Intrahepatic Interferon Research Articles

Immunomodulation of CXCL10 Secretion by Hepatitis C Virus: Could CXCL10 Be a Prognostic Marker of Chronic Hepatitis C?

Chemokine (C-X-C motif) ligand (CXCL)10 and other CXCR3 chemokines are involved in the pathogenesis of acute and “chronic hepatitis C virus (HCV) infection” (CHC). Here, we review the scientific literature about HCV and CXCL10. The combination of circulating CXCL10 and single nucleotide polymorphisms (SNPs) in IL-28B can identify patients with acute HCV infection most likely to undergo spontaneous HCV clearance and those in need of early antiviral therapy. In CHC, the HCV and intrahepatic interferon- (IFN-) γ drive a raised CXCL10 expression by sinusoidal endothelium and hepatocytes, thereby inducing the recruitment of CXCR3-expressing T cells into the liver; thus, CXCL10 plays an important role in the development of necroinflammation and fibrosis. Increased CXCL10 was significantly associated with the presence of active vasculitis in HCV-associated cryoglobulinemia, or with autoimmune thyroiditis in CHC. Pretreatment CXCL10 levels are predictive of early virological response and sustained virological response (SVR) to IFN-α and ribavirin and may be useful in the evaluation of candidates for therapy. The occurrence of SNPs adjacent to IL-28B (rs12979860, rs12980275, and rs8099917), and CXCL10 below 150 pg/mL, independently predicted the first phase viral decline and rapid virological response, which in turn independently predicted SVR. Directly acting antiviral agents-mediated clearance of HCV is associated with the loss of intrahepatic immune activation by IFN-α, associated by decreased levels of CXCL10. In conclusion, CXCL10 is an important marker of HCV clearance and successful therapy in CHC patients. Whether CXCL10 is a novel therapeutic target in CHC will be evaluated.

Anti-HBV response to toll-like receptor 7 agonist GS-9620 is associated with intrahepatic aggregates of T cells and B cells

GS-9620, an oral agonist of toll-like receptor 7, is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the chimpanzee and woodchuck models of CHB. Herein, we investigated the immunomodulatory mechanisms underlying these antiviral effects. Archived liver biopsies and paired peripheral blood mononuclear cell samples from a previous chimpanzee study were analyzed by RNA sequencing, quantitative reverse transcription PCR, immunohistochemistry (IHC) and in situ hybridization (ISH). GS-9620 treatment of CHB chimpanzees induced an intrahepatic transcriptional profile significantly enriched with genes associated with hepatitis B virus (HBV) clearance in acutely infected chimpanzees. Type I and II interferon, CD8+ T cell and B cell transcriptional signatures were associated with treatment response, together with evidence of hepatocyte death and liver regeneration. IHC and ISH confirmed an increase in intrahepatic CD8+ T cell and B cell numbers during treatment, and revealed that GS-9620 transiently induced aggregates predominantly comprised of CD8+ T cells and B cells in portal regions. There were no follicular dendritic cells or IgG-positive cells in these lymphoid aggregates and very few CD11b+ myeloid cells. There was no change in intrahepatic natural killer cell number during GS-9620 treatment. The antiviral response to GS-9620 treatment in CHB chimpanzees was associated with an intrahepatic interferon response and formation of lymphoid aggregates in the liver. Our data indicate these intrahepatic structures are not fully differentiated follicles containing germinal center reactions. However, the temporal correlation between development of these T and B cell aggregates and the antiviral response to treatment suggests they play a role in promoting an effective immune response against HBV. New therapies to treat chronic hepatitis B (CHB) are urgently needed. In this study we performed a retrospective analysis of liver and blood samples from a chimpanzee model of CHB to help understand how GS-9620, a drug in clinical trials, suppressed hepatitis B virus (HBV). We found that the antiviral response to GS-9620 was associated with accumulation of immune cells in the liver that can either kill cells infected with HBV or can produce antibodies that may prevent HBV from infecting new liver cells. These findings have important implications for how GS-9620 may be used in patients and may also help guide the development of new therapies to treat chronic HBV infection.

Interferon-alpha treatment rapidly clears Hepatitis E virus infection in humanized mice

Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immunological determinants of viral persistence remain largely unexplored. We studied the antiviral potency of pegylated interferon-α (pegIFNα) against HEV infections in humanized mice and modelled intrahepatic interferon stimulated gene (ISG) responses. Human gene expression levels in humanized mouse livers were analyzed by qPCR and Nanostring. Human CXCL10 was measured in mouse serum. HEV genotype 3 (gt3) infections were cleared from liver and feces within 8 pegIFNα doses in all mice and relapsed after a single pegIFNα injection in only half of treated animals. Rapid viral clearance by pegIFNα was confirmed in HEV gt1, but not in Hepatitis B Virus infected animals. No ISG induction was observed in untreated HEV gt3 and gt1 infected humanized livers compared to control chimeric mice, irrespective of the human hepatocyte donor, viral isolate or HEV infection duration. Human specific ISG transcript levels in mouse liver increased significantly after pegIFNα treatment and induced high circulating human CXCL10 in mouse serum. In conclusion, HEV gt1 and gt3 infections do not elicit innate intrahepatic immune responses and remain highly sensitive to pegIFNα in immunocompromised humanized mice.

