感染后内脏高敏感小鼠肠道黏膜固有层树突细胞对CD4~+T 细胞的作用

背景: 多项证据提示感染后肠易激综合征(PI-IBS)患者的肠道黏膜中存在T细胞介导的低度炎症。树突细胞(DC)是肠道黏膜免疫系统中最重要的抗原呈递细胞,目前关于DC在PI-IBS中作用的报道尚少。目的: 研究肠道黏膜固有层DC在急性肠道感染的不同阶段对CD4~+ T细胞的影响,探讨DC在肠道感染消退后维持肠道黏膜免疫系统持续激活中的作用。方法: 建立旋毛虫感染后内脏高敏感小鼠模型以模拟人类PI-IBS。肠道黏膜固有层DC与脾脏CD4~+T细胞于体外共培养120h,ELISA法检测细胞培养上清液中Th17、Th2、Th1相关细胞因子IL-17、IL-4、IFN-γ水平。结果: 与单独培养相比,CD4~+T细胞与DC共培养后,感染后8周组IL-17水平增加值明显高于对照组(P=0.001)和感染后2周组(P=0.279):感染后2周组IL-4水平增加值明显高于对照组(P=0.041)和感染后8周组(P=0.204);三组间IFN-γ水平增加值差异均无统计学意义。结论: 感染后内脏高敏感小鼠肠道黏膜固有层DC诱导CD4~+T细胞分化为Th17细胞并使之活化可能是肠道感染消退后维持肠道黏膜免疫系统持续激活的主要机制。     

胸腺基质淋巴细胞生成素对效应T细胞分化功能的影响

目的：探讨体外氧化型低密度脂蛋白（ ox-LDL）诱导人血管平滑肌细胞产生的胸腺基质淋巴细胞生成素（ TSLP）对效应T细胞分化功能的影响。方法分离并培养人血管平滑肌细胞、人树突状细胞（ DC）、初始CD4＋T细胞,随机分为4组,每组设5个样本。对照组、实验组人血管平滑肌细胞分别经PBS、ox-LDL处理后,取其上清液与DC、初始CD4＋T细胞共培养;TSLP中和抗体组、中和抗体对照组在实验组的基础上分别加入TSLP中和抗体、TSLP非特异性中和抗体与DC、初始CD4＋T细胞共培养。采用ELISA法检测各组细胞培养上清液中TSLP及Th17细胞因子IL-17、IL-22、TNF-α水平,流式细胞术检测Th17细胞构成比。结果与对照组比较,实验组培养上清液TSLP、IL-17、IL-22、TNF-α水平及Th17细胞构成比升高（ P均＜0．01）。与实验组、中和抗体对照组比较,TSLP中和抗体组TSLP、IL-17、IL-22、TNF-α水平及Th17细胞构成比降低（ P均＜0．01）。结论 Ox-LDL体外诱导人血管平滑肌细胞产生的TSLP可促进CD4＋T细胞向Th17细胞分化。     

肠系膜淋巴结Th1、Th17细胞在小鼠结肠炎模型发病中作用的研究

背景:大量研究表明细胞因子网络在溃疡性结肠炎(UC)的发病和疾病进程中起关键作用,但相关研究主要集中于肠黏膜免疫细胞方面。目的:探讨肠系膜淋巴结Th1、Th17细胞在模拟人类UC的小鼠DSS结肠炎模型发病中的作用。方法:C57BL/6小鼠饮用5%DSS溶液7 d诱导实验性结肠炎,实验过程中每天评估疾病活动指数(DAI)。于第8 d处死小鼠,ELISA法测定结肠组织IL-1β含量;分离肠系膜淋巴结细胞,以CD3/CD28单抗诱导淋巴细胞活化并以ELISA法测定细胞培养上清液中的Th1、Th17细胞因子含量,流式细胞术检测肠系膜淋巴结F4/80+CD11b+巨噬细胞和CD4+T细胞内Th1、Th17细胞因子表达。结果:结肠炎模型组DAI随实验进程而逐渐增加,于第7 d达峰值。与正常对照组相比,模型组结肠组织IL-1β蛋白表达显著上调(P<0.05),肠系膜淋巴结巨噬细胞浸润增加(P<0.001),淋巴细胞IL-17A分泌水平显著增高(P<0.05),IFN-γ分泌水平亦呈增高趋势(P>0.05),CD4+T细胞内IL-17A、IFN-γ表达显著上调(P<0.05)。结论:肠系膜淋巴结Th1、Th17细胞过度激活可能通过释放效应细胞因子诱导巨噬细胞等浸润、活化,参与介导小鼠DSS结肠炎模型的肠黏膜炎症反应和病理损伤。     

