POSTERS
Methods: pcDNA-HBV1.3 (pHBV) plasmid containing HBV
genome was injected into mice with hydrodynamics method.
HBV antigenemia and serum HBV-DNA were measured with
electrochemiluminescence and polymerase chain reaction (PCR)
respectively. HBsAg and HBcAg expression in liver tissue were
detected by immunohistochemical staining. Intrahepatic leukocytes
were isolated, and flow cytometry was used to detect the
percentage of gdT, CD25+ or CD69+ gdT, IFN-g- or TNF-a-producing
gdT cells, NK and T cells. Furthermore, total mRNA of liver tissue was
prepared and real-time quantitive PCR was applied to detect gene
expression involved in the innate immune responses, including
TLR3, TLR7, TLR9, IRF3, IRF7, IFN-b, IFN-a, TNF-a and IL12a.
Results: At day 1 post injection (p.i), serum HBsAg, HBeAg and
HBV-DNA were positive, and high percentage of HBsAg- or HBcAg-
positive hepatocytes showed in liver specimen. Then liver HBsAg
or HBcAg expression decreased sharply and showed negative after
7 days p.i. A murine acute HBV infection model was established.
On day 1 p.i, the percentage of liver gd T cells in pHBV group
significantly increased (8.3±4.3%) compared to the normal mice
(4.5±0.6%) and the pcDNA control group (3.8±1.6%). And liver gd
T also showed enhanced CD25 expression and IFN-g production.
At the same time, gene expression of IRF7, IFN-b or TNF-a in
liver biopsies from pHBV group was significantly higher than
pcDNA group. Furthermore, the percentage of liver NK cells sharply
increased at 1 d p.i.
Conclusions: The percentage and functions of liver gd T cells
enhanced in the early phase of acute HBV infection. And
these changes were correlated with hepatocyte HBV antigen
expression and the boosted innate immune responses. Our results
demonstrated that enhanced gd T cells along with boosted innate
immune responses might play important role in acute HBV infection
and viral clearance.
P0462
CXCR6 MEDIATES RECRUITMENT OF ACTIVATED NATURAL
KILLER CELLS IN CHRONIC LIVER DISEASE
R. Parker1 , C. Weston1 , G. Webb1 , G. Hirschfield1 , T. Schall2 ,
D. Adams1 . 1 Centre for Liver Research, University of Birmingham,
Birmingham, United Kingdom; 2 ChemoCentryx Inc., Mountain View,
United States
E-mail: richardparker@nhs.net
Background and Aims: The CXC-chemokine receptor CXCR6 is
widely expressed on lymphocytes where expression is linked with a
tissue-infiltrating effector phenotype. Previous work from our group
has shown CXCL16, the ligand for CXCR6, promotes lymphocyte
adhesion to cholangiocytes in inflamed human liver. In the present
study we examined expression of CXCR6 and investigated the role
of CXCR6 in mediating trafficking of leukocytes across hepatic
endothelium.
Methods: Explanted liver tissue was obtained from the transplant
program at University Hospitals Birmingham NHS Foundation
Trust. Expression of CXCR6 and CXCL16 were analysed by rt-PCR,
immunohistochemistry and flow cytometry. The role of CXCR6
in lymphocyte trafficking was analysed in a dynamic flow assay
system with CO335224–4B, a small molecule inhibitor of CXCR6
(ChemoCentryx Inc, USA).
Results: Gene expression of CXCL16 was increased on bile ductules
and endothelium in inflammatory liver disease, with the highest
expression seen in in PBC. Serum CXCL16 concentration did not
differ significantly between healthy controls and patients with
chronic liver disease, except for patients with PBC who had
significantly lower concentrations of circulating CXCL16 (p < 0.001).
CXCR6 expression was enriched on liver-infiltrating lymphocytes
(2-way ANOVA compared to blood p < 0.001). In cirrhosis, T
lymphocytes and NKT cells showed significantly reduced CXCR6
expression compared to normal liver, whereas NK cell CXCR6
expression tended to increase. NK CXCR6 expression was associated
S486 Journal of Hepatology 2015 vol. 62 | S263–S864
with CD56hi, Nkp30+ and CD16− expression, indicating an effector
phenotype. NK CXCR6 expression was highest in PBC (one-way
ANOVA p = 0.03). Inhibition of CXCR6 with a small molecule
inhibitor reduced transendothelial migration of NK cells across
hepatic endothelium under flow (Two-way ANOVA p < 0.001).
