CNI induced Th17/Treg imbalance and susceptibility to renal dysfunction in renal transplantation

Calcineurin inhibitors (CNI) prevent graft rejection by blocking interleukin-2 (IL-2), which was required for development and function of Foxp3⁺CD4⁺CD25⁺ regulatory T cells (Treg). Recently, IL-2 was reported to play a part in the inhibition of Th17 cells. The renal transplantation recipient who used CNI regularly might have Th17/Treg imbalance with increased Th17 cells and decreased Treg cells, which would cause renal dysfunction even rejection. To assess the effect of CNI on Th17 cells and Treg cells, we included 123 renal transplantation recipients (101 in a stable stage and 22 with renal dysfunction) and 27 healthy volunteers. Among all the recipients, 103 recipients used CNI and 20 recipients used sirolimus without CNI. The recipients who used CNI were further classified into four groups according to the blood levels of CNI: Of all these subjects, Th17 and Treg frequencies in the peripheral blood were analyzed by flow cytometry (FCM). Serums IL-17, IL-23, IL-6, IFN-r, and TGF-β were analyzed by ELISA. The results demonstrated that the transplantation recipient treated by CNI revealed an obvious increase in peripheral Th17 frequencies and a significant decrease in Treg frequencies when compared with the sirolimus group and healthy people (P < 0.05). Even more, the transplantation recipient with renal dysfunction had the highest level of Th17 cells (P < 0.05) while the lowest Treg cells compared with stable recipient and healthy control, with increased serums IL-6 and IL-17. Our results indicated that CNI was associated with Th17/Treg imbalance in peripheral blood, which supported the followed generation of renal dysfunction after transplantation.     

Obaculactone suppresses Th1 effector cell function through down-regulation of T-bet and prolongs skin graft survival in mice

Allograft rejection is a predominantly Th1 immune response. In this study, we showed that obaculactone, a natural compound derived from citrus fruit, prolonged skin graft survival in mice when treated after but not before transplantation. Furthermore, obaculactone inhibited alloantigen-specific production of Th1 cytokine IFN-γ as well as proinflammatory cytokine IL-2, TNFα and IL-6. In parallel, IL-10 production was markedly up-regulated. Obaculactone significantly enhanced the percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in the CD4⁺ splenocytes without any effect on their inhibitory function. In vitro and in vivo tests showed obaculactone down-regulated T-bet expression in Th1 effector cells. Taken together, the unique immunomodulatory properties might qualify obaculactone as a putative, therapeutic compound for the treatment of Th1-driven diseases, including transplant rejection.     

3, 3′-diindolylmethane alleviates steatosis and the progression of NASH partly through shifting the imbalance of Treg/Th17 cells to Treg dominance

This study was designed to discuss the effects of 3, 3′-diindolylmethane (DIM) on methionine–choline-deficient (MCD)-diet induced mouse nonalcoholic steatohepatitis (NASH) and the potential mechanisms. NASH mice were administrated with or without DIM at different concentrations for 8 weeks. Both the in-vivo and in-vitro effects of DIM on Treg/Th17 imbalance during NASH progression were analyzed. The in-vivo blocking of CD25 or IL-17 was performed to respectively deplete respective function of Treg or Th17 subset. Besides, with the assistance of AhR antagonist CH223191 and anti-TLR4 neutralizing antibody, we designed the in-vitro DIM-incubation experiments to discuss the roles of aryl hydrocarbon receptor (AhR) (CYP1A1, CYP1B1) and toll-like receptor 4 (TLR4) on DIM's effects when shifting Treg/Th17 imbalance. Notably, in NASH mouse models, DIM alleviated hepatic steatosis and inflammation, and shifted the Treg/Th17 imbalance from MCD diet-induced Th17 dominance to Treg dominance. In-vitro, DIM not only significantly up-regulated the mRNAs of Foxp3 (Treg-specific) in purified spleen CD4⁺ T cells, but also enhanced the immunosuppressive function of these Treg cells. Besides, DIM significantly up-regulated the proteins of CYP1A1 and CYP1B1 whereas down-regulated those of TLR4 on CD4⁺ T cells from MCD-diet mice. Moreover, blocking AhR attenuated while blocking TLR4 enhanced the effects of DIM when regulating Treg/Th17 imbalance. Conclusively, DIM could be used as a potential therapeutic candidate to treat NASH based on its dramatic induction of Treg dominance to alleviate intra-hepatic inflammation, suggesting us a clue that the dietary cruciferous vegetables (containing abundant DIM) might exist as a protective factor for patients with NASH-related liver diseases.     

