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GASTROENTEROLOGY 2007;132:733–744

efective Hepatic Response to Interferon and Activation of Suppressor ofytokine Signaling 3 in Chronic Hepatitis C

ING HUANG,* JORDAN J. FELD,* RONDA K. SAPP,* SANTOSH NANDA,* JIING-HUEY LIN,‡ LAWRENCE M. BLATT,‡

ICHAEL W. FRIED,§ KRISHNA MURTHY,� and T. JAKE LIANG*

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland; ‡InterMune, Inc,risbane, California; §Division of Gastroenterology and Hepatology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; and �Department of

irology and Immunology, Southwest Foundation for Biomedical Research, San Antonio, Texas

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ackground & Aims: Approximately half of hepati-is C virus (HCV)-infected patients do not respond tourrent interferon (IFN)-� combination therapy. Tonderstand IFN-� resistance in vivo, we examined theynamic responses to both type I and type II IFNs,uman IFN (hIFN)-�, -�, and consensus IFN, in thehimpanzee model. Methods: Naive and HCV-in-ected chimpanzees were treated with 3 forms ofIFNs in vivo. Quantitative real-time polymerasehain reaction was performed to evaluate the expres-ion of IFN-stimulated genes (ISGs) in both periph-ral blood mononuclear cells and liver to comparehe responses to hIFN between naive and infectedhimpanzees. The hepatic expression of IFN signal-ng components and inhibitory regulators includinguppressor of cytokine signaling 3 (SOCS3) were as-essed. SOCS3 expression was also evaluated in theiver of HCV-infected patients undergoing IFN treat-

ent. Results: The in vivo responses to all 3 hIFNsere much lower in the HCV-infected chimpanzees

han those in the naive chimpanzees. This defect wasarticularly evident in the liver because induction ofepatic ISGs was barely detectable in the infectednimals. Following IFN administration, the expres-ion of SOCS3 was significantly up-regulated, possi-ly through induction of interleukin-6, in the liver ofCV-infected chimpanzees. HCV-infected humans

lso showed a differential pattern of hepatic SOCS3xpression in response to IFN that is associated withreatment response. Conclusions: Our data indicatepredominantly defective hepatic response to IFN inCV-infected chimpanzees, which is probably medi-

ted through the activation of SOCS3 and may ex-lain the nonresponse of many HCV patients to IFN-ased therapy.

epatitis C virus (HCV) infection is one of the mostcommon blood-borne chronic infections with an

stimated 170 million infected people worldwide. In thenited States, approximately 3 million people are chron-

cally infected, and HCV is the leading cause of liver

ransplantation.1 No HCV vaccine is available to date,

nd the current antiviral therapy with pegylated inter-eron (IFN)-� (Peg-IFN-�) and ribavirin is expensive, ef-ective in approximately 50% of patients, and associatedith numerous adverse effects.2 Thus, there is a pressingeed for improvement of anti-HCV therapy.3 To achievehis goal, it is crucial to understand the mechanisms ofCV clearance and nonresponse to IFN-based therapy inCV patients.IFNs are naturally occurring proteins secreted by mam-alian cells that play a critical role in control of viral

nfection and provide a link between innate and adaptivemmunity. There are 3 types of IFN: type I IFNs includehe 14 nonallelic subtypes of IFN-� subtypes, as well asFN-�, -�, -�, -�, and -�, all of which bind to the type IFN receptor (IFNAR); type II IFN, IFN-�, binds to theype II IFN receptor; type III IFNs include 3 recentlyiscovered proteins called IFN-� that bind to a novel typeII receptor.4 Consensus IFN-� (IFN alfacon-1) is a syn-hetic type I IFN, whose sequence is derived from theonsensus sequences of various IFN-� subtypes.5 It haseen shown to be more potent than naturally occurringype I IFNs in cell culture models and more effective inlinical trials for the treatment of chronic hepatitis C.6,7

IFN induces an antiviral state in cells by activating theanus kinase (JAK)-signal transducers and activators ofranscription (STAT) pathway.8 Binding of IFN to itseceptor activates constitutively associated JAK proteins,hich leads to the docking of STAT molecules to the

Abbreviations used in this paper: HCV, hepatitis C virus; IFN, inter-eron; IFI15, IFN-induced protein 15; IFIT1, interferon-induced proteinith tetratricopeptide repeats 1; IFNAR, interferon-�/� receptor; IP10,

nterferon-�-inducible protein 10; IRF, interferon regulatory factor; ISG,nterferon-induced gene; ISGF, interferon-stimulated gene factor; ISRE,nterferon-stimulated response element; LMP, large multifunctionalrotease; MCP-1, membrane cofactor protein 1; MIP-1�, macrophage

nflammatory protein 1-�; MX1, myxovirus resistance 1; OAS, 2,5-ligoadenylate synthetase; PBMC, peripheral blood mononuclear cell;IAS, protein inhibitor of activated STAT; PP2A, protein phosphataseA; RR, rapid responders; SHP1, Src homology region 2-domain phos-hatase 1; SOCS, suppressor of cytokine signaling; SR, slow respond-rs; STAT, signal transducers and activators of transcription.

