TREM-1受体参与调控巨噬细胞内化革兰阴性细菌的机制研究

In the infection / endotoxemia of trauma or burn, Gram-negative bacteria internalizatioin by macrophages is closely related to activation and up-regulation of inflammatory signals, as well as killing and clearing pathogens. After the recently discovered TREM-1 receptor activated, the inflammatory signals can be significantly enlarged. But the roles and the mechanism of this receptor in the process in which Gram-negative bacteria was internalized by macrophages and how this receptor functioned haven't been systematic reported, which are the key scientific issues about anti-endotoxemia research for TREM-1 as a critical target or not. The project intends to detect the number of bacterial which were internalized by macrophages after TREM-1 was activated or its expression level was down-regulated by the Lentiviral interference. Then, the position of TREM-1 and TLR4 in macrophages will be observed by using multiple labeling technique when the Gram-negative bacteria was internalized by macrophages. After the expression level of TREM-1 receptor down-regulated, the transcription and expression level of TLR4 and MD-2 will also be further tested. At last, intracellular killing of internalized bacterial will be assessed after TREM-1 activated or down-regulated. These experiments aim to clarify the roles and mechanisms about TREM-1 in the process of Gram-negative bacteria internalization, to deepen the understanding about the pathogenesis of endotoxemia, and to provide theoretical basis for anti-endotoxemia treatment targeting on TREM-1.

吞噬细胞内化革兰阴性细菌与创（烧）伤感染/内毒素血症相关炎症信号的活化、放大及机体杀伤、清除病原菌均密切相关，新近发现TREM-1受体活化能够显著放大相关炎症信号，但其在介导吞噬细胞内化细菌过程中的作用及机制尚未见研究报道，而这是TREM-1受体能否作为抗内毒素血症关键靶分子研究亟待解决的关键科学问题。故本项目拟分别在活化TREM-1受体及通过慢病毒干扰下调TREM-1表达后，观测细胞吞噬细菌的变化；并进一步利用多重标记技术观察TREM-1、TLR4在巨噬细胞内化革兰阴性细菌过程中的空间定位；检测TREM-1下调后，TLR4、MD-2转录和表达水平变化；以及评估TREM-1受体状态对革兰阴性细菌细胞内杀伤的影响，旨在阐明TREM-1在巨噬细胞内化革兰阴性细菌中的作用及可能机制，深化革兰阴性细菌所致内毒素血症发病机制的认识，并为以TREM-1受体为靶点的抗内毒素血症治疗提供理论依据。

致病原感染机体后，其本身固有的特殊成分能够被机体先天免疫细胞的模式识别受体（pattern-recognition receptors, PRRs）识别，启动病原的吞噬、清除和杀伤，同时激活机体炎症反应。严重感染所致失控性炎症反应是引发感染患者多器官功能损伤、甚至衰竭的重要原因，目前还缺乏十分有效的防治手段。新近的研究证实表达于髓样细胞的TREM-1（triggering receptors expressed on myeloid cells-1，TREM-1）对炎症信号的级联放大具有重要调控作用，极有可能是抗过度炎症反应治疗研究的重要靶分子。但该受体是否对于先天免疫系统识别、吞噬、杀伤、清除革兰阴性细菌/LPS有作用以及作用的大小尚没有完全认识清楚，且TREM-1在双链RNA（double-stranded RNA，dsRNA）诱导的炎症反应中具有怎样的调控作用依然没有详细的研究报道。鉴于此，本研究通过体外实验证实了大肠杆菌能诱导巨噬细胞分泌促炎症介质，而TREM-1活化能显著放大此效应，并促进巨噬细胞内化并细胞内杀伤大肠杆菌，但细胞非特异胞饮能力并无差异性变化；编码乱序dsRNA的慢病毒感染小鼠巨噬细胞后，TREM-1表达上调，从而证实短片段dsRNA对TREM-1表达具有诱导作用，而敲减TREM-1表达并不影响TLR4、MD-2表达；Poly IC刺激RAW264.7细胞，可剂量依赖地上调TREM-1的表达，这一效应可被PI3K、MAPK信号通路抑制剂阻断，Poly IC刺激BMDMs可持续上调TREM-1表达，在刺激后36hrs达到高峰；TREM-1活化能使MAPK信号通路的JNK、ERK1/2和P38的磷酸化发生更早，磷酸化水平更高，而且持续时间更长；TREM-1活化能显著上调经Poly IC刺激的BMDMs 分泌IFN-β、TNF-α、IL-6的能力，同时，IL-1β、IL-10、MCP-1的mRNA转录显著增强；对于受Poly IC刺激的BMDMs细胞，TREM-1受体活化上调MDA5、TLR3和TLR7表达，而受到短片段dsRNA刺激后表现为RIG-I表达上调。本研究深化了对TREM-1在先天免疫中调节作用的重要性和广泛性的认识，为以TREM-1为靶分子的抗感染研究奠定理论基础。