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Innate Immunity and Steatohepatitis: A Critical Role of Another Toll (TLR-9)

2010; Elsevier BV; Volume: 139; Issue: 1 Linguagem: Inglês

10.1053/j.gastro.2010.05.018

1528-0012

Bin Gao,

Immune Cell Function and Interaction

See "Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice," by Miura K, Kodama Y, Inokuchi S, et al, on page 323.Worldwide, chronic viral hepatitis, alcoholic steatohepatitis (ASH), and nonalcoholic steatohepatitis (NASH) are 3 major causes of chronic liver diseases, leading to cirrhosis and hepatocellular carcinoma. Although hepatitis viruses themselves are not cytopathogenic and do not kill hepatocytes, the adaptive immune responses (eg, cytotoxic T cells) to viral infection are considered key in causing hepatocellular damage,1Rehermann B. Hepatitis C virus versus innate and adaptive immune responses: a tale of coevolution and coexistence.J Clin Invest. 2009; 119: 1745-1754Google Scholar despite recent data suggesting that natural killer cells are responsible also for liver damage in viral hepatitis infection.2Ahlenstiel G. Titerence R.H. Koh C. et al.Natural killer cells are polarized toward cytotoxicity in chronic hepatitis C in an interferon-alfa-dependent manner.Gastroenterology. 2010; 138: 325-335Google Scholar By contrast, innate immunity seems to play a key role in the pathogenesis of ASH and NASH, with adaptive immunity playing a less important role.3Maher J.J. Leon P. Ryan J.C. Beyond insulin resistance: Innate immunity in nonalcoholic steatohepatitis.Hepatology. 2008; 48: 670-678Google Scholar, 4Valenti L. Fracanzani A.L. Fargion S. The immunopathogenesis of alcoholic and nonalcoholic steatohepatitis: two triggers for one disease?.Semin Immunopathol. 2009; 31: 359-369Google Scholar, 5Pritchard M.T. McMullen M.R. Medof M.E. et al.Role of complement in ethanol-induced liver injury.Adv Exp Med Biol. 2008; 632: 175-186Google Scholar, 6Torres D.M. Harrison S.A. Diagnosis and therapy of nonalcoholic steatohepatitis.Gastroenterology. 2008; 134: 1682-1698Google ScholarInnate immunity is the first line of defense against microbial invasion, and includes physical and chemical barriers, humoral factors, lymphocytic and phagocytic cells, and a group of pattern-recognition receptors that identifies specific signature molecules expressed on invading pathogens. The best examples of pattern-recognition receptors include a group of Toll-like receptors (TLRs), which recognizes pathogen-associated molecular patterns to determine the presence of pathogens. Once pathogens are identified, TLRs then induce multiple signaling pathways that regulate the expression of proinflammatory cytokines and chemokines to mount protective responses against invading pathogens. Owing to constant exposure to intestinal-derived bacterial products in portal blood, the liver has strong innate immunity,7Gao B. Jeong W.I. Tian Z. Liver: An organ with predominant innate immunity.Hepatology. 2008; 47: 729-736Google Scholar which not only plays an important role in quickly removing invading pathogens from the gut but may also contribute to the pathogenesis of liver diseases, such as ASH and NASH.Currently, it is widely accepted that lipopolysaccharide (LPS), a gut bacteria-derived endotoxin, is important for the development and progression of ASH and NASH through TLR-4 activation and induction of Kupffer cell activity.8Seki E. Brenner D.A. Toll-like receptors and adaptor molecules in liver disease: update.Hepatology. 2008; 48: 322-335Google Scholar, 9Mencin A. Kluwe J. Schwabe R.F. Toll-like receptors as targets in chronic liver diseases.Gut. 2009; 58: 704-720Google Scholar, 10Szabo G. Dolganiuc A. Mandrekar P. Pattern recognition receptors: a contemporary view on liver diseases.Hepatology. 2006; 44: 287-298Google Scholar Experimental and clinical data have demonstrated that levels of circulating and hepatic LPS are elevated in both ASH and NASH. Increased LPS levels in ASH are likely owing to increased gut permeability caused by excessive alcohol intake,11Rao R. Endotoxemia and gut barrier dysfunction in alcoholic liver disease.Hepatology. 