Innate immunity


Concanavalin A-induced hepatitis: Gut-derived LPS, intestinal permeability, and TLR4

Beth L. Mallard1,4, Ayako Mabuchi1,4, Sachiko Akashi-Takamura2, Jacguie Harper3, Leo R. Quinlan1, Antony M. Wheatley1,4

1Physiology, NUI Galway, Galway, Ireland; 2Institute of Medical Sciences, University of Tokyo, Tokyo, Japan; 3Malaghan Institute of Medical Research, Wellington, New Zealand; 4Physiology, University of Otago, Dunedin, New Zealand

Background & Aims The plant lectin Concanavalin A (ConA) induces T cell-mediated acute hepatitis in mice that is manifested by histological changes and elevated plasma ALT up to 24 hrs after injection. TLR4 has been implicated in a wide range of liver patologies including ischaemia-reperfusion injury, hepatitis B and C and primary and secondary biliary cirrhosis. Here we tested the hypothesis that the LPS/TLR4 pathway played a role in ConA-induced hepatitis. Materials & Methods The effect of iv administration of ConA was tested in TLR4-/and wildtype C57/BL6, in C3H/HeN (LPS-sensitive) and C3H/HeJ mice (LPS-insensitive) and in C57/BL6 mice pretreated with the anti-TLR4 mAb, MTS510. Plasma levels of cytokine INF-γ, TNF-α and IL-6 were measured using the BioPlex suspension array system. The effect of ConA on TLR4 expression by T cells and Kupffer cells was FACS analysis. To test if gut-derived bacteria were involved, conA was given to mice treated for 5 days with antibiotics (Polymyxin B/Neomycin). Gut permeability was tested both in vivo (FITCalbumin by oral gavage) and in vitro (Ussing chamber experiment using native colon segments). Blood flow in the hepatic microcirculation was measured by laser Doppler flowmetry (LDF) and by intravital microscopy (IVM). Results ConA caused elevation of plasma ALT and histological sings of injury in C57/BL6 and C3H/HeN mice but not in TLR-/-, C3H/HeJ and MTS510-treated C57/BL6 mice. In mice depleted of Gram-negative bacteria, ConA did not induce liver injury. Plasma TNF-α (peak, 2 hr), INF-γ and IL-6 (peak, both 6 hr) were significantly elevated after ConA in wildtype but not in TLR4-/- or MTS510 treated mice. FACS analysis revealed that ConA administered in vivo induced increase increases in the percentage of TLR4positive CD3+ (T cells) and F4/80+ (Kupffer cells). ConA caused a 15-fold increased the permeability of the gut to FITCdextran, that was absent in TLR4-/- mice. In native colon, basolaterally applied ConA rapidly (<10min) reduced transepithelial electrical resistance (increased permeability) compared to control (∼20%). The lack of TLR4 was without impact on the ConA induced reduction in hepatic perfusion (LDF), but IVM revealed fewer rolling/adherent leukocytes in TLR4-/- mice than in control mice. Conclusions Our result indicate that ConA-induced hepatitis reguires (1)the presence of Gram-negative bacteria in the gut (2) ConA-induced increase in intestinal permeability and (3) the presence of a functionalTLR4. Thus Gram-negative bacteria or their products (eg, LPS) would appear to play a central role on ConA-induced acute hepatitis.


The following people have nothing to disclose: Beth L. Mallard, Ayako Mabuchi, Sachiko Akashi-Takamura, Jacguie Harper, Leo R. Quinlan, Antony M. Wheatley


The C5 receptor C5aR is profibrogenic during chronic liver injury but reguired for fibrosis resolution

Susanne N. Weber, Annika Bohner, Guzel R. Burganova, Frank Lammert

Department of Medicine II, Saarland University Medical Center, Homburg, Germany

Background: During complement activation complement factor C5 is cleaved and the small anaphylatoxin C5a binds to its receptors C5aR and C5L2, which have been shown to act proand anti-inflammatory, respectively. Since we identified C5 as a susceptibility gene that modifies liver fibrogenesis, the aim of this study was to dissect the critical downstream pathways and the roles of the two C5 receptors during the development and resolution of hepatic fibrosis. Methods: Mice deficient for C5aR and C5L2 on the Balb/cJ genetic background and wild-type (WT) controls were treated with carbon tetrachloride (CCl4; 0.7 ml/kg) for 24 hrs (acute model; 1 injection) and 6 wks (chronic model; CCl4 injections twice weekly), or underwent bile duct ligation (BDL) for 2 weeks to induce biliary fibrosis. In addition, we analyzed a resolution model (CCI4 injections twice weekly for 6 wks, followed by 6 wks regeneration time). Hepatic collagen contents were guantified, and steady-state mRNA levels of Th1 and Th2 cytokines were determined by gRTPCR. Results: After acute injury, the hepatic expression of proinflammatory markers IL6 (5.02 ± 1.75-fold) and TNFa (6.78 ± 2.71-fold) as well as the Th1 cytokine IL23 (116.49 ± 55.42) was induced significantly in C5L2-deficient mice as compared to WT controls, whereas C5aR-deficient mice did not show significant alterations. Chronic fibrosis was less pronounced in C5aR-deficient mice both after 6 weeks of CCl4 challenge and 2 weeks after BDL. Interestingly 6 weeks after CCl4, hepatic collagen contents were highest in C5aR-deficient mice, indicating ongoing injury even after termination of the hepatotoxin. This was also reflected by the cytokine expression profile, where IL12 and IL27 levels were upregulated in mice lacking C5aR. Conclusions: The data show that both C5a receptors are important modifiers of liver injury and chronic fibrosis. C5L2 seems to be profibrogenic during the early response, whereas C5aR appears to play a critical role during fibrosis resolution. As macrophages express C5aR and are relevant for fibrosis development and resolution, further studies are needed to identify the role of this complement receptor during the transition from fibrogenesis to hepatic wound healing.


The following people have nothing to disclose: Susanne N. Weber, Annika Bohner, Guzel R. Burganova, Frank Lammert


A novel modified cytokine modulates hepatic macrophage phenotype and augments liver growth after hepatectomy in mice with healthy and diseased liver

Ben Stutchfield1,2, Deborah Gow4, Alison MacKinnon1, Stephen Jenkins3, Luke Devey2,3, Davina Wojtacha1, Tak Yung Man1, David A. Hume4, Stephen J. Wigmore2,3, Stuart J. Forbes1

1MRC Centre for Regenerative Medicine, Edinburgh, United Kingdom; 2Hepatobiliary-Pancreatic Surgical Services and Edinburgh Transplant Unit, Royal Infirmary Edinburgh, Edinburgh, United Kingdom; 3Queen's Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom; 4The Roslin Institute, University of Edinburgh, Edinburgh, United Kingdom

Background: Therapies to augment liver regeneration in relation to liver surgery remain elusive. Macrophages are known to be central to liver regeneration but the pro-regenerative macrophage phenotype and potential for therapeutic manipulation have not been defined. We aimed to characterize the phenotype of the pro-regenerative macrophage following partial hepatectomy and explore the effects of manipulating macrophage phenotype using CSF1-Fc, a novel long- acting macrophage colony stimulating factor conjugate. Methods: Macrophage phenotype was assessed by flow cytometry and immunohistochemistry in wild type C57Bl/6 mice controls and after partial hepatectomy in the presence and absence of chronic liver injury induced by 8 weeks of carbon tetrachloride injection. CSF1-Fc (0.5mcg/g) was administered to normal C57Bl/6 mice and injury models compared with PBS control. The CSF1r-EGFP mouse was used to ascertain hepatic CSF1R expression using multiphoton microscopy. Results: Following partial hepatectomy the pro-regenerative macrophage phenotype included both infiltration and local proliferation (Ki67 and BRDU) of macrophages described as Ly6C / YM1 high infiltrating cells and F480 high / CD11B low resident macrophages. A novel CD45low CD206high phenotype of resident macrophages was identified in the normal liver which disappeared during regeneration. To test the function of macrophages in regeneration, a novel CSF1-Fc conjugate was developed demonstrating similar in vitro activity as CSF1 but with significantly increased in vivo half-life. CSF1-Fc administration to uninjured mice resulted in the same pro-regenerative macrophage phenotype as seen after partial hepatectomy, and induced widespread hepatocyte proliferation leading to a 27% increase in liver weight to body weight ratio (p=0.03). Multiphoton microscopy of CSF1r-EGFP mice demonstrated CSF1R expression was restricted to hepatic macrophages and was not expressed in hepatocytes. Liver weight to bodyweight ratio and hepatocyte proliferation (Ki67) were significantly increased at day 4 in both hepatectomy models following CSF1-Fc treatment while ALT levels were significantly reduced. In the chronic injury plus hepatectomy model there was a significant increase in body weight from day 3 suggestive of improved postoperative course with a trend to improved survival (p=0.07). Conclusion: The proregenerative macrophage phenotype seen following partial hepatectomy can be induced by administration of CSF1-Fc in wild type mice resulting in hepatic enlargement. Therapeutic potential of CSF1-Fc is suggested by enhanced regenerative parameters in two models of hepatic regeneration.


Ben Stutchfield - Patent Held/Filed: University of Edinburgh

The following people have nothing to disclose: Deborah Gow, Alison MacKinnon, Stephen Jenkins, Luke Devey, Davina Wojtacha, Tak Yung Man, David A. Hume, Stephen J. Wigmore, Stuart J. Forbes


Targeted deletion of fgl2 restores antiviral T cell response and enhances viral clearance in a murine model of chronic viral infection

Olga Luft, Gary A. Levy, Nazia Selzner

Multiorgan Transplant Program, University Health Network, Toronto, ON, Canada

Introduction: Chronic infections caused by HIV, hepatitis B and C viruses collectively present a major health problem affecting half a billion people worldwide. It is known that production of inhibitory factors may result in the inability of the host to clear viruses, resulting in chronic viral persistence. Fibrinogen-like protein 2 (FGL2), a novel immune suppressor molecule, regulates both innate and adaptive immune responses. FGL2 through binding to inhibitory Feγ receptors induces B cell apoptosis and inhibits maturation of bone marrow-derived dendritic cells leading to suppression of T cell proliferation. In this study, we explored whether deletion of FGL2 accelerates viral clearance using the murine model Lymphocytic Choriomeningitis Virus clone 13 (LCMV cl13), a model of chronic viral infection. Methods: C57BL/6 wild-type (Fgl2+/+) and Fgl2-/- mice were infected with 2x106 PFU of LCMV cl13. Plasma levels of FGL2 were measured weekly using the LEGEND MAX™ Mouse FGL2 ELISA Kit. Liver pathology was assessed by histology and levels of alanine transaminase (ALT). Viral load was determined using a plague assay. T cell response was analyzed in spleen by flow cytometry. Results: At day 7 (d7) post LCMV cl13 infection, plasma levels of FGL2 were elevated by 3-fold compared with non-infected mice (51.6U/L vs 135.6U/L, p<0.0001) and remained elevated until d56 post-infection (pi). At d28 pi, viral load in the heart, kidney, liver and lung tissue as well as in the plasma of Fgl2-/- mice were significant lower than the Fgl2+/+ mice. By d56 pi, LCMV cl13 titers were undetectable in the plasma and in the lung tissue of all Fgl2-/- mice while most Fgl2+/+ mice had detectable LCMV virus both in the plasma and lung (p<0.05). Fgl2-/- mice had significantly higher freguencies of LCMV specific CD8+ T cells at day 28 pi compared with Fgl2+/+ mice (3.2% vs 2.5%, p<0.05). Furthermore, increased production of IFNγ by LCMV specific CD8+ T was observed at d28 and d56 in Fgl2-/- mice compared with wildtype mice (d28 1.28 vs 0.71%, d56 1.5% vs 0.7%, p<0.05). Despite enhanced effector T cell responses following deletion of FGL2, liver histology and plasma ALT levels showed no evidence of increased immunopathology. Conclusion: Targeted deletion of Fgl2 restores adaptive immune T cell responses to LCMV cl13 leading to clearance of virus. These studies suggest that targeting FGL2 may be a novel therapeutic approach to treat patients with chronic viral hepatitis.


