2.1 Isolation and culture of mouse ADSCs
All animal procedures have been approved by the Institutional Animal Care and Use Committee of Nanfang Hospital, and all operations were performed in accordance with the National Health and Medical Research Council (People’s Republic of China) guidelines. All the surgical procedures were performed following the sterility principle. Female balb/c mice (6 weeks old, n=6) were purchased from Nanfang Hospital Animal Center (Guangzhou, China). After shaving, the mice were sacrificed and subcutaneous fat was harvested. About 1.5 g of fat was acquired from each mouse, and stored in a sterile 50 ml centrifuge tube. After removing red blood cell by washing 3 times with phosphate buffered saline (PBS), the isolated fat was cutting into small pieces. Then 0.2% type Ι collagenase (Sigma-Aldrich, St. Louis, Mo.) was used to digest the fat tissue for 45 minutes at 37°C with continuous stirring. After digestion, the stromal vascular fraction (SVF) was separated from the adipose tissue by centrifugation (200 ×g, 5 minutes), and resuspended with PBS, then filtered to remove large debris. The SVF suspension underwent an additional round of centrifugation (200 ×g,5 minutes) followed by resuspension in complete growth medium comprised of Dulbecco’s modified Eagle’s Medium (DMEM)-low glucose (GIBCO-BRL, Life Technologies, Gaithersburg, MD), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. The SVF suspension was then plated in T75 cell culture flasks at a density of approximately 5 × 105 cells/flask (P0) and incubated at 37℃ with 5% carbon dioxide. Medium was changed at a frequency of once every 3 days and cells were passaged at 90% confluence. ADSCs that were within the range of passage 3 to passage 5 were used for subcutaneous injection.
2.2 In vitro differentiation of ADSCs
To determine the multi-lineage differentiation potential of the ADSCs, they were induced into adipogenic, chondrogenic, and osteogenic lineages using the corresponding growth media, according to a previous study. The differentiated fat, cartilage, and bone cels were identified with Oil Red O, Alcian blue, and Alizarin red staining, respectively.
2.3 Murine Model of AD
Female balb/c mice (6 weeks of age, n = 24) were maintained at 25°C under pathogen-free conditions, with a 12-hour light-dark cycle, and were able to eat and drink freely. The mice were divided into 4 groups (n=6 per group) at random as follows: a. untreated group (normal control, NC); b. OVA-sensitized and PBS treated group (PBS group), c. OVA-sensitized and ADSCs-treated group (ADSCs group), and d. OVA-sensitized and cortisone cream-treated group (cortisone group).
A murine model of AD was prepared as described as previous study. Briefly, all mice except for those in the normal control group, were anesthetized with isoflurane and their dorsal skin was shaved, followed by the application of tape stripped six times with 3M tape (Tegaderm, Owens and Minor, Mechanicsville, VA, USA). Subsequently, each mouse was sensitized with OVA (100 µg), which was dissolved in 100 µl saline and added to a sterile patch. The patch was placed on the dorsal skin for 1 week and then removed. Two weeks later, the procedure described above was repeated for another 1 week. Each mouse was exposed to a total of three 1-week sensitizations, with 2-week intervals. (Figure 1a)
On days 28, 35 and 42 of sensitization and challenge by OVA, mice in the ADSCs group received subcutaneous administration of 1ml ADSCs suspension with the cell number of 1×106. The PBS group mice were subcutaneously injected with 1ml PBS at the same time points, as a negative control. In the cortisone group, 0.1 g of cortisone cream (Shufulin, Dinuo, Hunan, China) was daubed to dorsal skin of mice at the same time points, as a positive control. All mice were sacrificed at day 50, with half of the sensitized dorsal skin fixing in 10% formalin, and the the other half being stored at −80°C for further dectetion.