2009; 50: 638-644Google Scholar whereas in persons with NASH, it may be related to small intestinal bacterial overgrowth and alterations of the intestinal barrier.12Wigg A.J. Roberts-Thomson I.C. Dymock R.B. et al.The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumour necrosis factor alpha in the pathogenesis of non-alcoholic steatohepatitis.Gut. 2001; 48: 206-211Google Scholar Activation of TLR-4 by LPS, which requires co-receptors CD14 and MD-2, results in activation of MyD88-dependent and TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent (MyD88-independent) signaling pathways. The MyD88-dependent pathway induces inflammatory cytokines through activation of NF-κB, whereas the TRIF-dependent pathway activates interferon regulatory factor 3 (IRF-3) and NF-κB, through induction of interferons and inflammatory cytokines, respectively. Early studies conducted primarily by Dr Thurman's group have demonstrated clearly that TLR-4 signaling plays a critical role in the development of ASH.13Arteel G.E. Oxidants and antioxidants in alcohol-induced liver disease.Gastroenterology. 2003; 124: 778-790Google Scholar Recent findings that disruption of the TRIF gene, but not MyD88, abolished ethanol-induced liver injury suggest that the development of ASH in mice is more likely due to TLR-4 that has been activated downstream of the TRIF pathway, rather than through the MyD88-dependent pathway.14Hritz I. Mandrekar P. Velayudham A. et al.The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88.Hepatology. 2008; 48: 1224-1231Google Scholar, 15Zhao X.J. Dong Q. Bindas J. et al.TRIF and IRF-3 binding to the TNF promoter results in macrophage TNF dysregulation and steatosis induced by chronic ethanol.J Immunol. 2008; 181: 3049-3056Google Scholar The important role of TLR-4 in NASH has also been documented in rodent models16Spruss A.K.G. Wagnerberger S. Haub S. et al.Toll-like receptor 4 is involved in the development of fructose-induced hepatic steatosis in mice.Hepatology. 2009; 50: 1094-1104Google Scholar, 17Rivera C.A. Adegboyega P. van Rooijen N. et al.Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis.J Hepatol. 2007; 47: 571-579Google Scholar; however, the exact pathway downstream of TLR-4 that contributes to the pathogenesis of NASH is currently unknown. Although the role of bacterially derived LPS, and its receptor TLR-4, in ASH and NASH has been extensively investigated in the last 2 decades, understanding of the effect of other bacterial components on the pathogenesis of these 2 disorders have been nonexistent until recently. Reportedly, Szabo's group has determined that TLR-2, which recognizes lipoproteins and peptidoglycans from gram-positive bacteria, plays a protective role in NASH,18Szabo G. Velayudham A. Romics Jr., L. et al.Modulation of non-alcoholic steatohepatitis by pattern recognition receptors in mice: the role of toll-like receptors 2 and 4.Alcohol Clin Exp Res. 2005; 29: 140S-145SGoogle Scholar but has no role in the pathogenesis of ASH.14Hritz I. Mandrekar P. Velayudham A. et al.The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88.Hepatology. 2008; 48: 1224-1231Google ScholarIn this issue of Gastroenterology, Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar have identified TLR-9, which recognizes bacterial unmethylated CpG DNA, as another important player in the pathogenesis of NASH based on a murine model of NASH induced by a choline-deficient amino acid-defined (CDAA) diet. A member of the TLR group, TLR-9 is expressed intracellularly within endosomal compartments, and activates innate immune defenses against viral and bacterial infection by binding to DNA rich in CpG motifs. Upon activation of TLR-9 by CpG DNA within the endosome, MyD88 complexes with a group of proteins (IRAK-1, IRAK-4, IRF-7, TRAF-6), and then induces interferon expression and NF-κB activation that subsequently upregulates inflammatory cytokines including interleukin (IL)-1β. In this elegant paper, Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar provide clear evidence suggesting that the TLR-9–MyD88–IL-1β pathway plays a critical role in inducing hepatic steatosis, inflammation, and fibrosis in a model of NASH induced by the CDAA diet. Through a series of in vivo experiments using several strains of knockout mice and in vitro experiments of hepatocyte culture, Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar demonstrated that consumption of a CDAA diet activates TLR-9 signaling on Kupffer cells, thereby inducing IL-1β production via a MyD88-dependent pathway. Additionally, IL-1β then increases lipid accumulation in hepatocytes by up-regulating diacyglyceride acyltranferase 2 (DGAT-2) and subsequently induces steatotic hepatocyte death. Produced by Kupffer cells, IL-1β was also previously shown to play an important role in the development of hepatic steatosis via down-regulating peroxisome proliferator activated receptor (PPAR)-α in a murine model using a high-fat diet.20Stienstra R. Saudale F. Duval C. et al.Kupffer cells promote hepatic steatosis via interleukin-1beta-dependent suppression of peroxisome proliferator-activated receptor alpha activity.Hepatology. 