MicroRNAs are fine tuners of diverse biological responses and are expressed in various cell types of the liver. Here we hypothesized that circulating microRNAs (miRNAs) may serve as biomarkers of liver damage and inflammation. We studied miRNA-122 that is abundant in hepatocytes and miR-155, -146a and -125b that regulate inflammation in immune cells in mouse models of alcoholic liver disease (ALD), drug (acetaminophen; APAP)-induced liver injury (DILI), and Toll-like receptor (TLR) 9+4 ligands-induced inflammatory cell-mediated liver damage. We found that serum/plasma miR-122 correlated with ALT increases in the liver damage caused by alcohol, APAP and TLR9 (CpG)+4 (LPS) ligands. MiR-155, a regulator of inflammation, was increased in serum/plasma in alcoholic and inflammatory liver injury. Alcohol failed to increase serum miR-122 in TLR4-deficient and p47phox-deficient mice that were protected from ALD. We found the most robust increase in plasma miR-122 in DILI and it correlated with the highest ALT levels. Consistent with the massive inflammatory cell infiltration in the liver, plasma miR-155 and miR-146a were significantly elevated after CpG+LPS administration. We show for the first time that, depending on the type of liver injury, circulating miRNAs show association either with the exosome-rich or protein-rich compartments. In ALD and in inflammatory liver injury, serum/plasma miR-122 and miR-155 were predominantly associated with exosome-rich fraction while in DILI/APAP injury these miRNAs were present in the protein-rich fraction. In conclusion, our results suggest that circulating miRNAs may serve as biomarkers to differentiate between hepatocyte injury and inflammation and the exosome versus protein association of miRNAs may provide further specificity to mechanisms of liver pathology.

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediatedincrease in TNF␣ production has a central role in the pathogenesis of alcoholic liver disease. Micro-RNA (miR)-125b, miR-146a, and miR-155 can regulate inflammatory responses to LPS. Here we evaluated the involvement of miRs in alcoholinduced macrophage activation. Chronic alcohol treatment in vitro resulted in a time-dependent increase in miR-155 but not miR-125b or miR-146a levels in RAW 264.7 macrophages. Furthermore, alcohol pretreatment augmented LPS-induced miR-155 expression in macrophages. We found a linear correlation between alcohol-induced increase in miR-155 and TNF␣ induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNF␣ production in isolated KCs when compared with pairfed controls. The mechanistic role of miR-155 in TNF␣ regulation was indicated by decreased TNF␣ levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNF␣ production after miR-155 overexpression, respectively. We found that miR-155 affected TNF␣ mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNF␣ mRNA half-life. Using the NF-B inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-B activation mediated the up-regulation of miR-155 by alcohol in KCs. In conclusion, our novel data demonstrate that chronic alcohol consumption increases miR-155 in macrophages via NF-B and the increased miR-155 contributes to alcohol-induced elevation in TNF␣ production via increased mRNA stability.

The Toll-like receptor 4 (TLR4) that recognizes endotoxin, a trigger of inflammation in alcoholic liver disease (ALD), activates two signaling pathways utilizing different adapter molecules: the common TLR adapter, myeloid differentiation factor 88 (MyD88), or Toll/interleukin immuneresponse-domain-containing adaptor inducing interferon (IFN)-␤. The MyD88 pathway induces proinflammatory cytokine activation, a critical mediator of ALD. Here we evaluated the role of MyD88 in alcohol-induced liver injury in wild-type, TLR2-deficient, TLR4-deficient, or MyD88-deficient (knockout [KO]) mice after administration of the Lieber-De-Carli diet (4.5% volume/volume ethanol) or an isocaloric liquid control diet for 5 weeks. Alcohol feeding resulted in a significant increase in serum alanine aminotransferase levels, liver steatosis and triglyceride levels suggesting liver damage in WT, TLR2-KO, and MyD88-KO but not in TLR4-KO mice. Expression of inflammatory mediators (tumor necrosis factor-␣ and interleukin-6) and TLR4 coreceptors (CD14 and MD2) was significantly higher in livers of alcohol-fed WT, TLR2-KO, or MyD88-KO, but not in TLR4-KO mice, compared to controls. Reactive oxygen radicals produced by cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complexes contribute to alcoholic liver damage. Alcohol feeding-induced expression and activation of cytochrome P450 and the nicotinamide adenine dinucleotide phosphate complex were prevented by TLR4-deficiency but not by MyD88-deficiency. Liver expression of interferon regulatory factor 3 (IRF3), a MyD88-independent signaling molecule, was not affected by chronic alcohol treatment in whole livers of WT mice or in any of the KO mice. However, the induction of IRF7, an IRF3-inducible gene, was found in Kupffer cells of alcohol-fed WT mice. Alcohol feeding also induced nuclear factor-B activation in a TLR4-dependent MyD88-independent manner. Conclusion: While TLR4 deficiency was protective, MyD88 deficiency failed to prevent alcohol-induced liver damage and inflammation. These results suggest that the common TLR adapter, MyD88, is dispensable in TLR4-mediated liver injury in ALD. (HEPATOLOGY 2008;48:1224-1231 A lcoholic liver disease (ALD) is characterized by a spectrum of liver pathology ranging from fatty liver, steatohepatitis, to cirrhosis, and it represents the second leading cause for liver transplantation in the United States. 1 Gut-derived lipopolysaccharide (LPS), a component of the gram-negative bacterial wall, has been proposed as a key player in the pathogenesis of ALD. [2][3][4] Exposure to LPS during chronic alcohol consumption results in increased production of inflammatory mediators, as well as in induction of reactive oxygen species (ROS), leading to progression of liver injury. 5,6 Indeed, mice deficient in tumor necrosis factor-alpha (TNF␣) type I receptor were protected from alcohol-induced liver injury. 7 Of the different sources of ROS, the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation was suggested by atten...

