MMP9 Promotes EV71 Replication Via Binding IFNAR1 To Inhibit IFN/JAK/STAT Pathway

Enterovirus 71(EV71)and Coxsackievirus A 16(CA16)are the most important pathogens of Hand,foot and mouth disease(HFMD),all of which belong to belonging to the enterovirus A group from the Picornaviridae family.They can cause damage to the central nervous system(CNS),and poses a great threat to the health of infants and young children.However,the mechanism of their entry into the CNS remains unclear.In the previous study,we found that the expression of miR-1303 was inhibited after EV71 and CA16 infection,which target the regulation of matrix metalloproteinases 9(MMP9)and make significantly increase in MMP9.Then we confirmed that MMP9 can degrade the connecting molecules between cells thus change the permeability of the blood brain barrier(BBB),so that more viruses,inflammatory factors and inflammatory cells infiltrate,eventually leading to CNS damage.Based on previous research,we further explore whether MMP9 has an effect on viruses that have established infections in the CNS.We mainly explored the role of MMP9 in the innate immune response following EV71 infection.We mainly choose the immune cells in the nervous system,Human microglia(HM1900)as the research object and interfered with the expression of MMP9 by transfecting the siMMP9 plasmid,and then compared the differences in protein levels of interferon(IFN)pathway-associated molecules and inflammatory response-related molecules between cells infected with EV71 and the cells infected with EV71 after interfering with MMP9.The results showed that MMP9 was up-regulated after EV71 infection of HM1900 cells.VP1 of EV71 was significantly down-regulated in HM1900 cells that infection of EV71 after interference with MMP9,which indicates that MMP9 can promote the replication of EV71 in HM1900 cells.This result was also confirmed in human neuroblastoma cell(SHSY5Y)and human bronchial epithelial cells(16HBE).Subsequently,we detect the protein level of interferon(IFN).The results showed that there was no significant difference in protein levels of IFN-a between EV71 infected after interfered MMP9 and infected with EV71 alone in HM1900 cells,indicating that MMP9 does not affect the production of IFN-a.Next,we examined the protein levels of IFN-a/p receptor(IFNAR),which significantly increased the amount of IFNAR1 protein in cells infected with EV71 after interfered MMP9 compared with cells infected with EV71 alone.It was found that MMP9 and IFNAR1 co-localized by immunofluorescence and it was demonstrated that MMP9 binds to IFNAR1 by immunoprecipitation.These indicates that MMP9 acts by binding to IFNAR1.We further examine the levels of JAK/STAT pathway-related proteins in the downstream of IFNAR1,and found that the levels of STAT1,P-STAT2,P-JAK1,OAS1,and IFI44 proteins in cells infected with EV71 after interfered MMP9 were significantly higher than those in cells infected with EV71 alone.This indicates that MMP9 can inhibit the JAK/STAT pathway.In addition,we also explored the effect of MMP9 on the inflammatory response after EV71 infection.The results showed that levels of IL-1?,NLRP3 and Caspase-1 activity were significantly decreased in cells infected with EV71 after interfered MMP9,indicating that MMP9 can promote NLRP3-mediated inflammatory factor production.Taken together,our study demonstrates that MMP9 inhibits IFN/JAK/STAT/ISGs pathway via binding to IFNAR1 and ultimately promotes EV71 replication.MMP9 also promotes NLRP3-mediated activation of inflammasome,leading to the release of inflammatory factor IL-1?,which further aggravates the damage of the CNS.This study provides a candidate targets for clinical treatment of EV71 infection and early warning molecules for the diagnosis of patients with severe HFMD.