¹Section of Digestive Diseases, Yale University, New Haven, CT; ²2. Section of Digestive Diseases, Department of Veterans Affairs Connecticut Healthcare, West Haven, CT

Introduction: Inflammation is an important component of many forms of acute liver injury. Beta-hydroxybutyrate (BHB) and niacin are ligands for the plasma membrane receptor GPR109 which is expressed on Kupffer cells (KC). Some GPRs have immunomodulatory actions but a role for GPR109 in hepatic inflammation has not been investigated. Aim: Assess the role of GPR109 as a regulator of inflammation in acute liver injury, and the potential for therapy by its ligands BHB or niacin. Methods: WT (C57BL/6), Gpr109 null, and WT siRNA Gpr109 treated male mice were subject to LPS/d-Gal induced acute liver injury with/without sodium BHB or niacin (one i.p. dose 380 mcg and 30 mcg per g body weight respectively). WT mice were subject to APAP induced liver injury +/− saline, BHB, or niacin (two i.p. doses). Plasma ALT, H&E, neutrophil staining, liver inflammatory gene transcripts, and mortality were quantified. Inflammatory peritoneal macrophages and KC were isolated from WT and Gpr109 null mice and activated with LPS and ATP, +/− BHB or niacin. Pro-inflammatory gene transcripts, and IL1β release was quantified. NF-KB GFP reporter transgenic mice were treated with LPS and BHB or saline, livers isolated, and GFP expression quantified in KC. Results: Gpr109 null and Gpr109 siRNA treated WT mice had lethality from LPS/D-gal (5/7 and 6/6, respectively). No deaths in WT control, scramble siRNA treated animals (0/10 and 0/6, respectively). BHB or niacin versus saline supplementation protected WT mice from LPS/D-gal and APAP induced acute liver injury with reduced ALT (1010 +/− 905 and 1090 +/−1069 versus 8806 +/−1600 IU/L in APAP model P<0.05) and liver neutrophil infiltration (11.4 +/− 2.3 and 22.5 +/−5.8 versus 76.5 +/− 8.9 cells per 40 × magnified high power field P<0.05 in APAP model), as well as hepatic expression of nlrp3, casp1, and pro-il1 β. BHB did not protect Gpr109 null or Gpr109 siRNA treated mice. In macrophages, BHB and niacin dose dependently decreased LPS induced intracellular Pro- IL1β and IL1β release in a GPR109 dependent manner. BHB or niacin suppressed LPS mediated pro-inflammatory gene transcription in KC in vitro and in vivo in NF-KB GFP reporter mice. Conclusions: BHB and niacin have potent anti-inflammatory effects via the GPR109 plasma membrane receptor on Kupffer cells. This pathway is endogenously active, and can be additionally stimulated to provide hepatoprotection in a numbers of forms of liver disease.

¹Toronto Centre for Liver Disease, University Health Network, University of Toronto, Toronto, ON, Canada; ²Surgery, University Health Network, University of Toronto, Toronto, ON, Canada

Background Baseline upregulation of hepatic interferon-stimulated genes (ISGs) is associated with non-response to inter-feron-based antiviral therapy, however the cause of ISG preactivation remains unknown. Interferon lambda 4 (IFNL4) was recently described as a possible functional explanation for the association of the IL28B genotype and treatment response. Aim To determine whether expression of IFNL4 or other interferons correlates with intrahepatic ISG expression and treatment response. Methods RNA was extracted from pre-treatment liver biopsies from patients with known treatment outcome. Following DNAse 1 treatment, mRNA expression of IFN-alpha, IFN-beta, IFNL1 (IL29), IFNL2 (IL28A), IFNL3 (IL28B), IFNL4, the IFN-lambda receptor and selected ISGs (IP10, RSAD, IFI27, USP18, ISG15, MxA) were measured by qPCR. Genotyping for rs12979860 (CC vs non-CC) and ss469415590 (TT vs non-TT) was performed by sequencing. The correlation between expression of ISGs and the various interferons was assessed by the Spearman rank test and using non-hierarchical clustering. Results Of 66 patients, 17 (27%) achieved sustained virological response (SVR), 18 (28%) relapsed (REL) and 29 (45%) were non-responders (NRs) to peginterferon and ribavirin. The IFNL4 genotype was: TT (32%), TT/dG (50%) and dG/dG (18%). Although ISG expression was higher in future NRs than SVR/REL (p<0.0001), the expression levels of all interferons were similar by response. Similarly, ISG expression was higher in patients with a non-TT IFLN4 genotype but there were no differences in expression of interferons by genotype. There was no correlation between expression of ISGs and Type I interferons (alpha or beta). By cluster analysis, IL28a and IL29, but not IL28B, clustered with ISGs, but only in patients with the non-TT IFNL4 genotype or in NRs to therapy. Expression of IL28A and IL29 correlated most strongly with expression levels of ISG15 and UPS18, whereas there was a weak correlation between ISGs and IL28B. IFNL4 expression was very low in all patients and undetectable in 10 samples with no difference in expression level by IFNL4 genotype or treatment response. Expression of IFNL4 correlated with USP18 but only in patients with a non-TT IFNL4 genotype (non-TT: rho=0.48, p=0.0009 vs TT: rho=0.13, p=0.60) and in NRs (NRs: rho=0.58, p=0.0007, SVR/REL: rho=0.18, p=0.31). IFNL4 expression did not correlate with expression of other ISGs. Conclusion Despite similar levels of interferon expression, ISG preactivation appears to be driven by type III interferons, including IFNL4, but only in those with a non-TT IFNL4 genotype suggesting that it may be the functional explanation for the ‘IL28B’ genotype.

Disclosures:

Jordan J. Feld - Advisory Committees or Review Panels: Idenix, Merck, Janssen, Gilead, AbbVie, Merck, Theravance, Bristol Meiers Squibb; Grant/Research Support: AbbVie, Boehringer Ingelheim, Janssen, Gilead, Merck

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

The following people have nothing to disclose: Vera A. Cherepanov, Nicholas Anand, Sonya A. MacParland, Tawnya Hansen, Matthew Kowgier, Ian McGilvray

¹Liver Center of Excellence, Digestive Disease Institute, Seattle, WA; ²Benaroya Research Institute, Virginia Mason Medical Center, Seattle, WA

