Immunoregulatory Effects Of Interleukin-10 In Foot-and-mouth Virus Infection

Foot-and-mouth disease virus(FMDV) is a member of the family Picornaviridae. Many researchers have studied this disease,although the FMDV shows high genetic and antigenic variability,so that the traditionary vaccine can't control the epidemic and the explosion of FMD.The research presented that the bodies can secrete many cytokines when which affected by fmdvs,and the cytokines can regulate immune response of the bodies,so they played an important role in virus disease.For FMDV can cause the animals secreted many kinds of cytokines,and which have very important effects on immune state of animals.And Dendritic cells(DC) are the most potent antigen presenting cells(APC) of the immune system,the APCs could capable of stimulating resting T cells to generate an antigen specific primary immune response.So it has a special place in induce of immune response.We thought that the DC have the important affect through the process of viral infection.For that the bone marrow-derived dendritic cells were distributed in the epidermis,and we thought which was the major immunocyte for start the initial immune response,so they have the very important rules in control of the dieases.Now we thought that the changes of Th1/Th2 were very important for the research of pathogenic mechanism for FMDVs.The cell-surface maker would be lossed because of the virus' replication,which would caused the virus refrain from the bodies' immunological surveillance.Otherwise,the DCs could promote the antiviral immunologic response through regulate the balance of Th1/Th2.In addition to its effects on suppressing inflammatory and Th1 responses and IL-10 strongly inhibits the maturation of DCs and the cellullar immunologic response.Certain populations of DCs appear to produce IL-10,which may promote the generation of regulatory CD4~+ T cell populations.So,many factors which could regulated the secrete of IL-10 could affect the function of DCs and influence the development of disease.Therefore,we though that the regulatory factor which can affect the IL-10 would be a very important means to deal with the FMDV.For this reason,in the research,we used the IL-10 and IL-10 neutralizing antibody to regulate the immune state of bodies when they were infected by FMDVs.At frist,we used to generate dendritic cells(DCs) by culturing them in recombinant granulocyte macrophage-colony stimulating factor(GM-CSF) and recombinant interleukin-4(IL-4).We found that the fmdvs can infect the imDCs and mDCs.But we can't saw the cytopathic effects when used the light microscope:but we could saw from the indirect immunofluorescence microscope that the fmdvs could infect the imDCs and mDCs.And that the imDC have the powerful abilities to intake the virus,in the other hand,the mDC could stimulated the body to present the antigenes.If we added the recombination mouse IL-10 to the DCs which was infected by fmdvs,that the expressed of IL-12 and IFN-γwere decreased in transcriptional level,but the IL-4,IL-6,IL-10 were increased,if we added the IL-10 neutralizing antibody in the culture hole,the IL-I,IL-4,IL-6 and IL-10 became decreased,but the expression of IFN-γbecame higher.So we thought that IL-10 could affected the immunological response of the immune system when the immunological cell was infected by fmdvs.When we detected the cytokine of the supernatant of DCs by ELISA,that the secretory volume of IL-1,IL-4,IL-6,IL-12 and IFN-γhad no significant difference.But if we used the IL-10 neutralizing antibody to the DCs,we can seen that the secretory volume of IL-12 and IFN-γwere increased.The others had no significant difference.So we thought these were because of many factors which deal with the cells.The capabilities of antigen presentation were all have affected if we used the IL-10 or the IL-10 neutralizing antibody in the holes which were infected by fmdvs, and the proliferation index(SI) of lymphocyte which the IL-10 neutralizing antibody we used were higher than that used of IL-10.When detected the surface marker of MHCⅠ,MHCⅡand the CD11c of the DCs which were infected by fmdvs by flow cytometry,that the MHCⅠand MHCⅡwere all decreased when we used the IL-10 and the IL-10 neutralizing antibody,but if we used the IL-10 the CD11c could decreased,In contrast,the IL-10 neutralizing antibody caused the produced of the expression of CD11c became increased a little.So we designed the target of the IL-10 combined with the secondary structure prediction of mRNA.And cloned them into the expression vector behind of the promoter U6,mU6 and h7SK.Whereafter the DNA sequencing of the plasmids were detected by restriction enzyme digestion and sequence determination,which verified that we make successful construction of siRNA vectors which include of the gene of EGFP And the structure of which was Promotor,positive-sense strand of shRNA, loop,antisense strand of shRNA,terminate signal.The products can be transfected into the DCs by the transfection anget of FuGENE HD from Roch,but transfection efficiency was very low.After we injected the plasmids to the mices,then the virus were infected to the mice through belly cavity,that the plasmids could affect the immune states of the mice.And the secretory volume of IL-12 and IL-6 have somewhat changed after FMDV WFL was infected. Through the detection of the splenic lymphocyte by flow cytometry,we can known that the plasmid can affect the expressed of the CD3~+,CD4~+,CD8~+ of the mice which was infeted by fmdvs.So we thought that these researches can be revealed the immunological response and the mechanism of the viral infection.