Kombucha tea ameliorates experimental autoimmune encephalomyelitis in mouse model of multiple sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for multiple sclerosis (MS), in which an inflammatory demyelination and axonal damage occurs. Kombucha tea is a fermented beverage made from kombucha mushroom, brewed tea, and sugar. In recent years kombucha tea has attracted interest due to its pharmacological properties like antioxidant effects. The aim of the present research was to test the therapeutic effect of kombucha tea in EAE. We induced EAE model in 18 female C57BL/6 mice by inoculation of myelin oligodendrocyte glycoprotein-35-55 (MOG_35-55) in complete Freund’s adjuvant emulsion. Then, in order to ameliorate EAE symptoms, we used kombucha tea. During the course of study clinical evaluation was assessed, and on the day 21 post-immunization, for evaluation of nitric oxide (NO), total antioxidants capacity and tumor necrosis factor-alpha (TNF-α), blood samples were taken from the heart of mice. The mice were sacrificed and brains and cerebellums of mice were removed for histological analysis. Our findings demonstrated that kombucha tea had beneficial effects on EAE by lower incidence, attenuation in the severity, and also a delay in the onset of disease. Histological analysis showed that inflammatory criteria including the number of infiltrated immune cells and plaques as well as demyelination in kombucha tea dosed mice were significantly lower than the control group. Also, in comparison with control mice, the serum levels of NO and TNF-α in kombucha tea-treated mice were significantly decreased. Kombucha tea with its potential therapeutic effects and immunomodulatory properties might be proposed, after additional necessary tests and trials, for treatment of MS.     

Th1细胞和Th17细胞在多发性硬化和实验性变态反应性脑脊髓炎发生中的作用

多发性硬化(MS)是一种由效应性T细胞介导的中枢神经系统自身免疫性脱髓鞘疾病,其病因和发病机制迄今不明.实验性变态反应性脑脊髓膜炎(EAE)为MS的动物模型.传统观念认为Th1细胞是MS自身免疫反应中主要的效应性T细胞.然而,Th17细胞的发现使Th1细胞的核心作用受到挑战.     

Pathogenic and regulatory roles for B cells in experimental autoimmune encephalomyelitis

A dual role of B cells in experimental autoimmune encephalomyelitis (EAE), the animal model of the human autoimmune disease multiple sclerosis (MS), has been established. In the first role, B cells contribute to the pathogenesis of EAE through the production of anti-myelin antibodies that contribute to demyelination. On the contrary, B cells have also been shown to have protective functions in that they play an essential role in the spontaneous recovery from EAE. In this review, we summarize studies conducted in a number of species demonstrating the conditions under which B cells are pathogenic in EAE. We also discuss the phenotype and anti-inflammatory mechanisms of regulatory B cells.     

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.     

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.     

The effect of mesenchymal stem cells on dynamic changes of T cell subsets in experimental autoimmune uveoretinitis

Mesenchymal stem cells (MSCs) are being explored extensively as a promising treatment for autoimmune diseases. We have recently reported that MSCs could ameliorate experimental autoimmune uveoretinitis (EAU) in rats. In this study, we examined further the effects of MSCs on the dynamics of T cell subsets in both eye and spleen and their cytokine production during the course of EAU. We focused on when and where the MSCs had inhibitory effects on T helper type 1 (Th1) and Th17 cells and how long the inhibitory effect lasted, in order to provide more mechanistic evidence for MSCs on the treatment of uveitis. Compared to the control group, administration of MSCs decreased the production of Th1 and Th17 cytokines significantly, while the production of Th2 and regulatory T cell (T_reg) cytokines [interleukin (IL)‐10 and transforming growth factor (TGF)‐β] was elevated during the entire course of EAU. Correspondingly, the dynamic levels of IL‐17 in the aqueous humour (AqH) were reduced in MSC‐treated rats. Moreover, the ratio of Th17/T_reg cells in both spleen and eye was decreased. These results provide powerful evidence that MSCs can regulate negatively both Th1 and Th17 responses and restore the balance of Th17/T_regs in the whole course of EAU, which is important for the regression of the disease.     

