Markus H. Heim

Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study.

BACKGROUND & AIMS Hepatitis C virus (HCV) induces chronic infection in 50% to 80% of infected persons; approximately 50% of these do not respond to therapy. We performed a genome-wide association study to screen for host genetic determinants of HCV persistence and response to therapy. METHODS The analysis included 1362 individuals: 1015 with chronic hepatitis C and 347 who spontaneously cleared the virus (448 were coinfected with human immunodeficiency virus [HIV]). Responses to pegylated interferon alfa and ribavirin were assessed in 465 individuals. Associations between more than 500,000 single nucleotide polymorphisms (SNPs) and outcomes were assessed by multivariate logistic regression. RESULTS Chronic hepatitis C was associated with SNPs in the IL28B locus, which encodes the antiviral cytokine interferon lambda. The rs8099917 minor allele was associated with progression to chronic HCV infection (odds ratio [OR], 2.31; 95% confidence interval [CI], 1.74-3.06; P = 6.07 x 10(-9)). The association was observed in HCV mono-infected (OR, 2.49; 95% CI, 1.64-3.79; P = 1.96 x 10(-5)) and HCV/HIV coinfected individuals (OR, 2.16; 95% CI, 1.47-3.18; P = 8.24 x 10(-5)). rs8099917 was also associated with failure to respond to therapy (OR, 5.19; 95% CI, 2.90-9.30; P = 3.11 x 10(-8)), with the strongest effects in patients with HCV genotype 1 or 4. This risk allele was identified in 24% of individuals with spontaneous HCV clearance, 32% of chronically infected patients who responded to therapy, and 58% who did not respond (P = 3.2 x 10(-10)). Resequencing of IL28B identified distinct haplotypes that were associated with the clinical phenotype. CONCLUSIONS The association of the IL28B locus with natural and treatment-associated control of HCV indicates the importance of innate immunity and interferon lambda in the pathogenesis of HCV infection.

Interferon signaling and treatment outcome in chronic hepatitis C.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. The current standard therapy for chronic hepatitis C (CHC) consists of a combination of pegylated IFN alpha (pegIFNα) and ribavirin. It achieves a sustained viral clearance in only 50–60% of patients. To learn more about molecular mechanisms underlying treatment failure, we investigated IFN-induced signaling in paired liver biopsies collected from CHC patients before and after administration of pegIFNα. In patients with a rapid virological response to treatment, pegIFNα induced a strong up-regulation of IFN-stimulated genes (ISGs). As shown previously, nonresponders had high expression levels of ISGs before therapy. Analysis of posttreatment biopsies of these patients revealed that pegIFNα did not induce expression of ISGs above the pretreatment levels. In accordance with ISG expression data, phosphorylation, DNA binding, and nuclear localization of STAT1 indicated that the IFN signaling pathway in nonresponsive patients is preactivated and refractory to further stimulation. Some features characteristic of nonresponders were more accentuated in patients infected with HCV genotypes 1 and 4 compared with genotypes 2 and 3, providing a possible explanation for the poor response of the former group to therapy. Taken together with previous findings, our data support the concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection but also may impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway.

Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification

A method for genotyping poor metabolisers of debrisoquine is based on specific polymerase chain reaction (PCR) amplification of parts of mutant genes for hepatic cytochrome P450IID6. Analysis by restriction fragment length polymorphism allowed identification of only 25% of poor metabolisers, but when it was combined with allele-specific PCR over 95% of poor metabolisers could be identified. The PCR method also allowed the identification of heterozygous carriers of mutant alleles.

Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA.

Clearance of Chronic Virus The family of mRNA-editing enzymes, APOBEC, restricts hepatitis B virus (HBV) replication. Lucifora et al. (p. 1221, published online 20 February; see the Perspective by Shlomai and Rice) provide evidence that specific APOBECs mediate the anti-HBV effects of host cytokines, which in turn apparently induce nuclear deaminase activity without damaging host cells. Thus, there may be potential in these findings for developing a therapeutic route to curing chronic HBV infection. Cytokine induction renders viral DNA vulnerable and eliminates infection. Current antiviral agents can control but not eliminate hepatitis B virus (HBV), because HBV establishes a stable nuclear covalently closed circular DNA (cccDNA). Interferon-α treatment can clear HBV but is limited by systemic side effects. We describe how interferon-α can induce specific degradation of the nuclear viral DNA without hepatotoxicity and propose lymphotoxin-β receptor activation as a therapeutic alternative. Interferon-α and lymphotoxin-β receptor activation up-regulated APOBEC3A and APOBEC3B cytidine deaminases, respectively, in HBV-infected cells, primary hepatocytes, and human liver needle biopsies. HBV core protein mediated the interaction with nuclear cccDNA, resulting in cytidine deamination, apurinic/apyrimidinic site formation, and finally cccDNA degradation that prevented HBV reactivation. Genomic DNA was not affected. Thus, inducing nuclear deaminases—for example, by lymphotoxin-β receptor activation—allows the development of new therapeutics that, in combination with existing antivirals, may cure hepatitis B.