© 2007 by the AGA Institute0016-5085/07/$32.00

doi:10.1053/j.gastro.2006.11.045

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734 HUANG ET AL GASTROENTEROLOGY Vol. 132, No. 2

eceptor and subsequent STAT phosphorylation. The ac-ivated STATs dissociate from the receptor chain andorm dimers that translocate to the nucleus to modulateene transcriptional activity. For the type I IFNs, thenterferon-stimulated gene factor 3 (ISGF3) complex,onsisting of a STAT1 and STAT2 heterodimer and in-erferon regulatory factor 9 (IRF9), binds to the interfer-n-stimulated response element (ISRE). For the type IIFN, IFN-�, STAT1 homodimers bind directly to the-activated site element. Both types of IFNs induce thexpression of a large number of ISGs with substantialverlap and set up an antiviral, antiproliferative, and

mmunoregulatory state in the host cells. IFN-inducedntiviral activities have been extensively studied in theCV replicon system. Both IFN-� and IFN-� have been

hown to inhibit HCV replication,9,10 and type I/II IFNombinations resulted in a synergistic antiviral effect.11,12

owever, standard combination therapy with Peg-IFN-�nd ribavirin achieves sustained viral clearance in onlypproximately half of treated patients, and IFN-� as aingle agent appears to be ineffective in small clinicalrials.13 HCV appears to have developed strategies tonterfere with the IFN effector pathways, leading to non-esponse in many HCV-infected patients.

The mechanisms by which HCV interferes with IFN sig-aling and attenuates its antiviral efficacy have not been

ully elucidated. Various hypotheses have been proposed.14

mong them, 2 negative regulators, suppressor of cytokineignaling (SOCS) 3 and protein inhibitor of activated STATPIAS), have recently been reported to be induced by HCVroteins leading to inhibition of the JAK-STAT signaling.OCS3 can be induced by the HCV core protein and sup-ress JAK-STAT signaling to block the IFN-induced forma-ion of ISGF3 in cell culture.15 HCV protein expression iniver cells is associated with activation of PIAS and inhibi-ion of STAT function, possibly augmented by induction ofrotein phosphatase 2A (PP2A) expression.16

Despite significant advances in understanding of the ac-ions of IFN in the cell culture system, little is known abouthe inhibition of IFN signaling by HCV and nonresponse toFN therapy in vivo. The chimpanzee is the only animal

odel susceptible to HCV infection. In the present study,e conducted a comprehensive assessment of the antiviral

ffects of both type I and type II IFNs (IFN-�, -�, andonsensus IFN) in chimpanzees and demonstrated a poten-ial mechanism of IFN resistance. To confirm the clinicalelevance in human HCV infection, we also evaluated pa-ient samples before and during Peg-IFN therapy.

Materials and MethodsChimpanzees and InterferonsSix chimpanzees, X0176, X0284, X0101, X0233,

0142, and X0234, were maintained at the Southwestoundation for Biomedical Research, an Association for

ssessment and Accreditation of Laboratory Animal and v

are-accredited facility, and the study protocol was ap-roved by the Institutional Animal Care and Use Commit-ee at the Foundation and by the Interagency Animal Modelommittee at the National Institutes of Health (NIH). Two

himpanzees, X0142 and X0234, had persistent infectionith HCV genotype 1b derived from a homogenous sources described previously,17 whereas others were naive ani-als. Whole blood samples from 2 chimpanzees, X6394

nd X6475 (infected with genotype 1a viruses),18,19 wererovided by Dr. Stephen Feinstone at Center for Biologicsvaluation and Research (CBER), the Food and Drug Ad-inistration (FDA).Human IFN-�2a used for the in vitro study was pur-

hased from Fitzgerald Industries International, Inc.Concord, MA), and the product used for in vivo studyas purchased from Roche (Nutley, NJ). IFN-�1b and

onsensus IFN (Infergen, IFN alfacon-1) were providedy InterMune, Inc. (Brisbane, CA).

Patient SamplesPatient samples were derived from 2 sources.

reated patients were recruited from the Liver Diseases Unitt the University of North Carolina (UNC). Patients wereiven an initial dose of 180 g Peg-IFN-�-2a and underwentiver biopsy 24 hours later. Control samples came from liveriopsy specimens obtained from patients at the Clinicalenter of the NIH prior to undergoing antiviral therapyith Peg-IFN and ribavirin. Control patients were selected

o match treated patients in terms of age, gender, ethnicity,nitial viral load, and degree of histologic disease. Patientsigned informed consent, and the protocol was approved byhe Institutional Review Board of UNC and the NIH. Allatients had genotype 1 HCV infection. Biopsy samplesere snap frozen in liquid nitrogen and stored at �80°C.etails of the human study will be published elsewhere.20

uman liver biopsy tissue was handled similarly to chim-anzee liver tissue as described below. Both treated andontrol patients subsequently underwent a full course oftandard antiviral therapy consisting of Peg-IFN-�-2a 180g and weight-based ribavirin (1000 mg daily 75 kg and200 mg �75 kg for 48 weeks). Patients achieving �2-logopies/mL decrease in HCV RNA by 4 weeks of therapy wereeemed rapid responders (RR), and those with lesser de-reases in viral load were designated slow responders (SR).

Peripheral Blood Mononuclear Cells Isolationand IFN StimulationPeripheral blood mononuclear cells (PBMCs) were

solated from whole blood samples of chimpanzees andealthy human donors (informed consents were ob-ained) and stimulated with 3 different doses of humanFN (hIFN)-�, -�, and consensus IFN in RPMI mediumith 10% fetal bovine serum (FBS) (Cellgro, Herndon,A). After incubation for 6 or 24 hours, cells were har-

ested and subjected to RNA isolation.

