慢性阻塞性肺病稳定期患者外周血Th17/Treg失衡情况及与预后的关系

目的：观察慢性阻塞性肺病（chronic obstructive pulmonary disease,COPD）稳定期患者外周血中Th17与Treg细胞的水平.方法：选取本院2010年1月-2012年12月间确诊的40例COPD稳定期患者,选取同期40例健康体检健康人群作为对照组,收集两组人群的外周血后采用流式细胞术检测各个人群Th17与Treg细胞亚群的比例,采用ELISA法检测血清中的IL-17、TGF-β水平,采用RT-PCR检测转录因子RORγT与Foxp3 mRNA水平.结果：COPD稳定期患者外周血中Th17比例为（3.14±0.31）％,显著高于对照组人群的（1.23±0.24）％,差异有统计学意义（P＜0.05）;COPD稳定期患者外周血中Treg比例为（6.27±0.32）％,显著高于对照组人群的（3.43±0.32）％,差异有统计学意义（P＜0.01）;COPD稳定期患者外周血中IL-17水平为（89.36±4.13） pg/mL,显著高于对照组的（30.35±2.53） pg/mL,差异有统计学意义（P＜0.05）;COPD稳定期患者外周血中TGF-β水平为（681.45±41.65） pg/mL,显著高于对照组人群的（519.36±30.43） pg/mL,差异有统计学意义（P＜0.01）;COPD稳定期患者外周血转录因子Foxp3与RORγT mRNA水平均显著高于对照组人群,差异有统计学意义（均P＜0.01）.COPD稳定期患者Th17、Treg比例高水平的住院天数和病死率均高于对应低水平亚组（P＜0.05）.结论：Th17、Treg细胞及IL-17、TGF-β水平在COPD稳定期患者外周血表达增加,且转录因子Foxp3与RORγT mRNA水平表达增加,提示其与COPD患者的预后有关,其外周血中Th17与Treg细胞的不平衡可能参与了COPD发病和病程的进展.     

鼻敏片对寒证变应性鼻炎豚鼠辅助性T细胞17/调节性T细胞平衡的影响

目的:探讨鼻敏片对寒证变应性鼻炎(allergic rhinitis,AR)豚鼠辅助性T细胞17(Th17)/调节性T细胞(Treg)表达水平的影响及其作用机制.方法:将40只雄性豚鼠随机分为空白组、模型组、鼻敏片组和氯雷他定片组,以复方凉药+卵清蛋白致敏法建立寒证AR模型后给予相应的药物干预,治疗15 d后,取血清和...     

外源性LTB4对CIA小鼠Treg/Th17脾细胞分化的作用

目的探讨外源性白三烯B4(LTB4)对胶原诱导型关节炎(collagen-induced arthritis,CIA)小鼠脾细胞调节性T细胞(Treg)和Th17细胞分化的调节,进一步阐明LTB4在类风湿关节炎(RA)发病中的作用机制。方法建立CIA小鼠模型,取造模d28的脾细胞,体外实验分析外源性LTB4对Treg和Th17细胞分化的影响。应用流式细胞术检测CD4+CD25+Foxp3+细胞的数量,荧光定量PCR技术检测调控Treg和Th17细胞分化的特异性转录因子Foxp3和RORγt的mRNA的表达,酶联免疫吸附(ELISA)方法检测培养细胞上清IL-17的含量。结果成功建立CIA小鼠模型;造模d28分离小鼠脾细胞,体外培养加入鸡Ⅱ型胶原(CⅡ)共同孵育,随着LTB4浓度增加(0.01、0.1、1μmol·L-1),CD4+CD25+Foxp3+细胞数量相应减少,Foxp3 mRNA的表达相应降低;相反,IL-17的含量相应增加,RORγt mRNA的表达相应升高。结论LTB4抑制CIA模型脾细胞Treg细胞的分化,促进Th17细胞的分化,提示LTB4在CIA发病过程中具有一定的促炎活性。     

Mangiferin corrects the imbalance of Th17/Treg cells in mice with TNBS-induced colitis

