SSL5-PMPs促进单核细胞炎症反应的作用和机制研究
发布时间：2018-09-19 14:33
【摘要】：研究背景心血管疾病是全世界居首位的死亡原因,其最主要的病因是动脉粥样硬化。动脉粥样硬化的本质是由多种因素引起的血管慢性炎症,其中病原体的多重感染,即“感染负荷(infectious burden) ",被认为是动脉粥样硬化新的独立危险因素。感染因素可通过直接作用、触发全身性炎症和启动自身免疫反应等多种方式对动脉壁造成损害,导致动脉粥样硬化的发生发展,其中病原体激活血小板所导致的血管炎症和血栓形成具有重要作用。金黄色葡萄球菌(Staphylococcus aureus, S. aureus)是临床常见的致病菌之一。有研究显示,血行感染S. aureus会增加急性心肌梗死和脑卒中的风险,但机制尚不明确,目前认为可能与血小板激活、炎症反应增强以及血液的高凝状态有关。本课题小组前期研究发现,金黄色葡萄球菌超抗原样蛋白-5 (Staphylococcal superantigen-like protein 5, SSL5)可以激活血小板,一次性静脉注射SSL5还可导致小鼠肺动脉内形成血栓。血小板不仅在血栓和止血的过程中发挥作用,同时还是重要的免疫和炎症细胞,其可以通过激活产生的微粒或与白细胞相互作用导致炎症反应。因此,我们推测SSL5可能通过激活血小板而诱发炎症反应,探讨其机制可以为阐明感染负荷在动脉粥样硬化中的作用提供新的实验证据。研究内容1.采用流式细胞术,鉴定重组SSL5的生物学活性,检测外周血中PMPs数量以及SSL5激活的PMPs(SSL5-PMPs)表面分子的特征；利用扫描电镜观察SSL5-PMPs的形态学特征；体外制备SSL5-PMPs以及ADP激活的PMPs (ADP-PMPs)和凋亡产生的PMPs (ap-PMPs),作为后续实验的刺激物和对照。2.以人外周血单核细胞及THP-1细胞作为实验对象,利用CCK-8法检测SSL5对单核细胞活力的影响；扫描电镜观察SSL5-PMPs与单核细胞的结合,流式细胞术进一步检测两者结合的时效关系和差异性；以产生炎症介质(细胞因子、趋化因子和基质金属蛋白酶)及发生迁移作为单核细胞炎症效应的指标,实时定量PCR检测炎症介质mRNA的表达,ELISA法检测细胞因子及趋化因子释放的数量,明胶酶谱法检测蛋白酶的活性,transwell迁移试验检测细胞的迁移。同时一并观察对照PMPs (ADP-PMPs及ap-PMPs)和SSL5对单核细胞炎症介质mRNA表达的影响,以探讨PMPs致单核细胞炎症效应的差异性及SSL5可能的病理作用。3.以THP-1细胞作为实验对象,利用中和性抗体干扰SSL5-PMPs与THP-1细胞间CD40L/CD40和P-选择素/PSGL-1的结合,观察其对THP-1细胞产生细胞因子及迁移的影响,以明确介导SSL5-PMPs与THP-1细胞发生相互作用的分子基础；利用siRNA沉默THP-1细胞CD40或TRAF6的基因表达,观察其对THP-1细胞产生细胞因子的影响,同时采用Western blot和细胞免疫荧光等方法检测NFκB磷酸化及核转位的情况,以明确THP-1细胞产生炎症反应的胞内信号通路；进一步利用抑制剂阻断TLR-4信号通路,了解其对SSL5-PMPs促进THP-1细胞炎症反应的影响。研究结果1.SSL5激活人血小板并产生血小板(SSL5-PMPs).①重组SSL5具有生物学活性,可用于后续实验；②SSL5导致人外周血中PMPs的数量明显增加；③ SSL5-PMPs的直径为0.1～1 μm, PS外暴露于膜表面,表达血小板特异性的糖蛋白GPⅡb以及与细胞间相互作用的信号分子P-选择素和CD40L。2. SSL5-PMPs促进人单核细胞的致炎症效应。①SSL5不影响THP-1细胞的活力,排除因SSL5毒性作用而导致单核细胞产生炎症反应；② SSL5-PMPs与THP-1细胞的结合呈时间依赖性,且主要与外周血CD14++CD16+(中间型)的单核细胞结合；③ SSL5-PMPs时间和剂量依赖性的促进单核细胞产生炎症介质,包括表达和释放IL-1β和TNFα、MCP-1以及MMP-9,并增强MMP-9的活性；④ ADP-PMPs和ap-PMPs也可以促进THP-1细胞炎症介质mRNA的表达,其水平低于SSL5-PMPs刺激的结果,而SSL5对THP-1细胞炎症介质mRNA的表达没有影响；⑤ SSL5-PMPs促进MCP-1诱导的单核细胞发生迁移,而SSL5则抑制迁移；3. SSL5-PMPs促进人单核细胞炎症反应的机制。①阻断CD40L与CD40的相互作用,可以部分抑制SSL5-PMPs诱导单核细胞产生炎症介质,而抑制P-选择素与PSGL-1的相互作用则对炎症介质的产生没有影响；②阻断PSGL-1导致SSL5-PMPs介导的单核细胞迁移几乎完全被抑制,而抑制CD40L与CD40的相互作用则明显减少单核细胞迁移的比率；③下调单核细胞CD40或TRAF6基因的表达,导致 SSL5-PMPs诱导单核细胞炎症介质的产生减少,并抑制NFκB p65亚单位的磷酸化及核转位；④阻断TLR4信号通路对SSL5-PMPs诱导单核细胞释放炎症介质没有影响。研究结论SSL5,S.aureus分泌的一种毒素可以激活血小板并产生PMPs(SSL5-PMPs),导致外周血中的PMPs数量明显增加；SSL5-PMPs与单核细胞结合,且主要与外周血中的具有促炎功能的中间型单核细胞结合,并促进单核细胞产生广泛的炎症反应,包括增加炎性细胞因子和趋化因子的释放、增强基质金属蛋白酶的活性以及诱导迁移；在机制方面,SSL5-PMPs通过其来源的CD40L与单核细胞CD40发生直接的相互作用,激活单核细胞内CD40-TRAF6-NFκB信号通路,从而触发单核细胞的炎症反应；而SSL5本身则抑制白细胞的功能。本研究阐述了S.aureus导致机体产生炎症反应的一种新方式,以期从这一新的角度阐明感染负荷的致动脉粥样硬化机制。
[Abstract]:Background Cardiovascular disease is the leading cause of death in the world. Atherosclerosis is essentially chronic inflammation of blood vessels caused by a variety of factors. Multiple infections of pathogens, i.e. "infectious burden", are considered to be a new independent risk of atherosclerosis. Infectious factors can damage the arterial wall by direct action, triggering systemic inflammation and initiating autoimmune response, leading to the occurrence and development of atherosclerosis. S, S. aureus) is one of the common clinical pathogens. Studies have shown that S. aureus infection increases the risk of acute myocardial infarction and stroke, but the mechanism is not clear. At present, it is thought that it may be related to platelet activation, increased inflammation and blood hypercoagulability. Staphylococcal superantigen-like protein 5 (SSL5) can activate platelets, and a single intravenous injection of SSL5 can also cause thrombosis in the pulmonary arteries of mice. Therefore, we speculate that SSL5 may induce inflammation by activating platelets, and explore its mechanism to provide new experimental evidence for elucidating the role of infection load in atherosclerosis. Content 1. Using flow cytometry to identify the biological activity of recombinant SSL5, in vitro detection. The number of PMPs in peripheral blood and the surface molecules of SSL5-activated PMPs (SSL5-PMPs); the morphological characteristics of SSL5-PMPs were observed by scanning electron microscopy; SSL5-PMPs and ADP-activated PMPs (ADP-PMPs) and apoptosis-induced PMPs (ap-PMPs) were prepared in vitro as stimulants and controls in subsequent experiments. 