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IL-4 contributes to thrombocytopenia in AML
hematopoiesis.3,6-9 Utilizing a congenic AML mouse model, we recently investigated dynamic changes in nor- mal hematopoietic stem and progenitor cell (HSPC) num- bers and cell cycling.10 Our findings suggested that the AML microenvironment suppressed proliferation and dif- ferentiation of normal HSPC, in line with conclusions from another study based on a xenograft AML model.11 Notably, from among all the HSPC subpopulations inves- tigated, megakaryocytic-erythroid progenitors were the most affected. Subsequently, we observed the inhibitory effects of CCL3 from the AML microenvironment on ery- thropoiesis.12 Strikingly, CCL3 acted on megakaryocytic- erythroid progenitors rather than HSC, and specifically suppressed erythropoiesis without affecting megakaryo- poiesis.12 How the path from HSC to megakaryocytes (MK) was influenced by the AML BM microenvironment remains to be defined.
Thrombocytopenia is a major and sometimes even fatal complication of acute leukemia.3,13 It has been esti- mated that thrombocytopenia occurs in approximately 75% of all leukemia patients, with one-third of them having clinically significant bleeding.14,15 In healthy indi- viduals, functional platelets are produced on a daily basis by mature MK, which originate from multipotent HSPC. Thrombopoietin is recognized as the principal regulatory cytokine throughout the process of megakaryopoiesis.16 An elegant study by Rauch et al.17 attributed thrombocy- topenia in AML patients to thrombopoietin scavenging by MPLhi leukemic blasts and proposed the MPLhi state as an indicator for more severe thrombocytopenia at diag- nosis. However, despite its demonstrated effects in alle- viating chemotherapy-associated thrombocytopenia in patients with solid tumors, thrombopoietin did not work well for acute leukemia patients who underwent chemotherapy or hematopoietic stem cell transplanta- tion.18 These phenomena suggest that the mechanism of thrombocytopenia is much more complicated in AML, and cannot be simply explained by chemotherapy- induced injury or a paucity of thrombopoietin. Thrombocytopenia may be a prolonged state due to the profound influence of leukemia on the process of megakaryopoiesis and thrombopoiesis, and on the BM microenvironment which tightly regulates hematopoiesis. In fact, recent studies have indicated that the perturbation of the BM microenvironment of leukemic hosts cannot be completely cured by current therapies, even if the leukemic burden is substantially reduced.7,19 Thus, a thorough analysis of megakaryo- poiesis and thrombopoiesis during leukemia develop- ment is required to search for detrimental factors in the leukemic microenvironment that could serve as potential therapeutic targets for these processes. In the classical hierarchical model of hematopoiesis, MK and erythro- cytes share common progenitors.20 However, recent stud- ies suggest that MK can be generated directly by an upstream HSC subpopulation, independently of other lineage fates.21,22 The alterations along the path from HSC to MK in AML are still unknown.
In the present study, we used an MLL-AF9-induced AML mouse model to analyze the dynamic changes of HSPC with MK potential. We found that thrombocy- topenia in AML was caused primarily by reduced megakaryocytic differentiation of CD150+ HSC and defective maturation of MK. Furthermore, we detected elevated interleukin-4 (IL-4) levels in AML BM, which
were found to inhibit megakaryopoiesis and throm- bopoiesis in vivo. Finally, we observed that inhibition of IL-4 combined with chemotherapy attenuated leukemia progression as well as thrombocytopenia in our AML mouse model.
Methods
Mice
C57BL/6-Ly5.1 (Ly5.1) and C57BL/6-Ly5.2 (Ly5.2) mice were purchased from the State Key Laboratory of Experimental Hematology (SKLEH). R26-tdTomato mice23 were purchased from Jackson Laboratory and actin-eGFP mice24 were kindly pro- vided by Dr. Bing Liu (Academy of Military Medical Sciences, Beijing, China). Mice experiments were approved by the Institutional Animal Care and Use Committee of SKLEH.
Flow cytometry
Details of the staining and enrichment procedures for flow cytometry have been described previously.25 Intracellular von Willebrand factor (vWF) and Ki67 staining was performed using a BD IntraSureTM Kit (BD Biosciences, Franklin Lakes, NJ, USA). Cell surface markers for phenotypic analyses of hematopoietic and niche cells are listed in Online Supplementary Table S1. The antibodies used are listed in Online Supplementary Table S2.
Acquisition of bone marrow niche cells
Cells were isolated following a previously described proto- col.26 Immunophenotyping and antibodies are listed in Online Supplementary Table S1 and Online Supplementary Table S2, respectively.
Bone sectioning and immunofluorescence imaging
Femora were fixed in 4% paraformaldehyde and dehydrated in 30% sucrose at 4°C prior to being embedded in optimal cut- ting temperature compound and frozen. For two-dimensional imaging, frozen femora were then cut into 5 μm sections using a cryostat and stained according to the Kawamoto method.27 Images were acquired using a UltraView VOX confocal micro- scope (PerkinElmer, Waltham, MA, USA) and analyzed by Volocity.
Enzyme-linked immunosorbent assays
BM supernatants were prepared by flushing femora and tibiae of leukemic and control mice with 0.5 mL phosphate-buffered saline. Enzyme-linked immunosorbent assays for IL-4 (R&D Systems, Minneapolis, MN, USA) were performed according to the manufacturer’s instructions. The concentrations were calcu- lated using standard curves.
Isolation of bone marrow megakaryocytes
BM cells were flushed out and incubated with CD41-APC antibody for 15 min on ice. CD41+ cells were then enriched using anti-CD41 microbeads and incubated in Dulbecco modi- fied Eagle medium supplemented with 10% fetal bovine serum and 10 μg/mL Hoechst 33342 at 37°C for 1 h. MK were sorted as SSChigh, CD41high, ploidy ≥8N cells through a 100 μm nozzle.
Microarray analysis
The microarray was performed at Cnkingbio Company (Beijing, China). Gene set enrichment analysis was performed using GSEA v2.2.0. The microarray data have been deposited in the NCBI’s Gene Expression Omnibus under accession number GSE112942.
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