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.

The granulocyte-specific microRNA-223 (miR-223) has recently emerged as a negative regulator of NOD-like receptor 3 (NLRP3) expression, a central key player in chronic hepatic injuries such as fibrotic nonalcoholic steatohepatitis (NASH), as well as in other liver conditions including acute hepatitis. In this study, we evaluated the therapeutic effect of the synthetic miR-223 analog miR-223 3p in a murine model of lipopolysaccharide (LPS)/D-GalN-induced endotoxin acute hepatitis (EAH) or fibrotic NASH resultant of long-term feeding with a high-fat, fructose, and cholesterol (FFC) diet. miR-223 3p ameliorated the infiltration of monocytes, neutrophils, and early activated macrophages and downregulated the transcriptional expression of the pro-inflammatory cytokines Il6 and Il12 and the chemokines Ccl2, Ccl3, Cxcl1, and Cxcl2 in EAH. In fibrotic NASH, treatment with miR-223 3p led to a remarkable mitigation of fibrosis development and activation of hepatic stellate cells (HSCs). miR-223 3p disrupted the activation of the NLRP3 inflammasome by impairing the synthesis of cleaved interleukin-1β (IL-1β), mature IL-1β, and NLRP3, and the activation of caspase-1 p10 in both EAH and fibrotic NASH. Our data enlightens miR-223 3p as a post-transcriptional approach to treat acute and chronic hepatitis by silencing the activation of the NLRP3 inflammasome.

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.

Skeletal muscle plays an integral role in coordinating physiological homeostasis, where signaling to other tissues via myokines allows for coordination of complex processes. Here, we aimed to leverage natural genetic correlation structure of gene expression both within and across tissues to understand how muscle interacts with metabolic tissues. Specifically, we performed a survey of genetic correlations focused on myokine gene regulation, muscle cell composition, cross-tissue signaling, and interactions with genetic sex in humans. While expression levels of a majority of myokines and cell proportions within skeletal muscle showed little relative differences between males and females, nearly all significant cross-tissue enrichments operated in a sex-specific or hormone-dependent fashion; in particular, with estradiol. These sex- and hormone-specific effects were consistent across key metabolic tissues: liver, pancreas, hypothalamus, intestine, heart, visceral, and subcutaneous adipose tissue. To characterize the role of estradiol receptor signaling on myokine expression, we generated male and female mice which lack estrogen receptor α specifically in skeletal muscle (MERKO) and integrated with human data. These analyses highlighted potential mechanisms of sex-dependent myokine signaling conserved between species, such as myostatin enriched for divergent substrate utilization pathways between sexes. Several other putative sex-dependent mechanisms of myokine signaling were uncovered, such as muscle-derived tumor necrosis factor alpha (TNFA) enriched for stronger inflammatory signaling in females compared to males and GPX3 as a male-specific link between glycolytic fiber abundance and hepatic inflammation. Collectively, we provide a population genetics framework for inferring muscle signaling to metabolic tissues in humans. We further highlight sex and estradiol receptor signaling as critical variables when assaying myokine functions and how changes in cell composition are predicted to impact other metabolic organs.

The oxidation of dietary linoleic acid (LA) produces oxidized LA metabolites (OXLAMs) known to regulate multiple signaling pathways in vivo. Recently, we reported that feeding OXLAMs to mice resulted in liver inflammation and apoptosis. However, it is not known whether this is due to a direct effect of OXLAMs accumulating in the liver, or to their degradation into bioactive shorter chain molecules (e.g. aldehydes) that can provoke inflammation and related cascades. To address this question, mice were fed a low or high LA diet low in OXLAMs, or a low LA diet supplemented with OXLAMs from heated corn oil (high OXLAM diet). Unesterified oxidized fatty acids (i.e. oxylipins), including OXLAMs, were measured in liver after 8 weeks of dietary intervention using ultra-high pressure liquid chromatography coupled to tandem mass-spectrometry. The high OXLAM diet did not alter liver oxylipin concentrations compared to the low LA diet low in OXLAMs. Significant increases in several omega-6 derived oxylipins and reductions in omega-3 derived oxylipins were observed in the high LA dietary group compared to the low LA group. Our findings suggest that dietary OXLAMs do not accumulate in liver, and likely exert pro-inflammatory and pro-apoptotic effects via downstream secondary metabolites.

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.

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.