Adipose Derived Stem Cells Ameliorate Atopic Dermatitis by Down-regulating IL-17 Secretion of Th17 Cells in an Ovalbumin-induced Mouse Model


 Background: Mesenchymal stem cells (MSCs) has therapeutic potential for Atopic dermatitis (AD) due to their immunoregulatory effects. However, the underlying mechanisms for therapeutic efficacy of ADSCs on AD is still unclear. Objectives: We sought to investigate the therapeutic effect and mechanisms of adipose-derived stem cells (ADSCs) on AD using an ovalbumin-induced AD mouse model. Methods: AD mice were treated with mice-derived ADSCs, cortisone, or PBS. The therapeutic effect was determined via gross examination and additional in vitro assays using skin samples and blood. To further explore the underlying mechanisms, RNA sequencing analyses and co-culture assays were conducted.Results: ADSCs treatment attenuated the symptoms associated with AD, decreased the serum IgE level and mast cells infiltration. Tissue levels of T-cell relevant pro-inflammatory cytokine production, including IL-4R and IL-17A, were suppressed in both ADSCs and cortisone treatment groups. Genomics and bioinformatics analyses demonstrated a significant enrichment of certain of inflammation related pathway in the down regulated genes after the application of ADSCs and cortisone, specifically the IL-17 signaling pathway. Co-culture experiments revealed that ADSCs significantly suppressed the expression of pro-inflammation cytokines IL-17A and RORγT, as well as the proliferation of Th17 cells. Moreover, the expression of PD-L1, TGF-β, PGE2 was significantly upregulated in the co-cultured ADSCs compare to monocultured ADSCs. Conclusion: Taken together, our data may demonstrate that ADSCs ameliorate OVA- induced AD in mice by down-regulating IL-17 secretion of Th17 cells.


Background
Atopic dermatitis (AD) is a chronically recurrent skin disease that is characterized by a disruption of the epidermal barrie, immune dysregulation, and skin ora changes. It affects up to 20% of children and 10% of adults worldwide, and it signi cantly reduces the quality of life for affected individuals [1,2]. Since many years, it has been considered that the pathogenesis and development of AD are mainly due to the dysregulation of the helper T (Th) cell immune responses. Therefore, treatments that reduce the helpermediated immune response have been the mainstream therapeutic methods for AD [3]. These treatments include non-speci c anti-in ammatory and immunosuppressants drugs, such as corticosteroids, calcineurin inhibitors, leukotriene receptor antagonists, and antihistamines. However, these drugs only provide temporary and limited relief of symptoms and are often associated with adverse effects and drug resistance in long-term treatment [4,5]. In recent years, signi cant progress has been made toward increasing our understanding of the pathogenesis of AD. Consequently, the development of novel therapies that are safe and e cacious for AD has been intensely pursued.
Mesenchymal stem cells (MSCs) have been a promising cell-based treatment option for AD due to their unique tissue regenerative capacity and immunomodulatory ability [6,7]. In comparison to other types of MSCs, adipose-derived stem cells (ADSCs) have several advantages, since adipose tissue can be obtained easily through liposuction with a small incision, and ADSCs can be isolated in large amounts by relatively simple procedure. Although a few studies have demonstrated the use of ADSCs for the attenuation of AD [8][9][10], the mechanisms involved have yet to be elucidated. Recent studies demonstrated that in addition to classical Th2, other subsets of helper T cells also contribute to the pathogenesis of AD, such as Th1, Th17 and Th22 [11,12]. In this study, we investigated the therapeutic effect of autologous ADSCs on skin lesions in an ovalbumin (OVA)-induced AD mouse model. Furthermore, to verify the mechanistic mode of action, we employed transcriptome pro ling and pathway analyses to elucidate the therapeutic mechanism of ADSCs. 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 sacri ced 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 ltered 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 modi ed 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 asks at a density of approximately 5 × 10 5 cells/ ask (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% con uence. ADSCs that were within the range of passage 3 to passage 5 were used for subcutaneous injection.