Sequential control of hepatitis B virus in a mouse model of acute, self-resolving hepatitis B

The determinants of an immune response to the human hepatitis B virus (HBV) are poorly understood. As studies in man and chimpanzees are limited, we aimed at developing a model of self-limiting hepatitis B in mice that helps to dissect the control of HBV by humoral and cellular immune responses. Adenoviral vectors containing 1.3-fold HBV genomes allowed an efficient and reproducible transfer of HBV genomes into mouse livers and initiated HBV replication in mice. HBV transcripts were detected in mouse livers for more than 3 months. HBsAg and HBeAg peaked around day 6 and slowly declined thereafter. A two-phase mild to moderate liver inflammation with elevated serum alanine transaminase activities was observed around day 7 and around day 70 when the vast majority of HBV-specific T cells were detected in the liver. HBV was initially controlled when specific and nonspecific T cells infiltrated the liver and intrahepatic interferon γ levels peaked around day 7, but replicated again from day 10 to day 24 and persisted at low levels thereafter despite the presence of HBV-specific T cells. Finally, HBV replication was terminated after a sufficient B-cell response had been mounted- indicated by anti-HBs seroconversion around day 35. HBV-specific T cells infiltrated the liver a second time around day 70 postinfection. This demonstrates that the established mouse model allows studying the onset and termination of HBV infection and will help to dissect the determinants of HBV control and clearance by the immune response.

Bortezomib Inhibits Hepatitis B Virus Replication in Transgenic Mice

Pharmacological modulation of cellular proteins as a means to block virus replication has been proposed as an alternative antiviral strategy that may be less susceptible than others to the development of viral drug resistance. Recent evidence indicates that the ubiquitin-proteasome pathway interacts with different aspects of the hepatitis B virus (HBV) life cycle in cell culture models of virus replication. We therefore examined the effect of proteasome inhibition on HBV replication in vivo using HBV transgenic mice. The proteasome inhibitor bortezomib (Velcade) inhibits proteasome activity in vivo and is used therapeutically for the clinical treatment of multiple myeloma. We found that a single intravenous dose of 1 mg of bortezomib/kg of body weight reduced virus replication for as long as 6 days. The inhibition of HBV by bortezomib was dose dependent and occurred at a step in replication subsequent to viral RNA and protein expression. The reduction in HBV replication did not result from nonspecific hepatocellular toxicity and was not mediated indirectly through the induction of an intrahepatic interferon response. Thus, pharmacological manipulation of the ubiquitin-proteasome pathway may represent an alternative therapeutic approach for the treatment of chronic HBV infection.

Antifibrotic Effects of CXCL9 and Its Receptor CXCR3 in Livers of Mice and Humans

Fibrosis is the hallmark of chronic liver diseases, yet many aspects of its mechanism remain to be defined. Chemokines are ubiquitous chemotactic molecules that mediate many acute and chronic inflammatory conditions, and CXC chemokine genes colocalize with a locus previously shown to include fibrogenic genes. We investigated the roles of the chemokine CXCL9 and its receptor CXCR3 in liver fibrosis. The effects of CXCL variants on fibrogenesis were analyzed using samples from patients with hepatitis C virus infection and by induction of fibrosis in CXCR3(-/-) and wild-type mice. In mice, intrahepatic immune cell subsets were investigated and interferon gamma messenger RNA levels were measured at baseline and after injury. Human serum CXCL9 levels were measured and correlated with CXCL9 variant and fibrosis severity. The effects of stimulation with CXCL9 were investigated on human hepatic stellate cells (LX-2). Specific CXCL9 variants were associated with liver fibrosis in mice and humans; CXCL9 serum concentrations correlated with genotypes and levels of fibrosis in patients. In contrast to other chemokines, CXCL9 exerted antifibrotic effects in vitro, suppressing collagen production in LX-2 cells. CXCR3(-/-) mice had increased liver fibrosis; progression was associated with decreased numbers of intrahepatic interferon gamma-positive T cells and reduced interferon gamma messenger RNA, indicating that CXCL9-CXCR3 regulates Th1-associated immune pathways. This is the first description of a chemokine-based antifibrotic pathway in the liver; antifibrotic therapies might be developed to modulate CXC chemokine levels.

