IL-1β在真菌性角膜炎中的表达和调控机制研究

【作者】 车成业；

【导师】 赵桂秋；

【作者基本信息】 青岛大学 ， 眼科学， 2014， 博士

【摘要】 目的：探讨炎症因子白介素-1β(Interleukin-1β,IL-1β)在真菌性角膜炎中的表达及其分子调控机制。方法：采用基质内注射法建立小鼠真菌性角膜炎模型,以此作为大体水平的研究对象；获取小鼠骨髓中性粒细胞,以此作为细胞水平的研究对象。实验分为四部分：(1)建立C57BL/6小鼠真菌性角膜炎模型,采用流式细胞术和激光共聚焦显微镜观察IL-1β在角膜组织的表达,探索真菌性角膜炎时IL-1β的主要细胞来源；(2)获取C57BL/6、TLR4-/-以及TRIF-/-小鼠中性粒细胞,与烟曲霉菌株共同培养,采用酶联免疫吸附试验(ELISA)和Western-blot法检测IL-1β的表达,探索真菌性角膜炎时经典Signal1通路对IL-1β的调控作用；(3)建立C57BL/6、 NLRP3-/-、ASC-/-以及Caspase-1-/-小鼠真菌性角膜炎模型,获取C57BL/6、 Dectin-1-/-、NLRP3-/-、ASC-/-以及Caspase-1-/-小鼠中性粒细胞,与烟曲霉菌共同培养,采用ELISA和Western-blot法检测IL-1β的表达,探索真菌性角膜炎时经典Signal2通路对IL-1β的调控作用；(4)建立C57BL/6、Caspase-1-/-小鼠真菌性角膜炎模型,获取C57BL/6.TLR4-/-、TRIF-/-、Dectin-1-/-以及Caspase-1-/-小鼠中性粒细胞,与烟曲霉菌共同培养,采用Western-blot法检测IL-1β、Caspase-11和Caspase-1的表达,探索真菌性角膜炎时Caspase-11对IL-1β的调控作用。结果：(1)C57BL/6小鼠感染烟曲霉菌24h后,可见大量募集至角膜感染区域的中性粒细胞；所有能够表达IL-1β的细胞中,92.2%的是中性粒细胞；中性粒细胞中能够表达IL-1β的比例为85.4%。感染烟曲霉菌48h后,角膜区域出现较高比例的巨噬细胞；所有能够表达IL-1β的细胞中,29.7%的是巨噬细胞；巨噬细胞中能够表达IL-1β的比例为96.6%。(2)Signal1通路预激活后给予真菌刺激,C57BL/6小鼠中性粒细胞中pro-IL-1β的表达量明显增加,未预激活而只单纯给予真菌刺激,pro-IL-1β的表达量也明显增加,但与有预激相比其增加幅度略少；TLR4-1/-或TRIF-/-小鼠的中性粒细胞被真菌刺激4h或18h后,pro-IL-1β的表达较未感染时明显增多,但是与C57BL/6小鼠相比无明显差异。(3)Dectin-1-/--小鼠或者给予sky阻断剂的C57BL/6小鼠的中性粒细胞被真菌刺激4h后,pro-IL-1β的表达较C57BL/6小鼠明显减少；建立C57BL/6、NLRP3-/-、ASC-/-以及Caspase-1-/-小鼠真菌性角膜炎模型24h后,C57BL/6、NLRP3-/-和ASC-/-小鼠角膜中pro-IL-1β和mature IL-1β的表达量无明显差异,Caspase-1-/-小鼠角膜中pro-IL-1β的表达量无明显差异,但是mature IL-1β的表达量明显低于C57BL/6小鼠；NLRP3-/-和ASC-/-小鼠的中性粒细胞被真菌刺激4h后,pro-IL-1β和mature IL-1β的表达量与C57BL/6小鼠相比无明显差异；Caspase-1-/-小鼠或者给予Caspase-1阻断剂的C57BL/6小鼠的中性粒细胞被真菌刺激4h后,pro-IL-1β的表达量无明显差异,但mature IL-1β的表达量明显低于C57BL/6小鼠。(4)预先给予Caspase-11阻断剂的C57BL/6小鼠真菌性角膜炎模型,建模24h后其角膜中pro-IL-1β的表达量无明显差异,但是mature IL-1β的表达量明显低于未给予阻断剂的C57BL/6小鼠；给予Caspase-11阻断剂的C57BL/6小鼠的中性粒细胞被真菌刺激4h后,pro-IL-1β的表达量无明显差异,但mature IL-1β和mature Caspase-1的表达量明显低于单纯刺激组；随着真菌刺激时间的延长,C57BL/6小鼠中性粒细胞内Caspase-11的表达量逐渐增加；TLR4-/-以及TRIF-/-小鼠中性粒细胞被真菌刺激4h后,Caspase-11的表达量与C57BL/6小鼠无明显差异；Dectin-1-/-小鼠中性粒细胞被真菌刺激4h后,pro-Caspase-11的表达量略少于C57BL/6小鼠,给予syk阻断剂后C57BL/6小鼠中性粒细胞内pro-Caspase-11的表达明显低于未给予阻断剂者。结论：(1)IL-1β参与角膜抗真菌感染的固有免疫过程,中性粒细胞是该阶段主要的细胞来源；(2)真菌感染引起IL-1β的生成可以不通过经典Signal1通路的预激,TLR4/TRIF通路不参与pro-IL-1β的生成；(3)真菌感染时Dect in-1/syk通路参与IL-1β的生成,Caspase-1参与IL-1β由前体至成熟体的修饰,但炎性复合体NLRP3与ASC不参与IL-1β的生成；(4)Caspase-11通过调控Caspase-1由前体至成熟体的修饰过程进而参与IL-1β的生成。更多还原

