NLRP7炎症复合体在THP-1巨噬细胞防御牛分枝杆菌中的机制研究

发布时间：2017-12-31 18:32

本文关键词：NLRP7炎症复合体在THP-1巨噬细胞防御牛分枝杆菌中的机制研究　出处：《中国农业大学》2016年博士论文　论文类型：学位论文

更多相关文章： 牛分枝杆菌 IL-1β 炎症复合体 NLRP7

【摘要】：牛结核病是一种慢性消耗性传染病,致病菌是牛分枝杆菌,是结核分枝杆菌复合群的成员之一。每年全世界约有五千万头牛感染牛分枝杆菌,引起经济损失约三十亿美元。牛分枝杆菌能突破种间屏障感染包括人类在内的多种哺乳动物,全球2%左右的人结核病由牛分枝杆菌引起。由于该菌宿主范围广泛,在动物与人之间交叉感染,严重威胁了人类的健康,也极大的增加了牛分枝杆菌的清除和防控难度。巨噬细胞是机体防御病原菌的第一道防线。机体感染分枝杆菌后启动免疫应答释放细胞因子,体内外研究表明促炎因子IL-1β的释放对宿主细胞防御分枝杆菌的感染起着重要作用。牛分枝杆菌的细胞壁和细胞膜含有广谱的脂肽,并分泌MPB70/80等脂蛋白。NLRP7炎症复合体由NLRP7、ASC和caspase-1组成,能通过识别脂肽激活caspase-1,释放IL-1β,但牛分枝杆菌是否能激活NLRP7炎症复合体以及NLRP7炎症复合体在细胞防御牛分枝杆菌中的作用尚不清楚。本研究用牛分枝杆菌感染THP-1巨噬细胞2 h,然后用PBS洗去没有吞噬进入细胞的细菌,感染后3h发现IL-1β前体的蛋白表达量即显著升高,IL-1β活性形式的释放在3-50 h范围内逐渐增多。MOI为0.1-100感染牛分枝杆菌,caspase-1活性形式的释放不断增多,呈浓度依赖型,IL-1β的释放在MOI为0.1-10范围内不断增加,MOI=100时没有进一步增加。牛分枝杆菌诱导THP-1巨噬细胞分泌IL-1β依赖于caspase-1的活化以及细胞对牛分枝杆菌的吞噬作用。THP-1巨噬细胞感染牛分枝杆菌后NLRP7、ASC和 caspase-1发生聚集,NLRP7的转录水平发生上调,并与ASC和caspase-1共定位,干扰NLRP7或ASC的蛋白表达后caspase-1的活化以及IL-1β的释放受到显著抑制,表明NLRP7炎症复合体参与牛分枝杆菌诱导THP-1巨噬细胞释放IL-1β。牛分枝杆菌激活NLRP7炎症复合体有助于TNF-α、CCL3和IL-1β转录水平的升高,并促进依赖于caspase-1活化的细胞焦亡。分别干扰NLRP7或ASC的蛋白表达后,牛分枝杆菌在THP-1巨噬细胞内的存活率显著升高,表明NLRP7炎症复合体的激活可显著抑制牛分枝杆菌在胞内的存活。炎症复合体的激活受到多种因素的调控。为了阐明牛分枝杆菌在激活THP-1巨噬细胞NLRP7炎症复合体过程中受到的调控,本研究探索了高浓度钾离子、新蛋白的合成、去泛素化以及活性氧簇四种途径对牛分枝杆菌激活NLRP7炎症复合体的影响。试验分别使用高浓度钾离子、环己烯亚胺、PR-619和NAC四种抑制剂与牛分枝杆菌或NLRP7炎症复合体激动剂Pam3CSK4共处理THP-1巨噬细胞,检测IL-1β的释放以及NLRP7炎症复合体各组分的转录水平。结果表明,牛分枝杆菌激活NLRP7炎症复合体受到高浓度钾离子的抑制,并需要新蛋白的合成；牛分枝杆菌诱导IL-1β的分泌需要活性氧簇(mROS)的生成,而mROS对NLRP7炎症复合体的激活不起作用,表明mROS不参与调控牛分枝杆菌激活NLRP7炎症复合体；去泛素化促进牛分枝杆菌诱导IL-1体的分泌,抑制NLRP7炎症复合体的激活。在以上四种途径中,仅ASC的转录水平变化趋势与IL-1β的分泌水平变化一致。综上所述,牛分枝杆菌感染THP-1巨噬细胞后激活NLRP7炎症复合体,并受到高浓度钾离子和新蛋白合成的调控,mROS对其没有影响,去泛素化通过抑制NLRP7炎症复合体的激活负调控牛分枝杆菌诱导分泌IL-1β。
[Abstract]:Bovine tuberculosis is a chronic infectious disease, pathogen Mycobacterium bovis, is one of the members of the Mycobacterium tuberculosis complex. Around the world each year about fifty million cows infected with Mycobacterium bovis, causing economic losses of about $three billion. Mycobacterium bovis can break the species barrier to infect a variety of mammals including humans. About 2% of the world's human tuberculosis caused by Mycobacterium bovis bacteria. Due to the wide host range, cross infection between human and animal, a serious threat to human health, but also greatly increased the m.bovis removal and prevention difficult. Macrophages are the first line of host defense of pathogens infection of Mycobacterium. To initiate an immune response to release cytokines, in vitro and in vivo proinflammatory factor IL-1 beta release infection on host cell defense Mycobacterium plays an important role. Bovine Mycobacterium The lipopeptide bacteria cell wall and cell membrane with broad spectrum, and the secretion of MPB70/80 lipoprotein.NLRP7 inflammasome by NLRP7, ASC and caspase-1, can activate caspase-1 by identifying the lipopeptide, the release of IL-1 beta, but whether Mycobacterium bovis can activate the NLRP7 complex and NLRP7 complex in inflammatory inflammatory cells of Mycobacterium bovis in defense the role is not clear. This study used m.bovis-infected macrophages THP-1 2 h, and then use PBS to wash away not to enter the cell phagocytosis of bacteria, IL-1 beta precursor protein expression increased significantly 