Timea Csak

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;)

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.

Inflammasomes in liver diseases

Inflammation is a common element in the pathogenesis of most chronic liver diseases that lead to fibrosis and cirrhosis. Inflammation is characterized by activation of innate immune cells and production of pro-inflammatory cytokines IL-1α, IL-1β, and TNFα. Inflammasomes are intracellular multiprotein complexes expressed in both parenchymal and non-parenchymal cells of the liver that in response to cellular danger signals activate caspase-1, and release IL-1β and IL-18. The importance of inflammasome activation in various forms of liver diseases in relation to liver damage, steatosis, inflammation and fibrosis is discussed in this review.

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.

Hepatocyte-specific Hypoxia Inducible Factor-1α is a determinant of lipid accumulation and liver injury in alcohol-induced steatosis in mice

Chronic alcohol causes hepatic steatosis and liver hypoxia. Hypoxia‐regulated hypoxia‐inducible factor 1‐α, (HIF‐1α) may regulate liporegulatory genes, but the relationship of HIF‐1 to steatosis remains unknown. We investigated HIF‐1α in alcohol‐induced hepatic lipid accumulation. Alcohol administration resulted in steatosis, increased liver triglyceride levels, and increased serum alanine aminotransferase (ALT) levels, suggesting liver injury in wild‐type (WT) mice. There was increased hepatic HIF‐1α messenger RNA (mRNA), protein, and DNA‐binding activity in alcohol‐fed mice compared with controls. Mice engineered with hepatocyte‐specific HIF‐1 activation (HIF1dPA) had increased HIF‐1α mRNA, protein, and DNA‐binding activity, and alcohol feeding in HIF1dPA mice increased hepatomegaly and hepatic triglyceride compared with WT mice. In contrast, hepatocyte‐specific deletion of HIF‐1α [HIF‐1α(Hep−/−)], protected mice from alcohol‐ and lipopolysaccharide (LPS)‐induced liver damage, serum ALT elevation, hepatomegaly, and lipid accumulation. HIF‐1α(Hep−/−), WT, and HIF1dPA mice had equally suppressed levels of peroxisome proliferator‐activated receptor α mRNA after chronic ethanol, whereas the HIF target, adipocyte differentiation‐related protein, was up‐regulated in WT mice but not HIF‐1α(Hep−/−) ethanol‐fed/LPS‐challenged mice. The chemokine monocyte chemoattractant protein‐1 (MCP‐1) was cooperatively induced by alcohol feeding and LPS in WT but not HIF‐1α(Hep−/−) mice. Using Huh7 hepatoma cells in vitro, we found that MCP‐1 treatment induced lipid accumulation and increased HIF‐1α protein expression as well as DNA‐binding activity. Small interfering RNA inhibition of HIF‐1α prevented MCP‐1–induced lipid accumulation, suggesting a mechanistic role for HIF‐1α in hepatocyte lipid accumulation. Conclusion: Alcohol feeding results in lipid accumulation in hepatocytes involving HIF‐1α activation. The alcohol‐induced chemokine MCP‐1 triggers lipid accumulation in hepatocytes via HIF‐1α activation, suggesting a mechanistic link between inflammation and hepatic steatosis in alcoholic liver disease. (HEPATOLOGY 2011;)

STING-IRF3 pathway links endoplasmic reticulum stress with hepatocyte apoptosis in early alcoholic liver disease

Significance This paper provides previously undescribed evidence that STING, an endoplasmic reticulum (ER)-resident protein involved in DNA sensing, couples ER stress with apoptotic signaling in alcoholic liver disease. The proapoptotic role of STING is mediated by the interferon regulatory factor 3 (IRF3), and this is independent of inflammation or Type-I interferons. Activation of STING and IRF3, originally reported in the context of antiviral response, determines survival of hepatocytes in early alcoholic liver disease suggesting that innate immunity regulates hepatocyte pathophysiology independent of inflammation. 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.

Alcohol-induced IL-1β in the brain is mediated by NLRP3/ASC inflammasome activation that amplifies neuroinflammation

Alcohol‐induced neuroinflammation is mediated by proinflammatory cytokines, including IL‐1β. IL‐1β production requires caspase‐1 activation by inflammasomes—multiprotein complexes that are assembled in response to danger signals. We hypothesized that alcohol‐induced inflammasome activation contributes to increased IL‐1β in the brain. WT and TLR4‐, NLRP3‐, and ASC‐deficient (KO) mice received an ethanol‐containing or isocaloric control diet for 5 weeks, and some received the rIL‐1ra, anakinra, or saline treatment. Inflammasome activation, proinflammatory cytokines, endotoxin, and HMGB1 were measured in the cerebellum. Expression of inflammasome components (NLRP1, NLRP3, ASC) and proinflammatory cytokines (TNF‐α, MCP‐1) was increased in brains of alcohol‐fed compared with control mice. Increased caspase‐1 activity and IL‐1β protein in ethanol‐fed mice indicated inflammasome activation. TLR4 deficiency protected from TNF‐α, MCP‐1, and attenuated alcohol‐induced IL‐1β increases. The TLR4 ligand, LPS, was not increased in the cerebellum. However, we found up‐regulation of acetylated and phosphorylated HMGB1 and increased expression of the HMGB1 receptors (TLR2, TLR4, TLR9, RAGE) in alcohol‐fed mice. NLRP3‐ or ASC‐deficient mice were protected from caspase‐1 activation and alcohol‐induced IL‐1β increase in the brain. Furthermore, in vivo treatment with rIL‐1ra prevented alcohol‐induced inflammasome activation and IL‐1β, TNF‐α, and acetylated HMGB1 increases in the cerebellum. Conversely, intracranial IL‐1β administration induced TNF‐α and MCP‐1 in the cerebellum. In conclusion, alcohol up‐regulates and activates the NLRP3/ASC inflammasome, leading to caspase‐1 activation and IL‐1β increase in the cerebellum. IL‐1β amplifies neuroinflammation, and disruption of IL‐1/IL‐1R signaling prevents alcohol‐induced inflammasome activation and neuroinflammation. Increased levels of acetylated and phosphorylated HMGB1 may contribute to alcoholic neuroinflammation.

