HEPATOLOGY ELSEWHERE
Role of TWEAK in co-regulating liver progenitor cell and fibrogenic responses.
by
Janina E.E. Tirnitz-Parker1,2
, John K. Olynyk1,2,3,4
, Grant A. Ramm5,6
from
1School of Biomedical Sciences, CHIRI Biosciences Research Precinct, Curtin University, Bentley,
Australia; 2School of Medicine and Pharmacology, University of Western Australia, Fremantle,
Australia; 3Department of Gastroenterology, Fremantle Hospital, Fremantle, Australia;
4Institute for
Immunology and Infectious Diseases, Murdoch University, Murdoch, Australia; 5School of
Medicine, University of Queensland, Brisbane, Australia; 6The Hepatic Fibrosis Group, Department
of Cell and Molecular Biology, The Queensland Institute of Medical Research, Brisbane, Australia.
Kuramitsu K, Sverdlov DY, Liu SB, Csizmadia E, Burkly L, Schuppan D, et al. Failure of fibrotic
liver regeneration in mice is linked to a severe fibrogenic response driven by hepatic progenitor cell
activation. Am J Pathol 2013; doi: 10.1016/j.ajpath.2013.03.018 (Reprinted with permission.)
Abstract
Failure of fibrotic liver to regenerate after resection limits therapeutic options and increases demand for
liver transplantation, representing a significant clinical problem. The mechanism underlying regenerative
failure in fibrosis is poorly understood. Seventy percent partial hepatectomy (PHx) was performed in
C57Bl/6 mice with or without carbon tetrachloride (CCl4)-induced liver fibrosis. Liver function and
regeneration was monitored at 1 to 14 days thereafter by assessing liver mass, alanine aminotransferase
(ALT), mRNA expression, and histology. Progenitor (oval) cell mitogen tumor necrosis factor-like weak
inducer of apoptosis (TWEAK) and TWEAK-neutralizing antibody were used to manipulate progenitor
cell proliferation in vivo. In fibrotic liver, hepatocytes failed to replicate efficiently after PHx. Fibrotic livers
showed late (day 5) peak of serum ALT (3542 ± 355 IU/L compared to 93 ± 65 IU/L in nonfibrotic livers),
which coincided with progenitor cell expansion, increase in profibrogenic gene expression and de novo
collagen deposition. In fibrotic mice, inhibition of progenitor activation using TWEAK-neutralizing
antibody after PHx resulted in strongly down-regulated profibrogenic mRNA, reduced serum ALT levels
and improved regeneration. Failure of hepatocyte-mediated regeneration in fibrotic liver triggers
activation of the progenitor (oval) cell compartment and a severe fibrogenic response. Inhibition of
Hepatology
This article has been accepted for publication and undergone full peer review but has not beenthrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article asdoi: 10.1002/hep.26701
2
progenitor cell proliferation using anti-TWEAK antibody prevents fibrogenic response and augments
fibrotic liver regeneration. Targeting the fibrogenic progenitor response represents a promising strategy to
improve hepatectomy outcomes in patients with liver fibrosis.
Comment
In liver injury conditions with a chronic hepatocyte insult, and therefore continuous inhibition of
hepatocyte replication, a stem cell-like compartment of liver-resident progenitor cells is activated to
mediate regeneration. These liver progenitor cells (LPCs), also referred to as oval cells in rodents or
the Ductular Reaction in humans, are frequently observed to proliferate in a broad range of chronic
human liver diseases. Importantly, their numbers are directly proportional to fibrosis severity,
independent of the underlying pathology (1, 2). The common denominators of LPC-associated
chronic liver diseases are hepatocellular necrosis, release of immunomodulatory mediators by
recruited and resident inflammatory cells and a fibrogenic response. If the chronic injury stimulus is
controlled, LPCs differentiate into biliary epithelial cells and hepatocytes whilst fibrosis resolves to
restore structural and functional liver integrity. However, if the hepatic insult persists, regenerative
processes can spiral out of control and ultimately lead to hepatocellular carcinoma (HCC). This is
likely due to a number of different processes including: (i) hyperstimulation of inflammatory cells,
hepatic stellate cells (HSCs) and macrophages which continually secrete cytokines promoting
survival of proliferating hepatic cells, (ii) excessive matrix deposition by activated
HSCs/myofibroblasts leading to cirrhosis, and (iii) accumulation of mutations and epigenetic
aberrations due to the pro-survival, pro-proliferative microenvironment, which can lead to the
malignant transformation of preneoplastic lesions, potentially the generation of cancer stem cells,
and ultimately the formation of HCC.
