【作者】 王红；

【导师】 赵蔚明；

【作者基本信息】 山东大学 ， 病原生物学， 2012， 博士

【摘要】 衣原体(Chlamydia)是一类专性胞内寄生的原核细胞型微生物。沙眼衣原体泌尿生殖道感染是一种常见的性传播疾病,近年来在世界范围内发病率呈上升趋势。女性生殖道沙眼衣原体感染可引起宫颈炎、子宫内膜炎、输卵管炎等严重并发症进而导致不孕和异位妊娠,同时沙眼衣原体感染还能提高HIV感染以及HPV感染所致宫颈癌的机率。以往研究表明在宿主抗衣原体感染过程中,Th1细胞免疫应答,尤其是IFN-γ的产生对于宿主抗衣原体感染至关重要,但是目前对宿主抗衣原体感染的确切免疫机制还缺乏深入了解和认识。阐明衣原体感染过程中免疫保护和免疫病理损伤的机制是研制安全有效衣原体疫苗的首要条件。自然杀伤T细胞(Natural killer T cells, NKT细胞)是一类独特的T淋巴细胞亚群,同时表达T细胞和NK细胞的表面典型标志。NKT细胞的显著特点是当TCR受到CDld分子递呈的糖脂抗原的刺激后,能够迅速活化并产生一系列细胞因子。被激活的NKT细胞还能够活化其它细胞,如树突状细胞、NK细胞、巨噬细胞、B细胞以及T细胞。众多研究结果已证明了NKT细胞在自身免疫性疾病、变态反应、肿瘤的转移以及微生物感染中的重要作用,显示了其强大的免疫调节潜能。NKT细胞能够识别非经典的MHC Ⅰ类分子CDld递呈的糖脂抗原。α-galactosylceramide (a-GalCer,α-半乳糖神经酰胺)是一种海绵生物来源的化合物,可以与CD1d分子特异性结合,激活具有半恒定TCR的经典NKT细胞(以下简称为NKT细胞)。相反,拥有多变TCR的非经典NKT细胞不能被α-GalCer活化。大量研究结果表明NKT细胞在微生物感染中具有重要的免疫调节作用。研究显示不同种的衣原体,肺炎衣原体(C. pneumoniae)和鼠型衣原体(C.muridarum),在肺部感染模型中能诱导生成不同的NKT亚群,产生不同的细胞因子模式,从而导致不同的感染结局。此差异说明了NKT细胞群体作用的复杂性。要确切的揭示NKT在衣原体感染中的功能还需要更多的实验室证据。由于生殖道是沙眼衣原体的天然感染途径,生殖道又具有不同于呼吸道的特殊免疫微环境,因而有必要使用小鼠衣原体生殖道感染模型进行进一步的研究。C. muridarum是沙眼衣原体免疫学研究中最常使用的衣原体株,它是鼠类的天然病原体,有利于研究病原体与宿主间的关系。阴道接种C. muridarum后,小鼠感染的病理过程与女性生殖道衣原体急性感染在许多方面非常相似。因此,本研究利用C. muridarum小鼠生殖道感染模型,探讨了NKT细胞在衣原体生殖道感染中的作用。由于α-GalCer是NKT细胞的特异性配体,并已被广泛应用于观察NKT活化后的功能研究,因此我们对α-GalCer刺激的活化NKT细胞在抗C. muridarum生殖道感染中的作用进行了探讨。一、α-GalCer活化NKT细胞在小鼠C. muridarum生殖道感染中的作用1、α-GalCer能够增强生殖道局部淋巴结NKT细胞的活化首先检测了α-GalCer对小鼠C. muridarum生殖道感染时NKT细胞活化的影响。雌性BALB/c小鼠注射α-GalCer或者vehicle(溶媒对照)2小时后分别阴道接种衣原体,用四聚体染色方法对两组小鼠生殖道局部引流淋巴结NKT细胞的比例进行分析。结果表明,vehicle对照组小鼠与α-GalCer处理组小鼠的NKT(CDld tetramer+TCRβ+)细胞比例分别是1.55%和5.6%,两组之间有显著性差异(p<0.001)。CD69分子的表达,是NKT细胞的一个重要早期活化标志。因此,我们通过流式细胞术检测了NKT细胞CD69的表达水平,以进一步比较α-GalCer处理组和vehicle对照组小鼠NKT细胞的活化情况。尽管在感染前,NKT细胞已经有较高基础水平CD69的表达,但是感染后,CD69表达水平明显升高,而α-GalCer能够进一步上调衣原体感染局部NKT细胞CD69分子的表达。2、α-GalCer活化NKT细胞能减轻小鼠C. muridarum生殖道感染和炎症病理损伤我们对α-GalCer活化NKT细胞在C. muridarum生殖道感染过程的作用进行了观察分析。小鼠注射α-GalCer后阴道接种衣原体。感染后检测小鼠一般状况和体重变化,并在不同时间点取小鼠阴道拭子检测衣原体存活数(IFU)；对小鼠生殖道标本进行病理组织学分析。重复实验结果表明,与对照组小鼠相比,α-GalCer处理组小鼠一般状况较好,体重下降较少。自感染第3天开始,α-GalCer处理组小鼠IFU即较对照组轻度减少,第9天时两组的IFU差异显著(p<0.001)。在整个感染过程中α-GalCer处理组小鼠的阴道衣原体IFU明显低于对照组存活数较低(p<0.05)。肉眼生殖道组织大体观察结果表明,α-GalCer处理组小鼠较少发生子宫角扩张和输卵管积水。镜下观察病理组织切片,α-GalCer处理组小鼠的生嬗道局部组织炎症病变较轻,而对照组小鼠组织中的中性粒细胞等炎性细胞浸润严重、管腔内可见大量的脓性分泌物,可见部分上皮细胞发生炎性脱落。两组小鼠局部病变的差异在上生殖道(子宫角和输卵管)尤为明显。上述结果表明,在小鼠C.muridarum生殖道感染时,α-GalCer活化NKT细胞能够减轻C. muridarum的生殖道感染和炎性损伤,增强小鼠抗衣原体感染的保护性作用。二、活化NKT细胞在C. muridarum生殖道感染中发挥保护性作用的机制1、C.muridarum生殖道感染能够诱导局部淋巴结NKT细胞活化首先检测了NKT细胞对C. muridarum生殖道感染的反应。收集衣原体感染前和感染后不同时间点的生殖道局部引流淋巴结单个核细胞,通过免疫荧光染色和流式细胞术分析NKT细胞的数量变化,结果显示NKT细胞比例在感染后明显增加。我们还检测了NKT细胞表面的早期活化标志CD69的表达以确定感染后NKT细胞的活化状态,结果表明C. muridarum感染后NKT细胞CD69的表达快速上调。上述结果说明C. muridarum生殖道感染可以诱导NKT细胞的活化。2、C.muridarum生殖道感染能够诱导NKT细胞功能向IFN-γ方向极化为阐明C.muridarum生殖道感染时NKT细胞的功能,对NKT细胞分泌细胞因子的模式进行了检测。生殖道局部淋巴结细胞内细胞因子染色结果表明,未感染小鼠NKT细胞的IFN-γ和IL-4分泌水平均较低。衣原体感染后,NKT细胞产生的IFN-γ明显增加,感染前后有显著差异。尽管感染也促进了IL-4的分泌,但与IFN-y相比仍处于明显较低的水平。因此,小鼠C.muridarum生殖道感染能够诱导NKT细胞功能向IFN-γ方向极化。3、C.muridarum生殖道感染时,α-GalCer促使活化NKT细胞功能进一步向IFN-γ方向极化通过本论文的第一部分内容NKT细胞活化检测实验,已经证实了α-GalCer能够促进C. muridarum生殖道感染时NKT细胞的活化。