Defective Hepatic Response to Interferon and Activation of Suppressor of Cytokine Signaling 3 in Chronic Hepatitis C
2006; Elsevier BV; Volume: 132; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2006.11.045
ISSN1528-0012
AutoresYing Huang, Jordan J. Feld, Ronda K. Sapp, Santosh Kumar Nanda, Jiing-Huey Lin, Lawrence M. Blatt, Michael Fried, Krishna K. Murthy, T. Jake Liang,
Tópico(s)Systemic Lupus Erythematosus Research
ResumoBackground & Aims: Approximately half of hepatitis C virus (HCV)-infected patients do not respond to current interferon (IFN)-α combination therapy. To understand IFN-α resistance in vivo, we examined the dynamic responses to both type I and type II IFNs, human IFN (hIFN)-α, -γ, and consensus IFN, in the chimpanzee model. Methods: Naive and HCV-infected chimpanzees were treated with 3 forms of hIFNs in vivo. Quantitative real-time polymerase chain reaction was performed to evaluate the expression of IFN-stimulated genes (ISGs) in both peripheral blood mononuclear cells and liver to compare the responses to hIFN between naive and infected chimpanzees. The hepatic expression of IFN signaling components and inhibitory regulators including suppressor of cytokine signaling 3 (SOCS3) were assessed. SOCS3 expression was also evaluated in the liver of HCV-infected patients undergoing IFN treatment. Results: The in vivo responses to all 3 hIFNs were much lower in the HCV-infected chimpanzees than those in the naive chimpanzees. This defect was particularly evident in the liver because induction of hepatic ISGs was barely detectable in the infected animals. Following IFN administration, the expression of SOCS3 was significantly up-regulated, possibly through induction of interleukin-6, in the liver of HCV-infected chimpanzees. HCV-infected humans also showed a differential pattern of hepatic SOCS3 expression in response to IFN that is associated with treatment response. Conclusions: Our data indicate a predominantly defective hepatic response to IFN in HCV-infected chimpanzees, which is probably mediated through the activation of SOCS3 and may explain the nonresponse of many HCV patients to IFN-based therapy. Background & Aims: Approximately half of hepatitis C virus (HCV)-infected patients do not respond to current interferon (IFN)-α combination therapy. To understand IFN-α resistance in vivo, we examined the dynamic responses to both type I and type II IFNs, human IFN (hIFN)-α, -γ, and consensus IFN, in the chimpanzee model. Methods: Naive and HCV-infected chimpanzees were treated with 3 forms of hIFNs in vivo. Quantitative real-time polymerase chain reaction was performed to evaluate the expression of IFN-stimulated genes (ISGs) in both peripheral blood mononuclear cells and liver to compare the responses to hIFN between naive and infected chimpanzees. The hepatic expression of IFN signaling components and inhibitory regulators including suppressor of cytokine signaling 3 (SOCS3) were assessed. SOCS3 expression was also evaluated in the liver of HCV-infected patients undergoing IFN treatment. Results: The in vivo responses to all 3 hIFNs were much lower in the HCV-infected chimpanzees than those in the naive chimpanzees. This defect was particularly evident in the liver because induction of hepatic ISGs was barely detectable in the infected animals. Following IFN administration, the expression of SOCS3 was significantly up-regulated, possibly through induction of interleukin-6, in the liver of HCV-infected chimpanzees. HCV-infected humans also showed a differential pattern of hepatic SOCS3 expression in response to IFN that is associated with treatment response. Conclusions: Our data indicate a predominantly defective hepatic response to IFN in HCV-infected chimpanzees, which is probably mediated through the activation of SOCS3 and may explain the nonresponse of many HCV patients to IFN-based therapy. Hepatitis C virus (HCV) infection is one of the most common blood-borne chronic infections with an estimated 170 million infected people worldwide. In the United States, approximately 3 million people are chronically infected, and HCV is the leading cause of liver transplantation.1Liang T.J. Rehermann B. Seeff L.B. Hoofnagle J.H. Pathogenesis, natural history, treatment, and prevention of hepatitis C.Ann Intern Med. 2000; 132: 296-305Google Scholar No HCV vaccine is available to date, and the current antiviral therapy with pegylated interferon (IFN)-α (Peg-IFN-α) and ribavirin is expensive, effective in approximately 50% of patients, and associated with numerous adverse effects.2Feld J.J. Hoofnagle J.H. Mechanism of action of interferon and ribavirin in treatment of hepatitis C.Nature. 