Recent evidence implicates adaptive immunity as a key player in the mechanisms supporting hepatic inflammation during the progression of nonalcoholic fatty liver disease (NAFLD). In these settings, patients with NAFLD often show an increase in the circulating levels of antibodies against oxidative stress-derived epitopes (OSE). Nonetheless, the actual role of humoral immunity in NAFLD is still unclear. This study investigates the contribution of B-lymphocytes to NAFLD evolution. B-lymphocyte immunostaining of liver biopsies from NAFLD patients showed that B-cells were evident within cell aggregates rich in T-lymphocytes. In these subjects, B/T-lymphocyte infiltration positively correlated with both circulating IgG targeting oxidative stress-derived epitopes (OSE) and interferon-γ (IFN-γ) levels. Furthermore, high prevalence of lymphocyte aggregates identified patients with more severe lobular inflammation and fibrosis. In mouse models of NAFLD, the onset of steatohepatitis was characterized by hepatic B2-lymphocytes maturation to plasma cells and by an elevation in circulating anti-OSE IgG titers. B-cell responses preceded T-cell activation and were accompanied by the up-regulation in the hepatic expression of B-cell Activating Factor (BAFF). Selective B2-cell depletion in mice over-expressing a soluble form of the BAFF/APRIL receptor Transmembrane Activator and Cyclophilin Ligand Interactor (TACI-Ig) prevented plasma cell maturation and Th-1 activation of liver CD4 T-lymphocytes. Furthermore, TACI-Ig mice showed milder steatohepatitis and a decreased progression to fibrosis. Similarly, mice treatment with the BAFF-neutralizing monoclonal antibody Sandy-2 prevented hepatic B2-cell responses and ameliorated steatohepatitis. From these data we conclude that B2-lymphocyte activation is an early event in NAFLD evolution and contributes to the disease progression through the interaction with T-cells. Furthermore, combined clinical and experimental data suggest that elevated circulating anti-OSE IgG can identify a subset of NAFLD patients in whom adaptive immunity has a relevant role in the disease evolution toward fibrosis.

These results indicate that hepatocyte HIF-2α activation is a key feature in both human and experimental NAFLD and significantly contributes to the disease progression through the up-regulation of HRGP production. (Hepatology 2018;67:2196-2214).

SERPINB3 is a cysteine-proteases inhibitor up-regulated in a significant number of cirrhotic patients carrying hepatocellular carcinoma (HCC) and recently proposed as a prognostic marker for HCC early recurrence. SERPINB3 has been reported to stimulate proliferation, inhibit apoptosis and, similar to what reported for hypoxia, to trigger epithelial-to-mesenchymal transition (EMT) and increased invasiveness in liver cancer cells. This study has investigated whether SERPINB3 expression is regulated by hypoxia-related mechanisms in liver cancer cells.Exposure of HepG2 and Huh7 cells to hypoxia up-regulated SERPINB3 transcription, protein synthesis and release in the extracellular medium. Hypoxia-dependent SERPINB3 up-regulation was selective (no change detected for SERPINB4) and operated through hypoxia inducible factor (HIF)-2α (not HIF-1α) binding to SERPINB3 promoter, as confirmed by chromatin immuno-precipitation assay and silencing experiments employing specific siRNAs. HIF-2α-mediated SERPINB3 up-regulation under hypoxic conditions required intracellular generation of ROS. Immuno-histochemistry (IHC) and transcript analysis, performed in human HCC specimens, revealed co-localization of the two proteins in liver cancer cells and the existence of a positive correlation between HIF-2α and SERPINB3 transcript levels, respectively.Hypoxia, through HIF-2α-dependent and redox-sensitive mechanisms, up-regulates the transcription, synthesis and release of SERPINB3, a molecule with a high oncogenic potential.

SerpinB3 is a hypoxia- and hypoxia-inducible factor-2α-dependent cystein protease inhibitor that is up-regulated in hepatocellular carcinoma and in parenchymal cells during chronic liver diseases (CLD). SerpinB3 up-regulation in CLD patients has been reported to correlate with the extent of liver fibrosis and the production of transforming growth factor-β1, but the actual role of SerpinB3 in hepatic fibrogenesis is still poorly characterized. In the present study we analyzed the pro-fibrogenic action of SerpinB3 in cell cultures and in two different murine models of liver fibrosis. “In vitro” experiments revealed that SerpinB3 addition to either primary cultures of human activated myofibroblast-like hepatic stellate cells (HSC/MFs) or human stellate cell line (LX2 cells) strongly up-regulated the expression of genes involved in fibrogenesis and promoted oriented migration, but not cell proliferation. Chronic liver injury by CCl4 administration or by feeding a methionine/choline deficient diet to transgenic mice over-expressing human SerpinB3 in hepatocytes confirmed that SerpinB3 over-expression significantly increased the mRNA levels of pro-fibrogenic genes, collagen deposition and αSMA-positive HSC/MFs as compared to wild-type mice, without affecting parenchymal damage. The present study provides for the first time evidence that hepatocyte release of SerpinB3 during CLD can contribute to liver fibrogenesis by acting on HSC/MFs.