进一步利用细胞内细胞因子染色技术对NKT细胞产生细胞因子的模式进行检测。发现在衣原体生殖道感染时,与vehicle处理组相比,α-GalCer处理组小鼠NKT细胞产生的IFN-γ明显增加(16%比33%,p<0.001)。而两组小鼠NKT细胞的IL-4分泌均处在较低水平。值得注意的是,单纯α-GalCer注射而无衣原体感染组小鼠的NKT细胞分泌IFN-γ和IL-4均明显增加,展示了与α-GalCer处理合并感染小鼠截然不同的细胞因子模式。因此,α-GalCer处理组小鼠C.muridarum生殖道感染后,NKT细胞功能向IFN-γ方向明显极化,是衣原体感染诱导了极化作用的产生,α-GalCer处理则进一步加强了此极化作用。4、NKT细胞活化能够提高小鼠生殖道局部Thl保护性免疫应答为明确α-GalCer活化NKT细胞增强小鼠抗C.muridarum生殖道感染的保护性作用是否与Thl型免疫反应的加强有关,首先对感染小鼠生殖道组织细胞因子的表达进行了检测。荧光定量PCR结果表明,与对照组相比,α-GalCer处理小鼠生殖道组织的IFN-γ和IL-12mRNA水平升高,而IL-10水平下降。随后用ELISA方法检测了感染后小鼠的生殖道引流淋巴结和生殖道冲洗液的细胞因子模式。结果表明,a-GalCer处理组小鼠感染后局部淋巴结细胞的IFN-γ分泌水平明显高于vehicle对照组。尽管细胞因子IFN-γ在生殖道分泌物中的水平要低于淋巴结细胞,但是α-GalCer处理组和vehicle对照组之间差异仍然明显(p<0.05)。另一细胞因子IL-12p70在α-GalCer处理组小鼠生殖道分泌物和淋巴结中的表达也明显增加。IL-10的检测结果表明,衣原体生殖道感染后,α-GalCer处理组小鼠产生的IL-10比对照组明显减少(P<0.05)。上述ELISA结果与荧光定量PCR的检测结果吻合。C.muridarum生殖道感染后局部细胞因子的检测结果表明,NKT细胞有利于提高宿主的Th1保护性免疫应答反应。5、C.muridarum生殖道感染时,NKT细胞促进NK细胞和T细胞的IFN-γ分泌研究显示,NKT细胞TCR受CDld分子递呈的糖脂抗原的刺激活化后对其它细胞具有活化作用,是连接固有免疫和适应性免疫的桥梁。因此,利用细胞内细胞因子染色技术,检测了NKT细胞在衣原体生殖道感染过程中对NK细胞和传统T细胞的调节作用。检测结果发现,与对照组相比,α-GalCer处理组小鼠生殖道淋巴结中NK细胞分泌的IFN-γ显著增加。同时,α-GalCer处理组小鼠的CD4和CD8T细胞的IFN-γ也明显增加,而IL-4分泌却较对照组明显减少。上述结果表明NKT细胞活化对NK细胞和T细胞的功能具有调节作用,能促进其IFN-y的产生。综上所述,本研究首次对NKT细胞在C. muridarum生殖道感染中的作用进行了观察研究,并对其作用机制进行了初步探讨。结果显示,经特异性配体α-GalCer处理小鼠的生殖道感染和炎症病理损伤显著减轻,说明α-GalCer活化NKT细胞对C. muridarum的生殖道感染具有保护性作用。NKT细胞发挥保护性作用的机制与衣原体生殖道感染诱导NKT细胞活化并向IFN-γ方向极化有关。NKT细胞增强了宿主抗衣原体生殖道感染的Th1免疫反应,并对小鼠NK细胞和T细胞有调节作用,促进细胞因子IFN-γ的产生。这些发现不仅有助于我们对沙眼衣原体感染免疫机制的认识,而且可为衣原体感染防治和疫苗研究提供新思路和实验依据,具有不可低估的科学应用价值。更多还原

【Abstract】 Chlamydiae, an obligate intracellular bacterial pathogen, causes various human diseases. Chlamydia trachomatis genital tract infection is the most prevalent bacterial cause of sexually transmitted diseases in the world, with an estimated100million new cases each year in women. The genital tract chlamydial infection can result in severe complications, including pelvic inflammatory disease, ectopic pregnancy, and infertility. Moreover, chlamydial genital infection increases the risk of acquiring human immunodeficiency virus-related AIDS and human pappilloma virus-induced cervical carcinoma. Therefore, genital tract chlamydial infection is an important public health concern with a heavy financial burden. Previous studies have demonstrated that the Thl immune response, especially IFN-y production, is critical for protection against chlamydia infection. However, there is no clear and detail understanding of immune mechanisms to chlamydial infections.Natural killer T (NKT) cells are a unique lymphocyte subpopulation characterized by co-expression of surface markers with conventional T cells and NK cells. The most characteristic function of NKT cells is their ability to be rapidly activated and to produce cytokines in response to T-cell receptor engagement. Stimulation of NKT cells also results in the activation of other cells, including dendritic cells, NK cells, macrophages, B cells and conventional T cells. Because of their potent immuno-modulatory