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The role of Kupffer cells in hepatitis B and hepatitis C virus infections

  • Author Footnotes
    † These authors contributed equally to this work.
    Arjan Boltjes
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Dept. of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
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  • Author Footnotes
    † These authors contributed equally to this work.
    Dowty Movita
    Footnotes
    † These authors contributed equally to this work.
    Affiliations
    Dept. of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
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  • Author Footnotes
    ‡ These authors shared senior authorship.
    André Boonstra
    Footnotes
    ‡ These authors shared senior authorship.
    Affiliations
    Dept. of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
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  • Author Footnotes
    ‡ These authors shared senior authorship.
    Andrea M. Woltman
    Correspondence
    Corresponding author. Address: Dept. of Gastroenterology and Hepatology, Erasmus MC University Medical Center, ‘s Gravendijkwal 230, 3015 CE Rotterdam, The Netherlands. Tel.: +31 10 703 2759; fax: +31 10 703 2793.
    Footnotes
    ‡ These authors shared senior authorship.
    Affiliations
    Dept. of Gastroenterology and Hepatology, Erasmus MC University Medical Center, Rotterdam, The Netherlands
    Search for articles by this author
  • Author Footnotes
    † These authors contributed equally to this work.
    ‡ These authors shared senior authorship.
Open AccessPublished:May 02, 2014DOI:https://doi.org/10.1016/j.jhep.2014.04.026

      Summary

      Globally, over 500 million people are chronically infected with the hepatitis B virus (HBV) or hepatitis C virus (HCV). These chronic infections cause liver inflammation, and may result in fibrosis/cirrhosis or hepatocellular carcinoma. Albeit that HBV and HCV differ in various aspects, clearance, persistence, and immunopathology of either infection depends on the interplay between the innate and adaptive responses in the liver. Kupffer cells, the liver-resident macrophages, are abundantly present in the sinusoids of the liver. These cells have been shown to be crucial players to maintain homeostasis, but also contribute to pathology. However, it is important to note that especially during pathology, Kupffer cells are difficult to distinguish from infiltrating monocytes/macrophages and other myeloid cells. In this review we discuss our current understanding of Kupffer cells, and assess their role in the regulation of anti-viral immunity and disease pathogenesis during HBV and HCV infection.

      Abbreviations:

      HBV (Hepatitis B virus), HCV (Hepatitis C virus), KC (Kupffer cells), NK cells (natural killer cells), PRR (pattern recognition receptor), TLR (Toll-like receptor), RLR (RIG-like receptor), NLR (NOD-like receptor), CLR (C-type lectins), SR (Scavenger receptor), ROS (reactive oxygen species), LCMV (lymphocytic choriomeningitis virus), MCMV (murine cytomegalovirus), MHV (mouse hepatitis virus), IFN (Interferon), HCC (hepatocellular carcinoma), HBcAg (Hepatitis B core antigen), HBsAg (Hepatitis B surface antigen), HBeAg (Hepatitis B early antigen), HSPG (Heparan sulfate proteoglycan), MR (Mannose receptor), EGFR (Epidermal growth factor receptor), LDL (Low-Density Lipoprotein), ISG (Interferon-stimulated gene)

