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IL-6 pathway in the liver: From physiopathology to therapy

  • Dirk Schmidt-Arras
    Affiliations
    Institute of Biochemistry, Christian-Albrechts-University Kiel, Olshausenstrasse 40, Kiel, Germany
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  • Stefan Rose-John
    Correspondence
    Corresponding author. Address: Institute of Biochemistry, Christian-Albrechts-University Kiel, Olshausenstrasse 40, D-24098 Kiel, Germany. Tel.: +49 431 880 3336; fax: +49 431 880 5007.
    Affiliations
    Institute of Biochemistry, Christian-Albrechts-University Kiel, Olshausenstrasse 40, Kiel, Germany
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Open AccessPublished:February 08, 2016DOI:https://doi.org/10.1016/j.jhep.2016.02.004

      Summary

      Interleukin 6 (IL-6) is a pleiotropic four-helix-bundle cytokine that exerts multiple functions in the body. In the liver, IL-6 is an important inducer of the acute phase response and infection defense. IL-6 is furthermore crucial for hepatocyte homeostasis and is a potent hepatocyte mitogen. It is not only implicated in liver regeneration, but also in metabolic function of the liver. However, persistent activation of the IL-6 signaling pathway is detrimental to the liver and might ultimately result in the development of liver tumors. On target cells IL-6 can bind to the signal transducing subunit gp130 either in complex with the membrane-bound or with the soluble IL-6 receptor to induce intracellular signaling. In this review we describe how these different pathways are involved in the physiology and pathophyiology of the liver. We furthermore discuss how IL-6 pathways can be selectively inhibited and therapeutically exploited for the treatment of liver pathologies.

      Abbreviations:

      EGF (epidermal growth factor), EGFR (epidermal growth factor receptor), Fc (constant portion of an IgG antibody), gp130 (glycoprotein 130kDa), IL (interleukin), JAK1 (Janus kinase 1), JNK1 (Jun amino-terminal kinase 1), LPS (lipopolysaccharide), R (receptor), s (soluble), SOCS3 (suppressor of cytokine signaling 3), STAT (signal transducer and activator of transcription), TLR (Toll-like receptor), TNF-α (tumor necrosis factor α)

      Keywords

      Introduction

      Interleukin-6 and gp130 signal transduction

      Interleukin-6 (IL-6) is a four-helical cytokine of 184 amino acids [
      • Kishimoto T.
      IL-6: from its discovery to clinical applications.
      ]. The protein is synthesized by fibroblasts, monocytes, macrophages, T cells and endothelial cells. IL-6 synthesis and secretion is induced during inflammatory conditions such as upon stimulation of Toll-like receptor (TLR)-4 by lipopolysaccharide or upon stimulation of cells by IL-1 or tumor necrosis factor (TNF)-α [
      • Kishimoto T.
      IL-6: from its discovery to clinical applications.
      ]. When the IL-6 cDNA was molecularly cloned as B cell stimulating factor 2, it turned out that IL-6 was identical to the 26 kDa protein and to hybridoma growth factor [
      • Hirano T.
      • Yasukawa K.
      • Harada H.
      • Taga T.
      • Watanabe Y.
      • et al.
      Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin.
      ].
      On target cells, IL-6 binds to the 80 kDa Interleukin-6 receptor (IL-6R), which is not signaling competent. Signaling is initiated upon association of the IL-6/IL-6R complex with a second receptor protein, glycoprotein (gp) 130. Gp130 dimerization leads to the activation of the tyrosine kinase JAK1, which is constitutively bound to the cytoplasmic portion of gp130 [
      • Kishimoto T.
      Interleukin-6: from basic science to medicine–40 years in immunology.
      ]. After autophosphorylation, JAK1 phosphorylates five tyrosine residues within the cytoplasmic portion of gp130. This leads to the activation of several intracellular signaling pathways including the MAP kinase and PI3 kinase pathway and the signal transducer and activator of transcription (STAT) 1 and STAT3 pathway. Subsequently, STAT3 is able to upregulate suppressor of cytokine signaling (SOCS) 3, which leads to a downregulation of gp130 signals and thereby represents a negative feed-back loop [
      • Kishimoto T.
      Interleukin-6: from basic science to medicine–40 years in immunology.
      ,
      • Schaper F.
      • Rose-John S.
      Interleukin-6: biology, signaling and strategies of blockade.
      ].
      On target cells, IL-6 can signal via the IL-6 classic or IL-6 trans-signaling pathway.

      IL-6 trans-signaling

      Importantly, it was shown that IL-6 has only a measurable affinity for the IL-6R but not for gp130. Likewise, the IL-6R has no measurable affinity for gp130 [
      • Kishimoto T.
      Interleukin-6: from basic science to medicine–40 years in immunology.
      ]. Only when the IL-6/IL-6R complex is formed, binding to gp130 can be demonstrated [
      • Kishimoto T.
      Interleukin-6: from basic science to medicine–40 years in immunology.
      ]. This has important consequences. Whereas gp130 is expressed on all cells of the body, the IL-6R is only expressed on few cell types such as hepatocytes, some leukocytes and some epithelial (e.g. biliary epithelial cells) and non-epithelial cells (e.g. hepatic stellate cells) (Fig. 1A). Therefore, only IL-6R expressing cells can directly respond to the cytokine IL-6 [
      • Kishimoto T.
      Interleukin-6: from basic science to medicine–40 years in immunology.
      ].
      Figure thumbnail gr1
      Fig. 1Interleukin 6 (IL-6) signals via two distinct pathways on target cells. (A) Expression of IL-6R and gp130 on different cell types of the liver. HC: hepatocyte, BEC: biliary epithelial cell, HSC: hepatic stellate cell, KC: Kupffer cell, EC: endothelial cell (B) In the liver, IL-6 can directly bind to its cognate membrane-bound IL-6 receptor (mIL-6R) on hepatocytes (HC), leukocytes like KC and stellate cells (HSC). The complex of IL6/IL-6R subsequently recruits the signaling β-subunit gp130 and induces downstream signaling. This process is termed “IL-6 classic signaling”. Cells that do not express the mIL-6R, like endothelial cells (EC) can react to IL-6 via “IL-6 trans-signaling”: a soluble form of the IL-6R (sIL-6R) is proteolytically released by a disintegrin and metalloprotease 17 (ADAM17) from cells expressing the mIL-6R. The complex of IL-6/sIL-6R can bind to gp130 and induce downstream signaling. (C) Hyper-IL-6 is a fusion protein of IL-6 and the soluble IL-6 receptor. Hyper-IL-6 is a potent stimulator of gp130. D. sgp130Fc is a fusion protein of the extracellular portion of gp130 to the Fc part of human IgG1. sgp130Fc selectively inhibits IL-6 trans-signaling without interfering with IL-6 classic signaling (also see ).
      Interestingly, the IL-6R was shown to be cleaved at the cell surface by the metalloprotease a disintegrin and metalloprotease (ADAM) 17, a process called shedding [
      • Müllberg J.
      • Schooltink H.
      • Stoyan T.
      • Günther M.
      • Graeve L.
      • et al.
      The soluble interleukin-6 receptor is generated by shedding.
      ]. The shed soluble IL-6R (sIL-6R) could still bind its ligand IL-6. The complex of IL-6 and sIL-6R was able to associate with gp130 and initiate intracellular signaling [
      • Mackiewicz A.
      • Schooltink H.
      • Heinrich P.C.
      • Rose-John S.
      Complex of soluble human IL-6-receptor/IL-6 up-regulates expression of acute-phase proteins.
      ]. Strikingly, this could also be demonstrated to occur on cells, which did not express the membrane-bound IL-6R [
      • Mackiewicz A.
      • Schooltink H.
      • Heinrich P.C.
      • Rose-John S.
      Complex of soluble human IL-6-receptor/IL-6 up-regulates expression of acute-phase proteins.
      ]. This process has been named IL-6 trans-signaling (Fig. 1B). Therefore, IL-6 trans-signaling has the potential to largely increase the spectrum of target cells of IL-6 [
      • Rose-John S.
      • Heinrich P.C.
      Soluble receptors for cytokines and growth factors: generation and biological function.
      ].
      Hyper-IL-6 is an artificial fusion protein of IL-6 coupled to the sIL-6R via a flexible peptide linker [
      • Fischer M.
      • Goldschmitt J.
      • Peschel C.
      • Brakenhoff J.P.
      • Kallen K.J.
      • et al.
      I. A bioactive designer cytokine for human hematopoietic progenitor cell expansion.
      ] (Fig. 1C). It has been shown to be 100–1000 times more potent than the separate proteins IL-6 and sIL-6R. Hyper-IL-6 has been used to analyze the responsiveness of cells to IL-6 or to the trans-signaling complex IL-6/sIL-6R. It turned out that many cell types including neurons [
      • März P.
      • Cheng J.G.
      • Gadient R.A.
      • Patterson P.H.
      • Stoyan T.
      • et al.
      Sympathetic neurons can produce and respond to interleukin 6.
      ], glial cells [
      • März P.
      • Heese K.
      • Dimitriades-Schmutz B.
      • Rose-John S.
      • Otten U.
      Role of interleukin-6 and soluble IL-6 receptor in region-specific induction of astrocytic differentiation and neurotrophin expression.
      ], endothelial cells [
      • Romano M.
      • Sironi M.
      • Toniatti C.
      • Polentarutti N.
      • Fruscella P.
      • et al.
      Role of IL-6 and its soluble receptor in induction of chemokines and leukocyte recruitment.
      ], smooth muscle cells [
      • Klouche M.
      • Bhakdi S.
      • Hemmes M.
      • Rose-John S.
      Novel path to activation of vascular smooth muscle cells: up-regulation of gp130 creates an autocrine activation loop by IL-6 and its soluble receptor.
      ], hematopoietic stem cells [
      • Audet J.
      • Miller C.L.
      • Rose-John S.
      • Piret J.M.
      • Eaves C.J.
      Distinct role of gp130 activation in promoting self-renewal divisions by mitogenically stimulated murine hematopoietic stem cells.
      ] and embryonic stem cells [
      • Viswanathan S.
      • Benatar T.
      • Rose-John S.
      • Lauffenburger D.A.
      • Zandstra P.W.
      Ligand/receptor signaling threshold (LIST) model accounts for gp130-mediated embryonic stem cell self-renewal responses to LIF and HIL-6.
      ] do not express IL-6R and are therefore dependent on trans-signaling in their response to IL-6.
      A fusion protein of the entire extracellular portion of gp130 coupled to the Fc portion of a human IgG1 antibody (sgp130Fc) turned out to be a selective inhibitor of IL-6 trans-signaling [
      • Jostock T.
      • Müllberg J.
      • Ozbek S.
      • Atreya R.
      • Blinn G.
      • et al.
      Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses.
      ] (Fig. 1D). IL-6 signaling via the membrane-bound IL-6R was not affected by this protein. This surprising effect could be explained by the fact that neither IL-6 nor IL-6R alone showed a measurable affinity for gp130 [
      • Jostock T.
      • Müllberg J.
      • Ozbek S.
      • Atreya R.
      • Blinn G.
      • et al.
      Soluble gp130 is the natural inhibitor of soluble interleukin-6 receptor transsignaling responses.
      ].
      Under normal conditions, IL-6 levels in the blood are extremely low (1–5 pg/ml). Surprisingly, sIL-6R in the blood has been found at concentrations of 40–70 ng/ml. And a soluble form of gp130 (sgp130), which is generated by alternative splicing, is found at concentrations of around 400 ng/ml [
      • Rose-John S.
      IL-6 trans-signaling via the soluble IL-6 receptor: importance for the pro-inflammatory activities of IL-6.
      ]. We have argued that the sIL-6R and sgp130 constitute a buffer in the blood since IL-6, once secreted will bind to the sIL-6R with an affinity of 1 nM. Thereupon, the complex of IL-6/sIL-6R binds to sgp130 with a hundred times higher affinity (10 pM) and is neutralized. Only when IL-6 levels exceed the sIL-6R concentration, IL-6 can bind to membrane-bound IL-6R on target cells [
      • Scheller J.
      • Rose-John S.
      The interleukin 6 pathway and atherosclerosis.
      ,
      • Garbers C.
      • Aparicio-Siegmund S.
      • Rose-John S.
      The IL-6/gp130/STAT3 signaling axis: recent advances towards specific inhibition.
      ]. This concept might, however, not be fully valid in case of paracrine activity of IL-6 e.g. in the liver where activated Kupffer cells secrete IL-6 and neighboring hepatocytes respond to a strong local increase in IL-6.

