衣原体分泌糖脂对iNKT细胞的激活作用及IL-22在衣原体感染中的作用研究
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摘要
第一部分沙眼衣原体分泌性糖脂(GLXA)对iNKT细胞激活作用的研究
研究目的
衣原体(Chlamydiae)是一类专性细胞内寄生,有独特发育周期的原核细胞型微生物,衣原体感染与许多人类疾病有着密切联系。根据基因型的不同,衣原体可分为沙眼衣原体(Chlamydia trachomatis, Ct)和肺炎衣原体(Chlamydia pneumonias, Cpn)两个种属。其中沙眼衣原体主要引起呼吸道感染、眼部粘膜感染及性传播疾病。最近,沙眼衣原体中的鼠类变种株被单独划分出来,被命名为衣原体鼠肺炎株(Chlamydia muridarum, Cm),该株广泛用于建立小鼠肺部或生殖道感染模型,进行沙眼衣原体致病机制的理论研究。而肺炎衣原体主要引起支气管炎、鼻窦炎、肺炎等呼吸道疾病。虽然抗生素可有效控制衣原体的感染症状,但是衣原体感染的临床表现具有多样性,大多数以隐性感染的形式存在,被感染者不能及时就诊,使得抗生素对感染的控制力大大减弱,因此,研发安全有效的疫苗对控制衣原体感染意义重大,目前尚无有效的疫苗预防和(或)治疗衣原体感染。阐明衣原体感染过程中免疫保护和免疫病理的机制是合理研制安全有效衣原体疫苗的首要条件。
自然杀伤性T细胞(invariant natural killer T cell, iNKT)作为独特的T细胞亚群识别由非经典的MHCI类分子CD1d递呈的糖脂类抗原,而不是肽类抗原。研究表明,在衣原体感染中iNKT细胞被激活并产生细胞因子,通过其对获得性免疫应答的调节在衣原体感染的进程和转归中发挥重要作用。衣原体分泌性糖脂抗原(glycolipid exoantigen, GLXA)是目前已被报道的一种和衣原体感染相关的糖脂抗原,但GLXA能否活化NKT细胞,以及它是否是衣原体感染中对iNKT细胞作用的关键抗原尚不清楚。因此,本研究拟证实GLXA能否在体内外激活iNKT细胞,进一步明确衣原体感染的免疫机制,为以GLXA为靶点的衣原体疫苗的研制奠定理论基础。
研究方法
提取衣原体培养上清中的分泌性糖脂,并以相同的方法提取未感染细胞培养上清中的脂类(cell mock)作为实验对照,对糖脂成分进行银染显色和Western Blot鉴定分析。为验证该糖脂是否能够激活iNKT细胞,采用了体外实验和体内试验分别进行验证：
1.体外实验：
1) CDld蛋白+NKT杂交瘤共培养系统：用CDld融合蛋白包被96孔细胞培养板,然后在板中加入GLXA和NKT杂交瘤进行共培养,ELISA检测培养上清中IL-2的水平,以明确GLXA能否活化NKT杂交瘤细胞。
2)体外共培养系统：制备小鼠骨髓来源的树突状细胞(DC),经糖脂冲击树突状细胞24小时后,与肝脏单个核细胞(LMC)共培养,48小时后收集培养上清ELISA法检测细胞因子IFN-y和IL-4。并在共培养实验中采用iNKT缺陷小鼠以及CD1d抗体封闭系统进一步验证作用机制。
2.体内试验：
1)静脉注射：采用C57BL/6(WT)小鼠和iNKT缺陷小鼠,分别经尾静脉注射给予GLXA,注射后2h对小鼠取血,用ELISA检测血清中IL-4的表达水平,注射后12h取血,用ELISA检测血清中IFN-y的表达水平。
2)腹腔注射：小鼠腹腔注射GLXA,用流式细胞术分析其对iNKT表面活化标志及细胞因子分泌的影响。
研究结果
1.糖脂鉴定：银染显色和Western Blot分析显示,提取的衣原体分泌性糖脂与文献报道的GLXA条带一致,与cell mock实验对照组形成的条带明显不同。
2.体外实验：
1)CDld蛋白+NKT杂交瘤共培养系统：与对照组相比,GLXA可特异性活化NKT杂交瘤细胞,使其产生IL-2。
2)体外共培养系统：GLXA冲击组产生的IFN-γ和IL-4均高于cell mock冲击组及阴性对照组(DC未经抗原刺激),表明分泌性糖脂能够激活LMC产生细胞因子。为进一步验证LMC产生的IFN-γ和IL-4主要来源于iNKT细胞,在DC-LMC共培养实验中采用了iNKT缺陷小鼠以及CD1d抗体封闭,结果显示：(1)来源于iNKT缺陷小鼠LMC产生的IFN-γ和IL-4均显著低于野生型小鼠.(2)加入CD1d抗体封闭后,野生型小鼠LMC产生IFN-γ和IL-4也明显下降。由此证明衣原体分泌性糖脂诱导的细胞因子主要由iNKT细胞产生,而且此作用部分依赖于CDld分子。
3.体内试验：
1)静脉注射：在给予GLXA刺激的野生型小鼠血清中,可检测出较高水平的IFN-γ和IL-4,在iNKT缺陷小鼠中却没有发现这种现象,说明血清中较高水平的IFN-γ和IL-4主要是由iNKT细胞产生的,而且是GLXA促进iNKT细胞产生的细胞因子。
2)腹腔注射：通过流式细胞术检测,与对照组相比GLXA可明显提高iNKT细胞表面活化标志的表达,并促进其分泌细胞因子IFN-γ和IL-4,说明其对iNKT细胞有明显的激活作用。
研究结论
以上实验证明,衣原体分泌性糖脂GLXA能够激活iNKT细胞并产生细胞因子,并且该活化作用在一定程度上依赖CDld分子的递呈作用,GLXA可能是衣原体感染中iNKT细胞活化的关键抗原,有关GLXA激活iNKT细胞的作用机制尚需进一步研究。
研究意义
该研究进一步完善了我们对衣原体致病机制的认识,加深了衣原体感染引起的宿主免疫反应机制的了解,为以GLXA为靶点设计疫苗抵抗衣原体感染奠定了实验和理论基础。
第二部分白细胞介素-22在衣原体肺部感染中的生物学作用
研究目的
白细胞介素-22(IL-22)是IL-10细胞因子家族的一员,它是一种炎症因子,在调控组织局部免疫方面起着重要的作用。近期的研究证实,IL-22在抵抗细菌、真菌、病毒和寄生虫感染所引发的宿主免疫中起到一定保护作用。目前普遍接受的观点是IL-22在枸橼酸杆菌、克雷伯杆菌、分节丝状杆菌等胞外菌引起的感染中起到保护作用,但是IL-22在宿主抵抗胞内菌感染中的作用目前仍不明确。因此进一步在胞内菌感染模型中评估IL-22的作用是十分必要的,这有利于明确IL-22在胞内菌感染中的生物学效应,并为其提供更充分的理论学依据。
衣原体是专性胞内寄生菌,其中对人类致病的种属是沙眼衣原体(Chlamydia trachomatis, Ct)和肺炎衣原体(Chlamydia pneumonia, Cpn)。沙眼衣原体主要引起呼吸道、眼睛及性疾病的传播。肺炎衣原体主要引起支气管炎、鼻窦炎和肺炎。由于衣原体菌株具有一定的种属特异性,近些年引起小鼠肺炎的菌株被单独划分出来称为鼠肺炎衣原体(Chlamydia muridarum, Cm)。目前在实验研究中,鼠肺炎衣原体株被广泛用于小鼠呼吸道和生殖道感染模型中。在小鼠呼吸道感染模型中,目前的研究证实Th1型细胞免疫反应起到主要的保护作用,可有效控制衣原体感染,对疾病的转归起到决定性作用。我们前期的实验结果证实Th17在宿主防御衣原体感染中也起到重要的作用,它可以促进Thl型细胞免疫反应。因此,Thl和Th17细胞免疫反应对于宿主清除病原菌起到重要的促进作用。IL-22和IL-17是Th17细胞亚群主要产生的细胞因子,它们在许多感染模型中都起到相互协同的作用。迄今为止,没有实验研究证实IL-22在小鼠肺部衣原体感染中的作用。在本研究中,我们拟用抗小鼠IL-22单克隆抗体封闭小鼠体内固有产生的IL-22,从而进一步评估IL-22在小鼠肺部衣原体感染中的作用。
