PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of jvirolPermissionsJournals.ASM.orgJournalJV ArticleJournal InfoAuthorsReviewers
 
J Virol. 2016 October 15; 90(20): 9153–9162.
Published online 2016 September 29. Prepublished online 2016 August 3. doi:  10.1128/JVI.01051-16
PMCID: PMC5044824

Acute Liver Damage Associated with Innate Immune Activation in a Small Nonhuman Primate Model of Hepacivirus Infection

R. M. Sandri-Goldin, Editor
University of California, Irvine

ABSTRACT

Despite its importance in shaping adaptive immune responses, viral clearance, and immune-based inflammation, tissue-specific innate immunity remains poorly characterized for hepatitis C virus (HCV) infection due to the lack of access to acutely infected tissues. In this study, we evaluated the impact of natural killer (NK) cells and myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells on control of virus replication and virus-induced pathology caused by another, more rapidly resolving hepacivirus, GB virus B (GBV-B), in infections of common marmosets. High plasma and liver viral loads and robust hepatitis characterized acute GBV-B infection, and while viremia was generally cleared by 2 to 3 months postinfection, hepatitis and liver fibrosis persisted after clearance. Coinciding with peak viral loads and liver pathology, the levels of NK cells, mDCs, and pDCs in the liver increased up to 3-fold. Although no obvious numerical changes in peripheral innate cells occurred, circulating NK cells exhibited increased perforin and Ki67 expression levels and increased surface expression of CXCR3. These data suggested that increased NK cell arming and proliferation as well as tissue trafficking may be associated with influx into the liver during acute infection. Indeed, NK cell frequencies in the liver positively correlated with plasma (R = 0.698; P = 0.015) and liver (R = 0.567; P = 0.057) viral loads. Finally, soluble factors associated with NK cells and DCs, including gamma interferon (IFN-γ) and RANTES, were increased in acute infection and also were associated with viral loads and hepatitis. Collectively, the findings showed that mobilization of local and circulating innate immune responses was linked to acute virus-induced hepatitis, and potentially to resolution of GBV-B infection, and our results may provide insight into similar mechanisms in HCV infection.

IMPORTANCE Hepatitis C virus (HCV) infection has created a global health crisis, and despite new effective antivirals, it is still a leading cause of liver disease and death worldwide. Recent evidence suggests that innate immunity may be a potential therapeutic target for HCV, but it may also be a correlate of increased disease. Due to a lack of access to human tissues with acute HCV infection, in this study we evaluated the role of innate immunity in resolving infection with a hepacivirus, GBV-B, in common marmosets. Collectively, our data suggest that NK cell and DC mobilization in acute hepacivirus infection can dampen virus replication but also regulate acute and chronic liver damage. How these two opposing effects on the host may be modulated in future therapeutic and vaccine approaches warrants further study.

INTRODUCTION

Hepatitis C virus (HCV) infection has become a global health epidemic, with the virus infecting more than 170 million people worldwide (1) and resulting in 350,000 HCV-related liver disease deaths each year (2). HCV infection results in the following two disparate manifestations: (i) an acute, self-resolving infection and (ii) a chronic infection, which occurs in 60 to 80% of cases and can lead to liver fibrosis and cirrhosis and even, rarely, to hepatocellular carcinoma (3, 4). However, despite the global impact of chronic HCV infection, the viral and host immune factors leading to self-resolution or chronicity still remain unclear. Although limitations in access to acute or tissue samples make understanding early hepacivirus pathology and immunology challenging, other related hepaciviruses recapitulate many features of HCV. The most closely phylogenetically related virus, GB virus B (GBV-B), has a nearly identical genome organization (5, 6), and it is almost always cleared by the immune system (7,9). Most GBV-B infection studies are conducted using common marmosets, and the virus has previously been demonstrated to induce hepatitis and a T cell response very similar to those with HCV (5, 10,14). Thus, GBV-B can offer insight into both acute responses and, potentially, immune-mediated clearance.

Significant evidence indicates that at least some primary correlates of GBV-B and HCV control and prevention of disease progression are cellular immunology based. Hepacivirus infection induces a potent T cell response, and CD4+ T cells upregulate PD-1 acutely after GBV-B infection, which is indicative of activation; a similar PD-1+ T cell activation is observed in early HCV infection (15,18). Acute GBV-B infection is also characterized by an influx of CD8 T cells into the liver (9), and clearance of GBV-B has previously been associated with strong responses against the NS3 protein (14). Anti-NS3 responses may also be protective and important for clearance of HCV (19,21). Conversely, poor T cell responses in acute HCV infection have been associated with viral persistence and chronicity, and in chronic disease there is significant evidence of T cell exhaustion and dysfunction (22,26), but there is a lack of evidence for a similar phenomenon in GBV-B infection.

Recent burgeoning evidence also suggests that innate immunity may contribute to viral clearance or control of hepacivirus infections. Maintenance of normal dendritic cell (DC) phenotypes and functional repertoires has been shown to be critical for sustained antiviral responses and is necessary to augment antiviral therapies, including alpha interferon (IFN-α), which can block GBV-B infection in vitro (27,29). Conversely, GBV-B has evolved to inhibit IFN-α production, and HCV has similarly developed mechanisms to subvert plasmacytoid (pDCs) and myeloid (mDCs) DCs, decreasing the levels of IFN-α, interleukin-10 (IL-10), and IL-12 (30,32), all of which are necessary for initiation of T cell responses and mobilization of natural killer (NK) cells. In HCV infection, NK cell cytotoxicity in both the periphery and inflamed livers has been associated with lysis of infected hepatocytes and with viral clearance (33,35). Moreover, polyfunctional NK cells have been associated with resistance to infection in HCV-exposed health care workers (36). Although recent data from our lab and others have characterized marmoset NK cells and found them to be highly similar to those in humans (37, 38), the reciprocal effects of GBV-B infection and NK cells are unknown.

Despite advances in the HCV field, there are still limitations in the development of vaccines and immunotherapeutics, primarily due to a lack of access to acute infection and tissue samples. These knowledge gaps can be evaluated partially by studying the immune responses in other hepacivirus infections, particularly those that are resolved by the immune system, such as GBV-B infection. In this study, we aimed to describe the mobilization of innate immune factors, both cellular and soluble, which may influence GBV-B resolution and virus-induced pathology.

