studies

682A AASLD ABSTRACTS HEPATOLOGY, October, 2015
Christian Trautwein - Grant/Research Support: BMS, Novartis, BMS, Novartis;
Speaking and Teaching: Roche, BMS, Roche, BMS
Frank Tacke - Advisory Committees or Review Panels: Tobira; Grant/Research
Support: Novartis, Noxxon; Speaking and Teaching: BMS, Gilead, Falk, MSD,
Janssen, Abbvie
The following authors have nothing to disclose: Robert Schierwagen, Lara Maybüchen,
Sebastian Zimmer, Kanishka Hittatiya, Christer Baeck, Sabine Klein,
Frank E. Uschner, Winfried Reul, Peter Boor, Georg Nickenig, Jogchum Plat,
Dieter Luetjohann, Jonel Trebicka
962
TRPV4 deficiency enhances TLR4 recruitment to lipid
rafts exhibiting exacerbated stellate cell activation and
fibrosis in experimental nonalcoholic steatohepatitis
Suvarthi Das 1 , Ratanesh K. Seth 1 , Varun Chandrashekaran 1 , Firas
Alhasson 1 , Diptadip Dattaroy 1 , Gregory A. Michelotti 2 , Prakash
Nagarkatti 3 , Phillip D. Bell 4 , Wolfgang B. Liedtke 2 , Anna Diehl 2 ,
Saurabh Chatterjee 1 , Mitzi Nagarkatti 3 ; 1 Environmental Health
Sciences, University of South Carolina, Columbia, SC; 2 Duke University,
Durham, NC; 3 University of South Carolina, Columbia,
NC; 4 Medical University of South Carolina, Charleston, SC
Transient receptor potential vanilloid channel 4 (TRPV4) is a
nonselective cation channel that confers sensitivity to extracellular
osmolarity and regulates calcium inflow. We have observed
previously that TRPV4 protein levels are significantly higher in
NASH rodent models and human NASH patients. For the present
study we aimed to investigate the role of TRPV4 in stellate
cell activation and fibrosis in a methionine choline deficient
(MCD) diet-induced mouse model of NASH. Liver homogenates
from mice fed with MCD diet for 8 weeks (8 to 16 weeks
of age), were used for qRTPCR and Western Blots. Paraffin
embedded liver sections from the same mice were used for
immunostaining and histopathological analysis. The Results
showed that TRPV4 deficiency in mice fed with an MCD diet
for 8 weeks showed significantly higher -SMA immunoreactivity
in livers followed by significantly higher macro and micro
vesicular fibrosis (Picrosirius Red staining) when compared to
MCD wild type mice. TRPV4 deficiency led to inflammasome
activation as evidenced by significantly high Apoptosis-associated
speck-like protein containing a CARD (ASC II), cleaved
IL-1β and cleaved Caspase-1 immunoreactivity when assessed
by immunoblots as compared to wild type mice. Mice deficient
in TRPV4 showed higher mRNA expressions of MCP-1 and
IL-1β in the livers as compared to wildtype mice, an observation
that correlated well with histopathology where increased
infiltrating leukocytes were present in the liver. Since NASH
progression and fibrosis were associated with increased lipid
raft recruitment of TLR4 primarily mediated by NADPH oxidase,
we studied whether TRPV4 deficiency could lead to
enhanced NADPH oxidase driven TLR4 recruitment to lipid
rafts in these mice. Results showed that there was a significant
increase in NADPH oxidase activation as evidenced by colocalization
of membrane gp91phox and p47phox subunits in
TRPV4 knockout MCD mice as compared to wild type MCD
mice. This correlated well with a significant increase in the TLR4
colocalization in flotillin containing rafts in the cell membranes.
These dual labeled liver sections were imaged using immunofluorescence
microscopy. The TLR4 raft recruitment was also
accompanied by significant elevation in TLR4 protein in the
TRPV4 deficient mice. Finally, we conclude and propose that
MCD diet feeding might generate severe oxidative stress which
possibly leads to TRPV4 led calcium influx and might aid in
preservation of membrane integrity while lack of TRPV4 causes
severe loss of membrane structure causing leakage of damage
associated molecular patterns into cells causing enhanced
inflammation, stellate cell activation and fibrosis.
