DENV-2感染的HUVECs与调节性T细胞相互作用对主要炎性细胞因子产生的影响

Influences of interaction between DENV-2.infected HUVECs and regulatory T cells on major inflammatory cytokines

目的研究人原代脐静脉内皮细胞（primary human umbilical vein endothelial cells，HUVECs）被登革病毒2型（DENV-2）感染后，与调节性T细胞（regulatory T cells，Treg）共培养，HUVEC与Treg细胞相互作用对各自主要炎性细胞因子的影响。方法用密度梯度离心法从人浓缩白细胞分离外周血单个核细胞（peripheral blood mononuclear cell，PBMC），磁珠阴性分选Treg细胞，流式细胞术检测CD4＋CD25＋CD127＋细胞纯度。HUVECs经DENV-2感染后，用Transwell与Treg细胞共培养，对照组用鞘氨醇-1-磷酸（sphingosine-1-phosphate, S1P）1型特异性受体激动剂CYM-5442预处理24 h去除药物后感染病毒，并与Treg细胞共培养，real-time RT-PCR分别检测HUVECs的IL-6、IL-8、TNF-α和Treg细胞的IL-10、TGF-β的mRNA转录水平；双抗体夹心ELISA法检测培养上清中上述细胞因子表达。结果感染后HUVECs中DENV-2 NS1基因转录水平逐渐上升，在24 h达到峰值（3.03±0.26，P〈0.01）后下降。流式细胞术检测经免疫磁珠阴性分选的Treg细胞纯度为（84.3±0.5）%。DENV-2感染后HUVECs的IL-6、IL-8、TNF-α mRNA转录均有上调，感染后24 h IL-6、IL-8、TNF-α的mRNA转录量分别为16.64±2.64、26.80±5.81和5.25±0.42，而未感染组的转录水平分别为1.70±0.68、1.68±0.74、1.45±0.15，与Treg共培养后上述细胞因子在各时间点的转录有所降低，但仍高于未感染DENV-2的对照组。CYM-5442预处理后被感染的HUVECs IL-6、IL-8、TNF-α的mRNA转录水平显著下降；共培养的Treg细胞所产生的IL-10、TGF-β也下调。结论被DENV-2感染的原代HUVECs能增强Treg细胞IL-10与TGF-β的分泌，同时Treg细胞产生的抑制性细胞因子能降低被感染的HUVECs炎性细胞因子的产生。

ObjectiveTo investigate the influences on major inflammatory cytokines after co-culturing regulatory T cells （Treg） with human umbilical vein endothelial cells （HUVECs） that were infected with dengue virus type 2 （DENV-2）.MethodsPeripheral blood mononuclear cells （PBMC） were extracted from concentrated human leukocytes by density gradient centrifugation.Treg cells were sorted by immunomagnetic beads.Expression of CD4, CD25 and CD127 molecules on the membrane of Treg cells was detected by flow cytometry to identify the purity of Treg cells.HUVECs pretreated with or without sphingosine-1-phosphate S1P type 1 （S1P1）-specific receptor agonist CYM-5442 for 24 h were first infected with DENV-2 and then co-cultured with Treg cells.Expression of IL-6, IL-8, TNF-α, IL-10 and TGF-β at mRNA level was detected by real-time RT-PCR.Levels of IL-6, IL-8, IL-10 and TGF-β in the culture supernatants were detected by a double-antibody sandwich ELISA.ResultsThe purity of Treg cells was （84.3±0.5）%.Expression of NS1 at mRNA level in DENV-2-infected HUVECs first gradually increased and then decreased after reaching the peak at 24 h （3.03±0.26, P〈0.01）. Enhanced expression of IL-6, IL-8 and TNF-α at mRNA level in HUVECs was observed after DENV-2 infection （P〈0.01）. Expression of these cytokines at every time point was decreased after co-culturing DENV-2-infected HUVECs with Treg cells （P〈0.05）, but was still higher than that before infection.CYM-5442 pretreatment decreased the expression of IL-6, IL-8 and TNF-α at mRNA level in DENV-2-infected HUVECs and inhibited the secretion of IL-10 and TGF-β by Treg cells that were co-cultured with DENV-2-infected HUVECs.ConclusionPrimary HUVECs infected by DENV-2 can enhance the secretion of IL-10 and TGF-β by Treg cells, and the suppressive cytokines produced by Treg cells can reduce the production of inflammatory cytokines by DENV-2-infected HUVECs.