宿主对HBV感染反应的染色体组分析

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[B]Discussion  [/B]

The objective of this study was to identify liver genes that are transcriptionally regulated during HBV infection and are associated with either the entry and expansion of the virus or its immune-mediated clearance. The first group could include proviral genes that the virus might induce to optimize replication and antiviral genes that reflect the activation and/or effector functions of the innate immune response. The second group should include genes brought into the liver by cells of the adaptive immune response, plus hepatocellular genes that are activated by those cells and their products, e.g., IFN-.

These studies were modeled after a previous report from our laboratory (23) in which the genes associated with HCV spread and clearance were identified in the livers of chimpanzees that were acutely and chronically infected by HCV. Together, the results of the two studies display remarkable differences in the repertoire of genes that are induced during the infection by HBV in this study (no genes) and HCV in previous studies (23, 24) (27 genes, many of which are regulated by IFN-/), probably reflecting differences in the genome structure and the replication strategies of the two viruses and corresponding differences in their ability to induce an innate immune response. The results also reveal remarkable similarities in the repertoire of genes induced during viral clearance in both HBV and HCV infections (23, 24) and during T cell-mediated viral clearance in the HBV transgenic mouse model (11) (Table 1), probably reflecting similarities in the adaptive immune mechanisms responsible for their control. The results also illustrate that / T cells contribute to the inflammatory infiltrate during acute hepatitis B, and they identify a compact subset of candidate antiviral genes, some of which could mediate viral clearance by interrupting the HBV life cycle in infected cells.

IFN-/ and - are known to inhibit viral replication in the liver of HBV transgenic mice (8-10, 25). Both cytokines are produced during the innate immune response, IFN-/ primarily by infected cells and plasmacytoid dendritic cells (26, 27), and IFN- primarily by NK and NKT cells (28), which are abundant in the liver (28, 29). Thus, because we and others have shown that the expression of a large number of IFN-regulated genes is rapidly and consistently induced in the liver of HCV-infected chimpanzees and that the strength of their expression correlates with viremia (23, 24), we expected the same to occur in the HBV-infected animals. Surprisingly, not a single gene was induced or repressed by HBV in all three acutely infected animals. Furthermore, no genes were significantly up- or down-regulated in any of the animals in the lag phase of infection or the log phase of viral spread. These results imply that HBV did not induce an intrahepatic innate immune response that could be detected by gene chip analysis in all of the infected animals. The basis for this is not immediately clear. It may reflect the replication strategy of HBV whose DNA genome replicates within nucleocapsid particles (30) and thus is shielded from the cellular machinery that normally senses double-stranded RNA (31). It also implies that extracellular HBV virions and subviral products are not detected by the toll-like receptor system (32).

Many RNA viruses have developed elegant strategies to evade the antiviral effects of cytokines induced during the innate immune response (26). The need to develop these strategies exists for these viruses because they induce IFN-/ in infected cells or IFN- by cells of the innate immune system. We suggest that HBV achieves the same end by not inducing these cytokines in the first place. An interesting consequence of this is that by not needing to evade IFN early in the infection, the virus remains susceptible to IFN later in the infection when the adaptive immune response begins (6, 33).

In contrast to the apparent invisibility of HBV to the innate sensing machinery, when CD3+ MHC-restricted / receptor T cells appeared in the liver of the infected animals [Table 1 and Fig. 2B and 3 (shown in blue)], a temporal and functional cascade was initiated that correlated very strongly with the induction of IFN- and viral clearance. Because the IFN--inducible chemokine MIG was induced immediately before the onset of clearance in all three animals (data not shown), it is likely that IFN- played a major role in inducing most of the viral clearance-associated genes detected in this study, including 2'5'OAS, which is known to be inducible by IFN- (34). By using Ch1627 as a prototype, the early appearance of TCR/ receptor genes [Table 1 and Fig. 3 (shown in blue)] was accompanied by the induction of IFN--regulated genes (e.g., MHC class I, MHC class II, immunoproteasome components PA28 and PA28, and the TAP-binding protein tapasin), all of which may have facilitated antigen processing and presentation to the incoming T cells. In addition, the early induction of RANTES, MIG, and IP-10 probably enhanced the recruitment of additional inflammatory cells into the liver. The remaining early genes underlie a wide array of cellular functions, some of which might contribute to viral clearance. For example, ISG20 is an IFN-inducible RNase that increases the resistance to infection by RNA viruses (35) and is also induced during IFN-mediated suppression of viral replication in HBV transgenic mouse hepatocyte cell lines (11). Thus, ISG20 may also be able to destroy HBV RNA in infected hepatocytes. Additional studies are needed to test this hypothesis.

The early peak was followed by a second group of genes [Table 1 and Fig. 3 (shown in red)] reflecting expansion of the inflammatory infiltrate to include / T cells. / T cells do not express CD4 or CD8, do not recognize peptide antigens, and are MHC-unrestricted (36), but they have been shown to produce IFN- and contribute to the clearance of other viral infections (36, 37). Thus, further investigation of / T cell immunobiology in HBV infection appears to be warranted.

