γ干扰素和肿瘤坏死因子α被T细胞产生降低HBV持续性的形式

StephenW

Interferon-γ and Tumor Necrosis Factor-α Produced by T Cells Reduce the HBV Persistence Form, cccDNA, Without Cytolysis
Yuchen Xia∗
, Daniela Stadler∗
, Julie Lucifora
, Florian Reisinger
, Dennis Webb
, Marianna Hösel
, Thomas Michler
, Karin Wisskirchen
, Xiaoming Cheng
, Ke Zhang
, Wen-Min Chou
, Jochen M. Wettengel
, Antje Malo
, Felix Bohne
, Dieter Hoffmann
, Florian Eyer
, Robert Thimme
, Christine S. Falk
, Wolfgang E. Thasler
, Mathias Heikenwalder
, Ulrike Protzercorrespondenceemailemail
∗Authors share co-first authorship.
DOI: http://dx.doi.org/10.1053/j.gastro.2015.09.026 |
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Published Online: September 25, 2015Accepted: September 19, 2015Received: August 23, 2014

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Background & Aims

Viral clearance involves immune cell cytolysis of infected cells. However, studies of hepatitis B virus (HBV) infection in chimpanzees have indicated that cytokines released by T cells also can promote viral clearance via noncytolytic processes. We investigated the noncytolytic mechanisms by which T cells eliminate HBV from infected hepatocytes.
Methods

We performed a cytokine enzyme-linked immunosorbent assay of serum samples from patients with acute and chronic hepatitis B. Liver biopsy specimens were analyzed by in situ hybridization. HepG2-H1.3 cells, HBV-infected HepaRG cells, and primary human hepatocytes were incubated with interferon-γ (IFNγ) or tumor necrosis factor-α (TNF-α), or co-cultured with T cells. We measured markers of HBV replication, including the covalently closed circular DNA (cccDNA).
Results

Levels of IFNγ and TNF-α were increased in serum samples from patients with acute vs chronic hepatitis B and controls. In human hepatocytes with stably replicating HBV, as well as in HBV-infected primary human hepatocytes or HepaRG cells, IFNγ and TNF-α each induced deamination of cccDNA and interfered with its stability; their effects were additive. HBV-specific T cells, through secretion of IFNγ and TNF-α, inhibited HBV replication and reduced cccDNA in infected cells without the direct contact required for cytolysis. Blocking IFNγ and TNF-α after T-cell stimulation prevented the loss of cccDNA. Deprivation of cccDNA required activation of nuclear APOBEC3 deaminases by the cytokines. In liver biopsy specimens from patients with acute hepatitis B, but not chronic hepatitis B or controls, hepatocytes expressed APOBEC3A and APOBEC3B.
Conclusions

IFNγ and TNF-α, produced by T cells, reduce levels of HBV cccDNA in hepatocytes by inducing deamination and subsequent cccDNA decay.
Keywords:
Apolipoprotein B mRNA Editing Enzyme, HBV Persistence, T-Cell Receptor, Immune Regulation
Abbreviations used in this paper:
3D-PCR (differential DNA denaturation PCR), A3 (APOBEC3), ALT (alanine aminotransferase), AP (apurinic/apyrimidinic), cccDNA (covalently closed circular DNA), Cp (crossing point), CTL (cytotoxic T lymphocytes), dHepaRG (differentiated HepaRG), ETV (entecavir), HBeAg (hepatitis B e antigen), HBsAg (hepatitis B surface antigen), HBV (hepatitis B virus), IFN (interferon), LT (lymphotoxin), mRNA (messenger RNA), PHH (primary human hepatocyte), qPCR (quantitative polymerase chain reaction), rcDNA (relaxed circular DNA), S-CAR (hepatitis B virus–specific chimeric antigen receptor), TNF (tumor necrosis factor)

Current address of Y.X.: Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland.

Current address of J.L.: INSERM 1052, CNRS UMR 5286, Cancer Research Center of Lyon, University of Lyon, Lyon, France.

Conflicts of interest The authors disclose no conflicts.

