肝脏MAIT细胞在NAFLD进展为T2DM中的作用机制研究

Nonalcoholic fatty liver disease (NAFLD) is an independent risk factor for type 2 diabetes (T2DM). Recent studies demonstrate that the chronic inflammation resulted from immune response in liver, particularly M1 macrophage polarization, participates in the impaired glucose metabolism. However, the underlying provoking mechanism remains unclear. Mucosal-associated invariant T (MAIT) cells are a novel subset of innate-like immune cells, which are abundant in human liver and activated by host-derived small molecules. Our preliminary results indicated that as compared with NAFLD only, liver histology of patients with NAFLD and newly-diagnosed T2DM showed more leukocyte infiltration, and markers of M1 macrophage were upregulated. Meanwhile, nonalcoholic steatohepatitis (NASH) patients had higher HbA1c and fasting glucose levels than subjects with simple fatty liver. The hepatic gene expression of IL-18 was positively correlated with fructose 1, 6-bisphosphatase, a key enzyme involved in gluconeogenesis, in NASH. Importantly, circulating MAIT cells from T2DM patients compared with controls displayed a more-activated phenotype, as shown by a higher expression of CD69 and CD25. Based on these results, we propose that abnormal activation of intrahepatic MAIT cells may initiate glucose metabolism disorder through inducing M1 macrophage polarization. . In present study, we aim to clarify the central role of MAIT cells in hepatic immune and metabolism dysregulation, taking advantage of the NAFLD cohort and gene knockout mice. This study will provide clues into the new therapeutic target for NAFLD-associated T2DM.

非酒精性脂肪性肝病（NAFLD）是2型糖尿病（T2DM）发生的独立危险因素。近年研究发现肝脏M1型巨噬细胞极化引起的慢性炎症是糖代谢异常发生的重要病理机制，而其始动因素并不明确。粘膜相关恒定T细胞（MAIT）是一类肝脏优势分布、受局部微环境影响的非传统T细胞。我们前期通过肝组织病理发现NAFLD伴T2DM者肝脏炎症活动明显，M1巨噬细胞标志物基因表达上调。另一方面，非酒精性脂肪性肝炎患者糖化及空腹血糖升高，肝脏白介素-18与果糖1,6-二磷酸酶基因表达正相关。进一步发现，NAFLD伴T2DM者血MAIT细胞活性增强。据此我们推测MAIT细胞异常活化可能是促进T2DM发生的诱因，通过诱导M1型巨噬细胞极化引起糖代谢紊乱。本课题基于已建立的NAFLD队列及MAIT细胞缺陷小鼠模型，阐明肝脏MAIT细胞异常活化在NAFLD炎症进展及糖代谢紊乱发生中的始动作用，为T2DM防治策略提供新的干预靶点。

近年研究显示，肝脏组织区域免疫稳态在维持糖代谢稳态中发挥重要作用。粘膜相关恒定T（MAIT）细胞是一类肝脏优势分布的非传统 T细胞，而其在2型糖尿病发生发展中的作用尚不清楚。本研究纳入接受肝活检术的糖耐量正常受试者21例及新诊断2型糖尿病患者14例，采用流式细胞术检测肝脏及外周血MAIT细胞数量及细胞因子分泌水平。结果提示，新诊断2型糖尿病患者肝脏MAIT细胞比例较对照组显著升高，而两组间外周血MAIT细胞比例无明显变化。细胞功能分析发现，新诊断2型糖尿病患者肝脏MAIT细胞分泌干扰素γ及肿瘤坏死因子α水平增加，白介素-17、白介素-10、白介素-4分泌水平无差异；两组间外周血MAIT细胞分泌细胞因子水平均无差异。在高脂喂养糖尿病模型小鼠中，我们也观察到糖尿病小鼠肝脏MAIT细胞比例较对照小鼠升高，并主要分泌干扰素γ。进一步对人体肝脏MAIT细胞进行功能亚群分析发现，糖耐量正常人群肝脏MAIT细胞主要以IFN-γ-TNF-α-IL-17- MAIT细胞为主，新诊断2型糖尿病患者肝脏IFN-γ-TNF-α-IL-17- MAIT细胞比例下降，代之以IFN-γ+TNF-α+IL-17+/- MAIT细胞比例明显增加。相关性分析提示，IFN-γ+TNF-α+IL-17+/- MAIT细胞比例与空腹血糖、餐后2小时血糖呈正相关，与胰岛素敏感性呈负相关，这说明IFN-γ+TNF-α+IL-17+/- MAIT细胞可能参与肝脏糖代谢紊乱发生。进而我们将新诊断2型糖尿病患者肝脏MAIT细胞与HepG2细胞共培养，并在其中加入IFN-γ及TNF-α单克隆抗体或对照抗体，检测HepG2细胞中调节糖异生及糖原合成关键基因表达。结果表明，新诊断2型糖尿病患者肝脏MAIT细胞可上调HepG2细胞糖异生关键分子——丙酮酸羧化酶基因表达，加入IFN-γ及TNF-α单克隆抗体后，这一效应消失。这提示肝脏MAIT细胞可通过IFN-γ及TNF-α促进肝脏糖异生，参与2型糖尿病的发生发展。本研究为理解组织区域免疫系统对代谢稳态的调节作用提供了新的依据。