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). 作者: StephenW 时间: 2016-1-6 19:42
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.
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 作者: StephenW 时间: 2016-1-6 19:51