MATERIALS AND METHODS

Experimental animals.

Twelve common marmosets (Callithrix jacchus) of either sex and between 2 and 8 years of age were housed in biosafety level 2 (BSL2) biocontainment facilities at the New England Primate Research Center, Southborough, MA. These animal studies were performed in accordance with the guidelines of the local Institutional Animal Care and Use Committee and the Guide for the Care and Use of Laboratory Animals (39). A commercial New World nonhuman primate diet was supplemented with fruits, vegetables, eggs, and nuts, and water was provided ad libitum. Whole blood was collected prior to infection and every 3 days until day 14 postinfection (p.i.) for the acute study and at weekly intervals for the first 4 weeks followed by monthly collections until day 168 p.i. for the long-term study. Liver biopsies were also performed prior to infection and at day 14 p.i. in long-term study animals. Six animals were sacrificed at day 14 p.i., and six animals were sacrificed following a full infection, at day 168 p.i. On the day of sacrifice, animals were euthanized and evident postmortem pathology was recorded by the attending veterinarian and pathologist. The blood, liver, spleen, peripheral lymph nodes, and mesenteric lymph nodes (MLN) were collected at necropsy and processed using standard laboratory protocols for subsequent analyses. GBV-B-naive animal culls provided an additional source of normal tissue for some analyses.

Virus inoculum.

All marmosets were inoculated intravenously with 1.0 × 103 to 4.0 × 103 virus copy equivalents from one of two plasma-derived challenge stocks of related GBV-B isolates: (i) an uncloned GBV-B stock derived at the Southwest National Primate Research Center (SNPRC) (a kind gift from Robert Lanford) (40) and (ii) an uncloned GBV-B stock derived at the New England Primate Research Center (NEPRC) (9).

Histopathology.

Liver tissues were collected on the day of sacrifice, and all samples were formalin fixed, paraffin embedded, sectioned, and stained by Mass Histology Service, Inc. (Worcester, MA). Staining included hematoxylin and eosin (H&E) staining and trichrome staining for collagen deposition, using standard protocols. Semiquantitative histopathologic scoring for inflammation and fibrosis was performed independently by two blinded veterinary pathologists. The scores for hepatitis were reported as follows: 0, none; 1, minimal; 2, mild; 3, moderate; 4, moderate to severe; and 5, severe. The scores for fibrosis were reported as follows: 0, none; 1, mild; 2, moderate; and 3, severe.

Virus quantification.

Virus quantification was performed by quantitative reverse transcription-PCR (RT-PCR), using a modified assay as previously described (9). Briefly, cell-free plasma samples were processed to yield RNA by use of a QIAamp viral RNA minikit (Qiagen), and viral RNA was converted to cDNA by use of a high-capacity reverse transcription kit (Life Technologies). One-step RT-PCR amplification was performed with the forward primer CGCCGGTTGGCTCATC, the reverse primer GCCGCGTCAACGGTTATT, and the MGB probe CACAGGCTCTATACACC. RT-PCRs were conducted in an ABI 7900HT system, and the results were analyzed by use of ABI software. Quantitative standards were generated by in vitro transcription of a plasmid vector containing an NS3 insert. Serial dilutions ranging from 108 to 100 copies/reaction mix were used to generate a standard curve with a sensitivity threshold of 15 copies/reaction mix. Repeated measurements indicated that nominally 100 copies or more could be quantified reliably and reproducibly in each sample, regardless of volume. NS3 copy equivalents were extrapolated from the standard curve and expressed as copies per milliliter of plasma.

Isolation of mononuclear cells from tissues of infected animals.

Each liver was homogenized on a cell strainer with RPMI containing 5% fetal bovine serum (FBS) (R5). The filtrate was overlaid on 60% Percoll followed by 30% Percoll for density gradient centrifugation, and the interface layer containing the hepatic mononuclear cells was harvested. The spleen and lymph nodes were homogenized on a cell strainer and washed with R5. Contaminating red blood cells (RBCs) were lysed using hypotonic ammonium chloride solution. The mononuclear cells were washed with R5, resuspended in RPMI containing 10% FBS (R10), counted, and used for staining for flow cytometry or for enzyme-linked immunosorbent spot (ELISpot) assay.

ELISpot assay.

Virus-specific IFN-γ responses were assayed using an anti-human IFN-γ ELISpot kit (MAb Tech) on mononuclear cells of the liver and spleen. Responses were measured against GBV-B proteins—core (amino acids [aa] 1 to 156; 15 peptides), NS3 (aa 941 to 1250; 30 peptides), NS3/NS4A (aa 1241 to 1615; 37 peptides), and NS5B (aa 2275 to 2864; 58 peptides)—that were kindly provided by Chris Walker (Columbus Children's Research Institute, Columbus, OH). Cells were added at a concentration of 2 × 105 cells/well to antigens at a concentration of 1 μg/ml and phytohemagglutinin (PHA) at a concentration of 2 μg/ml and incubated at 37°C for 40 h. The cells were washed off, and the plate was developed according to the manufacturer's instructions. Spots were then counted by Zellnet Consulting Inc.

Flow cytometric staining of whole blood.

