结核分枝杆菌对中性粒细胞功能和共刺激分子表达的影响及相关机制

发布时间：2018-09-13 14:05

【摘要】：结核病是由结核分枝杆菌（Mycobacterium tuberculosis,Mtb）感染引起的以呼吸系统感染为主的慢性感染性疾病。由于结核病导致的组织损伤主要由机体的免疫系统介导，而且在结核病患者体内往往存在Th1型免疫应答受抑， Th2型免疫应答水平异常升高的现象，结核病也被认为是一种免疫紊乱性疾病，机体免疫应答的方向和水平在很大程度上决定着结核病的发展与转归。中性粒细胞作为机体重要的固有免疫效应细胞，是机体抵抗病原微生物感染的第一道防线。早期的研究认为中性粒细胞仅作为吞噬细胞在Mtb感染的早期参与抗结核病的固有免疫应答，但近年来的研究显示中性粒细胞还参与了结核病适应性免疫的诱导和调节，对结核病免疫的发展有重要影响。共刺激分子是一类表达于免疫细胞及免疫相关细胞上的蛋白，其介导的信号即为共刺激信号。共刺激信号对机体免疫应答的调节是双向的，通过正、负共刺激信号的调节使机体维持免疫平衡状态。正性共刺激分子CD28、CD80、CD86等能激活T细胞，使其增殖、分化为效应细胞，增强机体免疫应答反应，抵抗外来入侵的病原微生物；负性共刺激分子程序性死亡因子1（Programmeddeath1，PD-1）、程序性死亡因子1配体（Ligand of Programmed death1，PD-L1）则抑制T细胞增殖和产生细胞因子,抑制免疫应答反应,防止过度的免疫损伤和自身免疫性疾病的发生,有利于维持机体的免疫自稳。近年来有研究显示中性粒细胞表面也会有共刺激分子的表达，提示我们中性粒细胞表达共刺激分子可能对机体抗结核病免疫有一定影响。本研究通过对比分析活动性肺结核患者和健康人外周血中性粒细胞表面共刺激分子的表达水平及功能的差异，探讨结核病患者外周血中性粒细胞共刺激分子的表达特性及细胞功能的改变。并通过Mtb体外感染的方法验证了Mtb对中性粒细胞功能和共刺激分子表达的影响。在此基础上，本研究采用特异性单抗阻断PD-1/PD-L1信号途径的方法，初步探讨了该负性共刺激信号对中性粒细胞功能的调节作用。第一部分结核病患者外周血中性粒细胞的功能及共刺激分子的表达特性目的：通过分析结核病患者中性粒细胞共刺激分子的表达水平及细胞功能的变化，探讨结核病患者免疫功能异常与共刺激分子的关系。方法：收集30例活动性肺结核患者和20例健康体检者的外周血，应用流式细胞术检测中性粒细胞表面共刺激分子的表达水平，CD62L的表达水平以及呼吸爆发产生ROS的水平；用硝酸还原酶法检测中性粒细胞产生的NO；用Transwell装置检测中性粒细胞对IL-8的趋化能力。结果：与健康对照组相比，结核病患者外周血中性粒细胞具有以下特征：（1）CD86表达显著降低（P 0.05），PD-L1分子表达显著增加（P 0.01），而CD28、CD80、HLA-DR和PD-1均无显著性变化（P0.05）；（2）经PMA刺激后，呼吸爆发产生的ROS和NO水平显著提高（P0.05）（；3）CD62L表达无显著性差异（P0.05）；（4）对IL-8的趋化能力显著降低（P 0.05）。结论：结核病患者中性粒细胞正性共刺激分子CD86表达下降，负性共刺激分子PD-L1表达升高，产生ROS和NO的量明显增高，趋化能力有所降低。由此提示，共刺激分子表达失调可能会影响中性粒细胞功能。第二部分结核分枝杆菌感染对中性粒细胞的功能及共刺激分子表达的影响目的：分析Mtb感染后中性粒细胞共刺激分子的表达水平及细胞功能的变化，探讨Mtb感染对中性粒细胞的功能及共刺激分子表达的影响方法：收集20例健康体检者的外周血，应用流式细胞术检测感染Mtb后中性粒细胞表面共刺激分子的表达水平，呼吸爆发产生ROS和CD62L的表达水平以及中性粒细胞的凋亡率；用硝酸还原酶法检测感染Mtb后中性粒细胞产生的NO；使用Transwell装置检测感染Mtb后中性粒细胞对IL-8的趋化能力；用菌落计数（CFU）的方法观察中性粒细胞对Mtb的杀菌能力。结果：（1）与对照组相比，加入Mtb感染后中性粒细胞具有以下特征：①CD28、CD80、CD86、PD-1和PD-L1表达水平显著提高（P 0.01），两组中HLA-DR表达无显著性差异（P0.05）；②产生ROS和NO的水平显著提高（P 0.01）；③CD62L表达水平显著降低（P 0.01）；④对IL-8的趋化能力显著提高（P0.05）。（2）MS和Mtb感染均可显著上调中性粒细胞的凋亡水平，但与MS组相比，Mtb感染的中性粒细胞凋亡率更低（P 0.01）。（3）中性粒细胞能够有效地杀伤无致病性的MS，，却不能杀伤有毒性的Mtb,导致Mtb在细胞内繁殖。结论： 1、Mtb感染能有效活化中性粒细胞，同时伴随一系列共刺激分子的表达改变，但这些共刺激分子的变化是否与中性粒细胞的功能改变相关需要进一步的研究。 2、Mtb体外感染导致的中性粒细胞共刺激分子的改变与结核病患者外周血中性粒细胞表面共刺激分子的表达模式不完全一致，具体原因有待对结核病患者感染局部的中性粒细胞功能进行检测分析后确定。第三部分体外阻断PD-1/PD-L1通路对中性粒细胞功能的影响目的：通过在体外对PD-1/PD-L1信号通路进行干预，探讨该负性共刺激信号对中性粒细胞功能的调节作用。方法：收集15例健康体检者的外周血，加入抗PD-L1单克隆抗体阻断PD-1/PD-L1通路后，再加入Mtb感染30min，应用流式细胞术检测中性粒细胞ROS水平和CD62L的表达以及通过transwell趋化装置检测中性粒细胞趋化能力的改变。结果：与Mtb感染组相比，体外阻断PD-1/PD-L1信号通路后,中性粒细胞产生ROS水平显著增加（P 0.05），CD62L的表达水平显著降低（P 0.05），中性粒细胞对IL-8的趋化能力提高（P 0.05）。结论：体外阻断PD-1/PD-L1信号通路可以增强中性粒细胞的呼吸爆发功能，加速活化并增强其趋化能力，说明该负性共刺激信号会抑制中性粒细胞抗结核免疫的能力，这可能为临床治疗结核病开辟一条新途径。