Many cytokines are known to regulate LPC biology (3), however only recently the tumor necrosis
factor (TNF) family member TNF-like weak inducer of apoptosis (TWEAK) joined the ranks of
key LPC mediators. TWEAK was initially described as a weak, death domain-independent inducer
of apoptosis in a human adenocarcinoma cell line (4). Subsequent studies identified this
predominantly secreted type II-transmembrane protein as a versatile cytokine involved in regulating
a diverse range of cellular functions. TWEAK has been shown to act as a pro-angiogenic and pro-
inflammatory factor and has also been described as a mediator of proliferation, differentiation,
migration, cell survival and cell death (5). TWEAK is almost ubiquitously expressed in adult tissue
including the liver, with major cell sources being activated monocytes, natural killer cells and
macrophages. During chronic liver injury, LPCs as well as a subpopulation of activated HSCs
express the TWEAK receptor (6), Fn14, suggesting the potential for co-regulation of LPC
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proliferation and fibrogenesis, in part through the TWEAK/Fn14 signaling pathway. While it is
known that TWEAK is a direct mitogen to LPCs (7), mediating its effect via nuclear factor κ B
(NFκB) signaling, the role of Fn14 downstream signaling in HSCs remains to be elucidated. A
previous study provides some evidence for a link between TWEAK-regulated LPC and fibrogenic
responses by demonstrating that Fn14-deficient mice subjected to a choline-deficient, ethionine-
supplemented (CDE) diet showed diminished LPC proliferation as well as reduced collagen
deposition and transcript levels of tissue inhibitor of metalloproteinases (TIMP) 1 and 2 (6).
Expanding upon potential therapeutic roles of TWEAK signaling, authors of a recent publication in
the American Journal of Pathology suggest inhibition of the LPC and fibrogenic response by
blocking the TWEAK signaling pathway as a novel strategy to improve liver regeneration following
hepatectomy during liver fibrosis (8). To study regeneration in non-fibrotic versus fibrotic livers,
Kuramitsu et al. performed 70% partial hepatectomy (PHx) in normal and CCl4-treated fibrotic
mice. Compared to controls, fibrotic mice showed more profound liver weight loss after surgery
and significantly lower survival rates (68.9% vs. 90.8%) as well as slower liver weight gains (11%
vs. 38%) at day 10 post-PHx. These results are consistent with the generally held concept that liver
regeneration is impaired in subjects with advanced fibrosis/cirrhosis and suggest that hepatocyte-
mediated liver regeneration may be compromised even before progression to cirrhosis. The authors
then performed detailed histological and molecular analyses of the fibrotic liver tissue and
demonstrated apoptotic hepatocyte death and increased serum ALT levels, which coincided with the
activation of A6+/CKpan
+ LPCs. The LPC response went hand-in-hand with an increase in
circulating transforming growth factor (TGF) β1 levels, the upregulation of procollagen α 1, α-
smooth muscle actin (αSMA), TIMP-1 and TGFβ1 mRNA as well as increased de novo collagen
synthesis and deposition. This once again demonstrates that LPCs and HSCs are not only in close
spatio-temporal association but that fibrogenesis is intricately linked to the LPC response. When
LPCs were either stimulated to proliferate by recombinant TWEAK-Fc protein injection or
inhibited by TWEAK-neutralizing antibody treatment, the resulting LPC proliferation or inhibition,
respectively, was directly correlated with the activation or repression of pro-fibrogenic mediators
and consequential collagen deposition around ductular structures and parenchymally proliferating
LPCs. The authors conclude that in fibrotic, PHx-treated livers, hepatocyte replicative arrest and
subsequent apoptotic cell death triggers the activation of the LPC compartment, which in turn
provokes the aggravation of fibrogenesis and induces a negative feedback loop that further
compromises hepatocyte-mediated liver regeneration.
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Kuramitsu et al. clearly demonstrate a link between liver regeneration, LPC and fibrogenic
responses. Improved liver regeneration and fibrosis abrogation following manipulation of the
TWEAK pathway could be the result of either direct or indirect actions of TWEAK (Figure 1).
