The Role Of RelB Silenced Tolerogenic Dendritic Cells In Experimental Autoimmune Encephalomyelitis

Objective:Multiple sclerosis（MS）is an autoimmune disease characterized by inflammatory demyelination in central nervous system.The pathogenesis of MS is not clear and may be related to a variety of factors,such as genetics,environment and infection.MS is more prevalent in the 20⁴0 age group and millions of people are affected by it around the world.At present,medicine is the first-line treatment of MS,but traditional non-specific drugs only have limited therapeutic effect for some patients.Therefore,development of specific immunetreatment for MS is an urgent problem to be solved.Experimental autoimmune encephalomyelitis（EAE）is a classic animal model of MS.It has similar clinical and pathological features as MS,so it is widely used in the study about pathogenesis and treatments of MS.Dendritic cell（DC）is a kind of professional antigen-presenting cell.It can activate T cells and plays an important role in the induction and progression of adaptive immune response.Besides inducing autoimmune response,DC can also promote and maintain immune tolerance.This capacity of DC makes it a novel target for specific immunetreatment of MS and EAE.RelB is a member of the nuclear factor kappa B（NF-κB）transcription factor protein family.It is abundantly expressed in DC.Meanwhile,RelB participates in the growth,differentiation and apoptosis of DC.In the NF-κB family,RelB is the one who relates to the differentiation and maturation of DC most.Furthermore,RelB will up-regulate during the maturation of DC.In this study,in order to induce tolerogenic DC,we transfected DC by RelB short hairpin RNA（RelB-shRNA）lentivirus and reduced the expression of RelB in DC.After that,we transfered these DC infected by RelB-shRNA lentiviruses into EAE mice and explore their therapeutic effects.Methods:1.To establish the EAE animal model,mixed emulsion of MOG_35-55 peptide and complete Freund’s adjuvant was injected subcutaneously into the back of C57BL/6 mice.Then 300 ng pertussis toxin was injected intraperitoneally into each mouse on the day of model establishment and 48 hours later.Mononuclear cells in the femur and tibia of the mouse were isolated and induced into DC by adding interleukin-4（IL-4）and granulocyte-macrophage colony-stimulating factor（GM-CSF）into the culture medium.2.On the 8th day of DC culture,we extracted the RNA and protein in DC of EAE mice.Then the expression of RelB was measured.3.On the 6th day of DC culture,RelB-shRNA lentivirus was added to the culture medium.48 hours after the transfection,the tolerogenic DC（RelB-shRNA DC）was obtained.We extracted the RNA and protein inRelB-shRNA DC and the expression of RelB was measured.Besides,lipopolysaccharides（LPS）were added to the culture mediums of normal DC and RelB-shRNA DC.After 24 hours,the expressions of CD80,CD83,CD86,major histocompatibility complex-II（MHC-II）andprogrammed death-ligand 1（PD-L1）on thesurfaces of DC and RelB-shRNA DC weremeasured by flow cytometry.The secretions of cytokines incell culture supernatants,such as TNF,IL-2,IL-4,IL-6,IL-10 and IL-17A were detected using Cytometric Bead Array kits.4.On days 10,13 and 16 after the establishment of the EAE model,RelB-shRNA DC wastransfered into to EAE mice while the control groups received PBS or normal DCrespectively.The therapeutic effects of the treatments were compared among three groups.The secretions of cytokines（such as TNF,IL-2,IL-4,IL-6,IL-10 and IL-17A）in serum as well as the levels of spinal cord demyelination and inflammatory cell infiltration were also compared among three groups.Results:1.The EAE model could be established successfully by subcutaneous injection of mixed emulsion of MOG_35-55 peptide and complete Freund’s adjuvant as well as intraperitoneal injection of pertussis toxin.Bone marrow mononuclear cells could be induced into DC by adding IL-4 and GM-CSF into the culture medium.2.The expressions of RelB RNA and proteinin DC of EAE mice were higher than that of wide type mice.3.Transfection by RelB-shRNA lentivirus reduced the expression of RelB in DC.The expressions of CD83,CD86 and MHC-II on the surface of RelB-shRNA DCwere decreased.The secretion of IFN-γwas decreased while the secretions of IL-2and IL-4 were increased in RelB-shRNA DC.After stimulated by LPS,the expression of CD86 and MHC-II on the surface of RelB-shRNA DC as well as the secretions of IFN-γand IL-17A were significantly lower than normal DC.4.Compared with PBS,DC and RelB-shRNA DC alleviated the symptoms of EAE mice significantly.And the therapeutic effect of RelB-shRNA DC was better than DC.When compared with PBS group,the numbers of inflammatory cells in the spinal cord of EAE mice in DC group and the RelB-shRNA DC group were significantly decreased.Compared with the DC group,the number of inflammatory cells in the spinal cord of the RelB-shRNA DC group was reduced.The demyelination of the spinal cord was much severer in the EAE mice of PBS group and DC group than RelB-shRNA DC group.The secretion of IL-2 in serum of DC group mice was significantly lower than that of PBS group mice.Conclusion:1.EAE model could be established by MOG_35-55 peptide.2.The expression of RelB in DC of EAE mice was higher than normal DC.3.Transfecting DC by RelB-shRNA lentivirus could induce tolerogenic DC（RelB-shRNA DC）.4.Transferring RelB-shRNA DC into EAE mice could alleviate their clinical symptoms and reduce the inflammatory cell infiltration and demyelination in the spinal cord.