Intracellular accumulation of IFN-λ4 contributes to protection from liver cirrhosis by inducing ER stress and enhancing IRF1 signaling
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ABSTRACT: Chronic hepatitis C virus (HCV) infection and cirrhosis are major risk factors for developing hepatocellular carcinoma (HCC). Genetic polymorphisms in the IFNL3/IFNL4 locus have been associated both with poor clearance of HCV and protection from liver fibrosis, an early stage of cirrhosis. Here, we aimed to address the genetic and functional relationships between IFNL3/IFNL4 polymorphisms, cirrhosis, and HCC risk. We evaluated associations between IFNL4 genotype (presence of rs368234815-dG or rs12979860-T alleles) with cirrhosis and HCC risk in patients with chronic HCV - 2,931 from Taiwan and 3,566 from Japan, and with gene expression and somatic mutations in 370 HCC tumors in The Cancer Genome Atlas (TCGA). Functional analyses were performed in primary human hepatocytes and hepatic stellate cells and a panel of hepatoma HepG2 cell lines with gene-edited IFNLR1 and inducible expression of IFN-λ3 or IFN-λ4. We detected associations between IFNL4 genotype and decreased risk of cirrhosis (OR=0.66, 95%CI=0.46-0.93, P=0.018, in Taiwan), but increased risk of HCC in patients without HCV clearance (OR=1.28, 95%CI=1.07-1.52, P=0.0058, in Japan). IFNL4 genotype was also associated with reduced cell proliferation and enrichment of CTNNB1 mutations in HCC tumors in TCGA. Reduced proliferation in hepatic cells was contributed by intracellular accumulation of IFN-λ4 leading to induction of ER stress and enhanced IRF1 signaling. The strong anti-proliferative effects of IFN-λ4 in hepatic cells could explain the association of IFNL3/IFNL4 polymorphisms with decreased cirrhosis. However, by sustaining persistent HCV infection, IFN-λ4 may contribute to the development of CTNNB1 mutations and increased risk of HCC in patients without viral clearance.
Project description:Hepatitis C virus (HCV) is the most common chronic blood-borne infection in the United States with the majority of patients becoming chronically infected and a subset (20%) progressing to cirrhosis and hepatocellular carcinoma. Individual variations in immune responses may help define successful resistance to infection with HCV. We have examined the immune response in primary macrophages from patients who have spontaneously cleared HCV (viral load negative, VL-, n = 37) compared to HCV genotype 1 chronically infected (VL+) subjects (n=32) and found that macrophages from VL- subjects have an elevated baseline expression of Toll-like receptor 3 (TLR3). Macrophages from HCV patients were stimulated ex vivo through the TLR3 pathway and assessed using gene expression arrays and pathway analysis. We found elevated TLR3 response genes and pathway activity from VL- subjects. Furthermore, macrophages from VL- subjects showed higher production of IFN-b and related IFN response genes by Q-PCR, and increased phosphorylation of STAT-1 by immunoblot. Analysis of polymorphisms in TLR3 revealed a significant association of intronic TLR3 polymorphism (rs13126816) with the clearance of HCV and the expression of TLR3. Of note, PBMCs from the same donors showed opposite changes in gene expression, suggesting ongoing inflammatory responses in PBMCs from VL+ HCV patients. Our results suggest that an elevated innate immune response enhances HCV clearance mechanisms and may offer a potential therapeutic approach to increase viral clearance. Differential gene expression by primary human macrophages and PBMCs from patients with spontaneous clearance of HCV (VL-) and patients with chronic HCV infection (VL+) were generated by microarray.
