【作者】 王乐怡；

【导师】 吴欣怡；

【作者基本信息】 山东大学 ， 眼科学， 2014， 博士

【摘要】 真菌性角膜炎(fungal keratitis, FK)是最严重的眼部感染性疾病之一,治疗不当迅速恶化,数天内即可导致失明。诱发角膜真菌感染首要的原因在发展中国家为角膜创伤,在发达国家则为角膜接触镜的配戴。湿热的气候是所有地区的高危致病因素。镰刀菌和曲霉菌是真菌性角膜炎最常见的病原体。正常情况下,角膜对真菌感染有很强的抵抗力,当角膜上皮完整性遭到破坏,暴露其下方角膜基质层时,才会发生真菌性角膜炎。角膜基质细胞是角膜天然免疫防御体系的第二道屏障,参与真菌的快速识别、抵抗真菌入侵。Toll样受体(Toll like receptors, TLRs)是天然免疫的关键模式识别受体,通过识别微生物的固有成分病原相关分子模式(Pathogen-associated molecular patterns, PAMPs)发挥重要作用。多种TLRs在角膜基质细胞的胞膜上或胞质内都有表达,其中TLR4的特异性配体为脂多糖(lipopolysaccharide, LPS),TLR2则能够识别来源于革兰氏阴性和阳性细菌的多种脂肽和脂蛋白成分。我们前期研究发现TLR2及TLR4是角膜启动抗真菌天然免疫的主要受体,在烟曲霉菌(Aspergillus fumigatus, AF)角膜炎的发生发展过程中发挥决定性作用。TLR2、4的活化能激活下游信号转导通路,从而介导炎性因子的分泌,趋化炎症细胞向角膜基质层迁移,同时诱导抗菌因子产生,杀灭和清除致病原。然而,过度的炎症反应可导致组织损伤和破坏,诱发角膜穿孔或视力丧失,TLRs介导的角膜抗真菌免疫反应必须控制在适度范围内。TLRs通过与接头蛋白分子髓样分化因子88(myeloid differentiation factor88, Myd88)和TIR结构域接头分子(Toll-IL-1receptor domain-containing adaptor inducing interferon-beta, TRIF)结合激活下游信号转导级联反应。TLRs的炎症损伤效应主要与Myd88依赖的经典途径和Myd88依赖的促分裂素原活化蛋白激酶(mitogen-activated protein kinase, MAPK)途径有关,二者通过活化核转录因子-κB(nuclear factor κB, NF-κB)和激活蛋白-1(activator protein-1, AP-1),介导多种炎性细胞因子和趋化因子,如白介素(interleukin, IL)-6、IL-8、肿瘤坏死因子(tumor necrosis factor, TNF)-a等的分泌。与之相反,MyD88非依赖的TRIF途径能激活转录因子干扰素调节因子(interferon regulatory factor, IRF)-3,从而调节干扰素(interferon, IFN)-β和其他抗炎介质的表达。因此,TLRs的信号转导体系中存在着损伤与保护的微妙平衡,一旦打破将对角膜抗真菌天然免疫反应产生严重的影响。我们的前期研究证明,小剂量的LPS预处理能够诱导永生化人角膜基质细胞(telomerase-immortalized human stroma fibroblasts, THSFs)再编程,调控角膜抗烟曲霉菌天然免疫反应。小剂量LPS预处理THSFs一定时间后再用烟曲霉菌刺激,炎性细胞因子IL-6和IL-8的分泌量减少,抗菌肽CCL20和Tβ4的表达增加,多形核白细胞(polymorphonuclear leukocyte, PMN)的迁移受到抑制。所以,LPS预处理诱导的角膜基质细胞再编程为真菌感染提供了一种保护性机制,在避免过度角膜炎症反应的同时增强角膜先天抗感染能力。但是,关于LPS预处理诱导的烟曲霉菌刺激角膜基质细胞再编程的作用机制目前尚不明确。我们用小剂量LPS预处理12h后,再用大剂量烟曲霉菌刺激THSFs,观察TLR4、TLR2mRNA水平的变化,利用TLR4、2的特异性中和抗体进一步明确TLRs在角膜基质细胞再编程中的作用；继而研究了LPS预处理对TLRs下游Myd88依赖的经典途径、Myd88依赖的MAPK途径以及MyD88非依赖的TRIF信号转导通路的影响。探索脂多糖预处理诱导的基质细胞再编程调控角膜抗真菌天然免疫的分子机制,将为合理调控抗真菌炎症反应、避免角膜组织损伤与破坏以及内环境稳态的重建提供新的靶点和理论依据。第一部分TLR4介导LPS预处理诱导的角膜基质细胞再编程[目的]研究烟曲霉菌刺激下脂多糖预处理引起的角膜基质细胞再编程中的关键受体。[方法]1.AF菌种和菌丝的制备：AF菌株CCTCC93024购自中国典型培养物保藏中心。将菌种接种于沙氏葡萄糖琼脂培养基,37℃条件下200转每分钟(revolution per minute, rpm)摇床培养24h。收集孢子以108个/ml的终浓度接种于沙氏液体培养基,接种的试管26℃、500rpm摇床培养18h后1000×g离心10min。得到的AF菌丝体漂洗2次后悬浮于Dulbecco’s Hanks平衡盐溶液(Dulbecco’s Hanks balanced salt solution, D-Hanks)中,56℃加热60min灭活。然后Potter-Elvehjem组织匀浆器将菌丝研磨成20～40μm长的片段,浓度调整为1×106个菌丝体片段/ml,制成AF抗原刺激液。2.细胞的培养：THSFs由Fu-Shin X.Yu教授和Ilene K. Gipson教授慷慨提供。细胞在37℃、5%CO2的湿润条件下用含10%新生牛血清的改良Eagle培养基(Dulbecco’s Modified Eagle’s Medium, DMEM)培养。细胞以4×105个/孔的浓度接种于6孔微培养板并生长至80%融合(2天左右),不含血清的DMEM培养液饥饿16h后用于刺激试验。3.细胞的刺激和耐受的诱导：THSF细胞经LPS (10ng/ml)预处理12h后用不含血清的培养基冲洗2次,给予AF菌丝(106个/ml)再次刺激。第2次刺激后不同时间点(30min,1h,3h,6h)收取细胞,荧光定量实时聚合酶链反应(real time-polymerase chain reaction, RT-PCR)检测TLR4mRNA的水平,或于第2次刺激后4h收取细胞及细胞上清,RT-PCR检测TLR2的基因表达,酶联免疫吸附试验(enzyme-linked immunosorbent assay, ELISA)检测IL-6、 IL-8和TNF-α的分泌。4.抗体中和能力的测定和受体封闭实验：利用TLR2和TLR4的单克隆抗体孵育THSFs进行受体封闭试验。THSF细胞与抗人TLR4(100ug/ml)、抗人TLR2(100ug/ml)或抗人TLR4(100ug/ml)+TLR2(100ug/ml)单克隆抗体共孵育30min后,37℃条件下酵母聚糖(1mg/ml)或LPS (1μg/ml)刺激12h,或LPS (10ng/ml)预处理12h后AF菌丝(106个/ml)再次刺激4h。