      IL-6 and the acute phase response

      More than 25 years ago, it was found that Interleukin-6 was also identical with hepatocyte stimulating factor 2. Under inflammatory conditions, this factor was known to induce the liver to synthesize a group of proteins called acute phase proteins [
      • Gauldie J.
      • Richards C.
      • Harnish D.
      • Lansdorp P.
      • Baumann H.
      Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.
      ].
      In humans the acute phase proteins, which are most induced, include C-reactive protein (CRP), serum amyloid A (SAA), haptoglobin and fibrinogen. Functionally, some acute phase proteins are components of the complement system and of the coagulation cascade. Other acute phase proteins are protease inhibitors, transport proteins or participants in inflammatory responses, such as secreted phospholipase A2 [
      • Baumann H.
      • Gauldie J.
      The acute phase response.
      ,
      • Gabay C.
      • Kushner I.
      Acute-phase proteins and other systemic responses to inflammation.
      ]. As already mentioned, the major inducer of the hepatic acute phase proteins is the cytokine IL-6, which is secreted by neutrophils, monocytes and macrophages upon TLR stimulation by e.g. lipopolysaccharide [
      • Baumann H.
      • Gauldie J.
      The acute phase response.
      ,
      • Gabay C.
      • Kushner I.
      Acute-phase proteins and other systemic responses to inflammation.
      ]. Activated myeloid cells in addition release the inflammatory cytokines IL-1 and TNF-α, which can lead to massive production and secretion of IL-6 from other cells such as endothelial cells and fibroblasts thereby functioning as a positive feed-forward loop [
      • Bode J.G.
      • Albrecht U.
      • Häussinger D.
      • Heinrich P.C.
      • Schaper F.
      Hepatic acute phase proteins–regulation by IL-6- and IL-1-type cytokines involving STAT3 and its crosstalk with NF-κB-dependent signaling.
      ]. Interestingly, in IL-6 knockout mice, the acute phase response is largely inhibited upon injection of turpentine, whereas it is almost normal upon injection of lipopolysaccharide [
      • Kopf M.
      • Baumann H.
      • Freer G.
      • Freudenberg M.
      • Lamers M.
      • et al.
      Impaired immune and acute-phase responses in interleukin-6-deficient mice.
      ]. This might be due to compensation by other IL-6 type cytokines such as Interleukin-11, leukemia inhibitory factor, oncostatin M, ciliary neurotrophic factor, and cardiotrophin 1, which have been demonstrated to induce the synthesis of acute phase proteins in hepatocytes or hepatoma cells. It is therefore believed that members of this cytokine family are actors of liver physiopathology [
      • Schooltink H.
      • Stoyan T.
      • Roeb E.
      • Heinrich P.C.
      • Rose-John S.
      Ciliary neurotrophic factor induces acute-phase protein expression in hepatocytes.
      ,
      • Richards C.D.
      • Langdon C.
      • Pennica D.
      • Gauldie J.
      Murine cardiotrophin-1 stimulates the acute-phase response in rat hepatocytes and H35 hepatoma cells.
      ].
      Although not all functions of the acute phase proteins are known, their induced expression is thought to be beneficial for the response of the body to infectious insults and inflammation [
      • Gabay C.
      • Kushner I.
      Acute-phase proteins and other systemic responses to inflammation.
      ]. Clinically, the extent of the acute phase protein levels such as CRP is used to measure the extent of the inflammatory condition. 80–85% of patients who show CRP levels of more than 100 mg per liter are diagnosed with bacterial infections [
      • Morley J.J.
      • Kushner I.
      Serum C-reactive protein levels in disease.
      ].
      The duration of the acute phase response is normally 24–48 h, after which the organism returns to normal liver function. Under severe conditions, such as advanced cancer and the acquired immunodeficiency syndrome, the acute phase can convert to a chronic state of inflammation although the molecular mechanisms of such a chronicity are not completely understood [
      • Baumann H.
      • Gauldie J.
      The acute phase response.
      ,
      • Gabay C.
      • Kushner I.
      Acute-phase proteins and other systemic responses to inflammation.
      ].
      While IL-6 classic signaling is crucial for the induction of the acute phase response, IL-6 trans-signaling mediates strong mitogenic signals during liver regeneration.