Non-Alcoholic Fatty Liver Disease (NAFLD) is a major form of chronic liver disease in the general population in relation to its high prevalence among overweight/obese individuals and patients with diabetes type II or metabolic syndrome. NAFLD can progress to steatohepatitis (NASH), fibrosis and cirrhosis and end-stage of liver disease but mechanisms involved are still incompletely characterized. Within the mechanisms proposed to mediate the progression of NAFLD, lipotoxicity is believed to play a major role. In the present study we provide data suggesting that microvesicles (MVs) released by fat-laden cells undergoing lipotoxicity can activate NLRP3 inflammasome following internalization by either cells of hepatocellular origin or macrophages. Inflammasome activation involves NF-kB-mediated up-regulation of NLRP3, pro-caspase-1 and pro-Interleukin-1, then inflammasome complex formation and Caspase-1 activation leading finally to an increased release of IL-1β. Since the release of MVs from lipotoxic cells and the activation of NLRP3 inflammasome have been reported to occur in vivo in either clinical or experimental NASH, these data suggest a novel rational link between lipotoxicity and increased inflammatory response.

SerpinB3 has been recently described as an early marker of liver carcinogenesis, but the potential mechanistic role of this serpin in tumor development is still poorly understood. Overexpression of Myc often correlates with more aggressive tumour forms, supporting its involvement in carcinogenesis. Yes-associated protein (Yap), the main effector of the Hippo pathway, is a central regulator of proliferation and it has been found up-regulated in hepatocellular carcinomas. The study has been designed to investigate and characterize the interplay and functional modulation of Myc by SerpinB3 in liver cancer. Results from this study indicate that Myc was up-regulated by SerpinB3 through calpain and Hippo-dependent molecular mechanisms in transgenic mice and hepatoma cells overexpressing human SerpinB3, and also in human hepatocellular carcinomas. Human recombinant SerpinB3 was capable to inhibit the activity of Calpain in vitro, likely reducing its ability to cleave Myc in its non oncogenic Myc-nick cytoplasmic form. SerpinB3 indirectly increased the transcription of Myc through the induction of Yap pathway. These findings provide for the first time evidence that SerpinB3 can improve the production of Myc through direct and indirect mechanisms that include the inhibition of generation of its cytoplasmic form and the activation of Yap pathway.

Background: SerpinB3 (SB3) is a hypoxia and hypoxia-inducible factor (HIF)-2α-dependent cysteine-protease inhibitor up-regulated in hepatocellular carcinoma (HCC), released by cancer cells and able to stimulate proliferation and epithelial-to-mesenchymal-transition. Methods: In the study we employed transgenic and knock out SerpinB3 mice, liver cancer cell line, human HCC specimens, and mice receiving diethyl-nitrosamine (DEN) administration plus choline-deficient L-amino acid refined (CDAA) diet (DEN/CDAA protocol). Results: We provide detailed and mechanistic evidence that SB3 can act as a paracrine mediator able to affect the behavior of surrounding cells by differentially up-regulating, in normoxic conditions, HIF-1α and HIF-2α. SB3 acts by (i) up-regulating HIF-1α transcription, facilitating cell survival in a harsh microenvironment and promoting angiogenesis, (ii) increasing HIF-2α stabilization via direct/selective NEDDylation, promoting proliferation of liver cancer cells, and favoring HCC progression. Moreover (iii) the highest levels of NEDD8-E1 activating enzyme (NAE1) mRNA were detected in a subclass of HCC patients expressing the highest levels of HIF-2α transcripts; (iv) mice undergoing DEN/CDAA carcinogenic protocol showed a positive correlation between SB3 and HIF-2α transcripts with the highest levels of NAE1 mRNA detected in nodules expressing the highest levels of HIF-2α transcripts. Conclusions: These data outline either HIF-2α and NEDDylation as two novel putative therapeutic targets to interfere with the procarcinogenic role of SerpinB3 in the development of HCC.

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