应用RNAi技术探讨CAP10基因在新型隐球菌感染小鼠中对Th1-Th2型免疫的影响

目的 设计siRNA表达质粒干扰CAP10基因的合成,将siRNA干扰前与干扰后的新型隐球菌菌液经呼吸道感染小鼠分别建立动物模型,行组织病理学检查与细胞因子检测,探讨CAP10基因对Th1/Th2 免疫应答的影响。方法 建立小鼠吸入感染隐球菌模型,在感染后第7 d(急性期),PAS染色观察小鼠肺组织病理变化,并采用酶联免疫吸附试验定量检测小鼠血液中的Th1(IFN-γ、TNF-α)、Th2细胞因子(IL-10)水平。结果 急性期小鼠血液内IFN-γ、TNF-α及IL-10浓度测定分别为:对照组(uninfected组)(19.24±1.31)pg/mL,(36.94±2.04)pg/mL及(18.32±3.00)pg/mL,新型隐球菌未干扰组(WT组)(14.34±1.26)pg/mL,(25.37±1.37)pg/mL及(72.96±8.83)pg/mL,新型隐球菌干扰组(siRNA-CAP10组)(14.63±0.95)pg/mL,(26.22±1.55)pg/mL及(38.73±4.61)pg/mL。与对照组相比,WT组和siRNA-CAP10组Th1因子IFN-γ及TNF-α水平明显降低(P<0.01),Th2细胞因子IL-10水平明显增高。WT组与siRNA-CAP10组相比,IFN-γ及TNF-α水平未见明显增高而IL-10水平明显增高(P<0.01)。IFN-γ/IL-10及TNF-α/IL-10的比率分别为:对照组1.08±0.21,2.07±0.34,WT组0.20±0.03,0.35±0.05,siRNA-CAP10组0.38±0.04,0.69±0.09。WT组和siRNA-CAP10组IFN-γ/IL-10及TNF-α/IL-10的比率较对照组明显减少(P<0.01);WT组IFN-γ/IL-10及TNF-α/IL-10的比率亦明显低于siRNA-CAP10组(P<0.01)。结论 在新型隐球菌感染小鼠中,CAP10基因的表达与抗真菌的免疫反应有关,其下调有利于控制新型隐球菌播散,有利于Th1/Th2比率趋于平衡,在调节炎症反应方面有重要作用,有望成为新的分子治疗靶点。     

山羊Th1/Th2细胞因子实时荧光定量检测方法的建立

目的 在mRNA 水平对山羊Th1/Th2型细胞因子进行定量分析,建立山羊Th1/Th2型细胞因子实时荧光定量检测方法。方法 根据GenBank山羊Th1(IL-2和IFN-γ)和Th2(IL-4、IL-6和IL-10)细胞因子基因保守序列设计引物,以标准阳性质粒为模板分别进行荧光定量PCR反应的标准曲线、溶解曲线建立。结果 山羊Th1/Th2细胞因子实时荧光定量检测方法Ct值与标准品呈良好的线性关系,R²均大于0.985,所有稀释度标准品模板出现特异性熔解峰;利用N1蛋白缺失的重组山羊痘病毒(△N1L株)与山羊痘AV41株感染山羊外周血淋巴细胞进行IL-4和IFN-γ mRNA 表达水平检测,△N1L株与山羊痘AV41株均能能够刺激机体IL-4和IFN-γ细胞因子,但二者差异无统计学意义(t=3.333,P>0.5)。结论 本研究建立山羊Th1/Th2细胞因子实时荧光定量检测,为山羊痘基因工程疫苗的研究奠定基础。     