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February 2007 HEPATIC RESPONSE TO INTERFERON 735

Chimpanzee ExperimentChimpanzees used for in vivo study were treated

equentially with 10 million IU IFN-�, 400 g IFN-�, and0 g consensus IFN subcutaneously. Briefly, 4 animalsX0101, X0233, X0142, and X0234) were divided into 2roups, each comprising 1 naive and 1 infected chimpan-ee. The experiment was separated into 3 phases. For therst phase, group 1 chimpanzees (X0101 and X0142)ere administered with IFN-� and group 2 (X0233 and0234) with IFN-�. Blood samples (40 mL each) were

ollected at 9 time points (pretreatment, 8 hours, 24ours, 48 hours, 72 hours, 7 days, 14 days, 21 days, and8 days posttreatment), and liver biopsy samples wereollected at 5 time points (pretreatment, 8 hours, 24ours, 48 hours, and 72 hours posttreatment). All ani-als were rested for 6 weeks to avoid any residual drug

ffect. Next, group 1 was treated with IFN-� and group 2ith IFN-� for the second phase of the study. Similar

ample-collecting procedures were performed duringreatment up to 4 weeks. The animals were rested for 6eeks and started on the third and final phase of the

tudy, in which consensus IFN was given to both groups.ecause of a nonspecific infection during the rest periodfter the phase II study, one of the HCV-infected chim-anzees, X0142, was dropped from the phase III study.

Quantification of Serum HCV RNA andAlanine TransaminaseSera were isolated from serial blood samples of

he HCV-infected chimpanzees. HCV RNA was quantifiedy using the COBAS AMPLICOR HCV MONITOR TEST,2.0 (Roche Diagnostics, Branchburg, NJ), which has aetection limit of 600 IU/mL (1620 copies/mL). Alanineransaminase (ALT) values were measured by the Labo-atory Medicine Department of the Southwest Founda-ion for Biomedical Research.

TaqMan Real-Time PCR AnalysisTotal RNA was prepared from the PBMCs and

iver biopsy tissues with RNeasy Mini Kit according toanufacturer’s instructions (Qiagen, Valencia, CA).omplementary DNA (cDNA) was synthesized from totalNA with First-strand cDNA Synthesis System (Marligeniosciences, Ijamsville, MD). TaqMan real-time PCR anal-sis was used to quantify the mRNA expression levels ofenes of interest. The primers and probes used were Genexpression Assays (Applied Biosystems, Foster City, CA).ach reaction was performed in duplicate, and all sam-les were standardized using the internal control glycer-ldehyde-3-phosphate dehydrogenase (GAPDH) gene.eactions were set up using 12.5 L TaqMan universalCR master mix, cDNA template, and 1.25 L primersnd probe mix in a final volume of 25 L. Reactions wereerformed on an iCycler iQ Multicolor Real-Time Detec-ion System (Bio-Rad, Hercules, CA) with the following

eaction conditions: 95°C for 10 minutes, followed by 40 X

ycles of 95°C for 20 seconds, 60°C for 1 minute, anddditional incubation at 68°C for 10 minutes.

Western Blot AnalysisLiver biopsy tissues were lysed using mammalian

issue lysis/extraction reagent (CelLytic MT; Sigmahemical Co., St. Louis, MO) containing a Protease In-ibitor Cocktail Tablet (Roche, Indianapolis, IN) and arotein Phosphatase Inhibitor Set (Upstate Biotechnol-gy, Lake Placid, NY). The collection of supernatant

ysates, determination of protein concentration, andestern blot analysis have been described previously.21

ntibodies to STAT1, SOCS1, SOCS3, Src homologyegion 2-domain phosphatase (SHP)1, and �-actin wererom Abcam Inc. (Cambridge, MA). Antibodies to STAT2,TAT3, phosphorylated STAT1 (Y701), and proteinhosphatase 2A C subunit (PP2Ac) were from Upstateiotechnology. Anti-IRF9 antibody was from Santa Cruziotechnology (Santa Cruz, CA), and anti-IFNAR chain 2ntibody was from Fitzgerald Industries Internationalnc. (Concord, MA). The band intensities in the imagesere quantified by a public analysis program, ImageJ,ffered by the NIH (http://rsb.info.nih.gov/ij/).

Serum Cytokine AnalysisSerum cytokines (eotaxin, GM-CSF, IFN-�, inter-

eukin (IL)-1�, IL-1�, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,L-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, interferon-�-nducible protein (IP) 10, membrane cofactor proteinMCP)-1, macrophage inflammatory protein (MIP)-1�,umor necrosis factor (TNF)-�, and RANTES) were mea-ured simultaneously using Beadlyte Human 22-Plex Cy-okine Detection System (Upstate Biotechnology) accord-ng to the manufacturer’s instructions. Briefly, bothtandard and chimpanzee samples were diluted with hu-

an serum diluent, and 50 L was loaded onto the6-well filtration plate. Twenty-five-microliter beadsoated with target capture antibodies against cytokinesere added to each well and incubated overnight at 4°Cith low-speed shaking at 300 rpm in the dark. On theext day, after washing, the plate was supplied with 25L premixed biotin conjugate antibodies and incubated

or 2 hours at room temperature with shaking at 300pm. Finally, streptavidin-phycoerythrin was added, andhe results were read with Bioplex Luminex System (Bio-ad Laboratories Inc., Hercules, CA). The data were an-lyzed using Bio-Plex Manager software v.4.0 with Five-arameter Logistics curve fitting.