Whilst different models of fibrosis and liver regeneration may well have differences in underlying
pathogeneses, the close correlation between numbers of LPCs and fibrosis-driving HSCs during
progressive chronic liver injury, and their very close spatial association, suggest the potential for
cellular cross-talk (9). Indeed, recent studies have shown a role for the Notch ligand Jagged 1
expressed by myofibroblasts, in driving the biliary differentiation of LPCs via their expression of
Notch (10), potentially as part of the pro-fibrogenic Ductular Reaction (Figure 1). Others have
shown cellular cross-talk between LPCs, HSCs/myofibroblasts and inflammatory cells, leading to
increased expression of pro-fibrogenic proteins such as TGFβ and CTGF produced by LPCs (and
other cells), proposed as a mechanism for augmenting fibrogenesis (11, 12). Cholangiocytes as well
as Ductular Reaction cells and isolated parenchymal LPCs produce chemokines, such as monocyte
chemoattractant protein (MCP)-1, platelet-derived growth factor (PDGF) and endothelin-1 which
regulate chemotaxis of inflammatory cells and activated HSCs/myofibroblasts (Figure 1) as part of
the fibrogenic process (12-14).
In addition to TWEAK, lymphotoxin β (LTβ), another TNF-family member, has been shown to
play a role in inflammation and fibrosis associated with chronic liver injury (9). LTβ, produced by
inflammatory cells and importantly also by LPCs themselves, has been demonstrated to induce the
expression of chemotaxis-associated factors intercellular adhesion molecule 1 (ICAM-1) and
regulated upon activation, normal T-cell expressed and secreted (RANTES) in HSCs, which may
play a role in mediating recruitment of LPCs, other HSCs and leukocytes required for wound
healing, fibrogenesis and regeneration during liver injury (9) (Figure 1). LTβ has a well-
characterized role in mediating inflammatory cell responses. It signals as a cell surface-anchored
heterotrimer with LTα and thus is involved in autocrine and paracrine signaling to adjacent cells.
LTβ binds specifically to the LTβ receptor (LTβR) and once activated, LTβR initiates a signal
transduction cascade resulting in NFκB activation. Upregulated LTβ expression has been reported
following chronic liver injury induced by bile duct ligation (15) and the CDE diet (9) in animal
models, as well as in chronic HCV infection in humans, where there is a significant positive
correlation between hepatic LTβ mRNA and fibrosis severity (16). In these studies the close
proximity of LTβ+ cells to periportal fibrosis suggests a possible role for LTβR-initiated signaling
in controlling HSC function. Whilst the Kuramitsu et al. study is consistent with a role for the
TWEAK pathway in the regulation of hepatic fibrogenesis, it is possible that LTβ/LTβR interaction
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between LPCs and HSCs (and perhaps other inflammatory subpopulations such as CD45+ cells (9))
may represent an additional mechanism contributing to their observations. This could readily be
addressed by further studies in the PHx model as well as other models of hepatic injury,
inflammation, fibrogenesis and regeneration.
JANINA E.E. TIRNITZ-PARKER, PhD.
School of Biomedical Sciences,
Curtin Health Innovation Research Institute,
Biosciences Research Precinct,
Curtin University, Perth, Australia
JOHN K. OLYNYK, MD, FRACP.
Department of Gastroenterology,
Fremantle Hospital, Fremantle, Australia
GRANT A. RAMM, PhD.
Hepatic Fibrosis Group, Queensland Institute of Medical Research,
PO Royal Brisbane and Women’s Hospital,
Brisbane, Australia.
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Figure 1. Hypothetical schema showing proposed co-regulation of liver progenitor cell and
fibrogenic responses involving cellular cross-talk mediated by TWEAK/Fn14- and
LTββββ/LTββββR-induced signaling. Following hepatic insult, where normal hepatocyte replicative
repair is compromised, liver progenitor cell (LPC) expansion is induced via TWEAK produced by
monocytes, macrophages and natural killer (NK) cells, which aids in initiation of the Ductular
Reaction. Interaction between LPCs and activated hepatic stellate cells (HSCs)/myofibroblasts via
notch/jagged and other potential mediators may drive LPC differentiation to cholangiocytes via the
Ductular Reaction. Cellular cross-talk between LPCs, myofibroblasts (and inflammatory cells), with
the resultant increased expression of profibrogenic proteins such as TGF-β and CTGF produced by
LPCs (and other cells), is proposed as a mechanism for augmenting fibrogenesis. Cholangiocytes
as well as Ductular Reaction cells and isolated parenchymal LPCs produce chemokines, such as
MCP-1, PDGF and endothelin-1, which regulate chemotaxis of inflammatory cells and activated
HSCs/myofibroblasts. Interactions between lymphotoxin β (LTβ) expressed on LPCs and the LTβ
receptor (LTβR) on activated HSCs triggers an NFκB-driven signal transduction pathway,
upregulating the expression of chemotaxis-associated factors ICAM-1 and RANTES by HSCs,
which is proposed to play a role in mediating recruitment of LPCs, HSCs and leukocytes required
for wound healing, fibrogenesis and ultimately hepatic regeneration during liver injury (6, 9-14,
17).
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