Therapeutic Effects Of Tolerant DCs With Silencing MyD88 On EAE

Objective:Multiple sclerosis(MS)is a chronic inflammatory autoimmune disease that occurs in the Central nervous system(CNS).Its main pathological features are inflammatory cell infiltration and white matter demyelination.The disease often occurs in young adults and may be closely related to viral infection,heredity,environment and other factors.Since the current pathogenesis of the disease is not clear,and the disease brings irreversible physiological and psychological damage to the patients,there is no specific treatment.Only some non-specific drugs with high prices can be used in remission.Therefore,it is urgent to clarify the pathogenesis of MS.Experimental autoimmune encephalomyelitis(EAE)is one of the classical animal models through immunization of experimental animal via injection of antigen protein.Its symptoms and pathological manifestations are similar to MS,so it is widely used to study the pathogenesis of MS.Dendritic cells(DCs)are closely related to the immune response and tolerance of the body.DCs present antigen to T cells,activate the inflammatory response and mediate differentiation of T cells.DCs play a key role in the pathogenesis of MS.As tolerant DCs in immature state,tolerant DCs may be a new target for the treatment of MS.Myeloid differentiation factor 88(MyD88)connects downstream proteins through the Toll like receptor(TLR).It mediates adaptive immunity and promotes the expression of inflammatory proteins.MyD88 stimulates DC maturation by connecting TLR to nuclear factor kappa B(NF-κB).This study will explore the RNA and protein expression of MyD88 in DCs in EAE animal models.The short hairpin RNA(shRNA)is used to silence MyD88 to induce tolerant DCs.Then transfer the tolerant DCs into EAE animal models,to further explore the possible role of MyD88 in EAE animal models and the therapeutic value of tolerant DCs via silencing MyD88 in EAE.Methods:1.The female C57BL/6 mice were immunized with myelin oligodendrocyte glycolprotein(MOG)35-55 polypeptide mixed with complete freund’s adjuvant(CFA)and inactivated mycobacterium tuberculosis H37 Ra.Pertussis toxin(PTX)was injected into the animals immediately and 48 hours later.Then EAE animal model was constructed.2.Bone marrow derived single cell suspension was extracted from leg bones(tibia and femur)of mouse.Then granulocyte-macrophage colony stimulating factor(GM-CSF)and interleukin-4(IL-4)were added in medium to induce DC.3.RNA and proteins of DCs were extracted,and the expression level of MyD88 was detected by real-time quantitative polymerase chain reaction(RT-qPCR)and Western Blot.4.MyD88 gene in DCs were targeted and silenced by shRNA technology.We detected the expression of MyD88 by RT-qPCR to demonstrate the induction of tolerant DCs.The surface molecular expression and cytokine secretion of the tolerant DCs were detected by flow cytometry.At the same time,lipopolysaccharide(LPS)was added in medium to stimulate tolerant DCs.Then the expression of surface molecules and the secretion levels of cytokines were detected again.5.The acquired tolerant DCs were transferred to EAE animal models in three times and we observe the symptoms and score.During the peak period,serum cytokine secretion was detected.Meanwhile,we prepared the sections of spinal cords.Hematoxylin-eosin(H&E)staining and luxol fast blue(LFB)staining were performed to observe the infiltration of inflammatory cells in spinal cords and demyelination of spinal cords.Results:1.After the MOG35-55 polypeptide was well mixed with CFA and H37 Ra,it was injected into female C57BL/L mice,then PTX was injected into the animals immediately and 48 hours later.EAE animal models could be successfully constructed.2.The single cell suspension extracted from the bone marrow was cultured with GM-CSF and IL-4.The obtained cells were verified to be DCs by flow cytometry.3.In EAE animal models,the expression levels of MyD88 in DCs at the RNA and protein levels were higher than that of the wild-type DCs.4.After using shRNA interference technology to target and silence the MyD88 gene in DCs,the results demonstrated that the RNA expression of MyD88 was decreased.Moreover,the expression of molecules on the surface of DCs had certain changes: the expression of CD80 increased and the expression of CD86 and major histocompatibility complex II(MHC II)decreased.The level of IL-4 secreted by MyD88 shRNA induced DCs increased.After LPS stimulation,CD274,namely Programmed death 1 ligand(PD-L1),on MyD88-shRNA induced DCs was increased.But the increased level was lower than that of wild-type DCs.The secretion of cytokines of MyD88-shRNA induced DCs were not significantly different from that of wild-type DCs.5.After transferring the wild-type DCs and the MyD88-shRNA induced DCs to EAE animal models,the symptoms and scores of the experimental animals were alleviated.However,in the detection of serum cytokines at the peak periods,only the secretion level of IL-2 in the wild-type DC group than that in the PBS group has declined.The pathological staining results of the spinal cord showed that the infiltration of inflammatory cells and demyelination were less in the two groups than in the PBS group,but there was no significant difference between the two DC groups.Conclusion:1.Female C57BL/6 mice can be successfully immunized with MOG35-55 polypeptide to construct EAE animal model.2.In the DCs of EAE model mice,the expression of MyD88 in RNA and protein levels was higher than that of wild-type mice.3.shRNA lentiviral vector transfection technique can target MyD88 gene in DCs and successfully induce tolerant DCs.4.The infusion of MyD88-shRNA DCs to EAE animal models can reduce their symptoms,the infiltration of inflammatory cells in the spinal cord and the phenomenon of myelin loss.5.There was no significant difference in the therapeutic effect of the wild-type DCs and MyD88-shRNA induced DCs in EAE animal models.