POSTERS 785 INNATE SIGNALING BY HEPATITIS C VIRUS IS RIG-I AND MDA5 DEPENDENT AND MODULATED BY NS5A DOMAIN II M.-S. Hiet1 , O. Bauhofer1 , M. Zayas-Lopes1 , A. Ruggieri1 , F. Eberle1 , Y. Tanaka2 , P. Schirmacher3 , V. Lohmann1 , M. Binder1 , R. Bartenschlager1 . 1 Department of Infectious Diseases, University of Heidelberg, Heidelberg, Germany; 2 Department of Virology & Liver Unit, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan; 3 Institute of Pathology, University Hospital, Heidelberg, Germany E-mail:
[email protected] Background and Aims: One of the first line host defense against viral infection is the activation of an innate immune response. This event is trigger by viral RNA recognized by pattern recognition receptors (PRRs) such as the helicases retinoic acid-inducible gene I (Rig-I) and Myeloma differentiation associated factor 5 (Mda5) and leading to the activation of the interferon regulatory factor 3 (IRF3) as well as transcription of type I interferon (IFN). Although the hepatitis C virus (HCV) abrogates IRF3-dependent IFN induction in vitro, paradoxically expression of IFN stimulated genes (ISGs) is frequently found in the livers of hepatitis C patients. The aim of this study was to examine this apparent paradox and to determine the possible role of non-structural protein 5A (NS5A) in controlling ISG activation. Methods: Mechanism of activation of the IRF3 pathway by HCV as well as viral replication was determined in cell lines stably expressing Mda5 or Rig-I by using viral genomes expressing authentic NS5A or a mutant containing a deletion in domain II. Results were confirmed in immune competent primary human hepatocytes (PHH) in vitro and in an in vivo HCV-permissive mouse model. Results: We demonstrate that both Rig-I and Mda5 are sequentially activated upon HCV infection leading to activation of the innate immune response and control of viral replication in PHH. Importantly, the mutant lacking NS5A domain II activated the IFN-response to a much higher extent concomitant with a severe attenuation of viral replication both in PHH and in vivo, but not in the IFN-incompetent human hepatoma cell line Huh7.5. Conclusions: HCV activates innate immune response in IFNcompetent cells in a sequential manner via RIG-I and Mda5. This activation is suppressed by NS5A domain II. Thus, inspite of induction of an IFN response HCV persists arguing that IFN might promote persistence. 786 INCREASED LEVELS OF SOLUBLE LIGANDS OF THE ACTIVATING NATURAL KILLER RECEPTOR (NKG2D) ARE INDEPENDENT RISK FACTORS FOR ADVANCED FIBROSIS IN LIVER TRANSPLANT RECIPIENTS S. Iacob1,2,3 , V.R. Cicinnati1,4 , G. Wu1,4 , M. Lindemann5 , F.M. Heinemann5 , V. Rebmann5 , P.A. Horn5 , G.C. Sotiropoulos2 , H.H. Schmidt4 , A. Paul2 , G. Gerken1 , S. Beckebaum1,2 . 1 Department of Gastroenterology and Hepatology, 2 Department of General, Visceral and Transplant Surgery, University Hospital Essen, Essen, Germany; 3 Center of Gastroenterology & Hepatology, Fundeni Clinical Institute, Bucharest, Romania; 4 Department of Transplant Medicine, University Hospital M¨ unster, Munster, 5 Institute for Transfusion Medicine, University Hospital Essen, Essen, Germany E-mail:
[email protected] Introduction: Fibrosis progression in liver transplant (LT) patients has usually a multifactorial origin. Proinflammatory cytokines which can be induced by soluble ligands of the activating natural killer receptor (NKG2D) may sustain cell-mediated immune responses, thereby promoting hepatobiliary injury. The role of HLA mismatches and soluble NKG2D ligands (major MHC class I-related S308