研究方法
1、IL-22抗体封闭实验：在小鼠Cm肺感染模型中,用小鼠IL-22单克隆抗体对肺局部的IL-22进行封闭,以检测IL-22的作用。
1)流式细胞术：检测Th1、Th17、Treg等细胞亚群的变化情况。
2) ELISA:检测细胞培养上清中IL-6、IL-10、IL-12p40、IL-17、IFN-y等细胞因子的表达水平
3) RT-PCR:检测IL-22、IL-17、IFN-y、T-bet、RORyt在RNA水平上的变化情况。
4)HE染色：肺组织切片镜检。
2、IL-22蛋白给予实验：在小鼠Cm肺感染模型中,肺局部给予小鼠纯化IL-22蛋白,以检测IL-22对疾病转归的影响。
1)流式细胞术：检测Th1、Th17等细胞亚群的变化情况。
2) ELISA:检测培养上清中IL-17、IFN-γ等细胞因子的表达水平。
3)HE染色：肺组织切片镜检。
研究结果
1、IL-22抗体封闭实验：IL-22在小鼠Cm肺部感染的早期阶段就有升高表达,用抗体将其封闭后结果如下：与同型对照组相比,IL-22抗体封闭组的肺部炎症更加严重,表现为体重下降更明显,负菌量升高,病理组织切片炎症浸润更加明显。流式分析显示IL-22抗体封闭组的Th1细胞和Th17细胞免疫反应降低,而CD4+CD25+Foxp3+调节性T细胞群增高,此外IL-10的生成也随着IL-22的封闭而减少。
2、IL-22蛋白给予实验：将IL-22纯化蛋白给予Cm肺部感染的小鼠后,很好地控制了肺部炎症的发展和衣原体的增殖,并促进了肺部的Th17细胞免疫反应。
研究结论
IL-22产生并高表达于小鼠Cm肺部感染的早期阶段,可以保护机体减轻衣原体的肺部感染。IL-22这种保护性作用主要是通过促进Th17细胞免疫反应实现的。
研究意义
IL-22作为一种重要的炎症因子参与了机体防御衣原体肺部感染的进程,它对机体起到明显的保护作用,本研究为以IL-22为靶点研发新药控制衣原体肺部感染提供了理论依据和实验基础。
Part One:The Glycolipid Exoantigen (GLXA) Derived from Chlamydia muridarum Activates Invariant Natural Killer T Cells
Objective
Chlamydiae are obligate intracellular bacterial pathogens. The genus Chlamydia includes two species, Chlamydia trachomatis and Chlamydia pneumoniae, which cause various human diseases. C. trachomatis is a major cause of respiratory, ocular and sexually transmitted diseases. A murine biovar of C.trachomatis (known as mouse pneumonitis [MoPn] agent and now classified as a new species, Chlamydia muridarum) has been used to study the mechanisms of C. trachomatis pathogenesis and immunity in a mouse pulmonary and genital infection models. Further, C. pneumoniae causes respiratory diseases like bronchitis, sinusitis and pneumonia. To date, there is no effective vaccine available against human chlamydial diseases. Considering the public health significance of chlamydial diseases, it is highly desirable to have an effective and safe chlamydial vaccine. However, one of the main constraints in the way of vaccine development is poor understanding of the role of chlamydial components in host-chlamydial interactions. NKT (Natural killer T) cells represent an innate subset of T lymphocytes and recognize glycolipid antigens presented by CD1d, which is a nonclassical MHC class I molecule expressed on antigen presenting cells (APC). We have previously reported that iNKT are activated in vivo and play an important role in immune responses to chlamydial infections. GLXA is a glycolipid component of the chlamydial membrane and intracellular inclusion bodies, which has been implicated in chlamydial-host cell interaction. Taking account of these facts, we hypothesized that GLXA might specifically activate iNKT. To test the hypothesis, we investigated the role of GLXA derived from Chlamydia muridarum in iNKT activation using in vitro as well as in vivo settings.We want to further identify the immune mechnism of chlamydial infection and develop vaccine against chlamydial by targeting GLXA.
Methods
