Alteration of TLR3 pathways by glucocorticoids may be responsible for immunosusceptibility of human corneal epithelial cells to viral infections.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
These results indicate that DEX may increase the susceptibility of HCECs to viral infections by altering the TLR3 signaling pathways.
Hara
Yuko
Y
Department of Ophthalmology, Ehime University School of Medicine, Shitsukawa, Japan. yukoabc@m.ehime-u.ac.jp
Shiraishi
Atsushi
A
Kobayashi
Takeshi
T
Kadota
Yuko
Y
Shirakata
Yuji
Y
Hashimoto
Koji
K
Ohashi
Yuichi
Y
D002478:Cells, Cultured; D016572:Cyclosporine / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D003627:Data Interpretation, Statistical; D003907:Dexamethasone / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism / Q000821:virology*; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D005938:Glucocorticoids / Q000494:pharmacology*; D018259:Herpesvirus 1, Human / Q000502:physiology*; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D011070:Poly I-C / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D051196:Toll-Like Receptor 3 / Q000378:metabolism*
D016207:Cytokines; D005938:Glucocorticoids; C494232:IRF3 protein, human; D050838:Interferon Regulatory Factor-3; D016328:NF-kappa B; D012333:RNA, Messenger; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D003907:Dexamethasone; D016572:Cyclosporine; D011070:Poly I-C