Conclusions: CXCR6 is enriched on effector NK cells in the inflamed
human liver. This is particularly marked in PBC, where highest
CXCL16 expression was also observed. Interestingly patients with
PBC had lower peripheral CXCL16 concentration, which may be due
to sequestration in the liver. A small molecule inhibitor of CXCR6
inhibited NK cell migration demonstrating the ability of CXCR6 to
mediate NK cell recruitment though human hepatic endothelium.
CXCR6 may be a therapeutic target in chronic inflammatory liver
disease.
P0463
INFLUENCE OF KILLER CELL IMMUNOGLOBULIN-LIKE RECEPTORS
(KIRS) AND THEIR HLA LIGANDS ON VERTICAL TRANSMISSION
AND CHRONICITY OF HEPATITIS C VIRUS IN CHILDREN
E.-J. Pavon-Castillero1 , A. Ruiz-Extremera2,3,4 , M. Florido1 , P. Muñoz
de Rueda1,3 , J.-A. Muñoz-Gamez1 , J. Casado1 , A. Carazo1 , R. Quiles1,3 ,
A. Gila1,3 , S. Jimenez-Ruiz5 , A.B. Martin1 , J. León1,3 , J. Salmeron1,3,5 .
1
Clinical Management Unit of Digestive Diseases, Support unit for
research, University Hospital San Cecilio, 2 Pediatric unit, University
Hospital San Cecilio, University Hospital Virgen de las Nieves,
3
Ciberehd, Instituto de Salud Carlos III, 4 Pediatric deparment,
5
Medicine Department, Granada University, Granada, Spain
E-mail: estherjpavon@yahoo.es
Background and Aims: There is adequate evidence on the vertical
transmission (VT) of the maternal viral load of hepatitis C virus
(HCV) during delivery and on HIV coinfection, but they do not
explain all cases. The objective was to study the role of the
immunogenetic profile (HLA, KIR, and KIR-ligand binding) of
mothers and children in HCV-VT and chronicity
Methods: The study included 98 HCV-RNA(+) mothers and their
120 children. Among the 24 children infected, the disease became
chronic in 8 and the virus was cleared in 16. We determined
HLAclassI (A, B, Cw), HLAclassII (DRB1, DQA1, DQB1, DPA1, DPB1),
Bonferroni-corrected P-value (Pc), and 16 KIRs (KIR2DL1, 2DL2,
2DL3, 2DL4, 2DL5, 3DL1, 3DL2, 3DL3, 2DS1, 2DS2, 2DS3, 2DS4,
2DS5, and 3DS1), by Luminex.
Results: VT study: Children with KIR2DL3 have a lower risk of
infection (OR: 0.07, P = 0.019). The presence in the mother of
Cw*06 (OR: 5.8, P = 0.007, Pc = 0.07) and Cw*0602 (OR: 5.7, P = 0.007,
Pc = 0.08) increases the risk of infection of the child. Regarding C1
and C2 ligands, the presence of HLA-C1 in mothers and/or their
children reduced the risk of infection (mothers: OR: 0.2, P = 0.009,
children: OR: 0.3, P = 0.042), whereas the presence of HLA-C2 ligand
increased the risk (mothers: OR: 6.2, P = 0.019, children: OR: 3.3,
P = 0.042). Among the statistically significant associations found, we
highlight that KIR binding to C1 ligand protected against VT and
KIR binding to C2 ligand favored VT.
Chronicity: The presence in the mother of DQA1*01 (OR: 2.1,
P = 0.009, Pc = 0.036), KIR2DS1 (OR: 7.2, P = 0.042) or KIR3DS1
(OR = 13, P = 0.013) favors chronicity in the child. The presence
of allele DQB1*03 (OR: 0.05, P = 0.012, Pc = 0.048) and KIR2DS3
(OR: 0.5, P = 0.013) in the child and homozygosity for KIR3DL1/3DL1
(OR: 0.08, P = 0.0013) and for HLA-Bw4/Bw4 ligand (OR: 0.06,
P = 0.022) were associated with viral clearance, whereas the binding
of KIR3DS1 with Bw4 (OR = 7.2, P = 0.042), the binding of KIR2DS1
with C2 (OR = 7.2, P = 0.042), and heterozygosity for KIR3DL1/3DS1
(OR = 13, P = 0.013) favored viral chronicity.
