| Background:Aplastic anemia (AA) is a bone marrow disorder that is characterized by pancytopenia with hypocellular bone marrow, and the most common symptoms are anemia, haemorrhage and fever or infections repeatedly. According to guidelines for the diagnosis and management of AA (British), AA is classified into severe AA (SAA), very SAA and non-SAA (NSAA). SAA and VSAA have the highest fatality if untreated, with the majority of deaths 1-2 years after diagnosis, from infections and/or hemorrhagic complications. Immunosuppressive therapy and hematopoietic stem cell transplantation are the front-line treatment of AA But it seems these therapy fail to achieve ideal curative effect. It calls for another effective treatment for AA.MSCs transplantation has shown promise for many diseases, for their properties of self-renewal and the ability to differentiate into adipogenic, chondrogenic and osteogenic cells. MSCs are found to exert an immunomodulating capacity, exhibiting low expression of class â…¡ major histocompatibility complex (MHC-II) and co-stimulatory molecules CD40, CD40L, CD80, CD86 on cell surface. In the past several decades, mountains of data reported the regeneration of MSCs because of their self-replicative and secretory capacity.As well, many studies have shown that MSCs could suppress the maturation, differentiation, secretion and migration of lymphocytes including T cells, B cells, DCs, and NK cells. These make them attracted to be applied in clinical for several autoimmunity diseases, such as arthritis, systemic lupus erythematosus nephritis(SLE) and graft-versus-host disease(GVHD).Despite the remarkable application prospect of MSCs, there are still many obstacles to develop large scale clinical application of MSC-based cell therapy, the limitation of passage culture size. These call for a substitution of MSCs. As reported, conducting the co-culture system for MSCs and PBMCs showed that MSCs induced T cells differentiation into Regulatory T cell(Treg)as well as decreasing the secretion of proinflammatoryfactors such as TNF-a and IFN-y, and increasing the anti-inflammatory cytokines including IDO, TGF-p, IL-10 and NO. The anti-inflammatory Treg capacities of Treg seems offer new immunotherapeutic options for immune disease.Interestingly, MSCs conditioned medium or some extractive from the supernatant were used to treat kidney injury or myocardial damage in mouse model, and the results show that MSC-derived extra-cellular vesicles (MSC-EVs) may contribute to the outcome of MSC-based therapies Among the discovered EV, exosome arise the most interest due to the first cases reports of severe GVHD treated with MSCs-derived EV, which showed the effect of MSCs could be reproduced by MSCs-derived exosome treatment.Exosomes are nanovesicles with structure and function of biomembrane released from the parent cells to transfer of the bioactivator to the target, which including 857 unique gene products (www.exocarta.org) and 150 microRNAs. The exosome shares a feature of a flotation density of 1.10-1.18g/ml in a sucrose as well as the surface markers such as CD63.Our study focused on the immunomodulatory properties of exosome and try to examine whether MSC-derived exosome posses the immunosuppressive capacity like their cell source.Objective:we focused on the immunomodulatory properties of exosome and try to examine whether MSC-derived exosome posses the immunosuppressive capacity like their cell source.Methods:(1) Bone marrow (10 ml) was obtained from the iliac crest of voluntary donors from whom informed consent had been obtained. Bone marrow MSCs were isolated by density gradient centrifugation combined with adherent culture method. (2) Peripheral Blood from AA patients to collect serum to detect the level of TNF-a and IFN-y, and the PBMCs to detect the percentage of T cell subset.(3) At 80% confluence, the MSC Growth Medium was replaced MSCs Medium-serum free with Exosome Precipitation Solution to precipitated exosome released by MSCs. (4)Put the cells of the three passages into the differential environment to induce their differentiation potential to adipocytes and osteoblasts. (5) Exosome were evaluated by transmission (TEM) and NANOSIGHT. The protein concentrations were detected by BCA kit and maker CD63 was identified by Western blot. (6) PBMCs from healthy donors stimulated with ConA were co-culture with or without MSC-derived exosome for 3 days. Annexin V/propidium iodide (PI) (BD Biosciences, Franklin Lakes, NJ, USA) staining was performed to determine cell death and apoptosis according to the instructions provided by the manufacturer. (7) PBMCs pre-labeled with CFSE were analyzed by flow cytometry according to the manufacturer’s protocol. Briefly,200,000 cells were resuspended in RPMI,10% FBS,1% L-glutamine, labeled for 10 minutes at 37 ℃, 5% CO2 with CFSE (5 μM). After 6 days, an analysis of the CFSE staining on PBMCs and CD3+ T cells proliferation was performed by flow cytometry.