CD4+ Foxp3+ regulatory T cells induced by TGF-β, IL-2 and all-trans retinoic acid attenuate obliterative bronchiolitis in rat trachea transplantation

Obliterative bronchiolitis (OB) is the major obstacle for long-term allograft survival in lung transplantation, and the underlying mechanism is still not well understood. Regulatory T cells (Tregs) have been shown to be essential in the maintenance of immune tolerance. In this study we investigated the role of Tregs in protecting OB in rat. We show that the combination of TGF-β, Interleukin (IL)-2, and all-trans retinoic acid (atRA) could induce naïve rat CD4⁺CD25⁻ T cells to differentiate into CD4⁺CD25⁺Foxp3⁺ T cells in vitro, and they acquired suppressive function. In a rat orthotopic tracheal transplantation OB model, the adoptive transfer of the induced Tregs reduced symptoms of airway obliteration and fibrication of grafts when compared with adoptive transfer of control cells without suppressive property. Moreover, recipients treated with the induced Tregs secreted high level of immunosuppressive cytokine TGF-β and IL-10, and low level of pro-inflammatory cytokines IL-17, IFN-γ, IL-6, and MCP-1, and had fewer effector T cells including Th17 cells and Th1 cells in the graft. Taken together, these findings suggest that in vitro induced Tregs by the combination of TGF-β, IL-2, and atRA are effective in protecting rat trachea allograft rejection through the inhibition of effector T cells and their function. These datas implicate new therapies to prevent OB and allograft rejection in human lung transplantation.     

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.     

Increased IL-10/IL-17 ratio is aggravated along with the prognosis of patients with chronic lymphocytic leukemia

ObjectiveThis study is to investigate the association between the Treg/Th17 cells and prognosis of chronic lymphocytic leukemia (CLL).MethodsTotally 50 CLL patients and 20 Health controls were included in this study. Regulatory T (Treg) cells and the cell subset secreting IL-17 (Th17) in peripheral blood were detected with flow cytometry. Serum levels of IL-10 and IL-17 were determined with ELISA, and expression of Foxp3 and RORγt was assessed with quantitative real-time PCR.ResultsTreg and Th17 cell proportions in peripheral blood in the CLL patients were significantly higher than control. Serum levels of IL-10 and IL-17, and expression of Foxp3 and RORγt, were significantly increased in the CLL patients. Ratios of Treg/Th17 and IL-10/IL-17 were significantly elevated in the CLL patients. Compared with those before treatment, Treg/Th17 and IL-10/IL-17 ratios were declined in the CLL patients in remission. Compared with the non-remission group, Treg cells were significantly decreased, while Th17 cells were significantly increased, resulting in decreased Treg/Th17 ratio, in the remission group. Moreover, the serum IL-10 level was significantly decreased, while the serum IL-17 level was significantly increased, resulting in declined IL-10/IL-17 ratio, in the remission group. Correlation analysis showed that, Treg and Th17 cell counts were significantly associated with CD38 and ZAP-70 expression in the CLL patients. Moreover, the IL-10/IL-17 ratio was also significantly associated with CLL prognostic factors.ConclusionAltered Treg/Th17 and IL-10/IL-17 ratios in CLL would be aggravated along with the disease progression, which might be used as indicators for the disease prognosis.     

