羊种布鲁氏菌外膜蛋白介导的感染细胞免疫耐受或凋亡的分子机制

Brucellosis is one of the most severe zoonoses worldwide, with 500,000 new cases each year. About 85% human brucellosis were caused by Brucella melitensis from infected sheep and goats. An attenuated B. melitensis vaccine M5-90 has been currently used for vaccination of sheep and goats in China. However, this vaccines still remains residual virulence for humans and may result in pregnant sheep abortion. Addtionally the vaccinated animals were hardly distinguished from wild-type infected animals. In our latest study, we found that the vaccine M5-90 caused immunosuppression in vaccinated sheep, which might be one of the reasons for weak immune protection of vaccine. Innate immunity serves as the first line of defense against intracellular bacteria. The innate immune platform includes Toll-like receptors (TLRs). The most common PAMPs are lipopolysaccharide (LPS), peptidoglycan (PGN), bacterial lipoproteins, flagellin, and nucleic acids derived from viruses, bacteria, fungi, and protozoa, which can interact with the host innate immune system via a broad range of germ-line encoded pattern-recognition receptors (PRRs). TLR signaling is an important component bridging innate and adaptive immunity. Previous studies suggested that the level of HLA-G induced immune tolerance or immunosuppression, but it was unclear for function in Brucella infected cells, in particularly little known for apoptosis in Brucella infected cells. This proposal tries to analyze the inertactions between B. melitensis outer membrane proteins, HLA-G and TLR signal path proteins. The obtained data will further reveal the molecular mechanisms of immune tolerance and apoptosis inhibition from Brucella infected macrophages, or inversely promoting apoptosis from Brucella infected human trophoblast. Finally the results will answer why B. melitensis is persistence in the host cells and causes abortion of pregnant sheep and goats, and provide substantial help for molecular modification of the vaccine M5-90 in future.

布病是一种危害严重的人兽共患病，约85%人布病由羊种布菌感染所致。目前应用的羊布病疫苗M5-90仍存在毒力强,易导致孕畜流产，疫苗免疫和野生菌感染难以鉴别的缺陷。我们最新发现，M5-90接种羊后引发宿主的免疫抑制，免疫保护性反应低。天然免疫中重要的TLR信号通路可被布菌模式结构激活，并迅速启动炎症反应，是连接先天性和获得性免疫的桥梁。研究表明，HLA-G的表达水平可以诱导细胞免疫耐受或免疫抑制，但在布菌感染宿主细胞内的作用仍不清楚，尤其与细胞凋亡的关系未见报道。本研究拟利用过表达外膜蛋白的慢病毒，感染人巨噬细胞或滋养层细胞为模型，分析布菌外膜蛋白、HLA-G、TLR通路信号介质蛋白的相互作用与调控，揭示布菌感染巨噬细胞产生免疫耐受,抑制细胞凋亡，而感染胚胎滋养层细胞促凋亡的分子机制，阐明布菌感染羊引起的免疫抑制和孕畜流产的原因，进而为分子改造M5-90疫苗提供理论与技术支持。

本课题完成了计划，达到预期目标要求。.1. 课题成功获得原核和慢病毒过表达的外膜蛋白Omp19、Omp16、Omp10、Omp25、Bp26和Omp31，及相应的单抗，且经鉴定具有病原学和血清学检测的价值，可作为研究B. melitensis感染哺乳动物细胞模型，为布菌胞内感染与免疫研究提供了关键材料，建立的新型布病检测技术，可适用于人畜布病的检测，为布病治疗效果评价提供准确的参考。研究成果已发表。.2. 课题证实布菌感染过程中主要是布菌脂蛋白Omp19和Omp16的脂化形式引起宿主较强的炎症反应，是诱导THP-1细胞发生凋亡的关键成分。而与TLR-2免疫调控作用密切相关的外膜蛋白是Omp19和Omp31，其中L19可能是参与TLR-2通路对炎症反应进行负调控，并诱发宿主发生免疫抑制的关键成分。该成果已整理文章待发表，结题后2年内进一步深入研究新发现的外膜蛋白毒力作用及机制，揭示L19在布菌感染诱发的宿主细胞凋亡和焦亡转换中的角色及作用机制。.3. 首次调查布菌感染后宿主的免疫应答情况，评估Foxp3+ Treg介导的免疫抑制对布病治疗和疫苗免疫效果的影响。研究揭示了慢性期布病患者经过治疗，外周血 Foxp3+Treg细胞依然快速增殖并保持高位水平，患者呈现免疫抑制状态而导致布病易复发，难治愈；而过早的使用免疫增强剂可能不利于急性期布病的治疗。同时，经疫苗免疫的羊也呈现免疫抑制状态，导致疫苗保护效率的降低。研究认为，慢性期布病患者免疫抑制状态与M5-90疫苗诱发的免疫抑制相似，是由布菌本身免疫特性决定，因此课题建议，在未来的布菌疫苗研究中，必须考虑拮抗疫苗诱导的免疫抑制，实现增强疫苗保护性免疫反应。.4. 研究还证实布病对我国公共卫生安全危害日趋严重，建议布病重要流行地区应增强布菌的血液安全筛查。.5. 本课题产出研究论文7篇，其中SCI 3篇，分别发表在Vaccine、BMC Microbiology 和Transfusion等专业领域期刊；申请了国家发明专利4项；培养了2名中青年教师，均在完成课题期间晋升为高级实验师；培养研究生合计9名，其中博士4名、硕士生5名；课题负责人参加了第70届国际布病学术会议并作墙报展示。