Th22细胞在卵巢癌恶性腹水中募集的机制

目的 观察Th22细胞在卵巢癌恶性腹水(MA)中的分布情况,探讨其向腹腔内募集的可能机制。方法选择卵巢癌合并MA患者23例(观察组)、健康女性13例(对照组),用密度梯度离心法获得MA和外周血中单个核细胞,流式细胞仪检测MA和外周血中Th22、Th17细胞构成比及Th22细胞趋化因子表达水平,ELISA法检测腹水上清液和外周血中IL-17、IL-22的水平,用Spearman秩相关分析变量间关系。结果 观察组MA中Th22、Th17细胞构成比高于同组外周血和对照组外周血(P均<0.05);观察组MA中CCR4、CCR7、CD45RO型Th22细胞构成比高于外周血,CD45RA、CD62L型低于外周血(P均<0.05);MA中IL-17、IL-22水平高于同组外周血及对照组外周血(P均<0.05);经Spearman秩相关分析,MA中Th22细胞与Th17细胞构成比呈正相关(r=0.66,P<0.01),IL-22水平与Th22细胞、Th17细胞构成比呈正相关(r分别为0.55、0.54,P均<0.05)。结论 Th22细胞可能通过趋化因子与炎症细胞因子由外周血向卵巢癌MA中募集。     

嗜酸粒细胞在气道激活机制中的研究进展

支气管哮喘是一种以嗜酸粒细胞（eosinophils,EOS）和肥大细胞浸润、气道高反应性为主要病理生理特征的慢性气道炎症,其中EOS在气道的激活对疾病的发生发展起着关键作用。气道中的EOS主要来源于外周血,极小部分来源于气道内的嗜酸粒细胞前体祖细胞,前者早在外周血中便在内皮细胞的作用下开始启动激活,之后气道上皮细胞通过表达细胞因子IL-3、IL-6、IL-8、RANTES及EOS重要的趋化因子eotaxin等趋化其向气道迁移。两种来源的EOS在气道的激活都是各种炎症细胞、细胞因子、趋化因子相互作用的结果 ：一方面,气道内的Th2细胞、Th1细胞、Th17细胞、肥大细胞等通过分泌IL-4、IL-5、IL-17等细胞因子促进EOS的激活;另一方面,各种细胞因子如PAF、IL-33、IL-7等也通过各种途径激活EOS并维持EOS的活性。本文就目前国内外关于EOS在气道激活机制的研究情况作一综述。     

嗜中性粒细胞通过分泌IL-22促进结肠上皮细胞的修复研究

目的探讨嗜中性粒细胞通过分泌白介素-22(Interleukin-22,IL-22)刺激肠道上皮细胞分泌黏液蛋白的作用。方法分离小鼠骨髓来源的嗜中性粒细胞,不同浓度的IL-23刺激中性粒细胞,检测IL-22 mRNA及蛋白合成;收集经IL-23刺激后中性粒细胞上清,将其作为条件培养基培养小鼠肠道上皮细胞CMT-93,检测CMT-93合成Reg3及MUC2的变化,并用IL-22中和抗体验证IL-22是否在条件培养基中起主要作用。结果 50 ng/ml IL-23即可显著诱导嗜中性粒细胞合成IL-22 mRNA,ELISA检测培养6 h的嗜中性粒细胞上清,IL-22蛋白量为(92±19)pg/ml。条件培养基培养CMT-93细胞24 h,Reg3和MUC2的mRNA合成倍数与对照组相比分别上升(11±3)和(14±5)倍,与单用IL-22诱导无显著差异,但使用IL-22阻断抗体后,条件培养基诱导CMT-93细胞合成抗菌肽的倍数分别降低至(2.4±0.8)和(2.1±0.5)倍。结论嗜中性粒细胞通过分泌细胞因子IL-22在促进肠道上皮细胞分泌抗菌肽中起重要作用。     