白介素23受体在支气管哮喘小鼠脾源性CD4^＋T细胞中的表达及意义

-23R）的表达及在哮喘发病中的作用。方法建立急性哮喘小鼠模型,免疫磁珠分离小鼠脾源性CD4^＋T细胞,培养24h后,检测CD4^＋T细胞表面IL-23R的表达、Th17细胞的阳性率及细胞培养上清液中的IL-17水平。结果哮喘小鼠脾脏CD4^＋T细胞表面IL-23R的表达明显高于正常组（P〈0．01）;哮喘小鼠脾脏CD4^＋T细胞中Th17细胞的阳性率明显高于正常组（P〈0．01）;哮喘小鼠脾脏CD4^＋T细胞分泌IL-17浓度明显高于正常组（P〈O．01）;小鼠脾脏CD4^＋T细胞表面IL-23R的表达与Th17细胞的阳性率和IL-17的浓度呈正相关。结论IL-23R在急性哮喘的发病机制中可能起重要作用。     

Th17细胞与肝脏疾病

辅助性T细胞（Th细胞）根据产生细胞因子和生物学功能分为Th1和Th2细胞。最近研究发现了一种与Th1和Th2细胞亚群不同的活化CD4^＋T细胞亚群-Th17细胞。TGF-8与IL-6或IL-21的协同作用,诱导Th17细胞分化。IL-12家族的IL-23在促进IL-17分泌、增强Th17细胞效应功能方面发挥重要作用,而RORγt是其特异性转录因子。分化成熟的Th17细胞可以分泌IL-17A、IL-17F、IL-21、IL-22、IL-6、TNF—α等多种细胞因子,在介导炎性反应（防御病原菌感染）、自身免疫性疾病、肿瘤、移植排斥反应等过程中发挥重要作用。Th17细胞也为研究肝脏疾病的发病机制及防治策略提供了新思路和方向。     

Th17细胞分化及与疾病关系的研究新进展

Th17细胞是一类新型CD4^＋T细胞,它与Th1、Th2、Tregs细胞一起构成了CD4^＋T细胞的4个主要亚群。它由初始T细胞在转化生长因子（TGF）-β和白细胞介素（IL）-6联合诱导下分化而来。这一分化过程在细胞和分子水平都受到精细的调节。IL-17是Th17细胞最主要的分泌产物,在自身免疫性疾病、某些感染性疾病以及肿瘤中起到重要的调节作用。因此Th17细胞及其相关因子可能为此类疾病的治疗提供新的靶点和途径。     

