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

Hara Y, Shiraishi A, Kobayashi T, Kadota Y, Shirakata Y, Hashimoto K, Ohashi Y - Mol. Vis. (2009)

Bottom Line: The effects of DEX and CsA on HCECs exposed to HSV-1 (McKrae strain) were also examined.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ehime University School of Medicine, Shitsukawa, Japan. yukoabc@m.ehime-u.ac.jp

ABSTRACT

Purpose: 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.

Conclusion: These results indicate that DEX may increase the susceptibility of HCECs to viral infections by altering the TLR3 signaling pathways.

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Related in: MedlinePlus

Cytokines and chemokines secreted by HCECs stimulated with poly(I:C) and cultured with or without DEX or CsA for 24 h. Culture medium was collected 24 hours after poly(I:C) stimulation and analyzed for the presence of IL-6, IL-8, and IFN-β protein by ELISA. The p values were calculated using two-tailed paired-tests, (*p<0.05, **p<0.01, ***p<0.001).
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f5: Cytokines and chemokines secreted by HCECs stimulated with poly(I:C) and cultured with or without DEX or CsA for 24 h. Culture medium was collected 24 hours after poly(I:C) stimulation and analyzed for the presence of IL-6, IL-8, and IFN-β protein by ELISA. The p values were calculated using two-tailed paired-tests, (*p<0.05, **p<0.01, ***p<0.001).

Mentions: Incubation with DEX down-regulated the poly(I:C)-induced expression of the mRNA of IL-6 about 0.4 fold with 10-6 M and 0.5 fold with 10-5 M of DEX (Figure 4). ELISA also showed that the poly(I:C) induced IL-6 production was decreased about 0.6 fold with 10-6 M and 0.5 fold with 10-5 M of DEX (Figure 5). The poly(I:C)-induced expressions of the mRNA and proteins of IL-8 were more significantly down-regulated by DEX, and the decrease was dose-dependent. Real-time PCR showed that the expression of the mRNA of IL-8 was down-regulated about 0.4 fold with 10-6 M and 0.3 fold with 10-5 M of DEX. ELISA also showed a reduced production of IL-8 protein of about 0.5 fold with 10-6 M and 0.4 fold with 10-5 M of DEX (Figure 4 and Figure 5). DEX also down-regulated the poly(I:C)-induced mRNA expression of IFN-β by about 0.5 fold with 10-6 M and 10-5 M of DEX and decreased IFN-β production by about 0.6 fold with 10-6 M and 0.5 fold with 10-5 M of DEX (Figure 4 and Figure 5).


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

Hara Y, Shiraishi A, Kobayashi T, Kadota Y, Shirakata Y, Hashimoto K, Ohashi Y - Mol. Vis. (2009)

Cytokines and chemokines secreted by HCECs stimulated with poly(I:C) and cultured with or without DEX or CsA for 24 h. Culture medium was collected 24 hours after poly(I:C) stimulation and analyzed for the presence of IL-6, IL-8, and IFN-β protein by ELISA. The p values were calculated using two-tailed paired-tests, (*p<0.05, **p<0.01, ***p<0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683030&req=5

f5: Cytokines and chemokines secreted by HCECs stimulated with poly(I:C) and cultured with or without DEX or CsA for 24 h. Culture medium was collected 24 hours after poly(I:C) stimulation and analyzed for the presence of IL-6, IL-8, and IFN-β protein by ELISA. The p values were calculated using two-tailed paired-tests, (*p<0.05, **p<0.01, ***p<0.001).
Mentions: Incubation with DEX down-regulated the poly(I:C)-induced expression of the mRNA of IL-6 about 0.4 fold with 10-6 M and 0.5 fold with 10-5 M of DEX (Figure 4). ELISA also showed that the poly(I:C) induced IL-6 production was decreased about 0.6 fold with 10-6 M and 0.5 fold with 10-5 M of DEX (Figure 5). The poly(I:C)-induced expressions of the mRNA and proteins of IL-8 were more significantly down-regulated by DEX, and the decrease was dose-dependent. Real-time PCR showed that the expression of the mRNA of IL-8 was down-regulated about 0.4 fold with 10-6 M and 0.3 fold with 10-5 M of DEX. ELISA also showed a reduced production of IL-8 protein of about 0.5 fold with 10-6 M and 0.4 fold with 10-5 M of DEX (Figure 4 and Figure 5). DEX also down-regulated the poly(I:C)-induced mRNA expression of IFN-β by about 0.5 fold with 10-6 M and 10-5 M of DEX and decreased IFN-β production by about 0.6 fold with 10-6 M and 0.5 fold with 10-5 M of DEX (Figure 4 and Figure 5).

Bottom Line: The effects of DEX and CsA on HCECs exposed to HSV-1 (McKrae strain) were also examined.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Ehime University School of Medicine, Shitsukawa, Japan. yukoabc@m.ehime-u.ac.jp

ABSTRACT

Purpose: 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.

Conclusion: These results indicate that DEX may increase the susceptibility of HCECs to viral infections by altering the TLR3 signaling pathways.

Show MeSH
Related in: MedlinePlus