2005; 436: 967-972Google Scholar Thus, there is a pressing need for improvement of anti-HCV therapy.3Tan S.L. He Y. Huang Y. Gale Jr., M. Strategies for hepatitis C therapeutic intervention: now and next.Curr Opin Pharmacol. 2004; 4: 465-470Google Scholar To achieve this goal, it is crucial to understand the mechanisms of HCV clearance and nonresponse to IFN-based therapy in HCV patients. IFNs are naturally occurring proteins secreted by mammalian cells that play a critical role in control of viral infection and provide a link between innate and adaptive immunity. There are 3 types of IFN: type I IFNs include the 14 nonallelic subtypes of IFN-α subtypes, as well as IFN-β, -ε, -κ, -ω, and -τ, all of which bind to the type I IFN receptor (IFNAR); type II IFN, IFN-γ, binds to the type II IFN receptor; type III IFNs include 3 recently discovered proteins called IFN-λ that bind to a novel type III receptor.4Ank N. West H. Paludan S.R. IFN-λ: novel antiviral cytokines.J Interferon Cytokine Res. 2006; 26: 373-379Google Scholar Consensus IFN-α (IFN alfacon-1) is a synthetic type I IFN, whose sequence is derived from the consensus sequences of various IFN-α subtypes.5Blatt L.M. Davis J.M. Klein S.B. Taylor M.W. The biologic activity and molecular characterization of a novel synthetic interferon-α species, consensus interferon.J Interferon Cytokine Res. 1996; 16: 489-499Google Scholar It has been shown to be more potent than naturally occurring type I IFNs in cell culture models and more effective in clinical trials for the treatment of chronic hepatitis C.6Barbaro G. Barbarini G. Consensus interferon for chronic hepatitis C patients with genotype 1 who failed to respond to, or relapsed after, interferon α-2b and ribavirin in combination: an Italian pilot study.Eur J Gastroenterol Hepatol. 2002; 14: 477-483Google Scholar, 7Melian E.B. Plosker G.L. Interferon alfacon-1: a review of its pharmacology and therapeutic efficacy in the treatment of chronic hepatitis C.Drugs. 2001; 61: 1661-1691Google Scholar IFN induces an antiviral state in cells by activating the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway.8Platanias L.C. Mechanisms of type-I- and type-II-interferon-mediated signalling.Nat Rev Immunol. 2005; 5: 375-386Google Scholar Binding of IFN to its receptor activates constitutively associated JAK proteins, which leads to the docking of STAT molecules to the receptor and subsequent STAT phosphorylation. The activated STATs dissociate from the receptor chain and form dimers that translocate to the nucleus to modulate gene transcriptional activity. For the type I IFNs, the interferon-stimulated gene factor 3 (ISGF3) complex, consisting of a STAT1 and STAT2 heterodimer and interferon regulatory factor 9 (IRF9), binds to the interferon-stimulated response element (ISRE). For the type II IFN, IFN-γ, STAT1 homodimers bind directly to the γ-activated site element. Both types of IFNs induce the expression of a large number of ISGs with substantial overlap and set up an antiviral, antiproliferative, and immunoregulatory state in the host cells. IFN-induced antiviral activities have been extensively studied in the HCV replicon system. Both IFN-α and IFN-γ have been shown to inhibit HCV replication,9Frese M. Schwarzle V. Barth K. Krieger N. Lohmann V. Mihm S. Haller O. Bartenschlager R. Interferon-γ inhibits replication of subgenomic and genomic hepatitis C virus RNAs.Hepatology. 2002; 35: 694-703Google Scholar, 10Guo J.T. Bichko V.V. Seeger C. Effect of α interferon on the hepatitis C virus replicon.J Virol. 