内毒素血症大鼠肠黏膜免疫细胞的变化及通腑颗粒的干预作用

目的探讨内毒素血症大鼠肠黏膜免疫细胞的变化及中药通腑颗粒对其影响,为临床治疗提供理论依据。方法 120只雄性Wistar大鼠随机分为正常对照组（A组）、内毒素血症组（B组）及中药治疗组（C组）三组。B组及C组采取尾静脉一次性注射给药方法,注射1%LPS溶液（给药量为10 mg/kg）制作内毒素血症模型,A组注射等量生理盐水（1 mL/kg）;C组尾静脉注射给药后即刻给予50%通腑颗粒溶液（给药量为5 g/kg）灌胃,A组及B组给予等量蒸馏水灌胃。造模后在第2、6、12、24 h时间点分批（每次10只）麻醉后处死动物,取小肠组织进行检测。采用HE染色观察肠黏膜病理变化,采用免疫组织化学及TUNEL染色方法,分别标记并计数各组大鼠肠黏膜免疫细胞及凋亡淋巴细胞。结果与A组比较,B组肠黏膜M细胞、DC细胞、CD8＋T细胞及IgA＋B细胞计数明显减少,而CD4＋T细胞、Tr细胞及淋巴细胞凋亡计数明显增多;C组肠黏膜M细胞、CD8＋T及Tr细胞计数无明显改变,DC细胞、IgA＋B细胞及淋巴细胞凋亡计数明显减少,而CD4＋T细胞计数明显增多;与B组比较,C组肠黏膜M细胞无明显改变,Tr细胞及淋巴细胞凋亡计数明显减少,而DC细胞、CD4＋T细胞、CD8＋T细胞及IgA＋B细胞计数明显增多。结论内毒素血症时肠黏膜损伤,肠免疫不同阶段的免疫细胞均发生损害,Th1/Th2比例失调,淋巴细胞凋亡增多,导致肠黏膜局部免疫发生抑制。应用通腑颗粒可以减轻肠黏膜损伤,减轻免疫细胞损伤,改善Th1/Th2比例,减少淋巴细胞凋亡,保护局部免疫功能。     

溃疡性结肠炎肠黏膜损伤与白细胞介素-13调控

参与溃疡性结肠炎（UC）肠黏膜炎性反应的细胞和细胞因子不同于克罗恩病（CD）。在UC，聚集于肠黏膜固有层的CD4T淋巴细胞分泌大量白细胞介素（IL）-5等细胞因子，而IL-4、IL-12和γ干扰素（IFNγ）等却无显著变化；值得注意的是，CD1d标志的自然杀伤T细胞（NKT）分泌IL-13显著增高。UC的这种以IL-5和IL-13增高为特征的肠黏膜病变被称之为非典型Th2型免疫性炎症损伤。     

静脉注射丙种球蛋白无反应型川崎病Th17细胞的变化及意义

目的 探讨Th17细胞在静脉注射丙种球蛋白(IVIG)无反应型川崎病(KD)免疫发病机制中的作用.方法 选取KD患儿45例,其中IVIG敏感型KD 35例,IVIG无反应型KD 10例,同年龄健康对照组30名.KD患儿分别于IVIG治疗前后直接取血备检,IVIG无反应型KD分别在病程第8、9、11天取血备检.采用实时荧光定量聚合酶链反应(PCR)检测CD4~+T细胞白细胞介素(IL)-17A/F、转录因子ROR-γt mRNA表达;采用流式细胞术检测外周血Th17细胞占CD4~+T细胞比例.应用酶联免疫吸附试验(ELISA)检测Th17细胞相关的细胞因子IL-17A和IL-6的表达.结果 ①急性期KD患儿CD4~+T细胞IL-17A/F表达明显高于对照组(P<0.01);②急性期IVIG无反应型KD患儿CD4~+T细胞内IL-17蛋白、IL-17A/F mRNA、Th17细胞转录因子ROR-γt的基因表达明显高于IVIG敏感型KD,经IVIG治疗后敏感型KDTh17细胞相关因子表达明显降低(P<0.01),无反应型KD Th17细胞相关因子仍持续高表达(P>0.05);③急性期KD患儿治疗前IL-17A和IL-6血浓度明显高于对照组(P<0.01),其中IVIG无反应型KD活化细胞因子水平明显高于1VIG敏感型KD组(P<0.01).经治疗后均有下降趋势,但IVIG无反应型KD仍高于敏感型KD(P<0.01);④甲泼尼龙冲击治疗IVIG无反应型KD当天退热.血浆IL-6,IL-17A较前明显降低,C反应蛋白(CRP)恢复快,能够迅速控制血管炎性反应.结论 Th17细胞过度活化可能是导致IVIG无反应型KD的原因之一.     