2010; 51: 511-522Google Scholar Thus, the steatogenic effect of IL-1β in the pathogenesis of NASH is likely mediated via both induction of DGAT-2 and down-regulation of PPAR-α.In addition to Kupffer cells, many other cell types are reportedly targets for TLR-9 ligands, including T and NK T cells,21Jiang W. Sun R. Zhou R. et al.TLR-9 activation aggravates concanavalin A-induced hepatitis via promoting accumulation and activation of liver CD4+ NKT cells.J Immunol. 2009; 182: 3768-3774Google Scholar dendritic cells (DCs),22Bamboat Z.M. Ocuin L.M. Balachandran V.P. et al.Conventional DCs reduce liver ischemia/reperfusion injury in mice via IL-10 secretion.J Clin Invest. 2010; 120: 559-569Google Scholar neutrophils,23Bamboat Z.M. Balachandran V.P. Ocuin L.M. et al.Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury.Hepatology. 2010; 51: 621-632Google Scholar B cells,24Kikuchi K. Lian Z.X. Yang G.X. et al.Bacterial CpG induces hyper-IgM production in CD27(+) memory B cells in primary biliary cirrhosis.Gastroenterology. 2005; 128: 304-312Abstract Full Text Full Text PDF Scopus (129) Google Scholar and sinusoidal endothelial cells.25Martin-Armas M. Simon-Santamaria J. Pettersen I. et al.Toll-like receptor 9 (TLR9) is present in murine liver sinusoidal endothelial cells (LSECs) and mediates the effect of CpG-oligonucleotides.J Hepatol. 2006; 44: 939-946Google Scholar, 26Imaeda A.B. Watanabe A. Sohail M.A. et al.Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome.J Clin Invest. 2009; 119: 305-314Google Scholar Similar to how activation of TLR-9 on Kupffer cells leads to inflammation as reported by Miura et al,19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar activation of TLR-9 on T cells, NK T cells, neutrophils, and sinusoidal endothelial cells also results in secretion of proinflammatory cytokines and potentiation of liver inflammation in various rodent models.21Jiang W. Sun R. Zhou R. et al.TLR-9 activation aggravates concanavalin A-induced hepatitis via promoting accumulation and activation of liver CD4+ NKT cells.J Immunol. 2009; 182: 3768-3774Google Scholar, 23Bamboat Z.M. Balachandran V.P. Ocuin L.M. et al.Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury.Hepatology. 2010; 51: 621-632Google Scholar, 26Imaeda A.B. Watanabe A. Sohail M.A. et al.Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome.J Clin Invest. 2009; 119: 305-314Google Scholar By contrast, activation of TLR-9 on conventional DCs results in IL-10 secretion that subsequently attenuates inflammatory response and liver ischemia/reperfusion injury.22Bamboat Z.M. Ocuin L.M. Balachandran V.P. et al.Conventional DCs reduce liver ischemia/reperfusion injury in mice via IL-10 secretion.J Clin Invest. 2010; 120: 559-569Google Scholar Collectively, the data suggest that TLR-9 targets multiple cell types to promote inflammation, and to also stimulate conventional DCs to induce anti-inflammatory responses. Although the study by Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar mainly focused on TLR-9 signaling in Kupffer cells, the role of TLR-9–MyD88–IL-1β in other cell types in the pathogenesis of NASH remains unknown.Beside inducing fatty liver and inflammation, the TLR-9–MyD88–IL-1β pathway also likely contributes to fibrogenesis in this NASH model because disruption of either TLR-9, MyD88, or IL-1β signaling significantly attenuates liver fibrosis.19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar Additional studies from Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar suggest that TLR-9 signaling plays an important role in fibrogenesis via induction of IL-1β that stimulates collagen and TIMP-1 expression in hepatic stellate cells, rather than through direct activation of TLR-9 signaling in hepatic stellate cells. However, Gabele et al27Gabele E. Muhlbauer M. Dorn C. et al.Role of TLR9 in hepatic stellate cells and experimental liver fibrosis.Biochem Biophys Res Commun. 2008; 376: 271-276Google Scholar and Watanabe et al28Watanabe A. Hashmi A. Gomes D.A. et al.Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9.Hepatology. 2007; 46: 1509-1518Google Scholar have reported that TLR-9 ligands can directly target hepatic stellate cells via binding TLR-9 on these cells, followed by inhibition of stellate cell chemotaxis and induction of stellate cell activation, ultimately leading to liver fibrosis. The discrepancy among these studies requires clarification.Although the important role of TLR-9 in NASH has been clearly demonstrated in this study by Miura et al,19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar the ligands that activate TLR-9 in this model remain unknown. Because bacterial DNA was detected by polymerase chain reaction from blood taken from mice fed a CDAA diet,19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar it is plausible that DNA derived from gut bacteria may serve as an exogenous ligand that activates TLR-9 in this model. In addition, TLR-9 can also recognize DNA released by necrotic hepatocytes and subsequently contributes to liver inflammation and injury induced by acetaminophen or ischemia/reperfusion.23Bamboat Z.M. Balachandran V.P. Ocuin L.M. et al.Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury.Hepatology. 2010; 51: 621-632Google Scholar, 26Imaeda A.B. Watanabe A. Sohail M.A. et al.Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome.J Clin Invest. 2009; 119: 305-314Google Scholar, 28Watanabe A. Hashmi A. Gomes D.A. et al.Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9.Hepatology. 2007; 46: 1509-1518Google Scholar This leads us to consider that DNA from necrotic hepatocytes may also induce TLR-9 activation in the pathogenesis of NASH. Because excessive alcohol consumption induces hepatocyte necrosis, which can release DNA, increase bacterial DNA transfer from the gut into the liver,11Rao R. Endotoxemia and gut barrier dysfunction in alcoholic liver disease.Hepatology. 2009; 50: 638-644Google Scholar and up-regulate expression of TLR-9 in the liver,29Gustot T. Lemmers A. Moreno C. et al.Differential liver sensitization to toll-like receptor pathways in mice with alcoholic fatty liver.Hepatology. 2006; 43: 989-1000Google Scholar we speculate that necrotic hepatocyte DNA and bacterial DNA could serve as endogenous and exogenous ligands, respectively, to activate TLR-9 on Kupffer cells, contributing to the pathogenesis of ASH. However, this hypothesis requires experimental evidence to ensure its validity.Thus far, several TLRs are known to contribute to the pathogenesis of ASH and NASH, which are collectively illustrated in Figure 1. Among them, both TLR-4 and TLR-9 have been shown to play an important role in the pathogenesis of ASH and NASH14Hritz I. Mandrekar P. Velayudham A. et al.The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88.Hepatology. 2008; 48: 1224-1231Google Scholar, 16Spruss A.K.G. Wagnerberger S. Haub S. et al.Toll-like receptor 4 is involved in the development of fructose-induced hepatic steatosis in mice.Hepatology. 2009; 50: 1094-1104Google Scholar, 17Rivera C.A. Adegboyega P. van Rooijen N. et al.Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis.J Hepatol. 2007; 47: 571-579Google Scholar, 19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar; however, the interaction of these 2 receptors is unknown. Kupffer cells, as well as other liver cells, likely encounter bacteria-derived components such as LPS and CpG DNA simultaneously or sequentially, which then activate TLR-4 and TLR-9, respectively, on Kupffer cells. Interestingly, it has been reported that signaling cross-talk between TLR-4 and TLR-9 can induce either cross-tolerance or the priming of inflammatory response. For example, in vitro pretreatment with LPS for 20 hours resulted in cross-tolerance to subsequent CpG DNA stimulation in RAW264.7 macrophages, and vice versa.30Dalpke A.H. Lehner M.D. Hartung T. et al.Differential effects of CpG-DNA in Toll-like receptor-2/-4/-9 tolerance and cross-tolerance.Immunology. 2005; 116: 203-212Google Scholar, 31De Nardo D. De Nardo C.M. Nguyen T. et al.Signaling crosstalk during sequential TLR4 and TLR9 activation amplifies the inflammatory response of mouse macrophages.J Immunol. 2009; 183: 8110-8118Google Scholar By contrast, in vitro pretreatment with LPS for 2 or 12 hours primed the inflammatory response of mouse macrophages to subsequent CpG DNA stimulation.31De Nardo D. De Nardo C.M. Nguyen T. et al.Signaling crosstalk during sequential TLR4 and TLR9 activation amplifies the inflammatory response of mouse macrophages.J Immunol. 2009; 183: 8110-8118Google Scholar In vivo pretreatment with CpG DNA enhanced tumor necrosis factor-α production and liver damage upon subsequent challenge with LPS.30Dalpke A.H. Lehner M.D. Hartung T. et al.Differential effects of CpG-DNA in Toll-like receptor-2/-4/-9 tolerance and cross-tolerance.Immunology. 