Funding Supported in part by the German Research Foundation (grant CRC/TR 36), the German Center for Infection Research, and the Helmholtz-Alberta Initiative on Infectious Disease Research (HAI-IDR).

StephenW

γ干扰素和肿瘤坏死因子α被T细胞产生降低HBV持续性的形式，cccDNA的，无细胞溶解
雨辰霞*
，丹妮拉施泰德*
朱莉Lucifora
，弗洛里安·雷辛格
，丹尼斯·韦伯
，玛丽安娜杆颈
托马斯米氏
卡琳WISSKIRCHEN
，小明诚
，张恪
，文闵抽
，约亨·M。Wettengel
，安特耶·马洛
费利克斯Bohne
迪特尔·霍夫曼
，弗洛里安Eyer
罗伯特Thimme
，克里斯蒂娜S.福尔克
沃尔夫冈·E. Thasler
，马蒂亚斯Heikenwalder
，乌尔里克Protzercorrespondenceemailemail
*作者分享共同第一作者。
DOI：http://dx.doi.org/10.1053/j.gastro.2015.09.026 |
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出版历史
发布时间：9月25日，2015Accepted：9月19日，2015Received：2014年8月23日

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背景和目的

病毒清除涉及感染细胞的免疫细胞的细胞溶解。然而，B型肝炎病毒的研究（HBV）感染黑猩猩已表明由T细胞释放的细胞因子也可以促进经由noncytolytic工序的病毒清除。我们研究了通过其中的T细胞消除HBV的从感染的肝细胞的noncytolytic机制。
方法

我们进行血清样品的细胞因子酶联免疫吸附测定从急性和慢性乙型肝炎肝活检标本通过原位杂交进行分析。的HepG2-H1.3细胞，HBV感染HepaRG细胞和原代人肝细胞孵育干扰素γ（IFNγ）或肿瘤坏死因子α（TNF-α），或共培养的T细胞。我们测量HBV复制的标志物，包括共价闭合环状DNA（cccDNA的）。
结果

IFNγ和TNF-α水平的血清样品中的增加，从急性VS慢性乙型肝炎和控制。在人肝细胞与稳定地复制的HBV，以及在HBV感染的原代人肝细胞或HepaRG细胞，IFNγ和TNF-α的cccDNA的各诱导脱氨和干扰其稳定性;它们的作用是添加剂。 HBV特异性T细胞，通过分泌的IFNγ和TNF-α抑制HBV复制和感染细胞中降低的cccDNA而不需要细胞溶解的直接接触。后T细胞刺激阻断IFNγ和TNF-α抑制的cccDNA的损失。 cccDNA的剥夺需要激活核APOBEC3脱氨酶的细胞因子。在从急性乙肝，但没有慢性乙肝或控制肝活检，肝细胞表达APOBEC3A和APOBEC3B。
结论

IFNγ和TNF-α，由T细胞产生，通过诱导脱氨和随后的cccDNA衰变减少HBV的cccDNA的肝细胞中的水平。
关键词：
载脂蛋白B mRNA的编辑酶，乙肝病毒持续性，T细胞受体，免疫调节
在本文中使用的缩写：
3D-PCR（差分DNA变性PCR），A3（APOBEC3），ALT（谷丙转氨酶），AP（嘌呤/无嘧啶）的cccDNA（共价闭合环状DNA），CP（交叉点），CTL（细胞毒性T淋巴细胞），dHepaRG （分化HepaRG），ETV（恩替卡韦），e抗原（乙型肝炎e抗原），乙肝表面抗原（乙型肝炎表面抗原），HBV（乙肝病毒），干扰素（干扰素），LT（淋巴毒素），mRNA（信使核糖核酸），PHH （初级人肝细胞），定量PCR（定量聚合酶链式反应），rcDNA（放宽环状DNA），S-CAR（乙型肝炎病毒特异性嵌合抗原受体），TNF（肿瘤坏死因子）