收取细胞培养上清和细胞裂解液,ELISA检测IL-6、IL-8、TNF-α的分泌或RT-PCR检测TLR2的表达。[结果]1.单克隆抗体可有效封闭TLR2及TLR4:TLR2、4抗体对THSF中IL-8和TNF-α的基础分泌没有影响,TLR2配体酵母聚糖或TLR4配体脂多糖均刺激THSF细胞高表达IL-8和TNF-α,中和性抗体封闭相应TLR后这种刺激作用消失,抗体封闭组与空白对照组的炎性因子分泌量无明显统计学差异。因此,我们使用的特异性单克隆抗体能够有效的阻断相应TLRs的功能。2.LPS预处理对大剂量AF诱导的TLR4表达的影响：为了研究THSF细胞中LPS预处理对TLR4水平的调节作用,10ng/ml的LPS预处理12h后,烟曲霉菌菌丝再次刺激THSF, RT-PCR检测TLR4的表达。结果显示第二次刺激1小时、3小时后LPS预处理组TLR4mRNA的表达量均低于单纯AF刺激组。3.LPS预处理对烟曲霉菌刺激角膜基质细胞炎性因子分泌的抑制作用依赖于TLR4的功能：在前期研究中我们发现,10ng/ml LPS预处理能减少再次烟曲霉菌刺激诱导的角膜基质细胞IL-6和IL-8的分泌。在此我们进一步探讨这种炎性细胞因子表达的抑制现象是否依赖于TLR4的作用。在LPS预处理之前封闭TLR4,培养上清中IL-6、IL-8和TNF-α的含量较未封闭TLR4的LPS预处理组增加,与未封闭TLR4的无预处理烟曲霉菌刺激组之间没有明显差异。4.TLR2在LPS预处理诱导的角膜基质细胞再编程中的作用研究：实时荧光定量PCR检测TLR2的表达,结果显示LPS预处理对烟曲霉菌诱导的角膜基质细胞中TLR2mRNA的水平没有明显影响。虽然TLR4单克隆抗体对TLR2的基础表达没有刺激作用,但预处理前封闭TLR4, THSF细胞中烟曲霉菌刺激的TLR2表达明显上调。LPS预处理前利用特异性抗体将TLR2与TLR4共同封闭,IL-6、IL-8和TNF-α的分泌较无预处理对照组明显下降,接近基础分泌水平。[结论]1.在角膜基质细胞再编程中,TLR4作为关键的受体,能特异性的介导LPS预处理引起的炎性细胞因子抑制。2.LPS预处理对烟曲霉菌诱导的TLR2表达没有明显影响,但是封闭TLR4后TLR2是维持炎性细胞因子分泌水平的关键因素。第二部分LPS预处理诱导的角膜基质细胞再编程对TLR4下游信号转导通路的调控[目的]研究THSF细胞中LPS预处理对TLR4下游Myd88依赖的经典途径、Myd88依赖的MAPK途径以及MyD88非依赖的TRIF途径的影响。[方法]用小剂量LPS预处理12h后,再用大剂量AF刺激THSF,培养0.5、1、3、6、12小时分别收取细胞及培养上清,RT-PCR、ELISA及wenstern blot检测Myd88依赖的经典途径的关键因子MyD88、IκB-α、NF-Kb-p65, Myd88依赖的MAPK途径的关键因子MAPK3、AP-1、ERK1/2以及Myd88非依赖的TRIF途径的关键因子TRIF、IRF-3、IFN-β的基因和蛋白水平；细胞免疫荧光检测转录因子NF-Kb-p65向细胞核内的转移。[结果]1.Myd88依赖的经典途径的抑制：RT-PCR结果显示烟曲霉菌再次刺激1小时、3小时后LPS预处理组MyD88mRNA的表达低于非预处理组；IκB-α mRNA及蛋白的表达高于非预处理组,间接说明1小时、3小时LPS预处理组NF-κB的表达低于非预处理组；NF-κB-p65的蛋白表达低于非预处理组,向胞核内的转移也受到抑制。2.Myd88依赖的MAPK途径下游信号因子的表达下调：1小时、3小时LPS预处理组MAPK3、AP-1mRNA的表达均低于非预处理组,western blot结果显示1小时、3小时ERK1/2的蛋白表达量预处理组也明显低于无预处理组。3.Myd88非依赖的TRIF途径下游信号因子表达和细胞因子分泌上调：LPS预处理后再用烟曲霉菌菌丝刺激THSF细胞,RT-PCR结果显示真菌刺激3小时、6小时后TRIF、IRF3、IFN-β mRNA的表达增加,而LPS预处理组TRIF途径关键信号因子的水平较无预处理组进一步升高。IFN-β的ELISA检测显示了一致的结果。[结论]1.在角膜基质细胞再编程中,介导炎性细胞因子产生的Myd88依赖的经典途径以及Myd88依赖的MAPK途径作用受到抑制。2.LPS预处理促进MyD88非依赖的TRIF途径的功能,进一步增强其免疫保护作用。更多还原

【Abstract】 Fungal keratitis is among the most dangerous ocular infections. It can manifest fast and vicious progress and often leads to total sight loss in days if not properly controlled. The best known predisposing factors include trauma in underdeveloped countries or contact lens wear in developed countries. Hot and humid weather is a further risk factor in all countries. Fusarium spp. and Aspergillus spp. are the most common pathogens of fungal keratitis. Under normal conditions, the cornea is highly resistant to infection, and fungal keratitis always occur after the epithelial integrity of the cornea has been compromised, exposing underlying fibroblasts. The corneal fibroblasts are the second line of the host innate immunity defense and participate in immediate recognition and control of fungal invasion.Toll-like receptors (TLRs) are key players in innate immunity through their recognition of conserved pathogen-associated molecular patterns (PAMPs) in microbes and are expressed at the cell surface or in intracellular space in corneal