Hepatic mTORC2 Activates Glycolysis and Lipogenesis through Akt, Glucokinase, and SREBP1c

Mammalian target of rapamycin complex 2 (mTORC2) phosphorylates and activates AGC kinase family members, including Akt, SGK1, and PKC, in response to insulin/IGF1. The liver is a key organ in insulin-mediated regulation of metabolism. To assess the role of hepatic mTORC2, we generated liver-specific rictor knockout (LiRiKO) mice. Fed LiRiKO mice displayed loss of Akt Ser473 phosphorylation and reduced glucokinase and SREBP1c activity in the liver, leading to constitutive gluconeogenesis, and impaired glycolysis and lipogenesis, suggesting that the mTORC2-deficient liver is unable to sense satiety. These liver-specific defects resulted in systemic hyperglycemia, hyperinsulinemia, and hypolipidemia. Expression of constitutively active Akt2 in mTORC2-deficient hepatocytes restored both glucose flux and lipogenesis, whereas glucokinase overexpression rescued glucose flux but not lipogenesis. Thus, mTORC2 regulates hepatic glucose and lipid metabolism via insulin-induced Akt signaling to control whole-body metabolic homeostasis. These findings have implications for emerging drug therapies that target mTORC2.

Interferon-induced gene expression is a stronger predictor of treatment response than IL28B genotype in patients with hepatitis C.

BACKGROUND & AIMS The host immune response during the chronic phase of hepatitis C virus infection varies among individuals; some patients have a no interferon (IFN) response in the liver, whereas others have full activation of IFN-stimulated genes (ISGs). Preactivation of this endogenous IFN system is associated with nonresponse to pegylated IFN-α (pegIFN-α) and ribavirin. Genome-wide association studies have associated allelic variants near the IL28B (IFNλ3) gene with treatment response. We investigated whether IL28B genotype determines the constitutive expression of ISGs in the liver and compared the abilities of ISG levels and IL28B genotype to predict treatment outcome. METHODS We genotyped 109 patients with chronic hepatitis C for IL28B allelic variants and quantified the hepatic expression of ISGs and of IL28B. Decision tree ensembles, in the form of a random forest classifier, were used to calculate the relative predictive power of these different variables in a multivariate analysis. RESULTS The minor IL28B allele was significantly associated with increased expression of ISG. However, stratification of the patients according to treatment response revealed increased ISG expression in nonresponders, irrespective of IL28B genotype. Multivariate analysis of ISG expression, IL28B genotype, and several other factors associated with response to therapy identified ISG expression as the best predictor of treatment response. CONCLUSIONS IL28B genotype and hepatic expression of ISGs are independent predictors of response to treatment with pegIFN-α and ribavirin in patients with chronic hepatitis C. The most accurate prediction of response was obtained with a 4-gene classifier comprising IFI27, ISG15, RSAD2, and HTATIP2.

Genotype 3 is associated with accelerated fibrosis progression in chronic hepatitis C

BACKGROUND/AIMS While several risk factors for the histological progression of chronic hepatitis C have been identified, the contribution of HCV genotypes to liver fibrosis evolution remains controversial. The aim of this study was to assess independent predictors for fibrosis progression. METHODS We identified 1189 patients from the Swiss Hepatitis C Cohort database with at least one biopsy prior to antiviral treatment and assessable date of infection. Stage-constant fibrosis progression rate was assessed using the ratio of fibrosis Metavir score to duration of infection. Stage-specific fibrosis progression rates were obtained using a Markov model. Risk factors were assessed by univariate and multivariate regression models. RESULTS Independent risk factors for accelerated stage-constant fibrosis progression (>0.083 fibrosis units/year) included male sex (OR=1.60, [95% CI 1.21-2.12], P<0.001), age at infection (OR=1.08, [1.06-1.09], P<0.001), histological activity (OR=2.03, [1.54-2.68], P<0.001) and genotype 3 (OR=1.89, [1.37-2.61], P<0.001). Slower progression rates were observed in patients infected by blood transfusion (P=0.02) and invasive procedures or needle stick (P=0.03), compared to those infected by intravenous drug use. Maximum likelihood estimates (95% CI) of stage-specific progression rates (fibrosis units/year) for genotype 3 versus the other genotypes were: F0-->F1: 0.126 (0.106-0.145) versus 0.091 (0.083-0.100), F1-->F2: 0.099 (0.080-0.117) versus 0.065 (0.058-0.073), F2-->F3: 0.077 (0.058-0.096) versus 0.068 (0.057-0.080) and F3-->F4: 0.171 (0.106-0.236) versus 0.112 (0.083-0.142, overall P<0.001). CONCLUSIONS This study shows a significant association of genotype 3 with accelerated fibrosis using both stage-constant and stage-specific estimates of fibrosis progression rates. This observation may have important consequences for the management of patients infected with this genotype.

