Inhibition of Intrahepatic Gamma Interferon Production by Hepatitis C Virus Nonstructural Protein 5A in Transgenic Mice

0022-538X/09/$08.00⫹0 doi:10.1128/JVI.00751-09

Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Inhibition of Intrahepatic Gamma Interferon Production by Hepatitis C

Virus Nonstructural Protein 5A in Transgenic Mice

䌤

Tatsuo Kanda,

† Robert Steele,

Ranjit Ray,

and Ratna B. Ray

*

Departments of Pathology1_{and Internal Medicine}2_{and Liver Center,}3_{Saint Louis University, St. Louis, Missouri} Received 12 April 2009/Accepted 18 June 2009

Hepatitis C virus (HCV) utilizes strategies to suppress or evade the host immune response for establishment of persistent infection. We have shown previously that HCV nonstructural protein 5A (NS5A) impairs tumor necrosis factor alpha (TNF-␣)-mediated apoptosis. In this study, we have examined the immunomodulatory role of HCV NS5A protein in transgenic mouse (NS5A-Tg) liver when mice were challenged with an unrelated hepatotropic adenovirus as a nonspecific stimulus. Hepatotropic adenovirus was introduced intravenously into NS5A-Tg mice and control mice, and virus clearance from liver was compared over a time course of 3 weeks. The differential mRNA expression levels of 84 cytokine-related genes, signal pathway molecules, transcription factors, and cell surface molecules were determined using real-time reverse transcription-PCR array. NS5A-Tg mice failed to clear adenovirus from liver up to 3 weeks postinfection while control mice cleared virus within 1 to 2 weeks. Subsequent study revealed that gamma interferon (IFN-␥) expression is inhibited at both the mRNA and protein levels in NS5A-Tg mice, and an inverse expression of transcription factors Gata-3 and Tbx21 is observed. However, TNF-␣mRNA and protein expression were elevated in both NS5A-Tg and control mice. Together, our results suggested that HCV NS5A acts as an immunomodulator by inhibiting IFN-␥ production and may play an important role toward establishment of chronic HCV infection.

Hepatitis C virus (HCV), a member ofFlaviviridae, causes chronic infection associated with cirrhosis and hepatocellular carcinoma. The HCV genome containing positive-strand RNA is approximately 9.6 kb and encodes a polyprotein precursor of about 3,000 amino acids, which is cleaved by both viral and host proteases into structural (core, E1, E2, and p7) and non-structural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) pro-teins. HCV NS5A exists as two phosphoproteins, p56 and p58, phosphorylated at serine residues after the mature protein is released from the polyprotein (46). HCV NS5A has an anti-apoptotic effect (11, 27) and modulates immune responses (1, 39).

Interferons (IFNs) are key players of the innate immune response to virus infection. alpha IFN (IFN-␣) and IFN-␤ (type I IFNs) are secreted by almost all virus-infected cells and by specialized blood lymphocytes. In contrast, the production of IFN-␥(type II IFN) is restricted to cells of the immune system, such as natural killer (NK) cells, macrophages, and T cells. Although the antiviral effect of type I IFN on the repli-cation of HCV has been the subject of extensive research and although the endogenous type I IFN antiviral effector pathway is known to be broadly activated during chronic HCV disease (25), little is known about the role of other cytokines in cellular defense against HCV. Further, how HCV manages to evade the host defense is poorly understood.

In the liver of chimpanzees acutely infected with HCV, IFN-␥ was detected only in animals displaying sustained or

transient viral clearance in the context of an intrahepatic HCV-specific T-cell response (26, 41, 47, 52). An increase in the IFN-␥gene was not detected by microarray or by quantitative PCR in the liver of chimpanzees chronically infected with HCV compared with HCV-uninfected chimpanzees (4, 16). IFN-␥mRNA expression levels in peripheral blood mononu-clear cells of patients are significantly lower in nonresponders (15). During the first 6 months of onset of HCV infection, the number of IFN-␥-producing HCV-specific CD8⫹ T cells is associated with virus eradication (13). IFN-␥also inhibits HCV genome replication in vitro (9). On the other hand, tumor necrosis factor alpha (TNF-␣), a primary initiator of innate immune response, determines the magnitude and course of acquired immune responses. TNF-␣-induced apoptosis is im-portant for clearance of virus-infected cells, and IFN-␥ accel-erates the killing of virus-infected cells (17, 32, 48, 52).

