Female C57/BL6 mice (8 weeks) were provided by the Animal Institute of Shandong University and housed individually in separate cages with free access to water and food in a room with an alternating 12-h light/dark cycle and controlled temperature (22–25 °C) and humidity (40–60%).
Female C57/BL6 mice (4–6 weeks) were used for DC isolation.
2.2 Cell culture and exosome extraction
MSCs is kindly donated by Central Laboratory of Liaocheng People’s Hospital. HucMSCs at passage 4 were cultured in exosome-free medium for 48 h. The conditioned medium was collected (approximately 60 ml), centrifuged at 300×𝑔 for 10 minutes and 16,500×𝑔 for 30 minutes, and filtered through a 0.22 𝜇m pore size filter to remove cell debris. At each of these steps, the pellet was discarded, and the supernatant was used for the following step. The final supernatant was then ultracentrifuged at 100,000 × g for 70 minutes to pellet the small vesicles corresponding to exosomes. The pellet was filtered through a 0.22 𝜇m pore size filter to eliminate contaminating proteins and centrifuged one final time at the same high speed. The purified exosome pellets were resuspended in PBS and prepared for identification. Finally, the exosomes were filtered through a 0.22 𝜇m pore size filter for functional experiments.
2.3 Characterization of hucMSCs-Exo
The morphologic characteristics of hucMSCs-Exo were observed by transmission electron microscopy (TEM). The number of hucMSCs-Exo was quantified using nanoparticle tracking analysis. The phenotypic profile of hucMSCs-Exo was determined by Western blotting with antibodies specific for CD9, CD63 and CD81. The protein content of hucMSCs-Exo was quantified with a BCA Protein Assay kit.
2.4 In vivo experiments
2.4.1 Animal model
All mice were randomly divided into 4 groups (𝑛= 6 mice/group): control group (Normal), IMQ group (IMQ), PBS-treated IMQ group (PBS + IMQ), and hucMSCs-Exo-treated IMQ group (hucMSCs-Exo + IMQ). All experimental procedures were conducted in accordance with the Animal Use and Care Committee of Shandong University. To establish the IMQ model, the C57BL/6 mice were treated with a daily topical dose (62.5 mg) of 5% IMQ cream (Aldara, 3M Health Care Limited) on the shaved dorsal skin for 6 days. On days 0, 2 and 4 (Fig. 2(A)), the mice in the hucMSCs-Exo + IMQ group were subcutaneously injected with exosomes (50 µg/mouse), and the mice in the PBS + IMQ group were subcutaneously injected with PBS (50 µl/mouse). All mice were euthanized on day 7 for analysis. The dorsal skin of the mice was sectioned, and part of the skin was stained with haematoxylin and eosin (H&E) for histological evaluation. Another part was used for Western blotting.
2.4.2 Histological evaluation
Skin sections from the dorsal surface of the mice were stained with H&E for histological evaluation. Psoriasiform epidermal hyperplasia was assessed microscopically in the sections as reported earlier [26–27].
2.4.3 Mouse skin tissue test
We collected skin tissue from the dorsal surface of each group of mice and used Western blotting to detect STAT3/p-STAT3, IL-17, IL-23, and CCL20, with GAPDH as the internal control.
2.5 In vitro experiments
2.5.1 Generation and differentiation of bone marrow-derived DCs (BM–DCs) and hucMSCs-Exo co-cultures
We observed whether hucMSCs-Exo affected the activation and maturation of DCs and the release of cytokines. DCs were obtained from the BM of normal 4- to 6-week-old female C57BL/6 mice according to a previously described protocol . BM cell pellets were resuspended in RPMI-1640 medium (GIBCO) supplemented with 10% FBS (GIBCO, Australia), 1% penicillin-streptomycin, 20 ng/ml rmGM-CSF (PeproTech, USA), and 20 ng/ml rmIL-4 (PeproTech, USA) at 1 × 106 cells/ml and transferred into a 6-well plate. On day 3 of culture, non-adherent cells were gently removed, and fresh culture medium was added. Fresh culture medium was added every 2 days thereafter to induce DC differentiation. For DC maturation, on day 7, non-adherent cells were collected, and DCs were washed, resuspended at 2 × 105 cells/ml in culture medium with 10 ng/ml rmTNF-α (PeproTech, USA), and seeded in a 24-well plate, with or without hucMSCs-Exo (2.5 µg/ml) co-cultured for another 24 h. The different groups of cells were collected to detect the phenotype of DCs by flow cytometry, and the cell supernatants were collected and centrifuged at 1000 × g for 20 minutes at 4 °C. Then, the supernatants were stored at − 80 °C and utilized for IL-23p40 ELISAs within one month.
