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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

Effect of DEX and CsA on the expression of TLR3 by HCECs exposed to poly(I:C). Total RNA was isolated from HCECs at 6, 12, and 24 h after poly(I:C) stimulation (A), or from HCECs cultured with or without of DEX (B) or CsA (C) for 24 h and stimulated with poly(I:C) for 24 h. The expression of the mRNA of TLR3 was determined by real-time PCR. The relative level of expression of each cytokine and chemokine mRNA is normalized against G3PDH mRNA expression. The p values were calculated using two-tailed paired t-tests (*p<0.05, **p<0.01, ***p<0.001).
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f3: Effect of DEX and CsA on the expression of TLR3 by HCECs exposed to poly(I:C). Total RNA was isolated from HCECs at 6, 12, and 24 h after poly(I:C) stimulation (A), or from HCECs cultured with or without of DEX (B) or CsA (C) for 24 h and stimulated with poly(I:C) for 24 h. The expression of the mRNA of TLR3 was determined by real-time PCR. The relative level of expression of each cytokine and chemokine mRNA is normalized against G3PDH mRNA expression. The p values were calculated using two-tailed paired t-tests (*p<0.05, **p<0.01, ***p<0.001).

Mentions: The supernatants of the culture media were collected at 0, 6, 12, and 24 h, and the levels of MIP1-α, MIP1-β, IL-6, IL-8, RANTES, and IFN-β was evaluated using ELISA. The levels of MIP1-α, MIP1-β, and RANTES in the supernatant were elevated from undetectable levels at 0 h to 57.6 pg/ml, 630 pg/ml, and 1748.7 pg/ml, respectively, at 24 h after poly(I:C) stimulation. There was a slight but not significant elevation without poly(I:C) stimulation. The levels of IL-6 and IL-8 were slightly elevated without poly(I:C) stimulation, but were significantly elevated to 390 pg/ml and 920 pg/ml, respectively, at 24 h after poly(I:C) stimulation. The level of IFN-β was elevated to 43.8 pg/ml by poly(I:C) after 24 h, and no production of IFN-β was found without poly(I:C) stimulation (Figure 3).


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)

Effect of DEX and CsA on the expression of TLR3 by HCECs exposed to poly(I:C). Total RNA was isolated from HCECs at 6, 12, and 24 h after poly(I:C) stimulation (A), or from HCECs cultured with or without of DEX (B) or CsA (C) for 24 h and stimulated with poly(I:C) for 24 h. The expression of the mRNA of TLR3 was determined by real-time PCR. The relative level of expression of each cytokine and chemokine mRNA is normalized against G3PDH mRNA expression. The p values were calculated using two-tailed paired t-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

f3: Effect of DEX and CsA on the expression of TLR3 by HCECs exposed to poly(I:C). Total RNA was isolated from HCECs at 6, 12, and 24 h after poly(I:C) stimulation (A), or from HCECs cultured with or without of DEX (B) or CsA (C) for 24 h and stimulated with poly(I:C) for 24 h. The expression of the mRNA of TLR3 was determined by real-time PCR. The relative level of expression of each cytokine and chemokine mRNA is normalized against G3PDH mRNA expression. The p values were calculated using two-tailed paired t-tests (*p<0.05, **p<0.01, ***p<0.001).
Mentions: The supernatants of the culture media were collected at 0, 6, 12, and 24 h, and the levels of MIP1-α, MIP1-β, IL-6, IL-8, RANTES, and IFN-β was evaluated using ELISA. The levels of MIP1-α, MIP1-β, and RANTES in the supernatant were elevated from undetectable levels at 0 h to 57.6 pg/ml, 630 pg/ml, and 1748.7 pg/ml, respectively, at 24 h after poly(I:C) stimulation. There was a slight but not significant elevation without poly(I:C) stimulation. The levels of IL-6 and IL-8 were slightly elevated without poly(I:C) stimulation, but were significantly elevated to 390 pg/ml and 920 pg/ml, respectively, at 24 h after poly(I:C) stimulation. The level of IFN-β was elevated to 43.8 pg/ml by poly(I:C) after 24 h, and no production of IFN-β was found without poly(I:C) stimulation (Figure 3).

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