The Interaction Between DENV-2-infected HUVECs And Regulatory T Cells On Major Inflammatory Cytokines

Objective: To investigate the effect of the interaction between HUVEC and Treg cells on their major inflammatory cytokines when co-cultured with HUVEC and Treg cells.Methods: The DENV-2 virus was amplified with C6/36 cells.The virus was identified by RT-PCR.HUVECs cells were infected with DENV-2.Real-time RT-PCR was used to identify the expression level of viral NS1 fragment in infected HUVECs to reflect the infection of HUVECs..Density gradient centrifugation was used to isolate peripheral blood mononuclear cells(PBMCs)from human leukocytes and magnetic beads-negative Treg cells.The purity of CD4+CD25+CD127low cells was detected by flow cytometry.HUVECs were infected with DENV-2 and transwell was used to co-culture with Treg cells.The control group was treated with sphingosine-1-phosphate S1 P type 1 receptor-specific agonist CYM-5442 for 24 h and was used together with Treg cells.Culture,Real-time RT-PCR was used to detect the mRNA expression of IL-6,IL-8,TNF-? and the relative expression of IL-10 and TGF-? in the Treg.The double antibody sandwich ELISA was used to detect the content of the above cytokines in supernatant.Results: The purity of Treg cells was(84.3±0.5)%.The expression of NS1 gradually increased to 24h(3.03±0.26,P<0.01),decreased after reaching the peak.After infection,the expressin of IL-6?IL-8?TNF-? were up-regulated,and the expression of IL-6?IL-8?TNF-? at each timepoint was decreased after co-culturing with Treg(P<0.05).IL-6 of control group was significantly dropped,and expression of IL-8 dropped as also.Meanwhile,IL-10?TGF-? secreted by Treg also decreased.Conclusion: The primary HUVECs infected by DENV-2 can enhance the secretion of cytokines IL-10 and TGF-? by Treg cells,and the suppressive cytokines produced by Treg cells can reduce the production of inflammatory cytokines by DENV-infected HUVECs.