      IL-6 and liver regeneration

      The most important reaction of the liver to injury is liver regeneration [
      • Michalopoulos G.K.
      • DeFrances M.C.
      Liver regeneration.
      ,
      • Michalopoulos G.K.
      Liver regeneration after partial hepatectomy: critical analysis of mechanistic dilemmas.
      ] and it became clear from experiments with parabiotic animals that soluble extrahepatic factors provide the stimulus for hepatocyte proliferation [
      • Fisher B.
      • Szuch P.
      • Levine M.
      • Fisher E.R.
      A portal blood factor as the humoral agent in liver regeneration.
      ]. Only 2 h after hepatectomy, the level of TNF-α increased followed by a dramatic upregulation of IL-6 levels in the liver vein (Fig. 2A) [
      • Trautwein C.
      • Rakemann T.
      • Niehof M.
      • Rose-John S.
      • Manns M.P.
      Acute-phase response factor, increased binding, and target gene transcription during liver regeneration.
      ]. After hepatectomy or liver damage, gut-derived factors like lipopolysaccharide (LPS) activate liver-resident Kupffer cells resulting in a TNF-α-dependent secretion of IL-6 (Fig. 2B) [
      • Taub R.
      Liver regeneration: from myth to mechanism.
      ].
      Figure thumbnail gr2
      Fig. 2IL-6 is a major driver of liver regeneration. (A, B) Early after hepatocyte damage, Kupffer cells secrete TNF-α, which induces massive expression of IL-6 in an autocrine manner. IL-6 then stimulates hepatocyte proliferation. (C) IL-6 trans-signaling enhances liver regeneration. Mice injected with Hyper-IL-6 show accelerated hepatocyte proliferation and liver regeneration after partial hepatectomy in comparison to IL-6. Modified from
      [
      • Peters M.
      • Blinn G.
      • Jostock T.
      • Schirmacher P.
      • Meyer zum Büschenfelde K.H.
      • et al.
      Combined interleukin 6 and soluble interleukin 6 receptor accelerates murine liver regeneration.
      ]
      and
      [
      • Taub R.
      Liver regeneration: from myth to mechanism.
      ]
      . (D) IL-6 trans-signaling can boost IL-6 signaling on hepatocytes. IL-6 engages IL-6 receptor molecules but not all gp130 receptor molecules on the surface of hepatocytes. The presence of the soluble IL-6R enhances the possibility to engage gp130 receptor molecules on the cell surface. This leads to an amplification and prolongation of gp130 signals.
      Consistently, IL-6 knockout mice show impaired liver regeneration [
      • Cressman D.E.
      • Greenbaum L.E.
      • DeAngelis R.A.
      • Ciliberto G.
      • Furth E.E.
      • et al.
      Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice.
      ]. Also under cholestatic conditions, the number of regenerative liver progenitor cells is reduced if IL-6 signaling is blunted [
      • Yeoh G.C.T.
      • Ernst M.
      • Rose-John S.
      • Akhurst B.
      • Payne C.
      • et al.
      Opposing roles of gp130-mediated STAT-3 and ERK-1/ 2 signaling in liver progenitor cell migration and proliferation.
      ]. These experimental data pointed to an important role of IL-6 in liver regeneration.
      Mice transgenic for the human sIL-6R showed that this protein acts as a serum binding protein for IL-6 and prolongs the half-life of IL-6 [
      • Peters M.
      • Jacobs S.
      • Ehlers M.
      • Vollmer P.
      • Müllberg J.
      • et al.
      The function of the soluble interleukin 6 (IL-6) receptor in vivo: sensitization of human soluble IL-6 receptor transgenic mice towards IL-6 and prolongation of the plasma half-life of IL-6.
      ] and double transgenic mice expressing human IL-6 and human sIL-6R exhibited permanent hepatocyte proliferation [
      • Peters M.
      • Schirmacher P.
      • Goldschmitt J.
      • Odenthal M.
      • Peschel C.
      • et al.
      Extramedullary expansion of hematopoietic progenitor cells in interleukin (IL)-6-sIL-6R double transgenic mice.
      ,
      • Schirmacher P.
      • Peters M.
      • Ciliberto G.
      • Blessing M.
      • Lotz J.
      • et al.
      Hepatocellular hyperplasia, plasmacytoma formation, and extramedullary hematopoiesis in interleukin (IL)-6/soluble IL-6 receptor double-transgenic mice.
      ]. We concluded from these results that IL-6 in the presence of sIL-6R was a potent stimulus of liver regeneration [
      • Schirmacher P.
      • Peters M.
      • Ciliberto G.
      • Blessing M.
      • Lotz J.
      • et al.
      Hepatocellular hyperplasia, plasmacytoma formation, and extramedullary hematopoiesis in interleukin (IL)-6/soluble IL-6 receptor double-transgenic mice.
      ]. Therefore, the potential of Hyper-IL-6 (Fig. 1C) to accelerate liver regeneration was tested.
      When mice were treated with recombinant IL-6 or Hyper-IL-6 after 50% hepatectomy, it turned out that only Hyper-IL-6 significantly accelerated liver weight gain and led to a 36 h earlier peak of mitosis in hepatocytes (Fig. 2C) [
      • Peters M.
      • Blinn G.
      • Jostock T.
      • Schirmacher P.
      • Meyer zum Büschenfelde K.H.
      • et al.
      Combined interleukin 6 and soluble interleukin 6 receptor accelerates murine liver regeneration.
      ]. Likewise, Hyper-IL-6, but not IL-6, was shown to reverse D-galactosamine mediated liver toxicity and to significantly improve the survival rate of the animals [
      • Galun E.
      • Zeira E.
      • Pappo O.
      • Peters M.
      • Rose-John S.
      Liver regeneration induced by a designer human IL-6/sIL-6R fusion protein reverses severe hepatocellular injury.
      ]. Interestingly, it was shown that Hyper-IL-6 when genetically delivered via a recombinant adenovirus led to the survival of more than 90% of the mice whereas only 13% of the mock-treated animals survived the D-galactosamine treatment. Treatment with an adenovirus coding for IL-6 resulted only in survival of 21% of the animals [
      • Hecht N.
      • Pappo O.
      • Shouval D.
      • Rose-John S.
      • Galun E.
      • et al.
      Hyper-IL-6 gene therapy reverses fulminant hepatic failure.
      ]. These experiments demonstrated that stimulation of IL-6 trans-signaling via the sIL-6R dramatically accelerated and improved liver regeneration suggesting a physiologic role of the sIL-6R in this process [
      • Schaper F.
      • Rose-John S.
      Interleukin-6: biology, signaling and strategies of blockade.
      ,
      • Jones S.A.
      • Rose-John S.
      The role of soluble receptors in cytokine biology: the agonistic properties of the sIL-6R/IL-6 complex.
      ].
      Since hepatocytes express much more gp130 than IL-6R the presence of IL-6 and sIL-6R results in more gp130 activation and therefore to a higher amplitude of the IL-6 signal (Fig. 2D). Furthermore, it was shown that the complex of IL-6 and sIL-6R was internalized much less efficiently than IL-6 leading to longer duration of the IL-6 signal when mediated by trans-signaling [
      • Peters M.
      • Blinn G.
      • Solem F.
      • Fischer M.
      • Meyer zum Büschenfelde K.H.
      • et al.
      In vivo and in vitro activities of the gp130-stimulating designer cytokine Hyper-IL-6.
      ]. This explains why, in the absence of any liver insult, hepatocytes permanently proliferated in IL-6/sIL-6R double transgenic mice, while hepatocytes did not show any mitogenic response in IL-6 single transgenic mice. The signal induced via the membrane-bound IL-6R on hepatocytes was not sufficient to induce a proliferative response [
      • Peters M.
      • Schirmacher P.
      • Goldschmitt J.
      • Odenthal M.
      • Peschel C.
      • et al.
      Extramedullary expansion of hematopoietic progenitor cells in interleukin (IL)-6-sIL-6R double transgenic mice.
      ,
      • Schirmacher P.
      • Peters M.
      • Ciliberto G.
      • Blessing M.
      • Lotz J.
      • et al.
      Hepatocellular hyperplasia, plasmacytoma formation, and extramedullary hematopoiesis in interleukin (IL)-6/soluble IL-6 receptor double-transgenic mice.
      ].
      Since all IL-6 type cytokines use the gp130 receptor for signaling it is interesting to ask whether there is potential competition of members of this cytokine family for gp130 (see above). As depicted in Fig. 2D, hepatocytes have been shown to express far more gp130 than IL-6R making it unlikely that gp130 will be the limiting factor in the response to other members of the IL-6 type cytokine family [
      • Galun E.
      • Rose-John S.
      The regenerative activity of interleukin-6.
      ].
      These experiments demonstrated the potential of exogenous stimulation of IL-6 trans-signaling in the induction of liver regeneration but they did not directly show that IL-6 trans-signaling actually occurred in vivo. Therefore, experiments were conducted with the recombinant sgp130Fc protein, which specifically blocks IL-6 trans-signaling without affecting signaling via the membrane-bound IL-6R (Fig. 1D). Alternatively, transgenic mice were generated, which overexpress the sgp130Fc protein [
      • Rabe B.
      • Chalaris A.
      • May U.
      • Waetzig G.H.
      • Seegert D.
      • et al.
      Transgenic blockade of interleukin 6 transsignaling abrogates inflammation.
      ]. In these mice, trans-signaling is abrogated whereas IL-6 signaling via the membrane-bound IL-6R is intact.
      In these sgp130Fc transgenic mice, the response to D-galactosamine induced liver damage was shown to be compromised. Interestingly, the liver damage-induced glycogen consumption in the liver of the transgenic mice was strongly reduced indicating that glycogen consumption depended on IL-6 trans-signaling [
      • Drucker C.
      • Rabe B.
      • Chalaris A.
      • Schulz E.
      • Scheller J.
      • et al.
      Interleukin-6 trans-signaling regulates glycogen consumption after D-galactosamine-induced liver damage.
      ]. Upon acute CCl4 damage, blockade of IL-6 trans-signaling led to higher liver damage and to reduced refilling of hepatocyte glycogen stores [
      • Gewiese-Rabsch J.
      • Drucker C.
      • Malchow S.
      • Scheller J.
      • Rose-John S.
      Role of IL-6 trans-signaling in CCl₄ induced liver damage.
      ]. In the concanavalin A hepatitis model it was shown that IL-6 classic signaling via the membrane-bound IL-6R rather than IL-6 trans-signaling was responsible for the observed neutrophil accumulation and the induced liver damage [
      • Malchow S.
      • Thaiss W.
      • Jänner N.
      • Waetzig G.H.
      • Gewiese-Rabsch J.
      • et al.
      Essential role of neutrophil mobilization in concanavalin A-induced hepatitis is based on classic IL-6 signaling but not on IL-6 trans-signaling.
      ]. Collectively, these experiments demonstrate that IL-6 trans-signaling in the liver plays an important role in the regenerative response of this organ to injury [
      • Drucker C.
      • Gewiese J.
      • Malchow S.
      Scheller Jü and Rose-John S, Impact of interleukin-6 classic- and trans-signaling on liver damage and regeneration.
      ].
      Interestingly, IL-6 triggered the activation of YAP and Notch independently of the downstream effector STAT3 in primary hepatocytes and in mouse liver upon partial hepatectomy, suggesting an inflammation induced participation of the YAP and Notch pathways during liver regeneration [
      • Taniguchi K.
      • Wu L.-W.
      • Grivennikov S.I.
      • de Jong P.R.
      • Lian I.
      • et al.
      A gp130-Src-YAP module links inflammation to epithelial regeneration.
      ].
      There is a growing body of evidence that IL-6 is needed for the proper control of metabolic functions.

