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Danger signals in liver injury and restoration of homeostasis

  • Hui Han
    Affiliations
    Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., Suite 130 CSN, MC 847, Chicago, IL 60612, USA
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  • Author Footnotes
    These authors contributed equally.
    Romain Desert
    Footnotes
    † These authors contributed equally.
    Affiliations
    Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., Suite 130 CSN, MC 847, Chicago, IL 60612, USA
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  • Author Footnotes
    These authors contributed equally.
    Sukanta Das
    Footnotes
    † These authors contributed equally.
    Affiliations
    Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., Suite 130 CSN, MC 847, Chicago, IL 60612, USA
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  • Author Footnotes
    These authors contributed equally.
    Zhuolun Song
    Footnotes
    † These authors contributed equally.
    Affiliations
    Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., Suite 130 CSN, MC 847, Chicago, IL 60612, USA
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    These authors contributed equally.
    Dipti Athavale
    Footnotes
    † These authors contributed equally.
    Affiliations
    Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., Suite 130 CSN, MC 847, Chicago, IL 60612, USA
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    These authors contributed equally.
    Xiaodong Ge
    Footnotes
    † These authors contributed equally.
    Affiliations
    Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., Suite 130 CSN, MC 847, Chicago, IL 60612, USA
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  • Natalia Nieto
    Correspondence
    Corresponding author. Address: Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., Suite 130 CSN, MC 847, Chicago, IL 60612, USA. Tel.: +1 (312) 996-7316, fax: +1 (312) 355-0774.
    Affiliations
    Department of Pathology, University of Illinois at Chicago, 840 S. Wood St., Suite 130 CSN, MC 847, Chicago, IL 60612, USA

    Division of Gastroenterology and Hepatology, Department of Medicine, University of Illinois at Chicago, 840 S. Wood St., Suite 1020N, MC 787, Chicago, IL 60612, USA
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    These authors contributed equally.

      Summary

      Damage-associated molecular patterns are signalling molecules involved in inflammatory responses and restoration of homeostasis. Chronic release of these molecules can also promote inflammation in the context of liver disease. Herein, we provide a comprehensive summary of the role of damage-associated molecular patterns as danger signals in liver injury. We consider the role of reactive oxygen species and reactive nitrogen species as inducers of damage-associated molecular patterns, as well as how specific damage-associated molecular patterns participate in the pathogenesis of chronic liver diseases such as alcohol-related liver disease, non-alcoholic steatohepatitis, liver fibrosis and liver cancer. In addition, we discuss the role of damage-associated molecular patterns in ischaemia reperfusion injury and liver transplantation and highlight current studies in which blockade of specific damage-associated molecular patterns has proven beneficial in humans and mice.

      Keywords

      Introduction

      Detection of threats such as pathogens and cellular damage is critical to organismal survival. One mechanism of detection is the secretion of endogenous molecules to the extracellular environment, which cell-surface receptors recognise as a danger signals or “alarmins”, requiring initiation and persistence of innate immune responses. These relocated host cell-derived activators, called damage-associated molecular patterns (DAMPs), are a key aspect of inflammation.
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      The rapid increase in circulating levels of DAMPs reflects the severity of liver injury; therefore, these molecules could be promising biomarkers and/or potential therapeutic targets to prevent liver damage. However, the number of clinical trials targeting DAMPs, some of which are disease-specific, is still very limited; hence, a careful review of the main DAMPs that contribute to chronic liver disease is warranted.
      Damage-associated molecular patterns are signalling molecules involved in inflammatory responses and restoration of homeostasis.

