Female MRL/lpr mice(stock number 000485 and induced from Jackson laboratory, USA) were purchased from Shanghai SLAC Laboratory Animal Co. Ltd. and raised under the specific-pathogen-free facilities in the Laboratory Animal Center of the Second Xiangya Hospital, Central South University until they were used. All experimental applications were carried out in accordance with ARRIVE guidelines.The MRL/lpr mice used in this experiment is a classic animal model of SLE. The onset of autoimmune diseases in MRL/lpr mice was monitored by measuring proteinuria (100 mg/dl)[1, 16], and MRL/lpr mice have symptoms such as lymphoproliferation and skin damage, which are in line with the description in Jackson Laboratory. Please see the Jackson labratory website for details: https://www.jax.org/jax-mice-and-services. Female MRL/lpr mice were euthanized at age of 20 weeks and mononuclear cells were isolated from the spleen for in vitro experiments. Splenic mononuclear cells from the same mouse were separated into four groups(2×106cell/group) as phosphate-buffered saline (PBS)control group, MSC group, 25µg/ml hUCMSC-EVs group ,50µg/ml hUCMSC-EVs group. the Ethical approval was obtained from the Institutional Animal Care and Use Committee of the Second Xiangya Hospital, Central South University (Approval No. 2020344) for the study, animal experimentation guidelines were exactly followed.
hUCMSC-EVs extraction and identification
The hUCMSCs were subcultured and expanded by the primary hUCMSCs isolated from healthy human umbilical cord wharton’s jelly .The 3-8 passages of hUCMSCs were cultured in serum-free complete medium(Clin-Biotechnology, China). When the cells grew to 80%-90% fusion, the surface of the cells was cleaned with PBS solution, repeated twice, and then basic medium (Clin-Biotechnology, China) was added for 3 days of starvation culture. Next, the supernatant was collected and hUCMSC-EVs in supernatant was isolated by ultracentrifugation, specific steps were as follows: Centrifugation at 300g for 15min to remove residual cells; centrifugation was performed at 2,000g for 15min to remove large cell fragments; small fragments and apoptotic bodies were removed by centrifugation at 10, 000g for 30min. After that, the centrifuged supernatant was filtered with a needle filter of 0.22µm to remove smaller fragments and larger vesicles. Ultra-15 10-KDA MWCO test tubes (Merck Millipore, Ireland) were centrifugated at 4,000g for 30min to concentrate the supernatant after the above treatment and the concentrated supernatant was collected. Finally, centrifugation was performed at 120,000g for 2 hours (SW 41 Ti 41,000 RPM, Beckman Optima XPN-100, USA). The prepared EVs were ultimately resuspended in appropriate amount of PBS. All centrifugation processes were carried out under 4℃. The total protein concentration of EVs was measured by BCA protein quantitation method (Thermo Scientific, USA).
The purified EVs were diluted 10,000 times, and Nanoparticle Tracking Analysis (NTA) (ZetaView, Germany) was used to measure the particle size distribution of EVs. 10µl EVs were dropped onto the ultrathin carbon film copper mesh, then EVs were treated with glutaraldehyde, uranium oxalate, and methylcellulose -UA in turn, and after drying, the morphology of EVs was observed by the transmission electron microscope (Hitachi, Japan).
To detect EVs specific markers and negative markers, the samples were incubated with the antibodies of anti-CD9, anti-TSG101, and anti-Calnexin (Abcam,UK), and then incubated with enzyme-labeled anti-rabbit IgG antibody (Sigma, USA), and finally a western blot developer machine was used.
Mononuclear cells were co-cultured with hUCMSCs or hUCMSC-EVs
Spleen mononuclear cells were isolated by density gradient centrifugation with mouse ficoll according to the manufacturer's instructions. Briefly, female MRL/lpr mice for 20 weeks were euthanized and the spleen was removed aseptically. Spleen grinding liquid were layered over ficoll-paque PREMIUM 1.084g/ml(GE, catalog No. 17544602-1, USA) and centrifuged at 400g for 30-40min at 18-20℃. The mononuclear cells were collected at the interface, then the cells were mixed with 3 times the volume of PBS to wash twice by centrifugation at 60-100g for 10min at 18-20℃. Then the cells were resuspended in RPMI1640(Gibco, USA) supplemented with 10% fatal bovine serum(Gibco, USA). The final concentration of mononuclear cells was adjusted to 2×106/ml. hUCMSCs were pretreated with 10µg/ml mitomycin C(Sigma, USA) and inoculated at a density of 2×105/well into a 12-well plate (Corning, USA) and added with RPMI1640 medium (Gibco, USA) containing 10% fetal bovine serum, 100U/ml penicillin as well as 100 mg/ml streptomycin overnight. On the second day, 1ml suspension of 2×106/ml mouse splenic mononuclear cells was added into the well with hUCMSCs, and the ratio of hUCMSCs and splenic mononuclear cells =1:10. In the experimental well of hUCMSC-EVs immune response to T cells, to activate T cells under Th0 condition, 1.0µg/ml anti-CD3e and anti-CD28 were added to the co-culture mentioned above. In order to activate B cells, 6µg/ml ODN 1826 B cell activators were added to the above co-culture system. Then the final volume of each well was adjusted to 1.2ml with PBS and hUCMSCs were co-cultured with mononuclear cells for 3 days. In the hUCMSC-EVs group, fresh hUCMSC-EVs preparations of 25µg/ml or 50µg/ml were added to study the effect of hUCMSC-EVs in the same procedure and time. As 25µg/ml hUCMSC-EVs had no significant regulation effect on T cells, only 50µg/ml hUCMSC-EVs were used to study the regulation effect of hUCMSC-EVs on B cells. The control group was treated with PBS.
CD4+T cell subsets and CD19+B cells were detected by a flow cytometer
Suspended mononuclear cells were recovered from the culture medium, washed with PBS, and the proportions of CD4+T cell subsets and CD19+B cells were detected by a flow cytometer (Cytek, USA). Cells were stained with anti-mouse antibodies that are fluorescent-conjugated for CD4 (BD Pharmingen, USA), IFN-γ (BD Pharmingen, USA), IL-4 (BD Pharmingen, USA), IL-17a (BD Pharmingen, USA), CD25 (BD Pharmingen, USA), Foxp3 (BD Pharmingen, USA), CD185 (BD Pharmingen, USA), CD19 (BD Pharmingen, USA). CD4+IFN-γ+ T cells , CD4+IL-4+T cells ,CD4+IL-17+T cells, CD4+CD25+Foxp3+T cells and CD4+CD185+T cells were thought as Th1, Th2, Th17, Treg and Tfh cells, respectively. For intracellular staining, the cells were treated with cytokine stimulation blockers (BD Pharmingen, USA). The Flowjo10.5.3 program was used to analyze the data.
Cytokines were measured by ELISA
Cytokine levels in the co-culture supernatant were measured by commercial ELISA kits. Mice were tested for IFN-γ (USCN, catalog No. SEA049Mu, China), IL-4 (elabscience, catalog No. E-EL-M0043c, China), IL-17a (elabscience, catalog No. E-EL-M0043c, China), TGF-β1 (USCN, catalog No. SEA124Mu, China), and IL-10 (USCN, catalog No. SEA056Mu, China)in accordance with the manufacturer's instructions.
We used the One-way analysis of variance for statistical analysis for parametric dataand the Kruskal-Wallis test for non-parametric data. We performed statistical analyses and maps with GraphPad Prism 5 software and considered a P value less than 0.05 as significant. Data are shown as means±standard error of mean ( means±SEM).