1,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP)-Formulated, Immune-Stimulatory Vascular Endothelial Growth Factor A Small Interfering RNA (siRNA) Increases Antitumoral Efficacy in Murine Orthotopic Hepatocellular Carcinoma with Liver Fibrosis

Most experimental therapy studies are performed in mice that bear subcutaneous or orthotopic hepatoma but are otherwise healthy and nonfibrotic. The majority of hepatocellular carcinoma (HCC), however, develops in patients suffering from preexisting liver fibrosis. We investigated the efficacy of a standard experimental therapeutic approach to interrupt the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) cascade via VEGF-A silencing, with or without 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP; cationic lipid) formulation, in HCC mice with preexisting liver fibrosis. The data show that intraperitoneal treatment with naked VEGF-A small interfering RNA (siRNA; 200 microg/kg) was inefficient to treat HCC implanted into fibrotic livers. VEGF-A siRNA containing an immunostimulatory motif in combination with DOTAP formulation significantly reduced hepatic VEGF-A expression and additionally activated the innate and adapted immune system as shown by an increased intrahepatic interferon type 1 response (68-fold increased beta-interferon expression). DOTAP-formulated VEGF-A siRNA markedly improved VEGF-A siRNA uptake and enhanced the antitumor response. This study shows for the first time the therapeutic feasibility of using synergistic effects (gene silencing and activation of the immune system) united in one siRNA sequence to reduce HCC growth and metastasis in mice with preexisting liver fibrosis. We expect that these results will help to direct and improve future experimental gene-silencing approaches and establish more efficient antitumoral therapies against HCC.

Morphine Inhibits Intrahepatic Interferon‐α Expression and Enhances Complete Hepatitis C Virus Replication

Heroin addicts are a high-risk group for hepatitis C virus (HCV) infection and the development of chronic HCV disease. We thus examined whether morphine, the active metabolite of heroin, has the ability to inhibit intrahepatic interferon (IFN)- alpha expression, facilitating HCV replication in human hepatocytes. Morphine inhibited intrahepatic IFN- alpha expression, which was associated with an increase in HCV replication in hepatocytes. Moreover, morphine compromised the anti-HCV effect of recombinant IFN- alpha . Investigation of the mechanism responsible for the morphine action revealed that morphine inhibited expression of IFN regulatory factor 5 in the hepatocytes. In addition, morphine suppressed the expression of p38, an important signal-transducing molecule involved in IFN- alpha -mediated anti-HCV activity. These findings indicate that morphine plays a cofactor role in facilitating HCV persistence in human hepatocytes.

Hepatic Gene Expression Discriminates Responders and Nonresponders in Treatment of Chronic Hepatitis C Viral Infection

Pegylated interferon (IFN)-alpha plus ribavirin is the most effective treatment of chronic hepatitis C but has unpleasant side effects and high costs. A large proportion of patients do not respond to therapy for reasons that are unclear. We used gene expression profiling to investigate the molecular basis for treatment failure. Expression profiling was performed on percutaneous needle liver biopsy specimens taken before therapy. Gene expression levels were compared among 15 nonresponder, 16 responder, and 20 normal liver biopsy specimens. Differential gene expression was confirmed using real-time polymerase chain reaction. We identified 18 genes whose expression differed significantly between all responders and all nonresponders (P < .005). Many of these 18 genes are IFN sensitive and 2 (ISG15/USP18) are linked in a novel IFN-regulatory pathway, suggesting a possible rationale for treatment resistance. Using a number of independent classifier analyses, an 8-gene subset accurately predicted treatment response for 30 of 31 patients. The classifier analyses were applicable to patients with genotype 1 infection and were not correlated with viral load, disease activity, or fibrosis. Hepatic gene expression profiling identified consistent differences in patients who subsequently fail treatment with pegylated IFN-alpha plus ribavirin: up-regulation of a specific set of IFN-responsive genes predicts nonresponse to exogenous therapy. These data may be of use in predicting clinical responses to treatment.

The immunologic basis for hepatitis C infection

The host immune system is arguably involved in nearly every step of hepatitis C virus (HCV) infection. In patients, the outcome, whether it is a natural infection or results from an interferon-alpha-based treatment, is determined by a series of complex host-virus interactions. In this review, we focus on the state of research addressing the immune mechanisms critical for viral clearance and disease resolution. Additional discussion is devoted to the evasion and blockade tactics of HCV as well as to current efforts aimed at disrupting the replication cycle of this well-evolved virus. Current theories of immune-mediated injury of hepatocytes are also discussed. Strong and persistent CD8 and CD4 T-cell responses are critical in HCV clearance. Although each may play a unique role in the process, the intrahepatic interferon (IFN)-gamma produced by these cells is central to their antiviral action. IFN-alpha/beta alone, without triggering subsequent HCV-specific T-cell responses, may not lead to a sustained viral response in vivo. Synergism among several immune cells, including T, NK, and NKT cells is important for disease resolution. Additional data raise the possibility that viral clearance and liver injury are mediated through different effector mechanisms of T cells. HCV employs evasion and sabotage tactics to escape from the host's immune system. HCV NS3/4A serine protease can block viral activation of a key transcription factor in initiating cellular IFN response. A newly identified NS3 protease inhibitor can result in a reduction of viremia, illustrating the potential of the viral-enzyme-targeted drug in patients. Current data provide a rationale to further explore immune augmentation as a therapeutic intervention in HCV infection.

