studies

886A AASLD ABSTRACTS HEPATOLOGY, October, 2015
1379
Selective antibody targeting of lysyl oxidase-like 2
(LOXL2) suppresses hepatic fibrosis progression and
accelerates its reversal via crosslinking-dependent and
independent mechanisms
Naoki Ikenaga 1 , Susan B. Liu 1 , Zhen-Wei Peng 1 , Shuhei Yoshida 1 ,
Deanna Sverdlov 1 , Satyajit Karnik 2 , Amanda Mikels-Vigdal 2 , Victoria
Smith 2 , Detlef Schuppan 1 , Yury Popov 1 ; 1 Gatroenterology,
Beth Israel Deaconess Medical Center, Boston, MA; 2 Gilead Sciences,
Inc, Foster City, CA
BACKGROUND/AIMS: LOXL2 plays a role in collagen cross-linking
and epithelial cell differentiation. We studied the role of
LOXL2 in collagen cross-linking and hepatic progenitor cell
(HPC) differentiation, and the therapeutic efficacy of anti-LOXL2
mAb on liver fibrosis progression/reversal in mice. METHODS:
Anti-LOXL2 antibody (AB0023 mAb, 30mg/kg), control antibody
(M64, 30mg/kg) or placebo was administered i.p.
twice a week during thioacetamide (TAA)-induced fibrosis progression
(delayed treatment, week 6 to 12 of TAA) or during
recovery (1-12 weeks off TAA). Therapeutic efficacy in biliary
fibrosis was tested in BALB/c.Mdr2-/- and 3,5-diethoxycarbonyl-1,4-dihydrocollidine(DDC)-fed
mice. Collagen cross-linking
and fibrosis were assessed by histological and biochemical
methods. HPC differentiation was studied in primary EpCAM(+)
cells in vitro. RESULTS: LOXL2 was virtually absent from healthy
but strongly induced in fibrotic livers, with predominant localization
within fibrotic septa. Delayed anti-LOXL2 treatment
of pre-established TAA-induced fibrosis suppressed collagen
crosslinking, and histological signs of bridging fibrosis in the
anti-LOXL2-treated group improved, with a 53% reduction in
connective tissue deposition by morphometry. During recovery,
anti-LOXL2 mAb promoted fibrosis regression, histological
signs of fibrotic septa remodeling (splitting and thinning),
and a 45% decrease in connective tissue area (p=0.021) at
early recovery stages (4 weeks). In Mdr2-/- and DDC-induced
models of biliary fibrosis, anti-LOXL2 mAb achieved significant
antifibrotic efficacy but did not affect collagen cross-linking.
Instead, ductular reaction and proliferation was suppressed
in mice with LOXL2 inhibition, whereas hepatocyte replication
increased. The LOXL2-blocking antibody had a profound effect
on primary EpCAM(+) HPC in vitro, promoting their differentiation
towards a hepatocyte lineage while inhibiting ductal cell
lineage commitment. CONCLUSIONS: LOXL2 mediates collagen
cross-linking and fibrotic matrix stabilization during liver
fibrosis, and regulates hepatic progenitor cell differentiation. A
therapeutic anti-LOXL2 antibody inhibits the progression of liver
fibrosis and promotes fibrosis reversal.
Disclosures:
Satyajit Karnik - Employment: Gilead Sciences
Victoria Smith - Employment: Gilead Sciences Inc
Detlef Schuppan - Consulting: Boehringer Ingelheim, Conatus, GLG, Merck,
Mitsubishi-Tanabe, Takeda, Silence, Glenmark, Isis; Grant/Research Support:
Boehringer-Ingelheim
Yury Popov - Grant/Research Support: Gilead Sciences, Inc, Takeda
The following authors have nothing to disclose: Naoki Ikenaga, Susan B. Liu,
Zhen-Wei Peng, Shuhei Yoshida, Deanna Sverdlov, Amanda Mikels-Vigdal
1380
IL-22 enhances TGF-beta pro-fibrotic function in hepatic
stellate cells in a p38/MAPK dependent manner.
Thomas Fabre 1,2 , Manuel Flores Molina 1,2 , Naglaa H. Shoukry 1,3 ;
1 Centre de recherche du CHUM, Montrèéal, QC, Canada; 2 Département
de microbiologie, immunologie et infectiologie, Université
de Montréal, Montréal, QC, Canada; 3 Département de Médecine,
Université de Montréal, Montréal, QC, Canada
Background & Hypothesis: Activation of hepatic stellate
cells (HSCs) is a key event in liver fibrosis, characterized by
enhanced extracellular matrix (ECM) production and altered
degradation. The immune system may modulate activation of
HSCs through different cytokines. We have previously demonstrated
that IL-17A enhances the expression of profibrotic genes
through upregulation of the TGF-β receptor on hepatic stellate
cells in a JNK-dependent manner (Fabre, Journal of Immunology
2014). IL-22 is another Th17 enigmatic cytokine, from the
IL-10 family, with both pro- and anti-inflammatory properties.
IL-22 deficient mice develop high levels of hepatic inflammation
during acute injury compared to their wild type littermates.
In contrast, IL-22 is also elevated in the sera of patients with
liver cirrhosis and carcinoma. However, in a mouse model
of hepatitis B, IL-22 indirectly induces fibrosis by recruiting
the pro-fibrotic inflammatory Th17 cells. We hypothesized that
IL-22 may modulate activation and induction of the fibrogenic
process in HSCs. Methods & Results: The human HSC line
LX2 and primary human HSCs were stimulated with increasing
doses of IL-22 and compared to TGF-β- and PBS- treated
cells as positive and negative controls, respectively. IL-22 did
not induce activation of HSCs. However, IL-22 enhanced the
response of HSCs to suboptimal doses of TGF-β as observed by
strong induction of alpha-smooth muscle actin (-SMA), collagen
type I (COL1A1) and tissue inhibitor of matrix metalloproteinase
(TIMP-I). IL-22 stimulation, in contrast to IL-17A, did not
enhance cell surface expression of TGF-β-RII. To characterize
the cellular mechanisms by which IL-22 enhances the TGF-β
response we performed RNA-seq on primary human HSCs.
However, pre-treatment of HSCs with IL-22 led to increase
phosphorylation of SMAD2/3 in response to suboptimal doses
TGF-β. Gene expression analysis revealed an increased in activation
of p38/MAPK activity related pathways in IL-22 with
TGF-β lo - as compared to PBS- or TGF-β lo treated cells. Activity
was confirmed at the protein level and was associated with
increased phosphorylation of SMAD2/3. Chemical inhibition
of p38 significantly reduces HSCs activation in response to
IL-22 with TGF-β lo . Conclusion: Our results suggest that IL-22
enhances TGF-β signalling in a p38/MAPK dependent manner
leading to increased activation of HSCs.
Disclosures:
The following authors have nothing to disclose: Thomas Fabre, Manuel Flores
Molina, Naglaa H. Shoukry
1381
MeCP2 exerts global control over the myofibroblast
transcriptome and reveals new regulators of fibrosis.
EVA MORAN-SALVADOR, Pier P. Paoli, Graham R. Smith, Agata
Page, Rachel M. Howarth, Fiona Oakley, Jelena Mann, Derek
Mann; Institute of Cellular Medicine, Newcastle University, Newcastle
upon Tyne, United Kingdom
Background: Hepatic stellate cells (HSCs) are considered
the central extracellular matrix (ECM)-producing cells in the
wound-healing response process driven by a persistent liver
injury. Previous studies in our laboratory identified MeCP2
(methyl-CpG binding protein 2) as the epigenetics master reg-