Jan Petrasek

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.

VSL#3 probiotic treatment attenuates fibrosis without changes in steatohepatitis in a diet‐induced nonalcoholic steatohepatitis model in mice

Nonalcoholic fatty liver disease (NAFLD) and its advanced stage, nonalcoholic 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‐deficient (MCD) diet–induced mouse model of NASH. MCD diet resulted in NASH in C57BL/6 mice compared to methionine‐choline‐supplemented (MCS) diet feeding evidenced by liver steatosis, increased triglycerides, inflammatory cell accumulation, increased tumor necrosis factor α levels, and fibrosis. VSL#3 failed to prevent MCD‐induced liver steatosis or inflammation. MCD diet, even in the presence of VSL#3, induced up‐regulation of serum endotoxin and expression of the Toll‐like receptor 4 signaling components, including CD14 and MD2, MyD88 adaptor, and nuclear factor κB activation. In contrast, VSL#3 treatment ameliorated MCD diet–induced liver fibrosis resulting in diminished accumulation of collagen and α‐smooth muscle actin. We identified increased expression of liver peroxisome proliferator‐activated receptors and decreased expression of procollagen and matrix metalloproteinases in mice fed MCD+VSL#3 compared to MCD diet alone. MCD diet triggered up‐regulation of transforming growth factor beta (TGFβ), a known profibrotic 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. (HEPATOLOGY 2009.)

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.

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.

Inflammasome activation and function in liver disease

Inflammation contributes to the pathogenesis of most acute and chronic liver diseases. Inflammasomes are multiprotein complexes that can sense danger signals from damaged cells and pathogens and assemble to mediate caspase-1 activation, which proteolytically activates the cytokines IL-1β and IL-18. In contrast to other inflammatory responses, inflammasome activation uniquely requires two signals to induce inflammation, therefore setting an increased threshold. IL-1β, generated upon caspase-1 activation, provides positive feed-forward stimulation for inflammatory cytokines, thereby amplifying inflammation. Inflammasome activation has been studied in different human and experimental liver diseases and has been identified as a major contributor to hepatocyte damage, immune cell activation and amplification of liver inflammation. In this Review, we discuss the different types of inflammasomes, their activation and biological functions in the context of liver injury and disease progression. Specifically, we focus on the triggers of inflammasome activation in alcoholic steatohepatitis and NASH, chronic HCV infection, ischaemia–reperfusion injury and paracetamol-induced liver injury. The application and translation of these discoveries into therapies promises novel approaches in the treatment of inflammation in liver disease.

Gut-Liver Axis and Sensing Microbes

‘Detoxification’ of gut-derived toxins and microbial products from gut-derived microbes is a major role of the liver. While the full repertoire of gut-derived microbial products that reach the liver in health and disease is yet to be explored, the levels of bacterial lipopolysaccharide (LPS), a component of Gram-negative bacteria, is increased in the portal and/or systemic circulation in several types of chronic liver diseases. Increased gut permeability and LPS play a role in alcoholic liver disease where alcohol impairs the gut epithelial integrity through alterations in tight junction proteins. In addition, non-alcoholic fatty liver disease is also associated with increased serum LPS levels and activation of the pro-inflammatory cascade plays a central role in disease progression. Microbial danger signals are recognized by pattern recognition receptors such as the Toll-like receptor 4 (TLR4). Increasing evidence suggests that TLR4-mediated signaling via the MyD88-dependent or MyD88-independent pathways may play different roles in liver diseases associated with increased LPS exposure of the liver as a result of gut permeability. For example, we showed that in alcoholic liver disease, the MyD88-independent, IRF3-dependent TLR4 cascade plays a role in steatosis and inflammation. Our recent data demonstrate that chronic alcohol exposure in the liver leads to sensitization of Kupffer cells to LPS via a mechanism involving upregulation of microRNA-155 in Kupffer cells. Thus, understanding the cell-specific recognition and intracellular signaling events in sensing gut-derived microbes will help to achieve an optimal balance in the gut-liver axis and ameliorate liver diseases.

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.