Animals
All protocols used in the current study were approved by the Ethics and Research Committee of Shahid Beheshti University of Medical Sciences (Ethical approval code: IR.SBMU.MSP.REC.1400.142). C57BL/6 male mice (6–8 weeks old) were purchased from the Pasteur Institute of Iran.
Splenocytes Culture And Msc-ev Toxicity Assay
The MSC-EVs used in the present study had been isolated and characterized during a previous study [8]. Splenocytes were isolated from healthy C57BL/6 mice and were cultured in RPMI-1640 media (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). To remove the macrophages, the cells were first cultured for 24h in a treated flask (SPL, Korea). Consequently, the cells were incubated in plates coated with 2 µg/mL anti-CD3e antibody (eBioscience) and treated using 4 µg/m soluble anti-CD28 antibody (eBioscience).
To evaluate the probable cytotoxic effect of MSC-EV, the splenocytes were treated with 5, 15, 50, and 100 µg/mL of MSC-EV for 24h. Splenocytes with no MSC-EV treatment were used as a negative control. Moreover, a plate of cultured splenocytes was treated for 30 min using UV irradiation and incubated for 4 h at 37◦C as a positive control of apoptosis.
Flow Cytometry
To evaluate the viability of splenocytes, they were stained with annexin V-FITC (Biolegend,) and Propidium iodide (PI) (Biolegend). After 15 min incubation, the cells were evaluated using flow cytometry and the results were analyzed using FlowJo™ 7.6.1 software. The cells which were only positive for annexin V-FITC staining were considered apoptotic cells; the PI-positive cells were considered necrotic cells. Double negative cells represented live cells.
To estimate the effects of MSC-EV on the proliferation of splenocytes, the cells were first stained using CFSE (eBioscience). Afterward, the cells were treated with 1% PHA (Gibco, Thermo Fisher Scientific, Inc.) and 5, 15, and 50 µg/mL of MSC-EV; then were incubated for 5 days at 37◦ C. After the incubation time, the cells were stained using an anti-CD3 antibody conjugated with PE (Miltenyi Biotech) and evaluated using flow cytometry. Furthermore, splenocytes were activated using anti-CD3 and anti-CD28 antibodies and treated with 15 µg/mL of MCS-EV for 5 days; finally, the proliferation rate was evaluated using the protocol mentioned previously.
Treg cell's frequency among splenocytes was analyzed using a Treg detection kit (Miltenyi Biotech) and a BD FACSCalibur® instrument. The results were analyzed using the FlowJo™ 7.6.1 software.
Treg cells frequency among splenocytes was analyzed using Treg detection kit (CD4/CD25/FoxP3 (PE) (Miltenyi Biotech) according to the manufacturer’s instructions. The BD FACSCalibur® instrument was used to measure the regulatory T cells. The results were analyzed using FlowJo™ 7.6.1 software.
Cytokine Assay
The IFN-γ, IL-17A, and IL-10 cytokines levels were measured using the ELISA method (Mabtech, Sweden) according to the manufacturer's instructions. In addition, TGF-β level was measured following activation by 1 N HCl using the DuoSet ELISA kit according to the manufacturer's instructions (R&D Systems, UK).
Real-time PCR
The study of relative expression of genes was performed according to the previously reported study [8]. Briefly, the total RNA of the splenocytes was extracted (Favergen, Taiwan, then converted to cDNA using random primers (Yekta Tajhiz, Iran). The relative mRNA levels of Tbx21, Gata3, Foxp3, Rorc, Elf4 and β-actin as a housekeeping gene were evaluated using SYBR® Green Real-Time PCR (BioFact, Korea).
Statistical analysis
Statistical analyses were performed using SPSS (SPSS, Inc., Chicago, IL, USA, version 22). The data were expressed as mean ± standard error of the mean (SEM). The significant threshold was considered as P-value smaller than 0.05. The graphs were generated by Graph-Pad Prism (GraphPad Software, Inc., San Diego, CA version; 6).