studies

HEPATOLOGY, VOLUME 62, NUMBER 1 (SUPPL) AASLD ABSTRACTS 1251A
2143
Osteopontin promotes cholangiocyte chemokine secretion
and macrophage accumulation in mice
Jason D. Coombes 1 , Paul P. Manka 1,2 , Marzena Swiderska-Syn 3 ,
Antonio Riva 4 , Danielle Reid 5 , Lee C. Claridge 6 , Laurent Dollé 7 ,
Rasha Younis 1 , Marco A. Briones-Orta 1 , Naoto Kitamura 1 , Zhiyong
Mi 8 , Paul C. Kuo 8 , Roger Williams 1,4 , Anna Mae Diehl 3 ,
Shilpa Chokshi 4 , Bertus Eksteen 5 , Ali Canbay 2 , Wing-Kin Syn 1,8 ;
1 Regeneration and Repair Group, Institute of Hepatology, Foundation
for Liver Research, London, United Kingdom; 2 Department
of Gastroenterology and Hepatology, Essen University Hospital,
Essen, Germany; 3 Department of Medicine, Division of Gastroenterology,
Duke University, Durham, NC; 4 Viral Hepatitis and
Alcohol Research Group, The Institute of Hepatology, Foundation
for Liver Research, London, United Kingdom; 5 Snyder Institute for
Chronic Diseases, Health Research and Innovation Centre (HRIC),
University of Calgary, Calgary, AB, Canada; 6 Department of
Hepatology, Leeds Teaching Hospital NHS Trust, Leeds, United
Kingdom; 7 Liver Cell Biology Lab (LIVR), Department of Cell Biology
(CYTO),, Faculty of Medicine and Pharmacy, Vrije Universiteit
Brussel, Brussels, Belgium; 8 Department of Surgery, Loyola University
Chicago, Chicago, IL
Background/Aim: Liver disease progression is characterized
by recruitment and accumulation of inflammatory cells, which
cluster around the peri-portal regions (i.e. ductular inflammatory
response). We recently reported that repair-associated
Hedgehog (Hh) ligands could induce cholangiocytes to secrete
chemokines. The downstream Hh target Osteopontin (OPN)
is widely upregulated during tissue injury and repair, and
has been shown to regulate macrophage functions via pro-inflammatory
chemoattractant properties. Although OPN is recognised
as a key driver of liver fibrogenesis via activation of
hepatic stellate cells and progenitor cells, the role of OPN in
liver macrophage activation and recruitment remains unclear.
We hypothesized that OPN stimulates cholangiocyte production
of chemokines responsible for recruiting macrophage
subsets that enhance liver fibrogenesis. Methods: In vivo: the
role of OPN on total liver chemokine expression and macrophage
numbers were assessed in two models of liver fibrosis
(methionine-choline deficient diet and 3, 5,-diethoxycarbonyl-1,4-dihydrocollidine
diet) by qRTPCR and/or FACS analysis
(CD11b, Gr1, F4/80, CCR2) of liver infiltrating mononuclear
cells. In vitro: OPN knockdown was achieved in murine 603B
cholangiocytes using lentiviral mediated shRNA, and secreted
OPN neutralized using specific aptamers. To determine if OPN
modulates cholangiocyte chemokine production, conditioned
media and cell lysates were harvested. Cytokine secretion was
measured by cytometric bead array and mRNA by qRTPCR.
The RAW264.7 cell line was used to study macrophage migration
in a transwell assay. Results: At the end of treatment, MCD
and DDC-fed mice developed liver fibrosis, upregulated total
liver OPN, TGF-β, MCP-1, RANTES, and CXCL1 mRNA, and
accumulated liver CD11b/F480(+) CCR2 (hi) macrophages.
Conversely, MCD-fed mice that received OPN-aptamers (which
neutralizes circulating OPN) downregulated MCP-1, RANTES,
and CXCL1 mRNA, reduced liver CD11b/F4/80(+) CCR2 (hi)
by 30%, and developed less fibrosis. In vitro, OPN knockdown
reduced the secretion of chemokines RANTES, MCP-1
and CXCL1 (by 75%, 73% and 41%, respectively). There were
similar reductions in RANTES, MCP-1 and CXCL1 mRNA. Additionally,
RAW264.7 macrophages cultured with conditioned
media from 603B treated with OPN-neutralizing aptamer
exhibited a 35% decrease in migration. Conclusions: OPN is
overexpressed in progressive liver disease, enhances cholangiocyte
production of key macrophage chemokines MCP-1,
RANTES, and CXCL1, and promotes macrophage accumulation.
