POSTERS

P133

THE EXPRESSION OF THE SOLUBLE HFE CORRESPONDING

TRANSCRIPT IS UP-REGULATED BY INTRACELLULAR IRON AND

INHIBITS IRON ABSORPTION IN A DUODENAL CELL MODEL

B. Silva1, J. Ferreira1, V. Santos1, C. Baldaia2,3, F. Serejo2,3,

P. Faustino1. 1Departamento de Genetica Humana, Instituto Nacional

de Saude Dr. Ricardo Jorge, 2Gastrenterologia, Hospital de Santa

Maria, 3Faculdade de Medicina, Lisboa, Lisboa, Portugal

E-mail: cileniabaldaia@gmail.com

Background and Aims: Dietary iron absorption regulation is a

key-step for the maintenance of body iron homeostasis. Besides

the HFE full-length protein, the HFE gene codes for alternative

splicing variants responsible for the synthesis of a soluble form

of HFE protein (sHFE). Here we aimed to determine whether sHFE

transcript levels respond to different iron conditions in duodenal,

macrophage and hepatic cell models, as well, in vivo, in the liver.

Furthermore, we determined the functional effect of the sHFE

protein on the expression of iron metabolism-related genes in a

duodenal cell model.

Methods: The levels of sHFE transcripts were measured in HuTu-

80, activated THP1 and HepG2 cells, after holo-Tf stimulus, as well

as in RNA from liver biopsies of chronic HCV patients. Moreover,

the expression of iron metabolism-related genes was determined

by RT-qPCR after the overexpression of sHFE protein.

Results: Our results showed that the sHFE corresponding transcripts

expression is up-regulated by intracellular iron, despite the total

HFE levels not being affected. Peripheral blood iron biomarkers

do not correlate with sHFE levels at the liver of HCV patients.

Hephaestin and duodenal cytochrome b expressions are down-

regulated by both endogenous full-length HFE and sHFE proteins.

Exogenous sHFE stimulus also down-regulates hephaestin levels by

an endocytosis dependent mechanism.

Conclusions: sHFE is an important iron metabolism homeostasis

regulator which levels vary accordingly to intracellular iron. It

controls both hephaestin and duodenal cytochrome b expressions

and consequently dietary iron absorption by the duodenum.

Partially funded by FCT: PTDC/SAU-GMG/64494/2006, PTDC/SAU-

GMG/103307/2008, Pest-OE/SAU/UI00009/2011 and SFRH/BD/

60718/2009

P134

CYTOMEGALOVIRUS INFECTION MK2-DEPENDENTLY LEADS TO

A DECREASED IFN-g/IL-10 RATIO WHICH LIMITS MHC CLASS I/II

EXPRESSION AND PREVENTS FORMATION OF MONONUCLEAR

CELL INFILTRATES WITHIN THE LIVER

C. Ehlting1, M. Trilling2, C. Tiedje3, A. Zimmermann4, V.T.K. Le2,

M. Gaestel3, H. Hengel5, D. Haussinger1, J.G. Bode1. 1Clinic for

Gastroenterology, Hepatology and Infectious Diseases, Hospital of

the Heinrich-Heine-University Duesseldorf, Duesseldorf, 2Department

of Virology, Hospital of the University Duisburg Essen, Essen,3Department of Physiological Chemistry, Hannover Medical School,

Hannover, 4Institute of Virology, Hospital of the Heinrich-Heine-

University Duesseldorf, Duesseldorf, 5Institute of Virology, University

Medical Center of the Albert-Ludwigs-University Freiburg, Freiburg,

Germany

E-mail: christian.ehlting@uni-duesseldorf.de

Background and Aims: The anti-inflammatory cytokine IL-10

dampens the activity of immune cells, which are necessary

for pathogen clearance, but also contribute to tissue damage.

Moreover, IL-10 suppresses IFN-g-induced MHC surface expression

and diminishes the release of pro-inflammatory mediators. Murine

cytomegalovirus (MCMV) is thought to utilize the abilities of

IL-10 to escape from anti-viral effector mechanisms and to limit

tissue injury. However, the molecular mechanisms of MCMV-

induced IL-10 expression are unclear. The present study analyses

the relevance of MAPKAP kinase (MK)2 for MCMV-infection and

IL-10 synthesis in vitro and in vivo.

