β-Thalassemia Mouse Model: Macrophages and the Induction of Iron Overload.

Abstract Despite the use of transfusion and iron chelation therapy, patients with β-thalassemia major have a shortened life expectancy. Many of those deaths are attributable to cardiac iron overload. Nevertheless, the process by which cardiac iron overload occurs is not well understood. We have used the homozygous βmajor deletion [Hbbth-1] (THL) mouse model to assess hepatic and cardiac iron load. RBC indices for 3 THL mice and 2 C57BL/6 wildtype control mice prior to and post therapy with parenteral iron were evaluated with Advia. Intraperitoneal iron dextran injection at 10mg/25gm body weight daily 5 days per week for 12 days was performed and then switched to 1.25mg/25gm body weight of iron injection for another 10 days for a total of 4 weeks. Histological samples of liver and heart were stained with Prussian blue in mice prior to and post administration of parenteral iron. Immunohistochemistry with antibody to F4/80, specific for macrophages, was performed and counterstained with Prussian blue in livers and hearts of THL and C57 mice. The RBC indices in THL mice reveal an anemia (HCT 29.5±2.3 vs 45±2.1%, P=0.005) and reticulocytosis (2218±501 vs 406±101 x 109 cells, P=0.018) prior to therapy relative to the C57 mice (values presented as mean ± standard deviation). In THL mice after parenteral iron, HCT (41.8±6.8 vs 29.5±2.3%, P=0.04) and reticulocyte counts (2218±501 vs 3760±633 x 109 cells, P=0.03) increased significantly from pre-treatment values while in C57 mice, the HCT (53.8±6 vs 45±2.1%, NS) and reticulocyte count (406±101 vs 210±49 x109 cells, NS) did not change appreciably from baseline. Prior to therapy, the liver of THL mice exhibit 20–25% Kupffer cells staining with Prussian blue, with no Prussian blue staining in hepatocytes. The hearts of THL mice have no macrophages and no iron deposition at baseline. Prior to therapy, the livers of C57 mice had similar numbers of Kupffer cells compared to THL mice though none stain with Prussian blue. After treatment with parenteral iron, the livers of THL and C57 mice became significantly iron loaded (75–80% of Kupffer cells are positive for Prussian blue), the number of Kupffer cells increased 4-fold, and the majority of the Prussian blue staining was limited to Kupffer cells (90–95%). After treatment with parenteral iron, the hearts of THL and C57 mice became significantly iron loaded as well, but unlike the liver, most (90%) of the Prussian blue positive cells were myocytes. Only a small fraction of the myocytes in the heart was involved (5%). THL mice appear to be iron deficient and show bone marrow reserve with reticulocytosis significantly above baseline when excess iron is administered. Iron overload secondary to intraperitoneal iron dextran administration affects THL mice as well as C57 mice. In the liver of THL mice, Kupffer cells normally resident in the liver become laden with iron; little iron is deposited in hepatocytes. In the heart, an organ without resident macrophages and few macrophages migrating into the tissue during parenteral iron administration, both THL and C57 mice reveal myocyte deposition of iron. In conclusion, parenteral iron administration leads to a noticeable increase in RBCs in THL mice. Furthermore, both the livers and hearts of THL mice accumulate iron. Finally, these findings correlate well with the natural history of cardiac iron overload in human β-thalassemia major, leading to the conclusion that THL mice are a suitable model for the study of cardiac iron overload in thalassemia.