studies

706A AASLD ABSTRACTS HEPATOLOGY, October, 2015
Gastroenterology, 2014) shedding light on new pathways of
IFN. It is now clear that JAK/STAT and the hundreds of Interferon
Stimulated Genes (ISGs) it stimulates is not the only pathway
mediating IFN’s antiviral effects in the liver. One of the
genes we identified is IQ-motif containing GTPase activating
protein 2 (IQGAP2). IQGAP2 is predominantly expressed in
the liver and has been described as a tumor suppressor. Here
we show that IQGAP2 also mediates IFN anti-HCV response
in infected hepatoma cells. Indeed, IQGAP2 siRNA-mediated
knock-down rescues HCV infection from IFN inhibition. We
show that IQGAP2 is not itself an ISG and its knockdown
does not affect JAK/STAT activation, suggesting that IQGAP2
acts independently of the JAK/STAT pathway. We found that
IQGAP2 interacts with RelA/p65, a subunit of NF-κB transcription
factor. Interestingly, our data suggest that IFN alone
is sufficient to activate RelA and stimulate NF-κB-dependent
transcription in hepatoma cells; this process requires IQGAP2
expression. Furthermore, we found that RelA silencing mimics
IQGAP2 knock-down effects in rescuing HCV from IFN inhibition
in IFN-deficient cells. This suggests that IQGAP2 and RelA
mediate IFN anti-HCV response downstream of IFN binding to
its receptor, a mechanism different from the well characterized
role of NF-κB in IFN production. Finally, we investigated the
effect of IQGAP2 or RelA knock-down on the induction by
IFN of 23 ISGs with previously described anti-HCV properties.
We found that IQGAP2 and RelA are both required for the
full induction of two thirds of the tested genes suggesting that
IQGAP2 and RelA mediate IFN anti-HCV response by controlling
ISG induction in parallel of the JAK/STAT pathway.
Altogether, our data demonstrate a previously unrecognized
function for IQGAP2 in regulating innate antiviral response
against HCV, and probably other hepatotropic pathogens.
Since IQGAP2 is predominantly expressed in the liver, understanding
the mechanism of RelA activation by IQGAP2 may
open novel opportunities to prevent NF-kB associated liver disease.
Disclosures:
Stephane Chevaliez - Advisory Committees or Review Panels: Janssen; Speaking
and Teaching: Gilead, BMS
Dahlene Fusco - Grant/Research Support: Gilead
Raymond T. Chung - Grant/Research Support: Gilead, Mass Biologics, Abbvie,
Merck, BMS
The following authors have nothing to disclose: Cynthia Brisac, Shadi Salloum,
Victor Yang, Esperance A. Schaefer, Jian Hong, Charles Carlton-Smith, Nadia
Alatrakchi, Anna Lidofsky, Xiao Liu, Dachuan Cai, Lee F. Peng, Wenyu Lin
1016
Single-strand RNA-induced monocyte differentiation
generates pro-fibrogenic M2 macrophages in chronic
hepatitis C Virus infection
Banishree Saha, Karen Kodys, Gyongyi Szabo; Medicine, UMASS
Med School, Worcester, MA
Purpose: Innate immune responses, including monocyte and
macrophage activation, contribute to the pathogenesis of
chronic Hepatitis C Virus (HCV) infection and liver fibrosis.
However the mechanisms that lead to liver fibrosis during
HCV infection are not well understood. In response to the
tissue microenvironment, monocytes undergo differentiation
into polarized states including M1 (pro-inflammatory) or M2
(anti-inflammatory/pro-fibrotic) macrophages. We hypothesized
that during chronic HCV infection monocytes undergo
differentiation that affect liver disease progression. Methods:
Healthy human monocytes (n=10-15) were cultured with cellfree
HCV or exosomes isolated from HCV-infected Huh7.5
hepatoma cells for 5-7 days. Circulating monocytes and liver
macrophages from HCV-infected patients and controls were
analyzed by flow cytometry and western blotting. Results:
Co-culture of healthy monocytes with cell-free HCV or exosomes
derived from HCV-infected Huh7.5 cells resulted in differentiation
of monocytes into macrophages (MΦ) with high CD14
and CD68 expression. Exosomes from HCV-infected Huh7.5
cells contained HCV single stranded (ss) RNA. These MΦ displayed
M2 markers (CD206, CD163 and DC-SIGN) and produced
pro- and anti-inflammatory cytokines. The HCV-induced
M2-MΦ led to hepatic stellate cell (LX2) activation indicated
by increased expression of collagen, TIMP-1 and α-SMA and
this was prevented by anti-TGFβ blocking antibody administration.
The HCV-induced monocyte activation and differentiation
into M2-MΦ was prevented by TLR8 but not TLR3 or
TLR7 knock-down. TLR8 ligands, independent of HCV, caused
monocyte differentiation and M2-MΦ polarization suggesting
a role for TLR8 in this process. Furthermore, HCV ssRNA alone
induced monocyte to M2-MΦ differentiation and polarization,
demonstrating that HCV ssRNA signals via TLR8 induce monocyte
differentiation and polarization. In vivo in patients with
chronic HCV infection, we observed a significant increase in
the expression of M2 markers (CD206, CD163) on circulating
monocytes and in the liver. Furthermore, we found a significant
increase in collagen + CD206 + CD14 + circulating monocytes
and the increased frequency of collagen + CD206 + CD14 + circulating
monocytes correlated with liver fibrosis in HCV-infected
patients. Conclusion: Our data identify a new mechanism by
which cell-free and exosome-packaged HCV ssRNA interacts
with monocytes and induces TLR8-mediated differentiation to a
pro-fibrogenic, M2-MΦ. These M2-MΦ promote liver fibrosis.
We also identified M2-marker plus collagen-expressing circulating
monocytes as potential biomarkers of fibrosis in chronic
HCV infection.
Disclosures:
The following authors have nothing to disclose: Banishree Saha, Karen Kodys,
Gyongyi Szabo
1017
Elucidating Mechanisms Underlying Development of
Liver Disease in HIV/HCV Coinfection
Jinhee Hyun 3 , Masato Yoneda 1 , Eugene R. Schiff 1 , Emmanuel
Thomas 1,2 ; 1 Schiff Center for Liver Diseases, University of Miami
Miller School of Medicine, Miami, FL; 2 Sylvester Comprehensive
Cancer Center, Miami, FL; 3 Cell Biology, University of Miami
School of Medicine, Miami, FL
BACKGROUND: Patients with HIV that are coinfected with
HCV are at increased risk for rapidly progressive liver disease
and subsequently the development of Hepatocellular Carcinoma
(HCC). Specifically, HCC develops earlier in coinfected
patients and these patients are more symptomatic than those
with only HCV infection at diagnosis suggesting that both viruses
increase the propensity for malignant transformation. However,
the genetic and cellular based mechanisms underpinning how
HCV initiates and subsequently induces liver pathology and
why coinfection with HIV results in significantly worse hepatic
disease remains to be clarified. In addition, the specific cell
types that contribute to these clinical outcomes are unknown.
In this study, we specifically are testing the hypothesis that the
robust viral RNA dependent innate immune response that we
have characterized, that drives inflammation in HCV infected
livers, is augmented with HIV coinfection through activation
of viral DNA and RNA sensing pathways. METHODS: To this
end, we have developed novel in-vitro models that utilize HCV
infected primary human hepatocytes (PHHs) and HIV infected
primary kupffer cells (PKCs). Specifically, the JFH-1 strain was
used to study HCV while the BAL (R5) and IIIB (X4) strains were