      ROS and RNS induce DAMPs and events involved in chronic liver disease

      ROS and RNS are typically generated by healthy cells during biological and metabolic processes.
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      Reactive oxygen species: metabolism, oxidative stress, and signal transduction.
      Yet, the production of free radicals can also promote inflammatory disease. In the liver, excessive oxidative and nitrosative stress not only contributes to increased production of DAMPs, but also correlates with pathogenesis of chronic liver diseases such as alcohol-related liver disease (ALD), non-alcoholic steatohepatitis (NASH), fibrosis and hepatocellular carcinoma (HCC)
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      (Table 1 and Fig. 1). Thus, an initial injury response can promote subsequent chronic inflammatory processes and further cell and tissue damage.
      Table 1ROS and RNS induce events involved in chronic liver disease.
      Effect(s)Reference(s)
      ALD
       ROSMitochondrial dysfunction; Proinflammatory; Profibrogenic
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      • Horvath B.
      • Rajesh M.
      • Varga Z.V.
      • Gariani K.
      • Ryu D.
      • et al.
      PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis.
      ,
      • Araujo Junior R.F.
      • Garcia V.B.
      • Leitao R.F.
      • Brito G.A.
      • Miguel Ede C.
      • Guedes P.M.
      • et al.
      Carvedilol improves inflammatory response, oxidative stress and fibrosis in the alcohol-induced liver injury in rats by regulating Kuppfer cells and hepatic stellate cells.
      ,
      • Su X.
      • Wang Y.
      • Zhou G.
      • Yang X.
      • Yu R.
      • Lin Y.
      • et al.
      Probucol attenuates ethanol-induced liver fibrosis in rats by inhibiting oxidative stress, extracellular matrix protein accumulation and cytokine production.
       RNSONOO- induced liver injury
      • Urtasun R.
      • Cubero F.J.
      • Vera M.
      • Nieto N.
      Reactive nitrogen species switch on early extracellular matrix remodeling via induction of MMP1 and TNFalpha.
      ,
      • Chamulitrat W.
      • Spitzer J.J.
      Nitric oxide and liver injury in alcohol-fed rats after lipopolysaccharide administration.
      ,
      • Rubbo H.
      • Radi R.
      • Anselmi D.
      • Kirk M.
      • Barnes S.
      • Butler J.
      • et al.
      Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/alpha-tocopherol than alpha-tocopherol/ascorbate.
      NASH
       ROSLipid peroxidation; Proinflammatory
      • Rolo A.P.
      • Teodoro J.S.
      • Palmeira C.M.
      Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis.
      ,
      • Sutti S.
      • Jindal A.
      • Locatelli I.
      • Vacchiano M.
      • Gigliotti L.
      • Bozzola C.
      • et al.
      Adaptive immune responses triggered by oxidative stress contribute to hepatic inflammation in NASH.
      ,
      • Parola M.
      • Pinzani M.
      • Casini A.
      • Albano E.
      • Poli G.
      • Gentilini A.
      • et al.
      Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells.
      ,
      • Seki S.
      • Kitada T.
      • Yamada T.
      • Sakaguchi H.
      • Nakatani K.
      • Wakasa K.
      In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases.
       RNSDe novo lipogenesis; Proinflammatory
      • Navarro L.A.
      • Wree A.
      • Povero D.
      • Berk M.P.
      • Eguchi A.
      • Ghosh S.
      • et al.
      Arginase 2 deficiency results in spontaneous steatohepatitis: a novel link between innate immune activation and hepatic de novo lipogenesis.
      Fibrosis
       ROSTGFβ signalling; HSC activation
      • Torok N.J.
      Dysregulation of redox pathways in liver fibrosis.
      ,
      • Fabregat I.
      • Caballero-Diaz D.
      Transforming growth factor-beta-induced cell plasticity in liver fibrosis and hepatocarcinogenesis.
       RNSiNOS induces MMP9; DNA damage; Profibrogenic
      • Anavi S.
      • Eisenberg-Bord M.
      • Hahn-Obercyger M.
      • Genin O.
      • Pines M.
      • Tirosh O.
      The role of iNOS in cholesterol-induced liver fibrosis.
      ,
      • Aram G.
      • Potter J.J.
      • Liu X.
      • Torbenson M.S.
      • Mezey E.
      Lack of inducible nitric oxide synthase leads to increased hepatic apoptosis and decreased fibrosis in mice after chronic carbon tetrachloride administration.
      HCC
       ROSOxidative DNA damage; DNA adducts; Proinflammatory; Oncogenic; Increase telomerase activity, telomere length and HCC tumour growth; Protein oxidation
      • Ko E.
      • Kim J.S.
      • Ju S.
      • Seo H.W.
      • Chang Y.
      • Kang J.A.
      • et al.
      Oxidatively modified protein-disulfide isomerase-associated 3 promotes Dyskerin Pseudouridine synthase 1-mediated Malignancy and survival of hepatocellular carcinoma cells.
      • Ko E.
      • Seo H.W.
      • Jung G.
      Telomere length and reactive oxygen species levels are positively associated with a high risk of mortality and recurrence in hepatocellular carcinoma.
      • Lin C.Y.
      • Hu C.T.
      • Cheng C.C.
      • Lee M.C.
      • Pan S.M.
      • Lin T.Y.
      • et al.
      Oxidation of heat shock protein 60 and protein disulfide isomerase activates ERK and migration of human hepatocellular carcinoma HepG2.
       RNSiNOS promotes HCC stem cell phenotype
      • Wang R.
      • Li Y.
      • Tsung A.
      • Huang H.
      • Du Q.
      • Yang M.
      • et al.
      iNOS promotes CD24(+)CD133(+) liver cancer stem cell phenotype through a TACE/ADAM17-dependent Notch signaling pathway.
      ALD, alcohol-related liver disease; NASH, non-alcoholic steatohepatitis; HCC, hepatocellular carcinoma.
      Figure thumbnail gr1
      Fig. 1ROS and RNS induce DAMPs and events involved in chronic liver disease.
      ROS are produced mostly in hepatocytes and MFs by CYP2E1, mitochondrial injury and NOX. ROS participate in progression of chronic liver disease, causing hepatocyte damage, inflammation, HSC activation and CD4+ T cell apoptosis. Peroxisomal ROS and kinases contribute to HCC development and resolution, respectively. RNS are generated in hepatocytes and MFs due to activation of iNOS. Excess NO reacts with ROS to generate damaging RNS such as ONOO-. Enzymatic and non-enzymatic antioxidant defence systems balance the generation of ROS and play an important role in resolution of liver disease. 4-HNE, 4-hydroxynonenal; ACOX1, acetyl-CoA oxidase; CYP2E1, cytochrome P450 2E1; DAMP(s), damage-associated molecular pattern(s); EtOH, ethanol; FFAs, free fatty acids; GPx, glutathione peroxidase; GSR, glutathione-disulfide reductase; GST, glutathione S-transferase; HCC, hepatocellular carcinoma; HSC(s), hepatic stellate cell(s); iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; MF(s), macrophages; mtROS, mitochondrial ROS; NLRP3, NOD-like receptor protein-3; NO, nitric oxide; NOX, NADPH oxidase; [O]HMGB1, disulfide High-mobility group box-1; ONOO-, peroxynitrite; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase.
      Mitochondrial dysfunction is a key factor in the pathogenesis of fatty liver diseases.
      • Rolo A.P.
      • Teodoro J.S.
      • Palmeira C.M.
      Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis.
      ,
      • Garcia-Ruiz C.
      • Fernandez-Checa J.C.
      Mitochondrial oxidative stress and antioxidants balance in fatty liver disease.
      ,
      • Mukhopadhyay P.
      • Horvath B.
      • Rajesh M.
      • Varga Z.V.
      • Gariani K.
      • Ryu D.
      • et al.
      PARP inhibition protects against alcoholic and non-alcoholic steatohepatitis.
      Acetaldehyde, the end-product of alcohol metabolism, causes structural and functional alterations in mitochondria that lower production of ATP and increase generation of ROS.
      • Seitz H.K.
      • Bataller R.
      • Cortez-Pinto H.
      • Gao B.
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      • Lackner C.
      • et al.
      Alcoholic liver disease.
      Further, diets enriched in fructose and fat, together with insulin resistance, enhance flux of free fatty acids (FFAs) to the mitochondria for β-oxidation. This flux increases mitochondrial membrane permeability, proton leakage and ROS production, lowering ATP levels.
      • Mansouri A.
      • Gattolliat C.H.
      • Asselah T.
      Mitochondrial dysfunction and signaling in chronic liver diseases.
      In NASH, CD4+ T cells have increased mitochondrial mass, facilitating production of mitochondrial ROS (mtROS), although treatment with antioxidants increases CD4+ T cells, delaying the progression of NAFLD and HCC.
      • Ma C.
      • Kesarwala A.H.
      • Eggert T.
      • Medina-Echeverz J.
      • Kleiner D.E.
      • Jin P.
      • et al.
      NAFLD causes selective CD4(+) T lymphocyte loss and promotes hepatocarcinogenesis.
      ROS increase production of DAMPs, such as osteopontin (OPN)
      • Arriazu E.
      • Ge X.
      • Leung T.M.
      • Magdaleno F.
      • Lopategi A.
      • Lu Y.
      • et al.
      Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury.
      and high-mobility group box 1 (HMGB1), and induce oxidative modifications that enhance immunostimulatory properties.
      • Kazama H.
      • Ricci J.E.
      • Herndon J.M.
      • Hoppe G.
      • Green D.R.
      • Ferguson T.A.
      Induction of immunological tolerance by apoptotic cells requires caspase-dependent oxidation of high-mobility group box-1 protein.
      ,
      • Krysko D.V.
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      • Krysko O.
      • Garg A.D.
      • Bachert C.
      • Lambrecht B.N.
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      Emerging role of damage-associated molecular patterns derived from mitochondria in inflammation.
      In KCs and MFs, membrane-bound NOX is the major source of ROS and NOX-deficient (p47phox−/−) mice are protected from ALD.
      • Kono H.
      • Rusyn I.
      • Yin M.
      • Gabele E.
      • Yamashina S.
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      NADPH oxidase-derived free radicals are key oxidants in alcohol-induced liver disease.
      ,
      • McKim S.E.
      • Gabele E.
      • Isayama F.
      • Lambert J.C.
      • Tucker L.M.
      • Wheeler M.D.
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      Inducible nitric oxide synthase is required in alcohol-induced liver injury: studies with knockout mice.
      Alcohol stimulates cytoplasmic iNOS, the major source of RNS, and increases production of peroxynitrite (ONOO-) and, thus, oxidative and nitrosative stress.
      • Chamulitrat W.
      • Spitzer J.J.
      Nitric oxide and liver injury in alcohol-fed rats after lipopolysaccharide administration.
      ,
      • Rubbo H.
      • Radi R.
      • Anselmi D.
      • Kirk M.
      • Barnes S.
      • Butler J.
      • et al.
      Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/alpha-tocopherol than alpha-tocopherol/ascorbate.
      Indeed, iNos−/− mice are protected from ALD,
      • McKim S.E.
      • Gabele E.
      • Isayama F.
      • Lambert J.C.
      • Tucker L.M.
      • Wheeler M.D.
      • et al.
