studies

648A AASLD ABSTRACTS HEPATOLOGY, October, 2015
(a) low-density lipoprotein receptor deficient (Ldlr −/− ) mice fed
a high fat diet and treated with either antagomir-21, antagomir
control (without specific target) or phosphate buffer saline;
(b) microRNA-21 deficient and wild-type mice fed a methionine-choline-deficient
diet (MCD). Wild-type mice fed a chow
diet served as control. We quantified microRNA-21 levels and
assessed cell localization in the liver using real-time quantitative
PCR and in situ hybridization. To determine whether
the effect of inhibiting or deleting microRNA-21 involved
PPARα, a known microRNA-21 target implicated in NASH,
we also fed PPARα deficient mice a MCD diet and treated
them with either antagomir-21 or antagomir control. Finally,
microRNA-21 levels were also quantified in the liver of patients
with NASH or bland steatosis and in normal liver; its cellular
localization was also determined. Results: Inhibiting or deleting
liver microRNA-21 expression in both murine models of NASH
reduced liver cell injury, inflammation, and fibrogenesis without
affecting liver lipid accumulation or betaoxidation. PPARα
was decreased in the liver of mice with NASH and restored following
microRNA-21 inhibition or deletion. In PPARα deficient
mice fed a MCD diet, antagomir-21 had no effect on liver cell
injury, inflammation, and fibrogenesis. Liver microRNA-21 was
overexpressed, primarily in inflammatory and biliary cells, in
both mouse models as well as in patients with NASH, but not
in patients with bland steatosis. Conclusion: This study demonstrates
that microRNA-21 is increased in NASH in liver inflammatory
cells in mice and in patients. MicroRNA-21 inhibition
or deletion strongly decreases liver injury, inflammation and
fibrosis, through PPARα normalization. These findings underscore
the potential interest of antagomir-21 as a therapeutic
strategy for NASH.
Disclosures:
Jérémy Scetbun - Employment: Merck & Co.
Patrick Marcellin - Consulting: Roche, Gilead, BMS, Vertex, Novartis, Janssen,
MSD, Abbvie, Alios BioPharma, Idenix, Akron; Grant/Research Support: Roche,
Gilead, BMS, Novartis, Janssen, MSD, Alios BioPharma; Speaking and Teaching:
Roche, Gilead, BMS, Vertex, Novartis, Janssen, MSD, Boehringer, Pfizer,
Abbvie
Francois Durand - Advisory Committees or Review Panels: Astellas, Novartis,
BMS; Speaking and Teaching: Gilead
Bart Staels - Advisory Committees or Review Panels: MSD; Consulting: Genfit
Pierre-Emmanuel Rautou - Speaking and Teaching: Gilead
The following authors have nothing to disclose: Xavier Loyer, Valerie Paradis,
Carole Hénique, Anne-Clémence Vion, Nathalie Colnot, Coralie L. Guerin,
Cécile Devue, Sissi On, Mélissa Romain, Jean-Louis Paul, Marc E. Rothenberg,
Pierre Bedossa, Carina Prip-Buus, Eric Baugé, Chantal Boulanger, Alain Tedgui
888
Two different aspects of PD-1 expression on intrahepatic
CD4+ and CD8+ T cells in a High Fat High Cholesterol
Diet-induced murine NASH
Kanji Yamaguchi 1 , Akira Okajima 1 , Yuya Seko 1 , Hiroshi Ishiba 1 ,
Hiroyoshi Taketani 1 , Atsushi Umemura 1 , Taichiro Nishikawa 1 ,
Yoshio Sumida 1 , Hironori Mitsuyoshi 1 , Kohichiroh Yasui 1 , Takeshi
Okanoue 2 , Yoshito Itoh 1 ; 1 Gastroenterology, Kyoto Prefectural University
of Medicine, Kyoto, Japan; 2 Hepatology Center, Saiseikai
Suita Hospital, Osaka, Japan
Background: Programmed death (PD)-1 signaling plays an
important role of the negative regulation of immune responses
in chronic inflamed liver. Its ligand, PD-L1, is expressed on
hepatocytes in non-alcoholic steatohepatitis (NASH) livers, and
that hepatocytes induce apoptosis in T-cells via their interaction.
