Masked-hemolysis as novel cause of hepatic iron overload in ALD

Background:

Patients with alcoholic liver disease (ALD) develop prognostically unfavourable hepatic iron overload (HIO) and anemia, however, the underlying molecular mechanisms and the role of the systemic iron masterswitch hepcidin are poorly understood. Herein, we identify hemolysis as novel factor in disrupting hepcidin regulation and causing HIO.

Methods:

Iron-related parameters, hepcidin (mRNA and serum levels), molecular and laboratory markers were studied in a large cohort of Caucasian heavy drinkers (n = 831, age range 22 – 87 years, mean alcohol consumption 192 g/day). Severe and mild hemolysis was further studied in age-matched C57BL/6 mice using two different phenylhydrazine treatment regimens. Histology, iron stain, mRNA and protein expression were measured both in liver and spleen. Erythophagocytosis was finally studied with oxidized red blood cells differentiated human macrophages.

Results:

Indirect evidence for hemolysis (anemia, high ferritin, high LDH, high MCV) as cause for HIO was found in 16.4% of a large population of heavy drinkers. Notably, further indicators of hemolysis (haptoglobin, B12, folic acid) were not significantly changed most likely due to impaired liver synthesis. Despite higher ferritin levels as compared to controls, hepcidin was not adequately upregulated in the hemolysis group suggesting a suppressive effect of hemolysis. We next recapitulated suppression of hepcidin in a murine model of severe PHZ-induced hemolysis. PHZ induced severe anemia, elevated transaminases, transferrin saturation and LDH within 24 hours. In the same time period, hemoxygenase 1 was highly induced while hepcidin was significantly lowered by 50%. Histology confirmed an increased number of phagocytosed erythrocytes in the spleen and iron-loaded Kupffer cells in the liver. In contrast, mild hemolysis also induced by PHZ strongly upregulated hepcidin mRNA. We finally studied in vitro the process of erythrophagocytosis in human primary macrophages exposed to human oxidized red blood cells. In confirmation of the in vivo findings, hepcidin showed also a biphasic response. At physiological low levels of erythrocytes, hepcidin was induced while it was strongly suppressed at higher levels of hemolysis.

Conclusions:

Our data suggest that suppression of hepcidin by masked hemolysis seems to be an important mechanism of hepatic iron overload in patients with ALD.