      Inducible nitric oxide synthase is required in alcohol-induced liver injury: studies with knockout mice.
      while lack of iNOS decreases carbon tetrachloride (CCl4)-induced fibrosis.
      • Anavi S.
      • Eisenberg-Bord M.
      • Hahn-Obercyger M.
      • Genin O.
      • Pines M.
      • Tirosh O.
      The role of iNOS in cholesterol-induced liver fibrosis.
      ,
      • Aram G.
      • Potter J.J.
      • Liu X.
      • Torbenson M.S.
      • Mezey E.
      Lack of inducible nitric oxide synthase leads to increased hepatic apoptosis and decreased fibrosis in mice after chronic carbon tetrachloride administration.
      Alcohol is oxidised in hepatocytes by the microsomal cytochrome P450 2E1 (CYP2E1) and generates 1-hydroxyethyl radical, a major driver of alcohol-induced liver injury.
      • Lu Y.
      • Cederbaum A.I.
      CYP2E1 and oxidative liver injury by alcohol.
      ,
      • Knecht K.T.
      • Adachi Y.
      • Bradford B.U.
      • Iimuro Y.
      • Kadiiska M.
      • Xuang Q.H.
      • et al.
      Free radical adducts in the bile of rats treated chronically with intragastric alcohol: inhibition by destruction of Kupffer cells.
      Mice lacking Cyp2e1 display less alcohol-induced liver injury.
      • Lu Y.
      • Cederbaum A.I.
      CYP2E1 and oxidative liver injury by alcohol.
      ,
      • Leung T.M.
      • Nieto N.
      CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease.
      Moreover, ROS and RNS bind to proteins and generate neo-antigens that elicit immune responses.
      • Seitz H.K.
      • Bataller R.
      • Cortez-Pinto H.
      • Gao B.
      • Gual A.
      • Lackner C.
      • et al.
      Alcoholic liver disease.
      ,
      • Tuma D.J.
      • Thiele G.M.
      • Xu D.
      • Klassen L.W.
      • Sorrell M.F.
      Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during long-term ethanol administration.
      In NASH and ALD, lipid peroxidation end-products such as 4-hydroxynonenal and malondialdehyde bind DNA and proteins
      • Wang Y.
      • Millonig G.
      • Nair J.
      • Patsenker E.
      • Stickel F.
      • Mueller S.
      • et al.
      Ethanol-induced cytochrome P4502E1 causes carcinogenic etheno-DNA lesions in alcoholic liver disease.
      ,
      • Parola M.
      • Pinzani M.
      • Casini A.
      • Albano E.
      • Poli G.
      • Gentilini A.
      • et al.
      Stimulation of lipid peroxidation or 4-hydroxynonenal treatment increases procollagen alpha 1 (I) gene expression in human liver fat-storing cells.
      to form carcinogenic exocyclic etheno-DNA adducts
      • Mueller S.
      • Peccerella T.
      • Qin H.
      • Glassen K.
      • Waldherr R.
      • Flechtenmacher C.
      • et al.
      Carcinogenic etheno DNA adducts in alcoholic liver disease: correlation with cytochrome P-4502E1 and fibrosis.
      ,
      • Seki S.
      • Kitada T.
      • Yamada T.
      • Sakaguchi H.
      • Nakatani K.
      • Wakasa K.
      In situ detection of lipid peroxidation and oxidative DNA damage in non-alcoholic fatty liver diseases.
      and protein-adducts,
      • Tuma D.J.
      • Thiele G.M.
      • Xu D.
      • Klassen L.W.
      • Sorrell M.F.
      Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during long-term ethanol administration.
      ,
      • Ayala A.
      • Munoz M.F.
      • Arguelles S.
      Lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal.
      ,
      • Kharbanda K.K.
      • Shubert K.A.
      • Wyatt T.A.
      • Sorrell M.F.
      • Tuma D.J.
      Effect of malondialdehyde-acetaldehyde-protein adducts on the protein kinase C-dependent secretion of urokinase-type plasminogen activator in hepatic stellate cells.
      both of which enhance injury. ROS also regulate pro-angiogenic and profibrogenic responses in hepatic stellate cells (HSCs).
      • Nieto N.
      • Friedman S.L.
      • Cederbaum A.I.
      Stimulation and proliferation of primary rat hepatic stellate cells by cytochrome P450 2E1-derived reactive oxygen species.
      ,
      • Bataller R.
      • Lemon S.M.
      Fueling fibrosis in chronic hepatitis C.
      • Paik Y.H.
      • Schwabe R.F.
      • Bataller R.
      • Russo M.P.
      • Jobin C.
      • Brenner D.A.
      Toll-like receptor 4 mediates inflammatory signaling by bacterial lipopolysaccharide in human hepatic stellate cells.
      • Araujo Junior R.F.
      • Garcia V.B.
      • Leitao R.F.
      • Brito G.A.
      • Miguel Ede C.
      • Guedes P.M.
      • et al.
      Carvedilol improves inflammatory response, oxidative stress and fibrosis in the alcohol-induced liver injury in rats by regulating Kuppfer cells and hepatic stellate cells.
      • Su X.
      • Wang Y.
      • Zhou G.
      • Yang X.
      • Yu R.
      • Lin Y.
      • et al.
      Probucol attenuates ethanol-induced liver fibrosis in rats by inhibiting oxidative stress, extracellular matrix protein accumulation and cytokine production.
      • Fabregat I.
      • Caballero-Diaz D.
      Transforming growth factor-beta-induced cell plasticity in liver fibrosis and hepatocarcinogenesis.
      Importantly, peroxisomal ROS and kinases are implicated in HCC. The deacetylase sirtuin 5 suppresses activity of peroxisomal acetyl-CoA oxidase-1 (ACOX1), lowers generation of H2O2 and reduces oxidative DNA damage in in vivo models of HCC.
      • Chen X.F.
      • Tian M.X.
      • Sun R.Q.
      • Zhang M.L.
      • Zhou L.S.
      • Jin L.
      • et al.
      SIRT5 inhibits peroxisomal ACOX1 to prevent oxidative damage and is downregulated in liver cancer.
      In addition, liver-specific ablation of the stress-activated protein kinase p38α enhances ROS, whereas its re-introduction prevents fibrosis and HCC by limiting ROS.
      • Sakurai T.
      • Kudo M.
      • Umemura A.
      • He G.
      • Elsharkawy A.M.
      • Seki E.
      • et al.
      p38alpha inhibits liver fibrogenesis and consequent hepatocarcinogenesis by curtailing accumulation of reactive oxygen species.
      ROS and RNS do not bind receptors; instead, most cells react to them by transmitting signals to organelles including the nucleus.
      • Deshmukh P.
      • Unni S.
      • Krishnappa G.
      • Padmanabhan B.
      The Keap1-Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases.
      • He Y.
      • Hara H.
      • Nunez G.
      Mechanism and regulation of NLRP3 inflammasome activation.
      • Kelleher Z.T.
      • Matsumoto A.
      • Stamler J.S.
      • Marshall H.E.
      NOS2 regulation of NF-kappaB by S-nitrosylation of p65.
      • Kobayashi E.H.
      • Suzuki T.
      • Funayama R.
      • Nagashima T.
      • Hayashi M.
      • Sekine H.
      • et al.
      Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription.
      • Pantano C.
      • Reynaert N.L.
      • van der Vliet A.
      • Janssen-Heininger Y.M.
      Redox-sensitive kinases of the nuclear factor-kappaB signaling pathway.
      In MFs, the adaptor Kelch ECH associating protein 1 (KEAP1) senses ROS and transduces signals to nuclear factor erythroid 2-related factor-2 (NRF2) to regulate production of cytokines.
      • Deshmukh P.
      • Unni S.
      • Krishnappa G.
      • Padmanabhan B.
      The Keap1-Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases.
      ,
      • Kobayashi E.H.
      • Suzuki T.
      • Funayama R.
      • Nagashima T.
      • Hayashi M.
      • Sekine H.
      • et al.
      Nrf2 suppresses macrophage inflammatory response by blocking proinflammatory cytokine transcription.
      In myeloid and lymphoid cells, I-kappa-B kinase (IKK) senses ROS and transduces signals to activate NF-κB and regulate the inflammatory response.
      • Pantano C.
      • Reynaert N.L.
      • van der Vliet A.
      • Janssen-Heininger Y.M.
      Redox-sensitive kinases of the nuclear factor-kappaB signaling pathway.
      ,
      • Middleton G.
      • Hamanoue M.
      • Enokido Y.
      • Wyatt S.
      • Pennica D.
      • Jaffray E.
      • et al.
      Cytokine-induced nuclear factor kappa B activation promotes the survival of developing neurons.
      The NLRP3 inflammasome, a key player in chronic liver disease, is also stimulated by ROS.
      • He Y.
      • Hara H.
      • Nunez G.
      Mechanism and regulation of NLRP3 inflammasome activation.
      Pre-clinical and clinical trials have investigated the efficacy of antioxidants in acute and chronic liver disease, as the antioxidant defence is usually depleted.
      • Besse-Patin A.
      • Leveille M.
      • Oropeza D.
      • Nguyen B.N.
      • Prat A.
      • Estall J.L.
      Estrogen signals through peroxisome proliferator-activated receptor-gamma coactivator 1alpha to reduce oxidative damage associated with diet-induced fatty liver disease.
      ,
      • Videla L.A.
      • Rodrigo R.
      • Orellana M.
      • Fernandez V.
      • Tapia G.
      • Quinones L.
      • et al.
      Oxidative stress-related parameters in the liver of non-alcoholic fatty liver disease patients.
      Vitamin E alone or in combination with the lipid-lowering agent atorvastatin alleviates progression of steatosis to NASH in animal models.
      • Karimian G.
      • Kirschbaum M.
      • Veldhuis Z.J.
      • Bomfati F.
      • Porte R.J.
      • Lisman T.
      Vitamin E attenuates the progression of non-alcoholic fatty liver disease caused by partial Hepatectomy in mice.
      • Klaebel J.H.
      • Skjodt M.
      • Skat-Rordam J.
      • Rakipovski G.
      • Ipsen D.H.
      • Schou-Pedersen A.M.V.
      • et al.
      Atorvastatin and vitamin E accelerates NASH resolution by dietary intervention in a preclinical guinea pig model.
      • Presa N.
      • Clugston R.D.
      • Lingrell S.
      • Kelly S.E.
      • Merrill Jr., A.H.
      • Jana S.
      • et al.
      Vitamin E alleviates non-alcoholic fatty liver disease in phosphatidylethanolamine N-methyltransferase deficient mice.
      However, in a randomised clinical trial of patients with alcoholic hepatitis (AH), vitamin E alone or in combination with corticosteroids failed to confer a benefit.
      • Mezey E.
      • Potter J.J.
      • Rennie-Tankersley L.
      • Caballeria J.
      • Pares A.
      A randomized placebo controlled trial of vitamin E for alcoholic hepatitis.
      ,
      • Stewart S.
      • Prince M.
      • Bassendine M.
      • Hudson M.
      • James O.
      • Jones D.
      • et al.
      A randomized trial of antioxidant therapy alone or with corticosteroids in acute alcoholic hepatitis.
      Nonetheless, a clinical trial (NCT01792115) is currently evaluating the most effective dose of vitamin E for the treatment of NAFLD. N-acetylcysteine, a precursor of glutathione, is the only FDA-approved antioxidant for treatment of acetaminophen-induced hepatotoxicity.
      • Saito C.
      • Zwingmann C.
      • Jaeschke H.
      Novel mechanisms of protection against acetaminophen hepatotoxicity in mice by glutathione and N-acetylcysteine.
      Another option to reduce oxidative stress is dietary restriction, as high-calorie intake is associated with increased mtROS and reduced activity of antioxidant enzymes.
      • Walsh M.E.
      • Shi Y.
      • Van Remmen H.
      The effects of dietary restriction on oxidative stress in rodents.

