Signal Transduction Pathway Of Toll-like Receptor 4 In The Pathogenesis Of Acanthamoeba Keratitis

Part one:TLR4:the receptor bridging Acanthamoeba challenge and intracellular inflammatory responses in human corneal cell linesPurpose:To investigate the role of the Toll-like receptor (TLR) signaling pathway in human corneal cell lines challenged with Acanthamoeba.Methods:Human telomerase-immortalized corneal epithelial cells (HUCLs) and stromal fibroblasts (THSFs) were challenged with Acanthamoeba with or without pretreatment with anti-TLR antibodies or the specific inhibitors PDTC (for nuclear factor [NF]-kB) and U0126 (for extracellular signal-regulated kinase [ERK]). Real time-PCR was used to assess the mRNA expression of TLRs, myeloid differentiation protein 88 (MyD88), NF-kB, ERK1/2, c-Jun N-terminal kinase (JNK), and p38 mitogen activated protein kinase (P38). Western blot and immunofluorescence staining were used to examine the protein expression of TLR2 and 4, phospho-ERK1/2 and phospho-IkB. ELISA was used to detect the secretion of the inflammatory cytokines interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interferon (IFN)-β.Results:Acanthamoeba-treated cells showed a significant increase in the expressions of TLR4, MyD88, NF-kB, phospho-IkB and phospho-Erk1/2. Anti-TLR4 antibody attenuated the production of cytokines induced by Acanthamoeba infection. PDTC inhibited the production of IL-8 and TNF-a and U0126 the synthesis of IFN-β.Conclusion:TLR4 is a receptor for Acanthamoeba and acts through TLR4-MyD88-NF-KB and TLR4-ERK1/2 pathways in human corneal cell lines with Acanthamoeba infection.Part two:Evaluation of Three Different Methods to Establish Animal Models of Acanthamoeba Keratitis and the role of TLR4 signaling pathway in vivoPurpose:To produce animal models of Acanthamoeba keratitis and to evaluate the advantages and adaptation range of each of the three methods employed and to investigate the role of Toll-like receptor 4 in Wistar rats challenged by Acanthamoeba.Materials and methods:Mice and Wistar rats in three groups of 15 rats and 15 mice each were used to establish the models. Right corneas in group A were scratched and challenged with Acanthamoeba. Those in group B were scratched and covered with contact lenses incubated with Acanthamoeba. Those in group C received an intrastromal injection of Acanthamoeba. Five rats and 5 mice in each group were used for histopathological investigations and the other 10 in each group were used for clinical evaluation. The models were evaluated by slit lamp examination, microscope examination and culture of corneal scrapings, HE staining of corneal sections, and pathological scoring of the infections. The rat model of AK was established. Corneas were collected and analyzed by real time-PCR to assess the mRNA levels of TLR 2, TLR4, nuclear factor [NF]- kB, extracellular signal-regulated kinase [ERK], interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interferon (IFN)-β. Immunocytochemistry and Western blot was conducted to examine the proteins of TLR2, TLR4, p-Erk1/2, and p-IkB. The specific inhibitor PDTC and U0126 were used to pretreat the animals and then corneas were challenged and analyzed to determine the exact receptor (/s) and signaling pathway (/s) involved in pathogenesis.Result Four rats and 6 mice in group A,7 rats and 8 mice in group B,10 rats and 10 mice in group C developed typical Acanthamoeba keratitis. The method of scratching and challenging with Acanthamoeba could simulate the natural process of AK in human and the method of scratching, challenging with Acanthamoeba, and covering contact lenses could simulate the process of infection in eye-contact lenses wearers. Expressions of TLR4, all three cytokines, NF-kB, p-IkB and p-Erk1/2 were increased in Acanthamoeba-treated rat corneas. PDTC inhibited the production of IL-8 and TNF-α, while U0126 inhibited the synthesis of IFN-β.Conclusion Corneal scratching alone had the lowest infection rate, while scratching then covering with contaminated contact lenses had a moderate rate of infection and most closely mimics what happens in most human infections. Intrastromal injection of Acanthamoeba gave a much higher infection rate and more severe Acanthamoeba keratitis. TLR4 was involved in sensing the challenge of Acanthamoeba and inducing production of cytokines through TLR4-NF-kB and TLR4-Erkl/2 pathways in corneas of Wistar rat.Part Three:Mannose Inhibited Activation of Toll-like Receptor 4 Signaling Pathway in Human Corneal Epithelial Cells Challenged with AcanthamoebaPurpose:To investigate the role of mannose in the activation of Toll-like receptor 4 signaling pathway in human corneal epithelial cells challenged with AcanthamoebaMethods:Telomerase-immortalized human corneal epithelial cells (HUCLs) were challenged with Acanthamoeba with or without pretreatment by mannose and/or the specific inhibitors PDTC (for nuclear factor [NF]-kB) and U0126 (for extracellular signal-regulated kinase [ERK]). Real time-PCR was used to assess the mRNA expression of TLRs, NF-kB, and ERK1/2. Western blot and immunofluorescence staining were used to examine the protein expression of TLR 4, phospho-ERK1/2 and phospho-IkB. ELISA was used to detect the secretion of the inflammatory cytokines interleukin (IL)-8, tumor necrosis factor (TNF)-α, and interferon (IFN)-β.Result:Expressions of TLR4, NF-kB, p-ERK1/2, p-IkB and inflammatory cytokines were augmented by Acanthamoeba challenge. Mannose attenuated the production of cytokines mediated by TLR4 in HUCLs challenged with Acanthamoeba. PDTC inhibited the production of IL-8 and TNF-αand U0126 the synthesis of IFN-β. Combined use of mannose and pathway inhibitors could greatly inhibit the production of cytokines.Conclusion:Mannose could inhibit the activation of TLR4 signaling pathway to decrease the production of inflammatory cytokines. Combined use of mannose and signaling pathway inhibitor could significantly inhibit the production of cytokines.