ResultsResponse of Chimpanzees to hIFNs In VitroBefore performing the in vivo chimpanzee study,

e first determined the ability of chimpanzees to respondo hIFN. PBMCs from 2 naive chimpanzees (X0284 and

0176) and 2 healthy human donors were stimulated

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736 HUANG ET AL GASTROENTEROLOGY Vol. 132, No. 2

ith 3 different doses of IFN-�, IFN-�, and consensusFN. A panel of previously reported IFN-� or -� specificSGs were selected for analysis: myxovirus resistance 1MX1), 2,5-oligoadenylate synthetase (2,5-OAS), IFN-in-uced protein with tetratricopeptide repeats 1 (IFIT1),nd IFN-induced protein 15 (IFI15) were used as markersf IFN-�-induced genes, and the IP10, IRF1, and largeultifunctional protease (LMP) 2 and LMP7 were se-

ected as IFN-�-induced genes.22 All ISGs were induced athours after IFN treatment (data not shown) and

eached a peak at 24 hours (Figure 1). The overall genexpression patterns of the ISGs were comparable betweenuman and chimpanzee PBMCs. Consensus IFN wasore potent and induced a broader range of ISGs than

ither IFN-� or -�. All 3 IFNs induced higher levels of ISGxpression in humans than in chimpanzees (2–5 timesigher), suggesting that chimpanzees do not respond asell as humans to hIFN in vitro. Based on these data, 10illion IU IFN-�, 400 g IFN-�, and 30 g consensus

FNs were used for in vivo study in chimpanzees. Theseoses are 3– 4 times higher than the standard doses forhe treatment of human subjects (3 million IU IFN-�,00 g IFN-�, and 9 g consensus IFN) to compensate

igure 1. ISG induction of chimpanzee PBMCs to human IFNs in vitroonors (donors 1 and 2) were isolated and stimulated with 3 forms of IFonsensus IFN (0.3, 3, and 30 ng/mL). Fold inductions of selected ISGars indicate mean � SD of 3 experiments. conIFN, consensus IFN.

or the lower efficacy of hIFNs in chimpanzees. t

Impaired Response of HCV-InfectedChimpanzees to hIFN In Vivo

Four chimpanzees (2 naive: X0101 and X0233; 2nfected: X0142 and X0234) were divided into 2 groups inhe in vivo study. Each group consisted of 1 naive and 1nfected animal. The experiment was divided into 3hases: treatment with IFN-� (or IFN-�), treatment withFN-� (or IFN-�), and treatment with consensus IFN,espectively. With this design, each animal received all 3orms of IFN treatment, which allows for internal controlnd biologic duplication of the experiment. ConsensusFN was given last because consensus IFN is more potentnd induces a broader range of ISGs than either IFN-� or�. Similar to the results from the in vitro study, IFN-�nd consensus IFN treatment led to the induction of theFN-�-specific ISGs (MX1, OAS1, IFIT1, and IFI15) inoth PBMCs and liver tissues of the chimpanzees in vivo.ikewise, the IFN-�-specific ISGs (IP10, IRF1, LMP2, andMP7) were induced by IFN-� and consensus IFN in vivo.igure 2 shows the fold inductions of only 4 selectedSGs (MX1, IFI15, IP10, and IRF1), but the other 4IFIT1, OAS1, LMP2, and LMP7) behaved similarly. Al-

MCs from naive chimpanzees (X0284 and X0176) and healthy humanIFN-� (10, 100, and 1000 U/mL); (B) IFN-� (1, 10, and 100 ng/mL); (C)As were measured by real-time PCR at 24 hours posttreatment. Error

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February 2007 HEPATIC RESPONSE TO INTERFERON 737

anzees responded to all 3 forms of IFN, reaching a peakt 8 hours posttreatment and returning to basal levelsithin 48 hours, the levels of ISG induction in PBMCs

igure 2. Baseline and induction of ISG in naive and HCV-infected chimaive (X0101 and X0233) and HCV-infected (X0142 and X0234) chimpf all animals. The basal ISG levels, after normalized to GAPDH, were a

D) The hepatic expression levels of selected ISGs (MX1 and OAS1) in senit, defined as copy number normalized to GAPDH).

rom HCV-infected chimpanzees were much lower (�4 e

imes) than those in the naive animals (Figure 2A). All 3orms of IFN induced stronger ISG responses in the liverhan in PBMCs of naive chimpanzees (Figure 2B). How-

zees in vivo. Induction of ISG expression in PBMC (A) and liver (B) froms in vivo. conIFN, consensus IFN. (C) Basal ISG expression in the liverd by setting the value of naive chimpanzee (X0101) as 1 for each ISG.d animals (X0233 and X0234) following IFN administration (RU; relative

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ver, in the HCV-infected animals, little or no hepatic ISG

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738 HUANG ET AL GASTROENTEROLOGY Vol. 132, No. 2

nduction was observed. Although hepatic ISG inductionas severely blunted in the infected animals, the basal

evel of ISG expression in the liver were higher than thosen naive animals (Figure 2C), suggesting that HCV infec-ion resulted in endogenous IFN production. The higherasal hepatic ISG expression did not account for theailure to respond to exogenous IFN because the absoluteevels reached after treatment were still much lower innfected than naive chimpanzees (Figure 2D). These datandicate a deficiency of response, particularly in the liver,o hIFNs in HCV-infected chimpanzees.