      IL-6 in obesity and insulin resistance

      The role of IL-6 signaling on hepatic metabolism, obesity and insulin resistance is discussed controversially.
      The observation that serum levels of IL-6 correlate with the degree of obesity [
      • Weiss R.
      • Dziura J.
      • Burgert T.S.
      • Tamborlane W.V.
      • Taksali S.E.
      • et al.
      Obesity and the metabolic syndrome in children and adolescents.
      ] and the development of type 2 diabetes [
      • Pradhan A.D.
      • Manson J.E.
      • Rifai N.
      • Buring J.E.
      • Ridker P.M.
      C-reactive protein, interleukin 6, and risk of developing type 2 diabetes mellitus.
      ] suggested that IL-6 is causally linked to metabolic disease development. Proteomics analysis of TNF-α-stimulated adipocytes identified the adipokine progranulin (PGRN) as an inducer of IL-6 expression in adipose tissue during obesity [
      • Matsubara T.
      • Mita A.
      • Minami K.
      • Hosooka T.
      • Kitazawa S.
      • et al.
      PGRN is a key adipokine mediating high fat diet-induced insulin resistance and obesity through IL-6 in adipose tissue.
      ]. Furthermore, activation of JNK1 in adipose tissue of mice fed a high-fat diet (HFD) resulted in insulin resistance in the liver in an IL-6 dependent manner [
      • Sabio G.
      • Das M.
      • Mora A.
      • Zhang Z.
      • Jun J.Y.
      • et al.
      A stress signaling pathway in adipose tissue regulates hepatic insulin resistance.
      ]. Consistently, acute infusions of IL-6 in mice impaired insulin action on the liver and skeletal muscle in hyperinsulinemic-euglycemic clamp analysis [
      • Kim H.-J.
      • Higashimori T.
      • Park S.-Y.
      • Choi H.
      • Dong J.
      • et al.
      Differential effects of interleukin-6 and -10 on skeletal muscle and liver insulin action in vivo.
      ]. In the liver and skeletal muscle, IL-6 signaling leads to a STAT3-dependent upregulation of SOCS3 (Fig. 3A) which, in turn, impairs insulin-mediated phosporylation of insulin receptor substrates 1/2 (IRS-1/2) and subsequent protein kinase B (PKB/AKT) activation [
      • Ueki K.
      • Kondo T.
      • Kahn C.R.
      Suppressor of cytokine signaling 1 (SOCS-1) and SOCS-3 cause insulin resistance through inhibition of tyrosine phosphorylation of insulin receptor substrate proteins by discrete mechanisms.
      ,
      • Jorgensen S.B.
      • O’Neill H.M.
      • Sylow L.
      • Honeyman J.
      • Hewitt K.A.
      • et al.
      Deletion of skeletal muscle SOCS3 prevents insulin resistance in obesity.
      ,
      • Mashili F.
      • Chibalin A.V.
      • Krook A.
      • Zierath J.R.
      Constitutive STAT3 phosphorylation contributes to skeletal muscle insulin resistance in type 2 diabetes.
      ].
      Figure thumbnail gr3
      Fig. 3Multiple effects of IL-6 on metabolism. (A) Engagement of the insulin receptor (InsR) on agouti-related peptide (AgRP)-expressing neurons leads to the expression of IL-6 in the liver. Expression of IL-6 by Kupffer cells is negatively regulated by estrogen receptor α (ER-α) and nuclear receptor coactivator 5 (NCOA5). On hepatocytes IL-6 induces expression of insulin receptor substrate 2 (IRS-2), thereby enhancing insulin signaling. IL-6 dependent suppression of glucose-6 phosphatase (G6Pase) lowers peripheral blood glucose levels and increases glycogen stores in hepatocytes. Upregulation of suppressor of cytokine signaling 3 (SOCS-3) by IL-6 impairs gp130 and InsR signaling. (B) Loss of hepatic IL-6 signaling leads to hepatosteatosis, local inflammation, enhanced blood glucose levels and peripheral insulin resistance mediated by TNF-α. HC: hepatocyte, EC: endothelial cell, KC: Kupffer cell, NC: neuronal cell, AC: adipocyte.
      However, the role of IL-6 in control of metabolism seems to be more complex. The first hint that IL-6 might also exert beneficial roles on the metabolism came from the observation that mice deficient for IL-6 (IL-6−/−) developed mature-onset diabetes with increased leptin and insulin levels, liver inflammation and hepatosteatosis, in particular when fed a HFD [
      • Wallenius V.
      • Wallenius K.
      • Ahrén B.
      • Rudling M.
      • Carlsten H.
      • et al.
      Interleukin-6-deficient mice develop mature-onset obesity.
      ,
      • Matthews V.B.
      • Allen T.L.
      • Risis S.
      • Chan M.H.S.
      • Henstridge D.C.
      • et al.
      Interleukin-6-deficient mice develop hepatic inflammation and systemic insulin resistance.
      ]. Mice with a hepatocyte-specific deletion of the IL-6R also displayed insulin resistance in liver, skeletal muscle and white adipose tissue and exaggerated diet-induced liver inflammation, indicating a protective role of IL-6 on hepatocytes. Interestingly, glucose homeostasis in these mice could be restored after TNF-α-inhibition or Kupffer cell-depletion [
      • Wunderlich F.T.
      • Ströhle P.
      • Könner A.C.
      • Gruber S.
      • Tovar S.
      • et al.
      Interleukin-6 signaling in liver-parenchymal cells suppresses hepatic inflammation and improves systemic insulin action.
      ]. Consistently, hepatocyte-specific ablation of STAT3 or gp130 also led to insulin resistance and predisposed to diet-induced liver steatosis (Fig. 3B) [
      • Inoue H.
      • Ogawa W.
      • Ozaki M.
      • Haga S.
      • Matsumoto M.
      • et al.
      Role of STAT-3 in regulation of hepatic gluconeogenic genes and carbohydrate metabolism in vivo.
      ,
      • Kroy D.C.
      • Beraza N.
      • Tschaharganeh D.F.
      • Sander L.E.
      • Erschfeld S.
      • et al.
      Lack of interleukin-6/glycoprotein 130/signal transducers and activators of transcription-3 signaling in hepatocytes predisposes to liver steatosis and injury in mice.
      ]. Hepatic glucose production and release to the periphery, in particular the expression of the gluconeogenic enzyme glucose-6 phosphatase, is negatively regulated by IL-6 in a STAT3-dependent manner [
      • Wunderlich F.T.
      • Ströhle P.
      • Könner A.C.
      • Gruber S.
      • Tovar S.
      • et al.
      Interleukin-6 signaling in liver-parenchymal cells suppresses hepatic inflammation and improves systemic insulin action.
      ,
      • Inoue H.
      • Ogawa W.
      • Ozaki M.
      • Haga S.
      • Matsumoto M.
      • et al.
      Role of STAT-3 in regulation of hepatic gluconeogenic genes and carbohydrate metabolism in vivo.
      ,
      • Ramadoss P.
      • Unger-Smith N.E.
      • Lam F.S.
      • Hollenberg A.N.
      STAT3 targets the regulatory regions of gluconeogenic genes in vivo.
      ]. In addition, increase in insulin sensitivity through upregulation of IRS-2 by adiponectin, a well-recognized anti-diabetic cytokine has been shown to be IL-6 mediated (Fig. 3A) [
      • Awazawa M.
      • Ueki K.
      • Inabe K.
      • Yamauchi T.
      • Kubota N.
      • et al.
      Adiponectin enhances insulin sensitivity by increasing hepatic IRS-2 expression via a macrophage-derived IL-6-dependent pathway.
      ].
      These data show that IL-6 in the liver not only regulates glucose metabolism but is also necessary to maintain liver tissue homeostasis for proper control of metabolic functions. One of the hallmarks of obesity is the development of a chronic low-grade inflammatory state with increased infiltration of T lymphocytes and macrophages to adipose tissue. Interestingly, mice with a myeloid-specific ablation of the IL-6R also developed insulin resistance and increased inflammation in the liver under a HFD. The authors of that study could demonstrate that IL-6 mediates polarization of pro-inflammatory M1 macrophages to M2 macrophages in adipose tissue through upregulation of the IL-4 receptor (IL-4R) [
      • Mauer J.
      • Chaurasia B.
      • Goldau J.
      • Vogt M.C.
      • Ruud J.
      • et al.
      Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin.
      ]. The IL-4R is an integral component of the receptor complexes for the cytokines IL-4 and IL-13, which are needed for M2 macrophage differentiation. Therefore, in the context of obesity, elevated IL-6 serum levels rather seem to counterbalance obesity-associated hyperglycemia. Interestingly, also during physical exercise, IL-6 is produced by muscle cells, leading to an up to 100-fold increase in IL-6 plasma levels [
      • Pedersen B.K.
      • Febbraio M.A.
      Muscles, exercise and obesity: skeletal muscle as a secretory organ.
      ].
      One further has to consider that IL-6 plays an important role in the brain-liver axis. Insulin signaling in agouti-related peptide-expressing neurons induces hepatic IL-6 expression and concomitant downregulation of hepatic gluconeogenic enzymes (Fig. 3A) [
      • Könner A.C.
      • Janoschek R.
      • Plum L.
      • Jordan S.D.
      • Rother E.
      • et al.
      Insulin action in AgRP-expressing neurons is required for suppression of hepatic glucose production.
      ]. Furthermore, IL-6 action on the central nervous system stimulates energy expenditure [
      • Wallenius V.
      • Wallenius K.
      • Ahrén B.
      • Rudling M.
      • Carlsten H.
      • et al.
      Interleukin-6-deficient mice develop mature-onset obesity.
      ], again underlining a beneficial role of IL-6 in metabolism. Remarkably, we could recently show that most if not all IL-6 signaling in the brain is mediated by trans-signaling [
      • Campbell I.L.
      • Erta M.
      • Lim S.L.
      • Frausto R.
      • May U.
      • et al.
      Trans-signaling is a dominant mechanism for the pathogenic actions of interleukin-6 in the brain.
      ].
      Most of the studies so far used total ablation of IL-6 or tissue-specific inactivation of the IL-6R. However, future studies have to consider that IL-6 classic and IL-6 trans-signaling might differentially regulate metabolism. And indeed, a very recent report demonstrated that blockade of IL-6 trans-signaling impaired the recruitment of inflammatory macrophages to white adipose tissue under a HFD, while it did not alter insulin resistance in that model [
      • Kraakman M.J.
      • Kammoun H.L.
      • Allen T.L.
      • Deswaerte V.
      • Henstridge D.C.
      • et al.
      Blocking IL-6 trans-signaling prevents high-fat diet-induced adipose tissue macrophage recruitment but does not improve insulin resistance.
      ]. Furthermore, glycogen consumption and synthesis in hepatocytes was regulated by IL-6 trans-signaling in two different models of liver damage [
      • Drucker C.
      • Rabe B.
      • Chalaris A.
      • Schulz E.
      • Scheller J.
      • et al.
      Interleukin-6 trans-signaling regulates glycogen consumption after D-galactosamine-induced liver damage.
      ,
      • Gewiese-Rabsch J.
      • Drucker C.
      • Malchow S.
      • Scheller J.
      • Rose-John S.
      Role of IL-6 trans-signaling in CCl₄ induced liver damage.
      ].

