| Objective:1.To investigate the effect of cytokine production after treatment with S1P1 receptor specific agonist(CYM-5442)on the interaction between Human primary umbilical vein endothelial cells(HUVECs)infected with DENV-2 and the immune cells.2.CYM-5442 was used to treat Balb/c mice infected with DENV-2,the distributional characteristics of immune cells and the dynamic changes of serum cytokines in different organs were analyzed,and the possible immune mechanism was discussed.Method:1.The ultrastructure of HUVECs was observed after infection of HUVECs with DENV-2.2.PBMC were separated by density gradient centrifugation,Treg and monocytes were separated with magnetic beads,macrophage colony stimulating factor was used to induce monocytes to differentiate into macrophages.3.HUVEC was co-cultured with Treg,monocytes and macrophages by Transwell method.S1P1 receptor specific agonist intervention group was used to detect the level of inflammatory cytokines secreted by HUVECs and the level of anti-inflammatory cytokines secreted by Treg in each group.4.Balb/c mice were injected subcutaneously with multiple sites.Peripheral blood was collected after 72 hours of DENV infection,serum was isolated,and C6/36 cells were inoculated.The partial sequence of DENV-2 NS1 gene was detected by qPCR in cytopathic cells.5.5.The types and proportion of immune cells in peripheral blood and liver were detected by flow cytometry,the dynamic changes of inflammatory cytokines in serum were detected by double antibody sandwich ELISA method and the pathological changes of liver,spleen,kidney and brain of mice in groups were detected by HE staining.The liver and kidney function of groups were detected by automatic biochemical instrument.Results:1.The expression rate of CD31 on the surface of HUVECs was 98.96%by flow cytometry.The factor ? related antigen of HUVEC was detected by immunohistochemical method.The cytoplasm of HUVEC was brown,which was consistent with the characteristics of HUVECs.2.CD4+CD25+CD127+Treg cells were separated by magnetic beads,CD4 + CD25 +CD 127 + expression on Treg surface was determined by flow cytometry(84.2%),CD14+CD16-monocytes were sorted by magnetic beads,and CD14+CD16-expression on monocytes was determined by flow cytometry(94.8%).The expression of CD14+CD11b+(96.8%)on macrophages was detected after five days of monocyte culture induced by macrophage colony stimulating factor infected with DENV-2.3.HUVECs was co-cultured with Treg,monocytes,macrophages and S1P1 receptor specific agonists,respectively,and was divided into six different experimental groups.It was found that IL-8 in the infected group increased first and then decreased in comparison with the uninfected control group.4h(10.62±1.97),8h(22.28±3.85),12h(19.37±3.24),24h(26.80±5.81),36h(15.12±2.37),48h(10.71 ± 1.80),72h(8.81 ± 1.50)(P<0.001)The expression of IL-8 in S1P1 receptor specific agonist group was significantly lower than that in Treg monocyte macrophage co-culture group(P<0.05).The relative expression of IL-6 in the infected group was significantly higher than that in the uninfected control group(P<0.001).4h(16.55±2.68),8h(20.55±14.01),12h(31.37±4.18),24h(41.83±5.24),36h(28.83±4.25),48h(20.36±3.85),72h(13.52±1.97)(P<0.001);The expression of IL-6 in S1P1 receptor specific agonist group was significantly lower than that in Treg,monocyte and macrophage co-culture groups(P<0.05).The level of TNF-a?VCAM-1?ICAM-1?MCP-1 in S1P1 receptor specific agonist group was significantly lower than that in Treg,monocyte and macrophage co-culture group compared with that in Treg,monocyte and macrophage co-culture group(P<0.05).The IL-10,TGF-?production of Treg cells in HUVEC and Treg co-cultured group was significantly increased.4.The results of animal experiments confirmed that DENV-2 was injected into Balb/c mice subcutaneously to collect peripheral blood in 72h,then the virus was isolated and the NSI partial sequence of the virus nucleic acid was detected by qRT-PCR,the length of the agarose gel electrophoresis product was about 400bp,which was in accordance with the theoretical value.At the same time,the serum of each group was inoculated with C6/36 cells,typical vacuoles and wiredrawing were observed after five days,which confirmed that the virus infection model was successfully established.The mice infected with DENV-2 were affected by CYM-5442 as SI P1 receptor specific agonist,and the expression of inflammatory cytokines in IL-8,IL-6,TNF-a,MCP-1,VCAM-1 and CCL-2 in the intervention group was significantly lower than that of the DENV infection group at all time points(P<0.05).The proportion of peripheral blood and liver monocytes/macrophages,NK cells in the infection group was significantly higher than that in the control group(P<0.001),and the CYM-5442 intervention group was significantly lower than the infection group(P<0.05).The proportion of Treg cells in peripheral blood was not significantly different between the four groups(P>0.05).Staining HE in different organs of infected mices showed that the pathological damage of the infected group was the heaviest,and the pathological damage could be reduced after the intervention of CYM-5442.The liver function of the infected group was(62.31 ± 10.95 u/l),AST(52.33±11.58 u/l),LDH(398.17±38.24 u/l),TBIL(28.58±8.25 umol/1)which was significantly abnormal than that of the other groups(P<0.05),while the renal function was normal in the four groups.Conclusion:HUVECs was co-cultured with different immune cells after DENV2 infection,and the production of inflammatory cytokines was significantly changed under the action of CYM-5442.HUVECs infected with DENV-2 can induce Treg cells to up-regulate the secretion of anti-inflammatory cytokines.It is suggested that HUVEC may play an immunomodulatory role in different immune cells through the production of cytokines.An animal model of Balb/c mice infected with DENV-2 was established.The intervention of CYM-5442,a specific agonist of S1P1 receptor,can significantly down-regulate the expression of various inflammatory cytokines and protect the target organs from injury.After infection with DENV,vascular endothelial cells and immune cells can play an immunoregulatory role.S1P1 receptor specific agonists have a certain protective effect on tissue damage caused by DENV infection. |
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