Effect of IFN on Viral Level of HCV-InfectedChimpanzeesHCV RNA and ALT levels were analyzed from

erial serum samples of the 2 chronically HCV-infectedhimpanzees (X0142 and X0234) pre- and post-IFN ad-inistration. As shown in Figure 3, although chimpanzee0234 was initially inoculated with the week 2 serum

rom X0142 (ie, the same viral strain),17 the pattern ofnfection that ensued differed somewhat between the 2himpanzees. The viral load was much higher in X0142nd fluctuated in a range from 104 to 106 copies/mL asompared with a relatively stable viremia (approximately

� 104 copies/mL) seen in X0234. Notably, neitherFN-� nor IFN-� injection resulted in a significant de-rease in viral load in either chimpanzee, consistent withhe absence of hepatic ISG induction in both animals.owever, chimpanzee X0234 did have a transient de-

rease in viremia in response to IFNs with a reproducibleecrease in viral titer of 0.5–1 log copies/mL (IFN-� �FN-� � conIFN) at 8 –24 hours posttreatment. Interest-ngly, although the hepatic IFN response in this chim-anzee was blunted compared with naive animals, thereas low-level induction of hepatic ISGs in response to

igure 3. Serum HCV RNA titers of HCV-infected chimpanzees inesponse to IFN. HCV RNA levels of HCV-infected chimpanzees X0142A) and X0234 (B) were monitored for more than 1 year prior to thistudy. Arrows indicate time points of IFN administration. conIFN, con-ensus IFN.

FNs (eg, MX1 and IFI15 induced by IFN-�; IP10 and w

RF1 induced by IFN-�, Figure 2B and 2D). In contrast,himpanzee X0142 had no hepatic ISG induction afterreatment and actually had a transient increase in HCViremia within 24 – 48 hours posttreatment, which ap-eared to coincide with a transient ALT increase in thishimpanzee (data not shown). This effect could be ex-lained by an IFN-induced hepatotoxicity with release ofCV RNA from the injured hepatocytes resulting in a

ransient rise in viremia.

Comparable Induction of ISGs in PBMCsFrom Infected and Naive Chimpanzees ExVivoBecause of the apparently different ISG induction

n PBMCs between the naive and infected chimpanzees inivo, we sought to determine whether such a differenceersists ex vivo, free of any potential biologic interactionsith IFN that may be operational in vivo. Thus, we

valuated the actions of IFNs on PBMCs isolated fromaive and infected chimpanzees in vitro. Eight chimpan-ees, 4 naive and 4 HCV infected, were studied. The 4aive animals included the 2 used in the preliminarytudy to test the response of chimpanzees to hIFNsX0176 and X0284) and 2 others used in the in vivo studybove (X0101 and X0233). The 4 infected animals werehe 2 used for the study above (X0142 and X0234) and 2thers infected with a genotype 1a strain H77 (X6394nd X6475).18,19 Figure 4 summarizes the fold inductionsf representative ISGs in PBMCs from all 8 chimpanzeest 24 hours post-IFN stimulation. The overall ISG induc-ions of the HCV-infected animals were comparable withhose of the naive animals with the exception of IP10,hose induction by IFN-� was significantly lower in the

nfected chimpanzees. However, the difference in IP10nduction of PBMCs by IFN-� between naive and infectedhimpanzees ex vivo was much less than that in vivo (2-o 3-fold vs 4- to 6-fold, respectively). These data suggesthat the apparent difference in IFN response occurred

ostly in vivo, and, once the PBMCs were removed fromhe in vivo milieu of the infected animals, they respondedo IFN similarly to those from the naive animals.

Persistent Activation of the JAK-STATPathway in the Liver of HCV-InfectedChimpanzeesThe lack of hepatic ISG induction after IFN treat-

ent in HCV-infected chimpanzees suggests that the IFNignal transduction pathways are inhibited in the liver.o determine which step of IFN signaling was inhibited,arious components involved in the JAK-STAT pathwayere evaluated using Western blot analysis from liveriopsy samples of pre- and 8 hours post-IFN-� treat-ent. The 8-hour time point was chosen to coincide with

he peak hepatic ISG expression (Figure 2B). As shown inigure 5A for the naive chimpanzees, a slight increase ofhosphorylated STAT1 was detected with X0101,

hereas not with X0233. In these chimpanzees, the JAK-

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TAT pathway was likely activated by exogenous IFN butuickly returned to baseline by 8 hours. This time course

s consistent with the rapid onset and transient nature ofAK-STAT activation by IFN observed in vitro.23 In con-rast, all components of the JAK-STAT pathway werectivated even prior to IFN treatment in the infectedhimpanzees. In addition to a markedly elevated level ofhosphorylated STAT1, there was a general increase seen

n the total levels of STAT1, STAT2, STAT3, and IRF9roteins (Figure 5A). This effect was more dramatic inhimpanzee X0142. After IFN treatment, rather thanncreasing, the phosphorylated STAT1 level actually de-lined in X0142, which might be due to the activation ofegative regulator(s) of IFN signaling. This observation is

n keeping with the lack of ISG induction in the liver ofhese infected animals.

