The Immunomodulatory Effects And Mechanism Study Of MSCs Derived Exosomes On CD4~+T Lymphocytes In Patients With Primary Sjogren’s Syndrome

Background:Primary Sj（?）gren’s syndrome（p SS）is an autoimmune disease in which the immune disorder leads to the destruction of exocrine glands and multi-system lesions.Nowdays,there is still a lack of safe and effective treatments,which can regulate immune disorders at an early stage to stop disease progression and repair damaged tissues at an advanced stage.The abnormal proliferation,apoptosis and differentiation of CD4⁺T cells are key points in the pathogenesis of p SS,and are seriously affected the appearance,progress and outcome of the disease.Therefore,how to inhibit the abnormal proliferation,apoptosis and differentiation of CD4⁺T cells to prevent disease advance has become a new direction to explore the treatment of p SS.Mesenchymal stem cells（MSCs）have the ability of high proliferation,immunomodulatory and multi-directional differentiation.Previous studies have found that MSCs can inhibit T cell proliferation and pro-inflammatory factor secretion,reduce lymphocyte infiltration of salivary and lacrimal gland,and restore exocrine gland function in p SS patients and animal models.MSCs-derived exosomes（MSC-Exos）may effectively simulate immune regulation and tissue repair of MSCs while avoiding the risks involved in MSCs treatment,which is expected to become a new treatment of rheumatic diseases.However,whether MSC-Exos can regulate the abnormal proliferation,apoptosis and differentiation of CD4⁺T cells in p SS,and what the specific mechanisms involved remain to be elucidated.The results will provide an important theoretical and experimental foundation for exploring MSC-Exos as a new treatment for p SS and its application in clinical transformation.Objective:Human umbilical cord MSCs（UCMSCs）-derived exosomes（UCMSC-Exos）were extracted and identified.The study evaluated the immunomodulatory effects of UCMSC-Exos on peripheral blood CD4⁺T cells in p SS patients and clarified the specific mechanisms.Methods:1.The cell morphology,surface antigen phenotype,and adipogenic and osteogenic abilities of UCMSCs were examined.UCMSC-Exos were seperated by ultracentrifugation.The morphology,particle size,protein concentration,and surface labeling of UCMSC-Exos were examined.2.Peripheral blood CD4⁺T cells were sorted by immunomagnetic beads.The CD4⁺T cells proliferation was detected by CCK8 among healthy control（HC）,p SS and UCMSC-Exos（30μg/m L,60μg/m L,90μg/m L）groups to determine the optimal intervention concentration.Then,CD4⁺T cell cycle,apoptosis,cell subsets,and inflammatory factors were detected in HC,p SS,and UCMSC-Exos group by flow cytometry.3.The differential genes and enriched pathways of peripheral blood CD4⁺T cells between HC and p SS patients were analyzed by bioinformatics technology.Among HC,p SS,and UCMSC-Exos group,autophagosomes of CD4⁺T cells were detected by transmission electron microscope;autophagy related proteins were detected by western blot;and autophagy related genes were detected by RT-PCR.4.CD4⁺T cell cycle,apoptosis,and subsets were detected in p SS,UCMSC-Exos,autophagy inducer rapamycin（RAPA）,RAPA+UCMSC-Exos,autophagy inhibitor hydroxychloroquine（HCQ）,and HCQ+UCMSC-Exos group.Results:1.UCMSCs are plastic-adherent and have a fibrous shape.The cells highly express CD73,CD90,and CD105,low expression of CD45,CD34,and HLA-DR.They are able to differentiate into osteoblasts and adipocytes in vitro.UCMSC-Exos are vesicle-like structure,surrounded by a lipid bimolecular structure which have an average diameter of about 130 nm.NTA determined the diameter of the particles（144.4±44.0nm）,median diameter（137.7nm）,and their concentration(2.0×10¹¹particles/m L).The protein concentration detected by the BCA was 1.00μg/μL.Western blot analysis showed that the UCMSC-Exos express CD9,CD63,and TSG101,lack expression of calnexin;while the UCMSCs express CD9 and calnexin,lack expression of CD63 and TSG101.2.UCMSC-Exos exerts immunomodulatory effects in peripheral blood CD4⁺T cells in p SS patients.⑴The purity of CD4⁺T cells was 98.29%selected by immunomagnetic beads.⑵Compared with the HC group,the proliferation of CD4⁺T cells increased in the p SS group.Compared with the p SS group,the proliferation of CD4⁺T cells in UCMSC-Exos（60μg/m L,90μg/m L）groups reduced.The best concentration was90μg/m L,so it was used as the intervention concentration in subsequent experiments.⑶Compared with the HC group,flow cytometry detected that the proliferation of CD4⁺T cells increased,the G0/G1 phase cells ratio reduced,and the S phase cells ratio increased in the p SS group.Compared with the p SS group,the proliferation of CD4⁺T cells decreased,the proportion of the G0/G1 phase cells increased,and the proportion of the S phase cells decreased in the UCMSC-Exos group.There was no significant difference of G2/M phase cells during the three groups.