Background and Aim: We have previously demonstrated that hepatic iron overload in the reticuloendothelial system (RES) is associated with severe nonalcoholic steatohepatitis (NASH) and advanced fibrosis in nonalcoholic fatty liver disease. In this study, we hypothesized that iron loading of primary murine macrophages could influence their activation status and contribute to inflammation via promoting NF-KB activation and by diminishing M2 activation via impairing STAT6 activation. Methods: Mouse bone marrow derived macrophages (BMDM) were treated with either ferric ammonium citrate (FAC) alone, or cotreated with FAC/IL-4 (to prime macrophages for M2 polarization) for 4-24 hours to determine the effect of iron on iron homeostasis pathways, activation status of NF-KB, inducible nitric oxide synthase (iNOS) protein, phosphorylation of STAT-6 (a key regulator of M2 activation), and gene expression markers of proinflammatory (M1) or alternative (M2) pathways. Further, we assessed the effect of iron in concert with cytokines such as interferon-gamma, TNF-α or IL-1 β on M1 activation. Results: Treatment of BMDMs with iron led to an increased expression of iron homeostasis genes such as hepcidin, ferritin and ferroportin and downregulated the transferrin receptor gene expression. We assessed the effect of iron on M1 activation pathway. Iron-treated macrophages showed elevated levels of phospho-p65 (a subunit of NF-KB) and INOS protein and had increased gene expression of INOS, MCP-1, TNF-α, IL-6 and CD14. This effect could be reversed by Super Oxide Dismutase (SOD, an anti-oxidant), consistent with iron's known role as a pro-oxidant. We also observed increased levels of proinflammatory activation when iron was coadministered with cytokines such TNF-α or IL-1 β , leading to a synergistic upregulation of IL-6, TNF-α and iNOS genes. While IL-4 promoted activation of STAT6, iron treatment in conjunction with IL-4 decreased the phosphorylation of STAT6. It also reduced expression of IL-4-mediated upregulation of M2 markers such as Arginase-1, Ym1, Fizz-1/RELMα, TGF-β, IL-4R and Mgl-1. Interestingly, it also led to a downregulation in the fatty acid β-oxidation-related gene expression (the machinery that fuels M2 activation), evidenced by decreased PGC1β and Ppar gamma mRNA levels. Conclusions: Iron impacts macro-phage activation by enhancing NF-KB activation and promoting M1 activation, while impairing IL-4/p-STAT6-mediated M2 pathway in primary murine macrophages. This study highlights the direct role for iron in influencing critical innate M1 and M2 immune responses, providing useful insights into molecular pathways governing NASH pathogenesis.

Disclosures:

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

The following people have nothing to disclose: Priya Handa, Vicki Morgan-Stevenson

¹School of Natural Sciences, UC Merced, Merced, CA; ²School of Medicine, University of Alabama at Birmingham, Birmingham, AL; ³Department of Cardiovascular Physiology, Goethe Universität, Frankfurt am Main, Germany

Chronic Inflammation is an important factor in liver diseases. Toll-Like receptors (TLRs) play an important role in innate immunity, and TLR4 and intestinal microbiota have been associated with the promotion of hepatocellular carcinoma and other liver diseases. Studies indicate that TLR responses are modulated by NADPH oxidase (Nox) family enzymes in various cell types. Nox family enzymes consist of Nox proteins and dual oxidases (Duox) that catalyze the transfer of electrons from NAD(P)H to O₂ to produce reactive oxygen species (ROS). Nox enzymes are expressed in the liver, but whether these proteins function in TLR4 responses of hepatocytes is unknown. Therefore, we evaluated the role of Nox4 in TLR4 responses stimulated by lipopolysaccharides (LPS) in vitro, using Huh7 human hepatoma cells as well as hepatocytes isolated from control wildtype and Nox4 knockout (Nox4-/-) mice. In addition, hepatic responses to LPS were compared in the wildtype versus Nox4KO mice exposed to weekly i.p. injection of LPS (1mg/kg body weight) or saline only for up to six weeks. We found that LPS increased Nox activity and Nox4 as well as tumor necrosis factor alpha (TNFα) expression in Huh7 cells. In addition, diphenyleneiodonium, an inhibitor of Nox proteins and other flavoproteins, and Nox4 siRNA suppressed TNFα elevation induced by LPS in these cells. The LPS-induced TNFα elevation was MyD88-dependent. The LPS-stimulated increase in TNFα was also attenuated in primary hepatocytes isolated from Nox4KO mice, compared to control wildtype mice. In addition, LPS increased the level of proliferating cell nuclear antigen in Huh7 cells in a Nox4-dependent manner. With six weeks of repeated LPS stimulation in vivo, hepatic TNFα response also subsided more in the Nox4KO mice compared to the wildtype mice, such that TNFα mRNA was not elevated in the liver of LPS-injected Nox4KO mice compared to saline-injected controls, whereas the wildtype animals continued to show elevated TNFα with LPS at 24 hrs. Likewise, the LPS-stimulated increase in transforming growth factor beta1 mRNA in the liver was reduced in the Nox4KO group, compared to the wildtype control group. However, the effect of Nox4 deletion on in vivo LPS responses was time-dependent. Therefore, Nox4 mediates TLR4 responses in human hepatoma cells and murine hepatocytes in vitro. Nox4 may also play a role in hepatic responses to repeated LPS stimulation in vivo.

Disclosures:

The following people have nothing to disclose: Bhargav Koduru, Rui-Ming Liu, Nicole L. Corder, Katrin Schroder, Ralf P. Brandes, Jinah Choi

UMass Medical School, Worcester, MA

Purpose: MicroRNAs (miRNAs) are small non-coding regulatory RNAs that play an essential role in almost all cellular pro cesses. miRNA-155 is a multifunctional miRNA that regulates immune and non-immune biological processes. Previously, we showed increased expression of miR-155 in alcoholic liver disease (ALD), where it contributes to inflammation via TNF alpha. To elucidate the biological function of miR-155 in ALD, we employed miR-155 deficient (KO) mice. Methods: Wild type, WT (C57/BL6J) or miR-155 KO and TLR4 KO female mice (n=8-10) were fed the Lieber-DeCarli diet containing ethanol or control diet for 5 weeks. Results: We found that miR-155 KO mice were protected from alcohol-induced liver and intestinal inflammation. Alcohol metabolism was comparable between WT and miR-155 KO mice as measured by serum alcohol levels and hepatic Cyp2e1 expression. A significant attenuation in liver TNF alpha, IL-1 beta, and MCP1 was found in alcohol-fed miR-155 KO compared to WT mice administered with or without TLR4 ligand, LPS. Alcohol-induced increase in WT mice in hepatic CD68 (a macrophage activation marker) and MMP2, a remodeling protein, was dampened in miR-155 KO mice. Further, liver steatosis as evidenced by histological scores of H&E and Oil-Red-O staining was significantly decreased in alcohol-fed miR-155 KO compared to WT mice. Alcohol feeding resulted in a significant induction of oxidative stress (measured by TBAR assay) in the livers of WT mice, which was attenuated in miR-155 KO mice. Further, compared to WT, alcohol-induced decrease in hepatic miR-122 was prevented in miR-155 KO mice, suggesting a causative role for miR-155 in ALD. Remarkably, there was no increase in plasma endotoxin levels in miR-155 deficient mice after alcohol feeding, revealing intact gut barrier function. MiR-155 KO mice were also protected from alcohol induced increases in intestinal TNF alpha and NF-kappaB activation. Mechanistically, SHIP1, a miR-155 target that regulates inflammation was decreased in WT mice after alcohol feeding and this decrease in SHIP1 was prevented in miR-155 KO mice in the intestine. Also miR-155 KO mice were protected from alcohol-induced decrease in antimicrobial peptide, Reg3Beta in the intestine. Our results indicate that alcohol induces miR-155 via the TLR4 pathway. Compared to WT mice, TLR4 KO mice showed no increase in hepatic miR-155 expression after alcohol feeding. Conclusion: In conclusion, our results suggest that miR-155 plays an essential role in the gut-liver axis in ALD and that therapeutic inhibition of miRNA-155 might be an attractive strategy to ameliorate ALD.

