IL-4信号通路参与急性髓系白血病下巨核-血小板生成障碍及机制研究

Thrombocytopenia-associated bleeding is one of the major complications among acute myeloid leukemia (AML) patients, which leads to high mortality and dependence on blood transfusions. However, the specific mechanism is poorly understood. Our previous study discovered that normal hematopoietic stem cells (HSCs) were forced into quiescence and prevented from proliferation and differentiation in AML bone marrow; yet their changes were reversible, indicating that factors from microenvironment including cellular (e.g. mesenchymal stem cells) and extracellular (e.g. CCL3, TGF-β1) ingredients play a pivotal role in this process. Via cytokine array screening, we identified elevated IL-4 in AML bone marrow and validated by in vivo experiments that its inhibitory effects on megakaryocytic differentiation from HSCs corresponded with the alterations in AML. Thus we hypothesize that IL-4 is possibly involved in defective megakaryopoiesis and thrombopoiesis in AML, and could be used as a target in treatment. The current study will utilize IL-4 knock-out mice to investigate the curable effects of IL-4 targeted strategy for thrombocytopenia during AML development and post chemotherapy, as well as the cellular biological mechanism. And conditional knock-out of IL-4 in HSC niche cells will be performed to locate the origin of excessive IL-4 in AML. Moreover, we will analyze the relations between IL-4 level and AML subtypes, platelet counts at diagnosis and the duration of thrombocytopenia after chemotherapy among AML patients, in order to find out the appropriate timing and objects for anti-IL-4 strategy. Our study will provide a preclinical basis for IL-4 targeted strategy as an adjuvant therapy in AML treatment, to lower the risks of bleeding and to reduce the patients’ dependence on blood transfusions.

血小板减少和出血是急性髓系白血病（AML）患者的主要并发症之一，提高了患者死亡率和对血制品的依赖，但具体机制尚不完全清楚。我们研究发现，AML状态下，骨髓正常造血干细胞更多地进入静息期，而这个过程是可逆的，表明微环境因素在其中起了主导作用。我们前期研究发现AML骨髓IL-4水平的升高，而且IL-4抑制巨核-血小板发育的体内实验表型与白血病下的变化相符。因此提出假说，IL-4可能介导了AML状态下巨核-血小板一系生成障碍，且可能作为治疗的靶点。本研究拟运用IL-4基因敲除小鼠研究靶向IL-4的策略对AML化疗前后血小板减少的挽救作用以及细胞生物学机制，并通过特异性敲除微环境细胞中的IL-4探究其来源；通过检测AML患者骨髓IL-4的表达，分析与其白血病类型、初诊时血小板水平和化疗后血小板恢复正常所需时间之间的关系，寻找靶向IL-4策略适用的时机和对象，为AML的辅助治疗提供新思路。

我们前期研究发现AML小鼠骨髓IL-4水平升高,而且IL-4抑制巨核-血小板发育的体内实验表型与白血病下的变化相符。因此提出假说：IL-4可能介导了AML状态下巨核-血小板一系生成障碍,且可能作为治疗的靶点。本课题研究内容为：1）敲除或拮抗IL-4对AML化疗后血小板减少的挽救作用；2）AML骨髓升高的IL-4来源,以及微环境细胞特异性IL-4 敲除对AML小鼠化疗前后血小板减少的挽救作用；3）IL-4影响造血干细胞和血小板生成的作用机制。目前研究发现，IL-4中和抗体体外可逆转白血病上清对巨核集落的抑制作用； IL-4中和抗体联合化疗可显著恢复AML小鼠血小板数量 。进一步构建血小板减少症模型研究IL-4的体内作用，发现IL-4诱导CMPs 向GMPs分化,而不是向MEPs分化；IL-4使巨核细胞的成熟过程受阻。IL-4在造血干细胞水平的作用则是诱导细胞凋亡：IL-4RA 高表达的HSCs氧化应激通路和凋亡通路激活。IL-4体外刺激HSCs后促凋亡基因、p53 信号通路基因和应激反应基因表达显著上调；而巨核细胞相关转录因子下调。利用衣霉素（tunicamycin）体外孵育或给予小鼠2Gy放射来诱导内质网应激后，IL-4RA 高表达的 HSCs凋亡增多。IL-4体外刺激24小时后，HSCs表达蛋白酶体成分基因Psmd13明显下调，利用shRNA敲降Psmd13，Knockdown组相比scramble对照组, HSCs体外形成BFU-E、CFU-Mk、CFU-GM和CFU-mix数量显著减少。移植后4周，Psmd13 KD组T细胞、B细胞、粒细胞、巨核细胞和血小板重建比例显著低于scramble对照组；Psmd13 KD组HSCs凋亡比例显著增加。IL-4通过抑制蛋白酶体成分基因Psmd13的表达，降低了HSCs对内质网应激损伤的抗性而引起凋亡增加和向巨核-血小板分化受抑。