YX的现住址：肝病科，糖尿病研究所，消化和肾脏疾病，美国国立卫生研究院，美国马里兰州贝塞斯达。

JL的现住址：INSERM 1052，CNRS UMR 5286，里昂癌症研​​究中心，里昂，法国里昂大学。

利益冲突的作者公开冲突。

基金提供资助部分由德国研究基金会（授予CRC / TR 36），德国中心感染研究和亥姆霍兹艾伯塔省倡议传染病研究（HAI-IDR）。

StephenW

全文: http://www.gastrojournal.org/art ... %2901382-7/fulltext

StephenW

The Covalently Closed Circular Form of Hepatitis B Virus Genome: Is There Now an End in “Site”?
Timothy M. Blockcorrespondenceemail
, Ju-Tao Guocorrespondenceemail
Hepatitis B Foundation, Doylestown, Pennsylvania
Baruch S. Blumberg Institute, Doylestown, Pennsylvania
Article has an altmetric score of 1
DOI: http://dx.doi.org/10.1053/j.gastro.2015.11.032 |
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Published Online: November 22, 2015

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See “Interferon-γ and tumor necrosis factor-α produced by T cells reduce the HBV persistence form, cccDNA, without cytolysis,” by Xia Y, Stadler D, Lucifora J, et al, on page 194.

One of the major remaining mysteries about the pathogenesis of hepatitis B virus (HBV) infection is how the host immune system resolves the virus infection and eliminates the nuclear form of the viral genome, the covalently closed circular (ccc)DNA. The paper by Xia et al, in this issue of Gastroenterology, addresses this question.1

After entry of HBV into hepatocytes, the viral relaxed circular (rc)DNA genome is transported into the nucleus and converted into cccDNA with the help of cellular DNA repair machinery.2 cccDNA associates with histones and assembles into episomal minichromosome, which is the transcriptional template for viral messenger RNA.3 Persistent HBV infection relies on stable maintenance and proper functioning of cccDNA. Thus, resolution of infection depends on the elimination and/or transcriptional suppression of cccDNA and inability to repress cccDNA is the primary reason for the failure of current available antiviral medications to achieve a sustained, off-drug virologic response.4 Although previous work suggests that resolution of acute HBV infection involves both cytolysis and noncytolytic cure of infected hepatocytes,5, 6, 7 the question as to whether or not cccDNA can be degraded selectively by the host immune response without damaging the infected hepatocytes remains controversial.

The paper by Xia et al1 reports that cccDNA levels in HBV-infected primary human hepatocytes can be reduced by HBV-specific T lymphocytes via secreted inflammatory cytokines, interferon (IFN)-γ and tumor necrosis factor (TNF)-α, in a noncytopathic fashion. Similar to IFN-α and lymphotoxin-β receptor (LTβR) agonists,8 IFN-γ and TNF-α induce the expression of APOBEC3A and APOBEC3B, which are recruited to cccDNA to deaminate the cytidines in its minus-strand. The deamination of cccDNA results in uracil–DNA glycosylase cleavage of uracils to form apurinic/apyrimidinic sites, which are subsequently recognized and cleaved by apurinic/apyrimidinic endonucleases (Figure 1). To establish clinical relevance of the cell-based observation, the authors further demonstrate that IFN-γ and TNF-α are increased in the sera of patients with acute, but not chronic HBV infection.
Thumbnail image of Figure 1. Opens large image
Figure 1

Antiviral mechanism of inflammatory cytokines against hepatitis B virus (HBV). HBV infection of a hepatocyte begins by binding of virions to Na+-taurocholate cotransporting polypeptides (NTCP) on the cell surface and delivering nucleocapsids into the cytoplasm. Import of the cytoplasmic nucleocapsid DNA into the nucleus to form covalently closed circular (ccc)DNA, viral RNA transcription, pregenomic (pg) RNA encapsidation, DNA replication in the cytoplasmic nucleocapsids as well as virion assembly and secretion via multivesicle body (MVB) pathway are also depicted. Interferon (IFN)-α, IFN-γ, tumor necrosis factor (TNF)-α or lymphotoxin-β (LT-β) bind to their cognate receptors on cell surface to elicit signaling cascades leading to activation of APOBEC3A and/or APOBEC3B gene expression. The APOBEC3 proteins are subsequently recruited to cccDNA, through interaction with HBV capsid proteins, to deaminate the cytidines in the minus-strand of cccDNA. The deamination of cccDNA results in uracil–DNA glycosylase cleavage of uracils to form apurinic/apyrimidinic sites, which are recognized and cleaved by apurinic/apyrimidinic (AP) endonucleases. In addition, IFN-α also induces the expression of other IFN-stimulated genes (ISGs) that inhibit cccDNA transcription and pregenomic pgRNA encapsidation.