分子吸附再循环系统对肝衰竭患者血清Th1/Th2型细胞因子水平的影响

目的 观察分子吸附再循环系统（MARS）对肝衰竭患者Th1/Th2型细胞因子水平的影响,探讨MARS在肝衰竭治疗中的临床价值。方法 28例肝衰竭患者接受MARS治疗,采用ELISA法测定血清TNF-α、IFN-γ、IL-6、IL-10和IL-12水平,比较其在治疗前后的变化。结果 在MARS治疗结束时,患者血清TNF-α、IFN-γ、IL-6水平分别为（40.3±3.7） pg/ml 、（25.9±1.9） pg/ml、（33.6±2.5） pg/ml ,显著低于治疗前【（81.5±6.2） pg/ml、（49.3±2.2） pg/ml、（66.8±4.1） pg/ml,P均<0.01】;治疗结束时血清IL-10和IL-12水平分别为（39.3±2.6) pg/ml和（168.7±8.5) pg/ml ,显著高于治疗前【（37.1±1.9） pg/ml和（98.6±6.5）pg/ml,P<0.01】;在MARS治疗结束后7 d,患者TNF-α、IFN-γ、IL-6、IL-10和IL-12水平分别为（80.3±3.9） pg/ml、（48.6±2.1）pg/ml 、（65.1±2.4） pg/ml、（38.4±2.2） pg/ml、（99.8.6±7.9） pg/ml,与治疗前相比,差异无显著性（P>0.05)。结论 MARS治疗肝衰竭患者有利于纠正失衡的Th1/Th2型细胞因子水平,减轻免疫反应对肝组织的损伤,为肝细胞再生提供机会。     

慢性乙肝患者外周血中Th22、Th17细胞及其细胞因子浓度水平的变化与意义

目的探讨辅助T淋巴细胞(Th)22细胞、Th17细胞及其细胞因子在慢性乙型肝炎患者外周血中的表达及意义。方法选取2013年6月至2014年3月在我院就诊患者或体检者共101例,其中慢性乙型肝炎患者25例,乙肝病毒携带者21例,非病毒性肝炎35例。另选取同期20例健康体检者为正常对照组。用流式细胞术分别检测各组外周血中Th22细胞、Th17细胞所占比例。用酶联免疫吸附实验(ELISA)分别检测各组外周血中白细胞介素(IL)-22、IL-17的血清浓度。结果与正常对照组比较,慢性乙型肝炎组Th22细胞及IL-22、IL-17明显降低(P0.05),但Th17细胞无统计学差异(P0.05);与乙肝病毒携带者比较,慢性乙型肝炎组IL-22、IL-17明显降低(P0.05),Th17细胞明显升高(P0.05),但Th22细胞无统计学差异(P0.05);与非病毒性肝炎组相比,慢性乙型肝炎组Th22、Th17细胞及IL-22、IL-17均明显降低(P0.05);与正常组比较,乙肝病毒携带者Th22、Th17及IL-17均无统计学差异(P0.05),但IL-22明显降低(P0.05)。结论 Th22细胞数量减少或功能减退,IL-22浓度降低,使慢性乙型肝炎患者肝细胞失去IL-22的保护作用,是慢性乙型肝炎发病的重要因素;而Th17细胞及IL-17可能未参与慢性乙型肝炎的慢性期炎症。     

芽囊原虫致病机制的研究进展

芽囊原虫(Blastocystis)是常见的寄生于人和动物肠道的原生生物,但它是否具有致病性一直存在争议。临床调查发现,芽囊原虫感染与肠易激综合征(irritable bowel syndrome, IBS)、炎症性肠病(inflammatory bowel disease, IBD)以及免疫功能低下相关。芽囊原虫通过粪-口传播,主要寄居部位是盲肠和结肠,且芽囊原虫感染能引起细胞因子(IFN-γ、IL-12和TNF-α)上调及不同程度的肠道病理变化,但其致病机制尚不明确。近年来许多体外研究提出了芽囊原虫的几种致病机制,包括降解紧密连接蛋白引起肠上皮细胞通透性增加、细胞凋亡、上调肠道上皮细胞内的促炎细胞因子以及下调诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)。本文就芽囊原虫致病机制的最新研究进行综述。     