The following people have nothing to disclose: Olga Luft, Gary A. Levy, Nazia Selzner


Splenectomy causes hepatic inflammation and oxidative stress through activation of Kupffer cells in iron-overloaded mice

Sohji Nishina, Yuichi Hara, Yasuyuki Tomiyama, Keisuke Hino

Liver medicine, Kawasaki medical univercity, Kurashiki, Japan

Background and Aims: Spleen plays an important role in regulating iron homeostasis and inflammation in the whole body. Nevertheless, splenectomy is often performed prior to hepatocellular treatment such as hepatectomy or chemotherapy in expectation of increase in thrombocyte, and its effect on hepatic pathology remains unknown. The aim of this study was to investigate how splenectomy affects the liver in terms of hepatic iron metabolism or inflammation. Methods: Male C57BL/6N mice underwent splenectomy or sham operation (control) at the age of 8 weeks and were fed an excess-iron diet (carbonyl iron 225-mg/kg diet) at 4 weeks after operation. Mice in each group were assessed for molecules responsible for hepatic iron metabolism, inflammation and oxidative stress in addition to hepatic iron deposition and inflammation at 2 months after the commencement of feeding. Results: Compared to control mice, splenectomized mice showed higher ALT levels and more active hepatic inflammation. The liver tissue of splenectomized mice also showed abundant activated Kupffer cells indicated by F4/80, MCP-1 and Cd11c, and more nuclear translocation of NFkβ, so that mRNA expression of inflammatory cytokines such as IL-1, IL-6, TNF-α and IFN-γ was significantly greater in the liver of splenectomized mice. Additionally, NADPH oxidase expression and reactive oxygen species production were greater in splenectomized mice. Hepatic iron deposition was predominantly found in Kupffer cells rather than hepatocytes probably due to activation of Kupffer cells in splenectomized mice, but hepatic iron content was comparable between two groups. The latter result may be explained by the opposing effects of hepcidin-mediated negative iron accumulation and activated Kupffer cell-mediated positive iron accumulation. Indeed, the liver tissue of splenectomized mice showed IL-6-induced increase in phosphorylated STAT3 expression and subseguent increased expression of hepcidin. Conclusions: The present results indicated that splenectomy causes hepatic inflammation and oxidative stress through activation of Kupffer cells without inducing hepatic iron accumulation. Therefore, the risk of hepatic disease progression should be taken into consideration when splenectomy is performed in clinical settings.


The following people have nothing to disclose: Sohji Nishina, Yuichi Hara, Yasuyuki Tomiyama, Keisuke Hino


Features of immunomodulatory and therapeutic effects of mesenchymal stromal/stem cells on non-alcoholic steatohepatitis murine model in early and cirrhotic phases

Akihiro Seki1,Yoshio Sakai2,3, Mami Higashimoto1,Takuya Komura1, Alessandro Nasti1,Keiko Yoshida1,Takahiro Ochiya4, Takashi Wada2, Masao Honda3, Shuichi Kaneko1,3

1Disease control and homeostasis, Kanazawa university, Kanazawa, Japan; 2Department of Laboratory Medicine, Kanazawa University, Kanazawa, Japan; 3Department of Gastroenterology, Kanazawa University, Kanazawa, Japan; 4National Cancer Center, Tokyo, Japan

Adipose tissue derived stromal/stem cells (ADSCs) recently attract much attention in the context of the regeneration therapy, since they contain abundant pluripotent mesenchymal stem cells. In addition to their pluripotency, immune-modulation by ADSCs is the other important feature to be explored for their mechanism of regeneration/reparing effects on the impaired organs. In the mice fed with atherogenic-high fat diet, infiltration of inflammatory cells in the liver occur in early period after starting feeding, persist and ultimately develop cirrhosis. We examined the immunomodulatory effects of ADSCs in the liver of steatohepatitis mice in early phase as well as cirrhotic phase. [Materials and Methods] ADSCs isolated from adipose tissue of the GFP-Tg mouse are cultured and expanded. C57BI/6J mice (female, 8 weeks old) were initiated to be fed with high fat atherogenic (HF-AT) diet to develop steatohepatitis. After 12 weeks (n=4) or 32 weeks (n=4) feeding, 1 x 10^5 of ADSCs were injected twice every 2 weeks into the splenic subcapsule of the mice. One or 2 weeks after last injection, liver tissue, hepatic parenchymal cells and inflammatory cells were isolated for immunohistochemistry, gene expression analysis by RT-PCR and FACS. The liver tissues were immunohistochemically examined. CD11b+ cells were further isolated from inflammatory cells by magnetic-activated cell sorting. [Result] In the early phase of murine NASH model fed with HF-AT diet for 12 weeks, ballooning and inflammatory cells was confirmed in the non-cirrhotic liver. Fibrosis had been progressed with decreasing the albumin expression of hepatic parenchymal cells, and finally cirrhosis was established at 32weeks after feeding. Flow cytometry analysis and immunohistochemical analysis showed that ADSC injection decreased the number of CD11b+, Gr-1+ cells of the hepatic inflammatory cells in the early phase of NASH model mouse as well as in cirrhotic phase. RT-PCR gene expression analysis in hepatic inflammatory cells, and specific isolated subpopulation of CD11b+ cells of steatohepaitis mice treated with ADSCs showed that pro-inflammatory cytokines such as TNF-alpha, IFN-gamma, IL-1beta was decreased in the early as well as cirrhotic phase of murine NASH. In addition, ADSC injection decreased the fibrosis area and improved the albumin expression of the hepatic parenchymal cells, compared to control, when cirrhotic mice were treated (p<0.05). [Conclusion] ADSC treatment suppresses the inflammation mediated by CD11b+ cells even in the early stage of the steatohepatitis as well as in cirrhotic stage, suggesting that it is preventively and therapeutically efficacious to steatohepatitis.


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, MDS, Co., Inc, Chugai Pharma., Co., Inc, Toray Co., Inc, Daiichi Sankyo., Co., Inc, Dainippon Sumitomo, Co., Inc, Ajinomoto Co., Inc, Bayer Japan

The following people have nothing to disclose: Akihiro Seki, Yoshio Sakai, Mami Higashimoto, Takuya Komuma, Alessandro Nasti, Keiko Yoshida, Takahiro Ochiya, Takashi Wada, Masao Honda


Protein S exacerbates activation of liver natural killer T(NKT) cells during alcohol-induced liver injury

Hirohide Miyachi, Rumi Moroka, Naoki Fujita, Yoshinao Kobayashi, Motoh Iwasa, Yoshiyuki Takei

Gastroenterology, Mie University, Tsu, Japan

Background and Aims: It has been established that alcoholic liver disease is the conseguence not only of direct alcoholinduced hepatotoxicity but also the interactions between alcohol metabolism, the immune system, and hepatic cells. The liver is enriched with innate immune cells and particularly of NKT cells. Protein S(PS) is a Vitamin K-dependent anticoagulant glycoprotein. In addition to its anticoagulant activity, PS exerts anti-inflammatory and anti-apoptotic activities. In this study, we investigated the role of PS in acute alcoholic hepatitis. Methods: Study1: Human Protein S transgenic(hPS TG) mice and WT mice were received 3doses of ethanol every 12 hours. Six hours after the last injection, mice were sacrificed and the levels of plasma liver enzymes and hepatic concentration of inflammatory cytokines were measured. The liver tissues were also analyzed by flow cytometry. Study2: Liver NKT cells from hPS TG mice and WT mice were treated with 100nM of ethanol for 24 hours, and the activation of NKT cells was evaluated by the surface expression of FasL. Study3: NKT cells from WT mice were cultured in the presence of 10 ng/ml of a-GalCer and/or 20μg/ml of human PS for 48 hours. Cytokines in the supernatant were measured. Study4: Liver NKT cells from hPS TG mice or WT mice were cultured with 10ng/ml of a-GalCer for 24 hours,and activation and apoptosis of NKT cells were analyzed. Results: Study1: The plasma level of liver enzyme were increased in the hPS TG mice treated with ethanol compared to WT mice treated with ethanol. The hepatic concentration of cytokines in hPS TG mice treated with ethanol were significant increased. In the FACS analysis, there was a significant increase in the number of NKT cells in the liver of hPS TG mice that received ethanol when compared to ethanol-treated WT mice. And the expression of annexin-V in NKT cells from the liver of hPS TG was significantly decreased. Study2: FasL was significantly increased in NKT cells from hPS TG mice. Study3: Compared to untreated cells, significant levels of IFN-γ and osteopontin were detected in the supernatants from cells cultured in the presence of human PS. Study4: There was a significant increase in the number of NKT cells from hPS TG mice after α-Galcer stimulation compared to those from WT mice. In addition, after stimulation with α-Galcer, the expression of annexin-V was significantly decreased and that of FasL was significantly increased in NKT cells from hPS TG mice compared to cells from WT mice. Conclusion: PS exacerbates acute alcoholic-induced liver injury by enhancing the activation of NKT cells, suggesting that PS may be a molecular target for therapy of this disease.


