studies

876A AASLD ABSTRACTS HEPATOLOGY, October, 2015
exosome binding to HSC by RGD (p < 0.05) but not by RGE.
Binding of PKH26-stained HSC-derived exosomes to PKH67-
stained HSC, assessed over 12 hrs, was also dose-dependently
inhibited by EDTA (0-500 μM; p < 0.05). Since these data suggested
exosome-HSC interactions involve integrins and since
all five αV integrins (β1, β3, β5, β6, β8) and two β1 integrins
(α5, α8) are RGD-dependent, recipient HSC were transfected
for 24 hrs with siRNA to αV or β1 and then incubated with
PKH26-stained exosomes for 24 hrs. Each treatment alone was
effective in blocking >95% exosome binding. Finally, binding
of HSC-derived exosomes to recipient HSC was reduced by
heparin but not chondroitin sulfate (100μg/ml) while exosomal
miR-214-mediated suppression of CTGF 3’-UTR activity, accomplished
by 24-hr co-culture of miR-214-transfected donor HSC
with CTGF 3’-UTR luciferase reporter-transfected recipient HSC
was reversed in the presence of heparin but not chondroitin
sulfate (100μg/ml) (p < 0.05). Thus exosome-HSC interactions
or downstream gene activity are heparin- or integrin-dependent,
the latter of which requires expression of αV and/or β1
subunits by recipient HSC.
Disclosures:
David Brigstock - Stock Shareholder: FibroGen
The following authors have nothing to disclose: Li Chen, Ruju Chen, Sherri Kemper
1357
Potential of connective tissue growth factor as a novel
therapeutic target against hepatic fibrosis
Yuki Makino, Hayato Hikita, Yugo Kai, Yasutoshi Nozaki, Tasuku
Nakabori, Yoshinobu Saito, Satoshi Tanaka, Ryotaro Sakamori,
Naoki Hiramatsu, Tomohide Tatsumi, Tetsuo Takehara; Department
of Gastroenterology and Hepatology, Osaka University
Graduate School of Medicine, Suita, Japan
Background and aim: Connective tissue growth factor (CTGF)
is a multifunctional matricellular protein reported to be up-regulated
in the liver of patients with chronic liver diseases. However,
the association of CTGF and liver fibrosis has not been
fully studied. We analyzed the significance of CTGF and its
potential as a therapeutic target in hepatic fibrosis. Methods/
Results: Using clinical resected livers of 93 patients under the
approve of Research Ethics Committee, we revealed that CTGF
expression levels in non-tumorous liver tissue increased along
with the progression of fibrotic degrees of the liver and positively
correlated with those of other fibrosis-related genes such
as α-SMA, TGF-β1, and Col1a1. In a mouse experiment, CTGF
was up-regulated in the liver 3 weeks after bile duct ligation
(BDL). CTGF expression levels showed positive correlation with
those of other fibrosis-related genes listed above. Immunochemistry
of CTGF after BDL showed positive reaction both in parenchymal
cells (PC) and non-parenchymal cells (NPC). Thus, we
subsequently isolated PC and NPC from mice underwent BDL
or sham, and compared respective CTGF expression levels
between BDL and sham groups. CTGF was up-regulated both
in PC and NPC in BDL groups. In vitro experiments revealed
that treatment of TGF-β, an inducing factor of CTGF, increased
CTGF expression and secretion not only in hepatocyte cell
lines, Huh7 and PLC/PRF/5 cells but in hepatic stellate cell
(HSC) cell lines, LX-2 cells. These findings indicated that both
hepatocytes and HSC produce CTGF under the stimulation of
BDL. We finally analyzed the potential of CTGF as a therapeutic
target in liver fibrosis. In vitro CTGF treatment induced the
activation and collagen production in LX-2 cells, suggesting
CTGF has a promoting effect for liver fibrosis. We subsequently
evaluated the degree of fibrosis after BDL in 3 strains of conditional
CTGF knockout mice; HSC-specific CTGF deficient mice
(GFAP-Cre+ CTGF flox/flox mice; CTGFΔHSC mice), hepatocyte-specific
CTGF deficient mice (Alb-Cre+ CTGF flox/flox
mice; CTGFΔHep mice), and poly IC-induced PC and NPC
CTGF deficient mice (MX1-Cre+ CTGF flox/flox mice; CTG-
FΔPC+NPC mice). As a result, compared with Cre- littermates,
CTGF expression levels in the liver were significantly reduced
to 55.8% and 27.7% in CTGFΔHep and ΔPC+NPC mice,
respectively. No difference was observed in CTGFΔHSC mice.
