DGKαζ双缺失小鼠—— 一种模拟临床自身免疫性肝炎的小鼠模型

Human autoimmune hepatitis (AIH) is a form of chronic portal inflammation of unknown etiology. Progress in studying the pathogenesis of AIH has been hindered by the lack of a reliable animal model. The critical role of innate immune response in AIH has been reported, but the role for adaptive immunity in AIH remains unclear. Diacylglycerol (DAG) kinases (DGKs) play a synergistic and critical role in maintaining peripheral immune tolerance. We have found that DGK α or ζ negatively regulated T cell receptor (TCR)-mediated activation, and that T cells deficient in both DGKα and ζ were spontaneously activated, correlating with elevated Erk1/2 activation. Most importantly, DGKαζ-deficient mice developed spontaneous AIH that could be adoptively transferred to T cell deficient mice by DGKαζ-deficient T cells, but suppressed by wild-type Tregs. The chronic nature of AIH (including severe jaundice and liver fibrosis with elevation of serum ALT, AST, total and direct bilirubin levels and anti-DNA antibodies) and development of liver fibrosis into liver cirrhosis, observed in DGKαζ-deficient mice, have not been identified in most other animal models of AIH, and thus DGKαζ-deficient mice provide a unique new model for exploring the pathogenesis of both AIH and liver fibrosis (a common outcome of chronic liver inflammatory disease). We will address the hypothesis that DGKα and ζ synergistically regulate DAG metabolism and thereby play a pivotal role in hepatic self-tolerance, maintaining T-cell quiescence and promoting T cell anergy and Treg function in the liver. By use of conventional and conditional DGKαζ-deficient mice and parallel studies on clinical biopsies and blood samples from AIH patients, we will investigate the mechanisms by which DGKα and ζ regulate liver self-tolerance and the pathogenesis of AIH and thereby identify novel therapeutic strategies for effective prevention and treatment of AIH.

自身免疫性肝炎(AIH) 发病机制不清，缺乏准确模拟临床AIH的动物模型是目前必需突破的瓶颈。甘油二酯激酶(DGK)是调节外周免疫耐受的重要分子。我们发现：DGKαζ双缺失小鼠T细胞能自发激活；DGKαζ双缺失小鼠自发发生AIH，并慢性化发展；将AIH小鼠的T细胞过继转移至T细胞缺陷小鼠可诱发AIH，而野生型Treg则可抑制AIH发生；AIH小鼠呈现黄疸，以及谷丙转氨酶、谷草转氨酶和胆红素水平居高不下，自身抗核抗体阳性，并进展为肝纤维化、肝硬化。因此，我们提出“DGKα和ζ协同调节肝脏T细胞内DAG代谢，维持T细胞静息、诱导T 细胞无能和促进Treg 功能，在肝脏免疫耐受中起决定作用”的假说。本项目拟利用该AIH小鼠模型及其条件性缺陷小鼠模型，佐于AIH患者活检肝组织和外周血的临床验证研究，揭示DGKαζ作为关键节点维持肝脏免疫耐受和调控AIH发生发展的机制，为AIH有效防治提供新策略。

自身免疫性肝炎(AIH)发病机制不清，缺乏准确模拟临床AIH的动物模型是目前必需突破的瓶颈。甘油二酯激酶(DGK)是调节外周免疫耐受的重要分子。我们发现：DGKα和ζ双缺失小鼠肝脏功能明显下降，肝内大量淋巴细胞浸润证实其能自发诱导形成自身免疫性肝炎；DGKα和ζ双缺失小鼠脾脏中CD4+和CD8+T细胞比例都有所降低，尤其是CD8+T细胞的绝对数量明显减少，导致T细胞自发激活；延长DGKα和ζ双缺失小鼠生长时间证实6月龄DGKα和ζ双缺失小鼠肝内T细胞浸润和肝内胶原纤维含量较4月龄小鼠明显增加；DGKα和ζ双缺失T细胞主要通过CD8+T细胞诱导肝内炎症；DGKα和ζ双缺失小鼠模型胸腺、脾脏和外周淋巴结中DGKζ几乎不表达；DGKαζ条件性敲除小鼠上调CD44、CD25等T细胞活化标志，增加IFN-γ表达，介导外周CD4+T细胞免疫无能抵抗；DGKαζ双缺失的CD8+T细胞出现静息抵抗，mTOR复合物1相关信号通路的活参与了DGKαζ双缺失导致的T细胞静息状态打破；转染miR-223的骨髓间充质干细胞缓解小鼠自身免疫性肝炎；灌胃给予自身免疫性肝炎小鼠丁酸钠后，能够缓解自身免疫性肝炎相关的炎症关键基因和蛋白的变化。因此，我们提出“DGKα和ζ协同调节肝脏内T细胞的DAG代谢，维持T细胞静息、诱导T 细胞无能和促进Treg功能，在肝脏免疫耐受中起决定作用”假说。本项目拟利用该AIH小鼠模型及其条件性缺陷小鼠模型，佐于AIH患者活检肝组织和外周血的对照研究，揭示DGKα和ζ作为关键节点维持肝脏免疫耐受和调控AIH发生发展的机制，为AIH有效防治提供新策略。