Background & aims

Liver fibrosis is the most worrisome feature of non-alcoholic steatohepatitis (NASH). Growing evidence supports a link between hepatocyte apoptosis and liver fibrogenesis. Our aim was to determine the therapeutic efficacy and safety of liver Bid, a key pro-apoptotic molecule, suppression using RNA interference (RNAi) for the treatment of fibrosis.

Methods

First, we optimized the delivery system for Bid siRNA in mice using ten different stealth RNAi siRNAs and two lipid formulations -Invivofectamine2.0 and a newly developed Invivofectamine3.0 - that have been designed for high efficacy accumulation in the liver, assessed via real-time PCR of Bid mRNA. Next, C57BL/6 mice were placed on a choline-deficient L-amino acid defined (CDAA) diet. After 19weeks of the CDAA diet, a time point that results in severe fibrotic NASH, mice were injected with the selected Bid siRNA-Invivofectamine3.0 biweekly for three weeks. Additionally hepatocyte-specific Bid deficient (Bid(Δhep)) mice were placed on CDAA diet for 20weeks.

Results

A maximum Bid knockdown was achieved at 1.5mg/kg siRNA with Invivofectamine3.0, whereas it was at 7mg/kg with Invivofectamine2.0. In NASH mice, after 3weeks of treatment, BID protein was reduced to 10% and this was associated with an improvement in liver fibrosis and inflammation associated with a marked reduction in TUNEL positive cells, caspase 3 activation, and a reduction in mitochondrial BAX and BAK. Bid(Δhep) mice showed similar protection from fibrotic changes.

Conclusion

Our data demonstrate that liver Bid suppression by RNAi technology, as well as hepatocyte-specific Bid deficiency, improves liver fibrosis coupled with a reduction of inflammation in experimental NASH. These findings are consistent with existing evidence that hepatocyte apoptosis triggers hepatic stellate cell activation and liver fibrosis and suggest that Bid inhibition may be useful as an antifibrotic NASH therapy.

Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical "find-me" signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel "find me" signals that contributes to macrophage infiltration associated with obesity.

Unlabelled

Inflammasome activation plays a central role in the development of drug-induced and obesity-associated liver disease. However, the sources and mechanisms of inflammasome-mediated liver damage remain poorly understood. Our aim was to investigate the effect of NLRP3 inflammasome activation on the liver using novel mouse models. We generated global and myeloid cell-specific conditional mutant Nlrp3 knock-in mice expressing the D301N Nlrp3 mutation (ortholog of D303N in human NLRP3), resulting in a hyperactive NLRP3. To study the presence and significance of NLRP3-initiated pyroptotic cell death, we separated hepatocytes from nonparenchymal cells and developed a novel flow-cytometry-based (fluorescence-activated cell sorting; FACS) strategy to detect and quantify pyroptosis in vivo based on detection of active caspase 1 (Casp1)- and propidium iodide (PI)-positive cells. Liver inflammation was quantified histologically by FACS and gene expression analysis. Liver fibrosis was assessed by Sirius Red staining and quantitative polymerase chain reaction for markers of hepatic stellate cell (HSC) activation. NLRP3 activation resulted in shortened survival, poor growth, and severe liver inflammation; characterized by neutrophilic infiltration and HSC activation with collagen deposition in the liver. These changes were partially attenuated by treatment with anakinra, an interleukin-1 receptor antagonist. Notably, hepatocytes from global Nlrp3-mutant mice showed marked hepatocyte pyroptotic cell death, with more than a 5-fold increase in active Casp1/PI double-positive cells. Myeloid cell-restricted mutant NLRP3 activation resulted in a less-severe liver phenotype in the absence of detectable pyroptotic hepatocyte cell death.

Conclusions

Our data demonstrate that global and, to a lesser extent, myeloid-specific NLRP3 inflammasome activation results in severe liver inflammation and fibrosis while identifying hepatocyte pyroptotic cell death as a novel mechanism of NLRP3-mediated liver damage.

