studies

620A AASLD ABSTRACTS HEPATOLOGY, October, 2015
on biliary fibrosis in the Mdr2-/- mouse model, which develops
periportal fibrosis similar to human PSC. METHODS: Histology,
inflammatory cell infiltration, gene expression, and collagen
architecture and content were studied in livers from Mdr2-/-
;CD39-/- and Mdr2-/- mice. Liver infiltrating lymphocytes were
analyzed by flow cytometry. CD8-depleting antibody was
administered to cholic acid challenged Mdr2-/-;CD39-/- mice.
In vitro retinoic acid (RA)-induced gut-homing α4β7 expression
was assessed in the presence of extracellular nucleotides or
adenosine. RESULTS: Mdr2-/-;CD39-/- mice exhibited severe
liver injury and significantly worse portal fibrosis when compared
to Mdr2-/- mice. The Mdr2-/-;CD39-/- mice showed
more pronounced elevations in serum ALT levels, increase in
total collagen levels, as determined by hydroxyproline quantification,
and upregulated pro-fibrogenic mRNA levels in the
liver. Flow cytometry analysis of liver infiltrating lymphocytes
revealed a selective increase in CD8+ T cells in CD39 deficient
Mdr2-/- mice. Further, hepatic transcription of the b7 integrin, a
T cell gut tropism marker, was significantly increased in Mdr2-
/-;CD39-/- mice. Antibody-mediated CD8+ T cell depletion
in Mdr2-/-;CD39-/- mice improved outcomes with decreased
serum alkaline phosphatase and lower expression of liver fibrosis-related
genes, supporting the pathogenetic role of CD8+ T
cells in PSC. RA alone was able to induce the expression of the
α4β7 gut-homing integrin in naïve CD8+ T cells. Interestingly,
stimulation with ATP further enhanced the RA-mediated expression
of α4β7, whereas adenosine demonstrated the opposing
effect by downregulating integrin expression levels. CONCLU-
SION: Genetic deletion of CD39 exacerbates biliary injury
and fibrosis in Mdr2-/- mice. Disease progression occurs in a
CD8+ T cell-dependent manner and extracellular nucleotides
increase the expression of the gut-homing recpetor α4β7. Thus,
the purinergic system provides possible pharmacological targets
for the treatment of PSC.
Disclosures:
Yury Popov - Grant/Research Support: Gilead Sciences, Inc, Takeda
Simon C. Robson - Grant/Research Support: Pfizer, NIH, Dainippon; Independent
Contractor: Biolegend, EMD Millipore, Mersana; Management Position:
eBioscience; Speaking and Teaching: ACP, Elsevier, ATC; Stock Shareholder:
Nanopharma, Puretech
The following authors have nothing to disclose: Sonja Rothweiler, Zhen-Wei
Peng, Susan B. Liu, Naoki Ikenaga
827
Feedback regulation of autotaxin in mice: why cholestatic
mice don’t scratch
Ruth Bolier 1 , Dagmar Tolenaars 1 , Jacqueline Langedijk 1 , Piter J.
Bosma 1 , Ulrich Beuers 1,2 , Ronald Oude Elferink 1 ; 1 Tytgat Institute
for Liver and Intestinal Research, Academic Medical Center,
Amsterdam, Netherlands; 2 Department of Gastroenterology and
Hepatology, Academic Medical Center, Amsterdam, Netherlands
Introduction: Serum activity of autotaxin (ATX, which produces
lysophosphatidic acid, LPA) is increased in humans during
pregnancy and cholestasis and correlates with itch intensity
(Gastroenterology 2010;139:1008). In contrast, serum ATX in
mice is only marginally increased under these conditions and,
consistently, there is no increase in scratch behavior. Aim: To
study the difference in regulation of ATX expression between
humans and mice. Methods: Expression of LPA receptors
(LPAR 1-6
) and feedback repression of ATX expression trough
LPA receptor signaling was studied in human and mouse fibroblasts
by qPCR. LPA signaling was stimulated with the stable
LPA-analogue XY17 (1uM) or via endogenous ATX production
with lipid phosphate phosphatase 1 inhibitor XY14 (10uM), to
prevent the rapid breakdown of LPA. LPA production by endogenous
ATX was inhibited by ATX inhibitor HA115 (10uM) and
LPAR 1-3
signaling was blocked with the receptor antagonist
Ki16425 (25uM). Involvement of G-proteins and their downstream
pathways was investigated using the cAMP stimulator
forskolin (25uM), protein kinase A inhibitor RP-adenosine
(100uM), G α
i/o-inhibitor pertussis toxin (500ng/mL), inositosol
1,4,5-triphophate inhibitor U73122 (1uM) and protein
kinase C inhibitor Gö6983 (10uM). In addition, the established
induction of ATX by TNFα (10ng/mL) was compared
in fibroblasts of the two species. Results: TNFα stimulated
ATX expression 3- to 4-fold in fibroblasts from both species. In
murine cells, stimulation of LPAR-signaling by XY14 and XY17
repressed ATX mRNA expression by 70 and 82%, respectively
(both p