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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Figure 1

277x190mm (300 x 300 DPI)

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