轴突导向因子3A对脂多糖诱导的CD4+CD25+调节性T细胞稳定性的影响

Effect of semaphorin-3A on the cellular stability of CD4+CD25+regulatory T cells induced by lipopolysaccharide

目的探讨轴突导向因子3A(Sema3A)对脂多糖(LPS)诱导的CD4^(+)CD25^(+)调节性T细胞(Tregs)稳定性的作用及机制。方法采用体外免疫磁珠法分离和培养C57BL/6J小鼠脾脏CD4^(+)CD25^(+)Tregs,将分离的细胞按随机数字表法分为对照组(仅给予抗小鼠CD3e和CD28诱导细胞处于激活状态)、LPS组(在对照组的基础上给予LPS 100μg/L)、LPS+核转录因子-κB(NF-κB)抑制剂吡咯烷二硫代氨基甲酸酯(PDTC)组(给予LPS 100μg/L+PDTC 25 mg/L)、LPS+磷酸盐缓冲液(PBS)组(给予LPS 100μg/L+PBS 10μL)、LPS+PDTC+重组Sema3A(rSema3A)组(给予LPS 100μg/L+PDTC 25 mg/L+rSema3A 300μg/L)和LPS+PBS+rSema3A组(给予LPS 100μg/L+PBS 10μL+rSema3A 300μg/L)。各组培养24 h后,采用反转录-聚合酶链反应(RT-PCR)和免疫荧光法检测CD4^(+)CD25^(+)Tregs特异性标志物叉头翼状转录因子-3(Foxp-3)、细胞毒性T淋巴细胞相关抗原-4(CTLA-4)和膜相关转化型生长因子-β1(TGF-β1^(m+))的基因及蛋白表达,采用酶联免疫吸附试验(ELISA)检测细胞上清液中白细胞介素-10(IL-10)和分泌型转化生长因子-β1(sTGF-β1)的水平,采用免疫荧光法检测细胞凋亡,采用甲基化特异性聚合酶链反应(MSP)检测Foxp-3-Tregs特异性去甲基化区(Foxp-3-TSDR)的去甲基化程度,以反映CD4^(+)CD25^(+)Tregs的稳定性,采用电泳迁移率分析(EMSA)检测NF-κB信号通路的DNA结合活性,采用蛋白质免疫印迹试验(Western blotting)检测NF-κB信号通路活性。结果与对照组比较,LPS能够增加细胞稳定性,表现为Foxp-3、CTLA-4和TGF-β1^(m+)的基因及蛋白表达上调,IL-10和sTGF-β1分泌增加,细胞凋亡减少,Foxp-3-TSDR去甲基化程度增加;同时LPS可增加细胞内NF-κB信号通路的DNA结合活性,以及主要分子NF-κB抑制蛋白激酶β(IKKβ)和p65的磷酸化水平,说明LPS增加细胞稳定性的机制与NF-κB信号通路有关。与LPS组比较,PBS并未对细胞稳定性和NF-κB信号通路产生影响;但添加rSema3A后能进一步增加细胞稳定性,并激活NF-κB信号通路。而PDTC能够抑制rSema3A增加细胞稳定性的功能,表现为:与LPS+PBS+rSema3A组比较,LPS+PDTC+rSema3A组Foxp-3、CTLA-4和TGF-β1^(m+)的基因及蛋白表达明显下调〔Foxp-3基因(2^(-ΔΔCt)):8.092±1.117比18.509±1.068,Foxp-3蛋白(相对荧光强度):1.224±0.033比1.826±0.181;CTLA-4基因(2^(-ΔΔCt)):3.254±0.760比11.840±0.827,CTLA-4蛋白(相对荧光强度):1.305±0.058比1.842±0.111;TGF-β1^(m+)基因(2^(-ΔΔCt)):3.589±1.180比8.509±0.472,TGF-β1^(m+)蛋白(相对荧光强度):1.319±0.033比1.822±0.063,均P<0.01〕,IL-10和sTGF-β1分泌减少〔IL-10(ng/L):445.33±54.08比992.67±83.10,sTGF-β1(ng/L):1116.67±65.25比1494.67±94.45,均P<0.01〕,细胞凋亡明显增加(荧光强度:0.398±0.031比0.268±0.046,P<0.01),Foxp-3-TSDR去甲基化程度明显降低(灰度值:0.467±0.048比1.780±0.119,P<0.01),NF-κB信号通路的DNA结合活性被明显抑制(灰度值:1.23±0.02比3.95±0.06,P<0.01),IKKβ和p65的磷酸化水平降低〔p-IKKβ表达(p-IKKβ/IKKβ):0.97±0.07比1.97±0.04,p-p65(p-p65/p65):0.95±0.08比1.93±0.06,均P<0.01〕。结论LPS能通过NF-κB信号通路增加CD4^(+)CD25^(+)Tregs稳定性;Sema3A能够进一步增加CD4^(+)CD25^(+)Tregs稳定性,并且与NF-κB信号通路有关。

