羊种布氏菌诱导巨噬细胞M2极化介导其免疫逃逸的分子机制

The difficult-to-cure disease, Brucellosis, caused by Brucella melitensis (B.melitensis) is linked to its capacity to evade the immune responses in order to ensure its intracellular survival and replication in M2 macrophages. However, the key element and underlying mechanisms are not fully understood. According to our previous results, an important cell model of M2 polarization induced by B.melitensis infection. Moreover, the virulent strain M28 exhibited more stronger function to induce M1 to M2 polarization than the attenuated strain M5. In this program, base on the reported virulence-associated factors (especially T4SS, LPS, BvrR/BvrS, GntR, Omp31, Omp25 and etc) and the designed epitope belong to us, we aim to find out the key elements of B.melitensis mediating the M2 polarization. The techniques including gene knockout/knockdown, gene cloning and prepare the recombinant proteins will be carried out. Further, the mechanisms of M2 polarization implemented by the key elements would be explored. Finally, two strategies including interfering the expression of the key elements and blocking signal transduction of M2 polarization will be adopted to seek the novel target. The outcomes of our study may provide a novel clue in the treatment of human chronic Brucellosis and improvement host’s resistance to B.melitensis infection.

羊种布氏菌通过逃逸宿主免疫应答在M2巨噬细胞内长期存活与复制增殖是导致布病慢性化并难以治愈的最主要原因，但其诱导M2极化的关键成分及作用机制尚不清楚。我们前期研究成功建立了羊种布氏菌感染诱导巨噬细胞M2极化的细胞模型，发现羊种布氏菌慢性感染可明显导致M2极化，并且强毒株（M28株）诱导M1巨噬细胞向M2极化作用显著强于弱毒株（M5株）。据此，我们拟以羊种布氏菌已报道的毒力相关因子（如T4SS、LPS、BvrR/BvrS、GntR、Omp31、Omp25等）和前期自主筛选的一种模拟抗原肽为靶标，通过基因敲除/敲减或制备重组蛋白，筛选羊种布氏菌诱导M2极化的关键成分，进一步利用细胞和小鼠模型探讨这些关键成分作用的细胞和分子机制，进而通过干扰关键成分的表达和阻断M2极化信号通路两种策略干预其感染。本课题的实施有望为慢性布病治疗新方法和提高机体抗羊种布氏菌感染免疫功能等研究提供新的靶点和思路。

布鲁氏菌病（布病）是由布鲁氏菌感染引起的一种人畜共患传染病。布鲁氏菌为兼性细胞内寄生菌，感染牲畜后易引起母畜流产、死胎等，造成畜牧养殖业重大损失并增加人群感染的几率。布鲁氏菌通过逃逸宿主免疫应答并在巨噬细胞内长期存活与复制增殖是导致布病慢性化和难以治愈的最主要原因。研究提示布鲁氏菌诱导巨噬细胞M2极化可能与其免疫逃逸密切相关，但相关作用机制尚不清楚。本项目研究中，我们通过布病患者样本、布鲁氏菌感染小鼠模型和细胞模型进一步明确了布鲁氏菌感染可诱导巨噬细胞M2极化，并且强毒株（M28株）诱导M2极化的效应强于弱毒株（M5株），同时M28株感染组可诱导更高水平的IL-1β和TNF-α。通过构建多种不同突变株，发现M28△T4SS菌株诱导M2极化能力下降。在体外，rP39和rL7/L12两类重组蛋白可刺激巨噬细胞M1极化。有意思的是，研究发现布鲁氏菌感染会导致小鼠肺脏巨噬细胞数量减少，并且个别缺陷株感染可诱导巨噬细胞聚类发生变化。NLRP6在布鲁氏菌感染导致实验小鼠肺脏损伤中具有重要作用。布鲁氏菌感染小鼠模型中，给予Anti-Tim-3抗体或黄芪多糖治疗后，可以降低小鼠脾脏和肺脏的载菌量。综上，本研究通过体内外实验，进一步明确了巨噬细胞M2极化在布病中的作用，对其中发挥重要作用的成分及其机制作了有益探讨，取得了一些有意义的新发现。本课题的顺利完成可以为后续开展慢性布病防治等研究提供新的思路。