⑷The early,late,and overall apoptosis of CD4⁺T cells increased in the p SS group compared with the HC group.Compared with the p SS group,the apoptosis of CD4⁺T cells in the UCMSC-Exos group reduced,and there was no significant difference between the HC or UCMSC-Exos group.⑸There was no significant difference in the ratio of Th1 cells among the HC,p SS,or UCMSC-Exos group.Compared with the HC group,the Th2 and Treg cells ratio decreased in the p SS group,while the proportion of Th17 cells,Th1/Th2,and Th17/Treg increased.Compared with the p SS group,the ratio of Th17 cells,Th1/Th2,and Th17/Treg declined,while the Treg cells increased in the UCMSC-Exos group,the Th2cells ratio was increasing,but it was not statistically different.⑹Compared with the HC group,the expression of IFN-γ,TNF-α,IL-2,IL-6,IL-17A,and IL-17F increased in the p SS group,while the TGF-βdecreased;the IL-10expression was decreasing,but it was not statistically different.Compared with the p SS group,the expression of IFN-γ,TNF-α,IL-6,IL-17A,and IL-17F decreased,while IL-10and TGF-βexpression increased;the IL-2 expression was decreasing,but it was not statistically different.As for IL-4 and IL-22 among the HC,p SS,or UCMSC-Exos group there were no significant difference.3.UCMSC-Exos plays regulation effects on the autophagy levels of peripheral blood CD4⁺T cells in p SS patients.⑴The transcriptome data of peripheral blood CD4⁺T cells from HC and p SS patients in the GEO database was analyzed by bioinformatics technique.A total of 244differentially expressed genes were obtained,and the KEGG enrichment analysis found that the genes were enriched in the autophagy pathway.⑵The autolysosome was observed in peripheral blood CD4⁺T cells in the p SS group,while the HC and UCMSC-Exos group did not have this phenomenon.Compared with the HC group,Beclin1,Atg5,LC3Ⅱ/LC3Ⅰ,Beclin1 m RNA,Atg5 m RNA,and LC3Ⅱm RNA expression increased in the p SS group.Compared with the p SS group,these indexes decreased in the UCMSC-Exos group,and there was no significant difference between the HC or UCMSC-Exos group.4.UCMSC-Exos exerts its immunomodulatory effects on the peripheral blood CD4⁺T cells of p SS patients through the autophagy pathway.⑴Compared with the p SS group,the CD4⁺T cells proliferation was decreased in the UCMSC-Exos,RAPA,and RAPA+UCMSC-Exos group;compared with the RAPA group,the CD4⁺T cells proliferation was further decreased in the RAPA+UCMSC-Exos group.Compared with the p SS group,the CD4⁺T cells proliferation decreased in the UCMSC-Exos,HCQ,and HCQ+UCMSC-Exos group;compared with the HCQ group,the CD4⁺T cells proliferation further decreased in the HCQ+UCMSC-Exos group.⑵Compared with the p SS group,the proportion of early apoptosis of CD4⁺T cells decreased in the UCMSC-Exos group and increased in the RAPA group;compared with the RAPA group,the early apoptosis of CD4⁺T cells decreased in the RAPA+UCMSC-Exos group.Compared with the p SS group,the proportion of early apoptosis of CD4⁺T cells decreased in the UCMSC-Exos,HCQ,and HCQ+UCMSC-Exos group;compared with the HCQ group,the early apoptosis of CD4⁺T cells further decreased in the HCQ+UCMSC-Exos group.⑶There was no significant difference in the Th1 and Th2 cells.Compared with the p SS group,the ratio of Th17 cells,Th17/Treg declined,and the Treg cells increased in the UCMSC-Exos groups.However,the proportion of Treg cells decreased in the RAPA group compared with the p SS group,the proportion of Th17 cells and Th17/Treg were increasing,but no statistical difference between the p SS or the RAPA group.Compared with the RAPA group,the Th17 cells,Th17/Treg decreased,and the Treg cells increased in the RAPA+UCMSC-Exos group.Compared with the p SS group,the Th17 cells,and Th17/Treg decreased,and Treg cells increased in the UCMSC-Exos,HCQ,and UCMSC-Exos group;compared with the HCQ group,the Th17 cells and Th17/Treg further decreased,and the Treg cells further increased in the HCQ+UCMSC-Exos group.Conclusion:1.The UCMSCs meet the MSCs standards defined by the International Society for Cellular Therapy.UCMSC-Exos comply with the features of the MSCs derived Exos.2.UCMSC-Exos inhibit excessive proliferation of peripheral blood CD4⁺T cells in p SS patients,and block it in G0/G1 phase,inhibit it in S phase;suppress excessive apoptosis of CD4⁺T cells;reduce Th17 cells ratio,elevation Treg cells ratio,restoration of Th1/Th2 and Th17/Treg balance;inhibit CD4⁺T cell-associated pro-inflammatory factor IFN-γ,TNF-α,IL-6,IL-17A,and IL-17F secretion,promote anti-inflammatory factor IL-10 and TGF-βsecretion,exhibit immune regulation of CD4⁺T cells.3.The autophagy levels have increased in peripheral blood CD4⁺T cells of p SS patients,and UCMSC-Exos reduce the autophagy levels.4.UCMSC-Exos regulate the peripheral blood CD4⁺T cells proliferation and early apoptosis in p SS patients through the autophagy pathway,inhibit Th17 cells differentiation,promote Treg cells differentiation,and restore Th17/Treg balance.