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

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.

Figure 1

Expression of the mRNAs of cytokines and chemokines by HCEs exposed to poly(I:C), a TLR3 ligand. Total RNA was isolated from HCECs at 6, 12, and 24 h after poly(I:C) exposure, and the expressions of the mRNAs of MIP1-α, MIP1-β, IL-6, IL-8, RANTES, and IFN-β were determined by real-time PCR. The relative level of expression of each cytokine and chemokine mRNA is normalized to the level of G3PDH mRNA expression. The p values were calculated using two-tailed paired t-tests, (*p<0.05, **p<0.01, ***p<0.001).

Figure 2

Cytokines and chemokines secreted by HCECs treated with poly(I:C). Culture medium was collected at 6, 12, and 24 h after poly(I:C) stimulation and analyzed for MIP1-α, MIP1-β, IL-6, IL-8, RANTES, and IFN-β protein by ELISA. The p values were calculated using two-tailed paired t-tests (*p<0.05, **p<0.01, ***p<0.001).

Figure 3

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).

Figure 4

Effect of DEX and CsA on the expression of cytokines and chemokines by HCEs treated with poly(I:C). Total RNA was isolated from HCECs cultured with or without of DEX or CsA for 24 h and stimulated with poly(I:C) for 24 h. The expressions of the mRNAs of IL-6, IL-8, and IFN-β were determined by real-time PCR. The relative level of expression of each cytokine and chemokine mRNA is normalized to the level of G3PDH mRNA expression. The p values were calculated using two-tailed paired-tests (*p<0.05, **p<0.01, ***p<0.001).

Figure 5

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).

Figure 6

Immunohistochemical staining for NFκB and IRF3 in HCECs stimulated with poly(I:C) and cultured with or without of DEX or CsA for 24 h. NFκB p65 staining without poly(I:C, A), with poly(I:C, B), with DEX 10^-5M and poly(I:C, C), and with CsA 10^-5M and poly(I:C, D). IRF3 staining without poly(I:C, E), with poly(I:C, F), with DEX 10-5M and poly(I:C, G), and with CsA 10-5M and poly(I:C, H). Scale bar, 100 µm. Activated NFκB p65 and IRF-3 were clearly detected in the nuclei of most of cultured HCECs 3 h after stimulation by poly(I:C, B and F). In the presence of DEX, NFκB p65 and IRF-3 were detected in the nuclei of some HCECs but only in the cytosol of other HCECs (C, G). In the presence of CsA, NFκB p65 staining was detected in more HCEC nuclei after exposure to CsA than to DEX (D), while IRF3 was detected only in the nuclei of cultured HCECs (H).

Figure 7

Effect of DEX and CsA on Herpes simplex virus 1 (HSV-1) infection. HCECs were cultured in the presence or absence of DEX (10^-5 M), and inoculated with 50 MOI of HSV-1 for 48 h. The plaque area was increased when HCECs were pre-incubated with DEX (A). Real-time PCR results show a significantly higher level of HSV-1 DNA in the supernatant with DEX (B). (*p<0.05)

Figure 8

Effect of DEX and CsA on involvement of TLR3 signaling systems in HSV-1 infection of HCECs. HCECs were cultured in the presence or absence of DEX (10^-5 M) and inoculated with 1,000 MOI of HSV-1 for 24 h. Real-time PCR shows that IFN-β and TLR3 expression is down-regulated by DEX. IL-6 and IL-8 are also down-regulated, although the decrease of IL-8 was not statistically significant. (*p<0.05)