Extracellular vesicles (EVs) released during cell stress, or demise, can contain a barcode of the cell origin, including specific microRNAs (miRNAs). Here, we tested the hypothesis that during early alcoholic steatohepatitis (ASH) development, hepatocytes (HCs) release EVs with an miRNA signature that can be measured in circulation. A time-course experiment showed that after 2 weeks of intragastric infusion, a time point that results in isolated steatosis, there was no increase of blood EVs. After 4 weeks of infusion, mice developed features of early ASH accompanied by a marked increase in the level of EVs in blood (P < 0.05), as well as in culture media of isolated HCs (P < 0.001) and hepatic macrophages (P < 0.001), with HCs being the predominant source of EVs. The transcriptome analysis of HC-EVs from ASH mice detected differentially expressed miRNAs, including nine significantly up-regulated and four significantly down-regulated miRNAs. Target prediction and pathway analyses of the up-regulated miRNAs identified 121 potential target genes involved in inflammatory and cancer pathways, such as nuclear factor kappa B, EGF, Wnt, and B-cell lymphoma 2. Three miRNAs, let7f, miR-29a, and miR-340, were increased in blood EVs from ASH mice (P < 0.05), but not in blood EVs from three other models of chronic liver injury, including bile duct ligation, nonalcoholic steatohepatitis, and obese mice, as well as EVs released from hepatocytes exposed to ethanol. Blood EV level (P < 0.01) and three miRNAs (P < 0.05) were significantly increased in patients with ambulatory mild ALD as compared to nonalcoholics.

Conclusion

Damaged hepatocytes from ASH mice are a key EV source with a specific miRNA cargo, which are specific for ASH-related liver injury. These findings uncover EVs as a potentially novel diagnostic for ASH. (Hepatology 2017;65:475-490).

Background&aims

Hepatic stellate cells (HSCs) play a key role in liver fibrosis in various chronic liver disorders including nonalcoholic fatty liver disease (NAFLD). The development of liver fibrosis requires a phenotypic switch from quiescent to activated HSCs. The triggers for HSCs activation in NAFLD remain poorly understood. We investigated the role and molecular mechanism of extracellular vesicles (EVs) released by hepatocytes during lipotoxicity in modulation of HSC phenotype.

Methods

EVs were isolated from fat-laden hepatocytes by differential centrifugation and incubated with HSCs. EV internalization and HSCs activation, migration and proliferation were assessed. Loss- and gain-of-functions studies were performed to explore the potential role of PPAR-γ-targeting miRNAs carried by EVs into HSC.

Results

Hepatocyte-derived EVs released during lipotoxicity are efficiently internalized by HSCs resulting in their activation, as shown by marked up-regulation of pro-fibrogenic genes (Collagen-I, α-SMA and TIMP-2), proliferation, chemotaxis and wound healing responses. These changes were associated with miRNAs shuttled by EVs and suppression of PPAR-γ expression in HSC. Hepatocyte-derived EVs miRNA content included various miRNAs that are known inhibitors of PPAR-γ expression with miR-128-3p being the most effectively transferred. Furthermore loss- and gain-of-function studies identified miR-128-3p as a central modulator of the effects of EVs on PPAR-γ inhibition and HSC activation.

Conclusion

Our findings demonstrate a link between fat-laden hepatocyte-derived EVs and liver fibrosis and have potential implications for the development of novel anti-fibrotic targets for NAFLD and other fibrotic diseases.