We prepared GLXA antigen from the supernatant of C. muridarum infected cell. Supernatants harvested from uninfected cell monolayers were prepared in an identical manner and used as cell mock control. SDS-PAGE and Western blot analysis were done to identify GLXA. We investigated the role of GLXA in iNKT activation using in vitro as well as in vivo settings.
1、in vitro
1) CD1d fusion protein iNKT hybridoma culture:96-well plates were incubated with mouse CDld fusion protein, and GLXA and iNKT hybridoma cells were added to each well and co-culture. The culture supernatant was measured for IL-2by ELISA.
2) LMC-BMDC co-culture:DC were generated in vitro from bone marrow cells. BMDC were cultured in the presence of GLXA for24h. The treated BMDC were co-cultured with LMC for48h. The levels of IFN-y and IL-4in the culture supernatants were measured by ELISA. We use the iNKT knock out mice and CD1d antibody blocking system to do further identification in this co-culture system.
2、in vivo
1) Intravenous injection:C57BL/6(WT) and iNKT KO mice were intravenously treated with GLXA. The serum levels of IL-4and IFN-y were measured at2h and12h respectively after the treatment. The measurement of the cytokine levels was done by ELISA.
2) Intraperitoneal injection:Mice were injected with GLXA intraperitoneally. After72h, spleens and livers were harvested from the animals, and processed into single-cell suspensions. The cells were then stained with the appropriate Abs for flow cytometric analysis.
Results
1、Preparation of GLXA antigen:Silver staining and Western blot of the antigen sample demonstrated a banding pattern similar to GLXA as described previously, cell mock preparation failed to show the bands although.
2、in vitro
1) CD1d fusion protein iNKT hybridoma culture:On stimulation with GLXA-treated CD1d, in contrast to the cell mock or PBS, iNKT produced IL-2in a dose-dependent manner, suggesting that GLXA can specifically stimulate iNKT hybridoma.
2) LMC-BMDC co-culture:We found that GLXA stimulated LMC from WT mice to produce higher quantities of IFN-y and IL-4compared with cell mock. To confirm that the cytokines produced in the co-culture were secreted by iNKT, we isolated the LMC from iNKT KO and co-cultured it with BMDC. The data showed that the LMC from iNKT KO mice produced significantly lower quantities of IFN-y and IL-4compared with those from WT mice, indicating that the cytokines were mainly produced by iNKT. We further examined whether the cytokine production by the iNKT was CD1d-dependent by blocking CD1d using anti-CD1d Abs in the BMDC-LMC co-culture system. On addition of the Abs, the GLXA-treated co-culture showed a dramatic decrease in IFN-y and IL-4production compared with the controls, demonstrating the dependence of iNKT activation by GLXA on CD1d molecules
3、in vivo
1) Intravenous injection:GLXA stimulated WT mice to produce higher quantities of IFN-y and IL-4compared with cell mock, whereas there was no significant difference in the cytokine production between GLXA and cell mock in iNKT KO mice. These data suggested that the cytokines produced in the serum were largely secreted by iNKT, and that iNKT were activated by GLXA in this process.