The following people have nothing to disclose: Hirohide Miyachi, Rumi Moroka, Naoki Fujita, Yoshinao Kobayashi, Motoh Iwasa, Yoshiyuki Takei


Metabolic Analysis in Transgenic Mouse Models of Acute Intermittent Porphyria (AIP)

Sahil Mittal1, Makiko Yasuda3, Robert J. Desnick3, Karl E. Anderson2

1Gastroenterology and Hepotology, Baylor College of Medicine, Houston, TX; 2Preventive Medicine and Community Health, University of Texas Medical Branch, Galveston, TX; 3Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York, NY

AIP is an autosomal dominant disorder due to deficient activity of hydroxymethylbilane synthase (HMBS), third enzyme in the heme biosynthetic pathway. Environmental, nutritional, hormonal and metabolic factors are known to exacerbate disease, but differences in clinical severity among patients are often unexplained. This study in AIP mouse models explored metabolic differences between latent and active disease that might prove to be relevant to the human disease. Methods: Metabolic comparisons were made using 2 models.1) Homozygous knock-in mice with ∼5% residual HMBS activity and significant neuromotor impairment were compared to isogenic wild type controls.2) Mice with ∼30% residual HMBS activity and latent AIP were compared to those induced by phenobarbital treatment. Metabolomic profiles in 15 urine and 15 plasma samples were extracted (Metabolon Inc, Durham, NC) and then analyzed on gas chromatography/mass spectrometry and liguid chromatography/mass spectrometry platforms. Statistical analysis included Welch's two sample T-test applied to log-transformed data and differences were considered significant if P<0.05 and g< 0.10. Results: ln both models levels of porphobilinogen and its precursor 5-aminolevulinate (ALA) were significantly elevated in urine of mice with active AIP relative to controls.3-indoxyl sulfate and phenol sulfate are sulfated gut microbiome metabolites of tryptophan & tyrosine that were significantly elevated in plasma from mice with active AIP, relative to appropriate controls. The plasma levels of 2-hydroxy-3methylvalerate, 2-hydroxyisocaproate, and 2-hydroxyisovalerate (metabolites of branched chain amino acid catabolism) were also significantly elevated in mice with active AIP. Comparison of active AIP mice to controls revealed elevation of the lysine metabolite 2-aminoadipate in plasma and elevation of the downstream metabolite 2-oxoadipate in urine. These results indicate a defect in the conversion of 2-oxoadipate to glutarylCoA by 2-oxoglutarate dehydrogenase (OGDH). The OGDH complex shares enzymatic subunits with a-ketoglutarate dehydrogenase, a key participant in the tricarboxylic acid cycle (TCA) cycle. Conclusion: These early results reveal metabolic differences in mice with active AIP and controls not previously described. The results suggest changes in gut microbiome metabolism and/or composition, incomplete catabolism of branched chained amino acids, and decreased oxoglutarate dehydrogenase/ketoglutarate dehydrogenase activity. Further studies are needed in mice, and may guide human studies comparing metabolic profiles in active and latent AIP.


Makiko Yasuda - Patent Held/Filed: Alnylam Pharmaceuticals Robert J. Desnick - Consulting: Alnylam

The following people have nothing to disclose: Sahil Mittal, Karl E. Anderson


A high-fat diet results in nonalcoholic steatohepatitis, and adipose tissue hypoxia, macrophage infiltration and inflammatory M1 activation in obese diabetic mice

Priya Handa1,2, Vicki Morgan-Stevenson1,2, James E. Nelson1,2, Bryan D. Maliken1,2, Matthew M. Yeh3, Clinton T. Elfers4, Christian Roth4, Kris V. Kowdley1,2

1Liver Center of Excellence, Digestive Disease Institute, Seattle, WA; 2Virginia Mason Medical Center, Benaroya Research Institute, Seattle, WA; 3University of Washington, Seattle, WA; 4Seattle Children's Research Institute, Seattle, WA

Background and Aim: Nonalcoholic fatty liver disease (NAFLD) is linked to obesity and diabetes and a high-fat diet has been associated with development of nonalcoholic steatohepatitis (NASH) in murine models. We aimed to investigate development of NASH and changes in adipose tissue function in obese, diabetic mice given a high-fat diet. Methods: Leptin receptor deficient (Leprdb/db) mice were administered a 61% unsaturated fat diet (predominantly linoleic acid, an omega −6 fatty acid) or standard chow for 5 or 10 weeks. Histological assessment of liver tissue was performed for steatosis grade, hepatocellular ballooning, inflammation, fibrosis and the NAFLD activity score (NAS) and a diagnosis of NASH was rendered by a single blinded hepatopathologist. Mice were subseguently classified into DM (diabetes mellitus), NAFL (nonalcoholic fatty liver) and NASH (nonalcoholic steatohepatitis) groups. Serum was analyzed for metabolic changes, and epididymal white adipose tissue (EWAT) was analyzed using gRT-PCR and western blotting for changes related to lipid metabolism, oxidative stress, and macrophage levels and activation status. Results: The histological features of human NASH were recapitulated in 70% of Leprdb/db mice after 5 or 10 weeks of HF diet. In contrast, mild steatosis developed in only 27% of chow-fed Leprdb/db mice. Mice with NASH had elevated serum aminotransferases, insulin, triglycerides, glucose, and cytokines (IL-6, TNFα and MCP-1), compared to DM controls. Adipose tissue hypoxia as indicated by increased levels of Hif1α mRNA (p=0.01) and protein (p<0.05) was evident in the epididymal adipose tissue of NASH mice relative to DM group. Lipid metabolism genes such as Dgat2, Scd1 and Cebp/α (all p<0.05) were down regulated significantly in EWAT of NASH mice relative to DM. Relative mRNA expression of macrophage markers, CD68 and F480, and M1 inflammatory mediators, MCP-1 and TNFα; and protein levels of mac-2, an activated pan-macrophage marker, were found to be significantly upregulated (all p<0.05) in NASH mice relative to DM in the adipose tissue of these mice. Conclusions: We have developed an animal model that recapitulates the histological features of human NASH with ballooning and inflammation by feeding a diet rich in omega-6 fatty acids to Leprdb/db mice. Adipose tissue dysfunction characterized by hypoxia, inflammatory macrophage infiltration, and impaired lipid metabolism propels the development of NASH in this model. These findings highlight the role of adipose tissue dysfunction as an important mechanism contributing to NASH pathogenesis.


Kris V. Kowdley - Advisory Committees or Review Panels: Abbott, Gilead, Merck, Novartis, Vertex; Grant/Research Support: Abbott, Beckman, Boeringer Ingelheim, BMS, Gilead Sciences, Ikaria, Janssen, Merck, Mochida, Vertex

The following people have nothing to disclose: Priya Handa, Vicki MorganStevenson, James E. Nelson, Bryan D. Maliken, Matthew M. Yeh, Clinton T. Elfers, Christian Roth


Interleukin-33 break host immune tolerance via TFH cells in hepatitis B virus infection

Pingwei Zhao1, Xiguang Sun1, Cong Li1, Yanfang Jiang1,2

1First hospital, Jilin University, Changchun, china; 2Key Laboratory of Zoonosis Research, Ministry of Education, Jilin University, Changchun, China

Background: Chronic hepatitis B (CHB) patients are immunotolerant to the degree that they do not elicit an anti-HBV response sufficient to clear the virus or destroy infected cells. Our previous study has reported that IL-33 was an important factor in the pathogenesis and immune response of CHB. But it was still not clear how IL-33 played a role in HBV. The aim of this study was to investigate the effects of IL-33 on HBV in vivo and in vitro. Method: After stimulation of IL-33 in three different concentrations (Oμg/ml, 1μg/ml, 100μg/ml), the HepG2.2.15 RNA was extracted and the gene expression data were analyzed via Illumina's GenomeStudio Gene Expression Module. Three groups of mice were involved in the in vivo experiment: normal C57BL/6 (control), C57BL/6 HBV transgenic mice (Tg), and IL33 treated HBV-tg mice by intra-abdominal injection twice a week. The concentrations of serum HBV loads, ALT, AST and hepatic HBsAg, HBeAg, HBsAb and HBeAb in the HBV-tg mice were determined. Liver sections of HBV-tg mice were stained by anti-HBsAg, anti-HBsAb, anti-HBeAg, anti-HBeAb, and examined microscopically. The freguency of CD4+CXCR5+ TFH cells in the spleen and liver of HBV transgenic (HBV-tg) mice and Wild Type (WT) mice were characterized by flow cytometry analysis. Results: IFR4, AICDA and PRDM1 gene expressions were progressively increased and BCL-6 gene expression progressively decreased. In addition, CXCR5 and IL-21 gene expressions were increased. The Gene expression data revealed that IL-33 promoted the differentiation of B cells into Plasma cells. Moreover, the study found that IL-33 treatment obviously reduced the concentrations of serum HBV loads in vivo, but increased the concentrations of hepatic HBsAb, HBeAb in HBV-tg mice. It is also found that the freguencies of splenic and hepatic CD4+CXCR5+ TFH cells in HBV-tg mice treated with PBS were significantly higher than that of WT mice, but reduced significantly after IL-33 treatment. The concentrations of serum ALT were negatively correlated with the percentages of CD4+CXCR5+TFH cell expression in IL-33 HBV-tg mice. Conclusions: IL-33 could effectively break the host immune tolerance and induce HBeAb and HBsAb secretion via TFH cells in HBV-tg mice.


The following people have nothing to disclose: Pingwei Zhao, Xiguang Sun, Cong Li, Yanfang Jiang


Prednisolone treatment delays liver repair in hepatotoxininduced hepatitis by targeting glucocorticoid receptors in macrophages and neutrophils

Young-Suk Won, Hyo-Jung Kwon, Ogyi Park, Dechun Feng, Bin Gao

Laboratory of Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Rockville, MD

BACKGROUND & AIMS: Prednisolone is a corticosteroid that has been used to treat inflammatory liver diseases, such as autoimmune hepatitis and alcoholic hepatitis. However, the results have been controversial, and how prednisolone affects liver disease progression remains unknown. METHODS: In the current study, we examined the effect of prednisolone treatment on several models of liver injury, including T/NKT cell hepatitis induced by concanavalin A (ConA) and α-galactosylceramide (α-GalCer) and hepatotoxin-mediated hepatitis induced by carbon tetrachloride (CCl4). RESULTS: Prednisolone administration attenuated ConA- and α-GalCer-induced hepatitis and systemic inflammatory responses. Treating mice with prednisolone also suppressed inflammatory responses in a model of hepatotoxin (CCI4)-induced hepatitis, but surprisingly exacerbated liver injury and delayed liver repair. Immunohistochemical and flow cytometric analyses demonstrated that prednisolone treatment inhibited hepatic macrophage and neutrophil infiltration in CCl4-induced hepatitis and suppressed their phagocytic activities in vivo and in vitro. Macrophage and/or neutrophil depletion aggravated CCl4-induced liver injury and impeded liver regeneration. Finally, conditional disruption of glucocorticoid receptor in macrophages and neutrophils abolished prednisolone-mediated exacerbation of hepatotoxin-induced liver injury. CONCLUSIONS: Prednisolone treatment prevents T/NKT cell hepatitis but exacerbates hepatotoxin-induced liver injury by inhibiting macrophage- and neutrophil-mediated phagocytic and regenerative functions. These findings may not only increase our understanding of the steroid treatment mechanism but also help us to better manage steroid therapy in liver diseases.


