studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 843A
apeutic potential for the management of liver IRI in transplant
recipients.
Disclosures:
Ronald W. Busuttil - Grant/Research Support: Bayer, Fujisawa, Novartis, Roche/
Genentech, NIH
The following authors have nothing to disclose: Shi Yue, Changyong Li, Xingliang
Zhou, Qilong Ying, Jerzy Kupiec-Weglinski, Bibo Ke
1284
The long noncoding RNA VL30-1 is a key regulator of
liver injury via enhancement of HIF1a mediated inflammation
Xinshou Ouyang, Sheng-Na Han, Irma Garcia-Martinez, Luke
Cai, Richard A. Flavell, Wajahat Z. Mehal; Yale University, New
Haven, CT
Introduction: Inflammation is the key feature of liver injury in
response to many insults. The inflammatory gene expression
program is comprised of several specific transcription factors
involving long noncoding RNAs (lncRNAs). HIF1a is well
known regulator of inflammatory gene transcription, and is
required for persistent liver inflammation. LncRNAs play important
roles in diverse biological processes. However, the role of
lncRNAs in regulating hepatic inflammation is not known. Aim:
To identify lncRNA (s) that regulates liver inflammation and
injury. Methods: Primary mouse macrophages were treated
with bacterial lipopolysaccharide (LPS), and whole-transcriptome
analysis (RNA-seq) was conducted. Numerous lncRNAs
were differentially expressed including the short form of the
endogenous retrotransposon VL30-1. Further analysis determined
the functional role and molecular mechanisms of VL30-1
in regulation of immune genes. This was done by siRNA knockdown,,
transcriptome microarray, promoter luciferase assay,
ELISA, RT-PCR, confocal imaging and RNA immunoprecipitation
PCR (RIP)-PCR. The in vivo role of VL30-1 in liver injury
was demonstrated by knockdown in the LPS/D galactosamine
(LPS/D GlN) model. Results: VL30-1 is strongly induced by LPS
in mouse macrophages (fold > 90 vs control). Stable depletion
of VL30-1 in Raw264.7 cells by shRNA significantly down-regulated
LPS induced Il1b mRNA and protein levels. IL-1b secretion
was significantly reduced by VL30-1 depletion in mouse
peritoneal macrophages in response to typical inflammasome
activation (808+/-45 pg/ml in control vs 214+/-125 pg/ml
in VL30-1 siRNA). VL30-1 depletion also markedly reduced
mIL-1b luciferase activity under forced expression of HIF1a
and HIF2a. Conversely, forced VL30-1 expression dose-dependently
trans-activated mIL-1b reporter luciferase activity,
confirming that VL30-1 augments HIF1a mediated IL-1b
transcription. RIP-PCR assay showed a direct VL30-1 binding
with HIF1a. VL30-1 co-localized with HIF1a by confocal
imaging analysis. Depletion of VL30-1 in mice significantly
decreased LPS/ D-GalN induced serum IL-1b protein concentration
(188+/-76 pg/ml in control vs 23+/- 26 pg/ml) as
well as reduced liver damage as judged by morphology and
serum ALT level (418+/-297 in control vs 151+/- 76 in VL30-1
siRNA). Conclusions: These studies reveal a critical role of the
lncRNA VL30-1 as a broad-acting regulatory component in
the control of HIF1a induced inflammatory pathway activation
in mouse macrophages and liver injury. This occurs via direct
interaction of VL30-1 and HIF-1a, and up-regulation of HIF-1a
mediated inflammatory transcripts.
Disclosures:
The following authors have nothing to disclose: Xinshou Ouyang, Sheng-Na Han,
Irma Garcia-Martinez, Luke Cai, Richard A. Flavell, Wajahat Z. Mehal
1285
Galectin-3 mediates NLRP3 inflammasome signaling
and contributes to pathogenesis of primary biliary cirrhosis
Jijing Tian 1,2 , Guo-Xiang Yang 1 , Fu-Tong Liu 1 , M. Eric Gershwin
1 , Natalie J. Torok 1 , Joy Jiang 1 ; 1 UCDMC, Sacramento, CA;
2 Research Center for Eco-environmental Sciences, Chinese Academy
of Sciences, Beijing, China, Beijing, China
Macrophages play a significant role in early inflammatory
changes in primary biliary cirrhosis (PBC). However, their
role in modulating fibrogenic responses has not been evaluated.
We and others have shown that macrophages are
a major source of galectin-3, a profibrogenic lectin. As the
pathogenesis of PBC is not well understood, we hypothesized
that galectin-3 is required to activate inflammasome signaling
contributing to inflammation, fibrosis and progression in PBC.
Methods: Liver tissues from PBC patients and healthy controls;
and from the dnTGFβRII transgenic mouse model of PBC and
wt controls were collected for RT-PCR and Western blot to analyze
the expression of galectin-3, NLRP3, ASC and IL-1β. The
cleavage of caspase-1 and IL-1β in PBC patients was examined
by Western blots. Primary hepatic macrophages were
isolated from wt and the galectin3 -/- mice, and treated with
Deoxycholic Acid (DCA) with/without recombinant galectin-3.
RT-PCR was done to analyze the activation of inflammasome-related
transcripts; immunofluorescence (IF) to detect
the activation of inflammasomes; and immunoprecipitation (IP)
to detect the association of galectin-3 and NLRP3. DnTGFβRII/
gal3 -/- mice were generated by crossing the dnTGFβRII mice
with galectin3 -/- mice and the liver tissues were analyzed for
inflammasome activation and fibrosis. Results: The transcripts
of galectin-3, NLRP3, ASC and IL-1β significantly increased in
the livers of PBC patients (N=4, p