2. Human peripheral blood mononuclear cells and THP-1 were fine. Cells were used as experimental subjects to detect the effect of SSL5 on the viability of monocytes by CCK-8 method; the binding of SSL5-PMPs to monocytes was observed by scanning electron microscopy; the binding time-dependent relationship and difference between them were further detected by flow cytometry; the production of inflammatory mediators (cytokines, chemokines and matrix metalloproteinases) and migration were used as mononuclear cells. The expression of inflammatory mediators mRNA was detected by real-time quantitative PCR, the release of cytokines and chemokines by ELISA, the activity of protease by gelatin zymogram, and the migration of cells by Transwell migration assay. To investigate the difference of PMPs-induced inflammation in monocytes and the possible pathological role of SSL5. 3. Using THP-1 cells as experimental subjects, neutralizing antibodies interfered with the binding of CD40L/CD40 and P-selectin/PSGL-1 between SSL5-PMPs and THP-1 cells, and observed the effect of neutralizing antibodies on the production of cytokines and migration of THP-1 cells. To clarify the molecular basis of interaction between SSL5-PMPs and THP-1 cells, siRNA was used to silence the gene expression of CD40 or TRAF6 in THP-1 cells and observe its effect on the production of cytokines in THP-1 cells. Results 1. SSL5 activates human platelets and produces platelets (SSL5-PMPs). 1. Recombinant SSL5 has biological activity and can be used in subsequent experiments; 2. SSL5 induces PM in human peripheral blood. (3) SSL5-PMPs with a diameter of 0.1-1 micron were exposed to the membrane surface, and expressed platelet-specific glycoprotein GP II B and the signal molecules P-selectin and CD40L.2.SSL5-PMPs interacting with each other. SSL5 did not affect the activity of THP-1 cells and excluded the inflammatory effect of SSL5 on human monocytes. The toxicity of SSL5-PMPs induces inflammation of monocytes; the binding of SSL5-PMPs to THP-1 cells is time-dependent and mainly with peripheral blood CD14 +-CD16 + (intermediate type) monocytes; and the time-and dose-dependent effects of SSL5-PMPs promote the production of inflammatory mediators by monocytes, including the expression and release of IL-1beta and TNFalpha, and MCP-1 to ADP-PMPs and ap-PMPs could also promote the expression of inflammatory mediators mRNA in THP-1 cells, which was lower than that stimulated by SSL5-PMPs. SSL5 had no effect on the expression of inflammatory mediators mRNA in THP-1 cells. _SSL5-PMPs promoted MCP-1-induced migration of monocytes, while SSL5 inhibited migration. The mechanism of 5-PMPs promoting inflammation in human monocytes is: (1) Blocking the interaction between CD40L and CD40 partially inhibits the production of inflammatory mediators in monocytes induced by SSL5-PMPs, while inhibiting the interaction between P-selectin and PSGL-1 has no effect on the production of inflammatory mediators; (2) Blocking the interaction between PSGL-1 and SSL5-PMPs almost leads to the migration of monocytes mediated by PSGL-1. Inhibiting the interaction between CD40L and CD40 significantly reduced the rate of monocyte migration; 3) Downregulating the expression of