EAE诱导过程中脾脏及CNS浸润Treg/Teff的变化

探讨实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)诱导过程中脾脏及中枢神经系统(central nervous system,CNS)CD4+CD25+Foxp3+Treg、效应T细胞(effector T cell,Teff)的变化及作用。用髓鞘少突胶质细胞糖蛋白(myelin oligodendrocyte glycoprotein,MOG35-55)免疫C57BL/6小鼠诱导EAE;用流式细胞仪分析EAE发病初期及高峰期脾脏和CNS浸润Treg、Th1和Th17的变化。MOG35-55肽诱导产生典型的EAE症状,脊髓可见大量的单个核细胞浸润;EAE发病前,脾脏Treg、Th1、Th17数量增加,随着EAE临床症状的出现,脾脏Treg、Teff均减少;EAE发病前既有少量Teff迁移至CNS,主要为Th17;EAE发病高峰期,CNS内Th17比例降低,Th1比例及数量显著增加;EAE发病前CNS可见少量Treg存在,发病后大量Treg迁移至CNS,但不能抑制CNS的炎症反应。以上结果提示,Th17可能参与了CNS免疫损伤的启动,而CNS浸润的Thl与疾病的严重程度密切相关;EAE发病高峰期脾脏Treg伴随着Teff大量迁移至CNS,但不能控制EAE的进展。     

Th17细胞和调节性T细胞失衡参与实验性自身免疫性脑脊髓炎的发病

目的探讨Th17细胞和调节性T细胞(Treg)失衡在实验性自身免疫性脑脊髓炎(EAE)发病过程中的作用。方法建立EAE小鼠模型,在EAE发病的不同时期运用流式细胞术检测小鼠脾脏Treg比例变化,用实时定量PCR(qRT-PCR)分别检测小鼠脾淋巴细胞Foxp3、视黄酸相关的孤儿受体γt(RoR-γt)以及脑组织IL-17 mRNA表达,ELISA检测小鼠外周血IL-6、转化生长因子β(TGF-β)及IL-17A含量变化。结果与佐剂对照组比较,CD4+CD25+Foxp3+Treg比例和Foxp3 mRNA的表达,在EAE发病初期及高峰期明显下降,而在慢性期明显升高(P0.05);RoR-γt mRNA表达量在发病初期与佐剂对照组比较显著增加(P0.05),而高峰期及慢性期与佐剂对照组比较则无显著差异(P0.05)。EAE组小鼠外周血中TGF-β、IL-6浓度在发病初期和高峰期明显升高(P0.05);随病情发展,IL-6浓度逐渐降低,在发病慢性期与佐剂对照组比较无明显差异(P0.05),而TGF-β浓度在发病慢性期仍较高,与佐剂对照组比较明显升高(P0.05)。EAE组小鼠外周血中IL-17A含量在EAE不同时期均有明显升高,且高峰期升高最明显(P0.05)。结论 Th17细胞/Treg失衡参与了EAE发病过程。     

经鼻黏膜给予MBP68-86和87-99协同免疫预防Lewis大鼠EAE的实验研究

目的探讨鼻黏膜给予MBP68-86和87-99协同免疫预防Lewis大鼠实验性自身免疫性脑脊髓炎(EAE)的作用。方法合成3条不同的碱性髓鞘蛋白(MBP)多肽(MBP68-86、87-99和非致脑炎性肽段110-128),在用豚鼠MBP(gp-MBP)加弗氏完全佐剂免疫Lewis大鼠前的11、10、9、8和7d,经鼻黏膜分别给予MBP多肽,观察其对EAE的保护作用。结果致脑炎性肽段MBP68-86和87-99都有保护作用,其中MBP68-86的保护作用更强;而MBP110-128没有保护作用。鼻黏膜给予MBP68-86 87-99的混合物,在相对低的剂量可完全阻断gp-MBP引发的EAE。淋巴细胞增殖实验和IFN-γELISPOT检测显示,与鼻黏膜给予大鼠MBP110-128组相比,鼻黏膜给予大鼠MBP68-86 87-99可降低T细胞对于MBP的反应性,淋巴结单核细胞中表达IFN-γ和TNF-αmRNA的细胞数减少,而两组表达TGF-β及IL-4mRNA的淋巴细胞数都低。结论鼻黏膜给予致脑炎性MBP多肽能够导致抗原特异性T细胞耐受,对gp-MBP引发的EAE提供不完全的保护,MBP68-86和MBP87-99具有协同作用。鼻黏膜给予多肽引发的免疫耐受与非调节机制有关。     

