Oestrogen modulates experimental autoimmune encephalomyelitis and interleukin-17 production via programmed death 1

The mechanism by which oestrogens suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is only partially understood. We here demonstrate that treatment with 17β-oestradiol (E₂) in C57BL/6 mice boosted the expression of programmed death 1 (PD-1), a negative regulator of immune responses, in the CD4⁺ FoxP3⁺ regulatory T (Treg) cell compartment in a dose-dependent manner that correlated with the efficiency of EAE protection. Administration of E₂ at pregnancy levels but not lower concentrations also enhanced the frequency of Treg cells. Additionally, E₂ treatment drastically reduced the production of interleukin-17 (IL-17) in the periphery of immunized mice. However, E₂ treatment did not protect against EAE or suppress IL-17 production in PD-1 gene-deficient mice. Finally, E₂ failed to prevent Treg-deficient mice from developing spontaneous EAE. Taken together, our results suggest that E₂-induced protection against EAE is mediated by upregulation of PD-1 expression within the Treg-cell compartment.     

T cells in multiple sclerosis and experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a demyelinating inflammatory disorder of the central nervous system (CNS), which involves autoimmune responses to myelin antigens. Studies in experimental autoimmune encephalomyelitis (EAE), an animal model for MS, have provided convincing evidence that T cells specific for self‐antigens mediate pathology in these diseases. Until recently, T helper type 1 (Th1) cells were thought to be the main effector T cells responsible for the autoimmune inflammation. However more recent studies have highlighted an important pathogenic role for CD4⁺ T cells that secrete interleukin (IL)‐17, termed Th17, but also IL‐17‐secreting γδ T cells in EAE as well as other autoimmune and chronic inflammatory conditions. This has prompted intensive study of the induction, function and regulation of IL‐17‐producing T cells in MS and EAE. In this paper, we review the contribution of Th1, Th17, γδ, CD8⁺ and regulatory T cells as well as the possible development of new therapeutic approaches for MS based on manipulating these T cell subtypes.     

Regulatory T cells play a role in T-cell receptor CDR2 peptide regulation of experimental autoimmune encephalomyelitis

Eliciting T-cell receptor (TCR) -specific responsiveness has been known to provide an effective autoregulatory mechanism for limiting inflammation mediated by T effector cells. Our previous use of TCR peptides derived from the CDR3 regions of a pathogenic TCR effectively reversed ongoing experimental autoimmune encephalomyelitis (EAE) in a humanized TCR transgenic model. In this study, we use the TCR BV8S2 CDR2 peptide in the non-transgenic C57BL/6 EAE model to down-regulate the heterogeneous TCR BV8S2(+) MOG-35-55-specific pathogenic T-cell population and demonstrate successful treatment of EAE after disease onset. Suppression of disease was associated with reduced MOG-35-55-specific and non-specific T-cell production of interleukin-17a and interferon-γ in the central nervous system, as well as reduced numbers of CD4(+) and Foxp3(+) T cells in the central nervous system. With the use of Foxp3-GFP and Foxp3 conditional knockout mice, we demonstrate that the TCR CDR2 peptide treatment effect is dependent on the presence of Foxp3(+) regulatory T cells and that regulatory T cell numbers are significantly expanded in the periphery of treated mice. Hence, TCR CDR2 peptide therapy is effective in regulating heterogeneous, pathogenic T-cell populations through the activity of the Foxp3(+) regulatory T cell population.     

Peptide T does not ameliorate experimental autoimmune encephalomyelitis (EAE) in Lewis rats

Peptide T has been shown to inhibit T cell activation and cytokine production and function. Moreover, it has been reported to be a safe treatment in humans. We have studied the ability of peptide T to prevent or ameliorate EAE in Lewis rats. Peptide T was administered subcutaneously at different doses and phases of the disease according to several treatment protocols, but we could not observe a consistent effect of peptide T ameliorating the disease. Lymph node cell proliferation and IL-4 and interferon-gamma production were also studied. We conclude that peptide T neither prevents nor ameliorates EAE in Lewis rats.     