      Keywords

      The characteristics of Kupffer cells

      Kupffer cells (KC) are tissue-resident macrophages residing in the liver. They are located in the liver sinusoids, and are the largest population of innate immune cells in the liver [
      • Crispe I.N.
      The liver as a lymphoid organ.
      ,
      • Parker G.A.
      • Picut C.A.
      Liver immunobiology.
      ,
      • Jenne C.N.
      • Kubes P.
      Immune surveillance by the liver.
      ]. Due to their abundance and localization, KC are crucial cellular components of the intrahepatic innate immune system that are specialized to perform scavenger and phagocytic functions, thereby removing protein complexes, small particles, and apoptotic cells from blood [
      • Crispe I.N.
      The liver as a lymphoid organ.
      ,
      • Parker G.A.
      • Picut C.A.
      Liver immunobiology.
      ,
      • Jenne C.N.
      • Kubes P.
      Immune surveillance by the liver.
      ]. Together with the sinusoidal endothelial cells, KC are the first barrier for pathogens to enter the liver via the portal vein [
      • Vollmar B.
      • Menger M.D.
      The hepatic microcirculation: mechanistic contributions and therapeutic targets in liver injury and repair.
      ]. This is extremely important, since venous portal blood is rich in pathogen-derived products, such as lipopolysaccharide, and pathogens from the gut, which need to be eliminated from the circulation to avoid systemic immune activation.
      The specialized function of KC is reflected by the phenotype: they were identified in the early 1970s as peroxidase-positive cells with cytoplasm containing numerous granules and vacuoles, and occasional tubular, vermiform invaginations [
      • Fahimi H.D.
      The fine structural localization of endogenous and exogenous peroxidase activity in Kupffer cells of rat liver.
      ,
      • Klockars M.
      • Reitamo S.
      Tissue distribution of lysozyme in man.
      ,
      • Crofton R.W.
      • Diesselhoff-den Dulk M.M.
      • van Furth R.
      The origin, kinetics, and characteristics of the Kupffer cells in the normal steady state.
      ,
      • Widmann J.J.
      • Cotran R.S.
      • Fahimi H.D.
      Mononuclear phagocytes (Kupffer cells) and endothelial cells. Identification of two functional cell types in rat liver sinusoids by endogenous peroxidase activity.
      ]. At present, human KC are identified by immunohistochemistry or flow cytometry using antibodies directed against CD68, CD14, and CD16 [
      • Tu Z.
      • Bozorgzadeh A.
      • Pierce R.H.
      • Kurtis J.
      • Crispe I.N.
      • Orloff M.S.
      TLR-dependent cross talk between human Kupffer cells and NK cells.
      ,
      • Brown K.E.
      • Brunt E.M.
      • Heinecke J.W.
      Immunohistochemical detection of myeloperoxidase and its oxidation products in Kupffer cells of human liver.
      ,
      • Baldus S.E.
      • Zirbes T.K.
      • Weidner I.C.
      • Flucke U.
      • Dittmar E.
      • Thiele J.
      • et al.
      Comparative quantitative analysis of macrophage populations defined by CD68 and carbohydrate antigens in normal and pathologically altered human liver tissue.
      ]. However, it is important to mention that these markers are not unique for human KC and macrophages from other tissues, but are also expressed on monocytes, which are also considered a source of precursor cells for KC, and/or dendritic cells [
      • Gregori S.
      • Tomasoni D.
      • Pacciani V.
      • Scirpoli M.
      • Battaglia M.
      • Magnani C.F.
      • et al.
      Differentiation of type 1 T regulatory cells (Tr1) by tolerogenic DC-10 requires the IL-10-dependent ILT4/HLA-G pathway.
      ]. Different from their human counterpart, rat KC are commonly identified by antibodies against CD68 or CD163 (ED1 and ED2, respectively) [
      • Dijkstra C.D.
      • Dopp E.A.
      • Joling P.
      • Kraal G.
      The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2, and ED3.
      ], and mouse KC using the F4/80 marker [
      • Austyn J.M.
      • Gordon S.
      F4/80, a monoclonal antibody directed specifically against the mouse macrophage.
      ]. However, also the rat and mouse markers are not unique for KC, but are shared with other leukocytes.
      The ambiguity in the identification of KC that exists under steady state conditions is even more challenging under pathological conditions, in which cellular infiltrates are observed consisting of inflammatory monocytes and/or dendritic cells that share certain surface markers. In rat studies, large and small KC were shown to be present in a distinct area within the liver, i.e., in the peri-portal, and peri-venous and mid-zonal area, respectively [
      • Brown K.E.
      • Brunt E.M.
      • Heinecke J.W.
      Immunohistochemical detection of myeloperoxidase and its oxidation products in Kupffer cells of human liver.
      ,
      • Bouwens L.
      • Baekeland M.
      • de Zanger R.
      • Wisse E.
      Quantitation, tissue distribution and proliferation kinetics of Kupffer cells in normal rat liver.
      ,
      • Armbrust T.
      • Ramadori G.
      Functional characterization of two different Kupffer cell populations of normal rat liver.
      ,
      • Kono H.
      • Fujii H.
      • Asakawa M.
      • Yamamoto M.
      • Maki A.
      • Matsuda M.
      • et al.
      Functional heterogeneity of the Kupffer cell population is involved in the mechanism of gadolinium chloride in rats administered endotoxin.
      ,
      • Sleyster E.C.
      • Knook D.L.
      Relation between localization and function of rat liver Kupffer cells.
      ,
      • Dixon J.
      • Allan J.
      • Doherty P.
      • Hume D.
      Immunohistochemical analysis of the involvement of F4/80 and Ia-positive macrophages in mouse liver infected with lymphocytic choriomeningitis virus.
      ], and 2 subpopulations of KC have been isolated from rat liver tissue: ED1+ED2 and ED1+ED2+ cells [
      • Armbrust T.
      • Ramadori G.
      Functional characterization of two different Kupffer cell populations of normal rat liver.
      ,
      • Kono H.
      • Fujii H.
      • Asakawa M.
      • Yamamoto M.
      • Maki A.
      • Matsuda M.
      • et al.
      Functional heterogeneity of the Kupffer cell population is involved in the mechanism of gadolinium chloride in rats administered endotoxin.
      ]. Similarly, some studies have identified 2 subpopulations of mouse KC: F4/80+CD68+ and F4/80+CD11b+ cells from mouse liver tissue [
      • Kinoshita M.
      • Uchida T.
      • Sato A.
      • Nakashima M.
      • Nakashima H.
      • Shono S.
      • et al.
      Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice.
      ]. It is likely that these populations either illustrate distinct differentiation phases rather than distinct KC subpopulations, or that they identify infiltrating monocytes instead of resident tissue macrophages. In studies from our group, we defined only one KC population in mouse liver tissue on the basis of F4/80 and CD11b expression [
      • Movita D.
      • Kreefft K.
      • Biesta P.
      • van Oudenaren A.
      • Leenen P.J.
      • Janssen H.L.
      • et al.
      Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages.
      ]. This was in line with a study in humans where only a single population of KC was identified as CD14+, HLA-DR+, HLA-ABC+, CD86+, and DC-SIGN+ cells, with low expression of CD1b, CD40, and CD83 [
      • Tu Z.
      • Bozorgzadeh A.
      • Pierce R.H.
      • Kurtis J.
      • Crispe I.N.
      • Orloff M.S.
      TLR-dependent cross talk between human Kupffer cells and NK cells.
      ]. It is preferable to identify KC not solely based on the available markers, but also on their morphology and phagocytic ability as their hallmark function. In this review, KC are identified as CD68+, CD14+, and/or CD11b+ cells (human), ED1+ and/or ED2+ cells (rat) and CD68+, F4/80+ and/or CD11b+ cells (mouse), according to the original studies. Under steady state condition, the majority of tissue-resident macrophages in the mouse liver have a yolk sac origin and are self-maintained. Upon serious challenge, tissue resident KC can be replaced by precursor cells from bone marrow as well as monocytes, which develop into tissue-resident macrophages [
      • Wynn T.A.
      • Chawla A.
      • Pollard J.W.
      Macrophage biology in development, homeostasis and disease.
      ]. Since the distinction between tissue-resident KC and tissue-infiltrating monocyte/macrophages is difficult, and since most studies did not discriminate between these cells with a different origin, we will use the term “KC” to describe both cells.
      Studies on human KC are being performed using cells obtained from liver tissue or from liver graft perfusate. Liver graft perfusate is preserved in a different manner than liver tissue. Also, tissue-derived KC are commonly isolated using collagenase, a processing step not included for perfusate, which increases the amount of extracellular debris and may induce phenotypic and functional changes. The source of liver material as well as the method to process the samples are important to take into account when interpreting results on the phenotype and function of KC from the various studies.
      Macrophages are specialized in sensing and responding to pathogens and equipped with specific pattern recognition receptors, including scavenger receptors, Toll-like receptors (TLR), RIG-like receptors (RLR), NOD-like receptors (NLR) and C-type lectins. These receptors are expressed by tissue-derived as well as in vitro-generated macrophages (reviewed in [
      • Taylor P.R.
      • Martinez-Pomares L.
      • Stacey M.
      • Lin H.H.
      • Brown G.D.
      • Gordon S.
      Macrophage receptors and immune recognition.
      ]). However, only few of them have been described for KC and it is not clear whether the others are expressed by KC. Scavenger receptors and C-type lectins are important receptors mediating phagocytosis, which are expressed by human, rat, and mice KC [
      • Dominguez-Soto A.
      • Aragoneses-Fenoll L.
      • Gomez-Aguado F.
      • Corcuera M.T.
      • Claria J.
      • Garcia-Monzon C.
      • et al.
      The pathogen receptor liver and lymph node sinusoidal endotelial cell C-type lectin is expressed in human Kupffer cells and regulated by PU1.
      ,
      • Imaizumi T.
      • Sashinami H.
      • Mori F.
      • Matsumiya T.
      • Yoshi-Da H.
      • Nakane A.
      • et al.
      Listeria monocytogenes induces the expression of retinoic acid-inducible gene-I.
      ,
      • Reid D.M.
      • Montoya M.
      • Taylor P.R.
      • Borrow P.
      • Gordon S.
      • Brown G.D.
      • et al.
      Expression of the beta-glucan receptor, Dectin-1, on murine leukocytes in situ correlates with its function in pathogen recognition and reveals potential roles in leukocyte interactions.
      ]. The phagocytic ability of human KC has been shown in relation to removal of erythrocytes, apoptotic cells, and debris [
      • Clavien P.A.
      • Camargo Jr., C.A.
      • Cameron R.
      • Washington M.K.
      • Phillips M.J.
      • Greig P.D.
      • et al.
      Kupffer cell erythrophagocytosis and graft-vs.-host hemolysis in liver transplantation.
      ,
      • Dini L.
      • Pagliara P.
      • Carla E.C.
      Phagocytosis of apoptotic cells by liver: a morphological study.
      ]. In line with that notion, we and others have shown that rat and mouse KC are strongly phagocytic and possess a high level of basal reactive oxygen species (ROS) production [
      • Kinoshita M.
      • Uchida T.
      • Sato A.
      • Nakashima M.
      • Nakashima H.
      • Shono S.
      • et al.
      Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice.
      ,
      • Movita D.
      • Kreefft K.
      • Biesta P.
      • van Oudenaren A.
      • Leenen P.J.
      • Janssen H.L.
      • et al.
      Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages.
      ]. Upon in vivo administration of dextran particles, E. coli or gadolinium chloride, rat and mouse KC take up these particles, produce high levels of ROS, and demonstrate high lysosomal activity [
      • Kono H.
      • Fujii H.
      • Asakawa M.
      • Yamamoto M.
      • Maki A.
      • Matsuda M.
      • et al.
      Functional heterogeneity of the Kupffer cell population is involved in the mechanism of gadolinium chloride in rats administered endotoxin.
      ,
      • Sleyster E.C.
      • Knook D.L.
      Relation between localization and function of rat liver Kupffer cells.
      ,
      • Kinoshita M.
      • Uchida T.
      • Sato A.
      • Nakashima M.
      • Nakashima H.
      • Shono S.
      • et al.
      Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice.
      ,
      • Movita D.
      • Kreefft K.
      • Biesta P.
      • van Oudenaren A.
      • Leenen P.J.
      • Janssen H.L.
      • et al.
      Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages.
      ]. Human KC were shown to express TLR2, TLR3, and TLR4 [
      • Tu Z.
      • Bozorgzadeh A.
      • Pierce R.H.
      • Kurtis J.
      • Crispe I.N.
      • Orloff M.S.
      TLR-dependent cross talk between human Kupffer cells and NK cells.
      ,
      • Visvanathan K.
      • Skinner N.A.
      • Thompson A.J.V.
      • Riordan S.M.
      • Sozzi V.
      • Edwards R.
      • et al.
      Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.
      ]. The expression of other TLR, as well as NLR and RLR have not been described, but cannot be excluded since the murine counterparts were found to express functional TLR1-TLR9 and RIG-I [
      • Imaizumi T.
      • Sashinami H.
      • Mori F.
      • Matsumiya T.
      • Yoshi-Da H.
      • Nakane A.
      • et al.
      Listeria monocytogenes induces the expression of retinoic acid-inducible gene-I.
      ,
      • Chen J.
      • Wang X.M.
      • Wu X.J.
      • Wang Y.
      • Zhao H.
      • Shen B.
      • et al.
      Intrahepatic levels of PD-1/PD-L correlate with liver inflammation in chronic hepatitis B.
      ]. In human and rodents, ligation of TLR on tissue-derived and in vitro-generated macrophages resulted in cytokine production [
      • Murray P.J.
      • Wynn T.A.
      Obstacles and opportunities for understanding macrophage polarization.
      ]. However, to date, studies on the ability of KC to produce cytokines upon TLR ligation resulted in divergent conclusions. For instance, we and others show that KC from human liver tissue and perfusate release IL-10, IL-1β, IL-6, IL-12, IL-18, and TNF upon TLR2, TLR3, and TLR4 ligation ex vivo [
      • Tu Z.
      • Bozorgzadeh A.
      • Pierce R.H.
      • Kurtis J.
      • Crispe I.N.
      • Orloff M.S.
      TLR-dependent cross talk between human Kupffer cells and NK cells.
      ,
      • Knolle P.
      • Schlaak J.
      • Uhrig A.
      • Kempf P.
      • zum Büschenfelde K.-H.M.
      • Gerken G.
      Human Kupffer cells secrete IL-10 in response to lipopolysaccharide (LPS) challenge.
      ,
      • Fletcher N.F.
      • Sutaria R.
      • Jo J.
      • Barnes A.
      • Blahova M.
      • Meredith L.W.
      • et al.
      Activated macrophages promote hepatitis C virus entry in a tumor necrosis factor-dependent manner.
      ] and [Boltjes, unpublished data]. Similarly, Kono et al. showed that liver tissue-derived rat KC produce superoxide, TNF, and IL-6 upon TLR4 ligation ex vivo [
      • Kono H.
      • Fujii H.
      • Asakawa M.
      • Yamamoto M.
      • Maki A.
      • Matsuda M.
      • et al.
      Functional heterogeneity of the Kupffer cell population is involved in the mechanism of gadolinium chloride in rats administered endotoxin.
      ]. However, examination of mouse KC isolated from liver tissue by our group and others demonstrated weak induction of TNF and IL-12p40 upon ex vivo stimulation with agonist for TLR4, TLR7/8, or TLR9 [
      • Kinoshita M.
      • Uchida T.
      • Sato A.
      • Nakashima M.
      • Nakashima H.
      • Shono S.
      • et al.
      Characterization of two F4/80-positive Kupffer cell subsets by their function and phenotype in mice.
      ,
      • Movita D.
      • Kreefft K.
      • Biesta P.
      • van Oudenaren A.
      • Leenen P.J.
      • Janssen H.L.
      • et al.
      Kupffer cells express a unique combination of phenotypic and functional characteristics compared with splenic and peritoneal macrophages.
      ], whereas no data are available on the cytokine-producing ability of liver perfusate-derived rat or murine KC. Thus, more studies using highly purified KC with a well-defined phenotype need to be conducted to obtain conclusive data on the TLR responsiveness of KC.
      Figure thumbnail fx2