外源性TGF-β1对实验性自身免疫性脑脊髓炎模型小鼠中IL-23/Th17炎症轴的影响

目的:观察外源性TGF-β1对实验性自身免疫性脑脊髓炎模型小鼠IL-23/Th17炎症轴的影响,探讨其可能的作用机制。方法:以MOG35-55免疫C57BL/6小鼠制作EAE模型,多时间点皮下注射TGF-β1作为干预,RT-PCR和ELISA检测IL-23、IL-17和IL-6的表达。结果:与EAE组相比,干预组在发病初IL-23、IL-17、IL-6表达均升高(P0.05,P0.05,P0.05);在高峰期IL-23表达下降(P0.05),IL-17、IL-6表达升高(P0.05,P0.05);在慢性期IL-23、IL-17表达下降(P0.05,P0.05),而IL-6表达升高(P0.05)。结论:外源性的TGF-β1可能通过上调IL-23、IL-17的表达使EAE高峰期的症状加重并提前,而在慢性期相对地下调其表达,EAE症状趋于缓解。     

经腹腔诱导免疫耐受抑制EAE的实验研究

目的通过建立实验性自身免疫性脑脊髓炎(EAE)动物模型,研究经腹腔诱导免疫耐受的方法及机理。方法腹腔内注射可溶性髓鞘碱性蛋白(MBP)或转输可溶性MBP致敏的腹腔抗原提呈细胞(MBP-APC),观察各组动物EAE的发病情况,检测各组动物迟发型超敏反应(DTH)和淋巴细胞体外增殖应答。结果腹腔内注射MBP及MBP-APC的动物发病率明显低于EAE组,临床症状减轻,由MBP引起的DTH和体外特异的MBP淋巴细胞增殖反应也明显降低。结论腹腔注射MBP或MBP-APC可引起粘膜耐受,抑制EAE的发生。     

以EAE动物模型研究粘膜耐受

经粘膜诱导免疫耐受是免疫学领域迅速发展的一个分枝 ,用于研究并试验性治疗某些器官特异性自身免疫病。这些研究均首先从动物模型开始 ,应用最多的是实验性自身免疫性脑脊髓炎 (experimentalautoim muneencephalomyelitis ,EAE) ,或称实验性变态反应性脑脊髓炎 (experimentalallergicencephalomyelitis ,EAE) ,是人类多发性硬化症 (MS)的动物模型。本文简要介绍了以EAE研究粘膜耐受的概况 ,可能的机理及应用前景     

经消化道诱导昆明鼠耐受实验性自身免疫性脑脊髓炎

诱导特异性免疫耐受是多发性硬化治疗学的一个重要研究方向。实验性自身免疫性脑脊髓炎是研究多发性硬化常用的动物模型，在此介绍我们使用同源脊髓匀浆诱导昆明鼠耐受实验性自身免疫性脑脊髓炎的一些实验工作结果及体会。     

细胞因子在实验性自身免疫性脑脊髓炎耐受中的作用

细胞因子(CK)在实验性自身免疫性脑脊髓炎(EAE)的免疫机制中起着重要作用。Th细胞的不同转化决定EAE的发生、发展或抑制。由多种CK构成的免疫调节网络操纵着Th细胞的免疫应答。通过作用Th细胞使之向抑制EAE方向转化,从而寻找对EAE耐受的途径,是目前EAE研究的一个重要方面。以下就与EAE耐受相关的CK研究进行综述,探讨EAE免疫病理机制。     

口服MBP诱导免疫耐受预防实验性变态反应性脑脊髓炎的研究

目的：探讨口服耐受对实验性变态反应性脑脊髓炎(EAE)的预防作用。方法：给予健康Lewis大鼠口服MBP抗原诱导免疫耐受，随后以MBP CFA于足底免疫，检测口服耐受对实验大鼠EAE发病的预防作用及对MBP特异性淋转率、细胞因子和抗MBP抗体等免疫功能的变化情况。结果：致病组EAE发病率为91．7％，MBP特异性淋转率、IFN-γ与TNF-α含量升高，IL-10降低；口服耐受组EAE发病率为21．4％，MBP特异性淋转率、IFN-γ、TNF-α回复至正常，IL-10含量低于正常。MBP抗体水平两组均比正常对照组高。结论：口服耐受可降低宿主自身免疫反应性，减低EAE的发病率。     

EAE小鼠模型的构建及其免疫学机制的探讨

目的 用髓鞘少突胶质细胞糖蛋白35-55(MOG35-55) 多肽免疫C57BL/6小鼠建立实验性自身免疫性脑脊髓炎(EAE)模型并初步探讨EAE发病的免疫学机制.方法 用MOG35-55抗原免疫C57BL/6小鼠,观察实验动物的症状及中枢神经系统的病理改变;应用MTT法测定EAE 小鼠脾脏T淋巴细胞活化增殖程度;应用ELISA技术测定EAE小鼠血清IFN-γ浓度和抗MOG 35-55抗体水平.结果 EAE组发病率为100%,HE染色观察到脑脊髓炎性细胞浸润,白质脱髓鞘等病理改变;EAE小鼠脾脏MOG35-55反应性T淋巴细胞增殖活化,血清IFN-γ浓度和抗MOG35-55抗体随病情的加重而升高.结论 用MOG35-55多肽成功诱导了C57BL/6小鼠EAE模型;并推断EAE的发生可能与MOG 35-55反应性T淋巴细胞的活化、抗MOG35-55抗体和IFN-γ升高有关.     