2001; 75: 8516-8523Google Scholar and type I/II IFN combinations resulted in a synergistic antiviral effect.11Larkin J. Jin L. Farmen M. Venable D. Huang Y. Tan S.L. Glass J.I. Synergistic antiviral activity of human interferon combinations in the hepatitis C virus replicon system.J Interferon Cytokine Res. 2003; 23: 247-257Google Scholar, 12Tan H. Derrick J. Hong J. Sanda C. Grosse W.M. Edenberg H.J. Taylor M. Seiwert S. Blatt L.M. Global transcriptional profiling demonstrates the combination of type I and type II interferon enhances antiviral and immune responses at clinically relevant doses.J Interferon Cytokine Res. 2005; 25: 632-649Google Scholar However, standard combination therapy with Peg-IFN-α and ribavirin achieves sustained viral clearance in only approximately half of treated patients, and IFN-γ as a single agent appears to be ineffective in small clinical trials.13Soza A. Heller T. Ghany M. Lutchman G. Jake Liang T. Germain J. Hsu H.H. Park Y. Hoofnagle J.H. Pilot study of interferon γ for chronic hepatitis C.J Hepatol. 2005; 43: 67-71Google Scholar HCV appears to have developed strategies to interfere with the IFN effector pathways, leading to nonresponse in many HCV-infected patients. The mechanisms by which HCV interferes with IFN signaling and attenuates its antiviral efficacy have not been fully elucidated. Various hypotheses have been proposed.14Gale Jr, M. Foy E.M. Evasion of intracellular host defence by hepatitis C virus.Nature. 2005; 436: 939-945Google Scholar Among them, 2 negative regulators, suppressor of cytokine signaling (SOCS) 3 and protein inhibitor of activated STAT (PIAS), have recently been reported to be induced by HCV proteins leading to inhibition of the JAK-STAT signaling. SOCS3 can be induced by the HCV core protein and suppress JAK-STAT signaling to block the IFN-induced formation of ISGF3 in cell culture.15Bode J.G. Ludwig S. Ehrhardt C. Albrecht U. Erhardt A. Schaper F. Heinrich P.C. Haussinger D. IFN-α antagonistic activity of HCV core protein involves induction of suppressor of cytokine signaling-3.FASEB J. 2003; 17: 488-490Crossref Google Scholar HCV protein expression in liver cells is associated with activation of PIAS and inhibition of STAT function, possibly augmented by induction of protein phosphatase 2A (PP2A) expression.16Duong F.H. Filipowicz M. Tripodi M. La Monica N. Heim M.H. Hepatitis C virus inhibits interferon signaling through up-regulation of protein phosphatase 2A.Gastroenterology. 2004; 126: 263-277Abstract Full Text Full Text PDF Scopus (196) Google Scholar Despite significant advances in understanding of the actions of IFN in the cell culture system, little is known about the inhibition of IFN signaling by HCV and nonresponse to IFN therapy in vivo. The chimpanzee is the only animal model susceptible to HCV infection. In the present study, we conducted a comprehensive assessment of the antiviral effects of both type I and type II IFNs (IFN-α, -γ, and consensus IFN) in chimpanzees and demonstrated a potential mechanism of IFN resistance. To confirm the clinical relevance in human HCV infection, we also evaluated patient samples before and during Peg-IFN therapy. Six chimpanzees, X0176, X0284, X0101, X0233, X0142, and X0234, were maintained at the Southwest Foundation for Biomedical Research, an Association for Assessment and Accreditation of Laboratory Animal and Care-accredited facility, and the study protocol was approved by the Institutional Animal Care and Use Committee at the Foundation and by the Interagency Animal Model Committee at the National Institutes of Health (NIH). Two chimpanzees, X0142 and X0234, had persistent infection with HCV genotype 1b derived from a homogenous source as described previously,17Thomson M. Nascimbeni M. Gonzales S. Murthy K.K. Rehermann B. Liang T.J. Emergence of a distinct pattern of viral mutations in chimpanzees infected with a homogeneous inoculum of hepatitis C virus.Gastroenterology. 2001; 121: 1226-1233Google Scholar whereas others were naive animals. Whole blood samples from 2 chimpanzees, X6394 and X6475 (infected with genotype 1a viruses),18Major M.E. Dahari H. Mihalik K. Puig M. Rice C.M. Neumann A.U. Feinstone S.M. Hepatitis C virus kinetics and host responses associated with disease and outcome of infection in chimpanzees.Hepatology. 