chain A and B [sMICA/sMICB] and UL16 binding protein 2 [sULBP2]) in fibrosis progression has not been investigated in the LT setting. Aim: To investigate clinical, laboratory and immunological risk factors for occurrence of cirrhosis following LT. Methods: A total of 174 LT recipients were enrolled in the study. Patients with recurrent viral disease were excluded. Cytokines were assessed by flow cytometry with the “BD Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine Kit”. Assessment of sMICA, sMICB and sULBP2 was realized by enzyme linked immunosorbent assay. Screening for anti-HLA class I, class II or MICA antibodies (AB) was performed using Luminex technology. Chi-square or Fischer’s exact test was used for comparing categorical data and Student’s t-test or Mann-Whitney U test, when appropriate, was used for comparing continuous variables. Multivariate logistic regression analysis was performed to identify independent risk factors for development of cirrhosis after LT. Results: In the univariate analysis, donor age >50 years (p = 0.001), presence of anastomosis stenosis (p = 0.009), high levels of transaminases (aspartate transaminase (p < 0.0001), alanin aminotransferase (p = 0.001), total bilirubin (p = 0.0004) and alkaline phosphatase (p = 0.01) were associated with progression to cirrhosis. Immunological risk factors included high serum levels of inflammatory cytokines (TNF alpha (p = 0.01), interleukin (IL)10 (p = 0.004), IL6 (p < 0.0001)), presence of donor specific anti-HLA II AB (p = 0.01), high serum levels of sMICA (p = 0.04), sMICB (p = 0.004) and ULPB2 (p < 0.0001). In the multivariate analysis, high serum levels of total bilirubin, sMICB and ULPB2 were independent predictors for cirrhosis after LT. Conclusion: sMICB and ULPB2 molecules were associated with cirrhosis in LT patients. Screening of these biomarkers in LT patients may be useful for identifying patients at high risk of advanced fibrosis progression after LT. 787 A COMPREHENSIVE SUBCLASSIFICATION OF KUPFFER CELLS IN MICE M. Ikarashi1 , H. Nakashima1 , M. Kinoshita1 , A. Sato1 , M. Nakashima1 , H. Miyazaki2 , K. Nishiyama3 , J. Yamamoto3 , S. Seki1 . 1 Department of Immunology and Microbiology, 2 Division of Traumatology, National Defense Medical College Research Institute, 3 Department of Surgery, National Defense Medical College, Tokorozawa, Japan E-mail:
[email protected] Background and Aims: We recently reported that murine F4/80 positive Kupffer cells were classified into two subsets, cytokineproducing CD11b+ cells and phagocytic CD68+ cells (J. Hepatol. 53: 903–910, 2010). However, a substantial population with CD11b− CD68− still exists in the F4/80+ cells. This study sought an additional comprehensive classification using CD32 (FcgRII) Ab. Methods: Under the approval of the Institutional Review Board for the Care of Animal Subjects at the National Defense Medical College, Japan, the murine livers were removed under deep anesthesia. Liver mononuclear cells, which included Kupffer cells, were isolated using centrifugation with Percoll solution after collagenase treatment. CD11b, CD68 and CD32 expressions on the F4/80+ Kupffer cells were analyzed by flowcytometry. To assess phagocytosis, FITC-microsphere (beads) or FITC-E. coli were used. In addition, to assess phagolysosome formation, pHrodo-conjugated E. coli, which detected a decrease of pH in phagolysosomes by fluorescence emission after ingestion, were used. Results: The flowcytometry analysis showed that F4/80+ cells were subdivided into three subsets, CD11b+ (30%), CD68+ (40%) and CD32+ (50%) subsets, and approximately a half of CD68+ cells expressed CD32. Consistent with our recent report, in vivo administration of FITC-microsphere (beads) demonstrated that CD68+ cells had a potent phagocytic activity, and the most vigorous phagocytic cells were CD68+ CD32− . CD68+ CD32− cells increased in number
Journal of Hepatology 2012 vol. 56 | S225–S388