2) Intraperitoneal injection:Compared with those from the cell mock-treated, iNKT from GLXA-treated mice showed significantly higher expression of CD69. On flow cytometric analysis of the pattern of intracellular cytokine production by iNKT in vivo, we found that iNKT from the GLXA-treated mice produced large amounts of IFN-y and IL-4compared with the cell mock-treated mice. These data show that GLXA stimulates iNKT in vivo.
Conclusions
The findings from the present study show that GLXA, a glycolipid antigen derived from the chlamydia, can activate iNKT, and this activated function depend on CD1d molecules. GLXA may be responsible for activation of NKT cells as a mager antigen. The effect of GLXA on activation of iNKT cells remains to be elacidated.
Significance
This finding provides significant new knowledge on the molecular and cellular mechanisms of immune response to chlamydial infection. Our research laid the theoretical foundition using GLXA as a target to design vaccine against chlamydial infection.
Part Two:The Role of IL-22against Chlamydial Lung Infection
Objective
As a member of IL-10cytokine family, Interleukin (IL)-22is an inflammatory cytokine and it plays an important role in modulation tissue responses during inflammation. Recent studies have identified that IL-22is involved in the host defense mechanism against infections caused by bacteria, fungi, viruses and parasites. It is generally accepted that IL-22plays a protective role in extracellular bacterial infections, such as Citrobactor rodentium, Klebsiella pneumoniae, and segmented filamentous bacterium (SFB). However, the role of IL-22in the host defense against intracellular bacteria infections is not clear. Therefore, further investigation is required to evaluate the function of IL-22in an intracellular bacteria infection model. Chlamydiae are obligate intracellular bacterial pathogens. Chlamydia trachomatis and Chlamydia pneumonia are responsible for various human diseases. C. pneumoniae causes respiratory diseases such as bronchitis, sinusitis and pneumonia, and C. trachomatis is a major cause of respiratory, ocular and sexually transmitted diseases.The mouse pneumonitis agent strain is designated as Chlamydia muridarum (Cm). Cm has been widely used in mouse models of respiratory and genital tract infections in research laboratories. Studies from the respiratory chlamydial infection model have shown that the Thl-dominant and IFN-y-dependent immunity is a major host protective determinant for controlling chlamydial infection.Our previous studies indicated that Thl7also plays an important role in host defense against chlamydial infection by promoting Thl-type cell responses. Therefore, the development of Th1and Th17cell immune responses is important for host to effectively control the infection and clear the pathogens. IL-22and IL-17are both produced by the Th17lineage, and data to date suggest that IL-17and IL-22cooperate in some infection models. So far, there is no report on the functional role of IL-22during the development for chlamydial lung infection. In this report, we evaluated the role of IL-22using neutralized anti-IL-22mAb in chlamydial lung infection model.
Methods
1、IL-22neutralized:To assess whether IL-22is important for host defense against Cm infection in the lung, Anti-mouse IL-22mAb was used to neutralize endogenous IL-22after Cm lung infection.
1) Flow cytometry:detect the changes of Th, Th17and Treg cells.
2) ELISA:detect the levels of IL-6, IL-10, IL-12p40, IL-17and IFN-y in Cell culture supernatants.
3) RT-PCR:detect the changes of IL-22, IL-17, IFN-y, T-bet and RORyt at RNA levels.
4) HE staining:detect the pathological changes of lung tissue.
2、IL-22treated:To confirm the role of IL-22in the development of chlamydial pulmonary infection, we treated the C57BL/6mice with mouse recombinant IL-22or PBS following Cm lung infection.
1) Flow cytometry:detect the changes of Th1and Th17cells.
2) ELISA:detect the levels of IL-17and IFN-y in Cell culture supernatants.
3) HE staining:detect the pathological changes of lung tissue.
Results
1、IL-22neutralized:IL-22was produced in the early stages of infection in mice after an intranasal infection with C. muridarum. The mice treated with neutralizing anti-IL-22mAb exhibited severe disease, which showed significantly lower body weight, higher organism growth, and much more severe pathological changes in the lung compared to isotype control group. Immunological analysis showed lower Chlamydia-specific Thl and Th17responses with higher CD4+CD25+Foxp3+regulatory T cells responses in IL-22-neutrolized mice. Moreover, IL-10production was decreased in IL-22-deficient mice compared to WT mice.
2、IL-22treated:IL-22treatment in Chlamydia infected mice dramatically inhibited the development of pulmonary infection and the growth of bacteria. The exogenous IL-22promoted the production of Th17cells after Cm infection.
Conclusions
IL-22was produced in the early stages of infection in mice after an intranasal infection with C. muridarum. IL-22has a protective role during chlamydial lung infection. The protective role of IL-22in host defense against chlamydial infection was complied by promoting the Th17immune response.
