【作者】 陈丽丽；

【导师】 吴移谋； 钟光明；

【作者基本信息】 南华大学 ， 病原生物学， 2013， 博士

【摘要】 沙眼衣原体（Chlamydia trachomatis，Ct）是性病病原体之一，主要引起男性非淋球菌性尿道炎、附睾炎、前列腺炎；女性宫颈炎、子宫内膜炎、输卵管炎及盆腔炎，并可导致异位妊娠、不孕、不育等严重并发症，还可促进宫颈癌、卵巢癌的发生和HIV的感染。但迄今为止，Ct确切的致病机制及机体的抗感染免疫机制仍不十分清楚，尚无有效的疫苗来预防。因此，阐明Ct的致病及免疫机制，研究Ct治疗靶点对预防、控制Ct感染具有十分重要的意义。Ct为严格细胞内寄生的微生物，机体抗Ct免疫机制包括适应性免疫和固有免疫，其中以CD4+Th1细胞介导的免疫反应为主。这些免疫机制在发挥免疫保护作用的同时也引起相应的炎性病理损伤。髓样分化因子88（myeloid differentiationfactor88，MyD88）是Toll样受体（Toll-like receptor，TLR）信号转导途径中的主要接头蛋白，在天然免疫反应中发挥着极其重要的作用；LIGHT是T细胞活化过程中的协同刺激分子之一，与相应受体结合刺激T细胞增殖、分化及诱导Th1型细胞因子的产生；IL-12是重要的Th1型细胞因子，可促进Th1细胞的分化、增殖。因此，这些免疫分子在抗胞内寄生物Ct感染免疫中可能发挥重要作用。由于Ct人型株经阴道感染小鼠后，仅引起轻微的下生殖道炎症反应，而鼠衣原体（Chlamydia muridarum，mouse pneumonitis agent，MoPn）感染小鼠后出现的泌尿生殖道病理反应与人类Ct泌尿生殖道感染极其相似。因此，目前常用MoPn鼠生殖道感染模型来研究Ct的免疫保护和病理损伤机制。研究目的本研究采用MyD88KO、LIGHT KO、IL-12p35KO和IL-12p40KO小鼠为对象，分别建立小鼠MoPn生殖道感染模型，检测各组小鼠生殖道衣原体清除速度、泌尿生殖道病理反应及宿主的免疫反应，分析MyD88、LIGHT、IL-12和IL-23在小鼠抗衣原体感染免疫及病理损伤中的作用，为进一步阐明衣原体可能的致病机制提供依据。研究方法用1×104IFUs MoPn经阴道感染野生型（wild type，wt）、LIGHT KO、MyD88KO、IL-12p35KO和IL-12p40KO小鼠，每组一半小鼠于原发感染后，生殖道拭子MoPn检测为阴性后2周，再次感染相同剂量的MoPn。每隔3~4天取小鼠生殖道分泌物于500μl SPG中，冰浴超声，系列稀释后，感染HeLa细胞，测定其中衣原体包涵体的数量，比较生殖道衣原体清除速度。初次感染MoPn后80或143天，麻醉后断颈处死小鼠，眼眶取血，分离血清，用间接免疫荧光法测定其中抗体效价及抗体亚类；分离小鼠脾细胞，按5×106个/孔加入24孔板中，用1×106IFU/ml UV-MoPn EB刺激，72h后收集上清，测定其中的IL-4、IL-5、IL-17和IFN-γ等细胞因子水平。分离小鼠泌尿生殖道组织，肉眼观察输卵管、子宫角及肾脏等的异常情况，然后甲醛固定、病理切片，H&E染色后，显微镜下观察各组织炎性浸润程度和管腔水肿程度。若KO小鼠泌尿生殖道组织病变程度明显高于wt小鼠，则用免疫组化技术检测肾脏及上生殖道组织中的衣原体抗原并计数包涵体数量；并取组织匀浆，短暂超声后，感染HeLa细胞，计数相应组织中MoPn包涵体数量，同时将肾组织匀浆接种于血琼脂平板进行细菌计数。收集MyD88KO小鼠腹腔巨噬细胞，按2×105个/孔加入48孔板中，待细胞贴壁后，感染MoPn（MOI=5）后48h，收集培养上清液。ELISA测定小鼠巨噬细胞培养上清及生殖道分泌物中IL-1α、IL-6、TNF-α和MIP-2水平。研究结果1.与wt小鼠相比，MyD88KO小鼠下生殖道清除衣原体的速度没有明显区别，但上生殖道组织的大体特征和显微镜下病变程度评分，均显示MyD88KO小鼠上生殖道病理反应，尤其是输卵管水肿程度明显比wt小鼠严重（P＜0.01）。在感染后10天，MyD88KO和wt小鼠子宫颈、子宫角和输卵管组织匀浆中衣原体数量没有明显区别；但是在感染后23天，MyD88KO小鼠的上生殖道组织中检出的衣原体包涵体数量远远高于wt小鼠（P＜0.05）。组织切片中衣原体抗原检测结果也显示，在感染10和23天后，MyD88KO小鼠组织中的衣原体抗原显著高于wt小鼠（P＜0.05）。小鼠生殖道分泌物及腹腔巨噬细胞上清中IL-1α、IL-6、TNF-α和MIP-2等前炎症因子检测结果显示，MyD88KO小鼠产生水平显著低于wt小鼠（P＜0.05）。脾淋巴细胞受抗原再次刺激后，MyD88KO小鼠产生IFN-γ和IL-17的量明显低于wt小鼠，而IL-4和IL-5水平显著高于wt小鼠。血清特异性IgG抗体及抗体亚类检测，MyD88KO小鼠血清IgG2a/IgG1比值（小于1）明显低于wt小鼠（大于1）（P＜0.05），提示MyD88KO小鼠生殖道感染衣原体后，产生以Th2为主的免疫应答。2. LIGHT KO和wt小鼠感染MoPn后，生殖道衣原体清除速度大致相似，大部分小鼠均在原发感染后28天左右完全清除感染，且均产生对再次感染的免疫力。LIGHT KO和wt小鼠子宫角和输卵管均出现一定程度的病变，但无显著性差异。两组小鼠在原发和继发感染MoPn后，均产生高效价的特异性抗MoPn IgG抗体，总抗体及各IgG抗体亚类效价无显著性差异（P＞0.05），且IgG2a/IgG1比值均大于1。和wt小鼠一样，LIGHT KO小鼠脾淋巴细胞经衣原体再次刺激后均可产生较高水平的IFN-γ和IL-17，且未能检测到IL-4和IL-5。3.在MoPn原发感染中，IL-12p35KO和IL-12p40KO小鼠清除衣原体的速度相当，带菌时间均较wt小鼠明显延长；而再次感染后，三组小鼠带菌时间没有明显差异（P＞0.05）。所有小鼠均出现严重的上生殖道组织病变，KO小鼠和wt小鼠病变程度没有明显差异。几乎所有IL-12p40KO和部分IL-12p35KO小鼠均发生严重的肾脏病变，而wt小鼠肾脏未见明显异常。在原发和再次感染后，IL-12p40KO小鼠肾脏病变小鼠数显著高于wt小鼠（P<0.01）；且在原发感染后，也显著高于IL-12p35KO小鼠（P <0.05）。而未感染衣原体的年龄匹配的IL-12p35KO或IL-12p40KO对照组小鼠肾脏未见任何明显异常。在IL-12p35或IL-12p40KO小鼠病变的肾组织匀浆及切片中均能检测到衣原体抗原，包涵体数量均显著高于wt小鼠，且IL-12p40KO小鼠包涵体数量也明显高于IL-12p35KO小鼠（P<0.05）。IL-12p40KO和IL-12p35KO小鼠肾组织匀浆中可检出大量细菌，显著高于wt小鼠（P<0.05），但两组KO小鼠之间细菌数量没有显著性差异（P＞0.05）。所有小鼠在原发和继发感染衣原体后，均产生高效价的抗MoPn IgG抗体，而且IL-12p35KO或IL-12p40KO小鼠的IgG2a/IgG1比值显著低于wt小鼠（P＜0.05）。脾细胞体外经衣原体再次刺激后，wt小鼠产生高水平的IFN-γ，而IL-12p35KO和p40KO小鼠脾细胞几乎不产生IFN-γ （P＜0.01）；wt和IL-12p35KO小鼠脾细胞产生大量的IL-17，显著高于IL-12p40KO小鼠（P＜0.05）。各组小鼠脾淋巴细胞上清中均未检测到IL-4和IL-5。结论1. MyD88信号通路与小鼠下生殖道清除MoPn感染无关，但与MoPn引起的小鼠生殖道病理损伤及早期炎性细胞因子的产生密切相关，而且MyD88信号通路还可限制衣原体上行感染至上生殖道组织。2.小鼠抗MoPn生殖道感染及MoPn引起的生殖道病理损伤可能不依赖于LIGHT信号通路。3.抗MoPn原发感染依赖于IL-12介导的Th1型细胞免疫反应，MoPn导致的上生殖道病理损伤不完全依赖于IL-12和IL-23，但IL-12和IL-23可限制经生殖道感染的MoPn扩散到肾脏，IL-12或IL-23基因缺失后生殖道衣原体感染易并发肾脏炎症。更多还原

