肝脏III型固有淋巴细胞编程对肝纤维化进程的免疫调控及机制研究

Liver fibrosis is the intermediate link and reversible stage during the development of chronic liver disease to cirrhosis. The pathological immune responses can change hepatic immune homeostasis and immune microenvironment, which lead to the progression of liver fibrosis. Previously, the role of the homeostasis of hepatic CD4+T cells in the process of liver fibrosis had been illustrated and proved in our concluded project of NSFC (No.81070341). The balance of hepatic and peripheral regulatory T cells (Tregs)/T helper 17 cells (Th17) tended to Th17 dominance in the process of liver fibrosis along with the increased secretion of the associated cytokines by Th17 cells. Furthermore, the enhanced functions of Th17 cells promoted the pro-fibrotic functions of hepatic stellate cells (HSCs) and influenced the progression of liver fibrosis. On the other hand, innate immune functions play key roles in the regulation of liver fibrosis. Since the immune regulatory functions of the classical innate immune cells such as NK and NKT cells have been gradually elucidated, innate lymphoid cells (ILCs), which plays key roles in the regulation of tissue homeostasis and inflammatory infection, have been discovered due to the new understanding and classification of the phenotypes and functions of innate immune cells recently. Group III ILCs (RORγt+ILCs, ILC3s) not only own the same transcription factors as Th17 cells but also secret the similar Th17 associated cytokines to regulate the immune response, thus it may be an important link between hepatic innate and adaptive immune responses in the process of liver fibrosis. We have verified that the altered numbers and functions of hepatic ILC3s exist in the patients with chronic liver disease in the pre-experiments. Furthermore, cell programming is not only the results of the local microenvironment changes, but also the necessary conditions to obtain the specific phenotypic and functional characteristics and play the important immune regulatory functions at the specific sites. In this research, we will firstly observe the relationship between the hepatic subsets of ILC3s and pathological grades and states of liver fibrosis. And then, by using the techniques of flow cytometry and molecular biology, we will investigate the pro-chemotactic functions of Kupffer cells on hepatic ILC3s, identify specific programming of ILC3s under the microenvironment of liver fibrosis, and further testify the immune regulatory roles of CD4+T cells in programming of hepatic ILC3s to illustrate the adaptive regulation of innate lymphocytes responses and the maintenance of immune homeostasis through ILC3/T cell interactions under hepatic local microenvironment in the process of liver fibrosis. Finally, we will analyze the regulation of hepatic ILC3s on the fibrotic function of HSCs. Based on the above-mentioned research, we look forward to elaborating the network regulation and the specific mechanism between the immune cells of liver in the process of liver fibrosis. We hope these achievements can contribute to the basic theory and practical applications for the future clinical immunotherapy on liver fibrosis.

病理状态下的免疫应答是调控肝纤维化进程重要因素。我们NSFC课题研究表明：肝脏CD4+T细胞稳态改变影响肝纤维化进程，Th17表达及功能的增强调节HSCs促纤维化功能。新近发现固有淋巴细胞(ILCs)的重要免疫来源，其中III型ILCs(ILC3s)具有与Th17相同转录因子并分泌类似细胞因子，预实验证实慢性肝病患者肝脏ILC3s数量及功能的改变，而细胞编程不仅是局部微环境变化的结果，也是获得特异表型和功能特征、并在特定部位发挥免疫调控的必要条件。本研究将运用流式细胞术及分子生物学手段，以肝脏ILC3s及亚群与肝纤维化程度分析为基础，明确ILC3s在肝纤维化微环境中的特异性编程，论证CD4+T细胞对ILC3s编程的调控及对肝纤维化肝脏局部免疫稳态的协调，观察ILC3s对肝纤维化进程及对HSCs功能的影响，阐述肝纤维化时肝脏局部固有免疫与适应免疫应答网络调控，为免疫治疗肝纤维化提供理论基础。

病理状态下的免疫应答是调控肝纤维化进程重要因素。肝脏CD4+T细胞稳态改变影响肝纤维化进程，Th17表达及功能的增强调节肝星状细胞促纤维化功能。新近发现固有淋巴细胞(ILCs)的在多种疾病中发挥重要的作用，其中III型ILCs(ILC3s)具有与Th17相同转录因子并分泌类似细胞因子。本研究运用流式细胞术及分子生物学手段，证实慢性肝病患者肝脏ILC3s数量增多，同时细胞因子（IL-17A、IL-22、TNF-α、IFN-γ）表达也增加。细胞体外功能培养也证明ILC3s可通过分泌IL-17A及IL-22激活肝星状细胞。同样地，在四氯化碳诱导的肝纤维化小鼠模型中，我们也证明了ILC3s数量及功能与在人体中的有相同的改变。体外ILCs的敲除及ILC3s的过继转移进一步证明了ILC3s的促进纤维化的功能。本研究阐述了肝纤维化时肝脏局部固有免疫与适应免疫应答网络调控，为免疫治疗肝纤维化提供理论基础。