ABSTRACT
Objective To investigate the influences on major inflammatory cytokines after co-cul-turing 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 extrac-ted from concentrated human leukocytes by density gradient centrifugation. Treg cells were sorted by immu-nomagnetic beads. Expression of CD4,CD25 and CD127 molecules on the membrane of Treg cells was detec-ted 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 detec-ted 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 reac-hing 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.
ABSTRACT
Objective To investigate the effects of interaction between human umbilical vein endothelial cells (HUVECs) which were infected with dengue virus type 2 (DENV-2) and CD4+T cells on the expression of ICAM-1 (intercellular adhesion molecule 1),VCAM-1 (vascular cell adhesion molecule 1),IL-10 and TGF-β1 at mRNA level for further understanding the immunological mechanism of DENV infection.Methods HUVECs were treated with CYM-5442,a selective agonist for sphingosine-1-phosphate receptor 1 (S1P1),for 24 hours and then infected with 103 TCID50 (50% tissue culture infective dose) of DENV-2 before co-culturing with CD4+T cells.Changes in the expression of NS1 (DENV-2 nonstructural protein),SPHK1 (sphingosine kinase 1,phosphorylating sphingosine to S1P),ICAM-1,VCAM-1,IL-10 and TGF-β1 at mRNA level were detected by real-time PCR after 4,8,12,24,48 and 72 hours of co-culturing.Results There was a certain timeliness in the expression of NS1 at mRNA level after infecting HUVECs with DENV-2 and the expression reached a peak at 24 h.Treating HUVECs with or without CYM-5442 had no significant influence on the expression of DENV-2 NS1 at mRNA level.The expression of SPHK1 at mRNA level was significantly increased after treating HUVECs with CYM-5442 and DENV-2 (P<0.05).Compared with DENV-2-infected or untreated HUVECs,Co-culturing DENV-2-infected HUVECs with CD4+T cells increased the expression of ICAM-1 and VCAM-1 in HUVECs at mRNA level (P<0.01) as well as the expression of IL-10 in CD4+T cells at mRNA level (P<0.05),but had no significant influence on the expression of TGF-β1 in CD4+T cells at mRNA level.Conclusion This study shows that DENV-2 can replicate and proliferate in HUVECs,but CD4+T cells inhibit the replication and proliferation.CD4+T cells play an important role in promoting the expression of VCAM-1 and ICAM-1 in DENV-2-infected HUVECs at mRNA level,activating HUVECs and increasing inflammation,which may be associated with increased vascular permeability induced by DENV-2 infection.Co-culturing CD4+T cells with DENV-2-infected HUVECs promotes the expression of IL-10 in CD4+T cells at mRNA level,but has no significant effect on TGF-β1.
ABSTRACT
Objective:To reveal the primary mechanism of changing permeability in DENV-2 infected pHDMECs. Methods:pHDMECs was incubated by DENV-2 on the concentration of 103 TCID50 ,and the penetrability of the cell was detected by Transwell at 4,8,12,24,48 h,respectively. Then,the partial sequence of DENV-2 NS1 was analyzed by Real time-PCR,and NS1 protein was detected by immunofluorescence and flow cytometer (FCM). The apoptosis rate of pHDMECs was assayed by FCM. Finally,IL-6 and IL-8 secreted by pHDMECs were analyzed by Real time-PCR and double antibody sandwich ELISA. Results:The relative expression of NS1 gene was elevated but NS1 protein was not detected;the permeability of DENV-2 infected pHDMECs had dramatically increased both at 24,48 h,but the apoptosis rate has little changed even been influenced by DENV-2 at 72 h. However,the relative expression of IL-6/IL-8 mRNA was boosted at 8,24 h[(2. 49±0. 50) and (6. 82±1. 69) fold,respectively,P<0. 05]. In protein level,compared with control(869. 6±50. 70)pg/ml,IL-6 secreted by DENV-2 infected pHDMECs could reach by(1 248. 8±86. 9)pg/ml(P<0. 05),and IL-8 was(1 331. 0±86. 3)pg/ml(P<0. 05) while the control was (967. 6±156. 6)pg/ml. Conclusion:Indeed,pHDMECs can be infected by DENV-2;the increasing permeability of DENV-2 infected pHDMECs may not be caused by the pHDMECs′ apoptosis but the enhancing of pro-inflammatory cytokine IL-6 /IL-8.
ABSTRACT
Objective To investigate the effects of dengue type 2 virus(DENV-2)on the apopto-sis and autophagy of primary human hepatic sinusoidal endothelial cells(HHSECs)and the expression of ICAM-1 and Beclin-1 at mRNA level and to analyze the possible pathogenic mechanism of DENV-2. Meth-ods Immunohistochemistry(IHC)and flow cytometry analysis(FCM)were performed to identify HHSECs by detecting factor Ⅷ and CD31. The DENV-2 strain was identified by using PCR and HindⅢ. The 50%tissue culture infective dose(TCID50 )of DENV-2 was calculated after infecting C6 / 36 cells with DENV-2. Dynamic changes of DENV-2 NS1 were measured by real-time PCR after infecting HHSECs with DENV-2. CCK-8 was used to dynamically detect the cytotoxicity of DENV-2 to HHSECs. The transcriptional levers of Beclin-1 and ICAM-1 in DENV-2-infected HHSECs were detected by real-time PCR. FCM was performed to analyze the apoptosis of HHSECs and the expression of LC3B and ICAM-1. Results The cells in the exper-imental group were stained brown by DAB and the positive expression rate of CD31 reached 87. 1% . The TICD50 of DENV-2 to C6 / 36 cells was 10-6. 845 / 0. 1 ml. Compared with the uninfected cells,partial se-quences of NS1 gene were expressed in DENV-2-infected HHSECs. DENV-2 suppressed the cell activities of HHSECs. The suppression rates of DENV-2 to HHSECs at 12 h,24 h,36 h and 48 h were respectively (10. 90±1. 24)% ,(16. 40±0. 42)% ,(17. 00±0. 46)% and(29. 60±0. 26)%(P﹤0. 05). The tran-scriptional levels of Beclin-1 and ICAM-1 in HHSECs were significantly increased at the time point of 24 h after DENV-2 infection,the 2-△△Ct values of which were 46. 77±2. 55 and 40. 97±4. 91,respectively. The expression of LC3B and ICAM-1 in DENV-2-infected HHSECs were increased,the peaks of which were reached at 24 h(14. 7% )and 36 h(35. 5% ),respectively. The apoptosis of DENV-2-infected HHSECs was remarkably enhanced at 12 h with an apoptosis rate of 13. 17% . Conclusion HHSECs was susceptible to DENV-2. DENV-2 induced the upregulation of ICAM-1 and the activation of HHSECs. Moreover,autoph-agy and apoptosis of HHSECs could also be induced by DENV-2.