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Although this study provides an interesting answer for a long-standing question in HBV immunology, it also arises very important issues to be addressed in future studies. Specifically, the authors made a very strong case that APOBEC3A and APOBEC3B play an essential role in the cccDNA reduction induced by multiple inflammatory cytokines.1, 8 Although their studies suggest that HBV capsid protein interacts with and recruits the APOBEC3 proteins to cccDNA minichromosomes8 (Figure 1), the question is how the viral protein recruits the APOBEC3 specifically to cccDNA. In addition, despite evidence suggesting that cytidine deamination does occur on cccDNA, but not derive from HBV DNA in cytoplasmic nucleocapsids, it remains mysterious that the cytidine deamination occurs only on the minus strand of cccDNA. In contrast, it has been reported that APOBEC3 proteins deaminate other nuclear episomal viral DNA, such as human papilloma virus circular DNA genome, on both strands.9, 10 Importantly, to validate the role of the APOBEC3 enzymes in noncytolytic clearance of cccDNA, demonstration of cccDNA cytidine deamination by direct sequence of cccDNA extracted from HBV- or animal hepadnavirus-infected livers during the immune clearance phase is probably required. Finally, it should be pointed out that the modest reductions of cccDNA in hepatocytes by the inflammatory cytokines suggests that other mechanisms, such as suppression of cccDNA transcription11, 12 and modulation of nucleocapsid metabolism13, 14 (Figure 1), may also play an important role in the noncytolytic immune control of HBV infection. In conclusion, although the work of Xia et al offers a compelling explanation for how immune response could noncytolytically eliminate cccDNA, with regard to the controversy about whether and how cccDNA can be noncytolytically degraded by APOBEC3 enzymes, we have not yet seen an end.15, 16

StephenW

乙型肝炎病毒基因组的共价闭合环状形态：现在是否有在“网站”的终结？
蒂莫西·M。Blockcorrespondenceemail
，鞠涛Guocorrespondenceemail
乙型肝炎基金会，宾夕法尼亚州Doylestown
巴鲁克S. Blumberg的学院，宾夕法尼亚州Doylestown
文章的altmetric比分1
DOI：http://dx.doi.org/10.1053/j.gastro.2015.11.032 |
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发布时间：2015年11月22日

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看到夏Y，施泰德研发，Lucifora J，等“γ干扰素和T细胞产生的肿瘤坏死因子α降低HBV持续存在形式，cccDNA的，无细胞溶解”，第177页。

一个约乙型肝炎病毒（HBV）感染的发病机理的主要剩余的奥秘是宿主免疫系统如何解决该病毒感染，并消除病毒基因组中，共价闭合环状（CCC）的DNA的核形式。夏等人的论文，在这个问题上消化，解决了这个question.1

条目的HBV成肝细胞后，病毒松弛环状（RC）DNA基因组被输送至细胞核，并转换成cccDNA的与细胞DNA修复machinery.2 cccDNA的联营公司与组蛋白的帮助和组装成游离型微染色体，这是转录模板病毒信使RNA.3持续HBV感染者依靠稳定的维护和cccDNA的正常运作。因此，感染的分辨率取决于消除和/或cccDNA的转录抑制和无法抑制的cccDNA是主要原因当前可用抗病毒药物未能实现了持续，休药病毒学response.4尽管以前的研究表明，急性HBV感染分辨率既包括细胞溶解和感染的肝细胞，5，6的noncytolytic治愈，7的问题是，是否cccDNA的可以选择性地被宿主的免疫应答降低，而不会损坏感染的肝细胞仍存在争议。

