Vasoactive intestinal peptide induces regulatory T cells during experimental autoimmune encephalomyelitis

CD4(+)CD25(+) regulatory T cells (Treg) control the immune response to a variety of antigens, including self-antigens. Several models support the idea of the peripheral generation of CD4(+)CD25(+) Treg from CD4(+)CD25(-) T cells. Little is known about the endogenous factors and mechanisms controlling the peripheral expansion of CD4(+)CD25(+) Treg. In this study we report on the capacity of the vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide, to induce functional Treg in vivo during the development of experimental autoimmune encephalomyelitis (EAE), a multiple sclerosis model. The administration of VIP to EAE mice results in the expansion of the CD4(+)CD25(+), Foxp3-expressing T cells in the periphery and the nervous system, which inhibit encephalitogenic T cell activation. In addition to the increase in the number of CD4(+)CD25(+) Treg, VIP induces more efficient suppressors on a per cell basis. The VIP-generated CD4(+)CD25(+) Treg transfer suppression and significantly ameliorate the progression of the disease.     

Diversification and senescence of Foxp3+ regulatory T cells during experimental autoimmune encephalomyelitis

The fate of Foxp3⁺ regulatory T (Treg) cells responding during autoimmunity is not well defined. We observed a marked elevation in KLRG1⁺ (where KLRG1 stands for killer cell lectin‐like receptor G1) CNS‐infiltrating Treg cells in experimental autoimmune encephalomyelitis (EAE), and assessed their origin and properties. KLRG1⁺ Treg cells showed increased activation marker expression, Foxp3 and CD25 levels, and more rapid cell cycling than KLRG1^? cells. KLRG1^? Treg cells converted into KLRG1⁺ cells and this was increased in autoimmune inflammation. Conversion was unidirectional; KLRG1⁺ Treg cells did not revert to a KLRG1^? state. KLRG1⁺ but notKLRG1^?Treg cells survived poorly, indicative of terminal differentiation. This was associated with diminished BCL2 and increased apoptosis of isolated cells. KLRG1 was also upregulated on iTreg cells after transfer and EAE induction or on iTreg cells developing spontaneously during EAE. KLRG1⁺ Treg cells produced more IL‐10 and had altered effector cytokine production compared with their KLRG1^? counterparts. Despite their differences, KLRG1⁺ and KLRG1^? Treg cells proved similarly potent in suppressing EAE. KLRG1⁺ and KLRG1^? populations were phenotypically heterogeneous, with the extent and pattern of activation marker expression dependent both on cellular location and inflammation. Our results support an extensive diversification of Treg cells during EAE, and associate KLRG1 with altered Treg‐cell function and senescence.     

Epitope-specific immune tolerization ameliorates experimental autoimmune encephalomyelitis

The availability of glatiramer acetate (GA) for inducing immune tolerance is a significant advancement in the treatment of multiple sclerosis (MS). However, a sizable proportion of patients maintain active disease, regardless of treatment. Another approach to induce T-cell tolerance is therefore still an unmet medical need.     

