Collectively, these data suggest that increased hepatic IL-6 production may play an important role in NASH development, as well as in systemic insulin resistance and diabetes.

Background & AimNonalcoholic 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.DesignCholine 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.ResultsWe 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.ConclusionsThese findings suggest a potential for using specific circulating EVs as sensitive and specific biomarkers for the noninvasive diagnosis and monitoring of NAFLD.

Background & AimsHepatic 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 trigger for HSC 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.MethodsEVs were isolated from fat-laden hepatocytes by differential centrifugation and incubated with HSCs. EV internalization and HSC activation, migration, and proliferation were assessed. Loss- and gain-of-function studies were performed to explore the potential role of peroxisome proliferator-activated receptor-γ (PPAR-γ)-targeting microRNAs (miRNAs) carried by EVs into HSC.ResultsHepatocyte-derived EVs released during lipotoxicity are efficiently internalized by HSCs resulting in their activation, as shown by marked up-regulation of profibrogenic genes (collagen-I, α-smooth muscle actin, and tissue inhibitor of metalloproteinases-2), proliferation, chemotaxis, and wound-healing responses. These changes were associated with miRNAs shuttled by EVs and suppression of PPAR-γ expression in HSCs. The hepatocyte-derived EV miRNA content included various miRNAs that are known inhibitors of PPAR-γ expression, with miR-128-3p being the most efficiently 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.ConclusionsOur findings demonstrate a link between fat-laden hepatocyte-derived EVs and liver fibrosis and have potential implications for the development of novel antifibrotic targets for NAFLD and other fibrotic diseases.

Our study uncovers key roles for TNF and, to a lesser extent, IL-17 as mediators of liver inflammation and fibrosis induced by constitutive NLRP3 inflammasome activation in myeloid-derived cells. These findings may lead to therapeutic strategies aimed at halting the progression of liver injury and fibrogenesis in various liver pathogeneses driven by NLRP3 activation. (Hepatology 2017).

Nonalcoholic steatohepatitis (NASH) is associated with caspase activation. However, a role for pro-inflammatory caspases or inflammasomes has not been explored in diet-induced liver injury. Our aims were to examine the role of caspase-1 in high fat-induced NASH. C57BL/6 wild-type and caspase 1-knockout (Casp1-/-) mice were placed on a 12-week high fat diet. Wild-type mice on the high fat diet increased hepatic expression of pro-caspase-1 and IL-1β. Both wild-type and Casp1-/- mice on the high fat diet gained more weight than mice on a control diet. Hepatic steatosis and TG levels were increased in wild-type mice on high fat diet, but were attenuated in the absence of caspase-1. Plasma cholesterol and free fatty acids were elevated in wild-type, but not Casp1-/- mice, on high fat diet. ALT levels were elevated in both wild-type and Casp1-/- mice on high fat diet compared to control. Hepatic mRNA expression for genes associated with lipogenesis was lower in Casp1-/- mice on high fat diet compared to wild-type mice on high fat diet, while genes associated with fatty acid oxidation were not affected by diet or genotype. Hepatic Tnfα and Mcp-1 mRNA expression was increased in wild-type mice on high fat diet, but not in Casp1-/- mice on high fat diet. αSMA positive cells, Sirius red staining, and Col1α1 mRNA were increased in wild-type mice on high fat diet compared to control. Deficiency of caspase-1 prevented those increases. In summary, the absence of caspase-1 ameliorates the injurious effects of high fat diet-induced obesity on the liver. Specifically, mice deficient in caspase-1 are protected from high fat-induced hepatic steatosis, inflammation and early fibrogenesis. These data point to the inflammasome as an important therapeutic target for NASH.

Nonalcoholic steatohepatitis (NASH) is typically associated with pro-apoptotic caspase activation. A potential role for pro-inflammatory caspases remains incompletely understood. Our aims were to examine a potential role of caspase 1 in the development of liver damage and fibrosis in NASH. C57BL/6 wild-type (WT), developed marked steatohepatitis, HSC activation, fibrosis, and increased hepatic caspase 1 and IL-1β expression when placed on the methioninecholine deficient (MCD) diet. Marked caspase 1 activation was detected in the liver of MCD-fed mice. Hepatocyte and non-parenchymal fractionation of the livers further demonstrated that caspase 1 activation after MCD feeding was mainly localized to non-parenchymal cells. Caspase 1-knockout (Casp1−/−) mice on the MCD diet showed marked reduction in mRNA expression of genes involved in inflammation and fibrogenesis (TNFα was 7.6-fold greater in WT vs. Casp1−/−MCD-fed mice; F4/80 was 1.5-fold greater in WT vs. Casp1−/− MCD-fed mice; α-SMA was 3.2-fold greater in WT vs. Casp1−/− MCD-fed mice; Collagen 1-alpha was 7.6–fold greater in WT vs. Casp1−/− MCD-fed mice; TGFβ was 2.4-fold greater in WT vs. Casp1−/− MCD-fed mice; CRP2 was 3.2-fold greater in WT vs. Casp1−/− MCD-fed mice). Furthermore, Sirius red staining for hepatic collagen deposition was significantly reduced in Casp1−/− mice MCD-fed mice compared to WT MCD-fed animals. However, serum aminotransferase (ALT) levels, caspase 3 activity and TUNEL positive cells were similar in Casp1−/− and WT mice on the MCD diet. Selective Kupffer cell depletion by clodronate injection markedly suppressed MCD-induced caspase 1 activation and protected mice from fibrogenesis and fibrosis associated with this diet. Conclusion: this study uncovers a novel role for caspase 1 in inflammation and fibrosis during NASH development.