Significance
IL-22is an important inflammatory cytokine in host defense against chlamydial infection in the lung. Our study raises the possibility for new treatments to use IL-22to control or inhibit chlamydial lung infection.
研究目的
衣原体(Chlamydiae)是一类专性细胞内寄生,有独特发育周期的原核细胞型微生物,衣原体感染与许多人类疾病有着密切联系。根据基因型的不同,衣原体可分为沙眼衣原体(Chlamydia trachomatis, Ct)和肺炎衣原体(Chlamydia pneumonias, Cpn)两个种属。其中沙眼衣原体主要引起呼吸道感染、眼部粘膜感染及性传播疾病。最近,沙眼衣原体中的鼠类变种株被单独划分出来,被命名为衣原体鼠肺炎株(Chlamydia muridarum, Cm),该株广泛用于建立小鼠肺部或生殖道感染模型,进行沙眼衣原体致病机制的理论研究。而肺炎衣原体主要引起支气管炎、鼻窦炎、肺炎等呼吸道疾病。虽然抗生素可有效控制衣原体的感染症状,但是衣原体感染的临床表现具有多样性,大多数以隐性感染的形式存在,被感染者不能及时就诊,使得抗生素对感染的控制力大大减弱,因此,研发安全有效的疫苗对控制衣原体感染意义重大,目前尚无有效的疫苗预防和(或)治疗衣原体感染。阐明衣原体感染过程中免疫保护和免疫病理的机制是合理研制安全有效衣原体疫苗的首要条件。
自然杀伤性T细胞(invariant natural killer T cell, iNKT)作为独特的T细胞亚群识别由非经典的MHCI类分子CD1d递呈的糖脂类抗原,而不是肽类抗原。研究表明,在衣原体感染中iNKT细胞被激活并产生细胞因子,通过其对获得性免疫应答的调节在衣原体感染的进程和转归中发挥重要作用。衣原体分泌性糖脂抗原(glycolipid exoantigen, GLXA)是目前已被报道的一种和衣原体感染相关的糖脂抗原,但GLXA能否活化NKT细胞,以及它是否是衣原体感染中对iNKT细胞作用的关键抗原尚不清楚。因此,本研究拟证实GLXA能否在体内外激活iNKT细胞,进一步明确衣原体感染的免疫机制,为以GLXA为靶点的衣原体疫苗的研制奠定理论基础。
研究方法
提取衣原体培养上清中的分泌性糖脂,并以相同的方法提取未感染细胞培养上清中的脂类(cell mock)作为实验对照,对糖脂成分进行银染显色和Western Blot鉴定分析。为验证该糖脂是否能够激活iNKT细胞,采用了体外实验和体内试验分别进行验证：
1.体外实验：
1) CDld蛋白+NKT杂交瘤共培养系统：用CDld融合蛋白包被96孔细胞培养板,然后在板中加入GLXA和NKT杂交瘤进行共培养,ELISA检测培养上清中IL-2的水平,以明确GLXA能否活化NKT杂交瘤细胞。
2)体外共培养系统：制备小鼠骨髓来源的树突状细胞(DC),经糖脂冲击树突状细胞24小时后,与肝脏单个核细胞(LMC)共培养,48小时后收集培养上清ELISA法检测细胞因子IFN-y和IL-4。并在共培养实验中采用iNKT缺陷小鼠以及CD1d抗体封闭系统进一步验证作用机制。
2.体内试验：
1)静脉注射：采用C57BL/6(WT)小鼠和iNKT缺陷小鼠,分别经尾静脉注射给予GLXA,注射后2h对小鼠取血,用ELISA检测血清中IL-4的表达水平,注射后12h取血,用ELISA检测血清中IFN-y的表达水平。
2)腹腔注射：小鼠腹腔注射GLXA,用流式细胞术分析其对iNKT表面活化标志及细胞因子分泌的影响。
研究结果
1.糖脂鉴定：银染显色和Western Blot分析显示,提取的衣原体分泌性糖脂与文献报道的GLXA条带一致,与cell mock实验对照组形成的条带明显不同。
2.体外实验：
1)CDld蛋白+NKT杂交瘤共培养系统：与对照组相比,GLXA可特异性活化NKT杂交瘤细胞,使其产生IL-2。
2)体外共培养系统：GLXA冲击组产生的IFN-γ和IL-4均高于cell mock冲击组及阴性对照组(DC未经抗原刺激),表明分泌性糖脂能够激活LMC产生细胞因子。为进一步验证LMC产生的IFN-γ和IL-4主要来源于iNKT细胞,在DC-LMC共培养实验中采用了iNKT缺陷小鼠以及CD1d抗体封闭,结果显示：(1)来源于iNKT缺陷小鼠LMC产生的IFN-γ和IL-4均显著低于野生型小鼠.(2)加入CD1d抗体封闭后,野生型小鼠LMC产生IFN-γ和IL-4也明显下降。由此证明衣原体分泌性糖脂诱导的细胞因子主要由iNKT细胞产生,而且此作用部分依赖于CDld分子。
3.体内试验：
1)静脉注射：在给予GLXA刺激的野生型小鼠血清中,可检测出较高水平的IFN-γ和IL-4,在iNKT缺陷小鼠中却没有发现这种现象,说明血清中较高水平的IFN-γ和IL-4主要是由iNKT细胞产生的,而且是GLXA促进iNKT细胞产生的细胞因子。
2)腹腔注射：通过流式细胞术检测,与对照组相比GLXA可明显提高iNKT细胞表面活化标志的表达,并促进其分泌细胞因子IFN-γ和IL-4,说明其对iNKT细胞有明显的激活作用。
研究结论
以上实验证明,衣原体分泌性糖脂GLXA能够激活iNKT细胞并产生细胞因子,并且该活化作用在一定程度上依赖CDld分子的递呈作用,GLXA可能是衣原体感染中iNKT细胞活化的关键抗原,有关GLXA激活iNKT细胞的作用机制尚需进一步研究。
研究意义
该研究进一步完善了我们对衣原体致病机制的认识,加深了衣原体感染引起的宿主免疫反应机制的了解,为以GLXA为靶点设计疫苗抵抗衣原体感染奠定了实验和理论基础。
第二部分白细胞介素-22在衣原体肺部感染中的生物学作用
研究目的
白细胞介素-22(IL-22)是IL-10细胞因子家族的一员,它是一种炎症因子,在调控组织局部免疫方面起着重要的作用。近期的研究证实,IL-22在抵抗细菌、真菌、病毒和寄生虫感染所引发的宿主免疫中起到一定保护作用。目前普遍接受的观点是IL-22在枸橼酸杆菌、克雷伯杆菌、分节丝状杆菌等胞外菌引起的感染中起到保护作用,但是IL-22在宿主抵抗胞内菌感染中的作用目前仍不明确。因此进一步在胞内菌感染模型中评估IL-22的作用是十分必要的,这有利于明确IL-22在胞内菌感染中的生物学效应,并为其提供更充分的理论学依据。
衣原体是专性胞内寄生菌,其中对人类致病的种属是沙眼衣原体(Chlamydia trachomatis, Ct)和肺炎衣原体(Chlamydia pneumonia, Cpn)。沙眼衣原体主要引起呼吸道、眼睛及性疾病的传播。肺炎衣原体主要引起支气管炎、鼻窦炎和肺炎。由于衣原体菌株具有一定的种属特异性,近些年引起小鼠肺炎的菌株被单独划分出来称为鼠肺炎衣原体(Chlamydia muridarum, Cm)。目前在实验研究中,鼠肺炎衣原体株被广泛用于小鼠呼吸道和生殖道感染模型中。在小鼠呼吸道感染模型中,目前的研究证实Th1型细胞免疫反应起到主要的保护作用,可有效控制衣原体感染,对疾病的转归起到决定性作用。我们前期的实验结果证实Th17在宿主防御衣原体感染中也起到重要的作用,它可以促进Thl型细胞免疫反应。因此,Thl和Th17细胞免疫反应对于宿主清除病原菌起到重要的促进作用。IL-22和IL-17是Th17细胞亚群主要产生的细胞因子,它们在许多感染模型中都起到相互协同的作用。迄今为止,没有实验研究证实IL-22在小鼠肺部衣原体感染中的作用。在本研究中,我们拟用抗小鼠IL-22单克隆抗体封闭小鼠体内固有产生的IL-22,从而进一步评估IL-22在小鼠肺部衣原体感染中的作用。