The middle gene cluster [Table 1 and Fig. 3 (shown in red)] also displays further evidence of T and NK cells and macrophage activation in the liver, because CD38 and the T cell and NK cell death effectors granzyme A and granzyme K peaked at this time, as did a large number of IFN--regulated genes, consistent with the detection of IFN- mRNA and the decreasing viral DNA content of the liver (Fig. 1). For example, STAT1, which mediates IFN receptor signaling, was maximally induced at this time, as were several MHC class II genes, the chemokines IP-10, MIG and IP-9, the immunoproteasome components LMP2 and MECL-1, ubiquitin D, and the ubiquitin-conjugating enzymes E2C and E2L6, all of which likely contribute to the inflammatory process. In addition, the IFN--induced GTPases, GBP1 and -2, as well as IFI27 and IFI16, peaked at this time. Interestingly, the murine homologues of GBP1 and IFI27 inhibit vesicular stomatitis virus (12) and Sindbis virus infection in mice (38), respectively, and they are induced in IFN-treated murine hepatocyte cell lines when HBV replication is suppressed (Table 1). Thus, further investigation of the role these genes play in viral clearance during HBV infection appears to be warranted.

Last, the middle gene cluster contains several other genes that could play a role in viral clearance and/or disease pathogenesis during HBV infection. Among these are members of the Ras-related family of small GTPases (i.e., Rab20, -27a, and -31) (reviewed in ref. 39) that might inhibit HBV replication by virtue of their ability to regulate vesicular transport (39-41). Cathepsin B is a cysteine protease that is released into the cytoplasm (42) during hepatocyte apoptosis where it might also target HBV. Karyopherin (importin-) induction might also influence HBV replication because it is known to regulate HBV capsid nuclear import (43).

The last (late) wave consisted of Ig heavy and light chain genes [Table 1 and Fig. 3 (shown in green)], apparently reflecting an influx of B cells into the liver.

Although the results of this study elucidate and strongly underscore the role of the adaptive immune response and its products in viral clearance during acute HBV infection, they do not discriminate among genes that are expressed in the inflammatory cells or the hepatocytes. Although this should be clarified in future studies, some insight can be gained by comparing the current results with our previous analysis of the gene expression profiles of HBV transgenic murine hepatocyte cell lines in which HBV replication was suppressed by IFN- and - in vitro. As shown in Table 1, a relatively compact panel of genes [STAT1, IFI16, GBP1, GBP2, LMP2, MECL-1, PA28/, IP-10, MIG, ISG20, leucine aminopeptidase 3, placenta-specific 8, tryptophanyl-tRNA synthetase (TrpRS), -2-microglobulin, ubiquitin-conjugating enzyme E2L6 (UBE2L6), tapasin, and MHC class I] was induced during viral clearance in all three chimpanzees and in IFN-- and/or --treated mouse hepatocytes (11), suggesting that they may function as effector molecules that suppress viral replication and contribute noncytolytically to the control of HBV infection. The generality and potential importance of this subset of genes are underscored by the fact that several of them are also induced in the liver during viral clearance in HCV-infected chimpanzees (23, 24).

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[B]Conclusion  [/B]

The current results suggest that HBV acts like a stealth virus early in infection, remaining undetected and spreading until the onset of the adaptive immune response several weeks later. When MHC-restricted / T cells enter the liver and recognize antigen, they kill some of the infected cells and secrete IFN-, which induces the expression of a large number of genes that enhance antigen processing and presentation; recruit macrophages, NK cells, and / T cells that also produce IFN-; and amplify the process. Several of the IFN--induced genes have antiviral activity in other systems, raising the possibility that they may also inhibit HBV replication. These highly coordinated cellular and molecular events continue until the infection is terminated, at which point they rapidly subside. Because many of the same genes are also associated with clearance of HCV, they represent a starting point for further elucidation of the cellular and molecular immunobiology of these infections.


    Acknowledgements  

We thank M. Shapiro and Dr. M. St. Claire (Bioqual, Rockville, MD) for animal care, C. Steiger for coordination and handling of biopsies, and R. Koch and R. Engle for technical assistance. We thank The Scripps Research Institute DNA core facility (Director Dr. S. Head) for RNA labeling, microarray hybridization, and data acquisition; Dr. A. Su (Genomics Institute of the Novartis Research Foundation, La Jolla, CA) for helpful advice; and Dr. R. Lanford (Southwest Foundation for Biomedical Research, San Antonio, TX) for critical reading of the manuscript. This study was supported by National Institutes of Health Grants AI20001 and CA76403 and contracts N01-AI-52705, N01-AI-45180, and N01-CO-56000, and The Sam and Rose Stein Charitable Trust. R.T. was supported by Grants TH 719/1-1 and TH 719/2-1 (Emmy Noether Program) from the Deutsche Forschungsgemeinschaft, Bonn, and by a postdoctoral training fellowship from the Cancer Research Institute, New York. This is manuscript number 16438-MEM from The Scripps Research Institute.



    Footnotes  

Abbreviations: HBV, hepatitis B virus; STAT, signal transducer and activator of transcription; Chn, chimpanzee n; HCV, hepatitis C virus; 2'5'OAS, 2'5' oligoadenylate synthetase; sALT, serum alanine aminotransferase activity; NK, natural killer; TCR, T cell antigen receptor.

Present address: Abteilung Innere Medizin II, Medizinische Universitätsklinik Freiburg, 79106 Freiburg, Germany.

To whom correspondence should be addressed. E-mail: [email protected].

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