properties, NKT cells are considered to influence a wide range of diverse disease conditions such as autoimmune diseases, allergy and tumor rejection, as well as infections.NKT cells recognize lipid antigens that are presented by the nonclassical MHC I molecule CD1d. α-galactosylceramide (a-GalCer), a synthetic glycolipid ligand, which specifically binds to CD Id can stimulate classical NKT (invariant NKT) cells with semi-invariant TCR. In contrast, the nonclassical NKT cells with diverse TCR and CD1d-independent NKT-like cells do not respond to a-Galcer.Recent evidence suggests that NKT cells play an important immune regulatory role in the response to various microbial pathogens. Recent studies showed that NKT cells play a crucial protective role in the host defense against chlamydial infections. For example, NKT-knockout mice experience more severe disease and in vivo pathogen growth in lung C. pneumoniae infection. Moreover, the activation of NKT cells generated protection against C. trachomatis L2-induced arthritis. However, in a mouse model of C. muridarum lung infection, NKT cells was found to enhance the Th2immune response and promote chlamydial infection in vivo. These different observations reflect the functional complexity of NKT cells and the necessity for analyzing the role of NKT in different infection models including different infection routes.The genital tract is the natural route of sexually transmitted C. trachomatis infection, and genital tract mucosa has unique immunological features different from the mucosal immune system of lung or other organs. In the current study, we investigated the role of NKT cells in genital tract infection with C. muridarum for studying chlamydial genital infections in mouse models. Since α-GalCer is a specific ligand of NKT cells and has been shown in numerous studies for its stimulating effect on NKT cells, we used α-GalCer to enhance the function of NKT cells and investigated the mechanisms by which NKT cells modulate the immune responses against genital tract chlamydial infection.Ⅰ. The role of α-GalCer stimulated NKT cells in genital tract infection with C. muridarum1. α-GalCer enhance the activation of NKT cells during C. muridarum genital infection We first investigated whether α-GalCer could influence the activation of NKT cells. BALB/c mice were infected intravaginally with1×105IFUs of C. muridarum2h after α-Galcer or vehicle intravenous injection. The a-GalCer-treated mice showed a significant increase in percentages of NKT (CD1d tetramer+TCRβ+) cells compared with the vehicle control (5.6%vs1.55%,p<0.001).In addition, the expression of CD69, an early activation marker, was significantly upregulated in α-GalCer-treated mice than that of control (p<0.05). These data suggest that α-GalCer effectively promoted NKT cells activation during C. muridarum genital infection.2. α-Galcer-treated mice show enhanced clearance of C. muridarum genital infection and reduced inflammatory