Regulation of IL-10 and IL-17 mediated experimental autoimmune encephalomyelitis by S-nitrosoglutathione

In this study, we investigated IL-10 and IL-17 specific immunomodulatory potential of S-nitrosoglutathione (GSNO), a physiological nitric oxide carrier molecule, in experimental autoimmune encephalomyelitis (EAE). In active EAE model, GSNO treatment attenuated EAE severity and splenic CD4⁺ T cells isolated from these mice exhibited decreased IL-17 expression without affecting the IFN-γ expression compared to the cells from untreated EAE mice. Similarly, adoptive transfer of these cells to nave mice resulted in reduction in IL-17 expression in the spinal cords of recipient mice with milder EAE severity. CD4⁺ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, still expressed lower levels of IL-17 under T_H17 skewing conditions, but expressed similar levels of IFN-γ under T_H1 skewing condition. Interestingly, under both T_H17 and T_H1 skewing condition, CD4⁺ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, expressed higher levels of IL-10 and adoptive transfer of these T_H17 and T_H1 skewed cells seemingly exhibited milder EAE disease. In addition, adoptive transfer of CD4⁺ T cells from GSNO treated EAE mice to active EAE mice also ameliorated EAE disease with induction of spinal cord expression of IL-10 and reduction in of IL-17, thus suggesting the participation of IL-10 mechanism in GSNO mediated immunomodulation. GSNO treatment of mice passively immunized with CD4⁺ T cells either from GSNO treated EAE mice or untreated mice further ameliorated EAE disease, supporting efficacy of GSNO for prophylaxis and therapy in EAE. Overall, these data document a modulatory role of GSNO in IL-17/IL-10 axis of EAE and other autoimmune diseases.     

L-17与自身免疫性疾病关系的研究进展

IL-17是CD4 T细胞亚群Th17分泌的细胞因子。Th17的发育、扩增及分泌IL-17主要受TGF-β、IL-6、IL-15、IL-23等细胞因子的调控。IL-17调节前炎性因子、趋化因子等的产生及分泌,在白细胞的迁移、破骨细胞的活化和骨质的吸收等方面发挥重要作用。研究证实,IL-17在自身免疫性疾病的发生、发展中也具有一定的影响和作用。     

IL‐12‐polarized Th1 cells produce GM‐CSF and induce EAE independent of IL‐23

CD4⁺ T‐helper (Th) cells reactive against myelin antigens mediate the mouse model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL‐23 is universally required for the acquisition of pathogenic properties by myelin‐reactive T cells. We show that IL‐12‐modulated Th1 cells readily produce IFN‐γ and GM‐CSF in the CNS of mice and induce a severe form of EAE via an IL‐23‐independent pathway. Th1‐mediated EAE is characterized by monocyte‐rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2 dependent. Conversely, IL‐23‐modulated, stable Th17 cells induce EAE with a relatively mild course via an IL‐12‐independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4⁺ T‐helper‐cell subsets and polarizing factors.     

Protection from EAE in DOCK8 mutant mice occurs despite increased Th17 cell frequencies in the periphery

Mutation of Dedicator of cytokinesis 8 (DOCK8) has previously been reported to provide resistance to the Th17 cell dependent EAE in mice. Contrary to expectation, we observed an elevation of Th17 cells in two different DOCK8 mutant mouse strains in the steady state. This was specific for Th17 cells with no change in Th1 or Th2 cell populations. In vitro Th cell differentiation assays revealed that the elevated Th17 cell population was not due to a T cell intrinsic differentiation bias. Challenging these mutant mice in the EAE model, we confirmed a resistance to this autoimmune disease with Th17 cells remaining elevated systemically while cellular infiltration in the CNS was reduced. Infiltrating T cells lost the bias toward Th17 cells indicating a relative reduction of Th17 cells in the CNS and a Th17 cell specific migration disadvantage. Adoptive transfers of Th1 and Th17 cells in EAE‐affected mice further supported the Th17 cell‐specific migration defect, however, DOCK8‐deficient Th17 cells expressed normal Th17 cell‐specific CCR6 levels and migrated toward chemokine gradients in transwell assays. This study shows that resistance to EAE in DOCK8 mutant mice is achieved despite a systemic Th17 bias.     