Eotaxin-2 blockade ameliorates experimental autoimmune encephalomyelitis

AIM: To study the effect of blocking the eo-2 pathway on the development and severity of experimental autoimmune encephalomyelitis (EAE). METHODS: We produced mAb directed against eo-2, named D8. MOG35-55 induced-EAE mice were daily intravenously injected with either 25 μg or 100 μg D8, or with vehicle control alone [phosphate-buffered saline (PBS)], starting from day 0 post immunization and were monitored for EAE clinical score (n = 10 in each group). Mice were sacrificed on day 58 and their sera were assessed for the presence of anti-myelin oligodendrocyte glycoprotein (anti-MOG) antibodies autoantibodies, as well as for the profile of pro-inflammatory cytokines and chemokines. Histological analysis of brain sections was performed by hematoxylin and eosin staining. RESULTS: Daily treatment of EAE induced mice with D8 significantly decreased the severity of EAE symptoms. Treatment with both concentrations of D8 ameliorated EAE symptoms compared to PBS treated mice, starting from day 42 post immunization (0.89 ± 0.35 in D8 25 μg and D8 100 μg treated groups vs 2.11 ± 0.38 in the PBS treated group, P = 0.03). A significant improvement in EAE clinical score compared to total IgG treated mice was observed with the higher concentration of D8 (0.81 ± 0.38 in D8 100 μg treated group vs 2.11 ± 0.31 in IgG1 treated group, on day 56 post immunization, P = 0.04). D8 treated mice with EAE did not significantly exhibit lower sera levels of anti-MOG autoantibodies compared to IgG-treated mice. However, they expressed lower sera levels of the pro-inflammatory cytokines: tumor necrosis factor (7.8 ± 0.2 pg/mL in D8 100 μg treated mice vs 19.9 ± 3.4 pg/mL in IgG treated mice, P = 0.005) and interferon-gamma (1.4 ± 0.6 pg/mL in D8 100 μg treated mice vs 3.6 ± 0.4 pg/mL in IgG treated mice, P = 0.02), as well as reduced levels of the chemokine macrophage chemoattractant protein-1 (27.2 ± 3.1 pg/mL in D8 100 μg treated mice vs 63.7 ± 12.3 pg/mL in IgG treated mice, P = 0.03). These findings indicate that blocking the eo-2 pathway in EAE may affect not only eosinophil infiltration into the central nervous system (CNS), but also have an effect on monocytes and T cells, but not humoral, mediated responses. Histological analysis of the brains of D8 treated mice with EAE support that this treatment decreases immune cells infiltrates in the CNS. CONCLUSION: Taken together, these findings suggest a role for eo-2 in EAE pathogenesis and consequentially may support a therapeutic potential of anti-eo-2 neutralizing mAb in multiple sclerosis.     

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.     

滤泡辅助性T 细胞在实验性自身免疫性脑脊髓炎发病过程中的作用

目的：探讨滤泡辅助性T细胞在实验性自身免疫性脑脊髓炎中的作用机制。方法：〖HTSS〗随机将C57BL/6小鼠分为CFA组、EAE组、anti-ICOSL组和control 组。用流式细胞术检测不同时间点Tfh细胞的变化及T细胞的活化情况,石蜡切片染色观察炎细胞浸润、脱髓鞘及生发中心的改变,ELISA法检测髓鞘蛋白特异性抗体的分泌水平。结果： anti-ICOSL治疗组较EAE对照组临床评分低（ P <0.05）,炎症浸润减轻,无脱髓鞘病变,Tfh细胞比例降低,但活性不受影响,不能够形成生发中心,并且髓鞘蛋白特异性抗体的分泌减少。结论：滤泡辅助性T细胞通过与B细胞的相互作用促进生发中心形成,分泌抗神经髓鞘蛋白MOG35-55特异性抗体发挥致病性作用。     

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.     

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.     

Adoptive transfer of all-trans-retinal-induced regulatory T cells ameliorates experimental autoimmune arthritis in an interferon-gamma knockout model

