Indoles mitigate the development of experimental autoimmune encephalomyelitis by induction of reciprocal differentiation of regulatory T cells and Th17 cells

Background and Purpose

Dietary indole derivatives, indole-3-carbinol (I3C) and diindolylmethane (DIM), possess anti-cancer properties and exhibit the characteristics of aryl hydrocarbon receptor (AhR) ligands. Because AhR activation has recently been shown to regulate T cell differentiation, we tested the hypothesis that I3C and DIM may mediate anti-inflammatory properties by promoting regulatory T cell (T-regs) differentiation while inhibiting Th17 cells.

Experimental Approach

We investigated the therapeutic efficacy of I3C and DIM against experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis (MS). The efficacy was evaluated based on clinical scores of paralysis, histopathology, serum cytokines and infiltration of T cells in the CNS. We next studied the mechanism of induction of T cells against myelin oligodendrocyte glycoprotein (MOG_35–55) peptide, both in vivo and in vitro, specifically investigating the differentiation of T-regs and Th17 cells, and determined if indoles were acting through AhR.

Key Results

Pretreatment of EAE mice with I3C or DIM completely prevented the clinical symptoms and cellular infiltration into the CNS. Also, post-treatment of EAE with I3C or DIM proved highly effective in curtailing the overall severity of the disease. In addition, I3C or DIM promoted the generation of T-regs, while down-regulating the induction of MOG-specific Th17 cells. The regulation of FoxP3 induction and suppression of Th17 cells by indoles in vivo and in vitro were found to be AhR-dependent.

Conclusions and Implications

Together, our studies demonstrate for the first time that I3C and DIM may serve as novel therapeutics to suppress neuroinflammation seen during MS through activation of AhR.     

Pre-differentiated Th1 and Th17 effector T cells in autoimmune gastritis: Ag-specific regulatory T cells are more potent suppressors than polyclonal regulatory T cells

Naïve antigen-specific CD4⁺ T cells (TxA23) induce autoimmune gastritis when transferred into BALB/c nu/nu mice. Transfer of in vitro pre-differentiated Th1 or Th17 TxA23 effector T cells into BALB/c nu/nu recipients induces distinct histological patterns of disease. We have previously shown that co-transfer of polyclonal naturally occurring Treg (nTreg) suppressed development of Th1-, but not Th17-mediated disease. Therefore, we analysed the suppressive capacity of different types of Treg to suppress Th1- and Th17-mediated autoimmune gastritis. We compared nTreg with polyclonal TGFβ-induced WT Treg (iTreg) or TGFβ-induced antigen-specific TxA23 iTreg in co-transfer experiments with Th1 or Th17 TxA23 effector T cells. 6 weeks after transfer in vitro pre-differentiated TxA23 Th1 and Th17 effector cells induced destructive gastritis. Th1-mediated disease was prevented by co-transfer of nTreg and also antigen-specific iTreg, whereas WT iTreg did not show an effect. However, Th17-mediated disease was only suppressed by antigen-specific iTreg. Pre-activation of nTreg in vitro prior to transfer did not increase their suppressive activity in Th17-mediated gastritis. Thus, antigen-specific iTreg are potent suppressors of autoimmune gastritis induced by both, fully differentiated Th1 and Th17 effector cells.     

Rapamycin inhibits differentiation of Th17 cells and promotes generation of FoxP3+ T regulatory cells

Reciprocal differentiation of immunosuppressive CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) and proinflammatory IL-17-producing cells (Th17) from naïve CD4 cells is contingent upon the cytokine environment. Using MACS-purified CD4 cells, we found that rapamycin and cyclosporine A (CsA) potently inhibited the TGFβ and IL-6-induced generation of IL-17-producing cells. Intriguingly, rapamycin promoted, while CsA markedly inhibited, TGFβ-mediated generation of Tregs. The aforementioned effects of rapamycin and CsA were also observed for Flow-sorted CD4⁺CD25⁻ T cells, indicating that the effect of these two immunosuppressive agents was based on their action on de novo generation of Tregs and Th17 cells from naïve CD4 cells. Our observation suggests a distinct mode of immunosuppressive action and tolerance induction by rapamycin and CsA. The capacity of rapamycin to generate immunosuppressive Tregs and to suppress differentiation of pathogenic Th17 cells furthers our understanding of the basis for the therapeutic immunosuppressive effects of rapamycin in patients with autoimmune diseases and allo-transplantation reactions.     