Disclosures:

Gyongyi Szabo - Consulting: Idenix; Grant/Research Support: BMS, GSK, Conatus, Idera, Johnson&Johnson, Novartis, Ocera, Roche, Shering - Plough, Wyeth, Integrated Therapeutics, Idera The following people have nothing to disclose: Shashi Bala, Dora Lippai, Timea Csak, James V. Zatsiorsky, Donna Catalano, Karen Kodys

¹Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom; ²Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, United Kingdom

Background Mucosal associated invariant T (MAIT) cells represent 20% of CD3+ lymphocytes within the human liver. They are defined by an invariant T cell receptor (Va7.2+/Vb2 or 13.2) and innate-like activation by microbial-derived vitamin B metabolites presented by the highly evolutionarily conserved non-classical MHC class 1 related molecule (MR1). MAIT cells display marked cross-reactivity and express high levels of the multi-drug resistance protein 1 (MDR1). MAIT cells are resistant to and rapidly efflux chemotherapeutic MDR1 substrates such as duanorubicin. Tacrolimus, mycophenolic acid (MPA) and corticosteroids are all MDR1 substrates. The effect of these compounds on MAIT cell function, proliferation and apoptosis is not known. Aim The aim of this study was to establish the effect of exposure of MAIT cells in-vitro to tacrolimus, MPA and prednisolone on proliferation, apoptosis and innate effector function. Methods Peripheral blood mononucleocytes (PBMCs) from healthy donors obtained via NHS Blood and Transplant (Oxford, UK) were cultured in the presence of tacrolimus (20ng/ml;10ng/ml;5ng/ml), MPA (8mg/L;4mg/L;2mg/L) or prednisolone (100ng/ml;50ng/ml;25ng/ml) with cell trace violet (CTV) proliferation dye. On day 4 of culture a monocyte cell line (THP-1) was incubated over night with either formalin-fixed E.coli or a sterile control. On day 5 cultured PBMCs were either retained for annexin V/CTV staining or co-incubated for 5 hours with the E.coli exposed THP-1 line +/−anti-MR1 blocking antibody prior to intracellular cytokine staining and analysis by flow-cytometry. Results MAIT cells express very high levels of MDR-1 compared to non-MAIT T cell subsets (p<0.001). Amongst PBMCs MAIT cells uniquely produce IFNγ on co-culture with THP-1 monocytes pre-exposed to fixed E.coli in an MR1-dependent manner. Following 5 days of culture in the presence of tacrolimus, MPA and prednisolone such innate effector function of MAIT cells was maintained with no significant differences in IFNγ production observed on comparison between the drug treated, untreated and solvent-only treated controls. MAIT cells were equally inhibited in their ability to proliferate compared to other non-MAIT T cell subsets and underwent similar levels of apoptosis. Conclusions MAIT cells are an abundant hepatic human T cell subset and due to marked MR1-mediated cross reactivity may represent important mediators of allograft rejection. In this context it is a significant observation that their innate-effector function is maintained following exposure to agents used in standard immuno-suppression regimens.

Disclosures:

The following people have nothing to disclose: Joannah R. Fergusson, Lucy J. Walker, Paul Klenerman

Medicine, UMASS Med School, Worcester, MA

Introduction: Innate immune cells including monocytes and macrophages play an important role in the pathogenesis of chronic Hepatitis C Virus (HCV) infection. Pro-inflammatory cytokines; TNF-α, IL-α β and profibrotic cytokine, TGFβ are increased in chronic HCV patients. Though monocyte and macrophage activation contributes to the pathogenesis of HCV infection and liver fibrosis, mechanisms involved in the interactions between circulating monocytes/tissue macrophages, HCV and HCV-infected hepatocytes are not fully understood. We hypothesized that HCV signals circulating monocytes to differentiate into pathogenic, pro-fibrotic macrophages. Methods: Immunostaining of monocytes from HCV-infected patients and co-culture of healthy monocytes with HCV-infected Huh7.5 cells were performed. Results: In patients with chronic HCV infection we identified a unique population of circulating M2-po-larized monocytes expressing high levels of CD14, CD206, CD163 and collagen. The frequency of circulating pro-fibrotic CD14+ monocytes expressing M2 markers and collagen in HCV-infected patients positively correlated with the extent of liver fibrosis and with increased expression of M2 macrophage markers in the liver. To further gain mechanistic insights into the interactions of circulating monocytes and HCV, we performed long-term co-culture of healthy monocytes with HCV-infected Huh7.5 cells or HCV alone and observed an increased expression of collagen in the monocytes. Also we found that monocytes differentiated into macrophages in the presence of HCV and had increased expression of CD14, CD68 (macrophage markers), preferential expression of M2 markers (CD206, CD163 and DC-SIGN) and produced both pro- and anti-inflammatory cytokines. HCV-induced IL-1 β secretion promoted TGFβ production and polarization to an M2-like macrophage phenotype, which was prevented in the presence of the IL-1 receptor antagonist. Furthermore, IL-1 β stimulation, independent of HCV, led to polarization of monocytes to M2-like macrophages and increased expression of collagen. Monocytes stimulated with IL-1 β secreted large amounts of TGFβ that led stellate cell activation indicated by increased expression of collagen, α-SMA and TIMP-1. Conclusion: We identified the presence of a pro-fibrotic CD14+ monocyte population in the circulation of HCV-infected patients with liver fibrosis. These monocytes display the phenotypic characteristics of fibrocytes and alternatively activated M2 macrophages. We show that HCV induces monocyte differentiation into macrophages via autocrine IL-1 β secretion with mixed M1/M2 cytokine profile and M2 surface phenotype that promotes stellate cell activation via TGFβ .

