Background & Aims

Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus and how it affects disease progression are unclear.

Methods

We performed studies with BALB/c Rag2^–/–Il2rg^–/–Sirpa^NODAlb-uPA^tg/tg mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription polymerase chain reaction (PCR). Plasma levels of HBV DNA were quantified by PCR reaction, and antigen-specific antibodies were detected by immunocytochemistry of HBV-transfected BHK-21 cells.

Results

Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e (HBe), core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation.

Conclusion

In HIS-HUHEP mice, HBV infection completes a full life cycle and recapitulates some of the immunopathology observed in patients with chronic infection. Inoculation with different viral loads led to different immune responses and levels of virus control. We found HBV to infect liver progenitor cells, which could be involved in hepatocellular carcinogenesis. This is an important new system to study anti-HBV immune responses and screen for combination therapies against hepatotropic viruses.

中文翻译：

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病毒载量影响人源化免疫系统和肝脏小鼠对HBV的免疫反应

背景与目标

乙型肝炎病毒（HBV）感染肝细胞，但针对该病毒的免疫应答机制及其如何影响疾病进展的机制尚不清楚。

方法

我们进行研究，只BALB / c的Rag2 ^{- / -}的II2rg ^{- - /} SIRPA ^NOD ALB-uPA的^{TG / TG}具有或不具有人免疫系统（HIS）的小鼠，与人肝细胞（HUHEP）稳定地嫁接。给HUHEP和HIS-HUHEP小鼠腹膜内注射HBV。从脾脏和肝脏中分离出单核细胞，通过流式细胞术进行分析。通过免疫组织化学分析肝脏，并通​​过定量逆转录聚合酶链反应（PCR）测量mRNA水平。通过PCR反应定量测定HBV DNA的血浆水平，并通过HBV转染的BHK-21细胞的免疫细胞化学检测抗原特异性抗体。

结果

在HBV感染后，在HUHEP和HIS-HUHEP小鼠中观察到完整的病毒生命周期，并产生HBV DNA，乙型肝炎e（HBe），核心（HBc）和表面（HBs）抗原以及共价闭合的环状DNA。HBV在HUHEP小鼠中不受限制地复制，导致病毒滴度高而无病理影响。相比之下，感染HBV的HIS-HUHEP小鼠发生慢性肝炎，其滴度降低了10倍，并且抗原特异性IgG（抗HBs，抗HBc）与部分免疫控制相一致。HBV感染的HIS-HUHEP肝脏包含浸润的Kupffer细胞，成熟的活化自然杀伤细胞（CD69 +）和PD-1 +效应记忆T细胞（CD45RO +）。减少病毒接种物可实现更有效的免疫控制。来自HBV感染的HIS-HUHEP小鼠的血浆中炎症和免疫抑制细胞因子（CXC趋化因子配体10和白介素10）的水平升高，这与肝内CD4 + T细胞（CD45RO + PD-1 +）的种群有关。病毒血症高的小鼠感染了HBV肝祖细胞。给小鼠核苷类似物恩替卡韦减少病毒载量并减少肝脏炎症。

结论

在HIS-HUHEP小鼠中，HBV感染完成了整个生命周期，并概括了在慢性感染患者中观察到的一些免疫病理学。接种不同病毒载量会导致不同的免疫应答和病毒控制水平。我们发现HBV会感染肝祖细胞，而肝祖细胞可能与肝细胞癌变有关。这是一个重要的新系统，用于研究抗HBV免疫反应和筛选针对肝炎病毒的联合疗法。