2004; 39: 1709-1720Google Scholar, 19Logvinoff C. Major M.E. Oldach D. Heyward S. Talal A. Balfe P. Feinstone S.M. Alter H. Rice C.M. McKeating J.A. Neutralizing antibody response during acute and chronic hepatitis C virus infection.Proc Natl Acad Sci U S A. 2004; 101: 10149-10154Google Scholar were provided by Dr. Stephen Feinstone at Center for Biologics Evaluation and Research (CBER), the Food and Drug Administration (FDA). Human IFN-α2a used for the in vitro study was purchased from Fitzgerald Industries International, Inc. (Concord, MA), and the product used for in vivo study was purchased from Roche (Nutley, NJ). IFN-γ1b and consensus IFN (Infergen, IFN alfacon-1) were provided by InterMune, Inc. (Brisbane, CA). Patient samples were derived from 2 sources. Treated patients were recruited from the Liver Diseases Unit at the University of North Carolina (UNC). Patients were given an initial dose of 180 μg Peg-IFN-α-2a and underwent liver biopsy 24 hours later. Control samples came from liver biopsy specimens obtained from patients at the Clinical Center of the NIH prior to undergoing antiviral therapy with Peg-IFN and ribavirin. Control patients were selected to match treated patients in terms of age, gender, ethnicity, initial viral load, and degree of histologic disease. Patients signed informed consent, and the protocol was approved by the Institutional Review Board of UNC and the NIH. All patients had genotype 1 HCV infection. Biopsy samples were snap frozen in liquid nitrogen and stored at −80°C. Details of the human study will be published elsewhere.20Feld J.J. Nanda S. Susan P. Schweigler L. Theodore D. Dougherty K. Zacks S. Shrestha R. Liang T.J. Fried M.W. Hepatic gene expression profiles during treatment with peginterferon and ribavirin: identifying important molecular pathways for treatment response.Hepatology. 2006; 44: 315AGoogle Scholar Human liver biopsy tissue was handled similarly to chimpanzee liver tissue as described below. Both treated and control patients subsequently underwent a full course of standard antiviral therapy consisting of Peg-IFN-α-2a 180 μg and weight-based ribavirin (1000 mg daily ≤75 kg and 1200 mg >75 kg for 48 weeks). Patients achieving ≥2-log copies/mL decrease in HCV RNA by 4 weeks of therapy were deemed rapid responders (RR), and those with lesser decreases in viral load were designated slow responders (SR). Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood samples of chimpanzees and healthy human donors (informed consents were obtained) and stimulated with 3 different doses of human IFN (hIFN)-α, -γ, and consensus IFN in RPMI medium with 10% fetal bovine serum (FBS) (Cellgro, Herndon, VA). After incubation for 6 or 24 hours, cells were harvested and subjected to RNA isolation. Chimpanzees used for in vivo study were treated sequentially with 10 million IU IFN-α, 400 μg IFN-γ, and 30 μg consensus IFN subcutaneously. Briefly, 4 animals (X0101, X0233, X0142, and X0234) were divided into 2 groups, each comprising 1 naive and 1 infected chimpanzee. The experiment was separated into 3 phases. For the first phase, group 1 chimpanzees (X0101 and X0142) were administered with IFN-α and group 2 (X0233 and X0234) with IFN-γ. Blood samples (40 mL each) were collected at 9 time points (pretreatment, 8 hours, 24 hours, 48 hours, 72 hours, 7 days, 14 days, 21 days, and 28 days posttreatment), and liver biopsy samples were collected at 5 time points (pretreatment, 8 hours, 24 hours, 48 hours, and 72 hours posttreatment). All animals were rested for 6 weeks to avoid any residual drug effect. Next, group 1 was treated with IFN-γ and group 2 with IFN-α for the second phase of the study. Similar sample-collecting procedures were performed during treatment up to 4 weeks. The animals were rested for 6 weeks and started on the third and final phase of the study, in which consensus IFN was given to both groups. Because of a nonspecific infection during the rest period after the phase II study, one of the HCV-infected chimpanzees, X0142, was dropped from the phase III study. Sera were isolated from serial blood samples of the HCV-infected chimpanzees. HCV RNA was quantified by using the COBAS AMPLICOR HCV MONITOR TEST, v2.0 (Roche Diagnostics, Branchburg, NJ), which has a detection limit of 600 IU/mL (1620 copies/mL). Alanine transaminase (ALT) values were measured by the Laboratory Medicine Department of the Southwest Foundation for Biomedical Research. Total RNA was prepared from the PBMCs and liver biopsy tissues with RNeasy Mini Kit according to manufacturer's instructions (Qiagen, Valencia, CA). Complementary DNA (cDNA) was synthesized from total RNA with First-strand cDNA Synthesis System (Marligen