Project description:Hepatitis C virus (HCV) is the most common chronic blood-borne infection in the United States with the majority of patients becoming chronically infected and a subset (20%) progressing to cirrhosis and hepatocellular carcinoma. Individual variations in immune responses may help define successful resistance to infection with HCV. We have examined the immune response in primary macrophages from patients who have spontaneously cleared HCV (viral load negative, VL-, n = 37) compared to HCV genotype 1 chronically infected (VL+) subjects (n=32) and found that macrophages from VL- subjects have an elevated baseline expression of Toll-like receptor 3 (TLR3). Macrophages from HCV patients were stimulated ex vivo through the TLR3 pathway and assessed using gene expression arrays and pathway analysis. We found elevated TLR3 response genes and pathway activity from VL- subjects. Furthermore, macrophages from VL- subjects showed higher production of IFN-b and related IFN response genes by Q-PCR, and increased phosphorylation of STAT-1 by immunoblot. Analysis of polymorphisms in TLR3 revealed a significant association of intronic TLR3 polymorphism (rs13126816) with the clearance of HCV and the expression of TLR3. Of note, PBMCs from the same donors showed opposite changes in gene expression, suggesting ongoing inflammatory responses in PBMCs from VL+ HCV patients. Our results suggest that an elevated innate immune response enhances HCV clearance mechanisms and may offer a potential therapeutic approach to increase viral clearance.
Project description:The aim of this study is to compare post-hepatitis C virus (HCV) and post-alcoholism cirrhosis gene expression profiling. By transcriptome analysis with a cDNA array virtually covering every transcript in liver, we compared transcript levels in alcoholic- , HCV-cirrhosis and control liver. A stringent selection identified a list of 70 transcripts which completely separated the 3 groups of patients (7 HCV-cirrhosis, 7 alcoholic cirrhosis and 8 control livers). In contrast, in an hepatocellular carcinoma (HCC) context, comparison of 10 HCV-cirrhosis, 10 alcoholic cirrhosis and the 8 control livers failed to identify such transcripts. We report that dysregulations at the transcriptional level do exist in HCC-free cirrhosis, are transiently observed prior to detectable HCC. Keywords: etiology-dependent and HCC-dependent analysis
Project description:OBJECTIVE: The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. DESIGN: We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1?mg/dL) and platelet count (<100?000/mm3), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10?years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8?years). RESULTS: In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child-Turcotte-Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001). CONCLUSIONS: A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions. 145 liver tissue needle biopsy specimens from U.S. HCV cirrhosis cohort patients with histologically proven cirrhosis lacking evidence and a history of hepatic decompensation or HCC.
Project description:Lambda interferons IFNL1-3 mediate antiviral immunity by inducing interferon sensitive genes (ISGs) in epithelial tissues. Contrarily, a variant creating the functional gene IFNL4 is associated with impaired clearance of hepatitis C virus (HCV) despite of higher liver expression of ISGs in untreated HCV patients. We aimed to explore IFNL4 signaling mechanism by comparing expression profiles from human hepatic cell line clones with genetic modifications influencing the ISG signaling pathway (IFNLR1/IL10R2 knockouts, IFNL4/IFNL3 expression stimulation by transfection).
Project description:Chronic hepatitis C (CHC) is one of the major risk factor for the progressive development of end stage liver diseases including liver cirrhosis (LC) and HCC worldwide. A deep insight into the molecular mechanism of development and progression of liver fibrosis into cirrhosis and HCC following chronic HCV infection leads to characterization of multiple cellular processes and the underlying regulatory mechanisms. Non-coding RNAs (sncRNAs) including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are found to be the prime regulators of multiple cellular pathways. Using such tools several studies have identified myriads of differentially regulated non-coding RNAs and genes in HCV disease progression to HCC. Our study attempted to look into the integrative network of regulatory non-coding RNAs and target genes involved in HCV related HCC and thereby identify a potential diagnostic molecule as well as therapeutic target for HCC surveillance and management.
Project description:Background: Several studies have investigated the association of miRNAs with hepatocellular carcinoma (HCC) but the data are not univocal. Methods: We performed a microarray study of miRNAs in hepatitis C virus (HCV)-associated HCC and other liver diseases and healthy conditions. Results and Conclusions: The simultaneous comparison of different liver diseases and normal livers allowed the identification of 18 miRNAs exclusively expressed in HCV-associated HCC, with sensitivity and specificity values of diagnostic-grade. A total number of 76 liver specimens obtained from 43 patients were analyzed: 26 liver specimens obtained from 10 patients with HCV-associated HCC, including 9 specimens from the tumor area (HCC) and 17 specimens from the surrounding non-tumorous tissue affected by cirrhosis (HCC-CIR); 18 specimens from 10 patients with HCV-associated cirrhosis without HCC (CIR); 13 specimens from 4 patients with HBV-associated acute liver failure (ALF); 12 specimens from 12 liver donors (LD); and 7 from normal liver of 7 subjects who underwent hepatic resection for liver angioma (NL).
