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Blood, Vol. 109, Issue 11, 5027-5035, June 1, 2007
Ineffective erythropoiesis in β-thalassemia is characterized by increased iron absorption mediated by down-regulation of hepcidin and up-regulation of ferroportin
Blood Gardenghi et al.
109: 5027
Supplemental materials for: Gardenghi et al, Vol 109, Issue 11, 5027-2035
Files in this Data Supplement:
- Table S1. Iron distribution and content in the liver and spleen correlates with the level of IE (PDF, 46.4 KB) -
The large SD observed in the liver iron content of (th3/th3)tp-2M animals (Figure 3A) can be easily explained if IE or Hb levels are considered. The Hb level was measured the day that each animal was killed. The highest liver iron levels correspond to the animals that showed the highest percentage of early erythroid cells (CD71+/Ter119+) and lowest Hb levels. If the animals are arbitrarily separated into 2 groups based on their Hb levels (with group 1 > 3 g/dL 4.3 ± 0.6 g/dL, n = 4 and group 2 ≤ 3 g/dL 2.2 ± 0.7 g/dL, n = 5), group 2 shows a larger iron content in the liver than group 1 (group 1 = 1.6 ± 0.6 µg/mg of dry weight; group 2 = 5.1 ± 2.0 µg/mg). Spleen iron content also correlates with Hb levels, although to a lesser extent (group 1 = 1.4 ± 0.7 µg/mg of dry weight; group 2 = 2.9 ± 0.5 µg/mg). These data indicate that in the spleen of (th3/th3)tp-2M mice, on average, there was 2 times less total iron than in the liver, whereas in th3/+-2M and (th3/+)tp-2M mice the opposite (as shown in Figure 3).
- Figure S1. Mice affected by β-thalassemia intermedia and major show different levels of IE (PDF, 53.2 KB) -
We evaluated the fraction of spleen cells in discrete erythroid populations by flow cytometry using the transferrin-receptor CD71 and erythroid-specific TER119 costaining. The analysis was performed at 2 months after transplantation, when (th3/+)tp-2M and (th3/th3)tp-2M mice experienced anemia together with splenomegaly and hepatic EMH.1 The CD71+/Ter119+ population corresponds to early erythroid cells (basophilic erythroblasts and late basophilic and chromatophilic erythroblasts), whereas the CD71−/Ter119+ corresponds to orthochromatophilic erythroblasts and, mostly, to enucleated erythroid cells.2 The percentage of early erythroid cells (CD71+/Ter119+) in (th3/+)tp-2M and (th3/th3)tp-2M mice increased significantly compared to (+/+)tp-2M mice (79.8% and 87.7% versus 9%, respectively). These increases were inversely proportional to the Hb levels (Figure 1). The relative number of more differentiated erythroid cells (CD71−/Ter119+) decreased in (th3/+)tp-2M and (th3/th3)tp-2M mice (5.6% and 4.3 versus 29.2% respectively) compared to (+/+)tp mice. As a control, the same analyses were repeated on tissues from +/+-2M and th3/+-2M mice, obtaining similar results (not shown). The BM was also analyzed, showing a similar increase in CD71+/Ter119+ and reduction in CD71−/Ter119+ cells (not shown). In the spleen and BM, the amount of IE was estimated based on the ratio between early precursor (Ter119+/CD71+) and more mature erythroid cells (Ter119+/CD71−). On average, the ratios between Ter119+/CD71+ and Ter119+/CD71− cells were 0.3, 7, and 20, respectively, in (+/+)tp-2M, (th3/+)tp-2M, and (th3/th3)tp-2M mice. In aging th3/+ mice, the anemia and the ratio between Ter119+/CD71+ and Ter119+/CD71− increase over time (not shown). The same analyses were performed on transplanted and nontransplanted animals, the results being similar.
- Figure S2. In the liver of mice affected by β-thalassemia intermedia, iron preferentially accumulates in the Kupffer cells, whereas in mice affected by β-thalassemia major, it is stored mainly in parenchymal cells (PDF, 3.87 MB) -
Liver stained with Prussian blue to detect iron (blue granules; original magnification, ×132). (A) Mice 2 months after transplantation; (B) 5 months after transplantation; (C) 5 months after transplantation and transfused; and (D) 12 months old, no transplant. The red arrow indicates the presence of erythroid cells in the liver (extramedullary hematopoiesis EMH), a hallmark feature of  -thalassemia major. The images were captured on a Nikon Eclipse E800 microscope (Melville, NY), with a Retiga Exi camera (Qimaging, Burnaby, BC, Canada) and a Plan Fluor 40×/0.75 numerical aperture objective, then acquired using the IPLab 3.65a software (Scanalytics, Fairfax, VA). Brightness/contrast and color balance were adjusted using Adobe Photoshop 7.0.1 (Adobe Systems, San Jose, CA).
- Figure S3. The increased level of Tfr1 in the liver of th3/th3 mice is due to EMH (PDF, 31.8 KB) -
During erythropoiesis differentiation, only nucleated erythroid cells express the CD71 (Tfr1) marker. To evaluate the contribution of the nucleated erythroid cells present in the liver (EMH) to the expression of Tfr1, we measured the levels of  -globin and Tfr1 expression in samples generated mixing +/+ liver and th3/+ spleen tissues in predefined amounts. Knowing the proportion of CD71+ cells (which express Tfr1) in the spleen tissue and the relative amount of the two organs, we generated RNAs with 0.0%, 3.2%, 5.8%, 11.7%, 17.5%, and 23.3% of spleen-CD71+ tissue origin. (A) The corresponding values and regression curve obtained plotting the -globin CT (x-axis) versus the Tfr1 CT (y-axis) values. The percentage of erythroid cells is indicated underneath the corresponding -globin CT. (B) The -globin and Tfr1 values observed in the liver of four th3/+ and four th3/th3 mice are plotted using the same criteria and compared to the regression curve obtained in panel A. In particular, if Tfr1 is intrinsically overexpressed in cells of hepatic origin from th3/th3 mice, the intersection of the CT values should be localized above the regression curve. This would indicate that the corresponding Tfr1 RNAs are increased in the liver beyond the expected values due to the EMH. In contrast, if the intersections localize nearby or below the regression curve, most likely the Tfr1 RNA is exclusively of erythroid origin. An estimation of the statistical significance of our data was performed using the regression line (y = ax + b), plugging in the -globin values (x) in the graph for th3/+ and th3/th3 mice, respectively, and obtaining the Tfr1 (y) values that would be expected if the points fell on the line. The differences between the expected and actual values were estimated using the Wilcoxon test. P values of .125 and .625 were obtained for th3/+ and th3/th3 mice, respectively, indicating that our data are well modeled by the expected linear regression. In conclusion, although we observed that Tfr1 is up-regulated in the liver of th3/th3 mice, our assay indicates that erythroid contamination of the liver is sufficient to explain the increased expression of Tfr1.
REFERENCES
1. Rivella S, May C, Chadburn A, Riviere I, Sadelain M. A novel murine model of Cooley anemia and its rescue by lentiviral-mediated human beta-globin gene transfer. Blood. 2003;101:2932-2939.
2. Socolovsky M, Nam H, Fleming MD, Haase VH, Brugnara C, Lodish HF. Ineffective erythropoiesis in Stat5a(-/-)5b(-/-) mice due to decreased survival of early erythroblasts. Blood. 2001;98:3261-3273.
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