CD40 or TRAF6 gene in monocytes, resulting in the decrease of inflammatory mediators in monocytes induced by SSL5-PMPs, and inhibiting the phosphorylation and nuclear translocation of NF-kappa B p65 subunit; 4) Blocking the TLR4 signaling pathway to SSL5-PMPs. Conclusion SSL5, a toxin secreted by S. aureus, can activate platelets and produce PMPs (SSL5-PMPs), resulting in a marked increase in the number of PMPs in peripheral blood; SSL5-PMPs bind to monocytes, and mainly bind to intermediate monocytes with pro-inflammatory function in peripheral blood. Promote a wide range of inflammatory responses in monocytes, including increasing the release of inflammatory cytokines and chemokines, enhancing the activity of matrix metalloproteinases and inducing migration; in mechanism, SSL5-PMPs interact directly with monocyte CD40 through CD40L, activating CD40-TRAF6-NF-kappa B signaling in monocytes. This study elucidates a new way that S. aureus causes inflammation in the body, in order to elucidate the atherosclerotic mechanism of infection load from this new perspective.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54
本文编号：2250416
[Abstract]:Background Cardiovascular disease is the leading cause of death in the world. Atherosclerosis is essentially chronic inflammation of blood vessels caused by a variety of factors. Multiple infections of pathogens, i.e. "infectious burden", are considered to be a new independent risk of atherosclerosis. Infectious factors can damage the arterial wall by direct action, triggering systemic inflammation and initiating autoimmune response, leading to the occurrence and development of atherosclerosis. S, S. aureus) is one of the common clinical pathogens. Studies have shown that S. aureus infection increases the risk of acute myocardial infarction and stroke, but the mechanism is not clear. At present, it is thought that it may be related to platelet activation, increased inflammation and blood hypercoagulability. Staphylococcal superantigen-like protein 5 (SSL5) can activate platelets, and a single intravenous injection of SSL5 can also cause thrombosis in the pulmonary arteries of mice. Therefore, we speculate that SSL5 may induce inflammation by activating platelets, and explore its mechanism to provide new experimental evidence for elucidating the role of infection load in atherosclerosis. Content 1. Using flow cytometry to identify the biological activity of recombinant SSL5, in vitro detection. The number of PMPs in peripheral blood and the surface molecules of SSL5-activated PMPs (SSL5-PMPs); the morphological characteristics of SSL5-PMPs were observed by scanning electron microscopy; SSL5-PMPs and ADP-activated PMPs (ADP-PMPs) and apoptosis-induced PMPs (ap-PMPs) were prepared in vitro as stimulants and controls in subsequent experiments. 2. Human peripheral blood mononuclear cells and THP-1 were fine. Cells were used as experimental subjects to detect the effect of SSL5 on the viability of monocytes by CCK-8 method; the binding of SSL5-PMPs to monocytes was observed by scanning electron microscopy; the binding time-dependent relationship and difference between them were further detected by flow cytometry; the production of inflammatory mediators (cytokines, chemokines and matrix metalloproteinases) and migration were used as mononuclear cells. The expression of inflammatory mediators mRNA was detected by real-time quantitative PCR, the release of cytokines and chemokines by ELISA, the activity of protease by gelatin zymogram, and the migration of cells by Transwell migration assay. To investigate the difference of PMPs-induced inflammation in monocytes and the possible pathological role of SSL5. 