studies

666A AASLD ABSTRACTS HEPATOLOGY, October, 2015
was not changed. RNA sequencing showed 2314 expression
altered genes of total 38114 analyzed. When we focus on
iron regulatory proteins, we found decreased hepcidin and
increased BMP-SMAD signal related genes: Bmp4, Bmper and
Hfe2. In contrast, the other hepcidin inducing signal: transferrin
receptor signal and IL-6 signal, related genes were not
altered. Plasma hepcidin concentration in high-fat mice was
decreased and the hepcidin inducer SMAD phosphorylation
was decreased. BMP binding endothelial regulator (BMPER), a
BMP inhibiting protein, is increased in high-fat diet mice. Immunofluorescense
of frozen liver section showed BMPER expression
localized on sinusoid lumen. Cell isolation also showed
higher expression of BMPER from liver sinusoid endothelial
cells. [Discussions] Excessive irons in high-fat diet fed mice
were caused by decreased hepcidin. We identified that BMPER
is down-regulating hepcidin via BMP-SMAD signaling inhibition.
We also discovered BMPER is expressed by liver sinusoid
endothelial cells to work on hepcidin producer hepatocyte
extracellularly. This study shows importance of interaction with
hepatocytes and non-parenchymal cells in NAFL pathogenesis.
Disclosures:
Yutaka Kohgo - Grant/Research Support: Chugai Pharm., Novartis Japan, Asahikasei
Medical, Sapporo Beer Co.
The following authors have nothing to disclose: Takumu Hasebe, Koji Sawada,
Shunsuke Nakajima, Hiroki Tanaka, Takaaki Ohtake, Mikihiro Fujiya
928
Analysis of T and myeloid cells in patients with NASH
and diabetes mellitus
Luisa Vonghia 1 , Denis Mogilenko 2,3 , An Verrijken 4,5 , Luc van
Gaal 4,5 , Bart Staels 2,3 , Sven M. Francque 1,5 , David Dombrowicz
2,3 ; 1 Department of Gastroenterology and hepatology, University
Hospital, Antwerp, Belgium; 2 Inserm U1011, Lille, France;
3 Institut Pasteur, University of Lille, Lille, France; 4 Department of
Endocrinology, Diabetology and Metabolism, University Hospital
of Antwerp, Antwerp, Belgium; 5 Laboratory of Experimental Medicine
and Paediatrics, Faculty of Medicine and Health Sciences,
University of Antwerp, Antwerp, Belgium
BACKGROUND: The immune system potentially plays a pivotal
role in the onset of Non-Alcoholic Steatohepatitis (NASH) and
of the associated metabolic disturbances, including diabetes
mellitus (DM). AIM: To study the immune cells in peripheral
blood and differentially expressed genes in the liver of preselected
patients according to presence or absence of NASH and
DM. METHODS: We enrolled 32 patients who underwent liver
biopsy because of suspected NASH. The patients were divided
in 4 predefined groups according to liver biopsy and glucose
parameters: 1) control (NO NASH/NO DM), 2) NASH/NO
DM, 3) NASH/DM, 4) NO NASH/DM. A multicolour flow
cytometry analysis was performed in order to investigate T,
natural killer T cells (NKT), natural killer (NK) cells, monocytes
and dendritic cells. Gene expression in the liver was investigated
by microarray analysis in patients with similar clinical
characteristics. RESULTS: Significant differences between
groups of patients were found among T lymphocyte populations
but not in subsets of myeloid cells. Proportions of CD8 +
cells with enhanced production of pro-inflammatory cytokines
(TNF and INFγ) and cytotoxic molecules (granzyme A and B,
perforin) were increased in patients with NASH and/or DM.
The central memory CD8 + cells (CCR7 + CD45RA - ) were significantly
increased in NASH/DM compared to NASH/NO DM,
whereas the effector (CCR7 - CD45RA + ) and effector memory
(CCR7 - CD45RA - ) CD8 + cells were more potent to produce cytotoxic
molecules in all groups of patients with NASH and DM.