Inflammasome activation in multiple sclerosis and experimental autoimmune encephalomyelitis (EAE)

The aptly named inflammasomes are powerful signaling complexes that sense inflammatory signals under a myriad of conditions, including those from infections and endogenous sources. The inflammasomes promote inflammation by maturation and release of the pro‐inflammatory cytokines, IL‐1β and IL‐18. Several inflammasomes have been identified so far, but this review focuses mainly on the NLRP3 inflammasome. By still ill‐defined activation mechanisms, a sensor molecule, NLRP3 (NACHT, LRR and PYD domains‐containing protein 3), responds to danger signals and rapidly recruits ASC (apoptosis‐associated speck‐like protein containing a CARD) and pro‐caspase‐1 to form a large oligomeric signaling platform—the inflammasome. Involvement of the NLRP3 inflammasome in infections, metabolic disorders, autoinflammation, and autoimmunity, underscores its position as a central player in sensing microbial and damage signals and coordinating pro‐inflammatory immune responses. Indeed, evidence in patients with multiple sclerosis (MS) suggests inflammasome activation occurs during disease. Experiments with the mouse model of MS, experimental autoimmune encephalomyelitis (EAE), specifically describe the NLRP3 inflammasome as critical and necessary to disease development. This review discusses recent studies in EAE and MS which describe associations of inflammasome activation with promotion of T cell pathogenicity, infiltration of cells into the central nervous system (CNS) and direct neurodegeneration during EAE and MS.     

Ageing and recurrent episodes of neuroinflammation promote progressive experimental autoimmune encephalomyelitis in Biozzi ABH mice

Current therapies for multiple sclerosis (MS) reduce the frequency of relapses by modulating adaptive immune responses but fail to limit the irreversible neurodegeneration driving progressive disability. Experimental autoimmune encephalomyelitis (EAE) in Biozzi ABH mice recapitulates clinical features of MS including relapsing–remitting episodes and secondary‐progressive disability. To address the contribution of recurrent inflammatory events and ageing as factors that amplify progressive neurological disease, we examined EAE in 8‐ to 12‐week‐old and 12‐month‐old ABH mice. Compared with the relapsing–remitting (RREAE) and secondary progressive (SPEAE) EAE observed in young mice, old mice developed progressive disease from onset (PEAE) associated with pronounced axonal damage and increased numbers of CD3⁺ T cells and microglia/macrophages, but not B cells. Whereas the clinical neurological features of PEAE and SPEAE were comparable, the pathology was distinct. SPEAE was associated with significantly reduced perivascular infiltrates and T‐cell numbers in the central nervous system (CNS) compared with PEAE and the acute phase of RREAE. In contrast to perivascular infiltrates that declined during progression from RREAE into SPEAE, the numbers of microglia clusters remained constant. Similar to what is observed during MS, the microglia clusters emerging during EAE were associated with axonal damage and oligodendrocytes expressing heat‐shock protein B5, but not lymphocytes. Taken together, our data reveal that the course of EAE is dependent on the age of the mice. Younger mice show a relapsing–remitting phase followed by progressive disease, whereas old mice immediately show progression. This indicates that recurrent episodes of inflammation in the CNS, as well as age, contribute to progressive neurological disease.     

Artemisinin therapeutic efficacy in the experimental model of multiple sclerosis

Context: The immune system through T-helper 1 (Th1) and Th17 cells play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), whereas the Th2 responses inhibit myelin degeneration. Artemisinin, as an anti-malaria as its agent, has been used widely in the treatment of malaria, shifts the lymphocyte responses from Th1 to Th2.

Objective: In this study, we have investigated the therapeutic effects of artemisinin on the EAE treatment.

Materials and methods: EAE was induced in the inbred C57BL6 mice. High and low doses of prednisolone and artemisinin were injected daily with the control and test groups, respectively. The spleen and the brain of the mice were removed and used for ELISA and histological studies.

Results: The mean weight of mice was significantly (p value?p value p value?p value?Conclusions: Since, artemisinin can shift the immune responses from Th1 to Th2, therefore, it can be helpful in the treatment of MS after more investigation.     