Background & Aims Alcoholic liver disease (ALD) ranges from fatty liver to inflammation and cirrhosis. miRNA-155 is an important regulator of inflammation. In this study, we describe the in vivo role of miR-155 in ALD. Methods Wild type, WT (C57/BL6J) or miR-155 KO and TLR4 KO mice received Lieber-DeCarli diet for 5 weeks. Some mice received corn oil or CCl4 for 2 or 9 weeks. Results We found that miR-155 KO mice are protected from alcohol-induced steatosis and inflammation. The reduction in alcohol-induced fat accumulation in miR-155 KO mice was associated with increased PPRE and PPARα (miR-155 target) binding and decreased MCP1 production. Treatment with a miR-155 inhibitor increased PPARγ expression in naive and alcohol treated RAW macrophages. Alcohol increased lipid metabolism genes (FABP4, LXRα, ACC1 and LDLR) in WT mice and this was prevented in KO mice. Alcohol diet induced increase in the number of CD163+CD206+ infiltrating macrophages and neutrophils in WT was prevented in miR-155 KO mice. Kupffer cells isolated from miR-155 KO mice exhibited predominance of M2 phenotype when exposed to M1 polarized signals and this was due to increased C/EBPβ. Profibrotic genes were attenuated in miR-155 KO mice after alcohol diet or CCl4 treatment. Compared to WT attenuation in CCl4 induced hydroxyproline and α SMA was observed in KO mice. Finally, we show TLR4 signaling regulates miR-155 as TLR4 KO mice showed no induction of miR-155 after alcohol diet. Conclusions Collectively our results demonstrated the role of miR-155 in alcohol-induced steatohepatitis and fibrosis in vivo.

BackgroundIt has been well documented that alcohol and its metabolites induce injury and inflammation in the liver. However, there is no potential biomarker to monitor the extent of liver injury in alcoholic hepatitis patients. MicroRNAs (miRNAs) are a class of non-coding RNAs that are involved in various physiologic and pathologic processes. In the circulation, a great proportion of miRNAs is associated with extracellular vesicles (EVs)/exosomes. Here, we hypothesized that the exosome-associated miRNAs can be used as potential biomarkers in alcoholic hepatitis (AH).MethodsExosomes were isolated from sera of alcohol-fed mice or pair-fed mice, and plasma of alcoholic hepatitis patients or healthy controls by ExoQuick. The exosomes were characterized by transmission electron microscopy and Western blot and enumerated with a Nanoparticle Tracking Analysis system. Firefly™ microRNA Assay was performed on miRNA extracted from mice sera. TaqMan microRNA assay was used to identify differentially expressed miRNAs in plasma of cohort of patients with AH versus controls followed by construction of receiver operating characteristic (ROC) curves to determine the sensitivity and specificity of the candidates.ResultsThe total number of circulating EVs was significantly increased in mice after alcohol feeding. Those EVs mainly consisted of exosomes, the smaller size vesicle subpopulation of EVs. By performing microarray screening on exosomes, we found nine inflammatory miRNAs which were deregulated in sera of chronic alcohol-fed mice compared to controls including upregulated miRNAs: miRNA-192, miRNA-122, miRNA-30a, miRNA-744, miRNA-1246, miRNA 30b and miRNA-130a. The ROC analyses indicated excellent diagnostic value of miRNA-192, miRNA-122, and miRNA-30a to identify alcohol-induced liver injury. We further validated findings from our animal model in human samples. Consistent with the animal model, total number of EVs, mostly exosomes, was significantly increased in human subjects with AH. Both miRNA-192 and miRNA-30a were significantly increased in the circulation of subjects with AH. miRNA-192 showed promising value for the diagnosis of AH.ConclusionElevated level of EVs/exosomes and exosome-associated miRNA signature could serve as potential diagnostic markers for AH. In addition to the biomarker diagnostic capabilities, these findings may facilitate development of novel strategies for diagnostics, monitoring, and therapeutics of AH.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0623-9) contains supplementary material, which is available to authorized users.