| Objective:In this study,we tried to detect the effects of olfactory ecto-mesenchymal stem cell-derived extracellular vesicles(OE-MSC-EVs)on the development of collagen-induced arthritis(CIA).The regulation of OE-MSC-EVs on IL-10~+B cells and their molecular mechanisms were further explored.Methods:(1)OE-MSCs were got from the olfactory epithelium tissue after culture and purification,the surface markers of OE-MSCs were analyzed by flow cytometry(FCM),including CD44,CD90,CD45,and CD11b.OE-MSC-EVs were extracted from the supernatants of OE-MSCs.A transmission electron microscope(TEM)was used to monitor the morphology of OE-MSC-EVs,and the sizes of OE-MSC-EVs were detected by nanoparticle tracking analysis(NTA).CD9,CD63,and calnexin in OE-MSC-EVs were detected by western blotting.(2)Collagen-induced arthritis was established by using DBA/1 mice,then OE-MSC-EVs were injected into CIA mice via tail vein.We recorded the swelling of the toes and monitored the activity of the joints,then we calculated the clinical scores and the incidence of each group.Mice were sacrificed at day 42 since the first immunization,joint sections were made for histopathological assessment.The percentages of IL-10~+B cells in spleens and popliteal lymph nodes were detected by FCM,also were the proportions of plasma cells in spleens and bone marrow.The levels of serum IL-10 were detected via enzyme-linked immunosorbent assay(ELISA),and the numbers of CⅡ-specific B cells in spleens were measured by enzyme-linked immunospot assay(ELISPOT).In vitro,splenic CD19~+B cells from mice were cultured under the regulatory B cell(Breg)induction condition with the addition of OE-MSC-EVs.The percentages of IL-10~+B cells were detected by FCM.The levels of IL-10 m RNA in B cells and IL-10 in the supernatants were measured by quantitative real-time PCR(qRT-PCR)and ELISA,respectively.(3)Specific small-interfere RNA was used to knock down the expression of Epstein-Barr virus-induced gene 3(EBI3)in OE-MSCs for extracting si EBI3-OE-MSC-EVs,si NC-OE-MSC-EVs were used as controls.The effects of OE-MSC-EV-contained EBI3 on the generation of IL-10~+B cells were explored via detecting the proportions of IL-10~+B cells in si EBI3-OE-MSC-EV or si NC-OE-MSC-EV-treated B cells.Gp130,the receptor of EBI3 in B cells was blocked by anti-gp130 antibodies,then the percentages of IL-10~+B cells in OE-MSC-EV-treated B cells were detected by FCM.The levels of phosphorylated Janus kinase2(p-JAK2)and phosphorylated signal transducer and activator of transcription 3(p-STAT3)in OE-MSC-EV-treated B cells were detected via western blotting.(4)Collagen-induced arthritis was established and si EBI3-OE-MSC-EVs were injected into CIA mice via tail vein,si NC-OE-MSC-EV-treated mice were used as controls.We recorded the swelling of the toes and monitored the activity of the joints,then we calculated the clinical scores and the incidence of each group.Mice were sacrificed at day 42,joint sections were made for histopathological assessment.The percentages of IL-10~+B cells in spleens and popliteal lymph nodes,the proportions of plasma cells in spleens and bone marrow were detected by FCM.The levels of serum IL-10 and the numbers of CⅡ-specific B cells were measured via ELISA and ELISPOT,respectively.Results:(1)OE-MSCs expressed CD44,CD90,but not CD45 and CD11b,which were identified with the surface markers for defining MSCs.The results of TEM showed that OE-MSC-EVs were a cup-shaped structure with double invagination of the plasma membrane.The diameter of OE-MSC-EVs was about 50-150 nm.OE-MSC-EVs were positive for CD9 and CD63,but negative for calnexin.These results were in line with the criteria for defining EVs.(2)Compared to the control mice,the clinical scores of OE-MSC-EV-treated mice were decreased,the development of disease was suppressed and the joint space was normal with fewer inflammatory cell infiltration.The levels of serum anti-CⅡantibodies in OE-MSC-EV-treated mice were decreased(p<0.05).Mice were sacrificed at day 42,FCM analysis revealed that the proportions of IL-10~+B cells in spleens and popliteal lymph nodes of OE-MSC-EV-treated mice were increased than those in the control mice(p<0.05,p<0.01),accompanied by the increased levels of serum IL-10(p<0.01).The proportions of plasma cells and the numbers of CⅡ-specific B cells in spleens of OE-MSC-EV-treated mice were decreased than those in the control mice(p<0.05,p<0.05).In vitro,OE-MSC-EVs promoted the generation of IL-10~+B cells(p<0.001),accompanied by the increased expression of IL-10m RNA(p<0.01)and the production of IL-10(p<0.001).(3)Compared to the EV-free concentrated supernatants,the same volume of OE-MSC-EVs lysis from the same numbers of cells contained more EBI3,and EBI3 was carried into OE-MSC-EVs by CD81.EBI3 was knocked down in OE-MSCs for extracting EVs,the percentages of IL-10~+B cells in si EBI3-OE-MSC-EV-treated B cells were decreased than those in the si NC-OE-MSC-EV-treated B cells(p<0.05).When the receptor of EBI3 in B cells was blocked by anti-gp130 antibodies,the percentages of IL-10~+B cells in OE-MSC-EV-treated B cells were decreased than those pretreated with Ig G(p<0.05).OE-MSC-EV treatments increased the levels of p-JAK2 and p-STAT3 in B cells,while the levels of p-JAK2 and p-STAT3 in si EBI3-OE-MSC-EV-treated B cells were decreased than those in the si NC-OE-MSC-EV-treated B cells.In addition,when B cells were pretreated with anti-gp130antibodies to block the receptor of EBI3,the levels of p-JAK2 and p-STAT3 in OE-MSC-EV-treated B cells were decreased than those pretreated with Ig G.Thus,OE-MSC-EV-contained EBI3 promoted the generation of IL-10~+B cells through gp130-mediated JAK2-STAT3 signaling.(4)Compared to the si NC-OE-MSC-EV-treated mice,the clinical scores of si EBI3-OE-MSC-EV-treated mice were increased,the development of disease was accelerated and the joint space was narrow with more inflammatory cell infiltration.The levels of serum anti-CⅡantibodies in si EBI3-OE-MSC-EV-treated mice were increased(p<0.001).Mice were sacrificed at day 42,FCM analysis revealed that the proportions of IL-10~+B cells in spleens and popliteal lymph nodes of si EBI3-OE-MSC-EV-treated mice were decreased than those in the si NC-OE-MSC-EV-treated mice(p<0.05,p<0.01),accompanied by the decreased levels of serum IL-10(p<0.05).The proportions of plasma cells and the numbers of CⅡ-specific B cells in spleens of si EBI3-OE-MSC-EV-treated mice were increased than those in the si NC-OE-MSC-EV-treated mice(p<0.05,p<0.05).Conclusions:(1)OE-MSC-EVs suppress the development of CIA via EBI3-induced generation of IL-10~+B cells.In vitro,OE-MSC-EVs directly promote the generation of IL-10~+B cells.(2)OE-MSC-EV-contained EBI3 promotes the generation of IL-10~+B cells via gp130-mediated JAK2-STAT3 signaling. |
PDF Full Text Request