      The role of KC during LCMV infections

      Besides their barrier [
      • Vollmar B.
      • Menger M.D.
      The hepatic microcirculation: mechanistic contributions and therapeutic targets in liver injury and repair.
      ] and janitor function [
      • Bellone M.
      • Iezzi G.
      • Rovere P.
      • Galati G.
      • Ronchetti A.
      • Protti M.P.
      • et al.
      Processing of engulfed apoptotic bodies yields T cell epitopes.
      ,
      • Sitia G.
      • Iannacone M.
      • Aiolfi R.
      • Isogawa M.
      • van Rooijen N.
      • Scozzesi C.
      • et al.
      Kupffer cells hasten resolution of liver immunopathology in mouse models of viral hepatitis.
      ], KC have been shown to play a role in the response to pathogens, including viruses. Studies on the importance and anti-viral immune functions of KC in HBV and HCV infections are difficult to perform, since these viruses only infect and replicate in humans and non-human primates, and immunocompetent small animal models for viral hepatitis are not yet available (reviewed in [
      • Chayama K.
      • Hayes C.N.
      • Hiraga N.
      • Abe H.
      • Tsuge M.
      • Imamura M.
      Animal model for study of human hepatitis viruses.
      ,
      • Boonstra A.
      • van der Laan L.J.
      • Vanwolleghem T.
      • Janssen H.L.
      Experimental models for hepatitis C viral infection.
      ]). As an alternative approach several mouse infection models, including lymphocytic choriomeningitis virus (LCMV), murine cytomegalovirus (MCMV), mouse hepatitis virus (MHV) and adenovirus models, have provided information on the role of KC in viral infection. However, in contrast to HBV and HCV where infection and replication is restricted to hepatocytes, these hepatitis mouse models also infect other cells and even other organs. Of these models, MHV and LCMV have been shown to replicate in KC [
      • Keller F.
      • Schmitt C.
      • Kirn A.
      Interaction of mouse hepatitis virus 3 with Kupffer cells explanted from susceptible and resistant mouse strains. Antiviral activity, interleukin-1 synthesis.
      ,
      • Matloubian M.
      • Kolhekar S.R.
      • Somasundaram T.
      • Ahmed R.
      Molecular determinants of macrophage tropism and viral persistence: importance of single amino acid changes in the polymerase and glycoprotein of lymphocytic choriomeningitis virus.
      ]. LCMV, MHV, and adenovirus particles can be taken up from the circulation by murine KC via scavenger and complement receptors, which may limit infection [
      • Lang P.A.
      • Recher M.
      • Honke N.
      • Scheu S.
      • Borkens S.
      • Gailus N.
      • et al.
      Tissue macrophages suppress viral replication and prevent severe immunopathology in an interferon-I-dependent manner in mice.
      ,
      • Pereira C.A.
      • Steffan A.M.
      • Kirn A.
      Kupffer and endothelial liver cell damage renders A/J mice susceptible to mouse hepatitis virus type 3.
      ,
      • Smith J.S.
      • Xu Z.
      • Tian J.
      • Stevenson S.C.
      • Byrnes A.P.
      Interaction of systemically delivered adenovirus vectors with Kupffer cells in mouse liver.
      ,
      • Xu Z.
      • Tian J.
      • Smith J.S.
      • Byrnes A.P.
      Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement.
      ]. It has been shown that failure in clearing LCMV, MHV, and adenovirus particles during the acute phase results in “spill-over” infection of hepatocytes, prolonged infection, and exacerbated immunopathology [
      • Xu Z.
      • Tian J.
      • Smith J.S.
      • Byrnes A.P.
      Clearance of adenovirus by Kupffer cells is mediated by scavenger receptors, natural antibodies, and complement.
      ,
      • He J.Q.
      • Katschke Jr., K.J.
      • Gribling P.
      • Suto E.
      • Lee W.P.
      • Diehl L.
      • et al.
      CRIg mediates early Kupffer cell responses to adenovirus.
      ,
      • Bloch E.H.
      • Warren K.S.
      • Rosenthal M.S.
      In vivo microscopic observations of the pathogenesis of acute mouse viral hepatitis.
      ]. Studies using these mouse models have been instrumental in our understanding of the effects on KC during the early phases of virus infections. A number of studies have also evaluated KC during persistent infection in mice. These studies are conducted using specific isolates of LCMV, the clone 13 and WE strains. The development of persistent infection with a high rate of replication of LCMV is similar to HBV and HCV, and important mechanistic pathways identified in LCMV infected mice, were later confirmed to be operational during chronic viral infections in patients. However, in contrast to HBV and HCV, murine LCMV infections are not restricted to the liver, and LCMV replication can also be found in the spleen, lung, and kidney. The long-term consequences of human viral hepatitis, such as fibrosis, are absent in mice, although virus-induced liver damage is observed [
      • Lang P.A.
      • Recher M.
      • Honke N.
      • Scheu S.
      • Borkens S.
      • Gailus N.
      • et al.
      Tissue macrophages suppress viral replication and prevent severe immunopathology in an interferon-I-dependent manner in mice.
      ,
      • Oldstone M.B.
      Viral persistence: parameters, mechanisms and future predictions.
      ]. The effect of chronic LCMV infection on NK cells and virus-specific T cells has been extensively examined, however only few studies have focussed on KC. In contrast to HBV or HCV, active replication of LCMV in the liver, as evidenced by the detection of viral RNA and antigen, has been demonstrated in KC as well as in hepatocytes [
      • Matloubian M.
      • Kolhekar S.R.
      • Somasundaram T.
      • Ahmed R.
      Molecular determinants of macrophage tropism and viral persistence: importance of single amino acid changes in the polymerase and glycoprotein of lymphocytic choriomeningitis virus.
      ,
      • Lohler J.
      • Gossmann J.
      • Kratzberg T.
      • Lehmann-Grube F.
      Murine hepatitis caused by lymphocytic choriomeningitis virus I. The hepatic lesions.
      ,
      • Bergthaler A.
      • Merkler D.
      • Horvath E.
      • Bestmann L.
      • Pinschewer D.D.
      Contributions of the lymphocytic choriomeningitis virus glycoprotein and polymerase to strain-specific differences in murine liver pathogenicity.
      ]. During the first 2 weeks following LCMV infection, an increase of the number of F4/80+ cells is observed, followed by normalization of their numbers [
      • Dixon J.
      • Allan J.
      • Doherty P.
      • Hume D.
      Immunohistochemical analysis of the involvement of F4/80 and Ia-positive macrophages in mouse liver infected with lymphocytic choriomeningitis virus.
      ]. Although differences in MHC class-I expression levels were observed within the F4/80 population by immunohistochemistry, the relative contribution of infiltrating monocytes vs. enhanced activation of resident KC is difficult to determine.
      An elegant study by Lang et al. showed that clodronate-mediated depletion of KC resulted in rapid LCMV dissemination due to the inability to capture virus, which led to replication within hepatocytes and subsequently severe CD8+ T cell-mediated liver damage [
      • Lang P.A.
      • Recher M.
      • Honke N.
      • Scheu S.
      • Borkens S.
      • Gailus N.
      • et al.
      Tissue macrophages suppress viral replication and prevent severe immunopathology in an interferon-I-dependent manner in mice.
      ]. The study further showed that KC responded to type I IFN by inducing the expression of interferon-stimulated genes, and that mice lacking IFNAR specifically on macrophages exhibited strongly enhanced viral titers. However, recently a detrimental influence of granulocytes and macrophages in spleen and liver was reported by their ability to produce reactive oxygen species (ROS) following viral infection, although ROS production by liver F4/80+ cells was low [
      • Lang P.A.
      • Xu H.C.
      • Grusdat M.
      • McIlwain D.R.
      • Pandyra A.A.
      • Harris I.S.
      • et al.
      Reactive oxygen species delay control of lymphocytic choriomeningitis virus.
      ]. Importantly, the effect of ROS was an impairment of the immune response, and in the absence of ROS mice exhibited lower viral titers and less liver damage. In a different experimental mouse model, which makes use of transgenic intrahepatic expression of the HBV large envelope protein, ROS activity was observed in KC, and these mice exhibited a chronic necroinflammatory liver disease, resembling human chronic active hepatitis [
      • Hagen T.M.
      • Huang S.
      • Curnutte J.
      • Fowler P.
      • Martinez V.
      • Wehr C.M.
      • et al.
      Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma.
      ].
      The findings from the LCMV mouse model clearly show the complexity of the anti-viral response in the liver since KC can both contribute to promote and suppress viral eradication and liver pathology. In the following section, we will focus on the interaction of KC with HBV and HCV, and the functional consequences.

      The role of KC during HBV and HCV infections

      Both HBV and HCV are transmitted predominantly via percutaneous and sexual exposure, while perinatal exposure is often seen for HBV only [
      • Margolis H.S.
      • Alter M.J.
      • Hadler S.C.
      Hepatitis B evolving epidemiology and implications for control.
      ,
      • Lavanchy D.
      Evolving epidemiology of hepatitis C virus.
      ,
      • Alter M.J.
      Epidemiology of hepatitis C.
      ]. Infection with these viruses can either resolve spontaneously or develop into chronic liver disease with continuous viral replication in hepatocytes [
      • Lavanchy D.
      Evolving epidemiology of hepatitis C virus.
      ,
      • Alter M.J.
      Epidemiology of hepatitis C.
      ,
      • Ganem D.
      • Prince A.M.
      Hepatitis B virus infection–natural history and clinical consequences.
      ]. Chronic hepatitis poses an increased risk for liver fibrosis and cirrhosis, hepatic failure, and hepatocellular carcinoma (HCC) [
      • Ganem D.
      • Prince A.M.
      Hepatitis B virus infection–natural history and clinical consequences.
      ,
      • Lauer G.M.
      • Walker B.D.
      Hepatitis C virus infection.
      ]. Patients with a self-limiting HBV or HCV infection show sustained, vigorous, and multi-epitope-specific CD4+ or CD8+ T cell and B cell responses, whereas in chronic HBV and HCV these responses are weak and/or transient [
      • Thimme R.
      • Oldach D.
      • Chang K.M.
      • Steiger C.
      • Ray S.C.
      • Chisari F.V.
      Determinants of viral clearance and persistence during acute hepatitis C virus infection.
      ,
      • Day C.L.
      • Lauer G.M.
      • Robbins G.K.
      • McGovern B.
      • Wurcel A.G.
      • Gandhi R.T.
      • et al.
      Broad specificity of virus-specific CD4+ T-helper-cell responses in resolved hepatitis C virus infection.
      ,
      • Lauer G.M.
      • Ouchi K.
      • Chung R.T.
      • Nguyen T.N.
      • Day C.L.
      • Purkis D.R.
      • et al.
      Comprehensive analysis of CD8(+)-T-cell responses against hepatitis C virus reveals multiple unpredicted specificities.
      ,
      • Rehermann B.
      • Nascimbeni M.
      Immunology of hepatitis B virus and hepatitis C virus infection.
      ]. This demonstrates that clearance of the infection is dependent on strong multi-epitope-specific T and B cell responses, which is only possible following effective innate immune responses [
      • Rehermann B.
      • Nascimbeni M.
      Immunology of hepatitis B virus and hepatitis C virus infection.
      ,
      • Boonstra A.
      • Woltman A.M.
      • Janssen H.L.
      Immunology of hepatitis B and hepatitis C virus infections.
      ]. Here, we will firstly address the role of KC in the interaction and recognition of HBV and HCV, and their role in the induction of a pro-inflammatory response. Pro-inflammatory mediators are important for inhibition of viral replication, the induction of resistance to infection of neighboring cells, and attraction and activation of other immune cells, and consequently contribute to the development of effective virus-specific immunity. Secondly, we will discuss KC-virus interactions that may inhibit the development of effective viral immunity, facilitate viral persistence or promote liver damage.