Mother/child allele concordance: In the joint analysis of all HLAs,
higher concordance was found between mothers and children with
chronic infection vs those who had cleared the virus (67%±4.06 vs
57%±1.34, P = 0.045).

Conclusions: Studies of genetic factors in mothers and newborns
are necessary to understand the processes underlying VT and the
risk of chronic infection in the newborn. It can be affirmed that
type C1 and C2 HLA ligands and their binding to KIRs are related
to VT, while HLA-Bw4 ligands are associated with viral chronicity
P0464
KNOCKDOWN OF Gpbar1 (TGR5) RENDERS MICE HIGHLY
SUSCEPTIBLE TOWARDS Listeria monocytogenes INFECTION
M. Reich1 , P. Lang1 , D. Häussinger1 , V. Keitel1 . 1 Gastroenterology,
Hepatology and Infectious Diseases, Heinrich-Heine University,
Duesseldorf, Germany
E-mail: verena.keitel@med.uni-duesseldorf.de
Background and Aims: TGR5 (Gpbar1) is a membrane-bound bile
acid receptor expressed in macrophages of several organs such as
lung, liver and intestine as well as in peripheral blood mononuclear
cells [1–3]. Stimulation of TGR5 inhibits the nuclear translocation
of p65 and NF-úB transcriptional activation [3,4], thus reducing
LPS-mediated production of pro-inflammatory cytokines [1,2]. Aim
of the present study was to determine the role of TGR5 in an animal
model for pathogen defense.
Methods: Male, 8–12 week old TGR5 knockout (KO) and WT mice
were injected intravenously with 8×104 CFU/ml L. monocytogenes
and observed for 7 days. On the 3rd day after infection with
2.5×104 CFU/ml bacterial titers were determined in spleen and liver.
Serum levels of AST, ALT and LDH were measured using Spotchem-
analyzer. Cytokine levels were determined in serum using Luminex
cytometric bead assay. HE staining was carried out for all liver
samples.
Results: Following L. monocytogenes infection TGR5 KO displayed
a 50% mortality rate within a week of inoculation, while 90% of the
TGR5 WT animals survived. In addition higher L. monocytogenes
titers were detected in liver and spleen of TGR5 KO than in WT
animals and HE staining revealed significantly more inflammatory
infiltrates in livers from TGR5 KO mice. Accordingly, the levels of
AST/ALT and LDH were elevated in serum from TGR5 KO animals as
compared to the WT littermates. Furthermore, TGR5 KO mice had
increased serum cytokine levels after L. monocytogenes infection as
compared to WT mice.
Conclusions: Compared to WT littermates, TGR5 knockout mice
showed higher Listeria monocytogenes titers in liver and spleen
despite elevated serum inflammatory cytokines, such as TNFalpha.
Accordingly, TGR5 KO mice were unable to control bacterial
infection and systemic inflammation and thus displayed a
significantly increased mortality rate.
Reference(s)
[1] Kawamata Y et al. A G protein-coupled receptor responsive to bile acids.
J Biol Chem 2003; 278: 9435–9440.
[2] Keitel V et al. Expression and function of the bile acid receptor TGR5
in Kupffer cells. Biochem Biophys Res Commun 2008; 372: 78–84.
[3] Wang YD et al. The G-Protein-coupled bile acid receptor, Gpbar1
(TGR5), negatively regulates hepatic inflammatory response through
antagonizing nuclear factor kappa light-chain enhancer of activated B
cells (NF-kappaB) in mice. Hepatology 2011; 54: 1421–1432.
[4] Pols TW et al. TGR5 activation inhibits atherosclerosis by reducing
macrophage inflammation and lipid loading. Cell Metab 2011; 14: 747–
757.