Maintaining an appropriate balance between subsets of CD4⁺ helper T cells and T regulatory cells (Tregs) is a critical process in immune homeostasis and a protective mechanism against autoimmunity and inflammation. To identify the role of vitamin A-related compounds, we investigated the regulation of interleukin (IL)-17-producing helper T cells (Th17 cells) and Tregs treated with all-trans-retinal (retinal). CD4⁺T cells or total cells from the spleens of C57BL/6 mice were stimulated under Treg-polarizing (anti-CD3/CD28 and TGF-β) or Th17-polarizing (anti-CD3/CD28, TGF-β, and IL-6) conditions in the presence or absence of retinal. To analyze their suppressive abilities, retinal-induced Tregs or TGF-β-induced Tregs were co-cultured with responder T cells. Collagen-induced arthritis (CIA) was established in interferon (IFN)-γ knockout mice. On day 13, retinal-induced Tregs were adoptively transferred to mice with established CIA after second immunizations. Compared with TGF-β-induced Treg cells, retinal-induced Tregs showed increased Foxp3 expression and mediated stronger suppressive activity. Under Th17-polarizing conditions, retinal inhibited the production of IL-17 and increased the expression of Foxp3.Retinal-induced Tregs showed therapeutic effects in IFN-γ knockout CIA mice. Thus, we demonstrated that retinal reciprocally regulates Foxp3⁺ Tregs and Th17 cells. These findings suggest that retinal, a vitamin A metabolite, can regulate the balance between pro- and anti-inflammatory immunity. A better understanding of the manipulation of Foxp3 and Tregs may enable the application of this tremendous therapeutic potential in various autoimmune diseases.     

Adoptive transfer of all-trans-retinal-induced regulatory T cells ameliorates experimental autoimmune arthritis in an interferon-gamma knockout model

Maintaining an appropriate balance between subsets of CD4(+) helper T cells and T regulatory cells (Tregs) is a critical process in immune homeostasis and a protective mechanism against autoimmunity and inflammation. To identify the role of vitamin A-related compounds, we investigated the regulation of interleukin (IL)-17-producing helper T cells (Th17 cells) and Tregs treated with all-trans-retinal (retinal). CD4(+)T cells or total cells from the spleens of C57BL/6 mice were stimulated under Treg-polarizing (anti-CD3/CD28 and TGF-β) or Th17-polarizing (anti-CD3/CD28, TGF-β, and IL-6) conditions in the presence or absence of retinal. To analyze their suppressive abilities, retinal-induced Tregs or TGF-β-induced Tregs were co-cultured with responder T cells. Collagen-induced arthritis (CIA) was established in interferon (IFN)-γ knockout mice. On day 13, retinal-induced Tregs were adoptively transferred to mice with established CIA after second immunizations. Compared with TGF-β-induced Treg cells, retinal-induced Tregs showed increased Foxp3 expression and mediated stronger suppressive activity. Under Th17-polarizing conditions, retinal inhibited the production of IL-17 and increased the expression of Foxp3.Retinal-induced Tregs showed therapeutic effects in IFN-γ knockout CIA mice. Thus, we demonstrated that retinal reciprocally regulates Foxp3(+) Tregs and Th17 cells. These findings suggest that retinal, a vitamin A metabolite, can regulate the balance between pro- and anti-inflammatory immunity. A better understanding of the manipulation of Foxp3 and Tregs may enable the application of this tremendous therapeutic potential in various autoimmune diseases.     

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。     

Suppressor T cells prevent experimental autoimmune encephalomyelitis in mice.

Immune suppression (immunoprotection) in experimental autoimmune encephalomyelitis (EAE) was studied in (SJL X BALB/c)F1 mice using inocular of mouse spinal cord homogenate (MSCH), or mouse basic protein of myelin (M-BPM), in Freund's incomplete adjuvant (FIA). Such immunization specifically recruited lymphoid cells which markedly suppressed the capacity of effector lymph node cells from appropriately immunized syngeneic mice to transfer adoptively EAE. Suppression was demonstrable with transfer of bone marrow and spleen cells, but not with lymph nodes or thymus cells. Adoptively transferred suppression was maximal when cells were injected 9-30 days after the suppressive injection. Inhibition of EAE by suppressor cells was specific for the relavant antigen BPM, and required viable cells. Treatment of cells with anti-Thy-1 serum before transfer abolished their suppressor activity. After adoptive transfer of suppressor cells into syngeneic recipients subsequently immunized for EAE, there was inhibition of EAE and reduced cell-mediated immune response to BPM as judged by macrophage migration inhibition assays. Hence, in mice at least, immuno-protection against EAE is explicable by recruitment of suppressor T lymphocytes with the dual capacities of inhibiting development of effector T cells after antigenic stimulation, and of blocking their damaging effects on the antigen in the central nervous system.     