      IL-6 during liver tumorigenesis

      IL-6 is a major driver of hepatocellular carcinogenesis.

      IL-6 is crucial for the development of hepatocellular carcinoma

      Diabetes, obesity and male gender are associated with an increased risk to develop hepatocellular carcinoma (HCC). Additionally, high serum levels of IL-6 have been reported in several liver pathologies that predispose to the development of HCC, including acute and viral hepatitis [
      • Sun Y.
      • Tokushige K.
      • Isono E.
      • Yamauchi K.
      • Obata H.
      Elevated serum interleukin-6 levels in patients with acute hepatitis.
      ], alcoholic cirrhosis [
      • Deviere J.
      • Content J.
      • Denys C.
      • Vandenbussche P.
      • Schandene L.
      • et al.
      High interleukin-6 serum levels and increased production by leucocytes in alcoholic liver cirrhosis. Correlation with IgA serum levels and lymphokines production.
      ] and primary biliary cirrhosis [
      • Kakumu S.
      • Shinagawa T.
      • Ishikawa T.
      • Yoshioka K.
      • Wakita T.
      • et al.
      Interleukin 6 production by peripheral blood mononuclear cells in patients with chronic hepatitis B virus infection and primary biliary cirrhosis.
      ]. In a very recent prospective study, elevated IL-6 serum levels correlated with an increased risk to develop HCC [
      • Aleksandrova K.
      • Boeing H.
      • Nöthlings U.
      • Jenab M.
      • Fedirko V.
      • et al.
      Inflammatory and metabolic biomarkers and risk of liver and biliary tract cancer.
      ]. And in patients suffering from HCC, elevated serum levels of IL-6 and the sIL-6R have been detected [
      • Soresi M.
      • Giannitrapani L.
      • D’Antona F.
      • Florena A.-M.
      • La Spada E.
      • et al.
      Interleukin-6 and its soluble receptor in patients with liver cirrhosis and hepatocellular carcinoma.
      ].
      Extensive work on hepatocarcinogenesis has been performed using the murine diethylnitrosamine (DEN)-model of HCC. The cytotoxic effects of DEN are dependent on its metabolic activation by cytochrome P450 2E1 in hepatocytes. Once activated, DEN forms DNA adducts leading to hepatocyte damage. In this early phase of hepatocarcinogenesis, Kupffer cells become activated in a TLR- and EGFR-dependent manner to secrete TNF-α and IL-6 (Fig. 4) [
      • Maeda S.
      • Kamata H.
      • Luo J.-L.
      • Leffert H.
      • Karin M.
      IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis.
      ,
      • Naugler W.E.
      • Sakurai T.
      • Kim S.
      • Maeda S.
      • Kim K.
      • et al.
      Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production.
      ,
      • Lanaya H.
      • Natarajan A.
      • Komposch K.
      • Li L.
      • Amberg N.
      • et al.
      EGFR has a tumour-promoting role in liver macrophages during hepatocellular carcinoma∼formation.
      ]. IL-6 induces a compensatory proliferation of hepatocytes, which accumulate DNA damages due to DEN. Consistently, IL-6 deficient mice, as well as mice with liver-specific loss of gp130 show a lower incidence of HCC tumors and prolonged survival in the DEN model [
      • Naugler W.E.
      • Sakurai T.
      • Kim S.
      • Maeda S.
      • Kim K.
      • et al.
      Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production.
      ,
      • Hatting M.
      • Spannbauer M.
      • Peng J.
      • Al Masaoudi M.
      • Sellge G.
      • et al.
      Lack of gp130 expression in hepatocytes attenuates tumor progression in the DEN model.
      ]. In mulitdrug resistance 2-deficient mice, cholestasis leads to bile acid induced liver injury, inflammation and fibrosis and at a later stage to hepatocyte dysplasia and carcinoma formation [
      • Pikarsky E.
      • Porat R.M.
      • Stein I.
      • Abramovitch R.
      • Amit S.
      • et al.
      NF-kappaB functions as a tumour promoter in inflammation-associated cancer.
      ,
      • Mair M.
      • Zollner G.
      • Schneller D.
      • Musteanu M.
      • Fickert P.
      • et al.
      Signal transducer and activator of transcription 3 protects from liver injury and fibrosis in a mouse model of sclerosing cholangitis.
      ]. Interestingly, in these mice STAT3 or IL-6 ablation aggravated hepatocyte damage through impaired EGFR signaling and enhanced bile acid synthesis leading to enhanced liver fibrosis, predisposing to increased tumor formation [
      • Mair M.
      • Zollner G.
      • Schneller D.
      • Musteanu M.
      • Fickert P.
      • et al.
      Signal transducer and activator of transcription 3 protects from liver injury and fibrosis in a mouse model of sclerosing cholangitis.
      ].
      Figure thumbnail gr4
      Fig. 4IL-6 promotes multiple steps of hepatocarcinogenesis. Kupffer cells are activated by damaged hepatocytes in a TLR- and EGFR-dependent manner and secrete IL-6 and the soluble IL-6R, leading to a compensatory hepatocyte proliferation. Within a tumor-promoting microenvironment, hepatocytes transform into HCC progenitor cells (HcPCs) that aquire an autocrine IL-6 loop through upregulation of LIN28B. Hepatocyte transformation is facilitated by M2-type macrophages that also secrete IL-6. Polarization of M1 to M2 macrophages is promoted by IL-6. HcPCs egress from the tumor-promoting niche to form HCC nodules and eventually metastasis to distant organs.
      IL-6 expression is a major hallmark of the senescence-associated secretory phenotype. Senescent hepatocytes in chronic liver disease [
      • Aravinthan A.
      • Scarpini C.
      • Tachtatzis P.
      • Verma S.
      • Penrhyn-Lowe S.
      • et al.
      Hepatocyte senescence predicts progression in non-alcohol-related fatty liver disease.
      ,
      • Aravinthan A.D.
      • Alexander G.J.M.
      Hepatocyte senescence explains conjugated bilirubinaemia in chronic liver failure.
      ] or senescent cholangiocytes during primary sclerosing cholangitis [
      • Tabibian J.H.
      • O’Hara S.P.
      • Splinter P.L.
      • Trussoni C.E.
      • LaRusso N.F.
      Cholangiocyte senescence by way of N-ras activation is a characteristic of primary sclerosing cholangitis.
      ] therefore also contribute to increased IL-6 expression in the liver under diseased conditions.
      Interestingly, the incidence of HCC development is reduced in female mice as estrogen signaling via the estrogen receptor α, suppresses expression of IL-6 (Fig. 4) [
      • Naugler W.E.
      • Sakurai T.
      • Kim S.
      • Maeda S.
      • Kim K.
      • et al.
      Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production.
      ], which correlates well with the human situation. Very recently nuclear receptor coactivator 5 (NCOA5) has been shown to be another transcriptional repressor of IL-6 expression. Predisposition of NCOA5-haploinsufficient mice to insulin resistance, type 2 diabetes and HCC correlated with elevated IL-6 levels [
      • Gao S.
      • Li A.
      • Liu F.
      • Chen F.
      • Williams M.
      • et al.
      NCOA5 haploinsufficiency results in glucose intolerance and subsequent hepatocellular carcinoma.
      ]. HCC development in the DEN model was aggravated in mice with genetically induced or dietary-mediated obesity, which could be linked to elevated IL-6 levels [
      • Park E.J.
      • Lee J.H.
      • Yu G.-Y.
      • He G.
      • Ali S.R.
      • et al.
      Dietary and genetic obesity promote liver inflammation and tumorigenesis by enhancing IL-6 and TNF expression.
      ]. A very recent report suggested, however, that obesity-enhanced HCC formation was independent of the IL-6R on hepatocytes, suggesting that HCC formation might be independent of IL-6 or rely on IL-6 trans-signaling [
      • Gruber S.
      • Straub B.K.
      • Ackermann P.J.
      • Wunderlich C.M.
      • Mauer J.
      • et al.
      Obesity promotes liver carcinogenesis via Mcl-1 stabilization independent of IL-6Rα signaling.
      ]. Indeed, work from our laboratory shows that abrogation of IL-6 trans-signaling decreases tumor burden in the DEN model to a similar degree as in IL-6−/− mice, indicating that IL-6 trans-signaling significantly contributed to tumor formation in this model (personal communication).
      In the inflamed liver, IL-6 promotes the polarization of macrophages to a M2 phenotype through induction of the IL-4 receptor α chain [
      • Mauer J.
      • Chaurasia B.
      • Goldau J.
      • Vogt M.C.
      • Ruud J.
      • et al.
      Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin.
      ]. In human HCC, the amount of infiltrating tumor associated macrophages inversely correlates with prognosis in HCC patients and it could be shown that indeed, IL-6 secreted from tumor associated macrophages promoted the expansion of HCC progenitor cells (HcPCs) [
      • Wan S.
      • Zhao E.
      • Kryczek I.
      • Vatan L.
      • Sadovskaya A.
      • et al.
      Tumor-associated macrophages produce interleukin 6 and signal via STAT3 to promote expansion of human hepatocellular carcinoma stem cells.
      ]. Interestingly, at an early stage of hepatocarcinogenesis, HcPCs appeared in ectopic lymphoid structures containing M2 macrophages, B- and T lymphocytes creating a growth-promoting microenvironment [
      • Finkin S.
      • Yuan D.
      • Stein I.
      • Taniguchi K.
      • Weber A.
      • et al.
      Ectopic lymphoid structures function as microniches for tumor progenitor cells in hepatocellular carcinoma.
      ]. Albeit HcPCs have a liver progenitor cell phenotype, they originate through dedifferentiation from hepatocytes [
      • Tarlow B.D.
      • Pelz C.
      • Naugler W.E.
      • Wakefield L.
      • Wilson E.M.
      • et al.
      Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes.
      ,
      • Mu X.
      • Español-Suñer R.
      • Mederacke I.
      • Affò S.
      • Manco R.
      • et al.
      Hepatocellular carcinoma originates from hepatocytes and not from the progenitor/biliary compartment.
      ]. At a later stage, HcPCs aquire an IL-6 autocrine loop where expression of IL-6 is dependent on LIN28B-mediated degradation of the miRNA Let-7, a negative regulator of IL-6 expression (Fig. 4) [
      • He G.
      • Dhar D.
      • Nakagawa H.
      • Font-Burgada J.
      • Ogata H.
      • et al.
      Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling.
      ]. A similar epigenetic switch has been shown to be involved in the generation of breast cancer stem cells [
      • Iliopoulos D.
      • Hirsch H.A.
      • Wang G.
      • Struhl K.
      Inducible formation of breast cancer stem cells and their dynamic equilibrium with non-stem cancer cells via IL6 secretion.
      ]. However, the autocrine IL-6 loop alone is not sufficient to drive HCC formation, as HcPCs only formed tumors when transplanted to mice with a fibrotic liver [
      • He G.
      • Dhar D.
      • Nakagawa H.
      • Font-Burgada J.
      • Ogata H.
      • et al.
      Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling.
      ]. During the course of HCC development, HcPCs then egress from ectopic lymphoid structures to form HCC nodules in the liver and metastasis to distant organs.
      Taken together, while IL-6 secreted from myeloid cells is important during an early phase of HCC development, pre-neoplastic lesions are fed by an autocrine IL-6 loop at a later stage of tumorigenesis, but are still dependent on additional signals from the tumor microenvironment to develop full-blown HCC.