A number of viruses have been shown to encode proteins that have the potential to inhibit antiviral activity of innate and adaptive immune responses. Inflammatory cytokines contrib-ute to viral clearance during accontrib-ute viral hepatitis in humans, and the induction of these cytokines in the liver and other tissues of chronically infected patients may have therapeutic value (20). Disson et al. (8) have shown that transgenic mice expressing HCV polyprotein failed to clear an adenoviral in-fection. However, transgenic mice expressing either core or structural proteins do not appear to be immunologically impaired as they are fully competent to eliminate hepato-cytes infected with adenovirus (Ad) (24, 42). Together, these results suggest a role of HCV nonstructural protein(s) in the modulation of the immune system. In this present study, we have examined the role of HCV NS5A protein on intrahepatic cytokine production for clearance of an unre-lated hepatotropic Ad using a transgenic mouse model. Our results demonstrated that IFN-␥production is inhibited and

* Corresponding author. Mailing address: Department of Pathology, Saint Louis University, 1100 S. Grand Blvd., DRC-207, St. Louis, MO 63104. Phone: (314) 977-7822. Fax: (314) 771-3816. E-mail: rayrb@slu .edu.

† Present address: Chiba University, School of Medicine, Chiba, Japan.

䌤_{Published ahead of print on 24 June 2009.}

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infected liver in humans (29). NS5A-Tg mice or control (nontransgenic [NTg]) mice aged 10 to 12 weeks were used in this study.

Ad infection.A recombinant replication-deficient Ad designated AdGFP was kindly provided by Bert Vogelstein (Johns Hopkins University, Baltimore, MD). This virus system is based on the human Ad type 5 from which the E1a and E1b genes were deleted and is capable of infecting murine hepatocytes. Infecting cells with AdGFP expresses green fluorescent protein (GFP) under the control of a cytomegalovirus promoter. Stocks of AdGFP were grown in 293 cells and purified by two rounds of CsCl density gradient centrifugation as described previously (10). Virus titers were determined by plaque assay on 293 cells, and a single stock was used throughout this study. Mice (12 in each group and for each time point) were injected

with a single dose of AdGFP (1.0⫻109

PFU/mouse) or with 100␮l of saline as

control via tail vein. Infected mice were sacrificed at the time points indicated in Fig. 1. Livers were frozen with optimal cutting temperature medium for histological examination and snap frozen for RNA or protein analysis.

GFP expression in AdGFP-infected mouse liver.Cryostat-frozen liver sections

(5␮m) were examined by indirect fluorescence microscopy for in vivo expression

of GFP in AdGFP-infected mouse liver (Bio-Rad 1024; Bio-Rad Laboratories, Hercules, CA). One or two slides were examined for each liver specimen. GFP expression was also quantitated using a fluorometer (1420 Victor Multilabel Counter; Perkin Elmer, Wellesley, MA) as described earlier (2). We also exam-ined GFP expression using immunoblot analysis. Proteins were subjected to electrophoresis on a 12% polyacrylamide gel and transferred onto a nitro-cellulose membrane (Bio-Rad). The membrane was probed with a

monoclo-nal antibody to GFP or␤-tubulin (Santa Cruz Biotechnology, Santa Cruz,

CA). Protein bands were visualized using enhanced chemiluminescence (Pierce, Rockford, IL).

ELISA.Mouse sera were analyzed for circulating IFN-␥and TNF-␣using an enzyme-linked immunosorbent assay (ELISA; R & D System, Minneapolis, MN) following the manufacturer’s protocol. Briefly, serum samples were incubated in plates at 4°C overnight, followed by incubation with biotinylated monoclonal antibodies. Avidin-conjugated peroxidase was added to the plates, and enzyme activity was detected with an ELISA plate reader.

