Effect Of Interaction Between HUVECs Infected With DENV-2 And Macrophages On Production Of Major Inflammatory Cytokines

Objective:To study the primary human umbilical vein endothelial cells?HUVECs?infected by dengue virus type 2?DENV-2?,Co-culturing with macrophages,and the Effects of major inflammatory cytokines on the interaction between HUVEC and Macrophages.Methods:Density gradient centrifugation was used to isolate mononuclear cells from concentrated white blood cells in human peripheral blood and Monocyte were obtained from culture flasks and macrophages were obtained by stimulation of macrophage colony-stimulating factor?M-CSF?.Purity of macrophage surface molecule CD₁₄CD_11b was determined by flow cytometry.After HUVECs was infected with DENV-2,Transwell was used to co-culture with Macrophages.The control group was pretreated with sphingosine-1-phosphate specific receptor agonist CYM-5442 for 24h to remove the drug and infect the virus,Co-culture with macrophages.Real-time PCR was used to detect the reverse transcription levels of IL-6,IL-8,TNF-?and IL-1?in HUVECs and Macrophages respectively.ELISA double antibody sandwich assay was used to detect the expression of above cytokines in culture supernatants.Results:After HUVEC infection with denv-2,the transcription level of NS1 gene gradually increased and reached a peak at 24h?2.66±0.53,P<0.05?,and then decreased.The purity of macrophages detected by flow cytometry was 89.16±2.07%.The transcriptional levels of IL-6 and IL-8 in HUVECs infected with DENV-2 were up-regulated.The peak transcriptional levels of IL-6 and IL-8 reached 16.1±0.17 and 29.76±0.58 at 24h after infection,and the transcriptional levels of IL-6 and IL-8 in uninfected group were1.46±0.67 and 1.6±0.54 respectively.The expression of IL-6,IL-8,TNF-alpha and IL-1?in Macrophages infected with DENV-2 increased significantly.The mRNA expression of IL-6?IL-8?TNF-?and IL-1?at 24h were 45.82±3.72,52.34±1.69?12h?,8.94±1.75 and 30.96±1.44 respectively.The transcriptional levels of IL-6,IL-8,TNF-?and IL-1?in the uninfected group were 1.16±0.22,1.15±0.21,1.11±0.09 and1.47±0.31 respectively.The transcription level of these cytokines decreased at different time points after co-culture of HUVECs and Macrophages,and was still significantly higher than that of the uninfected control group.After pretreatment with CYM-5442,the mRNA transcription levels of IL-6 and IL-8 of the infected HUVEC in the co-culture group were significantly decreased?P<0.01?.The transcriptional levels of IL-6,IL-8,TNF-?and IL-1?in Macrophages were also down-regulated?P<0.01?.DENV-2 is able to infect primary HUVECs.The HUVECs infected with DENV-2 can activate Macrophages and stimulate their secretion of large amounts of IL-6,IL-8,TNF-?and IL-1?.At the same time,activated Macrophages can reduce the production of inflammatory cytokines in HUCECs to a certain extent.Compared with the control group,the secretion of IL-6,IL-8,TNF-a,IFN-a and IL-1?in the supernatant of HUVEC infected with DENV-2 increased significantly.Conclusion:DENV-2 can infect primary HUVECs,and HUVEC infected by DENV-2 can activate Macrophages promote the secretion of large amounts of IL-6,IL-8,TNF-?and IL-1?.At the same time,CYM-5442 pretreatment of HUVECs can reduce the secretion of major cytokines caused by HUVECs infected by virus and reduce the damage of endothelial cells to a certain extent.