【Abstract】 Chlamydia trachomatis is a leading cause of sexually transmitted diseases, whichcan induce urethritis, epididymitis and prostatitis among men and cervicitis,endometritis, salpingitis, and pelvic inflammatory disease among women, if untreated,which may lead to serious complications, including ectopic pregnancy, tubal infertility,infertility and so on. C. trachomatis infection can also increase the chance of cervicalcancer, ovarian cancer and HIV infection. But till now, the precise pathogenic andimmunologic mechanisms remain unclear and no effective vaccine is licenced. Sostuding on chlamydial pathogenesis and immunologic mechanisms and searching fortherapeutic targets will be of great importance.C. trachomatis is an obligate intracellular parasites, host anti-C. trachomatisimmune mechanism including adaptive and innate immunity, among which the CD4+Th1cell-dependent immunity represents a major protection mechanism. However, thesame immunity response may also contribute to the chlamydia-induced inflammatorypathologies. MyD88is a critical signaling adaptor molecule required by many Toll-likereceptors and plays important roles in innate immunity. LIGHT is a costimulatory factorfor T cells activation, which can enhance Th1cells differentiation, proliferation andTh1-type cytokines production after interacted with its receptor. Interleukin-12(IL-12)is an important Th1cytokine which can promote Th1cells differentiation andproliferation. So these immune molecules may develop significant effects in controllingthe intracellular C. trachomatis infection.Because mice only showed slight inflammation in lower genital tract after infectedwith human C. trachomatis strains, and mice intravaginally infected with a murine biovar of C. trachomatis (known as mouse pneumonitis [MoPn] agent and nowclassified as a new species, Chlamydia muridarum) organisms can developinflammatory pathologies including endometritis and hydrosalpinx similar to those inhumans. So MoPn has been used to study the mechanisms of C. trachomatispathogenesis and immunity in a mouse genital infection model.ObjectiveIn this research, mice deficient in MyD88, IL-12p35, IL-12p40and LIGHT wasused to intravaginally infected with MoPn. Numbers of Chlamydia inclusion bodies invaginal swab, pathologies of mice urogenital tract and host immune response werecompared with wild type mice to investigate the role of MyD88, IL-12and LIGHTplayed in MoPn urogenital infection. These study will lay the foundation for thepathogenesis research of C. trachomatis.MethodsEach of MyD88KO, IL-12p35KO, IL-12p40KO, LIGHT KO and wild type micewas inoculated intravaginally with1×104IFUs of live MoPn organisms. For some mice,a secondary infection was similarly carried out at two weeks later when MoPn wasnegative in vaginal swab after primary infection. To monitor live organism shedding,vaginal swabs were taken on different days after intravaginal infection (once every3to4days for the first50days and once per week thereafter). Each swab was suspended in500μl of SPG followed by sonication on ice and the released organisms were titrated onHeLa cell monolayers.On day80or143after primary infection, mice were sacrificed after anesthetized,and serum were collected for titrating and isotyping anti-MoPn antibodies by indirectimmuno-fluorescence assay. After mice spleen was isolated, splenocytes were harvestedand cultured in24-well plates at a density of5×106cells/well with or without stimulation