pathologic changesWe evaluated the role of NKT cells in host defense against the infection. The body weight loss of the α-GalCer-treated mice was less than that of control. The IFU level was significantly different between α-GalCer-treated group and vehicle-treated control group(p<0.05). Mice pretreated with α-GalCer showed clear reduction in chlamydial shedding at as early as day3postinfection. The difference expanded with time until day9post-infection when the α-GalCer-treated mice showed approximately10-folds lower chlamydial burden than the ones without α-GalCer-treatment.We further compared the histopathological changes in the genital tract between the two groups. Gross examination showed that the incidence of hydrosalpinx and dilatation of uterine horn was significantly reduced in the α-GalCer-treated mice. Detailed histopathological comparisons were performed by light microscopy. The a-GalCer-treated mice showed less severe tissue inflammation, in contrast, the vehicle-treated mice showed more severe inflammatory cellular, tissue inflammatory exudates and hyperemia infiltration.Therefore, NKT cells play a protective role in the host defense against C. muridarum genital infection in both pathogen clearance and pathological changes. Ⅱ. The mechanism by which activated a-GalCer-activated NKT cells play a promoting role in host resistance to C. muridarum genital tract infection1. NKT cells become activated during C. muridarum genital infection We investigated whether NKT cells could respond to chlamydial genital infection. At different time points after infection, the mice showed a significant increase in percentages of NKT (CD1d tetramer+TCRβ+) cells compared with the uninfection control. In addition, the expression of CD69was significantly upregulated after infection. These data suggest that C. muridarum genital infection effectively induce NKT cells activation.2. C. muridarum genital infection induces IFN-γ polarization of NKT cellsNext, we investigated the intracellular cytokine profile of NKT cells during genital chlamydial infection. Compared with the unfected control, the infected mice showed a significantly higher expression level of IFN-γ. Therefore, the pattern of cytokine production suggested that C. muridarum infection can skew the cytokine response of NKT cells to IFN-γ polarization.3. NKT activation by α-GalCer enhances IFN-γ polarization of NKT cells following genital chlamydial infectionWe further investigated the influence of α-GalCer on the cytokine pattern of NKT cells during chlamydial infection. Intracellular cytokine analysis showed that prior treatment with α-GalCer followed by C. muridarum infection enhanced the IFN-γ production by NKT after infection (p<0.001). Notably, NKT cells activated by α-GalCer alone (without C. muridarum infection) showed typically high levels of both IFN-γ and IL-4production. Therefore, the pattern of cytokine production by NKT cells was determined by C. muridarum genital tract