Biosciences, Ijamsville, MD). TaqMan real-time PCR analysis was used to quantify the mRNA expression levels of genes of interest. The primers and probes used were Gene Expression Assays (Applied Biosystems, Foster City, CA). Each reaction was performed in duplicate, and all samples were standardized using the internal control glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Reactions were set up using 12.5 μL TaqMan universal PCR master mix, cDNA template, and 1.25 μL primers and probe mix in a final volume of 25 μL. Reactions were performed on an iCycler iQ Multicolor Real-Time Detection System (Bio-Rad, Hercules, CA) with the following reaction conditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 20 seconds, 60°C for 1 minute, and additional incubation at 68°C for 10 minutes. Liver biopsy tissues were lysed using mammalian tissue lysis/extraction reagent (CelLytic MT; Sigma Chemical Co., St. Louis, MO) containing a Protease Inhibitor Cocktail Tablet (Roche, Indianapolis, IN) and a Protein Phosphatase Inhibitor Set (Upstate Biotechnology, Lake Placid, NY). The collection of supernatant lysates, determination of protein concentration, and Western blot analysis have been described previously.21Huang Y. Chen X.C. Konduri M. Fomina N. Lu J. Jin L. Kolykhalov A. Tan S.L. Mechanistic link between the anti-HCV effect of interferon gamma and control of viral replication by a Ras-MAPK signaling cascade.Hepatology. 2006; 43: 81-90Google Scholar Antibodies to STAT1, SOCS1, SOCS3, Src homology region 2-domain phosphatase (SHP)1, and β-actin were from Abcam Inc. (Cambridge, MA). Antibodies to STAT2, STAT3, phosphorylated STAT1 (Y701), and protein phosphatase 2A C subunit (PP2Ac) were from Upstate Biotechnology. Anti-IRF9 antibody was from Santa Cruz Biotechnology (Santa Cruz, CA), and anti-IFNAR chain 2 antibody was from Fitzgerald Industries International Inc. (Concord, MA). The band intensities in the images were quantified by a public analysis program, ImageJ, offered by the NIH (http://rsb.info.nih.gov/ij/). Serum cytokines (eotaxin, GM-CSF, IFN-γ, interleukin (IL)-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, interferon-γ-inducible protein (IP) 10, membrane cofactor protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, tumor necrosis factor (TNF)-α, and RANTES) were measured simultaneously using Beadlyte Human 22-Plex Cytokine Detection System (Upstate Biotechnology) according to the manufacturer's instructions. Briefly, both standard and chimpanzee samples were diluted with human serum diluent, and 50 μL was loaded onto the 96-well filtration plate. Twenty-five-microliter beads coated with target capture antibodies against cytokines were added to each well and incubated overnight at 4°C with low-speed shaking at 300 rpm in the dark. On the next day, after washing, the plate was supplied with 25 μL premixed biotin conjugate antibodies and incubated for 2 hours at room temperature with shaking at 300 rpm. Finally, streptavidin-phycoerythrin was added, and the results were read with Bioplex Luminex System (Bio-Rad Laboratories Inc., Hercules, CA). The data were analyzed using Bio-Plex Manager software v.4.0 with Five-Parameter Logistics curve fitting. Before performing the in vivo chimpanzee study, we first determined the ability of chimpanzees to respond to hIFN. PBMCs from 2 naive chimpanzees (X0284 and X0176) and 2 healthy human donors were stimulated with 3 different doses of IFN-α, IFN-γ, and consensus IFN. A panel of previously reported IFN-α or -γ specific ISGs were selected for analysis: myxovirus resistance 1 (MX1), 2,5-oligoadenylate synthetase (2,5-OAS), IFN-induced protein with tetratricopeptide repeats 1 (IFIT1), and IFN-induced protein 15 (IFI15) were used as markers of IFN-α-induced genes, and the IP10, IRF1, and large multifunctional protease (LMP) 2 and LMP7 were selected as IFN-γ-induced genes.22Der S.D. Zhou A. Williams B.R. Silverman R.H. Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays.Proc Natl Acad Sci U S A. 1998; 95: 15623-15628Google Scholar All ISGs were induced at 6 hours after IFN treatment (data not shown) and reached a peak at 24 hours (Figure 1). The overall gene expression patterns of the ISGs were comparable between human and chimpanzee PBMCs. Consensus IFN was more potent and induced a broader range of ISGs than either IFN-α or -γ. All 3 IFNs induced higher levels of ISG expression in humans than in chimpanzees (2–5 times higher), suggesting that chimpanzees do not respond as well as humans to hIFN in vitro. Based on these data, 10 million IU IFN-α, 400 μg IFN-γ, and 30 μg consensus IFNs were used for in vivo study in chimpanzees. These doses are 3–4 times higher than the standard doses for the treatment of human subjects (3 million IU IFN-α, 100 μg IFN-γ, and 9 μg consensus IFN) to compensate for the lower efficacy of hIFNs in chimpanzees. Four chimpanzees (2 naive: X0101 and X0233; 2 infected: X0142 and X0234) were divided into 2 groups in the in vivo study. Each group consisted of 1 naive and 1 infected animal. The experiment was divided into 3 phases: treatment with IFN-α (or IFN-γ), treatment with IFN-γ (or IFN-α), and treatment with consensus IFN, respectively. With this design, each animal received all 3 forms of IFN treatment, which allows for internal control and biologic duplication of the experiment. Consensus IFN was given last because consensus IFN is more potent and induces a broader range of ISGs than either IFN-α or -γ. Similar to the results from the in vitro study, IFN-α and consensus IFN treatment led to the induction of the IFN-α-specific ISGs (MX1, OAS1, IFIT1, and IFI15) in both PBMCs and liver tissues of the chimpanzees in vivo. Likewise, the IFN-γ-specific ISGs (IP10, IRF1, LMP2, and LMP7) were induced by IFN-γ and consensus IFN in vivo. Figure 2 shows the fold inductions of only 4 selected ISGs (MX1, IFI15, IP10, and IRF1), but the other 4 (IFIT1, OAS1, LMP2, and LMP7) behaved similarly. Although PBMCs from both naive and HCV-infected chimpanzees responded to all 3 forms of IFN, reaching a peak at 8 hours posttreatment and returning to basal levels within 48 hours, the levels of ISG induction in PBMCs from HCV-infected chimpanzees were much lower (∼4 times) than those in the naive animals (Figure 2A). All 3 forms of IFN induced stronger ISG responses in the liver than in PBMCs of naive chimpanzees (Figure 2B). However, in the HCV-infected animals, little or no hepatic ISG induction was observed. Although hepatic ISG induction was severely blunted in the infected animals, the basal level of ISG expression in the liver were higher than those in naive animals (Figure 2C), suggesting that HCV infection resulted in endogenous IFN production. The higher basal hepatic ISG expression did not account for the failure to respond to exogenous IFN because the absolute levels reached after treatment were still much lower in infected than naive chimpanzees (Figure 2D). These data indicate a deficiency of response, particularly in the liver, to hIFNs in HCV-infected chimpanzees. HCV RNA and ALT levels were analyzed from serial serum samples of the 2 chronically HCV-infected chimpanzees (X0142 and X0234) pre- and post-IFN administration. As shown in Figure 3, although chimpanzee X0234 was initially inoculated with the week 2 serum from X0142 (ie, the same viral strain),17Thomson M. Nascimbeni M. Gonzales S. Murthy K.K. Rehermann B. Liang T.J. Emergence of a distinct pattern of viral mutations in chimpanzees infected with a homogeneous inoculum of hepatitis C virus.Gastroenterology. 2001; 121: 1226-1233Google Scholar the pattern of infection that ensued differed somewhat between the 2 chimpanzees. The viral load was much higher in X0142 and fluctuated in a range from 104 to 106 copies/mL as compared with a relatively stable viremia (approximately 3 × 104 copies/mL) seen in X0234. Notably, neither IFN-α nor IFN-γ injection resulted in a significant decrease in viral load in either chimpanzee, consistent with the absence of hepatic ISG induction in both animals. However, chimpanzee X0234 did have a transient decrease in viremia in response to IFNs with a reproducible decrease in viral titer of 0.5–1 log copies/mL (IFN-γ < IFN-α < conIFN) at 8–24 hours posttreatment. Interestingly, although the hepatic IFN response in this chimpanzee was blunted compared with naive animals, there was low-level induction of hepatic ISGs in response to IFNs (eg, MX1 and IFI15 induced by IFN-α; IP10 and IRF1 induced by IFN-γ, Figure 2B and 2D). In contrast, chimpanzee X0142 had no hepatic ISG induction after treatment and actually had a transient increase