fibroblasts. Among these receptors, TLR4is capable of sensing LPS while TLR2recognizes a diverse set of microbial lipopeptides and lipoproteins that are present on gram-positive and gram-negative bacteria. Our previous study demonstrated that TLR2and TLR4represents two of the master switches initiating innate immunity in the cornea and have determinant roles in Aspergillus fumigates (AF) keratitis. Their activation leads to the initiation of downstream signal cascades, which regulate secretion of inflammatory cytokines that recruit inflammatory cells to the corneal stroma and the production of antimicrobial molecules that kill the invading pathogens. However, this response of TLRs must be stringently regulated, as excessive inflammation can cause tissue damage and have devastating effects on the host, which can result in vision loss or perforations.TLRs signal through the adaptor proteins myeloid differentiating factor88(MyD88) and Toll-IL-1receptor domain-containing adaptor inducing interferon-beta (TRIF) to activate downstream transduction cascades. The detrimental effect of TLR signaling is associated with the MyD88-dependent classical pathway and MyD88-dependent mitogen-activated protein kinase (MAPK) pathway that lead to transcription factor nuclear factor κB (NF-κB) and activator protein-1(AP-1) activation, which is essential for the induction of many cytokines and chemokines such as interleukin (IL)-6,-8and tumor necrosis factor (TNF)-α. In contrast, the MyD88-independent TRIF signaling pathway that activates interferon regulatory factor-3(IRF-3) can induce interferon (IFN)-β and anti-inflammatory mediators. Thus, in TLR signaling there is a fine balance between pathways leading to injury or protection, with even minor alterations of these fine-tuned endogenous pathways having profound effects on cellular responses to TLR engagement.We previously reported that telomerase-immortalized human stroma fibroblasts (THSFs) pretreated with lipopolysaccharide (LPS) develop a state of cellular reprogramming that modulates corneal antifungal innate immunity. Pretreatment of THSFs with low-dose LPS resulted in diminished production of cytokines IL-8and IL-6, elevated expression of antimicrobial peptides CC chemokine-ligand20and thymosin b, and suppression of polymorphonuclear leukocyte migration upon subsequent A. fumigatus challenge. Therefore, LPS pretreatment may induce protective mechanisms during fungal keratitis that prevent an excessive inflammatory response and provide an innate defense in the cornea.The underlying molecular mechanism by which LPS pretreatment induces cellular reprogramming of THSFs challenge by A. fumigatus hyphae through innate immunity pathways remains to be elucidated. In the present study, the effect of LPS pretreatment on TLR4and TLR2messenger RNA (mRNA) induction was evaluated in order to explore the signaling mechanisms for the resolution of inflammation, restoration of homeostasis and prevention