Disclosures:

Gyongyi Szabo - Consulting: Idenix; Grant/Research Support: BMS, GSK, Conatus, Idera, Johnson&Johnson, Novartis, Ocera, Roche, Shering - Plough, Wyeth, Integrated Therapeutics, Idera

The following people have nothing to disclose: Banishree Saha, Karen Kodys

Department of Gastroenterology, Juntendo University Graduate School of Medicine, Tokyo, Japan

Background: Glycine has been shown to be protective against a variety of liver injuries caused by ischemia-reperfusion, endotoxin, and ethanol, where macrophage activation through TLR4 is mainly involved. Recently, we demonstrated that dietary glycine prevents metabolic syndrome-related steatohepatitis, in which innate immune responses also play a pivotal role. In this study, we evaluated the effect of glycine on hepatic innate and autoimmune responses caused by double-stranded RNA, a TLR3 ligand. Methods: Female, 8 week-old C57Bl6 mice were fed a diet containing 5% glycine or casein as controls for 1 week, and then given a single, intraperitoneal injections of poly I:C (5 μg/g BW). Some mice were given repeated injections of poly I:C (twice/week) for 24 weeks in combination with glycine-diet. Serum alkaline phosphatase (ALP) levels were measured, and liver histology was assessed. Serum anti-mitochondria (AMA)-M2 antibody was detected by ELISA. Hepatic mRNA levels for TNF , IL-1 , IL-6, IFN , IFN , IFN , and TLR3 were quantified by real time RT-PCR. Results: A single injection of poly I:C caused trivial necro-inflammatory changes in the liver; however, poly I:C elicited swift elevations in hepatic TNF , IL-1 , and IL-6 mRNA levels in 1 hr. Further, mRNA levels for IFN and IFN were increased transiently in 6 hr after injection of poly I:C, while the levels for IFN were almost unchanged. All of these transient inductions in cytokines were significantly blunted in mice pre-fed a diet containing glycine. Hepatic expression levels of TLR3 mRNA were not altered by dietary pretreatment. On the other hand, long-term treatment with poly I:C resulted in development of autoimmune cholangitis with positive AMA-M2 antibody, resembling human primary biliary cirrhosis (PBC). Interestingly, simultaneous dietary treatment with glycine prevented the formation of cholangiopathy almost completely. Indeed, dietary glycine blunted not only elevations in serum ALP levels but also induction levels of AMA-M2. Moreover, poly I:C-induced cholangitis were markedly ameliorated even when glycine-diet were given only for 8 weeks, starting from 16 weeks after repeated poly I:C injections with control diet-feeding. Conclusions: These findings clearly indicated that dietary glycine attenuates TLR3-mediated innate immune responses elicited by double-stranded RNA. Further, glycine minimizes development of hepatic autoimmunity involving alterations in innate immune responses and molecular mimicry, thereby ameliorating induction of cholangitis. It is postulated that glycine is a promising immune-nutrient for prevention and treatment for autoimmune liver diseases including PBC.

Disclosures:

The following people have nothing to disclose: Akira Uchiyama, Kenichi Ikejima, Kumiko Arai, Kazuyoshi Kon, Kyoko Fukuhara, Tomonori Aoyama, Shunhei Yamashina, Sumio Watanabe

¹Institute of Liver Studies, King's College London, London, United Kingdom; ²Foundation for Liver Research, 69-75 Chenies Mews, London, United Kingdom; ³Hepatology Research Unit, St Mary's Hospital, Imperial College London, London, United Kingdom

Introduction: Acute alcoholic hepatitis (AAH) has a high mortality. Sepsis and resultant organ failure are frequently the cause of death. Fungal infection in this group of patients is increasingly recognized as a contributor to poor outcome. A balance between pro- and anti-inflammatory pathways are thought to be important but, overall, disease drivers in AAH and the subsequent impact on ability to survive infection are poorly understood. Neutrophil toll-like receptor (TLR) 9 detects fungal DNA and results in the release of pro-inflammatory cytokines that aid in the containment and clearance of fungi. Aims: We examined circulating neutrophil TLR9 expression and plasma lactoferrin (an antimicrobial lipopolysaccharide-binding protein released from neutrophils) in17 patients with AAH (discriminant factor >32), 20 patients with alcohol-related cirrhosis (ARC) and 10 healthy controls (HC). Methods: Neutrophil phenotype was characterized in peripheral blood using fluorochrome conjugated monoclonal antibodies anti-CD16, -CD11b, -TLR2, -TLR4 and TLR9. Plasma cytokines and lactoferrin were measured using CBA and ELISA, respectively. Results: Median MELD scores for the AAH and ARC cohorts were 25 and 11, respectively. 28-day mortality for the AAH cohort was 18%. Fungal or candida species were isolated from 6/17 AAH patients and 4/20 ARC patients. TLR9 expression was increased in both the AH and ARC patients compared to HC (p<0.05). Plasma lactoferrin was significantly elevated in AAH compared to cirrhotics and HC (p<0.01 and p<0.001, respectively). The circulating AAH neutrophils had reduced TLR2 expression (p=0.02) and showed a trend towards reduced TLR4 expression (p=0.05) compared to HC. Neutrophil TLR9 expression was found to inversely correlate with plasma lactoferrin (p=0.03; spearman r-0.7) in AAH supporting the role of TLR9 in 'non-sterile' inflammation thought to be of importance in AAH. There were no differences however in TLR9 or lactoferrin between those AAH patients that had confirmed fungal/candida infection compared to those who did not. Lactoferrin levels on day 7 (n=6) were significantly reduced compared to day 1 in the AAH patients (p=0.04). Conclusion: Neutrophil TLR9 expression is increased in AAH whereas expression of TLR2 and TLR4 is reduced. As TLR2 and TLR4 provide critical innate antifungal immune signals and TLR9 detects fungal DNA these findings may explain the susceptibility to fungal invasion in AAH. With regard to lipopolysaccharide-binding, lactoferrin may initially suppress endotoxin-related biological activity which may contribute to the reduction in TLR2 and 4 expression and account for the development of bacterial sepsis in AAH.