CD4＋CD25＋Treg调节性T细胞及其细胞因子在大鼠动脉粥样硬化中改变的研究

目的 研究CD4＋ CD25＋ Treg调节性T细胞及其分泌的细胞因子在大鼠动脉粥样硬化中的改变.方法 制备动脉粥样硬化模型,取雄性SD大鼠30只,随机分为正常饮食组（A组）10只及高脂饮食组（B组）20只,高脂饮食组再分为对照组与干预组各10只,干预组高脂饲料喂养前腹腔注射抗CD25＋单克隆抗体250μg,A组及对照组腹腔注射大鼠IgG抗体（250μg）,高脂饮食组定期用维生素D腹腔注射.上述各组喂养3个月后,各组随机抽取6只大鼠通过流式细胞学技术检测CD4＋ CD25 ＋Treg细胞及Foxp3＋的表达、检测炎症因子IL-10,IL-1β水平,处死各组大鼠做病理切片.结果 3组中干预组CD4＋ CD25＋ Treg细胞及Foxp3＋表达下降最明显,IL-10水平明显下降,IL-1β水平明显升高,A组CD4＋ CD25＋ Treg细胞及Foxp3＋表达最高,IL-10水平亦最高,IL-1β水平最低.而对照组在干预组及A组之间.结论 动脉粥样硬化形成中CD4＋ CD25＋ Treg调节性T细胞及其分泌的细胞因子受到抑制,而不是CD4＋ CD25＋ Treg调节性T细胞分泌的因子却显著升高,考虑动脉粥样硬化的形成有免疫反应参与.     

药物流产出血模型小鼠雌、孕激素水平与Th17/Treg亚群的关系#br#

摘要：目的　研究药物流产出血模型小鼠的雌、孕激素水平与Th17/Treg亚群的关系。方法　选取清洁级Balb/c雌性小鼠32只,通过随机数字表法分为正常妊娠组（A组）、小剂量米非司酮组（B组）、大剂量米非司酮组（C组）、米非司酮+宫清颗粒组（D组）,每组8只。B、C、D组小鼠于妊娠第6天时分别使用米非司酮0.88 mg·kg-1·d-1、1.5 mg·kg-1·d-1及米非司酮（1.5 mg·kg-1·d-1）+宫清颗粒组（308.16 mg·kg-1·d-1）灌胃。采用改良碱性正铁血红素法检测子宫出血量;采用放射免疫法检测血清雌二醇（E2）、孕酮（P）水平;采用流式细胞术双染色法检测子宫组织Th17（CD3+CD4+IL-17A+）、Treg（CD4+CD25+Foxp3+）及Th17/Treg亚群比值;采用逆转录聚合酶链式反应（RT-PCR）法检测子宫组织白细胞介素（IL）-17A、IL-10、转化生长因子（TGF）-β mRNA水平。结果　B、C、D组子宫出血量依次减少。药物流产后,A、B、C、D组小鼠E2水平依次升高,而P水平依次降低。Th17亚群依次升高,Treg亚群依次降低,Th17/Treg亚群比值依次升高（均P＜0.05）。IL-17A mRNA相对表达量依次升高,而IL-10、TGF-β mRNA相对表达量依次降低（均P＜0.05）。血清E2与Th17、Th17/Treg和IL-17A水平呈正相关,与Treg、IL-10和TGF-β水平呈负相关;血清P与Treg、IL-10和TGF-β水平呈正相关,与Th17、Th17/Treg和IL-17A水平呈负相关（均P＜0.05）。结论　药物流产后的异常子宫出血血清E2、P水平变化与Th17/Treg亚群比值变化关系密切,诱导Th17/Treg亚群向Th17偏移有助于减少子宫出血。     

The JAK2 inhibitor AG490 regulates the Treg/Th17 balance and alleviates DSS-induced intestinal damage in IBD rats