3h after infection, IL-1 beta activity in the form of 3-50 release in the range of H.MOI gradually increased 0.1-100 infection of Mycobacterium bovis coli, the activity of caspase-1 in the form of release increased in a concentration dependent type, IL-1 beta release of MOI in the range of 0.1-10 increase, no further increase of MOI=100 THP-1 induced by Mycobacterium bovis. Macrophages secrete IL-1 beta dependent activation of Caspase-1 cells and bovine Mycobacterium.THP-1 macrophage phagocytosis of Mycobacterium bovis infection after NLRP7, ASC and caspase-1 together, the transcriptional level of NLRP7 increased, and the co localization with ASC and caspase-1, the expression of NLRP7 protein after ASC interference or caspase-1 activation and IL-1 beta release by showed that NLRP7 significantly inhibited the inflammatory complex involved in Mycobacterium bovis IL-1 beta release of THP-1 macrophages induced by Mycobacterium bovis. Activation of NLRP7 inflammasome contributes to TNF- alpha, IL-1 beta and CCL3 increased the level of transcription and promote caspase-1 dependent activation of pyroptosis. Expression of NLRP7 or ASC protein interference respectively, significantly increased survival the rate of Mycobacterium bovis in THP-1 macrophages, suggesting that survival activation of NLRP7 complex can inhibit the inflammation of Mycobacterium bovis in cellular inflammation. Complex activation regulated by many factors. In order to elucidate the regulation of Mycobacterium bovis by macrophages in the activation of THP-1 inflammasome NLRP7 process, this study explored the high concentration of potassium ion, the synthesis of new proteins to affect ubiquitination and reactive oxygen species in four ways on the activation of NLRP7 of Mycobacterium inflammation complex. Experiments were used high concentration of potassium ion, cyclohexene imine, PR-619 and NAC four inhibitors with Mycobacterium bovis or inflammasome NLRP7 agonist Pam3CSK4 were THP-1 macrophages, the transcription level detection of IL-1 beta release and NLRP7 inflammatory complex components. The results showed that Mycobacterium bovis NLRP7 inhibited inflammatory activation of high concentration potassium ion complex the synthesis, and needs a new protein; IL-1 beta induced Mycobacterium bovis secretion to reactive oxygen species (mROS) generation, while the mROS of NLRP7 inflammatory compound The activation does not play a role, suggesting that mROS is not involved in the regulation of Mycobacterium bovis NLRP7 inflammasome activation; secretion to promote the ubiquitination of Mycobacterium bovis induced IL-1, inhibiting activation of inflammasome NLRP7. In the above four ways, only ASC expression level change trend and IL-1 beta secretion change. To sum up THP-1, infection of Mycobacterium bovis NLRP7 after activation of macrophage inflammation complex, which is regulated by high concentration of potassium ions and the synthesis of new proteins, mROS has no effect on the deubiquitination of NLRP7 by inhibiting the activation of inflammatory complex negative regulation of Mycobacterium bovis induced secretion of IL-1 beta.

【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61

【相似文献】