The Role Of Soluble PD-L1 In The Treatment Of EAE By Regulating Immune Tolerance Of Dendritic Cells

Background and Objective:Multiple sclerosis（MS）is an immune-mediated disease characterized by inflammatory demyelinating plaques in the central nervous system,mainly occurs in young and middle-aged adults with a high disability rate,bringing heavy burdens on the families and the society.The pathogenesis of MS is still not completely understood.Overactivation of adaptive immune response is thought to play a pivotal role in this disease.Currently,the effectiveness of the disease-modifying therapies for MS are limited,and the therapies may conduce to severe adverse effects.Therefore,it is of vital scientific significance and clinical value to explore highly efficient and specific immunoregulatory therapies for MS.Dendritic cells（DCs）are the most potent professional antigen-presenting cells.They can initiate self-reactive immune responses in MS.Meanwhile,it can differentiate into tolerogenic phenotype to induce and maintain immune tolerance,which is the potential therapeutic target for MS and its animal model experimental autoimmune encephalomyelitis（EAE）.Programmed cell death 1（PD-1）is an immunosuppressive receptor expressed on the surface of various types of immune cells.PD-L1,a ligand of PD-L1,is mainly expressed on antigen-presenting cells,such as DCs.The dysregulation of PD-1/PD-L1 signaling pathway plays a crucial role in the process of initiating and perpetuating MS.PD-L1 could be the new target of the immunomodulation of MS/EAE,while its detailed role and underlying mechanisms needs to be investigated.PD-L1 can trigger the PD-1 signaling,thereby inhibiting the activation and inducing the immune tolerance of DCs,which has not been studied in MS/EAE till now.The objective of this study is to investigate the immunoregulatory impact of soluble PD-L1（s PD-L1）on EAE,and to clarify whether s PD-L1 could induce the immune tolerance of DCs in vivo and in vitro.This study is supposed to provide a new strategy and corresponding theoretical basis for MS treatment.Materials and Methods:（1）To construct the EAE model,myelin oligodendrocyte glycoprotein（MOG）35-55 peptide emulsified with complete Freund’s adjuvant containing inactivated mycobacterium tuberculosis H37 Ra was subcutaneously injected into female C57BL/6 mice.Mice were injected with pertussis toxin intraperitoneally on the same day and day 2 postimmunization.After immunization,monitoring the changes of body weight and scores of clinical symptoms of the mice every day until peak of the disease.For mice of treatment group,s PD-L1 was given intraperitoneally once daily for 5 consecutive days.（2）Spinal cords of the mice were collected and cut into sections at the peak of disease activity.The hematoxylin-eosin staining and Luxol fast blue staining were performed to observe the infiltration of inflammatory cells and the demyelination of the spinal cords.Spleens were collected to prepare the single-cell suspensions.Flow cytometry was carried out to determine the proportion of DCs and the cell-surface expression of MHC-Ⅱ,CD40 and CD86 on DCs.（3）Bone marrow mononuclear cells were extracted from femurs and tibiae of EAE mice and cultured in vitro at peak disease.GM-CSF and IL-4 were added to the medium to induce the cells to differentiate into DCs.On day 6 post-culture,lipopolysaccharide was administrated to stimulate DCs maturation.DCs were incubated with s PD-L1 on day 7 and were harvested on day 8 post-culture.（4）A scanning electron microscope was used to observe the surface morphology of the bone marrow-derived DCs.The cell-surface expression of MHC-Ⅱ,CD40 and CD86 on cultured DCs were detected by flow cytometry.Results:（1）The EAE mice model was established successfully in female C57BL/6 mice by subcutaneous injection of mixed emulsion of MOG35-55 peptide,complete Freund’s adjuvant and inactivated mycobacterium tuberculosis H37 Ra,accompanied with intraperitoneal injection of pertussis toxin.（2）s PD-L1 treatment significantly reduced the clinical symptoms scores of EAE and decreased the immune cell infiltration and demyelination in spinal cords.（3）When compared to the normal control group,the expression of MHC-Ⅱ and CD86 on spleen DCs of EAE mice were elevated.The spleen DCs of EAE mice treated by s PD-L1 showed lower expression of CD86,compared to untreated EAE.（4）IL-4 and GM-CSF could induce bone marrow mononuclear cells to differentiate into DCs.Upon lipopolysaccharide stimulation,the expression of MHC-Ⅱ,CD40 and CD86 on cultured DCs showed a significant increase.s PD-L1 reduced the expression of MHC-Ⅱ and CD86 on LPS-stimulated DCs.Conclusions:（1）s PD-L1 had a significant treatment effect on EAE,which ameliorated inflammatory responses and demyelination in spinal cords.（2）The spleen DCs of EAE mice appeared more mature,with higher immunological activity.They expressed higher levels of MHC-Ⅱ and CD86 than did normal mice.（3）s PD-L1 could downregulate the expression of co-stimulatory molecule CD86 on spleen DCs,thus inducing the DCs differentiation toward tolerogenic phenotype in EAE.（4）In vitro,s PD-L1 could inhibit the maturation and activation of bone marrow-derived DCs from EAE mice.