Role of TWEAK in coregulating liver progenitor cell and fibrogenic responses

Authors

  • Janina E.E. Tirnitz-Parker Ph.D.,

    1. School of Biomedical Sciences, CHIRI Biosciences Research Precinct, Curtin University, Bentley, Australia
    2. School of Medicine and Pharmacology, University of Western Australia, Fremantle, Australia
    Search for more papers by this author
  • John K. Olynyk M.D.,

    1. School of Biomedical Sciences, CHIRI Biosciences Research Precinct, Curtin University, Bentley, Australia
    2. School of Medicine and Pharmacology, University of Western Australia, Fremantle, Australia
    3. Department of Gastroenterology, Fremantle Hospital, Fremantle, Australia
    4. Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, Australia
    Search for more papers by this author
  • Grant A. Ramm Ph.D.

    1. School of Medicine, University of Queensland, Brisbane, Australia
    2. The Hepatic Fibrosis Group, Department of Cell and Molecular Biology, QIMR Berghofer Medical Research Institute, Brisbane, Australia
    Search for more papers by this author

  • Potential conflict of interest: Nothing to report.

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;183:182-194. (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 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, while 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: (1) hyperstimulation of inflammatory cells, hepatic stellate cells (HSCs), and macrophages which continually secrete cytokines promoting survival of proliferating hepatic cells; (2) excessive matrix deposition by activated HSCs/myofibroblasts leading to cirrhosis; and (3) 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 proangiogenic and proinflammatory 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 coregulation of LPC 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 by way of 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 nonfibrotic versus fibrotic livers, Kuramitsu et al.[8] 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% versus 90.8%) as well as slower liver weight gains (11% versus 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 alanine aminotransferase (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 up-regulation of procollagen α1, α-smooth muscle actin (αSMA), TIMP-1, and TGFβ1 messenger RNA (mRNA) as well as increased de novo collagen synthesis and deposition. This once again demonstrates that LPCs and HSCs are not only in close spatiotemporal 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 profibrogenic 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.

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 (Fig. 1). While 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 crosstalk.[9] Indeed, recent studies have shown a role for the Notch ligand Jagged 1 expressed by myofibroblasts in driving the biliary differentiation of LPCs by way of their expression of Notch,[10] potentially as part of the profibrogenic Ductular Reaction (Fig. 1). Others have shown cellular crosstalk between LPCs, HSCs/myofibroblasts, and inflammatory cells, leading to increased expression of profibrogenic proteins such as TGFβ and connective tissue growth factor (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 (Fig. 1) as part of the fibrogenic process.[12-14]

Figure 1.

Hypothetical schema showing proposed coregulation of liver progenitor cell and fibrogenic responses involving cellular crosstalk mediated by TWEAK/Fn14- and LTβ/LTβR-induced signaling. Following hepatic insult, where normal hepatocyte replicative repair is compromised, LPC expansion is induced by way of TWEAK produced by monocytes, macrophages, and NK cells, which aids in initiation of the Ductular Reaction. Interaction between LPCs and activated HSCs/myofibroblasts by way of notch/jagged and other potential mediators may drive LPC differentiation to cholangiocytes by way of the Ductular Reaction. Cellular crosstalk 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 LTβ expressed on LPCs and the LTβR on activated HSCs triggers an NFκB-driven signal transduction pathway, up-regulating 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]

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] (Fig. 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. Up-regulated 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 hepatitis C virus (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. While 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 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, Ph.D.1,2

  • John K. Olynyk, M.D.1,2,3,4

  • Grant A. Ramm, Ph.D.5,6

  • 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, QIMR Berghofer Medical Research Institute, Brisbane, Australia

Ancillary