乙型肝炎和δ病毒如何建立肝细胞感染 研究人员使用新的细

StephenW

How hepatitis B and delta viruses establish infection of liver cells
Researchers explore infection using new cell culture system

Date:
    June 18, 2019
Source:
    Princeton University
Summary:
    Researchers have developed a new, scalable cell culture system that allows for detailed investigation of how host cells respond to infection with hepatitis B (HBV) and delta virus (HDV).
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Princeton University researchers have developed a new, scalable cell culture system that allows for detailed investigation of how host cells respond to infection with hepatitis B (HBV) and delta virus (HDV). The paper describing their findings was published online on June 18, 2019 in the journal Hepatology.

HBV causes an acute illness that is usually rapidly cleared by adults with intact immune systems, but young children and people with HIV are at particular risk of chronic HBV infection, which can lead to cirrhosis or cancer of the liver. Infection with HBV also renders a person vulnerable to infection with HDV, which can cause acute liver failure and/or accelerate the progression to cirrhosis or cancer. Fortunately, an effective vaccine exists for HBV, and because HDV requires HBV in order to reproduce, both can be considered preventable diseases. However, the expense and limited availability of the vaccine leaves millions at risk for infection worldwide.

A better understanding of how the viruses affect the cells they infect would assist in the development of drugs to combat or even cure infection, but HBV and HDV only infect liver cells (hepatocytes) from humans and chimpanzees. Such cells are difficult to obtain, and when grown outside the body in cell culture, they undergo a process called de-differentiation: over the course of just a few days, they lose their specialized characteristics and functionality. When this happens, they also lose the ability to be infected with HBV and HDV, creating a significant obstacle to studying chronic viral infection.

"Attempts have been made since the mid-1980s to establish robust -- and, importantly, persistent -- infection in primary human hepatocytes (PHHs) with limited success," said Alexander Ploss, an associate professor in the Department of Molecular Biology at Princeton and leader of the study.

Studies with other tissues have shown that specialized cell types sometimes require the support of other types of cells in order to maintain their differentiated status. Hepatocytes make up the majority of cells in the liver, but the tissue also contains many other cell types, which are collectively referred to as "non-parenchymal cells." Recently, graduate student Benjamin Winer, together with colleagues in Ploss' lab and at the Hurel Corporation, demonstrated that freshly isolated human hepatocytes can be grown together with non-parenchymal cells on a supporting surface made of collagen. In this cell culture system, which the researchers call a self-assembling co-culture of primary human hepatocytes (SACC-PHH), the hepatocytes retain their differentiated state and can support chronic HBV infection for up to 40 days.

"This system has created unprecedented opportunities to study host responses to hepatitis virus, especially in the context of persistent infection," Ploss said.

In the new study, Winer and colleagues investigated how hepatocytes respond to infection with HBV and HDV. First, they tested whether SACC-PHH can support infection with both viruses. HBV hijacks the cell's protein-making machinery to make viral proteins, and HDV co-opts HBV proteins to assemble itself. Therefore, HDV can only reproduce when it infects cells at the same time as HBV (co-infection) or in cells already chronically infected with HBV (super-infection). The researchers found that both of these scenarios can occur in SACC-PHH, even when the culture system has been scaled down to tiny 384 microwell culture plates -- a development that makes the system well suited for high-throughput screening of candidate drugs. Accordingly, the team showed that prophylactic treatment with two candidate antivirals, entecavir and Myrcludex B, could reduce levels of both hepatitis viruses in SACC-PHH.

Viral infection provokes many changes in the host cell, ranging from metabolic adaptations to the activation of innate immune defenses that can recognize and destroy a virus inside the cell. To explore what changes hepatocytes experience in response to HBV/HDV infection, the authors looked at which genes were being expressed using a technique called RNA-Seq analysis. The data showed that cells infected with HBV exhibit elevated expression of genes involved in oxidative phosphorylation and interaction with the extracellular environment. By contrast, cells additionally infected with HDV had similar gene expression patterns as uninfected cells.

Interestingly, even though the hepatocytes' innate immune signaling pathways were intact and could be stimulated by addition of chemicals such as poly(I:C), HBV infection did not activate these defenses. On the other hand, treatment with poly(I:C) helped suppress HBV growth, suggesting the virus flies under the radar of cells' defenses to establish persistent infection.

How innate immune defenses affect HBV/HDV co-infection appears more nuanced. Stimulation of innate immune pathways with poly(I:C) had little effect on HDV. HBV/HDV co-infection also failed to activate defense pathways in the hepatocytes of most human donors studied, but one donor showed innate immune activation upon co-infection, indicating a person's genetic makeup may influence their ability to combat the infection.

"We believe that our optimized, high-throughput SACC-PHH platform is a unique resource for investigating hepatotropic pathogens, and that our data will help advance understanding persistent infections by HBV and HDV," Ploss said.

The study was funded by the National Institutes of Health, the Burroughs Wellcome Fund, the National Institute of Diabetes and Digestive and Kidney Diseases, the National Institute of Allergy and Infectious Diseases, and the New Jersey Commission on Cancer Research.

Story Source:

Materials provided by Princeton University. Note: Content may be edited for style and length.

