MFHAS1在脓毒症器官损伤期单核细胞分化与功能失调中的作用与机制研究

Diminished monocyte to dendritic cell (DC) differentiation, and macrophage predominance, has been shown to correlate with adverse clinical outcomes such as organ dysfunction in sepsis. It is one of the several bottlenecks restricting clinical treatment of sepsis. None studies in above field are reported. We found that the expressions of MFHAS1 (Malignant fibrous histiocytoma amplified sequence 1) and Wnt5a in peripheral blood mononuclear cell of septic patients were significantly higher than healthy controls. We also found that recombinant Wnt5a and MFHAS1 could enhance the ability of DC to activate T cells, indicating that MFHAS1 might be involved in monocyte differentiation. Mechanistically, we found that MFHAS1 knockdown increased the protein expression of Dectin-1 downstream signaling pathway, alleviating inflammation. Hence, we assumed that MFHAS1 could regulate monocyte differentiation to DC and its function, through regulating Wnt/Dectin-1 signaling. Our project aimed to investigate the role of MFHAS1 in regulating monocyte differentiation and DC function, also to reveal the underlying mechanism in the regulation of Wnt/Dectin-1 signaling, from multi-dimensions of cells, molecules and whole animals, making the best use of mfhas1 knockdown zebrafish and Mfhas1 knock-out mouse. The goal of our project is to establish the foundation for the repression and target treatment of the excessive inflammatory response in the stage of organ dysfunction in septic patients.

脓毒症患者器官损伤期单核细胞向树突状细胞（DC）分化减少，而巨噬细胞分化增加及DC功能障碍的调控机制未明，是制约临床诊疗的关键瓶颈之一。我们前期研究发现脓毒症患者外周血单核细胞中Wnt5a和恶性纤维性组织细胞瘤扩增序列1（MFHAS1）表达显著高于非脓毒症患者，且重组Wnt5a和MFHAS1蛋白能增强DC活化T细胞的能力，而沉默MFHAS1使得DC中Dectin-1通路下游蛋白表达增加进而抑制炎性反应。基于此，我们假设MFHAS1参与调控Wnt/Dectin-1信号通路，继而引起单核细胞向DC分化及功能演变。本研究充分利用mfhas1基因沉默斑马鱼和Mfhas1基因敲除小鼠，从细胞、分子和整体动物多维度研究MFHAS1通过调控Wnt/Dectin-1信号通路影响单核细胞向DC分化及其功能的确切机制，为阻遏和靶向治疗脓毒症患者过度炎性反应造成的器官功能损伤提供科学依据。

脓毒症患者单核/巨噬细胞分化和功能障碍影响脓毒症进展。我们前期研究发现脓毒症患者外周血单核细胞中Wnt5a和ROCO蛋白家族分子恶性纤维性组织细胞瘤扩增序列1（MFHAS1）表达显著高于非脓毒症患者，MFHAS1可能调控单核细胞分化和功能，以及TLR信号通路。本研究从细胞、分子和整体动物多维度研究MFHAS1与单核细胞和树突状细胞（DC）分化和功能障碍之间的关系，以及MFHAS1对Dectin-1信号通路调控的影响。通过本项目研究，获得以下结果：①通过流式细胞仪检测，发现不同浓度LPS、β-glucan刺激人单核细胞THP-1后，单核细胞向CD209+细胞分化，且MFHAS1、pJNK表达随β-glucan浓度升高而上升；②通过诱导生成骨髓来源的巨噬细胞（BMDM），以及构建mfhas1基因沉默型（mfhas1-/-）斑马鱼脓毒症模型和内毒素休克多细菌感染脓毒症小鼠模型，结果发现：Fzd-5 mRNA和Wnt5a、MFHAS1在DC中表达高于巨噬细胞，重组Wnt5a和MFHAS1蛋白使得DC活化T细胞能力增强，MLR结果提示重组Wnt5a和MFHAS1蛋白使得DC活化T细胞能力增强；③MFHAS1抑制Dectin-1信号通路下游蛋白PIAS-1、SOCS-1表达；④单细胞转录组分析揭示了脓毒症患者外周血单核细胞失调，单核细胞占比显著降低，呈现显著地免疫功能抑制，其中经典单核细胞亚群的丰度和功能变化特征与个体症状和临床结局密切相关，具体表现为SLC39A8+ Mono比例降低，伴有免疫抑制的CCL3+ Mono比例增加，预示临床结局不佳。本项目研究获得的结果揭示了MFHAS1在脓毒症单核细胞和DC功能障碍中的作用，Dectin-1信号通路对单核细胞分化及其功能的调控作用，以及MFHAS1在Dectin-1信号通路调控，以及脓毒症小鼠单核细胞分化及其功能调节中的关键作用。研究结果将有助于进一步阐明脓毒症疾病进展的病理生理学特征及其分子机制，为脓毒症患者单核细胞、DC分化障碍和功能失调，以及过度炎性反应的阻遏和靶向治疗奠定科学依据。