The Role And Related Mechanisms Of Dendritic Cells In Multiple Sclerosis And Experimental Autoimmune Encephalomyelitis Mice

Multiple sclerosis（MS）is an inflammatory demyelinating disease of the central nervous system（CNS）.The pathological findings of MS are charac-terized by demyelination,inflammatory cell infiltration,axonal degeneration and glial cell proliferation in the central nervous system.The pathogenic factors of MS are complex,immune imbalance is considered to be the key of its pathogenesis.The initial T cells are abnormally activated to produce self-reactive T cells,which enter the CNS through the destroyed blood-brain barrier（BBB）and are reactivated by their own nerve myelin antigen to form myelin specific autoreactive T cells,which will secrete a large number of inflammatory substances,chemotaxis and recruit more inflammatory cells,leading to inflammatory cascade reaction.Finally,it causes serious damage to the nerve myelin sheath or axon.Therefore,the abnormal activation of T cells plays a crucial role in the occurrence and development of MS.Dendritic cells（DCs）are known to have the strongest antigen present-ation ability of professional APCs.As we all know,the activation of T cells is that antigen-presenting cells（APC）are processed and divided into different peptides after capturing antigens,and then interact with T cell receptors through major histocompatibility complexes（MHC）and other structures on their surface,and are completed under the synergistic action of multiple stimulation signals.Their induction and regulation of immune response depend on their own maturation and activation.On the one hand,mature and activated DCs itself can secrete a variety of inflammatory factors,such as IFN-γ,IL-2,IL-6,TNF-αand so on,which can induce inflammatory res-ponse.As APC,it not only plays a key role in T cell activation,but also regulates other types of immune cells,such as B cells,NK cells and so on.On the other hand,immature or semi-mature DCs can maintain immune tolerance and inhibit inflammatory response by expressing costimulatory factors at low levels,such as CD80,CD86,MHCⅡ,or at high levels,such as PD-L1,Fas L,ICOSL,etc.,which can inhibit the proliferation of reactive T cells,induce T tolerance or disability,promote the formation of Treg cells,and achieve the function of stable immune tolerance in inflammatory environment.The induction of DCs to maintain immune tolerance and inhibit inflammation can be achieved by anti-inflammatory cytokines and gene modification induction,such as IL-10,TGFβ,PD-L1 and so on,but the phenotype and function of DCs are easily affected by the in vivo environment,and even the same source of DCs may still lead to rejection,in addition,it is difficult to store DCs as living cells,so there are many factors interfering in the treatment of diseases.In this study,we will explore the effect of DCs on MS and its protective mechanism on experimental autoimmune encephalomyelitis（EAE）through the expression and function of PD-L1 and exosomes produced by gene modification DCs in MS and EAE.Part One Correlation analysis between serum s PD-1 and s PD-L1 levels and the degree of disease recurrence and disability in patients with multiple sclerosis in remissionObjective:Simoa technology was used to detect the expression levels of s PD-1 and s PD-L1 in serum of RRMS patients,and to explore the correlation between the expression levels in serum of RRMS patients in remission and their correlation with the course of disease,frequency of disease recurrence and degree of disability.Methods:1.27 RRMS patients and 26 healthy volunteers recruited during the same period matching the number of cases,gender and age of RRMS patients were selected as the healthy group.2.Clinical data were collected,including gender,age,date of first onset,course of disease,number of recurrence（total number of episodes from the first onset to the point of blood collection）,and neurological impairment was assessed using the Extended Disability Scale（EDSS）.3.The expression levels of s PD-1 and s PD-L1 in serum of RRMS patients and healthy group were detected by Simoa technique.And the correlation of the two with the course of disease,the number of attacks,and EDSS score were analyzedResults:1.Comparison of the general information of subjects in the two groups:The gender distribution and