The Pathogenic Role Of PLAC8 In EV71 Infection

Enterovirus 71(EV71)is one of the main pathogens causing severe cases of hand,foot and mouth disease in young children.EV71 infection can cause pathogenic changes like brainstem encephalitis and pulmonary edema,being a serious threat to the healthy growth of young children.Infiltration of neutrophils in brainstem,spinal cord and pulmonary interstitial were observed in EV71 infeced dead cases,suggesting that inflammatory damages of tissues played important roles in EV71 induced diseases.However,for now,there is little knowledge about the molecular mechanism of the series of inflammation cuased by EV71 infection and the reason for the age-dependent infection of EV71.In this study,EV71 infected mice brain were used for microarray analysis to identify the potential genes involved in brainstem encephalistis to reveal the mechanism of inflammatory damage in EV71 infection.EV71 infected mouse model was eastablished by intraperitoneal injection of EV71 to one-day-old suckling mice.After three and six days post infection,mouse brain were harvested from both infected and negative control mice and RNA were extracted to go though Affymetrix gene chips analysis to identify differentially expressed genes.The KEGG analysis and functional annotation of these differentially expressed genes revealed that these genes were mainly involved in antiviral immunity.Among these identified genes,PLAC8 were both up-regulated at three and six days post infection.For now,reports about PLAC8 function were mainly involved in promoting cell growth and cancer progression.However,differentially expression of PLAC8 was identified in the clearance of HBV infection and systemic inflammation.These results indicated that PLAC8 might have play important roles in antiviral immunity.Therefore,PLAC8 was chose for further study to illuminate the potential roles during EV71 infection.EV71 mainly infected infants and children under five years old and the severe cases were under three years old in primary.After EV71 infection in 1-day-old,2-day-old and 3-day-old suckling mice respectively,the 1-day-old mice showed symptoms of wasting,limb weakness and paralysis sequentially and were all sacrificed at seven days post infection(dpi).The 2-day-old mice showed remarkably decreased rates of lesion and death and the death rates reached twenty percent at 7 dpi.However,the 3-day-old mice showed no obvious lesions and death.These results indicated that the younger the mice,the more susceptible to EV71 infection,which meant the age-dependent of EV71 infection.Therefore,EV71 infected mouse model could be used for the research of the relationship between EV71 infection and age.In order to study the relationship between PLAC8 and the age-dependent infection of EV71,the m RNA and protein expression of PLAC8 was detected using the lungs of mice with different ages.PLAC8 expression turned out to be down-regulated along with the age.Moreover,peripheral blood from twelve to twenty healthy people whom aged less than three,three to twelve years and twelve to eighteen years respectively,were collected and RNA were extracted to detect the m RNA expression of PLAC8 in the leukocytes.As a result,PLAC8 expression in human was also significantly down-redulated with age.The results above suggested that PLAC8 might have close relationship with age-dependent infection of EV71.Then,the function of PLAC8 on promoting EV71 proliferation significantly was found by over-expression of PLAC8 in A549 cells.In addition,during the early stage of infection,like 6 hours post infection,the obvious expression of the structural protein VP1 of EV71 could be detected in PLAC8 over-expressed A549 cells,while no VP1 could be detected in control cells.These results illustrated that PLAC8 could facilitate EV71 proliferation during the early infection.The potential reasons behind this phenomenon could be concluded to three points.Firstly,PLAC8 favoured the expression of EV71 receptors SCARB2 and PSGL-1,increasing the virus numbers entered the cells.Secondly,PLAC8 regulated the activity of 5'UTR of EV71 genome,promoting the replication and translation of virus gene.Third,PLAC8 inhibited the antiviral immunity of host cells,facilitating virus proliferation.Therefore,we detected the m RNA expression of EV71 receptor,SCARB2 and PSGL-1,after PLAC8 expression.As a result,the over-expression of PLAC8 did not induce the expression of these receptors.Then,we inserted the 5'UTR of EV71 genome into the regulation sites of luciferase expression vector and PLAC8 did not increase the expression of luciferase gene,indicating that PLAC8 could not regulate the replication and translation of EV71 genome.However,the over-expression of PLAC8 could inhibit the expression of pattern recognition receptor TLR3 and interferon regulatory gene IRF3.These results showed that PLAC8 promoted EV71 replication in the early stage of infection was due to the repression of host antiviral immunity and provision of favorable host microevironment for the viruses.In addition,considering the inflammatory damage of organs,like brain and lung,during EV71 infection,the expression of IL-6 in THP-1 cells was detected.The overexpression of PLAC8 could also increase IL-6 production remarkably.The balance of T helper(Th)cells played important roles in immunity,antiviral and inflammatory response in infants.Therfore,to study the function of PLAC8 on the expression of Th cells supporting cytokines during EV71 infection,the mice model with over-expressed PLAC8 were constructed by injecting PLAC8-expression plasmids though the superficial temporal vein.Compared with the control mice,the PLAC8 overexpression mice expressed less Th1 surpporting cytokines,IFN-?,more Th2,Th17 and Treg supporting cytokines,IL-6,IL-4,IL-17 A and TGF-?.Therefore,the over-expression of PLAC8 could inhibit Th1 cell differentiation and the celluar antiviral immunity mediated by Th 1 cell.Meanwhile,PLAC8 could facilitate Th2,Th17 and Treg differentiation to exert their roles in inflammation and immunosuppression response.Meanwhile,the down-regulation of interferon gamma and up-regulation of inflammatory factor IL-6 suggested that PLAC8 over-expression mice do have repressed antiviral immunity and enhanced inflammation.After EV71 infection in PLAC8 over-expressed mice,the lung and brain tissue sections were underwent HE staining to observe the histopathological changes.Compared with the control mice,the mice with PLAC8 over-expression showed more significant thickened alveolar walls,alveolar disappear and lymphocytes infiltration in the lung.Similarly,the brain showed more decrease in the number of neurons and more severe of nuclear condensation in PLAC8 over-expression mice.Thses symptoms proved that EV71 could infect PLAC8 over-expressed mice and lead to lesions more efficiently.In conclusion,a gene microarray analysis was performed using EV71 infected mice brain and a potential protein,PLAC8,that might play important roles in EV71 infection,was identified in our study.Molecular biology experiments showed that PLAC8 could inhibit the expression of pattern recognition receptor TLR3 and interferon regulatory gene IRF3,thus,inhibiting antiviral immunity and providing favorable host microenvironment to promote viral proliferation.In addition,PLAC8 could facilitate the production of inflammatory factor IL-6.The animal experiments indicated that PLAC8 could regulate the expression of cytokines that inducing the differentiation of Th cells.PLAC8 overexpression lead Th cells to polarizing from Th1 to Th2,Th17 and Treg by inhibiting supporting cytokines IFN-? expression and promoting cytokines IL-6,IL-4,IL-17 A and TGF-? expression,resulting in the imbalance between the antiviral immunity and inflammatory response.Due to the expression of PLAC8 declined along with the age,suckling mice with high expression of PLAC8 would be more susceptible to EV71 infection and result in inflammatory injury.These results could provide important clues to reveal the reason for the age-dependent infection of EV71 in infants.