Th22细胞和IL-22在结核病人外周血中的变化及临床意义

目的探讨Th22细胞和IL-22在结核病人外周血中的变化及其临床意义。方法选取2015年1月至2017年5月到我院初次就诊的结核病患者作为研究对象,将其分为空洞型肺结核组(n=34)、浸润性肺结核组(n=39)和结核性胸膜炎组(n=36),另选择同期到我院体检的健康者作为对照组(n=35),采用流式细胞术检测四组外周血Th22细胞比例,采用酶联免疫吸附试验(ELISA)检测四组IL-22表达水平,分析各组Th22细胞比例和IL-22表达水平的变化,并进行二者的相关性分析。结果空洞型肺结核组、浸润性肺结核组、结核性胸膜炎组患者的外周血Th22细胞比例和IL-22表达水平均明显高于对照组,差异均具有统计学意义(均P 0. 05),空洞型肺结核组、浸润性肺结核组和结核性胸膜炎组三组间外周血Th22细胞比例及IL-22表达水平相比,差异均无统计学意义(均P 0. 05)。结核性胸膜炎组外周血Th22细胞比例与IL-22表达水平呈显著正相关(r=0. 683,P 0. 05),空洞型肺结核组、浸润性肺结核组及对照组外周血Th22细胞比例与IL-22表达水平之间无明显的相关性(均P 0. 05)。结论结核病人外周血中Th22细胞及IL-22水平显著升高,提示Th22细胞及IL-22可能参与了结核病的发病过程。     

结核分枝杆菌Rv3621c原核表达及免疫功能研究

目的 以结核分枝杆菌(Mycobacterium tuberculosis,M.tb)H37Rv基因组为模板,构建、纯化及鉴定原核表达质粒pPROEX-Rv3621c,通过人群、小鼠试验进行免疫原性评价。方法 构建重组质粒pPROEX-Rv3621c,并以全血干扰素释放分析技术(Whole-blood IFN-γ release assay,WBIA)检测其能否被安徽省淮南市M.tb感染者T细胞特异性识别。rRv3621c混合佐剂MTM[母牛分枝杆菌(M.vaccae),人工合成海藻糖-6'6,二分枝菌酸(TDB),单磷酰脂质A(MPLA)]免疫小鼠后,检测血清中特异性抗体分泌水平、脾细胞中抗原特异性Th1型细胞因子分泌水平及肺脏细胞因子mRNA表达水平。结果 成功构建重组质粒pPROEX-Rv3621c,并使之诱导表达、纯化和鉴定。在rRv3621c蛋白诱导下,活动性结核(Active tuberculosis, ATB)患者外周血淋巴细胞释放的IFN-γ水平明显较高(t=4.813, P<0.01),且ATB患者产生的IFN-γ水平高于潜伏性结核(Latent tuberculosis infection, LTBI)人群(t=4.442, P<0.01)。BCG+Rv3621c/MTM组小鼠产生的特异性抗体滴度水平明显高于Rv3621c/MTM组(P<0.01)和BCG组(P<0.01),Rv3621c/MTM组和BCG+Rv3621c/MTM组小鼠的IgG2a/IgG1比值大于1,明显高于MTM组和BCG组。BCG+Rv3621c/MTM组小鼠均分泌高水平IFN-γ、TNF-α和IL-2。Rv3621c/MTM组小鼠肺脏组织中IFN-γ、TNF-α及iNOS表达水平较高。结论 M.tb感染者外周血T细胞可特异性识别rRv3621c蛋白,rRv3621c混合佐剂MTM可以诱导较强烈的抗原特异性Th1型免疫应答。     

Antiviral Cytokine Response in Neuroinvasive and Non-Neuroinvasive West Nile Virus Infection

