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694A AASLD ABSTRACTS HEPATOLOGY, October, 2015 Methods: Cells were isolated from explants of 20 HCV-infected patients undergoing liver transplantation. Blood was obtained prior to surgery. Liver and peripheral blood mononuclear cells (PBMCs) were analyzed by CyTOF, FACS, microarray, and multiplex cytokine assays with and without 4 hr stimulation with R848 (TLR7/8 agonist/single-stranded RNA mimic) or Poly I:C (TLR9 agonist/double-stranded RNA analogue). Results: CyTOF delineated monocytes/macrophages, DC, NK, T and B cell subsets and revealed that pDCs are the only liver or blood cell that produces IFNα. Cytokine assays showed that liver pDCs were primed for robust IFNα production: secreted levels increased from 0.8 to 344.7 pg/mL after 4 hrs of R848 stimulation. This increase was much higher than that of blood pDCs, which increased production from 2.4 to 115 pg/mL, p
HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 695A as shown by increased ISG induction. Interestingly, SeP was found to regulate IFN signal transduction independent of the JAK-STAT pathway. In vivo, the ISGs Mx-1 and Oas2 were significantly increased in SepKO mice compared with WT mice following PolyI:C injection. To explore the clinical significance, hepatic gene expression was analyzed in 66 CHC patients; SeP was upregulated in the early stage of liver fibrosis and was significantly correlated with a poor outcome of IFN therapy. Conclusion: In CHC, SeP impaired innate immunity. The present findings would be useful for the development of novel therapeutic strategies for patients with chronic liver disease with various etiologies including NASH and CHC. Disclosures: Stanley M. Lemon - Advisory Committees or Review Panels: Merck, Santaris, Abbott, Gilead; Consulting: Achillion, Idenix; Grant/Research Support: Merck, Tibotec, Scynexis; Speaking and Teaching: Hoffman LaRoche Shuichi Kaneko - Grant/Research Support: MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bristol Myers Squibb., Inc, Pfizer., Co., Inc, Astellas., Inc, Takeda., Co., Inc, Otsuka„ÄÄPharmaceutical, Co., Inc, Eizai Co., Inc, Bayer Japan, Eli lilly Japan The following authors have nothing to disclose: Kazuhisa Murai, Masao Honda, Tetsuro Shimakami, Takayoshi Shirasaki, Hirofumi Misu, Toshinari Takamura, Seishi Murakami 991 Long noncoding RNAs regulate HCV replication and ISGs in a Jak-STAT independent manner Xiao Liu, Jian Hong, Dachuan Cai, Sae Hwan Lee, Dahlene Fusco, Esperance A. Schaefer, Cynthia Brisac, Anna Lidofsky, Wenyu Lin, Raymond T. Chung; Gastrointestinal Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA Background/ Aims: Long non-coding RNAs (lncRNAs) involve in many important cellular process regulations. However, their roles in the innate immune response including type-1 interferon (IFN) response to 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 antiviral effects (Lin et al, J Hepatology 2015, 62:1024). We sought to explore lncRNA regulators of HCV replication, the JAK-STAT pathway, and interferon-stimulated genes (ISGs). Methods: We performed SART1 siRNA knockdown and RNA- Seq in Huh7.5.1 cells with or without IFN treatment. Bioinformatic Homo sapiens Genome Browser Gateway analysis was performed to predict the location of lncRNAs and adjacent coding genes. Selected lncRNAs, Jak-STAT pathway genes and ISGs and their proteins, together with HCV replication, were monitored by qRT-PCR and Western blot in the JFH1 HCV infectious model. Results: We obtained 6699 lncRNAs regulated by SART1 and IFN in RNA-Seq. Our bioinformatic analysis of RNA-Seq data identified a cluster of 60 lncRNAs involved in IFN’s antiviral regulation. qRT-PCR confirmed that expression of 14 of these lncRNAs was consistent with the RNA-Seq data. We then identified 4 anti-sense lncRNA related to ISGs which negatively regulated the adjacent coding gene expression based on previous reports. Of these, we selected lncRNA RP11-670E13.5 (lncRNA-TRIM25) for further characterization of its regulation of HCV. We found that knockdown of lncRNA RP11-670E13.5 significantly increased HCV