BACKGROUND & AIMS Development of nonalcoholic steatohepatitis (NASH) involves the innate immune system and is mediated by Kupffer cells and hepatic stellate cells (HSCs). Toll-like receptor 9 (TLR9) is a pattern recognition receptor that recognizes bacteria-derived cytosine phosphate guanine (CpG)–containing DNA and activates innate immunity. We investigated the role of TLR9 signaling and the inflammatory cytokine interleukin-1β (IL-1β) in steatohepatitis, fibrosis, and insulin resistance. METHODS Wild-type (WT), TLR9−/−, IL-1 receptor (IL-1R)−/−, and MyD88−/− mice were fed a choline-deficient amino acid-defined (CDAA) diet for 22 weeks and then assessed for steatohepatitis, fibrosis, and insulin resistance. Lipid accumulation and cell death were assessed in isolated hepatocytes. Kupffer cells and HSCs were isolated to assess inflammatory and fibrogenic responses, respectively. RESULTS The CDAA diet induced NASH in WT mice, characterized by steatosis, inflammation, fibrosis, and insulin resistance. TLR9−/− mice showed less steatohepatitis and liver fibrosis than WT mice. Among inflammatory cytokines, IL-1β production was suppressed in TLR9−/− mice. Kupffer cells produced IL-1β in response to CpG oligodeoxynucleotide. IL-1β but not CpG-oligodeoxynucleotides, increased lipid accumulation in hepatocytes. Lipid accumulation in hepatocytes led to nuclear factor-κB inactivation, resulting in cell death in response to IL-1β. IL-1β induced fibrogenic responses in HSCs, including secretion of tissue inhibitor of metalloproteinase-1. IL-1R−/− mice had reduced steatohepatitis and fibrosis, compared with WT mice. Mice deficient in MyD88, an adaptor molecule for TLR9 and IL-1R signaling, also had reduced steatohepatitis and fibrosis. TLR9−/−, IL-1R−/−, and MyD88−/− mice had less insulin resistance than WT mice on the CDAA diet. CONCLUSIONS In a mouse model of NASH, TLR9 signaling induces production of IL-1β by Kupffer cells, leading to steatosis, inflammation, and fibrosis.

recruitment of macrophages promotes nonalcoholic steatohepatitis through CCR2.

Emerging data have shown a close association between compositional changes in gut microbiota and the development of nonalcoholic fatty liver disease (NAFLD). The change in gut microbiota may alter nutritional absorption and storage. In addition, gut microbiota are a source of Toll-like receptor (TLR) ligands, and their compositional change can also increase the amount of TLR ligands delivered to the liver. TLR ligands can stimulate liver cells to produce proinflammatory cytokines. Therefore, the gut-liver axis has attracted much interest, particularly regarding the pathogenesis of NAFLD. The abundance of the major gut microbiota, including Firmicutes and Bacteroidetes, has been considered a potential underlying mechanism of obesity and NAFLD, but the role of these microbiota in NAFLD remains unknown. Several reports have demonstrated that certain gut microbiota are associated with the development of obesity and NAFLD. For instance, a decrease in Akkermansia muciniphila causes a thinner intestinal mucus layer and promotes gut permeability, which allows the leakage of bacterial components. Interventions to increase Akkermansia muciniphila improve the metabolic parameters in obesity and NAFLD. In children, the levels of Escherichia were significantly increased in nonalcoholic steatohepatitis (NASH) compared with those in obese control. Escherichia can produce ethanol, which promotes gut permeability. Thus, normalization of gut microbiota using probiotics or prebiotics is a promising treatment option for NAFLD. In addition, TLR signaling in the liver is activated, and its downstream molecules, such as proinflammatory cytokines, are increased in NAFLD. To data, TLR2, TLR4, TLR5, and TLR9 have been shown to be associated with the pathogenesis of NAFLD. Therefore, gut microbiota and TLRs are targets for NAFLD treatment.

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Innate immune signaling associated with Toll like receptors (TLRs) is a key pathway involved in the progression of nonalcoholic steatohepatitis (NASH). Here we show that both TLR2 and palmitic acid are required for activation of the inflammasome, IL-1α and IL-1β, resulting in the progression of NASH. Wild type (WT) and TLR2−/− mice were fed a choline deficient amino acid defined (CDAA) diet for 22 weeks to induce NASH. Bone marrow transplanted-TLR2 chimeric mice were generated after the recipient mice were lethally irradiated. Kupffer cells and hepatic stellate cells (HSCs) were isolated from WT mice and stimulated with TLR2 ligand and/or palmitic acid. WT mice on the CDAA diet developed profound steatohepatitis and liver fibrosis. In contrast, TLR2−/− mice had suppressed progression of NASH. While both Kupffer cells and HSCs respond to TLR2 ligand, TLR2 bone marrow chimeric mice demonstrated that Kupffer cells were relatively more important than HSCs in TLR2-mediated progression of NASH. In vitro, palmitic acid alone did not increase TLR2 signaling-target genes including cytokines and inflammasome components in Kupffer cells and HSCs. The TLR2 ligand increased Nod-like receptor protein 3, an inflammasome component, in Kupffer cells, but not in HSCs. In the presence of TLR2 ligand, palmitic acid did induce caspase-1 activation and release of IL-1α and IL-1β in Kupffer cells, however, these effects were not observed in HSCs. In vivo, WT on the CDAA diet showed increased caspase-1 activation in the liver and elevated serum levels of IL-1α and IL-1β levels, which were suppressed in TLR2−/− mice. Conclusion: TLR2 and palmitic acid cooperatively activate inflammasome in Kupffer cells/macrophages in the development of NASH.