IL-21与NKT细胞相互作用参与慢性HBV感染的机制研究

Interleukin-21 (IL-21) plays a crucial role in the control of chronic viral infection by enhancing the function of CD8+ T cells. However, the role of IL-21 in chronic hepatitis B virus (HBV) infection is not fully elucidated. Our recently published data indicated that IL-21 was increased, whereas natural killer T (NKT) cells were increased in chronic hepatitis B (CHB) patients. Considering NKT cells are one of the major sources of IL-21, we hypothesize that NKT cells are activated during chronic HBV infection and produce IL-21, which in turn interacts with NKT cells and participates in the pathogenesis of CHB. To address this hypothesis, the correlation of IL-21 with NKT cells and the characteristics of NKT cells producing IL-21will be evaluated in CHB patients and HBV carriers enrolled in this project. Furthermore, NKT cells from individual subjects will be isolated and purified using fluorescence or magnetic activated cell sorting (FACS or MACS), and expanded in vitro. NKT cells will be co-cultured with HBV transfected hepatoma cell line HepG2.2.15 to investigate the influence of chronic HBV infection on IL-21 production of NKT cells and the role of IL-21 in the control of HBV replication mediated my NKT cells. Thereafter, purified B cells will be isolated by FACS or MACS and co-cultured with NKT cells to detect the role of IL-21 in the generation of HBV-specific antibodies induced by activated NKT cells. At last, the hypothesis will be further tested by performing experiments in vivo using HBV transgenic (THBV) mice. Alpha-galactosylceramide (α-GalCer), a CD1d-restricted antigen recognized specifically by NKT cells, will be injected intravenously into the THBV mice to activate NKT cells, then IL-21 or IL-21 receptor (IL-21R) specific antibodies or small interfere RNA (siRNA) will also be injected into the THBV mice to block the IL-21/IL-21R pathway or silence IL-21 and IL-21R gene expression. In some cases, THBV mice will be immunized with HBV-specific antigens. Mice will be sacrificed in different time points, then the circulating and intrahepatic NKT cells, IL-21, HBV viral load, inflammation status and HBV-specific antibodies will be analyzed to confirm the interaction between IL-21 and NKT cells. The completion of this project will help us to understand the immunopathogenesis of chronic HBV infection better, and provide a theoretical basis for the potential immune-based antiviral therapy.

白介素-21(IL-21)可增强CD8+T细胞的功能而在控制慢性病毒感染中发挥关键作用，但其在慢性HBV感染中的研究还极少。我们前期的研究发现慢性乙型肝炎患者外周血IL-21浓度升高而NKT细胞比例下降，机制未明。因NKT细胞是产生IL-21的主要细胞之一，故IL-21很可能通过与NKT细胞的相互作用而参与慢性HBV感染。本项目拟比较慢性HBV感染后不同时期患者及同一患者抗病毒治疗前后外周血及肝内NKT细胞分泌IL-21的差异，分析IL-21与NKT细胞频数及功能的关系。通过体外实验探讨HBV慢性感染对NKT细胞的功能、抗病毒活性及对HBV特异性抗体产生的影响；用抗体阻断实验阐明IL-21在其中的作用。通过HBV转基因小鼠实验阐明IL-21在NKT细胞抗HBV慢性感染中的作用。项目的完成将深化我们对慢性HBV感染免疫学发病机制的认识，并为慢性乙型肝炎的临床免疫学干预提供理论基。

中国是乙肝大国，每年有超过 50万的病人死于与 HBV感染相关的肝病，而每年用于抗病毒治疗的总花费达 9000 亿人民币，给乙肝患者、相关家庭及国家社会带来了沉重的负担。HBV 本身并不会导致肝细胞的损害，其所激发的宿主免疫反应才是引起肝细胞损伤和清除病毒的决定性因素。我们前期的研究发现慢性乙型肝炎患者外周血IL-21 浓度升高而 NKT 细胞比例下降，机制未明。因 NKT 细胞是产生 IL-21的主要细胞之一，故 IL-21 很可能通过与 NKT 细胞的相互作用而参与慢性 HBV 感染。本项目拟比较慢性HBV 感染后不同时期患者及同一患者抗病毒治疗前后外周血及肝内 NKT细胞分泌IL-21 的差异，分析IL-21与NKT细胞频数及功能的关系。通过体外实验探讨HBV慢性感染对NKT细胞的功能、抗病毒活性及对 HBV特异性抗体产生的影响。我们的研究结果显示CHB患者外周血中NKT细胞分泌IL-21的量与健康人无显著性差异；CHB患者外周血IL-21的浓度与NKT细胞的比例也无明显相关；用α-Galcer在体外刺激CHB患者PBMC 96小时, NKT细胞占T细胞的比例显著升高（0.23% ± 0.05% vs 0.71% ± 0.20%，P ＜ 0.01），而重组人 IL-21（100ng/ml）能在体外抑制 CHB 患者外周血 NKT 细胞的增值（0.71% ± 0.20% vs 0.22% ± 0.06%， P ＜ 0.01）；ELISPOT结果显示重组人 IL-21（100ng/ml）还能在体外抑制CHB患者外周血NKT细胞IFN-γ的分泌；抗体阻断实验提示IL-21对NKT细胞的抑制作用依赖于IL-10；体外用α-GalCer激活NKT细胞后不能促进HBeAb的产生。. 以上结果提示在HBV慢性感染过程中，患者外周血IL-21浓度会升高，而升高的IL-21能通过刺激IL-10的分泌进而抑制NKT细胞的增值及IFN-γ的分泌。