原代人肝细胞的增殖和乙型肝炎病毒再感染的预防有效地消

StephenW

                Hepatology


  
   
                Original article


  
   
                      Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo
        
  
  
   
                                      

• Lena Allweiss1,
• Tassilo Volz1,
• Katja Giersch1,
• Janine Kah1,
• Giuseppina Raffa2,
• Joerg Petersen3,
• Ansgar W Lohse1,4,
• Concetta Beninati5,
• Teresa Pollicino2,
• Stephan Urban4,6,
• Marc Lütgehetmann1,7,
• Maura Dandri1,4
  

Author affiliations

• Department of Medicine, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
• Department of Clinical and Experimental Medicine, University Hospital of Messina, Messina, Italy
• IFI Institute for Interdisciplinary Medicine at Asklepios Clinic St. Georg, Hamburg, Germany
• German Center for Infection Research (DZIF), Hamburg-Lübeck-Borstel and Heidelberg Partner Sites, Hamburg, Germany
• Department of Human Pathology, University Hospital of Messina, Messina, Italy
• Department of Infectious Diseases, Molecular Virology, University Hospital Heidelberg, Heidelberg, Germany
• Department of Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
  

• Correspondence to Professor Dr Maura Dandri, Department of Internal Medicine, Center for Internal Medicine, University Medical Center Hamburg-Eppendorf, Martinistr. 52, Hamburg D-20246, Germany; [email protected]
  

  
   
                Abstract

Objective The stability of the covalently closed circular DNA (cccDNA) in nuclei of non-dividing hepatocytes represents a key determinant of HBV persistence. Contrarily, studies with animal hepadnaviruses indicated that hepatocyte turnover can reduce cccDNA loads but knowledge on the proliferative capacity of HBV-infected primary human hepatocytes (PHHs) in vivo and the fate of cccDNA in dividing PHHs is still lacking. This study aimed to determine the impact of human hepatocyte division on cccDNA stability in vivo.

Methods PHH proliferation was triggered by serially transplanting hepatocytes from HBV-infected humanised mice into naïve recipients. Cell proliferation and virological changes were assessed by quantitative PCR, immunofluorescence and RNA in situ hybridisation. Viral integrations were analysed by gel separation and deep sequencing.

Results PHH proliferation strongly reduced all infection markers, including cccDNA (median 2.4 log/PHH). Remarkably, cell division appeared to cause cccDNA dilution among daughter cells and intrahepatic cccDNA loss. Nevertheless, HBV survived in sporadic non-proliferating human hepatocytes, so that virological markers rebounded as hepatocyte expansion relented. This was due to reinfection of quiescent PHHs since treatment with the entry inhibitor myrcludex-B or nucleoside analogues blocked viral spread and intrahepatic cccDNA accumulation. Viral integrations were detected both in donors and recipient mice but did not appear to contribute to antigen production.

Conclusions We demonstrate that human hepatocyte division even without involvement of cytolytic mechanisms triggers substantial cccDNA loss. This process may be fundamental to resolve self-limiting acute infection and should be considered in future therapeutic interventions along with entry inhibition strategies.

  
   
                          

  
   
                http://dx.doi.org/10.1136/gutjnl-2016-312162
  
   

StephenW

肝病学
来源文章
原代人肝细胞的增殖和乙型肝炎病毒再感染的预防有效地消耗体内核cccDNA

    Lena Allweiss1，Tassilo Volz1，Katja Giersch1，Janine Kah1，Giuseppina Raffa2，Joerg Petersen3，Ansgar W Lohse1,4，Concetta Beninati5，Teresa Pollicino2，Stephan Urban4,6，MarcLütgehetmann1,7，Maura Dandri 1,4

作者隶属

    德国汉堡汉堡Eppendorf大学医学中心内科医学系
    意大利墨西拿墨西拿大学医院临床和实验医学系
    德国汉堡Asklepios诊所St.GGI的IFI跨学科医学研究所
    德国感染研究中心（DZIF），汉堡吕贝克 - 博尔斯特尔和海德堡合作伙伴网站，汉堡，德国
    意大利墨西拿墨西拿大学医院人类病理学系
    德国海德堡海德堡大学医院分子病毒学传染病系
    德国汉堡汉堡Eppendorf大学医学中心医学微生物学，病毒学和卫生学系

    马丁斯顿大学医学中心大学医学中心内科医学系Maura Dandri教授的通讯。 52，德国汉堡D-20246; [email protected]

抽象

目的非分裂肝细胞核中共价闭合环状DNA（cccDNA）的稳定性是HBV持久性的关键决定因素。相反，动物肝炎病毒学研究表明，肝细胞周转可以降低cccDNA负荷，但仍然缺乏对分离PHHs中HBV感染的原代人肝细胞（PHH）体内增殖能力和cccDNA命运的认识。本研究旨在确定人肝细胞分化对体内cccDNA稳定性的影响。

