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X. Jin et al.
eration and GPX1 involved in ROS clearance in erythroid cells. Interestingly, the expression of NOX4 was signifi- cantly higher in RX291/FE mice than that of RX291/NS mice (Figure 5D), while the level of GPX1 was signifi- cantly lower in the RX291/FE group (Figure 5E), support- ing the view that iron overload can induce ROS to dam- age erythroid hematopoiesis. Previous studies have reported that TGF-b signaling is myelosuppressive and inhibits erythroid differentiation by induction of ROS and apoptosis in erythroblasts.15-17 We then examined the mRNA levels of TGF-b superfamily including GDF8, GDF11, GDF15, Activin A, Activin B, Acvr2b, ALK4, and ALK5, and found that GDF11 mRNA levels were signifi- cantly increased in RX291/FE mice compared to RX291/NS mice (Figure 5F). We further detected the GDF11 protein levels in serum, which showed similar results in these two groups (Figure 5G). Collectively, iron overload can damage erythroid hematopoiesis in MDS mice, which may partially be due to GDF11-induced ROS, leading to enhanced apoptosis of normal BM cells and inhibition of their function in MDS.
Iron overload shortens survival MDS mice
Given that iron overload results in a suppressive effect in the frequency and function of normal HSPCs in MDS mice, we postulated that iron overload can affect the median survival of these mice. The mice were observed for 360 days post transplantation and each group has more than ten mice. In our study, MDS mice have an obviously shorter median survival time than control mice (Figure 6), mostly due to AML transformation. For control mice, the survival time did not differ between Empty/NS and Empty/FE mice because the experiment was terminated at the end of day 360. However, RX291/FE mice exhibited a significantly shorter median survival than RX291/NS mice (Figure 6), suggesting that iron overload can shorten the survival time of MDS mice.
Discussion
Myelodysplastic syndrome is a heterogeneous group of clonal stem cell disorders characterized by multilineage dysplasia, ineffective hematopoiesis, pancytopenia, and with a high risk of developing into acute myeloid leukemia.18 Over recent years, investigators all over the world have been trying hard to establish an MDS model to further study this disease. However, only a few mouse models are available for human MDS. RUNX1 is a fre- quently mutated gene in MDS7 and RUNX1S291fs is one of the most common mutations in RUNX1. It has been demonstrated that transduction of this mutant into BM cells can be used to construct MDS mice.8 Indeed, our RUNX1S291fs-induced mice showed neutropenia, ane- mia, thrombocytopenia, multilineage dysplasia, together with less than 20% blasts, matching the criteria of MDS in the Bethesda proposal and phenotypically recapitulating human MDS.
Due to frequent blood transfusions and ineffective hematopoiesis, most MDS patients will eventually devel- op iron overload. Excessive iron deposits in the tissues and organs cause a series of complications, affecting the prognosis of MDS patients. In turn, iron overload has a negative role in hematopoiesis in MDS, further deteriorat- ing the situation of these patients. To understand how iron overload influences hematopoiesis in MDS, we first established an iron overload model in RUNX1S291fs- induced MDS mice by intraperitoneal injection of iron dextran. Our data presented more iron deposition in organs, and more significant liver and spleen enlargement in RX291/FE mice, supporting the successful establish- ment of an iron overload model in RUNX1S291fs-induced MDS mice. In the present study, although there was no statistical significance in the levels of WBC, PLT and HG in the PB between Empty/NS and Empty/FE, we observed a declining trend in Empty/FE relative to Empty/NS mice,
A
D
BC
EFG
Figure 5. Iron overload inhibits erythroid hematopoiesis in myelodysplastic syndrome (MDS) mice through reactive oxygen species (ROS). (A) Level of apoptosis in MDS mice. (B) ROS level in the bone marrow (BM) and erythrocytes. (C) ROS level in the erythrocytes. (D) mRNA expression of NADPH oxidase 4 (NOX4) gene. (E) mRNA expression of glutathione peroxidase 1 (GPX1) gene. (F) mRNA expression of growth differentiation factor 11 (GDF11) gene. (G) The concentration of GDF11 protein in serum detected by ELISA assay. *P<0.05, **P<0.01, ***P<0.001. BMMNC: bone marrow mononuclear cells.
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haematologica | 2018; 103(10)