Enhanced Hepatic SOCS3 Expression inResponse to IFN in HCV-InfectedChimpanzeesAt least 3 different classes of negative regulators

re known to contribute to the inhibition of IFN signal-ng24: the SHP1, SOCS, and PIAS families. To explorehether these suppressors were responsible for the IFN

esistance seen in the HCV-infected chimpanzees, weeasured the expression levels of regulatory factors

HP1, SOCS1, SOCS3, and PP2A in liver biopsy samplesre- and post-IFN-� induction (Figure 5A). Among these

igure 4. ISG induction of PBMCs from naive and HCV-infected chim-anzees ex vivo. PBMCs from naive (X0176, X0284, X0101, and0233) and HCV-infected (X0142, X0234, X6394, and X6475) chim-anzees were isolated and stimulated with IFN-� (10, 100, and 1000/mL) (A) and IFN-� (1, 10, and 100 ng/mL) (B), respectively. Fold

nductions of selected ISG mRNAs were measured by real-time PCR at4 hours posttreatment and compared between naive and infectedroups. Error bars indicate mean � SD for the 4 animals in each group.

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actors, the SOCS3 protein level increased significantly in n

CV-infected but not in naive animals after IFN treat-ent. This increase was also confirmed at the RNA level

y quantitative real-time PCR (Figure 5B). Although theepatic SOCS3 mRNA level also increased modestly inaive animals, the increase was much more dramatic inCV-infected animals (X0142 � X0234). These data in-icate that the SOCS3 up-regulation may be a potentialechanism for the defective IFN response in HCV-in-

ected chimpanzees.

igure 5. Expression of IFN signaling molecules and inhibitory regula-ors in the liver of chimpanzees. (A) Fifteen micrograms protein extractsf liver from chimpanzees with or without IFN-� treatment (8 hours)ere subjected to Western blot analysis with indicated antibodies.-actin was used as loading control. Band intensities were quantifiedith NIH ImageJ software. The relative values for each protein arehown below the bands with the baseline value of naive chimpanzeeX0101) setting as 100. (B) Liver SOCS3 mRNA expression levels mea-ured by real-time PCR (RU; relative unit, defined as copy number

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740 HUANG ET AL GASTROENTEROLOGY Vol. 132, No. 2

Induction of IL-6 by IFN in Serum and LiverTissue of HCV-Infected Chimpanzees

Because SOCS3 can be induced by a variety ofytokines including the proinflammatory cytokine IL-6,e determined the serum IL-6 levels following IFN ad-inistration in these chimpanzees. Although the serum

L-6 levels increased in response to IFN in all chimpan-ees, the infected chimpanzee X0142 showed a morexaggerated augmentation (Figure 6A). A similar patternas confirmed by real-time PCR of IL-6 mRNA in the

iver. This observation is consistent with the high level ofepatic SOCS3 induction in chimpanzee X0142. In ad-ition to IL-6, we also evaluated a panel of cytokine/hemokine levels in sera of HCV-infected chimpanzeesompared with those of naive animals in response to IFNreatment. Among the 22 cytokines/chemokines mea-ured, IP10, MCP-1, MIP-1�, and eotaxin were inducedy both type I and type II IFNs and behaved in a pat-ern similar to the ISG mRNA expression (Fig-

igure 6. Induction of IL-6 by IFN in the serum and liver of HCV-infectefined as copy number normalized to GAPDH) of IL-6 were determinedpanel of serum cytokines was measured. Selected cytokines (IP10, M

re 6B). q

Hepatic SOCS3 Expression in HCV-InfectedPatients Pre- and Post-IFN Treatment

Based on the findings in chimpanzees, we evalu-ted SOCS3 mRNA expression in HCV-infected patientsre- and post-IFN treatment. Two groups of genotype 1irus infected-patients were studied: a control group of1 patients in whom liver biopsies were performed prioro antiviral therapy and a treatment group of 13 patientsn whom liver biopsies were performed 24 hours after annitial dose of Peg-IFN-�2a. Two groups were matchedor age, gender, and race. All control patients were sub-equently treated, and their response to therapy isnown. Patients were categorized as rapid respondersRR) or slow responders (SR) depending on whether theychieved a 2-log copies/mL drop in HCV viral titer by 4eeks of therapy. This early virologic response has been

hown to be an accurate predictor of clinical treatmentesponse25 and probably a better marker of the antiviralffect of IFN in vivo because clinical end point is fre-

impanzees. (A) Serum protein and liver mRNA levels (RU; relative unit,detection limit of IL-6 mRNA level is 5 RU (dotted line) in this assay. (B)

�, MCP-1, and eotaxin) induced by IFN-� are shown.

ed ch. The

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nce or adverse effects. SOCS3 mRNA expression wasvaluated by real-time PCR for all patient samples (Fig-re 7).The treatment group had generally lower mean SOCS3

xpression than the control patients (Figure 7A). In theontrol group, patients who were considered as RR whenreated later had a higher SOCS3 expression than thoses SR, whereas the SOCS3 expression was similar be-ween RR and SR among the treated patients. In light ofhe inverse trend, the relative change in the RR and SRroups may be more biologically relevant. The foldhange of hepatic SOCS3 in response to IFN is deter-ined by taking the control group values as the baseline

xpression levels for the treatment group according toheir treatment response (Figure 7B). Although the SR inhe treatment group had a 1.7-fold higher absolute mean

igure 7. SOCS3 expression in human liver biopsy specimens pre-nd post-IFN treatment. (A) The mean SOCS3 expression (RU; relativenit, defined as copy number normalized to GAPDH) is shown for the 2ubgroups of both treatment (t) and control (c) groups as described inhe Materials and Methods section, respectively. RR, rapid responder;R, slow responder. (B) Fold change in SOCS3 expression is calculatedy dividing the mean SOCS3 expression of RR or SR in the controlroup (c) by that of RR or SR in the treatment group (t), respectively; the

old change is then shown as a negative value to account for the facthat SOCS3 expression declined with treatment. Error bars are showns mean � SD.

