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The critical function of Tfr1 in hematopoiesis
Loss of Tfr1 in HSC causes intracellular iron decrease in hematopoietic cells in postnatal mice
Given that the principal function of Tfr1 is iron uptake through the cell membrane, we reasoned that the loss of Tfr1 might cause cellular iron deficiency, thereby affecting systemic iron levels. Therefore we evaluated intracellular iron concentration in hematopoietic cells by loading BM derived HSPC with the iron-sensitive fluorophore calcein- AM, which is quenched upon binding ferrous iron (Fe2+) .24 Intracellular iron was significantly lower in cKO erythro- cytes compared to control cells at all five stages (Figure 5A-B). In addition, intracellular iron was lower in cKO Lin+ mature cells (Figure 5C), as well as LSK and HPC cells (Figure 5D), compared to their respective controls.
Next, we measured systematic iron levels and found that cKO pups have higher levels of serum iron (Figure 5E), transferrin saturation (Figure 5F), and serum hepcidin (Figure 5G). Considering smaller sizes of the livers and spleens in cKO pups compared to the controls (Online Supplementary Figure S4A-B), we found that liver non- heme iron was significantly higher than control measured either in mg/g wet weight or absolute amount of iron (Figure 5H-I). In contrast, splenic non-heme iron levels of cKO in mg/g wet weight didn’t change (Figure 5J), whereas the absolute amount of iron decreased (Figure 5K). In the muscle of cKO pups, non-heme iron (mg/g wet weight) increased (Figure 5L). In addition, whole body non-heme iron levels (mg/g wet weight) in cKO pups were signifi- cantly increased (Figure 5M), whereas the absolute amount of body iron (mg) remained unchanged (Figure 5N). Taken together, these data suggest that hematopoiet- ic Tfr1 deficiency significantly impaired iron uptake of hematopoietic cells, in turn led to iron redistribution to other organs (e.g. serum, liver and muscle).
Loss of Tfr1 in HSC affects iron homeostasis in mature cells but not HSPC in the FL
Next, we investigated whether Tfr1 affects iron home- ostasis differently between prenatal and postnatal stages of development. Intracellular iron levels were lower in cKO Lin+ cells at E16.5 (Figure 5O), which is consistent with decreased numbers of mature cells. Interestingly, intracellular iron levels and ferritin protein levels (Online Supplementary Figure S5A) in both LSK and HPC cells were similar between cKO and control embryos at E16.5 (Figure 5P), indicating that loss of Tfr1 does not affect intracellular iron in HSPC during prenatal development. Meanwhile, we found no difference between cKO and control HPC cells with respect to the mRNA levels of various mem- brane iron transporters and Alas2, catalyzing the rate-lim- iting step of heme biosynthesis in erythroid cells (Online Supplementary Figure S5B). Therefore, we hypothesized that hematopoietic Tfr1 is required for differentiation into lineages in which iron plays an essential role. In addition, we quantified expression levels of critical transcription factors required for cell fate determination of HSC.1 We found significant decreases of a subset of transcription fac- tors involved in the development and differentiation of HPC, including Gli1, C/EBPa, Fli, PU.1, Gata2, Notch1, and Gata3 (Online Supplementary Figure S5C).
Loss of Tfr1 in HSC impairs the regeneration capacity of HSPC
The maintenance and survival of HSPC requires an interplay between these cells and their niche within the
BM.25 Therefore, we speculated whether loss of Tfr1 in cKO HSPC disrupts this niche, leading to a feedback loop that impairs hematopoiesis. To test this possibility, we performed a transplantation assay to measure the regen- erative capacity of HSPC in recipient BM. We used FL cells obtained from E14.5 embryos, as cells at this stage were only mildly affected in the cKO mice.
To determine their short-term regenerative capacity, FL cells were isolated from cKO and control mice expressing the alloantigen CD45.2 and injected into the tail vein in lethally irradiated CD45.1 recipient mice. We found that recipient mice that received control cells had 100% sur- vival, whereas mice that received cKO cells all died with- in 12 days of transplantation (Figure 6A). Next, we per- formed competitive BM transplants in order to assess the long-term regenerative capacity and found that recipient mice that received 50% cKO cells and 50% CD45.1 com- petitor cells had virtually no donor-derived cKO cells in their peripheral blood by four weeks post-transplantation (Figure 6B). In addition, virtually no contribution of cKO HSPC or committed cell lineages in the recipient mice was measured 16 weeks after transplantation (Figure 6C- E). Finally, we found virtually no contribution of cKO HSPC to myeloid cells, B cells in the spleen, or T cells in the thymus of the recipient mice (Figure 6F). These results indicate that Tfr1 is required for both the short-term and long-term regeneration of FL cells.
Upon transplantation, HSPC migrate to the recipient’s BM, where they home to their proper niche. When we analyzed BM cells in lethally irradiated CD45.1 recipient mice 40 hours after transplanted with CD45.2 cKO or control FL cells, no difference was found (Figure 6G), indi- cating that the homing process is not impaired in cKO FL cells. Taken together, these results suggest that the reduced regenerative capacity in Tfr1-deficient HSPC is due to a cell-autonomous mechanism.
Hemin treatment rescues the proliferation and differentiation defects in Tfr1-deficient HSPC
The results described above indicate that the composi- tion of cKO HSPC is normal in the early stages of embry- onic development. Thus, other modes of iron delivery might be presented in order to support the demand of FL HSPC for iron. To examine whether an alternative modes of iron deliver is able to bypass the loss of Tfr1 in cKO HSPC, we performed various in vitro rescue experiments using a methylcellulose colony formation assay with FL cells obtained from E14.5 embryos. cKit+ cells were sorted and then cultured in a methylcellulose medium contain- ing IL-6, IL-3, stem cell factor, and erythropoietin. We found that colony-forming units (CFU) were formed by control cells, whereas cKO cells failed to develop any of these CFU (Figure 7A). These results are consistent with our in vivo data and suggest that the differentiation of both myeloid and erythrocyte cell lineages are blocked in cKO mice.
Next, we treated the cells with 5 mM holo-transferrin (holo-Tf), 10 mM ferric ammonium citrate (FAC), 10 mM hemin, or 0.25 mM Fe (III)/8-hydroxyquinoline (Fe/8-HQ), a highly membrane-permeable complex that delivers iron directly into the cells,26 thereby bypassing Tfr1. Interestingly, at these concentrations of both hemin and Fe/8-HQ partially rescued the colony-forming capacity of cKO cells, whereas holo-Tf and FAC had no effect (Figure
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