Gyongyi Szabo

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)

IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice

Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of proinflammatory cytokines, including IL-1β. IL-1β, type I IL-1 receptor (IL-1R1), and IL-1 receptor antagonist (IL-1Ra) are all important regulators of the IL-1 signaling complex, which plays a role in inflammation. Furthermore, IL-1β maturation is dependent on caspase-1 (Casp-1). Using IL-1Ra-treated mice as well as 3 mouse models deficient in regulators of IL-1β activation (Casp-1 and ASC) or signaling (IL-1R1), we found that IL-1β signaling is required for the development of alcohol-induced liver steatosis, inflammation, and injury. Increased IL-1β was due to upregulation of Casp-1 activity and inflammasome activation. The pathogenic role of IL-1 signaling in ALD was attributable to the activation of the inflammasome in BM-derived Kupffer cells. Importantly, in vivo intervention with a recombinant IL-1Ra blocked IL-1 signaling and markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. Furthermore, physiological doses of IL-1β induced steatosis, increased the inflammatory and prosteatotic chemokine MCP-1 in hepatocytes, and augmented TLR4-dependent upregulation of inflammatory signaling in macrophages. In conclusion, we demonstrated that Casp-1-dependent upregulation of IL-1β and signaling mediated by IL-1R1 are crucial in ALD pathogenesis. Our findings suggest a potential role of IL-1R1 inhibition in the treatment of ALD.

Fatty acid and endotoxin activate inflammasomes in mouse hepatocytes that release danger signals to stimulate immune cells

The pathogenesis of nonalcoholic steatohepatitis (NASH) and inflammasome activation involves sequential hits. The inflammasome, which cleaves pro–interleukin‐1β (pro–IL‐1β) into secreted IL‐1β, is induced by endogenous and exogenous danger signals. Lipopolysaccharide (LPS), a toll‐like receptor 4 ligand, plays a role in NASH and also activates the inflammasome. In this study, we hypothesized that the inflammasome is activated in NASH by multiple hits involving endogenous and exogenous danger signals. Using mouse models of methionine choline–deficient (MCD) diet–induced NASH and high‐fat diet–induced NASH, we found up‐regulation of the inflammasome [including NACHT, LRR, and PYD domains–containing protein 3 (NALP3; cryopyrin), apoptosis‐associated speck‐like CARD‐domain containing protein, pannexin‐1, and pro–caspase‐1] at the messenger RNA (mRNA) level increased caspase‐1 activity, and mature IL‐1β protein levels in mice with steatohepatitis in comparison with control livers. There was no inflammasome activation in mice with only steatosis. The MCD diet sensitized mice to LPS‐induced increases in NALP3, pannexin‐1, IL‐1β mRNA, and mature IL‐1β protein levels in the liver. We demonstrate for the first time that inflammasome activation occurs in isolated hepatocytes in steatohepatitis. Our novel data show that the saturated fatty acid (FA) palmitic acid (PA) activates the inflammasome and induces sensitization to LPS‐induced IL‐1β release in hepatocytes. Furthermore, PA triggers the release of danger signals from hepatocytes in a caspase‐dependent manner. These hepatocyte‐derived danger signals, in turn, activate inflammasome, IL‐1β, and tumor necrosis factor α release in liver mononuclear cells. Conclusion: Our novel findings indicate that saturated FAs represent an endogenous danger in the form of a first hit, up‐regulate the inflammasome in NASH, and induce sensitization to a second hit with LPS for IL‐β release in hepatocytes. Furthermore, hepatocytes exposed to saturated FAs release danger signals that trigger inflammasome activation in immune cells. Thus, hepatocytes play a key role in orchestrating tissue responses to danger signals in NASH. (HEPATOLOGY 2011;)

A Recent Perspective on Alcohol, Immunity, and Host Defense

BACKGROUND Multiple line of clinical and experimental evidence demonstrates that both acute, moderate, and chronic, excessive alcohol use result in various abnormalities in the functions of the immune system. METHODS Medline and PubMed databases were used to identify published reports with particular interest in the period of 2000-2008 in the subject of alcohol use, infection, inflammation, innate, and adaptive immunity. RESULTS This review article summarizes recent findings relevant to acute or chronic alcohol use-induced immunomodulation and its consequences on host defense against microbial pathogens and tissue injury. Studies with in vivo and in vitro alcohol administration are both discussed. The effects of alcohol on lung infections, trauma and burn injury, liver, pancreas, and cardiovascular diseases are evaluated with respect to the role of immune cells. Specific changes in innate immune response and abnormalities in adaptive immunity caused by alcohol intake are detailed. CONCLUSION Altered inflammatory cell and adaptive immune responses after alcohol consumption result in increased incidence and poor outcome of infections and other organ-specific immune-mediated effects.

