Role of NADPH oxidases and NFκB signaling in mediating cancerogenic iron overload in alcoholic liver disease (ALD)
Final Report Abstract
Alcoholic liver disease (ALD) is one of the leading health problems worldwide. Hepatic iron overload is frequently observed in ALD patients and is an independent risk factor for disease progression, survival and development of hepatocellular carcinoma (HCC). At the systemic level, iron homeostasis is controlled by hepcidin. Hepcidin regulation is complex and still not completely understood, but modulated by many pathophysiological conditions associated with ALD, such as inflammation, anemia, oxidative stress/H2O2 or hypoxia. Major aim of this research project was to study and unravel new underlying mechanisms and involved signaling molecules that lead to hepatic iron overload in the context of ALD. In this project we first have clearly shown, that low O2 levels (1-5 O2%) drastically enhance the H2O2-mediated induction of hepcidin, strongly suggesting the role of intracellular oxidases as powerful upstream regulators of hepcidin. We have further identified NADPH oxidases 4-generated H2O2 as hepcidin inducer over STAT3 signaling pathway and most interestingly independent of HIF-1α in hepatocytes. Moreover, since we have shown that other oxidases, such as urate oxidase (UOX) were also able to induce hepcidin expression, various oxidases or a concerted action of oxidases may be potential upstream regulator of hepcidin. The essential role of oxidases in regulating hepcidin could be transferred to another cell type, namely differentiated macrophages. We next demonstrated that macrophage differentiation led to 60-fold upregulation of macrophage hepcidin as well as increased NOX2 mRNA in both, THP-1 cells or primary isolated human monocytes. Cytokines such as IL-1β were also highly induced during macrophage differentiation and this induction could be completely blocked by inhibiting H2O2-generating NOX2. We further elucidated the macrophage-hepatocyte crosstalk in regulating hepatocellular hepcidin and found that co-culture of macrophages with hepatocytes cells strongly induced hepcidin mRNA expression under low O2 levels. Hepatocyte hepcidin mRNA was also upregulated during exposure to the macrophage-conditioned hypoxic medium. Detailed cytokine analysis and subsequent investigation of signaling pathways revealed that macrophage-secreted IL-1β is the major inducer of hepcidin in hepatocytes through activation of STAT3 and C/EBPδ, but independently of IL-6 signaling. Finally, by analyzing prospectively enrolled ALD patients as compared to healthy controls, we demonstrated in contrast to earlier reports and studies on alcohol animal models that hepcidin is upregulated in chronic drinkers and varies considerably suggesting the involvement of multiple coexisting pathological situations, such as hemolysis and inflammation during alcohol-induced liver injury. In addition, enhanced NOX4 expression and inflammation present in ALD patients act in concert for increasing hepcidin expression via STAT3 signaling, nevertheless STAT3 does not play a role in inducing hepcidin in patients with advanced stages of alcoholic liver injury. In summary, this work demonstrates an efficient induction of hepcidin by intracellular oxidases as NOX4 in hepatocytes and NOX2 in macrophages, via STAT3 signaling pathway. The activation of NOX2 during monocyte to macrophage differentiation is able to induce hepatocyte hepcidin mainly through secretion of IL-1β but also through STAT3 activation. All together, these mechanisms are important contributors to the disruption of iron homeostasis in ALD and may be a promising approach for clinical applications after further detailed investigations.
Publications
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Primary Liver Injury and Delayed Resolution of Liver Stiffness after Alcohol Detoxification in Heavy Drinkers with the PNPLA3 Variant I148M. Journal of Hepatology, 64(2), S244.
Rausch, V.; Peccerella, T.; Lackner, C.; Yagmur, E.; Seitz, H.-K.; Longerich, T. & Mueller, S.
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Caspase‐cleaved keratin‐18 fragments increase during alcohol withdrawal and predict liver‐related death in patients with alcoholic liver disease. Hepatology, 66(1), 96-107.
Mueller, Sebastian; Nahon, Pierre; Rausch, Vanessa; Peccerella, Tessa; Silva, Ines; Yagmur, Eray; Straub, Beate K.; Lackner, Carolin; Seitz, Helmut K.; Rufat, Pierre; Sutton, Angela; Bantel, Heike & Longerich, Thomas
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Does Hypoxia Cause Carcinogenic Iron Accumulation in Alcoholic Liver Disease (ALD)?. Cancers, 9(11), 145.
Silva, Inês; Rausch, Vanessa; Seitz, Helmut-Karl & Mueller, Sebastian
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Sensitive and non-invasive assessment of hepatocellular iron using a novel room-temperature susceptometer. Journal of Hepatology, 67(3), 535-542.
Mueller, Johannes; Raisi, Hanna; Rausch, Vanessa; Peccerella, Teresa; Simons, David; Ziener, Christian Herbert; Schlemmer, Heinz-Peter; Seitz, Helmut Karl; Waldburger, Nina; Longerich, Thomas; Straub, Beate Katharina & Mueller, Sebastian
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Hypoxia enhances H2O2-mediated upregulation of hepcidin: Evidence for NOX4-mediated iron regulation. Redox Biology, 16, 1-10.
Silva, Inês; Rausch, Vanessa; Peccerella, Teresa; Millonig, Gunda; Seitz, Helmut-Karl & Mueller, Sebastian
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Peroxiredoxin-2: A Novel Regulator of Iron Homeostasis in Ineffective Erythropoiesis. Antioxidants & Redox Signaling, 28(1), 1-14.
Matte, Alessandro; De Falco, Luigia; Federti, Enrica; Cozzi, Anna; Iolascon, Achille; Levi, Sonia; Mohandas, Narla; Zamo, Alberto; Bruno, Mariasole; Lebouef, Christophe; Janin, Anne; Siciliano, Angela; Ganz, Tom; Federico, Giorgia; Carlomagno, Francesca; Mueller, Sebastian; Silva, Ines; Carbone, Carmine; Melisi, Davide ... & De Franceschi, Lucia
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IL-1 beta-mediated macrophage-hepatocyte crosstalk upregulates hepcidin under physiological low oxygen levels. Redox Biology, 24, 101209.
Silva, Inês; Peccerella, Teresa; Mueller, Sebastian & Rausch, Vanessa