infection, while α-GalCer treatment enhanced the IFN-γ polarization of NKT cells during genital chlamydial infection.4. α-GalCer-treated mice show enhanced type1immune response during C. muridarum genital tract infectionTo explore whether the improved protective effect in host defense against C. muridarum genital infection in α-GalCer-treated mice was associated with an enhanced Chlamydia-specific Thl immune response, we evaluated cytokine production in genital tract tissues and local lymph node cells of the different groups of mice. First, real-time PCR results suggested α-GalCer-treated mice showed a significantly higher expression level of IFN-y and IL-12mRNA but decreased IL-10expression in genital tissues compared with the control.We next determined the level of cytokine protein by ELIS A. IFN-y production by lymph node cells and in genital secretions from a-GalCer-treated mice was significantly higher than those from vehicle-treated mice post-infection. Similarly, a-GalCer-treated mice showed high levels of IL-12production. In contrast, IL-10production was significantly lower in a-GalCer-treated mice than in vehicle-treated mice.Therefore, NKT cells activated by a-GalCer significantly increase Thl immune response in the genital infection model.5. α-GalCer-treated mice show increased IFN-y production by NK and T cells during chlamydial infectionWe further examined the influence of NKT cell activation on NK and T cells during genital chlamydial infection. Compared with cells from vehicle-treated mice, NK cells from lymph nodes isolated from α-GalCer-treated mice showed significantly enhanced IFN-γ production after infection. This suggested that enhanced NKT cells activation by α-GalCer promoted IFN-γ production of NK cells. Next, we compared intracellular IFN-γ and IL-4production by CD4+T and CD8+T cells in splenocytes from α-GalCer-treated mice and control mice. CD4+T and CD8+T cells from α-GalCer treatment mice produced a greater amount of IFN-γ than did untreated mice after infection. In contrast, the control mice induced more IL-4production by CD4and CD8T cells. Therefore, NKT cell activation by α-GalCer contributed to enhanced type1T cell response during C. muridarum genital tract infection.In conclusion, our results demonstrate that enhanced activation of NKT cells by the administration of a-Galcer led to an enhancement of protection against C. muridarum genital tract infection The beneficial effect of NKT cells is associated with a shift in cytokine profile of NKT cells to IFN-y polarization induced by C. muridarum genital tract infection. The results also suggested that activated NKT cells have an promoting effect on IFN-y production by NK and conventional T cells in this infection. These findings are helpful to enrich our understanding on the mechanism of chlamydial infection, moreover, the investigation provides novel strategy and experiment foundation for developing prophylactic vaccine against chlamydial infection.更多还原