白介素23受体在支气管哮喘小鼠脾源性CD4+T细胞中的表达及意义

目的 探讨急性支气管哮喘(简称哮喘)小鼠CD4+T细胞表面白介素23受体(IL-23R)的表达及在哮喘发病中的作用.方法 建立急性哮喘小鼠模型,免疫磁珠分离小鼠脾源性CD4+T细胞,培养24 h后,检测CD4+T细胞表面IL-23R的表达、Th17细胞的阳性率及细胞培养上清液中的IL-17水平.结果 哮喘小鼠脾脏CD4+T细胞表面IL-23R的表达明显高于正常组(P＜0.01);哮喘小鼠脾脏CD4+T细胞中Th17细胞的阳性率明显高于正常组(P＜0.01);哮喘小鼠脾脏CD4+T细胞分泌IL-17浓度明显高于正常组(P＜0.01);小鼠脾脏CD4+T细胞表面IL-23R的表达与Th17细胞的阳性率和IL-17的浓度呈正相关.结论 IL-23R在急性哮喘的发病机制中可能起重要作用.     

Characteristics of intestinal lamina propria dendritic cells in a mouse model of postinfectious irritable bowel syndrome

Background and Aim: Postinfectious irritable bowel syndrome (PI‐IBS), which results from inflammation has been emphasized a lot recently. Dendritic cells (DCs) may contribute to intestinal mucosal immune activation in the pathogenesis of PI‐IBS. This study tested the hypothesis that phenotype and function of intestinal lamina propria DCs (LPDCs) changed in the development of a PI‐IBS mouse model. Methods: Mice infected with Trichinella spiralis underwent abdominal withdrawal reflex (AWR) to evaluate visceral sensitivity. LPDCs were isolated and purified by intestine digestion and magnetic label‐based technique. Surface markers were analyzed by flow cytometry. Endocytic activity, mixed lymphocyte reaction (MLR) and chemotaxis were studied. Cytokine production of the LPDCs cocultured with CD4⁺ T cells was determined. Results: Intestinal inflammation resolved after 8 weeks infection with sustained visceral hyperalgesia. Surface markers CD86 and MHCII were lower in the acute infection group, but increased in the PI‐IBS stage. Enhanced ability of endocytic activity and decreased abilities to attract and stimulate CD4⁺ T cell proliferation were in the acute infection group. However, LPDCs in the PI‐IBS stage showed weakened endocytic ability with enhanced abilities to attract and stimulate CD4⁺ T cell proliferation. Cocultured LPDCs with CD4⁺ T cells showed a predominant Th2 response in the acute infection stage, and more important roles of Th1, Th17 responses in the PI‐IBS stage. Conclusions: The hypothesis was supported that the phenotype and function of LPDCs changed in the development of PI‐IBS, which induced the maintenance of intestinal mucosal immune activation and might provide a clue for the treatment of the disease.     

The Effect of Beta Interferon on Dendritic Cells and Cytokine Synthesis by CD4+ T Cells

Background: Dendritic cells (DC) are a key regulator of the immune response, and interferon- beta (IFN-β) is considered an immunomodulatory molecule for DC. Objective: The purpose of this study was to evaluate the ability of IFN-β treated DC to induce cytokine secretion by CD4+ T cells. Methods: Dendritic cells were generated from blood monocytes with granulocyte-monocyte colony-stimulating factor and interleukin-4 with or without IFN-β. We analyzed the production of CD4+ T helper cytokines (IL-17, IFN- γ and IL-10) in the supernatant of the dendritic cell-T cell co- cultures by ELISA. We also studied the effects of HLA-G and costimulatory molecules on immature and mature DC. Results: IFN-γ and IL-17 decreased significantly in the presence of HLA-Gbearing DC compared to control cultures (p<0.05). Conclusion: Using the mixed leukocyte reaction, we found that DC treated with IFN-β mediated the inhibition of T cell activation via cytokine production. We conclude that this is important for preventing overactivation of the immune system.     