RNA extraction and real-time PCR.Total RNA was extracted from mouse liver (day 14 after Ad infection) using a Purescript RNA isolation kit (Gentra Systems, Minneapolis, MN). We isolated RNA from individual livers of six mice (i.e., not pooled samples) from each group. Each mouse liver RNA sample was used in each PCR array. cDNA synthesis was performed using a random hex-amer. Comparison of the relative expression levels of 84 inflammatory cytokines and T-cell marker-related genes was performed with a mouse PCR array (Su-perarray Bioscience Corporation, Frederick, MD). For RNA quantitation, real-time PCR was conducted using SYBR Green I (ABI Prism 7700; Applied Biosystems, Foster City, CA). The two housekeeping genes (glyceraldehyde-3-phosphate dehydrogenase and actin) were used for normalization, and data were

analyzed by the comparative threshold cycle (CT) method. We have done the

assay on day 14 from each group following Ad infection. Relative quantification

of gene expression using the 2⫺⌬⌬Ct_{method correlated with the absolute gene}

quantification obtained using a standard curve (40). Data were analyzed with

RT2 _{profiler PCR array data analysis software (http://www.superarray.com}

/pcrarraydataanalysis.php). Genes were annotated using Mouse Genome Infor-matics (http://www.inforInfor-matics.jax.org/). The relative differences were calculated by comparing untreated mice with day 14 Ad-infected mice, and results were then compared between normal and transgenic mouse groups.

RESULTS

Impaired clearance of Ad from HCV NS5A-Tg mouse liver.

To investigate the effect of HCV NS5A on unrelated virus clearance from mouse liver, age- and sex-matched NS5A-Tg

and NTg mice (12 in each group) were injected with⬃1⫻109

PFU of AdGFP via tail vain. Livers were collected from mice at 3, 7, 14, and 21 days postinfection. Recombinant Ad infec-tion was monitored from GFP marker expression. Hepatocytes (⬎95%) displayed GFP expression from the recombinant Ad in NS5A-Tg and NTg mouse livers at day 3 (Fig. 1A). GFP-positive cells were also readily detectable at days 7, 14, and 21 following Ad infection in NS5A-Tg mouse livers in contrast to the NTg controls. Liver extracts were prepared from both NS5A-Tg and NTg mice at days 3, 7, 14, and 21 as described

FIG. 1. Impaired clearance of Ad from NS5A-Tg mouse liver. (A) NTg (panels a, c, e, and g) and NS5A-Tg mice (panels b, d, f, and h) were infected with replication-deficient recombinant Ad expressing GFP by injection through the tail vein (1⫻ 109_{PFU). Mice were} sacrificed at day 3 (a and b), day 7 (c and d), day 14 (e and f), or day 21 (g and h), and liver sections were evaluated microscopically for GFP expression. (B) GFP expression was quantitated from the crude liver extracts of NS5A-Tg and NTg mice at different days. GFP expression from day 3 postinfection mouse liver was chosen arbitrarily as 100%. (C) GFP expression from liver extracts (day 21) of NS5A-Tg and NTg mice was analyzed using specific antibody. The blot was reprobed with an antibody to␤-tubulin for comparison of protein load.

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previously (28), and GFP expression was quantitated using a fluorometer (Fig. 1B). GFP expression was normalized to that from mouse livers on day 3. GFP expression was also examined by Western blot analysis (Fig. 1C). Together, our results re-vealed that the NS5A-Tg mice fail to clear Ad from liver up to 21 days postinfection.