The following people have nothing to disclose: Young-Suk Won, Hyo-Jung Kwon, Ogyi Park, Dechun Feng, Bin Gao


Patients with severe alcoholic hepatitis have expanded CD14+CD16+ monocytes with an anti-inflammatory phenotype

Ashwin D. Dhanda1,2, Baoying Liu3, Robert Nussenblatt3, Richard W. Lee1,Peter Collins2

1Department of Live; Medicine, University Hospitals Bristol NHS Foundation Trust, Bristol, United Kingdom; 2School of Clinical Sciences, University of Bristol, Bristol, United Kingdom; 3National Eye Institute, National Institutes of Health, Bethesda, MD

Background and Aim Alcoholic hepatitis (AH) is a serious complication of alcohol misuse and is associated with a high mortality of 40%. In AH, inflammation in the liver is triggered by activation of monocytes leading to tissue damage from proinflammatory T cells. Monocytes interact with other immune cells and have the capability to drive T cell proliferation and polarisation. We therefore aimed to characterise different subsets of monocytes in patients with AH and investigate their role in shaping T cell phenotype. Methods Peripheral blood was drawn from 26 healthy volunteers (HV) and 10 patients with severe AH at initial presentation. PBMCs were isolated by density gradient centrifugation, and monocytes stained with a broad panel of cell surface markers (CD14, CD16, CCR2, CCR5, CX3CR1, CD206, CD80 and HLA-DR) and fluorescence intensity determined by flow cytometry. In addition PBMCs were sorted by FACS into CD14+CD16-, CD14+CD16+, CD14IoCD16+ monocytes and memory CD4+ T cells. RNA was extracted from each subset and analysed for cytokine expression. Also, each subset was cultured for 5 days with CFSE labelled memory T cells and the T cell proliferation determined by the proportion of CFSEIo and phenotype by interleukin (IL)−17 and interferon gamma (IFNγ) production by flow cytometry. Results CD14+CD16+ monocytes are significantly expanded in patients with severe AH compared to HV (17.4% v 7.0% total monocytes; p<0.0001). Compared to CD14+CD16- monocytes this subset expressed more IL-10 (3.1 fold difference; p=0.02) and less TNFα (0.6 fold; p=0.002) mRNA and, after LPS stimulation, less IL-1β (0.6 fold; p=0.007) and IL-8 (0.4 fold; p=0.02) protein by flow cytometry. In AH patients this subset had significantly higher CCR5 (p=0.0004) and HLA-DR (p=0.005) expression than CD14+CD16- monocytes. Compared to HV CD14+CD16+ monocytes, this subset in AH patients have greater CCR2 (p=0.049) and CD206 (p=0.006) expression. In HV co-cultures CD14+CD16- monocytes preferentially drive T cell proliferation (38% v 29%; p=0.009) and IL17 production (12.5% v 9.0%; p=0.0004) while IFNγ was similar (26% v 29%; p=0.14) compared to CD14+CD16+ monocytes. Conclusion For the first time we have demonstrated an expanded population of CD14+CD16+ monocytes in AH. Higher HLA-DR and CCR2 expression suggest greater activation and improved recruitment to sites of inflammation; more CD206 and IL-10 mRNA and less pro-inflammatory cytokine production suggest they are alternatively activated (anti-inflammatory). In addition the CD14+CD16+ subset are less able to drive T cell proliferation and fail to induce the Th17 phenotype.


The following people have nothing to disclose: Ashwin D. Dhanda, Baoying Liu, Robert Nussenblatt, Richard W. Lee, Peter Collins


Type III IFN Levels Correlate with Differential Expression of IFN-Stimulated Genes during Acute HCV Infection

Marion Depla1, Camille Brunaud1,2, Julie Bruneau1,3, Naglaa H. Shoukry1,4

1Hopital Saint Luc, CRCHUM, Monteal, QC, Canada; 2Département de microbiologie et immunologie, Faculté de Médecine, Université de Montreal, Montreal, QC, Canada; 3Départemernt de médecine familiale, Faculté de Médecine, Universite de Montreal, Montreal, QC, Canada; 4Departement de médecine, Faculté de Médecine, Universite de Montréal, Montreal, QC, Canada

Background: Single nucleotide polymorphisms (SNPs) (rs12979860) located in the region of the IL28B gene encoding the type Ill interferon, IFN-λ3, can predict spontaneous resolution of acute hepatitis C virus (HCV) infection. The favourable genotype is defined as CC and the unfavourable genotype as CT or TT (*T). The predictive value of this SNP is enhanced when combined with polymorphisms in the killer cell immunoglobulin-like receptor (KIR) genes controlling the activity of natural killer (NK) cells and when combined with plasma levels of the interferon gamma-induced protein 10 (IP-10), a chemokine that regulates lymphocyte migration to different tissues. This suggests that the IL28B polymorphism may act at the level of NK cell activation and lymphocyte recruitment. Hypothesis: We hypothesized that polymorphism in the IL28B region may modulate the expression of type Ill IFNs during acute HCV, that may in turn modulate the induction and cross-talk between innate and adaptive immunity including the induction of innate immune genes, as well as activation and recruitment of NK cells and T cells to the liver. Methods: We performed longitudinal guantification of type Ill IFNs (IL28B, IL29) by ELISA in plasma samples collected from a cohort of injection drug users (n=31)during acute HCV infection that progressed to either spontaneous resolution or chronic infection. We used gRT-PCR to monitor expression of genes previously associated with response to interferon following HCV infection in in vitro models (IFI6, IFIT1, Mx1, USP18, IP-10) and with NK cell activity (NKp30, CD94, NKG2A, NKG2D). Results: Plasma levels of IL29 were higher than those of IL28B in all HCV infected individuals (p=0.0426) irrespective of infection outcome. In chronic individuals carrying the favourable genotype, both IL28B and IL29 plasma levels correlated positively with expression of USP18 (p=0.0213 and p=0.0091, respectively), a negative regulator of the IFN signaling pathway, and with expression of the inhibitory NK cell receptor NKG2A (p=0.0148 and p=0.0379, respectively). In chronic individuals carrying the unfavourable genotype, we observed a positive correlation between IL29 plasma levels and expression of the activating ISG IFIT1 (p=0.0058) and with expression of the NK cell activating receptor NKG2D (p=0.0238). In contrast, in individuals who resolved spontaneously we observed a negative correlation between IL28B plasma levels and expression of IFIT1 (p=0.0415) and IFI6 (p=0.0152). Conclusion: These results suggest that type Ill IFN levels and IL28B genotype may modulate induction of ISGs and pathways associated with regulation of NK cells activity during acute HCV.


The following people have nothing to disclose: Marion Depla, Camille Brunaud, Julie Bruneau, Naglaa H. Shoukry


Aspartic Acid Protects from TLR4 and NLRP3 Inflammasome Dependent Hepatitis via NMDA Receptor and beta-arrestin 2

Ahmad Faroog, Rafaz Hogue, Ahsan Faroog, Md Kaimul Ahsan, Ayaz Ghani, Xinshou Ouyang, Wajahat Z. Mehal

Disgestive Diseases, Yale University, New Haven, CT

Background: TLR4 and NLRP3 inflammasome activation are responsible for many inflammatory liver disease but little is known about their regulation. The NMDA receptor is known to be present on macrophages and its role in immune regulation has not been investigated. We used the NMDA ligand aspartic acid (AA) to test the role of NMDA activation in liver inflammation. Aims: To test if AA can modulate TLR4 and NLRP3 inflammasome signaling and liver injury via its known NMDA receptor. Methods: The NLRP3 inflammasome was activated by LPS and ATP in primary mouse macrophages, Kupffer cells and human peripheral monocytes in the presence and absence of AA and production of pro-II1 beta and IL-1 beta assayed. NMDA receptor and beta-arrestin 2 dependence of AA effects was examined in the RAW 264.7 cells using siRNA knockdown. AA was supplemented in vivo in the presence or absence of beta-arrestin 2 knockdown in the LPS/d-Gal hepatitis and acetaminophen hepatotoxicity. Liver tissue was examined for injury and inflammation by histological grading and serum transaminases. Results: AA suppresses in vitro TLR4 and NLRP3 inflammasome dependent inflammation in human peripheral monocytes, mouse peritoneal macrophages and Kupffer cells as assessed by levels of pro-II1 beta and IL-1 beta. AA immunosuppressive effects reguire NMDA and beta-arrestin 2. In vivo AA supplementation decreases liver inflammation and injury in the LPS/d-GaIN hepatitis (hemorrhage 1.03 /0.3 versus 3.89 +/- 0.2, ALT 744 +/- 406 versus 12560 +/5295, P < 0.05), and acetaminophen hepatotoxicity (necrosis 0.1 +/- 0.1 versus 1.4 +/- 0.1, hemorrhage 1.77 +/- 0.2 versus 2.5 +/- 0.6, liver transcript for pro-IL-1 beta and Nlrp3 caspase 1 and serum IL-1 beta release, P < 0.01). AA induced in vivo protection is dependent on NMDA and beta-arrestin 2. Conclusions: Aspartic Acid acts through NMDA and betaarrestin 2 to suppress TLR4 and NLRP3 mediated pro-inflammatory signaling and hepatitis. Aspartic acid has potential as a therapeutic agent in the treatment of acute liver failure. Keywords: Aspartic Acid, acute liver injury, inflammasome, acetaminophen Supported by NIH R01DK076674-02, vA Merit award and Yale Liver Center


Wajahat Z. Mehal - Management Position: Gloabl BioReserach Partners

The following people have nothing to disclose: Ahmad Faroog, Rafaz Hogue, Ahsan Faroog, Md Kaimul Ahsan, Ayaz Ghani, Xinshou Ouyang


PD-L1+MDSCs were induced by hepatocellular carcinoma: ex vivo and in vitro analysis

Tomoaki Iwata, Yasuteru Kondo, Osamu Kimura, Takayuki Kogure, Eiji Kakazu, Masashi Ninomiya, Tatsuki Morosawa, Yasuyuki Fujisaka, Tooru Shimosegawa

Tohoku University, Gastroenterology, Sendai, Japan

Background: Hepatocellular carcinoma (HCC) is the fifth-most common cancer in the world and globally the third highest cause of cancer-related mortality. Various types of immunosuppressive networks exist in the microenvironment of HCC. Recently, it has been reported that myeloid-derived suppressor cells (MDSCs) suppress the function of NK cells and tumor-specific T cells in the tumor-bearing host. In patients with hepatocellular carcinoma, MDSCs are described as CD33+HLA-DRlowCD11b+CD14+ cells. We showed that MDSCs expresses surface PD-L1 molecules in peripheral blood mononuclear cells (PBMCs) from patients with HCC. The aim of this study is to analyze in ex vivo and in vitro the relationship between HCC and PD-L1+ MDSCs. Material and method: (i) We harvested the blood from 19 healthy donors and 54 patients with various stages of HCC who were hospitalized in our institute, and subjected them to multicolor flow cytometric analysis (FACS) of the percentages of PD-L1+MDSCs. (ii) We collected tumor infiltrating lymphocytes (TILs), normal liver infiltrating lymphocytes (LILs) and PBMCs from HCC patients who were seen in our Hospital. The percentage of PD-L1+MDSCs was analyzed by multicolor FACS analysis. (iii) In order to investigate the function of soluble factor from HCC, we used a trans-well co-culture system with PBMCs and several different liver cancer cell lines such as HepG2, Huh7, Hep3B, Li7, PLC. After 72 hours co-incubation, multicolor FACS analysis was performed. Results: (i) Our results demonstrated that PBMCs from HCC patients contained significantly higher percentages of PD-L1+MDSCs in comparison to those from healthy subjects. PBMCs from TNM IV HCC patients had significantly higher percentages of PD-L1+MDSCs in comparison to those of TNM I, II and III HCC patients. However, this increase was not correlated with the Child-Pugh grade, tumor size, tumor number or serum concentrations of cancer biomarkers (AFP and PIVKA-II). (ii) TILs contained remarkably high percentages of PD-L1+MDSCs in comparison to those of PBMCs and LILs from HCC patients. (iii) After 72 hours co-incubation with the liver cancer cell lines, the percentages of PD-L1+MDSCs were significantly increased compared with control. Conclusion: The differentiation of PDL1+MDSCs was induced by soluble factor from hepatocellular carcinoma, and the peripheral blood from HCC patients had significantly higher percentages of PD-L1+MDSCs in comparison to those of healthy subjects. These results suggest that we might use PD-L1+MDSCs as a new biomarker of hepatocellular carcinoma.


