Effect Of VP1 Proteins Of Enterovirus EV71 And CA16 On Expression Of Innate Immunity-associated Signaling Molecules And Immune Response Of T Cells In Human Bronchial Epithelial Cells

Enterovirus 71(EV-A71)and Coxsackievirus A 16(CV-A16)[1]are the main pathogens of hand,foot and mouth disease(HFMD),which can cause similar clinical symptoms,but the difference of infection mechanism makes the research of CV-A16 vaccine lag behind EV-A71.Therefore,further exploring the relationship between the two viruses and the host immune system is still of great theoretical and practical significance.Previous studies have shown that both EV-A71 and CV-A16 can infect the human body through the respiratory tract,and that there are systemic pattern recognition receptors(PRR)in and out of the respiratory epithelial cells.These PRR have the ability to recognize various pathogen-associated molecular patterns(PAMP),and then initiate the regulation of the expression of innate immune-associated signaling molecules[2].So far,it remains unknown that the interaction between enterovirus and bronchial epithelial cells and its effect on the changing trend of innate immune-associated signaling.Therefore,this study designed a separate expression system of VP1 protein as the main antigen of enterovirus in bronchial epithelial cells 16HBE to explore its effect on the expression of innate immune-associated signaling molecules and T cell immune response[2].Firstly,the VP1 protein sequences of EV-A71 and CV-A16 were amplified,and inserted into the vector pcDNA3.1(+)respectively to construct the eukaryotic recombinant plasmids pcDNA-EV-A71-VP1 and pcDNA-CV-A16-VP1.Restriction analysis and sequencing proved that these two recombinant plasmids were correctly constructed.And western blot test proved that the VP1 proteins expressed correctly[2].Then,the two plasmids were transfected to human bronchial epithelial cell line 16HBE respectively.The cells were collected 24,48,and 72h after transfection and determined for the expression of innate immune-associated signaling molecules by Real-time quantitative PCR[2].The results show that,on the one hand,the signaling molecules expression profiles of EV-A71 and CV-A16-VP1 experimental groups are quite different in time and quantity.From 24 hours to 72 hours after transfection,the expression of EV-A71-VP1 showed a jump-like fluctuation,especially IFN-0,OX40L,IFN-a,IFN-y,RANKL and IL-2.At 24 hours,the expressions of EV-A71-VP1 group were 10-350 times higher than that of baseline control,while at 48 hours,these were dramatically below 10 times.However,the expressions of CV-A16-VP1 group tended to be stable in time,and the quantities were always below 10 times.On the other hand,we found that those signal molecules were up-regulated in CV-A16-VP1 group and down-regulated in EV-A71-VP1 group were anti-inflammatory factors,such as IL-4,IL-13,GITRL;on the contrary,those signal molecules were down-regulated in CV-A16-VP1 group and up-regulated in EV-A71-VP1 group were pro-inflammatory factors.Therefore,the results showed that VP1 of EV-A71 activated the natural immune response to some extent,but VP1 of CV-A16 had no obvious effect.Subsequently,in order to evaluate the synthetical effects of the above innate immune-associated signaling molecules,we carried out the human IFN-? specific Elispot test and found that the results of the two viral experimental groups were similar,that is,the innate immune-associated signaling molecules produced by the above treatment had a significant stimulating effect on the non-specific proliferation of T cells,but not in the specific proliferation of T cells presented by DC cells.In conclusion,after the expression of enterovirus EV-A71 and CV-A16 VP1 proteins in 16HBE,the expression of innate immune-associated signaling molecules is different[2],but the effect on the non-specific and specific proliferation of T cells is similar.Thus,this study provides experimental basis for further exploring the mechanism of early respiratory tract infection by different enteroviruses.