Disclosures:

John G. O'Grady - Advisory Committees or Review Panels: Astellas, Novartis; Speaking and Teaching: Astellas, Roche

Debbie Shawcross - Advisory Committees or Review Panels: Norgine; Grant/ Research Support: Norgine; Speaking and Teaching: Norgine

The following people have nothing to disclose: Jennifer M. Ryan, Godhev K. Manakkat Vijay, (Robin) Daniel Abeles, Thomas Tranah, Laura J. Blackmore, Victoria T. Kronsten, Lee J. Markwick, Antonio Riva, Nikhil Vergis, Nicholas J. Taylor, Shilpa Chokshi, Yun Ma

Pathology, LABioMed at Harbor UCLA, Torrance, CA

Activation of Toll-like receptor (TLR) signaling which stimulates inflammatory and proliferative pathways is the key element in the pathogenesis of Mallory-Denk bodies (MDBs) in mice fed DDC. However, little is known as to how TLR signaling regulates MDB formation during chronic liver disease development. The first systematic study of transcript regulation in human archived formalin-fixed, paraffin-embedded (FFPE) liver biopsies and frozen liver sections from DDC re-fed mice with MDB formation is presented here. When compared to the activation of Toll-like signaling of Kupffer cells in alcoholic liver disease, striking similarities and obvious differences were observed. Similar TLRs (TLR3 and TLR4, etc.), TLR downstream adaptors (MyD88 and TRIF, etc.) and transcript factors (NF-κB and IRF7, etc.) were all up regulated, in both DDC re-fed mice livers and patients' livers. MyD88, TLR3 and TLR4, however, were significantly induced in alcoholic hepatitis (AH) and Non alcoholic steatohepatitis (NASH) compared to normal subjects, while TRIF and IRF7 mRNA were only slightly up regulated. This is a different pathway from the induction of the TLR4-MyD88-in-dependent pathway in the AH and NASH patients with MDBs present. The subunits of the NF-κB family p65 and p50 which induce the proinflammatory cytokines were also significantly up regulated both in the AH and NASH biopsies. Importantly, chemokine receptor 4 and 7 (CXCR4/7) mRNA were found to be significantly up regulated both in the patients livers and mice fed DDC in FAT10 positive hepatocytes. The CXCR7 pathway was up regulated in patients with AH and the CXCR4 was up regulated in patients with NASH, indicating that CXCR4/7 is crucial in liver MDB formation. This data constitutes the first demonstration of the up regulation of the MyD88-dependent TLR4/NFkB pathway in AH and NASH where MDBs formed, via the MyD88- NF-κB -CXCR4/7 pathway, and provides further insight into the investigation of the human liver disease development mechanism of MDB formation.

Disclosures:

The following people have nothing to disclose: Samuel W. French, Hui Liu, Barbara A. French, Brittany C. Tillman, Jun Li

¹Division of Digestive Diseases, Emory University, Atlanta, GA; ²Emory Vaccine Center, Emory University School of Medicine, Atlanta, GA

Background and Methods: Natural Killer (NK) cells comprise 30-50% of the lymphocyte pool in the human liver, yet their role in maintaining hepatic immune homeostasis remains poorly understood. Their abundance in the liver - compared to 10-15% frequency in the blood - suggests an important role for these cells during hepatic inflammatory responses. As NK cells have been proposed as a therapeutic option for liver fibrosis and cancer, we sought to define the effects of hepatic inflammation on NK cell function. B6 mice were given a singe dose of the hepatotoxic drug carbon tetrachloride (CCl4) to induce liver inflammation and injury. Three days post injection, hepatic NK cell function was evaluated by assessing cytokine secretion and cytotoxic capacity. To demonstrate the in vivo consequences of hepatic inflammation and injury on NK cell function, mice were given an I.V. injection of GFP bearing B16 melanoma cells, which have previously been shown to metastasize to the liver in the setting of global NK cell depletion. Results: Three days post injection of CCl4 the frequency of (CD3-NK1.1+) NK cells in the liver remained unaltered. However, functional analysis revealed significantly reduced cytotoxic potential as assessed by expression of CD107a, a marker of degranulation. Furthermore, we observed a significant decrease in IFNγ and TNF-α production when NK cells were stimulated with PMA/ Ionomycin directly ex vivo. As B16 melanoma cells metastasis to the liver in the setting of NK cell depletion, we sought to investigate whether the observed ex vivo compartmental defect in NK function allowed for increased metastatic disease. Accordingly, mice that received a single dose of CCl4 had more GFP bearing B16 metastatic lesions compared to control animals. Our data demonstrates that hepatic inflammation and injury leads to hypofunctional NK cells with consequent failure to prevent tumor metastasis. Conclusions: While the timing and capacity for functional NK cells to repopulate the liver after inflammation and injury will need to be further explored, our work demonstrates that a prolonged compartmental defect in NK cell function - as measured by inadequate cytokine secretion and cytotoxic capacity - leads to increased potential for metastatic disease. Understanding the mechanisms leading to NK dysfunction secondary to inflammation from hepatitis and acute injury may help reveal novel pathways to prevent tumor metastasis and development of hepatocellular carcinoma.

Disclosures:

The following people have nothing to disclose: Justin B. Mendel, Aryn Price, Arash Grakoui

¹Edelman Lab, Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, MA, USA, Cambridge, MA; ²Cardiovascular Division, Brigham and Women's Hospital, Harvard Medical School, Boston, MA

Background and aims: Liver regeneration is a complex process that requires the sequential activation of multiple pathways and different cell types. Understanding the regulation of the interactions that allow hepatocyte expansion is of vital importance for developing clinical approaches toward liver repair. However the role of recruited monocytes in vascular and hepatic regeneration is not yet well-defined. We aimed to investigate the role of circulating monocytes in vascular and liver regeneration after partial hepatectomy in mice. Methods: Vascular architecture and the location of macrophages were analyzed using simultaneous angiography and macrophage staining with Texas-red dextran 70 kDa in the whole mouse liver by fluorescent multiphoton microscopy throughout the regenerative process. Tri-dimensional segmentation of z-stack images was used to determine macrophage position and contacts in vascular network. CD14 staining in liver sections followed the rate of recruited monocytes. The gene expression profiles of monocyte adhesion molecules, monocyte chemotactic protein type 1 (MCP-1) and inducible nitric oxide synthase (iNOS) were quantified by Real-Time PCR from 16h to 7 days after hepatectomy. To investigate the role of infiltrated monocytes in priming sprouting, Wnt5a, Notch1 and angiopoitein-1 (Ang-1), together with F4/80 as macrophage marker, were examined by immunofluorescence. Moreover protein phosphorylation of vascular endothelial (VE)-cadherin was compared to the amount of monocyte-vessel interactions. Results: Vasodila-tion begins after 16 hours of hepatectomy and correlates with iNOS expression. Kupffer cells in the space of Disse do not migrate to interact with vessels while infiltrating monocytes surround initial sprouting points. Infiltrated macrophages deliver Wnt5a, angiopoietin 1 and Notch-1 in contact points and are positively correlated with phosphorylation and disruption of VE-cadherin. MCP-1 and the intercellular adhesion molecule 1 expression are sequentially up-regulated at 16h and 72h after hepatectomy preceding the waves of hepatocyte proliferation. Conclusions: Direct vascular interactions of infiltrating monocytes, but not Kupffer cells, are responsible for driving vascular sprouting preceding parenchymal expansion. These outcomes provide new mechanistic insight and potential targets to develop new strategies for hepatic regeneration.

