Tofacitinib is Effective in Treating Refractory Immune Checkpoint Inhibitor Hepatitis.

Immune checkpoint inhibitors (ICI) have improved metastatic melanoma outcomes; however, toxicities, such as hepatitis, can be dose-limiting or even fatal.1 Systemic glucocorticoids and antimetabolite immunosuppressive medications remain the mainstay of treatment for ICI-hepatitis, but options for patients refractory to these therapies are limited.2 Herein we present 3 cases of glucocorticoid-refractory ICI-hepatitis treated with tofacitinib, an inhibitor of Janus kinase (JAK) 1 and 3. These patients represent consecutive patients referred to the Massachusetts General Hospital Severe Immunotherapy Complications service who were determined by our experts to have treatment failure with systemic glucocorticoid and antimetabolite combination therapy between August 2022 and September 2023.3 These were the only 3 patients managed by the Severe Immunotherapy Complications service who were treated with tofacitinib for ICI-hepatitis during that time.

Serum Fibroblast Growth Factor-21 Discriminates Between Decompensated Alcohol-Associated Cirrhosis and Severe Alcohol-Associated Hepatitis.

INTRODUCTION: We hypothesized that fibroblast growth factor-21 (FGF-21) would be highly expressed in patients with alcohol-associated hepatitis (AH) and could be a novel and biologically relevant predictive biomarker to reliably distinguish severe AH and decompensated alcohol-associated cirrhosis (AC). METHODS: We identified a discovery cohort of 88 subjects with alcohol-associated liver disease (ALD) of varying disease severity from our ALD repository. Our validation cohort consisted of 37 patients with a biopsy-proven diagnosis of AH, AC, or absence of ALD with Model for End-Stage Liver Disease scores ≥10. Serum from both groups during index hospitalization was assayed for FGF-21 by ELISA. We performed receiver operating characteristic analysis and prediction modeling in both cohorts to discriminate between AH and AC in high Model for End-Stage Liver Disease (≥20) patients. RESULTS: In both cohorts, FGF-21 concentrations were highest in subjects with moderate to severe AH compared with those having alcohol use disorder or AC (mean: 2,609 pg/mL, P < 0.0001). The discovery cohort area under the curve of FGF-21 between AH and AC was 0.81 (95% confidence interval: 0.65-0.98, P < 0.01). In the validation cohort, FGF-21 levels were higher in severe AH compared with AC (3,052 vs 1,235 pg/mL, P = 0.03), and the area under the curve was 0.76 (95% confidence interval: 0.56-0.96, P < 0.03). A survival analysis showed that patients with FGF-21 serum levels in the second interquartile had the highest survival compared with all other quartiles. DISCUSSION: FGF-21 performs well as a predictive biomarker to distinguish severe AH from AC and may be helpful in the management and clinical investigation of patients with severe alcohol-associated liver diseases.

Hepatitis B and Hepatitis C Virus Infection Promote Liver Fibrogenesis through a TGF-ß1-Induced OCT4/Nanog Pathway.

Hepatitis B virus (HBV)/hepatitis C virus (HCV) coinfection accelerates liver fibrosis progression compared with HBV or HCV monoinfection. Octamer binding transcription factor 4 (OCT4) and Nanog are direct targets of the profibrogenic TGF-ß1 signaling cascade. We leveraged a coculture model to monitor the effects of HBV and HCV coinfection on fibrogenesis in both sodium taurocholate cotransporting polypeptide-transfected Huh7.5.1 hepatoma cells and LX2 hepatic stellate cells (HSCs). We used CRISPR-Cas9 to knock out OCT4 and Nanog to evaluate their effects on HBV-, HCV-, or TGF-ß1-induced liver fibrogenesis. HBV/HCV coinfection and HBx, HBV preS2, HCV Core, and HCV NS2/3 overexpression increased TGF-ß1 mRNA levels in sodium taurocholate cotransporting polypeptide-Huh7.5.1 cells compared with controls. HBV/HCV coinfection further enhanced profibrogenic gene expression relative to HBV or HCV monoinfection. Coculture of HBV and HCV monoinfected or HBV/HCV coinfected hepatocytes with LX2 cells significantly increased profibrotic gene expression and LX2 cell invasion and migration. OCT4 and Nanog guide RNA independently suppressed HBV-, HCV-, HBV/HCV-, and TGF-ß1-induced α-SMA, TIMP-1, and Col1A1 expression and reduced Huh7.5.1, LX2, primary hepatocyte, and primary human HSC migratory capacity. OCT4/Nanog protein expression also correlated positively with fibrosis stage in liver biopsies from patients with chronic HBV or HCV infection. In conclusion, HBV and HCV independently and cooperatively promote liver fibrogenesis through a TGF-ß1-induced OCT4/Nanog-dependent pathway.