Regulatory T cells in experimental allergic encephalomyelitis. I. Frequency and specificity analysis in normal and immune rats of a T cell subset that inhibits disease

We have shown previously that administration of myelin basic protein (MBP)-reactive T cells to naive Lewis rats induces not only autoimmune encephalomyelitis (EAE) but also a near total resistance to subsequent disease. By isolating the effector cells that are responsible for the resistance, we demonstrated that disease protection paralleled with increased numbers of a CD8+ regulatory T cell (RTC) subset and that co-injection of this RTC subset with encephalitogenic T cells aborted the pathogenic activity of the latter cells. Here, we show that a radio-sensitive splenic population of RTC also exists in naive rats that can be recruited and activated to inhibit the onset of secondary episodes of adoptive EAE. In co-transfer experiments, this protective RTC subpopulation can be isolated to neutralize the pathogenic activity of stimulatory MBP-reactive T cells in vivo. We show that the frequency of RTC with specificity for MBP-reactive T cells in naive rats is two orders of magnitude higher than the frequency of MBP-specific precursors, the activity of RTC increases substantially with age and RTC frequencies increase as a consequence of immunization with MBP-reactive cells lines. In specificity studies, we show that RTC isolated from naive rats and RTC from animals primed with one MBP-reactive cell line show cross-reactive responses to a variety of different MBP-reactive T cell lines. However, following repeated stimulation with a given MBP line, these RTC display a more limited, clonotypic response to the selecting line and assume a uniform CD8 phenotype. Finally, functional studies with RTC indicate that proliferative and lytic specificities do not necessarily correlate and that activated rat RTC are especially lytic for a Fas-sensitive murine cell line.     

实验性变态反应性脑脊髓炎大鼠单核细胞趋化蛋白-1的表达与临床评分的相关性

目的：探讨实验性变态反应性脑脊髓炎（EAE）大鼠脑和脊髓中单核细胞趋化蛋白-1（MCP-1）的表达及其与临床评分的关系。方法：将Wistar大鼠分为正常组、佐剂（CFA）组和EAE组,取脑和脊髓制成石蜡切片,进行HE染色和MCP-1mRNA的原位杂交,并与各项临床指标比较。结果：EAE组的体重减轻、MCP-1 mRNA表达的阳性细胞百分数与正常大鼠、佐剂组相比显著性增加。EAE组大鼠MCP-1 mRNA的表达呈动态性变化,其MCP-1 mRNA先于临床症状高表达,并随临床评分的升高而升高,与临床评分呈正相关。结论：MCP-1是参与EAE发病的重要的炎性介质。     

雷帕霉素通过下调Th17细胞/调节性T细胞比值减轻实验性自身免疫性重症肌无力大鼠症状

目的 初步研究雷帕霉素(RAPA)对实验性自身免疫性重症肌无力(EAMG)大鼠的治疗作用,并研究相关免疫机制.方法 应用鼠源性乙酰胆碱受体α亚基97～116肽段(R97-116)免疫Lewis大鼠,建立EAMG动物模型,随机分为完全Freund佐剂对照组(CFA组)、EAMG模型对照组、RAPA处理组[1 mg/(kg...     

Pre‐existing central nervous system lesions negate cytokine requirements for regional experimental autoimmune encephalomyelitis development