      Interaction of KC with HBV and HCV

      HBV is a 3.2 kb partially double-stranded DNA envelope-virus which replicates via RNA intermediates. Hepatitis B core protein (HBcAg)-encapsulated viral DNA and hepatitis B envelope protein (HBsAg) form a complete viral or Dane particle. HBV particles, HBsAg, and hepatitis B early antigen (HBeAg; a truncated form of HBcAg) are secreted by infected hepatocytes and can be detected in serum of HBV patients [
      • Ganem D.
      • Prince A.M.
      Hepatitis B virus infection–natural history and clinical consequences.
      ,
      • Locarnini S.
      Molecular virology of hepatitis B virus.
      ].
      Evidence for productive HBV infection of cells other than hepatocytes is lacking. Also, detailed information on the presence of HBV (proteins) in KC in vivo or the uptake of HBV or its proteins by human KC ex vivo has not been reported. Although no information is available on KC, studies using THP-1 monocytic cells, monocytes, and dendritic cells have shown binding of HBV or HBV proteins, leading to their activation. For instance, TLR2 and heparan sulfate proteoglycan (HSPG) were suggested to be responsible for HBcAg recognition on THP-1 cells, and HBcAg-induced activation of THP-1 cells resulted in production of IL-6, IL-12p40, and TNF [
      • Cooper A.
      • Tal G.
      • Lider O.
      • Shaul Y.
      Cytokine induction by the hepatitis B virus capsid in macrophages is facilitated by membrane heparan sulfate and involves TLR2.
      ]. However, since HBcAg is only found within infected hepatocytes or viral particles, it is unclear whether HBcAg interacts with KC, via HSPG and/or another extracellular receptor like TLR2. Also, other receptors expressed by KC are known to interact with HBV proteins as demonstrated in other cell-systems (Table 1). For instance, HBsAg can interact with human blood monocytes in a CD14-dependent fashion [
      • Vanlandschoot P.
      • Van Houtte F.
      • Roobrouck A.
      • Farhoudi A.
      • Stelter F.
      • Peterson D.L.
      • et al.
      LPS-binding protein and CD14-dependent attachment of hepatitis B surface antigen to monocytes is determined by the phospholipid moiety of the particles.
      ], and with dendritic cells via the mannose receptor [
      • Op den Brouw M.L.
      • Binda R.S.
      • Geijtenbeek T.B.
      • Janssen H.L.
      • Woltman A.M.
      The mannose receptor acts as hepatitis B virus surface antigen receptor mediating interaction with intrahepatic dendritic cells.
      ], which are both receptors known to be also expressed on KC [
      • Ono K.
      • Nishitani C.
      • Mitsuzawa H.
      • Shimizu T.
      • Sano H.
      • Suzuki H.
      • et al.
      Mannose-binding lectin augments the uptake of lipid A, Staphylococcus aureus, and Escherichia coli by Kupffer cells through increased cell surface expression of scavenger receptor A.
      ]. Finally, complex formation of HBsAg with albumin may lead to enhanced uptake of HBsAg from the circulation by KC and endothelial cells [
      • Wright T.L.
      • Roll F.J.
      • Jones A.L.
      • Weisiger R.A.
      Uptake and metabolism of polymerized albumin by rat liver. Role of the scavenger receptor.
      ].
      Table 1Surface molecules and secreted inflammatory mediators facilitating KC roles in HBV/HCV infection.
      HCV contains a 9.6 kb positive-strand RNA genome that translates into the structural proteins, core, and E1 and E2 envelope proteins, and the non-structural proteins NS1–NS5. After replication, they form a small-enveloped virus particle containing the newly synthesized RNA genome [
      • Ashfaq U.A.
      • Javed T.
      • Rehman S.
      • Nawaz Z.
      • Riazuddin S.
      An overview of HCV molecular biology, replication and immune responses.
      ,
      • Brass V.
      • Moradpour D.
      • Blum H.E.
      Molecular virology of hepatitis C virus (HCV): 2006 update.
      ].
      Compared to HBV, there is a better understanding of the entry receptors on hepatocytes used by HCV. In addition to claudin1, occludin, epidermal growth factor receptor (EGFR), and ephrin type-A receptor-2, HCV infects hepatocytes by attaching to HSPG, low-density lipoprotein (LDL) receptor, scavenger receptor (SR)-B1 and CD81. Some, but not all, receptors are expressed by KC (Table 1) [
      • Lupberger J.
      • Zeisel M.B.
      • Xiao F.
      • Thumann C.
      • Fofana I.
      • Zona L.
      • et al.
      EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy.
      ,
      • Ploss A.
      • Evans M.J.
      • Gaysinskaya V.A.
      • Panis M.
      • You H.
      • de Jong Y.P.
      • et al.
      Human occludin is a hepatitis C virus entry factor required for infection of mouse cells.
      ,
      • Evans M.J.
      • von Hahn T.
      • Tscherne D.M.
      • Syder A.J.
      • Panis M.
      • Wolk B.
      • et al.
      Claudin-1 is a hepatitis C virus co-receptor required for a late step in entry.
      ,
      • Scarselli E.
      • Ansuini H.
      • Cerino R.
      • Roccasecca R.M.
      • Acali S.
      • Filocamo G.
      • et al.
      The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus.
      ,
      • Agnello V.
      • Abel G.
      • Elfahal M.
      • Knight G.B.
      • Zhang Q.X.
      Hepatitis C virus and other Flaviviridae viruses enter cells via low density lipoprotein receptor.
      ,
      • Pileri P.
      • Uematsu Y.
      • Campagnoli S.
      • Galli G.
      • Falugi F.
      • Petracca R.
      • et al.
      Binding of hepatitis C virus to CD81.
      ,
      • Laskin J.D.
      • Dokidis A.
      • Gardner C.R.
      • Laskin D.L.
      Changes in sulfated proteoglycan production after activation of rat liver macrophages.
      ,
      • Kleinherenbrink-Stins M.F.
      • van de Boom J.H.
      • Schouten D.
      • Roholl P.J.
      • van der Heyde M.N.
      • Brouwer A.
      • et al.
      Visualization of the interaction of native and modified lipoproteins with parenchymal, endothelial and Kupffer cells from human liver.
      ,
      • Kamps J.A.
      • Kruijt J.K.
      • Kuiper J.
      • Van Berkel T.J.
      Uptake and degradation of human low-density lipoprotein by human liver parenchymal and Kupffer cells in culture.
      ,
      • Terpstra V.
      • van Berkel T.J.
      Scavenger receptors on liver Kupffer cells mediate the in vivo uptake of oxidatively damaged red blood cells in mice.
      ]. It has been reported that incubation of human liver cells with HCV-E2 resulted in HCV-E2 binding to KC in a CD81-dependent manner [
      • Petracca R.
      • Falugi F.
      • Galli G.
      • Norais N.
      • Rosa D.
      • Campagnoli S.
      • et al.
      Structure-function analysis of hepatitis C virus envelope-CD81 binding.
      ], but also DC-SIGN, a C-type lectin not expressed by hepatocytes, has been demonstrated to bind HCV on KC [
      • Tu Z.
      • Pierce R.H.
      • Kurtis J.
      • Kuroki Y.
      • Crispe I.N.
      • Orloff M.S.
      Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.
      ,
      • Lai W.K.
      • Sun P.J.
      • Zhang J.
      • Jennings A.
      • Lalor P.F.
      • Hubscher S.
      • et al.
      Expression of DC-SIGN and DC-SIGNR on human sinusoidal endothelium: a role for capturing hepatitis C virus particles.
      ,
      • Pohlmann S.
      • Zhang J.
      • Baribaud F.
      • Chen Z.
      • Leslie G.J.
      • Lin G.
      • et al.
      Hepatitis C virus glycoproteins interact with DC-SIGN and DC-SIGNR.
      ].
      Although it is unlikely that HCV can replicate in KC, activation of KC by HCV and its proteins has been demonstrated. HCV core and NS3 stimulate human liver perfusate-derived CD14+ KC and monocyte-derived macrophages via TLR2 to produce pro-inflammatory IL-1β, IL-6, and TNF and immunosuppressive IL-10 [
      • Tu Z.
      • Pierce R.H.
      • Kurtis J.
      • Kuroki Y.
      • Crispe I.N.
      • Orloff M.S.
      Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.
      ,
      • Chang S.
      • Dolganiuc A.
      • Szabo G.
      Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins.
      ]. Recently, it was shown that TLR4, in density gradient- and adherence-isolated liver-derived human KC, mediates NS3 recognition, resulting in TNF production [
      • Hosomura N.
      • Kono H.
      • Tsuchiya M.
      • Ishii K.
      • Ogiku M.
      • Matsuda M.
      • et al.
      HCV-related proteins activate Kupffer cells isolated from human liver tissues.
      ]. However, HCV core and NS3 are not secreted at significant levels by infected hepatocytes, posing little relevance to extracellular recognition of HCV by KC via these TLR. Alternatively, phagocytosis of infected hepatocytes by KC may allow intracellular exposure to viral RNA, but so far no evidence exists.