In the previous study, 80% ethanol extract of the rhizome mixture of Anemarrhena asphodeloides and Coptidis chinensis (AC) and its main constituent mangiferin improved TNBS-induced colitis in mice by inhibiting macrophage activation related to the innate immunity. In the preliminary study, we found that AC could inhibit Th17 cell differentiation in mice with TNBS-induced colitis. Therefore, we investigated whether AC and it main constituent mangiferin are capable of inhibiting inflammation by regulating T cell differentiation related to the adaptive immunity in vitro and in vivo. AC and mangiferin potently suppressed colon shortening and myeloperoxidase activity in mice with TNBS-induced colitis. They also suppressed TNBS-induced Th17 cell differentiation and IL-17 expression, but increased TNBS-suppressed Treg cell differentiation and IL-10 expression. Moreover, AC and mangiferin strongly inhibited the expression of TNF-α and IL-17, as well as the activation of NF-κB. Furthermore, mangiferin potently inhibited the differentiation of splenocytes into Th7 cells and increased the differentiation into Treg cells in vitro. Mangiferin also inhibited RORγt and IL-17 expression and STAT3 activation in splenocytes and induced Foxp3 and IL-10 expression and STAT5 activation. Based on these findings, mangiferin may ameliorate colitis by the restoration of disturbed Th17/Treg cells and inhibition of macrophage activation.     

Butyrate inhibit collagen-induced arthritis via Treg/IL-10/Th17 axis

Rheumatoid arthritis (RA) is a chronic autoimmune disorder demanding the development of novel therapeutic strategy. Butyrate is a functional short-chain fatty acid produced by the anaerobic intestinal microbiota. This study aimed to investigate the attenuation of butyrate on RA. The collagen-induced arthritis (CIA) mouse model was established and butyrate was administered in drinking water along with the collagen immunization. The histopathological features, clinical score, paw swelling, as well as the production of pro-inflammatory cytokines including interleukin (IL)-1β, IL-6 and IL-17A were measured to determine the amelioration of butyrate on arthritis. The differentiation of Treg cells and Th17 cells in the splenic cells was assessed by flow cytometry. The expression of Foxp3, IL-10, Rorγt and IL-17A were detected by RT-PCR and FACS immunostaining. Anti-IL10R antibody was used in the CIA and CD4⁺ cell cultures to mediate the effects of butyrate. Butyrate significantly inhibited expressions of IL-1β, IL-6 and IL-17A, but promoted the expression of IL-10. Butyrate also increased systematical Treg cells and reduced Th17 cells. Mechanism study revealed that butyrate directly enhanced the polarization of Treg cells but not Th17 cells. All effects of butyrate on RA were inversed by the co-administered anti-IL10R antibody. This study showed that butyrate administration inhibited arthritis in CIA mice model, suppressed the expression of inflammatory cytokines. The modulation may be mediated the differentiation of CD4 T cells towards Treg cells, which produce anti-inflammatory cytokine IL-10, and thus influenced the function of Th17 cells.     

Thymol as a reciprocal regulator of T cell differentiation: Promotion of regulatory T cells and suppression of Th1/Th17 cells

Regulatory T cells (Tregs) are critical for maintaining immune response and enhancing their differentiation has therapeutic implications for autoimmune diseases. In this study, we investigated the effects of thymol a well-known monoterpene from Thyme on differentiation and function of Tregs. In vitro generation of Tregs from purified naïve CD4⁺CD25⁻ T cells in the presence of thymol was carried out. Suppressor activity of generated Tregs was examined by changes in the proliferation of CFSE-labeled conventional T cells. Thymol promotes differentiation of naïve CD4⁺CD25⁻ T cells to CD4⁺CD25⁺Foxp3⁺ Tregs [66.9–71.8% vs. control (47%)] and increased intensity of Foxp3 expression on Tregs (p < 0.01). In functional assay, an increased immune suppression by thymol-induced Tregs (≈2.5 times of untreated Tregs) was detected. For in vivo study, thymol was intraperitoneally administered to ovalbumin (Ova)-immunized mice. Flow cytometry assessment of spleens from thymol-treated Ova-immunized mice showed increased number of CD4⁺ Foxp3⁺ Tregs (>8%, p < 0.01(and decreased levels of CD4⁺T-bet⁺ Th1 and CD4⁺RORγt⁺ Th17 cells resulted in significant decreased Th1/Treg and Th17/Treg ratios. In ex vivo Ova challenge of splenocytes from thymol-treated Ova-immunized mice, similarly higher levels of CD4⁺ Foxp3⁺ Tregs, and also elevated TGF-β expression in CD4⁺Foxp3⁺ population (48.1% vs. 18.9% in untreated Ova-immunized group) and reduced IFN-γ-producing CD4⁺T-bet⁺ T cells and IL-17-producing CD4⁺RORγt⁺ T cells were detected. This led to marked decreased ratios of IFNγ/TGF-β and IL-17/TGF-β expressions. In conclusion, this study revealed thymol as a compound with enhancing effects on Treg differentiation and function, which may have potential benefits in treatment of immune-mediated diseases with Th1/Th17 over-activation.     

