The Role Of Vagal Nerveregulated And α 7nAChR Medicated Negative Inflammatory Regulation In Severe EV71 Infection

Hand,foot and mouth disease(HFMD)has become one of the major concerns for children healthy care worldwide since its outbreak in population mainly under the age of 5 in last decade.As the main etiological agent of HFMD,EV71 is considered to be one of the neurotropic viruses,and the severe EV71 infection has been associated with high incidence of mortality and morbidity.Moreover,no effective antiviral agent is currently available for patients with severe EV71 infection.Therefore,to understand the mechanism of EV71 infection in CNS and to develop effective therapeutic drugs against EV71 infection is still imminent.Both CA16 and EV71 are members of the family Picornaviridae belonging to the group of human Enterovirus species A,and they share plenty of the construct similarities,while severe neurological diseases caused by CA16 infection were barely reported.In the first part of this study,non-neuronal cells(Vero and RD)and neuronal cells(U251 and SY5Y cells)were used and infected with nine virus strains which of those were isolated from children with HFMD presented different clinical symptoms from mild to vere diseases,respectively.The pathogenicity of EV71 and CA16 strains in the four types of cells were compared according to characterizes of infected cells in aspects of cell viability,cytopathic effect(CPE),virus loading and mRNA expression of apoptins.The difference between CA16 and EV71 infections in the four cell lines,especially in SY5Y cells,was analyzed to further understand the relationship between pathogenicities of viruses and the clinical outcome of viral infection.It was showed that all of the cells were able to be infected by these nine virus strains.Pathogenicities of the three kinds of virus strains were extremely similar and no significant difference in between was observed by comparing CPEs,the level of viral replication and cell viabilities caused by virus infections within the same cell line in all of the four cell lines.Both CA16 and EV71 infection in the three types of human cancer cells were found to induce similar apoptotic response in both neuronal cells and non-neuronal cells in vitro,and the apoptotic pathways stimulated by viral infections were same in three host cells.No enormous viral replication and cell death had been found in neurons and glial cells after infected by EV71 strains,especially by EV71 neruovirulent strain,comparing with infection in Vero and RD cells,indicating glial cell was not a sensitive cell for EV71 infection.The data indicated that pathogenicity of EV71neruovirulent strain in neurons may not be the definitive factor for the neurotropism of EV71.Unusually elevated plasma proinflammtory cytokine levels in patients with severe EV71 infection were reported clinically,and the abnormal immune response from host was considered to be closely related to severity of EV71 infection.The issue regarding the possible interplay between neuronal damage and abnormal immune response from host in the presence of EV71 infection was explored.The factors which may have impact on production and release of inflammatory factors during viral infection in vitro were investigated.Then the influences of exogenous inflammatory factors to cell viabilities of the four types of cells as well as to the pathogenicities of EV71 infections were observed.Results indicated that the levels of inflammatory factors were not only closely related to the level of viral replication in cells,but also related to the type of cells which was stimulated by viral infections.In the presence of exogenous inflammatory factors,degree of neuronal death induced by inflammatory factors was increased more than the degree of the other three types of cells.Moreover,in the presence of exogenous inflammatory factors,degree of cell death in neuronal induced by viral infection was increased more than other three types of cells,suggested that the inflammatory microenvironment of neuron may have significant contributed to neuronal damage during EV71 infection.In the third part of this study,the role of inflammatory regulation mediated by a7nAChR in EV71 infection was assessed by comparing the changes of cell viabilities and viral replication levels in viral infected cells before and after using special a7nAChR agonist and special a7nAChR antagonist.Treatment of PNU-282987 which is ana7nAChR agonist improved cell viability,inhibited viral replication and decreased inflammatory factors level in EV71 infected cells,suggested its anti-inflammatory activities and antiviral activities against EV71 infection.In contrast,treatment of special a7nAChR antagonist(MLA)increased viral replication in EV71 infected cells,suggested its benefit for EV71 replication.Those results supported our hypothesis that the selective a7nAChR medicated inflammatory regulation play a role in antiviral activities and cell protective effect against EV71 infection in vitro.Further investigation revealed that inhibition of EV71 production by PNU-282987 is likely associated with events of RNA replication.By up-regulating the down-regulated JAK-STAT pathway and down-regulating the up-regulated NF-kB pathway in virus infected cells,PNU-282987 increased INFs and mainly their downstream antiviral proteins,and therefore changed the inflammatory microenvironment of virus infected cells,and improved cell viability of viral infected cells eventually.In recent years,researches have paid great attention to the cholinergic anti-inflammatory pathway which is regulated by vagal nerve and medicate by a7nAChR,as this pathway plays an important role in the negative regulation of inflammation by significantly reducing the release of a variety of pro-inflammatory cytokines.A study on effect of negative inflammatory regulation to neuronal injure were applied,and we found that the a7nAChR mediated negative inflammatory regulation had considerable impact on EV71 infected cells.Those findings may provide further understand to the mechanism of severe viral infection.And the selective a7nAChR-agonist could be a viable candidate for future therapeutic treatment of severe EV71 infection,even for the potential treatment of neurotropic viral infection.