具核梭杆菌脂多糖诱导THP-1细胞M2极化及对低浓度白介素6产生的作用  

Fusobacterium nucleatum lipopolysaccharide induces THP-1 cells to polarize towards M2 and produce a low concentration of IL-6

作　　者：岳晔 薛松 季林华[2] 范殊璇 贺正文[3] 傅翔 其力格尔 马伟[1] 邓子新[1] YUE Ye;XUE Song;JI Linhua;FAN Shuxuan;HE Zhengwen;FU Xiang;BAO Qelger;MA Wei;DENG Zixin(Key Laboratory of Microbial Metabolism,School of Life Sciences and Biotechnology,Shanghai Jiao Tong University,Shanghai 200240,China;Renji Hospital Affiliated to School of Medicine of Shanghai Jiao Tong University,Shanghai 200127,China;Institute of Schistosomiasis Control and Prevention,Gong’an County Center for Disease Control and Prevention,Jingzhou City,Hubei Province,Jingzhou,Hubei 434399,China)

机构地区：[1]上海交通大学生命科学技术学院微生物代谢国家重点实验室,上海200240 [2]上海交通大学医学院附属仁济医院,上海200127 [3]湖北省荆州市公安县疾病预防控制中心血吸虫病预防控制所,湖北荆州434399

出　　处：《微生物学通报》2021年第12期4817-4827,共11页Microbiology China

基　　金：上海交通大学医工交叉项目(YG2021QN38)。

摘　　要：【背景】在结直肠肿瘤等多种肿瘤中普遍存在的具核梭杆菌(Fusobacterium nucleatum)与结直肠肿瘤发生、预后不良、复发及化疗耐药等密切相关。其引发炎症、对肿瘤微环境中巨噬细胞等免疫细胞作用与机制尚待阐明。【目的】对比分析F.nucleatum脂多糖(Lipopolysaccharide,LPS)与嗜黏蛋白阿克曼氏菌(Akkermansia muciniphila)、大肠杆菌(Escherichia coli)的LPS诱导单核细胞极化、炎性细胞因子表达等活性差异,探讨F.nucleatum在诱发慢性炎症、致癌等过程中的作用与机制。【方法】分别用A.muciniphila、E.coli、F.nucleatum LPS或联合干扰素γ(Interferon-γ,IFN-γ)处理后,观察THP-1、THP-1 M0细胞的细胞形态变化,然后检测M0(CD11B)、M1(CD40、CD86)和M2(CD163、CD206)巨噬细胞标志基因、TLR3、TLR4、IL-6、IL-10等基因转录水平,以及IL-6、IL-10、C反应蛋白(C Reactive Protein,CRP)翻译水平的表达变化。【结果】聚丙烯酰胺凝胶电泳(Polyacrylamide Gel Electrophoresis,PAGE)分析显示,A.muciniphila、E.coli、F.nucleatum这3种细菌的LPS条带位置、数量存在明显差异。F.nucleatum LPS在具有较强诱导THP-1细胞贴壁的同时,对经佛波肉豆蔻醋酸(Phorbol Myristate Acetate,PMA)处理贴壁的THP-1细胞,无论是单独或是联合IFN-γ处理,诱导形成伪足数、伪足长度及形成梭形细胞比例(M1型巨噬细胞)等均低于A.muciniphila和E.coli LPS。进一步转录水平检测巨噬细胞标志基因表达发现,M1标志基因中,CD40分别上调5011.0%(P<0.001)、6048.9%(P<0.001)和1011.6%(P=0.0094),CD86分别上调637.3%(P<0.001)、657.9%(P<0.001)和194.1%(P>0.05);M2标志基因中,CD163分别下调39.5%(P=0.0011)、53.7%(P<0.001)和5.9%(P>0.05),CD206下调18.6%(P>0.05)、88.4%(P=0.0055)和24.8%(P>0.05)。TLR、白介素家族基因转录水平分析发现,TLR3分别下调32.3%(P=0.0447)、311.5%(P=0.0019)、9.6%(P>0.05);IL-6分别上调17763.2%(P<0.001)、35458.2%(P<0.001)、1123.6%(P>0.05);IL-10分别上调7[Background]Fusobacterium nucleatum identified in many kinds of tumors is closely related to tumor initiation,poor prognosis,recurrence,and chemotherapy resistance in colorectal tumor.However,the mechanisms of F.nucleatum inducing inflammation and affecting immune cells such as macrophages in tumor microenvironment remain to be elucidated.[Objective]We explored the role and mechanism of F.nucleatum in the process of inducing chronic inflammation and cancer by comparing the monocyte polarization and inflammatory cytokine expression induced by Fusobacterium nucleatum-,Akkermansia muciniphila-,and Escherichia coli-derived lipopolysaccharides(LPSs).[Methods]After the treatment with A.muciniphila LPS,E.coli LPS,F.nucleatum LPS alone or combined with interferon-γ(IFN-γ),we observed the morphological changes of THP-1 and THP-1(M0)cells.Further,we determined the mRNA levels of macrophage marker genes[including M0(CD11 B),M1(CD40,CD86),and M2(CD163,CD206)],TLR3,TLR4,IL-6,and IL-10 as well as the protein levels of IL-6,IL-10,and C-reactive protein.[Results]PAGE results showed that the LPSs from the three microbial species were significantly different in position and number of bands.F.nucleatum LPS possessed stronger activity of inducing adhesion of THP-1 cells.Meanwhile,the group treated with F.nucleatum LPS alone or in combination with IFN-γhad shorter pseudopodia and lower proportion of cells with pseudopodia and spindle-shaped cells(M1 cells)than the groups treated with A.muciniphila LPS and E.coli LPS.The LPSs from A.muciniphila,E.coli,and F.nucleatum up-regulated the m RNA level of CD40 by 5011.0%(P<0.001),6048.9%(P<0.001),and 1011.6%(P=0.0094)and that of CD86 by 637.3%(P<0.001),657.9%(P<0.001),and 194.1%(P>0.05),respectively.The LPSs down-regulated the m RNA level of CD163 by 39.5%(P=0.0011),53.7%(P<0.001),and 5.9%(P>0.05)and that of CD206 by 18.6%(P>0.05),88.4%(P=0.0055),and 24.8%(P>0.05),respectively.They down-regulated the m RNA level of TLR3 by 32.3%(P=0.0447),311.5%(P=0.0019),and 9.6%(P>0.05),up-regulated tha

关 键 词：具核梭杆菌 脂多糖 THP-1 白介素6 慢性炎症 单核细胞 

分 类 号：R378[医药卫生—病原生物学]