Study On The Mechanism Underlying The Permeability Change Of Vascular Endothelial Cells By Dengue Virus Type 2 Infection

Objective:Dengue virus(DENV)infection can lead to significantly increased permeability of vascular endothelial cells(VECs)with unknown reasons.Study on this phenomenon will contribute to the prevention and treatment of severe diseases caused by DENV infection.The present study evaluated whether DENV inoculation of primary human umbilical vein endothelial cells(PHUVEC)would result in cell infection,apoptosis and increased permeability;and discussed the effects of IFN-β and IL-29 on the defense of VECs against apoptosis and permeability changes caused by DENV infection.Furthermore,this study also evaluated whether IFNLRl could inhibit VECs permeability increase during DENV infection in vivo and in vitro experiments.It is expected that this study will provide an insight into the causes of cell permeability change during direct DENV infection of VECs.Methods:1.PHUVEC model in vitro was constructed based on physiological stiffness by application of polyacrylamide hydrogels.The expression levels of VECs markers and cell size were analyzed by immunofluorescence and flow cytometry.Cell count and BrdU methods were used to measure the cell proliferation.The expression of connexin-40(CX40)was analyzed by real-time PCR and Western blotting assays,and the sugar content of the cell surface was analyzed by glycosaminoglycan detection kit.2.PHUVEC which was cultured on hydrogels was directly infected with DENV,and the flow cytometry was used to assess cell infection and apoptosis levels after DENV inoculation.The gene expression profile of PHUVEC after virus infection was determined by gene chip technique to analyze the biological behavior and signal transduction pathways involved in cell activation after DENV infection.Flow cytometry was used to assess the cell infection status and apoptosis after using antibody to neutralize IFN-β and IL-29.The cell culture method was used to determine the virus titer in the culture supernatant.3.Immunofluorescence and Western blotting were performed to examine the presence of Interferon lambda receptor 1(IFNLRl)on PHUVEC,and the immunohistochemical technique was used to evaluate the presence of this receptor in C57BL/6 mice.After confirming the presence of this receptor,IL-29 was applied to PHUVEC,the TEER value and HRP leakage were measured to assess the potential effect of IFNLRl on monolayer permeability after IL-29 stimulation.The monoclonal antibody was used to block IFNLRl,and the TEER value and HRP leakage were measured to assess the effect of this receptor on cell permeability after PHUVEC was infected with DENV.Western blotting was performed to evaluate the correlation between permeability of IFNLRl andmonitoring RhoA enzyme activity.C57BL/6 mice were injected with IFNLRl monoclonal antibody and then infected with DENV.The virus load of animal serum,liver and spleen was measured by cell culture method.The changes of vascular permeability were monitored after Evanslan tail vein injection on the mice.Results:Compared with those of traditional culture method,the expression of CD31 and factor VID in PHUVEC cells cultured on hydrogels were not different.But the cells were smaller and the proliferation rate was slower.CX40 expression and glycosaminoglycan content was relatively higher.This indicated that the biological traits of PHUVEC at different base elasticity had some significant differences.Whether cultured on TCP or hydrogel,PHUVEC could not be effectively infected and induced apoptosis after DENV inoculation.The results of microarray showed that PHUVEC was in an immunopathic state.IL-29 and other genes were significantly expressed.Upon application of antibodies to neutralize IFN-p,the cell infection,level of apoptosis and the amount of virus cultured in supernatant increased significantly.After neutralization with IL-29,these changes could not be noticed.The presence of IFNLRl was confirmed in PHUVEC and in multiple organ VECs of C57BL/6 mice.TEER value of PHUVEC cell monolayer was increased after treating with IL-29.Presence of IFNLRl helped DENV infected PHUVEC to resist increased cell permeability due to viral interaction.Upon IFNLR1 monoclonal antibody injection in mice infected with DENV,Evans blue staining level in organs were significantly increased,but the viral load of serum,liver and spleen did not show any significant difference before and after antibody administration.There was no significant difference between in vivo and in vitro experimental results.IFNLRl resistance to permeability enhancement of PHUVEC after DENV infection is closely related to its expression regulation of downstream RhoA protein.Conclusion:Even though DENV Ⅱ NGC strains could not infect PHUVEC effectively and cause apoptosis,obvious immune activation was noticed after DENV inoculation.Abundant secretion of IFN-β contributed to resistance of DENV infection and consequently apoptosis of the cell.In the VECs,IFNLRl exhibited was functional distribution,and IL-29 produced by VECs after DENV infection was acted on this specific receptor which could subsequently resist the increase in VECs permeability caused by DENV infection.This biological effect showed a positive correlation with the inhibition of RhoA kinase activity.