Transmembrane protein 173 (TMEM173 or STING) signaling by macrophage activates the type I interferon-mediated innate immune response. The innate immune response contributes to hepatic steatosis and non-alcoholic fatty liver disease (NAFLD). We investigated wither STING regulates diet-induced in hepatic steatosis, inflammation, and liver fibrosis in mice. Mice with disruption of Tmem173 (STING^gt) on a C57BL/6J background, mice without disruption of this gene (controls), and mice with disruption of Tmem173 only in myeloid cells were fed a standard chow diet, a high-fat diet (HFD, 60% fat calories), or a methionine- and choline-deficient diet (MCD). Liver tissues were collected and analyzed by histology and immunohistochemistry. Bone marrow cells were isolated from mice, differentiated into macrophages, and incubated with DMXAA (an activator of STING) or cGAMP. Macrophages or their media were applied to mouse hepatocytes or human hepatic stellate cells (LX2) cells, which were analyzed for cytokine expression, protein phosphorylation, and fat deposition (oil red O staining following incubation with palmitate). We obtained liver tissues from patients with and without NAFLD and analyzed these by immunohistochemistry. Non-parenchymal cells of liver tissues from patients with NAFLD had higher levels of STING than liver tissues from patients without NAFLD. STING^gt mice and mice with disruption only in myeloid cells developed less severe hepatic steatosis, inflammation, and/or fibrosis following a HFD or MCD than control mice. Levels of phosphorylated JNK and p65 and the mRNAs encoding TNF, IL1B, and IL6 (markers of inflammation) were significantly lower in liver tissues from STING^gt mice vs control mice after a HFD or MCD. Transplantation of bone marrow cells from control mice to STING^gt mice restored the severity of steatosis and inflammation following a HFD. Macrophages from control, but not STING^gt mice, increased markers of inflammation in response to lipopolysaccharide and cGAMP. Hepatocytes and stellate cells co-cultured with STING^gt macrophages in the presence of DMXAA, or incubated with the medium collected from these macrophages, had decreased fat deposition and markers of inflammation compared to hepatocytes incubated with control macrophages. We found levels of STING to be increased in liver tissues from patients with NAFLD mice with HFD-induced steatosis. In mice, loss of STING from liver macrophages reduces the severity of fibrosis and the inflammatory response. STING might be a therapeutic target for NAFLD.

Whether or not cholangiocytes or their hepatic progenitors undergo an epithelial-to-mesenchymal transition (EMT) to become matrix-producing myofibroblasts during biliary fibrosis is a significant ongoing controversy. To assess whether EMT is active during biliary fibrosis, we used Alfp-Cre × Rosa26-YFP mice, in which the epithelial cells of the liver (hepatocytes, cholangiocytes, and their bipotential progenitors) are heritably labeled at high efficiency with yellow fluorescent protein (YFP). Primary cholangiocytes isolated from our reporter strain were able to undergo EMT in vitro when treated with transforming growth factor-β1 alone or in combination with tumor necrosis factor-α, as indicated by adoption of fibroblastoid morphology, intracellular relocalization of E-cadherin, and expression of α-smooth muscle actin (α-SMA). To determine whether EMT occurs in vivo , we induced liver fibrosis in Alfp-Cre × Rosa26-YFP mice using the bile duct ligation (BDL) (2, 4, and 8 weeks), carbon tetrachloride (CCl4) (3 weeks), and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC; 2 and 3 weeks) models. In no case did we find evidence of colocalization of YFP with the mesenchymal markers S100A4, vimentin, α-SMA, or procollagen 1α2, although these proteins were abundant in the peribiliary regions. Conclusion: Hepatocytes and cholangiocytes do not undergo EMT in murine models of hepatic fibrosis. (Hepatology 2011;)

Epigenetic changes are associated with the regulation of transcription of key cell regulatory genes [micro RNAs (miRNAs)] during different types of liver injury. This study evaluated the role of methylation-associated miRNA, miR-34a, in alcoholic liver diseases. We identified that ethanol feeding for 4 weeks significantly up-regulated 0.8% of known miRNA compared with controls, including miR-34a. Treatment of normal human hepatocytes (N-Heps) and cholangiocytes [human intrahepatic biliary epithelial cells (HiBECs)] with ethanol and lipopolysaccharide induced a significant increase of miR-34a expression. Overexpression of miR-34a decreased ethanol-induced apoptosis in both N-Heps and HiBECs. In support of the concept that the 5'-promoter region of miR-34a was noted to be embedded within a CpG island, the expression level of miR-34a was significantly increased after demethylation treatment in N-Heps and HiBECs. By methylation-specific PCR, we confirmed that miR-34a activation is associated with ethanol-linked hypomethylation of the miR-34a promoter. A combination of bioinformatics, dual-luciferase reporter assay, mass spectrometry, and Western blot analysis revealed that caspase-2 and sirtuin 1 are the direct targets of miR-34a. Furthermore, modulation of miR-34a also altered expression of matrix metalloproteases 1 and 2, the mediators involved in hepatic remodeling during alcoholic liver fibrosis. These findings provide the basis for an exciting field in which the epigenomic microRNAs of hepatic cells may be manipulated with potential therapeutic benefits in human alcoholic liver diseases.