Toll-like Receptor4Mediated Human Corneal Inflammatory Responses Against Acanthamoeba And The Regulatory Effects Of Hypoxia

BackgroundAcanthamoeba keratitis (AK) is a vision-threatening disease caused by a free-living, pathogenic Acanthamoeba. In the past few decades, with the wide-spread use of contact lenses, the incidence of AK has steadily increased. The United States Centers for Disease Control and Prevention found that several important risk factors are associated with AK, such as corneal injuries and contact with contaminated water, of which contact lens wear is the predominant risk factor. A study revealed that the incidence of AK is1.36per million contact lens weares in the United States,3.06in the Netherlands and17.53-21.14in England. In China, it has been reported the first case of AK was caused by contact lens wear in1992. Then another report showed that about30.8%of AK patients were associated with contact lenses. Acanthamoeba cysts have strong environmental adaptability resistance and long-term slow growth in the cornea, leading to poor results of drug treatment of the disease, postoperative recurrence. AK has become a corneal infection which is difficult to treat and with high rate of blind. Therefore, a better understanding of the pathogenesis and mechanisms responsible for the host defense against Acanthamoeba infection is of crucial importance.Corneal epithelia cells serve as the first-line defensive barrier of cornea and participate in the innate immune response to invasive pathogens through its pattern recognition receptors (PRRs). This rapid recognition triggers the production of inflammatory cytokines, chemokines, and the antimicrobial peptide, ultimately resulting in the clearance of invading pathogens. Toll-like receptors (TLRs) are important PRR, which recognize distinct pathogen-associated molecular patterns (PAMPs) that are shared by many pathogenic microorganisms. Recognition of these patterns by TLRs initiates cell signal cascade, induces the synthesis and secretion of inflammatory cytokines, triggers neutrophils infiltration, activates the inflammatory and immune responses, which plays an important role in the defense of bacteria, fungi, viruses and other pathogens invasion.Toll-like receptor4(TLR4) is the first discovered TLR, which mainly express on immune cells, such as lymphocytes, monocytes and macrophages, and also express on the epithelial cells at the host-environmental surface, such as airway epithelial and intestinal epithelial cells. On ligand binding, TLR4activates different downstream signaling through myeloid differentiation primary response gene88(MyD88)-dependent or MyD88-independent pathways, which leads to the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and interferon regulatory factor-3(IRF-3), induces the secretion of inflammatory cytokines and thus triggers the inflammation and innate immunity. In our previous study, we showed that human corneal epithelial cells expressed TLR4. When exposed to Acanthamoeba challenge, among TLR1-10, the expression of TLR4was significantly increased, indicating that TLR4may be involved in the inflammatory response of AK.As we know, oxygen is the key substance of metabolism and life activities, and the changes of oxygen pressure in tissue are also important environment signals involved in many physiological and pathological processes, such as inflammation, infection, hypoxia and so on. About80%of the oxygen which is needed to corneal metabolism is from air. Attached by contact lenses, the cornea is subjected to a hypoxic condition, thus affecting the biological properties of the corneal epithelial cells, such as edema, apoptosis, necrosis, shedding and so on, destructing the epithelial barrier function and reducing the natural defensive capacity. A study revealed that hypoxia can promote the adhesion of Pseudomonas aerugionsa on corneal epithelial cells, reduce the proliferation of epithelial cells, accelerate cells apoptosis and increase the susceptibility of corneal epithelial cells against P. aeruginosa. A study comparing high-vs low-Dk contact lenses showed increased incidence of P. aeruginosa keratitis (0:30%). It is suggested that incidence of the most severe adverse events is actually increased with low-Dk lenses when compared with high-Dk lenses. Recent reports showed that hypoxia can regulate the expression of TLR4. It was found that hypoxia also diminished TLR4expression in human umbilical vein endothelial cells for48-72h exposure. While, it has been demonstrated that hypoxia increased TLR4expression in microglia cells after8h exposure. These studies indicate that different cell types and hypoxic exposure durations lead to different cellular responses of TLR4expression. There is no report about the effect of hypoxia on human corneal epithelial cells. Since TLR4is involved in the inflammatory process of AK, it is necessary to investigate the effect of hypoxia on TLR4and its signaling and whether hypoxia affect the susceptibility of corneal epithelial cells to Acanthamoeba through regulating TLR4, which may help in designing more efficient strategies in prevention of AK.The research is divided into two parts:the one is through the use of RNA interference (RNAi) to silence the TLR4gene expression to investigate the effects of TLR4inhibition on inflammatory responses of human corneal epithelial cells against Acanthamoeba. The other is to determine whether hypoxia affects the corneal susceptibility to Acanthamoeba through regulating TLR4and its signaling, and to explain why contact lens use is one of the prominent risk factors for AK. This may help to develop strategies aimed at establishing rational prevention and useful treatment.Part I:Toll-Like Receptor4Mediates Innate Responses of