In region‐specific forms of experimental autoimmune encephalomyelitis (EAE), lesion initiation is regulated by T‐cell‐produced interferon‐γ (IFN‐γ) resulting in spinal cord disease in the presence of IFN‐γ and cerebellar disease in the absence of IFN‐γ. Although this role for IFN‐γ in regional disease initiation is well defined, little is known about the consequences of previous tissue inflammation on subsequent regional disease, information vital to the development of therapeutics in established disease states. This study addressed the hypothesis that previous establishment of regional EAE would determine subsequent tissue localization of new T‐cell invasion and associated symptoms regardless of the presence or absence of IFN‐γ production. Serial transfer of optimal or suboptimal doses of encephalitogenic IFN‐γ‐sufficient or ‐deficient T‐cell lines was used to examine the development of new clinical responses associated with the spinal cord and cerebellum at various times after EAE initiation. Previous inflammation within either cerebellum or spinal cord allowed subsequent T‐cell driven inflammation within that tissue regardless of IFN‐γ presence. Further, T‐cell IFN‐γ production after initial lesion formation exacerbated disease within the cerebellum, suggesting that IFN‐γ plays different roles at different stages of cerebellar disease. For the spinal cord, IFN‐γ‐deficient cells (that are ordinarily cerebellum disease initiators) were capable of driving new spinal‐cord‐associated clinical symptoms more than 60 days after the initial acute EAE resolution. These data suggest that previous inflammation modulates the molecular requirements for new neuroinflammation development.     

Dendritic cells exposed in vitro to TGF-beta1 ameliorate experimental autoimmune myasthenia gravis

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG), characterized by an autoaggressive T-cell-dependent antibody-mediated immune response directed against the acetylcholine receptor (AChR) of the neuromuscular junction. Dendritic cells (DC) are unique antigen-presenting cells which control T- and B-cell functions and induce immunity or tolerance. Here, we demonstrate that DC exposed to TGF-beta1 in vitro mediate protection against EAMG. Freshly prepared DC from spleen of healthy rats were exposed to TGF-beta1 in vitro for 48 h, and administered subcutaneously to Lewis rats (2 x 10(6)DC/rat) on day 5 post immunization with AChR in Freund's complete adjuvant. Control EAMG rats were injected in parallel with untreated DC (naive DC) or PBS. Lewis rats receiving TGF-beta1-exposed DC developed very mild symptoms of EAMG without loss of body weight compared with control EAMG rats receiving naive DC or PBS. This effect of TGF-beta1-exposed DC was associated with augmented spontaneous and AChR-induced proliferation, IFN-gamma and NO production, and decreased levels of anti-AChR antibody-secreting cells. Autologous DC exposed in vitro to TGF-beta1 could represent a new opportunity for DC-based immunotherapy of antibody-mediated autoimmune diseases.     

Antigen clearance at the peak of the primary immune response induces experimental autoimmune encephalomyelitis

Autoimmune demyelinating diseases can be induced by an immune response against myelin peptides; however, the exact mechanism underlying the development of such diseases remains unclear. In experimental autoimmune encephalomyelitis, we found that the clearance of exogenous myelin antigen at the peak of the primary immune response is key to the pathogenesis of the disease. The generation of effector T cells requires continuous antigen stimulation, whereas redundant antigen traps and exhausts effector T cells in the periphery, which induces resistance to the disease. Moreover, insufficient antigenic stimulation fails to induce disease efficiently owing to insufficient numbers of effector T cells. When myelin antigen is entirely cleared, the number of effector T cells reaches a peak, which facilitates infiltration of more effector T cells into the central nervous system. The peripheral antigen clearance initiates the first wave of effector T cell entry into the central nervous system and induces chronic inflammation. The inflamed central nervous system recruits the second wave of effector T cells that worsen inflammation, resulting in loss of self-tolerance. These findings provide new insights into the mechanism underlying the development of autoimmune demyelinating diseases, which may potentially impact future treatments.     

Inconsistence between number and function of autoreactive T cells in the course of experimental autoimmune encephalomyelitis

Background: Mouse experimental autoimmune encephalomyelitis (EAE) is widely used model of multiple sclerosis (MS). The role of autoreactive CD4⁺ and CD8⁺ T cells in the development of mouse EAE has been demonstrated. However, little information is available about the relation between the frequency and reactivity of myelin antigen-reactive CD4⁺ and CD8⁺ T cells in secondary lymphoid organs and their relevance with the inflammation and pathological lesion of CNS during the course of EAE mouse model.