Disclosures:

The following people have nothing to disclose: Pedro Melgar-Lesmes, Elazer R. Edelman

Division of Gastroenterology and Hepatology, Niigata university Graduate School of Medical and Dental Sciences, Niigata, Japan

BACKGROUND/AIM: The functional profile and preferential localization of mucosal-associated invariant T (MAIT) cells in the gut and liver indicate that these cells may play a major role in controlling the pathogens that could escape the gut mucosal barrier. MAIT cells constitute a unique subset of innate-like T lymphocytes characterized by a semi-invariant T cell receptor (TCR) repertoire (made of an invariant V 7.2-J 33 TCR chain) capable of recognizing bacterial products. Although MAIT cells are abundant in the human liver, the characteristics of human MAIT cells in the liver remains to be further elucidated. METHODS: Heparinized peripheral blood and surgically removed liver tissues were collected from 8 patients with metastatic liver tumors and 2 patients with focal nodular hyperplasia. All the patients did not have any previous liver disease, and the liver tissues distant from the tumor were examined. Mononu-clear cells were separated by Ficoll-gradient, and then various surface markers were investigated by flow cytometry. mRNA expression was quantified by real-time PCR. Cytokine production was investigated using peripheral blood MAIT cells after stimulation with anti-CD3/CD28-coupled beads in the presence or absence of IL-7. We also investigated the distribution of V 7.2+ CD161+ cells in the liver by immunohistochemical staining. RESULTS: CD3+ TCR- - CD161high V 7.2+ MAIT cells comprised 6.8% (median) (range 1.1-17.9) of the total T cells in the liver but only 1.6% (0.1-6.7) of the total T cells in the blood. The proportions of activated CD69+ MAIT cells were significantly increased in the liver (82.8%, 27.6-99.4) compared to the blood (25.2%, 7.1-42.9). Although the expression of the NK cell receptor, NKG2D, was similar between the liver (44.8%, 27.6-89.2) and blood (55.8%, 39.4-70.9), an inhibitory NK cell receptor, NKG2A, was significantly higher in the liver (12.4%, 3.-32.30) than in the blood (0.3%, 0-6.1). The median proportion of IL-18 receptor (IL-18R) + and IL-7R+ cells in the MAIT cells were 97.9% (94.6-100) and 75.5% (23.1-93.7) in the liver but 99.0% (64.5-99.8) and 90.8% (38.5-98.6) in the blood, respectively. Although MAIT cells exhibited high levels of the chemokine receptor CCR6, the expression of CCR5 was heterogeneous. We also confirmed that the functions of MAIT cells were dynamically regulated by the presence of IL-7. CONCLUSIONS: Intrahepatic MAIT cells exhibit an activated phenotype and preferentially express the Th17-associated chemokine receptor CCR6. Our results indicate that MAIT cells are a specialized cell population highly adapted to exert specific immune functions in the liver.

Disclosures:

The following people have nothing to disclose: Kentaro Tominaga, Toru Setsu, Satoshi Yamagiwa, Naruhiro Kimura, Hiroki Honda, Hiroteru Kamimura, Masaaki Takamura, Minoru Nomoto

¹Gastroenterology,Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; ²Transplant Immunology, IFB-Tx, Hannover Medical School, Hannover, Germany

Infections with hepatotropic viruses can cause acute and chronic hepatitis. The detailed immuno-pathophysiological events leading to inflammation and fibrosis progression are not well understood. In order to investigate the effects of different hepatitis viruses on systemic cytokine and chemokine expression, we performed a large scale multianalyte profiling of samples from 163 individuals with acute hepatitis (12 acute HAV, 5 acute HBV, 21 acute HCV) or chronic viral hepatitis (12 HBV, 14 HBV/HDV coinfection, 51 HCV). In addition, 25 subjects who had recovered from acute hepatitis C and 22 healthy controls were studied. 50 serum cytokine, chemokines and angiogenic factors were studied using multiplex technology (Bio-Plex System). Moreover, various clinical parameters were included in the statistical analysis including viral load, ALT,AST, gamma GT and blood counts. Principal component analysis (PCA) was performed by ANOVA/t-test testing using Qlucore Omics Explorer software(Qlucore Lund, Sweden). Results: Profiling revealed distinct signatures by of 31 markers differentiating acute hepatitis from healthy controls as well as chronic hepatitis patients from controls (32 parameters). Similarly, acute and chronic infections could be separated by biomarkers (9 parameters). Interestingly, signatures differed for distinct viruses as HAV infections caused a much more upregulation of various pro-inflammatory cytokines as compared to acute hepatitis C. In contrast, chronic hepatitis C was associated with a broad upregulation of various cytokines and chemokines including IP-10, IL-18, IL12, SDF-1a, and VCAM-1 compared to chronic hepatitis B patients while RANTES (CCL5) appears as the only cytokine/chemokine upregulated in HBV and HBV/HDV. No significant markers could be identified to differentiate between HBV monoinfection and hepatitis delta. Interestingly both groups showed a cluster of markers which are clearly less expressed compared to controls including IL-2RA,IL-3,IL-5, IL-15, IL-17 and G-CFS. Discussion: Acute and chronic liver inflammation is characterized by distinct cytokine signatures which differ between different hepatitis viruses. This large-scale profiling reveals novel insights in the immunopathogenesis and host pathogen interactions.

Disclosures:

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, Abbvie, Janssen Cilag, BMS; Grant/Research Support: Merck (MSD Germamny), Roche; Speaking and Teaching: Merck (MSD Germamny), Roche, Gilead, BMS, Novartis, Falk, Abbvie Heiner Wedemeyer - Advisory Committees or Review Panels: Transgene, MSD, Roche, Gilead, Abbott, BMS, Falk, Abbvie, Novartis, GSK; Grant/Research Support: MSD, Novartis, Gilead, Roche, Abbott; Speaking and Teaching: BMS, MSD, Novartis, ITF, Abbvie, Gilead The following people have nothing to disclose: Svenja Hardtke, Julia Hengst, Katja Deterding, Falk S. Christine, Verena Schlaphoff

¹Department of Disease control and Homeostasis, Kanazawa University, Kanazawa city, Japan; ²Department of Immunology, University of Toyama, Toyama city, Japan