We have examined the T-cell infiltration in both NTg and NS5A-Tg mice at day 7 following Ad infection. For this, the presence of CD3⫹ cells was examined in liver sections by immunohistochemistry. Slides were immunostained with anti-CD3 antibody and counterstained with hematoxylin. anti-CD3⫹ cells from at least five fields of each slide were counted in a blinded manner. We did not observe a significant difference in the level of CD3 marker between the NS5A-Tg and NTg mice (Fig. 2). The slides were also stained with hematoxylin and eosin to examine inflammation. We have observed a slightly higher level of inflammation in NTg mice than in HCV NS5A-Tg mice following Ad infection. A representative illus-tration is shown in Fig. 2 (bottom panel). Our results are in agreement with the earlier study of transgenic mice expressing the entire HCV genome (8), suggesting that the presence of the HCV transgene does not impair intrahepatic T-cell infil-tration. We also examined the alanine aminotransferase (ALT) level from serum of NS5A-Tg and NTg mice at different time points. The ALT level was increased approximately five-to eightfold on day 7 and day 14 in NTg mice compared five-to levels in NS5A-Tg mice (Table 1).

Intrahepatic cytokine gene expression during Ad infection in HCV NS5A-Tg mice. CD4⫹T cells are a major player in the induction of protective immune response to HCV infection. Th17 is a newly defined subset of CD4⫹T cells. The discovery of the Th17 lineage has revealed additional complexity in the fates chosen by helper T cells and has begun to reshape our view of how signaling and transcriptional networks generate appropriate and inappropriate immunity (37). We have exam-ined cytokine-related gene expression profiles using real-time PCR-based focused microarrays for intrahepatic inflammatory genes related to the Th17 regulatory network. A comparison of intrahepatic cytokines between NS5A-Tg and NTg mice at day 14 of Ad infection is shown in Fig. 3A. Out of 84 cytokine-related genes examined, 63 (75.0%) were upregulated by

2.0-FIG. 2. Liver specimens from day 7 Ad-infected NTg and NS5A-Tg mice were immunostained with CD3⫹antibody and counterstained with hematoxylin to determine infiltrating T cells (top panels). Liver sections were also stained with hematoxylin and eosin (H&E) to examine inflammatory infiltrates and liver histopathology (bottom panels).

TABLE 1. Serum ALT level in NTg and NS5A-Tg mice

Time point (day)

ALT level (U/liter)a

NTg mice NS5A-Tg mice

0 120⫾31 122⫾23

7 696⫾218 188⫾65

14 1071⫾206 298⫾104

24 ND 144⫾56

a

Data for each time point represent the sera of six animals, and values are

means⫾standard deviations. ND, not done (AdGFP was cleared from mouse

liver).

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fold or greater in NTg mice while 38 (45%) genes were up-regulated 2.0-fold or higher in NS5A-Tg mice (Fig. 3B). The response to Ad infection was partly related to NS5A expres-sion, with 32 genes unique to the NTg mice, 7 unique to NS5A-Tg mice, and 31 expressed in both NS5A-Tg and NTg mice. Eleven genes (TNF-␣, interleukin-23 receptor [IL-23R], CCL20, granulocyte colony-stimulating factor, IL-21, CXCL5, IL-12RB2, IL-10, IFN-␥, Toll-like receptor 4 [TLR4], and IL-17␣) were upregulated fivefold or more in NTg mice, and only TNF-␣was upregulated in NS5A-Tg mice following Ad infec-tion. The results of the genes upregulated twofold or greater are summarized in Fig. 3. On the other hand, out of 84 genes, 5 (5.9%) genes were downregulated 2.0-fold or more in both groups of mice (Fig. 3A). Among these, four genes were unique to the NTg mice (IL-17, PBST-1, PB1, and calcyclin binding protein) and NS5A-Tg mice (IL-23, CD127, NF-E1, and Stat3), and one gene was expressed in both types of mice. CCAAT/enhancer binding protein beta, which has antiapop-totic function, was the only gene downregulated in both animal groups.

Further analysis revealed that among the Th1 cytokines, IFN-␥, granulocyte colony-stimulating factor, and IL-2 mRNA expression levels were suppressed significantly in NS5A-Tg mice compared to NTg control mice (Fig. 4A). On the other hand, TNF-␣ and IL-12␤ mRNA inductions were similar in

both NS5A-Tg and NTg mice infected with Ad (Fig. 4A). Expression of chemokine genes, CCL7 (MCP-3) and CX3CR1, was upregulated to approximately 2.0-fold in NS5A-Tg mice. Importantly, CCL7 has been implicated with portal inflamma-tion in liver disease (49) while CX3CR1 is involved in liver fibrosis due to chronic HCV infection (51).