2005; 116: 203-212Google Scholar The in vivo synergistic effect could be due to different expression profiles of TLR-4 and TLR-9 on various immune cells and liver cells. Whether TLR-4 and TLR-9 signaling also have synergistic effects in vivo in inducing steatosis, inflammation, and fibrosis in ASH and NASH remains unknown. Future studies to understand the cross-talk of TLR-4 and TLR-9 signaling in ASH and NASH may help us to design novel therapeutic strategies to target these TLRs to treat these 2 disorders. See "Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice," by Miura K, Kodama Y, Inokuchi S, et al, on page 323. See "Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice," by Miura K, Kodama Y, Inokuchi S, et al, on page 323. See "Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice," by Miura K, Kodama Y, Inokuchi S, et al, on page 323. Worldwide, chronic viral hepatitis, alcoholic steatohepatitis (ASH), and nonalcoholic steatohepatitis (NASH) are 3 major causes of chronic liver diseases, leading to cirrhosis and hepatocellular carcinoma. Although hepatitis viruses themselves are not cytopathogenic and do not kill hepatocytes, the adaptive immune responses (eg, cytotoxic T cells) to viral infection are considered key in causing hepatocellular damage,1Rehermann B. Hepatitis C virus versus innate and adaptive immune responses: a tale of coevolution and coexistence.J Clin Invest. 2009; 119: 1745-1754Google Scholar despite recent data suggesting that natural killer cells are responsible also for liver damage in viral hepatitis infection.2Ahlenstiel G. Titerence R.H. Koh C. et al.Natural killer cells are polarized toward cytotoxicity in chronic hepatitis C in an interferon-alfa-dependent manner.Gastroenterology. 2010; 138: 325-335Google Scholar By contrast, innate immunity seems to play a key role in the pathogenesis of ASH and NASH, with adaptive immunity playing a less important role.3Maher J.J. Leon P. Ryan J.C. Beyond insulin resistance: Innate immunity in nonalcoholic steatohepatitis.Hepatology. 2008; 48: 670-678Google Scholar, 4Valenti L. Fracanzani A.L. Fargion S. The immunopathogenesis of alcoholic and nonalcoholic steatohepatitis: two triggers for one disease?.Semin Immunopathol. 2009; 31: 359-369Google Scholar, 5Pritchard M.T. McMullen M.R. Medof M.E. et al.Role of complement in ethanol-induced liver injury.Adv Exp Med Biol. 2008; 632: 175-186Google Scholar, 6Torres D.M. Harrison S.A. Diagnosis and therapy of nonalcoholic steatohepatitis.Gastroenterology. 2008; 134: 1682-1698Google Scholar Innate immunity is the first line of defense against microbial invasion, and includes physical and chemical barriers, humoral factors, lymphocytic and phagocytic cells, and a group of pattern-recognition receptors that identifies specific signature molecules expressed on invading pathogens. The best examples of pattern-recognition receptors include a group of Toll-like receptors (TLRs), which recognizes pathogen-associated molecular patterns to determine the presence of pathogens. Once pathogens are identified, TLRs then induce multiple signaling pathways that regulate the expression of proinflammatory cytokines and chemokines to mount protective responses against invading pathogens. Owing to constant exposure to intestinal-derived bacterial products in portal blood, the liver has strong innate immunity,7Gao B. Jeong W.I. Tian Z. Liver: An organ with predominant innate immunity.Hepatology. 2008; 47: 729-736Google Scholar which not only plays an important role in quickly removing invading pathogens from the gut but may also contribute to the pathogenesis of liver diseases, such as ASH and NASH. Currently, it is widely accepted that lipopolysaccharide (LPS), a gut bacteria-derived endotoxin, is important for the development and progression of ASH and NASH through TLR-4 activation and induction of Kupffer cell activity.8Seki E. Brenner D.A. Toll-like receptors and adaptor molecules in liver disease: update.Hepatology. 2008; 48: 322-335Google Scholar, 9Mencin A. Kluwe J. Schwabe R.F. Toll-like receptors as targets in chronic liver diseases.Gut. 2009; 58: 704-720Google Scholar, 10Szabo G. Dolganiuc A. Mandrekar P. Pattern recognition receptors: a contemporary view on liver diseases.Hepatology. 2006; 44: 287-298Google Scholar Experimental and clinical data have demonstrated that levels of circulating and hepatic LPS are elevated in both ASH and NASH. Increased LPS levels in ASH are likely owing to increased gut permeability caused by excessive alcohol intake,11Rao R. Endotoxemia and gut barrier dysfunction in alcoholic liver disease.Hepatology. 2009; 50: 638-644Google Scholar whereas in persons with NASH, it may be related to small intestinal bacterial overgrowth and alterations of the intestinal barrier.12Wigg A.J. Roberts-Thomson I.C. Dymock R.B. et al.The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumour necrosis factor alpha in the pathogenesis of non-alcoholic steatohepatitis.Gut. 2001; 48: 206-211Google Scholar Activation of TLR-4 by LPS, which requires co-receptors CD14 and MD-2, results in activation of MyD88-dependent and TIR-domain-containing adapter-inducing interferon-β (TRIF)-dependent (MyD88-independent) signaling pathways. The MyD88-dependent pathway induces inflammatory cytokines through activation of NF-κB, whereas the TRIF-dependent pathway activates interferon regulatory factor 3 (IRF-3) and NF-κB, through induction of interferons and inflammatory cytokines, respectively. Early studies conducted primarily by Dr Thurman's group have demonstrated clearly that TLR-4 signaling plays a critical role in the development of ASH.13Arteel G.E. Oxidants and antioxidants in alcohol-induced liver disease.Gastroenterology. 