MicroRNAs in liver disease

Small, noncoding microRNAs (miRNAs) regulate diverse biological functions in the liver and increasing evidence suggests that they have a role in liver pathology. This Review summarizes advances in the field of miRNAs in liver diseases, inflammation and cirrhosis. MicroRNA-122, the most abundant miRNA in hepatocytes, has well-defined roles in HCV replication, and data indicate that it also serves as a viable therapeutic target. The role of miR-122 is also emerging in other liver diseases. Ample evidence exists for the important regulatory potential of other miRNAs in conditions associated with liver inflammation related to alcohol use, the metabolic syndrome or autoimmune processes. In addition, a broad array of miRNAs have been associated with the development of liver fibrosis both in animal models and human studies. The significance of the function and cellular distribution of miRNAs in the liver and the potential of miRNAs as a means of communication between cells and organs is discussed as well as the emerging utility of circulating miRNAs as biomarkers of different forms of liver damage and as early markers of disease and progression in hepatocellular carcinoma. Importantly, miRNA modulation in the liver represents a new therapeutic approach in the treatment armamentarium of hepatologists in the future.

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.)

Signalling pathways in alcohol-induced liver inflammation☆

The pathogenesis of alcoholic liver injury involves interactions of several intracellular signalling pathways in different cell types of the liver. Alcohol-induced sensitization of liver macrophages to portal endotoxin/lipopolysaccharide (LPS) is considered a hallmark of alcoholic liver disease (ALD). Intracellular mechanisms associated with LPS-induced signalling play a crucial role in the initiation and progression of alcoholic liver injury, and are being extensively explored. LPS recognition by Toll-like receptor 4 (TLR4) on macrophages and other cell types in the liver, activation of downstream signalling pathways culminating in activation of transcription factors such as NFkappaB, AP-1 leads to increased inflammatory cytokine production in ALD. In addition, LPS-induced MAPK such as ERK and p38 also contribute to liver injury. The importance of alcohol-induced reactive oxygen species and interactions with TLR pathways in macrophages leading to inflammation is becoming increasingly evident. Collectively, these signalling pathways induce pro- and anti-inflammatory cytokines that play an important role in ALD. In this review we describe the key signalling intermediates leading to alcohol-induced inflammation in alcoholic liver disease.

Hepatitis C Virus Core and Nonstructural Protein 3 Proteins Induce Pro- and Anti-inflammatory Cytokines and Inhibit Dendritic Cell Differentiation

Antiviral immunity requires recognition of viral pathogens and activation of cytotoxic and Th cells by innate immune cells. In this study, we demonstrate that hepatitis C virus (HCV) core and nonstructural protein 3 (NS3), but not envelope 2 proteins (E2), activate monocytes and myeloid dendritic cells (DCs) and partially reproduce abnormalities found in chronic HCV infection. HCV core or NS3 (not E2) triggered inflammatory cytokine mRNA and TNF-α production in monocytes. Degradation of I-κBα suggested involvement of NF-κB activation. HCV core and NS3 induced production of the anti-inflammatory cytokine, IL-10. Both monocyte TNF-α and IL-10 levels were higher upon HCV core and NS3 protein stimulation in HCV-infected patients than in normals. HCV core and NS3 (not E2) inhibited differentiation and allostimulatory capacity of immature DCs similar to defects in HCV infection. This was associated with elevated IL-10 and decreased IL-2 levels during T cell proliferation. Increased IL-10 was produced by HCV patients’ DCs and by core- or NS3-treated normal DCs, while IL-12 was decreased only in HCV DCs. Addition of anti-IL-10 Ab, not IL-12, ameliorated T cell proliferation with HCV core- or NS3-treated DCs. Reduced allostimulatory capacity in HCV core- and NS3-treated immature DCs, but not in DCs of HCV patients, was reversed by LPS maturation, suggesting more complex DC defects in vivo than those mediated by core or NS3 proteins. Our results reveal that HCV core and NS3 proteins activate monocytes and inhibit DC differentiation in the absence of the intact virus and mediate some of the immunoinhibitory effects of HCV via IL-10 induction.

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.

Hepatitis C Virus (HCV) core protein-induced, monocyte-mediated mechanisms of reduced IFN-α and plasmacytoid dendritic cell loss in chronic HCV infection

IFN-α production by plasmacytoid dendritic cells (PDCs) is critical in antiviral immunity. In the present study, we evaluated the IFN-α-producing capacity of PDCs of patients with chronic hepatitis C virus (HCV) infection in treatment-naive, sustained responder, and nonresponder patients. IFN-α production was tested in PBMCs or isolated PDCs after TLR9 stimulation. Treatment-naive patients with chronic HCV infection had reduced frequency of circulating PDCs due to increased apoptosis and showed diminished IFN-α production after stimulation with TLR9 ligands. These PDC defects correlated with the presence of HCV and were in contrast with normal PDC functions of sustained responders. HCV core protein, which was detectable in the plasma of infected patients, reduced TLR9-triggered IFN-α and increased TNF-α and IL-10 production in PBMCs but not in isolated PDCs, suggesting HCV core induced PDC defects. Indeed, addition of rTNF-α and IL-10 induced apoptosis and inhibited IFN-α production in PDCs. Neutralization of TNF-α and/or IL-10 prevented HCV core-induced inhibition of IFN-α production. We identified CD14+ monocytes as the source of TNF-α and IL-10 in the HCV core-induced inhibition of PDC IFN-α production. Anti-TLR2-, not anti-TLR4-, blocking Ab prevented the HCV core-induced inhibition of IFN-α production. In conclusion, our results suggest that HCV interferes with antiviral immunity through TLR2-mediated monocyte activation triggered by the HCV core protein to induce cytokines that in turn lead to PDC apoptosis and inhibit IFN-α production. These mechanisms are likely to contribute to HCV viral escape from immune responses.