雷公藤内酯醇治疗实验性自身免疫性脑脊髓炎的免疫调节机制研究

目的:探讨雷公藤内酯醇治疗实验性自身免疫性脑脊髓炎(EAE)的免疫调节机制。方法:用MOG35-55肽段免疫C57/BL6小鼠,建立EAE动物模型。免疫后将小鼠随机分为治疗组和对照组,治疗组在开始发病后每天给予雷公藤内酯醇(100μg/kg)治疗,对照组于同一天开始每天给予相同体积生理盐水,每天观察小鼠临床症状。疾病高峰期处死小鼠,HE染色检测脊髓炎性细胞浸润;ELISA检测血清中细胞因子含量,3H掺入法检测淋巴细胞增殖;FACS检测中枢神经系统中Th1/Th17和Treg细胞数量。结果:雷公藤内酯醇治疗能够缓解EAE发病,减轻中枢神经系统炎性细胞浸润;抑制EAE小鼠MOG特异性T细胞增殖;减少血清中炎症细胞因子含量;减少病灶处CD4+T细胞IFNγ-和IL-17的分泌;上调CD4+T细胞Foxp3的表达。结论:雷公藤内酯醇通过减少致病细胞Th1和Th17的浸润,增加Treg细胞的数量来治疗EAE。     

银杏提取物对实验性自身免疫性脑脊髓炎小鼠的影响

目的:探讨银杏提取物(GBE)对实验性自身免疫性脑脊髓炎(EAE)小鼠炎症脱髓鞘病变的影响。方法:应用髓鞘少突胶质细胞糖蛋白33-55(MOG33-55)配以完全弗氏佐剂(CFA)免疫小鼠,诱发EAE模型。将小鼠分为CFA对照组、EAE模型组和GBE治疗组(每日腹腔注射GBE70mg/kg)。通过神经功能评分、行为学实验以及免疫荧光染色,观察GBE对EAE小鼠的影响。结果:GBE组小鼠各时间段神经功能评分均低于EAE组(P0.05),行为学检测显示发病高峰期falling latency时间较EAE组延长10s;GBE组较EAE组视神经髓鞘碱性蛋白(MBP)表达水平增高,可见MBP阳性髓鞘结构包绕轴突;海马伞矢状切片免疫荧光染色证实GBE组CD11b阳性小胶质细胞较EAE组明显减少,但是GFAP阳性星形胶质细胞数量与EAE组无明显差别。结论:GBE可能通过抑制小胶质细胞激活从而延缓EAE小鼠脱髓鞘进程,提示GBE对多发性硬化具有一定的治疗作用。     

Methylprednisolone induces reversible clinical and pathological remission and loss of lymphocyte reactivity to myelin oligodendrocyte glycoprotein in experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is an animal model of human multiple sclerosis (MS). EAE, induced by immunisation with myelin-associated autoantigens, is characterised by an inflammatory infiltrate in the central nervous system (CNS) associated with axonal degeneration, demyelination and damage. We have recently shown in an experimental mouse model of autoimmune gastritis that methylprednisolone treatment induces a reversible remission of gastritis with regeneration of the gastric mucosa. Here, we examined the effect of oral methylprednisolone on the mouse EAE model of human MS induced by immunisation with myelin oligodendrocyte glycoprotein peptide (MOG_35–55). We examined the clinical scores, CNS pathology and lymphocyte reactivity to MOG_35–55 following treatment and withdrawal of the steroid. Methylprednisolone remitted the clinical signs of EAE and the inflammatory infiltrate in the CNS, accompanied by loss of lymphocyte reactivity to MOG_35–55 peptide. Methylprednisolone withdrawal initiated relapse of the clinical features, a return of the CNS inflammatory infiltrate and lymphocyte reactivity to MOG_35–55 peptide. This is the first study to show that methylprednisolone induced a reversible remission in the clinical and pathological features of EAE in mice accompanied by loss of lymphocyte reactivity to the encephalitogen. This model will be useful for studies directed at a better understanding of mechanisms associated with steroid-induced disease remission, relapse and remyelination and also as an essential adjunct to an overall curative strategy.     