The aim of this study is to clarify the pathophysiological
role of PD-1 expressing CD4+ and CD8+ T cells in murine
NASH livers. Methods: Eight-week-old male C57/BL6 mice
were fed either chow or high fat high cholesterol (HFHC) diets
for 24 weeks. Hepatic lymphocytes were isolated by density
gradient centrifuge with 35% isotonic Percoll solution after the
perfusion with 10 ml PBS via portal vein and expression of
PD-1 was analyzed by FACS at the time points of 8, 16, and
24 weeks. Moreover, after sorting the fractions of CD4+/PD-1-,
CD4+/PD-1+, CD8+/PD-1-, CD8+/PD-1+ T cells, mRNA levels
of TNFα, IL-4, IFNγ, IL-6, foxp3, IL-10 in each group of CD4+ T
cells and TNFα, IFNγ, granzyme B, perforin, IL-6 in each group
of CD8+ T cells were assessed by real-time PCR. For further
study of PD-1 expressing T cells, we analyzed the blocking
effect of PD-1 signaling on liver injury by continuously using
PD-1 and PDL-1 neutralizing antibody in HFHC-fed mice for
24 weeks. Results: The number of PD-1 expressing CD4+ or
CD8+ T cells was higher in HFHC diet-fed mouse livers and
increased in a time and the degree of hepatic injury dependent
manner. Furthermore, HFHC diets significantly induced
hepatic PD-L1 mRNA at 24 weeks as previously reported. Realtime
PCR analysis revealed that whereas PD-1 expressions on
CD4+ T cells suppressed TNFα and IL-6 expression but rather
increased IL-10 expression, those on CD8+ T cells significantly
increased granzyme B and perforin despite of decreasing IL-6
expression. Finally, blockade of PD-1 signaling for 24 weeks
greatly ameliorated plasma levels of AST and ALT. Conclusion:
PD-1 expressing T cells were increased in murine NASH livers
with the elevation of hepatic PD-L1 expression. This regulatory
molecule may act as a suppressor in CD4+ T cells but an activator
in CD8+ T cells in this murine NASH model and blockade
of these PD-1 signaling led to the amelioration of liver injury.
Disclosures:
Yoshito Itoh - Grant/Research Support: MSD KK, Bristol-Meyers Squibb, Dainippon
Sumitomo Pharm. Co., Ltd., Otsuka Pharmaceutical Co., Chugai Pharm
Co., Ltd, Mitsubish iTanabe Pharm. Co.,Ltd., Daiichi Sankyo Pharm. Co.,Ltd.,
Takeda Pharm. Co.,Ltd., AstraZeneca K.K.:, Eisai Co.,Pharm.Ltd, FUJIFILM Medical
Co.,Ltd., Gelaed Sciences Co., GlaxoSmithKline
The following authors have nothing to disclose: Kanji Yamaguchi, Akira Okajima,
Yuya Seko, Hiroshi Ishiba, Hiroyoshi Taketani, Atsushi Umemura, Taichiro Nishikawa,
Yoshio Sumida, Hironori Mitsuyoshi, Kohichiroh Yasui, Takeshi Okanoue
889
DNA Methylation Profiles of Fibrosis-Associated Gene in
Blood Reflects Liver Fibrosis Severity in Human Nonalcoholic
Fatty Liver Disease
Cynthia A. Moylan 1,2 , Susan K. Murphy 3 , Carole Grenier 3 , Manal
F. Abdelmalek 1 , Anna Mae Diehl 1 ; 1 Duke University Medical Center,
Durham, NC; 2 Medicine, Durham Veterans Affairs Medical
Center, Durham, NC; 3 Obstetrics & Gynecology, Duke University,
Durham, NC
Background: Nonalcoholic fatty liver disease (NAFLD) can lead
to cirrhosis, but disease progression is highly variable. Fibrosis
severity is the only independent predictor of cirrhosis risk in
NAFLD. DNA methylation and other epigenetic mechanisms
play a role in instructing fibrosis progression. We reported
that DNA methylation at specific loci of certain liver genes correlated
with their expression in liver and with fibrosis severity
in human NAFLD. Aim: We sought to determine if DNA methylation
profiles of fibrosis-associated genes can be measured in
blood and whether they reflect fibrosis severity (i.e., cirrhosis
risk) in NAFLD patients. Methods: Patients with near-normal histology
and biopsy-proven NAFLD were selected from the Duke
NAFLD Biobank. The cohort was stratified into 3 groups as
shown in the Table: Advanced NAFLD, Mild NAFLD, and Controls.
DNA was isolated from liver and blood, bisulfite modified
using the Zymo EZ DNA Methylation Kit, and then analyzed
by pyrosequencing (PS). Site-specific methylation levels (%met)
for two genes of interest (FGFR2, cg1385327; CASP1,
cg13802966) were correlated by tissue type and compared
by NAFLD severity using Pearson correlation and non-para-