从Th1/Th2、Treg/Th17之间的平衡研究轻、中度慢性乙型肝炎患者CD4~+T淋巴细胞的免疫失衡状态

目的从Th17及其相关细胞因子与Th1、Th2、Treg的相互关系,探讨轻、中度CHB患者CD4~+T淋巴细胞的整体免疫失衡状态。方法选择轻、中度CHB患者,与健康志愿者进行比较,观察其外周血各免疫细胞(Th17、Th1、Th2、Treg、Th1/Th2、Treg/Th17)、相关细胞因子(IL-17、IL-6、TGF-β、IFN-γ、IL-4)、主要效应分子(穿孔素、颗粒酶B)和核转录因子(孤独核受体RORγt、Foxp3等)的变化及其相互关系。结果与健康志愿者相比,CHB患者CD8~+T淋巴细胞比例更高,CD4~+/CD8~+比例显著下降,差异有统计学意义(P0.01);与健康志愿者相比,CHB患者Th1细胞比例更高,Th1/Th2显著升高,差异有统计学意义(P0.01);与健康志愿者相比,CHB患者Th17细胞比例更高,Treg/Th17比例显著下降,差异有统计学意义(P0.01);与健康志愿者相比,CHB患者穿孔素和颗粒酶B的表达更高,差异有统计学意义(P0.01);与健康志愿者相比,CHB患者FOXP3、IL17mRNA和RORγt的表达差异无统计学意义(P0.05);与健康志愿者相比,CHB患者TGF-B1、IL-6、IL-17显著增高,差异有统计学意义(P0.01);IFN-γ和IL-4的表达差异无统计学意义(P0.05)。结论轻、中度CHB患者在免疫细胞及其相关细胞因子、效应分子等各环节,尤其是Treg/Th17比例,均处于严重的免疫失衡状态。     

T Helper Cells Profile and CD4+CD25+Foxp3+Regulatory T Cells in Polycystic Ovary Syndrome

Background: Polycystic ovary syndrome (PCOS) is considered as the most common cause of female infertility that affects 4-10% of women in the reproductive age. Previous studies have shown the role of a balanced immune response in a successful pregnancy and fertility. Objective: To investigate the T helper cells type 1 (Th1) /Th2/Th17/Treg paradigms in peripheral blood of infertile PCOS compared with normal fertile women. Methods: Peripheral blood mononuclear cells (PBMCs) were isolated at the late follicular phase from 10 PCOS and 10 fertile women. PBMCs were stimulated with PMA and ionomycin in the presence of Berefeldin A as Golgi stop agent to detect intracellular cytokine production (IFN-γ, IL-17, and IL-4) from CD3+CD4+T cells population indicating T helper (Th) cells subsets by flowcytometry. Moreover, regulatory T cells were enumerated using CD25 and Foxp3 markers. Results: In this study, we report that the frequency of Th1 cells was increased compared to Th2 cells in infertile PCOS when considering Th1/Th2 ratio (P=0.05). Analysis of Th17/Th2 ratio showed a significant difference with a bias toward Th17 dominancy in PCOS (P=0.02). The proportion of CD4+CD25+Foxp3+ regulatory T cells was significantly lower in PCOS patients than that of healthy fertile women (P=0.02). Conclusion: In summary, Th1 and Th17 bias and reduction of Treg and Th2 cells as regulators of immune responses might be involved in the pathogenesis of PCOS. These results are suggestive of an altered immune response to inflammatory status in PCOS patients, likely causing some complications such as infertility in these patients.     

Spontaneous and aging-dependent development of arthritis in NADPH oxidase 2 deficiency through altered differentiation of CD11b+ and Th/Treg cells

Emerging evidence indicates that NADPH oxidase (NOX) and its reactive oxygen species (ROS) products modulate a variety of cellular events, including proliferation, differentiation, and apoptosis. In this study, we investigated the functions of NOX2 and ROS in immune modulation using NOX2 knockout (KO) mice. Interestingly, NOX2 KO mice spontaneously developed arthritis with onset at 6-7 wk of age and high incidence (60%) at 15-18 wk of age. Arthritis severity in NOX2 KO mice was proportionally increased with age and higher in females than in males. Bone destruction was confirmed by microcomputed tomography scanning and histological analyses of joints. Inflammatory factors, including TNF-α, IL-1β, and RANKL, and serum level of anti-type II collagen IgG were significantly increased in NOX2 KO mice. In addition, NOX2 deficiency perturbed the immune system upon aging. NOX2 KO mice demonstrated preferred development of CD11b+Gr-1+ myeloid cells with profound production of proinflammatory cytokines and augmented expression of IL-17 through the activation of STAT3 and RORγt in vivo. NOX2 deficiency increased differentiation of effector Th cells in vitro and decreased CD25+FoxP3+ Treg cells both in vitro and in vivo. Furthermore, adoptive transfer of NOX2-deficient CD4(+) T cells into RAG KO mice increased arthritic inflammation compared with WT cells. These results demonstrated that NOX2 deficiency affected the development of CD11b+ myeloid cells and Th17/Treg cells, and thus promoted inflammatory cytokine production and inflammatory arthritis development, strongly supporting a crucial role for ROS generation in the modulation of Th17/Treg cell development and its related inflammatory immune response upon aging.     

