PDL1融合蛋白抑制CD8^(+)T细胞过度活化对小鼠脑型疟的保护性作用  

Protective effect of PDL1 fusion protein on experimental cerebral malaria by inhibiting overactivation of CD8^(+)T cells

作　　者：王旭 张宁宁 沈燕 赵晓庆 梁姣 王军 WANG Xu;ZHANG Ning-ning;SHEN Yan;ZHAO Xiao-qing;LIANG Jiao;WANG Jun(Second Student Brigade,School of Basic Medical Sciences,Air Force Medical University,Xi'an,Shaanxi 710032;Department of Medical Microbiology and Parasitology,Air Force Medical University,Xi'an,Shaanxi 710032;Department of Cardiology and Nephrology,Xi'an Electric System Center Hospital,Xi'an,Shaanxi 710000,China)

机构地区：[1]空军军医大学基础医学院学员二大队,陕西西安710032 [2]空军军医大学基础医学院微生物与病原生物学教研室,陕西西安710032 [3]西安电力医院心肾内科,陕西西安710000

出　　处：《热带医学杂志》2022年第3期322-326,338,447,共7页Journal of Tropical Medicine

基　　金：国家自然科学基金(82002158,81702019);陕西省自然科学基础研究计划(2019JQ-408)

摘　　要：目的探讨免疫检查点程序性死亡受体-1(PD-1)/PDL1通路在脑型疟(CM)中的作用,研究人为强化CD8^(+)T细胞PD-1/PDL1通路对小鼠CM模型的保护性作用。方法建立小鼠实验型脑型疟(ECM)模型,对其脑组织切片进行HE染色和免疫荧光染色,评价ECM小鼠脑部病理变化;分离ECM小鼠脾脏CD8^(+)T细胞,流式细胞术检测其PD-1分子表达,评价脾脏CD8^(+)T细胞活化情况;对ECM小鼠脾脏CD8^(+)T细胞进行羧基荧光素二醋酸盐琥珀酰亚胺酯(CFSE)染色,评价其增殖情况;分离小鼠原代脑血管内皮细胞(BMECs),添加γ干扰素(IFN-γ)和感染疟原虫的红细胞活化BMECs,建立CD8^(+)T细胞-BMECs共孵育模型,模拟ECM小鼠脑部免疫微环境,使用乳酸脱氢酶(LDH)释放实验评价活化的脾脏CD8^(+)T细胞对BMECs的杀伤效果;利用CD8^(+)T细胞-BMECs共孵育模型和小鼠脑组织切片免疫荧光实验,检测BMECs、小胶质细胞、星形胶质细胞的PDL1表达情况;体外表达具有生物学活性的PDL1-IgG1Fc融合蛋白和IgG1Fc对照蛋白;使用PDL1-IgG1Fc融合蛋白干预ECM小鼠,通过记录小鼠存活时间、检测血清细胞因子含量、以及CD8^(+)T细胞-BMECs共孵育模型,评价PDL1-IgG1Fc融合蛋白降低CD8^(+)T细胞过度活化状态,降低脑型疟症状的作用。结果ECM小鼠脑部血脑屏障开放,CD8^(+)T细胞浸润至脑实质,且部分CD8^(+)T细胞与神经元存在“共定位”现象,ECM小鼠脾脏CD8^(+)T细胞PD-1表达水平上调,活化增殖程度升高;CD8^(+)T细胞-BMECs共孵育模型证实,活化的CD8^(+)T细胞可有效杀伤BMECs,导致BBB开放和中枢神经系统损伤;CD8^(+)T细胞-BMECs共孵育模型表明,脑部免疫微环境可诱导内皮细胞高表达PDL1分子,脑组织切片免疫荧光实验也证实ECM小鼠的小胶质细胞和星形胶质细胞高表达PDL1分子;与IgG1Fc对照蛋白相比,PDL1-IgG1Fc融合蛋白干预ECM小鼠,可抑制其CD8^(+)T细胞过度活化以及对BMECs的杀伤功能,缓解ECM小鼠体内过Objective To investigate the role of immune checkpoint programmed death 1(PD-1)/PDL1 pathway in cerebral malaria(CM),and the protective effect of artificially enhanced CD8^(+)T cells programmed death 1(PD-1)/PDL1 inhibition pathway on mouse CM model.Methods Experimental cerebral malaria(ECM)model was established,brain sections were stained with HE and immunofluorescence to evaluate the pathological changes of ECM mice.Splenic CD8^(+)T cells of ECM mice were isolated,PD-1 expression was detected by flow cytometry to evaluate the activation of these cells,proliferation of splenic CD8^(+)T cells was evaluated by carboxyfluorescein succinimidyl ester(CFSE)staining.Primary brain microvascular endothelial cells(BMECs)were isolated from mice brain,activated with interferon-γ(IFN-γ)and parasitized-RBCs(pRBCs),then co-incubation with splenic CD8^(+)T cells from ECM mice to mimic the pro-inflammatory microenvironment observed in the brain of ECM mice,the killing effect of activated splenic CD8^(+)T cells on BMECs was evaluated by the lactic dehydrogenase(LDH)release.CD8^(+)T cell-BMECs co-incubated model and mouse brain sections immunofluorescence assay were used to evaluate the expression of PDL1 on BMECs,microglia and astrocytes.PDL1-IgG1Fc fusion protein with biological activity and IgG1Fc control protein were expressed in vitro.ECM mice were treated with PDL1-IgG1Fc fusion protein,the effects of PDL1-IgG1Fc fusion protein were evaluated by recording the survival time of ECM mice,the serum cytokine content,and with CD8^(+)T cells-BMECs co-incubation model.Results In ECM mice,the blood-brain barrier was open,and CD8^(+)T cells infiltrated into the brain parenchyma and co-located with neurons.The expression of PD-1 in splenic CD8^(+)T cells,as well as the activation and proliferation of splenic CD8^(+)T cells in ECM,were increased.CD8^(+)T cellBMECs co-incubation model confirmed that activated CD8^(+)T cells can effectively kill BMECs,resulting in BBB opening and central nervous system damage.CD8^(+)T cell-BMECs co-incu

关 键 词：PD-1/PDL1信号通路 PDL1融合蛋白 脑型疟 CD8^(+)T细胞 免疫病理损伤 

分 类 号：R531.3[医药卫生—内科学]