研究方法
1、IL-22抗体封闭实验：在小鼠Cm肺感染模型中,用小鼠IL-22单克隆抗体对肺局部的IL-22进行封闭,以检测IL-22的作用。
1)流式细胞术：检测Th1、Th17、Treg等细胞亚群的变化情况。
2) ELISA:检测细胞培养上清中IL-6、IL-10、IL-12p40、IL-17、IFN-y等细胞因子的表达水平
3) RT-PCR:检测IL-22、IL-17、IFN-y、T-bet、RORyt在RNA水平上的变化情况。
4)HE染色：肺组织切片镜检。
2、IL-22蛋白给予实验：在小鼠Cm肺感染模型中,肺局部给予小鼠纯化IL-22蛋白,以检测IL-22对疾病转归的影响。
1)流式细胞术：检测Th1、Th17等细胞亚群的变化情况。
2) ELISA:检测培养上清中IL-17、IFN-γ等细胞因子的表达水平。
3)HE染色：肺组织切片镜检。
研究结果
1、IL-22抗体封闭实验：IL-22在小鼠Cm肺部感染的早期阶段就有升高表达,用抗体将其封闭后结果如下：与同型对照组相比,IL-22抗体封闭组的肺部炎症更加严重,表现为体重下降更明显,负菌量升高,病理组织切片炎症浸润更加明显。流式分析显示IL-22抗体封闭组的Th1细胞和Th17细胞免疫反应降低,而CD4+CD25+Foxp3+调节性T细胞群增高,此外IL-10的生成也随着IL-22的封闭而减少。
2、IL-22蛋白给予实验：将IL-22纯化蛋白给予Cm肺部感染的小鼠后,很好地控制了肺部炎症的发展和衣原体的增殖,并促进了肺部的Th17细胞免疫反应。
研究结论
IL-22产生并高表达于小鼠Cm肺部感染的早期阶段,可以保护机体减轻衣原体的肺部感染。IL-22这种保护性作用主要是通过促进Th17细胞免疫反应实现的。
研究意义
IL-22作为一种重要的炎症因子参与了机体防御衣原体肺部感染的进程,它对机体起到明显的保护作用,本研究为以IL-22为靶点研发新药控制衣原体肺部感染提供了理论依据和实验基础。
Part One:The Glycolipid Exoantigen (GLXA) Derived from Chlamydia muridarum Activates Invariant Natural Killer T Cells
Objective
Chlamydiae are obligate intracellular bacterial pathogens. The genus Chlamydia includes two species, Chlamydia trachomatis and Chlamydia pneumoniae, which cause various human diseases. C. trachomatis is a major cause of respiratory, ocular and sexually transmitted diseases. A murine biovar of C.trachomatis (known as mouse pneumonitis [MoPn] agent and now classified as a new species, Chlamydia muridarum) has been used to study the mechanisms of C. trachomatis pathogenesis and immunity in a mouse pulmonary and genital infection models. Further, C. pneumoniae causes respiratory diseases like bronchitis, sinusitis and pneumonia. To date, there is no effective vaccine available against human chlamydial diseases. Considering the public health significance of chlamydial diseases, it is highly desirable to have an effective and safe chlamydial vaccine. However, one of the main constraints in the way of vaccine development is poor understanding of the role of chlamydial components in host-chlamydial interactions. NKT (Natural killer T) cells represent an innate subset of T lymphocytes and recognize glycolipid antigens presented by CD1d, which is a nonclassical MHC class I molecule expressed on antigen presenting cells (APC). We have previously reported that iNKT are activated in vivo and play an important role in immune responses to chlamydial infections. GLXA is a glycolipid component of the chlamydial membrane and intracellular inclusion bodies, which has been implicated in chlamydial-host cell interaction. Taking account of these facts, we hypothesized that GLXA might specifically activate iNKT. To test the hypothesis, we investigated the role of GLXA derived from Chlamydia muridarum in iNKT activation using in vitro as well as in vivo settings.We want to further identify the immune mechnism of chlamydial infection and develop vaccine against chlamydial by targeting GLXA.
Methods