Ad-infected NS5A-Tg mice display altered expression of intrahepatic lymphoid cell markers. One of the earliest mani-festations of a cell-mediated immune response in the infected liver is an increase in lymphoid cells. To examine cell infiltra-tion, liver from mice injected with Ad was harvested and ana-lyzed for lymphoid cell surface molecules using real-time PCR (Fig. 4B.). Among the T-cell markers, the greatest suppression was seen in CD8␣(synonymous for CD8 in human) mRNA, while CD4 mRNA was suppressed to a smaller extent in NS5A-Tg animals. Among the B-cell markers, CD40lg was suppressed in NS5A-Tg animals (Fig. 4B). ICAM-1, a well-known cell adhesion molecule, was upregulated in NS5A-Tg mice. Expression of Gata-3, Stat6, Socs3, Syk, Socs1, Jak1, Myd88, TLR4, Stat4 and T-bet were upregulatedⱖ2.0-fold in NS5A-Tg mouse livers. Therefore, these results suggested that NS5A-Tg mouse liver exhibits decreased expression of CD4 and CD8 cell surface molecules. This could be the result of the impact of NS5A expression on the overall ratio of CD4 to CD8 cells and awaits further in-depth analysis. An inverse expres-sion of IFN-␥and TLR in NS5A-Tg mouse liver is in agree-ment with chronically infected HCV individuals who failed to respond to IFN therapy (14). Tbx21 modulates IFN-␥ expres-sion in Th1 cells (45). Our results suggested that Tbx21 mRNA expression is suppressed in Ad-infected NS5A-Tg mice. This

FIG. 3. Differential expression of intrahepatic cytokine genes in NS5A-Tg and NTg mice following Ad infection. (A) NTg and NS5A-Tg mice were infected with Ad or left untreated. Liver RNA was isolated at day 14 postinfection and used in focused microarray analysis for 84 genes. All gene expression levels were corrected by comparison with the level of two housekeeping genes (glyceralde-hyde-3-phosphate dehydrogenase and actin) and compared. Six sets of real-time PCR arrays were performed, and the results were analyzed using RT2_{profiler PCR array data analysis software} (Su-perarray). (B) The details of genes upregulated 2.0-fold or greater in NTg mice and HCV NS5A-Tg mice are shown.

FIG. 4. Differential expression of intrahepatic cytokine mRNA lev-els (A) and presence of lymphoid cell markers (B) in Ad-infected the liver of NTg and NS5A-Tg mice. Gene expression was measured by quantitative real-time PCR, and results are presented as induction relative to basal levels in each of the uninfected mice. Six mice in each group were examined.

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result is in agreement with the previous observation that the Tbx21 mRNA level is reduced in HCV-infected T cells (19).

Ad infection of NS5A-Tg mice does not enhance IFN-␥ se-cretion.TNF-␣ determines the magnitude and course of ac-quired immune responses. TNF-␣-induced apoptosis is impor-tant for lysis of virus-infected cells, and the activity of killing virus-infected cells is accelerated by IFN-␥(17, 32, 53). Mod-ulation of TNF-␣ and IFN-␥has been observed in HCV-in-fected patients (16). Since IFN-␥and TNF-␣have also been implicated in clearance of Ad from mouse liver (17, 43), the modulation of these two cytokines involved in innate signaling of NS5A-Tg mice was verified at the protein level by ELISA. Our results demonstrated that TNF-␣expression was upregu-lated in both NTg and NS5A-Tg mice (Fig. 5). On the other hand, IFN-␥production was significantly lower in Ad-infected NS5A-Tg mice. These results are in agreement with our quan-titative reverse transcription-PCR data described above and the observations from chronically HCV-infected humans (35).