      Stimulatory effects of HBV or HCV on KC function

      There are only few publications that show a stimulatory effect of HBV or HBV proteins on the function of KC. Hösel et al. showed that HBV particles and HBsAg induce IL-1β, IL-6, CXCL8, and TNF production by human CD68+ cell-enriched non-parenchymal cells via NF-кB activation [
      • Hösel M.
      • Quasdorff M.
      • Wiegmann K.
      • Webb D.
      • Zedler U.
      • Broxtermann M.
      • et al.
      Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection.
      ] and subsequently inhibit HBV replication in primary hepatocytes. This inhibitory effect was mainly ascribed to IL-6, but also TNF inhibited HBV replication in a non-cytopathic manner [
      • Phillips S.
      • Chokshi S.
      • Riva A.
      • Evans A.
      • Williams R.
      • Naoumov N.V.
      CD8(+) T cell control of hepatitis B virus replication: direct comparison between cytolytic and noncytolytic functions.
      ]. In contrast, Li et al. demonstrated that rat ED1+ adherent KC exposed to HBV virions hardly expressed IL-1β, IL-6, or TNF, but produced the immunoregulatory cytokine TGFβ [
      • Li H.
      • Zheng H.W.
      • Chen H.
      • Xing Z.Z.
      • You H.
      • Cong M.
      • et al.
      Hepatitis B virus particles preferably induce Kupffer cells to produce TGF-beta1 over pro-inflammatory cytokines.
      ].
      During chronic HCV infection, KC are increased in numbers in the liver [
      • Khakoo S.I.
      • Soni P.N.
      • Savage K.
      • Brown D.
      • Dhillon A.P.
      • Poulter L.W.
      • et al.
      Lymphocyte and macrophage phenotypes in chronic hepatitis C infection. Correlation with disease activity.
      ,
      • McGuinness P.H.
      • Painter D.
      • Davies S.
      • McCaughan G.W.
      Increases in intrahepatic CD68 positive cells, MAC387 positive cells, and proinflammatory cytokines (particularly interleukin 18) in chronic hepatitis C infection.
      ], and exhibit an activated phenotype with higher mRNA expression levels of the activation markers CD163 and CD33 in livers of chronic HCV patients vs. controls [
      • Burgio V.L.
      • Ballardini G.
      • Artini M.
      • Caratozzolo M.
      • Bianchi F.B.
      • Levrero M.
      Expression of co-stimulatory molecules by kupffer cells in chronic hepatitis of hepatitis C virus etiology.
      ,
      • Dolganiuc A.
      • Norkina O.
      • Kodys K.
      • Catalano D.
      • Bakis G.
      • Marshall C.
      • et al.
      Viral and host factors induce macrophage activation and loss of toll-like receptor tolerance in chronic HCV infection.
      ]. Recently, it was reported that in response to HCV human KC release IL-1β and IL-18 in vitro [
      • Shrivastava S.
      • Mukherjee A.
      • Ray R.
      • Ray R.B.
      Hepatitis C virus induces IL-1beta/IL-18 in circulatory and resident liver macrophages.
      ]. In line with these findings, stimulation of CD14+CD68+ cells from liver perfusate with UV irradiated cell culture-derived HCV induced IL-1β production. To support this data, in vivo co-expression of IL-1β and CD68 was observed using immunofluorescence on liver tissues from patients with chronic HCV [
      • Negash A.A.
      • Ramos H.J.
      • Crochet N.
      • Lau D.T.
      • Doehle B.
      • Papic N.
      • et al.
      IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease.
      ]. Besides intrahepatic IL-1β, also elevated serum IL-1β levels were detected in patients as compared to healthy individuals [
      • Negash A.A.
      • Ramos H.J.
      • Crochet N.
      • Lau D.T.
      • Doehle B.
      • Papic N.
      • et al.
      IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease.
      ].
      Although a direct effect of HCV-exposed KC on HCV replication is unknown, it was recently reported that KC-derived TNF increased the permissivity of hepatoma cells to HCV. In this study, LPS as well as HCV induced KC to produce TNF, thereby indirectly promoting HCV infection [
      • Fletcher N.F.
      • Sutaria R.
      • Jo J.
      • Barnes A.
      • Blahova M.
      • Meredith L.W.
      • et al.
      Activated macrophages promote hepatitis C virus entry in a tumor necrosis factor-dependent manner.
      ]. On the other hand, HCV- or TLR-ligand-induced KC-derived cytokines, such as IL-6, IL-1β, and IFNβ [
      • Tu Z.
      • Pierce R.H.
      • Kurtis J.
      • Kuroki Y.
      • Crispe I.N.
      • Orloff M.S.
      Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.
      ,
      • Chang S.
      • Dolganiuc A.
      • Szabo G.
      Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins.
      ,
      • Negash A.A.
      • Ramos H.J.
      • Crochet N.
      • Lau D.T.
      • Doehle B.
      • Papic N.
      • et al.
      IL-1beta production through the NLRP3 inflammasome by hepatic macrophages links hepatitis C virus infection with liver inflammation and disease.
      ,
      • Broering R.
      • Wu J.
      • Meng Z.
      • Hilgard P.
      • Lu M.
      • Trippler M.
      • et al.
      Toll-like receptor-stimulated non-parenchymal liver cells can regulate hepatitis C virus replication.
      ], were found to inhibit HCV replication in the HCV replicon model [
      • Broering R.
      • Wu J.
      • Meng Z.
      • Hilgard P.
      • Lu M.
      • Trippler M.
      • et al.
      Toll-like receptor-stimulated non-parenchymal liver cells can regulate hepatitis C virus replication.
      ,
      • Zhu H.
      • Liu C.
      Interleukin-1 inhibits hepatitis C virus subgenomic RNA replication by activation of extracellular regulated kinase pathway.
      ,
      • Zhu H.
      • Shang X.
      • Terada N.
      • Liu C.
      STAT3 induces anti-hepatitis C viral activity in liver cells.
      ], implying that KC are also capable of displaying antiviral activity upon HCV exposure.
      In addition, release of chemokines and cytokines by KC has an indirect effect on the immune response in the liver by recruitment and activation of infiltrating leukocytes, as also discussed by Heydtmann et al. [
      • Heydtmann M.
      Macrophages in hepatitis B and hepatitis C virus infections.
      ]. This may result in a complex interaction between factors produced by liver parenchymal cells, liver resident immune cells including KC, and infiltrating leukocytes. KC are able to activate NK cells and NKT cells, both present at relatively high numbers in the liver, via the production of pro-inflammatory cytokines [
      • Tu Z.
      • Bozorgzadeh A.
      • Pierce R.H.
      • Kurtis J.
      • Crispe I.N.
      • Orloff M.S.
      TLR-dependent cross talk between human Kupffer cells and NK cells.
      ]. In turn, NK and NKT cells produce cytokines such as TNF and IFNγ and are cytotoxic in nature [
      • Tu Z.
      • Bozorgzadeh A.
      • Pierce R.H.
      • Kurtis J.
      • Crispe I.N.
      • Orloff M.S.
      TLR-dependent cross talk between human Kupffer cells and NK cells.
      ,
      • Dao T.
      • Mehal W.Z.
      • Crispe I.N.
      IL-18 augments perforin-dependent cytotoxicity of liver NK-T cells.
      ]. Upon HBV exposure, KC were found to produce CXCL8 [
      • Hösel M.
      • Quasdorff M.
      • Wiegmann K.
      • Webb D.
      • Zedler U.
      • Broxtermann M.
      • et al.
      Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection.
      ], which potentially attracts NK and NKT cells during the early phase of HBV infection. KC are also able to recruit dendritic cells to the liver, which involved C-type lectins interactions [
      • Uwatoku R.
      • Suematsu M.
      • Ezaki T.
      • Saiki T.
      • Tsuiji M.
      • Irimura T.
      • et al.
      Kupffer cell-mediated recruitment of rat dendritic cells to the liver: roles of N-acetylgalactosamine-specific sugar receptors.
      ]. This enhanced dendritic cell recruitment may initiate and promote virus-specific T cell responses. In contrast to dendritic cells, KC are less efficient in priming naïve T cells. Nevertheless, mouse KC have been shown to present antigen to CD4+ and CD8+ T cells, inducing these to proliferate and produce IFNγ [
      • Ebrahimkhani M.R.
      • Mohar I.
      • Crispe I.N.
      Cross-presentation of antigen by diverse subsets of murine liver cells.
      ,
      • You Q.
      • Cheng L.
      • Kedl R.M.
      • Ju C.
      Mechanism of T cell tolerance induction by murine hepatic Kupffer cells.
      ]. The relatively high expression of CD40, CD80, and MHC class II found on CD68+ cells in chronic HCV patients [
      • Burgio V.L.
      • Ballardini G.
      • Artini M.
      • Caratozzolo M.
      • Bianchi F.B.
      • Levrero M.
      Expression of co-stimulatory molecules by kupffer cells in chronic hepatitis of hepatitis C virus etiology.
      ] might point towards possible antigen presentation by intrahepatic macrophages.
      Although lymphocytes such as NK cells and CD8+ T cells are potent effector cells responsible to kill virus infected cells, KC have been reported to express cytotoxic molecules such as TRAIL, Fas-ligand, granzyme B, perforin, and ROS, enabling them to lyse infected hepatocytes [
      • Tordjmann T.
      • Soulie A.
      • Guettier C.
      • Schmidt M.
      • Berthou C.
      • Beaugrand M.
      • et al.
      Perforin and granzyme B lytic protein expression during chronic viral and autoimmune hepatitis.
      ,
      • Tang T.J.
      • Kwekkeboom J.
      • Laman J.D.
      • Niesters H.G.
      • Zondervan P.E.
      • de Man R.A.
      • et al.
      The role of intrahepatic immune effector cells in inflammatory liver injury and viral control during chronic hepatitis B infection.
      ,
      • Kolios G.
      • Valatas V.
      • Kouroumalis E.
      Role of Kupffer cells in the pathogenesis of liver disease.
      ]. However, since KC act in an antigen-nonspecific manner and hence can lyse hepatocytes irrespective of their infection state, it is tempting to speculate that KC cause more damage to the organ due to their cytotoxic capacity than that they provide protective immunity to the host.
      In summary, only limited information exists on the direct interaction between HBV and HCV with KC in vivo and ex vivo. Macrophages are able to bind HBV or HCV or virus-related proteins in vitro, triggering surface and/or intracellular receptors. However, receptors used for these purposes need to be further investigated. Several studies indicate that KC may play a role in controlling HBV and HCV infections by inhibiting viral replication, either directly via the production of cytokines or via their interaction with other cells, as well as in shaping the inflammatory response towards the induction of virus-specific immunity. However, more research is required to get a better insight into the role of KC in regulating intrahepatic immunity.