Pathogenic mechanisms and experimental models of multiple sclerosis

Multiple sclerosis (MS) is a devastating autoimmune disease that affects more than 1 million people worldwide and severely compromises motor and sensory function through demyelination and axonal loss. This review covers current therapies, lessons learned from failed clinical trials, genetic susceptibility, key cell types involved, animal models, gene expression, and biomarker information. The current first-line therapies for MS include the type I interferons (IFN-I) and glatiramer acetate (GA) but because of their limited effectiveness new therapeutic modalities are required. Tysabri is an anti very late antigen-4 antibody that antagonizes the migration of multiple cell types and appears more efficacious as compared to the IFNs or GA. Tysabri blocks the transmigration of T cells and monocytes, which indicates that blocking multiple cell types may increase the effectiveness of the therapy. However, this therapy may increase the risk of progressive multifocal leukoencephalopathy. The major cell types hypothesized to be pathogenic include T cells and antigen-presenting cells, including B cells. The correlation of the animal model experimental autoimmune encephalomyelitis (EAE) of MS and its predictive value to determine efficacy in the clinic appears limited. However, all current therapies do demonstrate efficacy in EAE models. There are also examples of mechanisms that have worked in EAE but have failed in the clinic, such as the TNFα antagonists and anti-p40 (a subunit of IL-12 and IL-23). The MS field would benefit if clinical biomarkers were available to monitor clinical efficacy. The etiology of MS remains elusive but additional understanding of mechanisms involved in the pathogenesis of MS may guide us to more effective treatment and management of this autoimmune disease.     

Erythropoietin enhances endogenous haem oxygenase-1 and represses immune responses to ameliorate experimental autoimmune encephalomyelitis

Both erythropoietin (EPO) and haem oxygenase-1 (HO-1), an anti-oxidative stress protein, have proven protective roles in experimental autoimmune encephalomyelitis (EAE), a reliable animal model of multiple sclerosis. In this study, EPO delivered intraperitoneally could reduce disease severity in myelin oligodendrocyte glycoprotein (MOG)–EAE mice. To assess the effect of EPO on endogenous HO-1 in EAE, we investigated expression of HO-1 mRNA by real-time polymerase chain reaction (RT–PCR), protein expression centrally and peripherally by Western blot and immunohistochemistry and mean fluorescence intensity of splenic HO-1 by flow cytometry. A significantly higher expression of HO-1 in both the central nervous system (CNS) and spleen was shown in EPO-treated MOG–EAE mice than in controls.We further examined the immunomodulatory effect of EPO in EAE, and via RT–PCR demonstrated significantly lower expression of interferon-γ, interleukin (IL)-23, IL-6 and IL-17 mRNA, and significantly higher expression of IL-4 and IL-10 mRNA in CNS of EPO-treated MOG–EAE mice than in controls. Using flow cytometry, we also observed a significantly decreased ratio of both T helper type 1 (Th1) and Th17 lymphocyte subsets isolated from CNS and a significantly increased ratio of splenic regulatory CD4 T cells in EPO-treated MOG–EAE mice. In addition, we demonstrated that MOG-specific T cell proliferation was lower in the EPO-treated group than in controls and showed amelioration of EAE by adoptive transfer of splenocytes from EPO-treated MOG–EAE mice. Together, our data show that in EAE, EPO induction of endogenous HO-1 and modulation of adaptive immunity both centrally and peripherally may involve the repression of inflammatory responses.     

地塞米松对实验性自身免疫性脑脊髓炎小鼠髓鞘保护作用的研究

目的:分析地塞米松对实验性自身免疫性脑脊髓炎(EAE)小鼠炎症反应、髓鞘脱失及髓鞘再生的影响,探讨地塞米松治疗多发性硬化的新作用。方法:应用MOG35-55免疫C57BL/6小鼠建立EAE模型。小鼠随机分为正常对照组、EAE组及地塞米松组,观察各组临床症状;采用HE染色、LFB染色、透射电镜扫描及免疫组化染色方法,检测免疫后第13、20、30 d各组小鼠脊髓组织炎症反应、髓鞘脱失及髓鞘再生情况。结果:地塞米松显著降低EAE小鼠发病率、延缓起病时间、减轻疾病严重程度。各个时间点地塞米松组脊髓组织炎性细胞浸润、髓鞘脱失及轴索变性程度较EAE组明显减轻。免疫后第20、30 d,EAE组Olig2阳性细胞数较正常对照组明显增加;免疫后各时间点,地塞米松组Olig2阳性细胞数较正常对照组均明显增加,第13、20 d较EAE组明显增加。结论:地塞米松可增加脊髓组织Olig2表达、促进髓鞘再生,这可能为地塞米松治疗EAE及多发性硬化的效应途径。     

Tracking of myelin‐reactive T cells in experimental autoimmune encephalomyelitis (EAE) animals using small particles of iron oxide and MRI