Hepatitis C virus kinetics and host responses associated with disease and outcome of infection in chimpanzees.

To study determinants of clinical outcome following HCV infection, viral kinetics, immune events, and intrahepatic cytokine markers were compared in 10 naive chimpanzees. Four of the animals cleared HCV; 6 developed persistent infections. All animals developed similar acute infections with increasing viremia from 1 to 2 weeks, followed by alanine aminotransferase (ALT) elevations and seroconversion. This viremia pattern consisted of a biphasic increase, a rapid slope (mean doubling time [t(2)] = 0.5 days) followed by a slower slope after the second week (t(2) = 7.5 days). This slowing of virus replication correlated in all animals with increased intrahepatic 2'5' oligoadenylate synthetase 1 (2OAS-1) messenger RNA (mRNA) levels and was independent of disease outcome. An effective control of virus replication was observed following increases in intrahepatic interferon gamma (IFN-gamma) mRNA and ALT levels. Although this control was associated in all animals with a 2-log decrease in virus titer, the timing occurred approximately 2 weeks later in the chronic group (P <.05). Additionally, while cleared infections were characterized by a continual decrease in virus titer, the titers in the persistent infections reached a steady state level of 10(4) to 10(5) RNA copies/mL. This inability of the immune response to sustain viral clearance in the persistent infections was associated with a reduced intrahepatic CD3e and monocyte-induced protein 1alpha (MIP-1alpha) mRNA induction. In conclusion, these data indicate that, regardless of outcome, chimpanzees generate responses that control HCV replication during the early and late acute phase. However, the pathogenesis of HCV may be determined by a more rapid onset of the induced response and the cell population that migrates to the liver.

Upregulation of endogenous intrahepatic interferon stimulated genes during chronic hepatitis C virus infection.

The success of interferon-alpha and ribavirin combination therapy for the treatment of chronic hepatitis C viral infection differs between patients. In an attempt to identify predictors of host response to therapy, the levels of mRNA for interferon (IFN) stimulated genes: MxA, PKR, 2'5' OAS, ISG15, and interleukin 8 (IL-8), were examined in liver by real-time RT-PCR prior to commencement of therapy. The levels of intrahepatic classical IFN stimulated genes, but not IL-8, in chronic HCV disease (n = 44) were found to be significantly upregulated (P < 0.001) compared to the control cohort (n = 12). The genotype of the infecting HCV strain did not influence IFN stimulated gene expression. These results suggest that the endogenous type 1 IFN antiviral effector pathway is broadly activated during chronic HCV disease, although the levels of mRNA for any of the IFN-stimulated genes tested did not predict the outcome of combination therapy.

Intrahepatic MxA expression is correlated with interferon-alpha expression in chronic and fulminant hepatitis.

Interferon-alpha (IFN-alpha) has potent pro-inflammatory and anti-viral functions. It exerts its effects by inducing intracellular proteins such as MxA. To analyse the role of intrahepatic interferon activation, IFN-alpha and MxA expression was studied by immunohistochemistry in explant livers of 20 patients with fulminant hepatic failure (FHF), 41 patients with chronic liver disease (CLD), and ten normal controls (NCs). In NCs only small numbers of Kupffer cells, but no hepatocytes, showed IFN-alpha and MxA expression. In contrast, significantly enhanced numbers of IFN-alpha- and MxA-positive Kupffer cells, along with small numbers of MxA-positive and larger numbers of IFN-alpha-positive lymphocytes, were found in CLD and in FHF. MxA protein was also expressed on hepatocytes and bile ducts in the vicinity of IFN-alpha-positive inflammatory infiltrates (hepatocytes: NCs: 0%, CLD: 8%, FHF: 68%; bile ducts: NCs: 19%, CLD: 46%, FHF: 83%). A significant correlation was found between the numbers of IFN-alpha- and MxA-positive cells (r=0.67, p<0.001). Thus, large amounts of IFN-alpha are released in the livers of patients with FHF, which is likely to contribute to immune-mediated liver cell damage. Intrahepatic MxA expression corresponds to IFN-alpha produced particularly by infiltrating inflammatory cells, rather than by hepatocytes themselves.