BACKGROUND/AIM: In the past decade, several clinical studies of immunotherapy for hepatocellular carcinoma (HCC) using tumor-associated antigens (TAAs) have proceeded. In these studies, the advents of TAA-specific T-cells in the patients were monitored using cytokine-secretion assays or peptide-MHC multimers. However, functional features of the antigen-specific T-cell receptors (TCRs) in HCC immunotherapy remain unclear. In this study, we revealed the TCR repertoire landscape of the vaccinated patients and its role on the clinical response. METHODS: Fifteen patients who underwent more than 3 times of vaccinations were analyzed among the par ticipants of the phase I clinical trial using HLA-A24-restricted -fetoprotein (AFP)-derived peptide vaccines for advanced HCC (trial registration: UMIN000003514). AFP-specific cytotoxic T lymphocytes (CTLs) were induced and analyzed using the hTEC10 (human TCR efficient cloning within 10 days) system that we had developed for rapid TCR cloning (Kobayashi et al. Nat Med. 2013). Finally, the avidities of obtained TCRs were estimated by comparing the cytotoxicity against C1R-A24 cells loaded with various concentrations of peptides and the EC50 values were calculated. RESULTS: Of 15 vaccinated patients (male, 60%; median age, 73; LCSGJ stage III/IVa/ IVb, 9/2/4), one patient (6.7%) achieved complete response (CR), and stable disease (SD) and progressive disease (PD) were recorded in 8 patients (55.3%) and in 6 patients (40%), respectively. Median time to progression (TTP) was 79 days (Mizukoshi et al. AASLD2013). AFP-specific CTLs were induced in 4 patients whose clinical responses were CR (no recurrence until day 1,336), long SD (TTP, 812 days), SD (91 days) and PD (46 days), respectively (long SD is defined as SD over 6 months). Totally, 347 specific TCR clones that consisted of 10 kinds of TCR gene rearrangements (3 from the CR patient; 4 from the long SD patient; 2 from the SD patient and 1 from the PD patient) were amplified. From the CR patient and the long SD patient, TCRs possessing higher avidities (0.0058 μM and 0.0168 μM, respectively) were obtained; meanwhile the SD patient and the PD patient had weaker TCRs (0.2811 μM and 0.0613 μM, respectively). CONCLUSION: AFP-derived peptide vaccine treatment for advanced hepatocellular carcinoma induced antigen-specific CTLs with diverse TCR repertoires in the vaccinated individuals. Furthermore, the inductions of TCRs with high avidities reflected the favorable clinical responses. TCR repertoire analysis could be a potent tool for immunomonitoring in HCC immunotherapy.

Disclosures:

Atsushi Muraguchi - Consulting: SCW

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: Hidetoshi Nakagawa, Eishiro Mizukoshi, Eiji Kobayashi, Takeshi Terashima, Masaaki Kitahara, Noriho Iida, Hiroyuki Kishi

Kanazawa Univercity, Kanazawa, Japan

BACKGROUND: Dendritic cell (DC)-based immunotherapies are expected to contribute to the eradication of the residual and recurrent tumor including hepatocellular carcinoma (HCC). We have developed the combined therapy of radiofrequency ablation (RFA) with infusion of OK432, a Streptococcus-derived anticancer immunotherapeutic agent, stimulated monocyte-derived DCs (MoDCs) for HCC, and indicated that patients treated with RFA and OK432-stimulated DC transfer had prolonged recurrence-free survival compared with the historical controls that had been treated with RFA alone (Hepatology 58(S1):1265,2013). In the present study, we analyzed immunobiological responses in peripheral blood monocytes (PBMCs) before and after DC infusion into HCC following RFA. METHODS: MoDCs were derived from PBMCs of hepatitis C-related HCC patients (n=30) in the presence of 50ng/ml IL-4 and 100ng/ml GM-CSF for five days. The cells were cultured for two additional days in the medium and stimulated with 0.1 KE/ml OK432. On day 7, DCs were harvested for injection, 5×106 cells suspended in 5ml normal saline containing 1% autologous plasma, and injected into HCC with a needle percutaneously after RFA. The immune responses were evaluated by NK cell activity, intracellular cytokine production (IFN-γ and IL-4) and IFN-γ enzyme-linked immunospot (ELISPOT) assay using PBMCs. NK cell cytotoxity against K562 erythroleukaemia target cells measured using the 51Cr-release assay. ELIS-POT assay was performed in HLA-A24 positive patients using HLA-A24 restricted peptides derived from AFP, MRP3, SART2, SART3 and hTERT, which we previously identified as HCC-specific tumor associated antigen (TAA). RESULTS: The level of NK cell activity was unaltered following treatment. There were no significant changes in terms of cytokine production capacity in the CD4+, CD8+ and CD56+ subsets in the patients following treatment. After HCC treatment, positive T cell responses against at least one TAA-derived peptide were observed in 6 of 17 (35%) patients. The increase of the frequency of TAA-specific T cells after treatment was detected in 5 of 9 (56%) peptides. In addition, the length of HCC recurrence-free survival in patients with T cell responses against 2 and more peptides after treatment was longer than that of the patients without the T cell responses (p=0.042). CONCLUSIONS: OK432-stimulated DC infusion into HCC following RFA newly induced immune responses to unprimed tumor antigens, implying that antigen-non-specific DC injection into the treated tumor enhanced tumor immunity.

Disclosures:

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: Masaaki Kitahara, Eishiro Mizukoshi, Hidetoshi Nakagawa, Noriho Iida, Hajime Sunagozaka, Kuniaki Arai, Tatsuya Yamashita

¹Second Department of Internal Medicine, University of Fukui, Fukui, Japan; ²Division of Cancer Immunotherapy, Exploratory Oncology Research & Clinical Trial Center, National Cancer Center, Kashiwa, Chiba, Japan

BACKGROUND: Metastatic liver tumors are originated from various malignancies including lung. Since the phenotypes of metastatic tumor cells are often altered from the original cancer cells, we observe the difference in susceptibility to chemotherapy at metastatic lesions. The efficacy of EGFR-tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, against non-small cell lung cancer (NSCLC) with activating EGFR-mutation is dramatic. However, almost all patients developed acquired drug resistance to EGFR-TKI. EGFR T790M mutation is the most common acquired resistance mutation in patients with NSCLC. Mutation-derived antigens associated with tumor cell progression and drug resistance may provide a candidate target in the future strategies of immunotherapy. Here, we evaluated the immunogenicity of antigen derived from EGFR T790M point mutation that is associated with drug resistance. METHODS: The computer-based epitope prediction programs BIMAS was used for the prediction of peptides binding to HLA-A2. T2 binding assay was done to evaluate the binding capability of selected candidate peptides to HLA-A2. To the induction of peptide-specific cytotoxic T lymphocytes (CTL), CD8 ⁺ cells of healthy donors were stimulated by peptide-pulsed DCs for one week and subsequently, stimulated twice per week by peptide-pulsed artificial-APC-A2 (K562/HLA-A2/CD80/CD83). By IFN-γ ELISPOT assay, CD107a assay, and cytotoxic assay, reactivity of established peptide specific CTLs against peptide-pulsed or non-pulsed target cells was assessed. RESULTS: 9 or 10-mer five peptides were selected as candidate sequences. Peptide binding assay showed that EGFR T790M-derived five peptides had relatively high affinity to HLA-A2 molecules. By stimulation with peptide-pulsed DCs and artificial APC-A2, T790M-A _(789-797) (IMQLMPFGC)-specific CTLs were induced from PBMCs of all four donors, and peptide-specific CTL lines were established. T790M-A specific CTL line was able to specifically recognize T2 cells pulsed with T790M-A peptide but not pulsed with T790M-A _(789-797), wild-type (ITQLMPFGC) peptide. Finally, this CTL line showed the reactivity against NCSLC cell line, H1975-A2 (HLA-A2+, T790M+), but not H1975 (HLA-A2-, T790M+) by IFN-γ ELISPOT assay and CD107a assay. This CTL line also demonstrated the peptide-specific cytotoxicity against H1975-A2. CONCLUSIONS: We determined the immunogenicity of an HLA-A2 restricted, EGFR T790M mutation-derived antigen. Immunotherapy targeting to the current mutation epitope may be a treatment option for patients with metastatic liver tumors originated from EGFR-TKI resistant NSCLC.

