studies

710A AASLD ABSTRACTS HEPATOLOGY, October, 2015
duced HCV (JFH-1; genotype 2a). IRF9, STAT1, and STAT2
were overexpressed by lentiviral transduction, or their expression
was silenced with siRNAs. The expression of the negative
regulators of type I IFN signaling, USP18 and ISG15, was
studied in HCV-infected liver and IFN-λ 3
treated hepatoma
cells, and their regulation by U-ISGF3 was analyzed. Results
The level of U-ISGF3, but not tyrosine phosphorylated STAT1,
is significantly elevated in response to IFN-λ and IFN-β during
chronic HCV infection. U-ISGF3 prolongs the expression of a
subset of ISGs and restricts HCV chronic replication. However,
paradoxically, high levels of U-ISGF3 also conferred unresponsiveness
to IFN-α therapy. As a mechanism of U-ISGF3-induced
resistance to IFN-α, we found that ISG15, a U-ISGF3-induced
protein, sustains the abundance of USP18. Silencing ISG15
decreased the level of USP18 in IFN-λ 3
treated hepatoma cells
and restored USP18-mediated attenuation of STATs phosphorylation
after treatment of IFN-α. Conclusions Our data demonstrate
that U-ISGF3 induced by IFN-λs and -β drives prolonged
expression of a set of ISGs, leading to chronic activation of
innate responses and conferring a lack of response to IFN-α in
HCV-infected liver.
Disclosures:
The following authors have nothing to disclose: Pil Soo Sung, HyeonJoo Cheon,
Do Youn Park, Hyung-Il Seo, Su-Hyung Park, Seung Kew Yoon, George R Stark,
Eui-Cheol Shin
1026
The expression of Immune-miRs could contribute to the
immunopathogenesis of HCV and be modified by 1(OH)
vitamin D3 supplementation
Yasuteru Kondo 1 , Tatsuki Morosawa 1 , Masashi Ninomiya 1 ,
Yasuyuki Fujisaka 1 , Yasuhito Tanaka 2 , Takayuki Kogure 1 , Jun
Inoue 1 , Teruyuki Umetsu 1 , Tooru Shimosegawa 1 ; 1 Gastroenterology,
Tohoku University Hospital, Sendai, Japan; 2 Nagoya City
University, Nagoya, Japan
[Background] We reported that the expression profiles of serum
miRNAs could be important biomarkers for the diagnosis of
various liver diseases (PLoS One 2013). It has been reported
that various kinds of miRNAs known as immune-miRs could contribute
to the immune regulation. [Aim] The aim of this study is
to analyze the biological significance of immune-miRs expression
on specific immune cells in chronic hepatitis C (CH-C)
patients. [Material and Methods] Patients: Permission for the
study was obtained from the ethics committee at our institute
(2013-1-268). Twenty CH-C patients, 20 chronic hepatitis B
patients (CH-B), and 10 healthy subjects were examined by
deep sequencing analysis (Illumina G2X). Moreover, 50 CH-C
patients (genotype 1b and high viral load) and control patients
(CH-B, NASH, and healthy subjects) were examined by validation
analysis. Deep sequencing analysis: Illumina deep
sequencing for the initial screening to determine the read numbers
of miRNAs expression in serum and PBMCs was carried
out. Immune cells isolation: CD3 + T cells, CD14 + monocytes,
CD19 + B cells and CD56 + NK cells were isolated using the
magnetic beads method. Transfection of HCV individual protein
expressing plasmids: The HCV individual expression plasmids
(HCV-E1, E2, Core, NS3, NS4 and NS5A) were transfected
into immune cells to identify the HCV proteins responsible for the
suppression of miR146b-5p. Silencing and forced expression of
miR146b-5p: Transfections of miRNA mimic (#MC10105) and
miRNA inhibitor into THP-1, Jurkat, Molt-4, Raji and human
primary lymphoid cells using 4D-nucleofector TM were carried
out. [Results] The expressions of five miRNAs (miR146b-5p,
miR-181a-2-3p, miR-204-5p, miR374a-3p and miR374a-5p)
in the serum were significantly lower than those of the control
groups. Among them, miR146b-5p was abundantly expressed
in PBMCs. The expression of miR146b-5p in CD3 + T cells and
CD14 + monocytes in CH-C patients was significantly lower
than those in control groups. The silencing of miR146b-5p in
the monocytes could significantly enhance the expression of
CXCL10, TGF-β and IL10 under the stimulation (p