Background & aims

Toll-like receptor 4 (TLR4) signaling is activated through 2 adaptor proteins: MyD88 and TIR-domain containing adaptor-inducing interferon-β (TRIF). TLR4 and MyD88 are crucial in nonalcoholic steatohepatitis (NASH) and fibrosis. However, the role of TRIF in TLR4-mediated NASH and fibrosis has been elusive. This study investigated the differential roles of TRIF in hepatic steatosis and inflammation/fibrosis.

Methods

A choline-deficient amino acid defined (CDAA) diet was used for the mouse NASH model. On this diet, the mice develop hepatic steatosis, inflammation, and fibrosis. TLR4 wild-type and TLR4^-/- bone marrow chimeric mice and TRIF^-/- mice were fed CDAA or a control diet for 22 weeks. Hepatic steatosis, inflammation, and fibrosis were examined.

Results

In the CDAA diet-induced NASH, the mice with wild-type bone marrow had higher alanine aminotransferase and hepatic tumor necrosis factor levels than the mice with TLR4^-/- bone marrow. The nonalcoholic fatty liver disease activity score showed that both wild-type and TLR4^-/- bone marrow chimeras had reduced hepatic steatosis, and that both types of chimeras had similar levels of inflammation and hepatocyte ballooning to whole-body wild-type mice. Notably, wild-type recipients showed more liver fibrosis than TLR4^-/- recipients. Although TRIF^-/- mice showed reduced hepatic steatosis, these mice showed more liver injury, inflammation, and fibrosis than wild-type mice. TRIF^-/- stellate cells and hepatocytes produced more C-X-C motif chemokine ligand 1 (CXCL1) and C-C motif chemokine ligand than wild-type cells in response to lipopolysaccharide. Consistently, TRIF^-/- mice showed increased CXCL1 and CCL3 expression along with neutrophil and macrophage infiltration, which promotes liver inflammation and injury.

Conclusions

In TLR4-mediated NASH, different liver cells have distinct roles in hepatic steatosis, inflammation, and fibrosis. TRIF promotes hepatic steatosis but it inhibits injury, inflammation, and fibrosis.

The MAP kinase kinase kinase TGFβ-activated kinase 1 (TAK1) is activated by TLRs, IL-1, TNF, and TGFβ and in turn activates IKK-NF-κB and JNK, which regulate cell survival, growth, tumorigenesis, and metabolism. TAK1 signaling also upregulates AMPK activity and autophagy. Here, we investigated TAK1-dependent regulation of autophagy, lipid metabolism, and tumorigenesis in the liver. Fasted mice with hepatocyte-specific deletion of Tak1 exhibited severe hepatosteatosis with increased mTORC1 activity and suppression of autophagy compared with their WT counterparts. TAK1-deficient hepatocytes exhibited suppressed AMPK activity and autophagy in response to starvation or metformin treatment; however, ectopic activation of AMPK restored autophagy in these cells. Peroxisome proliferator-activated receptor α (PPARα) target genes and β-oxidation, which regulate hepatic lipid degradation, were also suppressed in hepatocytes lacking TAK1. Due to suppression of autophagy and β-oxidation, a high-fat diet challenge aggravated steatohepatitis in mice with hepatocyte-specific deletion of Tak1. Notably, inhibition of mTORC1 restored autophagy and PPARα target gene expression in TAK1-deficient livers, indicating that TAK1 acts upstream of mTORC1. mTORC1 inhibition also suppressed spontaneous liver fibrosis and hepatocarcinogenesis in animals with hepatocyte-specific deletion of Tak1. These data indicate that TAK1 regulates hepatic lipid metabolism and tumorigenesis via the AMPK/mTORC1 axis, affecting both autophagy and PPARα activity.