3. Using THP-1 cells as experimental subjects, neutralizing antibodies interfered with the binding of CD40L/CD40 and P-selectin/PSGL-1 between SSL5-PMPs and THP-1 cells, and observed the effect of neutralizing antibodies on the production of cytokines and migration of THP-1 cells. To clarify the molecular basis of interaction between SSL5-PMPs and THP-1 cells, siRNA was used to silence the gene expression of CD40 or TRAF6 in THP-1 cells and observe its effect on the production of cytokines in THP-1 cells. Results 1. SSL5 activates human platelets and produces platelets (SSL5-PMPs). 1. Recombinant SSL5 has biological activity and can be used in subsequent experiments; 2. SSL5 induces PM in human peripheral blood. (3) SSL5-PMPs with a diameter of 0.1-1 micron were exposed to the membrane surface, and expressed platelet-specific glycoprotein GP II B and the signal molecules P-selectin and CD40L.2.SSL5-PMPs interacting with each other. SSL5 did not affect the activity of THP-1 cells and excluded the inflammatory effect of SSL5 on human monocytes. The toxicity of SSL5-PMPs induces inflammation of monocytes; the binding of SSL5-PMPs to THP-1 cells is time-dependent and mainly with peripheral blood CD14 +-CD16 + (intermediate type) monocytes; and the time-and dose-dependent effects of SSL5-PMPs promote the production of inflammatory mediators by monocytes, including the expression and release of IL-1beta and TNFalpha, and MCP-1 to ADP-PMPs and ap-PMPs could also promote the expression of inflammatory mediators mRNA in THP-1 cells, which was lower than that stimulated by SSL5-PMPs. SSL5 had no effect on the expression of inflammatory mediators mRNA in THP-1 cells. _SSL5-PMPs promoted MCP-1-induced migration of monocytes, while SSL5 inhibited migration. The mechanism of 5-PMPs promoting inflammation in human monocytes is: (1) Blocking the interaction between CD40L and CD40 partially inhibits the production of inflammatory mediators in monocytes induced by SSL5-PMPs, while inhibiting the interaction between P-selectin and PSGL-1 has no effect on the production of inflammatory mediators; (2) Blocking the interaction between PSGL-1 and SSL5-PMPs almost leads to the migration of monocytes mediated by PSGL-1. Inhibiting the interaction between CD40L and CD40 significantly reduced the rate of monocyte migration; 3) Downregulating the expression of CD40 or TRAF6 gene in monocytes, resulting in the decrease of inflammatory mediators in monocytes induced by SSL5-PMPs, and inhibiting the phosphorylation and nuclear translocation of NF-kappa B p65 subunit; 4) Blocking the TLR4 signaling pathway to SSL5-PMPs. Conclusion SSL5, a toxin secreted by S. aureus, can activate platelets and produce PMPs (SSL5-PMPs), resulting in a marked increase in the number of PMPs in peripheral blood; SSL5-PMPs bind to monocytes, and mainly bind to intermediate monocytes with pro-inflammatory function in peripheral blood. Promote a wide range of inflammatory responses in monocytes, including increasing the release of inflammatory cytokines and chemokines, enhancing the activity of matrix metalloproteinases and inducing migration; in mechanism, SSL5-PMPs interact directly with monocyte CD40 through CD40L, activating CD40-TRAF6-NF-kappa B signaling in monocytes. This study elucidates a new way that S. aureus causes inflammation in the body, in order to elucidate the atherosclerotic mechanism of infection load from this new perspective.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R54
【参考文献】
相关期刊论文 前1条
1 Rosa Sessa;Marisa Di Pietro;Simone Filardo;Ombretta Turriziani;;Infectious burden and atherosclerosis: A clinical issue[J];World Journal of Clinical Cases;2014年07期
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