Development of portal hypertension (PHT) is a central prognostic factor in patients with cirrhosis. Circulating microparticles (MPs) are released by hepatocytes in a caspase-dependent manner, are increased in circulation of patients with cirrhosis, and contribute to PHT via induction of impaired vasoconstrictor responses. Here, we tested the hypothesis that emricasan, a pan-caspase inhibitor, ameliorates PHT and reduction in release of MPs. We used a short-term and long-term protocol following common bile-duct ligation (BDL) in C57BL/6 mice (10 and 20 days, respectively). Mice were treated daily via intraperitoneal injection with 10 mg/kg/day of emricasan or placebo. Circulating MP levels were analyzed using flow cytometry and function via ex vivo angiogenesis assays. In contrast to BDL-placebo group, nearly all BDL-emricasan-treated mice survived after long-term BDL. Assessment of portal pressure showed a significant increase in BDL-placebo mice compared to sham-placebo mice. In contrast, BDL-emricasan mice had significantly lower levels of portal pressure compared to BDL-placebo mice. Although emricasan treatment resulted in a decrease in fibrosis, the changes did not reach statistical significance, suggesting that the effects on PHT are at least in part independent of the anti-fibrotic effects of the drug. Following short-term BDL, hepatocellular cell death as well as liver fibrosis had improved and circulating MPs were significantly reduced in BDL-emricasan mice compared to BDL-placebo. Circulating MPs from BDL-placebo mice induced endothelial cell activation, and this was significantly reduced in MPs from BDL-emricasan mice. Our results indicate that emricasan treatment improves survival and PHT in a murine model of long-term BDL. Emricasan is a promising agent for the treatment of PHT. KEY MESSAGE:Emricasan, a pan-caspase inhibitor, improves survival and portal hypertension induced by long-term bile-duct ligation (BDL) in mice Emricasan reduces liver damage, hepatocyte death, and fibrosis, following short-term BDL in mice, and these changes are associated with a decrease in circulating microparticle (MPs) Circulating MPs from BDL-placebo but not from BDL-emiricasan-treated mice activate endothelial cells ex vivo.

Circulating oxidized linoleic acid (LA) metabolites (OXLAMs) are increased in patients with nonalcoholic steatohepatitis (NASH) and their levels correlate with disease severity. However, the mechanisms by which OXLAMs contribute to NASH development are incompletely understood. We tested the hypothesis that LA or OXLAMs provided directly through the diet are involved in the development of hepatic injury. C57BL/6 mice were fed an isocaloric high-fat diet containing low LA, high LA, or OXLAMs for 8 weeks. The livers of OXLAM-fed mice showed lower triglyceride concentrations, but higher FA oxidation and lipid peroxidation in association with increased oxidative stress. OXLAM-induced mitochondrial dysfunction was associated with reduced Complex I protein and hepatic ATP levels, as well as increased mitochondrial biogenesis and cytoplasmic mitochondrial DNA. Oxidative stress increased thioredoxin-interacting protein (TXNIP) in the liver and stimulated the activation of mitochondrial apoptosis signal-regulating kinase 1 (ASK1) leading to apoptosis. We also found increased levels of NOD-like receptor protein 3 (NLRP3) inflammasome components and Caspase-1 activation in the livers of OXLAM-fed mice. In vitro, OXLAMs induced hepatocyte cell death, which was partly dependent on Caspase-1 activation. This study identified key mechanisms by which dietary OXLAMs contribute to NASH development, including mitochondrial dysfunction, hepatocyte cell death, and NLRP3 inflammasome activation.

We recently reported that stressed adipocytes release extracellular vesicles (EVs) that act as "find-me" signals to promote macrophage migration and activation. In this study, we performed a comprehensive characterization of stressed adipocyte-derived EVs, assessing their antigenic composition, lipidomics, and RNA profiles. Perilipin A was identified as one of the adipose-specific proteins and studied as a potential novel biomarker to detect adipocyte-derived EVs in circulation. Circulating EVs were significantly increased in mice with diet-induced obesity (DIO) and in obese humans with metabolic syndrome compared to lean controls. This increase was associated with decreased glucose tolerance in the DIO mice and metabolic dysfunction, elevated insulin, and homeostatic model assessment of insulin resistance (HOMA-IR) in the obese humans. EVs from both DIO mice and obese humans were enriched in perilipin A, a central gatekeeper of the adipocyte lipid storehouse and a marker of adipocyte differentiation. In obese humans, circulating levels of EVs enriched in perilipin A were dynamic, decreasing 35 % (p < 0.05) after a 3-month reduced calorie diet intervention. This translational study provides an extensive characterization of adipocyte-derived EVs. The findings identify perilipin A as a novel biomarker of circulating EVs of adipocyte origin and support the development of circulating perilipin A-positive EVs as indicators of adipose tissue health.

