studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 697A
hand, iron loss has been reported to trigger PINK1/Parkin-independent
mitophagy. The aim of this study was to determine
whether iron chelation restores mitophagy suppressed by HCV.
Methods: HCV-JFH1 infected cells were treated for 24h with a
final concentration of 1mM deferiprone (DFP), iron chelator.
Transgenic mice expressing the HCV polyprotein were administered
0.075g of DFP dissolved in water through gastric tube
for 2 months. The effect of DFP on mitophagy and mitochondrial
function was examined in vitro and in vivo. Results: As
reported previously, suppressed mitophagy was confirmed in
HCV-JFH1 infected cells. DFP treatment increased the expression
of microtubule-associated protein light chain 3 (LC3)-II and
decreased the expression of p62 in dose-dependent manner,
but did not affect translocation of Parkin to mitochondria in
HCV-JFH1 infected cells. Electron microscopy also revealed
that DFP significantly increased the number of mitophagosomes
in HCV-JFH1 infected cells and liver specimens from transgenic
mice expressing the HCV polyprotein. These results showed
that DFP restored mitophagy suppressed by HCV core protein.
DFP also decreased mitochondrial membrane potential
and reactive oxygen species (ROS) production, but not ATP
production in HCV-JFH1 infected cells. Next, we measured
mitochondrial ferrous iron content, using a highly specific
turn-on fluorescent probe (Ac-MT-FluNox1) that is specific to
labile ferrous iron in mitochondria. Mitochondrial ferrous iron
content was significantly higher in cells with HCV-JFH1 infection
than in those without HCV-JFH1 infection in the absence of
DFP treatment. DFP decreased mitochondrial ferrous iron content
in dose dependent manner regardless of HCV infection.
Conclusion: These results indicated that iron chelation restores
mitophagy suppressed by HCV. The mechanisms underlying
mitophagy induced by iron chelation remains to be unknown,
but decrease in mitochondrial ferrous iron and disrupted mitochondrial
function may be critical for inducing mitophagy.
Disclosures:
The following authors have nothing to disclose: Yuichi Hara, Sohji Nishina,
Tasuku Hirayama, Hideko Nagasawa, Keisuke Hino
996
Cell-specific peripheral innate immune responses to
immunomodulatory stimulation in chronic HCV
Jacinta A. Holmes 1,2 , Narelle A. Skinner 2 , Mario Congiu 2 , Rosemary
M. Millen 2 , Lijia Yu 2 , William Sievert 3 , Paul V. Desmond 1 ,
Kumar Visvanathan 2 , Alex J. Thompson 1,2 ; 1 Gastroenterology, St
Vincent’s Hospital, Fitzroy, VIC, Australia; 2 Immunology Research
Centre, St Vincent’s Hospital, Melbourne, VIC, Australia; 3 Gastroenterology,
Monash Health, Monash University, Melbourne, VIC,
Australia
INTRODUCTION: Our preliminary data showed chronic HCV
(CHC) patients (pts) who achieve SVR have potent and early
immune responses, with chemokine, cytokine and interferon-stimulated
gene (ISG) induction. The pattern is similar in CC
IFNL3 genotype (gt) pts. There are no data regarding which
cell types contribute most to immunomodulatory responses or
which immune pathways are important in CHC. We hypothesised
monocytes (MC) would display the greatest induction
of ISGs post-stimulation, particularly in CC pts, and viral sensing
TLR pathways would generate the greatest responses. We
performed a pilot study to characterise cell-specific ISGs in
CHC PBMCs pre/post immunomodulatory stimulation. METH-
ODS: Treatment-naïve HCV1 F0-3 pts were enrolled. PBMCs
were stimulated with TLR2, 3, 4, 7/8, 9 ligands and IFNa.
PBMCs were sorted into NKs, NKTs and MC using a cell
sorter. RNA was extracted, and ISG expression (MX1/ISG15)
measured (rtPCR). Levels were compared between cell types,
pre/post-stimulation (fold-change), and according to IFNL3 gt.
RESULTS: 10 pts were included: 50% CC, 40% male, median
age 53 years. At baseline non-CC pts demonstrated higher ISG
mRNA, predominantly from NK/NKT cells (Fig.1a). Post-stimulation,
ISG induction was significantly higher in MC (131 to
168-fold increase) than NK/NKT cells (maximum 60-fold-increase)
following TLR7/8 and IFNa stimulation (Fig.1b). MC
from CC pts demonstrated a 2-3 fold-increase in MX1 than
non-CC pts following stimulation (Fig.1c). CONCLUSIONS:
The data identify MC as key immunomodulatory cells, and
demonstrate the importance of the TLR7/8 pathway in immune
responses in CHC, supporting clinical development of TLR7
agonists. Furthermore, MC from CC IFNL3 gt pts demonstrated
greater immune reactivity, and may contribute to the more
potent immune responses observed during therapy, leading to
greater viral clearance. Unraveling these differences in immune
responses may lead to new therapeutic targets, particularly for
patients who fail IFN-free therapies.
Disclosures:
Jacinta A. Holmes - Grant/Research Support: Roche, Merck, AASLD, St Vincent’s
Hospital Research Endowment Fund
William Sievert - Advisory Committees or Review Panels: Gilead Sciences, Bristol
Myers Squibb, Merck; Speaking and Teaching: Merck, Bristol Myers Squibb
Alex J. Thompson - Advisory Committees or Review Panels: Gilead, Abbvie,
BMS, Merck, Spring Bank Pharmaceuticals, Arrowhead, Roche; Grant/Research
Support: Gilead, Abbvie, BMS, Merck; Speaking and Teaching: Roche, Gilead,
Abbvie, BMS
The following authors have nothing to disclose: Narelle A. Skinner, Mario Congiu,
Rosemary M. Millen, Lijia Yu, Paul V. Desmond, Kumar Visvanathan
997
Role of very-low-density lipoprotein (VLDL) biogenesis
in hepatitis C virus (HCV) production: a reassessment in
primary human adult hepatocytes
Veronique Pene 1 , Maxime Villaret 1 , Matthieu Lemasson 1 , Lynda
Aoudjehane 2,3 , Jean-François Méritet 1,4 , Filomena Conti 3,5 , Yvon
Calmus 3,5 , Arielle R. Rosenberg 1,4 ; 1 EA 4474 “Hepatitis C Virology”,
Université Paris Descartes, Paris, France; 2 Human HepCell,
Hôpital Saint-Antoine, Paris, France; 3 UMRS 938 “CDR Saint-Antoine”,
Inserm, Paris, France; 4 Service de Virologie, AP-HP, Hôpital
Cochin, Paris, France; 5 Unité de Transplantation Hépatique,
AP-HP, Hôpital Pitié-Salpêtrière, Paris, France
Background and Aim. In the blood of HCV-infected patients,
infectivity is mainly supported by viral particles associated
with triglyceride-rich lipoproteins containing apolipoprotein B
(ApoB) and ApoE. These complexes are believed to assemble
within the hepatocyte, which is both the primary replication site