with UV-inactivated MoPn elementary bodies at a final concentration of1×106IFU/ml. After culture for72hours, the culture supernatants were collected forIL-4, IL-5, IL-17and IFN-γ measurements with ELISA.Urogenital tract tissues and kidneys were harvested from mice after sacrificed.Before the tissues were removed from mouse bodies, an in situ gross examination wasperformed for evidence of hydrosalpinx and enlarged, swollen or necrotic kidneys orany other related abnormalities of uterine horn, oviducts and kidneys. The excisedtissues, after photographing, were fixed in10%neutral formalin and embedded inparaffin and serially sectioned longitudinally. The sections were stained withhematoxylin and eosin (H&E). The H&E stained sections were assessed by pathologistsblinded to mouse treatment and scored for severity of inflammation and pathologies. Ifthe difference of urogenital tract tissues and kidneys was significant between theknockout and wild type mice, the chlamydial Ags in tissues sections were detected withimmunohistochemisty. And the whole kidney, cevix, uterine horn and oviduct tissueswere homogenized separately in SPG and briefly sonicated. The released organismswere titrated on HeLa cell monolayers and the chlamydia inclusion bodies were counted.The kidney homogenates also were serially diluted and plated on blood agar plates fortitrating bacteria.For MyD88knockout mice group, Mφs were collected from peritoneal cavity, and2×105cells were added to each well of48-well plates. After washing away nonadherentcells, the adherent Mφs were infected with MoPn at a multiplicity of infection of5. Theinfected cultures were incubated for48h at37°C in a CO2incubator and measuredIL-1α, IL-6, TNF-α and MIP-2by ELISA.Results1. The chlamydia shedding time of MyD88KO mice was similar to wild type mice, butboth the gross appearance and the dilation and inflammation scores under microscope of the isolated genital tracts showed that the genital tract pathology especiallyhydrosalpinx degree from the MyD88-deficient mice was much more severe thanwild type mice (P＜0.01). After titrating the chlamydia IFUs from genital tract tissuehomogenates, we found that genital tissues from both wild-type and MyD88KO micecontained similar amounts of live organisms on day10. However, by day23, liveorganisms were detected in the upper genital tract tissues (uterine horn and oviduct)of the MyD88KO but not wild-type mice. The similar result also showed in directdetection of chlamydial Ags in mouse genital tissue sections. There weresignificantly higher numbers of chlamydial inclusions in the cervix, uterine horn, andoviduct tissue sections of MyD88KO than wild-type mice on day10and day23post-infection (P＜0.05).The proinflammatory cytokines IL-1a, IL-6, TNF-a and MIP-2in the urogenitaltract or macrophage supernatant from the MyD88KO mice were much lower thanthat from wild type mice (P＜0.05). Splenocytes from wild-type mice produced highlevels of IFN-γ and IL-17but low levels of IL-4and IL-5upon restimulation withchlamydial Ags. However, splenocytes from the MyD88KO mice produced onlyminimal levels of IFN-γ or IL-17but significantly high levels of IL-4and IL-5. Allmice developed robust anti-C. muridarum organism IgG antibody, but the ratio ofChlamydia specific IgG2a versus IgG1is＞1(indicative of Th1dominance) inwild-type and＜1(reflective of Th2dominance) in MyD88KO mice.2. The chlamydia shedding time of LIGHT KO mice was similar