Abstract
Objective To 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). Methods Peripheral 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. Results The 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. Conclusion Primary 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. Key words: DENV-2; HUVEC; Treg cell; Cytokine; Immunomodulation
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