Methods: In this study, an EAE model with a clinical course containing acute onset, peak and chronic remission stages was established in C57BL/6J mice by myelin oligodendrocyte protein (MOG)_35–55 peptide immunization, and followed by the monitoring of clinical and pathological parameters and autoreactive T cells at different stages during the course.

Results: The dynamic changes of inflammatory infiltration, myelin loss, and astrocyte proliferation in brain and spinal cord were highly consistent with clinical severity observed in EAE course. However, the frequencies of both MOG-specific CD4⁺ and CD8⁺ T cells in secondary lymphoid organs presented different dynamic trends from the IFN-γ production by MOG-reactive T cells. Meanwhile, the IL-17 production by MOG-reactive CD4⁺ T cells was consistent with the proliferation of MOG-specific CD4⁺ T cells.

Conclusions: Both CD4⁺ and CD8⁺ T cells were most sensitive to MOG antigen stimulation for IFN-γ production during the early stage of EAE, but then rapidly lost the function despite their vigorous proliferation at the peak stage and later.     

β-榄香烯对自身免疫性脑脊髓炎小鼠早期炎性损伤的治疗作用及其免疫学机制

目的探讨β-榄香烯对实验性自身免疫性脑脊髓炎(experimental autoimmune encephalomyelitis,EAE)小鼠早期炎性损伤的治疗作用及可能的免疫学机制。方法 6～8周龄健康雌性野生型C57BL/6小鼠(SPF15)共64只,均分为对照组、模型组和β-榄香烯治疗组。采用髓鞘少突胶质细胞糖蛋白(myelinoligodendrocyte glycoprotein peptide35-55,MOG35-55)多肽作为抗原诱发EAE小鼠模型。于免疫后15日剥离脊髓组织进行病理学研究,MTT实验检测EAE小鼠MOG抗原特异性增殖反应及β-榄香烯(β-elemene)对MOG抗原特异性增殖反应的影响,定量PCR技术分别检测各组小鼠脊髓及淋巴结细胞白细胞介素17(interleukin-17,IL-17)、转录因子孤儿核受体(retinoid acid-related orphan receptors-γt,RORγt)、白细胞介素6(interleukin-6,IL-6)、白细胞介素23(interleukin-23,IL-23)、转化生长因子β(transforming growth factor-β,TGF-β)以及叉头蛋白3(Foxp3)的mRNA的表达。结果模型组的神经功能评分高于治疗组(<0.05),治疗组小鼠脊髓炎症及轴索损伤较模型组减轻。免疫后11d,与模型组比较,治疗组小鼠脊髓IL-17、RORγt、IL-6、IL-23的mRNA的表达减少(<0.05),Foxp3mRNA的表达增加(<0.05),并持续至免疫后15d。体外研究发现模型组T淋巴细胞增殖较对照组更明显(<0.05),治疗组β-榄香烯对T淋巴细胞增殖抑制呈现明显的量效关系(<0.05);与模型组比较,治疗组体外培养的淋巴细胞IL-17、RORγt、IL-6、IL-23mRNA表达水平增加,Foxp3mRNA表达水平减少(<0.05)。结论β-榄香烯减轻EAE小鼠早期炎性损伤,可能与其抑制髓鞘少突胶质细胞糖蛋白多肽反应性T细胞的激活以及维持Th17细胞/调节T细胞亚群的平衡密切相关。     

Loss of β‐arrestin 2 exacerbates experimental autoimmune encephalomyelitis with reduced number of Foxp3+ CD4+ regulatory T cells

β-Arrestins are well-known regulators and mediators of G protein-coupled receptor signalling, and accumulating evidence reveals that they are functionally involved in inflammation and autoimmune diseases. Of the two β-arrestins, β-arrestin 1 is documented to play regulatory roles in an animal model of multiple sclerosis (MS), whereas the role of β-arrestin 2 is less clear. Here, we show that β-arrestin 2-deficient mice displayed the exacerbated and sustained symptoms of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. At the cellular level, deficiency of β-arrestin 2 led to a decreased number of Foxp3⁺ CD4⁺ regulatory T (Treg) cells in peripheral lymphoid organs of EAE mice. Consistently, our in vitro observations also revealed that loss of β-arrestin 2 impaired the conversion of Foxp3⁻ CD4⁺ T cells into Foxp3⁺ CD4⁺ inducible Treg cells. Taken together, our data suggest that β-arrestin 2 plays a regulatory role in MS, that is opposite to that of β-arrestin 1, in autoimmune diseases such as MS, which is at least partially through regulation of iTreg cell differentiation.     

