免疫抑制性受体LILRB2促进新型冠状病毒刺突蛋白介导的炎症过程  

Immune inhibitory receptor LILRB2 enhances SARS-CoV-2 spike protein-mediated immune inflammation

作　　者：杨文倩 陈迟琪 赵路 曹力元 夏一秋 卢智刚 郑俊克 YANG Wenqian;CHEN Chiqi;ZHAO Lu;CAO Liyuan;XIA Yiqiu;LU Zhigang;ZHENG Junke(Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education,Shanghai Jiao Tong University School of Medicine,Shanghai 200025,China;Institutes of Biomedical Sciences,Fudan University,Shanghai 200032,China)

机构地区：[1]上海交通大学医学院细胞分化与凋亡教育部重点实验室,上海200025 [2]复旦大学生物医学研究院,上海200032

出　　处：《上海交通大学学报（医学版）》2022年第9期1188-1196,共9页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81825001,32030030);上海市科学技术委员会科学基金(19XD1422100,20JC1410100)。

摘　　要：目的·探究免疫抑制性受体白细胞免疫球蛋白样受体亚家族B成员2(leukocyte immunoglobulin-like receptor subfamily B member 2,LILRB2)在新型冠状(新冠)病毒感染所导致的免疫细胞炎症因子释放中的作用及机制,为新冠病毒肺炎治疗提供潜在靶点。方法·收集包含刺突蛋白胞外段(S-ECD)的细胞上清液,分别使用Western blotting及流式细胞术检测上清液中蛋白表达情况及活性;通过流式细胞术及免疫共沉淀技术检测该上清液中S-ECD与LILRB2的结合;用刺突蛋白处理人单核细胞系THP1或人外周血单个核细胞(peripheral blood mononuclear cell,PBMC),24 h后收集细胞,使用实时定量PCR技术检测相关炎症因子基因mRNA水平改变,同时使用酶联免疫吸附法检测细胞培养液中白细胞介素6(interleukin-6,IL-6)及IL-1β含量;将si LILRB2通过Lipofectamine 3000试剂转染至人外周血CD33~+髓系细胞中,24 h后使用流式细胞术检测基因干扰效果;在对照组及LILRB2敲低的CD33~+髓系细胞中加入刺突蛋白培养24 h,使用酶联免疫吸附试剂盒检测细胞培养液中IL-6的含量变化。结果·实验成功构建可分泌具有生物活性的新冠病毒S-ECD的293T细胞转染体系;免疫共沉淀实验显示刺突蛋白与LILRB2蛋白存在相互作用,且流式细胞术结果提示刺突蛋白胞外段能够与细胞表面的LILRB2结合;与对照组相比,经刺突蛋白处理24 h的THP1细胞中IL-6、IL-8、精氨酸酶(arginase 1)及IL-2基因表达水平均有明显上调(均P<0.05);PBMC经刺突蛋白处理24 h后,IL-6、转化生长因子-β(transforming growth factor-β,TGF-β)、IL-8、IL-10及IL-1β的mRNA水平均显著上升(均P<0.05);酶联免疫吸附实验结果显示,在PBMC培养液中加入刺突蛋白能够提高上清液中IL-6及IL-1β的浓度(均P<0.05);使用S-ECD或刺突蛋白处理CD33~+髓系细胞,IL-1β及IL-6含量随之升高(均P<0.05);并且过表达LILRB2的THP1细胞经刺突蛋白刺激后展现了更强的IL-6分Objective·To explore the possible roles of immune inhibitory receptor leukocyte immunoglobulin-like receptor subfamily B member 2(LILRB2) in the immune inflammation after severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) infection, and provide a potential therapeutic way for the coronavirus disease 2019(COVID-19). Methods·The supernatants containing the extracellular domain of spike protein(S-ECD) were collected, and the detection of the protein expression and activity in the conditional medium by Western blotting and flow cytometric analysis was followed by. The binding of S-ECD with LILRB2 was measured by co-immunoprecipitation and flow cytometric analysis. The mRNA expression levels of several inflammation genes in a human mononuclear cell line(THP1) or peripheral blood mononuclear cells(PBMC) were measured after spike protein stimulation for 24 h by quantitative RT-PCR. The protein levels of interleukin-6(IL-6) and interleukin-1β(IL-1β) in the conditional medium were examined by enzyme-linked immunosorbent assay(ELISA). The si LILRB2 was transferred into CD33myeloid cells purified from human peripheral blood with Lipofectamine 3000 reagents. The knockdown efficiency was detected 24 h after transfection by flow cytometric analysis. The difference in the protein levels of IL-6 between the control cells and LILRB2-knocked-down cells after spike protein treatment was evaluated by ELISA. Results·The study established a transfection system with 293T cells by which the SARSCoV-2 S-ECD could be secreted to supernatants with normal biological activities. The interaction and the binding of spike protein with LILRB2 were evaluated by a co-immunoprecipitation assay and flow cytometric analysis, respectively. The mRNA expression levels of IL-6, IL-8, arginase 1 and IL-2 in THP1 cells were significantly up-regulated 24 h after spike protein treatment compared to the control cells(all P<0.05). Consistently, the mRNA levels of IL-6, transforming growth factor-β(TGF-β), IL-8, IL-10 and IL-1β in PBMC were not

关 键 词：新型冠状病毒 刺突蛋白 白细胞免疫球蛋白样受体亚家族B成员2 炎症因子 髓系细胞 

分 类 号：R363.1[医药卫生—病理学]