白细胞介素-23在炎症性肠病的表达升高并诱导促炎细胞因子分泌

目的 通过分析白细胞介素(IL)-23p19及其受体(IL-23R)在炎症性肠病(IBD)患者中的表达,研究IL-23对IBD患者外周血T细胞激活和效应应答的影响,探讨其在疾病发生过程中的免疫病理作用.方法 收集12例克罗恩病(CD)患者、25例溃疡性结肠炎(UC)患者和20名健康者外周血和肠黏膜组织活检标本,使用免疫组化染色和逆转录(RT)-PCR分析IL-23p19表达,分离外周血单个核细胞(PBMC)和肠黏膜固有层内单个核细胞(LPMC),利用流式细胞仪检测IL-23R在CD4+、CD8+T细胞和自然杀伤(NK)细胞表面表达.体外培养PBMC,使用IL-23和抗CD3单抗体外刺激,使用酶联免疫吸附试验检测肿瘤坏死因子(TNF)-α、干扰素(IFN)-γ和白细胞介素(IL)-2分泌.结果 IBD患者炎症肠黏膜组织内IL-23p19蛋白和mRNA表达水平比健康对照者显著升高(P<0.05).IL-23R在IBD患者外周血和肠黏膜固有层组织内CD4+、CD8+T细胞和NK细胞表达水平也比健康者显著升高(P<0.05).体外培养PBMC,使用IL-23刺激,发现IL-23可显著诱导IBD患者,尤其是CD患者PBMC激活,分泌高水平TNF-α、IFN-γ和IL-2(P<0.05).结论 IL-23及其受体在IBD患者炎症肠黏膜组织中表达升高,IL-23可诱导IBD患者淋巴细胞分泌高水平的炎性介质,提示IL-23参与了肠黏膜炎症损伤,阻断IL-23生物学效应可能治疗IBD.     

Increased CD4~+CD45RA~-FoxP3~(low) cells alter the balance between Treg and Th17 cells in colitis mice

AIM To investigate the role of regulatory T cell(Treg) subsets in the balance between Treg and T helper 17(Th17) cells in various tissues from mice with dextran sulfate sodium-induced colitis.METHODS T r e g c e l l s, T r e g c e l l s u b s e t s, T h 1 7 c e l l s, a n d CD4+CD25+FoxP 3+IL-17+ cells from the lamina propria of colon(LPC) and other ulcerative colitis(UC) mouse tissues were evaluated by flow cytometry. Forkhead box protein 3(FoxP 3), interleukin 17A(IL-17A), and RORC m RNA levels were assessed by real-time PCR, while interleukin-10(IL-10) and IL-17 A levels were detected with a Cytometric Beads Array.RESULTS In peripheral blood monocytes(PBMC), mesenteric lymphnode(MLN), lamina propria of jejunum(LPJ) and LPC from UC mice, Treg cell numbers were increased(P 0.05), and FoxP 3 and IL-10 mR NA levels were decreased. Th17 cell numbers were also increased in PBMC and LPC, as were IL-17 A levels in PBMC, LPJ, and serum. The number of FrI subset cells(CD4+CD45RA+FoxP 3low) was increased in the spleen, MLN, LPJ, and LPC. FrI I subset cells(CD4+CD45RA-Fox P3high) were decreased among PBMC, MLN, LPJ, and LPC, but the number of Fr III cells(CD4+CD45RA-FoxP 3low) and CD4+CD25+FoxP 3+IL-17A+ cells was increased. Fox P3 m RNA levels in CD4+CD45RA-Fox P3 low cells decreased in PBMC, MLN, LPJ, and LPC in UC mice, while IL-17 A and RORC mR NA increased. In UC mice the distribution of Treg, Th17 cells, CD4+CD45RA-FoxP 3high, and CD4+CD45RA-FoxP 3low cells was higher in LPC relative to other tissues.CONCLUSION Increased numbers of CD4+CD45RA-FoxP 3low cells may cause an imbalance between Treg and Th17 cells that is mainly localized to the LPC rather than secondary lymphoid tissues.     

Methyl-cpg-binding Domain Protein 2 Silencing Inhibits Th17 Differentiation of CD4+T cells Induced by Ovalbumin