The pathogenesis of inflammatory bowel disease (IBD) remains unclear, and it is currently believed that an imbalance in regulatory T (Treg) cells/T helper 17 cells (Th17 cells) is related to the occurrence and development of IBD. Recently, the JAK2 inhibitor AG490 has been used in animal models such as rheumatoid arthritis and bronchial asthma models and shown to exert immunoregulatory functions that improve disorder in the Treg/Th17 cell balance. This study aimed to evaluate the effect of AG490 on the intestinal inflammatory process in an IBD rat model. A dextran sulfate sodium (DSS)-induced IBD rat model was established, and disease activity index (DAI) scores were calculated. The histopathological damage score was determined by haematoxylin-eosin (H&E) staining. Treg/Th17 cells in the spleen were detected by flow cytometry. The levels of interleukin (IL)-10, IL-6 and IL-17A were detected by enzyme-linked immunosorbent assay (ELISA). AG490 attenuated DSS-induced IBD injury by regulating the Treg/Th17 balance and related cytokine secretion to reduce the DAI and colonic tissue damage. Thus, AG490 may be a new method for effective treatment of IBD.     

Th17/Treg平衡在类风湿关节炎中作用的研究进展

类风湿性关节炎(rheumatoid arthritis,RA)是一种以慢性多关节滑膜炎为主要特征的自身免疫性疾病。虽然目前对于RA的确切发病机制尚不明确,但一般认为和T细胞相关。最近研究发现调节性T细胞(regulatory T cell,Treg)和Th17细胞在RA的发生发展中发挥重要作用。Th17细胞能够分泌促炎症因子IL-17,通过诱导基质金属蛋白酶(ma-trix metallo proteinases,MMPs)和破骨细胞生成,促进骨滑膜炎症、骨和关节损伤;而Treg则通过释放抑制性细胞因子IL-10和TGF-β发挥免疫效应,调控RA中的炎症性免疫应答过程。单独TGF-β作用下诱导初始T细胞分化为Treg,而在TGF-β和IL-6共同作用下诱导初始T细胞分化为Th17细胞,因此,Th17和Treg细胞在特定的细胞因子微环境下可以相互转化。调节Th17/Treg之间的平衡可能成为治疗RA的新方法。该文将对Th17/Treg平衡在RA发生发展中的调节作用作一综述。     

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.     

Alloantigen specific T regulatory cells in transplant tolerance

CD4⁺CD25⁺Foxp3⁺T cells are regulatory/suppressor cells (Treg) that include non-antigen(Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naïve CD4⁺CD25⁺Treg develop into specific Tregs is unknown. We have studied DA rats tolerant to fully allogeneic PVG cardiac grafts that survived with out immunosuppression for over 100 days and identified the cellular basis of alloantigen specific tolerance. Key observations from our studies will be reviewed including how CD4⁺CD25⁺Tregs were first identified and the cytokine dependence of CD4⁺T cells that transfer alloantigen specific transplant tolerance which died in culture unless stimulated with both cytokine rich ConA supernatant and specific donor alloantigen. Both the tolerant CD4⁺CD25⁺ and CD4⁺CD25⁻ T cell populations are required to transfer tolerance, yet alone the CD4⁺CD25⁻ T cell effect rejection. Tolerance transfer occurs with a low ratio of CD4⁺CD25⁺T cells (< 1:10), whereas to induce tolerance with naive CD4⁺CD25⁺T cells requires both a ratio of > 1:1 and is not alloantigen specific.Recent findings on how naïve CD4⁺CD25⁺T cells developed into two separated pathways of alloantigen specific Tregs, by culturing them with alloAg with either IL-2 or IL-4 and donor alloantigen are described. IL-2 enhances IFN-γR and IL-5 mRNA while IL-4 induced a reciprocal profile with de novo IL-5Rα and increased IFN-γ mRNA expression. Both IL-2 and IL-4 alloactivated CD4⁺CD25⁺Tregs within 3–4 days of culture can induce alloantigen specific tolerance at ratios of 1:10. Long term, CD4⁺CD25⁺T cells from tolerant hosts given IL-2 cultured cells have increased IL-5 and IFN-γR mRNA; whereas hosts given IL-4 cultured cells had enhanced IL-5Rα mRNA expression and IL-5 enhanced their proliferation to donor but not third party alloAg.These findings suggest that Th1 and Th2 responses activate two pathways of alloantigen specific Tregs that can mediate transplant tolerance but are dependent upon cytokines produced by ongoing Th1 and/or Th2 immune responses.