Myelin‐reactive T cells are responsible for initiating the cascade of autoreactive immune responses leading to the development of multiple sclerosis. For better insights into the disease mechanism, it is of major importance to have knowledge on the sites at which these cells are active during disease progression. Herein, we investigated the feasibility of tracking myelin‐reactive T cells, upon labelled with SPIO particles, in the central nervous system (CNS) of experimental autoimmune encephalomyelitis (EAE) animals by MRI. First, we determined the optimal labelling condition leading to a high particle uptake and minimal SPIO–Poly‐l‐lysine (PLL) aggregate formation using Prussian blue staining and inductively coupled plasma spectroscopy measurements. Results from labelling of myelin reactive T cells with low concentrations of SPIO particles (i.e. 25 µg/ml) combined with different concentrations of PLL (0–1.5 µg/ml) showed that increasing amounts of PLL led to augmented levels of free remnant SPIO‐PLL aggregates. In contrast, a low PLL concentration (i.e. 0.5 µg/ml) combined with high concentrations of SPIO (i.e. 400 µg Fe/ml) led to a high labelling efficiency with minimal amounts of aggregates. Second, the labelled myelin‐reactive T cells were transferred to control rats to induce EAE. At the occurrence of hindlimb paralysis, the SPIO labelled myelin‐reactive T cells were detected in the sacral part of the spinal cord and shown to be highly confined to this region. However, upon transfer in already primed rats, T cells were more widely distributed in the CNS and shown present in the spinal cord as well as in the brain. Our study demonstrates the feasibility of tracking SPIO labelled myelin‐reactive T cells in the spinal cord as well as the brain of EAE rats upon systemic administration. Furthermore, we provide data on the optimal labelling conditions for T cells leading to a high particle uptake and minimal aggregate formation. Copyright © 2010 John Wiley & Sons, Ltd.     

Complement activation and expression during chronic relapsing experimental autoimmune encephalomyelitis in the Biozzi ABH mouse

Chronic relapsing experimental autoimmune encephalomyelitis (crEAE) in mice recapitulates many of the clinical and histopathological features of human multiple sclerosis (MS), making it a preferred model for the disease. In both, adaptive immunity and anti‐myelin T cells responses are thought to be important, while in MS a role for innate immunity and complement has emerged. Here we sought to test whether complement is activated in crEAE and important for disease. Disease was induced in Biozzi ABH mice that were terminated at different stages of the disease to assess complement activation and local complement expression in the central nervous system. Complement activation products were abundant in all spinal cord areas examined in acute disease during relapse and in the progressive phase, but were absent in early disease remission, despite significant residual clinical disease. Local expression of C1q and C3 was increased at all stages of disease, while C9 expression was increased only in acute disease; expression of the complement regulators CD55, complement receptor 1‐related gene/protein y (Crry) and CD59a was reduced at all stages of the disease compared to naive controls. These data show that complement is activated in the central nervous system in the model and suggest that it is a suitable candidate for exploring whether anti‐complement agents might be of benefit in MS.     

Macrophage CD40 signaling drives experimental autoimmune encephalomyelitis

The costimulatory CD40L–CD40 dyad plays a major role in multiple sclerosis (MS). CD40 is highly expressed on MHCII⁺ B cells, dendritic cells and macrophages in human MS lesions. Here we investigated the role of the CD40 downstream signaling intermediates TNF receptor-associated factor 2 (TRAF2) and TRAF6 in MHCII⁺ cells in experimental autoimmune encephalomyelitis (EAE). Both MHCII–CD40–Traf2^−/− and MHCII–CD40–Traf6^−/− mice showed a reduction in clinical signs of EAE and prevented demyelination. However, only MHCII–CD40–Traf6^−/− mice displayed a decrease in myeloid and lymphoid cell infiltration into the CNS that was accompanied by reduced levels of TNF-α, IL-6 and IFN-γ. As CD40–TRAF6 interactions predominantly occur in macrophages, we subjected CD40^flflLysM^cre mice to EAE. This myeloid-specific deletion of CD40 resulted in a significant reduction in EAE severity, reduced CNS inflammation and demyelination. In conclusion, the CD40–TRAF6 signaling pathway in MHCII⁺ cells plays a key role in neuroinflammation and demyelination during EAE. Concomitant with the fact that CD40–TRAF6 interactions are predominant in macrophages, depletion of myeloid CD40 also reduces neuroinflammation. CD40–TRAF6 interactions thus represent a promising therapeutic target for MS. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.