Potential conflict of interest: Nothing to report.
Hepatitis C virus (HCV) infection causes acute and chronic liver disease often leading to liver cirrhosis and hepatocellular carcinoma. Numerous studies have shown that despite induction of virus specific immunity, a curative response is often not attained; this has led to the hypothesis that HCV genes modulate immunity, thereby enabling chronic infections. This study examined the effects on immune-mediated liver injury in transgenic mice expressing core protein throughout the body and bone marrow chimeras expressing core protein in either the lymphoid compartment or liver parenchyma. Presence of core protein in the liver parenchyma but not in lymphoid cells protects from autoimmune hepatitis induced by mitogen concanavalin A (ConA). Consistent with this observation, core transgenic hepatocytes are relatively resistant to death induced by anti-Fas antibody and tumor necrosis factor α (TNFα). This protective effect is associated with preferential activation of signal transducer and activation of transcription factor 3 (STAT3) versus STAT1 in livers of ConA-injected animals. In agreement with this effect of core protein on the Janus kinase (JAK)-STAT signaling pathway, transgenic mice accelerate liver regeneration after partial hepatectomy but are not protected from hepatocyte death. In conclusion, HCV core inhibits STAT1 and stimulates STAT3 activation, which protects infected hepatocytes from attack by the cell-mediated immune system and promotes their proliferation. (HEPATOLOGY 2006;44:936–944.)
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Viral liver infections induce host immune responses, associated with hepatic inflammation, mononuclear cell infiltration and increase of liver enzymes in the serum. While some infections evoke strong immune responses and severe liver injury, others are mild, resulting in chronic infections that persist over years. Hepatitis C virus (HCV) is an example of the latter1 and its ability to induce liver cirrhosis and hepatomas is cause for serious health concerns. Therefore, an important goal is to elucidate the mechanisms by which this virus maintains persistent infections despite the well-documented humoral- and cell-mediated immune responses to this pathogen.2
An attractive hypothesis has been that HCV genes modulate antiviral immune responses and much effort has been expensed to elucidate the mechanisms involved. However, a coherent picture as to the actions of HCV genes in establishing chronic infections has yet to emerge. Among the many reports on HCV genes and their effects on antiviral responses, a number have shown that the HCV core can modulate interferon (IFN) induced signal transducer and activation of transcription factor 1 (STAT1) signaling.3–5 However, the question as to whether this alters cell-mediated immune responses to the infection was not resolved. In this study, we observed transgenic mice expressing the HCV core under control of the elongation factor-1α (EF-1α) promoter in many tissues, including the lymphoid compartment and liver. Here we show that expression of the core in the liver parenchyma conveys resistance to autoimmune liver injury, induced by T cell mitogen concanavalin A (ConA).6 This resistance is explained by relative inhibition of STAT1 and stimulation of STAT3 activation in mice expressing the core protein and induced for autoimmune liver injury. Consistent with the stimulatory effect of the core on STAT3 signaling, we show that liver regeneration following partial hepatectomy (PHx) is accelerated in core transgenic mice, leading to the conclusion that the core gene by protecting cells from destruction by the immune system and stimulating their proliferation promotes long lasting infections.
Mouse Strains, Preparation of Bone Marrow Chimeras, Partial Hepatectomy, Assays for Liver Regeneration and Caspase 3 Activity.