Journal of Hepatology 2015 vol. 62 | S263–S864
POSTERS
P0465
PERSISTENT INTRAHEPATIC Vg9Vd2 T-CELLS IMPAIRMENT IN
HCV-INFECTED PERSONS
E. Cimini1 , V. Bordoni1 , U. Visco-Comandini2 , M. Montalbano2 ,
R. Lionetti2 , A. Sacchi1 , R. Casetti1 , N. Tumino1 , M.R. Capobianchi3 ,
F. Martini1 , C. Agrati1 . 1 Cellular Immunology Laboratory, 2 Clinical
Department, 3 Virology Laboratory, National Institute for Infectious
Diseases “Lazzaro Spallanzani”, Rome, Italy
E-mail: eleonora.cimini@inmi.it
Background and Aims: dgHepatitis C virus (HCV) persistence in the
host results from inefficiency of both innate and adaptive immune
responses to eradicate the infection. Among innate immune cells, a
functional impairment of peripheral Vg9Vd2 T-cells was described
during chronic HCV infection; this functional defect can be partially
restored by using IFN-a, opening the possibility to boost antiviral
innate immune response. Unfortunately, no data are available
on intrahepatic Vg9Vd2 T-cells that instead, may represent the
key actors in the anti-HCV response. Aim of our work was to
compare intrahepatic and peripheral Vg9Vd2 T-cells in chronic
HCV-infected patients (HCV) and in healthy donors (HD) subjected
to gut resection surgery.
Methods: Phenotypic and functional features of intrahepatic and
peripheral Vg9Vd2 T-cells were analyzed in 17 chronic HCV patients
and in 14 HD by flow cytometry and ELISA assay.
Results: Irrespective of HCV infection, intrahepatic compartment
was characterized by a lower frequency of Vg9Vd2 T-cells than
in the peripheral blood. Although expressing an effector/activated
phenotype, intrahepatic Vg9Vd2 T-cells showed a drastic reduction
of their ability to produce IFN-g after specific stimulation both in
HCV and in HD subjects. Nevertheless, while intrahepatic Vg9Vd2
T-cells from HD can be functionally restored by using IFN-a co-
stimulation, intrahepatic Vg9Vd2 T-cells from HCV-patients are
refractory also to IFN-a co-stimulation, suggesting an exhaustion
profile. Accordingly, Vg9Vd2 T-cells from HCV-patients presented a
higher PD-1 expression than HD both in peripheral and intrahepatic
compartments, probably due to persistent antigenic stimulation.
Conclusions: Overall, intrahepatic Vg9Vd2 T-cells from HCV
patients presented an exhaustion phenotype and are functional
impaired also after IFN-a co-stimulation. New studies are
mandatory in order to identify molecular mechanisms inducing
this functional anergy and to discover pathway able to restore
Vg9Vd2 T-cell functionality.
P0466
IL-33 KNOCK OUT MICE ARE SENSITIZED TO SEVERE ConA LIVER
INJURY BUT NOT CCl4 -MEDIATED LIVER INJURY
M. Imran Arshad1 , G. Noel2 , A. Filliol2 , V. Genet2 , C. Lucas-Leclerc3 ,
J.-P. Girard4 , C. Piquet-Pellorce2 , M. Samson2 . 1 Université de Rennes
1, U.1085 Inserm, 2 Université de Rennes 1, IRSET – U.1085 Inserm,
3
Université de Rennes 1, CHU Rennes, Rennes, 4 IPBS-CNRS, Université
de Toulouse, Toulouse, France
E-mail: michel.samson@univ-rennes1.fr
Background and Aims: IL-33/ST2 axis play a protective role during
acute hepatitis but little is known about the functional role of
endogenous IL-33 in liver patho-physiology. We aimed to decipher
the functional role of IL-33 by using IL-33 deficient mice during
immune cell mediated and hepatotoxic driven liver injury.
Methods: We used a genetic model of acute hepatitis by using
IL-33 deficient mice in ConA (a T cell-mediated hepatitis) and
CCl4 (a hepatotoxic agent-induced hepatitis) induced acute liver
injury. The liver functions (AST/ALT), signature of cytokines and
characterization of infiltrate cell in WT and IL-33−/− mice were
carried out by biochemistry, qPCR and flow cytometry analyses.
Results: Our results demonstrated that IL-33−/− mice exhibited more
severe ConA liver injury than WT mice evidencing a protective
effect of IL-33 in this hepatic model while no difference was
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