Fatty Acids Activate the Transcriptional Coactivator YAP1 to Promote Liver Fibrosis via p38 Mitogen-Activated Protein Kinase.

BACKGROUND & AIMS: Patients with simple steatosis (SS) and nonalcoholic steatohepatitis can develop progressive liver fibrosis, which is associated with liver-related mortality. The mechanisms contributing to liver fibrosis development in SS, however, are poorly understood. SS is characterized by hepatocellular free fatty acid (FFA) accumulation without lobular inflammation seen in nonalcoholic steatohepatitis. Because the Hippo signaling transcriptional coactivator YAP1 (YAP) has previously been linked with nonalcoholic fatty liver disease (NAFLD)-related fibrosis, we sought to explore how hepatocyte FFAs activate a YAP-mediated profibrogenic program. METHODS: We analyzed RNA sequencing data from a GEO DataSet (accession: GSE162694) consisting of 143 patients with NAFLD. We also performed immunohistochemical, immunofluorescence, immunoblot, and quantitative reverse-transcription polymerase chain reaction analyses (qRT-PCR) in liver specimens from NAFLD subjects, from a murine dietary NAFLD model, and in FFA-treated hepatic spheroids and hepatocytes. RESULTS: YAP-target gene expression correlated with increasing fibrosis stage in NAFLD patients and was associated with fibrosis in mice fed a NAFLD-inducing diet. Hepatocyte-specific YAP deletion in the murine NAFLD model attenuated diet-induced fibrosis, suggesting a causative role of YAP in NAFLD-related fibrosis. Likewise, in hepatic spheroids composed of Huh7 hepatoma cells and primary human hepatic stellate cells, Huh7 YAP silencing reduced FFA-induced fibrogenic gene expression. Notably, inhibition of p38 mitogen-activated protein kinase could block YAP activation in FFA-treated Huh7 cells. CONCLUSIONS: These studies provide further evidence for the pathological role of YAP in NAFLD-associated fibrosis and that YAP activation in NAFLD may be driven by FFA-induced p38 MAPK activation.

A Deep Ravine Rather Than a Shallow Gap: Many More Bridges Needed to Improve Care of Chronic Hepatitis B in the United States.

ABSTRACT: In the United States, improved screening of those who are at highest risk of chronic hepatitis B (CHB) has been a major focus of public health efforts, as has facilitating access to care for those with chronic infection. Despite this, data suggest that less than half of those at risk are tested, and another minority of those who harbor chronic infection receive longitudinal care for the disease. In this study by Tran et al., the authors find that even among those being treated for CHB, a vast minority receive basic testing and screening for staging and complications of CHB.

Macrophage Activation Marker Soluble CD163 Is a Dynamic Marker of Liver Fibrogenesis in Human Immunodeficiency Virus/Hepatitis C Virus Coinfection.