DISCUSSION

We have examined whether HCV NS5A interferes with the innate antiviral signaling pathway in liver. For this, unrelated hepatotropic Ad clearance from NS5A-Tg mouse liver was investigated. The Ad-dependent antiviral effect occurs in two distinct phases and has been eloquently described by Ca-vanaugh et al. (5). The first phase of induction occurs within 12 to 24 h of infection and is mediated by IFN-␣/␤. The second phase occurs after 3 to 7 days of infection and is associated with intrahepatic induction of IFN-␣/␤and TNF-␣as well as IFN-␥for induction of the cellular immune response to the invading virus. The production of these cytokines was also seen in human infection from Ad and plays an important role in viral clearance (30, 33, 43). An earlier report suggested that transgenic mice expressing the entire HCV genome delay Ad clearance from liver (8). On the other hand, mice expressing HCV structural proteins do not impair Ad clearance (24, 42). Here, we have shown that NS5A-Tg mice modulate the innate immune response in the liver and impair Ad clearance. Inter-estingly, both NTg and NS5A-Tg mice displayed elevation of TNF-␣ while the level of IFN-␥ was significantly lower in NS5A-Tg mice following Ad infection. However, HCV NS5A blocks TNF-␣-mediated apoptosis (29), and a high level of

TNF-␣expression is observed in the liver of chronically HCV-infected individuals (22). Together, our results suggested that inhibition of IFN-␥expression in NS5A-Tg mice and blockade of TNF-␣-mediated apoptosis by NS5A contribute to delayed Ad clearance. IFN-␥is not expressed in hepatocytes, and we therefore believe that a secondary event may modulate IFN-␥ production since other resident cells cross talk in the liver. IFN-␥with potential antifibrotic action is associated with the blockage of stellate cell activation, proliferation, and synthesis of collagen. Experimental studies have demonstrated that IFN-␥ inhibits extracellular matrix mRNA expression (47). Suppression of IFN-␥by NS5A may also be associated with the progression of hepatic fibrosis in chronically infected HCV patients. Our data support the earlier observation that patients with self-limited hepatitis C display the strongest virus-specific CD4⫹T-cell (IFN-␥) reactivity while persistently infected sub-jects initially show a weak antiviral CD4⫹T-cell (IFN-␥) re-sponse (14). We did not observe significant poly(ADP-ribose) polymerase cleavage in Ad-infected NS5A-Tg mouse liver ex-tract, suggesting an absence of apoptosis.

Gata-3 is essential for NK cell homing to the liver. While Gata-3 antagonizes IFN-␥production in differentiating T cells, Gata-3-deficient NK cells paradoxically produce less IFN-␥ than control NK cells (37, 38). Gata-3 serves as a principal switch in determining Th1/Th2 responses (54). Optimal induc-tion of IFN-␥expression also requires genetic interaction be-tween Tbx21 (T-bet) and its target, the homeoprotein Hlx (34). Tbx21 expression correlates with IFN-␥expression in Th1 and NK cells (45). Tbx21 mRNA was 2.2-fold upregulated in NS5A-Tg mice and 8.7-fold upregulated in NTg mice following Ad infection. These results are in agreement with the obser-vation that the Tbx21 mRNA level is suppressed in HCV-infected T cells (19). Tbx21 is required for protection against human viral infection (6, 21, 31, 44). The principal function of Tbx21 in developing Th1 cells is to negatively regulate Gata-3 (50). T lymphocytes have an essential role in the clearance of acute HCV infection (7, 23). Individuals who are able to suc-cessfully clear HCV seem to have not only specific but also

FIG. 5. Ad infection in NS5A-Tg mice does not enhance IFN-␥ secretion. IFN-␥and TNF-␣levels were determined by ELISA using sera from NTg and NS5A-Tg mice._*,P⬍0.001 by the Student’sttest from comparison between sera of NTg and NS5A-Tg mice.

FIG. 6. A schematic diagram depicting the highlights of observa-tions following Ad challenge in NTg and NS5A-Tg mice.

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ACKNOWLEDGMENTS

We thank Brendan Kappas for PCR arrays and John E. Sagartz for helping us to evaluate the immunohistochemistry slides.

This work was supported by research grants AI45144 and AI065535 (R.B.R.) from the National Institutes of Health and by the Blue Rib-bon Fund from Saint Louis University.

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