We prepared GLXA antigen from the supernatant of C. muridarum infected cell. Supernatants harvested from uninfected cell monolayers were prepared in an identical manner and used as cell mock control. SDS-PAGE and Western blot analysis were done to identify GLXA. We investigated the role of GLXA in iNKT activation using in vitro as well as in vivo settings.
1、in vitro
1) CD1d fusion protein iNKT hybridoma culture:96-well plates were incubated with mouse CDld fusion protein, and GLXA and iNKT hybridoma cells were added to each well and co-culture. The culture supernatant was measured for IL-2by ELISA.
2) LMC-BMDC co-culture:DC were generated in vitro from bone marrow cells. BMDC were cultured in the presence of GLXA for24h. The treated BMDC were co-cultured with LMC for48h. The levels of IFN-y and IL-4in the culture supernatants were measured by ELISA. We use the iNKT knock out mice and CD1d antibody blocking system to do further identification in this co-culture system.
2、in vivo
1) Intravenous injection:C57BL/6(WT) and iNKT KO mice were intravenously treated with GLXA. The serum levels of IL-4and IFN-y were measured at2h and12h respectively after the treatment. The measurement of the cytokine levels was done by ELISA.
2) Intraperitoneal injection:Mice were injected with GLXA intraperitoneally. After72h, spleens and livers were harvested from the animals, and processed into single-cell suspensions. The cells were then stained with the appropriate Abs for flow cytometric analysis.
Results
1、Preparation of GLXA antigen:Silver staining and Western blot of the antigen sample demonstrated a banding pattern similar to GLXA as described previously, cell mock preparation failed to show the bands although.
2、in vitro
1) CD1d fusion protein iNKT hybridoma culture:On stimulation with GLXA-treated CD1d, in contrast to the cell mock or PBS, iNKT produced IL-2in a dose-dependent manner, suggesting that GLXA can specifically stimulate iNKT hybridoma.
2) LMC-BMDC co-culture:We found that GLXA stimulated LMC from WT mice to produce higher quantities of IFN-y and IL-4compared with cell mock. To confirm that the cytokines produced in the co-culture were secreted by iNKT, we isolated the LMC from iNKT KO and co-cultured it with BMDC. The data showed that the LMC from iNKT KO mice produced significantly lower quantities of IFN-y and IL-4compared with those from WT mice, indicating that the cytokines were mainly produced by iNKT. We further examined whether the cytokine production by the iNKT was CD1d-dependent by blocking CD1d using anti-CD1d Abs in the BMDC-LMC co-culture system. On addition of the Abs, the GLXA-treated co-culture showed a dramatic decrease in IFN-y and IL-4production compared with the controls, demonstrating the dependence of iNKT activation by GLXA on CD1d molecules
3、in vivo
1) Intravenous injection:GLXA stimulated WT mice to produce higher quantities of IFN-y and IL-4compared with cell mock, whereas there was no significant difference in the cytokine production between GLXA and cell mock in iNKT KO mice. These data suggested that the cytokines produced in the serum were largely secreted by iNKT, and that iNKT were activated by GLXA in this process.
