PKM2激活NLRP3引发mDC细胞焦亡在SAA免疫发病机制中的作用

Pathogenesis of severe aplastic anemia (SAA) is closely related to the damage of the hematopoietic cells of the bone marrow caused by the hyperfunctional T lymphocytes immune. Recently, expansion of autoimmune inflammatory response aroused by the apoptosis of antigen-presenting cells has become a hotspot in the research of immune diseases. Our previous study had shown that the dysfunction of myeloid dendritic cells (mDC) in SAA patients might be an important factor leading to the occurrence of a series of immune responses. In recent years, applicants have mainly participate in the studies on the immune pathogenesis of SAA, and early researches in proteomics had indicated that the expression of pyruvate kinase M2 subtypes (PKM2) was significantly associated with the abnormal function of mDCs, in addition, a later research showed that its expression was obviously correlated with the high expression level of NLRP3, Caspase-1 in mDC apoptosis pathways. On the basis of the previous research work and taking SAA patients as the research object, this study purpose to analyze the regulation as well as the mechanism of the apoptosis of mDC cells induced by PKM2 through NLRP3 pathway. Cell model in vitro and SAA mouse model are established to elucidate the influences, including the action mechanisms of PKM2 on the apoptosis of mDC cells and activating the subsequent immune responses. Furthermore, to explore the effects resulting from the PKM2 inhibition on the apoptosis of mDC cells and the immune pathogenesis of SAA. Through above research, we are aimed at providing an experimental basis for the application of blocking cells apoptosis to treat SAA.

重型再生障碍性贫血（SAA）发病与T细胞免疫功能亢进损伤自身骨髓造血细胞密切相关。近期抗原呈递细胞焦亡引发自身免疫炎症反应扩大成为免疫性疾病研究热点。我们既往研究显示SAA患者髓系树突细胞（mDC）功能异常，可能是导致一系列免疫反应的重要因素。申请人近年来主要参与SAA免疫发病机制的研究，前期蛋白质组学研究中发现丙酮酸激酶M2亚型（PKM2）表达与mDC功能异常明显相关，进一步研究显示其表达与mDC细胞焦亡通路NLRP3、Caspase-1的表达增高明显相关。本课题拟在前期研究工作的基础上，以SAA患者为研究对象，分析PKM2通过NLRP3调控mDC细胞焦亡及其机制；建立体外细胞模型及SAA小鼠模型阐明PKM2对mDC细胞焦亡及其激活进一步免疫反应的影响及其机制；探讨抑制PKM2对mDC细胞焦亡及SAA免疫发病的影响。通过上述研究为应用基于阻断细胞焦亡治疗SAA提供实验依据。

重型再生障碍性贫血（SAA）是一种由多种病因导致的重度骨髓造血衰竭性疾病。SAA免疫学发病机制表现为交叉网络免疫的特点，即起抑制作用的细胞功能增强、比例增多，负性调控因子增多，而诱导免疫耐受的细胞不足。具体表现为以下几个方面，未知抗原激活髓系树突状细胞(mDC)，导致辅助性T细胞Th0偏向于向Th1分化，因此Th1相关的细胞因子分泌相应增多，杀伤性T细胞功能亢进、数目增多，同时Th17增多，调节性T细胞（Treg）功能减弱，自然杀伤细胞（NK）数目减少，加重炎症反应。以上研究证实mDC的功能亢进有可能是SAA免疫瀑布的起点。PKM2作为调节机体代谢的重要物质，我们的前期通过蛋白组学研究显示，PKM2在SAA患者mDC及效应细胞功能激活中发挥重要作用，通过调控及影响SAA免疫瀑布的上游始动环节参与疾病的发生发展。但PKM2激活mDC的具体机制并不清楚，因此，探索诱发SAA患者mDC细胞激活的原因，对于深入揭示SAA的免疫发病机制，明确其发病机理具有重大意义。PKM2在NLRP3炎症小体所介导的细胞焦亡中发挥了重要作用。减少巨噬细胞中PKM2的表达能够通过抑制NLRP3和减少IL-1β的产生而减轻小鼠的脓毒血症。总之，我们进一步明确了SAA患者mDC细胞焦亡水平，分析NLRP3与SAA免疫状态的相关性。建立体外细胞模型和AA小鼠模型检测 PKM2 调控 NLRP3 对mDC 细胞焦亡状态及其激活机体免疫炎症反应的影响，通过干扰 PKM2 与 NLRP3探索细胞焦亡对CD8+T细胞及CD4+T细胞功能的影响。同时追溯上游短链脂肪酸对mDC细胞焦亡状态影响，为完善SAA免疫发病机制提供实验依据。