Pathogen-free female C57BL/6 (B6) mice, were obtained from the Jackson Laboratory (Bar Harbor, ME). Transgenic mice, expressing the HCV core under control of the EF-1α promoter7 were backcrossed onto the B6 strain for ten generations at the USC animal facility. Only mice aged 6 to 8 weeks were used to avoid age related effects of the core gene and of immune functions. Bone marrow chimeras were prepared by irradiation of nontransgenic littermates or core transgenic mice 900 rads, immediately followed by intravenous injection of 2 × 107 bone marrow cells and 3 ×107 spleen cells. Mice were used after 2 weeks.8 For PHx, mice were anesthetized with ketamine/xylazine and 70% of the liver, i.e., the left, right median and left lateral hepatic lobes were removed surgically after ligation.9, 10 Sham-operated mice received anesthesia and abdominal incisions. Liver regeneration was monitored by determination of liver weights and incorporation of bromodeoxyuridine (BrdU). Mice received intraperitoneally 100 μg/g BrdU (Sigma, St. Louis, MO), livers were harvested 2 hours later, fixed in 4% paraformaldehyde and sections stained with an BrdU immunostaining kit (BD Biosciences, San Diego, CA). Stained cells were counted in a blinded fashion. Caspase-3 activity was measured in liver lysates using the Caspase-3/CPP32 Colorimetric Assay Kit (BioVision, Palo Alto, CA).11 Liver tissue was homogenized, suspended in lysis buffer for 10 min on ice, centrifuged at 14,000 rpm, 5 minutes at 4°C and protein concentrations in the supernatant determined with the DC protein assay kit (Bio-Rad, Hercules, CA). Caspase 3 substrates Asp-Glu-Val-Asp-p-nitroaniline (pNA) or Ile-Glu-Thr-Asp-pNA (200 μmol/L, final concentration) were incubated with 200 μg protein extract and the optical density read at 405 nm.
Induction of Hepatitis by ConA, TNFα, anti-Fas and Assays for Liver Enzymes and Liver Histology.
To induce hepatitis, animals were injected 10 mg/kg ConA (Sigma).6, 12 To test for sensitivity to tumor necrosis factor α (TNFα) mediated liver injury, animals received by intraperitoneal injection 800 mg/kg D-galactosamine, followed by an intraperitoneal dose of 0.5-1.5 mg/kg recombinant TNFα (rTNFα) (Sigma). To induce Fas-mediated liver injury, mice received intraperitoneal 500 μg/kg hamster anti-mouse Fas monoclonal antibody (mAb) (Jo2) (BD Biosciences). Serum alanine aminotransferase (ALT) levels were assayed using a commercial assay kit (Sigma).13 For histological examination, liver tissue samples were fixed in 4% paraformaldehyde, sections stained with hematoxylin-eosin and examined by light-microscopy.13
Isolation of Hepatocytes and Liver Mononuclear Cells (MNC).
Hepatocytes were prepared as previously described13 and only cells exceeding 89% viability were used. To test sensitivity to Fas and TNFα, hepatocytes were cultured for 4 hours with anti-Fas mAb in presence of 100 ng/mL protein A (Amersham Biosciences, Uppsala, Sweden), rTNFα and 1 μg/mL actinomycin D (Sigma). Actinomycin D was added 1 hour before rTNFα and anti-Fas mAb. Supernatants were assayed for LDH by Cytotox96 Non-Radioactive Cytotoxicity Assay kit (Promega, Madison, WI). Liver MNC were isolated from exsanguinated mice as described previously.6, 13, 14 For flow cytometric analysis, MNC were preincubated with anti-mouse CD16/CD32 (2.4G2) mAb from BD Biosciences to block Fc γ receptors, followed by various mAbs for 30 minutes at 4°C.8, 15 For analysis of natural killer T (NKT) cells, labeled anti-mouse CD3ϵ (145-2C11) and anti-NK1.1 (PK-136) mAbs (BD Biosciences) were used. For intracellular IFNγ and IL-4 staining, cells were fixed and permeabilized (Cytofix/Cytoperm kit, BD Biosciences) then stained with PE-labeled anti-IFNγ and IL-4 mAb (BD Biosciences). Cells were analyzed by FACSCalibur (BD Biosciences).
Immunoblots for HCV Core, Assays for Signaling Molecules and TUNEL Staining.