Interleukin-17A Serves a Priming Role in Autoimmunity by Recruiting IL-1β-Producing Myeloid Cells that Promote Pathogenic T Cells

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Th17及其细胞因子IL-17参与脑缺血损伤机制的研究

目的 检测Th17细胞在缺血脑组织中的表达及其细胞因子IL-17在厌氧条件下对神经元损伤机制的研究.方法 线栓法建立小鼠持续性大脑中动脉栓塞模型(pMCAO),免疫荧光技术检测pMCAO小鼠缺血侧脑组织中Th17细胞的表达;体外原代培养新生小鼠海马神经元,建立体外氧糖剥夺模型(OGD)模拟体内脑缺血缺氧微环境,观察不同厌氧时间对神经元的损伤作用,同时进行不同浓度的IL-17干预以及IL-17受体(IL-17R)封闭实验,探讨IL-17在厌氧条件下对神经元损伤过程中的作用.结果 成功建立小鼠右侧大脑中动脉栓塞模型,HE染色可见患鼠缺血侧脑组织空洞坏死样改变并有大量淋巴细胞浸润,TTC染色呈现白色缺血灶.免疫荧光染色可见缺血灶周边大量Th17细胞(CD4+IL-17+).体外实验中,外源性IL-17的干预加剧了OGD条件对海马神经元的损伤作用,并且这种作用能够被IL-17 R封闭剂所抑制.结论 小鼠缺血脑组织中存在Th17细胞的浸润,Th17细胞分泌的IL-17与厌氧条件下神经元的损伤有关.     

IL-23/IL-17轴在银屑病免疫发病机制中的作用

银屑病是一种以T淋巴细胞异常活化和浸润为主要特征的慢性炎性反应皮肤病。Th17细胞及IL-23/IL-17轴在银屑病的发病机制中可能处于关键地位,并成为新的治疗靶标。IL-23诱导Th17细胞分化增殖,分化成熟的Th17可以分泌IL-17、IL-21、IL-22等多种细胞因子,Th17类细胞因子在银屑病等多种自身免疫疾病和炎性疾病中起重要作用。     

成人哮喘患者Treg/Th17细胞失衡与呼出气一氧化氮的相关性研究

目的:探讨成人哮喘患者Treg/Th17细胞失衡与呼出气一氧化氮(FeNO)的相关关系。方法:募集成人哮喘患者17例,慢性咳嗽非哮喘患者16例。所有患者均检测FeNO值,并用ELISA法检测血浆IL-17的表达水平,流式细胞术检测外周血中Treg、Th17细胞占CD4~+T淋巴细胞比例。比较两组患者FeNO水平的差异及其与Treg/Th17的相关关系。结果:与慢性咳嗽组相比,哮喘组血浆IL-17浓度升高(P<0.05)、外周血Th17/CD4~+T细胞比值升高(P<0.001)、FeNO水平升高(P<0.001)、Treg/Th17细胞的比值降低(P<0.001),而Treg/CD4~+细胞比值无明显变化(P>0.05)。哮喘组FeNO值与外周血Treg/CD4~+T细胞比值无明显相关性,而与Th17细胞占CD4~+T淋巴细胞比例呈正相关(r=0.663,P=0.01),与Treg/Th17细胞比值呈负相关(r=-0.757,P=0.002)。结论:哮喘患者FeNO水平升高与Treg/Th17失衡有关,主要与Th17细胞表达升高有关。     

Targeting pre-ligand assembly domain of TNFR1 ameliorates autoimmune diseases - an unrevealed role in downregulation of Th17 cells

The pre-ligand assembly domain (PLAD) of tumor necrosis factor receptors mediates specific ligand-independent receptor assembly and subsequent signaling. However, the physiological role of PLAD in the regulation of TNFR-mediated immune responses in autoimmunity is still unclear. By using the recombinant PLAD.Fc protein to block TNFR1 assembly, we demonstrated that PLAD.Fc treatment significantly reduced the TNFR1-driving proinflammatory cytokines and protected NOD mice from diabetes. Strikingly, Th17 differentiation was significantly inhibited in PLAD.Fc-treated NOD and TNFR1-deficient mice, indicating a TNFR1-dependent Th17 development. PLAD.Fc-modulated effects on DCs, in terms of the downregulation of Th17-inducing cytokines, IL-6 and TGF-β, explained the potential mechanism for Th17 suppression. Finally, we provided an additional result that PLAD.Fc administration diminished the infiltration of Th17 cells in the central nervous system and ameliorated the experimental autoimmune encephalomyelitis in mice. Collectively, these data demonstrated that targeting PLAD of TNFR1 provides protection from autoimmune diseases through the downregulation of Th17 and suggested a therapeutic potential of PLAD-modulation in TNF-involved inflammatory diseases.