通过夏等纸张AL1报告的cccDNA水平HBV感染的原代人肝细胞可通过HBV特异性T淋巴细胞经由分泌的炎性细胞因子，干扰素（IFN）-γ和肿瘤坏死因子（TNF）-α，在一个noncytopathic减小时尚。类似于IFN-α和淋巴毒素β受体（LTβR）激动剂，8的IFN-γ和TNF-α诱导APOBEC3A和APOBEC3B，该被招募到的cccDNA到脱氨在其负链的胞苷的表达。的cccDNA的结果在尿嘧啶的尿嘧啶DNA糖基化酶裂解脱氨以形成脱嘌呤/脱嘧啶位点，其随后承认和脱嘌呤/脱嘧啶内切核酸酶切割（图1）。要建立临床的基于细胞的观察相关性，作者进一步证明IFN-γ和TNF-α治疗急性血清增加，但不是慢性HBV感染。
图1.缩略图打开大图
图1

炎性细胞因子对乙型肝炎病毒（HBV）的抗病毒机制。 HBV感染肝细胞的开始由病毒颗粒在细胞表面上结合的Na + -taurocholate cotransporting多肽（NTCP）和提供核壳进入细胞质。细胞质核衣壳的DNA进入细胞核的导入，形成共价闭合环状（CCC）的DNA，病毒RNA转录，前基因组（PG）RNA包壳，通过multivesicle体（MVB）途径在细胞质核衣壳DNA的复制以及病毒粒子组装和分泌还描绘。干扰素（IFN）-α，IFN-γ，肿瘤坏死因子（TNF）-α或淋巴毒素β（LT-β）结合到其同源受体在细胞表面上以引发信号级联从而激活APOBEC3A和/或APOBEC3B基因表达。所述APOBEC3蛋白质随后被招募到cccDNA的，通过与HBV的衣壳蛋白相互作用，以脱氨在负链的cccDNA的胞苷。的cccDNA的结果在尿嘧啶的尿嘧啶DNA糖基化酶裂解脱氨以形成脱嘌呤/脱嘧啶位点，这是公认的，并通过脱嘌呤/脱嘧啶（AP）内切核酸酶切割。另外，IFN-α也诱导的其他干扰素刺激基因（的ISG），该抑制的cccDNA转录和前基因组pgRNA壳体化的表达。

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尽管本研究提供了在HBV免疫学的长期问题的有趣的答案，这也出现在未来的研究要处理非常重要的问题。具体来说，作者提出了一个非常强烈的情况下APOBEC3A和APOBEC3B发挥着重要作用，诱发多种炎症cytokines.1，8 cccDNA的降低虽然他们的研究表明，乙肝病毒衣壳蛋白相互作用并募集APOBEC3蛋白cccDNA的minichromosomes8（图1），问题是如何病毒蛋白募集APOBEC3专门cccDNA的。此外，尽管有证据表明胞苷脱氨基确实发生于cccDNA的，但是从HBV DNA在细胞质核衣壳不派生，它仍然是神秘的胞苷脱氨仅发生在cccDNA的的负链。与此相反，它已经报道APOBEC3蛋白脱氨其他核游离型病毒DNA，例如人类乳头状瘤病毒的环状DNA基因组，在两个strands.9，10重要的是，以验证APOBEC3酶中的cccDNA的noncytolytic间隙的作用，示范对cccDNA的胞嘧啶脱氨通过的cccDNA在免疫清除期的HBV-或动物嗜肝DNA病毒感染的肝脏中提取的直接序列可能是必需的。最后，应该指出的是的cccDNA中肝细胞的适度减少由炎症性细胞因子表明其他机制，如抑制的cccDNA transcription11，12和核衣壳metabolism13的调制，14（图1），也可发挥重要的作用在HBV感染的noncytolytic免疫控制。总之，尽管夏等人的工作提供了一个令人信服的解释如何免疫反应可能noncytolytically消除cccDNA的，关于是否以及如何的cccDNA可以通过APOBEC3酶来降解noncytolytically的争议，我们还没有看到一个end.15 ，16

zgct

讲的好有深度