To assay expression of HCV core protein, tissue slices were homogenized in lysis buffer (30 mmol/L Tris pH 7.5, 150 mmol/L sodium chloride, 1 mmol/L PMSF, 1 mmol/L sodium orthovanadate, 1% Nonidet P-40, 10% glycerol). Suspensions of bone marrow and spleen cells were extracted in the same buffer. After centrifugation at 14,000 × g and 4°C for 10-minute protein concentrations were determined using the Bio-Rad protein assay kit. Proteins (25 μg per lane) were separated on 12.5% SDS-PAGE and transfered to nitrocellulose membranes (Hybound C; Amersham Biosciences). The membranes were reacted with polyclonal rabbit or monoclonal mouse anti-HCV core antibodies (Anogen, Mississauga, ON).
Anti-STAT1α p91 mAb, anti-STAT3 mAb, anti-αTubulin mAb, anti-suppressors of cytokine signaling (SOCS)-1 Ab and anti-SOSC-3 Ab were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). Antiphospho-STAT1 (Tyr701) and antiphospho-STAT3 (Tyr705), were obtained from Cell Signaling (Beverly, MA). Liver tissue was homogenized in lysis buffer at 4°C, and centrifuged at 14,000 rpm at 4°C for 10 minutes. Tissue extracts (30 μg protein per lane) were separated on 10% SDS-PAGE and blotted onto PVDF membranes (BIO-RAD). After blocking with buffer (LI-COR Bioscience, Lincoln, NE), the membrane was incubated with primary antibodies, followed by incubation with Alexis Fluor 680-conjugated secondary antibodies (Molecular Probe, Eugene, OR). Protein bands were visualized by an infrared image system (LI-COR). To visualize hepatocyte death, tissue sections were stained with a terminal dUTP nick-end labeling (TUNEL) kit (Roche Diagnostics, Indianopolis, IN) and positive cells counted in several random quadrants at 40× magnification.
Data are shown as means ± SEM. For comparisons of means between 2 experimental groups a Student unpaired t test was used. Values of P less than .05 were considered significant.
HCV Core Transgenic Mice Are Relatively Resistant to ConA-Induced Hepatitis.
To investigate effects of the HCV core on immune-mediated liver injury, the core gene was expressed under control of the EF-1α promoter in transgenic mice.7 Fig. 1A shows the core protein is demonstrable by immunoblot in many tissues, among which are liver, spleen, lungs, kidney, skin and brain. As expected, it is not demonstrable in nontransgenic littermates. This mouse model was therefore deemed suitable to examine regulatory effects of the core on cell-mediated liver injury. To explore this, a mild form of autoimmune hepatitis was induced by injection of the T cell mitogen ConA.6, 8, 12 In this model the STAT1 and STAT3 signaling pathways exert opposing effects.16 Therefore, for the case that the core modulates STAT1 signaling,3,4,5effects on liver injury should be demonstrable.
Results in Fig. 2A show that 10 mg/kg ConA induces a substantial increase of serum ALT values in nontransgenic littermates. ALT values also increase in core transgenic mice but are significantly lower than in nontransgenic littermates. Consistent with the increase in ALT levels in ConA-injected mice, caspase 3 levels also increase in the liver, but are significantly lower in core transgenic mice compared to control littermates (Fig. 2B). Histological examination of liver sections documents concordant results. There is heavy mid-zonal necrosis in livers of ConA-injected control mice, whereas livers from mice expressing the core protein show much reduced injury (Fig. 2C). These results show that expression of the core correlates with relative resistance to ConA-induced hepatitis.
Expression of Core in the Liver Parenchyma But Not the Lymphoid Compartment Is Required for Resistance to ConA-Induced Liver Injury.
In our transgenic mouse model, the core protein is demonstrable in many tissues (Fig. 1A). Therefore, we asked whether expression in a specific tissue could convey the observed effects. We first considered the possibility that the core inhibits the ConA-induced immune response. Because NKT cells are activated by injection of ConA and perform a pivotal role in liver injury prior to undergoing cell death,6 it was important to examine whether activation of these cells is inhibited in core transgenic animals. This was done by staining NKT cells in liver MNC for intracellular cytokines. Figure 3A shows that two hours after ConA injection there is a substantial and similar decrease of NKT cells in livers of transgenic mice and control littermates as expected.6 Staining of the remaining NKT cells for IFNγ and IL-4 reveals a significant increase after ConA injection, which however does not differ in cells from core transgenic mice and control littermates. It therefore appears that the core does not modulate the response of NKT cells to activation by ConA.
