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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,1 despite recent data suggesting that natural killer cells are responsible also for liver damage in viral hepatitis infection.2 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.3–6
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,7 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.8–10 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,11 whereas in persons with NASH, it may be related to small intestinal bacterial overgrowth and alterations of the intestinal barrier.12 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.13 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.14,15 The important role of TLR-4 in NASH has also been documented in rodent models16,17; 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,18 but has no role in the pathogenesis of ASH.14
In this issue of Gastroenterology, Miura et al19 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 al19 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 al19 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.20 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,21 dendritic cells (DCs),22 neutrophils,23 B cells,24 and sinusoidal endothelial cells.25,26 Similar to how activation of TLR-9 on Kupffer cells leads to inflammation as reported by Miura et al,19 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.21,23,26 By contrast, activation of TLR-9 on conventional DCs results in IL-10 secretion that subsequently attenuates inflammatory response and liver ischemia/reperfusion injury.22 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 al19 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.19 Additional studies from Miura et al19 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 al27 and Watanabe et al28 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,19 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,19 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.23,26,28 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,11 and up-regulate expression of TLR-9 in the liver,29 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 NASH14,16,17,19; 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.30,31 By contrast, in vitro pretreatment with LPS for 2 or 12 hours primed the inflammatory response of mouse macrophages to subsequent CpG DNA stimulation.31 In vivo pretreatment with CpG DNA enhanced tumor necrosis factor-α production and liver damage upon subsequent challenge with LPS.30 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.
Conflicts of interest
The author discloses no conflicts.