Disclosures:
The following authors have nothing to disclose: Suvarthi Das, Ratanesh K. Seth,
Varun Chandrashekaran, Firas Alhasson, Diptadip Dattaroy, Gregory A. Michelotti,
Prakash Nagarkatti, Phillip D. Bell, Wolfgang B. Liedtke, Anna Diehl,
Saurabh Chatterjee, Mitzi Nagarkatti
963
β 7
-Integrins and MAdCAM-1 have opposing functions in
development and progression of Non-alcoholic steatohepatitis
(NASH)
Hannah K. Drescher 1 , Angela Schippers 2 , Thomas Clahsen 2 ,
Hacer Sahin 1 , Norbert Wagner 2 , Christian Trautwein 1 , Konrad L.
Streetz 1 , Daniela C. Kroy 1 ; 1 Department of Internal Medicine III,
University Hospital, RWTH Aachen, Germany, Aachen, Germany;
2 Department of Pediatrics, University Hospital RWTH Aachen,
Aachen, Germany
Introduction: Non-alcoholic steatohepatitis (NASH) is the third
most common reason for liver transplantations in developing
countries and one of the fastest growing medical problems.
Aim: The significance of infiltrating leukocytes and how they
affect NASH development and progression remains unclear.
Therefore, this study investigates the role of the homing receptor
pair MAdCAM-1/β 7
in two different mouse NASH-models.
Methods: Constitutive β 7
-Integrin and MAdCAM-1 knockout
mice were fed either MCDdiet (methionine and choline deficient)
for 4 weeks or HF-diet (high fat) for 26 weeks. Results:
After 4 weeks of MCD treatment β 7
-deficient animals displayed
earlier and faster progressing steatosis reflected by significant
histomorphological changes while MAdCAM-1-KO mice
showed an intact liver architecture, both compared to wildtype
controls. Consistent with severe disease progression β 7
-Integrin
deficient animals showed an increased oxidative stress
response (DHE (Dihydroethidium)-staining) with simultaneous
down regulation of the expression of Treg markers (FoxP3,
IL-2, TGFβ). In contrast, MAdCAM-1-KO mice displayed an
up-regulation of anti-oxidative stress proteins involved and an
enhanced number of infiltrating macrophages tending to provide
a strengthening of the anti-inflammatory response. The
β 7
-KO group exhibited a stronger hepatic infiltration of inflammatory
cells reflecting an earlier onset of NASH. Especially
Th17 positive T cells were increased leading to elevated numbers
of infiltrating neutrophils. Those changes finally resulted
in an earlier and stronger collagen accumulation in these animals
while MAdCAM-1-KO mice were protected from fibrosis
progression. The results suggest a direct effect of the homing
receptor pair MAdCAM-1/β 7
on the gut-liver-axis in NASH
development by an infiltration blockade of cells which have
a positive immuno-modulatory effect on the liver. In a second
model, better reflecting the metabolic changes during NASH
development, HF-diet was fed to mice for 26 weeks. Preliminary
results show comparable results to those in the MCD
model, namely a significantly increased bodyweight and a
worsened glucose tolerance in β 7
-KO animals indicating the
occurrence of the metabolic syndrome in this group compared
to MAdCAM-1-KO and WT. Conclusion: MAdCAM-1 and β 7
Integrin
deficiency leads to opposing effects in the MCD and the
HF model, with protection in MAdCAM-1-KO animals and a
more severe phenotype and significantly stronger fibrosis progression
in β 7
-Integrin-KO mice. Therefore, the interaction of
β 7
Integrins and their receptor MAdCAM-1 provide a potential
novel target for therapeutic interventions during NASH development.
Disclosures:
Christian Trautwein - Grant/Research Support: BMS, Novartis, BMS, Novartis;
Speaking and Teaching: Roche, BMS, Roche, BMS