Background: Little is known about MBD2’s epigenetic regulation in the immune pathogenesis of CD4+T cell differentiation.Objective: This study attempted to explore the mechanism of methyl-cpg-binding domain protein 2 (MBD2) in CD4+T cell differentiation stimulated by environmental allergen ovalbumin (OVA).Methods: Mononuclear cells were separated from the spleen tissues of male C57BL/6 mice. The OVA interfered with the differentiation of splenic mononuclear cells and CD4+T cells. The CD4+T cells were obtained by magnetic beads and identified by CD4 labeled antibody. CD4+T cells were transfected with lentivirus to silence MBD2 gene. A methylation quantification kit was used to detect 5-mC levels.Results: The purity of CD4+T cells reached 95.99% after magnetic beads sorting. Treatment with 200 μg/mL OVA stimulated the CD4+T cells differentiation to Th17 cells and promoted the secretion of IL-17. After being induced, the Th17 cell ratio increased. 5-Aza inhibited the Th17 cell differentiation and the IL-17 level in a dose-dependent manner. Under the intervention of the Th17 induction and 5-Aza, MBD2 silencing inhibited the differentiation of Th17 cell, and decreased the IL-17 and 5-mC levels in the cell supernatants. MBD2 silencing reduced the scale of the Th17 cell and IL-17 levels in the OVA-treated CD4+T cells.Conclusion: MBD2 affected IL-17 and 5-mC levels by mediating the Th17 cell differentiation in splenic CD4+T cells that were interfered with 5-Aza. OVA induced Th17 differentiation and increased IL-17 levels, inhibited by MBD2 silencing.     

Monoclonal anti-interleukin 23 reverses active colitis in a T cell-mediated model in mice

BACKGROUND & AIMS: Interleukin (IL)-23 supports a distinct lineage of T cells producing IL-17 (Th17) that can mediate chronic inflammation. This study was performed to define the role of IL-23 and Th17 cells in chronic colitis in mice. METHODS: Colitis was induced by transfer of a cecal bacterial antigen-specific C3H/HeJBir (C3Bir) CD4(+) T-cell line to C3H/HeSnJ SCID mice. Cytokines were measured by flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction. Monoclonal anti-IL-23p19 was administered at the same time as or 4 weeks after pathogenic CD4 T-cell transfer. A histopathology colitis score was assessed in a blinded fashion. RESULTS: The pathogenic C3Bir CD4(+) T-cell line contained more cells producing IL-17 than those producing interferon-gamma and these were distinct subsets; after adoptive transfer to SCID recipients, Th17 cells were predominant in the lamina propria of mice with colitis. Bacteria-reactive CD4(+) Th1 and Th17 lines were generated. The Th17 cells induced marked inflammation in a dose-dependent manner. Even at a dose as low as 10(4) cells/mouse, Th17 cells induced more severe disease than Th1 cells did at 10(6) cells/mouse. Monoclonal anti-IL-23p19 prevented and treated active colitis, with down-regulation of a broad array of inflammatory cytokines and chemokines in the colon. Anti-IL-23p19 induced apoptosis in colitogenic Th17 cells in vitro and in vivo. CONCLUSIONS: Bacterial-reactive CD4(+) Th17 cells are potent effector cells in chronic colitis. Inhibition of IL-23p19 was effective in both prevention and treatment of active colitis. IL-23 is an attractive therapeutic target for inflammatory bowel disease.     

Prostaglandin E2-dependent IL-23 production in aged murine dendritic cells

CD4+ T cells of the Th17 subtype are over-represented in the aged immune system. Dendritic cells (DC) play a critical role in naïve CD4+ T cell differentiation. However, expression of cytokines by aged DC that promote differentiation or survival of Th17 cells has not been extensively investigated. Using bone marrow-derived DC from C57BL/6 mice of different ages we compared cytokine production after DC activation by Toll-like receptor agonists for TLR4 and/or TLR7/8. DC-derived TNF-α and IL-12p70 production and expression of DC co-stimulatory molecules did not vary significantly by age indicating that TLR expression, function and signal transduction were intact in aged DC. There were relatively minor age-related changes in TGF-β and IL-6 which promote Th17 differentiation, but IL-23, a Th17-suvival cytokine, increased more than 40-fold across the lifespan. DC-derived prostaglandin E2 (PGE2) also increased with age and the up-regulation of IL-23 expression by aged DC was blocked by indomethacin that prevents PGE2 production, and by antagonists of PGE2 receptors. Exogenous PGE2 added to DC cultures further enhanced IL-23 production from aged but not young DCs. These data indicate that age-related changes in DC PGE2 production are necessary, but not sufficient to induce DC IL-23 production. Such changes may play a role in the expansion of Th17 cells in the aged immune system.