地塞米松改善胶原性关节炎大鼠免疫功能与其血药浓度的相关性研究

目的研究地塞米松对胶原诱导性关节炎(Collagen induced arthritis,CIA)大鼠炎症和免疫的调节作用。方法32只Wistar大鼠,分为3组,正常组10只、模型组11只,给药组11只。建立CIA模型后,给药组腹腔注射地塞米松(1 mg/kg),每周3次。连续给药4周后,处死并计算脾脏指数、胸腺指数。采用Real-time PCR方法检测脾淋巴细胞炎症因子IL-1β、IL-6、IL-17、TNF-α,转录因子T-bet、GATA3、RORγT、Foxp3mRNA的表达。HPLC法测大鼠地塞米松血药浓度,并将血药浓度与脾脏指数、Th亚型淋巴细胞亚群特异性转录因子做相关性分析。结果腹腔注射地塞米松能显著缓解CIA足爪肿胀、预防踝关节变形,降低脾脏指数、胸腺指数(P<0.01)。PCR结果显示,地塞米松抑制CIA大鼠脾淋巴细胞中炎症因子,并改善Th1/Th2、Th17/Treg的失衡(P<0.05)。HPLC法检测大鼠地塞米松平均血药浓度为0.1738μg/ml。血药浓度与脾脏指数呈负相关,与T-bet、RORγT呈负相关,与GATA3、Foxp3呈正相关。结论地塞米松对CIA大鼠具有强大的抗炎和免疫调节作用。     

布鲁氏菌病患者外周血树突状细胞、Th1／Th2细胞因子含量的检测及意义

目的：观察布鲁氏菌病患者外周血树突状细胞表型、Th1／Th2细胞含量的检测及意义。方法选取诊治的布鲁氏菌病患者50例为病例组,另选取同期进行健康体检的正常人50例作为正常组。采用Real time-PCR测定2组Th1相关转录因子T细胞表达的T盒（T-bet）、GATA连接蛋白3（GATA-3）、维A酸相关核孤儿受体γt（RORγt）、叉头蛋白3（Foxp3）及Th1／Th2细胞因子肿瘤坏死因子α（TNF-α）、干扰素γ（IFN-γ）、白介素6（IL-6）、白介素10（IL-10）mRNA含量。酶联免疫吸附实验（ELISA）测定TNF-α、IFN-γ、IL-6、IL-10蛋白表达及补体C3、C4含量。流式细胞术测定树突状细胞表型、Th1、Th2及T淋巴细胞亚群（ CD4＋T细胞、CD8＋T细胞、NK细胞）含量。结果病例组外周血Th1细胞相关转录因子T-bet、RORγt、Foxp3及Th1细胞、Th1／Th2、TNF-α、IFN-γ含量较对照组显著降低,Th2细胞及IL-6、IL-10含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;病例组CD8＋3、CD8＋0、CD8＋6阳性的树突状细胞比例、CD4＋T细胞、NK细胞及补体C3、C4含量较对照组显著降低,CD8＋T细胞含量较对照组显著升高,差异有统计学意义（ P ＜0．05）;TNF-α、IFN-γ含量与CD4＋T细胞、NK细胞、C3、C4含量呈正相关性（ r值分别为2．879、3．214、3．255和2．978, P ＜0．05）,与CD8＋T细胞含量呈负相关性（ r值分别为－3．146和－3．011, P ＜0．05）。 IL-6、IL-10含量与CD4＋T细胞、NK细胞、C3、C4含量呈负相关性（ r值分别为－2．124、－2．343、－3．423、－2．789、－2．993、－2．566、－3．758, P ＜0．05）,与CD8＋T细胞含量呈正相关性（ r值分别为3．465、3．129, P ＜0．05）。结论布鲁氏菌病患者外周血成熟树突状细胞数目减少,同时Th1／Th2细胞及相关细胞因子失衡,且与机体天然免疫和细胞免疫功能降低有关,这可能在布鲁氏菌病发生发展过程中发挥重要作用。     