2.4 Measurement of clinical severity
The severity of dorsal skin lesions was assessed according to six symptoms: erythema/hemorrhage, oozing/crust, erosion/excoriation, swelling/oedema, lichenification and dryness). Scores of 0 to 3 (absent to severe) were given by two independent investigators. The sum of the scores was defined as the clinical severity (scale 0-18).
2.5 Histological examination
For histological analyses, skin samples were obtained from the dorsal skins of mice in the four groups at day 50 (24 h after the patch was removed). Skins that soaked in 10% formalin were prepared into paraffin samples and then cut into 4 µm sections for haematoxylin-eosin (H&E) staining or toluidine blue staining. A microscope (Olympus, Tokyo, Japan) was used to obtain photomicrographs (magnification, × 200). Ten H&E staining regions of each group were selected randomly for epidermal thickness analysis using ImageJ software. The infiltrated mast cells were assessed by toluidine blue (TB) staining (10 random regions of each group), and counted by ImageJ software.
2.6 Measurement of serum Immunoglobulin E (IgE)
On the day of sacrifice (day 50), the eyeballs of mice were removed to collect whole blood after anesthesia. After clotting at room temperature for 30 minutes, the blood samples were centrifuged at 1000 ×g for 10 minutes at 4℃. Serum was collected and preserved at −80°C until use. Serum IgE was detected using mouse IgE enzyme-linked immunosorbent assay (ELISA) kit (BioLegend, USA) according to the manufacturer’s instructions.
2.7 RNA Sequencing and Analyses
The total RNA was extracted from dorsal skin samples from the 4 groups (n=3 per group) using TRIzol (Invitrogen, USA) as shown in the manufacturer’s instructions. The quantification of RNAs were implemented using the Quant-IT RiboGreen (Invitrogen, USA), and qualification of RNAs were assessed through the Bioanalyzer 2100 system (Agilent, USA). After the quality inspection, mRNAs were purified, fragmented and converted to first strand complementary DNA (cDNA) with reverse transcriptase. The first cDNA strand was subsequently converted to second cDNA strand, and then end-repair, A-tailing, and adapter ligation were carried out. The AMPure XP system (Beckman Coulter, USA) was used to purify the library fragments and screen out 370-420 bp cDNA fragments. After the libraries were constructed, polymerase chain reaction (PCR) was performed, and the PCR products were purified and qualified using AMPure XP system (Beckman Coulter, USA) and the Agilent Bioanalyzer 2100 system, respectively. Then, a cluster of the index-coded samples were generated using TruSeq PE Cluster Kit v3-cBot-HS (Illumia, USA) on a cBot Cluster Generation System according to the manufacturer’s instructions. After that, Novaseq platform (Illumina, USA) was used to sequence the prepared libraries in a 150 bp paired-end to generate raw reads. Through the in-house perl scripts, the raw reads of fastq format were processed into clean reads, and then aligned to the reference genome sequence of Mus musculus (mm10) using the Hisat2 v2.0.5. Finally, the reads numbers of the genes in each group were calculated using FeatureCounts v1.5.0-p3.
For RNA sequencing analyses, we identified the differentially expressed genes (DEGs) between each group using DESeq2 R package (1.20.0) with a significancy threshold for a relative expression fold change ≤ − 1 or ≥ 1 (|log2FC| ≥ 1) and adjusted p (Padj) ≤ 0.05. The ClusterProfiler R software package was used for the Gene Ontology (GO) enrichment analysis and the Gene and Genome Kyoto Encyclopedia (KEGG) enrichment analysis. The GO terms and reactome pathways with Padj ≤ 0.05 were considered significantly enriched.