Background: Coinfection with human immunodeficiency virus (HIV) accelerates hepatitis C virus (HCV)-related liver fibrosis. Macrophages are triggered during both viral infections and are critical in liver inflammation/fibrogenesis. Liver fibrosis strongly associates with serum soluble CD163 (sCD163, a macrophage activation marker); comprehensive evaluation in HIV/HCV coinfection is lacking. Methods: We retrospectively analyzed sCD163 (enzyme-linked immunosorbent assay) and hepatic CD163 (immunofluorescent CD163/CD68 costaining) in patients infected with HIV/HCV, HCV, or HIV, pre- and post-antiviral therapy. Results: sCD163 was significantly higher in HIV/HCV compared to either monoinfection, and decreased following successful antiviral therapy, although did not fully normalize. In HIV/HCV, sCD163 was associated with necroinflammation, Ishak fibrosis scores, and noninvasive fibrosis scores. We observed a novel trend whereby sCD163 levels progressively increase with increasing Ishak fibrosis score, peaking at stage 4, above which levels plateaued. Periportal CD163+ macrophage frequency was also higher with increasing fibrosis score. When stratified by fibrosis stage, sCD163 levels were higher in HIV/HCV than HCV but only in individuals with mild to moderate fibrosis. Conclusions: In HIV/HCV, increasing sCD163 levels accompanied periportal CD163+ macrophage enrichment in mild to moderate fibrosis, but not in established cirrhosis, suggesting that sCD163 is a dynamic biomarker of fibrogenesis rather than accumulated fibrosis. Our findings implicate HIV-related macrophage activation in accelerated fibrosis progression in HIV/HCV coinfection.

MicroRNA 130a Regulates both Hepatitis C Virus and Hepatitis B Virus Replication through a Central Metabolic Pathway.

Hepatitis C virus (HCV) infection has been shown to regulate microRNA 130a (miR-130a) in patient biopsy specimens and in cultured cells. We sought to identify miR-130a target genes and to explore the mechanisms by which miR-130a regulates HCV and hepatitis B virus (HBV) replication. We used bioinformatics software, including miRanda, TargetScan, PITA, and RNAhybrid, to predict potential miR-130a target genes. miR-130a and its target genes were overexpressed or were knocked down by use of small interfering RNA (siRNA) or clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 guide RNA (gRNA). Selected gene mRNAs and their proteins, together with HCV replication in OR6 cells, HCV JFH1-infected Huh7.5.1 cells, and HCV JFH1-infected primary human hepatocytes (PHHs) and HBV replication in HepAD38 cells, HBV-infected NTCP-Huh7.5.1 cells, and HBV-infected PHHs, were measured by quantitative reverse transcription-PCR (qRT-PCR) and Western blotting, respectively. We selected 116 predicted target genes whose expression was related to viral pathogenesis or immunity for qPCR validation. Of these, the gene encoding pyruvate kinase in liver and red blood cell (PKLR) was confirmed to be regulated by miR-130a overexpression. miR-130a overexpression (via a mimic) knocked down PKLR mRNA and protein levels. A miR-130a inhibitor and gRNA increased PKLR expression, HCV replication, and HBV replication, while miR-130a gRNA and PKLR overexpression increased HCV and HBV replication. Supplemental pyruvate increased HCV and HBV replication and rescued the inhibition of HCV and HBV replication by the miR-130a mimic and PKLR knockdown. We concluded that miR-130a regulates HCV and HBV replication through its targeting of PKLR and subsequent pyruvate production. Our data provide novel insights into key metabolic enzymatic pathway steps regulated by miR-130a, including the steps involving PKLR and pyruvate, which are subverted by HCV and HBV replication.IMPORTANCE We identified that miR-130a regulates the target gene PKLR and its subsequent effect on pyruvate production. Pyruvate is a key intermediate in several metabolic pathways, and we identified that pyruvate plays a key role in regulation of HCV and HBV replication. This previously unrecognized, miRNA-regulated antiviral mechanism has implications for the development of host-directed strategies to interrupt the viral life cycle and prevent establishment of persistent infection for HCV, HBV, and potentially other viral infections.

Vascular Injury Characterizes Doxycycline-induced Upper Gastrointestinal Tract Mucosal Injury.