【Abstract】 Objective To investigate the expression and molecular mechanisms of inflammatory cytokine IL-1β in fungal keratitis.Methods Mouse model of fungal keratitis established by release Aspergillus fumigatus spores into the corneal stroma with injection is used for in vivo experiments. Bone marrow cells isolated from mice are used for in vitro experiments.(1) To investigate the expression of IL-1β in the fungal keratitis model of C57BL/6mice with flow cytometry and confocal microscope and to explore the major cellular source of IL-1β in fungal keratitis.(2) To investigate the expression of IL-1β in the neutrophils of C57BL/6, TLR4-/-and TRIF-/-mice treated with Aspergillus fumigatus by ELISA and Western-blot and to explore the regulation for IL-1β by the classic Signal1pathway in fungal keratitis.(3) To investigate the expression of IL-1β in the fungal keratitis model of C57BL/6, NLRP3-/-, ASC-/-and Caspase-1-/-mice and in the neutrophils of C57BL/6, Dectin-1-/-, NLRP3-/-, ASC-/-and Caspase-1-/-mice treated with Aspergillus fumigatus by ELISA and Western-blot and to explore the regulation for IL-1β by the classic Signal2pathway in fungal keratitis.(4) To investigate the expression of IL-1β, Caspase-11and Caspase-1in the fungal keratitis model of C57BL/6and Caspase-1-/-mice and in the neutrophils of C57BL/6, Dectin-1-/-, TLR4-/-, TRIF-/-and Caspase-1-/-mice treated with Aspergillus fumigatus by Western-blot and to explore the regulation for IL-1β by Caspase-11in fungal keratitis.Results (1) There is a pronounced neutrophil infiltration to the corneal stroma of mouse fungal keratitis model in24h.92.2%cells in cornea of fungal keratitis which can express IL-1β are neutrophils and85.4%neutrophils can express IL-1β. There are many macrophages recruitment to the corneal stroma in48h.29.7%cells in cornea which can express IL-1β are macrophages and96.6%macrophages can express IL-1β.(2) The expression of pro-IL-1β in bone marrow neutrophils of C57BL/6mice treated with Aspergillus fumigatus spores increased when the priming signal was activated in advance. The expression of pro-IL-1β also increased without the priming, but was slightly less than priming group. The expression of pro-IL-1β in neutrophils of TLR4-/-or TRIF-/-mice treated with Aspergillus fumigatus spores increased in4h and18h, but was not different from C57BL/6mice.(3) The expression of pro-IL-1β in neutrophils of Dectin-1-/-mice or C57BL/6mice added syk inhibitor in advance treated with Aspergillus fumigatus spores was less than C57BL/6mice in4h. The expression of pro-IL-1β and mature IL-1β in the corneas of fungal keratitis model of NLRP3-/-or ASC-/-mice was not different from C57BL/6mice in24h. The expression of pro-IL-1β in the corneas of fungal keratitis model of Caspase-1-/-mice was not different from C57BL/6mice, but mature IL-1β was significantly less than C57BL/6mice in24h. The expression of pro-IL-1β and mature IL-1β in neutrophils of NLRP3-/-or ASC-/-mice treated with Aspergillus fumigatus spores was not different from C57BL/6mice in4h. The expression of pro-IL-1β in neutrophils of Caspase-1-/-mice or C57BL/6mice added Caspase-1inhibitor in advance treated with Aspergillus fumigatus spores was not different from C57BL/6mice, but mature IL-1β was significantly less than C57BL/6mice in4h.(4) The expression of pro-IL-1β in the corneas of fungal keratitis model of C57BL/6mice added Caspase-11inhibitor in advance was not different from C57BL/6mice, but mature IL-1β was significantly less than C57BL/6mice in24h. The expression of pro-IL-1β in neutrophils of C57BL/6mice added Caspase-11inhibitor in advance treated with Aspergillus fumigatus spores was not different from C57BL/6mice, but mature IL-1β and mature Caspase-1were significantly less than C57BL/6mice in4h. The expression of Caspase-11was time-dependent in neutrophils of C57BL/6mice treated with Aspergillus fumigatus spores. The expression of Caspase-11in neutrophils of TLR4-/-or TRIF-/-mice treated with Aspergillus fumigatus spores was not different from C57BL/6mice in4h. The expression of pro-Caspase-11in neutrophils of Dectin-1-/-mice treated with Aspergillus fumigatus spores was slightly less than C57BL/6mice in4h. But the expression of pro-Caspase-11in neutrophils of C57BL/6mice added Caspase-11inhibitor in advance treated with Aspergillus fumigatus spores was significantly less than C57BL/6mice in4h. Conclusions (1) IL-1β has a role in the innate immunity of fungal keratitis, and neutrophil is its major cellular source.(2) IL-lp can be expressed to against fungal infection without the priming signal, and the expression of pro-IL-1β is independent of TLR4/TRIF.(3) The expression of IL-1β is dependent on Dectin-1/syk in the innate immunity of fungal keratitis. Caspase-1leads to maturation of the proinflammatory cytokines IL-1β in response to fungal infection, but NLRP3inflammasome and ASC have no roles in this activation process.更多还原