[Abstract]:Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). Tissue damage caused by tuberculosis is mainly mediated by the body's immune system. Th1 immune response is often suppressed in patients with tuberculosis. Th2 immune response level Tuberculosis is also considered to be an immune disorder, and the direction and level of immune response to a large extent determines the development and outcome of tuberculosis.
Neutrophils, as important innate immune effector cells, are the first line of defense against pathogenic microbial infections. Early studies suggested that neutrophils, as phagocytes, participate only in the innate immune response to tuberculosis in the early stage of MTB infection, but recent studies have shown that neutrophils also participate in tuberculosis. Induction and regulation of adaptive immunity play an important role in the development of tuberculosis immunity.
Co-stimulatory molecule is a kind of protein expressed in immune cells and immune-related cells, and its mediated signal is co-stimulatory signal. Co-stimulatory signal regulates the immune response of the body in two directions. The body maintains immune balance through the regulation of positive and negative co-stimulatory signals. Cells, which proliferate and differentiate into effector cells, enhance the body's immune response and resist invasive pathogenic microorganisms; negative costimulatory molecule programmed death factor 1 (PD-1), programmed death factor 1 ligand (PD-L1) inhibits T cell proliferation and production of cytokines, inhibits immune response. Recent studies have shown that the expression of costimulatory molecules on the surface of neutrophils also suggests that the expression of costimulatory molecules in neutrophils may have some effect on the anti-tuberculosis immunity of the body.
In this study, the expression and function of costimulatory molecule on peripheral blood neutrophils were compared between active pulmonary tuberculosis patients and healthy people, and the expression characteristics and cell function of costimulatory molecule on peripheral blood neutrophils in patients with tuberculosis were investigated. On this basis, the regulation of the negative costimulatory signal on neutrophil function was preliminarily investigated by blocking the PD-1/PD-L1 signaling pathway with specific monoclonal antibodies.
Part one: function of peripheral blood neutrophils and expression characteristics of costimulatory molecules in TB patients
Objective:
To investigate the relationship between abnormal immune function and costimulatory molecule in tuberculosis patients by analyzing the expression level and cell function of neutrophil costimulatory molecule.