      Suppressive effects of HBV and HCV on KC function

      Besides the contribution of KC to viral clearance, viruses may actively interfere with the pro-inflammatory functions of KC to evade host immunity. Various studies show that HBV and HCV are able to interfere with TLR pathways, RIG-I signaling and subsequent pro-inflammatory activities of hepatocytes and immune cells [
      • Lin W.
      • Kim S.S.
      • Yeung E.
      • Kamegaya Y.
      • Blackard J.T.
      • Kim K.A.
      • et al.
      Hepatitis C virus core protein blocks interferon signaling by interaction with the STAT1 SH2 domain.
      ,
      • Meylan E.
      • Curran J.
      • Hofmann K.
      • Moradpour D.
      • Binder M.
      • Bartenschlager R.
      • et al.
      Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.
      ,
      • Miyazaki M.
      • Kanto T.
      • Inoue M.
      • Itose I.
      • Miyatake H.
      • Sakakibara M.
      • et al.
      Impaired cytokine response in myeloid dendritic cells in chronic hepatitis C virus infection regardless of enhanced expression of Toll-like receptors and retinoic acid inducible gene-I.
      ,
      • Woltman A.M.
      • Boonstra A.
      • Janssen H.L.
      Dendritic cells in chronic viral hepatitis B and C: victims or guardian angels?.
      ,
      • Woltman A.M.
      • Op den Brouw M.L.
      • Biesta P.J.
      • Shi C.C.
      • Janssen H.L.
      Hepatitis B virus lacks immune activating capacity, but actively inhibits plasmacytoid dendritic cell function.
      ], but studies describing the effect on human KC are limited. Only one study described that type I IFN production and TRAIL expression by human perfusate-derived KC were suppressed by HCV core protein via disruption of the TLR3/TRIF/TRK1/IRF3 pathway [
      • Tu Z.
      • Pierce R.H.
      • Kurtis J.
      • Kuroki Y.
      • Crispe I.N.
      • Orloff M.S.
      Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.
      ]. In addition, numerous studies on monocytes have demonstrated modulation of cytokine production by HCV proteins, and altered TLR responsiveness of monocytes obtained from chronic HCV patients [
      • Abe T.
      • Kaname Y.
      • Hamamoto I.
      • Tsuda Y.
      • Wen X.Y.
      • Taguwa S.
      • et al.
      Hepatitis C virus nonstructural protein 5A modulates the toll-like receptor-MyD88-dependent signaling pathway in macrophage cell lines.
      ,
      • Chung H.
      • Watanabe T.
      • Kudo M.
      • Chiba T.
      Hepatitis C virus core protein induces homotolerance and cross-tolerance to Toll-like receptor ligands by activation of Toll-like receptor 2.
      ,
      • Szabo G.
      • Dolganiuc A.
      Hepatitis C and innate immunity: recent advances.
      ].
      Concerning HBV, pretreatment of non-parenchymal cells including KC, with HBV-Met cell-derived supernatants, HBsAg, HBeAg, or hepatitis B virions almost completely abrogated TLR-induced anti-viral activity, i.e., IFNβ production, interferon-stimulated gene (ISG) induction, IRF3, NF-κB, and ERK1/2 expression [
      • Wu J.
      • Meng Z.
      • Jiang M.
      • Pei R.
      • Trippler M.
      • Broering R.
      • et al.
      Hepatitis B virus suppresses toll-like receptor–mediated innate immune responses in murine parenchymal and nonparenchymal liver cells.
      ]. Accordingly, incubating human monocytes with HBeAg or HBsAg inhibited TLR2-induced phosphorylation of p38 MAPK and JNK MAPK, and subsequent production of IL-6, TNF, and IL-12 [
      • Visvanathan K.
      • Skinner N.A.
      • Thompson A.J.V.
      • Riordan S.M.
      • Sozzi V.
      • Edwards R.
      • et al.
      Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.
      ,
      • Riordan S.M.
      • Skinner N.
      • Kurtovic J.
      • Locarnini S.
      • Visvanathan K.
      Reduced expression of toll-like receptor 2 on peripheral monocytes in patients with chronic hepatitis B.
      ,
      • Wang S.
      • Chen Z.
      • Hu C.
      • Qian F.
      • Cheng Y.
      • Wu M.
      • et al.
      Hepatitis B virus surface antigen selectively inhibits TLR2 ligand-induced IL-12 production in monocytes/macrophages by interfering with JNK activation.
      ]. In vivo, TLR2 expression by KC and peripheral blood monocytes in HBeAg-positive chronic HBV-infected individuals was lower than that in HBeAg-negative patients and controls. Moreover, TLR2 ligation induced less IL-6 and TNF in those HBeAg-positive patients [
      • Visvanathan K.
      • Skinner N.A.
      • Thompson A.J.V.
      • Riordan S.M.
      • Sozzi V.
      • Edwards R.
      • et al.
      Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.
      ]. These alterations may be related to the inhibitory effect of HBeAg on TLR2 signaling demonstrated in vitro. In addition, also TLR3 expression was found to be lower on PBMC from chronic HBV patients compared to control patients as well as on liver cells, including KC [
      • Huang Y.W.
      • Lin S.C.
      • Wei S.C.
      • Hu J.T.
      • Chang H.Y.
      • Huang S.H.
      • et al.
      Reduced toll-like receptor 3 expression in chronic hepatitis B patients and its restoration by interferon therapy.
      ]. Antiviral therapy of chronic HBV patients with entecavir or pegylated IFN-α partially restored TLR3 expression, but it is unclear whether this is a direct viral effect.

      Tolerogenic effects of HBV and HCV related to KC

      As mentioned above, KC are constantly exposed to pathogen-derived products from the gut. To prevent excessive inflammation and pathology of the liver, continuous activation of KC is avoided as these cells become refractory to subsequent endotoxin challenge, a phenomenon known as endotoxin-tolerance [
      • Biswas S.K.
      • Lopez-Collazo E.
      Endotoxin tolerance: new mechanisms, molecules and clinical significance.
      ,
      • Thomson A.W.
      • Knolle P.A.
      Antigen-presenting cell function in the tolerogenic liver environment.
      ]. This contributes to the well-described tolerogenic milieu in the liver. Besides modulation of TLR-signaling pathways, also expression of anti-inflammatory mediators, such as IL-10 and TGFβ, and other soluble and membrane-bound inhibitory molecules are underlying the intrahepatic tolerance [
      • Roth S.
      • Gong W.
      • Gressner A.M.
      Expression of different isoforms of TGF-beta and the latent TGF-beta binding protein (LTBP) by rat Kupffer cells.
      ,
      • You Q.
      • Cheng L.
      • Kedl R.M.
      • Ju C.
      Mechanism of T cell tolerance induction by murine hepatic Kupffer cells.
      ,
      • Thomson A.W.
      • Knolle P.A.
      Antigen-presenting cell function in the tolerogenic liver environment.
      ,
      • Mengshol J.A.
      • Golden-Mason L.
      • Arikawa T.
      • Smith M.
      • Niki T.
      • McWilliams R.
      • et al.
      A crucial role for Kupffer cell-derived galectin-9 in regulation of T cell immunity in hepatitis C infection.
      ].
      A number of studies have reported that HBV and HCV components affect the production of immunoregulatory cytokines, and consequently promote the tolerogenic milieu of the liver. In this respect, it has been reported that HBV particles preferably induced TGFβ production by rat KC instead of pro-inflammatory cytokines [
      • Li H.
      • Zheng H.W.
      • Chen H.
      • Xing Z.Z.
      • You H.
      • Cong M.
      • et al.
      Hepatitis B virus particles preferably induce Kupffer cells to produce TGF-beta1 over pro-inflammatory cytokines.
      ]. One of the activities of TGFβ is that it plays a role in maintaining tolerance towards self-antigens by selectively supporting the differentiation of FoxP3+ regulatory T cells [
      • Oertelt S.
      • Lian Z.X.
      • Cheng C.M.
      • Chuang Y.H.
      • Padgett K.A.
      • He X.S.
      • et al.
      Anti-mitochondrial antibodies and primary biliary cirrhosis in TGF-beta receptor II dominant-negative mice.
      ,
      • Gandhi R.
      • Anderson D.E.
      • Weiner H.L.
      Cutting edge: immature human dendritic cells express latency-associated peptide and inhibit T cell activation in a TGF-beta-dependent manner.
      ]. Furthermore, HCV core protein induces IL-10 production by human KC [
      • Tu Z.
      • Pierce R.H.
      • Kurtis J.
      • Kuroki Y.
      • Crispe I.N.
      • Orloff M.S.
      Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.
      ,
      • Chang S.
      • Dolganiuc A.
      • Szabo G.
      Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins.
      ]. Elevated intrahepatic IL-10 levels may suppress pro-inflammatory cytokine production by intrahepatic cells, frustrate KC-NK cell interaction [
      • Tu Z.
      • Bozorgzadeh A.
      • Pierce R.H.
      • Kurtis J.
      • Crispe I.N.
      • Orloff M.S.
      TLR-dependent cross talk between human Kupffer cells and NK cells.
      ,
      • Lassen M.G.
      • Lukens J.R.
      • Dolina J.S.
      • Brown M.G.
      • Hahn Y.S.
      Intrahepatic IL-10 maintains NKG2A+Ly49− liver NK cells in a functionally hyporesponsive state.
      ] and antigen presentation to T cells and their activation [
      • You Q.
      • Cheng L.
      • Kedl R.M.
      • Ju C.
      Mechanism of T cell tolerance induction by murine hepatic Kupffer cells.
      ,
      • Bamboat Z.M.
      • Ocuin L.M.
      • Balachandran V.P.
      • Obaid H.
      • Plitas G.
      • DeMatteo R.P.
      Conventional DCs reduce liver ischemia/reperfusion injury in mice via IL-10 secretion.
      ,
      • Brooks D.G.
      • Walsh K.B.
      • Elsaesser H.
      • Oldstone M.B.
      IL-10 directly suppresses CD4 but not CD8 T cell effector and memory responses following acute viral infection.
      ,
      • Ellett J.D.
      • Atkinson C.
      • Evans Z.P.
      • Amani Z.
      • Balish E.
      • Schmidt M.G.
      • et al.
      Murine Kupffer cells are protective in total hepatic ischemia/reperfusion injury with bowel congestion through IL-10.
      ,
      • Wilson E.B.
      • Brooks D.G.
      The role of IL-10 in regulating immunity to persistent viral infections.
      ,
      • Ye Z.
      • Huang H.
      • Hao S.
      • Xu S.
      • Yu H.
      • Van Den Hurk S.
      • et al.
      IL-10 has a distinct immunoregulatory effect on naive and active T cell subsets.
      ,
      • Bamboat Z.M.
      • Stableford J.A.
      • Plitas G.
      • Burt B.M.
      • Nguyen H.M.
      • Welles A.P.
      • et al.
      Human liver dendritic cells promote T cell hyporesponsiveness.
      ,
      • Breous E.
      • Somanathan S.
      • Vandenberghe L.H.
      • Wilson J.M.
      Hepatic regulatory T cells and Kupffer cells are crucial mediators of systemic T cell tolerance to antigens targeting murine liver.
      ]. Interestingly, chronic HBV and HCV patients showed higher plasma levels of IL-10 than uninfected individuals [
      • Sandler N.G.
      • Koh C.
      • Roque A.
      • Eccleston J.L.
      • Siegel R.B.
      • Demino M.
      • et al.
      Host response to translocated microbial products predicts outcomes of patients with HBV or HCV infection.
      ,
      • Liu B.S.
      • Groothuismink Z.M.
      • Janssen H.L.
      • Boonstra A.
      Role for IL-10 in inducing functional impairment of monocytes upon TLR4 ligation in patients with chronic HCV infections.
      ], which could be the result of a direct viral effect on KC and/or other cells, or the result of a negative feedback mechanism resulting from ongoing liver inflammation. Recently, the role of KC was examined in an established HBV-carrier mouse model. In this model, KC as well as IL-10 were involved in the establishment of antigen-specific tolerance towards peripheral HBsAg vaccination [
      • Xu L.
      • Yin W.
      • Sun R.
      • Wei H.
      • Tian Z.
      Liver type I regulatory T cells suppress germinal center formation in HBV-tolerant mice.
      ].
      KC express membrane-bound inhibitory ligands that could facilitate a tolerogenic milieu in the liver. For instance, under steady state conditions, KC are known to express PD-L1, which is a ligand for PD-1 and known to impede T cell function by inhibiting proliferation and cell division [
      • Iwai Y.
      • Terawaki S.
      • Ikegawa M.
      • Okazaki T.
      • Honjo T.
      PD-1 inhibits antiviral immunity at the effector phase in the liver.
      ]. Immunohistochemical analyses of liver biopsies from chronic viral hepatitis patients revealed that CD68+ macrophages expressed increased levels of PD-L2 compared to control liver tissue [
      • Chen J.
      • Wang X.M.
      • Wu X.J.
      • Wang Y.
      • Zhao H.
      • Shen B.
      • et al.
      Intrahepatic levels of PD-1/PD-L correlate with liver inflammation in chronic hepatitis B.
      ,
      • Mengshol J.A.
      • Golden-Mason L.
      • Arikawa T.
      • Smith M.
      • Niki T.
      • McWilliams R.
      • et al.
      A crucial role for Kupffer cell-derived galectin-9 in regulation of T cell immunity in hepatitis C infection.
      ,
      • Nebbia G.
      • Peppa D.
      • Schurich A.
      • Khanna P.
      • Singh H.D.
      • Cheng Y.
      • et al.
      Upregulation of the Tim-3/galectin-9 pathway of T cell exhaustion in chronic hepatitis B virus infection.
      ]. Similar results were reported for galectin-9 with enhanced expression by CD68+ cells by immunohistochemistry, which was confirmed by flow cytometry [
      • Nebbia G.
      • Peppa D.
      • Schurich A.
      • Khanna P.
      • Singh H.D.
      • Cheng Y.
      • et al.
      Upregulation of the Tim-3/galectin-9 pathway of T cell exhaustion in chronic hepatitis B virus infection.
      ]. Interestingly, enhanced serum levels of galectin-9 were observed in patients with biochemical evidence of highly active chronic HBV-related liver disease (ALT >100 U/L) as compared to patients with relatively low ALT levels (<50 IU/L) or healthy controls. Also comparison of plasma galectin-9 levels in patients with chronic HCV showed higher levels in patients compared to healthy individuals [
      • Mengshol J.A.
      • Golden-Mason L.
      • Arikawa T.
      • Smith M.
      • Niki T.
      • McWilliams R.
      • et al.
      A crucial role for Kupffer cell-derived galectin-9 in regulation of T cell immunity in hepatitis C infection.
      ]. Furthermore, co-localization of CD68 and galectin-9 was observed in the peri-portal regions of the livers of virtually all the patients with HCV infection, regardless of grade of inflammation or stage of fibrosis, but not in normal control livers [
      • Mengshol J.A.
      • Golden-Mason L.
      • Arikawa T.
      • Smith M.
      • Niki T.
      • McWilliams R.
      • et al.
      A crucial role for Kupffer cell-derived galectin-9 in regulation of T cell immunity in hepatitis C infection.
      ].
      These inhibitory ligands are known to inhibit T cell function upon cell-cell contact via interaction with PD-1 and Tim-3, respectively [
      • Larsson M.
      • Shankar E.M.
      • Che K.F.
      • Saeidi A.
      • Ellegard R.
      • Barathan M.
      • et al.
      Molecular signatures of T-cell inhibition in HIV-1 infection.
      ], which is of relevance since both PD-1 and Tim-3 are reported to be upregulated on HBV- and HCV-specific intrahepatic and peripheral blood-derived CD8+ T cells and associated with T cell dysfunction and exhaustion during chronic viral hepatitis [
      • Mengshol J.A.
      • Golden-Mason L.
      • Arikawa T.
      • Smith M.
      • Niki T.
      • McWilliams R.
      • et al.
      A crucial role for Kupffer cell-derived galectin-9 in regulation of T cell immunity in hepatitis C infection.
      ,
      • Radziewicz H.
      • Ibegbu C.C.
      • Fernandez M.L.
      • Workowski K.A.
      • Obideen K.
      • Wehbi M.
      • et al.
      Liver-infiltrating lymphocytes in chronic human hepatitis C virus infection display an exhausted phenotype with high levels of PD-1 and low levels of CD127 expression.
      ,
      • Penna A.
      • Pilli M.
      • Zerbini A.
      • Orlandini A.
      • Mezzadri S.
      • Sacchelli L.
      • et al.
      Dysfunction and functional restoration of HCV-specific CD8 responses in chronic hepatitis C virus infection.
      ]. Intrahepatic expression levels of PD-L1, PD-L2, and PD-1 correlated with liver inflammation in chronic HBV [
      • Chen J.
      • Wang X.M.
      • Wu X.J.
      • Wang Y.
      • Zhao H.
      • Shen B.
      • et al.
      Intrahepatic levels of PD-1/PD-L correlate with liver inflammation in chronic hepatitis B.
      ]. Although it has been shown that HCV core protein can induce PD-L1 expression on human perfusate-derived KC [
      • Tu Z.
      • Pierce R.H.
      • Kurtis J.
      • Kuroki Y.
      • Crispe I.N.
      • Orloff M.S.
      Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.
      ], it is not clear whether the upregulation of inhibitory ligands on intrahepatic macrophages and its correlation with inflammation are direct effects of HBV or HCV, or are components of negative feedback mechanisms that develop as a consequence of persistent inflammation.
      Thus, several studies indicate that both HBV and HCV compromise anti-viral immunity to a certain extent by (1) interfering with signaling of pathogen recognition receptors and the production of pro-inflammatory cytokines by KC and (2) increasing the tolerogenic capacities of KC resulting in the elevated expression of anti-inflammatory mediators. As persistent inflammation in general is accompanied by negative feedback mechanisms, the KC-related anti-inflammatory signals observed during chronic viral hepatitis could be explained by direct viral effects, immune regulation as part of the ongoing inflammatory response, or a combination. However, also immune activating functions of KC have been described upon HBV/HCV interaction. These seemingly contradictory functions probably indicate a critical balance influenced by the extent to which receptors are triggered (or over-triggered) and also by the type of KC receptors that are triggered. Therefore, not only the concentration of virus (proteins), but also the time since infection may strongly affect KC function. Whether also age influences KC function as one of the mechanisms explaining the self-limiting hepatitis often seen in HBV-infected adults, whereas young children usually develop chronic infection, has to be investigated.

