Effect Of The Interaction Between CD4~+ T Cells And HUVECs Infected With DENV-2 On The Expression Of Inflammatory Cytokines

Objective: To investigate the effect of interaction of the cytokines expression between CD4~+ T cells and human umbilical vein endothelial cells(HUVECs)infected with dengue virus type 2(DENV-2).Methods: C6/36 cells was used to proliferat the DENV-2.The NS1 partial sequence(413bp)of DENV-2 was identified by RT-PCR,TCID50 was used to determine the titration of DENV-2.The expression of CD31 on HUVECs surface and the factor ? related antigen in cytoplasmic were identified by flow cytometry and immunohistochemistry.Density gradient centrifugation was used to extract PBMC from the white blood cells.CD4~+ T cells were sorted by immunomagnetic beads,and the expression of CD3 and CD4 molecules on the cell surface was detected by flow cytometry to identify the purity of CD4~+ T cells.After anti-CD3 and anti-CD28 monoclonal antibodies were used to stimulate the cells,the expression of CD69 on the surface of CD4~+ T cells was detected by flow cytometry,to detecte the activity of CD4~+ T cells.First,HUVECs was treated with the specific-S1P1 receptor agonist,CYM-5442.Second,HUVECs infected with DENV-2 on the titer of 10P3 P TCID50.Last,co-culturing with CD4~+ T cells.Real-time PCR detected the expression of m RNA of NS1 partial sequence,IL-6,IL-8,IL-4,IL-17,TNF-? and IFN-?.The IL-6 and IL-8 in supernatant analyzed by ELISA.Results: RT-PCR amplification of NS1 partial sequence by agarose electrophoresis,the length of product about 400 bp,with the expected 413 bp,confirmed that the virus was DENV-2.The CD31 surface molecule expression rate was 99.86%.The factor VIII related antigen of HUVECs was detected by immune-histochemical,visible in the brown patina,consistent with the characteristics of HUVECs.The purity of CD4~+ T cells was about 98.02%,and the expression of CD69 was increased from 2.08% to 75.24% after being stimulated by anti-CD3 and anti-CD28 monoclonal antibodies for 12 h.The expression of NS1 gradually increased to 24h(3.03±0.26,P<0.001),decreased after reaching the peak.The relative expression of NS1 in the group of co-culturing with CD4P+ PT cells was lower than before.After infecting,the expression of IL-6 and IL-8 were up-regulated,and the expression of IL-6 and IL-8 at each time was significantly increased after co-culturing with CD4~+ T(P<0.001).The expression of IL-6 in the group of pretreating with CYM-5442,significantly decreased at 24h(28.91±2.34,P<0.05),36h(19.36±0.1,P<0.05),72h(13.84±0.82,P<0.05).The expression of IL-8 at 8h(9.91±1.24,P<0.05),12h(12.24±2.06,P<0.05),24h(4.9±0.61,P<0.05),48h(2.11±0.25,P<0.05)decreased significantly.The m RNA expression of IL-4,IL-17,TNF-? and IFN-? in CD4~+ T cells was significantly increased after co-culturing with HUVECs.After treating with CYM-5442,the expression was decreased in different degree.Conclusion: DENV-2 could infect the primary HUVECs,and the expression of NS1 was inhibited after co-culturing with CD4P+ T cells.CD4~+ T cells can not only enhance the activation of HUVECs,but also can be activated by the infected HUVECs.