OCS3 expression than that of RR, they had a much less b

elative change than the RR (22.6-fold vs 268-fold; P �037).

DiscussionAlthough new therapies based on small molecule

nhibitors of HCV protease and polymerase look prom-sing for HCV infection, it is likely that they will be usedn combination with, rather than instead of, IFN-�-basedegimens. Therefore, an improved understanding of the

echanisms of action and resistance to IFN in HCVnfection will be critical to improve outcomes as well asor the development of new therapeutic approaches. Inhis study, we have performed an extensive examinationf the responses to 3 different forms of IFN in theBMCs and liver of the chimpanzee, which is the onlyeliable animal model of HCV infection. We showed that,lthough less responsive than humans to hIFN, chim-anzees are able to respond to hIFN-�, -�, and consensusFN in both PBMCs and liver. The patterns of ISG in-uction in response to the various IFNs were similar tohose previously reported in cell culture,22 chimpan-ees,26,27 and humans.12 Although consensus IFN is de-ived from type I IFNs, it has been shown to induce bothype I- and type II-specific ISGs and was shown to be

ore potent than either IFN-� or -� in ISG induction.his effect is a result of the higher binding affinity ofonsensus IFN to IFNAR than IFN-�5,28 and the crossnteraction of the signal transduction pathways betweenhe 2 types of IFNs.

Our data are consistent with the recently reportedicroarray data showing strong ISG induction after

FN-� treatment of naive chimpanzees.29 More impor-antly, for the first time, we showed that HCV-infectedhimpanzees had a blunted response to IFN therapy.lthough a lower ISG induction by all 3 forms of IFNas observed in PBMCs, the most notable difference was

een in the liver. Despite high doses of all 3 forms of IFN,ittle or no hepatic ISG induction was seen in the infectednimals. Consistent with the lack of detectable IFN re-ponse, there was little or no change in HCV viral load.he basal levels of ISG and phosphorylated STAT1 ex-ression were higher in infected than naive chimpanzees,

ndicating that HCV infection led to a type I IFN re-ponse. This observation is consistent with previous stud-es in chimpanzees26,27 and humans30,31 and raises theossibility that the ISGs were already maximally induced

n infected chimpanzees such that exogenous IFNs didot lead to any further ISG induction. However, the

ncreased basal level in the infected chimpanzees does notccount entirely for the reduced ISG induction becausehe absolute levels of ISGs after IFN treatment wereubstantially lower in infected animals than those inaive animals (10- to 100-fold lower). This suggests that,

n infected chimpanzees, there is interference with abilityo respond to IFN. Two possibilities could explain this

lunted IFN response. During chronic infection, the con-

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742 HUANG ET AL GASTROENTEROLOGY Vol. 132, No. 2

inuous but ineffective IFN action leads to a relativelyesistant state of the liver to further exposure of IFN.ontinuous IFN action could lead to IFN receptor down-

egulation, a phenomenon indeed observed in ligand-eceptor interaction. We found no decrease in IFNAR inither chimpanzees or humans (data not shown) beforer during IFN treatment, consistent with previous initro data.32 Alternatively, chronic HCV infection, eitherhrough a direct viral mechanism or indirect actions, mayctivate negative regulator(s) of IFN leading to an IFNesistant state in the liver. This concept is supported byhe observation that chronic hepatitis C patients may be

ore susceptible to other hepatic viral infections.33,34

The observation that hepatic phosphorylated STAT1howed a paradoxical decline after IFN treatment in thenfected animals strongly supports the activation of anFN-inhibitory pathway, as postulated above. By assess-ng the status of the various IFN-signaling inhibitors inhe liver biopsy samples, we found that, following IFNdministration, the expression of SOCS3 was signifi-antly up-regulated. It appears that the induction ofOCS3 expression was much more dramatic at theRNA (Figure 5B) than the protein level (Figure 5A) for

oth infected chimpanzees. This difference might be dueo the unstable nature of the SOCS proteins and the lackf samples at earlier time points, which made it difficulto compare the 2 levels. SOCS3 acts by interacting withhe JAKs, resulting in impaired STAT1 and STAT3 phos-horylation. This leads to reduced STAT1 nuclear trans-

ocation, lack of binding to the ISRE, and ultimatelyecreased ISG expression.24 Being IFN inducible, SOCS3xpression is also induced by IL-6, IL-10, TNF-�, andoll-like receptors 4 and 9.35 A previous study using anCV transgenic mouse model showed a similar inhibi-

ion of JAK-STAT signaling by HCV expression; however,o induction of SOCS1 or SOCS3 was detected.36 On thether hand, HCV core protein has been shown to induceOCS3 expression in several cell lines, resulting in im-aired IFN and specifically STAT1 signaling.15,37,38 Inddition, Zhu et al32 also showed that cells harboringCV replicons resistant to IFN therapy produced higher

evels of SOCS3. With silencing of SOCS3, IFN sensitivityas partially restored.32 Our in vivo finding of increasedOCS3 expression and reduced phosphorylated STAT1fter IFN treatment is consistent with the known func-ional mechanism of this IFN-inhibitory pathway. Thisathway could provide a plausible explanation for a di-ect viral mechanism of IFN resistance in vivo.