自身免疫性甲状腺炎小鼠发病中调节性T细胞、Th17细胞的变化

目的观察高碘诱发自身免疫性甲状腺炎(AIT)动物模型中CD4+CD25+调节性T细胞、Th17细胞的变化。方法选取NOD.H-2h4雌鼠饮0.005%碘化钠水,HE染色观察淋巴细胞浸润情况并进行甲状腺炎症程度评分;测定血清甲状腺球蛋白抗体(TgA b)水平;免疫荧光染色流式细胞仪分析CD4+CD25+Foxp3+调节性T细胞、Th17细胞比例的变化;实时定量RT-PCR检测Foxp3 mRNA、IL-17 mRNA、RORγt mRNA表达水平。结果 NOD.H-2h4小鼠高碘饮水后,甲状腺炎的发生率明显高于对照组,甲状腺组织有不同程度的淋巴细胞浸润,甲状腺相对重量及血清TgA b水平均较对照组小鼠明显升高(P<0.05)。脾细胞中CD4+CD25+Foxp3+调节性T细胞所占比例和Foxp3 mRNA表达量均较对照组明显降低(P<0.01);脾细胞中Th17细胞所占比例和IL-17 mRNA表达量、RORγt mRNA表达水平均较对照组明显升高(P<0.05)。结论 NOD.H-2h4小鼠在高碘诱导发生甲状腺炎时,脾脏CD4+CD25+调节性T细胞比例明显降低,Th17细胞比例明显升高。     

Th17 and regulatory T cell subsets in diseases and clinical application

In the past decade it has been established that regulatory T cells (Tregs) control all immune responses. As the induction and effector mechanisms used by Tregs are being unraveled, it is emerging that a reciprocal population of CD4(+) T lymphocytes exists in the immune system that produces inflammatory cytokine IL-17, and coined "Th17 cells". Th17 cells have been implicated in the pathogenesis of many forms of human disease. The development, function, mechanism of action, and homeostasis of Tregs and Th17 cells, and the reciprocal control between Tregs and Th17 cells were presented at the Second International Conference on Regulatory T Cells and Th17 Cells and Clinical Application in Human Diseases in Shanghai on 17-20 July 2010 (China Tregs/Th17 2010). In this Special Issue of International Immunopharmacology, several paper submitted to the conference will highlight the biology of Tregs and Th17 cells, and their clinical application in human disease.     

Ursolic acid ameliorates autoimmune arthritis via suppression of Th17 and B cell differentiation

Aim:

Ursolic acid (UA) is a pentacyclic triterpenoid found in most plant species, which has been shown anti-inflammatory and anti-oxidative activities. In this study, we examined the effects of UA on collagen-induced arthritis (CIA) in mice, and to identify the mechanisms underlying the effects.

Methods:

CIA was induced in mice. Two weeks later, the mice were treated with UA (150 mg/kg, ip, 3 times per week) for 4 weeks. The expression of cytokines and oxidative stress markers in joint tissues was measured with immunohistochemistry. The numbers of CD4⁺IL-17⁺, CD4⁺CD25⁺Foxp3⁺ and pSTAT3 cells in spleens were determined using confocal immunostaining or flowcytometric analyses. Serum antibody levels and B cell-associated marker mRNAs were analyzed with ELISAs and qRT-PCR, respectively. CD4⁺ T cells and CD19⁺ B cells were purified from mice spleens for in vitro studies.

Results:

UA treatment significantly reduced the incidence and severity of CIA-induced arthritis, accompanied by decreased expression of proinflammatory cytokines (TNF-α, IL-1β, IL-6, IL-21 and IL-17) and oxidative stress markers (nitrotyrosine and iNOS) in arthritic joints. In CIA mice, UA treatment significantly decreased the number of Th17 cells, while increased the number of Treg cells in the spleens, which was consistent with decreased expression of pSTAT3, along with IL-17 and RORγt in the splenocytes. In addition, UA treatment significantly reduced the serum CII-specific IgG levels in CIA mice. The inhibitory effects of UA on Th17 cells were confirmed in an in vitro model of Th17 differentiation. Furthermore, UA dose-dependently suppressed the expression of B cell-associated markers Bcl-6, Blimp1 and AID mRNAs in purified CD19⁺ B cells pretreated with IL-21 or LPS in vitro.