of corneal destruction. Then these TLRs were inhibited using monoclonal antibodies to investigate the role of TLR2and TLR4in LPS-induced tolerance using the THSF cell line. In addition, the downstream influence of LPS pretreatment was examined for the purpose of identifying the TLRs signaling pathways involved in THSFs, including the MyD88-dependent classical pathway, MyD88-dependent MAPK pathway and MyD88-independent TRIF signaling pathway.Part I TLR4mediates cellular reprogramming induced by lipopolysaccharide pretreatment in corneal fibroblasts[Purpose]To determine the key receptors in cellular reprogramming induced by lipopolysaccharide pretreatment in corneal fibroblasts challenged with Aspergillus fumigates.[Methods]1. Aspergillus fumigatus strain and preparation of hyphae:The A. fumigatus strain CCTCC93024, purchased from the China Centre for Type Culture Collection, was grown on Sabouraud glucose agar for24h at37℃on a shaking table with a rotation speed of200rpm. Conidia were harvested and planted into Sabouraud fluid media, at a final concentration of108micro-organisms/ml. The tubes were shaken at26℃,500rpm for18h and then centrifuged at1000g for10minutes. The mycelia were washed twice, suspended in phosphate-buffered saline (PBS) and sterilized by heat treatment at56C for60min. Then, the mycelia were disrupted into20-40mm pieces in a Potter-Elvehjem Tissue Grinde and yielded1×106pieces/ml as A. fumigates hyphae antigen.2. Cell cultures:THSFs (kindly provided by Dr. Fu-Shin X. Yu and produced by Dr. Ilene K. Gipson) were cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with10%newborn bovine serum at37℃in a humidified atmosphere with5%CO2. For experiments, cells were seeded at4×105cells/well in6-well microculture plates and grown until80%confluence (-2days). To prepare cells for stimulation, the THSFs were then starved in serum-free DMEM for16h.3. Cell treatment and tolerance induction:The THSFs were preconditioned with a TLR4ligand, LPS at a density of10ng/ml for12h. Then the cells were washed twice with serum-free medium and re-stimulated with A. fumigatus hyphae (106pieces/ml) for various periods (30min,1h,3h,4h,6h). The Cell culture supernatants and cells were collected for measurement of TLR4, TLR2mRNA expression and IL-6, IL-8, TNF-α protein secretion.4. Evaluation of neutralizing efficiency of antibodies and blocking experiment of receptors:TLR blocking experiments were conducted by incubating THSFs with monoclonal Abs against TLR2or TLR4. THSF cells were incubated with antihuman TLR2(100mg/ml) or anti-human TLR4(100mg/ml), or both anti-human TLR2(100mg/ml) and anti-human TLR4(100mg/ml) monoclonal Abs for30min. They were then challenged with TLR2ligand zymosan at a density of1mg/ml or TLR4ligand LPS (1μg/ml) for12h at37℃, or pretreated with LPS for12h and re-challenged with A. fumigatus hyphae for4h. The cell culture media and cell lysates were harvested and used for IL-6, IL-8and TNF-a protein ELISA and TLR2mRNA Real-time reverse transcriptase polymerase chain reaction (RT-PCR).