Ling Yang

Inflammatory cell infiltration in the liver is a hallmark of nonalcoholic steatohepatitis (NASH). The chemokine-chemokine receptor interaction induces inflammatory cell recruitment. CC-chemokine receptor (CCR)2 is expressed on hepatic macrophages and hepatic stellate cells. This study aims to investigate the therapeutic potential of CCR2 to NASH. Twenty-two weeks on a choline-deficient amino acid-defined (CDAA) diet induced steatosis, inflammatory cell infiltration, and liver fibrosis with increased CCR2 and monocyte chemoattractant protein (MCP)-1 expression in the wild-type livers. The infiltrated macrophages expressed CD68, CCR2, and a marker of bone marrow-derived monocytes, Ly6C. CCR2(-/-) mice had less steatosis, inflammatory cell infiltration, and fibrosis, and hepatic macrophages expressing CD68 and Ly6C were decreased. Toll-like receptor (TLR)4(-/-), TLR9(-/-), and MyD88(-/-) mice had reduced hepatic macrophage infiltration with decreased MCP-1 and CCR2 expression because TLR signaling is a potent inducer of MCP-1. To assess the role of Kupffer cells at the onset of NASH, Kupffer cells were depleted by liposomal clodronate. The Kupffer cell depletion ameliorated steatohepatitis with a decrease in the MCP-1 expression and recruitment of Ly6C-expressing macrophages at the onset of NASH. Finally, to test the therapeutic potential of targeting CCR2, a CCR2 inhibitor was administered to mice on a CDAA diet. The pharmaceutical inhibition of CCR2 prevented infiltration of the Ly6C-positive macrophages, resulting in an inhibition of liver inflammation and fibrosis. We concluded that CCR2 and Kupffer cells contribute to the progression of NASH by recruiting bone marrow-derived monocytes.

The spectrum of non-alcoholic fatty liver disease (NAFLD) ranges from simple hepatic steatosis, commonly associated with obesity, to non-alcoholic steatohepatitis, which can progress to fibrosis, cirrhosis and hepatocellular carcinoma. NAFLD pathophysiology involves environmental, genetic and metabolic factors, as well as changes in the intestinal microbiota and their products. Dysfunction of the intestinal barrier can contribute to NAFLD development and progression. Although there are technical limitations in assessing intestinal permeability in humans and the number of patients in these studies is rather small, fewer than half of the patients have increased intestinal permeability and translocation of bacterial products. Microbe-derived metabolites and the signalling pathways they affect might play more important roles in development of NAFLD. We review the microbial metabolites that contribute to the development of NAFLD, such as trimethylamine, bile acids, short-chain fatty acids and ethanol. We discuss the mechanisms by which metabolites produced by microbes might affect disease progression and/or serve as therapeutic targets or biomarkers for NAFLD.

UNLABELLED: 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, interleukin (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. Although 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 mice 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 the inflammasome in Kupffer cells and/or macrophages in the development of NASH.

UNLABELLED: Transforming growth factor beta (TGF-β) signaling activates Smad- and TGF-β-activated kinase 1 (TAK1)-dependent signaling to regulate cell survival, proliferation, fibrosis, and tumorigenesis. The effects of TGF-β signaling on metabolic syndrome, including nonalcoholic fatty liver disease, remain elusive. Wild-type (WT) and hepatocyte-specific TGF-β receptor type II-deficient (Tgfbr2ΔHEP) mice were fed a choline-deficient amino acid (CDAA)-defined diet for 22 weeks to induce NASH. WT mice fed a CDAA diet displayed increased activation of Smad2/3 and had marked lipid accumulation, inflammatory cell infiltration, hepatocyte death, and fibrosis; in comparison, Tgfbr2ΔHEP mice fed a CDAA diet had suppressed liver steatosis, inflammation, and fibrosis. Both palmitate-induced steatotic hepatocytes and hepatocytes isolated from WT mice fed a CDAA diet had increased susceptibility to TGF-β-mediated death. TGF-β-mediated death in steatotic hepatocytes was inhibited by silencing Smad2 or blocking reactive oxygen species (ROS) production and was enhanced by inhibiting TAK1 or nuclear factor kappa B. Increased hepatic steatosis in WT mice fed a CDAA diet was associated with the increased expression of lipogenesis genes (Dgat1 and Srebp1c), whereas the decreased steatosis in Tgfbr2ΔHEP mice was accompanied by the increased expression of genes involved in β-oxidation (Cpt1 and Acox1). In combination with palmitate treatment, TGF-β signaling promoted lipid accumulation with induction of lipogenesis-related genes and suppression of β-oxidation-related genes in hepatocytes. Silencing Smad2 decreased TGF-β-mediated lipid accumulation and corrected altered gene expression related to lipid metabolism in hepatocytes. Finally, we confirmed that livers from patients with nonalcoholic steatohepatitis (NASH) displayed phosphorylation and nuclear translocation of Smad2/3. CONCLUSIONS: TGF-β signaling in hepatocytes contributes to hepatocyte death and lipid accumulation through Smad signaling and ROS production that promote the development of NASH.