G-MDSCs Exosomes Alleviated CIA Mouse By Regulating Breg Cells

ObjectiveThis study aimed to explore an isolation method of myeloid derived suppressor cells(MDSCs)shed exosomes(G-exosomes)and the effect of G-MDSC-exosomes(G-exosomes)on the mouse model of collagen-induced arthritis(CIA).Then,we preliminary explore the mechanism of IL-10 production。Methods(1)Fisrtly,we separated G-MDSC cells from the spleen of tumor-bearing mice.Then,isolated G-MDSCs were added into culture fluid up to 16 hours.Thirdly,we collected the supernatant of culture system.Next,we extracted G-exosomes by combination use of Membrane ultrafiltration and ExoQuick-TC.Finally,we determined the characteristic of G-exosomes by the means of Western blot and NTA(2)The first immunization with the emulsion of type II bovine collagen(C-II)and CFA,and the second immunization with the emulsion of type II bovine collagen(C-II)and FIA,we built CIA model.After injecting G-exosomes into mice via caudal vein,we then observed the swelling of the toes,recoreded the severity of joints and calculated mean arthritis index(MAI)of each mouse.At the days of 42,the mice were sacrificed for experimental study.We collected paw to make pathological section and observed the infiltration of inflammatory cells.We detected the percentage of Breg cells、Treg cells、plasma cell Tfh cells and Tfh cells in the lymph nodes and spleen through FCM,and the level of antibody from blood serun of mice were detected by ELISA.(3)We firstly sorted B cells from the spleen of normal mice and inoculated 2×10~6 cells on the 24-well plate.In the culture system,PBS,G-exosomes(60μg/m L)alone,LPS(10μg/mL),LPS and G-exosomes(60μg/m L)were separately added.we detected the percentage of Breg cells by FCM.Moreover,We treated LPS-activated B cells by the use of 60μg/m L G-exosomes.Then,we detected the level of IL-10mRNA by qRT-PCR.Finally,we detected the phosphorylation level of GSK-3βand CREB in B cells.Results(1)The purity of G-MDSCs is 96.4%.Then,we extracted G-exosomes from the culture supernatant of G-MDSCs.We observed the size of G-exosomes were round or oval,which ranged from 50 to 200nm.G-exosomes highly expressed CD63 without expressing calnexin while G-MDSCs expressed calnexin without expressing CD63.(2)In the group of G-exosomes treated CIA mouse model,the mouse was in good condition without the appearance of action obstacles.The result of the HE staining in toes showed that joint structure was relatively complete,the joint space was normal and the number of infiltrating lymphocytes were less.When come to the days of 42,the mice sacrificed for research.The percentage of Breg cells in lymph nodes were increased(P<0.05)while no change in spleen.The percentage of Treg cells in lymph nodes and spleen were no change.The percentage of plasma cells in lymph nodes were reduced(P<0.05)while no change in spleen.The percentage of Tfh cells in lymph nodes and spleen were redueced(P<0.05).The level of antibody in G-exosomes treated groups were reduced.(3)By analysis of FCM,we found compared with the group of LPS,the percentage of Breg cells in the group of G-exosomes alone showed a rising trend.The percentage of Breg cells treated by combination use of LPS and G-exosomes were more than the group of LPS treatment.The result of qRT-PCR demenstrated the high level of IL-10mRNA in LPS-activated B cells which were treated by G-exosomes.The result of Western blot showed that G-exosomes treatment increased the phosphorylation level of GSK-3βand CREB in B cells.Conclusion(1)G-exosomes alleviated the severity of CIA,which may be associated with the increase in Breg cells,decrease in plasma cells and Tfh cells and the level of antibody in serum of mice.(2)G-exosomes increased the percentage of Breg cells in vitro,which may be associated with increased phosphorylation level of GSK-3βand CREB in B cells.