Page 96 - 2020_08-Haematologica-web

S. Wang et al.
ment and proliferation of lymphocytes.14 A recent study found that patients who carry a homozygous mutation in the TFR1 gene have reduced T-cell and B-cell proliferation, as well as reduced antibody production.15
Several key questions remain, however, regarding the function of Tfr1 in hematopoiesis. First, because previous studies focused on mature hematopoietic lineages, whether Tfr1 plays a role in upstream of hematopoiesis (for example, in stem cells, progenitors, and precursor cells) is unknown. Second, Tfr1 plays a role in signal trans- duction pathways other than iron uptake and is required for maintaining intestinal epithelial homeostasis,16 thus raising the question of whether the function of Tfr1 in hematopoiesis is independent of its iron-uptake function. Finally, because Tfr1 knockout embryos die at embryonic stage E10.5-E12.5, the embryo’s iron demands at later stages of development have not been investigated.
To address these key questions, we generated mice that lack Tfr1 expression selectively in HSC and used this model to study the role of Tfr1 in hematopoiesis. We found that loss of Tfr1 in HSC does not affect the produc- tion of hematopoietic stem/progenitor cells (HSPC) in the fetal liver (FL) but markedly impairs the expansion of func- tional HSC in the bone marrow (BM). Mechanistically, iron uptake rather than signal transduction appears to be the key function of Tfr1 in hematopoiesis, and iron uptake mediated by Tfr1 is required for the differentiation of HSPC, particularly in mid-gestation.
Methods
All animal experiments were approved by the Institutional Animal Care and Use Committee of Zhejiang University. Tfr1fl/fl mice on the C57BL6/J background were obtained from Dr. Ying Shen.17 To generate HSC-specific Tfr1 knockout mice ( Tfr1fl/fl;Vav-Cre, referred to hereafter as cKO mice), we crossed female Tfr1fl/fl mice with male Tfr1fl/wt;Vav-Cre transgenic mice; the Vav-Cre line we used was B6.Cg-Commd10Tg(Vav1-icre)A2Kio, the Vav-Cre transgene is expressed mainly in hematopoietic cells.18 Hematological parameters, blood smears, embryo dissection and single-cell isolation, flow cytometric analysis, evaluation of intracellular iron status, iron parameters, colony formation assays, and transplantation assays are described in the Online Supplementary Materials and Methods. Except where indicated otherwise, all summary data are presented as the mean ± stan- dard deviation. The Student’s t-test was used to compare two groups, multiple groups were subjected to analysis of variance (ANOVA) with Bonferroni post hoc test comparison, and the log- rank (Mantel-Cox) test for survival curve analysis (GraphPad version 7). P-values <0.05 were considered statistically signifi- cant.
Results
Trf1 is expressed at higher levels in hematopoietic progenitor cells than in HSC and mature cell lineages
First, we measured Tfr1 expression in mouse FL HSPC and the different stages of erythroid differentiation using multiparametric flow cytometry and the gating strategies shown in Figure 1A-B. As shown in Figure 1C-D, Tfr1 expression was extremely low in Lin–Sca1+cKit+ subsets, virtually undetectable on the surface of long-term HSC, short-term HSC, multipotent progenitors, and common
myeloid progenitors. In contrast, Tfr1 expression was rela- tively high in granulocyte/monocyte progenitors and com- mon lymphoid progenitors, and was highest in megakary- ocyte/erythrocyte progenitors. In erythroid lineage, Tfr1 expression was high in pro-erythroblast, and gradually decreased with the differentiation of erythroblast. These findings at the protein level were generally consistent with RNA sequencing data (Online Supplementary Figure S1) that we extracted from previous studies.19,20 In addition, Tfr1 expression was higher in progenitors/precursors (early T- cell progenitors, progenitor B cells, and granulocyte mono- cyte progenitors) compared to their mature counterparts (T cells, B cells, NK cells, granulocytes, and monocytes) (Online Supplementary Figure S1), suggesting that Tfr1 may play a more important role in the progenitor/precursor stages than in stem cells.
HSC-specific loss of Tfr1 causes early postnatal lethality
To examine further the role of Tfr1 in hematopoiesis, we generated HSC-specific Tfr1 cKO mice. The cKO off- spring were born at the expected Mendelian ratio but smaller and paler than control (Tfr1fl/fl) littermates (Figure 2A). Importantly, although cKO pups were born at the expected Mendelian ratio, homozygous cKO pups failed to thrive and died by postnatal day 7 (P7), whereas both heterozygous and control littermates grew normally (Figure 2B-C). We confirmed the knockout efficiency in both mRNA and protein level. Tfr1 mRNA was virtually non-detectable in the BM of cKO mice (Figure 2D) and Trf1 (CD71) expression in cKO HSPC significantly decreased compared to control littermates by flow cytometry analysis (Figure 2E-F).
Tfr1-deficient HSC have multiple lineage defects in hematopoiesis
A possible explanation for the early postnatal mortality and paleness of the cKO mice is that the Tfr1-deficient HSC may perturb erythropoiesis. To test this possibility, we examined various hematopoietic organs. In peripheral blood, cKO mice had significantly fewer red blood cells, fragmented and irregularly shaped (Online Supplementary Figure S2), as well as reduced erythrocyte mean corpuscu- lar volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and hemoglobin concentration (Online Supplementary Table S1) compared to control, indicating that cKO mice had microcytic hypochromic anemia. In addition, cKO pups had reduced numbers of white blood cells and reticulocytes, and increased numbers of platelets (Online Supplementary Table S1).
To examine the role of Tfr1 in the development of proerythroblasts into mature red blood cells, we per- formed flow cytometry using the markers Ter119 and CD44.21,22 The number of early basophilic erythroblasts (R2), polychromatophilic erythroblasts (R3), and orthochromatophilic erythroblasts (R4) decreased signifi- cantly in cKO mice compared to controls (Figure 3A). In addition, orthochromatophilic erythroblasts (R4) and mature erythrocytes (R5) decreased significantly in the spleen of cKO mice (Online Supplementary Figure S3A). These data indicate that erythrocyte development is severely blocked in the early stages in the absence of Tfr1. Moreover, cKO mice had fewer Mac1+Gr1+ (mature macrophage/granulocyte) cells in the spleen and liver
2072
haematologica | 2020; 105(8)