These results pointed to the possibility that the core-induced protective effect against liver injury takes place on the level of the liver parenchyma, rather than the lymphoid system. To examine this, bone marrow chimeras were generated in which either the lymphoid compartment or residual tissues, including the liver express the core. It is shown in Fig. 1A that transgenic mice express the core protein in the liver. Spleen cells also express the core as do bone marrow cells but at much reduced level (Fig. 1B). Therefore to generate chimeras, bone marrow and spleen were transplanted into irradiated animals. Normal littermates or core transgenic mice were lethally irradiated and bone marrow and spleen cells from normal littermates or core transgenic mice injected. Fig. 1C shows that transgenic mice reconstituted with nontransgenic bone marrow and spleen do not express the core protein in spleen cells, whereas spleens from nontransgenic mice reconstituted with transgenic bone marrow and spleen express the core protein. After two weeks, animals were challenged with ConA and tested for ALT levels. Results in Fig. 3B show that normal littermates reconstituted with littermate marrow and spleen express high ALT values, as do normal littermates reconstituted with spleen and marrow from core transgenic mice. In contrast, core transgenic mice reconstituted with littermate marrow and spleen cells show low ALT values. These results suggest that resistance to ConA-induced liver injury is due to resistance of the liver parenchyma to immune attack, rather than caused by suppression of the cell-mediated immune response.
Phosphorylation of STAT1 Is Inhibited and That of STAT3 Is Increased in Livers of Core Transgenic Mice Injected With ConA.
The finding that expression of the core in the liver rather than lymphoid compartment conveys resistance to hepatitis, suggests that core-expressing hepatocytes may be resistant to cell-mediated immune attack. Injection of ConA activates transcription factor STAT1, and inhibition of its activation protects from ConA-induced liver injury.17 We therefore examined whether STAT1 activation is inhibited in livers of core transgenic mice.
Animals were injected with ConA, livers harvested 1 hour later and analyzed for phosphorylated and nonphosphorylated STAT1 and STAT3 by immunoblot. Figure 4 shows that livers from core transgenic mice and normal littermates express equal levels of STAT1 and STAT3. In livers of ConA-injected core transgenic mice, phosphorylation of STAT1 is inhibited, whereas that of STAT3 is stimulated. Consistent with this, there is a decrease in SOCS-1 in core transgenic versus normal littermates injected with ConA, but there is no change in expression of SOCS-3. Therefore, the increased resistance of core transgenic mice to ConA-induced liver injury correlates with a preferential activation of STAT3 and inhibition of STAT1 activation. This result agrees with our recent finding that infection of B cells with HCV or expression of the core protein alone induces STAT3 phosphorylation.18
Core Expressing Hepatocytes Are Relatively Resistant to Cell Death by TNFα and Anti-Fas mAb.
Constitutively activated STAT3 protects from Fas-mediated liver injury.19 Because Fas and TNFα cooperate in cell-mediated liver injury,13 it was interesting to examine whether the core modulated STAT activation changes the sensitivity of hepatocytes to TNFα and Fas induced death. Fig. 5A shows that injection of 0.5 or 1.5 mg/kg rTNFα increases ALT levels in both nontransgenic and core transgenic animals. ALT levels are lower, however, in core transgenic mice, compared to control littermates, which was repeatable in numerous experiments. We therefore assayed TNFα production in transgenic mice and nontransgenic littermates under various conditions. In response to injection of bacterial lipopolysaccharide both sets of mice produced identical serum levels of TNFα and in response to ConA injection, NKT cells showed identical increases in TNFα staining (data not shown). Therefore, TNFα production does not appear to be affected by the core. Assay of sensitivity to liver injury by anti-Fas mAb revealed slightly lower values in transgenic mice, which did not reach statistical significance (Fig. 5B).