2) Intraperitoneal injection:Compared with those from the cell mock-treated, iNKT from GLXA-treated mice showed significantly higher expression of CD69. On flow cytometric analysis of the pattern of intracellular cytokine production by iNKT in vivo, we found that iNKT from the GLXA-treated mice produced large amounts of IFN-y and IL-4compared with the cell mock-treated mice. These data show that GLXA stimulates iNKT in vivo.
Conclusions
The findings from the present study show that GLXA, a glycolipid antigen derived from the chlamydia, can activate iNKT, and this activated function depend on CD1d molecules. GLXA may be responsible for activation of NKT cells as a mager antigen. The effect of GLXA on activation of iNKT cells remains to be elacidated.
Significance
This finding provides significant new knowledge on the molecular and cellular mechanisms of immune response to chlamydial infection. Our research laid the theoretical foundition using GLXA as a target to design vaccine against chlamydial infection.
Part Two:The Role of IL-22against Chlamydial Lung Infection
Objective
As a member of IL-10cytokine family, Interleukin (IL)-22is an inflammatory cytokine and it plays an important role in modulation tissue responses during inflammation. Recent studies have identified that IL-22is involved in the host defense mechanism against infections caused by bacteria, fungi, viruses and parasites. It is generally accepted that IL-22plays a protective role in extracellular bacterial infections, such as Citrobactor rodentium, Klebsiella pneumoniae, and segmented filamentous bacterium (SFB). However, the role of IL-22in the host defense against intracellular bacteria infections is not clear. Therefore, further investigation is required to evaluate the function of IL-22in an intracellular bacteria infection model. Chlamydiae are obligate intracellular bacterial pathogens. Chlamydia trachomatis and Chlamydia pneumonia are responsible for various human diseases. C. pneumoniae causes respiratory diseases such as bronchitis, sinusitis and pneumonia, and C. trachomatis is a major cause of respiratory, ocular and sexually transmitted diseases.The mouse pneumonitis agent strain is designated as Chlamydia muridarum (Cm). Cm has been widely used in mouse models of respiratory and genital tract infections in research laboratories. Studies from the respiratory chlamydial infection model have shown that the Thl-dominant and IFN-y-dependent immunity is a major host protective determinant for controlling chlamydial infection.Our previous studies indicated that Thl7also plays an important role in host defense against chlamydial infection by promoting Thl-type cell responses. Therefore, the development of Th1and Th17cell immune responses is important for host to effectively control the infection and clear the pathogens. IL-22and IL-17are both produced by the Th17lineage, and data to date suggest that IL-17and IL-22cooperate in some infection models. So far, there is no report on the functional role of IL-22during the development for chlamydial lung infection. In this report, we evaluated the role of IL-22using neutralized anti-IL-22mAb in chlamydial lung infection model.
Methods
1、IL-22neutralized:To assess whether IL-22is important for host defense against Cm infection in the lung, Anti-mouse IL-22mAb was used to neutralize endogenous IL-22after Cm lung infection.
1) Flow cytometry:detect the changes of Th, Th17and Treg cells.
2) ELISA:detect the levels of IL-6, IL-10, IL-12p40, IL-17and IFN-y in Cell culture supernatants.
3) RT-PCR:detect the changes of IL-22, IL-17, IFN-y, T-bet and RORyt at RNA levels.
4) HE staining:detect the pathological changes of lung tissue.
2、IL-22treated:To confirm the role of IL-22in the development of chlamydial pulmonary infection, we treated the C57BL/6mice with mouse recombinant IL-22or PBS following Cm lung infection.
1) Flow cytometry:detect the changes of Th1and Th17cells.
2) ELISA:detect the levels of IL-17and IFN-y in Cell culture supernatants.
3) HE staining:detect the pathological changes of lung tissue.
Results
1、IL-22neutralized:IL-22was produced in the early stages of infection in mice after an intranasal infection with C. muridarum. The mice treated with neutralizing anti-IL-22mAb exhibited severe disease, which showed significantly lower body weight, higher organism growth, and much more severe pathological changes in the lung compared to isotype control group. Immunological analysis showed lower Chlamydia-specific Thl and Th17responses with higher CD4+CD25+Foxp3+regulatory T cells responses in IL-22-neutrolized mice. Moreover, IL-10production was decreased in IL-22-deficient mice compared to WT mice.
2、IL-22treated:IL-22treatment in Chlamydia infected mice dramatically inhibited the development of pulmonary infection and the growth of bacteria. The exogenous IL-22promoted the production of Th17cells after Cm infection.
Conclusions
IL-22was produced in the early stages of infection in mice after an intranasal infection with C. muridarum. IL-22has a protective role during chlamydial lung infection. The protective role of IL-22in host defense against chlamydial infection was complied by promoting the Th17immune response.
Significance
IL-22is an important inflammatory cytokine in host defense against chlamydial infection in the lung. Our study raises the possibility for new treatments to use IL-22to control or inhibit chlamydial lung infection.
引文
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