To enumerate and phenotype NK cells, dendritic cells, and T cells, polychromatic flow cytometry was performed using whole blood. Antibodies used include CD3 (allophycocyanin [APC]-Cy7; clone SP34-2; BD Biosciences), NKp46 (phycoerythrin [PE]; clone BAB281; Immunotech), HLA-DR (ECD; clone Immu-357; Beckman-Coulter), CXCR3 (PE-Cy5; clone 1C6; BD Biosciences), CD56 (PE-Cy7; clone NCAM16.2; BD Biosciences), NKG2a (APC; clone Z199; Beckman Coulter), CD16 (Alexa Fluor 700; clone 3G8; Invitrogen), CD4 (PE; clone L-200; BD Biosciences), CD45RA (PE-Cy5; clone 5H9; BD Biosciences), CD95 (APC; clone DX2; BD Biosciences), CD11c (PE; clone S-HCL-3; BD Biosciences), CD20 (peridinin chlorophyll protein [PerCP]-Cy5.5; clone L27; BD Biosciences), and CD14 (PE-Cy7; clone MoP9; BD Biosciences) antibodies. The samples were incubated with antibody for 30 min at room temperature and then lysed with fluorescence-activated cell sorter (FACS) lysis buffer (BD Biosciences) according to the manufacturer's recommended protocol. Cells were then fixed using a 1% formaldehyde solution. Intracellular staining was performed using Fix/Perm reagents (Caltag Laboratories) and included staining for Ki67 (fluorescein isothiocyanate [FITC] clone B56; BD Biosciences) and perforin (Pacific Blue clone dG9; BioLegend). Flow cytometry acquisitions were performed on an LSR II flow cytometer (BD Biosciences), and data were analyzed with FlowJo software (version 9.6.4; Tree Star).

Flow cytometric staining of tissue mononuclear cells.

Single-cell suspensions were subjected to multicolor immunostaining for flow cytometric analysis. Briefly, 1 × 106 cells were resuspended in phosphate-buffered saline (PBS) containing 2% FBS. Cells were then stained, and flow cytometric acquisitions were performed as described above.

Multiplex cytokine analysis of plasma.

Plasma samples were analyzed for marmoset cytokines and chemokines by Luminex methodology, using established protocols for New World primates (42). Evaluation of the analytes IL-1β, IL-1Ra, tumor necrosis factor alpha (TNF-α), IL-2, RANTES, macrophage inflammatory protein 1α (MIP-1α), MIP-1β, IL-12 (p40), IL-8, IFN-α, IL-13, IFN-γ, IL-17, TNF-β, and vascular endothelial growth factor (VEGF) was included in this assay. Only data for analytes quantifiable above the limit of detection are presented.

Statistical analyses.

Statistical evaluations of differences between groups included the Mann-Whitney U test, the Kruskal-Wallis test followed by Dunn's multiple-comparison posttest, and Student's t test and were performed using GraphPad Prism 6.0 software. Differences between the mean ranks of different time points compared to the mean rank on day 0 were considered significant when the P value was <0.05.

RESULTS

GBV-B infection of marmosets.

To first confirm the take of infection and to evaluate the kinetics of viremia, virus loads in plasma samples and livers of all animals were quantified at multiple time points. Virus was detectable as early as 3 days p.i. in approximately 50% of infected animals, and all animals were viremic at day 14 (Fig. 1A and andB).B). In longitudinally evaluated animals, plasma levels peaked between 106 and 107 copies/ml, generally by day 14. The duration of detectable viremia was highly variable, with viremia being controlled between day 28 and day 112. Five of six animals had high levels of viremia in the liver at day 14 p.i. (range, 104 to 5.0 × 106 copy equivalents/μg) (Fig. 1C), while one outlier (animal 1-09) had an undetectable load in the liver, below the limit of the assay, but also had low levels of virus in the more sensitive plasma assay. By the time of sacrifice at day 168 p.i., only one animal had detectable virus replication in the liver (Fig. 1D), and although this animal had cleared plasma virus by this time point, it did experience the longest duration of plasma viremia. For completeness and to verify the development and evolution of adaptive responses, we systemically quantified the CD8+ T cell responses to GBV-B antigens—core, NS3, NS3/4a, and NS5—by IFN-γ ELISpot assay at day 14 and day 168. As expected, the magnitudes of the CD8+ T cell responses to all antigens were higher at day 168 than at day 14 (Fig. 2). Also, responses were higher in the liver, where the virus predominantly replicates, than in the spleen. Similar to previous observations, the dominant response was observed against NS3/4a peptides (14).

FIG 1
Plasma and tissue viral loads in GBV-B-infected marmosets. GBV-B viral loads in cell-free plasma samples were quantified at multiple time points following GBV-B challenge in acute (A) and longitudinal (B) study cohorts. Tissue viral loads in liver samples ...
FIG 2
T cell ELISpot responses in acute and chronic GBV-B infections. The graphs show the frequencies of IFN-γ-secreting cells among isolated tissue mononuclear cells from the liver (A) and spleen (B) against overlapping peptide pools from the NS5a, ...

Histopathological changes in GBV-B-infected animals.

Next, we evaluated both acute and chronic measures of liver pathology and fibrosis in GBV-B-infected animals. Histopathology analysis by H&E staining indicated multifocal lymphocyte aggregation, mild to moderate inflammation, and focal necrosis of hepatocytes in acutely infected animals compared to healthy controls (Fig. 3A; Table 1). Not surprisingly, hepatitis was more advanced in animals sacrificed at day 168, even though the virus had already been cleared. Trichrome staining revealed little to no indication of fibrosis for acute infection, but extensive fibrosis around the portal triad regions was found in animals sacrificed at day 168 (Fig. 3B; Table 1). Collectively, these data indicate that significant hepatitis occurs acutely but that advanced hepatitis and fibrosis occur later and persist even after viral clearance, and thus may continue to have impacts on liver homeostasis.

FIG 3
Hepatitis and liver fibrosis in GBV-B-infected marmosets. Representative photomicrographs show H&E (magnification, ×400) (A) and trichrome (B) (magnification, ×100) staining of tissues from healthy and GBV-B-infected animals, as ...
TABLE 1
Histopathological changes in GBV-B-infected animals

Elevated serum liver enzymes are often used as surrogate indicators of liver damage, but their association with actual hepatitis is not always consistent. Similarly, although alanine aminotransferase (ALT), aspartate transaminase (AST), and alkaline phosphatase (ALP) levels became elevated in some animals by day 14 or 28 p.i., and continued to stay above baseline values (Fig. 4), these changes were not significant and did not correlate with viral load (data not shown). Similar to findings for HCV-infected humans, these serum chemistry measurements may not always be effective surrogate indicators of liver damage (43, 44).

FIG 4
Liver function enzyme levels during GBV-B infection. The serum biochemistry of alanine aminotransferase (ALT) (A), aspartate transaminase (AST) (B), and alkaline phosphatase (ALP) (C) was examined in GBV-B-infected marmosets. All values are shown in units ...