The following people have nothing to disclose: Tomoaki Iwata, Yasuteru Kondo, Osamu Kimura, Takayuki Kogure, Eiji Kakazu, Masashi Ninomiya, Tatsuki Morosawa, Yasuyuki Fujisaka, Tooru Shimosegawa


Lectin pathway proteins are associated with survival in patients with acute liver failure

Tea L. Laursen1, Thomas D. Sandahl1, Sidsel Støy1, Frank V. Schioedt2, William M. Lee3, Steffen Thiel4, Henning Grønbæk1;

1Medicine V - Hepatology and Gastroenterology, Aarhus University Hospital, Aarhus C, Denmark; 2Gastroenterology and Endocrinology, Bispebjerg Hospital, Bispebjerg, Denmark; 3Division of Digestive and Liver Diseases, University of Texas, Southwestern Medical Center, Dallas, TX; 4Biomedicine - Medical Microbiology and Immunology, Aarhus University, Aarhus, Denmark

Background: The complement system is activated in several liver diseases including acute liver failure (ALF). Previous studies have focused on the classical and alternative pathway of the complement system, but the role of the lectin pathway has never been investigated in the course of ALF. The lectin pathway is initiated by liver-derived soluble pattern recognition molecules, i e. mannan-binding lectin (MBL), H- and L-ficolin and CollectinLiver 1 (CL-L1) and by M-ficolin that originates from granulocytes and monocytes. We hypothesized that the lectin pathway is involved in the massive hepatic injury by activation of the complement system as demonstrated in other liver diseases. Therefore, we aimed to investigate the lectin levels in patients with ALF and their associations with clinical outcome. Methods: Serum samples from 75 patients prospectively enrolled in the U. S. ALF Study Group were collected on days 1 and 3 and 75 healthy blood donors were included as controls. MBL, H- and M-ficolin and CL-L1 levels were guantified by time-resolved immunofluorometric assays. L-ficolin levels were guantified by ELISA. Results: At day 1, the MBL level in the ALF patients was nearly 40% lower compared to controls (median (interguartile range)) (718.0 ng/ml (910.6) vs.1154.0 ng/ml (1928.0) (p=0.02)). The MBL level increased over time and was significantly higher by day 3 (832.3 ng/ml (940.5) (p=0.01)). Patients with a superinfection had twice as high MBL levels as non-infected patients by day 3 (p=0.02). The level of M-ficolin was decreased with more than 60% at day 1 compared to controls (540.7 ng/ml (501.9) vs.1484.6 ng/ml (1012.8) (p<0. 0001)). In contrast, the level of CL-L1 at day 1 was slightly higher in the ALF patients than in controls (1229.3 mU/ml (913.3) vs.1014.4 mU/ml (276.4) (p=0.11)); this difference was significantly greater at day 3 (1286.9 mU/ml (708.7) p=0.006)). The levels of H- and L-ficolin were similar to the controls. ALF patients with spontaneous survival had a higher level of MBL at day 1(955.8 ng/ml (1146.4) vs.596.2 ng/ml (601.3) (p=0.02)) and a lower level of L-ficolin by day 3 (1611.8 ng/ml (1193.8) vs.2171.4 ng/ml (2193.5) (p=0.02) compared to patients who died or were transplanted. Conclusion: We observed significant differences in both the liver- and monocyte/granulocyte-derived lectin levels in ALF patients compared to controls. We demonstrated impact on the lectin pathway in ALF, which may suggest that this pathway is involved in the pathogenesis of ALF. A low level of MBL and a high level of L-ficolin were associated with fatal outcome and might be used with other parameters to determine prognosis.


William M. Lee - Consulting: Eli Lilly, Novartis; Grant/Research Support: Gilead, Roche, Vertex, BI, Anadys, BMS, merck; Speaking and Teaching: Merck

Henning Grønbæk - Advisory Committees or Review Panels: Novartis; Grant/Research Support: NOVO Nordisk; Speaking and Teaching: Eli Lilly, Ipsen

The following people have nothing to disclose: Tea L. Laursen, Thomas D. Sandahl, Sidsel Støy, Frank V. Schioedt, Steffen Thiel


Role of NAD(P)H oxidases in the RIG-I/MDA5-mediated interferon response in hepatocytes

Seung Bum Park, Jinah Choi

University of California, Merced, Merced, CA

The first line of defense against viruses largely depends on restriction of virus replication by pathogen recognition receptors such as retinoic acid inducible gene inhibitor (RIG-I) and melanoma differentiation associated gene 5 (MDA5) that transmit signals to induce antiviral genes. Although recent studies have increased our understanding of how antiviral signaling mechanisms operate in various systems, much remains to be known concerning molecular mechanisms involved in the transduction of signals from viral recognition to antiviral gene expression. Aside from the established roles in innate immune functions of macrophages and other cells, NAD(P)H oxidase family enzymes, which consists of Nox and Duox proteins, function as an important source of regulated production of reactive oxygen species (ROS) in cell signaling and regulation of gene expression. Previous studies indicate that Nox enzymes play an important role in viral infections. In this study, we show that a viral pathogen associated molecular pattern (PAMP) and a synthetic double strand RNA, poly(IC), which are recognized by RIG-I and/or MDA5, elevate Nox1, Nox4, Nox5, Duox1, and Duox2 mRNA levels in Huh7 human hepatoma cells. Nox/Duox mRNAs were also increased by poly(IC) in telomerase-reconstituted primary human hepatocytes. Furthermore, IFNgamma but not IFNalpha increased Nox5 mRNA in Huh7 cells, particularly in the presence of hepatitis C virus. The PAMPstimulated increases in IFNbeta1 could be modulated by diphenyleneiodonium, an inhibitor of flavoproteins such as Nox. Therefore, Nox/Duox enzymes are regulated by intracellular RNA PAMP in hepatocytes. These data suggest that Nox/Duox enzymes function in the RIG-I/MDA5-mediated IFN signaling in host innate immune response during viral infections.


The following people have nothing to disclose: Seung Bum Park, Jinah Choi


The tricarboxylic acid (TCA) cycle of dendritic cells was disturbed by imbalance of plasma amino acid observed in cirrhosis

Eiji Kakazu1,2, Yasuteru Kondo2, Takayuki Kogure2, Masashi Ninomiya2, Osamu Kimura1,2, Tatsuki Morosawa2, Tomoaki Iwata2, Yoshiyuki Ueno3, Tooru Shimosegawa2

1Community Medical Supports, Tohoku Medical Megabank Organization, Tohoku University, Aoba-ku Sendai, Japan; 2Gastroenterology, Tohoku University Graduate School of Medicine, Sendai, Japan; 3Gastroente terology, Faculty of Medicine, Yamagata University, Yamagata, Japan

Background/Aims: An imbalance of plasma amino acids is commonly observed in patients with cirrhosis. Previously, we reported that an imbalance in the plasma amino acids of patients with advanced cirrhosis suppresses the maturation of dendritic cells (DCs) (J Immunol.2007, Hepatology.2009). The mechanisms that underlie these phenomena are largely unknown. The aim of this study was to investigate the influence of the extracellular amino acid imbalance observed in patients with cirrhosis on the function of DCs and on energy metabolism. Methods: We used serum-free culture media consistent with the average concentration of the plasma amino acids from a healthy volunteer (HCM) and that from patients with advanced cirrhosis (ACM). Monocyte-derived DCs (MoDCs) were generated from the CD14-positive monocytes with GM-CSF and IL-4 under these media. For examination of the DCs function, the phenotypes and mitochondrial membrane potential were determined by flow cytometry with JC-1 dye. Phagocytosis (uptake of fluorescent latex beads) and CCL19-chemotaxis were determined by Time-lapse fluorescence microscopy. The intracellular ATP level was measured by luminometric assay. The intracellular metabolites were determined by capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Tricarboxylic acid cycle (TCA cycle) and oxidative phosphorylation (OXPHOS) related genes were compared under each medium by real-time PCR. Furthermore, we compared these genes of CD1c+ DCs and CD14+ monocytes between healthy volunteers (n=4) and patients with advanced cirrhosis (Child-Pugh grade B or C; n=8). Results: The maturation (phenotype, IL12 production and CCL19 chemotaxis) and mitochondrial membrane potential of immature DCs were impaired under ACM in spite of the enhancement of mitochondrial respiratory chain complex genes. The amino acid consumption of DCs was significantly decreased but the glucose consumption of DCs was significantly increased under ACM. Metabolomics revealed that the tricarboxylic acid (TCA) cycle metabolites, fumarate, and 2oxoglutarate, were increased, and the nucleic acid metabolites were decreased in ACM. Consistent with the in vitro data, fumarate hydratase mRNA was higher, and succinate dehydrogenase complex subunit C and dihydrolipoamide S-succinyltransferase were lower in CD1c+ or CD14+ cells of cirrhotic patients than in healthy controls. Conclusions: An imbalance of the plasma amino acids in cirrhosis suppresses the maturation of dendritic cells by reducing the intracellular ATP due to interference with the mitochondrial TCA cycle, especially 2-oxogIutarate-succinate-fumarate transition.


Yoshiyuki Ueno - Advisory Committees or Review Panels: Jansen

The following people have nothing to disclose: Eiji Kakazu, Yasuteru Kondo, Takayuki Kogure, Masashi Ninomiya, Osamu Kimura, Tatsuki Morosawa, Tomoaki Iwata, Tooru Shimosegawa


Hepatitis C Virus Core protein inhibits Interferon production in plasmacytoid Dendritic Cells through suppression of Interferon Regulatory Factor 7 (IRF-7) protein

Amy Stone1,2, Angela Mitchell1,2, Daniel J. Miklin1, Lucy GoldenMason1,2, Hugo R. Rosen1,2

1GI/ Hepatology, University of Colorado Denver, Aurora, CO; 2lntegrated Department of Immunology, University of Colorado Denver and National Jewish Health, Denver, CO

Background Understanding the mechanisms of how HCV interacts with the Interferon (IFN) system may lead to novel treatments. Plasmacytoid dendritic cells (pDCs) act as sentinels for virus infections and make IFNs upon recognition of viral particles. pDCs are targeted by HCV resulting in reduced cell numbers and dysfunctional IFN production. We hypothesized that pDCs were targeted through HCV Core protein (Core) to disrupt IFN production, thus attenuating the pDC-driven IFN response during infection. Methods: Using a pDC cell line and recombinant Core we characterized the inhibition of Toll-Like Receptor (TLR) and RIG-I-Like Receptor induced IFN production by HCV Core protein. Results: Pre-treatment with Core blocks the induction of Type I and III IFN mRNA of GEN2.2-pDCs after TLR stimulation [Loxoribine - TLR7 agonist (IFNb p<0.0001) and CpGA - TLR9 agonist (IFNb p<0.05)], as well as the robust induction of IFN mRNA after stimulation with the pU/UC RNA (HCV 3' UTR RNA; Figure). Additionally, IFNa and TNFa protein production was significantly inhibited by Core pretreatment followed by stimulation with CpGA (IFNa p<0.001; TNFa p<0.05) or pU/UC RNA (IFNa p<0.05; TNFa p<0.001). IFNL1 levels were also inhibited by the presence of Core prior to stimulation (p<0.01). Exposure to Core prior to stimulation also induced a decrease of IRF7 (Western blotting; p<0.05) but not IRF3 protein levels. Conclusions: Core directly acts upon pDCs to induce reduced IFN mRNA levels following stimulation through TLRs via suppression of IRF-7. Through understanding how Core disrupts the pDC IFN response, we can develop novel methods of subverting the virus and inducing an antiviral state in patients.