Data on the immune response to West Nile virus (WNV) are limited. We analyzed the antiviral cytokine response in serum and cerebrospinal fluid (CSF) samples of patients with WNV fever and WNV neuroinvasive disease using a multiplex bead-based assay for the simultaneous quantification of 13 human cytokines. The panel included cytokines associated with innate and early pro-inflammatory immune responses (TNF-α/IL-6), Th1 (IL-2/IFN-γ), Th2 (IL-4/IL-5/IL-9/IL-13), Th17 immune response (IL-17A/IL-17F/IL-21/IL-22) and the key anti-inflammatory cytokine IL-10. Elevated levels of IFN-γ were detected in 71.7% of CSF and 22.7% of serum samples (p = 0.003). Expression of IL-2/IL-4/TNF-α and Th1 17 cytokines (IL-17A/IL-17F/IL-21) was detected in the serum but not in the CSF (except one positive CSF sample for IL-17F/IL-4). While IL-6 levels were markedly higher in the CSF compared to serum (CSF median 2036.71, IQR 213.82–6190.50; serum median 24.48, IQR 11.93–49.81; p < 0.001), no difference in the IL-13/IL-9/IL-10/IFN-γ/IL-22 levels in serum/CSF was found. In conclusion, increased concentrations of the key cytokines associated with innate and early acute phase responses (IL-6) and Th1 type immune responses (IFN-γ) were found in the CNS of patients with WNV infection. In contrast, expression of the key T-cell growth factor IL-2, Th17 cytokines, a Th2 cytokine IL-4 and the proinflammatory cytokine TNF-α appear to be concentrated mainly in the periphery.     

Generation and regulation of human CD4+ IL-17-producing T cells in ovarian cancer

Despite the important role of Th17 cells in the pathogenesis of many autoimmune diseases, their prevalence and the mechanisms by which they are generated and regulated in cancer remain unclear. Here, we report the presence of a high percentage of CD4⁺ Th17 cells at sites of ovarian cancer, compared with a low percentage of Th17 cells in peripheral blood mononuclear cells from healthy donors and cancer patients. Analysis of cytokine production profiles revealed that ovarian tumor cells, tumor-derived fibroblasts, and antigen-presenting cells (APCs) secreted several key cytokines including IL-1β, IL-6, TNF-α and TGF-β, which formed a cytokine milieu that regulated and expanded human IL-17-producing T-helper (Th17) cells. We further show that IL-1β was critically required for the differentiation and expansion of human Th17 cells, whereas IL-6 and IL-23 may also play a role in the expansion of memory Th17 cells, even though IL-23 levels are low or undetectable in ovarian cancer. Further experiments demonstrated that coculture of naïve or memory CD4⁺ T cells with tumor cells, APCs, or both could generate high percentages of Th17 cells. Treatment with anti-IL-1 alone or a combination of anti-IL-1 and anti-IL-6 reduced the ability of tumor cells to expand memory Th17 cells. Thus, we have identified a set of key cytokines secreted by ovarian tumor cells and tumor-associated APCs that favor the generation and expansion of human Th17 cells. These findings should accelerate efforts to define the function of this important subset of CD4⁺ T cells in the human immune response to cancer.     

2型固有淋巴细胞在过敏性疾病发生中的作用

过敏性疾病尤其是支气管哮喘(简称哮喘)一直被认为是由Th2细胞介导的炎症反应.近期研究发现,2型固有淋巴细胞(type-2 innate lymphoid cell,ILC2)作为一种先天性免疫细胞,同样参与哮喘发生的始动环节.该细胞可产生Th2型细胞因子IL-13、IL-5从而应答受损上皮组织释放的IL-33及IL-25,不仅仅作用于固有免疫的初级阶段,还介导获得性免疫相关功能,这使得先天性及获得性两种免疫系统之间存在了某种特殊的联系,也给研究过敏性哮喘发生机制带来了新的思路.     