Mobilization of innate immune responses during GBV-B infection.

Dendritic and NK cells are among the first cells to respond to viral infections, but they are poorly described for acute HCV infection, generally due to a lack of access to samples. To begin to address some of this deficit, we characterized the innate cellular response following GBV-B infection. In GBV-B-infected animals, the frequency of circulating mDCs was generally unaltered, but pDC frequencies increased following infection and then declined to normal levels by day 28 p.i. (Fig. 5A to toC).C). Subsequent analyses of necropsy tissues found that both mDCs and pDCs were elevated in the liver during acute infection and remained after viral clearance, but they were generally unchanged in extrahepatic tissues (Fig. 5D and andE).E). These are the first observations documenting an accumulation of DCs in livers from GBV-B-infected animals, but they are consistent with observations of other altered DC phenotypes in HCV-infected humans (30, 32, 45).

FIG 5
Acute mobilization of circulating and liver mDCs and pDCs during GBV-B infection. (A) Representative gating strategy used to delineate DC populations in marmosets. (B and C) Individual data points represent percentages of mDCs (B) and pDCs (C) following ...

Similar to the observations for DCs, there were no overt changes in circulating NK cell frequencies or subpopulations (Fig. 6A to toC).C). Interestingly, however, the phenotype of peripheral NK cells was altered. Intracellular perforin expression was upregulated acutely (Fig. 6D) (P = 0.0284; Kruskal-Wallis test), while the tissue homing marker CXCR3 (Fig. 6E) (P < 0.0001; Kruskal-Wallis test) and the proliferation marker Ki67 (Fig. 6F) were both upregulated early in infection and remained elevated until viral clearance. This suggested that while no net numerical change in peripheral NK cells was observed, the effects of GBV-B infection were not silent and induced increases in tissue homing, turnover, and cytotoxic arming related to ongoing virus replication. In line with these observations, the frequency of NK cells in the liver increased significantly during acute GBV-B infection (Fig. 7A), and CD16+ cytotoxic NK cell, perforin, and Ki67 levels were all increased, albeit not significantly (Fig. 7B to toD).D). IFN-γ production by hepatic NK cells also increased significantly during acute GBV-B infection (Fig. 7E). NK cell frequencies in the liver correlated with plasma viral load (R = 0.698; P = 0.015) and liver viral load (R = 0.567; P = 0.057), indicating that GBV-B replication likely induces NK cell recruitment. Moderate increases in NK cell frequencies were also observed in the spleen and in MLN that drain the liver, but these did not reach statistical significance.

FIG 6
Mobilization of circulating NK cells during GBV-B infection. (A) Representative gating strategy used to delineate NK cells and NK cell subpopulations in marmosets, as previously described (37). (B) Individual data points represent percentages of NK cells ...
FIG 7
Liver NK cells in GBV-B infection. (A) Individual data points represent individual animal samples analyzed at necropsy; horizontal bars represent median percentages of NK cells. (B) Medians and ranges are shown for NK cell subpopulations at the indicated ...

Modulation of the inflammatory cytokine milieu during GBV-B infection.

Since cellular innate immune responses were perturbed during acute GBV-B infection, we next evaluated soluble innate factors by using optimized protocols specific to detection of neotropical primate analytes by Luminex methodology. Not surprisingly, inflammatory mediators were found at low concentrations in naive animals but were selectively increased in individual animals during acute infection (Fig. 8). For example, the median serum concentration of RANTES was 11 pg/ml at baseline, but it increased to 37 and 70 pg/ml in two animals by day 14 p.i. and to >20 pg/ml in longitudinally evaluated animals by day 28 p.i. Similarly, the baseline IFN-γ level in serum was low (median, 1 pg/ml) but increased up to 9-fold by day 28 p.i. Both IFN-γ (R = 0.417; P = 0.025) and RANTES (R = 0.326; P = 0.085) levels correlated with plasma viral loads. IL-12, MIP-1β, and IFN-α levels were unchanged by day 14, but all increased longitudinally in individual animals before rapidly returning to baseline levels. Although these analyses were severely limited by the volume of plasma/serum recoverable from longitudinal samples inherent to this species, collectively these data suggest that transient increases in soluble inflammatory mediators are a characteristic of acute GBV-B infection.

FIG 8
Elevated cytokines and chemokines in plasma samples from GBV-B-infected animals. Analytes in plasma samples from acutely (A) and longitudinally (B) GBV-B-infected animals were quantified by a Luminex assay using validated marmoset cross-reactive antibody ...

DISCUSSION

GBV-B, a hepacivirus similar to HCV, has previously been shown to cause hepatitis in marmosets, and differences in viral immunity have been related to clearance, control, and disease progression (5, 10,13). Despite the fact that innate immunity is a well-studied correlate for both modulation of adaptive responses and indirect pathology in other disease models, its role in hepacivirus infections remains unclear. This study highlights innate immune and pathological responses to acute GBV-B infection and following virus clearance.

In general, the acute NK cell response to GBV-B infection was characterized by increased cytotoxic arming, IFN-γ production, cell proliferation, tissue homing, and accumulation in the liver. These features persisted in the postacute period, but most functions resolved to baseline levels by the time of virus clearance. Empirical HCV studies suggest that enhanced NK cell function during acute infection can lead to viral containment (46,48), and in persons accidentally exposed to HCV, NK cells exert upregulated effector functions (36). Collectively, these data may suggest that the robust NK cell response we observed in acute GBV-B infection is related to the virus clearance normally observed (40, 49). Further, CXCR3 expression on circulating NK cells was upregulated soon after GBV-B infection, which may indicate recruitment of immunocompetent cells into the liver. CXCR3 expression on liver NK cells has previously been associated with liver cell damage and fibrosis (50, 51), and biomarkers such as increased CXCR3 or NK cell perforin may be indicative of acute hepacivirus infection and hepatitis. Indeed, the kinetics and accumulation of NK cells and other innate immune functions coincided with acute hepatitis (Fig. 3). Similarly, hepatocytes upregulate NK cell NKG2D ligands in a murine model of HBV infection, rendering the hepatocytes susceptible to increased lysis (52), and NKG2D-mediated hepatocyte killing has been described for nonalcoholic steatohepatitis (53). NK cell upregulation of TRAIL, FAS, and other granzymes and perforins has been shown to be involved in both hepatocellular damage and HCV clearance (54,56). Taken together, our data suggest that although NK cells may be a correlate of virus clearance, they may also be a cause of liver pathology.