CD4+白细胞介素-17+辅助性T细胞对香烟暴露小鼠肺部炎症及肺气肿的作用

目的 观察香烟烟雾暴露小鼠肺实质中CD4+白细胞介素(IL)-17+辅助性T细胞(Th17)数量及活性的表达,探讨其在香烟暴露小鼠肺部CD4+ γ-干扰素+(Th1)炎症及肺气肿中的作用及相关机制.方法 将40只雄性Balb/c小鼠按随机数字表法分为4组:对照12周(C12)组、对照24周(C24)组、烟雾暴露12周(S12)组、烟雾暴露24周(S24)组,每组10只.香烟烟雾暴露法建立小鼠肺气肿模型.HE染色观察小鼠肺气肿的改变,计算平均内衬间隔和肺泡破坏指数(DI);流式细胞术检测小鼠肺实质中CD4+IL-17+T(Th17)细胞、CD4+γ-干扰素+T(Th1)细胞、CD4+IL-17+γ-干扰素+T(Th17/Th1)细胞、CD8+γ-干扰素+T(Tc1)细胞、CD8+IL-21R+细胞及CD4+IL-17+IL-21+细胞比例;荧光定量PCR法检测小鼠肺实质中维甲酸相关孤独受体(RORγt)和IL-17的mRNA表达,并分析这些指标的相互关系.结果 S12组和S24组的平均内衬间隔[(39±4)μm和(47±7)μm]和DI(39.1±1.6和45.2±3.1)明显高于C12组[(32±4)μm和28.2±1.6]和C24组[(33±3)μm和28.9±2.1],且以S24组的增高更为明显,差异均有统计学意义(t值为4.378～15.188,均P＜0.05);S12组和S24组Th17细胞比例[(3.3±1.1)%和(7.2±2.2)%]均明显高于C12组和C24组[(1.8±0.8)%和(2.0±0.6)%];S12组和S24组RORγtmRNA表达量[(25±4)和(35±3)]及IL-17的mRNA表达量[(26±3)和(36±3)]亦明显高于C12组[(10±5)和(13±5)]和C24组[(11±7)和(8±6)],以S24组增高更为明显,差异均有统计学意义(P＜0.05);S12组和S24组Th1细胞比例[(10.0±3.7)%和(26.2 ±6.0)%]、Th17/Th1细胞比例[(0.61±0.30)%和(1.82±0.52)%]及Tc1细胞比例[(17.0±4.5)%和(26.8±8.5)%]均明显高于C12组[(3.8±1.7)%、(0.27±0.17)%和(4.8±1.9)%]和C24组[(4.2±1.3)%、(0.28±0.11)%和(5.2±1.0)%],以S24组增高更为明显,差异均有统计学意义(P＜0.05);S12组和S24组小鼠Th17细胞与Th1、Tc1细胞比例、平均内衬间隔、DI值均呈显著正相关(r值为0.519～0.797,均P＜0.01);Th17/Th1细胞比例与平均内衬间隔、DI值呈显著正相关(r值分别为0.742和0.802,均P＜0.01);S12组和S24组CD4+IL-17+IL-21+细胞比例[(0.19±0.04)和(0.55±0.24)]明显高于C12组和C24组[(0.07±0.03)和(0.08±0.03)],S24组增高更为明显,差异均有统计学意义(P＜0.05).S12组和S24组的CD8+IL-21R+细胞比例[(2.94±1.26)和(4.12±2.26)]高于C12组和C24组[(1.22±0.31)和(1.34±0.18)](P＞0.05);S12组及S24组小鼠CD4+IL-17+IL-21+细胞比例与Th1、Tc1细胞比例、平均内衬间隔和DI值均呈显著正相关(r值为0.694～0.754,均P＜0.05);S12及S24组小鼠CD8+IL-21R+细胞比例与平均内衬间隔和DI呈显著正相关(r值分别为0.516和0.725均P＜0.05).结论 香烟暴露导致肺气肿小鼠肺内Th17细胞数量及活性上调,并随烟雾暴露时间延长而增强;Th17细胞通过IL-21及IL-21R在肺部Th1/Tc1炎症中起重要促进作用;这对探讨COPD肺部炎症和肺气肿发生机制以及新的治疗靶点具有重要意义.