Doxycycline is an oral tetracycline antibiotic that has been associated with upper gastrointestinal (GI) mucosal injury. Recently, characteristic vascular degeneration has been reported in the stomach and duodenum in patients with doxycycline-induced injury. Fourteen patients who underwent upper GI endoscopy for nonspecific symptoms and were found to have doxycycline-induced gastric and esophageal injury are described. Most patients showed characteristic vascular injury. A control group of gastric erosions and esophageal ulcers showed no cases with the characteristic vascular changes. Clinical, endoscopic, and pathologic features of doxycycline-induced upper GI tract injury are reviewed, with an emphasis on vascular injury.

Apolipoprotein B100 is required for hepatitis C infectivity and Mipomersen inhibits hepatitis C.

AIM: To characterize the role of apolipoprotein B100 (apoB100) in hepatitis C viral (HCV) infection. METHODS: In this study, we utilize a gene editing tool, transcription activator-like effector nucleases (TALENs), to generate human hepatoma cells with a stable genetic deletion of APOB to assess of apoB in HCV. Using infectious cell culture-competent HCV, viral pseudoparticles, replicon models, and lipidomic analysis we determined the contribution of apoB to each step of the viral lifecycle. We further studied the effect of mipomersen, an FDA-approved antisense inhibitor of apoB100, on HCV using in vitro cell-culture competent HCV and determined its impact on viral infectivity with the TCID50 method. RESULTS: We found that apoB100 is indispensable for HCV infection. Using the JFH-1 fully infectious cell-culture competent virus in Huh 7 hepatoma cells with TALEN-mediated gene deletion of apoB (APOB KO), we found a significant reduction in HCV RNA and protein levels following infection. Pseudoparticle and replicon models demonstrated that apoB did not play a role in HCV entry or replication. However, the virus produced by APOB KO cells had significantly diminished infectivity as measured by the TCID-50 method compared to wild-type virus. Lipidomic analysis demonstrated that these virions have a fundamentally altered lipidome, with complete depletion of cholesterol esters. We further demonstrate that inhibition of apoB using mipomersen, an FDA-approved anti-sense oligonucleotide, results in a potent anti-HCV effect and significantly reduces the infectivity of the virus. CONCLUSION: ApoB is required for the generation of fully infectious HCV virions, and inhibition of apoB with mipomersen blocks HCV. Targeting lipid metabolic pathways to impair viral infectivity represents a novel host targeted strategy to inhibit HCV.

Exposure to human immunodeficiency virus/hepatitis C virus in hepatic and stellate cell lines reveals cooperative profibrotic transcriptional activation between viruses and cell types.

Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) coinfection accelerates progressive liver fibrosis; however, the mechanisms remain poorly understood. HCV and HIV independently induce profibrogenic markers transforming growth factor beta-1 (TGFß1) (mediated by reactive oxygen species [ROS]) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) in hepatocytes and hepatic stellate cells in monoculture; however, they do not account for cellular crosstalk that naturally occurs. We created an in vitro coculture model and investigated the contributions of HIV and HCV to hepatic fibrogenesis. Green fluorescent protein reporter cell lines driven by functional ROS (antioxidant response elements), NFκB, and mothers against decapentaplegic homolog 3 (SMAD3) promoters were created in Huh7.5.1 and LX2 cells, using a transwell to generate cocultures. Reporter cell lines were exposed to HIV, HCV, or HIV/HCV. Activation of the 3 pathways was measured and compared according to infection status. Extracellular matrix products (collagen type 1 alpha 1 (CoL1A1) and tissue inhibitor of metalloproteinase 1 (TIMP1)) were also measured. Both HCV and HIV independently activated TGFß1 signaling through ROS (antioxidant response elements), NFκB, and SMAD3 in both cell lines in coculture. Activation of these profibrotic pathways was additive following HIV/HCV coexposure. This was confirmed when examining CoL1A1 and TIMP1, where messenger RNA and protein levels were significantly higher in LX2 cells in coculture following HIV/HCV coexposure compared with either virus alone. In addition, expression of these profibrotic genes was significantly higher in the coculture model compared to either cell type in monoculture, suggesting an interaction and feedback mechanism between Huh7.5.1 and LX2 cells. CONCLUSION: HIV accentuates an HCV-driven profibrogenic program in hepatocyte and hepatic stellate cell lines through ROS, NFκB, and TGFß1 up-regulation; coculture of hepatocyte and hepatic stellate cell lines significantly increased expression of CoL1A1 and TIMP1; and our novel coculture reporter cell model represents an efficient and more authentic system for studying transcriptional fibrosis responses and may provide important insights into hepatic fibrosis. (Hepatology 2016;64:1951-1968).