(8) Supernatants were harvested from cell cultures at 72 h and frozen at -80℃ until the assay was performed. Cytokine analysis was determined by commercially available ELISA kits for human IL-1β,TNF-a and TGF-β.(9) Flow cytometry was used to determine the expression of CTLA-4-PE, CD3-APC, CD4-PerCP-Cy5, CD25-APC, CD127-FITC, CD4/Thl/Thl7-complex-antibody in PBMCs. And CD4+/CD25+/CD1271ow cells were considered Treg cells. The levels of IFN--γ for TH1, IL-4 for TH2 and IL-17 for TH17 were determined.(10)We evaluated the biological activity of IDO by measuring the level of kynurenine in culture supernatants from lymphocyte in the presence of MSC-derived exosome as previous reportedStatistical methods:use SPSS 20.0 statistical software to analyze, measurement data are expressed in (x±s), multiple factors analysis using linear regression; More comparison using single factor analysis of variance between groups, when the variance is approximate F inspection Welch method. Together using Turkey multiple comparison when variance method, the variance is not using Dunnett’T3. Inspection level ofa= 0.05, double side inspection.Results:(1) The bone marrow MSCs were isolated by density gradient centrifugation combined with adherent separation and cultured to three passages to obtain an ample amount of the bone marrow MSCs with a uniform, fibroblast-like and adherent appearance. (2) The concentration of TNF-a and IFN-y of AA patinets are higher than healthy control. The detection of T cells subgroup shows Thl of AA patinets and control groub is (8.67±3.1 vs.4.42±0.82, p<0.05); Th2 is decreased in AApatients(3.2±1.24 vs.6.57±0.87, p<0.05). In addition, Thl7 is increased (7.1±2.3 vs 3.8±1.42,, p<0.05) along with a decline of Treg (1.56±0.8 vs.5.7±1.0, p<0.05). (3)Flow cytometry analyzed that the cells positively expressed CD29 (100%), CD44 (99.99%) CD73(99.97%), CD90(99.98%),CD105 (99.89%), and negatively expressed CD34 (0.40%)ã€CD45 (0.28%).(4) Transmission electron microscopy showed round shaped membranous vesicles. NANOSIGHT showed that the vesicles measuring from 30 to 250 nm in diameter with a mean size of 121 nm. MSCs derived exosome had immuno-reactivity with a specific antibody against CD63, CD9 and CD 81, the key exosomal membrane proteins.(5) the result showed that the concentration of IL-1β were 2021±126 vs.1796±84,1649±63,1403±102pg/ml (P<0.05), and the concentration of TNF-α were 439±68 vs.269±44,177±27,111±15 pg/ml (P< 0.05). For TGF-β, they were 530±67.8 vs.616.3±166.9,702±116,767±92, showed the significant difference in the 10μg and 20μg exosome group (P< 0.05). (6) Different concentration of exosomeas before were used to treat PBMCs, CFSE pre-labeled results showed that MSCs-derived exosome had no effect on the proliferation of PBMCs(51.37%±1.58% vs.50.67%±2.39%, 49.81%±3.18%,51.04%±3.07%, p=ns) as well as CD3+T cells (56.85%±3.27% vs. 55.1%±2.53%,56%±2.43%,55.03%±3.12%,p=ns).(7)We demonstrated that MSCs-derived exosome treatment increased the rate of Treg (CD4+/CD25+/CD1271ow) without a dose-dependent (10.49%±0.79%vs. 14.57±3.52,15.3±3.12,p=ns)(10.49%±0.79% vs.12.7±0.96, p<0.05) Moreover, CTLA-4expressionin Treg was elevated after stimulated with MSC-derived exosome. (2.2%±0.3% vs.4.43%±1.0%,5.5%±1.67%,7.0%±1.08%, p<0.05.(8) In the presence of MSC-derived exosome, there was a significant decrease of IFN-y produced by TH1(25.58%±1.88% vs.22.32%±1.69%,11.25%±1.19%,8.28%±1.49%, p<0.05) along with a increase of IL-4 in TH2 cells(5.49%±0.98%vs.7.15%±0.55%, 9.37%±1.02%,10.44%±1.25%, p<0.05). In addition, it suppressed the secretion IL-17 from TH17 cells (28.34±1.78% vs.26.78%±2.63%, 10.09%±1.46%,7.45%±1.05%, p<0.05). MSCs-derived exosome increased the apoptosis rate of PBMCs (17%±1.48% vs.22.53±4.56%,25.16±2.55%,28.18±1.57%, p<0.05), as well as CD3+T cells. (31.51%±3.67%vs.43.03±2.17%,46.65±2.40%, 51.74±2.84%, p<0.05).(9) Supernatants from MSCs-PBMCs co-culture system was the positive control. No IDO activity was detected in conditioned medium of PBMCs treated with MSCs-derived exosome, but a significantly difference for MSCs-PBMCs co-culture.Conclusion:The immune system of AA patients is disequilibrated. The inflammatory factor is high expression and the balance among T cell subset is breaking among Thl,Th2,Thl7 and Treg. MSCs-derived exosome suppressed the secretion of pro-inflammatory factor TNF-a and IL-1β but increased the concentration of anti-inflammatory factor TGF-β in culture medium. In addition, the results showed exosome may induce TH1 transfer into TH2 and decreased T cell differentiation into TH17. Moreover, it increased the ratio of Treg as well as the expression of Cytotoxic T lymphocyte-associated protein 4 (CTLA-4) in it. However, it showed no effects on the proliferation of PBMCs or CD3+ T cells. IDO showed no significant changes in PBMCs exposed to MSCs-derived exosome.It seems that exosome regulated immune activity, sharing the different manner with MSCs. |
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