Orally administered myelin basic protein in neonates primes for immune responses and enhances experimental autoimmune encephalomyelitis in adult animals

Antigen-driven tolerance is an effective method for suppression of autoimmune diseases. Adult animals can be tolerized against the induction of experimental autoimmune encephalomyelitis (EAE) by both oral and parenteral administration of myelin basic protein (MBP). We have found that in contrast to previous studies of neonatal tolerance in which parenterally administered autoantigens induced tolerance, the oral administration of MBP in neonatal rats did not result in tolerization to MBP, but instead, primed for immunologic responses. Proliferative responses to MBP and its encephalitogenic epitope were present in animals fed with MBP as neonates and co-culture of encephalitogenic T cells with cells from neonatal rats fed with MBP were associated with enhanced MBP responses rather than the suppression observed with cells from adult rats fed with MBP. Furthermore, neonates fed with MBP and immunized 6–8 weeks later with MBP in adjuvant to induce EAE revealed enhancement of disease severity, and were not protected from a second attack upon active reinduction of EAE. Subcutaneous injection of soluble MBP into neonates had no effect on EAE induction as adults, whereas intraperitoneal injection of MBP in neonates was associated with marked suppression of disease in adults. Suppression of EAE began to appear in animals fed with MBP at 4 weeks of age, and was similar to oral tolerance in adult animals when animals were fed at 6 weeks of age. These results suggest that immaturity of the immunoregulatory network associated with oral tolerance and sensitization to autoantigens via the gut in the neonatal period may contribute to the pathogenesis of autoimmune diseases.     

红景天苷对实验性自身免疫性脑脊髓炎小鼠的干预作用与机制

目的:观察不同浓度红景天苷(salidroside)对实验性自身免疫脑脊髓炎(experimental autoimmuneencephalomyelitis,EAE)小鼠模型的作用,并探讨初步的作用机制.方法:建立实验性自身免疫性脊髓炎小鼠模型,免疫当天起给予高中低剂量的红景天苷灌胃,1次/d,共给药35 d.给药完成后,进行神经功能评分,TUNEL染色检测脊髓髓鞘组织的细胞凋亡情况,ATP水平检测试剂盒检测脊髓组织ATP含量水平,免疫印迹法检测线粒体内细胞色素c氧化酶(mito cyto-c)、胞浆中细胞色素c氧化酶(cyto cyto-c)、活化的含半胱氨酸的天冬氨酸蛋白水解酶-3(cleaved cas-3)与活化的含半胱氨酸的天冬氨酸蛋白水解酶-9(cleaved cas-9)蛋白表达水平.结果:红景天苷降低EAE小鼠的神经功能评分,后延发病时间;随红景天苷浓度升高,脊髓髓鞘组织的细胞凋亡数逐渐降低,脊髓组织ATP水平显著升高;EAE小鼠脊髓组织细胞胞浆中的cyto-c蛋白表达显著升高,同时cleavedcas-3与cleaved cas-9蛋白表达显著升高,红景天苷干预后,胞浆中cyto-c蛋白表达显著下降,同时cleaved cas-3与cleaved cas-9蛋白表达下降.结论:红景天苷对缓解实验性自身免疫性脊髓炎症状具有良好的效果,可能通过抑制内源性线粒体通路而降低脊髓组织细胞的凋亡而发挥作用.     

