IL-27预处理后的T细胞在干预自身免疫性肝病小鼠模型中的研究

Objective: to study the roles of IL-27-primed T cells in inducing immune tolerance as a result of preventing experimental Autoimmune Liver Disease (AILD). Backgrounds: AILD is a group of immunologically induced hepatic damage that is either hepatocellular or cholestatic. Genetic predisposition, auto-reactive T cells, and defects in numbers and function of regulatory cells (Tregs) have all been demonstrated in AILD as the possible mechanisms of loss of self-tolerance. Recently, a key area of investigation concerns that the altered balance of pro- and anti-inflammatory CD4+ T cell subsets, namely Th17 and Treg, might be responsible in the induction and perpetuation of aberrant immune response. In this setting, targeting Treg/Th17 balance may represent an encouraging tool for AILD treatment in the next future. IL-27 is a heterodimeric cytokine with broad pro- and anti-inflammatory properties that belongs to the IL-6/IL-12 family. Our previous study demonstrated that IL-27-primed T cells prevent mice from severe Experimental Autoimmune Encephalomyelitis (EAE). In the present study, we aim to explore immunosuppressive roles of IL-27 in AILD. Methods: To generate animal models that mainly reflect the hepatocellular and cholestatic features of AILD, we developed a model in which male RAG-/- mice will be reconstituted with pathological liver cells from Scurfy mice. Scurfy is an X-linked recessive mouse mutant that suffers from an aggressive T cell-mediated autoimmune disease with multi-organ inflammation resulting from. The nature of this model manifests select immunological and clinical features of AILD in humans. Our central hypothesis states that donor liver's auto-reactive cells will be able to home to the recipient liver and therefore induce the autoimmune inflammation phenotype in recipient liver. This model will be subsequently studied in comparison to T-cell mitogenic agent Concanavalin A (Con-A)-induced liver inflammation. Next, we will isolate highly purified populations of both non-Treg (GFP-) and Treg CD4 T cells (GFP+) from mice expressing a GFP-Foxp3 fusion protein. Following IL-27 pretreatment and adoptive transfer to recipient animals, we will examine whether some of the na?ve conventional CD4+GFP- cells can differentiate into GFP+ iTreg cells, and thus play therapeutic role in experimental AILD model. Aims: to explore possible roles of the action of IL-27 on generation of suppressive T cells and inhibition of Th17 cell-mediated pathology during liver injury. We will further characterize the phenotype of suppressive T cells in liver, exam their inhibitory function in vitro; and also identify accessory function of host liver cells, such as hepatic stellate cells on liver-associated Treg induction. Finally, the gene expression profile of IL-27-primed T cells before, during and after induction of liver injury will be analyzed to understand molecular and genetic mechanisms underlying effects of IL-27 on the course of experimental AILD.

白细胞介素-27 (IL-27)是IL-6/IL-12家族的新成员，具有促进炎性反应和抗炎性反应的双重作用。我们既往的研究表明, 经IL-27预处理过后的初始T淋巴细胞能有效的预防小鼠实验性自身免疫性脑脊髓炎(EAE)的发生，预示其免疫调节功能在临床上存在潜在应用价值。据报道，自身免疫性肝病(AILD)在中国的发病率逐年增高，临床治疗方法仍有待改进。本课题将分别用刀豆蛋白A(Con-A)诱导的及Scurfy小鼠肝脏自身反应性细胞诱导的肝损伤作为AILD模型，将观察IL-27 预处理过后的初始T细胞对AILD的干预效果；通过跟踪它在宿主肝组织及外周淋巴结内的转归，探讨①作为治疗用途的T细胞输入宿主后，在炎症过程中的表型和转归；②是否通过干预宿主体内Treg/Th17细胞比例来发挥免疫调节功能；③进一步通过免疫学、分子生物学、高通量基因组学等手段阐明其作用机制；争取为找到治疗AILD的新靶点。

项目背景：自身免疫性肝病(AIH)在中国的发病率逐年增高，临床治疗方法仍有待改进。白介素-27(interleukin-27,IL-27)是近年来新发现的IL-12家族成员,主要由激活的抗原递呈细胞、巨噬细胞和树突状细胞产生,在自身免疫疾病和宿主抗感染过程中起着重要作用。我们我们既往的研究表明, 经IL-27预处理过后的初始T淋巴细胞能有效的预防小鼠实验性自身免疫性脑脊髓炎(EAE)的发生，预示其免疫调节功能在临床上存在潜在应用价值。..主要研究内容：本课题分别用刀豆蛋白A(Con-A)诱导的、CYP2D6腺病毒感染诱导的及Scurfy小鼠肝脏自身反应性细胞诱导的肝损伤作为AIH模型，来观察IL-27 预处理过后的初始T细胞对AIH的干预效果；并跟踪它在宿主肝组织及外周淋巴结内的转归。..方法与结果：在本次研究中，我们先用IL-27 预处理初始T细胞，再观察其对用刀豆蛋白A(Con-A)及CYP2D6腺病毒感染诱导AIH的干预效果。我们发现IL-27 预处理初始T细胞可有效抑制CYP2D6腺病毒感染诱导的AIH模型。并抑制小鼠肝脏的炎性反应及肝纤维化的进程。我们接下借助与华大基因新的LHC-BS检测－分析平台。我们对比了正常肝组织、AIH模型的肝脏组织及IL－27处理AIH模型后的肝脏组织。经过纯化分离的组织后，通过基于Illumina的液相杂交捕获的亚硫酸氢盐修饰结合直接测序法（the optimized liquid hybridization capture-based bisulfite sequencing (LHC-BS) approach），由8对组织分析1.86百万个CpG位点（启动子区），建立整体的甲基化谱。并通过结合亚硫酸氢钠处理和酶解分析法及亚硫酸氢盐测序，对8个在肝纤维化中显著高甲基化的基因进行验证。另外，我们发现了一个抑癌基因（CSMD2）、一个致癌基因（EXT1）。..科学意义：综上所述，IL-27除了促进Th1型免疫反应和抑制Th17细胞以外，还可以通过调节Treg细胞，以及对基因表观遗传调节作用，从而可能具有抗肿瘤的作用。