2003; 124: 778-790Google Scholar Recent findings that disruption of the TRIF gene, but not MyD88, abolished ethanol-induced liver injury suggest that the development of ASH in mice is more likely due to TLR-4 that has been activated downstream of the TRIF pathway, rather than through the MyD88-dependent pathway.14Hritz I. Mandrekar P. Velayudham A. et al.The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88.Hepatology. 2008; 48: 1224-1231Google Scholar, 15Zhao X.J. Dong Q. Bindas J. et al.TRIF and IRF-3 binding to the TNF promoter results in macrophage TNF dysregulation and steatosis induced by chronic ethanol.J Immunol. 2008; 181: 3049-3056Google Scholar The important role of TLR-4 in NASH has also been documented in rodent models16Spruss A.K.G. Wagnerberger S. Haub S. et al.Toll-like receptor 4 is involved in the development of fructose-induced hepatic steatosis in mice.Hepatology. 2009; 50: 1094-1104Google Scholar, 17Rivera C.A. Adegboyega P. van Rooijen N. et al.Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis.J Hepatol. 2007; 47: 571-579Google Scholar; however, the exact pathway downstream of TLR-4 that contributes to the pathogenesis of NASH is currently unknown. Although the role of bacterially derived LPS, and its receptor TLR-4, in ASH and NASH has been extensively investigated in the last 2 decades, understanding of the effect of other bacterial components on the pathogenesis of these 2 disorders have been nonexistent until recently. Reportedly, Szabo's group has determined that TLR-2, which recognizes lipoproteins and peptidoglycans from gram-positive bacteria, plays a protective role in NASH,18Szabo G. Velayudham A. Romics Jr., L. et al.Modulation of non-alcoholic steatohepatitis by pattern recognition receptors in mice: the role of toll-like receptors 2 and 4.Alcohol Clin Exp Res. 2005; 29: 140S-145SGoogle Scholar but has no role in the pathogenesis of ASH.14Hritz I. Mandrekar P. Velayudham A. et al.The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88.Hepatology. 2008; 48: 1224-1231Google Scholar In this issue of Gastroenterology, Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar have identified TLR-9, which recognizes bacterial unmethylated CpG DNA, as another important player in the pathogenesis of NASH based on a murine model of NASH induced by a choline-deficient amino acid-defined (CDAA) diet. A member of the TLR group, TLR-9 is expressed intracellularly within endosomal compartments, and activates innate immune defenses against viral and bacterial infection by binding to DNA rich in CpG motifs. Upon activation of TLR-9 by CpG DNA within the endosome, MyD88 complexes with a group of proteins (IRAK-1, IRAK-4, IRF-7, TRAF-6), and then induces interferon expression and NF-κB activation that subsequently upregulates inflammatory cytokines including interleukin (IL)-1β. In this elegant paper, Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar provide clear evidence suggesting that the TLR-9–MyD88–IL-1β pathway plays a critical role in inducing hepatic steatosis, inflammation, and fibrosis in a model of NASH induced by the CDAA diet. Through a series of in vivo experiments using several strains of knockout mice and in vitro experiments of hepatocyte culture, Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar demonstrated that consumption of a CDAA diet activates TLR-9 signaling on Kupffer cells, thereby inducing IL-1β production via a MyD88-dependent pathway. Additionally, IL-1β then increases lipid accumulation in hepatocytes by up-regulating diacyglyceride acyltranferase 2 (DGAT-2) and subsequently induces steatotic hepatocyte death. Produced by Kupffer cells, IL-1β was also previously shown to play an important role in the development of hepatic steatosis via down-regulating peroxisome proliferator activated receptor (PPAR)-α in a murine model using a high-fat diet.20Stienstra R. Saudale F. Duval C. et al.Kupffer cells promote hepatic steatosis via interleukin-1beta-dependent suppression of peroxisome proliferator-activated receptor alpha activity.Hepatology. 