IL-33/ST2途径活化小胶质细胞在广州管圆线虫感染所致脑炎中的作用及其机制研究

Angiostrongylus cantonensis (A. cantonensis) infection induces eosinophilic meningitis and meningoencephalitis. Eosinophil infiltration is the pathology foundation of angiostrongyliasis, but the mechanism is not fully understood. IL-33 is a newly discovered cytokine from IL-1 family which is closely related to the Th2 immune response and its receptor ST2 can be expressed by microglia in the brain. We found that the expression of IL-33 and ST2 in mononuclear cells increased and microglia were constantly activated in the mice brain infected by A. cantonensis ; after we used the inhibitor of microglia, central nervous system inflammation was down-regulated significantly. We think that microlgia activation induced by IL-33/ST2 pathway is the key factor for eosinophil chemotaxis in encephalitis caused by A. cantonensis infection. Therefore, in this study, we will use BALB/c and ST2-/- mice as the model, combining with the experiments in vitro, research microglia activation characteristics induced by IL-33; after blocking IL-33/ST2 pathway, study the changes of microglia and eosinophil chemokine by flow cytometry, immunohistochemistry, etc; investigate the signal pathway of eosinophil chemokine production from microglia induced by IL-33/ST2 pathway by DNA chips and RNAi. Finally, we will reveal the role and mechanism of microglia activation induced by IL-33/ST2 pathway in encephalitis induced by A. cantonensis infection, which will provide a scientific basis for optimization of current treatment options.

广州管圆线虫（AC）感染引起嗜酸性粒细胞(EOS)增高性脑膜炎和脑膜脑炎，EOS浸润是病理学基础，但发生机制尚不完全清楚。IL-33是新发现的IL-1家族细胞因子，与Th2反应密切相关，其受体ST2在脑内可由小胶质细胞(MG)表达。我们发现，感染AC小鼠脑内IL-33和单个核细胞表达的ST2均升高，且MG不断活化；用米诺环素抑制MG后，脑炎明显减轻。我们认为，IL-33/ST2途径活化MG是AC感染所致脑炎中EOS趋化的关键因素。为此，本研究以BALB/c和ST2-/-小鼠为模型，结合体外实验，采用流式细胞术、免疫组化等技术研究MG活化特征及阻断IL-33/ST2后MG和EOS趋化因子的变化；采用DNA芯片和RNAi技术研究IL-33/ST2活化MG诱导主要EOS趋化因子表达的通路，揭示IL-33/ST2途径活化MG在AC感染所致脑炎发生过程中的作用及机制，为优化现行治疗方案提供科学依据。

广州管圆线虫（AC）是重要的食源性寄生虫，人和小鼠感染后引起嗜酸性粒细胞(EOS)增高性脑炎。目前，机制尚不明确。IL-33/ST2L途径是趋化、活化EOS的重要因素。我们前期试验表明，AC感染可引起IL-33及其受体ST2L高表达，但该途径如何发挥作用尚不清楚。通过本项目的开展，发现小鼠感染AC后，脑内星形胶质细胞和小胶质细胞（MG）活化产生IL-33，MG高表达ST2L，并诱导细胞产生与EOS密切相关的IL-13和Ym1。体外实验证实，IL-33刺激BV2细胞，ST2L高表达，产生IL-6、TNF-α、IL-13。AC可溶性虫体抗原（Ag）可协同IL-13刺激MG产生Ym1。Ym1可与单个核细胞特异性标志物CD11b共标记，位于胞浆，分布于血管周围，小脑区多于大脑区。它可以与MG/巨噬细胞（MΦ）特异性标记物Iba-1共标，位于胞浆。Ym1阳性细胞的特征为SSClowFSClowCD11bhighCD45highF4/80pos。通过对Ym1阳性细胞类型的鉴定，我们发现正常小鼠脑单个核细胞中，MG/MΦ所在区域SSClowFSClow含有大量细胞，且该群细胞中大部分表现为CX3CR1highCCR2dim。此外，MG/MΦ群可分为Ly-6Cneg、Ly-6Cdim和Ly-6Chigh，大部分CX3CR1highCCR2dim细胞表型为Ly-6Cneg。感染AC小鼠注射ST2L抗体后，脑部EOS浸润和出血减少，IL-6、TNF-α、iNOS、Eotaxin、IL-13、Ym1等水平降低，炎症缓解。为了研究Ym1产生的信号通路，用IL-13诱导MΦRAW 264.7产生高水平Ym1；此外，还可以诱导BV2产生一定水平的Ym1。细胞内STAT6蛋白水平的降低可以显著下调细胞内Ym1的水平，且随着培养体系中STAT6抑制剂AS1517499浓度的增加而降低。当细胞受到Ag与IL-13的联合刺激后，胞内p-STAT6-Y641的水平显著上升，并且大量集中在细胞核内。本项目的研究成果明确了AC感染引起IL-33/ST2L活化在AC感染所致脑炎中发挥的重要作用，鉴定了产生Ym1的细胞类型，研究了IL-33/ST2L-IL-13-Ym1这条通路的关键信号分子。本项目的开展为揭示AC感染引起EOS脑炎的机制提供了实验数据，为优化现有治疗方案提供了新的思路。