      DAMPs in alcohol-related liver disease

      In ALD, the type of cell death determines the release of DAMPs.
      • Barnes M.A.
      • Roychowdhury S.
      • Nagy L.E.
      Innate immunity and cell death in alcoholic liver disease: role of cytochrome P4502E1.
      Apoptosis is the most common and is associated with the release of DAMPs from hepatocytes.
      • Elmore S.
      Apoptosis: a review of programmed cell death.
      Necrosis is typically observed in severe acute AH,
      • Crawford J.M.
      Histologic findings in alcoholic liver disease.
      where hepatocytes undergo swelling, autolysis and death without significant signal transduction.
      • Galluzzi L.
      • Vitale I.
      • Aaronson S.A.
      • Abrams J.M.
      • Adam D.
      • Agostinis P.
      • et al.
      Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.
      Necroptosis, which resembles necrosis, is the regulated version of necrotic cell death through the RIPK1-RIPK3 heterodimer scaffold complex that leads to the release of intracellular contents.
      • Vanden Berghe T.
      • Hassannia B.
      • Vandenabeele P.
      An outline of necrosome triggers.
      In both necrosis and necroptosis, multiple DAMPs are secreted into the extracellular space and initiate an inflammatory response
      • Vanden Berghe T.
      • Hassannia B.
      • Vandenabeele P.
      An outline of necrosome triggers.
      ,
      • Malhi H.
      • Guicciardi M.E.
      • Gores G.J.
      Hepatocyte death: a clear and present danger.
      (Table 2 and Fig. 2).
      Chronic release of these molecules promotes inflammation in the context of liver disease.
      Table 2DAMPs are involved in chronic liver injury and restoration of homeostasis.
      DAMPReceptor(s)Effect(s)Reference(s)
      ALD
       mtDNATLR9Proinflammatory; Profibrogenic
      • Lemasters J.J.
      • Zhong Z.
      Mitophagy in hepatocytes: types, initiators and role in adaptive ethanol metabolism.
       Uric acidNLRProinflammatory
      • Petrasek J.
      • Iracheta-Vellve A.
      • Saha B.
      • Satishchandran A.
      • Kodys K.
      • Fitzgerald K.A.
      • et al.
      Metabolic danger signals, uric acid and ATP, mediate inflammatory cross-talk between hepatocytes and immune cells in alcoholic liver disease.
       ATPP2RX7Proinflammatory
      • Iracheta-Vellve A.
      • Petrasek J.
      • Satishchandran A.
      • Gyongyosi B.
      • Saha B.
      • Kodys K.
      • et al.
      Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice.
       HSP90Oxidative stress; Prosteatotic; Proinflammatory
      • Ambade A.
      • Catalano D.
      • Lim A.
      • Kopoyan A.
      • Shaffer S.A.
      • Mandrekar P.
      Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury.
       HMGB1RAGE/TLR4Prosteatotic
      • Ge X.
      • Arriazu E.
      • Magdaleno F.
      • Antoine D.J.
      • Dela Cruz R.
      • Theise N.
      • et al.
      High mobility group box-1 drives fibrosis progression signaling via the receptor for advanced glycation end products in mice.
       Hyaluronic acidTLRsProfibrogenic
      • Naveau S.
      • Raynard B.
      • Ratziu V.
      • Abella A.
      • Imbert-Bismut F.
      • Messous D.
      • et al.
      Biomarkers for the prediction of liver fibrosis in patients with chronic alcoholic liver disease.
       LCN2LCN2RProinflammatory
      • Dubuquoy L.
      Lipocalin 2 highlights the complex role of neutrophils in alcoholic liver disease.
       PGE2PGE2 receptorImmunosuppression; Prosteatotic
      • Arroyo V.
      • Moreau R.
      Tying up PGE2 with albumin to relieve immunosuppression in cirrhosis.
      NASH
       mtDNATLR9Prosteatotic; Profibrogenic
      • Handa P.
      • Vemulakonda A.
      • Kowdley K.V.
      • Uribe M.
      • Mendez-Sanchez N.
      Mitochondrial DNA from hepatocytes as a ligand for TLR9: drivers of nonalcoholic steatohepatitis?.
      ,
      • Mridha A.R.
      • Haczeyni F.
      • Yeh M.M.
      • Haigh W.G.
      • Ioannou G.N.
      • Barn V.
      • et al.
      TLR9 is up-regulated in human and murine NASH: pivotal role in inflammatory recruitment and cell survival.
       ssRNATLR7Monocyte-derived macrophage activation; IFNγ and TNFα production; T cell recruitment
      • Roh Y.S.
      • Kim J.W.
      • Park S.
      • Shon C.
      • Kim S.
      • Eo S.K.
      • et al.
      Toll-like receptor-7 signaling promotes nonalcoholic steatohepatitis by inhibiting regulatory T cells in mice.
      ,
      • Kim S.
      • Park S.
      • Kim B.
      • Kwon J.
      Toll-like receptor 7 affects the pathogenesis of non-alcoholic fatty liver disease.
       AGERAGEIncrease during NASH progression
      • Mehta R.
      • Shaw G.
      • Masschelin P.
      • Felix S.
      • Otgonsuren M.
      • Baranova A.
      • et al.
      Polymorphisms in the receptor for advanced glycation end-products (RAGE) gene and circulating RAGE levels as a susceptibility factor for non-alcoholic steatohepatitis (NASH).
       mtROSNLRP3Prosteatotic; Profibrogenic
      • Bettaieb A.
      • Jiang J.X.
      • Sasaki Y.
      • Chao T.I.
      • Kiss Z.
      • Chen X.
      • et al.
      Hepatocyte nicotinamide adenine dinucleotide phosphate reduced oxidase 4 regulates stress signaling, fibrosis, and insulin sensitivity during development of steatohepatitis in mice.
      ,
      • Moon J.S.
      • Nakahira K.
      • Chung K.P.
      • DeNicola G.M.
      • Koo M.J.
      • Pabon M.A.
      • et al.
      NOX4-dependent fatty acid oxidation promotes NLRP3 inflammasome activation in macrophages.
       BiglycanTLR2/4Increase during NASH progression
      • van Koppen A.
      • Verschuren L.
      • van den Hoek A.M.
      • Verheij J.
      • Morrison M.C.
      • Li K.
      • et al.
      Uncovering a predictive molecular signature for the onset of NASH-related fibrosis in a translational NASH mouse model.
       Galectin-3TLR2/4Promote fibrosis and hepatocyte ballooning
      • Pejnovic N.
      • Jeftic I.
      • Jovicic N.
      • Arsenijevic N.
      • Lukic M.L.
      Galectin-3 and IL-33/ST2 axis roles and interplay in diet-induced steatohepatitis.
      ,
      • Chalasani N.
      • Abdelmalek M.F.
      • Garcia-Tsao G.
      • Vuppalanchi R.
      • Alkhouri N.
      • Rinella M.
      • et al.
      Effects of belapectin, an inhibitor of galectin-3, in patients with nonalcoholic steatohepatitis with cirrhosis and portal hypertension.
       FibrinogenTLR4Form deposits to promote fatty liver disease
      • Kopec A.K.
      • Abrahams S.R.
      • Thornton S.
      • Palumbo J.S.
      • Mullins E.S.
      • Divanovic S.
      • et al.
      Thrombin promotes diet-induced obesity through fibrin-driven inflammation.
       Cholesterol crystalsNLRP3Prosteatotic
      • Sheedy F.J.
      • Grebe A.
      • Rayner K.J.
      • Kalantari P.
      • Ramkhelawon B.
      • Carpenter S.B.
      • et al.
      CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation.
      ,
      • Ioannou G.N.
      • Van Rooyen D.M.
      • Savard C.
      • Haigh W.G.
      • Yeh M.M.
      • Teoh N.C.
      • et al.
      Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH.
      Fibrosis
       HMGB1RAGEHepatic stellate cell activation; Collagen type I production; Endoplasmic reticulum stress
      • Arriazu E.
      • Ge X.
      • Leung T.M.
      • Magdaleno F.
      • Lopategi A.
      • Lu Y.
      • et al.
      Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury.
      ,
      • Ge X.
      • Arriazu E.
      • Magdaleno F.
      • Antoine D.J.
      • Dela Cruz R.
      • Theise N.
      • et al.
      High mobility group box-1 drives fibrosis progression signaling via the receptor for advanced glycation end products in mice.
      ,
      • He Q.
      • Fu Y.
      • Ding X.
      • Li D.
      • Wang Z.
      • Tian D.
      • et al.
      High-mobility group box 1 induces endoplasmic reticulum stress and activates hepatic stellate cells.
       OPNIntegrin αVβ3Increases HMGB1; HSC activation via PI3K/pAkt/NF-κB; Ductular reaction
      • Arriazu E.
      • Ge X.
      • Leung T.M.
      • Magdaleno F.
      • Lopategi A.
      • Lu Y.
      • et al.
      Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury.
      ,
      • Wang X.
      • Lopategi A.
      • Ge X.
      • Lu Y.
      • Kitamura N.
      • Urtasun R.
      • et al.
      Osteopontin induces ductular reaction contributing to liver fibrosis.
       HSP90TLR2/TLR4HSC activation
      • Yu Z.
      • Jv Y.
      • Cai L.
      • Tian X.
      • Huo X.
      • Wang C.
      • et al.
      Gambogic acid attenuates liver fibrosis by inhibiting the PI3K/AKT and MAPK signaling pathways via inhibiting HSP90.
      ,
      • Zhang F.
      • Hao M.
      • Jin H.
      • Yao Z.
      • Lian N.
      • Wu L.
      • et al.
      Canonical hedgehog signalling regulates hepatic stellate cell-mediated angiogenesis in liver fibrosis.
       HSP47TLRsProfibrogenic
      • Ito S.
      • Ogawa K.
      • Takeuchi K.
      • Takagi M.
      • Yoshida M.
      • Hirokawa T.
      • et al.
      A small-molecule compound inhibits a collagen-specific molecular chaperone and could represent a potential remedy for fibrosis.
      ,
      • Ito S.
      • Nagata K.
      Biology of Hsp47 (Serpin H1), a collagen-specific molecular chaperone.
       IL-33IL-33RProfibrogenic via NF-κB and MAPKs; Proinflammatory (Th2 cytokines)
      • Pichery M.
      • Mirey E.
      • Mercier P.
      • Lefrancais E.
      • Dujardin A.
      • Ortega N.
      • et al.
      Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain.
      • Tan Z.
      • Liu Q.
      • Jiang R.
      • Lv L.
      • Shoto S.S.
      • Maillet I.
      • et al.
      Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells.
      • Liu J.
      • Yang Y.
      • Zheng C.
      • Chen G.
      • Shen Z.
      • Zheng S.
      • et al.
      Correlation of interleukin-33/ST2 receptor and liver fibrosis progression in biliary atresia patients.
      • McHedlidze T.
      • Waldner M.
      • Zopf S.
      • Walker J.
      • Rankin A.L.
      • Schuchmann M.
      • et al.
      Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis.
       ATP adenosineP2rX7, A2AR, A2BRMF release of IL1β and HMGB1; Profibrogenic
      • Toki Y.
      • Takenouchi T.
      • Harada H.
      • Tanuma S.
      • Kitani H.
      • Kojima S.
      • et al.
      Extracellular ATP induces P2X7 receptor activation in mouse Kupffer cells, leading to release of IL-1beta, HMGB1, and PGE2, decreased MHC class I expression and necrotic cell death.
      ,
      • Ferrari D.
      • Gambari R.
      • Idzko M.
      • Muller T.
      • Albanesi C.
      • Pastore S.
      • et al.
      Purinergic signaling in scarring.
      HCC
       HMGB1RAGE, TLR9Tumour initiation and progression
      • Yan W.
      • Chang Y.
      • Liang X.
      • Cardinal J.S.
      • Huang H.
      • Thorne S.H.
      • et al.
      High-mobility group box 1 activates caspase-1 and promotes hepatocellular carcinoma invasiveness and metastases.
      ,
      • Chen R.C.
      • Yi P.P.
      • Zhou R.R.
      • Xiao M.F.
      • Huang Z.B.
      • Tang D.L.
      • et al.
      The role of HMGB1-RAGE axis in migration and invasion of hepatocellular carcinoma cell lines.
      ,
      • Liu Y.
      • Yan W.
      • Tohme S.
      • Chen M.
      • Fu Y.
      • Tian D.
      • et al.
      Hypoxia induced HMGB1 and mitochondrial DNA interactions mediate tumor growth in hepatocellular carcinoma through Toll-like receptor 9.
       OPNIntegrins, CD44Tumour growth, metastasis and immune escape
      • Zhu Y.
      • Yang J.
      • Xu D.
      • Gao X.M.
      • Zhang Z.
      • Hsu J.L.
      • et al.
      Disruption of tumour-associated macrophage trafficking by the osteopontin-induced colony-stimulating factor-1 signalling sensitises hepatocellular carcinoma to anti-PD-L1 blockade.
      ,
      • Zhao J.
      • Dong L.
      • Lu B.
      • Wu G.
      • Xu D.
      • Chen J.
      • et al.
      Down-regulation of osteopontin suppresses growth and metastasis of hepatocellular carcinoma via induction of apoptosis.
       S100A1RAGEIncreases in HCC and correlates with poor survival
      • Guo Q.
      • Wang J.
      • Cao Z.
      • Tang Y.
      • Feng C.
      • Huang F.
      Interaction of S100A1 with LATS1 promotes cell growth through regulation of the Hippo pathway in hepatocellular carcinoma.
       S100A4RAGETumour growth and metastasis
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      Hepatocellular carcinoma-associated mesenchymal stem cells promote hepatocarcinoma progression: role of the S100A4-miR155-SOCS1-MMP9 axis.
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      miR-187-3p inhibits the metastasis and epithelial-mesenchymal transition of hepatocellular carcinoma by targeting S100A4.
       S100A8RAGETumour growth and metastasis
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      • et al.
      S100A8 and S100A9 are novel nuclear factor kappa B target genes during malignant progression of murine and human liver carcinogenesis.
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      Methylation of S100A8 is a promising diagnosis and prognostic marker in hepatocellular carcinoma.
       S100A9RAGETumour initiation and progression
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      A pro-tumorigenic function of S100A8/A9 in carcinogen-induced hepatocellular carcinoma.
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      • et al.
      S100A8 and S100A9 are novel nuclear factor kappa B target genes during malignant progression of murine and human liver carcinogenesis.
       mtDNATLR9HMGB1 binding to TLR9
      • Liu Y.
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      • Tohme S.
      • Chen M.
      • Fu Y.
      • Tian D.
      • et al.
      Hypoxia induced HMGB1 and mitochondrial DNA interactions mediate tumor growth in hepatocellular carcinoma through Toll-like receptor 9.
       Extracellular ATPP2HCC cell migration
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      • Fromont G.
      • et al.
      Carcinoma-specific expression of P2Y11 receptor and its contribution in ATP-induced purinergic signalling and cell migration in human hepatocellular carcinoma cells.
       CalreticulinN/ATumour growth and invasion
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      • Zhou C.
      • Qi L.
      • Fu Z.
      • Yan B.
      • et al.
      Calreticulin down-regulation inhibits the cell growth, invasion and cell cycle progression of human hepatocellular carcinoma cells.
       HistonesTLR4HCC metastasis
      • Chen R.
      • Xie Y.
      • Zhong X.
      • Fu Y.
      • Huang Y.
      • Zhen Y.
      • et al.
      Novel chemokine-like activities of histones in tumor metastasis.
      IRI & LT
       ATPP1, P2IRI; Graft rejection
      • Kim H.Y.
      • Kim S.J.
      • Lee S.M.
      Activation of NLRP3 and AIM2 inflammasomes in Kupffer cells in hepatic ischemia/reperfusion.
      ,
      • Yoshida O.
      • Dou L.
      • Kimura S.
      • Yokota S.
      • Isse K.
      • Robson S.C.
      • et al.
      CD39 deficiency in murine liver allografts promotes inflammatory injury and immune-mediated rejection.
       DNATLRsAccumulates following machine perfusion
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      • Zhu M.
      • Song M.
      • Yerxa J.
      • Gao Q.
      • Davis R.P.
      • et al.
      Damage-associated molecular patterns induce inflammatory injury during machine preservation of the liver: potential targets to enhance a promising technology.
       HistonesTLR9, NLRP3IRI
      • Huang H.
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      • Yan W.
      • Nace G.
      • Zhang L.
      • Ross M.
      • et al.
      Endogenous histones function as alarmins in sterile inflammatory liver injury through Toll-like receptor 9 in mice.
      ,
      • Huang H.
      • Chen H.W.
      • Evankovich J.
      • Yan W.
      • Rosborough B.R.
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      • et al.
      Histones activate the NLRP3 inflammasome in Kupffer cells during sterile inflammatory liver injury.
       HMGB1TLR4, RAGECirculating HMGB1 exacerbates hepatic IRI; Intracellular HMGB1 protects from IRI
      • Tsung A.
      • Sahai R.
      • Tanaka H.
      • Nakao A.
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      • et al.
      The nuclear factor HMGB1 mediates hepatic injury after murine liver ischemia-reperfusion.
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      • Huang H.
      • Nace G.W.
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      • Tai S.
      • Klune J.R.
      • Rosborough B.R.
      • et al.
      Hepatocyte-specific high-mobility group box 1 deletion worsens the injury in liver ischemia/reperfusion: a role for intracellular high-mobility group box 1 in cellular protection.
       HSP 70