age of subjects in the two groups showed no statistical significance（P=0.766,P=0.159）,so the two groups were compara-ble.2.The statistical analysis results showed the levels of s PD-1 and s PD-L1in RRMS group were significantly higher than those in healthy control group（P<0.05）.3.The serum levels of s PD-1 and s PD-L1 in RRMS patients were correlated with the course of disease,the number of attacks and EDSS score,and the results showed that s PD-1 and s PD-L1 were positively correlated（P<0.001）.Serum s PD1 level in RRMS patients was positively correlated with the number of attacks（P=0.030）,while serum s PD-1 and s PD-L1 levels were not significantly correlated with the course of disease and EDSS score（P>0.05）.Conclusions:The expression levels of s PD-1 and s PD-L1 in the serum of RRMS patients were significantly higher than those in the healthy control group,but the s PD-1 and s PD-L1 levels were not significantly correlated with disease duration and EDSS score.Part Two Extraction and identification of exosomes from immature dendritic cells transfected with FOXP3 gene overexpression adenovirusObjective:This part aimed to amplify the target gene of mouse FOXP3gene and construct an adenovirus vector with overexpression of FOXP3 gene.The Ad was proliferated and amplified from the infected HEK293 cells of AD,and the high titer adenovirus was finally purified.the purified FOXP3 overex-pression adenovirus was co-cultured with immature dendritic cells,and the exosome was extracted from the culture medium.The exosome was further identified and analyzed to determine the expression of FOXP3.Methods:1.Using FOXP3 gene as template,the target gene was amplified by PCR and connected with the vector.2.FOXP3 overexpression adenovirus was used to transfect HEK293 cells,and the virus was collected and purified after amplification,then the activity and titer were detected.3.Clean-grade C57BL/6 female mice were selected.The intact femurs of the mice were isolated in aseptic environment,and the muscle tissue was removed.The bone marrow cavity of the femur was gently washed with RPMI-1640 culture medium.After the cells were collected,the erythrocytes were lysed,and the cells suspension were washed with RPMI-1640 culture medium before centrifugation.The centrifuged cells were treated with RPMI-1640suspension containing 10%fetal bovine serum,10ng/ml interleu-kin-4（interleukin-4,IL-4）and 10ng/ml granulocyte-macrophage stimulating factor（GM-CSF）.The cell concentration was adjusted to 1×106/ml and inoculated on a six-well plate.In the process of cell culture,the morphology and growth state of cells were dynamically observed.4.The immature dendritic cells（im DCs）were purified by Miltenyi mag-netic beads sorting system.The purified im DCs and purified FOXP3 overex-pressed adenoviruses were co-cultured according to the proportion of infected complex MOI=500.After 24 hours,the culture medium was adjusted to 10%of fetal bovine serum with exosome removal,mixed with RPMI-1640.5.Im DCs and FOXP3 overexpressed adenoviruses were co-cultured for 72hours,then cell suspension was collected.Cell fragments were removed by centrifugation,and mixed evenly by adding the exosome extraction kit accor-ding to the proportion.After standing at room temperature for 12 hours,the exosome was collected by centrifugation.6.The morphology of im DCs and exosomes were observed by electron microscope,the purified exosome was analyzed by particle size analysis,and the expression of landmark protein and FOXP3 in exosome was detected by Western-blot.Results:1.After the adenovirus was purified according to the experimental steps,the virus titer was calculated according to the formula,and the virus titer was determined to be:HBAD-m-foxp3-3*flag-EGFP1.58*10^10 PFU/m L HBAD-m-foxp3-3*flag-EGFP1.99*10^10 PFU/m L HBAD-EGFP overexpression control 1.58*10^10 PFU/m L2.Virus quality detection:The cell culture medium was clear and trans-parent,there were no obvious particles in the intercellular space,and no signs of bacterial or fungal infection were found.3.The protein expression of FOXP3 in im DCs after transfection was detected by Western blot.The results showed that the FOXP3 expression of im DCs in Ad-FOXP3 transfection group was significantly different from that in Ad-Con transfection group（P<0.05）.4.Under electron microscope,the EXOs were round