Abstract：

Objective To evaluate the expression and the role of Th 17 in cigarette smoke-induced lung inflammation and emphysema in mice.Methods Forty male BALB/c mice were randomly divided into 4 groups, including a control group C12, a control group C24, a smoke-exposure 12 week group (S12) and a smoke-exposure 24 week group S24 (n = 10 each).Morphological changes were evaluated by mean linear intercepts and destructive index (DI).The proportion of CD4+ IL-17 + Th17, CD4+ IFN-γ+ Th1, CD4+ IL-17 +IFN-γ+ T( Th17/ Th1 ), CD8+ IFN-γ+ Tc1, CD8+ IL-21R + and CD4+ IL-17 + IL-21 + T cells in lungs of mice was determined by flow cytometry.The mRNA expressions of RORγt and IL-17 were evaluated by real-time PCR.Results Mean linear intercepts and DI were significantly higher in S12 and S24 groups [(39 ± 4)μm, (47 ±7) μm], (39.1 ± 1.6, 45.2 ±3.1 ) as compared to C12[(32 ±4) μm,28.2 ± 1.6] and C24groups [(33 ± 3 ) μm ,28.9 ± 2.1], all P ＜ O.05.The percentage of Th17 of S12 and S24 groups [(3.3 ±1.1 )%, (7.2 ±2.2)%] was significantly increased as compared with that of C12 and C24 groups [( 1.8± 0.8) %, (2.0 ± 0.6) %], all P ＜ 0.05.The mRNA levels of RORγt [( 25 ± 4), ( 35 ± 3 )] and IL-17 [(26 ± 3), (36 ± 3 )] in S12 and S24 groups were higher than in C12 [(10 ± 5 ), (13 ± 5 )] and C24 groups [( 11 ± 7 ), (8 ± 6)], all P ＜ 0.05.The percentage of Th 1, Th17/Th1 and Tc1 cells of S12 and S24 groups [(10.0 ±3.7)%, (26.2 ±6.0)%], [(0.61 ±0.30)%, (1.82 ±0.52)%], [(17.0±4.5 ) %, ( 26.8 ± 8.5 ) %] was significantly increased as compared with that of C12 [( 3.8 ± 1.7 ) %,(0.27±0.17)%, (4.8 ±1.9)%] and C24 groups [(4.2±1.3)%, (0.28±0.11)%, (5.2±1.0)%], all P＜0.05.Moreover, the frequency of Th17 cells had a positive correlation with Th1, Tc1 cells and emphysematous lesions ( r =0.519 - 0.797, all P ＜ 0.01 ).In addition, a positive correlation between Th17/Th1 cells and emphysematous lesions was also found (r =0.742, 0.802, all P ＜0.01 ).The percentage of CD4+ IL-17+ IL-21 +T cells was significantly increased in S12 and S24 groups [(0.19 ±0.04) %, (0.55 ± 0.24) %] compared to controls [(0.07 ± 0.03 ) %, (0.08 ± 0.03 ) %], all P ＜ 0.05.Meanwhile, as compared with that of the controls [( 1.22 ± 0.31 ), ( 1.34 ± 0.18 )], the percentage of CD8+ IL-21 R + T cells was also increased in SI 2 and S24 groups [( 2.94 ± 1.26 ), (4.12 ± 2.26 )], but there were no differences among smoke-exposure groups ( P ＞0.05 ).The frequency of CD4+ IL-17 + IL-21 + T cells had a positive correlation with Th 1, Tc1 cells and emphysematous lesions (r = 0.694 -0.754, all P ＜0.05).And the frequency of CD8+ IL-21R+ T cells also had a positive correlation with emphysematous lesions ( r = 0.516, 0.725, all P ＜ 0.05).Conclusions Cigarette smoke increased the expression and the activity of Th17 in mice.Th17 may play a potential (active) role in the development of lung inflammation through IL-21/IL-21R pathway.     