2010; 51: 511-522Google Scholar Thus, the steatogenic effect of IL-1β in the pathogenesis of NASH is likely mediated via both induction of DGAT-2 and down-regulation of PPAR-α. In addition to Kupffer cells, many other cell types are reportedly targets for TLR-9 ligands, including T and NK T cells,21Jiang W. Sun R. Zhou R. et al.TLR-9 activation aggravates concanavalin A-induced hepatitis via promoting accumulation and activation of liver CD4+ NKT cells.J Immunol. 2009; 182: 3768-3774Google Scholar dendritic cells (DCs),22Bamboat Z.M. Ocuin L.M. Balachandran V.P. et al.Conventional DCs reduce liver ischemia/reperfusion injury in mice via IL-10 secretion.J Clin Invest. 2010; 120: 559-569Google Scholar neutrophils,23Bamboat Z.M. Balachandran V.P. Ocuin L.M. et al.Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury.Hepatology. 2010; 51: 621-632Google Scholar B cells,24Kikuchi K. Lian Z.X. Yang G.X. et al.Bacterial CpG induces hyper-IgM production in CD27(+) memory B cells in primary biliary cirrhosis.Gastroenterology. 2005; 128: 304-312Abstract Full Text Full Text PDF Scopus (129) Google Scholar and sinusoidal endothelial cells.25Martin-Armas M. Simon-Santamaria J. Pettersen I. et al.Toll-like receptor 9 (TLR9) is present in murine liver sinusoidal endothelial cells (LSECs) and mediates the effect of CpG-oligonucleotides.J Hepatol. 2006; 44: 939-946Google Scholar, 26Imaeda A.B. Watanabe A. Sohail M.A. et al.Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome.J Clin Invest. 2009; 119: 305-314Google Scholar Similar to how activation of TLR-9 on Kupffer cells leads to inflammation as reported by Miura et al,19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar activation of TLR-9 on T cells, NK T cells, neutrophils, and sinusoidal endothelial cells also results in secretion of proinflammatory cytokines and potentiation of liver inflammation in various rodent models.21Jiang W. Sun R. Zhou R. et al.TLR-9 activation aggravates concanavalin A-induced hepatitis via promoting accumulation and activation of liver CD4+ NKT cells.J Immunol. 2009; 182: 3768-3774Google Scholar, 23Bamboat Z.M. Balachandran V.P. Ocuin L.M. et al.Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury.Hepatology. 2010; 51: 621-632Google Scholar, 26Imaeda A.B. Watanabe A. Sohail M.A. et al.Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome.J Clin Invest. 2009; 119: 305-314Google Scholar By contrast, activation of TLR-9 on conventional DCs results in IL-10 secretion that subsequently attenuates inflammatory response and liver ischemia/reperfusion injury.22Bamboat Z.M. Ocuin L.M. Balachandran V.P. et al.Conventional DCs reduce liver ischemia/reperfusion injury in mice via IL-10 secretion.J Clin Invest. 2010; 120: 559-569Google Scholar Collectively, the data suggest that TLR-9 targets multiple cell types to promote inflammation, and to also stimulate conventional DCs to induce anti-inflammatory responses. Although the study by Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar mainly focused on TLR-9 signaling in Kupffer cells, the role of TLR-9–MyD88–IL-1β in other cell types in the pathogenesis of NASH remains unknown. Beside inducing fatty liver and inflammation, the TLR-9–MyD88–IL-1β pathway also likely contributes to fibrogenesis in this NASH model because disruption of either TLR-9, MyD88, or IL-1β signaling significantly attenuates liver fibrosis.19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar Additional studies from Miura et al19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar suggest that TLR-9 signaling plays an important role in fibrogenesis via induction of IL-1β that stimulates collagen and TIMP-1 expression in hepatic stellate cells, rather than through direct activation of TLR-9 signaling in hepatic stellate cells. However, Gabele et al27Gabele E. Muhlbauer M. Dorn C. et al.Role of TLR9 in hepatic stellate cells and experimental liver fibrosis.Biochem Biophys Res Commun. 2008; 376: 271-276Google Scholar and Watanabe et al28Watanabe A. Hashmi A. Gomes D.A. et al.Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9.Hepatology. 2007; 46: 1509-1518Google Scholar have reported that TLR-9 ligands can directly target hepatic stellate cells via binding TLR-9 on these cells, followed by inhibition of stellate cell chemotaxis and induction of stellate cell activation, ultimately leading to liver fibrosis. The discrepancy among these studies requires clarification. Although the important role of TLR-9 in NASH has been clearly demonstrated in this study by Miura et al,19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar the ligands that activate TLR-9 in this model remain unknown. Because bacterial DNA was detected by polymerase chain reaction from blood taken from mice fed a CDAA diet,19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar it is plausible that DNA derived from gut bacteria may serve as an exogenous ligand that activates TLR-9 in this model. In addition, TLR-9 can also recognize DNA released by necrotic hepatocytes and subsequently contributes to liver inflammation and injury induced by acetaminophen or ischemia/reperfusion.23Bamboat Z.M. Balachandran V.P. Ocuin L.M. et al.Toll-like receptor 9 inhibition confers protection from liver ischemia-reperfusion injury.Hepatology. 