CpG ODN调控巨噬细胞极化保护烧伤继发肺部感染的作用与机理

Severe burns lead to the disorder of immuno-responses upon infection, and the secondary lung infection is life-threatening. Macrophage polarization palys key roles in immune responses during lung infection. CpG-oligodeoxynucleotide (CpG ODN) was reported to be capable of regulating the expression of immune-response related genes dynamically. By establishing severely burned mice model with secondary lung infection mimicing the clinical situation, we found beneficial effects of CpG ODN treatment on the mice model. Only one time subcutaneous injection of CpG ODN improves the survival remarkably and significantly changes the PD-L1 expression in macrophages of the infected lung. According to the literature that PD1/PD-L1 regulates macrophage polarization, we speculate that PD-L1 is involved in the regulation of CpG ODN on macrophage polarization, exerting beneficial effects. In this study, we try to analyze the following systematically and dynamically by means of single-cell proteomics: 1. The effects of CpG ODN treatment on the subpopulations, polarization and function of macrophages as well as related molecules; 2. Differential regulation of CpG ODN on the activation status of PD1/PD-L1 signaling in each macrophage subpopulation; 3. The molecular mechanisms of PD1/PD-L1 signaling underlying CpG ODN-regulated macrophage polarization. All these could help finding the target of CpG ODN treatment and providing new insight on how CpG ODN regulates macrophages at single-cell level and globally. Thus, we provide new ideas for the treatment of related diseases.

严重烧伤继发肺部感染威胁生命, 巨噬细胞（Mφ）极化在肺部炎症应答中发挥重要作用。研究表明CpG寡聚脱氧核苷酸(CpG ODN)能动态调节免疫应答基因表达，项目组发现致伤后单次皮下注射CpG ODN可有效调控严重烧伤继发肺部感染模型动物肺部Mφ中PD-L1水平，显著降低病死率。结合PD1/PD-L1参与Mφ极化调控的报道，本项目以单细胞蛋白组技术为主要手段，用系统免疫学方法动态分析如下问题：1.CpG ODN对严重烧伤继发肺部感染各阶段肺泡和间质Mφ亚群、极化状态、功能及所涉及分子的作用；2.各Mφ亚群中PD1/PD-L1-STAT1表达及活化状态；3.PD1/PD-L1参与CpG ODN调控Mφ极化的分子机制。项目以Mφ极化为主线、PD1/PD-L1为切入点在整体和单细胞水平提供CpG ODN调控Mφ新知，揭示其对烧伤继发肺部感染的保护机制，为其临床转化奠定基础并为相关疾病救治提供新策略

本项目成功构建了小鼠严重烧伤继发肺部感染的小鼠模型，并证明CpG ODN治疗有效提高严重烧伤合并感染动物的存活率。CpG ODN 治疗帮助机体有效清除肺组织中的肺炎链球菌，下调血清中IL-10水平，下调脾脏中的IL-10、IL-6、TNF-α、IL-12p40水平，上调肺组织中IL-33水平和下调肺泡灌洗液中IL-17， IL-6，IL-12p40，从而减轻机体的炎症反应水平。CpG ODN治疗也抑制烧伤早期引起的肺泡灌洗液中B 细胞，NK 细胞和巨噬细胞水平的上调。单细胞质谱分析鉴定肺泡灌洗液中共发现36个细胞亚群，其中有B 细胞, NK细胞, CD4+T细胞，CD8+ T细胞, 嗜酸性粒细胞,和6个单核细胞亚群，12个中性粒细胞亚群，其中总共有20个细胞亚群的丰度在烧伤后发生显著变化。烧伤显著上调CD11c+PDL-1+MHCII+M2丰度, CpG ODN治疗则可抑制上述M2细胞亚群的丰度增加，令其丰度接近假伤组水平。此外烧伤还导致MHCIIhighCD11c-PDL-1-巨噬细胞 CXCR4表达水平上调，且肺泡中该亚群细胞数量增加，CpG ODN 治疗可下调CXCR4水平并减少该细胞数量；除此之外，烧伤可导致肺泡巨噬细胞表面CD11c表达下调，而CpG ODN 治疗 令CD11c表达水平回复到假伤对照组水平。体内外实验均说明CpG治疗可以调节巨噬细胞的极化状态，同时，CpG ODN 还可调节部分因烧伤引起中性粒细胞和单核细胞及嗜酸性粒细胞丰度及其表面分子表达水平。经PD-1条件敲除发现巨噬细胞水平未发生显著变化，PD-1敲除减弱模型小鼠IL-12p40，IL-10，MCP-1和IL-17的释放水平。进一步分析CpG ODN受体TLR9对其诱导巨噬细胞极化的影响，发现TLR9受体介导了CpG ODN诱导骨髓来源的巨噬细胞的极化，CpG ODN特异性活化细胞中STAT-1 和STAT-6信号通路，促进巨噬细胞向M1极化。且仅STAT-6是TLR9依赖的。