IQGAP2 is a novel interferon-alpha antiviral effector gene acting non-conventionally through the NF-κB pathway.

BACKGROUND & AIMS: Type I interferons (IFN) provide the first line of defense against invading pathogens but its mechanism of action is still not well understood. Using unbiased genome-wide siRNA screens, we recently identified IQ-motif containing GTPase activating protein 2 (IQGAP2), a tumor suppressor predominantly expressed in the liver, as a novel gene putatively required for IFN antiviral response against hepatitis C virus (HCV) infection. Here we sought to characterize IQGAP2 role in IFN response. METHODS: We used transient small interfering RNA knockdown strategy in hepatic cell lines highly permissive to JFH1 strain of HCV infection. RESULTS: We found that IQGAP2 acts downstream of IFN binding to its receptor, and independently of the JAK-STAT pathway, by physically interacting with RelA (also known as p65), a subunit of the NF-κB transcription factor. Interestingly, our data reveal a mechanism distinct from the well-characterized role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in IFN production. Indeed, IFN alone was sufficient to stimulate NF-κB-dependent transcription in the absence of viral infection. Finally, both IQGAP2 and RelA were required for the induction by IFN of a subset of IFN-stimulated genes (ISG) with known antiviral properties. CONCLUSIONS: Our data identify a novel function for IQGAP2 in IFN antiviral response in hepatoma cells. We demonstrate the involvement of IQGAP2 in regulating ISG induction by IFN in an NF-κB-dependent manner. The IQGAP2 pathway may provide new targets for antiviral strategies in the liver, and may have a wider therapeutic implication in other disease pathogeneses driven by NF-κB activation. LAY SUMMARY: In this study, we identify a novel mechanism of action of interferon involving the IQGAP2 protein and the NF-κB pathway that is ultimately protective against hepatitis C virus infection. This newly identified pathway functions independently of the well-known STAT pathway and may therefore provide new targets for antiviral strategies in the liver.

HCV induces transforming growth factor ß1 through activation of endoplasmic reticulum stress and the unfolded protein response.

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1, and PERK. HCV increases TGF-ß1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-ß1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2, and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-ß1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-ß1 expression. ROS, ER stress, NFκB, and TGF-ß1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-ß1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-ß1 activation. We conclude that HCV activates NFκB and TGF-ß1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-ß1.

EFTUD2 Is a Novel Innate Immune Regulator Restricting Hepatitis C Virus Infection through the RIG-I/MDA5 Pathway.

