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...

Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice. Am J Physiol Gastrointest Liver Physiol 300: G433-G441, 2011. First published January 13, 2011; doi:10.1152/ajpgi.00163.2009.-Toll-like receptor 4 (TLR4) and its coreceptor, myeloid differentiation factor-2 (MD-2), are key in recognition of lipopolysaccharide (LPS) and activation of proinflammatory pathways. Here we tested the hypothesis that TLR4 and its coreceptor MD-2 play a central role in nonalcoholic steatohepatitis (NASH) and liver fibrosis in nonalcoholic fatty liver disease. Mice of control genotypes and those deficient in MD-2 or TLR4 [knockout (KO)] received methionine choline-deficient (MCD) or methionine choline-supplemented (MCS) diet. In mice of control genotypes, MCD diet resulted in NASH, liver triglycerides accumulation, and increased thiobarbituric acid reactive substances, a marker of lipid peroxidation, compared with MCS diet. These features of NASH were significantly attenuated in MD-2 KO and TLR4 KO mice. Serum alanine aminotransferase, an indicator of liver injury, was increased in MCD diet-fed genotype controls but was attenuated in MD-2 KO and TLR4 KO mice. Inflammatory activation, indicated by serum TNF-␣ and nictoinamide adenine dinucleotide phosphate oxidase complex mRNA expression and activation, was significantly lower in MCD diet-fed MD-2 KO and TLR4 KO compared with corresponding genotype control mice. Markers of liver fibrosis [collagen by Sirius red and ␣-smooth muscle actin (SMA) staining, procollagen-I, transforming growth factor-␤1, ␣-SMA, matrix metalloproteinase-2, and tissue inhibitor of matrix metalloproteinase-1 mRNA] were attenuated in MD-2 and TLR4 KO compared with their control genotype counterparts. In conclusion, our results demonstrate a novel, critical role for LPS recognition complex, including MD-2 and TLR4, through NADPH activation in liver steatosis, and fibrosis in a NASH model in mice.

Non-alcoholic fatty liver disease (NAFLD) and its advanced stage, non-alcoholic steatohepatitis (NASH), are the most common causes of chronic liver disease in the United States. NASH features the metabolic syndrome, inflammation, and fibrosis. Probiotics exhibit immunoregulatory and anti-inflammatory activity. We tested the hypothesis that probiotic VSL#3 may ameliorate the methionine-choline-defficient (MCD) diet-induced mouse model of NASH. MCD diet resulted in NASH in C57Bl6 mice compared to methionine-choline supplemented (MCS) diet feeding evidenced by liver steatosis, increased triglycerides, inflammatory cell accumulation, increased TNFα, and fibrosis. VSL#3 failed to prevent MCD-induced liver steatosis or inflammation. MCD diet, even in the presence of VSL#3, induced upregulation of serum endotoxin and expression the TLR4 signaling components, including CD14 and MD2, MyD88 adaptor, and NF-κB activation. In contrast, VSL#3 treatment ameliorated MCD diet-induced liver fibrosis resulting in diminished accumulation of collagen and α-SMA. We identified increased expression of liver PPARs and decreased expression of pro-collagen and matrix metalloproteinases in mice fed MCD+VSL#3 compared to MCD diet alone. MCD diet triggered up-regulation of TGFβ, a known pro-fibrotic agent. In the presence of VSL#3 the MCD diet-induced expression of TGFβ was maintained, however, the expression of Bambi, a TGFβ pseudoreceptor with negative regulatory function, was increased. In summary, our data indicate that VSL#3 modulates liver fibrosis but does not protect from inflammation and steatosis in NASH. The mechanisms of VSL#3-mediated protection from MCD diet-induced liver fibrosis likely include modulation of collagen expression and impaired TGFβ signaling owed to modulation of TGFβ signaling.

Background Alcoholic and non-alcoholic steatohepatitis are leading causes of liver diseases worldwide. While of different etiology, these share common pathophysiological mechanisms and feature abnormal fat metabolism, inflammation and fibrosis. Micro-RNAs (miRNA) are highly conserved non-coding RNAs that control gene expression at the post-transcriptional level either via the degradation of target mRNAs or the inhibition of translation. Each miRNA controls the expression of multiple targets; miRNAs have been linked to regulation of lipid metabolism and inflammation. Methods We fed Lieber-DeCarli alcohol or methionine-choline-deficient (MCD) diets to C57Bl6 and analyzed livers for histopathology, cytokines by ELISA, ALT by biochemical assay, and microRNA profile by microarray. Results Both Lieber-DeCarli and MCD diets lead to development of liver steatosis, liver injury, indicated by increased ALT, and elevated levels of serum TNFα, suggesting that animal models portray the pathophysiological features of alcoholic and non-alcoholic fatty liver, respectively. We identified that Lieber-deCarli diet up-regulated 1% and down-regulated 1% of known miRNA; MCD diet up-regulated 3% and down-regulated 1% of known miRNA, compared to controls. Of miRNAs that changed expression levels, five miRNAs were common in alcoholic and non-alcoholic fatty livers: the expression of both miR-705 and miR-1224 was increased after Lieber-DeCarli or MCD diet feeding. In contrast, miR-182, miR-183, and miR-199a-3p were downregulated in Lieber-deCarli feeding, while MCD diet lead to their up-regulation, compared to corresponding controls. Conclusions Our findings indicate etiology-specific changes in miRNA expression profile during steatohepatitis models, which opens new avenues for research in the pathophysiology of alcoholic and non-alcoholic fatty liver diesase.

Toll-like receptors (TLR) recognize pathogen-derived molecules and induce downstream activation of inflammatory pathways. Fatty liver has been shown to result in increased sensitivity to lipopolysaccharide (LPS), a TLR4 ligand. In this study, we investigated the roles of TLR2 and TLR4 in liver damage and on cytokine induction in a methionine-choline deficient (MCD) diet-induced model of nonalcoholic steatohepatitis. We found that mice with nonalcoholic fatty liver had increased liver injury and inflammatory cytokine induction after challenge with a TLR4 but not with a TLR2 ligand. TLR2 deficient mice were not protected against the development of steatohepatitis after MCD diet feeding. On the contrary, TLR2 mice had significantly higher levels of serum ALT and greater TNF-alpha levels after LPS challenge suggesting increased liver injury. This was associated with reduced production of IL-6, a cytokine with hepatoprotective effects in fatty liver. Increased liver injury in the MCD diet-fed TLR2 mice was associated with reduced baseline and LPS-induced NF-kB and PPRE binding compared to MCS controls. These results demonstrate that TLR2 deficiency results in increased liver injury in association with nonalcoholic steatohepatitis and may suggest a protective role for TLR2-mediated signals in liver injury.