To follow up these results, hepatocytes were cultured with different doses of rTNFα and anti-Fas mAb, then assayed for cell death. Incubation with 0.1 or 1 ng/mL rTNFα induces a small degree of cell lysis in hepatocytes from normal littermates but not in cells from transgenic mice (Fig. 5C). A virtually identical result is seen in cultures incubated with 0.1 or 1μg/mL anti-Fas mAb. Here again cell lysis is higher in hepatocytes from normal littermates compared to those from transgenic mice. Addition of anti-Fas mAb to cultures containing rTNFα also causes higher cell death in littermate compared to transgenic hepatocytes. These data are consistent with the earlier observation that Fas and TNFα signals cooperate in the induction of hepatocyte death.13 In addition, they show that core transgenic hepatocytes express relative resistance to Fas and TNFα-induced cell death.
Core Transgenic Mice Show Increased Liver Regeneration.
The finding that STAT1 phosphorylation is decreased while that of STAT3 is increased in livers of ConA-injected transgenic mice, raises the question whether this shift in STAT signaling exerts effects other than protection from cell-mediated immunity. STAT3 had been reported to stimulate hepatocyte proliferation induced by PHx.20 Moreover, constitutive activation of STAT3 was observed in livers of core transgenic mice.21, 22 Although this effect was not seen in our experiments (Fig. 4), we reasoned that liver regeneration might be stimulated via more efficient activation of STAT3. To find out, transgenic mice and control littermates underwent PHx by surgical removal of 70% of liver tissue. At various times thereafter mice were sacrificed and liver weights determined. Figure 6A shows that liver weights are significantly higher in transgenic mice at day three, compared to normal littermates. To examine whether this is reflected in circulating liver enzyme levels, animals were assayed for ALT levels but no differences were demonstrable (data not shown). This suggested that PHx induced hepatocyte death is identical in transgenic mice and normal littermates. To examine this further, liver sections were stained for fragmented DNA by terminal dUTP nick-end labeling (TUNEL). Figure 6B reveals a comparable proportion of TUNEL staining hepatocytes in transgenic mice and normal littermates and counts of staining cells did not show any differences (littermates: 105+10.8 per quadrant; transgenic mice: 101+8.5 per quadrant). Therefore, the core protein does not appear to modulate apoptotic hepatocyte death induced by PHx. Consequently the accelerated increase in liver weights in transgenic mice should be caused by increased hepatocyte proliferation. To examine this, transgenic mice and littermates underwent PHx and were injected with BrdU to assess DNA synthesis in dividing hepatocytes. Figure 6C-D shows that there is a fourfold higher number of BrdU staining hepatocytes in livers of core transgenic mice compared to littermates 48 hours after PHx. Differences at other time points did not reach statistical significance. These data show that core transgenic mice undergo accelerated liver regeneration after PHx.
Here we show that HCV core transgenic mice induced for autoimmune hepatitis by injection of ConA are relatively resistant to liver injury. This resistance is associated with a shift from STAT1 to STAT3 activation in liver tissue of transgenic mice versus normal littermates. We also show that expression of the core in the liver parenchyma and not in the lymphoid compartment conveys resistance to ConA-induced hepatitis. These results support the contention that immune-mediated liver injury is ameliorated by ability of the core to inhibit STAT1 in favor of STAT3 activation in hepatocytes.
Previous experiments had demonstrated that expression of STAT1 under control of the CD2 promoter increases sensitivity to ConA-induced liver injury, whereas deletion of STAT1 conveys resistance.17 Moreover, in STAT1 deficient mice, STAT3 activation is stimulated by injection of ConA, leading to the conclusion that STAT1 and STAT3 mediate opposing roles.16 Our result that activation of STAT3 is stimulated in ConA-injected core transgenic mice compared to littermate controls, is consistent with these findings and suggests that suppression of STAT1 and increased activation of STAT3 provides the mechanism by which the core inhibits liver injury. In agreement, we recently found that HCV infection of B cells or expression of the core alone induces phosphorylation of STAT3.18
A number of reports have provided support for the notion that effects of the core on STAT signaling are caused by direct interaction with STAT1 and STAT3.3, 5, 21 Others proposed that these effects are caused by modulation of SOCS-1 and SOCS-3 induction, which in turn regulates activation of STATs.22, 23 Our finding that there is a decrease in SOCS-1 induction in livers of transgenic mice injected with ConA compared to controls is consistent with the latter possibility, without excluding the former.