2010; 51: 621-632Google Scholar, 26Imaeda A.B. Watanabe A. Sohail M.A. et al.Acetaminophen-induced hepatotoxicity in mice is dependent on Tlr9 and the Nalp3 inflammasome.J Clin Invest. 2009; 119: 305-314Google Scholar, 28Watanabe A. Hashmi A. Gomes D.A. et al.Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9.Hepatology. 2007; 46: 1509-1518Google Scholar This leads us to consider that DNA from necrotic hepatocytes may also induce TLR-9 activation in the pathogenesis of NASH. Because excessive alcohol consumption induces hepatocyte necrosis, which can release DNA, increase bacterial DNA transfer from the gut into the liver,11Rao R. Endotoxemia and gut barrier dysfunction in alcoholic liver disease.Hepatology. 2009; 50: 638-644Google Scholar and up-regulate expression of TLR-9 in the liver,29Gustot T. Lemmers A. Moreno C. et al.Differential liver sensitization to toll-like receptor pathways in mice with alcoholic fatty liver.Hepatology. 2006; 43: 989-1000Google Scholar we speculate that necrotic hepatocyte DNA and bacterial DNA could serve as endogenous and exogenous ligands, respectively, to activate TLR-9 on Kupffer cells, contributing to the pathogenesis of ASH. However, this hypothesis requires experimental evidence to ensure its validity. Thus far, several TLRs are known to contribute to the pathogenesis of ASH and NASH, which are collectively illustrated in Figure 1. Among them, both TLR-4 and TLR-9 have been shown to play an important role in the pathogenesis of ASH and NASH14Hritz I. Mandrekar P. Velayudham A. et al.The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88.Hepatology. 2008; 48: 1224-1231Google Scholar, 16Spruss A.K.G. Wagnerberger S. Haub S. et al.Toll-like receptor 4 is involved in the development of fructose-induced hepatic steatosis in mice.Hepatology. 2009; 50: 1094-1104Google Scholar, 17Rivera C.A. Adegboyega P. van Rooijen N. et al.Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis.J Hepatol. 2007; 47: 571-579Google Scholar, 19Miura K. Kodama Y. Inokuchi S. et al.Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1β in mice.Gastroenterology. 2010; 139: 323-334Google Scholar; however, the interaction of these 2 receptors is unknown. Kupffer cells, as well as other liver cells, likely encounter bacteria-derived components such as LPS and CpG DNA simultaneously or sequentially, which then activate TLR-4 and TLR-9, respectively, on Kupffer cells. Interestingly, it has been reported that signaling cross-talk between TLR-4 and TLR-9 can induce either cross-tolerance or the priming of inflammatory response. For example, in vitro pretreatment with LPS for 20 hours resulted in cross-tolerance to subsequent CpG DNA stimulation in RAW264.7 macrophages, and vice versa.30Dalpke A.H. Lehner M.D. Hartung T. et al.Differential effects of CpG-DNA in Toll-like receptor-2/-4/-9 tolerance and cross-tolerance.Immunology. 2005; 116: 203-212Google Scholar, 31De Nardo D. De Nardo C.M. Nguyen T. et al.Signaling crosstalk during sequential TLR4 and TLR9 activation amplifies the inflammatory response of mouse macrophages.J Immunol. 2009; 183: 8110-8118Google Scholar By contrast, in vitro pretreatment with LPS for 2 or 12 hours primed the inflammatory response of mouse macrophages to subsequent CpG DNA stimulation.31De Nardo D. De Nardo C.M. Nguyen T. et al.Signaling crosstalk during sequential TLR4 and TLR9 activation amplifies the inflammatory response of mouse macrophages.J Immunol. 2009; 183: 8110-8118Google Scholar In vivo pretreatment with CpG DNA enhanced tumor necrosis factor-α production and liver damage upon subsequent challenge with LPS.30Dalpke A.H. Lehner M.D. Hartung T. et al.Differential effects of CpG-DNA in Toll-like receptor-2/-4/-9 tolerance and cross-tolerance.Immunology. 2005; 116: 203-212Google Scholar The in vivo synergistic effect could be due to different expression profiles of TLR-4 and TLR-9 on various immune cells and liver cells. Whether TLR-4 and TLR-9 signaling also have synergistic effects in vivo in inducing steatosis, inflammation, and fibrosis in ASH and NASH remains unknown. Future studies to understand the cross-talk of TLR-4 and TLR-9 signaling in ASH and NASH may help us to design novel therapeutic strategies to target these TLRs to treat these 2 disorders.