Liver fibrosis was attenuated only in CTGFΔPC+NPC mice, evidenced
by reduced Collagen expression and Sirius Red-stained
area. Conclusion: CTGF is produced from both PC and NPC
and promotes hepatic fibrosis. CTGF is a promising therapeutic
target in liver fibrosis.
Disclosures:
Hayato Hikita - Grant/Research Support: Bristol-Myers Squibb
Tetsuo Takehara - Grant/Research Support: Chugai Pharmaceutical Co., MSD
K.K., Bristol-Meyer Squibb, Mitsubishi Tanabe Pharma Corparation, Toray Industories
Inc. ; Speaking and Teaching: MSD K.K., Bristol-Meyer Squibb, Janssen
Pharmaceutical Companies
The following authors have nothing to disclose: Yuki Makino, Yugo Kai, Yasutoshi
Nozaki, Tasuku Nakabori, Yoshinobu Saito, Satoshi Tanaka, Ryotaro Sakamori,
Naoki Hiramatsu, Tomohide Tatsumi
1358
Exosome-mediated activation of TLR3 in stellate cells
stimulates IL-17A production of γδ T cells in liver fibrosis
of mice
Wonhyo Seo, Hyuk-Soo Eun, So Yeon Kim, Seol-Hee Park, Jong-
Min Jeong, Won-Mook Choi, Myung-Ho Kim, Won-IL Jeong;
KAIST, Daejeon, Korea (the Republic of)
Background: During liver injury, hepatocytes secrete numerous
exosomes including diverse types of self-RNAs. Recently,
self-noncoding RNA has been considered as an activator to tolllike
receptor 3 (TLR3). However, the roles of hepatic exosome
and its detection by TLR3 have not been fully understood in the
pathogenesis of liver fibrosis. In the present study, we identified
that exosome-induced activation of TLR3 in hepatic stellate cells
(HSCs) stimulated production of interleukin-17A (IL-17A) in γδ
T cells, subsequently leading to acceleration of liver fibrosis
in mice. Methods: Liver injury and fibrosis was induced with
single or 2-week injection of carbon tetrachloride (CCl 4
) in wild
type (WT) and TLR3-deficient (TLR3 -/- ) mice. Liver immune cells,
HSCs and hepatocytes were freshly isolated for flow cytometry
and in vitro experiments. Results: In acute and chronic liver
injuries, increased IL-17A production was mainly detected in
hepatic γδ T cells of WT mice, whereas those of TLR3 -/- mice did
not show any increase of IL-17A production in both liver injuries.
In addition, liver fibrosis of TLR3 -/- mice was significantly
attenuated compared with WT mice. More interestingly, IL-17A
producing γδ T cells were in close contact with activated HSCs,
suggesting implication of HSCs and TLR3 in IL-17A production
of γδ T cells. Furthermore, in vitro treatments of exosomes
derived from CCl 4
- or ethanol-treated hepatocytes significantly
increased expression of IL-1β, IL-23 and IL-17A in HSCs, and
remarkable increase of IL-17A production in γδ T cells was
observed as they were co-cultured with exosome-treated WT
HSCs or conditioned medium from TLR3-activated WT HSCs.
However, all these finding were not detected as γδ T cells were
co-cultured with exosome-treated HSCs of IL-17A -/- or TLR3 -
/-
mice. Using reciprocal bone marrow transplantation of WT
and TLR3 -/- mice, we demonstrated TLR3 deficiency in HSCs
contributed to decreased liver fibrosis and IL-17A production
in γδ T cells. Conclusion: In liver injury, exosome-mediated activation
of TLR3 in HSCs accelerates liver fibrosis by enhancing
IL-17A production of γδ T cells, which might be associated
with IL-17A production of HSCs by unknown self-TLR3 ligands