Project description:OBJECTIVE: The number of patients with HCV-related cirrhosis is increasing, leading to a rising risk of complications and death. Prognostic stratification in patients with early-stage cirrhosis is still challenging. We aimed to develop and validate a clinically useful prognostic index based on genomic and clinical variables to identify patients at high risk of disease progression. DESIGN: We developed a prognostic index, comprised of a 186-gene signature validated in our previous genome-wide profiling study, bilirubin (>1 mg/dL) and platelet count (<100 000/mm3), in an Italian HCV cirrhosis cohort (training cohort, n=216, median follow-up 10 years). The gene signature test was implemented using a digital transcript counting (nCounter) assay specifically developed for clinical use and the prognostic index was evaluated using archived specimens from an independent cohort of HCV-related cirrhosis in the USA (validation cohort, n=145, median follow-up 8 years). RESULTS: In the training cohort, the prognostic index was associated with hepatic decompensation (HR=2.71, p=0.003), overall death (HR=6.00, p<0.001), hepatocellular carcinoma (HR=3.31, p=0.001) and progression of Child-Turcotte-Pugh class (HR=6.70, p<0.001). The patients in the validation cohort were stratified into high-risk (16%), intermediate-risk (42%) or low-risk (42%) groups by the prognostic index. The high-risk group had a significantly increased risk of hepatic decompensation (HR=7.36, p<0.001), overall death (HR=3.57, p=0.002), liver-related death (HR=6.49, p<0.001) and all liver-related adverse events (HR=4.98, p<0.001). CONCLUSIONS: A genomic and clinical prognostic index readily available for clinical use was successfully validated, warranting further clinical evaluation for prognostic prediction and clinical trial stratification and enrichment for preventive interventions. 90 liver tissue needle biopsy specimens from patients with histologically proven cirrhosis lacking evidence and a history of hepatic decompensation or HCC.
Project description:BACKGROUND & AIMS: Although patients infected by genotype 1b hepatitis C virus (HCV) with Q(70) and/or M(91)core gene mutations have an almost five-fold increased risk of developing hepatocellular carcinoma (HCC) and increased insulin resistance, the absence of a suitable experimental system has precluded direct experimentation on the effects of these mutations on cellular gene expression. METHODS: HuH7 cells were treated long-term with human serum to induce differentiation and to produce a model system for testing high-risk and control HCV. For clinical validation, profiles of infected cells were compared to each other and to those of liver biopsies of patients with early-stage HCV-related cirrhosis followed prospectively for up to 23 years (n=216). RESULTS: Long-term culture in human serum produced growth-arrested, hepatocyte-like cells whose gene profile overlapped significantly with that of primary human hepatocytes. High-risk (Q(70)/M(91)) and control (R(70)/L(91)) viruses had dramatically different effects on gene expression of these cells. The high-risk virus enhanced expression of pathways associated with cancer and type II diabetes, while the control virus enhanced pathways associated with oxidative phosphorylation. Of special clinical relevance, the transcriptome of cells replicating the high-risk virus correlated significantly with an HCC high-risk profile in patients (Bonferroni-corrected p=0.03), whereas no such association was observed for non-HCC-related clinical outcomes. CONCLUSIONS: The cell-based system allowed direct head-to-head comparison of HCV variants, and provided experimental support for previous clinical data indicating an oncogenic effect of core gene mutations. This simple experimental system distinguished HCV variants and will enable future mechanistic analysis and exploration of interventional approaches.
Project description:In this study we used a high-throughput method for assaying methylation of CpG sites simultaneously in a single sample for identifying differences in methylation observed in tissues ranging from normal liver to pre-neoplastic (cirrhosis) and neoplastic (HCC) states. Since there are important clinical and prognostic differences among HCC patients due to etiology, this study was designed to focus on HCC due to HCV-infection, a more common etiology of HCC among Western countries cross-sectional: 20 cirrhosis, 20 HCC, and 16 normal patients, 87 arrays