乳腺癌患者外周血CD4^＋CD25^＋ Treg细胞及Th1/Th2类细胞因子水平测定及意义

目的探讨乳腺癌患者外周血CD4＋CD2＋5调节性T细胞（CD4＋CD2＋5Treg）水平变化在肿瘤发生、发展中的作用及机制。方法流式细胞术检测45例健康人（对照组）、44例乳腺良性增生患者（增生组）、61例乳腺癌患者（乳腺癌组）外周血CD4＋CD2＋5Treg水平,ELISA法检测三组Th1类细胞因子IL-2、IFN-γ、TNF-α和Th2类细胞因子IL-6水平;分析CD4＋CD2＋5Treg与肿瘤临床病理特征及细胞因子的关系。结果乳腺癌组TNM分期Ⅲ、Ⅳ期患者外周血CD4＋CD2＋5Treg细胞水平显著高于0~Ⅱ期患者、对照组和增生组,且术后水平显著低于术前（P均〈0.05）;乳腺癌组TNM分期Ⅲ、Ⅳ期患者血清IL-2、IFN-γ、TNF-α水平明显低于0~Ⅱ期患者、对照组和增生组,IL-6水平则反之（P均〈0.05）;乳腺癌组外周血CD4＋CD2＋5Treg细胞水平与IL-2、IFN-γ、TNF-α水平呈负相关（r=-0.579、-0.607、-0.691）,与IL-6呈正相关（r=0.804）,P均〈0.05。结论外周血CD4＋CD2＋5Treg细胞水平与乳腺癌发生、发展有关,可能机制为通过调节Th1/Th2细胞因子平衡参与机体免疫应答。     

Feeding Administration of Daikenchuto Suppresses Colitis Induced by Naive CD4+ T Cell Transfer into SCID Mice

Background and Aims

Daikenchuto, a traditional Japanese herbal medicine, suppresses bacterial translocation by improvement of gastrointestinal motility and blood flow. As Daikenchuto reportedly reduces gastrointestinal inflammatory activity by these mechanisms, we analyzed whether Daikenchuto suppresses experimental colitis and reduces inflammatory cytokine expression in a mouse model.

Methods

Colitis was induced by transfer of naive CD4⁺ T cells of BALB/c mice into SCID mice, and mice were given either control or 2.7?% Daikenchuto-containing feed. We investigated body weight, clinical symptoms, histological changes, and Th1- and Th17-cytokine expression. Cytokine mRNA expression was analyzed using real-time RT-PCR. The ratio of IL-17⁺ and IFN-γ⁺ CD4⁺ T cells were analyzed by flow cytometry.

Results

Daikenchuto delayed the development of colitis and significantly reduced the histological inflammation scores. Analyses of cytokine mRNA revealed that Th17 cytokines were significantly decreased in colons of mice that received Daikenchuto. Absolute numbers of IL-17⁺ or IFN-γ⁺ CD4⁺ T cells per colon were less in mice receiving Daikenchuto than in mice that received control feed, as both groups received naive CD4⁺ T cells to induce colitis.

Conclusions

We demonstrated that feeding administration of Daikenchuto suppresses colitis induced by naive CD4⁺ T cell transfer into SCID mice. Daikenchuto may show clinical benefit in the treatment of human inflammatory bowel disease and further studies are warranted.     

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。     

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.     

成纤维滑膜细胞p53基因表达对类风湿关节炎患者CD4+T淋巴细胞的调节

目的 研究人成纤维滑膜细胞(FLS)p53基因表达对类风湿关节炎(RA)患者CD4+T淋巴细胞的调节作用.方法 采用小分子干扰RNA(siRNA)转染抑制FKS内p53基因表达,并与RA患者外周血CD4+T淋巴细胞共同培养.检测转染后FLS中骨保护素(OPG)表达及白细胞介素(IL)-6分泌.并对与之共培养的CD4+细胞膜胞质内蛋白干扰素(IFN)-γ、IL-17、IL-4和CD25以及IFN-γ孤儿核受体γt(RORγt)、IL-17、Foxp3 RNA水平进行测定.结果 p53基因被抑制后,FLS分泌IL-6减少,但OPG表达未受影响.p53基因被抑制的FLS使共培养的CD4+T淋巴细胞内IL-17及IFN-γ蛋白和RNA表达上调,但对CD4+T淋巴细胞RORγt RNA影响不大.虽可上调Foxp3表达,但CD4+CD25high细胞阳性率并无明显变化.结论 FKS内D53表达对RA外周血Th1、Th17有调节作用.     

Regulatory Role of CD4+CD25+Foxp3+ Regulatory T Cells on IL-17-Secreting T Cells in Chronic Hepatitis B Patients

Background

Both interleukin (IL)-17-secreting CD4⁺ T (Th17) and CD4⁺CD25⁺Foxp3⁺ T regulatory (Treg) cells have been shown to be associated with disease progression or liver damage in chronic hepatitis B (CHB) patients. However, the relationship between Treg cells and IL-17-secreting T cells in hepatitis B virus (HBV) infections is unclear.