If SOCS3 expression were induced by viral proteins,nly infected cells would be resistant to IFN. However,ur in vivo data suggest a general IFN-resistant state inhe infected liver (Figure 2B). Alternatively, activation ofFN inhibitory pathway(s) may occur through the pro-uction of soluble factor(s) that act in an autocrine/aracrine fashion in the liver and to a lesser extent

BMCs as they traverse the liver in blood. To examine the p

ossible soluble factor(s), we studied a panel of cytokine/hemokine levels in the serum of HCV-infected and naivehimpanzees in response to IFN treatment. Several cyto-ines have been reported to be induced by HCV proteins,uch as IL-6, IL-8, TNF-�, MCP-1, and RANTES.39 – 43

mong them, IL-8 has been shown to have anti-IFNctivity through inhibition of ISGF3 assembly and inter-ction with the ISRE. We found no induction of IL-8 byFN in infected chimpanzees. However, IL-6 was inducedn response to IFN in both serum and liver tissue of thenfected chimpanzees with a time course similar toOCS3 induction. Other cytokines known to modulateOCS3 expression, such as IL-10 and TNF-�, were not

nduced by IFN in the infected animals. IL-6 mRNA wasot detectable in the livers of treated patients in ourohort; however, biopsies were performed 24 hours afterFN treatment compared with 8 hours in the chimpan-ees, and, consequently, IL-6 expression may have beenissed, particularly given the low levels seen in chimpan-

ees suggesting that the peak may already have beenissed at 8 hours.Chen et al reported that human nonresponsders to

FN-�-based therapy had a higher expression of numer-us ISGs in pretreatment liver biopsy samples as com-ared with those who achieved a sustained virologic re-ponse.44 The opposite pattern was seen with the hepaticOCS3 expression in our cohort, with �11-fold higherxpression in control patients who responded to treat-ent (RR group) compared with the nonresponders (SR

roup). As a negative regulator of IFN, high SOCS3 levelsrior to treatment would block ISG expression. WithFN-� treatment, SOCS3 declines, allowing for ISG in-uction. The degree of ISG induction may be more im-ortant than the absolute level of ISG expression for virallearance. Consequently, the baseline level of SOCS3 ex-ression and the degree to which SOCS3 changes afterFN treatment determine the pretreatment level of ISGxpression and the degree to which ISGs can be inducedy IFN-� therapy. In our human study, the SOCS3 ex-ression declined much less in the SR than the RR inesponse to IFN-�. This difference could contribute to alunted ISG induction in the human nonresponders.nfected chimpanzees, perhaps as an extreme example ofhe human nonresponders, actually have a rise in hepaticOCS3 with IFN therapy and therefore exhibit little oro ISG induction and thus no decline in viremia.Based on these observations, we propose a model (Fig-

re 8) in which HCV infection leads to endogenous IFNroduction and also to an increased expression ofOCS3, either directly by a viral mechanism or indirectlyy the induction of a soluble factor, possibly IL-6. As aonsequence of SOCS3 induction and other mechanismseg, interaction of HCV NS5A or E2 protein with double-trand RNA-dependent protein kinase [PKR]), the antivi-al activity of IFN is impaired, allowing HCV to establish

ersistent infection. In chimpanzees, IFN treatment leads

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February 2007 HEPATIC RESPONSE TO INTERFERON 743

o further SOCS3 induction, which prevents ISG activa-ion and markedly blunts the IFN response. As describedbove in humans, SOCS3 declines but much greater inR than in SR after IFN treatment. This proposed inhib-

tory pathway is variably operational in HCV-infectedumans because of the genetic heterogeneity in the hu-an population, thus accounting for the diverse infec-

ion outcomes and treatment responses.In summary, we have shown that chimpanzees respond

o hIFNs with ISG production in both PBMCs and liver.CV infection leads to the production of type I IFN

esulting in sustained activation of the JAK-STAT path-ay and ISG induction in the liver. Typical ISG responsesere seen in treated naive chimpanzees; however, ISGroduction was reduced in PBMCs and almost com-letely abrogated in the liver of HCV-infected animals.OCS3 expression was markedly increased in the livers of

nfected chimpanzees after IFN treatment and was asso-iated with a decrease in phosphorylated STAT1. A sim-lar pattern of SOCS3 expression was seen in patientsssociated with treatment response. This negative regu-ator of IFN, possibly mediated by the production of IL-6,

ay be an important mechanism of IFN nonresponse inhronic HCV infection. Further studies to elucidate theiologic importance of this interaction may have crucial

mplications for the improvement of current HCV ther-py.

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Received October 2, 2006. Accepted November 8, 2006.Address requests for reprints to: T. Jake Liang, MD, Building 10,

oom 9B16, 10 Center Drive, National Institutes of Health, Bethesda,aryland 20892. e-mail: jliang@nih.gov; fax: (301) 402-0491.Supported in part by the Intramural Research Program of the Na-

ional Institute of Diabetes and Digestive and Kidney Diseases, NIH,nd under contract N01-HB-27091 of NHLBI.The authors thank Stephen Feinstone and Marian Major (CBER,

DA) for providing the chimpanzee blood samples and the NIH Clinical

enter Department of Transfusion Medicine for the virologic testing.