Induction of a type 1 regulatory CD4 T cell response following V beta 8.2 DNA vaccination results in immune deviation and protection from experimental autoimmune encephalomyelitis

DNA vaccination has been used to generate effective cellular as well as humoral immunity against target antigens. Here we have investigated the induction and involvement of regulatory T cell (T(reg)) responses in mediating prevention of experimental autoimmune encephalomyelitis (EAE), following vaccination with plasmid DNA encoding the TCR V(beta)8.2 chain predominantly displayed on disease-causing lymphocytes. Vaccination with DNA encoding the wild-type TCR results in priming of type 1 CD4 T(reg) and skewing of the global response to myelin basic protein in a T(h)2 direction, leading to significant protection from disease. In contrast, vaccination with mutant DNA encoding altered residues critically involved in recognition by the T(reg) results in priming of a type 2 regulatory response which fails to mediate immune deviation or protection from EAE. Control mice immunized with DNA, encoding TCR with changes at an irrelevant site, were protected from antigen-induced disease. Furthermore, protection can be transferred into naive recipients with CD4 T(reg) from wild-type DNA-immunized mice but not from animals vaccinated with the mutant DNA. These data suggest that vaccination with plasmid DNA encoding one or multiple V(beta) genes can be exploited to enhance natural regulatory responses for intervention in autoimmune conditions.     

IL‐15‐dependent CD8+CD122+ T cells ameliorate experimental autoimmune encephalomyelitis by modulating IL‐17 production by CD4+ T cells

Interleukin‐15 (IL‐15) is an inflammatory cytokine whose role in autoimmune diseases has not been fully elucidated. Th17 cells have been shown to play critical roles in experimental autoimmune encephalomyelitis (EAE) models. In this study, we demonstrate that blockade of IL‐15 signaling by TMβ‐1 mAb treatment aggravated EAE severity. The key mechanism was not NK‐cell depletion but depletion of CD8⁺CD122⁺ T cells. Adoptive transfer of exogenous CD8⁺CD122⁺ T cells to TMβ‐1‐treated mice rescued animals from severe disease. Moreover, transfer of preactivated CD8⁺CD122⁺ T cells prevented EAE development and significantly reduced IL‐17 secretion. Naïve effector CD4⁺CD25^? T cells cultured with either CD8⁺CD122⁺ T cells from wild‐type mice or IL‐15 transgenic mice displayed lower frequencies of IL‐17A production with lower amounts of IL‐17 in the supernatants when compared with production by effector CD4⁺CD25^? T cells cultured alone. Addition of a neutralizing antibody to IL‐10 led to recovery of IL‐17A production in Th17 cultures. Furthermore, coculture of CD8⁺CD122⁺ T cells with effector CD4⁺ T cells inhibited their proliferation significantly, suggesting a regulatory function for IL‐15 dependent CD8⁺CD122⁺ T cells. Taken together, these observations suggest that IL‐15, acting through CD8⁺CD122⁺ T cells, has a negative regulatory role in reducing IL‐17 production and Th17‐mediated EAE inflammation.     

天花粉蛋白合成肽通过诱导CD8抑制性T细胞治疗实验性自身反应性脑脊髓炎

为了探讨天花粉蛋白合成肽(M-Tk)治疗实验性自身反应性脑脊髓炎(EAE)的可能性及其作用机制,应用自身抗原MBP衍生肽MOG35-55免疫C57BL/6小鼠成功地诱发EAE后,以流式细胞术测定小鼠淋巴结细胞及脾细胞T亚群及细胞因子的表达,并通过临床评分观察M-Tk治疗EAE的有效性,包括以HE和LFB染色观察髓鞘病变。结果发现,M-Tk可抑制MOG35-55特异性T细胞的增殖反应,选择性诱导CD8+CD28-T调节细胞的扩增,明显提高IL-10的分泌和降低IFN-γ的产生,并有效改善EAE小鼠神经功能评分。采用M-Tk诱导的CD8 T细胞作体内输注可取得相似甚至更好的治疗效果(P>0.01)。提示M-Tk在体内诱导产生CD8+CD28-抑制性T细胞并上调IL-10分泌降低IFN-γ产生,是对EAE取得疗效的关键。