       HSP 27
      TLRs, LOX-1Protects from hepatic IRI; Inhibits graft rejection
      • Wu H.H.
      • Huang C.C.
      • Chang C.P.
      • Lin M.T.
      • Niu K.C.
      • Tian Y.F.
      Heat shock protein 70 (HSP70) reduces hepatic inflammatory and oxidative damage in a rat model of liver ischemia/reperfusion injury with Hyperbaric oxygen preconditioning.
      ,
      • Chen S.W.
      • Park S.W.
      • Kim M.
      • Brown K.M.
      • D'Agati V.D.
      • Lee H.T.
      Human heat shock protein 27 overexpressing mice are protected against hepatic ischemia and reperfusion injury.
      ,
      • Flohe S.
      • Speidel N.
      • Flach R.
      • Lange R.
      • Erhard J.
      • Schade F.U.
      Expression of HSP 70 as a potential prognostic marker for acute rejection in human liver transplantation.
       IL-33ST2IRI; NETs
      • Barrientos L.
      • Bignon A.
      • Gueguen C.
      • de Chaisemartin L.
      • Gorges R.
      • Sandre C.
      • et al.
      Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells.
      ,
      • Yazdani H.O.
      • Chen H.W.
      • Tohme S.
      • Tai S.
      • van der Windt D.J.
      • Loughran P.
      • et al.
      IL-33 exacerbates liver sterile inflammation by amplifying neutrophil extracellular trap formation.
       PGE2PG receptorInduced in recipients with good graft function
      • Motino O.
      • Frances D.E.
      • Casanova N.
      • Fuertes-Agudo M.
      • Cucarella C.
      • Flores J.M.
      • et al.
      Protective role of hepatocyte cyclooxygenase-2 expression against liver ischemia-reperfusion injury in mice.
      ALD, alcohol-related liver disease; NASH, non-alcoholic steatohepatitis; HCC, hepatocellular carcinoma; IRI, ischaemia reperfusion injury; LT, liver transplantation; AGE, advanced glycation end-products.
      Figure thumbnail gr2
      Fig. 2DAMPs promote inflammation, steatosis and hepatocyte injury in ALD.
      Ethanol-induced hepatocyte injury causes release of DAMPs, including mitochondrial DAMPs (mtDNA and ATP), uric acid, HSPs and HMGB1 from damaged hepatocytes. Most of these DAMPs are recognised by MFs through RAGE, TLRs and P2RX7 and activate NF-κB and the NLRP3 inflammasome. These result in release of proinflammatory cytokines that trigger cellular injury and steatosis. HSCs release HA and are responsive to mtDNA which activates them. MFs produce PGE2 that causes steatosis via cAMP activation. Neutrophils produce LCN2 and respond to it by infiltrating the liver to exacerbate cellular injury by releasing proinflammatory cytokines. cAMP, cyclic adenosine monophosphate; COX2, cyclooxygenase-2; DAMP(s), damage-associated molecular pattern(s); EtOH, ethanol; HA, hyaluronic acid; HMGB1, high-mobility group box-1; HSC(s), hepatic stellate cell(s); HSPs, heat shock proteins; LCN2, lipocalin-2; MF(s), macrophage(s); mtDNA, mitochondrial DNA; mtROS, mitochondrial ROS; NFkB, nuclear factor kappa B; NLRP3, NOD-like receptor protein-3; P2RX7, purinergic receptor P2X7; PGE2, prostaglandin E2; RAGE, receptor for advanced glycation end-products; ROS, reactive oxygen species; TLR9, toll-like receptor 9.

      Mitochondrial DAMPs

      Mitochondrial DNA (mtDNA) and ATP maintain the mitochondrial structure and aid in energy metabolism.
      • Osellame L.D.
      • Blacker T.S.
      • Duchen M.R.
      Cellular and molecular mechanisms of mitochondrial function.
      ,
      • West A.P.
      • Shadel G.S.
      Mitochondrial DNA in innate immune responses and inflammatory pathology.
      Chronic alcohol abuse increases mtROS and causes mtDNA oxidation.
      • Fromenty B.
      • Grimbert S.
      • Mansouri A.
      • Beaugrand M.
      • Erlinger S.
      • Rotig A.
      • et al.
      Hepatic mitochondrial DNA deletion in alcoholics: association with microvesicular steatosis.
      ,
      • Lemasters J.J.
      • Zhong Z.
      Mitophagy in hepatocytes: types, initiators and role in adaptive ethanol metabolism.
      Moreover, alcohol depolarises mitochondria, disrupts mitophagy and leads to the release of mitochondrial DAMPs (mtDAMPs) into the cytosol, before they are eventually secreted from hepatocytes into the extracellular space.
      • Lemasters J.J.
      • Zhong Z.
      Mitophagy in hepatocytes: types, initiators and role in adaptive ethanol metabolism.
      Once released, mtDAMPs promote proinflammatory and profibrotic events that lead to ALD progression.
      • Lemasters J.J.
      • Zhong Z.
      Mitophagy in hepatocytes: types, initiators and role in adaptive ethanol metabolism.

      Metabolic DAMPs

      Alcohol-induced hepatocyte damage leads to the release of metabolic DAMPs, such as uric acid (following the degradation of nucleic acids) and ATP.
      • Nagy L.E.
      • Ding W.X.
      • Cresci G.
      • Saikia P.
      • Shah V.H.
      Linking pathogenic mechanisms of alcoholic liver disease with clinical phenotypes.
      Uric acid acts as an antioxidant by scavenging ROS and ONOO- in the plasma.
      • Sautin Y.Y.
      • Johnson R.J.
      Uric acid: the oxidant-antioxidant paradox.
      • Fabbrini E.
      • Serafini M.
      • Colic Baric I.
      • Hazen S.L.
      • Klein S.
      Effect of plasma uric acid on antioxidant capacity, oxidative stress, and insulin sensitivity in obese subjects.
      • Ames B.N.
      • Cathcart R.
      • Schwiers E.
      • Hochstein P.
      Uric acid provides an antioxidant defense in humans against oxidant- and radical-caused aging and cancer: a hypothesis.
      Uric acid and ATP levels are elevated in serum and liver tissue from alcoholic patients and alcohol-fed mice
      • Petrasek J.
      • Iracheta-Vellve A.
      • Saha B.
      • Satishchandran A.
      • Kodys K.
      • Fitzgerald K.A.
      • et al.
      Metabolic danger signals, uric acid and ATP, mediate inflammatory cross-talk between hepatocytes and immune cells in alcoholic liver disease.
      ; both uric acid and ATP mediate cross-talk between hepatocytes and immune cells, enhancing inflammation.
      • Petrasek J.
      • Iracheta-Vellve A.
      • Saha B.
      • Satishchandran A.
      • Kodys K.
      • Fitzgerald K.A.
      • et al.
      Metabolic danger signals, uric acid and ATP, mediate inflammatory cross-talk between hepatocytes and immune cells in alcoholic liver disease.
      Further, pharmacological depletion of uric acid and blockade of ATP protect against ALD,
      • Iracheta-Vellve A.
      • Petrasek J.
      • Satishchandran A.
      • Gyongyosi B.
      • Saha B.
      • Kodys K.
      • et al.
      Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice.
      suggesting they are candidate targets to prevent disease progression.

      Stress-induced DAMPs

      Cellular stress increases expression of heat shock proteins (HSPs), which act as chaperones for refolding, disaggregation and degradation of polypeptides.
      • Mandrekar P.
      Signaling mechanisms in alcoholic liver injury: role of transcription factors, kinases and heat shock proteins.
      When the chaperone activity of HSP90 is abnormal, it promotes alcohol-induced injury by enhancing hepatic lipid accumulation, MF-mediated inflammation and cellular stress.
      • Saha B.
      • Momen-Heravi F.
      • Furi I.
      • Kodys K.
      • Catalano D.
      • Gangopadhyay A.
      • et al.
      Extracellular vesicles from mice with alcoholic liver disease carry a distinct protein cargo and induce macrophage activation through heat shock protein 90.
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      • Kiebler Z.
      • Ickes B.R.
      • Petersen D.R.
      Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease.
      • Smathers R.L.
      • Galligan J.J.
      • Stewart B.J.
      • Petersen D.R.
      Overview of lipid peroxidation products and hepatic protein modification in alcoholic liver disease.
      Pharmacological inhibition of HSP90 promotes reversal of alcohol-induced liver injury.
      • Ambade A.
      • Catalano D.
      • Lim A.
      • Kopoyan A.
      • Shaffer S.A.
      • Mandrekar P.
      Inhibition of heat shock protein 90 alleviates steatosis and macrophage activation in murine alcoholic liver injury.
      HMGB1 is an architectural protein that plays a physiological role. It binds chromatin to facilitate bending and participates in nucleosome formation, DNA replication and DNA repair.
      • Lange S.S.
      • Vasquez K.M.
      HMGB1: the jack-of-all-trades protein is a master DNA repair mechanic.
      ,
      • Lange S.S.
      • Mitchell D.L.
      • Vasquez K.M.
      High mobility group protein B1 enhances DNA repair and chromatin modification after DNA damage.
      HMGB1 also acts as a DAMP, serving as a ligand for the receptor for advanced glycation end-products (RAGE) and for toll-like receptor 4 (TLR4).
      • Ge X.
      • Antoine D.J.
      • Lu Y.
      • Arriazu E.
      • Leung T.M.
      • Klepper A.L.
      • et al.
      High mobility group box-1 (HMGB1) participates in the pathogenesis of alcoholic liver disease (ALD).
      ,
      • Ge X.
      • Arriazu E.
      • Magdaleno F.
      • Antoine D.J.
      • Dela Cruz R.
      • Theise N.
      • et al.
      High mobility group box-1 drives fibrosis progression signaling via the receptor for advanced glycation end products in mice.
      Liver biopsies from alcoholic patients show a robust increase in HMGB1 expression and translocation, which correlate with disease stage. Similar findings are observed in chronic ethanol-fed mice.
      • Ge X.
      • Antoine D.J.
      • Lu Y.
      • Arriazu E.
      • Leung T.M.
      • Klepper A.L.
      • et al.
      High mobility group box-1 (HMGB1) participates in the pathogenesis of alcoholic liver disease (ALD).
      Further, ablation of Hmgb1 in hepatocytes protects mice from alcohol-induced liver injury by elevating LDL and VLDL export and increasing the levels of carnitine palmitoyltransferase-1, phosphorylated 5′ AMP-activated protein kinase-α and phosphorylated peroxisome proliferator-activated receptor-α.
      • Ge X.
      • Antoine D.J.
      • Lu Y.
      • Arriazu E.
      • Leung T.M.
      • Klepper A.L.
      • et al.
      High mobility group box-1 (HMGB1) participates in the pathogenesis of alcoholic liver disease (ALD).
      Non-parenchymal cells also release DAMPs in ALD. For instance, hyaluronic acid (HA) produced by HSCs and hepatocytes is abundant in the extracellular matrix (ECM) of alcoholic patients.
      • Saikia P.
      • Roychowdhury S.
      • Bellos D.
      • Pollard K.A.
      • McMullen M.R.
      • McCullough R.L.
      • et al.
      Hyaluronic acid 35 normalizes TLR4 signaling in Kupffer cells from ethanol-fed rats via regulation of microRNA291b and its target Tollip.
      ,
      • He Y.
      • Gao B.
      A small specific-sized hyaluronic acid ameliorates alcoholic liver disease by targeting a small RNA: new hope for therapy?.
      Individuals with ALD have increased serum HA levels, which correlate with progression of ALD and fibrosis.
      • Stickel F.
      • Poeschl G.
      • Schuppan D.
      • Conradt C.
      • Strenge-Hesse A.
      • Fuchs F.S.
      • et al.
      Serum hyaluronate correlates with histological progression in alcoholic liver disease.
      ,
      • Naveau S.
      • Raynard B.
      • Ratziu V.
      • Abella A.
      • Imbert-Bismut F.
      • Messous D.
      • et al.
      Biomarkers for the prediction of liver fibrosis in patients with chronic alcoholic liver disease.
      In addition, lipocalin-2 (LCN2), an acute-phase protein increased in patients with AH,
      • Das S.
      • Maras J.S.
      • Hussain M.S.
      • Sharma S.
      • David P.
      • Sukriti S.
      • et al.
      Hyperoxidized albumin modulates neutrophils to induce oxidative stress and inflammation in severe alcoholic hepatitis.
      acts as an alarmin by recruiting neutrophils to the liver.
      • Wieser V.
      • Tymoszuk P.
      • Adolph T.E.
      • Grander C.
      • Grabherr F.
      • Enrich B.
      • et al.
      Lipocalin 2 drives neutrophilic inflammation in alcoholic liver disease.
      • Dubuquoy L.
      Lipocalin 2 highlights the complex role of neutrophils in alcoholic liver disease.
      • Cai Y.
      • Jogasuria A.
      • Yin H.
      • Xu M.J.
      • Hu X.
      • Wang J.
      • et al.
      The detrimental role played by lipocalin-2 in alcoholic fatty liver in mice.
      Prostaglandin E2 (PGE2) is a potent vasodilator. In patients with advanced AH, upregulation of cyclooxygenase-2 (COX2) in MFs and KCs elevates plasma levels of PGE2, which causes immunosuppression and thus increased susceptibility to infection.
      • O'Brien A.J.
      • Fullerton J.N.
      • Massey K.A.
      • Auld G.
      • Sewell G.
      • James S.
      • et al.
      Immunosuppression in acutely decompensated cirrhosis is mediated by prostaglandin E2.
      • Arroyo V.
      • Moreau R.
      Tying up PGE2 with albumin to relieve immunosuppression in cirrhosis.
      • Choe W.H.
      • Baik S.K.
      Prostaglandin E2 -mediated immunosuppression and the role of albumin as its modulator.
      Moreover, KC-derived PGE2 increases cAMP in hepatocytes and triglyceride accumulation in livers from alcoholic patients.
      • Enomoto N.
      • Ikejima K.
      • Yamashina S.
      • Enomoto A.
      • Nishiura T.
      • Nishimura T.
      • et al.
      Kupffer cell-derived prostaglandin E(2) is involved in alcohol-induced fat accumulation in rat liver.