2.5.2 HaCat cell and hucMSCs-Exo co-cultures
We aimed to observe whether hucMSCs-Exo affect inflammatory factors and chemokines in keratinocytes. HaCat cells (Zhongqiao Xinzhou,China) were used as the cell model system. When the cells had grown to nearly 90% confluency, they were seeded in 6-well flat-bottom plates (2 × 105 cells/ml). When the cells had grown to nearly 80% confluency, they were starved overnight in serum-free and antibiotic-free medium and were then stimulated with 100 ng/ml IL-17A (PeproTech, USA) for the indicated times with or without hucMSCs-Exo (2.5 µg/ml) for 24 h. The different groups of HaCat cells were harvested, and cellular proteins were extracted for Western blotting to detect STAT3/p-STAT3 and IL-17, with GAPDH as the internal control. The cell supernatants were collected and centrifuged at 1000 × g for 20 minutes at 4 °C. Then, the supernatants were stored at − 80 °C and utilized for IL-23 and CCL20 ELISAs within one month.
2.6. Western blot analysis
The different groups of HaCat cells and mouse skin tissue were harvested and lysed in modified RIPA buffer with combined protease and phosphatase inhibitors. Protein samples (30 µg/lane) were separated by 12% SDS-PAGE and transferred to PVDF membranes (Millipore, USA). Membranes were blocked with 5% BSA in TBST for 2 h at room temperature (RT) and incubated with primary rabbit monoclonal antibodies specific for CD9 (1:2000, Abcam, England), CD63 (1:1000, Abcam, England), CD81 (1:1000, Abcam, England), GAPDH (1:1000, Abcam, England), STAT3 (1:1000, Abcam, England), p-STAT3 (1:1000, Abcam, England), IL-17A (2 µg/ml, Abcam, England), IL-23 (1 µg/ml, Abcam, England), and CCL20 (0.2 µg/ml, Abcam, England) at 4 °C overnight. The next day, membranes were incubated with secondary antibodies for 1 h at room temperature and visualized using chemiluminescence. The data were analysed with imaging software.
2.7 Measurement of inflammatory cytokines by ELISA
Supernatants of in vitro cell cultures were analysed via ELISA using a commercially available ELISA kit. The protein level of IL-23p40 (Elabscience, China) was evaluated in the DC supernatant. The protein levels of IL-23p19 (Elabscience, China) and CCL20 (Elabscience, China) were evaluated in the HaCat cells supernatant. The absorbance at 450 nm was measured using an ELISA plate reader (Bio-Rad, USA).
2.8 Flow cytometric analysis
To analyse the DC phenotype, cytofluorimetric analysis of DCs was performed by triple-colour staining. Cells were incubated with the following fluorochrome-conjugated antibodies for 30 minutes at 4 °C: FITC-anti-mouse CD11c (N418, hamster IgG, eBioscience, USA), APC-anti-mouse CD86 (GL1, rat IgG2a, eBioscience, USA), and PE-anti-mouse MHCII (M5/114.15.2, rat IgG2b, eBioscience, USA). Then, cells were centrifuged at 300 × g for 5 minutes at room temperature, washed, resuspended in PBS, and analysed in a FACSCalibur equipped with Cell Quest software (BD Biosciences).
2.9 Statistical analysis
Data are presented as the means plus or minus the SD. All experiments were performed at least three times. Statistical analysis was performed using GraphPad Prism 7.00 (GraphPad Software, La Jolla, CA, USA.). P values of < 0.05 were considered statistically significant, and the level of significance is indicated as follows: *, P < 0.05 and **, P < 0.01.