Method:
Peripheral blood samples from 30 patients with active pulmonary tuberculosis and 20 healthy subjects were collected. The expression of costimulatory molecules on the surface of neutrophils, the expression of CD62L and ROS were detected by flow cytometry, the production of NO by neutrophils was detected by nitrate reductase method, and neutrophils were detected by Transwell apparatus. The chemotaxis ability of cells to IL-8.
Result:
Compared with the healthy control group, the peripheral blood neutrophils of tuberculosis patients had the following characteristics: (1) CD86 expression was significantly decreased (P 0.05), PD-L1 molecule expression was significantly increased (P 0.01), while CD28, CD80, HLA-DR and PD-1 were not significantly changed (P 0.05); (2) ROS and NO levels in respiratory burst were significantly increased (P 0.05) after PMA stimulation (P 0.05); There was no significant difference in expression (P0.05); (4) the chemotactic ability of IL-8 was significantly decreased (P 0.05).
Conclusion:
The positive co-stimulatory molecule CD86 expression decreased, the negative co-stimulatory molecule PD-L1 expression increased, the production of ROS and NO increased, and the chemotaxis decreased in patients with tuberculosis.
The second part is the effect of Mycobacterium tuberculosis infection on the function of neutrophils and the expression of costimulatory molecules.
Objective:
To analyze the expression of costimulatory molecules in neutrophils and the changes of cell function after Mtb infection, and to explore the effect of Mtb infection on the function of neutrophils and the expression of costimulatory molecules.
Method:
The expression of costimulatory molecules on the surface of neutrophils, the expression of ROS and CD62L in respiratory burst and the apoptosis rate of neutrophils were detected by flow cytometry in peripheral blood of 20 healthy subjects. The chemotactic ability of neutrophils to IL-8 was tested after Mtb infection, and the bactericidal ability of neutrophils to Mtb was observed by colony count (CFU).
Result:
(1) Compared with the control group, the expression of CD28, CD80, CD86, PD-1 and PD-L1 increased significantly (P 0.01), but there was no significant difference in HLA-DR expression between the two groups (P 0.05); the expression of ROS and NO increased significantly (P 0.01); the expression of CD62L decreased significantly (P 0.01); and the chemotaxis of IL-8 decreased significantly (P 0.01). Ability was significantly improved (P0.05).
(2) Both MS and MTb infection significantly increased the level of apoptosis of neutrophils, but the apoptosis rate of neutrophils infected with Mtb was lower than that of MS group (P 0.01).
(3) Neutrophils can kill non-pathogenic MS effectively, but can not kill toxic Mtb, leading to the proliferation of Mtb in cells.
Conclusion:
1. Mtb infection can effectively activate neutrophils, accompanied by a series of co-stimulatory molecule expression changes, but whether these co-stimulatory molecule changes are related to the functional changes of neutrophils needs further study.
2. The changes of neutrophil costimulatory molecule induced by Mtb infection in vitro are inconsistent with the expression pattern of neutrophil surface costimulatory molecule in peripheral blood of tuberculosis patients. The specific reasons need to be determined after the detection and analysis of the local neutrophil function of tuberculosis patients.
The third part is the effect of blocking PD-1/PD-L1 pathway on neutrophil function in vitro.
Objective:
By interfering with the PD-1/PD-L1 signaling pathway in vitro, the regulatory effect of the negative costimulatory signal on neutrophil function was investigated.
Method:
After blocking PD-1/PD-L1 pathway with anti-PD-L1 monoclonal antibody and adding Mtb for 30 minutes, the expression of CD62L and ROS of neutrophils were detected by flow cytometry, and the chemotactic ability of neutrophils was detected by Transwell chemotactic apparatus.
Result:
Compared with Mtb infection group, the level of ROS production by neutrophils increased significantly (P 0.05), the expression of CD62L decreased significantly (P 0.05), and the chemotactic ability of neutrophils to IL-8 increased (P 0.05) after blocking PD-1/PD-L1 signaling pathway in vitro.
Conclusion:
Blocking the PD-1/PD-L1 signaling pathway in vitro can enhance the respiratory burst function of neutrophils, accelerate activation and enhance their chemotaxis. This indicates that the negative costimulatory signal can inhibit the anti-tuberculosis immunity of neutrophils, which may open up a new way for clinical treatment of tuberculosis.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R378.911

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