Similar to the observations for NK cells, GBV-B infection induced a partial accumulation of DCs in the liver. In patients with chronic HCV infection, there is a preferential migration of mDCs to areas of liver inflammation (57, 58), coinciding with a reduction in circulating DCs driven by HCV E2 protein-mediated RANTES and MIP-1α secretion (58). Subsequently, dysfunctional DCs are trapped inside the liver, unable to migrate to lymph nodes. A similar mechanism may occur in GBV-B infection, which would explain the accumulation of mDCs and pDCs in the liver without changes in the lymph nodes. Perturbation of the dendritic cell compartment may lead to altered antigen processing and skew the T cell response, both of which are well documented for HCV (41, 59), and accumulation may lead to overproduction of inflammatory mediators and subsequent liver damage.

The overaccumulation of innate immune cells in the livers of infected animals may also be an indirect source of inflammation and hepatitis mediated by soluble factors. Indeed, RANTES, MIP-1β, and IFN-γ are all produced by NK cells, mDCs, or both, and they may be associated with generalized activation. Similarly, activated pDCs and mDCs may contribute to increased IFN-α and IL-12 levels. Each of these factors has some association with virus control but also has well-described mechanisms for hepatocyte death, particularly IFN-γ (58). This may suggest that in acute hepatitis, DC- and NK cell-mediated inflammation and hepatocyte death are indirect mechanisms via cytokine production.

In summary, modeling acute hepacivirus infection in GBV-B-infected marmosets can aid in clarifying mechanisms of both virus clearance and pathology. Innate responses were robust during acute infection, whereas adaptive T cell responses, not surprisingly, were weak; previous observations suggest that humoral responses also develop much later (60). We surmise that this increased innate inflammation can have a bifurcated effect on the host: it (i) dampens virus replication but (ii) also regulates acute and chronic liver damage. The full contribution of these responses and how they can be targeted in therapeutic or vaccine modalities will need to be elucidated in further studies.

ACKNOWLEDGMENTS

This work was supported by NIH grants R21 AI118468 and R21 AI101170 to R.K.R. and by NIH Office of Research Infrastructure Programs grants supporting the NEPRC (grant P51 OD011103) and SNPRC (grant P51 OD011133).

We thank the flow cytometry cores of the NEPRC and CVVR (supported by the Harvard Center for AIDS Research [grant P30 AI060354]) for technical support.

