团头鲂甘露糖受体在Ⅰ型巨噬细胞（M1）极化中的作用机制研究

Macrophages are important innate immune cells and play central immunological roles through their functional polarization. TypeⅠ macrophages（M1） are classically activated macrophages with powerful pro-inflammatory and antigen presenting activities. Mannose receptor (MR) is an important pattern recognition receptor as well as an endocytic receptor on macrophages. Earlier research by the applicant has demonstrated that MR of Megalobrama amblycephala (maMR) not only took part in recognizing aquatic bacterial pathogen, but also specifically recognized oligochitosan, and induced pro-inflammatory responses of macrophages. However, the mechanism of macrophage activation is not clear. Due to the lack of signaling motif in the cytoplasmic tail, MR has to act in synergy with other pattern recognition receptors in the activation of downstream signaling pathways. Studies have proven that mammal MR synergized with TLR2 or TLR4 signaling for both pro-inflammatory and anti-inflammatory macrophage polarization. On the basis of the above, in this study the head kidney macrophages of M. amblycephala will be purified and cultured in vitro, M1 model with certain M1 molecular markers will be established after oligochitosan stimulation, and the hypothesized signaling pathways of maMR-maTLR2 or maMR-maTLR4 will be investigated for the M1 polarization mechanism. The results of the study will provide important basis for the understanding of fish macrophage polarization, and for the development of fish MR-targeted vaccine and immunostimulants as well.

巨噬细胞是重要的天然免疫细胞，通过不同方向的极化发挥免疫中枢作用。Ⅰ型巨噬细胞（M1）是经典激活的巨噬细胞，具有很强的促炎和抗原提呈能力。甘露糖受体（MR）是巨噬细胞重要的模式识别受体和内吞受体。申请人前期研究发现，团头鲂甘露糖受体（maMR）不仅参与识别水产病原细菌，而且特异性地识别壳寡糖，并诱导团头鲂巨噬细胞的促炎反应，但激活机制不清楚。由于缺乏胞内信号传导基序，MR需要与其他模式识别受体协同作用，才能启动下游信号级联转导。有研究证明，哺乳动物MR通过与TLR2和TLR4协同作用介导巨噬细胞向促炎或抑炎方向极化。据此，本项目拟以壳寡糖刺激体外纯化培养的团头鲂头肾巨噬细胞，确定M1分子标记，建立M1极化模型，对maMR在M1极化中的maMR-maTLR2或maMR-maTLR4信号途径进行研究。研究结果将为理解鱼类巨噬细胞极化机制和鱼类MR靶向免疫制剂的研发提供重要依据。

巨噬细胞是重要的天然免疫细胞，甘露糖受体（mannose receptor, MR）是巨噬细胞重要的模式识别受体和内吞受体。研究MR介导的鱼类巨噬细胞M1极化机制，是鱼类免疫学领域重要的研究课题，也是研发鱼类MR靶向免疫制剂的理论基础。本项目通过建立壳寡糖刺激团头鲂巨噬细胞M1极化模型，研究和解析团头鲂甘露糖受体（maMR）在特异性识别配体、激活促炎信号通路过程中的作用机制。本项目执行四年，已经按计划完成所有项目研究工作。重要结果包括（1）构建了壳寡糖COS6刺激团头鲂巨噬细胞M1极化模型；（2）maMR的CTLD4-8结构域特异性识别和结合COS6，并介导其内化，内化依赖网格蛋白或小窝蛋白参与的途径；（3）COS6激活团头鲂巨噬细胞后，通过促进MR与TLR4共同向脂筏中易位，最终MR与TLR4协同作用，通过TLR4-MyD88-NF-κB信号通路调控团头鲂巨噬细胞的炎性应答；（4）COS6与干扰素IFN-γ联合激活团头鲂巨噬细胞向M1方向极化，产生过度炎症效应。