Conclusion:

UA treatment significantly ameliorates CIA in mice via suppression of Th17 and differentiation. By targeting pathogenic Th17 cells and autoantibody production, UA may be useful for the treatment of autoimmune arthritis and other Th17-related diseases.     

Immunoregulatory Cordyceps sinensis increases regulatory T cells to Th17 cell ratio and delays diabetes in NOD mice

Cordyceps sinensis (CS) is a parasitic fungus, and it has been used widely in traditional Chinese medicines (TCM) for centuries. Many studies have shown that CS has immunoregulatory activity in many disease models, but the underlying mechanism remains elusive. We studied whether CS could suppress the onset of diabetes by altering T lymphocyte subsets in non-obese diabetic (NOD) mice. We found that the onset of type1 diabetes in NOD mice was associated with an imbalance of CD4⁺CD25⁺FoxP3⁺ regulatory T (Treg) cells and IL-17 producing Th17 cells. Oral administration of CS resulted in reduction in the overall incidence of diabetes, and this was due to an increase in the ratio of Treg cells to Th17 in the spleen and pancreatic lymph nodes (PLNs). Taken together, these data imply that CS is able to modulate Treg to Th17 cell ratio in vivo, thus contributing to the inhibition of diabetes.     

类风湿关节炎患者外周血Th1、Th2和Th17细胞亚群表达比例变化的特点

目的研究类风湿关节炎（rheumatoidarthritis,RA）患者外周血中Th1、Th2和Th17细胞亚群表达比例变化,分析RA免疫学发病机制。方法运用流式细胞术检测RA患者及对照组外周血单个核细胞（PBMCs）中Th1、Th2、Th17细胞表达比例。并用酶联免疫吸附法检测RA患者及对照组血清中IFN-γ、IL-4、IL-17的浓度。结果RA患者CD4＋T细胞明显高于健康对照组（P〈0．05）;活动期RA患者Th1、Th17及其分泌的细胞因子含量明显高于健康对照组（P〈0．05）;非活动期RA患者Th17也明显高于健康对照组（P〈0．05）。结论类风湿关节炎Th1、Th2、Th17平衡改变与其疾病的活动度有关,处于活动期Th1、Th17反应增强,非活动期Th1、Th2、Th17趋于平衡。通过调节Th1、Th2、Th17平衡,有可能为RA的治疗提供一条新思路。     

Dual effect of T helper cell 17 (Th17) and regulatory T cell (Treg) in liver pathological process: From occurrence to end stage of disease

Liver disease is a complicated pathological status with acute or chronic progressions, causing a series of damages to liver and massive burden to public health and society. Th17 and Treg, two subsets of CD4⁺ T helper cells, seem to keep a subtle balance in the maintenance of organic immune homeostasis including liver. The dysfunction of Th17/Treg balance in liver has been proved associated with hepatic injury and disease. Herein, we summarized the research advance of Th17 and Treg cells in different phenotypes of liver diseases in the past decade. It is known to all that hepatic diseases start from stimulations or infections like virus, autoimmune, alcohol and so on in the early stage, which would cause inflammation. With the disease consistently existed, severe outcomes like cirrhosis and hepatocellular carcinoma appear finally. In conclusion, it is found that Th17 and Treg cells serve as an important role in the immune response imbalance of liver diseases from the beginning to the end stage. However, the effect of these two subsets of CD4⁺ T helper cells is not a stereotype. Pathological role which exacerbates the disease and protective character which inhibits damage to liver are co-existed in the effect of Th17 and Treg cells. Still, more studies should be carried out to enrich the understandings of liver disease and Th17/Treg immune balance in the future.     

