Zhuang Chenchen, Hong Xuezhi, Liu Jia, Luo Xiaohong, Mo Hanyou
Department of Clinical Immunology and Rheumatology, Affiliated Hospital of The Guilin Medical University, Guilin 541004, P.R. China.
Biomed Rep. 2017 Feb;6(2):206-210. doi: 10.3892/br.2017.836. Epub 2017 Jan 5.
The study aimed to investigate the relationship between tumor necrosis factor receptor-associated factor 6 (TRAF6) and a differentially mature dendritic cell (mDC) in collagen-induced arthritis (CIA) mice and to determine whether or not TRAF6 regulates the activation of an immature dendritic cell (iDC) and inhibits iDC maturation to induce immune tolerance. The mouse bone marrow stem cells were induced with recombinant granulocyte-macrophage colony-stimulating factor (rmGM-CSF) and recombinant interleukin-4 (rmIL-4) to differentiate immature dendritic cells (DCs), which were divided into four groups with different maturation states: rmGM-CSF, rmIL-4; TNF-α; LPS; and FK506 group. The levels of the cell surfaces of CD80, CD86, and MHI-II were analyzed by flow cytometry to prove DCs at different levels of maturity. The expression of IL-12 in DCs at different maturation states was detected by enzyme-linked immunosorbent assay (ELISA). The expression of TRAF6 mRNA and protein in each group of DCs was detected by a reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis. The results revealed that the differentiation of bone marrow cells into iDCs was significantly induced by cytokines (rmGM-CSF, IL-4). CD80, CD86, MHC-II were expressed in the four groups, and the difference between them was statistically significant (P<0.05). A higher degree of DC differentiation led to a gradual increase of IL-12 secretion in the four groups. The difference was statistically significant (P<0.05) for this secretion (group D, 10,620.73±276.73 pg/ml). The expression levels of TRAF6 mRNA were significantly higher in group D than those in the other three groups (P<0.01). Although there was no significant difference in the expression levels of TRAF6 mRNA between groups B and C, the expression levels of TRAF6 mRNA between groups B and C were higher than those of the control group. The TRAF6 protein expression was higher in group D than that in the other three groups (P<0.01), and the difference was statistically significant. There was a statistically significant difference in the TRAF6 protein expression between group A and groups B and C, but the expression in group C was higher than that in group B (P<0.01). In conclusion, the expression of co-stimulatory molecules gradually increased in the DCs of different maturation states, and the expression of IL-12, TRAF6 mRNA, and TRAF6 protein positively correlated with the degree of DC maturation. TRAF6 is important in iDC polarity and maturation.
本研究旨在探讨肿瘤坏死因子受体相关因子6（TRAF6）与胶原诱导性关节炎（CIA）小鼠中差异成熟的树突状细胞（mDC）之间的关系，并确定TRAF6是否调节未成熟树突状细胞（iDC）的活化并抑制iDC成熟以诱导免疫耐受。用重组粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）和重组白细胞介素-4（rmIL-4）诱导小鼠骨髓干细胞分化为未成熟树突状细胞（DCs），将其分为具有不同成熟状态的四组：rmGM-CSF、rmIL-4组；TNF-α组；LPS组；和FK506组。通过流式细胞术分析CD80、CD86和MHI-II细胞表面水平，以证实不同成熟水平的DCs。采用酶联免疫吸附测定（ELISA）检测不同成熟状态DCs中IL-12的表达。通过逆转录-聚合酶链反应（RT-PCR）和蛋白质印迹分析检测每组DCs中TRAF6 mRNA和蛋白的表达。结果显示，细胞因子（rmGM-CSF、IL-4）显著诱导骨髓细胞分化为iDCs。四组均表达CD80、CD86、MHC-II，且它们之间的差异具有统计学意义（P<0.05）。DC分化程度越高，四组中IL-12分泌逐渐增加。该分泌差异具有统计学意义（P<0.05）（D组，10620.73±276.73 pg/ml）。D组TRAF6 mRNA表达水平显著高于其他三组（P<0.01）。虽然B组和C组之间TRAF6 mRNA表达水平无显著差异，但B组和C组的TRAF6 mRNA表达水平高于对照组。D组TRAF6蛋白表达高于其他三组（P<0.01），差异具有统计学意义。A组与B组和C组之间TRAF6蛋白表达存在统计学差异，但C组表达高于B组（P<0.01）。总之，不同成熟状态DCs中共刺激分子表达逐渐增加，IL-12、TRAF6 mRNA和TRAF6蛋白表达与DC成熟程度呈正相关。TRAF6在iDC极化和成熟中起重要作用。