Journal Reference:

    Benjamin Y. Winer, Jenna M. Gaska, Gabriel Lipkowitz, Yaron Bram, Amit Parekh, Lance Parsons, Robert Leach, Rohit Jindal, Cheul H. Cho, Anil Shrirao, Eric Novik, Robert E. Schwartz, Alexander Ploss. Analysis of host responses to hepatitis B and delta viral infections in a micro-scalable hepatic co-culture system. Hepatology, 2019; DOI: 10.1002/hep.30815

StephenW

乙型肝炎和δ病毒如何建立肝细胞感染
研究人员使用新的细胞培养系统探索感染

日期：
2019年6月18日
资源：
普林斯顿大学
摘要：
研究人员开发了一种新的，可扩展的细胞培养系统，可以详细研究宿主细胞如何应对乙型肝炎（HBV）和δ病毒（HDV）感染。
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完整的故事

普林斯顿大学的研究人员开发了一种新的，可扩展的细胞培养系统，可以对宿主细胞如何应对乙型肝炎病毒（HBV）和δ病毒（HDV）进行详细调查。描述他们发现的论文于2019年6月18日在线发表在肝脏病学杂志上。

HBV引起急性疾病，通常由具有完整免疫系统的成年人迅速清除，但幼儿和HIV患者特别容易患上慢性HBV感染，这可能导致肝硬化或肝癌。 HBV感染也使人容易感染HDV，这可能导致急性肝功能衰竭和/或加速进展为肝硬化或癌症。幸运的是，HBV存在有效的疫苗，并且由于HDV需要HBV才能繁殖，因此两者都可以被认为是可预防的疾病。然而，疫苗的费用和有限的可用性使全世界有数百万人感染风险。

更好地了解病毒如何影响它们感染的细胞将有助于开发抗击甚至治愈感染的药物，但HBV和HDV仅感染来自人类和黑猩猩的肝细胞（肝细胞）。这些细胞难以获得，并且当在细胞培养中在体外生长时，它们经历称为去分化的过程：在短短几天的过程中，它们失去其特有的特征和功能。当这种情况发生时，它们也失去了感染HBV和HDV的能力，从而成为研究慢性病毒感染的重大障碍。

普林斯顿大学分子生物学系副教授亚历山大普洛斯说：“自20世纪80年代中期以来，人们已经尝试在原代人肝细胞（PHHs）中建立稳健且重要的持续性感染，但效果有限。”该研究的领导者。

对其他组织的研究表明，特殊细胞类型有时需要其他类型细胞的支持以维持其分化状态。肝细胞构成肝脏中的大部分细胞，但组织中还含有许多其他细胞类型，最近被研究为“非实质细胞”。最近，研究生Benjamin Winer和Ploss实验室以及Hurel公司的同事证明，新鲜分离的人肝细胞可以与支持表面上的非实质细胞一起生长在细胞培养系统中，研究人员称之为原代人肝细胞（SACC-PHH）的自组装共培养，肝细胞保持其分化状态并且可以支持长达40天的慢性HBV感染。

Ploss说：“该系统为研究宿主对肝炎病毒的反应创造了前所未有的机会，特别是在持续感染的情况下。”

在这项新研究中，Winer及其同事研究了肝细胞如何应对HBV和HDV感染。首先，他们测试了SACC-PHH是否可以支持两种病毒的感染。 HBV劫持了细胞的蛋白质制造机器来制造病毒蛋白，而HDV则将HBV蛋白质联合起来组装起来。 HDV只能在HBV（共感染）和已经慢性感染HBV（超感染）的细胞中同时感染细胞时繁殖。研究人员发现，这两种情况都可以在SACC-PHH中发生，即使培养系统已经缩小到384微孔培养板 - 这一系统的开发使得该系统非常适合候选药物的高通量筛选。该研究小组表明，使用两种候选抗病毒药物恩替卡韦和Myrcludex B进行预防性治疗可以降低SACC-PHH中两种肝炎病毒的水平。

病毒感染引起宿主细胞的许多变化，从代谢适应到能够识别和破坏细胞内病毒的先天免疫防御的激活。为了探究肝细胞对HBV / HDV感染的反应有何变化，作者数据显示感染HBV的细胞表现出参与氧化磷酸化和与细胞外环境相互作用的基因表达升高。相比之下，另外感染HDV的细胞具有与未感染细胞相似的基因表达模式。
有趣的是，尽管肝细胞的先天免疫信号通路是完整的并且可以通过添加化学物质如聚（I：C）来刺激，但HBV感染并未激活这些防御。另一方面，用聚（I：C）治疗有助于抑制HBV的生长，这表明该病毒在细胞防御的雷达下飞行以建立持续感染。

先天免疫防御如何影响HBV / HDV合并感染似乎更加细致入微。用聚（I：C）刺激先天免疫途径对HDV几乎没有影响。 HBV / HDV共感染也未能激活所研究的大多数人类供体的肝细胞中的防御途径，但是一个供体在共感染时显示出先天免疫激活，表明人的基因构成可能影响其抵抗感染的能力。

“我们相信，我们优化的高通量SACC-PHH平台是研究嗜肝性病原体的独特资源，我们的数据将有助于推进对HBV和HDV持续感染的了解，”Ploss说。

该研究由美国国立卫生研究院，Burroughs Wellcome基金会，国家糖尿病和消化和肾脏疾病研究所，国家过敏和传染病研究所以及新泽西州癌症研究委员会资助。

故事来源：

材料由普林斯顿大学提供。注意：可以根据样式和长度编辑内容。

期刊参考：

    Benjamin Y. Winer，Jenna M. Gaska，Gabriel Lipkowitz，Yaron Bram，Amit Parekh，Lance Parsons，Robert Leach，Rohit Jindal，Cheul H. Cho，Anil Shrirao，Eric Novik，Robert E. Schwartz，Alexander Ploss。在微型可扩展肝脏共培养系统中分析宿主对乙型肝炎和δ病毒感染的反应。肝病学，2019年; DOI：10.1002 / hep.30815