Methods

The frequencies of Treg cells and IL-17-secreting T cells in hepatitis B e antigen (HBeAg)-positive CHB patients and healthy subjects were measured by flow cytometric analysis. The role of Treg cells on the differentiation of Ag-specific IL-17-secreting T cells was determined by removing the Treg cells from peripheral blood mononuclear cells (PBMCs) in HBeAg-positive CHB patients.

Results

The frequencies of both Th17 (1.71 ± 0.58 vs. 1.08 ± 0.36 %; P < 0.01) and Treg cells (8.92 ± 4.11 vs. 6.45 ± 1.56 %; P < 0.01) were increased in the peripheral blood of HBeAg-positive CHB patients compared with healthy controls, but in not the IL-17-secreting CD8⁺ T (Tc17) cells. The frequency of Treg cells was significantly associated with that of Th17 cells (r = 0.625, P = 0.001) in CHB patients. Spearman analysis showed a positive correlation between the frequency of Treg cells and HBV DNA load (r = 0.508, P = 0.008), as well as between the frequency of Th17 cells and serum alanine aminotransferase level (r = 0.516, P = 0.007). The deletion of Treg cells significantly enhanced both Th17 and Tc17 cell development in PBMCs following recombinant HBV core antigen stimulation.

Conclusions

Our data indicate a clear inverse relationship between Th17 cells and Treg cells and that Treg cells can inhibit Th17 cell development in CHB patients.     

慢性乙型肝炎患者应用拉米夫定前后T淋巴细胞亚群、CD4+CD25+调节性T淋巴细胞及白细胞介素-10、干扰素-γ的变化

目的 观察慢性乙型肝炎(CHB)患者应用拉米夫定抗病毒治疗前后外周血T淋巴细胞亚群、CD4+CD25+调节性T淋巴细胞(CD4+CD25+Treg)及IL-10、IFN-γ水平的变化.方法 选择CHB患者90例,在应用拉米夫定抗病毒治疗前及治疗后52周时,用流式细胞仪检测患者外周血T淋巴细胞亚群;在治疗前及治疗后12、24、36、52周时,用流式细胞仪检测外周血CD4+CD25+Treg频率,用双抗体夹心ELISA法检测IL-10、IFN-γ水平.组间及组内总体均数比较采用方差齐性的单因素方差分析或Dunnett's检验,组内治疗前后两时段均数比较采用配对t检验.结果 在90例CHB患者中,完全应答的32例患者的CD4+T淋巴细胞、CD8+T淋巴细胞及CD4+/CD8+较治疗前升高(t=4.055、3.267、2.328,均P＜0.05),外周血CD4+CD25+Treg频率在0、12、24、36、52周时分别为(5.40±0.60)%、(4.99±0.59)%、(4.54±0.72)%、(3.86±0.95)%、(3.44±0.76)%;IFN-γ水平、IFN-γ/IL-10逐步上升,IL-10水平逐步下降.部分应答的43例患者的CD4+T淋巴细胞及CD4+/CD8+较治疗前升高(t=3.484、2.018,均P＜0.05),CD8+T淋巴细胞较治疗前无明显差异,外周血CD4+CD25+Treg频率在0、12、24、36、52周时分别为(5.65±0.60)%、(5.23±0.63)%、(4.65±0.98)%、(4.42±0.97)%、(4.32±0.82)%,IFN-γ水平、IFN-γ/IL-10升高,IL-10水平降低.无应答的15例患者的外周血CD4+T淋巴细胞、CD8+T淋巴细胞及CD4+/CD8+、外周血CD4+CD25+Treg频率及IFN-y水平、IFN-γ/IL-10、IL-10水平较治疗前无明显变化.结论 应用拉米夫定治疗过程中,获得满意应答的CHB患者的外周血CD4+CD25+Treg频率下降,CD4+/CD8+、IFN-γ/IL-10的比例升高.