[Results]1. Efficient neutralization of TLR2or TLR4by monoclonal antibodies:TLR2-or TLR4-antibody had no apparent effect on the basal release of IL-8and TNF-α in THSFs. While THSFs secreted significantly high levels of IL-8and TNF-α in response to TLR2ligand zymosan or TLR4ligand LPS challenge, this elevated production was significantly inhibited in cells treated with TLR2-or TLR4-antibody, and there is no significant difference between blank control and the ligand-treated THSFs with previous TLRs antibodies inhibition. Thus, our monoclonal antibodies specific for TLR2and TLR4can efficiently neutralize the function of TLR2and TLR4, respectively.2. Effect of LPS pretreatment on TLR4expression induced by A. fumigatus in THSFs:To examine whether TLR4expression is affected by LPS pretreatment in THSFs, we pre-stimulated THSFs with10ng/ml LPS for12h and assessed the response of TLR4expression to subsequent challenge with A. fumigatus hyphae. RT-PCR revealed that LPS pretreatment significantly down-regulated A. fumigatus hyphae-induced mRNA expression of TLR4after1h and3h of re-stimulation.3. LPS pretreatment induced down-regulation of pro-inflammatory cytokines secretion in A. fumigates antigens-challenged THSFs depends on TLR4:In the previous study, we demonstrated that pretreatment of THSFs withlO ng/ml LPS resulted in impaired production of IL-6and IL-8in response to a secondary A. fumigatus hyphae challenge. We next sought to determine whether the reduction of pro-inflammatory cytokines secretion induced by LPS pretreatment in THSFs is dependent on TLR4. THSFs pre-incubated with TLR4antibodies before LPS pretreatment showed increased IL-6, IL-8and TNF-α accumulation in culture medium compared to LPS-pretreated THSFs without the TLR4antibody, and there was no significant difference of IL-6, IL-8and TNF-a accumulation in culture medium compared to non-LPS-pretreated controls challenged with Aspergillus fumigates without TLR4antibody inhibition.4. Involvement of TLR2in LPS-induced cellular reprogramming to secondary A. fumigatus hyphae stimulation:Real-time PCR was used to assess expression of TLR2. It showed that LPS pretreatment had no apparent effect on TLR2expression in THSFs challenged with A. fumigatus hyphae. However, whereas the TLR4antibody had no effect on basal TLR2mRNA expression, THSFs treated with TLR4neutralizing antibody before LPS pretreatment exhibited significantly increased levels of TLR2after the secondary fungi stimulation compared with the pretreated controls. In addition, pre-incubation of THSFs with both TLR2and TLR4antibodies before LPS pre-stimulation resulted in dramatic reduction of common pro-inflammatory cytokines, including IL-6, IL-8and TNF-α expression compared with the non-pretreated THSFs.