      DAMPs in non-alcoholic steatohepatitis

      NASH is characterised by increased steatosis, lobular inflammation and the presence of chicken-wire fibrosis.
      • Kleiner D.E.
      • Brunt E.M.
      • Van Natta M.
      • Behling C.
      • Contos M.J.
      • Cummings O.W.
      • et al.
      Design and validation of a histological scoring system for nonalcoholic fatty liver disease.
      ,
      • Younossi Z.
      • Anstee Q.M.
      • Marietti M.
      • Hardy T.
      • Henry L.
      • Eslam M.
      • et al.
      Global burden of NAFLD and NASH: trends, predictions, risk factors and prevention.
      During NASH progression, excessive lipid accumulation, ROS generation and endoplasmic reticulum (ER) stress damage hepatocytes. This damage triggers regulated cell death primarily through apoptosis and pyroptosis, which involves the formation of plasma membrane pores by the gasdermin family of proteins, largely induced by activation of proinflammatory caspases.
      • Lebeaupin C.
      • Proics E.
      • de Bieville C.H.
      • Rousseau D.
      • Bonnafous S.
      • Patouraux S.
      • et al.
      ER stress induces NLRP3 inflammasome activation and hepatocyte death.
      ,
      • Galluzzi L.
      • Vitale I.
      • Aaronson S.A.
      • Abrams J.M.
      • Adam D.
      • Agostinis P.
      • et al.
      Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018.
      ,
      • Kim J.Y.
      • Garcia-Carbonell R.
      • Yamachika S.
      • Zhao P.
      • Dhar D.
      • Loomba R.
      • et al.
      ER stress drives lipogenesis and steatohepatitis via caspase-2 activation of S1P.
      ,
      • Wree A.
      • Eguchi A.
      • McGeough M.D.
      • Pena C.A.
      • Johnson C.D.
      • Canbay A.
      • et al.
      NLRP3 inflammasome activation results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.
      Regulated cell death results in secondary necrosis and release of intracellular materials into the extracellular space, where they act as DAMPs recognised by PRRs.
      • Takeuchi O.
      • Akira S.
      Pattern recognition receptors and inflammation.
      TLRs and NLRs sense multiple DAMPs (Table 2 and Fig. 3) that mediate inflammation and fibrosis during NASH progression.
      • Aragones G.
      • Colom-Pellicer M.
      • Aguilar C.
      • Guiu-Jurado E.
      • Martinez S.
      • Sabench F.
      • et al.
      Circulating microbiota-derived metabolites: a “liquid biopsy?.
      • Cengiz M.
      • Ozenirler S.
      • Elbeg S.
      Role of serum toll-like receptors 2 and 4 in non-alcoholic steatohepatitis and liver fibrosis.
      • Mridha A.R.
      • Haczeyni F.
      • Yeh M.M.
      • Haigh W.G.
      • Ioannou G.N.
      • Barn V.
      • et al.
      TLR9 is up-regulated in human and murine NASH: pivotal role in inflammatory recruitment and cell survival.
      • O'Neill L.A.
      • Golenbock D.
      • Bowie A.G.
      The history of Toll-like receptors - redefining innate immunity.
      • Mridha A.R.
      • Wree A.
      • Robertson A.A.B.
      • Yeh M.M.
      • Johnson C.D.
      • Van Rooyen D.M.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      Figure thumbnail gr3
      Fig. 3Intrahepatic and extrahepatic DAMPs contribute to NASH.
      Damaged hepatocytes are the major source of intrahepatic DAMPs (mtDNA and ssRNA). ECM components such as biglycan, fibrinogen and galectin-3 can also act as DAMPs to active TLRs. MFs and dendritic cells recognise DAMPs through RAGE, TLRs and NLRP3 signalling. Extrahepatic DAMPs (AGE, FFAs and oxidised LDLs) are delivered via circulation and can bind RAGE and CD36, contributing to steatohepatitis. AGE, advanced glycation end-products; CHO, cholesterol; DAMP(s), damage-associated molecular pattern(s); ER, endoplasmic reticulum; FAO, fatty acid oxidation; FFA(s), free fatty acid(s); IRF, interferon-regulatory factor; MF(s), macrophage(s); mtDNA, mitochondrial DNA; mtROS, mitochondrial ROS; NFkB, nuclear factor kappa B; NLRP3, NOD-like receptor protein-3; NOX4, NADPH oxidase 4; oxLDL, oxidized low-density lipoproteins; ssRNA, single-stranded RNA; RAGE, receptor for advanced glycation end-products; ROS, reactive oxygen species; TLR(s), Toll-like receptor(s).

      Intrahepatic DAMPs

      Mitochondrial damage and subsequent cell death release immunogenic mtDNA.
      • Garcia-Martinez I.
      • Santoro N.
      • Chen Y.
      • Hoque R.
      • Ouyang X.
      • Caprio S.
      • et al.
      Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9.
      TLR9, a mtDNA receptor, is internalised in intracellular organelles such as endosomes and recognises phagocytosed unmethylated CpG DNA fragments,
      • Mridha A.R.
      • Haczeyni F.
      • Yeh M.M.
      • Haigh W.G.
      • Ioannou G.N.
      • Barn V.
      • et al.
      TLR9 is up-regulated in human and murine NASH: pivotal role in inflammatory recruitment and cell survival.
      ,
      • Garcia-Martinez I.
      • Santoro N.
      • Chen Y.
      • Hoque R.
      • Ouyang X.
      • Caprio S.
      • et al.
      Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9.
      which are rare in host genomic DNA but abundant in mtDNA.
      • Garcia-Martinez I.
      • Santoro N.
      • Chen Y.
      • Hoque R.
      • Ouyang X.
      • Caprio S.
      • et al.
      Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9.
      Unmethylated CpG DNA fragments are elevated in serum from obese patients, together with upregulated TLR9 expression.
      • Garcia-Martinez I.
      • Santoro N.
      • Chen Y.
      • Hoque R.
      • Ouyang X.
      • Caprio S.
      • et al.
      Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9.
      Mice with global or myeloid cell-specific ablation of Tlr9 fed either a high-fat (HF) diet or a high-fat, fructose and cholesterol (HFHC) diet show reduced liver steatosis, inflammation and fibrosis.
      • Mridha A.R.
      • Haczeyni F.
      • Yeh M.M.
      • Haigh W.G.
      • Ioannou G.N.
      • Barn V.
      • et al.
      TLR9 is up-regulated in human and murine NASH: pivotal role in inflammatory recruitment and cell survival.
      ,
      • Garcia-Martinez I.
      • Santoro N.
      • Chen Y.
      • Hoque R.
      • Ouyang X.
      • Caprio S.
      • et al.
      Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9.
      Likewise, treatment with the TLR9 antagonist IRS954 attenuates NASH, suggesting a possible therapeutic avenue.
      • Garcia-Martinez I.
      • Santoro N.
      • Chen Y.
      • Hoque R.
      • Ouyang X.
      • Caprio S.
      • et al.
      Hepatocyte mitochondrial DNA drives nonalcoholic steatohepatitis by activation of TLR9.
      Further, single-stranded RNA (ssRNA) binds TLR7 and triggers an inflammatory response in MFs and dendritic cells.
      • Roh Y.S.
      • Kim J.W.
      • Park S.
      • Shon C.
      • Kim S.
      • Eo S.K.
      • et al.
      Toll-like receptor-7 signaling promotes nonalcoholic steatohepatitis by inhibiting regulatory T cells in mice.
      Ablation of Tlr7 attenuates progression of NASH in a methionine and choline-deficient diet mouse model by suppressing TNFα and interferon-γ (IFNγ) production and CD4+ T cell recruitment.
      • Roh Y.S.
      • Kim J.W.
      • Park S.
      • Shon C.
      • Kim S.
      • Eo S.K.
      • et al.
      Toll-like receptor-7 signaling promotes nonalcoholic steatohepatitis by inhibiting regulatory T cells in mice.
      ,
      • Kim S.
      • Park S.
      • Kim B.
      • Kwon J.
      Toll-like receptor 7 affects the pathogenesis of non-alcoholic fatty liver disease.
      Reactive oxygen species and reactive nitrogen species induce damage-associated molecular patterns.
      mtROS also act as DAMPs and contribute to NASH progression. Hepatocyte-specific ablation of Nox4 attenuates inflammation and fibrosis in the HF and choline-deficient L-amino acid-defined (CDAA) murine models.
      • Bettaieb A.
      • Jiang J.X.
      • Sasaki Y.
      • Chao T.I.
      • Kiss Z.
      • Chen X.
      • et al.
      Hepatocyte nicotinamide adenine dinucleotide phosphate reduced oxidase 4 regulates stress signaling, fibrosis, and insulin sensitivity during development of steatohepatitis in mice.
      In MFs, NOX4 accelerates β-oxidation of long-chain FFAs causing oxidative stress and polarisation toward a more proinflammatory phenotype.
      • Moon J.S.
      • Nakahira K.
      • Chung K.P.
      • DeNicola G.M.
      • Koo M.J.
      • Pabon M.A.
      • et al.
      NOX4-dependent fatty acid oxidation promotes NLRP3 inflammasome activation in macrophages.
      NLRP3 is the intracellular PRR that responds to these ROS
      • Moon J.S.
      • Nakahira K.
      • Chung K.P.
      • DeNicola G.M.
      • Koo M.J.
      • Pabon M.A.
      • et al.
      NOX4-dependent fatty acid oxidation promotes NLRP3 inflammasome activation in macrophages.
      ; it is upregulated in the livers of patients with NASH and ablation of Nlrp3 prevents NASH progression in mice.
      • Mridha A.R.
      • Wree A.
      • Robertson A.A.B.
      • Yeh M.M.
      • Johnson C.D.
      • Van Rooyen D.M.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.
      Likewise, treating mice with GKT137831, a NOX1/4 inhibitor currently being tested in clinical trials, reduces ROS and activation of NLRP3 in palmitate-treated bone marrow-derived MFs and decreases inflammation in the CDAA murine model of NASH.
      • Bettaieb A.
      • Jiang J.X.
      • Sasaki Y.
      • Chao T.I.
      • Kiss Z.
      • Chen X.
      • et al.
      Hepatocyte nicotinamide adenine dinucleotide phosphate reduced oxidase 4 regulates stress signaling, fibrosis, and insulin sensitivity during development of steatohepatitis in mice.
      ,
      • Moon J.S.
      • Nakahira K.
      • Chung K.P.
      • DeNicola G.M.
      • Koo M.J.
      • Pabon M.A.
      • et al.
      NOX4-dependent fatty acid oxidation promotes NLRP3 inflammasome activation in macrophages.
      Notably, MCC950, an NLRP3 inhibitor, improves NAFLD and fibrosis in obese diabetic mice.
      • Mridha A.R.
      • Wree A.
      • Robertson A.A.B.
      • Yeh M.M.
      • Johnson C.D.
      • Van Rooyen D.M.
      • et al.
      NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice.