CD4+CD25+调节性T细胞及Th17细胞与Graves病机制研究

目的：探讨CD4+CD25+调节性T细胞以及Th17细胞与Graves病的关系。方法：检测GD患者和对照组外周血单个核细胞CD4+CD25+Tregs、Th17细胞的数量,PBMC中TGF-β、IL-10和FoxP3 mRNA的表达水平以及血清TGF-β、IL-10和IL-17水平。结果：GD组外周血Th17数量高于对照组,血清中IL-17水平也高于对照组（P＜0.05）;而CD4+CD25+Tregs的数量低于对照组,而且TGF-β、FoxP3表达水平低于对照组（P＜0.05）;GD组血清中TGF-β、IL-10水平高于对照组（P＜0.05）。结论：CD4+CD25+Tregs数量的减少或功能障碍以及Th17细胞数量的升高,可能参与GD的发病过程。     

系统性红斑狼疮Th17细胞分子调控机制研究进展

系统性红斑狼疮(SLE)是一种典型的多系统炎症性自身免疫性疾病,发病机制复杂。Th17细胞是一种以分泌IL-17为主的CD4⁺T细胞亚群,与肿瘤、感染、过敏以及自身免疫性疾病等相关。Th17细胞生物学特性及其相关的细胞因子与其致病机制密切相关,研究发现Th17细胞在SLE的发生发展中发挥重要作用。归纳近年来Th17细胞的研究进展,探讨其在SLE可能的发病机制,为治疗提供新的依据。     

Neem leaf glycoprotein suppresses regulatory T cell mediated suppression of monocyte/macrophage functions

We have shown that neem leaf glycoprotein (NLGP) inhibits the regulatory T cell (Tregs) induced suppression of tumoricidal functions of CD14(+)CD68(+) monocyte/macrophages (MO/Mφ) from human peripheral blood. Cytotoxic efficacy of MO/Mφ toward macrophage sensitive cells, U937, is decreased in presence of Tregs (induced), however, it was increased further by supplementation of NLGP in culture. Associated Treg mediated inhibition of perforin/granzyme B expression and nitric oxide release from MO/Mφ was normalized by NLGP. Altered status of signature cytokines, like, IL-12, IL-10, IL-6, TNFα from MO/Mφ under influence of Tregs is also rectified by NLGP. Tregs significantly enhanced the expression of altered marker, mannose receptor (CD206) on CD68(+) cells that was downregulated upon NLGP exposure. In addition to tumoricidal functions, antigen presenting ability of MO/Mφ is hampered by Treg induced downregulation of CD80, CD86 and HLA-ABC. NLGP upregulated these molecules in MO/Mφ even in the presence of Tregs. Treg mediated inhibition of MO/Mφ chemotaxis in contact dependent manner was also normalized partially by NLGP, where participation of CCR5 was documented. Overall results suggest that Treg influenced pro-tumor MO/Mφ functions are rectified in a significant extent by NLGP to create an anti-tumor immune environment.     

血清MK和Th17/调节性T细胞对类风湿关节炎患者病情发生发展的影响

目的观察辅助性T细胞17(Th17)/调节性T细胞(Terg)免疫失衡在来风湿关节炎患者发病时的作用,探讨血清中期因子(MK)和Th17/调节性T细胞对类风湿关节炎患者病情发生发展的影响。方法选取2013年3月~2016年1月在我院接受治疗的类风湿关节炎患者58例,分为活动组和非活动组,DAS28≤3.2为非活动组,DAS28>3.2为活动组,另将30例健康患者作为对照组,活动组患者32例,非活动组患者26例,采用酶联免疫吸附法对3组患者的血清MK、白介素-17(IL-17)、白介素-6(IL-6)、白介素-23(IL-23)水平进行检测,利用流式细胞术检测调节性T细胞和外周血Th17细胞的比例变化,并对其比例变化和血清MK、IL-17、IL-6、IL-23的水平进行比较。结果活动组Th17、调节性T细胞比例分别与非活动组相比较差异具有统计学意义(P<0.05),活动组与非活动组的Th17、调节性T细胞比例分别与对照组相比较异具有统计学意义(P<0.05)。活动组与非活动组的IL-17、IL-6和IL-23水平分别与对照组相比较异具有统计学意义(P<0.05)。血清MK与Th17、T细胞的百分比和平衡相关的相关性结果显示,Th17和Treg均与血清MK无关,P>0.05,而与STAT5成负相关(P<0.05)。结论类风湿性关节炎患者的血清MK上升,且类风湿性关节炎患者Th17/Terg平衡中Th17相对增高,而Terg会相对降低,MK的上升和Th17/Terg平衡失调都促进了RA的发生发展。     

