Donna Catalano

Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases

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, which is abundant in hepatocytes, and miR‐155, ‐146a, and ‐125b, which 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 ligand‐induced inflammatory cell‐mediated liver damage. We found that serum/plasma miR‐122 correlated with alanine aminotransferase (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 are associated 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 the exosome‐rich fraction, whereas in DILI/APAP injury these miRNAs were present in the protein‐rich fraction. 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. (HEPATOLOGY 2012;56:1946–1957)

The critical role of toll-like receptor (TLR) 4 in alcoholic liver disease is independent of the common TLR adapter MyD88

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 immune‐response–domain‐containing adaptor inducing interferon (IFN)‐β. 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–α 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–κB 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.)

Up-regulation of microRNA-155 in macrophages contributes to increased tumor necrosis factor {alpha} (TNF{alpha}) production via increased mRNA half-life in alcoholic liver disease

Activation of Kupffer cells (KCs) by gut-derived lipopolysaccharide (LPS) and Toll-Like Receptors 4 (TLR4)-LPS-mediated increase in TNFα 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 alcohol-induced 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 TNFα induction. In a mouse model of alcoholic liver disease, we found a significant increase in both miR-155 levels and TNFα production in isolated KCs when compared with pair-fed controls. The mechanistic role of miR-155 in TNFα regulation was indicated by decreased TNFα levels in alcohol-treated macrophages after inhibition of miR-155 and by increased TNFα production after miR-155 overexpression, respectively. We found that miR-155 affected TNFα mRNA stability because miR-155 inhibition decreased whereas miR-155 overexpression increased TNFα mRNA half-life. Using the NF-κB inhibitors, MG-132 or Bay11-7082, we demonstrated that NF-κB 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-κB and the increased miR-155 contributes to alcohol-induced elevation in TNFα production via increased mRNA stability.

MicroRNA expression profile in Lieber-DeCarli diet-induced alcoholic and methionine choline deficient diet-induced nonalcoholic steatohepatitis models in mice.

BACKGROUND Alcoholic and nonalcoholic 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. MicroRNAs (miRNA) are highly conserved noncoding 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, alanine aminotransferase (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 TNFalpha, suggesting that animal models portray the pathophysiological features of alcoholic and nonalcoholic 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, 5 miRNAs were common in alcoholic and nonalcoholic 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 down-regulated 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 nonalcoholic fatty liver disease.

An essential role for monocyte chemoattractant protein‐1 in alcoholic liver injury: Regulation of proinflammatory cytokines and hepatic steatosis in mice

The importance of chemokines in alcoholic liver injury has been implicated. The role of the chemokine, monocyte chemoattractant protein‐1 (MCP‐1), elevated in patients with alcoholic liver disease is not yet understood. Here, we evaluated the pathophysiological significance of MCP‐1 and its receptor, chemokine (C‐C motif) receptor 2 (CCR2), in alcoholic liver injury. The Leiber‐DeCarli diet containing alcohol or isocaloric control diets were fed to wild‐type (WT) and MCP‐1‐deficient knockout (KO) mice for 6 weeks. In vivo and in vitro assays were performed to study the role of MCP‐1 in alcoholic liver injury. MCP‐1 was increased in Kupffer cells (KCs) as well as hepatocytes of alcohol‐fed mice. Alcohol feeding increased serum alanine aminotransferase in WT and CCR2KO, but not MCP‐1KO, mice. Alcohol‐induced liver steatosis and triglyceride were attenuated in alcohol‐fed MCP‐1KO, but high in CCR2KO mice, compared to WT, whereas serum endotoxin was high in alcohol‐fed WT and MCP‐1KO mice. Expression of liver proinflammatory cytokines tumor necrosis factor alpha, interleukin (IL)‐1β, IL‐6, KC/IL‐8, intercellular adhesion molecule 1, and cluster of differentiation 68 was induced in alcohol‐fed WT, but inhibited in MCP‐1KO, mice independent of nuclear factor kappa light‐chain enhancer of activated B cell activation in KCs. Oxidative stress, but not cytochrome P450 2E1, was prevented in chronic alcohol‐fed MCP‐1KO mice, compared to WT. Increased expression of peroxisome proliferator‐activated receptor (PPAR)α and PPARγ was accompanied by nuclear translocation, DNA binding, and induction of fatty acid metabolism genes acyl coenzyme A oxidase and carnitine palmitoyltransferase 1A in livers of alcohol‐fed MCP‐1KO mice, compared to WT controls. In vitro assays uncovered an inhibitory effect of recombinant MCP‐1 on PPARα messenger RNA and peroxisome proliferator response element binding in hepatocytes independent of CCR2. Conclusion: Deficiency of MCP‐1 protects mice against alcoholic liver injury, independent of CCR2, by inhibition of proinflammatory cytokines and induction of genes related to fatty acid oxidation, linking chemokines to hepatic lipid metabolism. (HEPATOLOGY 2011)

Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice

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-α 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 α-smooth muscle actin (SMA) staining, procollagen-I, transforming growth factor-β1, α-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.

The Opposite Effects of Acute and Chronic Alcohol on Lipopolysaccharide-Induced Inflammation Are Linked to IRAK-M in Human Monocytes

Impaired host defense after alcohol use is linked to altered cytokine production, however, acute and chronic alcohol differently modulate monocyte/macrophage activation. We hypothesized that in human monocytes, acute alcohol induces hyporesponsiveness to LPS, resulting in decreased TNF-α, whereas chronic alcohol increases TNF-α by sensitization to LPS. We found that acute alcohol increased IL-1R-associated kinase-monocyte (IRAK-M), a negative regulator of IRAK-1, in human monocytes. This was associated with decreased IκBα kinase activity, NFκB DNA binding, and NFκB-driven reporter activity after LPS stimulation. In contrast, chronic alcohol decreased IRAK-M expression but increased IRAK-1 and IKK kinase activities, NFκB DNA binding, and NFκB-reporter activity. Inhibition of IRAK-M in acute alcohol-exposed monocytes using small interfering RNA restored the LPS-induced TNF-α production whereas over-expression of IRAK-M in chronic alcohol macrophages prevented the increase in TNF-α production. Addition of inhibitors of alcohol metabolism did not alter LPS signaling and TNF-α production during chronic alcohol exposure. IRAK-1 activation induces MAPKs that play an important role in TNF-α induction. We determined that acute alcohol decreased but chronic alcohol increased activation of ERK in monocytes and ERK inhibitor, PD98059, prevented the chronic alcohol-induced increase in TNF-α. In summary, inhibition of LPS-induced NFκB and ERK activation by acute alcohol leads to hyporesponsiveness of monocytes to LPS due to increased IRAK-M. In contrast, chronic alcohol sensitizes monocytes to LPS through decreased IRAK-M expression and activation of NFκB and ERK kinases. Our data indicate that IRAK-M is a central player in the opposite regulation of LPS signaling by different lengths of alcohol exposure in monocytes.

Acute binge drinking increases serum endotoxin and bacterial DNA levels in healthy individuals

Binge drinking, the most common form of alcohol consumption, is associated with increased mortality and morbidity; yet, its biological consequences are poorly defined. Previous studies demonstrated that chronic alcohol use results in increased gut permeability and increased serum endotoxin levels that contribute to many of the biological effects of chronic alcohol, including alcoholic liver disease. In this study, we evaluated the effects of acute binge drinking in healthy adults on serum endotoxin levels. We found that acute alcohol binge resulted in a rapid increase in serum endotoxin and 16S rDNA, a marker of bacterial translocation from the gut. Compared to men, women had higher blood alcohol and circulating endotoxin levels. In addition, alcohol binge caused a prolonged increase in acute phase protein levels in the systemic circulation. The biological significance of the in vivo endotoxin elevation was underscored by increased levels of inflammatory cytokines, TNFα and IL-6, and chemokine, MCP-1, measured in total blood after in vitro lipopolysaccharide stimulation. Our findings indicate that even a single alcohol binge results in increased serum endotoxin levels likely due to translocation of gut bacterial products and disturbs innate immune responses that can contribute to the deleterious effects of binge drinking.