The NLRP3 inflammasome plays an important role in liver fibrosis development. However, the mechanisms involved in NLRP3-induced fibrosis are unclear. Our aim was to test the hypothesis that the NLRP3 inflammasome in hepatic stellate cells (HSC) can directly regulate their activation and contribute to liver fibrosis. Primary HSC isolated from WT, Nlrp3 , or Nlrp3 knock-in crossed to inducible (estrogen receptor Cre - CreT) mice were incubated with LPS and ATP, or 4OH-tamoxifen, respectively. HSC-specific Nlrp3 -knock-in mice were generated by crossing transgenic mice expressing lecithin retinol acyltransferase (Lrat)-driven Cre and maintained on standard rodent chow for 6 months. Mice were then sacrificed; liver tissue and serum were harvested. Nlrp3 inflammasome activation along with HSC phenotype and fibrosis were assessed by RT-PCR, Western blot, FACS, ELISA, immunofluorescence and immunohistochemistry. Stimulated WT HSC displayed increased levels of NLRP3 inflammasome-induced ROS production and Cathepsin B activity, accompanied by an upregulation of mRNA and protein levels of fibrotic makers, an effect abrogated in Nlrp3 HSC. Nlrp3 CreT HSC also showed elevated mRNA and protein expression of fibrotic markers 24h after inflammasome activation induced with 4OH-tamoxifen. Protein and mRNA expression levels of fibrotic markers were also found to be increased in isolated HSC and whole liver tissue from Nlrp3 Lrat Cre mice compared to WT. Liver sections from 24 week-old Nlrp Lrat Cre mice showed fibrotic changes with increased αSMA and desmin positive cells and collagen deposition, independent of inflammatory infiltrates; these changes were also observed after LPS challenge in 8 week-old Nlrp Lrat Cre mice. Conclusion Our results highlight a direct role for the NLRP3 inflammasome in the activation of HSC directly triggering liver fibrosis. This article is protected by copyright. All rights reserved.

Only a subset of hepatitis C virus (HCV)-infected patients develop progressive hepatic fibrosis after liver transplantation (LT). Hepatic stellate cell (HSC) activation is a pivotal step in hepatic fibrosis and precedes clinically apparent fibrosis. We determined whether early HSC activation, measured in 4-month protocol post-LT biopsies, is predictive of subsequent development of more histologically severe recurrence of HCV. Early (4 month) post-LT HSC activation, as measured by ␣-smooth muscle actin (␣-SMA) staining, was determined in liver biopsies from recipients with severe (fibrosis score > 2, n ‫؍‬ 13) and with mild (fibrosis score of 0, n ‫؍‬ 13) recurrence of HCV at one-year post-LT. Immunohistochemical staining for ␣-smooth muscle actin (␣-SMA) was used to generate HSC activation scores (regional and total). Total HSC activation scores at 4 months were similar in patients with severe and mild HCV recurrence (3.9 ؎ 2.0 vs. 2.7 ؎ 2.2, P ‫؍‬ 0.2). Regional HSC activation, assessed as parenchymal (zones 1, 2, and 3) or mesenchymal (portal tracts and fibrous septa), was different between the study groups, with higher mesenchymal scores predictive of progression. No patients in the mild recurrence group had detectable mesenchymal ␣-SMA staining vs. 46% (6/13) of patients with severe recurrence (P < 0.01). Mesenchymal activation of HSC had a specificity and positive predictive value of 100% for development of progressive fibrosis in liver allografts of patients with hepatitis C. In conclusion, early activation of mesenchymal HSCs is a marker for progressive fibrosis in patients with hepatitis C post-LT and may help select patients who would benefit from HCV or HSC-targeted therapy. (Liver Transpl 2005;11:1207-1213.)