      Role of KC in viral hepatitis-related liver damage

      Liver fibrosis

      One of the consequences of sustained low-grade injury induced by persistence of HBV and HCV in the liver is fibrosis, which is characterized by excess collagen deposition and accumulation of extracellular matrix. HBV and HCV may induce fibrinogenesis by activating hepatic stellate cells directly or indirectly by inducing cellular injury, apoptosis, and necrosis, which triggers a wound healing response. KC are thought to be involved in fibrogenesis by the release of various pro-fibrinogenic factors, such as ROS and certain cytokines, such as IL-6, TNF, IL-1, PDGF, and TGFβ, that induce activation of hepatic stellate cells [
      • Wallace K.
      • Burt A.D.
      • Wright M.C.
      Liver fibrosis.
      ]. In addition, KC produce enzymes that are important or the breakdown of matrix, such as collagenases and metalloproteinases, but they also regulate the production of these factors by other cells, leading to disturbance of the homeostatic mechanisms involved in extracellular matrix deposition [
      • Xidakis C.
      • Ljumovic D.
      • Manousou P.
      • Notas G.
      • Valatas V.
      • Kolios G.
      • et al.
      Production of pro- and anti-fibrotic agents by rat Kupffer cells; the effect of octreotide.
      ]. Recent studies in experimental animal models demonstrate that these activities are only partially conducted by liver-resident macrophages, but largely depend on recruitment of monocytes as precursors of macrophages into the inflamed and damaged liver [
      • Imamura M.
      • Ogawa T.
      • Sasaguri Y.
      • Chayama K.
      • Ueno H.
      Suppression of macrophage infiltration inhibits activation of hepatic stellate cells and liver fibrogenesis in rats.
      ,
      • Karlmark K.R.
      • Weiskirchen R.
      • Zimmermann H.W.
      • Gassler N.
      • Ginhoux F.
      • Weber C.
      • et al.
      Hepatic recruitment of the inflammatory Gr1+ monocyte subset upon liver injury promotes hepatic fibrosis.
      ].
      Although, in patients with viral hepatitis, no causative role has been demonstrated for KC in the development of liver fibrosis, increased numbers of CD14+CD68+ KC were found around the regions of damage and fibrosis [
      • Sandler N.G.
      • Koh C.
      • Roque A.
      • Eccleston J.L.
      • Siegel R.B.
      • Demino M.
      • et al.
      Host response to translocated microbial products predicts outcomes of patients with HBV or HCV infection.
      ]. These increased numbers were associated with liver injury [
      • McGuinness P.H.
      • Painter D.
      • Davies S.
      • McCaughan G.W.
      Increases in intrahepatic CD68 positive cells, MAC387 positive cells, and proinflammatory cytokines (particularly interleukin 18) in chronic hepatitis C infection.
      ,
      • Wallace K.
      • Burt A.D.
      • Wright M.C.
      Liver fibrosis.
      ,
      • Marrogi A.J.
      • Cheles M.K.
      • Gerber M.A.
      Chronic hepatitis C. Analysis of host immune response by immunohistochemistry.
      ,
      • Dioguardi N.
      • Dell’Oca M.
      • Arosio E.
      A computer-assisted study of macrophage behavior in HBV and nAnB related infectious chronic active hepatitis.
      ]. A detailed study by Liaskou et al. observed that in liver tissue from non-viral hepatitis patients with end-stage liver disease a specific monocyte subpopulation accumulated in the liver, which was able to conduct phagocytic activity and to release inflammatory and profibrinogenic cytokines [
      • Liaskou E.
      • Zimmermann H.W.
      • Li K.K.
      • Oo Y.H.
      • Suresh S.
      • Stamataki Z.
      • et al.
      Monocyte subsets in human liver disease show distinct phenotypic and functional characteristics.
      ]. Interestingly, a study in HBV replication-competent transgenic mice showed an opposite effect of KC by demonstrating that they did not contribute to liver damage, but prevented liver injury by removal of apoptotic hepatocytes during viral hepatitis [
      • Sitia G.
      • Iannacone M.
      • Aiolfi R.
      • Isogawa M.
      • van Rooijen N.
      • Scozzesi C.
      • et al.
      Kupffer cells hasten resolution of liver immunopathology in mouse models of viral hepatitis.
      ]. In this model, clodronate-mediated depletion of KC resulted in higher numbers of necrotic hepatocytes and elevated serum ALT levels. In line with this, in a different mouse model, liver-infiltrating monocyte/macrophages mediated regression of fibrosis via phagocytosis of cellular debris [
      • Ramachandran P.
      • Pellicoro A.
      • Vernon M.A.
      • Boulter L.
      • Aucott R.L.
      • Ali A.
      • et al.
      Differential Ly-6C expression identifies the recruited macrophage phenotype, which orchestrates the regression of murine liver fibrosis.
      ].
      Liver damage and ultimately the induction of fibrosis may, at least in part, be attributed to cytokines produced by KC. Moreover, during viral hepatitis KC have also been found to express cytotoxic molecules, like TRAIL, Fas-ligand, granzyme B, perforin, and ROS, that enable them to kill infected as well as non-infected “bystander” hepatocytes [
      • Tordjmann T.
      • Soulie A.
      • Guettier C.
      • Schmidt M.
      • Berthou C.
      • Beaugrand M.
      • et al.
      Perforin and granzyme B lytic protein expression during chronic viral and autoimmune hepatitis.
      ,
      • Tang T.J.
      • Kwekkeboom J.
      • Laman J.D.
      • Niesters H.G.
      • Zondervan P.E.
      • de Man R.A.
      • et al.
      The role of intrahepatic immune effector cells in inflammatory liver injury and viral control during chronic hepatitis B infection.
      ,
      • Kolios G.
      • Valatas V.
      • Kouroumalis E.
      Role of Kupffer cells in the pathogenesis of liver disease.
      ]. Fas-ligand expression by KC was increased in chronic HBV patients and associated with elevated ALT levels, while granzyme B and perforin expression by KC was increased in both chronic HBV and HCV patients [
      • Tordjmann T.
      • Soulie A.
      • Guettier C.
      • Schmidt M.
      • Berthou C.
      • Beaugrand M.
      • et al.
      Perforin and granzyme B lytic protein expression during chronic viral and autoimmune hepatitis.
      ,
      • Tang T.J.
      • Kwekkeboom J.
      • Laman J.D.
      • Niesters H.G.
      • Zondervan P.E.
      • de Man R.A.
      • et al.
      The role of intrahepatic immune effector cells in inflammatory liver injury and viral control during chronic hepatitis B infection.
      ]. Interestingly, a direct contribution of KC to the pathogenesis of hepatitis has also been reported for viral infections by viruses that infect other organs and are not detected in the liver itself [
      • Polakos N.K.
      • Cornejo J.C.
      • Murray D.A.
      • Wright K.O.
      • Treanor J.J.
      • Crispe I.N.
      • et al.
      Kupffer cell-dependent hepatitis occurs during influenza infection.
      ]. In influenza infection, KC were indicated as the effector cells killing hepatocytes in an as yet unidentified manner, leading to damage-associated hepatitis. KC can kill hepatocytes either directly via Fas-dependent apoptotic pathways or indirectly by interacting with CD8+ (and possibly CD4+) T cells through stimulation of cytokine secretion and other mediators, such as ROS [
      • Polakos N.K.
      • Cornejo J.C.
      • Murray D.A.
      • Wright K.O.
      • Treanor J.J.
      • Crispe I.N.
      • et al.
      Kupffer cell-dependent hepatitis occurs during influenza infection.
      ].

