以B型肝炎病毒小鼠模式探討核心蛋白引發免疫反應之機制

Induction of Immune Response by Hepatitis B virus Core Protein in a Mouse Model

曾鴻泰(Horng-Tay Tzeng)

指導教授 ： 許秉寧

國立臺灣大學/醫學院/免疫學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.00712

摘要

B型肝炎持續性感染會造成慢性肝炎，肝纖維化和肝癌。近來在動物模式中研究病毒感染的報告指出淋巴球上的抑制性受體 (PD-1) 和配體 (PD-L1) 間的相互作用對於抑制T細胞的活性扮演重要角色。我們利用最近發展的B型肝炎小鼠模式來探討T細胞exhaustion現象及阻斷PD-1/PD-L1作用後對於肝浸潤T細胞的效應。在此小鼠模式中，我們證明了B型肝炎的持續性和肝浸潤T細胞的PD-1表現成正相關，在帶原小鼠中肝浸潤CD8 T細胞表現較高的PD-1和較低的CD127 exhaustion現象，並且利用單株抗體阻斷PD-1/PD-L1作用可增加病毒核心蛋白專一性的IFN-γ產生，而且可以反轉T淋巴球的exhaustion及增加小鼠對病毒的清除能力。我們的結果指出，在小鼠模式中阻斷PD-1的途徑可恢復對B肝病毒的免疫反應及增進對病毒的清除。
先天與後天免疫反應的調和才能有效的控制病毒感染，為了瞭解對抗B肝病毒的先天免疫反應，我們使用了一系列與先天免疫反應相關受體之基因缺陷小鼠來測試，結果發現除了腫瘤壞死因子缺失的小鼠外，其餘包括IFN-α/β receptor, RIG-I, MDA5, MYD88, NLRP3, ASC, 和 IL-1R基因缺失小鼠都與控制組相當。我們進一步發現，給予小鼠腫瘤壞死因子受體阻斷劑可促使CD8 T細胞PD-1表現、肝臟中病毒複製、核心抗原和表面抗原及血清病毒量增加；純化後的病毒核心蛋白可在小鼠引發腫瘤壞死因子及IL-6表現，但IFN-β則否。我們也發現病毒核心蛋白造成肝臟中白血球引發的腫瘤壞死因子是清除病毒的重要途徑，這些結果提供了腫瘤壞死因子相關的先天免疫反應對抗病毒的機制，也解釋了臨床上使用腫瘤壞死因子阻斷劑造成B肝病毒再活化的機制。

關鍵字

B型肝炎病毒 ； PD-1阻斷 ； 病毒持續性 ； 先天免疫力 ； 腫瘤壞死因子

並列摘要

Persistent hepatitis B viral (HBV) infection results in chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Recent studies in animal models of viral infection indicate that the interaction between the inhibitory receptor, programmed death (PD)-1, on lymphocytes and its ligand (PD-L1) play a critical role in T-cell exhaustion by inducing T-cell inactivation. We studied T-cell exhaustion and the effects of PD-1 and PD-L1 blockade on intrahepatic infiltrating T-cells in our recently developed mouse model of HBV persistence. In this mouse animal model, we demonstrated that the chronicity of hepatitis B virus infection was associated with PD-1 expression on intrahepatic lymphocytes. The Intrahepatic CD8+ T-cells expressed higher levels of PD-1 and lower levels of CD127 in carrier mice. Blockade of PD-1/PD-L1 interactions by an anti-PD-1 monoclonal antibody (mAb) increased HBcAg-specific interferon (IFN)-γ production in intrahepatic T lymphocytes and reversed the exhausted phenotype in intrahepatic T lymphocytes and viral persistence to clearance of HBV in vivo. Our results indicated that PD-1 blockage reverses immune dysfunction and viral persistence of HBV infection in a mouse animal model, suggesting that the anti-PD-1 mAb might be a good therapeutic candidate for chronic HBV infection.
An efficient control of virus infections requires the coordinated actions of both innate and adaptive immune responses. In order to define the role of innate immunity effectors against HBV, viral clearance was studied in a panel of immunodeficient mouse strains. Our results demonstrate that HBV viral clearance is not changed in IFN-α/β receptor (IFNAR), RIG-I, MDA5, MYD88, NLRP3, ASC, and IL-1R knockout mice. In contrast, HBV persists in the absence of tumor necrosis factor-alpha (TNF-α) or in mice treated with the soluble TNF receptor blocker, Etanercept. In these mice, there was an increase in PD-1-expressing CD8+ T-cells and an increase of serum HBV DNA, HBV core, and surface antigen expression as well as viral replication within the liver. Furthermore, the induction of TNF-α in clearing HBV is dependent on the HBV core, and TNF blockage eliminated HBV core-induced viral clearance effects. Finally, the intra-hepatic leukocytes (IHLs), but not the hepatocytes, are the cell source responsible for TNF-α production induced by HBcAg. These results provide evidences for TNF-α mediated innate immune mechanisms in HBV clearance and explain the mechanism of HBV reactivation during therapy with TNF blockage agents.

並列關鍵字

Hepatitis B virus ； PD-1 blockage ； viral persistence ； innate immunity ； Tumor necrosis factor

參考文獻

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