Gingival Mesenchymal Stem Cell-derived Exosomes Ameliorate Collagen Induced Arthritis Via Regulating CD4~+T Cell Subsets And Suppressing IL-17RA-NF-κB Pathway

Objective:Rheumatoid arthritis(RA)is a systemic autoimmune disease,with synovitis as the main pathological feature,and further invasion of adjacent joint structures,resulting in pannus formation,cartilage damage,bone erosion,and ultimately leading to joint deformity,affecting the performance of joint functions.Its pathogenesis is closely related to the CD4~+T cell subsets,mainly including Th1,Th17 and Treg cells.And the role of Th17 cells has attracted increasing attention,which is regarded as the key factor during the formation of synovial osteoclasts.So the role of IL-17 secreted by Th17 in joint inflammation and destruction was decisive.The purpose of this study was to investigate whether exosomes derived from gingival mesenchymal stem cells(GMSC-EXO)have therapeutic effects on the mouse model of RA--collagen-induced arthritis(CIA),and to explore the potential therapeutic mechanism from the perspective of CD4~+T cells and IL-17 related signaling pathways.Methods:The study contains the following two parts.In vivo experiment:CIA model was successfully constructed using DBA/1J mice,and randomly set up three groups,including PBS group,GMSC group and GMSC-EXO group.Then responding treatment was given timely.Changes in paw thickness,arthritis score and body weight of mice during the development of inflammation were observed and recorded.After 56 days,mice were sacrificed to assemble samples.The joint bone erosion was detected by Micro-CT scanning.Western Blot was used to detect the expression of paw proteins(IL-17RA,Act1,TRAF6,p65,p50)to explore whether GMSC-EXO could play an immunosuppressive role through IL-17RA-NF-κB signaling pathway.HE staining was used to compare the pathological changes of foot sections.The distribution range of inflammatory protein was compared by immunohistochemical detection.The proportion of CD4~+T cell subsets(containing Th1,Th17,Treg)in spleen and lymph node cells was detected by flow cytometry.ELISA was used in the detection of serum levels of inflammation-related cytokines(IFN-γ,IL-17A,IL-10,IL-6,TNF-α).In vitro experiment:The influence of GMSC-EXO on the proportion of CD4~+T cell subsets(containing Th1,Th17 and Tr1)was investigated by direct co-culture method,and the changes of IFN-γ,IL-17A and IL-10 levels in the cultured supernatant were detected by ELISA.Results:In vivo,data displaied that,contrasted with the PBS group,GMSC-EXO group and GMSC group had a significant improvement in inflammation;the condition of bone erosion significantly reduced;the expression of IL-17 RA,Act1,TRAF6,p65,p50 of IL-17RA-NF-κB signal pathway have cut in paws.Results of HE staining found the joint synovial inflammatory cell infiltration is obviously ameliorated,and immunohistochemical detection displayed obviously decrease at the range of inflammatory protein distribution.The proportion of pro-inflammatory Th1 and Th17cells decreased,while the proportion of anti-inflammatory Treg cells increased in the spleen and lymph nodes.Pro-inflammatory cytokines,including IFN-γ,TNF-α,IL-17A and IL-6 were declined in serum,and the levels of anti-inflammatory cytokines IL-10were rised.In vitro,results showed that CD4~+T cell subsets increased after co-culture with GMSC and GMSC-EXO.And GMSC-EXO increased the proportion of Tr1 subsets in a dose-dependent manner.Both GMSC-EXO and GMSC significantly suppressed the proportion of Th1 and Th17 subsets.ELISA results showed that the level of IL-10 was significantly up-regulated after GMSC-EXO and GMSC treatment(similar to the Treg subpopulation).IFN-γand IL-17A levels were significantly down-regulated,and the level of IL-17A decreased in a dose-dependent manner with the does increasing of GMSC-EXO,while IFN-γlevels did not.Conclusion:1.GMSC-EXO and GMSC have similar immunomodulatory effects,both of which can effectively improve CIA.2.GMSC-EXO can alleviate inflammation by promoting Treg differentiation and suppressing Th1 and Th17 differentiation.3.GMSC-EXO can inhibit IL-17RA-NF-κB pathway to alleviate inflammation and reduce bone erosion.In conclusion,GMSC-EXO can ameliorate CIA by regulating CD4~+T cell subsets and suppressing IL-17RA-NF-κB pathway.