Type I Interferons Protect From Toll-Like Receptor 9–Associated Liver Injury and Regulate IL-1 Receptor Antagonist in Mice

BACKGROUND & AIMS Liver inflammation and injury are mediated by the innate immune response, which is regulated by Toll-like receptors (TLR). Activation of TLR9 induces type I interferons (IFNs) via the interferon regulatory factor (IRF)-7. We investigated the roles of type I IFNs in TLR9-associated liver injury. METHODS Wild-type (WT), IRF7-deficient, and IFN-α/β receptor 1 (IFNAR1)-deficient mice were stimulated with TLR9 or TLR2 ligands. Findings from mice were verified in cultured hepatocytes and liver mononuclear cells (LMNCs) as well as in vivo experiments using recombinant type I IFN and interleukin-1 receptor antagonist (IL-1ra). RESULTS Type I IFNs were up-regulated during TLR9-associated liver injury in WT mice. IRF7- and IFNAR1-deficient mice, which have disruptions in type I IFN production or signaling, respectively, had increased liver damage and inflammation, decreased recruitment of dendritic cells, and increased production of tumor necrosis factor α by LMNCs. These findings indicate that type I IFNs have anti-inflammatory activities in liver. IL-1ra, which is produced by LMNCs and hepatocytes, is an IFN-regulated antagonist of the proinflammatory cytokine IL-1β; IRF7- and IFNAR1-deficient mice had decreased levels of IL-1ra compared with WT mice. IL-1ra protected cultured hepatocytes from IL-1β-mediated sensitization to cytotoxicity from tumor necrosis factor α. In vivo exposure to type I IFN, which induced IL-1ra, or administration of IL-1ra reduced TLR9-associated liver injury; the protective effect of type I IFNs therefore appears to be mediated by IFN-dependent induction of IL-1ra. CONCLUSIONS Type I IFNs have anti-inflammatory effects mediated by endogenous IL-1ra, which regulates the extent of TLR9-induced liver damage. Type I IFN signaling is therefore required for protection from immune-mediated liver injury.

Chronic alcohol-induced microRNA-155 contributes to neuroinflammation in a TLR4-dependent manner in mice

Introduction Alcohol-induced neuroinflammation is mediated by pro-inflammatory cytokines and chemokines including tumor necrosis factor-α (TNFα), monocyte chemotactic protein-1 (MCP1) and interleukin-1-beta (IL-1β). Toll-like receptor-4 (TLR4) pathway induced nuclear factor-κB (NF-κB) activation is involved in the pathogenesis of alcohol-induced neuroinflammation. Inflammation is a highly regulated process. Recent studies suggest that microRNAs (miRNAs) play crucial role in fine tuning gene expression and miR-155 is a major regulator of inflammation in immune cells after TLR stimulation. Aim To evaluate the role of miR-155 in the pathogenesis of alcohol-induced neuroinflammation. Methods Wild type (WT), miR-155- and TLR4-knockout (KO) mice received 5% ethanol-containing or isocaloric control diet for 5 weeks. Microglia markers were measured by q-RTPCR; inflammasome activation was measured by enzyme activity; TNFα, MCP1, IL-1β mRNA and protein were measured by q-RTPCR and ELISA; phospho-p65 protein and NF-κB were measured by Western-blotting and EMSA; miRNAs were measured by q-PCR in the cerebellum. MiR-155 was measured in immortalized and primary mouse microglia after lipopolysaccharide and ethanol stimulation. Results Chronic ethanol feeding up-regulated miR-155 and miR-132 expression in mouse cerebellum. Deficiency in miR-155 protected mice from alcohol-induced increase in inflammatory cytokines; TNFα, MCP1 protein and TNFα, MCP1, pro-IL-1β and pro-caspase-1 mRNA levels were reduced in miR-155 KO alcohol-fed mice. NF-κB was activated in WT but not in miR-155 KO alcohol-fed mice. However increases in cerebellar caspase-1 activity and IL-1β levels were similar in alcohol-fed miR-155-KO and WT mice. Alcohol-fed TLR4-KO mice were protected from the induction of miR-155. NF-κB activation measured by phosphorylation of p65 and neuroinflammation were reduced in alcohol-fed TLR4-KO compared to control mice. TLR4 stimulation with lipopolysaccharide in primary or immortalized mouse microglia resulted in increased miR-155. Conclusion Chronic alcohol induces miR-155 in the cerebellum in a TLR4-dependent manner. Alcohol-induced miR-155 regulates TNFα and MCP1 expression but not caspase-dependent IL-1β increase in neuroinflammation.