Angela Dolganiuc

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;)

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.)

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.

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.

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.)

Toll-like receptors 1 and 6 are involved in TLR2-mediated macrophage activation by hepatitis C virus core and NS3 proteins

Hepatitis C virus (HCV) is a leading cause of end‐stage liver disease through sustained inflammation of the liver produced by the hosts immune system. The mechanism for HCV evasion or activation of the immune system is not clear. TLRs are cellular activators of the innate immune system. We recently reported that TLR2‐mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo‐ or heterodimerization with TLR1 or TLR6. Here, we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing small interfering RNAs targeted to TLR2, TLR1, and TLR6, we showed that knockdown of each of these receptors impairs pro‐ and anti‐inflammatory cytokine activation by TLR‐specific ligands as well as by HCV core and NS3 proteins in human embryonic kidney‐TLR2 cells and in primary human acrophages. We found that HCV core and NS3 proteins induced TNF‐α and IL‐10 production in human monocyte‐derived macrophages, which was impaired by TLR2, TLR1, and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1, or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein‐induced peritoneal macrophage activation. In conclusion, TLR2 may use TLR1 and TLR6 coreceptors for HCV core‐ and NS3‐mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins.

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.

Pattern recognition receptors: A contemporary view on liver diseases

Pattern recognition receptors (PRRs) function as sensors of microbial danger signals enabling the vertebrate host to initiate an immune response. PRRs are present not only in immune cells but also in liver parenchymal cells and the complexity of the cell populations provide unique aspects to pathogen recognition and tissue damage in the liver. This review discusses the role of different PRRs in pathogen recognition in the liver, and focuses on the role of PRRs in hepatic inflammation, cholestasis, ischemia, repair and fibrosis. PRRs as novel therapeutic targets are evaluated. (HEPATOLOGY 2006;44:287–298.)

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.

TLR4, Ethanol, and Lipid Rafts: A New Mechanism of Ethanol Action with Implications for other Receptor-Mediated Effects

Ethanol (EtOH) is the most widely abused substance in the United States, and it contributes to well-documented harmful (at high dosages) and beneficial (at low dosages) changes in inflammatory and immune responses. Lipid rafts have been implicated in the regulation and activation of several important receptor complexes in the immune system, including the TLR4 complex. Many questions remain about the precise mechanisms by which rafts regulate the assembly of these receptor complexes. Results summarized in this review indicate that EtOH acts by altering the LPS-induced redistribution of components of the TLR4 complex within the lipid raft and that this is related to changes in actin cytoskeleton rearrangement, receptor clustering, and subsequent signaling. EtOH provides an example of an immunomodulatory drug that acts at least in part by modifying lipid rafts, and it could represent a model to probe the relationships between rafts, receptor complexes, and signaling.