Abstract：

Objective To explore the correlation between the efficacy of lamivudine (LAM)therapy and changes of T lymphocyte subsets,CD4+ CD25+ regulatory T lymphocytes (Treg),and levels of interleukin-10 (IL-10)and interferon-gamma (IFN-γ)in the peripheral blood of patients with chronic hepatitis B (CHB).Methods Ninety CHB patients were enrolled in this study.T lymphocyte subsets in the peripheral blood were detected by flow cytometry at baseline and week 52 of LAM therapy.The frequencies of CD4+ CD25+ Treg in the peripheral blood were detected by flow cytometry and levels of IL-10 and IFN-γ were detected by enzyme-linked immunosorbent assay (ELISA)at baseline,week 12,24,36 and 52.The comparisons of overall means between groups and within groups were done by analysis of variance or Dunnett's test.The comparison of means before and after LAM therapy was done by paired t test.Results In 32 complete-responders of 90 CHB patients,the proportions of CD4+ T lymphocytes,CD8+ T lymphocytes and CD4+/CD8+ ratio were increased significantly after LAM therapy (t=4.055、3.267、2.328,all P＜0.05); the frequencies of CD4+CD25+ Treg at baseline,week 12,24,36 and 52 were (5.40±0.60)%,(4.99±0.59)%,(4.54± 0.72)%,(3.86±0.95)% and (3.44±0.76)%,respectively; the levels of IFN-γ,IFN-γ/IL-10 ratio were increased,while the IL-10 level was decreased after LAM therapy.In 43 partial-responders,the proportion of CD4+T lymphocytes and ratio of CD4+/CD8+ were increased after LAM therapy (t= 3.484,2.018,both P＜0.05); the proportion of CD8+ T lymphocytes was not changed significantly after therapy; the frequencies of CD4+ CD25+ Treg at baseline,week 12,24,36 and 52 were (5.65±0.60)%,(5.23±0.63)%,(4.65±0.98)%,(4.42±0.97)% and (4.32±0.82)%,respectively;IFN-γ level,IFN-γ/IL-10 ratio were increased,while IL-10 level was decreased.In 15 non-responders,the proportion of T lymphocyte subsets,the frequency of CD4+ CD25+ Treg,the levels of IFN-γ and IL-10 were not changed significantly after LAM treatment.Conclusions In CHB patients who have achieved response after LAM therapy,the frequency of CD4+ CD25+ Treg in the peripheral blood is decreased,while ratios of CD4+/CD8+ and IFN-γ/IL-10 in the peripheral blood are increased.     

Increased IL-4- and IL-17-producing CD8+ cells are related to decreased CD39+CD4+Foxp3+ cells in allergic asthma

Objective: In allergic asthma, regulatory T cell (Treg) number and function are decreased. Antigen-primed CD8⁺ T cells play an indispensable role in the full development of airway inflammation and airway hyper-responsiveness (AHR) occurring in asthma. In this study, we investigated the relationship between subpopulations of CD8⁺ T cells and CD39⁺ Tregs. Methods: Female C57BL/6 mice were used to develop the model of allergic asthma. Experimental mice were immunized with ovalbumin (OVA) by intra-peritoneal (i.p) injection and then challenged with OVA by intra-tracheal administration. Control mice were immunized with vehicle by i.p injection and challenged with OVA. Airway inflammation was determined by histology and AHR was measured by an invasive method. Levels of interferon (IFN)-γ, IL-4, and IL-17 in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay. The frequencies of CD8⁺IFN-γ⁺ cells (Tc1), CD8⁺IL-4⁺ cells (Tc2), CD8⁺IL-17⁺cells (Tc17), and CD39⁺Tregs were measured by flow cytometry. The correlation between CD39⁺Tregs and Tc subsets was analyzed by Pearson’s test. Results: Experimental mice displayed phenotypes of allergic asthma, including inflammatory cell infiltration into the lungs, goblet cell hyperplasia, increased airway resistance, and increased IL-4 and IL-17 in BALF. Compared to control mice, experimental mice displayed lower CD39⁺Tregs and Tc1 but higher Tc2 and Tc17. There was a negative correlation between CD39⁺Tregs and Tc2 or Tc17. Conclusion: In allergic asthma, increased Tc2 and Tc17 are possibly related to insufficient CD39⁺Tregs.     

Restoring homeostasis of CD4~+ T cells in hepatitis-B-virusrelated liver fibrosis