      ECM-derived DAMPs

      The ECM is dynamic and supports tissue homeostasis.
      • van Koppen A.
      • Verschuren L.
      • van den Hoek A.M.
      • Verheij J.
      • Morrison M.C.
      • Li K.
      • et al.
      Uncovering a predictive molecular signature for the onset of NASH-related fibrosis in a translational NASH mouse model.
      ,
      • Hennig E.E.
      • Mikula M.
      • Goryca K.
      • Paziewska A.
      • Ledwon J.
      • Nesteruk M.
      • et al.
      Extracellular matrix and cytochrome P450 gene expression can distinguish steatohepatitis from steatosis in mice.
      Active ECM remodelling is observed in both patients with NASH and mouse models of NASH.
      • van Koppen A.
      • Verschuren L.
      • van den Hoek A.M.
      • Verheij J.
      • Morrison M.C.
      • Li K.
      • et al.
      Uncovering a predictive molecular signature for the onset of NASH-related fibrosis in a translational NASH mouse model.
      ,
      • Decaris M.L.
      • Li K.W.
      • Emson C.L.
      • Gatmaitan M.
      • Liu S.
      • Wang Y.
      • et al.
      Identifying nonalcoholic fatty liver disease patients with active fibrosis by measuring extracellular matrix remodeling rates in tissue and blood.
      The deposition of fibrin and fibrinogen into the ECM occurs in the liver of patients with NASH and mice fed a HF diet. Additionally, mice overexpressing mutated fibrinogen are protected from fatty liver disease.
      • Kopec A.K.
      • Abrahams S.R.
      • Thornton S.
      • Palumbo J.S.
      • Mullins E.S.
      • Divanovic S.
      • et al.
      Thrombin promotes diet-induced obesity through fibrin-driven inflammation.
      Although no functional studies were performed, proteomics analysis revealed a sustained increase in biglycan, a potential ligand for TLRs, in hepatic ECM from mouse models of NASH.
      • van Koppen A.
      • Verschuren L.
      • van den Hoek A.M.
      • Verheij J.
      • Morrison M.C.
      • Li K.
      • et al.
      Uncovering a predictive molecular signature for the onset of NASH-related fibrosis in a translational NASH mouse model.
      Further, galectin-3, a secreted lectin regulating matrix-to-cell interactions, promotes progression of NASH by interacting with the interleukin-33 (IL33)/ST2 axis.
      • Pejnovic N.
      • Jeftic I.
      • Jovicic N.
      • Arsenijevic N.
      • Lukic M.L.
      Galectin-3 and IL-33/ST2 axis roles and interplay in diet-induced steatohepatitis.
      Although a clinical trial (NCT02462967) of belapectin, an inhibitor of galectin-3, did not improve fibrosis in patients with NASH, a significant decrease in hepatocyte ballooning was observed.
      • Chalasani N.
      • Abdelmalek M.F.
      • Garcia-Tsao G.
      • Vuppalanchi R.
      • Alkhouri N.
      • Rinella M.
      • et al.
      Effects of belapectin, an inhibitor of galectin-3, in patients with nonalcoholic steatohepatitis with cirrhosis and portal hypertension.

      Extrahepatic DAMPs

      Cholesterol species act as surfactants to maintain the plasma membrane and excessive cholesterol intake and hypercholesterolemia are risk factors for NASH.
      • McGettigan B.
      • McMahan R.
      • Orlicky D.
      • Burchill M.
      • Danhorn T.
      • Francis P.
      • et al.
      Dietary lipids differentially shape nonalcoholic steatohepatitis progression and the transcriptome of Kupffer cells and infiltrating macrophages.
      Cholesterol crystals are delivered by oxidised LDLs through CD36 and activate the NLRP3 inflammasome in MFs.
      • Sheedy F.J.
      • Grebe A.
      • Rayner K.J.
      • Kalantari P.
      • Ramkhelawon B.
      • Carpenter S.B.
      • et al.
      CD36 coordinates NLRP3 inflammasome activation by facilitating intracellular nucleation of soluble ligands into particulate ligands in sterile inflammation.
      Moreover, the cholesterol-lowering drugs ezetimibe and atorvastatin suppress NLRP3 expression and inflammation in an HFHC mouse model of NASH, while targeting CD36 protects mice from NASH.
      • Ioannou G.N.
      • Van Rooyen D.M.
      • Savard C.
      • Haigh W.G.
      • Yeh M.M.
      • Teoh N.C.
      • et al.
      Cholesterol-lowering drugs cause dissolution of cholesterol crystals and disperse Kupffer cell crown-like structures during resolution of NASH.
      ,
      • Zhao L.
      • Zhang C.
      • Luo X.
      • Wang P.
      • Zhou W.
      • Zhong S.
      • et al.
      CD36 palmitoylation disrupts free fatty acid metabolism and promotes tissue inflammation in non-alcoholic steatohepatitis.
      Advanced glycation end-products (AGEs) are generated via the non-enzymatic Amadori reaction between a reducing sugar (e.g., glucose) and proteins, lipids or nucleic acids.
      • Fu M.X.
      • Wells-Knecht K.J.
      • Blackledge J.A.
      • Lyons T.J.
      • Thorpe S.R.
      • Baynes J.W.
      Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction.
      Diabetic patients have increased AGEs due to hyperglycaemia.
      • Fu M.X.
      • Wells-Knecht K.J.
      • Blackledge J.A.
      • Lyons T.J.
      • Thorpe S.R.
      • Baynes J.W.
      Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction.
      In addition, population genetics suggest that a polymorphism in the AGE receptor (RAGE) gene and circulating soluble RAGE (encoded by AGER) are associated with the risk of NASH.
      • Mehta R.
      • Shaw G.
      • Masschelin P.
      • Felix S.
      • Otgonsuren M.
      • Baranova A.
      • et al.
      Polymorphisms in the receptor for advanced glycation end-products (RAGE) gene and circulating RAGE levels as a susceptibility factor for non-alcoholic steatohepatitis (NASH).
      In addition, a HFHC mouse model shows that dietary supplementation with AGEs aggravates inflammation and ROS production in KCs, exacerbating NASH-induced liver injury.
      • Leung C.
      • Herath C.B.
      • Jia Z.
      • Andrikopoulos S.
      • Brown B.E.
      • Davies M.J.
      • et al.
      Dietary advanced glycation end-products aggravate non-alcoholic fatty liver disease.
      However, global knockout of Ager in Ldlr−/− mice minimally affects progression of NASH under short- or long-term HFHC diet feeding.
      • Bijnen M.
      • Beelen N.
      • Wetzels S.
      • Gaar J.V.
      • Vroomen M.
      • Wijnands E.
      • et al.
      RAGE deficiency does not affect non-alcoholic steatohepatitis and atherosclerosis in Western type diet-fed Ldlr(-/-) mice.
      The role of RAGE in NASH remains inconclusive as these studies used mice of different sex.
      • Leung C.
      • Herath C.B.
      • Jia Z.
      • Andrikopoulos S.
      • Brown B.E.
      • Davies M.J.
      • et al.
      Dietary advanced glycation end-products aggravate non-alcoholic fatty liver disease.
      ,
      • Bijnen M.
      • Beelen N.
      • Wetzels S.
      • Gaar J.V.
      • Vroomen M.
      • Wijnands E.
      • et al.
      RAGE deficiency does not affect non-alcoholic steatohepatitis and atherosclerosis in Western type diet-fed Ldlr(-/-) mice.