UNLABELLED: The elongation factor Tu GTP binding domain-containing protein 2 (EFTUD2) was identified as an anti-hepatitis C virus (HCV) host factor in our recent genome-wide small interfering RNA (siRNA) screen. In this study, we sought to further determine EFTUD2's role in HCV infection and investigate the interaction between EFTUD2 and other regulators involved in HCV innate immune (RIG-I, MDA5, TBK1, and IRF3) and JAK-STAT1 pathways. We found that HCV infection decreased the expression of EFTUD2 and the viral RNA sensors RIG-I and MDA5 in HCV-infected Huh7 and Huh7.5.1 cells and in liver tissue from in HCV-infected patients, suggesting that HCV infection downregulated EFTUD2 expression to circumvent the innate immune response. EFTUD2 inhibited HCV infection by inducing expression of the interferon (IFN)-stimulated genes (ISGs) in Huh7 cells. However, its impact on HCV infection was absent in both RIG-I knockdown Huh7 cells and RIG-I-defective Huh7.5.1 cells, indicating that the antiviral effect of EFTUD2 is dependent on RIG-I. Furthermore, EFTUD2 upregulated the expression of the RIG-I-like receptors (RLRs) RIG-I and MDA5 to enhance the innate immune response by gene splicing. Functional experiments revealed that EFTUD2-induced expression of ISGs was mediated through interaction of the EFTUD2 downstream regulators RIG-I, MDA5, TBK1, and IRF3. Interestingly, the EFTUD2-induced antiviral effect was independent of the classical IFN-induced JAK-STAT pathway. Our data demonstrate that EFTUD2 restricts HCV infection mainly through an RIG-I/MDA5-mediated, JAK-STAT-independent pathway, thereby revealing the participation of EFTUD2 as a novel innate immune regulator and suggesting a potentially targetable antiviral pathway. IMPORTANCE: Innate immunity is the first line defense against HCV and determines the outcome of HCV infection. Based on a recent high-throughput whole-genome siRNA library screen revealing a network of host factors mediating antiviral effects against HCV, we identified EFTUD2 as a novel innate immune regulator against HCV in the infectious HCV cell culture model and confirmed that its expression in HCV-infected liver tissue is inversely related to HCV infection. Furthermore, we determined that EFTUD2 exerts its antiviral activity mainly through governing its downstream regulators RIG-I and MDA5 by gene splicing to activate IRF3 and induce classical ISG expression independent of the JAT-STAT signaling pathway. This study broadens our understanding of the HCV innate immune response and provides a possible new antiviral strategy targeting this novel regulator of the innate response.

The spliceosome factor SART1 exerts its anti-HCV action through mRNA splicing.

BACKGROUND &/AIMS: The broadly used antiviral cytokine interferon-α (IFNα)'s mechanisms of action against HCV infection are not well understood. We previously identified SART1, a host protein involved in RNA splicing and pre-mRNA processing, as a regulator of IFN's antiviral effects. We hypothesized that SART1 regulates antiviral IFN effector genes (IEGs) through mRNA processing and splicing. METHODS: We performed siRNA knockdown in HuH7.5.1 cells and mRNA-sequencing with or without IFN treatment. Selected gene mRNA variants and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in HCV OR6 replicon cells and the JFH1 HCV infectious model. RESULTS: We identified 419 genes with a greater than 2-fold expression difference between Neg siRNA and SART1 siRNA treated cells in the presence or absence of IFN. Bioinformatic analysis identified at least 10 functional pathways. SART1 knockdown reduced classical IFN stimulating genes (ISG) mRNA transcription including MX1 and OAS3. However, SART1 did not affect JAK-STAT pathway gene mRNA expression and IFN stimulated response element (ISRE) signaling. We identified alternative mRNA splicing events for several genes, including EIF4G3, GORASP2, ZFAND6, and RAB6A that contribute to their antiviral effects. EIF4G3 and GORASP2 were also confirmed to have anti-HCV effect. CONCLUSIONS: The spliceosome factor SART1 is not IFN-inducible but is an IEG. SART1 exerts its anti-HCV action through direct transcriptional regulation for some ISGs and alternative splicing for others, including EIF4G3, GORASP2. SART1 does not have an effect on IFN receptor or canonical signal transduction components. Thus, SART1 regulates ISGs using a novel, non-classical mechanism.

Kinetic differences in the induction of interferon stimulated genes by interferon-α and interleukin 28B are altered by infection with hepatitis C virus.

