丁酸盐对骨髓来源树突状细胞中STING信号通路的影响  

Regulation of butyrate on STING signaling pathway in bone marrow-derived dendritic cells

作　　者：刘烁 厉雨婷 李宏慧 陈少聃 陶泽华 蒋雅澜 俞宛君 姚雪 邵启祥 夏圣 LIU Shuo;LI Yu-ting;LI Hong-hui;CHEN Shao-dan;TAO Ze-hua;JIANG Ya-lan;YU Wan-jun;YAO Xue;SHAO Qi-xiang;XIA Sheng(Department of Immunology,School of Medicine,Jiangsu University,Zhenjiang 212013,China)

机构地区：[1]江苏大学医学院免疫学教研室,镇江212013

出　　处：《现代免疫学》2022年第6期457-465,共9页Current Immunology

基　　金：国家自然科学基金面上项目(81871234;31570879);江苏省社会发展重点项目(BE2017696)。

摘　　要：为探究丁酸盐(butyrate, BU)对环鸟苷酸-腺苷酸(cyclic GMP-AMP,cGAMP)激活的骨髓来源树突状细胞(bonemarrow-derived dendritic cell, BMDC)中干扰素基因刺激因子(stimulator of interferon gene, STING)信号通路的调控效应,体外诱导、培养制备BMDC后,将细胞分为对照组、BU组、cGAMP组和BU+cGAMP组及聚脱氧腺苷-脱氧胸苷[Poly (deoxyadenylic-deoxythymidylic),Poly(dA:dT)]/lip2000组、BU+Poly(dA:dT)/lip2000组。光学显微镜下观察各组BMDC形态;FACS检测BMDC表面标志CD80、CD86和MHCⅡ类分子的表达情况,并检测BU及cGAMP处理后BMDC对特异性CD4~+T细胞活化、增殖及分化能力的影响;Griess偶联反应及qPCR分别检测BMDC合成一氧化氮(nitric oxide, NO)和表达诱生型一氧化氮合酶(inducible nitric oxide synthase, iNOS)的能力;ELISA检测对照组、BU组、cGAMP组、BU+cGAMP组、Poly(dA:dT)/lip2000组及BU+Poly(dA:dT)/lip2000组细胞培养上清液中IFN-β、IL-6和IL-12的含量;qPCR检测BMDC中IFN-β、IL-6和IL-12的mRNA相对表达量;Western blotting检测STING信号通路相关蛋白的表达,如STING、TANK结合激酶1(TANK binding kinase 1,TBK1)和p-TBK1等。结果显示,BU可显著上调BMDC表面CD80、CD86和MHCⅡ类分子的表达,但BU抑制STING激动剂cGAMP引起的BMDC表面CD80、CD86和MHCⅡ类分子的表达,抑制NO及IFN-β、IL-6的合成和分泌(均P<0.05);BU显著抑制cGAMP促进CD4~+T细胞的增殖,对初始CD4~+T细胞向Th1分化无明显影响;STING激动剂Poly(dA:dT)引起BMDC IFN-β、IL-6和IL-12分泌的增加也可被BU抑制(均P<0.05)。由此,BU可在BMDC中抑制因STING信号通路激活上调的CD80、CD86和MHCⅡ类分子及NO、IFN-β、IL-6的合成和分泌,并抑制其对CD4~+T细胞增殖的促进作用。To explore the regulation of butyrate(BU) on the stimulator of interferon gene(STING) signaling pathway in bone marrow-derived dendritic cell(BMDC) stimulated by cyclic GMP-AMP(cGAMP), BMDCs were induced and cultured in vitro, and subsequently divided into the control group, BU group, cGAMP group, BU+cGAMP group, poly(deoxyadenylic-deoxythymidylic)(Poly [dA:dT])/lip2000 group, and BU+Poly(dA:dT)/lip2000 group. The morphology of BMDCs in each group was observed under a light microscope. The fluorescence intensity of surface markers CD80, CD86, and MHCⅡon BMDCs, and the efficacy of BU and/or cGAMP on CD4T cell priming were analyzed using FACS. The concentration of nitric oxide(NO) and the expression of inducible nitric oxide synthase(iNOS) in cultured BMDCs were detected by Griess coupling analysis and qPCR, respectively. The mRNA levels and protein concentrations of IFN-β, IL-6, and IL-12 in BMDC culture supernatant were detected using qPCR and ELISA. Western blotting was used to detect the expression of proteins related to the STING signaling pathway, including STING, TANK binding kinase 1(TBK1), and p-TBK1. The results showed that BU up-regulated the expression of surface markers CD80, CD86, and MHCⅡon BMDCs, but inhibited the STING agonist cGAMP stimulated expression of CD80, CD86, and MHCⅡ. Moreover, BU treatment inhibited the synthesis and secretion of NO, IL-6, and IFN-β in cGAMP-stimulated BMDCs(all with P<0.05). BU inhibited CD4T cell proliferation in cGAMP-activated BMDCs but had no obvious effect on the differentiation of na6 ve CD4T cell to Th1. In addition, STING agonist Poly(dA:dT) induced secretion of IFN-β, IL-6, and IL-12 in BMDC was inhibited by BU(all with P<0.05). Therefore, BU inhibits the expression levels of CD80, CD86 and MHC Ⅱ on cell membrane and the priming ability on CD4T cell proliferation of cGAMP activated BMDC. Moreover, the secretions of NO, IL-6 and IFN-β in these BMDCs were also suppressed.

关 键 词：丁酸盐 干扰素基因刺激因子信号通路 骨髓来源树突状细胞 CD4~+T细胞 环鸟苷酸-腺苷酸 聚脱氧腺苷-脱氧胸苷 

分 类 号：R392.12[医药卫生—免疫学]