Human Corneal Epithelial Cells against AcanthamoebaPurpose:To further determine the potential role of Toll-like receptor4(TLR4) in the inflammatory responses of human corneal epithelial cells against Acanthamoeba.Methods:1. Telomerase-immortalized human corneal epithelial cells (THCEs) were challenged by different concentrations (1×103/ml,1×104/ml,1×105/ml,1×106/ml) of Acanthamoeba with30min and after12h incubation, cells were collected. Real-time PCR was used to assess the mRNA level of TLR4, and Western blot was used to examine the protein level of TLR4. Then THCEs were stimulated with Acanthamoeba at the concentration of1×106/ml. Cells and culture media collected at different time points (1h,3h,6h,12h) were subjected to Real-time PCR, Western blot and enzyme-linked immunosorbent assay (ELISA) to detect the expression of TLR4, interleukin-6(IL-6) and interleukin-8(IL-8).2. The sequence of siRNA targeting TLR4was designed and synthesized. The sequences used for targeting TLR4are5'-GGCAAUUCUUUCCAGGAAATT-3'(for sense) and5'-UUUCCUGGAAAGAAUUGCCAG-3'(for antisense). The sequences of negative control siRNA (NC-siRNA) are5'-UUCUCCGAACGUGUCACGUTT-3'(for sense) and5'-ACGUGACACGUUCGGAGAATT-3'(for antisense). THCEs were transfected with siRNA using LipofectamineTM2000as instructed by the manufacturer. Forty-eight hours after transfection, the expression of TLR4was assessed by Real-time PCR and Western blot. After the transfected cells were challenged with Acanthamoeba, the production of IL-6and IL-8were measured by ELISA.Results:1. We observed that the mRNA and protein levels of TLR4were significantly upregulated after stimulation and this regulation effect was further enhanced with increasing severity of Acanthamoeba exposure. After Acanthamoeba challenge at the concentration of1×106/ml, the mRNA and protein levels of TLR4were upregulated in a time-dependent manner, with a significantly increase from6h after stimulation. And exposure of THCEs to Acanthamoeba resulted in the upregualtion of IL-6and IL-8from6h after stimulation, which continued to increase with prolonged incubation. 2. We found that TLR4mRNA and protein levels were evidently reduced after TLR4siRNA treatment in THCEs, compared with NC-siRNA. The secretion of IL-6and IL-8was obviously increased in response to Acanthamoeba challenge, while the secretion was reduced after pretreatment with TLR4-siRNA compared with NC-siRNA.Conclusions:Our results indicated that Acanthamoeba-induced inflammatory response was mediated by TLR4in human corneal epithelial cells. TLR4siRNA treatment reduced inflammatory response of AK by the downregulation of inflammatory cytokines, which may provide a novel gene therapeutic strategy for the treatment of AK. Part Ⅱ:Hypoxia Inhibits Acanthamoeba-Induced Inflammatory Responses of Human Corneal Epithelial Cells via Toll-Like Receptor4SignalingPurpose:To investigate the role of hypoxia in the expression of toll-like receptor4signaling and in the regulation of inflammatory response of human corneal epithelial cells against Acanthamoeba.Methods:1. THCEs challenged with Acanthamoeba (1×106/ml) were incubated at37℃for24h under normoxic (21%O2) or hypoxic (1%O2) conditions. Cell viability was analyzed by flurescence activating cell sorter (FACS) using the Annexin V-PE/7-AAD staining.2. After exposure to normoxia or hypoxia for24h, THCEs were challenged with Acanthamoeba resuspension for30min under normoxic condition followed by incubation with culture medium for an additional6h under normoxia or hypoxia, depending on the exposure condition. Real-time PCR was used to assess the mRNA of TLR4, MyD88, IL-8and IFN-p. Western blot was used to examine the protein level of TLR4, p-IκBα, p-ERK1/2and IκBα ELISA was used to detect the secretion of the inflammatory cytokines IL-8and IFN-(3. 3. THCEs were incubated under normoxia and hypoxia for6h,12h,24h and48h. The mRNA and protein levels of TLR4were measured by Real-time PCR and Western blot analysis.4. After exposure to hypoxia for24h, THCEs were challenged with TLR4ligand LPS (1μg/ml) for6h. Real-time PCR was used to assess the mRNA level of MyD88, IL-6and IL-8. Western blot was used to examine the protein level of p-IμBα and IκBα. ELISA was used to detect the secretion of the inflammatory cytokines IL-6and IL-8.Results:1. The data showed that there was no significant change in the percentage of cells undergoing apoptosis and necrosis when the cells were exposed to hypoxia, Acanthamoeba challenge or both compared with the untreated control, indicating that the viability of THCEs was maintained under hypoxia and Acanthamoeba challenge.2. Real-time PCR results showed that the mRNA levels of Acanthamoeba-induced TLR4, MyD88, IL-8and IFN-β in THCEs under hypoxia was remarkably decreased. Western blot analysis results demonstrated that the protein level of TLR4, p-IκBa and p-ERK1/2were significantly diminished, while the IκBα protein expression was increased. The secretions of IL-8and IFN-β under hypoxia were also reduced.3. The results of Real-time PCR and Western blot analysis showed that the expression of TLR4significantly decreased at24h and48h after hypoxic exposure, respectively. Moreover, the decrease of TLR4mRNA level occurred in a time-dependent manner.4. Hypoxia also inhibited LPS-induced IL-6and IL-8secretion, MyD88expression and NF-κB activation.Conclusions:Our results demonstrated that hypoxia attenuated the host immune and inflammatory response against Acanthamoeba infection by suppressing TLR4signaling, indicating that hypoxia might impair the host cell's ability to eliminate the Acanthamoeba invasion and could enhance cell susceptibility to Acanthamoeba infection. These results may explain why contact lens use is one of the most prominent risk factors for AK.