in HCV viremia within 24–48 hours posttreatment, which appeared to coincide with a transient ALT increase in this chimpanzee (data not shown). This effect could be explained by an IFN-induced hepatotoxicity with release of HCV RNA from the injured hepatocytes resulting in a transient rise in viremia. Because of the apparently different ISG induction in PBMCs between the naive and infected chimpanzees in vivo, we sought to determine whether such a difference persists ex vivo, free of any potential biologic interactions with IFN that may be operational in vivo. Thus, we evaluated the actions of IFNs on PBMCs isolated from naive and infected chimpanzees in vitro. Eight chimpanzees, 4 naive and 4 HCV infected, were studied. The 4 naive animals included the 2 used in the preliminary study to test the response of chimpanzees to hIFNs (X0176 and X0284) and 2 others used in the in vivo study above (X0101 and X0233). The 4 infected animals were the 2 used for the study above (X0142 and X0234) and 2 others infected with a genotype 1a strain H77 (X6394 and X6475).18Major M.E. Dahari H. Mihalik K. Puig M. Rice C.M. Neumann A.U. Feinstone S.M. Hepatitis C virus kinetics and host responses associated with disease and outcome of infection in chimpanzees.Hepatology. 2004; 39: 1709-1720Google Scholar, 19Logvinoff C. Major M.E. Oldach D. Heyward S. Talal A. Balfe P. Feinstone S.M. Alter H. Rice C.M. McKeating J.A. Neutralizing antibody response during acute and chronic hepatitis C virus infection.Proc Natl Acad Sci U S A. 2004; 101: 10149-10154Google ScholarFigure 4 summarizes the fold inductions of representative ISGs in PBMCs from all 8 chimpanzees at 24 hours post-IFN stimulation. The overall ISG inductions of the HCV-infected animals were comparable with those of the naive animals with the exception of IP10, whose induction by IFN-γ was significantly lower in the infected chimpanzees. However, the difference in IP10 induction of PBMCs by IFN-γ between naive and infected chimpanzees ex vivo was much less than that in vivo (2- to 3-fold vs 4- to 6-fold, respectively). These data suggest that the apparent difference in IFN response occurred mostly in vivo, and, once the PBMCs were removed from the in vivo milieu of the infected animals, they responded to IFN similarly to those from the naive animals. The lack of hepatic ISG induction after IFN treatment in HCV-infected chimpanzees suggests that the IFN signal transduction pathways are inhibited in the liver. To determine which step of IFN signaling was inhibited, various components involved in the JAK-STAT pathway were evaluated using Western blot analysis from liver biopsy samples of pre- and 8 hours post-IFN-α treatment. The 8-hour time point was chosen to coincide with the peak hepatic ISG expression (Figure 2B). As shown in Figure 5A for the naive chimpanzees, a slight increase of phosphorylated STAT1 was detected with X0101, whereas not with X0233. In these chimpanzees, the JAK-STAT pathway was likely activated by exogenous IFN but quickly returned to baseline by 8 hours. This time course is consistent with the rapid onset and transient nature of JAK-STAT activation by IFN observed in vitro.23Radaeva S. Jaruga B. Hong F. Kim W.H. Fan S. Cai H. Strom S. Liu Y. El-Assal O. Gao B. Interferon-α activates multiple STAT signals and down-regulates c-Met in primary human hepatocytes.Gastroenterology. 2002; 122: 1020-1034Abstract Full Text Full Text PDF Scopus (123) Google Scholar In contrast, all components of the JAK-STAT pathway were activated even prior to IFN treatment in the infected chimpanzees. In addition to a markedly elevated level of phosphorylated STAT1, there was a general increase seen in the total levels of STAT1, STAT2, STAT3, and IRF9 proteins (Figure 5A). This effect was more dramatic in chimpanzee X0142. After IFN treatment, rather than increasing, the phosphorylated STAT1 level actually declined in X0142, which might be due to the activation of negative regulator(s) of IFN signaling. This observation is in keeping with the lack of ISG induction in the liver of these infected animals. At least 3 different classes of negative regulators are known to contribute to the inhibition of IFN signaling24Yasukawa H. Sasaki A. Yoshimura A. Negative regulation of cytokine signaling pathways.Annu
Referência(s)