      Activation of the IL-6/JAK/STAT3 pathway in inflammatory hepatocellular adenoma

      Inflammatory hepatocellular adenomas (IHCA) are rare benign tumors of the liver predominantly found in women and frequently associated with oral contraception, obesity and alcohol abuse. They are characterized by constitutive activation of the acute phase genes in hepatocytes and highly polymorphous inflammatory infiltrates in the liver [
      • Rebouissou S.
      • Amessou M.
      • Couchy G.
      • Poussin K.
      • Imbeaud S.
      • et al.
      Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours.
      ].
      The discovery of activating mutations in gp130 and downstream signaling molecules, including JAK1, STAT3, Fyn-related kinase (FRK) and G-protein G(s) subunit alpha (GNAS) (Fig. 5) in IHCA highlighted the importance of the IL-6 pathway for the pathogenesis of benign liver tumors. While novel activating STAT3 mutations are found in 12% of IHCA cases [
      • Pilati C.
      • Amessou M.
      • Bihl M.P.
      • Balabaud C.
      • Nhieu J.T.V.
      • et al.
      Somatic mutations activating STAT3 in human inflammatory hepatocellular adenomas.
      ], small in-frame deletions within the coding region of gp130 are present in 60% of IHCA cases [
      • Rebouissou S.
      • Amessou M.
      • Couchy G.
      • Poussin K.
      • Imbeaud S.
      • et al.
      Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours.
      ]. These deletions vary in length but always cluster within a loop of the extracellular domain D2 which represents an IL-6 contact site. As a consequence stabilizing hydrophobic interactions between domain D2 and D3 are lost and the extracellular domain adopts an active conformation, resulting in constitutive downstream signaling [
      • Schütt A.
      • Zacharias M.
      • Schneider N.
      • Horn S.
      • Grötzinger J.
      • et al.
      Gp130 activation is regulated by D2–D3 interdomain connectivity.
      ]. Persistent activation of IL-6 trans-signaling has already previously been shown to induce hepatocellular adenoma formation in IL-6/sIL-6R double transgenic but not in IL-6 single transgenic mice [
      • Maione D.
      • Di Carlo E.
      • Li W.
      • Musiani P.
      • Modesti A.
      • et al.
      Coexpression of IL-6 and soluble IL-6R causes nodular regenerative hyperplasia and adenomas of the liver.
      ]. These findings suggest that enhanced gp130 activation by IL-6 trans-signaling is needed to induce oncogenic transformation of hepatocytes. Interestingly, the described in-frame deletion mutants of gp130 display a delayed biosynthesis and are therefore predominantly localized within the endoplasmic reticulum, potentially resulting in altered signal transduction [
      • Schmidt-Arras D.
      • Müller M.
      • Stevanovic M.
      • Horn S.
      • Schütt A.
      • et al.
      Oncogenic deletion mutants of gp130 signal from intracellular compartments.
      ] contributing to oncogenic transformation (Fig. 5).
      Figure thumbnail gr5
      Fig. 5The IL-6/gp130 pathway is consitutively activated in inflammatory hepatocellular adenoma (IHCA). Activating deletion mutations in gp130 are the most frequently occurring mutations in IHCA. Constitutively active gp130 can induce STAT3 phosphorylation from the endoplasmic reticulum and endosomes, while Ras-activation occurs at the plasma membrane. Activating mutations are also found in STAT3, the Fyn-related kinase (FRK) and G-protein G(s) subunit alpha (GNAS).
      Albeit IL-6 has been shown to be critically involved in the development of hepatocellular carcinoma, only 1–2% of HCC cases harbor gp130 deletion mutations [
      • Rebouissou S.
      • Amessou M.
      • Couchy G.
      • Poussin K.
      • Imbeaud S.
      • et al.
      Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours.
      ]. These findings support the notion that hepatocellular adenomas only very rarely progress to HCC and that persistent activation of the IL-6 pathway alone is not sufficient to trigger malignant transformation of hepatocytes [
      • Rebouissou S.
      • Amessou M.
      • Couchy G.
      • Poussin K.
      • Imbeaud S.
      • et al.
      Frequent in-frame somatic deletions activate gp130 in inflammatory hepatocellular tumours.
      ].