The following people have nothing to disclose: Amy Stone, Angela Mitchell, Daniel J. Miklin, Lucy Golden-Mason, Hugo R. Rosen


Differentiational status of circulating B cells and their responses to the TLR9 signals are associated with chronic HBV infection at different clinical stages

Ruitian Yi, Hongli Liu, Yinghua Niu, Yu Zhang, Li Jin, Yingren Zhao

Infectious Diseases, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China

Background: The mechanism of how HBV clearance or persistence in HBV infection is not very clear. Although the role of T cells in host against HBV infection have been well studied, the contribution of B cells to HBV clearance is largely under investigated. Studies have showed that abnormal expression of TLR9 leads to dysfunction of immune cells in CHB patients. In order to explore the relationship between the B cell response to the TLR9 signaling and the clinic outcome of the disease, we systematically investigated the differentiational status of circulating B cells and their reponses to the TLR9 signals in CHB patients. Methods: We studied 111 CHB patients at different clinic stages (26 moderate-CHB, 23 severe-CHB, 28 liver cirrhosis (LC), 16 acute-on-chronic liver failure (ACLF), 19 HBV-related HCC and 20 healthy controls. PBMCs from the subjects were isolated, Bcell differentiational phenotypes and TLR9 expression of B cells were assessed by flow cytometry using antibodies specific for CD19, CD23, CD27, CD38 and TLR9. B cell activation associated markers (CD86, CD69, CD40) and chemokine receptor CXCR3 were also analyzed before and after stimulation with CpG ODN2006 as innate immunity signal. Results: The proportions of B cells and subsets (naïve, memory, plasma) as well as TLR9+B cells in ACLF patients were significantly lower than that in the other CHB patients and controls, and they were positively correlated with the levels of CD86 and CD40 on B cells. The ratios of CD86+ and CD40+ B cells in CHB patients (7.31 ±0.72%, 7.58±0.68%) were higher, although those in ACLF patients (2.4±0.22%, 2.9±0.3%) were significantly lower compared with that in the controls. The CD69 expression on B cells was decreased significantly in all the patients compared to the controls. After stimulating with CpG ODN2006, the population of TLR9+B cells was increased obviously in patients with severe-CHB (13.3% to 26.9%) and ACLF (4.0 % to 11%), and the expression of activity markers such as CD86 and CD40 was also increased correspondingly in LC and ACLF. We also noticed that the level of CXCR3 on B cells was significantly increased both before and after stimulation in ACLF patients (2.06% to 22.42%). The results suggest differentiational status of circulating B cells and the degree of inflammation are associated with the clinical stages in CHB patients. Conclusion: The TLR9 signaling mediates and regulates differentiational characteristics and expression of the activity markers of B cells in chronic HBV infection, which may be correlated to B cell dysfunction in CHB patients and implicate its potential as a therapeutic target for this disease.


The following people have nothing to disclose: Ruitian Yi, Hongli Liu, Yinghua Niu, Yu Zhang, Li Jin, Yingren Zhao


Single-nucleotide polymorphisms (SNPs) in IL18BP and IFN-γ genes as biomarker candidates for spontaneous HCV clearance prediction

Paolo Faria1, Fernanda Malta1, Ana Catharina S. Nastri1, Ketti G. Oliveira1, Flair J. Carrilho1, João R. Pinho1,2

1Gastroenterology, School of Medicine, University of São Paulo, São Paulo, Brazil; 2Albert Einstein Medicina Diagnóstica, Hospital Israelita Albert Einstein, São Paulo, Brazil

BACKGROUND AND AIMS: A hallmark of HCV infection is the extraordinary ability of the virus to persist in most of the infected people. Soluble immune factors or other biomarkers associated with acute HCV pathogenesis are not well defined and there is a lack on the knowledge about the early events in virus-host interactions that are keypoints indetermining spontaneous HCV clearance or the progression to chronic HCV infection. Interleukin (IL)−18 is a pleiotropic cytokine involved in both innate and adaptive immune responses that is widely expressed in monocytes/macrophages and Kupffer cells. Originally identified as an interferon (IFN-γ) inducing factor, it stimulates IFN-γ production in T lymphocytes and natural killer (NK) cells. IL-18 activity is partially determined by the action of an intrinsic inhibitor, IL-18 binding protein (IL18BP) which prevents IL18-induced IFN-γ production. The aim of this study was to determine the genotype of five SNPs located in the following genes: IL-18 (−607 C/A and −137 G/C), IL18BP (rs1541304 and rs2298455) and IFN-γ (+874 T/A). These genes participate in the immune response and are associated with the outcome of HCV infection. METHODS: Fifty-one individuals with spontaneous HCV clearance and 51 with chronic HCV infection were enrolled. DNA was isolated from peripheral blood of all individuals. Allelic discrimination was performed for all these genes using Tagman SNP genotyping assay on 7500 Fast Real-Time PCR System (Life Technologies, Foster City, CA, USA). RESULTS: SNPs located in IL-18 (−607 C/A and −137 G/C) and one SNP located in IL18BP (rs1541304) did not show differences in the genotypic profiles between the groups. On the other hand, rs2298455, located in the IL18BP gene, and the IFN-y (+8/4 T/A), showed significant differences in genotype freguencies between the two groups: the AA genotype of IL18BP gene (rs2298455) was more freguent in individuals with spontaneous HCV clearance, while most individuals with chronic HCV infection showed CC genotype (p< 0.0001; OR 21.14; IC 95% 5,487 to 81.47). Regarding the polymorphism in the IFN-γ gene, the AA genotype was observed in higher freguency in the group with spontaneous clearance, whilst the group of chronic HCV infection showed a higher freguency of genotype TA (p<0.0107; OR 0.307; IC 95% 0.1295 to 0.7308). CONCLUSION: Polymorphisms in both IFN-γ and IL18BP gene have been implicated in HCV infection and it is possible that variants in IL18BP could affect the activity or production of IL-18 and IFN-γ. Our study supports the role of IL18BP AA genotype (rs2298455) and the IFN-γ +874 AA genotype as genetic determinant of spontaneous HCV clearance.


João R. Pinho - Employment: Albert Einstein Medicina Diagn√≥stica

The following people have nothing to disclose: Paola Faria, Fernanda Malta, Ana Catharina S. Nastri, Ketti G. Oliveira, Flair J. Carrilho


Increased Acute Hepatitis in Axl and Mer Tyrosine Kinase Receptor Knockout Mice Through Up Regulation of TNF-α

Ayaz Ghani1, Ahmad Faroog1, Xinshou Ouyong1, Leonel Joannas2, Carla Rothlin2, Wajahat Z. Mehal1

1Department of Digestive Diseases, Yale University, New Haven, CT; 2Department of Immunobiology, Yale University, New Haven, CT

Background: Tyro-3, Axl, and Mer constitute the TAM family of receptor tyrosine kinases that are involved in multiple biologic processes including cell proliferation/survival, cell adhesion, migration, blood clot stabilization, and regulation of inflammatory cytokine release. Aim: To identify the role of TAM receptor tyrosine kinases and their downstream signaling in acute hepatitis. Methods: A single intra-peritoneal injection of LPS (4mg/kg) + D-Gal (250mg/kg) was given to WT, Tyro-3-/-, Axl-/-, and Mer-/- mice to induce acute hepatitis. Mice were euthanized 6 hrs post injection to collect liver tissue for histology and cytokine analysis and serum for cytokine analysis. For in-vitro analysis, peritoneal macrophages were collected 3 days after a single i. p injection of 4% thioglycolate from WT, Axl-/-, and Mer-/- mice. Cells were then treated with LPS (100 ng/ml) for 6 hrs to check cytokine analysis by guantitative PCR. Results: All three TAM receptors were expressed in the liver, with a highest level of expression in Kupffer cells. There was significantly more LPS/D-Gal induced inflammation and injury in the Axl-/- and Mer-/- mice (but not in Tyro-3-/ -) compared to WT as evidenced by hemorrhage scoring (3+/- 0.5 in Axl-/-, 2.9+/- 0.5 in Mer-/-, 1.5+/-0.5 in Tyro-3-/- vs 1.2+/-0.5 in WT), serum ALT (5000U/L +/-1000 in Axl-/-, 4500U/L +/1500 in Mer -/- 1500U/L +/- 500 in Tyro-3-/- vs 1200 U/L +/300 in WT), and serum IL-1β (700pg/ml +/- 200 in Axl-/-, 750+/-150 in Mer-/-, 500 +/-100 in Tyro-3-/- vs 450 pg/ml +/-100 in WT). The gene expression for pro-IL-1β, TNF-α, IL-6, and Nalp3 in whole liver was also significantly more in the Axl-/- and Mer-/- mice compared to WT (p < 0.05). There was also a significant increase in the transcript for TNF-α in the LPS treated peritoneal macrophages from Axl-/-, and Mer-/- mice compared to WT (p < 0.05). Conclusion: The TAM receptors Axl and Mer are highly expressed on Kupffer cells and limit the degree of acute liver injury by down-regulating pro-inflammatory cytokines including TNF-α


Wajahat Z. Mehal - Management Position: Gloabl BioReserach Partners

The following people have nothing to disclose: Ayaz Ghani, Ahmad Faroog, Xinshou Ouyang, Leonel Joannas, Carla Rothlin


Innate Lymphoid Cells (ILC) Modulate T Cell Response and Liver Injury in Viral Hepatitis

Yuejin Liang, Zuliang Jie, Jiaren Sun

Microbiology and Immunology, Univ of Texas Med Branch, Galveston, TX

Molecules containing damage-associated molecular patterns (DAMP) play an important role in many pathogenic processes. The purpose of the study was to investigate the role of IL-33, a DAMP molecule, in lymphocytic choriomeningitis virus (LCMV)induced liver inflammation. Method: Mice were infected with LCMV and immune responses were analyzed with flow cytometry and other technigues. Results: Infected animals exhibited a steadily increased IL-33 and its receptor ST2 expression in the liver during the first week of the infection. Treatment of exogenous IL-33 resulted in a great decrease in the serum alanine aminotransferase (ALT) levels and the number of Councilman bodies in the liver. Attenuated liver injury by IL-33 correlated with an increase in T regulatory (Treg) cells but with a decrease in macrophages, dendritic cells and NK cells in the liver. IL-33 enhanced both type 1 (IL-2 and IFN-γ) and type 2 (IL-5 and IL-13) immune responses in infected mice. However, IL-33 inhibited TNF-α expression in hepatic T cells and macrophages, and significantly reduced TNF-α levels in the liver. In addition to direct effects of IL-33, we found that IL-33 strongly induced novel innate lymphoid cells 2 (ILC2) in the liver and spleen of infected mice. When co-cultured with ILC2, hepatic T cells and macrophages expressed lower levels of TNF-α. In conclusion, this study reveals that IL-33 acts as a potent immune stimulator and a hepatoprotective cytokine in viral hepatitis. Its direct immunoregulatory functions and ability to induce novel ILC2 further suggest that it may be a potentially promising therapeutic candidate for the management of viral hepatitis.


