studies

220A AASLD ABSTRACTS HEPATOLOGY, October, 2015
26
Elevation of Galectin-9 is associated with the cytotoxicity
of NK cells in chronic hepatitis
Akira Nishio, Tomohide Tatsumi, Takatoshi Nawa, Takahiro
Suda, Atsuo Takigawa, Tadashi Kegasawa, Yoshiki Onishi, Seiichi
Tawara, Teppei Yoshioka, Satoshi Aono, Minoru Shigekawa,
Hayato Hikita, Ryotaro Sakamori, Naoki Hiramatsu, Tetsuo Takehara;
Department of Gastroenterology and Hepatology, Osaka
University Graduate School of Medicine, Suita, Japan
Background & Aims: NK cells play an essential role in the
pathogenesis of chronic hepatitis C (CHC). NK cells are activated
in CHC and activated NK cells exhibit elevated cytotoxicity
leading to liver injury. Recently, the activated NK cells
contribute to exhaustion of virus-specific T cells, which is associated
with persistent viral infection. However, the mechanism
of NK cell activation has not been clearly understood. The
immunoregulatory protein Galectin-9 (Gal-9) is elevated in the
serum and liver in CHC patients, suggesting that Gal-9 might
be involved in the pathogenesis of CHC. In this study, we investigated
the effect of Gal-9 on the activation of NK cells in CHC.
Methods: Serum Gal-9 levels were analyzed in 39 healthy
donors, 50 CHC patients and 24 IFN-treated patients achieving
sustained viral response (SVR) by ELISA. The cytotoxicity of
NK cells and Gal-9 levels in the supernatant were evaluated by
flow cytometry and ELISA. Immunofluorescence staining was
performed in CHC liver tissue. Results: Serum Gal-9 levels in
CHC patients were significantly higher than those in healthy
donors, and those in SVR patients were significantly lower
than those in CHC patients (median, 0-90 percentile, CHC:
146 pg/ml, 0-508, healthy donors: 0, 0-55.4, SVR: 53.6,
0-185.6). In CHC patients, serum levels of Gal-9 in elevated
ALT patients were significantly higher than those in normal ALT
patients, suggesting that elevated Gal-9 might be associated
with liver injury. Serum Gal-9 levels were not associated with
HCV viral loads. In vitro study revealed that addition of recombinant
Gal-9 resulted in significant increase of the cytotoxicity
of naïve NK cells in a dose dependent manner, independent of
Tim-3. Gal-9-activated NK cells showed the cytotoxicity against
CD4+ or CD8+ T cells, which might contribute to persistent
infection by depleting T cell. We found that a large amount of
Gal-9 production was induced in the co-culture of PBMCs with
JFH-1/Huh7.5.1 cells, but not with uninfected Huh7.5.1 cells.
A transwell system revealed that cell-cell contact was required
for Gal-9 production. In vitro depletion study revealed CD14+
monocytes were required for Gal-9 in the co-culture, and both
Gal-9 and CD14 positive cells were observed in immunofluorescence
staining, indicating that Gal-9 was produced by
Kupffer cells in the liver. The cytotoxicity of NK cells was significantly
elevated when co-cultured with JFH-1 cells compared
with control. Conclusion: Monocytes-derived Gal-9 induces the
cytotoxicity of NK cells, which might be involved in liver injury
and persistent HCV infection. These results suggest the role of
Gal-9 in the pathogenesis of chronic hepatitis C.
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: Akira Nishio, Tomohide Tatsumi,
Takatoshi Nawa, Takahiro Suda, Atsuo Takigawa, Tadashi Kegasawa, Yoshiki
Onishi, Seiichi Tawara, Teppei Yoshioka, Satoshi Aono, Minoru Shigekawa,
Ryotaro Sakamori, Naoki Hiramatsu
27
De novo formation and maturation of hepatitis C virus
replication membranes visualized by pulse-chase imaging
Hongliang Wang 1 , Andrew W. Tai 1,2 ; 1 Internal Medicine, University
of Michigan, Ann Arbor, MI; 2 VA Ann Arbor Healthcare
System, Ann Arbor, MI
Hepatitis C virus (HCV) replicates on an altered host membrane
structure called the “membranous web” (MW). Little is known
regarding temporal events in MW formation and association
with putative sites of virus assembly at or near lipid droplets
(LDs). For example, it is not known whether membranous webs
are stable structures that are continually renewed by newly
synthesized viral proteins, or whether newly synthesized viral
proteins are directed to form new MWs de novo. The HCV
nonstructural protein NS5A is believed to be a component of
MWs. We have developed pulse-chase fluorescent labeling
and imaging approaches that allow us to discriminate ‘old’
from ‘new’ NS5A-positive membranous structures (NS5A foci).
With this system, we find that ‘new’ NS5A-positive foci form at
sites distinct from ‘old’ NS5A foci, supporting a model of de
novo MW formation instead of renewal of previously formed
MWs. We also find that the phosphatidylinositol 4-kinase
PI4KA and oxysterol-binding protein, which are known to be
required for normal MW morphogenesis, are required to maintain
the normal morphology of both ‘old’ and ‘new’ foci, and
that these two proteins appear to play a role in de novo NS5A
focus formation. Finally, we observe increasing colocalization
of NS5A-positive foci with HCV core protein and LDs over time.
Overall, these data support a model in which HCV MWs are
formed de novo rather than renewed. We hypothesize that
newly-formed NS5A foci undergo a process of maturation and
movement towards sites of putative virion assembly at or near
LDs. This may have implications for our understanding of how
replication membranes are formed for other positive-sense RNA
viruses.
Disclosures:
The following authors have nothing to disclose: Hongliang Wang, Andrew W.
Tai
28
Rapid normalization of antiviral and pro-inflammatory
transcriptional signatures in peripheral blood of patients
and livers of humanized mice treated with DAAs
Matthew A. Burchill 1 , Lucy Golden-Mason 1 , Megan Wind-Rotolo 2 ,
Michael Gale 3 , Hugo R. Rosen 1 ; 1 GI-Hepatology, University of
Colorado-Denver, Aurora, CO; 2 Exploratory Clinical and Translational
Research, Bristol-Myers Squibb, Lawrenceville, NJ; 3 Immunology,
University of Washington, Seattle, WA
Chronic HCV infection results in sustained immune activation
in both the periphery and hepatic tissue. Recently, novel
direct acting antiviral (DAA) regimens have been shown to
induce rapid and sustained clearance of HCV. DAAs have
provided the unique opportunity to determine whether successful
treatment-induced eradication of viral RNA normalizes
the dysregulated antiviral innate immune response in patients
chronically infected with HCV. Methods: We used both a
prospective cohort of patients and a novel humanized mouse
model. Results: First, in 35 patients receiving DAAs, we found
that a regimen of daclatasvir, asunaprevir, and BMS-791325
induced not only rapid viral clearance, but also a re-setting
of antiviral responses in the peripheral blood. Specifically,
we observed that following treatment with DAAs, there was
a significant reduction in IFITM1 and the chemokines CXCL10