Objective To investigate the effect and mechanism of semaphorin-3A(Sema3A)in maintaining the cellular stability of CD4^(+)CD25^(+)regulatory T cells(Tregs)induced by lipopolysaccharide(LPS).Methods In vitro,using immunomagnetic beads,splenic CD4^(+)CD25^(+)Tregs of C57BL/6J mice were isolated and cultured.According to the random number table,the isolated cells were divided into control group(treated with anti-CD3e and anti-CD28 for polyclonal activation),LPS group(on the basis of control group,treated with LPS at the dose of 100μg/L),LPS+nuclear factor kappa B(NF-κB)inhibitor pyrrolidine dithiocarbamate(PDTC)group(treated with LPS at the dose of 100μg/L and PDTC at the dose of 25 mg/L),LPS+phosphate buffer solution(PBS)group(treated with LPS at the dose of 100μg/L and PBS at the volume of 10μL),LPS+PDTC+recombinant Sema3A(rSema3A)group(treated with LPS at the dose of 100μg/L,PDTC at the dose of 25 mg/L and rSema3A at the dose of 300μg/L),and LPS+PBS+rSema3A group(treated with LPS at the dose of 100μg/L,PBS at the volume of 10μL and rSema3A at the dose of 300μg/L).mRNA and protein expressions of the specific markers of CD4^(+)CD25^(+)Tregs,including forkhead box protein P-3(Foxp-3),cytotoxic T lymphocyte-associated antigen-4(CTLA-4)and membrane-associated transforming growth factor-β1(TGF-β1^(m+))were detected by reverse transcription-polymerase chain reaction(RT-PCR)and immunofluorescence methods after 24 hours.The supernatant interleukin-10(IL-10)and secretory type TGF-β1(sTGF-β1)were detected by enzyme-linked immunosorbent assay(ELISA).The apoptotic level was detected by immunofluorescence.The demethylation of Foxp3-Tregs-specific demethylated region(Foxp-3-TSDR)was detected by methylation specific PCR(MSP)in order to reflect the cellular stability of CD4^(+)CD25^(+)Tregs.DNA binding activity of NF-κB signaling pathway was determined by electrophoretic mobility shift assay(EMSA),and activity of NF-κB signaling pathway was determined by Western blotting.Results Compared with control group,LPS could increase the cellular stability,including an increase in the mRNA and protein expressions of Foxp-3,CTLA-4 and TGF-β1^(m+)and secretion of IL-10 and sTGF-β1,decrease in the level of apoptosis and increase in the methylation of Foxp-3-TSDR.At the same time,LPS increased DNA binding activity of NF-κB signaling pathway and phosphorylation levels of the major molecules of NF-κB,including inhibitory protein kinaseβ(IKKβ)and p65,suggesting that the mechanism of enhancing cellular stability by LPS was related to the NF-κB signaling pathway.Compared with LPS group,PBS had no effect on cellular stability and NF-κB signaling pathway.However,administration of rSema3A further promoted cellular stability and activated NF-κB signaling pathway.Compared with LPS+PBS+rSema3A group,PDTC inhibited the function of rSema3A to increase cellular stability,as the mRNA and protein expressions of Foxp-3,CTLA-4 and TGF-β1^(m+)were significantly down-regulated in the LPS+PDTC+rSema3A group[Foxp-3 mRNA(2^(-ΔΔCt)):8.092±1.117 vs.18.509±1.068,Foxp-3 protein(relative fluorescence intensity):1.224±0.033 vs.1.826±0.181;CTLA-4 mRNA(2^(-ΔΔCt)):3.254±0.760 vs.11.840±0.827,CTLA-4 protein(relative fluorescence intensity):1.305±0.058 vs.1.842±0.111;TGF-β1^(m+)mRNA(2^(-ΔΔCt)):3.589±1.180 vs.8.509±0.472,TGF-β1^(m+)protein(relative fluorescence intensity):1.319±0.033 vs.1.822±0.063,all P<0.01],the secretion of IL-10 and sTGF-β1 were significantly decreased[IL-10(ng/L):445.33±54.08 vs.992.67±83.10,sTGF-β1(ng/L):1116.67±65.25 vs.1494.67±94.45,both P<0.01],the apoptosis was significantly increased(fluorescence intensity:0.398±0.031 vs.0.268±0.046,P<0.01),the methylation of Foxp-3-TSDR was significantly decreased(grey value:0.467±0.048 vs.1.780±0.119,P<0.01),the DNA binding activity of NF-κB signaling pathway was significantly inhibited(grey value:1.23±0.02 vs.3.95±0.06,P<0.01),and the phosphorylation levels of IKKβand p65 were significantly decreased[p-IKKβ(p-IKKβ/IKKβ):0.97±0.07 vs.1.97±0.04,p-p65(p-p65/p65):0.95±0.08 vs.1.93±0.06,both P<0.01].Conclusion LPS increases the cellular stability of CD4^(+)CD25^(+)Tregs through the NF-κB signaling pathway,and Sema3A further increases the cellular stability of CD4^(+)CD25^(+)Tregs,and is related to the NF-κB signaling pathway.