Immune-mediated liver injury is widely seen during hepatitis B virus(HBV) infection. Unsuccessful immune clearance of HBV results in chronic hepatitis and increases the risk of liver cirrhosis and hepatocellular carcinoma. HBV-related liver fibrosis(HBVLF),occurring as a result of HBV-induced chronic hepatitis,is a reversible,intermediate stage of chronic hepatitis B(CHB) and liver cirrhosis. Therefore,defining the pathogenesis of HBVLF is of practical significance for achieving better clinical outcomes. Recently,the homeostasis of CD4+ T cells was considered to be pivotal in the process of HBVLF. To better uncover the underlying mechanisms,in this review,we systematically retrospect the impacts of different CD4+T-cell subsets on CHB and HBVLF. We emphasize CD4+ T-cell homeostasis and the important balance between regulatory T(Treg) and T helper 17(Th17) cells. We discuss some cytokines associated with Treg and Th17 cells such as interleukin(IL)-17,IL-22,IL-21,IL-23,IL-10,IL-35 and IL-33,as well as surface molecules such as programmed cell death protein 1,cytotoxic T lymphocyte-associated antigen 4,T cell immunoglobulin domain and mucin domain-containing molecule 3 and cannabinoid receptor 2 that have potential therapeutic implications for the homeostasis of CD4+ T cells in CHB and HBVLF.     

Cell-contact-dependent activation of CD4+ T cells by adhesion molecules on synovial fibroblasts

Objective: To determine how cell–cell contact with synovial fibroblasts (SF) influence on the proliferation and cytokine production of CD4^+?T cells.

Methods: Naïve CD4^+?T cells were cultured with SF from rheumatoid arthritis patients, stimulated by anti-CD3/28 antibody, and CD4^+?T cell proliferation and IFN-γ/IL-17 production were analyzed. To study the role of adhesion molecules, cell contact was blocked by transwell plate or anti-intracellular adhesion molecule-1 (ICAM-1)/vascular cell adhesion molecule-1(VCAM-1) antibody. To study the direct role of adhesion molecules for CD4^+?T cells, CD161^+?or CD161^- naïve CD4^+?T cells were stimulated on plastic plates coated by recombinant ICAM-1 or VCAM-1, and the source of IFN-γ/IL-17 were analyzed.

Results: SF enhanced naïve CD4^+?T cell proliferation and IFN-γ/IL-17 production in cell-contact and in part ICAM-1-/VCAM-1-dependent manner. Plate-coated ICAM-1 and VCAM-1 enhanced naïve CD4^+?T cell proliferation and IFN-γ production, while VCAM-1 efficiently promoting IL-17 production. CD161^+?naïve T cells upregulating LFA-1 and VLA-4 were the major source of IFN-γ/IL-17 upon interaction with ICAM-1/VCAM-1.

Conclusion: CD4^+?T cells rapidly expand and secrete IFN-γ/IL-17 upon cell-contact with SF via adhesion molecules. Interfering with ICAM-1-/VCAM-1 may be beneficial for inhibiting RA synovitis.     

CD4~+CD25~+调节性T细胞在约氏疟原虫感染小鼠应答早期中的作用

目的探讨CD4+CD25+调节性T细胞在约氏疟原虫感染早期的作用及意义。方法用约氏疟原虫(致死型)感染DBA/2和BALB/c小鼠,计数红细胞感染率;在感染后第0d、3d、4d、5d和6d提取脾细胞应为,流式细胞术检测两种小鼠感染不同时间脾细胞悬液中CD4+T细胞和CD4+CD25+T细胞百分含量;ELISA法检测脾细胞培养上清IFNγ、IL10和TGFβ1水平。结果DBA/2小鼠的IFNγ水平在感染后第3d迅速升高后缓慢下降(P<0.01),CD4+CD25+T细胞数仅在感染后第5d出现有意义的升高(P<0.05),而IL10水平和CD4+T细胞数量都无明显改变。BALB/c小鼠的IFNγ水平在感染后第3d出现有意义的升高后迅速下降(P<0.01),CD4+CD25+T细胞数在感染后第3d开始明显升高,于感染后第5d出现下降(P<0.05),而IL10水平自感染后第3～6d持续维持高水平(P<0.05),CD4+T细胞数量于感染后第6天明显下降(P<0.05)。结论CD4+CD25+调节性T细胞在致死型约氏疟原虫感染BALB/c小鼠早期可能通过抑制性细胞因子IL10发挥免疫抑制作用。     

辅助性T细胞17和白细胞介素17在急性铜绿假单胞菌肺炎早期免疫应答中的变化及作用

目的:探究辅助性T细胞17(Th17细胞)在急性铜绿假单胞菌肺炎宿主早期免疫应答中的变化及白细胞介素17(IL-17)在此类感染中的作用。方法:应用酶联免疫吸附试验(ELISA)测定铜绿假单胞菌感染模型小鼠肺泡灌洗液(BALF)和磷酸盐缓冲液(PBS)对照组中IL-17、干扰素γ(IFN-γ)、IL-4、角质形成细胞趋化物(KC)、粒细胞集落刺激因子(G-CSF)、巨噬细胞炎症蛋白(MIP)-1α及MIP-2的浓度变化;流式分析IL-17的来源细胞;应用抗IL-17抗体探究IL-17的作用。结果:在小鼠感染模型中,IL-17在感染后4 h明显增高,且在8 h达到峰值,与PBS对照组相比具有显著的统计学差异(均P     

