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

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5 011.0%（P<0.001）、6 048.9%（P<0.001）和1 011.6%（P=0.009 4），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=0.001 1）、53.7%（P<0.001）和5.9%（P>0.05），CD206下调18.6%（P>0.05）、88.4%（P=0.005 5）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=0.044 7）、311.5%（P=0.001 9）、9.6%（P>0.05）；IL-6分别上调17 763.2%（P<0.001）、35 458.2%（P<0.001）、1 123.6%（P>0.05）；IL-10分别上调729.3%（P<0.001）、1 223.3%（P<0.001）、124.4%（P>0.05）。翻译水平上，A.muciniphila、E.coli、F.nucleatum LPS单独或联合IFN-γ处理时，THP-1细胞产生IL-6分别为0.16、6.17、0 pg/mL与410.03、1 334.40、46.20 pg/mL。[结论] F.nucleatum LPS不仅具有较强招募单核细胞并诱导其向M2极化的作用，同时，具有诱导巨噬细胞分泌低浓度IL-6的特性，说明其在引发慢性炎症及肿瘤免疫应答、逃逸等过程中发挥重要作用。综合上述信息，对致癌、免疫激活及肿瘤治疗相关细菌LPS的结构、活性、分子机制等研究将有助于明确革兰氏阴性细菌在慢性炎症、肿瘤发生、免疫调控等中的作用，以期为相关疾病预防与治疗提供新的策略与靶点。

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

Abstract:

[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 (CD11B), 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-γ had 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 mRNA level of CD40 by 5 011.0% (P<0.001), 6 048.9% (P<0.001), and 1 011.6% (P=0.009 4) 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 mRNA level of CD163 by 39.5% (P=0.001 1), 53.7% (P<0.001), and 5.9% (P>0.05) and that of CD206 by 18.6% (P>0.05), 88.4% (P=0.005 5), and 24.8% (P>0.05), respectively. They down-regulated the mRNA level of TLR3 by 32.3% (P=0.044 7), 311.5% (P=0.001 9), and 9.6% (P>0.05), up-regulated that of IL-6 by 17 763.2% (P<0.001), 35 458.2% (P<0.001), and 1 123.6% (P>0.05), and up-regulated that of IL-10 by 729.3% (P<0.001), 1 223.3% (P<0.001), and 124.4% (P>0.05), respectively. The THP-1 cells treated with the LPSs of A. muciniphila, E. coli, and F. nucleatum alone produced IL-6 at 0.16 pg/mL, 6.17 pg/mL, and 0 pg/mL , and those treated with the LPSs in combination with IFN-γ produced IL-6 at 410.03 pg/mL, 1 334.40 pg/mL, and 46.20 pg/mL, respectively. [Conclusion] F. nucleatum LPS possessed a strong activity of recruiting monocytes and inducing them to polarize toward M2. It induced macrophages to produce a much lower amount of IL-6 than the LPSs of A. muciniphila and E. coli, which may play a role in triggering chronic inflammation and tumor immune response and escape. These findings suggest that studying the structure, activity, and mechanism of LPS from carcinogenic, immunomodulating or tumor therapy-associated bacteria will facilitate the elucidation of the role of these bacteria in chronic inflammation and tumorigenesis, which will provide new targets and strategies for the prevention and treatment of these diseases.

Key words:Fusobacterium nucleatum;lipopolysaccharide;THP-1;IL-6;chronic inflammation;monocyte