膜上皮细胞（HCECs）暴露于聚（I:C），使用实时逆转录-聚合酶链反应（RT-PCR）测定细胞因子/趋化因子巨噬细胞炎性蛋白1α（MIP1-α）、巨噬细胞炎性蛋白1β（MIP1-β）、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）、活化调节正常T细胞表达和分泌因子（RANTES）、干扰素-β（IFN-β）以及TLR3的mRNA表达。还使用实时RT-PCR测定地塞米松（DEX，10⁻⁶或10⁻⁵M）和环孢素A（CsA，10⁻⁶或10⁻⁵M）对这些细胞因子和TLR3表达的影响。使用酶联免疫吸附测定（ELISA）测量MIP1-α、MIP1-β、IL-6、IL-8、RANTES和IFN-β的水平。通过免疫组织化学染色评估HCECs中核因子κB（NFκB）和干扰素调节因子3（IRF3）的激活情况。还检测了DEX和CsA对暴露于HSV-1（McKrae株）的HCECs的影响。
暴露于聚（I:C）的HCECs中，MIP1-α、MIP1-β、IL-6、IL-8、RANTES、IFN-β和TLR3的表达上调。DEX和CsA均下调了聚（I:C）诱导的IL-6和IL-8的表达，而仅DEX抑制了IFN-β和TLR3的表达。同样，DEX和CsA均降低了聚（I:C）诱导的NFκB的激活，而仅DEX降低了IRF3的激活。当HCECs接种HSV-1时，DEX导致IL6、IFN-β和TLR3的表达降低以及噬斑形成的扩大。
这些结果表明，DEX可能通过改变TLR3信号通路增加HCECs对病毒感染的易感性。