REFERENCES

1. Alter HJ, Seeff LB 2000. Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin Liver Dis 20:17–35. doi:.10.1055/s-2000-9505 [PubMed] [Cross Ref]
2. Sagnelli E, Santantonio T, Coppola N, Fasano M, Pisaturo M, Sagnelli C 2014. Acute hepatitis C: clinical and laboratory diagnosis, course of the disease, treatment. Infection 42:601–610. doi:.10.1007/s15010-014-0608-2 [PubMed] [Cross Ref]
3. Micallef JM, Kaldor JM, Dore GJ 2006. Spontaneous viral clearance following acute hepatitis C infection: a systematic review of longitudinal studies. J Viral Hepat 13:34–41. doi:.10.1111/j.1365-2893.2005.00651.x [PubMed] [Cross Ref]
4. Seeff LB. 2002. Natural history of chronic hepatitis C. Hepatology 36:S35–S46. [PubMed]
5. Muerhoff AS, Leary TP, Simons JN, Pilot-Matias TJ, Dawson GJ, Erker JC, Chalmers ML, Schlauder GG, Desai SM, Mushahwar IK 1995. Genomic organization of GB viruses A and B: two new members of the Flaviviridae associated with GB agent hepatitis. J Virol 69:5621–5630. [PMC free article] [PubMed]
6. Stapleton JT, Foung S, Muerhoff AS, Bukh J, Simmonds P 2011. The GB viruses: a review and proposed classification of GBV-A, GBV-C (HGV), and GBV-D in genus Pegivirus within the family Flaviviridae. J Gen Virol 92:233–246. doi:.10.1099/vir.0.027490-0 [PMC free article] [PubMed] [Cross Ref]
7. Bright H, Carroll AR, Watts PA, Fenton RJ 2004. Development of a GB virus B marmoset model and its validation with a novel series of hepatitis C virus NS3 protease inhibitors. J Virol 78:2062–2071. doi:.10.1128/JVI.78.4.2062-2071.2004 [PMC free article] [PubMed] [Cross Ref]
8. Iwasaki Y, Mori K, Ishii K, Maki N, Iijima S, Yoshida T, Okabayashi S, Katakai Y, Lee YJ, Saito A, Fukai H, Kimura N, Ageyama N, Yoshizaki S, Suzuki T, Yasutomi Y, Miyamura T, Kannagi M, Akari H 2011. Long-term persistent GBV-B infection and development of a chronic and progressive hepatitis C-like disease in marmosets. Front Microbiol 2:240. doi:.10.3389/fmicb.2011.00240 [PMC free article] [PubMed] [Cross Ref]
9. Jacob JR, Lin KC, Tennant BC, Mansfield KG 2004. GB virus B infection of the common marmoset (Callithrix jacchus) and associated liver pathology. J Gen Virol 85:2525–2533. doi:.10.1099/vir.0.80036-0 [PubMed] [Cross Ref]
10. Bukh J, Apgar CL, Govindarajan S, Purcell RH 2001. Host range studies of GB virus-B hepatitis agent, the closest relative of hepatitis C virus, in New World monkeys and chimpanzees. J Med Virol 65:694–697. doi:.10.1002/jmv.2092 [PubMed] [Cross Ref]
11. Bukh J, Apgar CL, Yanagi M 1999. Toward a surrogate model for hepatitis C virus: an infectious molecular clone of the GB virus-B hepatitis agent. Virology 262:470–478. doi:.10.1006/viro.1999.9941 [PubMed] [Cross Ref]
12. Deinhardt F, Peterson D, Cross G, Wolfe L, Holmes AW 1975. Hepatitis in marmosets. Am J Med Sci 270:73–80. doi:.10.1097/00000441-197507000-00011 [PubMed] [Cross Ref]
13. Simons JN, Pilot-Matias TJ, Leary TP, Dawson GJ, Desai SM, Schlauder GG, Muerhoff AS, Erker JC, Buijk SL, Chalmers ML, Van Sant CL, Mushahwar IK 1995. Identification of two flavivirus-like genomes in the GB hepatitis agent. Proc Natl Acad Sci U S A 92:3401–3405. doi:.10.1073/pnas.92.8.3401 [PubMed] [Cross Ref]
14. Woollard DJ, Haqshenas G, Dong X, Pratt BF, Kent SJ, Gowans EJ 2008. Virus-specific T-cell immunity correlates with control of GB virus B infection in marmosets. J Virol 82:3054–3060. doi:.10.1128/JVI.01153-07 [PMC free article] [PubMed] [Cross Ref]
15. Gruner NH, Gerlach TJ, Jung MC, Diepolder HM, Schirren CA, Schraut WW, Hoffmann R, Zachoval R, Santantonio T, Cucchiarini M, Cerny A, Pape GR 2000. Association of hepatitis C virus-specific CD8+ T cells with viral clearance in acute hepatitis C. J Infect Dis 181:1528–1536. doi:.10.1086/315450 [PubMed] [Cross Ref]
16. Kasprowicz V, Schulze Zur Wiesch J, Kuntzen T, Nolan BE, Longworth S, Berical A, Blum J, McMahon C, Reyor LL, Elias N, Kwok WW, McGovern BG, Freeman G, Chung RT, Klenerman P, Lewis-Ximenez L, Walker BD, Allen TM, Kim AY, Lauer GM 2008. High level of PD-1 expression on hepatitis C virus (HCV)-specific CD8+ and CD4+ T cells during acute HCV infection, irrespective of clinical outcome. J Virol 82:3154–3160. doi:.10.1128/JVI.02474-07 [PMC free article] [PubMed] [Cross Ref]
17. Neumann-Haefelin C, Spangenberg HC, Blum HE, Thimme R 2007. Host and viral factors contributing to CD8+ T cell failure in hepatitis C virus infection. World J Gastroenterol 13:4839–4847. doi:.10.3748/wjg.v13.i36.4839 [PMC free article] [PubMed] [Cross Ref]
18. Urbani S, Amadei B, Fisicaro P, Tola D, Orlandini A, Sacchelli L, Mori C, Missale G, Ferrari C 2006. Outcome of acute hepatitis C is related to virus-specific CD4 function and maturation of antiviral memory CD8 responses. Hepatology 44:126–139. [PubMed]
19. Arribillaga L, de Cerio AL, Sarobe P, Casares N, Gorraiz M, Vales A, Bruna-Romero O, Borras-Cuesta F, Paranhos-Baccala G, Prieto J, Ruiz J, Lasarte JJ 2002. Vaccination with an adenoviral vector encoding hepatitis C virus (HCV) NS3 protein protects against infection with HCV-recombinant vaccinia virus. Vaccine 21:202–210. doi:.10.1016/S0264-410X(02)00456-5 [PubMed] [Cross Ref]
20. Diepolder HM, Zachoval R, Hoffmann RM, Wierenga EA, Santantonio T, Jung MC, Eichenlaub D, Pape GR 1995. Possible mechanism involving T-lymphocyte response to non-structural protein 3 in viral clearance in acute hepatitis C virus infection. Lancet 346:1006–1007. doi:.10.1016/S0140-6736(95)91691-1 [PubMed] [Cross Ref]
21. Satake S, Nagaki M, Kimura K, Naiki T, Hayashi H, Sugihara J, Tomita E, Moriwaki H 2008. Significant effect of hepatitis C virus specific CTLs on viral clearance in patients with type C chronic hepatitis treated with antiviral agents. Hepatol Res 38:491–500. doi:.10.1111/j.1872-034X.2007.00291.x [PubMed] [Cross Ref]