2.8 Isolation of naïve mouse CD4+ T Cells and differentiation of Th17 cells in vitro
The spleens and lymph nodes of balb/c female mice (5-10 weeks old) were obtained under aseptic conditions. These two tissues were then placed in PBS supplemented with 1% penicillin/streptomycin (PBS+), followed by grinding into a suspension using two slides. After filtration through a 120 µm pore size nylon mesh, the suspension was replenished using PBS+ and centrifuged at 475 ×g at 4℃ for 5 min. The lymph node cell pellet was resuspended with 2 ml PBS+. The splenic cell pellet was resuspended with ACK lysis buffer (1ml per spleen) for 1 min to lyse red blood cells, then 10 ml of PBS+ was added and centrifugated at 475 ×g at 4℃ for 5 min. The splenic cell pellet was resuspended with 10ml PBS+ again, and mixed with the lymph node cells suspension. After a final round of centrifugation at 475 ×g at 4℃ for 5 min, the combined cell pellet was used for CD4 enrichment.
T-cell enrichment columns (R&D Systems) and mouse CD4+ T cell isolation kits (Miltenyi Biotech,, CA, USA) were used for Naive CD4+ T cells purification through negative selection. For in vitro differentiation, the naive CD4+ T cells were then place in a 6-well plate coated with anti-CD3 and anti-CD28 (2 µg/mL) antibody. The cells was cultured in Roswell Park Memorial Institute (RPMI) -1640 (GIBCO, USA), supplemented with 10% FBS, 2 mmol / L-glutamine, 1% penicillin/streptomycin and Th17 polarizing cytokines (10 ng/mL TGF-β, 20 ng/mL IL-6, 10 µg/mL anti-IL-4 antibody, and 10 µg/mL anti-IFN-γ antibody). All cytokines and antibodies applied for differentiation of CD4+ T cell were purchased from BD Biosciences.
2.9 Co-culture of ADSCs and Th17 cells
The co-culture experiment consisted of three groups: the direct co-culture group (ADSCs + Th17 cells) as an experimental group, and monoculture of Th17 cells and ADSCs respectively as two control groups. For the co-culture group, 1×105 ADSCs were placed into a 6-well plate and cultured overnight in 2 ml DMEM-low glucose, supplemented with 10% FBS and 1% penicillin/streptomycin (DMEM-low glucose +), until they became adherent. Then, the medium was changed with 5 ml RPMI-1640 supplemented with 10% FBS, 2 mmol / L-glutamine and 1% penicillin/streptomycin (RPMI-1640 +). Th17 cells (2×106 cells/well) were added to reach a ratio of 20:1 with ADSCs. For the monoculture of Th17 cells, an equal amount of Th17 cells were added to a blank well and cultured with 5ml RPMI-1640 +. For the monoculture of ADSCs, 1×105 ADSCs were placed into a blank well and cultured with 5 ml DMEM-low glucose +. The ADSCs and Th17 cells of each group were collected after 72 h of culturing for further experiments.
2.10 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
The total RNA was extracted from the skin samples and cell samples with Trizol Reagent (Tiangen Biotech, Beijing, China.), then reverse transcripted into cDNA using a Reverse-Transcription kit (DBI Bioscience, Germany), according to the manufacturer’s instructions. FastStart Universal SYBR Green Master (atalogue no. 04913850001; Roche, Switzerland) was used to perform the qRT-PCR. The skin samples were tested for the following genes: interleukin (IL)-13, IL-4, IL-4 receptor (IL-4R), IL-17A, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Th17 cell samples were tested for IL-17A, IL-17F and RAR-related orphan receptor γT (RORγT), and ADSCs samples were tested for programed death ligand 1 (PD-L1), transforming growth factor-β (TGF-β) and prostaglandin E2 (PGE2). The primer-probe sequences of the genes above were shown Supplementary Table 1. Expression levels were determined by the 2−ΔΔCt method, and β-actin was used as an endogenous reference gene.
2.11 Statistical Analysis
For RNA sequencing, statistical analysis was implemented by R software (www.R-project.org), and R packages were acquired on Bioconductor (www.bioconductor.org). Regarding the experimental data, they were expressed as mean ± standard deviation (SD), and analyzed using SPSS Version 21.0 (IBM Corp., USA.). Comparisons was performed using one-way ANOVA for multiple groups, and t tests two groups. P≤ 0.05 were considered statistically significant.