类风湿关节炎患者Th1、Th17细胞的表达

目的 分析类风湿关节炎(RA)患者外周血和关节滑液中Th极化细胞(Th1、Th17细胞)的表达.方法 流式细胞技术测定RA患者外周血、关节滑液中CD4T细胞上 IFN-γ~+ IL-17~- (Th1)、IL-17~1 IFN-γ(Th17)的表达,并与健康人比较,分析疾病活动指数(DAS28)和Th极化细胞表达量、Th1/Th17比值之间的关系.结果 RA患者关节滑液Th1、Th17细胞的平均百分比与RA外周血、健康人外周血比较,差异有统计学意义(P＜0.01).RA患者关节滑液中Th1细胞、Th1/Th17细胞比值与DAS28指数呈正相关(P＜0.01).结论 在RA疾病部位关节液中是Th1细胞占优势,IL-Th1细胞的表达与疾病活动性密切相关.     

滋肾通络方对胶原性关节炎小鼠Th17/Treg细胞分化及平衡的影响

目的观察滋肾通络方(ZTF)对胶原性关节炎(CIA)小鼠Th17/Treg细胞分化平衡的影响,探讨其治疗类风湿关节炎的机制。方法 DBA/1小鼠随机分为正常对照组、模型组和ZTF组(8.4 g生药·kg~(-1)),造模21 d后每日灌胃给药,连续4周。检测关节炎指数、关节病理改变、血浆IL-10和IL-17水平、外周血和脾脏Th17/Treg细胞比例、CD4~+T细胞IL-10、IL-17 mRNA水平。结果 CIA小鼠踝关节病变明显,血浆IL-17水平、外周血和脾脏Th17细胞比例、CD4~+T细胞IL-17 mRNA表达明显升高;血浆IL-10水平、外周血和脾脏Treg细胞比例、CD4~+T细胞IL-10 mRNA表达明显降低。ZTF能明显减轻CIA小鼠关节病变,降低血浆IL-17水平、外周血和脾脏Th17细胞比例、CD4~+T细胞IL-17 mRNA表达;升高血浆IL-10水平、外周血和脾脏Treg细胞比例、CD4~+T细胞IL-10 mRNA水平。结论 CIA小鼠存在Th17/Treg细胞失平衡,ZTF可调节Th17/Treg细胞比例,重构Th17/Treg细胞平衡,调节相关细胞因子释放,这可能是ZTF治疗类风湿关节炎的机制之一。     

宫颈癌中单核细胞对Th17细胞分化的影响

目的 检测宫颈癌病人外周血中Th17细胞的比例及相关细胞因子白细胞介素（IL）-1β、IL-6的表达水平,探讨宫颈癌外周血中单核细胞对Th17细胞分化的影响及机制。方法 2019年6月至2020年12月在潍坊市人民医院妇科收治的宫颈癌病人（宫颈癌组）39例,对照组（30例）为健康志愿者及因良性疾病在潍坊市人民医院妇科就诊的病人,流式细胞仪检测宫颈癌组、对照组外周血中Th17细胞的比例;实时荧光定量逆转录聚合酶链反应（qRT-PCR）检测宫颈癌病人、对照组外周血中IL-1β、IL-6的表达水平;分离健康志愿者外周血中的初始CD4+T细胞,分别与对照组、宫颈癌组外周血中分离出的单核细胞进行共培养,并分别加入anti-IL-1β、anti-IL-6中和抗体,流式细胞仪检测Th17细胞的比例。结果 宫颈癌组外周血中,Th17细胞比例（3.5±1.4）%、IL-1β 0.457(0.078,1.193)、IL-6 0.094(0.043,0.272)的表达水平明显高于对照组（2.3±1.6）%、0.279(0.017,0.686)、0.038(0.019,0.112)。初始CD4+T细胞与宫颈癌...