T细胞定向迁移诱导实验性自身免疫性脑脊髓炎小鼠胸腺萎缩

目的 初步探讨实验性自身免疫性脑脊髓炎(EAE)小鼠胸腺萎缩的机制.方法 髓鞘少突胶质细胞糖蛋白(MOG)免疫C57BL/6小鼠诱导EAE,卵清白蛋白(OVA)免疫的小鼠作为对照;不同时间点计数胸腺、脾脏、淋巴结细胞总数,检测脾脏中胸腺来源细胞及中枢神经系统(CNS)浸润细胞.结果 MOG肽成功诱导EAE动物模型,小鼠出现典型的肢体运动功能障碍,脊髓可见大量炎性细胞浸润;MOG和OVA免疫均诱导胸腺细胞增加,第5天达到高峰,随后逐渐下降;EAE发病后胸腺细胞迅速减少,发病高峰期几乎完全消失,胸腺严重萎缩;MOG和OVA免疫后脾脏和淋巴结细胞总数持续升高,新近胸腺来源的T细胞增加尤其明显;EAE发病后脾脏T细胞总数减少,CNS浸润淋巴细胞总数增加.结论 大量T细胞在胸腺发育成熟并释放到外周,进而定向迁移至CNS诱导EAE是胸腺萎缩的主要原因.     

Oestrogen-mediated protection of experimental autoimmune encephalomyelitis in the absence of Foxp3+ regulatory T cells implicates compensatory pathways including regulatory B cells

Oestrogen (17β‐oestradiol, E₂) is a highly effective treatment for experimental autoimmune encephalomyelitis (EAE) that may potentiate Foxp3⁺ regulatory T (Treg) cells, which in turn limit the expansion of encephalitogenic T‐cell specificities. To determine if Treg cells constitute the major non‐redundant protective pathway for E₂, we evaluated E₂ protection of EAE after targeted deletion of Foxp3 expression in Foxp3‐DTR mice. Unexpectedly, E₂‐treated Foxp3‐deficient mice were completely protected against clinical and histological myelin oligodendrocyte glycoprotein (MOG)‐35‐55 peptide‐induced EAE before succumbing to diphtheria toxin‐induced mortality. This finding indicated the presence of alternative E₂‐dependent EAE‐protective pathways that could compensate for the lack of Treg cells. Further investigation revealed that E₂ treatment inhibited proliferation and expression of CCL2 and CXCL2, but enhanced secretion of interleukin‐10 (IL‐10) and IL‐13 by MOG‐35‐55‐specific spleen cells. These changes occurred concomitantly with increased expression of several chemokines and receptors, including CXCL13 and CXCR5, and the negative co‐activation molecules, PD‐L1 and B7.2, by B cells and dendritic cells. Furthermore, E₂ treatment resulted in higher percentages of spleen and lymph node T cells expressing IL‐17, interferon‐γ and tumour necrosis factor‐α, but with lower expression of CCR6, suggesting sequestration of MOG‐35‐55 peptide‐specific T cells in peripheral immune organs. Taken together, these data suggest that E₂‐induced mechanisms that provide protection against EAE in the absence of Foxp3⁺ Treg cells include induction of regulatory B cells and peripheral sequestration of encephalitogenic T cells.     