The expression of Foxp3 and ROR gamma t in lung tissues from normal smokers and chronic obstructive pulmonary disease patients

Foxp3- and ROR gamma t-expressing cells are involved in acquired immune responses. The change in Foxp3 and ROR gamma t expression in lung tissue and their role in emphysema has not been studied for COPD patients and normal smokers. In the present study, Foxp3 and ROR gamma t were assessed using real-time quantitative polymerase chain reaction and western blotting, and the expression and distribution of Foxp3, IL-17, IL-23R and CCR6 were measured by immunohistochemistry in peripheral lung tissue (10 smokers with COPD, 10 smokers and 10 nonsmokers with normal lung function). Foxp3 expression was lower and ROR gamma t expression was higher in COPD patients when compared with smokers and nonsmokers (all P values were less than 0.001). The ratios of Foxp3/ROR gamma t mRNA and protein were positively correlated to FEV1%pred and negatively correlated to the mean alveoli area. Foxp3⁺ cell numbers were decreased, while the number of IL-17⁺ cells, IL-23R⁺ cells and CCR6⁺ cells were increased in the lung alveolar walls of COPD patients compared with normal smokers and nonsmokers (all P values were less than 0.001). The IL-17⁺ cell numbers were positively correlated to both CCR6⁺ and IL-23R⁺ cells. Our data show a decreased Foxp3 expression and an increased ROR gamma t expression in COPD patients and normal smokers that parallels the aggravation of the disease. The IL-17⁺-cell-related cytokines receptors CCR6 and IL-23R had an association with the mechanism of IL-17⁺ cell number increasing, which will provide a new immuno-therapeutic target for COPD.     

IL-17+ Foxp3+ T 细胞的研究进展

近来有文献报道复杂的细胞因子环境下, 调节性 T 细胞（regulatory T cells, Tregs）可以转变为表型和功能上酷似 Th17 的新的 T 细胞亚群, 即白细胞介素 （IL） -17 + Foxp3+T 细胞。IL-17 + Foxp3+T 细胞分泌 IL-17 并表达维甲酸受体相关孤儿受体γt（RORγt）, 在免疫系统中表现出双重特性。IL-17 + Foxp3 +T 细胞的发现为理解 Tregs 和 Th17的关系提供了新的思路。本文重点介绍了 IL-17 + Foxp3 +T 细胞的表型特征、 分化来源和多效性功能, 并进一步总结了炎症性疾病和肿瘤微环境中 IL-17+ Foxp3+T 细胞的作用。     

Regulatory effect of daphnetin, a coumarin extracted from Daphne odora, on the balance of Treg and Th17 in collagen-induced arthritis

Daphnetin extracted from Daphne odora Var. marginata contains coumarin compounds, which possess properties of analgesic and anti-inflammatory effects. In this study, we investigated the therapeutic effect of daphnetin on anti-arthritis and its role on the balance of Tregs and Th17, using a collagen-induced arthritis rat model. Collagen-induced arthritis rats were treated with daphnetin for 21 days. The therapeutic effects of daphnetin were evaluated by clinical symptoms and histopathology. The levels of Th17-, Treg-, Th2-, Th1-type cytokines in serum were determined by ELISA. The expression levels of related receptors RORγt, NF-κB, Foxp3 and CD77 in joint tissues were detected by immunohistochemistry. Our results showed that administration of daphnetin significantly alleviated the severity of the arthritis, as evidenced by the reduction of arthritis scores, suppression of the infiltration of inflammatory cells and prevention of synovial hyperplasia, thereby resulting in the joint destruction in the arthritis rats. Additionally, daphnetin treatment reduced the serum level of Th17-, Th2- and Th1-type in collagen-induced arthritis rats. Correspondingly, the expression of RORγt, NF-κB and CD77 in joint tissue of collagen-induced arthritis rats was remarkably decreased, while the expression of Foxp3 and IL-10 was remarkably increased after being administered with daphnetin. Collectively, this study demonstrated that administration of daphnetin attenuated the clinical symptoms and pathological destruction of arthritis joints. The therapeutic effects were associated with the up-regulation of Tregs, down-regulation of Th17-, Th2- and Th1-type cell responses. The results provide novel evidence that daphnetin has therapeutic effects on autoimmune arthritis through modulating the balance of Tregs and Th17.