Karen Kodys

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.

UNLABELLED: 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.

UNLABELLED: 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.

Emerging evidence suggests that innate immunity drives alcoholic liver disease (ALD) and that the interferon regulatory factor 3 (IRF3),a transcription factor regulating innate immune responses, is indispensable for the development of ALD. Here we report that IRF3 mediates ALD via linking endoplasmic reticulum (ER) stress with apoptotic signaling in hepatocytes. We found that ethanol induced ER stress and triggered the association of IRF3 with the ER adaptor, stimulator of interferon genes (STING), as well as subsequent phosphorylation of IRF3. Activated IRF3 associated with the proapoptotic molecule Bax [B-cell lymphoma 2 (Bcl2)-associated X protein] and contributed to hepatocyte apoptosis. Deficiency of STING prevented IRF3 phosphorylation by ethanol or ER stress, and absence of IRF3 prevented hepatocyte apoptosis. The pathogenic role of IRF3 in ALD was independent of inflammation or Type-I interferons. Thus, STING and IRF3 are key determinants of ALD, linking ER stress signaling with the mitochondrial pathway of hepatocyte apoptosis.

UNLABELLED: 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)-beta. 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-alpha 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-kappaB 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.