Methods: For in vivo studies wild-type and MK2-deficient mice

were infected with MCMV-Dm157. For in vitro studies hepatocytes

were isolated or bone marrow was prepared and differentiated into

macrophages.

Results: MK2 is crucial for MCMV-induced IFN-b expression, which

represents an important mediator of sustained IL-10 production.

Furthermore, MK2 and its downstream target tristetraproline

(TTP) are involved in MCMV-induced IL-10 transcript stability.

Moreover, MK2 plays a pivotal role for IL-6 and TNF-a, but

it is dispensable for IFN-g production. Consequently, MK2-

deficiency results in an increased IFN-g/IL-10 ratio, which may

be responsible for enhanced MHC protein expression in vivo,

resulting in the formation of mononuclear infiltrates and apoptotic

cell death observable in the liver tissue of MCMV-infected MK2-

deficient mice. Most interestingly, despite of extensive alterations

of the mediator pattern viral clearance is not affected by

MK2-deficiency.

Conclusions: We show for the first time that in the context of

CMV-infection MK2 exerts a protective effect on the liver in vivo.

P135

DEPLETION OF H2O2 BY CATALASE INDUCES MYELOID-DERIVED

SUPPRESSOR CELLS – A NOVEL MECHANISM OF

IMMUNE-REGULATION BY HEPATIC STELLATE CELLS

Y.J. Resheq1, K.-K. Li1, S. Wards1, A. Wilhelm1, A. Garg1,

S.M. Curbishley1, H.W. Zimmermann2, R. Jitschin3, D. Mougiakakos3,

C. Weston1, D.H. Adams1. 1Division of Immunity and Infection,

Centre for Liver Research and National Institute for Health Research

Biomedical Research Unit, College of Medicine and Dentistry,

University of Birmingham, Birmingham, United Kingdom; 2Medical

Department III, University of Aachen, Aachen, 3Department of

Internal Medicine 5, Hematology and Oncology, University of

Erlangen-Nuremberg, Erlangen, Germany

E-mail: y.resheq@bham.ac.uk

Background and Aims: Hepatic stellate cells (HSC) represent

a population of stromal cells with unique immune-modulator

properties. Contact-dependent induction of myeloid-derived

suppressor cells (MDSC) from mature monocytes by HSC has

been reported. Understanding the underlying mechanism is crucial

as MDSC induce potent immunosuppression in diseases such as

hepatocellular carcinoma. Hence, we sought to investigate into the

molecular interaction of monocytes and HSC.

Methods: As activated monocytes undergo oxidative burst, the role

of hydrogen-peroxide and its detoxifying enzyme, hepatic catalase,

was analysed. Monocytes were coincubated with purified hepatic

catalase and analysed for phenotype, function and transcriptome.

The role of catalase in HSC-driven induction of MDSC was assessed

by qPCR, western-blot and functional analysis.

Results: Coincubation of monocytes with purified hepatic catalase

led to a significant induction of functional MDSC (p < 0.001 at

day 5) compared to media alone. Frequency of induced MDSC

inversely correlated with hydrogen-peroxide-levels (r = −0.6555,

p < 0.05). Increased catalase-expression was found in HSC and LX2-

cell-line compared to activated liver-myofibroblasts and addition

of the competitive catalase-inhibitor hydroxylamine resulted in

a dose-dependent decrease of MDSC by HSC. The NADPH-

oxidase-subunit gp91 was increased in catalase-induced MDSC

on transcriptome-analysis and while IL-6 was identified on

proteome-profiling to be potentially involved in this mechanism

blocking of the IL6-receptor did not impact on catalase-dependent

MDSC induction.

Conclusions: HSC mediate a novel mechanism of MDSC-

induction allowing a precise control of inflammation dependent

S110 Journal of Hepatology 2014 vol. 60 | S67–S214