      Hepatocellular carcinoma

      Chronic HBV/HCV and cirrhosis are major risk factors for the development of hepatocellular carcinoma [
      • Perz J.F.
      • Armstrong G.L.
      • Farrington L.A.
      • Hutin Y.J.
      • Bell B.P.
      The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide.
      ]. Although HCC development has been extensively studied in mice and rat, only few studies have directly assessed the importance of KC in HCC development in chronic HBV settings, and no studies are available from chronic HCV settings. Dying hepatocytes, likely resulting from anti-viral activities since HBV and HCV are considered non-cytopathic, will activate neighboring cells, including KC [
      • Wu J.
      • Li J.
      • Salcedo R.
      • Mivechi N.F.
      • Trinchieri G.
      • Horuzsko A.
      The proinflammatory myeloid cell receptor TREM-1 controls Kupffer cell activation and development of hepatocellular carcinoma.
      ], to produce cytokines and growth factors, such as hepatocyte growth factor, IL-6, and TNF, which will further amplify the inflammatory response and drive the compensatory proliferation of surviving hepatocytes [
      • Maeda S.
      • Kamata H.
      • Luo J.L.
      • Leffert H.
      • Karin M.
      IKKbeta couples hepatocyte death to cytokine-driven compensatory proliferation that promotes chemical hepatocarcinogenesis.
      ]. Ongoing cycles of hepatocyte death and regeneration increase the chances of spontaneous mutations and DNA damage [
      • Arzumanyan A.
      • Reis H.M.
      • Feitelson M.A.
      Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma.
      ] eventually resulting in HCC. In HBV-transgenic mice, KC and/or infiltrating macrophages produced high levels of ROS, resulting in extensive oxidative DNA damage in neighboring proliferating hepatocytes and development of HCC [
      • Hagen T.M.
      • Huang S.
      • Curnutte J.
      • Fowler P.
      • Martinez V.
      • Wehr C.M.
      • et al.
      Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma.
      ]. HBV/HCV also activate KC to produce these types of pro-inflammatory mediators, which may support the development of HCC [
      • Tu Z.
      • Pierce R.H.
      • Kurtis J.
      • Kuroki Y.
      • Crispe I.N.
      • Orloff M.S.
      Hepatitis C virus core protein subverts the antiviral activities of human Kupffer cells.
      ,
      • Hösel M.
      • Quasdorff M.
      • Wiegmann K.
      • Webb D.
      • Zedler U.
      • Broxtermann M.
      • et al.
      Not interferon, but interleukin-6 controls early gene expression in hepatitis B virus infection.
      ]. Additionally, the immunoregulatory mediators expressed by KC, either as a direct virus-KC interaction or as a consequence of the inflammatory response, may also inhibit tumor-specific immune responses. For instance, galectin-9 expressed on intrahepatic macrophages caused senescence of CD4+ and CD8+ Tim3+ T cells, and may explain part of the mechanism leading to the development of HCC [
      • Li H.
      • Wu K.
      • Tao K.
      • Chen L.
      • Zheng Q.
      • Lu X.
      • et al.
      Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma.
      ]. Furthermore, one of the HBV-derived proteins, HBxAg, also has direct tumorigenic effects [
      • Wang C.
      • Yang W.
      • Yan H.X.
      • Luo T.
      • Zhang J.
      • Tang L.
      • et al.
      Hepatitis B virus X (HBx) induces tumorigenicity of hepatic progenitor cells in 3,5-diethoxycarbonyl-1,4-dihydrocollidine-treated HBx transgenic mice.
      ]. Hepatocyte regeneration, either influenced by KC or not, allows HBxAg integration in DNA of hepatocytes, which is one of the processes involved in the development of HCC (reviewed in [
      • Arzumanyan A.
      • Reis H.M.
      • Feitelson M.A.
      Pathogenic mechanisms in HBV- and HCV-associated hepatocellular carcinoma.
      ]). Whether HBxAg directly interacts with KC is not described.
      In conclusion, KC play a central role in liver damage during hepatitis, having all the tools to induce inflammation, cell death, fibrosis, and ultimately HCC, but further research during HBV/HCV infection remains to be carried out to determine the exact contribution of KC to liver damage in viral hepatitis.

      Perspectives

      Currently, our understanding of the role of KC in viral hepatitis is incomplete. The detailed contributions of liver-resident KC vs. liver-infiltrating macrophages to various processes of disease pathogenesis are difficult to determine, because of the highly overlapping characteristics of these cells. Nevertheless, we can appreciate several possible anti-viral roles of KC, including binding and/or uptake of virus leading to immune recognition and the production of pro-inflammatory mediators resulting in (1) inhibition of viral replication in hepatocytes, (2) activation of neighboring cells, and (3) attraction, activation, and interaction with other immune cells, which will further increase the anti-viral and inflammatory response (Fig. 1). These immune activating roles of KC are beneficial to combat HBV and HCV in the early phases after infection, but may also contribute to tissue damage and the development of fibrosis, cirrhosis, and HCC during chronic viral hepatitis (Fig. 1). Furthermore, also immune regulatory functions of KC have been described, either as a consequence of direct virus-KC interaction, or as part of the complex tolerogenic liver environment and the ongoing inflammatory response upon HBV and HCV-infection, which may counteract the development of effective anti-viral immunity and support viral persistence and related disease pathogenesis (Fig. 2).
      Figure thumbnail gr1
      Fig. 1The role of KC in anti-viral immunity and tissue damage during HBV and HCV infection. Exposure of KC to HBV or HCV will lead to direct activation of KC that, together with infected hepatocytes, release cytokines and chemokines, which are responsible for the attraction of other leukocytes. Activation of infiltrating immune cells leads to further production of cytokines that indirectly activate KC. The secreted cytokines may inhibit viral replication (green text). However, persistent exposure of KC to HBV or HCV will continuously activate KC leading to the ongoing release of cytokines and chemokines attracting and activating more leukocytes. Likewise, continuous activation of infiltrating leukocytes leads to ongoing production of cytokines that indirectly activate KC. Some of the cytokines secreted are pro-fibrotic factors. Additionally, KC and other immune cells are able to induce apoptosis of infected as well as uninfected hepatocytes, and release cytokines, which drive compensatory proliferation of hepatocytes. The ongoing cycles of hepatocyte death and regeneration increase the chances of spontaneous mutations and DNA damage, which may eventually result in HCC (red text).
      Figure thumbnail gr2
      Fig. 2Role of KC in immune regulation and viral persistence during HBV and HCV infection. Exposure of KC to HBV or HCV will lead to their activation and the release of anti-inflammatory cytokines and expression of inhibitory molecules. Combined with impaired antigen presentation by KC, these regulatory mechanisms will interfere with KC function and that of other immune cells, frustrating anti-viral immunity.
      With our growing appreciation of the roles of intrahepatic macrophages in both protective and harmful responses, intrahepatic macrophages form an interesting but complex cellular target for treatment options in viral hepatitis. The versatile features assigned to KC may partly belong to infiltrating monocytes/macrophages and therefore future efforts should focus on identifying phenotypical and/or functional characteristics discriminating KC from infiltrating macrophages. Furthermore, the function of KC and other intrahepatic macrophages will largely depend on type, level, and duration of receptors triggered pushing the balance towards either protective or harmful responses. Identification of receptors and underlying molecular mechanisms involved in virus-cell interactions and insight into mechanisms involved in wanted and unwanted responses of the different macrophage populations that exert distinctive functions during the early and later phases of HBV/HCV infection are needed to move the field forward.

      Financial support

      This work was supported by the Virgo consortium , funded by the Dutch government project number FES0908 , by the Netherlands Genomics Initiative (NGI) project number 050-060-452 , and by The Netherlands Organization for Scientific Research (NWO VIDI Grant 91712329 to AMW).

      Conflict of interest

      The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

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