From the Cover: IL-22–producing neutrophils contribute to antimicrobial defense and restitution of colonic epithelial integrity during colitis

IL-22 plays an important role in mucosal epithelial cell homeostasis. Using a dextran sodium sulfate-induced mouse model of acute colitis, we observed an IL-23–dependent up-regulation of IL-22 in the middle and distal colon at the onset of epithelial cell damage. This heightened IL-22 correlated with an influx of innate immune cells, suggesting an important role in colonic epithelial protection. Freshly isolated colon-infiltrating neutrophils produced IL-22 contingent upon IL-23 signaling, and IL-22 production was augmented by TNF-α. Importantly, the depletion of neutrophils resulted in diminished IL-22 levels in the colon, and the transfer of IL-22–competent neutrophils to Il22a-deficient mice protected the colonic epithelium from dextran sodium sulfate-induced damage. In addition, IL-22–producing neutrophils targeted colonic epithelial cells to up-regulate the antimicrobial peptides, RegIIIβ and S100A8. This study establishes a role for neutrophils in providing IL-22–dependent mucosal epithelial support that contributes to the resolution of colitis.     

Expansion of human NK-22 cells with IL-7, IL-2, and IL-1β reveals intrinsic functional plasticity

Natural killer-22 (NK-22) cells are a human NK cell subset situated in mucosal-associated lymphoid tissues that specialize in IL-22 secretion in response to IL-23. Here we investigated the cytokine requirements for NK-22 cell expansion. IL-7 maintained the sur-vival of NK-22 cells and IL-22 production in response to IL-23 but was insufficient to induce robust expansion. Proliferation of NK-22 cells was increased markedly by adding either IL-1β or IL-2 to IL-7 and was even stronger in the presence of IL-1β plus IL-2. In contrast to IL-7, continuous culture in IL-1β and IL-2 modified NK-22 cytokine profiles. IL-1β promoted constitutive IL-22 secretion rather than acute IL-22 production in response to IL-23 and induced IL-17 in some cells. IL-2 reduced secretion of IL-22 and IL-17, increasing production of IFN-γ and leukemia inhibitory factor. Functional deviation toward IFN-γ production also was induced by continuous culture in IL-23. These results demonstrate the functional plasticity of NK-22 cells, which may allow flexible responses to different pathogens. Finally, we found that NK-22 cells released the B-cell survival factor, B-cell activating factor belonging to the TNF family (BAFF), suggesting a potential role of NK-22 cells in promoting B-cell–mediated mucosal immunity.     

Functional Alterations of Proinflammatory Monocytes by T Regulatory Cells: Implications for the Prevention and Reversal of Type 1 Diabetes

The onset and development of type 1 diabetes (T1D) occurs in genetically predisposed individuals, and is attributed to autoimmune destruction of pancreatic β-cells involving a multitude of immune mechanisms. Defects in immune regulation may play a central role in T1D, involving impaired function and communication of both myeloid and lymphoid cells of the innate and adaptive immune compartments. Dendritic cells and regulatory T (Treg) cells are part of this network, which seem to be hampered in their quest to control and regulate tissue-destructive autoimmunity. Recent studies have shown that in vivo activated CD16- blood monocytes exhibiting proinflammatory features are present in diabetic subjects. These monocytes may govern T cell-mediated immune responses towards the development of tissue-destructive Th1 and Th17 subtypes, and give rise to inflammatory macrophages in tissues. Differential effects of cytokines IFN-γ and IL-4 in the development of inflammatory macrophages, and the distinct developmental pathways of proinflammatory or tissue-repair-associated monocytes suggest that controlling the activity of these monocytes could be part of an immune intervention strategy to prevent T1D. Similarly, strategies to target autoantigens to immature, steady-state dendritic cells could guide the immune response away from Th1 and Th17 immune effectors. This review examines potential approaches to this goal by manipulation of myeloid and lymphoid cell regulatory networks in T1D.