TDO调节芳烃受体通路对诱导移植肝脏低免疫应答的影响及机制研究

The liver is known as an immunologically privileged organ in solid organ transplants. However, its mechanism is to be further understanding. Recently, it shows that the tryptophan metabolism take an important role in the induction of the womb immune tolerance and tumor immune-escape. Our preliminary study found that the expression of the tryptophan catabolism speed limit enzyme, Tryptophan 2,3-dioxygenase (TDO), is effected by reaction degree of the graft rejection in allogenic rat transplant liver tissue. The data also showed that this result was closely related to the inhibition of cell toxicity T lymphocyte proliferation. Moreover, we observed that applying a tryptophan metabolite, kynurenine (Kyn) derivant could alleviate the extent of graft rejection in rat liver transplantation model. A study in 2011 《NATURE》 firstly reported that Kyn is an endogenous ligand of aryl hydrocarbon receptor (AhR). The combination can activate aryl hydrocarbon receptor path to induce tumor immune escape. These indicate the existence of TDO - Kyn - AhR signal pathway. Therefore, we speculate that TDO which only express in hepatocytes participates in graft immune preference. The mechanism might be regulating tryptophan metabolism and kynurenine generation, control of TDO - Kyn - AhR pathway, suppressive of T cell proliferation and induction of Treg cells generation resulted in graft low immune response or immune tolerance. We propose to confirm our speculation by using TDO knockout rat as donor in liver transplantation model combined with human liver sample. This study is going to clarify the effects and its mechanism of TDO in inducing immune tolerance in liver transplantation. The results may help to better understanding of immune preferential phenomenon of liver graft, which probably bring new prospective for inducing clinical immune tolerance.

移植肝脏免疫特惠现象机制仍待研究。已知色氨酸（Try）分解代谢与免疫应答密切相关。我们前期研究发现大鼠移植肝Try分解代谢限速酶色氨酸-2,3-双加氧酶（TDO）表达与移植排斥反应程度相关，使肝细胞高表达TDO可抑制细胞毒性T细胞增殖；且加用Try分解代谢产物犬尿氨酸（Kyn）的衍生物可显著减轻大鼠肝移植排斥反应，大幅减少环孢霉素应用剂量。新据Nature报道 Kyn 与芳烃受体（AhR）结合参与肿瘤免疫逃逸。由此推测，作为仅在肝细胞内表达的Try代谢酶，TDO参与移植肝免疫特惠，机制为调节Try代谢和Kyn产生，调控TDO-Kyn-AhR通路，致移植肝免疫低应答或无应答。我们拟采用CRISPR/Cas9技术构建TDO基因敲除大鼠肝移植模型，基因芯片分析Kyn-AhR调控T细胞增殖分化靶点，结合临床病理标本，研究并明确TDO在肝脏免疫特惠中的作用及分子机制，为开发致耐受新药提供理论依据。

背景：临床上移植肝脏免疫特惠现象机制仍待研究。我们前期研究发现色氨酸（Try）与其分解代谢限速酶色氨酸-2,3-双加氧酶（TDO）高表达可抑制细胞毒性T细胞增殖，加用Try分解代谢产物犬尿氨酸（Kyn）的衍生物可显著减轻大鼠肝移植排斥反应。Kyn为芳烃受体（Ahr）内源性配体，结合后参与肿瘤免疫逃逸。综上，调控TDO-Kyn-Ahr通路，可能致移植肝免疫低应答或无应答。 研究内容：（1） Kamada“二袖套法” 建立大鼠原位肝移植免疫排斥及耐受模型。利用TDO抑制剂680C91进行处理；（2）敲除/增加 TDO 基因表达对自发免疫耐受/排斥的影响；（3）研究 Kyn-Ahr 途径在 TDO 介导的免疫抑制反应中的作用；（4）探索 Kyn-Ahr 调控T细胞免疫耐受的机制；（5）临床标本研究 TDO 酶及 Ahr 靶基因表达变化与排斥反应相关性。重要结果：(1) 成功建立大鼠原位肝移植模型。(2) 在排斥组中，移植肝组织病理结果表明：术后免疫排斥反应RAI 评分在 72h 逐渐增强，抑制TDO后，排斥反应加重，同时生存时间减少。检测Try及Kyn表达情况，发现排斥组TDO表达增加后，Kyn含量增加，而抑制TDO后，Kyn水平降低，导致排斥反应加剧。进一步检测Ahr的靶基因CYP1A1、CYP1B1，我们发现表达趋势与Kyn一致。(3) 体外细胞实验中，抑制TDO表达，Kyn明显降低，CYP1A1、CYP1B1表达减少，SI值降低。抑制Ahr表达后，CYP1A1、CYP1B1表达降低，而SI值增加，而额外补充Kyn后，SI值降低。敲减 Ahr 后与组对照相比，SI皆明显降低，各处理组之间亦符合Kyn变化趋势。关键数据极其科学意义：（1）术后 TDO、CYP1A1、CYP1B1 表达及 Kyn、Trp 浓度变化表明，TDO 介导 Trp-Kyn-Ahr 通路能一定程度上影响大鼠肝移植术后免疫排斥反应，TDO 在大鼠肝移植术后肝组织中表达增加，抑制 TDO 酶活性能一定程度上加重大鼠肝移植免疫排斥反应。（2）体外实验证明高浓度 Kyn 能抑制淋巴细胞增殖，诱导肝细胞免疫耐受。下调 Ahr、降低 Kyn 浓度都可以导致抑制淋巴细胞增殖功能降低。本项目研究成果在以Cell Death & Disease，Oncogene等为代表的期刊发表中英文论文12篇。