UNLABELLED: Several genome-wide association studies (GWAS) have identified a genetic polymorphism associated with the gene locus for interleukin 28B (IL28B), a type III interferon (IFN), as a major predictor of clinical outcome in hepatitis C. Antiviral effects of the type III IFN family have previously been shown against several viruses, including hepatitis C virus (HCV), and resemble the function of type I IFN including utilization of the intracellular Janus kinase signal transducer and activator of transcription (JAK-STAT) pathway. Effects unique to IL28B that would distinguish it from IFN-α are not well defined. By analyzing the transcriptomes of primary human hepatocytes (PHH) treated with IFN-α or IL28B, we sought to identify functional differences between IFN-α and IL28B to better understand the roles of these cytokines in the innate immune response. Although our data did not reveal distinct gene signatures, we detected striking kinetic differences between IFN-α and IL28B stimulation for interferon stimulated genes (ISGs). While gene induction was rapid and peaked at 8 hours of stimulation with IFN-α in PHH, IL28B produced a slower, but more sustained increase in gene expression. We confirmed these findings in the human hepatoma cell line Huh7.5.1. Interestingly, in HCV-infected cells the rapid response after stimulation with IFN-α was blunted, and the induction pattern resembled that caused by IL28B. CONCLUSION: The kinetics of gene induction are fundamentally different for stimulations with either IFN-α or IL28B in hepatocytes, suggesting distinct roles of these cytokines within the immune response. Furthermore, the observed differences are substantially altered by infection with HCV.

The AMPK-related kinase SNARK regulates hepatitis C virus replication and pathogenesis through enhancement of TGF-ß signaling.

BACKGROUND & AIMS: Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. The biological and therapeutic importance of host cellular cofactors for viral replication has been recently appreciated. Here we examined the roles of SNF1/AMP kinase-related kinase (SNARK) in HCV replication and pathogenesis. METHODS: The JFH1 infection system and the full-length HCV replicon OR6 cell line were used. Gene expression was knocked down by siRNAs. SNARK mutants were created by site-directed mutagenesis. Intracellular mRNA levels were measured by qRT-PCR. Endogenous and overexpressed proteins were detected by Western blot analysis and immunofluorescence. Transforming growth factor (TGF)-ß signaling was monitored by a luciferase reporter construct. Liver biopsy samples from HCV-infected patients were analyzed for SNARK expression. RESULTS: Knockdown of SNARK impaired viral replication, which was rescued by wild type SNARK but not by unphosphorylated or kinase-deficient mutants. Knockdown and overexpression studies demonstrated that SNARK promoted TGF-ß signaling in a manner dependent on both its phosphorylation and kinase activity. In turn, chronic HCV replication upregulated the expression of SNARK in patients. Further, the SNARK kinase inhibitor metformin suppressed both HCV replication and SNARK-mediated enhancement of TGF-ß signaling. CONCLUSIONS: Thus reciprocal regulation between HCV and SNARK promotes TGF-ß signaling, a major driver of hepatic fibrogenesis. These findings suggest that SNARK will be an attractive target for the design of novel host-directed antiviral and antifibrotic drugs.

Ceestatin, a novel small molecule inhibitor of hepatitis C virus replication, inhibits 3-hydroxy-3-methylglutaryl-coenzyme A synthase.

BACKGROUND: Hepatitis C virus (HCV) chronically infects >170 million persons worldwide and is a leading cause of cirrhosis and hepatocellular carcinoma. The identification of more effective and better-tolerated agents for treating HCV is a high priority. We have reported elsewhere the discovery of the anti-HCV compound ceestatin using a high-throughput screen of a small molecule library. METHODS: To identify host or viral protein targets in an unbiased fashion, we performed affinity chromatography, using tandem liquid chromatography/mass spectrometry to identify specific potential targets. RESULTS. Ceestatin binds to 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase and irreversibly inhibits HMG-CoA synthase in a dose-dependent manner. Ceestatin's anti-HCV effects are reversed by addition of HMG-CoA, mevalonic acid, or geranylgeraniol. Treatment with small interfering RNA against HMG-CoA synthase led to a substantial reduction in HCV replication, further validating HMG-CoA synthase as an enzyme essential for HCV replication. CONCLUSIONS: Ceestatin therefore exerts its anti-HCV effects through inhibition of HMG-CoA synthase. It may prove useful as an antiviral agent, as a probe to study HCV replication, and as a cholesterol-lowering agent. The logical stepwise process employed to discover the mechanism of action of ceestatin can serve as a general experimental strategy to uncover the targets on which novel uncharacterized anti-HCV compounds act.