Inhibition of Myeloid Dendritic Cell Accessory Cell Function and Induction of T Cell Anergy by Alcohol Correlates with Decreased IL-12 Production

Alcohol consumption inhibits accessory cell function and Ag-specific T cell responses. Myeloid dendritic cells (DCs) coordinate innate immune responses and T cell activation. In this report, we found that in vivo moderate alcohol intake (0.8 g/kg of body weight) in normal volunteers inhibited DC allostimulatory capacity. Furthermore, in vitro alcohol treatment during DC differentiation significantly reduced allostimulatory activity in a MLR using naive CD4+ T cells, and inhibited tetanus toxoid Ag presentation by DCs. Alcohol-treated DCs showed reduced IL-12, increased IL-10 production, and a decrease in expression of the costimulatory molecules CD80 and CD86. Addition of exogenous IL-12 and IL-2, but not neutralization of IL-10, during MLR ameliorated the reduced allostimulatory capacity of alcohol-treated DCs. Naive CD4+ T cells primed with alcohol-treated DCs showed decreased IFN-γ production that was restored by exogenous IL-12, indicating inhibition of Th1 responses. Furthermore, CD4+ T cells primed with alcohol-treated DCs were hyporesponsive to subsequent stimulation with the same donor-derived normal DCs, suggesting the ability of alcohol-treated DCs to induce T cell anergy. LPS-induced maturation of alcohol-treated immature DCs partially restored the reduced allostimulatory activity, whereas alcohol given only during DC maturation failed to inhibit DC functions, suggesting that alcohol primarily impairs DC differentiation rather than maturation. NFκB activation, a marker of DC maturation was not affected by alcohol. Taken together, alcohol both in vitro and in vivo can impair generation of Th1 immune responses via inhibition of DC differentiation and accessory cell function through mechanisms that involve decreased IL-12 induction.

Inhibition of superantigen-induced T cell proliferation and monocyte IL-1 beta, TNF-alpha, and IL-6 production by acute ethanol treatment.

Alcohol use has been shown to decrease monocyte antigen presentation capacity and inflammatory cytokine production, thereby increasing susceptibility to infections. Here, we demonstrate that in vitro acute treatment of normal monocytes with pharmacological doses of ethanol can decrease superantigen [Staphylococcus enterotoxins B (SEB) and A (SEA)]‐induced T cell proliferation. Furthermore, ethanol treatment (25–100 mM) significantly inhibited SEA‐ or SEB‐induced production of tumor necrosis factor‐alpha (TNF‐α), interleukin‐1β (IL‐1β), and IL‐6 in monocytes. Ethanol‐induced down‐regulation of monocyte TNF‐α, IL‐1β, and IL‐6 occurred at both the protein and mRNA levels. Additional data suggest that ethanol can decrease IL‐1β mRNA stability. Furthermore, experiments using cycloheximide indicate that de novo protein synthesis is required for the inhibitory effect of ethanol on SEB‐induced IL‐1β mRNA production. Finally, ethanol treatment decreased HLA‐DR expression in monocytes, suggesting that ethanol treatment can compromise monocyte stimulation by down‐regulating the SEB‐binding capacity of monocytes. These results suggest that acute ethanol treatment can interfere with monocyte activation by SEB at multiple steps. Consequently, decreased superantigen‐induced polyclonal T cell activation and inflammatory monokine production would contribute to an impaired immune response to bacterial challenge with superantigens after acute alcohol intake.