Gene set enrichment analysis evenso revealed dysregulation of
genes associated with activation of CD8 + cells in the liver in
NASH/DM compared to NASH/NO DM. We found a trend
to increased proportion of NKT cells in patients with NASH.
CD4 + T regulatory (Treg) and Th22 cells were increased in
NASH/DM compared to NASH/NO DM patients, while Tregs
where decreased in NASH patients without DM. CONCLU-
SION: T-cell, bot not myeloid cell, alterations are associated
with the presence of NASH regardless of the presence of diabetes.
Both flow cytometry and gene expression demonstrate
that increased effector functions of cytotoxic CD8 + T cells and
CD4 + Th22 and Treg cells are found when NASH is associated
with DM, suggesting that these cells are specifically involved
in the crosstalk between liver inflammation and metabolic dysfunctions
and contribute both to hepatotoxic and hepatoprotective
mechanisms.
Disclosures:
Bart Staels - Advisory Committees or Review Panels: MSD; Consulting: Genfit
The following authors have nothing to disclose: Luisa Vonghia, Denis Mogilenko,
An Verrijken, Luc van Gaal, Sven M. Francque, David Dombrowicz
929
Regulation of hepatocellular senescence by melatonin
during alcoholic liver injury
Jessica S. Garner 1 , Yuyan Han 3,2 , Tiaohao Zhou 3,2 , Kelly McDaniel
2,3 , Ying Wan 1,2 , Tami Annable 2,1 , Nan Wu 3,2 , Julie Venter 3,2 ,
Haibo Bai 1,2 , Shannon S. Glaser 3,2 , Heather L. Francis 1,2 , Fanyin
Meng 2,1 , Gianfranco Alpini 3,2 ; 1 Scott & White Hospital, Texas
A&M HSC College of Medicine, Temple, TX; 2 Central Texas Veterans
Healthcare System, Temple, TX; 3 Texas A&M HSC College of
Medicine, Temple, TX
Background: Chronic alcohol consumption leads to hepatic
DNA damage, cellular senescence and permanent cell cycle
arrest. Melatonin, an endogenously produced neurohormone
secreted by the pineal gland as well as the liver, has a variety
of protective effects during organ injury. However, its effect
and mechanism on the hepatic tissues and cells during alcoholic
liver injury remains to be explored. The objective of this
study was to evaluate the role of melatonin regulated hepatic
senescence phenotype during alcoholic liver injury. Methods:
The mRNA expression of melatonin, its downstream miRNA
(miR-34a) and its upstream enzyme serotonin N-acetyltransferase
(AANAT), was assessed in ethanol and LPS-treated
human hepatocytes (N-Heps), as well as in 5 weeks chronic
alcohol feeding mouse liver specimen (with or without antimiR-34a
Vivo-Morpholino treatment) and control liver tissue
by PCR array and real-time PCR assay. The secretion of melatonin
was verified by ELISA assay. Cellular senescence and
proliferation was evaluated by β-gal activity and MTS assays.
The hepatic expressions of the senescence genes (EGR1, PAI-1
and CCL2), clock circadian genes (PER1, BMAL1 and CRY1),
and miR-34a were also determined by real-time PCR assay.
Results: The total liver histopathology score, beta-gal activity
and miR-34a expression increased after 5 weeks chronic ethanol
feeding relative to control mice, along with the significant
reduction of melatonin and AANAT in isolated liver tissues.
Enhanced expression of the senescence markers EGR1, PAI-1
and CCL2 was demonstrated by senescence PCR array in vivo.
Treatment with ethanol (20 mM) and LPS (20 μg/ml) for 7
days induced significant increases of beta-gal staining, along
with the enhanced expression of cellular senescence markers
EGR1, PAI-1 and CCL2 in cultured human hepatocytes. Treatment
of N-Heps with melatonin (10 -11 M for 7 days) also prevented
alcohol-induced cellular senescence and death, and
subsequently reduced senescence markers EGR1 and CCL2,
as well as miR-34a expression. Furthermore, the expression