Investigation On Toll-Like Receptors Mediated Inflammatory Responses In Corneal Fungal Infection And Treatment By Gene Silencing

PartⅠ:TLRs-mediated innate responses of corneal epithelial cells against Aspergillus fumigatus challengePurpose. To determine the responses of Toll-like receptor (TLR) 2 and TLR4 to their ligands in corneal epithelial cells and their roles in mediating inflammatory responses against Aspergillus fumigatus challenge.Methods. Telomerase-immortalized human corneal epithelial cells (THCE) were challenged by TLR2 ligand zymosan, TLR4 ligand LPS and Aspergillus fumigatus hyphae, respectively. Culture media collected at different time points (1h,3h,6h,12h) was subjected to cytokine ELISA to detect the levels of IL-1βand IL-6. THCEs treated with TLR2- or TLR4-siRNA plasmid to knock down TLR2 and TLR4 expression were challenged with Aspergillus fumigatus hyphae. Culture media were collected at different time points (1h,3h,6h,12h) for ELISA to detect IL-1βand IL-6 levels.Results. THCEs responded to the challenge of TLR2 or TLR4 ligand by expressing and secreting inflammatory cytokines IL-1βand IL-6. Zymosan-induced cytokines level began to increase at 6h in a time-dependent manner. LPS caused significant increase of IL-1βand IL-6 at 12h after challenge but exhibit lower levels than that of zymosan. And exposure of THCEs to Aspergillus fumigatus hyphae resulted in the upregulation of IL-1βand IL-6 since 3h after stimulation which continued to increase with prolonged incubation. Knockdown of TLR2 or TLR4 expression by specific siRNA caused a significant decrease in Aspergillus fumigatus-induced production of IL-1βand IL-6.Conclusions. THCEs can respond to TLR2 and TLR4 ligands challenge by secreting IL-1βand IL-6. They recognize Aspergillus fumigatus hyphae via TLR2 and TLR4 receptors and initiate innate immune responses against Aspergillus fumigatus challenge. Corneal epithelial cells play a role in innate defense against fungal infection through producing inflammatory cytokines.Part II:Toll-like receptor 2 mediates anti-inflammatory cytokine expression in human corneal fibroblasts in response to Fusarium soluPurpose To determine the role of Toll-like receptor (TLR) 2 in the expression of proinflammatory and anti-inflammatory cytokines in corneal fibroblasts challenged by fungi.Methods The sequence of siRNA targeting TLR2 was designed and cloned into the p-silencer 2.0 U6 vector to be a combined expression vector that expresses TLR2-siRNA. The cultured telomerase-immortalized human stroma fibroblasts (THSF) were transfected with the plasmid containing TLR2-siRNA and the expression of TLR2 was assessed by immunocytochemistry, RT-PCR, and Western blotting analyses. The transfected cells were stimulated by hyphae or conidia of Fusarium solu, respectively, and mRNA levels of IL-1βand IL-10 were measured by real time RT-PCR.Results THSF transfected with TLR2-siRNA exhibited a reduced level of TLR2 when compared with the control cells transfected with empty plasmid. TLR2-siRNA exhibited a more dramatic reduction in mRNA level of about 60% and TLR2 protein was decreased over controls approximately 65%. There is no difference between the group of control siRNA, empty vector and blank controls. The IL-lbeta and IL-10 levels increased dramatically in blank controls after stimulation of Fusarium solu and the levels induced by hyphae were much higher than by conidia. In contrast, THSF treated with TLR2-siRNA compared with control exhibited reduced levels of IL-1beta and IL-10 after fungi stimulation. After RNA interference, IL-10 level significantly decreased over controls approximately 82% in hyphae stimulation (P＜0.01) and 70% in conidia stimulation(P＜0.01). IL-lbeta level showed a reduction of 60%(P＜0.01) in hyphae stimulation and 54% (P＜0.01) in conidia stimulation, respectively. Fusarium solu-stimulated IL-10 production by THSF transfected with TLR2-siRNA was severely impaired while IL-iβproduction was partially inhibited.Conclusions TLR2 appears to be a major pattern recognition receptor to detect Fusarium solu in vitro and may play the anti-inflammatory role through the induction of IL-10. This may lead to a better understanding of the pathogenesis of fungal infections in cornea and may help in designing more efficient strategies in inhibiting the fungi-triggered inflammatory reaction.PartⅢ:Effect of RNA interfering of Toll-like receptor (TLR) 2 in rat cornea on Aspergillus fumigatus keratitisPurpose To investigate the effect of RNA interfering of Toll-like receptor (TLR) 2 in rat cornea on Aspergillus fumigatus keratitis.Methods The rat corneas were wounded and Aspergillus fumigatus strain was applied to the cornea surface. Slit lamp, clinical scoring and histopathological analysis were performed to monitor disease. Control or TLR2 siRNA was injected subconjunctively before infection and applied topically to cornea. TLR2 expression was measured by immunofluorescence staining to determine the feasibility and efficiency of TLR2 siRNA delivery. Production of inflammatory cytokines such as TNFa, IL-1β,IL-4, IL-6, IL12p40 and chemokines such as MCP-1 and MIP-2 were determined by real time quantitative PCR. PMN infiltration was assessed by myeloperoxidase (MPO) activity assay.Results Rat corneas treated with TLR2 siRNA showed a significant reduction of TLR2 expression which distributed in clusters through the corneal epithelium. Keratitis occurred and developed progressively after inoculation of Aspergillus fumigatus. Corneal ulcers were evident at day 3 p.i. with rough surface and obscure boundary. The ulceration expanded deeper and larger at day 4-5 p.i. accompanied by the destruction and degradation of cornea tissue. Descementocele, corneal staphyloma and perforation were observed in several cases. In contrast, TLR2 siRNA-treated corneas displayed mild infiltration at 1-3 days p.i. and only slight corneal opacity at day 5 p.i. with preserved corneal integrity. Compared to the control siRNA-treated corneas, mRNA levels of inflammatory cytokines TNFa, IL-1β, IL-6, IL12p40 and chemokines MCP-1, MIP-2 in the corneas of TLR2 siRNA-treated rats was dramatically reduced whereas IL-4 level was unchanged. While there was no measurable MPO activity in non-infected corneas (data not shown), MPO activity was detected in Aspergillus fumigatus-infected corneas at day 1 p.i. and upregulated at day 3 and 5 p.i. in the control corneas. This increase in PMN infiltration, however, was dramatically attenuated by TLR2 siRNA treatment, suggesting an enhanced resolution of inflammation.Conclusions TLR2 siRNA treatment reduces inflammation of Aspergillus fumigatus keratitis by the downregulation of inflammatory cytokines and cell infiltration, which may suggest a novel avenue to control infection and reduce damage caused by excessive inflammation.