An important question is which cytolytic pathway mediated by immune effector cells, is inhibited by the core in hepatocytes? Numerous experiments have shown that among pathways, TNFα and Fas induced cell death are particularly effective in the liver, whereas perforin is much less effective.24–26 In agreement, we show that hepatocytes from transgenic mice are relatively resistant to Fas and TNFα-mediated death and that injection of TNFα into transgenic mice leads to decreased liver injury compared to littermate controls. It therefore appears that the core protects hepatocytes against the two major lytic mechanisms operating in the liver. One would therefore expect that base levels of activated STAT3 should be higher in livers of transgenic mice compared to littermates. While we were not able to show this, there are results in the literature demonstrating that this is the case.21, 22
The finding in core transgenic mice that STAT1 and SOCS-1 activation are suppressed, while STAT3 activation is stimulated, is in agreement with a number of reports. HCV proteins including the core had been shown to inhibit type I interferon signaling by selectively inducing STAT1 degradation and inhibiting its nuclear translocation,3, 4, 5, 27 an effect that was explained by direct binding of the core to STAT1.5 STAT1 is believed to play a critical role in IFN-mediated control of HCV, which is supported by the demonstration that STAT1 activation causes inhibition of HCV gene expression.5 Published data has also shown that the core protein stimulates STAT3 activation and that STAT3 protects against Fas-mediated liver injury.19, 21 Moreover in HCV core transgenic mice, injected with IL-6, gene expression of SOCS-1 is inhibited.23 It is therefore not unexpected that in ConA-injected core transgenic mice, STAT1 activation is inhibited in favor of STAT3 activation. This report shows that HCV core-induced resistance to cell-mediated liver injury correlates with changes in STAT signaling in the liver parenchyma rather than the lymphoid compartment.
It had been demonstrated that livers of HCV core transgenic mice treated with diethylnitrosamine express higher cyclin D1 levels and increased phosphorylation of STAT3, consistent with the notion that the core stimulates STAT3 activation and cell proliferation by direct binding to STAT3.21 This observation together with the demonstration that STAT3 is rapidly induced after PHx and that it promotes cell cycle progression and cell proliferation20,21,28raised the possibility that liver regeneration may be stimulated in core transgenic mice. Indeed, we show here for the first time that liver regeneration is accelerated in transgenic mice, demonstrable by BrdU labeling and increases in liver weights.
Increased liver regeneration during a viral liver infection could be of benefit to the virus as it promotes host survival and viral spread. In addition inhibition of STAT1 and stimulation of STAT3 activation in infected cells protects from cell-mediated immunity, which could promote more efficient spread of the infection. Our data show that expression of the core in the lymphoid compartment of bone marrow chimeras has no detectable effect on ConA-induced liver injury. In order to convey protection the core has to be expressed in the liver parenchyma. These data lead to the conclusion that the core does not modulate cell-mediated immunity, a conclusion that was also reached in experiments in which induction of cytotoxic T cells to viral liver infections were examined in HCV core transgenic mice.3, 7, 29 We therefore conclude that the core does not modulate cell-mediated immunity, but rather protects the liver from attack by the immune system. This effect is demonstrable in the relatively moderate response induced by a low dose of ConA, but apparently not in models in which strong immune responses to viral infections are generated.3, 7, 29 While these data are consistent with the possibility that the core can act by these pathways to promote chronic HCV infections, our experiments do not prove that it does. Liver injury in this model is relatively mild and may not reflect the scenario of acute and chronic HCV infections in humans.