22. Aberle JH, Formann E, Steindl-Munda P, Weseslindtner L, Gurguta C, Perstinger G, Grilnberger E, Laferl H, Dienes HP, Popow-Kraupp T, Ferenci P, Holzmann H 2006. Prospective study of viral clearance and CD4(+) T-cell response in acute hepatitis C primary infection and reinfection. J Clin Virol 36:24–31. doi:.10.1016/j.jcv.2005.12.010 [PubMed] [Cross Ref]
23. Badr G, Bedard N, Abdel-Hakeem MS, Trautmann L, Willems B, Villeneuve JP, Haddad EK, Sekaly RP, Bruneau J, Shoukry NH 2008. Early interferon therapy for hepatitis C virus infection rescues polyfunctional, long-lived CD8+ memory T cells. J Virol 82:10017–10031. doi:.10.1128/JVI.01083-08 [PMC free article] [PubMed] [Cross Ref]
24. Boettler T, Spangenberg HC, Neumann-Haefelin C, Panther E, Urbani S, Ferrari C, Blum HE, von Weizsacker F, Thimme R 2005. T cells with a CD4+ CD25+ regulatory phenotype suppress in vitro proliferation of virus-specific CD8+ T cells during chronic hepatitis C virus infection. J Virol 79:7860–7867. doi:.10.1128/JVI.79.12.7860-7867.2005 [PMC free article] [PubMed] [Cross Ref]
25. Harcourt G, Hellier S, Bunce M, Satsangi J, Collier J, Chapman R, Phillips R, Klenerman P 2001. Effect of HLA class II genotype on T helper lymphocyte responses and viral control in hepatitis C virus infection. J Viral Hepat 8:174–179. doi:.10.1046/j.1365-2893.2001.00289.x [PubMed] [Cross Ref]
26. Wedemeyer H, He XS, Nascimbeni M, Davis AR, Greenberg HB, Hoofnagle JH, Liang TJ, Alter H, Rehermann B 2002. Impaired effector function of hepatitis C virus-specific CD8+ T cells in chronic hepatitis C virus infection. J Immunol 169:3447–3458. doi:.10.4049/jimmunol.169.6.3447 [PubMed] [Cross Ref]
27. Chavez D, Guerra B, Lanford RE 2009. Antiviral activity and host gene induction by tamarin and marmoset interferon-alpha and interferon-gamma in the GBV-B primary hepatocyte culture model. Virology 390:186–196. doi:.10.1016/j.virol.2009.05.005 [PMC free article] [PubMed] [Cross Ref]
28. Rana D, Chawla YK, Duseja A, Dhiman R, Arora SK 2012. Functional reconstitution of defective myeloid dendritic cells in chronic hepatitis C infection on successful antiviral treatment. Liver Int 32:1128–1137. doi:.10.1111/j.1478-3231.2011.02754.x [PubMed] [Cross Ref]
29. Rodrigue-Gervais IG, Rigsby H, Jouan L, Willems B, Lamarre D 2014. Intact dendritic cell pathogen-recognition receptor functions associate with chronic hepatitis C treatment-induced viral clearance. PLoS One 9:e102605. doi:.10.1371/journal.pone.0102605 [PMC free article] [PubMed] [Cross Ref]
30. Anthony DD, Yonkers NL, Post AB, Asaad R, Heinzel FP, Lederman MM, Lehmann PV, Valdez H 2004. Selective impairments in dendritic cell-associated function distinguish hepatitis C virus and HIV infection. J Immunol 172:4907–4916. doi:.10.4049/jimmunol.172.8.4907 [PubMed] [Cross Ref]
31. Chen Z, Benureau Y, Rijnbrand R, Yi J, Wang T, Warter L, Lanford RE, Weinman SA, Lemon SM, Martin A, Li K 2007. GB virus B disrupts RIG-I signaling by NS3/4A-mediated cleavage of the adaptor protein MAVS. J Virol 81:964–976. doi:.10.1128/JVI.02076-06 [PMC free article] [PubMed] [Cross Ref]
32. Simone O, Tortorella C, Zaccaro B, Napoli N, Antonaci S 2010. Impairment of TLR7-dependent signaling in dendritic cells from chronic hepatitis C virus (HCV)-infected non-responders to interferon/ribavirin therapy. J Clin Immunol 30:556–565. doi:.10.1007/s10875-010-9387-4 [PubMed] [Cross Ref]
33. Ahlenstiel G, Titerence RH, Koh C, Edlich B, Feld JJ, Rotman Y, Ghany MG, Hoofnagle JH, Liang TJ, Heller T, Rehermann B 2010. Natural killer cells are polarized toward cytotoxicity in chronic hepatitis C in an interferon-alfa-dependent manner. Gastroenterology 138:325.e2–335.e2. doi:.10.1053/j.gastro.2009.08.066 [PMC free article] [PubMed] [Cross Ref]
34. Kramer B, Korner C, Kebschull M, Glassner A, Eisenhardt M, Nischalke HD, Alexander M, Sauerbruch T, Spengler U, Nattermann J 2012. Natural killer p46High expression defines a natural killer cell subset that is potentially involved in control of hepatitis C virus replication and modulation of liver fibrosis. Hepatology 56:1201–1213. doi:.10.1002/hep.25804 [PubMed] [Cross Ref]
35. Oliviero B, Varchetta S, Paudice E, Michelone G, Zaramella M, Mavilio D, De Filippi F, Bruno S, Mondelli MU 2009. Natural killer cell functional dichotomy in chronic hepatitis B and chronic hepatitis C virus infections. Gastroenterology 137:1151.e7–1160.e7. doi:.10.1053/j.gastro.2009.05.047 [PubMed] [Cross Ref]
36. Werner JM, Heller T, Gordon AM, Sheets A, Sherker AH, Kessler E, Bean KS, Stevens M, Schmitt J, Rehermann B 2013. Innate immune responses in hepatitis C virus-exposed healthcare workers who do not develop acute infection. Hepatology 58:1621–1631. doi:.10.1002/hep.26353 [PMC free article] [PubMed] [Cross Ref]
37. Carville A, Evans TI, Reeves RK 2013. Characterization of circulating natural killer cells in neotropical primates. PLoS One 8:e78793. doi:.10.1371/journal.pone.0078793 [PMC free article] [PubMed] [Cross Ref]
38. Yoshida T, Saito A, Iwasaki Y, Iijima S, Kurosawa T, Katakai Y, Yasutomi Y, Reimann KA, Hayakawa T, Akari H 2010. Characterization of natural killer cells in tamarins: a technical basis for studies of innate immunity. Front Microbiol 1:128. doi:.10.3389/fmicb.2010.00128 [PMC free article] [PubMed] [Cross Ref]
39. National Research Council. 2011. Guide for the care and use of laboratory animals, 8th ed National Academies Press, Washington, DC.
40. Weatherford T, Chavez D, Brasky KM, Lanford RE 2009. The marmoset model of GB virus B infections: adaptation to host phenotypic variation. J Virol 83:5806–5814. doi:.10.1128/JVI.00033-09 [PMC free article] [PubMed] [Cross Ref]