      IL-6 directed therapy in liver pathologies

      Acceleration of liver regeneration

      As described above, different preclinical rodent models of liver regeneration demonstrated that Hyper-IL-6 strongly enhances liver regeneration. Transient infusions of Hyper-IL-6 might therefore be beneficial to patients after partial liver resection to boost hepatocyte-mediated regeneration. However, these patients very often underwent tumor resection and up to now it is not clear if transient IL-6/Hyper-IL-6 therapy could cause recurrence of tumor growth.
      A more elegant way would be to enhance ex vivo expansion of bi-potential liver progenitor cells (LPC) by IL-6 or Hyper-IL-6 prior to its infusion to patients. Indeed IL-6 and even more Hyper-IL-6 were able to enhance proliferation of LPCs in vivo and in vitro and infusion of expanded LPCs were able to regenerate impaired liver function in vivo [
      • Yeoh G.C.T.
      • Ernst M.
      • Rose-John S.
      • Akhurst B.
      • Payne C.
      • et al.
      Opposing roles of gp130-mediated STAT-3 and ERK-1/ 2 signaling in liver progenitor cell migration and proliferation.
      ,
      • Lu W.-Y.
      • Bird T.G.
      • Boulter L.
      • Tsuchiya A.
      • Cole A.M.
      • et al.
      Hepatic progenitor cells of biliary origin with liver repopulation capacity.
      ].

      Neutralization of IL-6 and IL-6R

      IL-6 has been previously recognized as growth factor in multiple myeloma and use of a neutralizing anti-IL-6 antibodies (Fig. 6) was effective in multiple myeloma patients [
      • Klein B.
      • Wijdenes J.
      • Zhang X.G.
      • Jourdan M.
      • Boiron J.M.
      • et al.
      Murine anti-interleukin-6 monoclonal antibody therapy for a patient with plasma cell leukemia.
      ]. In a very recent clinical phase I/II study, however, monotherapy with the anti-IL6 antibody siltuximab did not show clinical activity against a series of solid tumors [
      • Angevin E.
      • Tabernero J.
      • Elez E.
      • Cohen S.J.
      • Bahleda R.
      • et al.
      A phase I/II, multiple-dose, dose-escalation study of siltuximab, an anti-interleukin-6 monoclonal antibody, in patients with advanced solid tumors.
      ]. Furthermore, it turned out that anti-IL-6 antibody treatment led to the formation of high molecular weight antibody-IL-6 complexes and thereby prevented IL-6 clearance from the circulation leading to massive systemic IL-6 elevations [
      • Lu Z.Y.
      • Brochier J.
      • Wijdenes J.
      • Brailly H.
      • Bataille R.
      • et al.
      High amounts of circulating interleukin (IL)-6 in the form of monomeric immune complexes during anti-IL-6 therapy. Towards a new methodology for measuring overall cytokine production in human in vivo.
      ].
      Figure thumbnail gr6
      Fig. 6Strategies to specifically block IL-6 signaling. (A) Neutralizing antibodies to IL-6 block both, IL-6 classic and IL-6 trans-signaling. (B) Antibodies directed against the IL-6R inhibit binding of IL-6 to IL-6R and block both, IL-6 classic and IL-6 trans-signaling. (C) Recombinant sgp130Fc selectively blocks IL-6 trans-signaling by sequestration of the IL-6/sIL-6R complex. Therefore, the beneficial effects of IL-6 like acute phase response and infection defense are not impaired by the sgp130Fc protein. (D) Small molecule inhibitors of JAK kinases prevent phosphorylation of gp130 and downstream molecules like STAT3 of both IL-6 classic and IL-6 trans-signaling. (E) Small molecule inhibitors of STAT3 prevent either dimerization of STAT3 through binding to its SH2 domain or prevent binding of STAT3 to DNA. STAT3-dependent effects of both IL-6 classic and IL-6 trans-signaling are blocked. However, STAT3-independent effects of IL-6 like activation of the gp130-YAP-Notch module during liver regeneration are not altered.
      This led to the development of tocilizumab, a humanized anti-IL-6R antibody (Fig. 6). Tocilizumab binds to the IL-6 binding site of the IL-6R and thereby prevents IL-6 binding and also high molecular weight antibody-IL-6 complex formation. Tocilizumab blocks IL-6 classic- and trans-signaling. A series of clinical studies have shown its therapeutic benefit in rheumatoid arhtritis [
      • Gabay C.
      • Emery P.
      • van Vollenhoven R.
      • Dikranian A.
      • Alten R.
      • et al.
      Tocilizumab monotherapy versus adalimumab monotherapy for treatment of rheumatoid arthritis (ADACTA): a randomised, double-blind, controlled phase 4 trial.
      ], juvenile idiopathic arthritis [
      • Nishimoto N.
      • Kishimoto T.
      Interleukin 6: from bench to bedside.
      ] and Castleman’s disease [
      • Nishimoto N.
      • Kanakura Y.
      • Aozasa K.
      • Johkoh T.
      • Nakamura M.
      • et al.
      Humanized anti-interleukin-6 receptor antibody treatment of multicentric Castleman disease.
      ]. A phase I/II clinical trial also showed that tocilizumab might be also benefical to prevent graft-versus host disease after bone marrow transplantation [
      • Kennedy G.A.
      • Varelias A.
      • Vuckovic S.
      • Le Texier L.
      • Gartlan K.H.
      • et al.
      Addition of interleukin-6 inhibition with tocilizumab to standard graft-versus-host disease prophylaxis after allogeneic stem-cell transplantation: a phase 1/2 trial.
      ].
      Preclinical studies using murine xenograft models showed that anti-IL-6R therapy suppresses tumor angiogenesis and tumor growth in colon cancer and oral squamous cell carcinoma [
      • Shinriki S.
      • Jono H.
      • Ota K.
      • Ueda M.
      • Kudo M.
      • et al.
      Humanized anti-interleukin-6 receptor antibody suppresses tumor angiogenesis and in vivo growth of human oral squamous cell carcinoma.
      ,
      • Nagasaki T.
      • Hara M.
      • Nakanishi H.
      • Takahashi H.
      • Sato M.
      • et al.
      Interleukin-6 released by colon cancer-associated fibroblasts is critical for tumour angiogenesis: anti-interleukin-6 receptor antibody suppressed angiogenesis and inhibited tumour-stroma interaction.
      ]. Also, in an HCC xenograft model, tocilizumab reduced HCC growth by blunting IL-6 signaling from tumor associated macrophages to HCC cells and therefore preventing the formation of cancer stem cells [
      • Wan S.
      • Zhao E.
      • Kryczek I.
      • Vatan L.
      • Sadovskaya A.
      • et al.
      Tumor-associated macrophages produce interleukin 6 and signal via STAT3 to promote expansion of human hepatocellular carcinoma stem cells.
      ]. The fact that expansion of HcPCs is dependent on IL-6 [
      • He G.
      • Dhar D.
      • Nakagawa H.
      • Font-Burgada J.
      • Ogata H.
      • et al.
      Identification of liver cancer progenitors whose malignant progression depends on autocrine IL-6 signaling.
      ], that IL-6 promotes M2 macrophage polarization [
      • Mauer J.
      • Chaurasia B.
      • Goldau J.
      • Vogt M.C.
      • Ruud J.
      • et al.
      Signaling by IL-6 promotes alternative activation of macrophages to limit endotoxemia and obesity-associated resistance to insulin.
      ] and that growth of HCC seems to be promoted by M2 macrophages [
      • Li X.
      • Yao W.
      • Yuan Y.
      • Chen P.
      • Li B.
      • et al.
      Targeting of tumour-infiltrating macrophages via CCL2/CCR2 signalling as a therapeutic strategy against hepatocellular carcinoma.
      ] suggest that HCC patients might benefit from an IL-6 directed therapy. Beside its direct effect on tumor growth, anti-IL-6R therapy has been considered for the treatment of tumor cachexia. In preclinical models of cancer cachexia, genetic loss of IL-6 or the use of anti-IL-6 antibodies prevented white adipose tissue browning and therefore counterbalanced cancer cachexia in these models [
      • Petruzzelli M.
      • Schweiger M.
      • Schreiber R.
      • Campos-Olivas R.
      • Tsoli M.
      • et al.
      A switch from white to brown fat increases energy expenditure in cancer-associated cachexia.
      ]. Consequently, in a study with a single cancer patient, tocilizumab was able to reverse cancer cachexia symptoms [
      • Ando K.
      • Takahashi F.
      • Motojima S.
      • Nakashima K.
      • Kaneko N.
      • et al.
      Possible role for tocilizumab, an anti-interleukin-6 receptor antibody, in treating cancer cachexia.
      ].
      As outlined above, classic IL-6 receptor signaling is believed to control some crucial homeostatic mechanisms such as the hepatic acute phase response [
      • Gauldie J.
      • Richards C.
      • Harnish D.
      • Lansdorp P.
      • Baumann H.
      Interferon beta 2/B-cell stimulatory factor type 2 shares identity with monocyte-derived hepatocyte-stimulating factor and regulates the major acute phase protein response in liver cells.
      ,
      • Baumann H.
      • Gauldie J.
      The acute phase response.
      ,
      • Gabay C.
      • Kushner I.
      Acute-phase proteins and other systemic responses to inflammation.
      ,
      • Hoge J.
      • Yan I.
      • Jänner N.
      • Schumacher V.
      • Chalaris A.
      • et al.
      IL-6 controls the innate immune response against Listeria monocytogenes via classical IL-6 signaling.
      ]. In contrast, IL-6 trans-signaling seems to play a role in overshooting immunological reactions resulting in autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease [
      • Hunter C.A.
      • Jones S.A.
      IL-6 as a keystone cytokine in health and disease.
      ].
      One therefore has to consider several caveats when using anti-IL-6R antibody therapy. Anti-IL-6R antibodies prevent both, classic IL-6 and IL-6 trans-signaling (Fig. 6). Classic IL-6 signaling however is important for the induction of the acute phase response as a first-line defense to infection and as a prevention of sepsis [
      • Vandevyver S.
      • Dejager L.
      • Vandenbroucke R.E.
      • Libert C.
      An acute phase protein ready to go therapeutic for sepsis.
      ,
      • Kaner Z.
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      • Shahaf G.
      • Baranovski B.M.
      • Bahar N.
      • et al.
      Acute phase protein α1-antitrypsin reduces the bacterial burden in mice by selective modulation of innate cell responses.
      ]. Classic IL-6 signaling indeed has been shown to be important for the control of bacterial and viral, in particular hepatitis B virus (HBV) infections [
      • Hoge J.
      • Yan I.
      • Jänner N.
      • Schumacher V.
      • Chalaris A.
      • et al.
      IL-6 controls the innate immune response against Listeria monocytogenes via classical IL-6 signaling.
      ,
      • Hösel M.
      • Quasdorff M.
      • Wiegmann K.
      • Webb D.
      • Zedler U.
      • et al.
      Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection.
      ]. Also a significant reduction in the number of peripheral neutrophils [
      • Berti A.
      • Boccalatte F.
      • Sabbadini M.G.
      • Dagna L.
      Assessment of tocilizumab in the treatment of cancer cachexia.
      ] and an increase in body weight and triglyceride levels has been observed as a side effect of tocilizumab treatment [
      • Nishimoto N.
      • Kanakura Y.
      • Aozasa K.
      • Johkoh T.
      • Nakamura M.
      • et al.
      Humanized anti-interleukin-6 receptor antibody treatment of multicentric Castleman disease.
      ]. These parameters therefore have to be tightly monitored under anti-IL-6R treatment.
      Given the fact that sgp130Fc transgenic mice do not show a negative metabolic phenotype, while possessing anti-inflammatory properties in various preclinical models [
      • Kraakman M.J.
      • Kammoun H.L.
      • Allen T.L.
      • Deswaerte V.
      • Henstridge D.C.
      • et al.
      Blocking IL-6 trans-signaling prevents high-fat diet-induced adipose tissue macrophage recruitment but does not improve insulin resistance.
      ], sgp130Fc treatment might represent a more advantageous therapy option in diverse disease settings. A recent phase I study has shown that sgp130Fc is well tolerated in humans [
      • Calabrese L.H.
      • Rose-John S.
      IL-6 biology: implications for clinical targeting in rheumatic disease.
      ]. Further studies have to reveal if sgp130Fc therapy is an option for the treatment of HCC and other liver pathologies. A phase IIa clinical trial in patients with IL-6 related inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis is planned for 2016.
      Patients with liver pathologies might benefit from selective inhibition of IL-6 pathways.

