E. Morello

SerpinB3 promotes pro-fibrogenic responses in activated hepatic stellate cells

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.

Serpinb3 is overexpressed in the liver in presence of iron overload

Iron overload results in cellular toxicity, tissue injury, organ fibrosis and increased risk of neoplastic transformation. SerpinB3 is a serine protease inhibitor overexpressed in the liver in oxidative stress conditions, able to induce fibrosis and increased risk of malignant transformation. Aim of the present study was to assess the effect of iron overload on SerpinB3 expression in the liver using in vivo and in vitro models. The expression of Serpinb3 was assessed in the liver of hemojuvelin knockout mice (Hjv−/−), an established model of hereditary hemochromatosis, and of wild type control mice, following dietary or pharmacological iron manipulation. To assess the direct effect of iron in vitro, cell lines were treated with different concentration of hemin or with an iron chelator. Hepatic Serpinb3 mRNA and protein were highly expressed in Hjv−/− mice, but not in wild type controls fed with a standard diet. Serpinb3 became detectable in wild type mice fed with a high iron diet or injected with iron dextran; these treatments further induced Serpinb3 expression in Hjv−/− mice. Livers expressing Serpinb3 showed a positive staining also for HIF-2α in the same areas. Hemin promoted induction of SerpinB3 mRNA in HeLa and HA22T/VGH cells, but a mild stimulation of SerpinB3 promoter activity in HeLa and Huh7 cells. In conclusion, Serpinb3 is strongly induced by iron in the mouse liver. The molecular link between iron, ROS and SerpinB3 seems to be HIF-2α, which is induced by iron overload and was previously found capable of up-regulating SerpinB3 at the transcriptional level.

B2-Lymphocyte responses to oxidative stress-derived antigens contribute to the evolution of nonalcoholic fatty liver disease (NAFLD)

Abstract 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‐&ggr; (IFN‐&ggr;) 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. Graphical abstract Figure. No Caption available. HighlightsLiver B/T‐cell infiltration identify NAFLD patients with advanced disease.Oxidative stress‐derived antigens promote IgG production in NAFLD patients.Hepatic B2‐cells activation to plasma cells characterizes the onset of steatohepatitis.Selective B2‐cell depletion prevents Th‐1 activation of liver CD4+ T‐lymphocytes.Interference with B2‐cells improve NAFLD evolution to fibrosis.