The following people have nothing to disclose: Yuejin Liang, Zuliang Jie, Jiamen Sun


IFNα and IFNλ differentially regulate TLR-activated human macrophages

Rik A. de Groen, Arjan Boltjes, Jun Hou, Bisheng Liu, Harry L. Janssen, Andre Boonstra

Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, Netherlands

Associations between single nucleotide polymorphisms upstream of the interferon lambda (IFNλ)3 gene and spontaneous as well as therapy-induced clearance of the hepatitis C virus (HCV) increased interest in this particular family of cytokines. Although IFNλs have been extensively studied for their antiviral activity, there is limited knowledge of IFNλ's immunological functions and how they differ from other interferons. With pegylated-IFNλ1 being investigated as an alternative option to IFNα in the therapy of chronic HCV infection, the basic biology of IFNλ, specifically its effect on cell populations of the immune system, remains to be determined. As a first step to identify potential cellular targets of IFNλ, various human leukocyte populations were screened for expression of IFNλ receptor 1 (IFNλR1) using flow cytometry and real-time PCR analysis. Further assays were then performed on monocytederived and primary macrophages, one of the principal populations found to be positive for IFNλR1. Human monocytes, monocyte-derived macrophages, and Kupffer cells were stimulated with IFNα or IFNλ1 and further challenged with toll-like receptor (TLR) agonists. Modulation of the phenotype and function of each cell type was evaluated by flow cytometry, enzymelinked immunosorbent assay, and microarray transcriptional profiling. Analysis showed that B cells, T cells, and monocytederived macrophages were all positive for IFNλR1 mRNA expression, whereas NK cells, monocytes, and Kupffer cells showed limited or no expression. FACS analysis for IFNλR1 protein expression only showed positivity on monocyte-derived macrophages. In functional assays, IFNα and IFNλ1 differed in their ability to modulate the production of pro-inflamatory cytokines IL-12p40 and TNFα upon TLR-activation in monocytes and monocyte-derived macrophages. Monocytes, which lack IFNλ1 expression, were unaffected by IFNA1 stimulation, whereas macrophage TLR-induced IL-12p40 was enhanced in the presence of IFNλ1 and inhibited in the presence of IFNa. Kupffer cells, similar to monocytes, were unaffected by IFNλ1 stimulation alongside TLR challenge in their production of various pro-inflammatory cytokines. Microarray analysis of resting and TLR-challenged macrophages pretreated with IFNα or IFNλ1 showed distinct differences between these IFNs in the regulation of multiple members of the IL-12 family of cytokines, including the upregulation of EBI3, IL-27, and IL-12p40 expression by IFNλ. Further investigation of IFNλ's immunological distinctions may provide insight on its effects as a therapeutic, and the potential benefits or disadvantages it may have to conventional IFNα-based therapy.


Harry L. Janssen - Consulting: Abbott, Bristol Myers Sguibb, Debio, Gilead Sciences, Merck, Medtronic, Novartis, Roche, Santaris; Grant/Research Support: Anadys, Bristol Myers Sguibb, Gilead Sciences, Innogenetics, Kirin, Merck, Medtronic, Novartis, Roche, Santaris

Andre Boonstra - Grant/Research Support: BMS, Janssen Pharmaceutics, Merck

The following people have nothing to disclose: Rik A. de Groen, Arjan Boltjes, Jun Hou, Bisheng Liu


IL28B genotype is not associated with differential gene expression in a large cohort of patients without known HCV infection

Nikolaus Jilg1,Ida J. Hatoum1,2, Danielle M. Greenawalt3, Wenyu Lin1, Lee M. Kaplan1,2, Raymond T. Chung1

1GI Unit, Department of Medicine, Massachusetts General Hospital, Boston, MA; 2Obesity, Metabolism & Nutrition Institute, Massachusetts General Hospifol, Boston, MA; 3Merck Research Laboratories, Boston, MA

Background and Aims: Recent genome-wide association studies (GWAS) have demonstrated that genetic variation in the area of the gene for interleukin 28B (IL28B), a type Ill interferon (IFN), is a strong predictor for therapeutic response to IFN as well as for spontaneous clearance of hepatitis C virus (HCV). The underlying biological mechanism, however, has not been fully elucidated. In an attempt to better understand the biology of type Ill IFN, we sought to clarify whether or not the IL28B genotype is linked to differential gene expression in a large patient collective not known to be infected with HCV by using a comprehensive, unbiased transcriptome-wide association study. Methods: Samples were obtained from more than 1,000 patients with severe obesity during Roux-en-Y gastric bypass surgery at a single academic healthcare center. Total RNA was extracted from liver (n=651), omental (n=848) and subcutaneous adipose tissue (SCF; n=701), and DNA was isolated from the liver samples (n=950). RNA was reverse-transcribed to fluorescence labeled cDNA that was hybridized to custom 44K DNA oligonucleotide microarrays to assess gene expression. DNA samples were directly genotyped using the Illumina HumanHap 650Y BeadChip array. After strict guality control measures, 525,054 single nucleotide polymorphisms (SNPs) were available for analysis in 922 individuals. Results: We examined the retrieved transcriptomic data from liver, omental and SCF tissue for potential associations with the IL28B related genotype. In this large cohort, allelic distribution of the rs8099917 SNP, previously found to be highly predictive for the clinical outcomes in patients with hepatitis C, was not significantly associated with differential expression of any gene when using a p-value significance cutoff of 5 x 10-6. Moreover, we tested several other SNPs known to be in linkage diseguilibrium with rs8099917, none of them exhibited a significant correlation with differential gene expression. As a positive control, we examined the effect of rs6051702, a previously described marker for inosine triphosphatase (ITPA) deficiency, observing a significant association between this SNP and ITPA expression. Conclusions: In this cohort without known HCV infection, we were not able to detect a relationship between the IL28B genotype and gene expression in the liver or adipose tissue. These observations suggest that there is no physiological effect of the IL28B polymorphism in the absence of HCV infection. Given the powerful clinical impact of this polymorphism, it would be useful to determine its correlation with hepatic gene expression in a large cohort of patients infected with HCV.


Raymond T. Chung - Advisory Committees or Review Panels: Idenix; Consulting: Enanta; Grant/Research Support: Gilead, Merck, Mass Biologic, Gilead

The following people have nothing to disclose: Nikolaus Jilg, Ida J. Hatoum, Danielle M. Greenawalt, Wenyu Lin, Lee M. Kaplan


Worsening severity of cirrhosis is associated with increased toll-like receptor mediated inflammatory responses by monocytes

Rohf Sawhney1,2, Jessica Howell1,2, Adam Testro1, Norelle A. Skinner3, Peter W. Angus1,2, Paul Gow1,2, Kumar Visvanathan2,3

1Gastroenterology/Liver Transplant Unit, Austin Health, Melbourne, VIC, Australia; 2Medicine, The University of Melbourne, Melbourne, VIC, Australia; 3lnnate Immunity Laboratory, St Vincent's Hospital, Melbourne, VIC, Australia

Background: Toll-like receptors (TLRs) are a critical component of the innate immune response to infections and inflammation. Patients with cirrhosis are at increased risk of infection, leading to systemic inflammation, decompensated liver disease and death. Changes in TLR mediated immune function could contribute to the altered inflammatory responses and worse clinical outcomes seen in more advanced cirrhosis. The aim of this study was to investigate changes in TLR2 and TLR4 mediated immune function with increasing severity of cirrhosis. Methods: Peripheral blood was collected from 31 patients with cirrhosis of varying etiology and degree of severity according to ChildPugh (CP) classification (10 CP A, 10 CP B, 11 CP C; range 5-14) or Model of End Stage Liver Disease (MELD) score (range 6-26). Intracellular cytokine production of TNF and IL6 by CD14+ monocytes was measured by flow cytometry at baseline and following stimulation with TLR2 (P3C) and TLR4 (LPS) specific ligands. Results were compared with 5 healthy controls. Results: The fold increase in TNF and IL6 production by monocytes following both TLR2 and TLR4 ligand stimulation strongly correlated with increasing MELD score (TLR2 TNF p=0.0006, IL6 p=0.02; TLR4 TNF p=0.003, IL6 p=0.005). There was also a strong correlation between TLR2 and TLR4 mediated cytokine production and increasing CP score (TLR2 TNF p<0.0001, IL6 p=0.009; TLR4 TNF p=0.0003, IL6 p=0.016). TLR2 and TLR4 mediated TNF and IL6 production correlated with increasing bilirubin (TLR2 TNF p=0.0002, IL6 0.007; TLR4 TNF p=0.001, IL6 p=0.0009) and TNF production was inversely correlated with albumin (TLR2 p=0.0015, TLR4 p=0.002) but there was no association with creatinine levels. In comparison with healthy controls, patients with more advanced cirrhosis (CP C) had greater TLR2 and TLR4 mediated TNF production (TLR2 p=0.0015, TLR4 p=0.06). Conclusions: Severity of liver disease, measured by either MELD or CP score, strongly correlates with increased TLR2 and TLR4 mediated cytokine production by monocytes. Patients with advanced cirrhosis have an exaggerated inflammatory response to TLR2 and TLR4 stimulation compared with healthy controls. Increased TLR mediated responses to infection or injury may therefore contribute to dysregulated systemic inflammation and poor clinical outcomes in advanced cirrhosis.


Peter W. Angus - Grant/Research Support: Gilead Sciences

The following people have nothing to disclose: Rohit Sawhney, Jessica Howell, Adam Testro, Narelle A. Skinner, Paul Gow, Kumar Visvanathan


NKP30-B7-H6 recognition aggravates hepatocyte damage through up-regulation of interleukin-32 expression in hepatitis B virus-related acute-on-chronic liver failure

Yong Zou1, Jun-jie Bao2, Guo-Ying Wang1

1The Third Affiliated Hospital of Sun Yat-sen university, Guangzhou, China; 2Guangzhou Women and Children's Medical Center, Guangzhou, China

Background and aims: Through it is known that natural killer (NK) cells play a pivotal role in the pathogenesis of hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF), the underlying mechanisms remain obscure. In the present study, we aimed to discover the role of NKP30-B7-H6 recognition in liver NK cells-mediated hepatocyte damage in HBV-ACLF. Methods: Hepatic expressions of the NKp30 ligand B7-H6 and the proinflammatory cytokine interleukin-32 (IL-32) from patients with HBV-ACLF and mild chronic hepatitis B (CHB) were examined by immunochemistry staining. The cytotoxic activities of human natural killer cell line NK-92 against target cells (Huh-7, HepG2 and LO2) were evaluated by cell counting Kit-8 (CCK8) assay. IL-32 secreted by NK-92 cells was detected by intracellular cytokine staining. Results: Remarkably enhanced expression of hepatic B7-H6 and IL-32 was associated with the severity of liver injury in HBV-ACLF patients. In vitro cytotoxicity assays showed NKP30-expressing NK-92 cells robustly killed B7-H6 highly expressed hepatoma cell line Huh7, and this cytotoxic effect was remarkably inhibited by the soluble NKP30-Fc proteins. Furthermore, NK-92 cells exhibited marked elevated IL-32 expression when stimulated with anti-NKP30 antibody or co-cultured with Huh7 cells. Conclusion: NKP30-B7-H6 recognition aggravates hepatocyte damage through up-regulation IL32 expression in HBV-ACLF. This work was supported by National Natural Science Fund for Young Scholars of China (81000180).