Key message

• Extensive characterization of 3T3L1 EVs identified perilipin A in their composition. • Circulating EVs are elevated in obese mice and associated with glucose intolerance. • Circulating EVs are elevated in obese human and correlated with metabolic factors. • Perilipin A and EV levels are increased in the circulation of obese mice and human. • Circulating EV and perilipin A levels decrease with low calorie intervention.

Background & aim

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in both adult and children. Currently there are no reliable methods to determine disease severity, monitor disease progression, or efficacy of therapy, other than an invasive liver biopsy.

Design

Choline Deficient L-Amino Acid (CDAA) and high fat diets were used as physiologically relevant mouse models of NAFLD. Circulating extracellular vesicles were isolated, fully characterized by proteomics and molecular analyses and compared to control groups. Liver-related microRNAs were isolated from purified extracellular vesicles and liver specimens.

Results

We observed statistically significant differences in the level of extracellular vesicles (EVs) in liver and blood between two control groups and NAFLD animals. Time-course studies showed that EV levels increase early during disease development and reflect changes in liver histolopathology. EV levels correlated with hepatocyte cell death (r2 = 0.64, p<0.05), fibrosis (r2 = 0.66, p<0.05) and pathological angiogenesis (r2 = 0.71, p<0.05). Extensive characterization of blood EVs identified both microparticles (MPs) and exosomes (EXO) present in blood of NAFLD animals. Proteomic analysis of blood EVs detected various differentially expressed proteins in NAFLD versus control animals. Moreover, unsupervised hierarchical clustering identified a signature that allowed for discrimination between NAFLD and controls. Finally, the liver appears to be an important source of circulating EVs in NAFLD animals as evidenced by the enrichment in blood with miR-122 and 192--two microRNAs previously described in chronic liver diseases, coupled with a corresponding decrease in expression of these microRNAs in the liver.

Conclusions

These findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.

Innate immune mechanisms play an important role in inflammatory chronic liver diseases. In this study, we investigated the role of type I or invariant NKT (iNKT) cell subsets in the progression of nonalcoholic steatohepatitis (NASH). We used α-galactosylceramide/CD1d tetramers and clonotypic mAb together with intracytoplasmic cytokine staining to analyze iNKT cells in choline-deficient l-amino acid-defined (CDAA)-induced murine NASH model and in human PBMCs, respectively. Cytokine secretion of hepatic iNKT cells in CDAA-fed C57BL/6 mice altered from predominantly IL-17⁺ to IFN-γ⁺ and IL-4⁺ during NASH progression along with the downmodulation of TCR and NK1.1 expression. Importantly, steatosis, steatohepatitis, and fibrosis were dependent upon the presence of iNKT cells. Hepatic stellate cell activation and infiltration of neutrophils, Kupffer cells, and CD8⁺ T cells as well as expression of key proinflammatory and fibrogenic genes were significantly blunted in Jα18^-/- mice and in C57BL/6 mice treated with an iNKT-inhibitory RAR-γ agonist. Gut microbial diversity was significantly impacted in Jα18^-/- and in CDAA diet-fed mice. An increased frequency of CXCR3⁺IFN-γ⁺T-bet⁺ and IL-17A⁺ iNKT cells was found in PBMC from NASH patients in comparison with nonalcoholic fatty liver patients or healthy controls. Consistent with their in vivo activation, iNKT cells from NASH patients remained hyporesponsive to ex-vivo stimulation with α-galactosylceramide. Accumulation of plasmacytoid dendritic cells in both mice and NASH patients suggest their role in activation of iNKT cells. In summary, our findings indicate that the differential activation of iNKT cells play a key role in mediating diet-induced hepatic steatosis and fibrosis in mice and its potential involvement in NASH progression in humans.