replication compared to Neg siRNA. We also found that siRNA lncRNA RP11- 670E13.5 significantly reduced TRIM25, MX1, ISG15 mRNA compared to Neg siRNA in JFH1-infected Huh7.5.1 cells. Interestingly, STAT1, JAK1, OAS3, and RIG-I mRNA expression were not affected by the knock-down of lncRNA RP11- 670E13.5. Moreover, we found that siRNA to STAT1 had no effect on lncRNA RP11-670E13.5 expression. These data suggest that lncRNA RP11-670E13.5 regulation of HCV is independent of the Jak-STAT signaling pathway. Conclusion: We found that lncRNA RP11-670E13.5 has important regulatory functions of antiviral innate immunity in the JFH1 HCV model. Our data suggested that lncRNA RP11-670E13.5 regulates HCV replication independently of the JAK-STAT pathway. We speculate that RP11-670E13.5 exerts its regulatory function on the enhancer and promoter activation of nearby protein-coding (target) genes such as TRIM25, MX1, and ISG15. Further characterization of the mechanism by which selected lncRNAs that associate with these genes regulate HCV replication and ISGs are warranted. Disclosures: Dahlene Fusco - Grant/Research Support: Gilead Raymond T. Chung - Grant/Research Support: Gilead, Mass Biologics, Abbvie, Merck, BMS The following authors have nothing to disclose: Xiao Liu, Jian Hong, Dachuan Cai, Sae Hwan Lee, Esperance A. Schaefer, Cynthia Brisac, Anna Lidofsky, Wenyu Lin 992 Evaluation of protective and therapeutic strategies against hepatitis C virus in a syngenic transplantation mouse model Matti Sällberg 1 , Sepideh Levander 1 , Fredrik Holmstom 1 , Gustaf Ahlén 1 , Lars Frelin 1 , Gang Long 2 , Daniel Rupp 2 , Ralf Bartenschlager 2 ; 1 Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; 2 Molecular Virology and Infectious Diseases, University of Heidelberg, Heidelberg, Germany Key features of the hepatitis C virus (HCV) are its high genetic plasticity and its ability to establish chronic infection in hepatocytes. Development of prophylactic vaccines is hampered by the lack of immune competent small animal models supporting robust HCV replication. Herein, we devised an immune competent mouse model that is based on the syngenic transplantation of H-2 b -restricted Hep56 cells containing a self-replicating subgenomic HCV replicon RNA of genotype (gt) 2a. To allow non-invasive in vivo monitoring of tumor growth, cells were also stably transfected with a luciferase (Luc) reporter gene. As controls, Hep56 cells stably expressing an enzymatically active HCV NS3/4A (gt2a) protease complex were used. Upon subcutaneous injection, all cell lines generated palpable tumors with sizes peaking around day 8-16 post inoculation. Tumor growth correlated with an accumulation of inflammatory cells and central necrotic areas, concomitant with a decrease of HCV RNA and Luc DNA copy numbers. DNA vaccination resulted in a robust NS3/4A(gt2a)-specific CD4+ and CD8+ T cell response protecting wild type, but not HCV NS3/4A(gt1a)-transgenic (Tg) mice against HCV replicon cell tumors suggesting cross-genotypic tolerance in NS3/4A(gt1a)-Tg mice. Hep56 cells stably expressing gt2a NS3/4A primed and boosted a strong CTL-dominated response, whereas the HCV replicon cells did not, despite comparable levels of antigen expression. Thus, similar to human infection, hepatocytes with replicating HCV RNA appear to be poorly immunogenic arguing that the replicon-cell based syngenic transplantation model shares features of infected hepatocytes in patients. Unlike other models, syngenic transplantation of HCV replicon cells can be rapidly applied to genetically modified immune competent mice of the widely used C57BL/6J background for the study of HCV-specific cellular immune responses. This model will be highly useful in vaccine development for hepatitis C Disclosures: Matti Sällberg - Consulting: Chrontech Pharma AB, Abbvie The following authors have nothing to disclose: Sepideh Levander, Fredrik Holmstom, Gustaf Ahlén, Lars Frelin, Gang Long, Daniel Rupp, Ralf Bartenschlager
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1312A AUTHOR INDEX HEPATOLOGY, Octo
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