The following people have nothing to disclose: Yong Zou, Jun-jie Bao, Guo-Ying Wang


Ribavirin does not alter NK cell function in patients with chronic hepatitis C

Antoaneta A. Markova1, Ulrike Mihm2, Eva Herrmann2, Birgit Bremer1, Sebastian Lunemann1, Verena Schlaphoff1,Thomas Berg3, Stefan Zeuzem2, Michael P. Manns1, Markus Cornberg1,Heiner Wedemeyer1

1Gastroenterology, Hepatology and Endocrinology, MHH Hannover, Hanover, Germany; 2Department of Medicine1, JW Goethe University Hospital, Frankfurt am Main, Germany; 3Department of Internal Medicine, Devision of Gastroenterology and Rheumatology, University of Leipzig, Leipzig, Germany

Background: Ribavirin (RBV) remains part of several interferonfree treatment strategies even though the mechanisms of action of RBV are still not fully understood. One hypothesis is that RBV increases responsiveness to type I interferons. Pegylated Interferon alpha (PEG-IFNα) has recently been shown to alter phenotype and function of NK cells which correlates with control of HCV infection. However, the effects of ribavirin alone or in combination with IFNα on peripheral NK cells are unknown. The aim of this study was to investigate the effects of ribavirin on NK cells in vitro and directly ex vivo in patients receiving RBV monotherapy, PEG-IFNα monotherapy or combination treatment. Methods: We first studied the effect of ribavirin on NK cell function in vitro by determination of degranulation (CD107 expression) and cytokine production after co-culture with K562 or Huh7.5 cells. In addition, extensive ex vivo phenotyping and functional analysis of NK cells was performed in 30 individuals with chronic hepatitis C infection at 10 timepoints (including day 2 of treatment). Patients were treated for 6 weeks with RBV monotherapy (n=11, group A), placebo (Group B, n=13) or PEG-IFNa-2a alone (group C n=6) followed by standard PEG-IFNa-2a/Ribavirin combination therapy. Results: In vitro, PEG-IFNα but not RBV stimulation of NK cells increased degranulation as well as IFN-γ and TNF expression after co-culture with target cells, which was not altered by additional RBV co-stimulation. Ex vivo in samples from treated patients, ribavirin monotherapy had no obvious effects on expression levels of CD56, NKp30, NKp46, NKG2A and CD57. In contrast, PEG-IFNa-2a therapy was associated with a relative increase of CD56bright cells, an activated NK cell phenotype and a loss of terminally differentiated NK cells. Increased functionality (degranulation, IFNg production and NKp30 expression) was associated with lower HCV viral loads. Some NK cell properties before therapy (enhanced expression of CD57 and lower NK cells cytotoxicity) correlated with subseguent anitiviral response. Conclusions: Antiviral activities of RBV alone or in combination with PEG-IFNα cannot be linked to obvious phenotypical or functional changes of NK cells. PEGIFNa activates NK cells possibly contributing to virological responses independently of RBV. The role of NK cells during future IFN-free combination therapies including RBV remains to be determined.


Thomas Berg - Advisory Committees or Review Panels: Gilead, BMS, Roche, Tibotec, Vertex, Jannsen, Novartis, Abbott, Merck; Consulting: Gilead, BMS, Roche, Tibotec; Vertex, Janssen; Grant/Research Support: Gilead, BMS, Roche, Tibotec; Vertex, Jannssen, Schering Plough, Boehringer Ingelheim, Novartis; Speaking and Teaching: Gilead, BMS, Roche, Tibotec; Vertex, Janssen, Schering Plough, Novartis, Merck, Bayer

Stefan Zeuzem - Consulting: Abbvie, Achillion Pharmaceuticals, Boehringer Ingelheim GmbH, Bristol-Myers Sguibb Co., Gilead, Novartis Pharmaceuticals, Merck & Co., Idenix, Janssen, Roche Pharma AG, Vertex Pharmaceuticals, Presidio, Santaris, Inc

Michael P. Manns - Consulting: Roche, BMS, Gilead, Boehringer Ingelheim, Novartis, Idenix, Achillion, GSK, Merck/MSD, Janssen, Medgenics; Grant/Research Support: Merck/MSD, Roche, Gilead, Novartis, Boehringer Ingelheim, BMS; Speaking and Teaching: Merck/MSD, Roche, BMS, Gilead, Janssen, GSK, Novartis

Markus Cornberg - Advisory Committees or Review Panels: Merck (MSD Germamny), Roche, Gilead, Novartis; Grant/Research Support: Merck (MSD Germamny), Roche; Speaking and Teaching: Merck (MSD Germamny), Roche, Gilead, BMS, Novartis, Falk

Heiner Wedemeyer - Advisory Committees or Review Panels: Transgene, MSD, Roche, Gilead, Abbott, BMS, Falk; Grant/Research Support: MSD, Novartis, Gilead, Roche, Abbott; Speaking and Teaching: BMS, MSD, Novartis, ITF

The following people have nothing to disclose: Antoaneta A. Markova, Ulrike Mihm, Eva Herrmann, Birgit Bremer, Sebastian Lunemann, Verena Schlaphoff


Differential expression of microRNAs and their role in the tolerogenic effects of hepatic plasmacytoid dendritic cells

Audrey H. Lau1,2, Liang Wei2, Xiumei Qu2, Sheri M. Krams2; 1Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, UCSF, San Francisco, CA; 2Surgery, Division of Transplantation, Stanford, Stanford, CA

Liver allografts are well tolerated and other solid organ allografts, such as small intestine (SI) and kidney, when transplanted concurrently with livers, show improved graft outcomes. However, the mechanisms underlying “hepatic tolerance” have yet to be elucidated. Previous data show that hepatic (H) dendritic cells (DC) can regulate immune responses and have diminished antigen presenting and immune stimulatory function compared with those in lymphoid tissue. Recent focus to explain this has been that functional differences between DC subsets including plasmacytoid (p)DC and myeloid (m)DC exist. It has been hypothesized that immature pDC are inherently tolerogenic. Indeed, recent data show that pDC play a unigue and important role in the generation of tolerance including a study examining patients rejecting SI transplant have a higher ratio of mDC to pDC, supporting a tolerogenic role for pDC. Our work confirms that hepatic pDC prolong cardiac allograft survival (p<0.01) in a murine model of cardiac transplantation compared to bulk and mDC. Using microarray analysis and confirmation with guantitative polymerase chain reaction we investigated changes in specific microRNA (mir) - short RNA molecules which can post-transcriptionally regulate messenger (m)RNA transcripts - in HpDC. mir-23b (p<0.01; Figure 1a) and −181a (p<0.005; Figure 1b) are significantly elevated in HpDC compared to HmDC and splenic DC (mDC and pDC). Using bioinformatic analysis, predicted mRNA targets (p < 0.05) for these microRNA include SP1, MAPK1, JAK1, and IL12B. These data suggest that therapeutics that enhance mir-23 and mir-181a expression or decrease expression of specific targets may promote tolerance.

Figure 1. Elevated mir-23b (A) and mir-181a (B) in hepatic plasmacyfoid (p) dendritic cells (DC) compared fo hepatic myeloid (m)DC and splenic DC. Immature pDC (CD11clowPDCA1+CD11b-) and mDC (CD11c+PDCA-CD11b+) from livers and spleens of C57BL/10J mice were flow-sorted fo >92% purity.

RNA was isolated using Invifrogen™ mirVana kif and guantitative polymerase chain reaction performed using primers from Applied Biosysfems. Results are from 5-9 samples with p values determined using Student's unpaired t-test.



Audrey H. Lau - Grant/Research Support: Salix Pharmaceuticals

The following people have nothing to disclose: Liang Wei, Xiumei Qu, Sheri M. Krams


Distribution of Natural Killer Cells in Liver Biopsies from Patients with Chronic Hepatitis C and Chronic Hepatitis B

Emel Ahishali1, Munhsetseg Banzragch2, Faruk Erdem Kombak3, Naziye Ozkan3, Ender G. Yegin2, Can Dolapcioglu1,Cigdem Ataizi Celikel3, Osman C. Ozdogon2

1Department of Gastroenterology Dr. Lutfi Kirdar Kartal Education and Research Hospital, Istanbul, Turkey; 2Department of Gastroenterology, Marmara University, Faculty of Medicine, Istanbul, Turkey; 3Department of Pathology, Marmara University, Faculty of Medicine, Istanbul, Turkey

Introduction: Innate immune response plays an important role in chronic hepatitis C (HCV) and chronic hepatitis B virus (HBV) infections. The aim of this study was to evaluate the distribution of natural killer (NK) cells in parenchymal and portal areas in liver in correlation with fibrosis development in patients with chronic HBV and HCV. Methods: Liver biopsies were obtained from 20 patients with chronic HCV and 19 patients with chronic HBV who had not received specific treatment. Normal liver tissue obtained from hepatic resections carried out for metastasectomy for colon cancer from 19 patients served as controls. Paraffin sections were immunostained for CD16, CD56, CD57 and IgG and the number of NK cells were counted in parenchymal and portal areas. The degree of fibrosis was also assessed according to Ishak scoring system. The mean numbers of NK cells in both hepatic regions in chronic HCV and HBV patients were compared with those in controls using one-way ANOVA test and the relationship between NK cell distribution and degree of fibrosis was evaluated by Spearman correlation test. Results: The mean numbers of NK cells positively stained for CD16, CD56 and IG-G in parenchymal and portal areas in patients with chronic HCV and HBV were significantly higher than those in controls, while immunostaining for CD57 yielded a significant increase only in parenchymal area (Table 1). A negative correlation was observed between the increase in the distribution of NK cells and degree of fibrosis by CD57 immunostaining in parenchymal areas in chronic HCV and HBV patients (rs:-0.404 and −0.589, respectively) while the relationship between these two parameters was found to be significant in patients with chronic HBV infection (p = 0.01). Conclusion: The distribution of NK cells in liver tissue during chronic HBC and HBV infections is not well known in humans. Our data suggest that the number of NK cells increases in parenchymal and portal areas in liver biopsies from chronic HCV and HBV patients.

The distribution of NK cells in liver biopsies with chronic HCV and HBV patients and control subjects

ControlChronic HCVChronic HBVP
Parenchymal Areas
Portal Areas
IgG4.35±4.0129.55±18.8432.21 ±17.030.000


The following people have nothing to disclose: Emel Ahishali, Munhsetseg Banzmagch, Famuk Erdem Kombak, Naziye Ozkan, Ende; G. Yegin, Can Dolapcioglu, Cigdem Ataizi Celikel, Osman C. Ozdogan