to wild type mice,which cleared the organisms within28days after primary infection, and acquiredprotective immunity against MoPn reinfection. All mice regardless of genotypesdeveloped severe upper genital tract pathology and showed no significant differencebetween LIGHT KO and wild type mice. All mice developed robust anti-MoPnorganism IgG antibody responses and the ratios of IgG2a versus IgG1showed nosignificant difference between LIGHT KO and wild type mice. Splenocytes fromMoPn-infected LIGHT KO and wild type mice produced high levels of IFN-γ and IL-17, but IL-4and IL-5couldn’t be detected.3. Compared with wild type mice, mice deficient in either IL-12p35or p40bothdeveloped similar but prolonged infection time courses after primary infection, butsimilar time couses after secondary infection. All mice regardless of genotypesdeveloped severe upper genital tract pathology, and no significant difference wasfound among the mouse groups.Almost all kidneys from IL-12p40KO mice and some IL-12p35KO mice wereabnormal, which exhibited pathology of pyelonephritis, but no obvious abnormalitywas noted in any of the kidneys from wild type mice. The number of mice withabnormal kidneys in the p40KO group was significantly higher than that of wildtype mice after both primary and secondary infections and than that of p35KO groupafter primary infection. Neither the age-matched IL-12p35KO nor p40KO micedeveloped any gross kidney pathology in the absence of chlamydial infection. MoPninclusions could be detected only in kidneys with gross pathology from eitherIL-12p35or p40KO groups, while no inclusion was ever detected in kidneys fromwild type mice. The number of chlamydial inclusions of p40KO mice was also muchhigher than that from the p35KO mice. Live bacteria were detected in mice deficientin either p35or p40KO mice but not wild type mice. However, there was nosignificant difference in the number of live bacteria recovered from p35versus p40KO mice.All mice regardless of genotypes developed robust anti-MoPn organism IgGantibody responses during the course of both primary and secondary infections. Theratios of IgG2a versus IgG1revealed that wild type mice had a significantly higherratio than either IL-12p35or p40KO mice. Splenocytes from MoPn-infected wildtype mice produced high levels of IFN-γ upon restimulation with chlamydialantigens while mice deficient in either IL-12p35or p40failed to do so. Splenocytesfrom both wild type and IL-12p35KO mice produced significantly high amounts ofIL-17while the splenocytes from IL-12p40KO mice failed to do so. IL-4and IL-5 couldn’t be detected.Conclusions1. MyD88is dispensable for protection against MoPn urogenital tract infection, but isnecessary for inflammatory cytokine production in the early phase of host response tochlamydial infection, preventing urogenital tract pathology and limitting theorganisms to ascend into the upper genital tract following intravaginal infection.2. LIGHT signal pathway may be not correlated with protection against MoPnurogenital tract infection and urogenital tract pathology induced by MoPn.3. Protection against MoPn urogenital tract primary infection rely on IL-12dependentTh1immune response, and MoPn induced urogenital tract pathology is notcompletely dependent on IL-12and IL-23. IL-12and IL-23can limit intravaginallyinfected MoPn to spread into kidneys, and chlamydia infection may correlated withnephritis when IL-12or IL-23is deficient.更多还原