Augmenting DAF levels in vivo ameliorates experimental autoimmune encephalomyelitis

Recent studies in experimental autoimmune encephalomyelitis (EAE) have found that CNS injury in Daf1^−/− mice is much greater than in wild types (WTs), suggesting that upregulating DAF levels in vivo might ameliorate disease. To test this, we generated a Daf1 transgenic (Tg) mouse which had elevated DAF levels on its cell surfaces. In by-stand C3b uptake assays, Daf1 Tg mouse erythrocytes took up less C3b on their surfaces than WT erythrocytes. When co-cultured with OT-II CD4⁺ T cells together with OVA_323–339 peptide, Daf1 Tg mouse bone marrow derived dendritic cells (BM-DCs) produced less C5a and C3a than WT BM-DCs and stimulated a lesser T cell response. In MOG_35–55 immunization induced EAE model, Daf1 Tg mice exhibited delayed disease onset and decreased clinical scores compared to WTs. Histological analyses showed that there were less inflammation and demyelination in spinal cords in Daf1 Tg mice than those in WTs. In accordance with these results, Daf1 Tg mice had decreased MOG_35–55 specific Th1 and Th17 responses. These data provide further evidence that DAF suppresses autoreactive T cell responses in EAE, and indicate that augmenting its expression levels could be effective therapeutically in treating multiple sclerosis as well as other T cell mediated diseases.     

Edaravone, a free radical scavenger, ameliorates experimental autoimmune encephalomyelitis

Reactive oxygen species (ROS) are implicated in the pathogenesis of multiple sclerosis (MS) and its murine model experimental autoimmune encephalomyelitis (EAE). The effect of edaravone, a free radical scavenger, on EAE was investigated in this study. Treatment with edaravone significantly ameliorated the clinical severity of EAE, and a reduced infiltration of lymphocytes was observed based on a histological analysis. The expression of inducible NO synthase (iNOS) in the spinal cords appeared to be reduced by the treatment with edaravone and this effect was confirmed in vitro. A reduction of both the cellular infiltration and the expression of iNOS may therefore underlie the mechanisms of the beneficial effect of edaravone on EAE.     

Cytokine secretion by deltagamma and alphabeta T cells in monophasic experimental autoimmune encephalomyelitis

Mononuclear cells were isolated from the central nervous system (CNS), lymph nodes (LN), spleen and blood, over the course of murine monophasic experimental autoimmune encephalomyelitis (EAE). Individual cytokine secreting T cells were enumerated. IL-2-secreting alphabeta T cells were numerous at all sites at disease onset. By disease peak their numbers had fallen profoundly; they remained low thereafter. IL-2 secreting gammadelta T cells were rare throughout. IFN-gamma-secreting cells were plentiful at all sites at disease onset. gammadelta T cells comprised 7% of total and 20% of IFN-gamma-secreting CNS-derived cells at disease onset; values at disease peak were 12 and 40% respectively. IL-4-secreting alphabeta T cells were rare in the CNS and LN throughout and did not increase in the spleen from baseline values. In contrast, splenic IL-4-secreting gammadelta T cells had increased to four-fold baseline values at disease onset and seven-fold at disease peak. Recovery from EAE is associated with a global inhibition of IL-2-secreting alphabeta T cells and to a lesser extent with IFN-gamma-secreting alphabeta and gammadelta T cells, whereas IL-4-secreting gammadelta T cells increase in the spleen as disease evolves.     

Inhibition of glial cell activation ameliorates the severity of experimental autoimmune encephalomyelitis

Activated microglia and astrocytes have been implicated in the course of multiple sclerosis (MS) and its animal model: experimental autoimmune encephalomyelitis (EAE). MW01-5-188WH is a novel drug that selectively inhibits glial activation in the central nervous system (CNS). We report here that MW01-5-188WH is effective to ameliorate the severity of myelin oligodendrocyte glycoprotein (MOG)-induced EAE. Daily oral administration of MW01-5-188WH at 5mg/kg body weight reduced the clinical scores of EAE mice while having no influence on the disease incidence or animal mortality. Pathological examination revealed reduced numbers of microglia and astrocytes in the spinal cord of MW01-5-188WH-treated EAE mice. Moreover, MW01-5-188WH suppressed the release of key chemokines, which are involved in MS pathology, from cultured microglia and astrocytes. Taken together, our results indicate that treatments that suppress the activation of microglia and astrocytes should be pursued in future research for their potential as avenues for the treatment of MS.