41. Lee J, Suh WI, Shin EC 2010. T-cell dysfunction and inhibitory receptors in hepatitis C virus infection. Immune Netw 10:120–125. doi:.10.4110/in.2010.10.4.120 [PMC free article] [PubMed] [Cross Ref]
42. Giavedoni LD. 2005. Simultaneous detection of multiple cytokines and chemokines from nonhuman primates using Luminex technology. J Immunol Methods 301:89–101. doi:.10.1016/j.jim.2005.03.015 [PubMed] [Cross Ref]
43. Bacon BR. 2002. Treatment of patients with hepatitis C and normal serum aminotransferase levels. Hepatology 36:S179–S184. [PubMed]
44. Marcellin P, Martinot M, Boyer N, Levy S 1999. Treatment of hepatitis C patients with normal aminotransferases levels. Clin Liver Dis 3:843–853. doi:.10.1016/S1089-3261(05)70242-7 [PubMed] [Cross Ref]
45. Swiecki M, Wang Y, Vermi W, Gilfillan S, Schreiber RD, Colonna M 2011. Type I interferon negatively controls plasmacytoid dendritic cell numbers in vivo. J Exp Med 208:2367–2374. doi:.10.1084/jem.20110654 [PMC free article] [PubMed] [Cross Ref]
46. Amadei B, Urbani S, Cazaly A, Fisicaro P, Zerbini A, Ahmed P, Missale G, Ferrari C, Khakoo SI 2010. Activation of natural killer cells during acute infection with hepatitis C virus. Gastroenterology 138:1536–1545. doi:.10.1053/j.gastro.2010.01.006 [PMC free article] [PubMed] [Cross Ref]
47. Kokordelis P, Kramer B, Korner C, Boesecke C, Voigt E, Ingiliz P, Glassner A, Eisenhardt M, Wolter F, Kaczmarek D, Nischalke HD, Rockstroh JK, Spengler U, Nattermann J 2014. An effective interferon-gamma-mediated inhibition of hepatitis C virus replication by natural killer cells is associated with spontaneous clearance of acute hepatitis C in human immunodeficiency virus-positive patients. Hepatology 59:814–827. doi:.10.1002/hep.26782 [PubMed] [Cross Ref]
48. Pelletier S, Drouin C, Bedard N, Khakoo SI, Bruneau J, Shoukry NH 2010. Increased degranulation of natural killer cells during acute HCV correlates with the magnitude of virus-specific T cell responses. J Hepatol 53:805–816. doi:.10.1016/j.jhep.2010.05.013 [PMC free article] [PubMed] [Cross Ref]
49. Manickam C, Reeves RK 2014. Modeling HCV disease in animals: virology, immunology and pathogenesis of HCV and GBV-B infections. Front Microbiol 5:690. doi:.10.3389/fmicb.2014.00690 [PMC free article] [PubMed] [Cross Ref]
50. Harvey CE, Post JJ, Palladinetti P, Freeman AJ, Ffrench RA, Kumar RK, Marinos G, Lloyd AR 2003. Expression of the chemokine IP-10 (CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation. J Leukoc Biol 74:360–369. doi:.10.1189/jlb.0303093 [PubMed] [Cross Ref]
51. Helbig KJ, Ruszkiewicz A, Semendric L, Harley HA, McColl SR, Beard MR 2004. Expression of the CXCR3 ligand I-TAC by hepatocytes in chronic hepatitis C and its correlation with hepatic inflammation. Hepatology 39:1220–1229. doi:.10.1002/hep.20167 [PubMed] [Cross Ref]
52. Chen Y, Wei H, Sun R, Dong Z, Zhang J, Tian Z 2007. Increased susceptibility to liver injury in hepatitis B virus transgenic mice involves NKG2D-ligand interaction and natural killer cells. Hepatology 46:706–715. doi:.10.1002/hep.21872 [PubMed] [Cross Ref]
53. Kahraman A, Schlattjan M, Kocabayoglu P, Yildiz-Meziletoglu S, Schlensak M, Fingas CD, Wedemeyer I, Marquitan G, Gieseler RK, Baba HA, Gerken G, Canbay A 2010. Major histocompatibility complex class I-related chains A and B (MIC A/B): a novel role in nonalcoholic steatohepatitis. Hepatology 51:92–102. doi:.10.1002/hep.23253 [PubMed] [Cross Ref]
54. Dunn C, Brunetto M, Reynolds G, Christophides T, Kennedy PT, Lampertico P, Das A, Lopes AR, Borrow P, Williams K, Humphreys E, Afford S, Adams DH, Bertoletti A, Maini MK 2007. Cytokines induced during chronic hepatitis B virus infection promote a pathway for NK cell-mediated liver damage. J Exp Med 204:667–680. doi:.10.1084/jem.20061287 [PMC free article] [PubMed] [Cross Ref]
55. Ochi M, Ohdan H, Mitsuta H, Onoe T, Tokita D, Hara H, Ishiyama K, Zhou W, Tanaka Y, Asahara T 2004. Liver NK cells expressing TRAIL are toxic against self hepatocytes in mice. Hepatology 39:1321–1331. doi:.10.1002/hep.20204 [PubMed] [Cross Ref]
56. Stegmann KA, Bjorkstrom NK, Veber H, Ciesek S, Riese P, Wiegand J, Hadem J, Suneetha PV, Jaroszewicz J, Wang C, Schlaphoff V, Fytili P, Cornberg M, Manns MP, Geffers R, Pietschmann T, Guzman CA, Ljunggren HG, Wedemeyer H 2010. Interferon-alpha-induced TRAIL on natural killer cells is associated with control of hepatitis C virus infection. Gastroenterology 138:1885–1897. doi:.10.1053/j.gastro.2010.01.051 [PubMed] [Cross Ref]
57. Kunitani H, Shimizu Y, Murata H, Higuchi K, Watanabe A 2002. Phenotypic analysis of circulating and intrahepatic dendritic cell subsets in patients with chronic liver diseases. J Hepatol 36:734–741. doi:.10.1016/S0168-8278(02)00062-4 [PubMed] [Cross Ref]
58. Nattermann J, Zimmermann H, Iwan A, von Lilienfeld-Toal M, Leifeld L, Nischalke HD, Langhans B, Sauerbruch T, Spengler U 2006. Hepatitis C virus E2 and CD81 interaction may be associated with altered trafficking of dendritic cells in chronic hepatitis C. Hepatology 44:945–954. doi:.10.1002/hep.21350 [PubMed] [Cross Ref]
59. Averill L, Lee WM, Karandikar NJ 2007. Differential dysfunction in dendritic cell subsets during chronic HCV infection. Clin Immunol 123:40–49. doi:.10.1016/j.clim.2006.12.001 [PMC free article] [PubMed] [Cross Ref]
60. Netski DM, Mosbruger T, Depla E, Maertens G, Ray SC, Hamilton RG, Roundtree S, Thomas DL, McKeating J, Cox A 2005. Humoral immune response in acute hepatitis C virus infection. Clin Infect Dis 41:667–675. doi:.10.1086/432478 [PubMed] [Cross Ref]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)