      DAMPs in liver fibrosis

      Chronic liver injury leads to pathological scarring and fibrosis.
      • Sun M.
      • Kisseleva T.
      Reversibility of liver fibrosis.
      ,
      • Friedman S.L.
      Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver.
      DAMPs such as HMGB1, OPN, HSPs, IL33 and ATP activate HSCs, the main source of fibrillar collagen, the main ECM component in fibrosis
      • Mederacke I.
      • Hsu C.C.
      • Troeger J.S.
      • Huebener P.
      • Mu X.
      • Dapito D.H.
      • et al.
      Fate tracing reveals hepatic stellate cells as dominant contributors to liver fibrosis independent of its aetiology.
      ,
      • Iwaisako K.
      • Jiang C.
      • Zhang M.
      • Cong M.
      • Moore-Morris T.J.
      • Park T.J.
      • et al.
      Origin of myofibroblasts in the fibrotic liver in mice.
      (Table 2 and Fig. 4).
      Figure thumbnail gr4
      Fig. 4DAMPs activate HSCs and contribute to fibrosis.
      In addition to being a significant source of ROS, hepatocytes produce adenosine, OPN and HMGB1, which target HSCs through A2AR/A2BR, αvβ3 integrin and RAGE, respectively and activate HSCs to promote scar deposition. MFs are also a significant source of ROS due to NOX activation and they produce HMGB1, OPN, IL33 and HSPs, which signal through RAGE, αvβ3 integrin, IL33R and TLRs, respectively, in HSCs to magnify the fibrogenic response. The contribution of biliary epithelial cells to HSC activation is significant as they produce TGFβ, which enhances collagen type I synthesis. HMGB1, OPN, IL-33, HSPs, ATP and adenosine, through interaction with their receptors on HSCs, signal via MEK1/2/c-Jun, PI3k/pAKT/NF-κB and TGFBR/Smad4 pathways to enhance collagen type I. Ab, antibody; DAMP(s), damage-associated molecular pattern(s); HMGB1, high-mobility group box-1; HSC(s), hepatic stellate cell(s); HSF, heat shock factor; HSPs, heat shock proteins; IL1, interleukin-1; IL33R, IL33 receptor; MF(s), macrophage(s); NOX, NADPH oxidase; OPN, osteopontin; RAGE, receptor for advanced glycation end-products; ROS, reactive oxygen species; TGFβ, transforming growth factor β; TGFBR, transforming growth factor beta receptor; TLR(s), Toll-like receptor(s).
      Hepatic expression and serum levels of HMGB1 correlate with fibrosis stage in patients with chronic HCV or HBV infection, primary biliary cirrhosis and AH, as well as in mouse models of fibrosis based on administration of CCl4 or thioacetamide and in the bile duct ligation model.
      • Ge X.
      • Arriazu E.
      • Magdaleno F.
      • Antoine D.J.
      • Dela Cruz R.
      • Theise N.
      • et al.
      High mobility group box-1 drives fibrosis progression signaling via the receptor for advanced glycation end products in mice.
      ,
      • Inkaya A.C.
      • Demir N.A.
      • Kolgelier S.
      • Sumer S.
      • Demir L.S.
      • Ural O.
      • et al.
      Is serum high-mobility group box 1 (HMGB-1) level correlated with liver fibrosis in chronic hepatitis B?.
      ,
      • Hu Y.B.
      • Hu D.P.
      • Fu R.Q.
      Correlation between high mobility group box-1 protein and chronic hepatitis B infection with severe hepatitis B and acute-on-chronic liver failure: a meta-analysis.
      HMGB1 activates HSCs
      • He Q.
      • Fu Y.
      • Ding X.
      • Li D.
      • Wang Z.
      • Tian D.
      • et al.
      High-mobility group box 1 induces endoplasmic reticulum stress and activates hepatic stellate cells.
      and induces ER stress (unpublished observations). Our laboratory demonstrated that ablation of Hmgb1 in hepatocytes and myeloid cells as well as neutralisation of HMGB1 and RAGE protects mice from fibrosis.
      • Ge X.
      • Arriazu E.
      • Magdaleno F.
      • Antoine D.J.
      • Dela Cruz R.
      • Theise N.
      • et al.
      High mobility group box-1 drives fibrosis progression signaling via the receptor for advanced glycation end products in mice.
      In addition, HMGB1 signals through RAGE in HSCs to upregulate collagen type I expression via the pMEK1/2/pERK1/2/pc-Jun signalling pathway. We showed that pMEK1/2 is upstream of pAkt and enhances collagen type I as well.
      • Arriazu E.
      • Ge X.
      • Leung T.M.
      • Magdaleno F.
      • Lopategi A.
      • Lu Y.
      • et al.
      Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury.
      In addition, nilotinib, a tyrosine kinase inhibitor, ameliorates CCl4-induced fibrosis in rats by attenuating Hmgb1/Rage expression and oxidative stress.
      • Khanjarsim V.
      • Karimi J.
      • Khodadadi I.
      • Mohammadalipour A.
      • Goodarzi M.T.
      • Solgi G.
      • et al.
      Ameliorative effects of nilotinib on CCl4 induced liver fibrosis via attenuation of RAGE/HMGB1 gene expression and oxidative stress in rat.
      OPN, a matrix-bound protein sensitive to oxidant stress and highly induced upon liver damage emerges as a key DAMP in the pathogenesis of fibrosis by increasing HMGB1 and collagen type I expression in HSCs through RAGE.
      • Arriazu E.
      • Ge X.
      • Leung T.M.
      • Magdaleno F.
      • Lopategi A.
      • Lu Y.
      • et al.
      Signalling via the osteopontin and high mobility group box-1 axis drives the fibrogenic response to liver injury.
      OPN itself upregulates collagen type I through integrin αVβ3 engagement and PI3K/pAkt/NFκB signalling. Moreover, OPN drives ductular reaction and contributes to periportal scarring and fibrosis via TGFβ signalling.
      • Wang X.
      • Lopategi A.
      • Ge X.
      • Lu Y.
      • Kitamura N.
      • Urtasun R.
      • et al.
      Osteopontin induces ductular reaction contributing to liver fibrosis.
      Specific damage-associated molecular patterns participate in pathogenesis of chronic liver diseases such as alcohol-related liver disease, non-alcoholic steatohepatitis, liver fibrosis and liver cancer.
      HSP90 is involved in the activation and survival of HSCs.
      • Yu Z.
      • Jv Y.
      • Cai L.
      • Tian X.
      • Huo X.
      • Wang C.
      • et al.
      Gambogic acid attenuates liver fibrosis by inhibiting the PI3K/AKT and MAPK signaling pathways via inhibiting HSP90.
      ,
      • Zhang F.
      • Hao M.
      • Jin H.
      • Yao Z.
      • Lian N.
      • Wu L.
      • et al.
      Canonical hedgehog signalling regulates hepatic stellate cell-mediated angiogenesis in liver fibrosis.
      The HSP90 inhibitor 17-AAG induces apoptosis and reduces activation of HSCs in a thioacetamide model of fibrosis.
      • Myung S.J.
      • Yoon J.H.
      • Kim B.H.
      • Lee J.H.
      • Jung E.U.
      • Lee H.S.
      Heat shock protein 90 inhibitor induces apoptosis and attenuates activation of hepatic stellate cells.
      HSP47, a collagen-specific chaperone, plays a key role in the deposition of collagen around fibrotic areas and is thus involved in fibrosis.
      • Ito S.
      • Ogawa K.
      • Takeuchi K.
      • Takagi M.
      • Yoshida M.
      • Hirokawa T.
      • et al.
      A small-molecule compound inhibits a collagen-specific molecular chaperone and could represent a potential remedy for fibrosis.
      ,
      • Ito S.
      • Nagata K.
      Biology of Hsp47 (Serpin H1), a collagen-specific molecular chaperone.
      Moreover, inhibitors of HSP47 such as lactoferrin and silymarin prevent HSC activation.
      • Rizk F.H.
      • Sarhan N.I.
      • Soliman N.A.
      • Ibrahim M.A.A.
      • Abd-Elsalam M.
      • Abd-Elsalam S.
      Heat shock protein 47 as indispensible participant in liver fibrosis: possible protective effect of lactoferrin.
      Overexpression of heat shock factor 1 (HSF1) in HSCs activates them and increases cell proliferation by inducing HSP47 and upregulating the TGFβ/SMAD4 signalling pathway. Notably, miR-455-3p alleviates HSC activation and fibrosis by suppressing its target gene, Hsf1.
      • Wei S.
      • Wang Q.
      • Zhou H.
      • Qiu J.
      • Li C.
      • Shi C.
      • et al.
      miR-455-3p alleviates hepatic stellate cell activation and liver fibrosis by suppressing HSF1 expression.
      IL33 is constitutively present in the nucleus and binds DNA.
      • Pichery M.
      • Mirey E.
      • Mercier P.
      • Lefrancais E.
      • Dujardin A.
      • Ortega N.
      • et al.
      Endogenous IL-33 is highly expressed in mouse epithelial barrier tissues, lymphoid organs, brain, embryos, and inflamed tissues: in situ analysis using a novel Il-33-LacZ gene trap reporter strain.
      Hepatic IL33 expression is increased in mice with portal fibrosis and in liver biopsies from fibrotic patients.
      • Tan Z.
      • Liu Q.
      • Jiang R.
      • Lv L.
      • Shoto S.S.
      • Maillet I.
      • et al.
      Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells.
      ,
      • Liu J.
      • Yang Y.
      • Zheng C.
      • Chen G.
      • Shen Z.
      • Zheng S.
      • et al.
      Correlation of interleukin-33/ST2 receptor and liver fibrosis progression in biliary atresia patients.
      In chronic liver injury, IL33 binds the IL33 receptor (IL33R) and activates NF-κB and MAPKs to enhance profibrogenic responses.
      • McHedlidze T.
      • Waldner M.
      • Zopf S.
      • Walker J.
      • Rankin A.L.
      • Schuchmann M.
      • et al.
      Interleukin-33-dependent innate lymphoid cells mediate hepatic fibrosis.
      IL33 binding to its receptor also produces proinflammatory and T helper 2 (Th2) cytokines. Recombinant IL33 increases hepatic inflammation and activates HSCs – an effect abrogated by ablation of Il33r or pharmacological inhibition of MAPK signalling.
      • Tan Z.
      • Liu Q.
      • Jiang R.
      • Lv L.
      • Shoto S.S.
      • Maillet I.
      • et al.
      Interleukin-33 drives hepatic fibrosis through activation of hepatic stellate cells.
      ,
      • Gao Y.
      • Liu Y.
      • Yang M.
      • Guo X.
      • Zhang M.
      • Li H.
      • et al.
      IL-33 treatment attenuated diet-induced hepatic steatosis but aggravated hepatic fibrosis.
      To fuel various processes, cells transport ATP into the extracellular space via pannexin-1, converting ATP to AMP and adenosine. Extracellular ATP activates MFs through the P2X7 receptor; activated MFs release IL1β and HMGB1 that trigger inflammation and fibrogenesis.
      • Toki Y.
      • Takenouchi T.
      • Harada H.
      • Tanuma S.
      • Kitani H.
      • Kojima S.
      • et al.
      Extracellular ATP induces P2X7 receptor activation in mouse Kupffer cells, leading to release of IL-1beta, HMGB1, and PGE2, decreased MHC class I expression and necrotic cell death.
      Extracellular adenosine interacts with the A2A (A2AR) or A2B (A2BR) G-coupled protein receptors to directly stimulate fibroblast production of ECM and increase fibrosis.
      • Ferrari D.
      • Gambari R.
      • Idzko M.
      • Muller T.
      • Albanesi C.
      • Pastore S.
      • et al.
      Purinergic signaling in scarring.
      Deletion of Cd73 or Cd9, involved in adenosine production and blockade of A2A or A2B prevents fibrosis in mice.
      • Feig J.L.
      • Mediero A.
      • Corciulo C.
      • Liu H.
      • Zhang J.
      • Perez-Aso M.
      • et al.
      The antiviral drug tenofovir, an inhibitor of pannexin-1-mediated ATP release, prevents liver and skin fibrosis by downregulating adenosine levels in the liver and skin.
      In addition, mice lacking adenosine deaminase have a marked increase in extracellular adenosine and develop fibrosis, which is prevented by blockade of A2A and A2B.
      • Wang G.
      • Wang H.
      • Singh S.
      • Zhou P.
      • Yang S.
      • Wang Y.
      • et al.
      ADAR1 prevents liver injury from inflammation and suppresses interferon production in hepatocytes.