Mesenchymal stem cells improve the outcomes of liver recipients via regulating CD4+ T helper cytokines in rats

BACKGROUND:Bone marrow mesenchymal stem cells(BMMSCs) exert immunosuppressive activities in transplantation.This study aimed to determine whether BMMSCs reduce acute rejection and improve outcomes of liver transplantation in rats.METHODS:Orthotopic liver transplantation from Lewis to Brown Norway rats was performed,which was followed by the infusion of BMMSCs through the penile superficial dorsal vein.Normal saline infusion was used as a control.Animals were sacrificed at 0,24,72,or 168 hours after BMMSCs infusion.Liver grafts,and recipient serum and spleen tissues were obtained.Histopathology,apoptosis,serum liver enzymes,serum cytokines,and circulating regulatory T(Treg),Th1,Th2 and Th17 cells were assessed at each time point.RESULTS:BMMSCs significantly attenuated acute rejection and improved the survival rate of allogeneic liver transplantation recipients.Liver enzymes and liver apoptosis were significantly alleviated.The levels of the Th1/Th2 ratio-associated cytokines such as IL-2 and IFN-γ were significantly reduced and IL-10 was significantly increased.The levels of the Th17/Tregs axis-associated cytokines such as IL-6,IL-17,IL-23,and TNF-α were significantly reduced,whereas TGF-β concentration was significantly increased.Moreover,flow cytometry analysis showed that the infusion of BMMSCs significantly increased Th2 and Treg cells and decreased Th1 and Th17 cells.CONCLUSION:BMMSCs had immunomodulatory effects,attenuated acute rejection and improved outcomes of allogeneic liver transplantation in rats by regulating the levels of cytokines associated with Th1/Th2 and Th17/Treg ratios.     

The levels of IL-17A and of the cytokines involved in Th17 cell commitment are increased in patients with chronic immune thrombocytopenia

Th17 cells have been associated with immune-mediated diseases in humans but it has still not been determined whether they play a role in immune thrombocytopenia. We evaluated representative cytokines of the Th17, Th1, Th2 and Treg cell commitment in the serum of patients with chronic immune thrombocytopenia, as well as the cell source of IL-17A. Higher levels of IL-17A and Th17-related cytokines, and an increased percentage of IL-17A producing CD4+ and neutrophils were observed in patients. The levels of cytokines involved in Th1 cell commitment IFN-γ, IL-2, IL12-p70 and the percentages of Th1 cells were also increased, but IL-4 was not detected. Although the concentrations of IL-10 were higher, the levels of TGF-β were similar in both groups. In conclusion, our results point to a putative role for Th-17 cells/IL-17A cytokine in the pathogenesis of chronic immune thrombocytopenia.     

Human RORγt+ TH17 cells preferentially differentiate from naive FOXP3+Treg in the presence of lineage-specific polarizing factors

RORγt(+) T(H)17 cells are a proinflammatory CD4(+) T-cell population associated with autoimmune tissue injury. In mice, priming of T(H)17 requires TGF-β, which alone directs the priming of FOXP3(+) regulatory T cells (Treg), in association with inflammatory cytokines. Priming of human T(H)17 cells from conventional naive CD4(+) T cells under similar conditions, however, has proved difficult to achieve. Here, we report that differentiation of human T(H)17 cells preferentially occurs from FOXP3(+) naive Treg (NTreg) in the presence of IL-2 and IL-1β and is increased by IL-23 and TGF-β. IL-1β-mediated differentiation correlated with IL-1RI expression in stimulated NTreg and was accompanied by induction of RORγt along with down-regulation of FOXP3. IL-17-secreting cells in NTreg cultures cosecreted TNF-α and IL-2 and contained distinct subpopulations cosecreting or not cosecreting IFN-γ and other T(H)17-associated cytokines. Polarized NTreg contained significant subpopulations of CCR6-expressing cells that were highly enriched in IL-17-secreting cells. Finally, analysis of CCR6 expression with respect to that of IL-1RI identified distinct IL-17-secreting subpopulations that had maintained or lost their suppressive functions. Together our results support the concept that priming of human T(H)17 from naive CD4(+) T cells preferentially takes place from FOXP3(+) Treg precursors in the presence of lineage-specific polarizing factors.