H fe Gene Knock-Out in a Mouse Model of Hereditary Hemochromatosis Affects Bodily Iron Isotope Compositions

Abstract

Hereditary hemochromatosis is a genetic iron overload disease related to a mutation within the HFE gene that controls the expression of hepcidin, the master regulator of systemic iron metabolism. The natural stable iron isotope composition in whole blood of control subjects is different from that of hemochromatosis patients and is sensitive to the amount of total iron removed by the phlebotomy treatment. The use of stable isotopes to unravel the pathological mechanisms of iron overload diseases is promising but hampered by the lack of data in organs involved in the iron metabolism. Here, we use Hfe ^-/- mice, a model of hereditary hemochromatosis, to study the impact of the knock-out on iron isotope compositions of erythrocytes, spleen and liver. Iron concentration increases in liver and red blood cells of Hfe ^-/- mice compared to controls. The iron stable isotope composition also increases in liver and erythrocytes, consistent with a preferential accumulation of iron heavy isotopes in Hfe ^-/- mice. In contrast, no difference in the iron concentration nor isotope composition is observed in spleen of Hfe ^-/- and control mice. Our results in mice suggest that the observed increase of whole blood isotope composition in hemochromatosis human patients does not originate from, but is aggravated by, bloodletting. The subsequent rapid increase of whole blood iron isotope composition of treated hemochromatosis patients is rather due to the release of hepatic heavy isotope-enriched iron than augmented iron dietary absorption. Further research is required to uncover the iron light isotope component that needs to balance the accumulation of hepatic iron heavy isotope, and to better understand the iron isotope fractionation associated to metabolism dysregulation during hereditary hemochromatosis.

Keywords: hereditary hemochromatosis (HFE); iron isotope; iron overloaded; mouse model; organs.

Figure 1

Boxplots of the biological parameters commonly investigated in hereditary hemochromatosis disease measured in the Hfe^−/− mouse model and compared to the WT group. (A), Fe plasma concentration. (B), Transferrin Saturation. (C), Hepcidin Expression. The P-values of Wilcoxon test and associated significance (***P < 0.001, **P < 0.01, *P < 0.05) are given.

Figure 2

Boxplots of Fe concentration in organs of the Hfe^−/− mouse model compared to the WT group (A–C) and of Fe isotope composition in organs of the Hfe^−/− mouse model compared to the WT group (D–F). RBC stands for erythrocytes. The P-values of Wilcoxon test and associated significance (***P < 0.001, **P < 0.01, *P < 0.05) are given.

Figure 3

Schematic representation of the mechanisms leading to iron overloaded during Hfe-related hemochromatosis according to general knowledge and conclusions of this study. Tr-Sat and NTBI stand for Transferrin saturation level and Non-Transferrin Bond Iron, respectively. In normal situation, the regulation of hepcidin expression allows control of Tr-Sat (<45%). Genetic hemochromatosis results in iron release in plasma from the dietary absorption through the duodenum and the recycling of erythrocytes by macrophages during the erythrophagocytosis process. The Tr-Sat increases above 45%, leading to the appearance of abnormal biochemical form of iron in plasma, the NTBI. The present study shows that Fe concentrations and isotope compositions of liver and erythrocytes, but not spleen, increase in a mouse model of HH compare to wild type. Our results suggest that the altered erythrocyte Fe isotope composition of HH patients originates from the disease and is aggravated by the cure. The heavy isotopes iron accumulation in liver could be related to the NTBI whose role should be confirmed by further studies.

Figure 4

Scatterplot of the spleen Fe isotope composition as a function of the erythrocytes Fe concentration for the WT (A) and Hfe-/- (B) mouse groups. RBC stands for erythrocytes. The ρ coefficient and P-values of the Spearman test with associated significance (***P < 0.001, **P < 0.01, *P < 0.05) are given.