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IL-4 contributes to thrombocytopenia in AML
megakaryocytic colony formation of human CD34+ BM cells37 and to have relevance in the thrombocytopenic state of idiopathic thrombocytopenic purpura and allo- geneic hematopoietic stem cell transplantation patients.38,39 We confirmed the elevated level of IL-4 in the AML group using enzyme-linked immunosorbent assays (Figure 4A). Our in vitro colony-forming cell assays showed that IL-4 imposed a selective inhibitory effect on colony-forming unit-MK formation from BM cells (Figure 4B) without affecting other myeloid and erythroid lineag- es (Figure 4C). Interestingly, upon in vitro IL-4 stimulation, HSC-enriched LKS+ cells exhibited an even more promi- nent response than myeloid progenitors (Figure 4D), as indicated by intracellular phosphorylation of Stat6 (Figure 4E) which has been recognized as a downstream trans- ducer of IL-4 signaling.40 In response to exposure to IL-4, all MK-associated transcription factors except for Gata2 were universally downregulated in LKS+ cells (Figure 4f), suggesting the possible effects of this cytokine on MK dif- ferentiation of primitive hematopoietic cells. We next analyzed the transcriptome of LKS+ cells from AML BM (GSE52506)10 and found significant upregulation of IL-4 signaling genes and predicted Stat6-bound genes (Figure 4Gg). As BM immune cells have been reported to be the main source of IL-4,41 we first quantified the IL-4 mRNA expression in T lymphocytes, B lymphocytes, mono- cytes, macrophages, natural killer cells and eosinophils. However, we did not detect upregulation of IL-4 in these cells from AML BM (Online Supplementary Figure S6A). The level of expression of IL-4 by AML blasts was similar to that by normal hematopoietic cells (Online Supplementary Figure S6B). We then quantified IL-4 expression in HSC niche cells including mesenchymal stem cells, endothelial cells and osteoblasts. We detected reproducible significant upregulation of IL-4 in endothe- lial cells but not in the other niche cells (Figure 4H). Thus, BM endothelial cells produced excessive IL-4 in AML to activate IL-4 signaling in HSC-enriched LKS+ subsets.
To further understand the effects of IL-4 on MK differen- tiation, we intraperitoneally injected IL-4 into wildtype mice at 48 h intervals and analyzed the alterations of MK as well as HSPC with MK potential in mice BM (Figure 5A). In order to prolong the half-life of IL-4 and enhance its biological activity in vivo, we pre-associated the IL-4 with its specific monoclonal antibody as previously described42 and injected the IL-4 complex (IL-4cx) into the mice. We observed a remarkable decrease of platelets in peripheral blood after the injection of two doses, while erythrocytes and leukocytes were barely affected (Figure 5B). Simultaneously, though the total number of BM cells was not changed (Figure 5C), the number of MK in the BM of IL-4-treated mice was severely reduced by ~84% (Figure 5D). The ploidy distribution of MK exhibited a left shift with high-ploidy (≥32N) cells being more severely decreased (Figure 5E), indicating that IL-4 treatment sup- pressed MK maturation. Among HSPC with MK potential, we observed an ~54% loss of PreMegE and ~37% loss of MkP (Figure 5F), whereas the proportion of LT-HSC was unaltered, and that of MPPs increased ~4 fold (Figure 5G). These results suggest that the downward differentiation of MPP2 was severely hampered by IL-4, especially in the route via PreMegE. As expected, vWF expression was sig- nificantly reduced in MPP2 from IL-4-treated mice (Figure 5H, I). Interestingly, we observed a remarkable increase of vWF+ cells among LT-HSC (Figure 5H, I), indicating that IL-
4 did not have an obvious inhibitory effect on their MK dif- ferentiation in our setting; on the contrary, LT-HSC gave rise to MK more actively as compensation for the reduced contribution of MPP2 to the MK pool, which limited the loss of MkP to a relatively small extent. Given the smaller reduction (~37%) of MkP and the considerable decrease of MK (~84%) (Figure 5D, F), thrombocytopenia induced by IL-4 administration was largely caused by a drastic inhibi- tion of MkP maturation. Notably, IL-4 receptor (IL-4Rα) expression on MPP2 and MkP was higher than that on LT- HSC and PreMegE subsets (Figure 5J), in accordance with the more prominent response of MPP2 and MkP to in vivo IL-4 treatment.
Targeting interleukin-4 in conjunction with chemotherapy enhances platelet recovery in acute myeloid leukemia mice
Lastly, we tested whether targeting IL-4 could ameliorate thrombocytopenia in AML mice. To do this, AML mice were treated with anti-mIL-4 on days 7, 9, and 11 after injection of leukemic cells and were sacrificed on day 13 for analysis (Online Supplementary Figure S7A). Administration of anti-mIL-4 alone to leukemic mice nei- ther significantly decreased leukemia load nor increased platelet count in the peripheral blood (Online Supplementary Figure S7B, C). Intensive induction chemotherapy is cur- rently used for patients with AML as a general therapeutic strategy.43,44 Because cytarabine (AraC) together with an anthracycline remains the mainstay of induction therapy,44 we set up a treatment protocol in which AML mice were treated daily with 60 mg/kg of AraC for 1 week (Online Supplementary Figure S7D). This treatment significantly reduced the leukemic burden in the peripheral blood (Online Supplementary Figure S7E) and prolonged the sur- vival of AML mice (Online Supplementary Figure S7F), but it was noted that the animals developed severe thrombocy- topenia (Online Supplementary Figure S7G). To test the hypothesis that thrombocytopenia may be more mitigated by anti-IL-4 given during or after AraC treatment (Online Supplementary Figure S7H), we next established a treatment protocol in which AML mice were intraperitoneally inject- ed daily with 10 mg/kg of anti-mIL-4 during AraC chemotherapy (Figure 6A). Interestingly, this treatment not only significantly reduced the leukemic burden (Figure 6B) but also enhanced the recovery of platelets (~4.7-fold increase) and erythrocytes (~1.9-fold increase). In contrast, leukocytes (Figure 6C) and serum thrombopoietin concen- tration (Online Supplementary Figure S7I) were not signifi- cantly affected. As a result, this new strategy of combining AraC with anti-IL-4 also significantly extended the dura- tion of remission of AML mice (Figure 6D). Together, these data demonstrate that anti-mIL-4 combined with chemotherapy could improve the therapeutic response compared to that achieved with standard chemotherapy for AML.
Discussion
Thrombocytopenia is a frequent complication among AML patients: It can lead to a strong dependence on platelet transfusions and even fatal bleeding. Using an MLL-AF9-induced AML mouse model, we demonstrated that thrombocytopenia in AML was accompanied by a progressive loss of mature MK in the BM. A systematic
haematologica | 2019; 104(10)
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