or oval vesicles about100nm in diameter.The particle size analysis of the extracted EXOs showed that the diameter of the vesicles was about 100nm.5.The EXOs obtained by FOXP3 overexpression adenovirus infection with im DCs stably expressed exosome landmark proteins CD63,HSP70 and FOXP3.In the control group,the EXOs obtained from adenoviruses infected with im DCs only expressed exosome landmark proteins CD63,HSP70.Conclusions:1.After transfection of FOXP3 overexpression adenovirus,the expres-sion of FOXP3 in im DCs was detected.2.FOXP3 pression was confirmed by exosomes extracted from im DCs transfected with FOXP3 overexpression adenovirus.Part Three Protective effect of exosomes transfected with immature dendritic cells by FOXP3 gene overexpressed adenovirus on mice with experimental autoimmune encephalomyelitisObjective:In the FOXP3 overexpressed adenovirus transfected imma-ture dendritic cells’exosomes（FOXP3-EXOs）intervention EAE,the neurolo-gical function score and the pathological changes of brain and spinal cord tissues of mice were observed,the proliferation of CD4⁺T cells in vitro and the changes of T cell subtypes in vivo were observed.Methods:1.Establishment of animal model of EAE:C57BL/6 mice were randomly divided into blank control group,EAE group,Con-EXOs group and FOXP3-EXOs group.Mice in EAE group,Con-EXOs group and FOXP3-EXOs group were immunized.Myelin oligodendrocyte glycoprotein（MOG）_35-55 polypep-tide was diluted with normal saline and prepared into 10mg/ml solution.Com-plete Freund’s adjuvant of equal volume was added,and Mycobacterium tuberculosis H37Ra was added to reach the concentration of 4mg/ml.The mixture was fully emulsified.Subcutaneous injection was implemented at four evenly distributed points on both sides of the spine of mice,0.1ml for each mouse.The mice were then given 0.5ml pertussis toxin intraperitoneal injec-tion at 0h and 48h respectively.2.On the 3^th,6^th,9^th,12^th and 15^th days after immunization,mice in each group were injected with normal saline,Con-EXOs and FOXP3-EXOs thr-ough caudal vein,respectively.Then,the four groups of mice were evaluated daily by using the Weaver’s method（15 points）,and weight of mice were evaluated daily from immunization day to day 23 after immunization.3.HE staining was used to observe the inflammatory cell infiltration in the spinal cord,and LFB staining was used to observe the myelin detachment.4.The effects of FOXP3-EXOs and Con-EXOs on the proliferation of spleen CD4+T lymphocytes in C57BL/6 mice were detected by CCK8 kit.And the levels of IFN-γ,IL-4,IL-6,IL-17 and IL-10 in supernatant of spleen CD4+T lymphocytes were detected by ELISA.5.The concentrations of Th1,Th2,Th17 and Treg cells in spleen of mice in each group were detected by flow cytometry.6.The levels of IFN-γ,IL-4,IL-6,IL-17 and IL-10 in serum of mice in the intervention group and control group were detected by ELISA.Results:1.FOXP3-EXOs can reduce the neurological damage of EAE and delay the onset of the disease,and the disease can aggravate the weight loss after the onset of EAE mice.2.Compared with the control group,HE staining of the spinal cord of EAE mice after the intervention of FOXP3-EXOs showed the degree of infla-mmatory cell infiltration was reduced,and LFB staining showed the degree of nerve myelin loss was reduced.3.FOXP3-EXOs significantly decreased the levels of Th1 and Th17 cells and increased the levels of Treg cells in EAE mice compared with the control group and Con-EXOs group.4.Compared with the control group and Con-EXOs,FOXP3-EXOs signi-ficantly decreased the levels of IFN-γ,IL-6 and IL-17 in serum of EAE mice,and increased the concentration of IL-10.5.FOXP3-EXOs significantly inhibited CD4+T cell proliferation in dose dependence;Con-EXOs group had no significant inhibitory effect on CD4+T cells.Based on the above results,we further evaluated CD4+T cell differen-tiation using 2ug/m L exosomes（Con-EXOs,FOXP3-EXOs）,and found that FOXP3-EXOs significantly inhibited the secretion of INF-γ,IL-6 and IL-17,and promoted the production of IL-10.Conclusions:1.FOXP3-EXOs inhibited the production of Th1 and Th17 cells,promo-ted the differentiation of Treg cells,and improved the pathological process of EAE in vitro and in vivo.2.The neuroprotective effect of FOXP3-EXOs on EAE may be realized due to the regulation of Th/Treg balance.