Expression of hepatitis c virus proteins inhibits interferon α signaling in the liver of transgenic mice

UNLABELLED BACKGROUND & AIMS Hepatitis C virus (HCV) is a major cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma worldwide. The majority of patients treated with interferon alpha do not have a sustained response with clearance of the virus. The molecular mechanisms underlying interferon resistance are poorly understood. Interferon-induced activation of the Jak-STAT (signal transducer and activator of transcription) signal transduction pathway is essential for the induction of an antiviral state. Interference of viral proteins with the Jak-STAT pathway could be responsible for interferon resistance in patients with chronic HCV. METHODS We have analyzed interferon-induced signal transduction through the Jak-STAT pathway in transgenic mice that express HCV proteins in their liver cells. STAT activation was investigated with Western blots, immunofluorescence, and electrophoretic mobility shift assays. Virus challenge experiments with lymphocytic choriomeningitis virus were used to demonstrate the functional importance of Jak-STAT inhibition. RESULTS STAT signaling was found to be strongly inhibited in liver cells of HCV transgenic mice. The inhibition occurred in the nucleus and blocked binding of STAT transcription factors to the promoters of interferon-stimulated genes. Tyrosine phosphorylation of STAT proteins by Janus kinases at the interferon receptor was not inhibited. This lack in interferon response resulted in an enhanced susceptibility of the transgenic mice to infection with a hepatotropic strain of lymphocytic choriomeningitis virus. CONCLUSIONS Interferon-induced intracellular signaling is impaired in HCV transgenic mice. Interference of HCV proteins with interferon-induced intracellular signaling could be an important mechanism of viral persistence and treatment resistance.

The Liver May Act as a Firewall Mediating Mutualism Between the Host and Its Gut Commensal Microbiota

The liver forms a firewall that protects against vascular-borne gut microbes and is commonly impaired in liver disease. Breaching Barriers Premature death from chronic liver disease is a rising global trend. Opportunistic bacterial infections caused by beneficial microbes that have breached the gut and its immune barrier often lead to death in liver cirrhosis patients. Balmer et al. now show that the liver forms a second vascular barrier for eliminating commensal bacteria that have escaped from the gut. In animal models of liver disease and gut dysfunction and in patients with nonalcoholic steatohepatitis, the liver is unable to capture escaped gut commensal bacteria, which then leak into the systemic circulation, resulting in a robust host nonmucosal immune response and the breakdown of mutualism between the host and its gut microbiota. Mutualism breakdown is an important complication of liver disease. A prerequisite for establishment of mutualism between the host and the microbial community that inhabits the large intestine is the stringent mucosal compartmentalization of microorganisms. Microbe-loaded dendritic cells trafficking through lymphatics are arrested at the mesenteric lymph nodes, which constitute the firewall of the intestinal lymphatic circulation. We show in different mouse models that the liver, which receives the intestinal venous blood circulation, forms a vascular firewall that captures gut commensal bacteria entering the bloodstream during intestinal pathology. Phagocytic Kupffer cells in the liver of mice clear commensals from the systemic vasculature independently of the spleen through the liver’s own arterial supply. Damage to the liver firewall in mice impairs functional clearance of commensals from blood, despite heightened innate immunity, resulting in spontaneous priming of nonmucosal immune responses through increased systemic exposure to gut commensals. Systemic immune responses consistent with increased extraintestinal commensal exposure were found in humans with liver disease (nonalcoholic steatohepatitis). The liver may act as a functional vascular firewall that clears commensals that have penetrated either intestinal or systemic vascular circuits.

Efficacy of the Protease Inhibitor BI 201335, Polymerase Inhibitor BI 207127, and Ribavirin in Patients With Chronic HCV Infection

BACKGROUND & AIMS Therapeutic regimens are being developed for patients with hepatitis C virus (HCV) infection that do not include the combination of peginterferon alfa and ribavirin. We investigated the antiviral effect and safety of BI 201335 (an inhibitor of the NS3/4A protease) and BI 207127 (an inhibitor of the NS5B non-nucleoside polymerase) with ribavirin. METHODS Thirty-two treatment-naïve patients with chronic HCV genotype 1 infection were randomly assigned to groups that were given 400 mg or 600 mg BI 207127 3 times daily plus 120 mg BI 201335 once daily and 1000 to 1200 mg/day ribavirin for 4 weeks. The primary efficacy end point was virologic response (HCV RNA level <25 IU/mL at week 4). Thirty-two patients received treatment; 31 completed all 4 weeks of assigned combination therapy. RESULTS In the group given BI 207127 400 mg 3 times daily, the rates of virologic response were 47%, 67%, and 73% at days 15, 22, and 29; a higher rate of response was observed in patients with genotype-1b compared with genotype-1a infections. In the group given BI 207127 600 mg 3 times daily, the rates of virologic response were 82%, 100%, and 100%, respectively, and did not differ among genotypes. One patient in the group given 400 mg 3 times daily had virologic breakthrough (≥1 log(10) rebound in HCV RNA) at day 22. The most frequent adverse events were mild gastrointestinal disorders, rash, and photosensitivity. There were no severe or serious adverse events; no patients discontinued therapy prematurely. CONCLUSIONS The combination of the protease inhibitor BI 201335, the polymerase inhibitor BI 207127, and ribavirin has rapid and strong activity against HCV genotype-1 and did not cause serious or severe adverse events.