      Inhibition of IL-6 downstream signaling

      As described above, IL-6 binding to IL-6R and gp130 results in the activation of cytoplasmic tyrosine kinases of the JAK and Src kinase family and subsequent phosphorylation of STAT3. Inhibtion of JAK and Src kinases would therefore blunt intracellular IL-6 signaling. Ruxolitinib and tasocitinib are small molecule inhibitors of JAK kinases (Fig. 6D) and have been clinically approved for the treatment of myeloproliferative neoplasms and rheumatoid arthritis [
      • Quintás-Cardama A.
      • Verstovsek S.
      Molecular pathways: Jak/STAT pathway: mutations, inhibitors, and resistance.
      ]. Other compounds are currently under development and in clinical trials for hematological malignancies, solid tumors and rheumatoid arthritis [
      • Quintás-Cardama A.
      • Verstovsek S.
      Molecular pathways: Jak/STAT pathway: mutations, inhibitors, and resistance.
      ,
      • Geyer H.L.
      • Mesa R.A.
      Therapy for myeloproliferative neoplasms: when, which agent, and how?.
      ]. Activated STAT3 can also be directly targeted by small molecule inhibitors binding to its SH2 or DNA binding domain (Fig. 6E). These compounds however haven’t entered yet clinical trials [
      • Wang X.
      • Crowe P.J.
      • Goldstein D.
      • Yang J.-L.
      STAT3 inhibition, a novel approach to enhancing targeted therapy in human cancers (review).
      ].
      There are preclinical data available that both JAK and STAT3 inhibition abrogates the proliferation of HCC cell lines and the growth of orthotopic HCC tumors [
      • Wang X.
      • Crowe P.J.
      • Goldstein D.
      • Yang J.-L.
      STAT3 inhibition, a novel approach to enhancing targeted therapy in human cancers (review).
      ,
      • Wilson G.S.
      • Tian A.
      • Hebbard L.
      • Duan W.
      • George J.
      • et al.
      Tumoricidal effects of the JAK inhibitor Ruxolitinib (INC424) on hepatocellular carcinoma in vitro.
      ,
      • Mohan C.D.
      • Bharathkumar H.
      • Bulusu K.C.
      • Pandey V.
      • Rangappa S.
      • et al.
      Development of a novel azaspirane that targets the Janus kinase-signal transducer and activator of transcription (STAT) pathway in hepatocellular carcinoma in vitro and in vivo.
      ]. Consequently, the use of JAK inhibitors for the treatment of IHCA has been suggested [
      • Poussin K.
      • Pilati C.
      • Couchy G.
      • Calderaro J.
      • Bioulac-Sage P.
      • et al.
      Biochemical and functional analyses of gp130 mutants unveil JAK1 as a novel therapeutic target in human inflammatory hepatocellular adenoma.
      ] and its use for the treatment of HCC might be considered. However, JAK kinase inhibition would not only abrogate both, IL-6 classic and IL-6 trans-signaling, but also interfere with the downstream signaling of several cytokines. One therefore would need to carefully monitor potential side effects.

      Conclusions and perspectives

      IL-6 is a cytokine with pleiotropic functions in the body. Under physiological conditions it is essential for proper hepatic tissue homeostasis, liver regeneration, infection defense and fine tuning of metabolic functions. Persistent activation of the IL-6 pathway however seems to be detrimental and can even lead to the development of liver cancer.
      Although much progress has been made, there are still many open questions concerning the implication of IL-6 in physiology and pathology of the liver. In order to efficiently target only the detrimental effects of IL-6 on the liver, we need to better dissect the effects of IL-6 of different cell types of the liver.
      Most of the murine models used so far investigated effects in the complete absence of IL-6. However, in order to better understand the complex nature of IL-6 in the liver and other tissues, a cell type-specific analysis of IL-6 signals is warranted. Deletion of the IL-6R from selected cell types would be informative to analyze the effect of IL-6 classic signaling on different cell types in hepatic pathologies. These experiments would also help to unravel which cells of the liver provide the soluble IL-6R to induce IL-6 trans-signaling. Another elegant way to dissect the different effects of IL-6 signaling in the liver would be the use of mice with an inducible, cell-autonomous and ligand-independent activation of gp130.
      Albeit both, classic and trans-signaling pathways lead to activation of the signaling subunit gp130, the effects on intracellular signaling, but also biological effects seem to differ between IL-6 classic and IL-6 trans-signaling. We therefore need a more detailed spatial and temporal cell biological analysis to better explain these effects and to be able to identify cells in complex tissues that underwent IL-6 trans-signaling.
      There is now a growing body of evidence that IL-6 trans-signaling is also implicated in liver pathologies. Selective inhibition of IL-6 trans-signaling rather than complete blockade of both IL-6 signaling pathway might therefore be more effective in the treatment of liver pathologies.

      Financial support

      The work described in this review was supported by grants from the Deutsche Forschungsgemeinschaft Bonn, Germany ( SFB 841 , projects C1, SFB 877, project A1 and the Cluster of Excellence ‘Inflammation at Interfaces’).

      Conflict of interest

      S. R-J is an inventor of patents owned by CONARIS Research Institute, which develops the sgp130Fc protein together with Ferring Pharmaceuticals and he has stock ownership in CONARIS. No conflicts of interest, financial or otherwise, is declared by D. S-A.

      Authors’ contributions

      Both authors contributed equally to this article.

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