OPN-neutralization leads to attenuated fibrogenic outcomes
and is a promising anti-fibrotic strategy.
Disclosures:
The following authors have nothing to disclose: Jason D. Coombes, Paul P.
Manka, Marzena Swiderska-Syn, Antonio Riva, Danielle Reid, Lee C. Claridge,
Laurent Dollé, Rasha Younis, Marco A. Briones-Orta, Naoto Kitamura, Zhiyong
Mi, Paul C. Kuo, Roger Williams, Anna Mae Diehl, Shilpa Chokshi, Bertus
Eksteen, Ali Canbay, Wing-Kin Syn
2144
Liver Enzymes Elevation In The Setting Of Chronic
Graft-Versus-Host Disease Is A Non Specific Marker Of
Inflammation That Does Not Accurately Predict Disease
Related Hepatic Injury
Ma Ai Thanda Han 4 , Niharika Samala 4 , Bisharah S. Rizvi 2 , Kenneth
J. Wilkins 4 , Ohad Etzion 4 , Elizabeth C. Jones 5 , Christopher
Koh 4 , David E. Kleiner 1 , Steven Pavletic 3 , Theo Heller 4 ; 1 LABORA-
TORY OF PATHOLOGY, NCI, Bethesda, MD; 2 Internal Medicine,
Canton medical education foundation, Canton, OH; 3 NIH-NCI,
Bethesda, MD; 4 Liver Diseases Branch, NIH/NIDDK, Bethesda,
MD; 5 Radiology, NIH-Clinical Center, Bethesda, MD
Introduction: Diagnosis of chronic hepatic graft-versus-host disease
(HcGVHD) is challenging, as the current scoring system
(the NIH Scoring System or NSS) is based on total bilirubin
(TB), alanine aminotransferase (ALT) and alkaline phosphatase
(ALP). The challenge is that liver associated enzyme (LAE) and
bilirubin elevations may be nonspecific. Once diagnosed with
HcGVHD, patients are treated with immunosuppressive medications
that have risk. Aims: To stratify patterns of LAE that could
mimic HcGVHD, to assess for cytokine associations, and to
assess the accuracy of the NSS for HcGVHD. Method: Patients
with stable chronic GVHD (cGVHD) at any site were enrolled at
variable times from bone marrow transplant. LAE, PT, TB, albumin,
platelets, inflammatory markers (ESR, ferritin, C3), and
cytokines were measured. Patients underwent liver biopsy at
the physicians’ discretion. Box-Cox power transformation and t
test were used for testing strength of associations. Results: 302
patients (58% men, median age 43 y, ALT=39 IU/ml, ALP=96
IU/ml, platelet=245 x 10 3 /ml) with cGVHD were included.
.Abnormal ALT and AST were significantly associated with
lower platelets, and higher TB and PT, in addition to higher ferritin,
C3, MDC, TGFα, IFNα, IL15 and amyloid A (p= 0.0009,
0.02, 0.007, 0.02, 0.04, 0.02, 0.04 respectively). Abnormal
γ glutamyl transferase (GGT) was associated higher TB and
PT but not with lower platelets. In addition, higher GGT was
associated with elevated ESR (p=0.04) and C3 (p=0.0002),
as well as with MDC, TGFα, IL 6, IP10 and IFNγ (p= 0.009,
0.02, 0.008, 0.04, 0.02 respectively). Abnormal ALT, AST
and GGT together were associated with abnormal platelet,
TB, PT, C3 and ferritin (p=0.03, 0.01, 0.0003, 0.0007,
0.02 respectively), as well as with MDC, and IL15 (p=0.008,
0.01 respectively). 151/302 patients were diagnosed with
HcGVHD by NSS. 24/302 patients underwent liver biopsy.
Among them, 12 patients were diagnosed with HcGVHD by
NSS of whom only 5 had histologic features that were consistent
with HcGVHD, while others had characteristics of non-alcoholic
steatohepatitis or nodular regenerative hyperplasia.
Of the non-HcGVHD group by NSS, 4 had HcGVHD. Overall
sensitivity and specificity for HcGVHD was 55% and 53%.
There was no statistical differences between biopsy proven
HcGVHD versus non-HcGVHD for AST, ALT, ALP, GGT, platelet,
TB and albumin. Conclusion: Abnormal LAE in cGVHD is a
marker of inflammation rather than a reflection of any specific
pattern of liver injury. The NSS is neither sensitive nor specific
for HcGVHD diagnosis. Hepatic histology may therefore be