方法通过将HBV感染的人源化小鼠的肝细胞连续移植到幼稚的接受者中引发PHH增殖。通过定量PCR，免疫荧光和RNA原位杂交来评估细胞增殖和病毒学变化。通过凝胶分离和深度测序分析病毒整合。

结果PHH增殖强烈减少所有感染标志物，包括cccDNA（中值2.4log / PHH）。显着地，细胞分裂似乎导致子细胞中的cccDNA稀释和肝内cccDNA损失。然而，HBV在散发性非增殖性人肝细胞中存活，因此随着肝细胞扩张的退化，病毒学标志物反弹。这是由于静止PHH的再次感染，因为用入口抑制剂myrcludex-B或核苷类似物治疗阻断病毒扩散和肝内cccDNA积累。在供体和受体小鼠中检测到病毒整合，但似乎没有促成抗原生成。

结论我们证明人肝细胞分裂甚至不涉及细胞溶解机制会引起大量的cccDNA损失。这个过程可能是解决自身限制性急性感染的基础，并且在未来的治疗干预中应该考虑入侵抑制策略。

http://dx.doi.org/10.1136/gutjnl-2016-312162

StephenW

Significance of this study
What is already known on this subject?
▸ HBV chronic infection is guaranteed by the
persistence of the viral genome forming a
minichromosome, called covalently closed
circular DNA (cccDNA), in hepatocyte nuclei.
▸ HBV polymerase inhibitors do not target the
cccDNA. Thus, HBV surface antigen
seroconversion rates remain low and virus
elimination is not achieved.
▸ Cytolytic and cytokine-mediated mechanisms
were shown to lower cccDNA loads. However,
the fast recovery from acute self-limiting
infection suggests that additional mechanisms
may be involved to explain cccDNA clearance
while the liver remains functional.
▸ Experiments with HBV-related animal viruses
and non-human hepatocytes indicated that cell
division can lower cccDNA contents. However,
knowledge about the capacity of nuclear
cccDNA to survive human hepatocyte division
is still lacking.
What are the new findings?
▸ In vivo proliferation of HBV-infected human
hepatocytes promotes strong cccDNA
destabilisation and its clearance in the great
majority of cells.
▸ Nevertheless, the proliferative capacity of
human hepatocytes displaying high HBV
replication levels appears reduced in
comparison to cells negative for HBV
replication markers.
▸ In the absence of immune-mediated cell killing,
persistence of very few HBV-producing and
apparently non-proliferating human
hepatocytes serves as virus reservoir.
▸ Treatment with HBV entry or polymerase
inhibitors efficiently blocks reinfection of
hepatocytes that have cleared cccDNA through
cell division. Both treatments also hinder
intrahepatic cccDNA accumulation.
How might it impact on clinical practice in the
foreseeable future?
▸ The strong cccDNA destabilisation caused by cell division
suggests that curative therapeutic approaches should
suppress HBV replication and involve controlled destruction
of infected cells (ie, by boosting immune responses). This
would accelerate cccDNA clearance also in surviving
proliferating hepatocytes, while strategies aiming at
preventing HBV entry and/or suppressing HBV replication
would prevent reinfection of cured hepatocytes.

StephenW

这项研究的意义
这个问题已经知道了什么？
▸HBV慢性感染得到保证
持续的病毒基因组形成a
微染色体称为共价闭合
循环DNA（cccDNA），在肝细胞核中。
▸HBV聚合酶抑制剂不针对
cccDNA。因此，HBV表面抗原
血清学转换率仍然很低，病毒
淘汰是没有实现的。
▸细胞溶解和细胞因子介导的机制
显示降低cccDNA负载。然而，
急性自限性恢复快
感染意味着额外的机制
可能涉及到解释cccDNA清除
而肝脏保持功能。
▸HBV相关动物病毒的实验
和非人肝细胞表明细胞
分裂可以降低cccDNA含量。然而，
有关核能力的知识
cccDNA能够存活人类肝细胞分裂
仍然缺乏

什么是新发现？
▸HBV感染者的体内增殖
肝细胞促进强cccDNA
不稳定和清除伟大
大多数细胞。
▸然而，增殖能力
人肝细胞显示高HBV
复制级别减少
与HBV阴性的细胞比较
复制标记。
▸在没有免疫介导的细胞杀伤的情况下，
持续非常少的HBV产生和
显然是不增殖的人
肝细胞作为病毒库。
▸用HBV进入或聚合酶治疗
抑制剂有效地阻止了再感染
通过cccDNA清除的肝细胞
细胞分裂。两种治疗方式也都阻碍了
肝内cccDNA积累。

如何影响临床实践
可以预见的将来？
▸由细胞分裂引起的强cccDNA不稳定
建议治疗方法应该
抑制HBV复制并涉及受控的破坏
的感染细胞（即通过增强免疫应答）。这个
会加速cccDNA清除也存活
增殖肝细胞，而策略瞄准
预防HBV进入和/或抑制HBV复制
将阻止再次感染固化的肝细胞。