[Conclusion]1. As the key receptor in cellular reprogramming, TLR4mediates attenuated cytokine production induced by LPS pretreatment in THSFs.2. Levels of TLR2mRNA induced by A. fumigatus were not affected by LPS pretreatment. However, after TLR4blockage, pro-inflammatory cytokines secretion is mediated by TLR2in LPS-pretreated THSFs. Part II Influence of cellular reprogramming induced by LPS pretreatment on TLR4signaling pathways involved in THSFs[Purpose]To examine the downstream influence of LPS pretreatment for the purpose of identifying the TLR4signaling pathways involved in THSFs, including the MyD88-dependent classical pathway, MyD88-dependent MAPK pathway and MyD88-independent TRIF signaling pathway.[Methods]THSFs were pretreated with10ng/ml of LPS for12h and then re-challenged with A. fumigatus hyphae for various period(30min,1h,3h,4h,6h). The cell culture media and cell lysates were harvested and used for analysis of key signaling factors MyD88, IκB-α, NF-Kb-p65in MyD88-dependent classic pathway, MAPK3, AP-1, ERK1/2in MyD88-dependent MAPK pathway and TRIF, IRF-3, IFN-p in MyD88-independent TRIF pathway of gene and protein levels. Immunofluorescence staining were applied to determine the expression and location of NF-Kb-p65.[Results]1. Impaired response of MyD88-dependent classical signaling pathway:mRNA expressions of MyD88were proved to be inhibited after1h and3h of A. fumigates re-stimulation according to the PCR analysis, and an increase in gene and protein expression of inhibitory NF-κB (IκB)-α was observed1h and3h after fungus treatment. NF-icB-p65protein expression was diminished after1h and3h of stimulation with A. fumigatus hyphae, and the translocation of NF-κB-p65into cell nuclei was significantly attenuated by LPS pretreatment.2. Down-regulated expression of downstream molecules in MyD88-dependent MAPK signaling pathway:Expression of MAPK3and AP-1mRNA was lowered by LPS pretreatment1h and3h after the secondary A. fumigates stimulation, and Western blot analysis showed that protein level of phosphorylated-extracellular signal-regulated kinase (ERK)1/2was decreased after1h and3h of treatment with A. fumigatus hyphae.3. Up-regulation of downstream molecular expression and cytokine secretion of the MyD88-independent TRIF signaling pathway:THSFs pretreated with LPS were challenged with A. fumigatus hyphae, and TRIF, IRF3and IFN-β expression were measured by real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA). In non-pretreated controls, THSFs express relatively high levels of TRIF, IRF3and IFN-β mRNA in response to A. fumigates hyphae challenge for3h and6h, and this elevated expression was significantly increased in cells pretreated with LPS. IFN-β secretion levels were consistent with trends observed for IFN-p mRNA.[Conclusion]1. The impaired activity of the MyD88-dependent classical pathway and MyD88-dependent MAPK pathway may contribute to the suppressed secretion of pro-inflammatory cytokines induced by LPS pretreatment in THSFs.2. TRIF-mediated immunological protection was augmented in LPS-pretreated THSFs.更多还原