Disclosures:

Tetsuya Nakatsura - Consulting: Ono Pharmaceutical co.Ltd.; Grant/Research Support: Ono Pharmaceutical co.Ltd., MEDINET co.Ltd.

The following people have nothing to disclose: Kazuya Ofuji, Toshiaki Yoshikawa, Yoshitaka Tada, Manami Shimomura, Yasunari Nakamoto

Infectious Disease, the First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi'an, China

Aims: Overwhelming evidence suggests that acute-on-chronic liver failure (ACLF) is an inflammatory disease mediated by innate and adoptive immune systems. Cytokines are important mediators during immune responses. The purpose of this study was to quantify Th1, Th2 and Th17 cytokines in serum of patients with ACLF and to analyze their dynamic changes in the progression of ACLF. Methods: Dynamic blood samples were taken from 15 patients with HBV related ALCF, whose liver and anticoagulation function recovered during the follow-up. 27 patients with chronic HBV infection (CHB) were enrolled as control. Th1 cytokines (IL-2, IFN-γand TNF), Th2 cytokines (IL-4, IL-6 and IL-10) and Th17 cytokine (IL-17A) were detected in serum using cytometric bead array. Results: The concentrations of IFN-γ, TNF, IL-6, IL-10 and IL17A as well as MELD scores were higher in ACLF patients than those in CHB patients on admission. With the development of ACLF, the concentrations of Th1, Th2 and Th17 cytokines were slightly raised during the peak ACLF severity (MELD score peak), although the differences were not statistically significant except MELD scores (P=0.004). During the 3th week of follow-up, all the concentrations of the cytokines as well as MELD scores were declined. The concentrations of IFN-γ, TNF, IL-10 and IL17A were even declined to the same levels as the CHB patients (p>0.05), while IL-6 and MELD scores were still higher than CHB patients (p=0.000). In addition, on the peak ACLF severity, the concentrations of IFN-γ, IL-6 and IL-10 were positive correlation with MELD scores (R=0.514, P=0.05; R=0.518, P=0.048; R=0.629, P=0.012, respectively), while the rates of IFN-γto IL-10 and IFN-γto IL-6 were negative correlation to MELD scores (R=-0.564, P=0.028; R=-0.531, P=0.042, respectively). Conclusions: Th1/Th2/Th17 the cytokines were raised in the acute phase of ACLF and exhausted quickly, which were paralleled with MELD scores. Th2 cytokines such as IL6 and IL10 were associated with aggravation of ACLF.

Disclosures:

The following people have nothing to disclose: Li Jin, Yingli He, Dan Du, Yuanyuan Li, Ruitian Yi, Jing Wang, Tianyan Chen, Yingren Zhao

¹Pediatrics, Emory University, Atlanta, GA; ²Pediatrics, Baylor College of Medicine, Houston, TX

The interplay of resident hepatic macrophages (Kupffer Cells, KCs) and neighboring hepatocytes in the liver plays a major role in innate and adaptive immune responses by mutual responses to small molecules shuttling between the cell types, and affecting the release of various cytokines and acute-phase proteins. The full extent of the roles and intercellular communications between hepatocytes and KCs as a model for hepatic inflammation has not been explored in detail. To address this gap, RNA microarray analysis was performed on livers of mice treated with 2 mg/kg LPS or saline by ip for 16 hrs. We discovered that Slc10a6 (an incompletely characterized member of the SLC10A Na+-dependent bile acid [BA] transporter family) was the most-induced transcript (∼500-fold), confirmed independently by qPCR (>50 fold) in the array. Slc10a6 RNA levels were upregulated in mouse liver at 2 hrs (7-fold) and 4 hrs (100-fold) after LPS treatment and 35-fold after treatment with the cytokine IL-1 p (5mg/kg) for 4 hrs. In silico promoter analysis suggests the presence of adjacent binding sites for NFkB and RXRa heterodimers, whereby previous studies indicate that RXRa agonists and NFkB/JNK inhibitors can serve as anti-inflammatory agents. Upregulation of Slc10a6 RNA by LPS (16 hrs) was attenuated by ∼60% when mice were pretreated for 5 days with the synthetic RXRa ligand LG268 (30 mg/kg/day). In vitro, Slc10a6 RNA was induced 30-fold (p<0.05) by LPS in macrophages (mouse RAW264.7 cells) in a time-dependent manner (max. at 6-8 hrs), but not in multiple hepatocyte, cholangiocyte, stellate and endothelial cell lines. This induction was abrogated in the presence of either NFkB or JNK inhibitors. Slc10a6 differs from well-characterized Slc10a family members, the BA-uptake transporters Slc10a1 (Ntcp) and Slc10a2 (Asbt), as its substrates are sulfated steroids. The Slc10a6 substrate dehydroxyepiandrosterone sulfate (DHEAS) enhanced the LPS-induced CCL5 expression in Raw264.7 cells after 24h by 50% (p<0.05), which was abrogated in the presence of the RXRα ligand 9cisRA (p<0.05). Upregulation of Slc10a6 RNA by LPS and enhancement of the LPS-mediated upregulation of CCL5, a pro-inflammatory neutrophil-attracting chemokine, by the Slc10a6 substrate DHEAS in macrophages, suggests that Slc10a6 is a significant component of the inflammatory response of KCs and suggests the presence of DHEAS in an ongoing inflammatory state has additional pro-inflammatory effects. Further characterization of regulation of expression and transporter function of Slc10a6 in KCs under inflammatory conditions will expand our understanding of, and provide means for curtailing the hepatic immune response.

Disclosures:

The following people have nothing to disclose: Astrid Kosters, Demesew Abebe, Julio Felix, Saul J. Karpen