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 [13]. The differentiated fat, cartilage, and bone cels were identi ed with Oil Red O, Alcian blue, and Alizarin red staining, respectively.

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. OVAsensitized and cortisone cream-treated group (cortisone group).
A murine model of AD was prepared as described as previous study [14]. Brie y, all mice except for those in the normal control group, were anesthetized with iso urane 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 1×10 6 . 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 sacri ced at day 50, with half of the sensitized dorsal skin xing in 10% formalin, and the the other half being stored at −80°C for further dectetion.

Measurement of clinical severity
The severity of dorsal skin lesions was assessed according to six symptoms: erythema/hemorrhage, oozing/crust, erosion/excoriation, swelling/oedema, licheni cation and dryness). Scores of 0 to 3 (absent to severe) were given by two independent investigators. The sum of the scores was de ned as the clinical severity (scale 0-18).

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 para n 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 (magni cation, × 200). Ten H&E staining regions of each group were selected randomly for epidermal thickness analysis using ImageJ software. The in ltrated mast cells were assessed by toluidine blue (TB) staining (10 random regions of each group), and counted by ImageJ software.

Measurement of serum Immunoglobulin E (IgE)
On the day of sacri ce (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.

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 quanti cation of RNAs were implemented using the Quant-IT RiboGreen (Invitrogen, USA), and quali cation of RNAs were assessed through the Bioanalyzer 2100 system (Agilent, USA). After the quality inspection, mRNAs were puri ed, fragmented and converted to rst strand complementary DNA (cDNA) with reverse transcriptase. The rst 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 puri ed and quali ed 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 identi ed the differentially expressed genes (DEGs) between each group using DESeq2 R package (1.20.0) with a signi cancy threshold for a relative expression fold change ≤ − 1 or ≥ 1 (|log2FC| ≥ 1) and adjusted p (Padj) ≤ 0.05. The ClusterPro ler 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 signi cantly enriched.

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 ltration 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 nal round of centrifugation at 475 ×g at 4℃ for 5 min, the combined cell pellet was used for CD4 enrichment.

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, 1×10 5 ADSCs were placed into a 6-well plate and cultured overnight in 2 ml DMEMlow 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 (2×10 6 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.

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 signi cant.

The cultured ADSCs exhibited the potential of multilineage differentiation
ADSCs were rst subjected to chondrogenic, osteogenic and adipogenic differentiation condition respectively in vitro to test their multi-lineage differentiation potential. As shown in Supplementary Figure  1, the cells were successfully stained with Alcian blue, Alizarin red and Oil Red O respectively, indicating that the ADSCs process capacity of multi-lineage differentiation.

Subcutaneous administration of ADSCs ameliorates OVA-induced AD-like skin lesions
During the OVA-sensitized period, AD-like lesions were successfully induced on the dorsal skin of mice in each group after OVA application. ADSCs (1×10 6 ), PBS, and cortisone were administrated to OVA-induced AD mice on days 28, 35 and 42 for 1 week, and the dorsal skin was assessed to evaluate the therapeutic e cacy of each treatment on day 50. In the PBS group, serious erythema and dryness was observed on the dorsal skin, accompanied by licheni cation and excoriation. In comparison with the PBS group, skin lesions were signi cantly improved in mice that received either ADSCs or cortisone treatment (Figure 1b). Speci cally, the clinical severity of the skin lesions in the PBS group (13.11 ± 0.2606), was signi cantly lower in groups treated with ADSCs (9.500 ± 0.4629, P < 0.0001) and cortisone (9.875 ± 0.4407, P < 0.001) on day 50 (Figure 1c).
In comparison to the normal control (16.51 ± 0.4562 µm), the epidermis of dorsal skin in PBS-treated mice (66.80 ± 1.415 µm, P < 0.0001) exhibited remarkable thickening, whereas the epidermal thickness was decreased after administration of ADSCs (29.97 ± 1.000 µm, P < 0.0001) and cortisone (36.39 ± 2.727 µm, P < 0.0001). Furthermore, epidermal thickness of the ADSC-treated group is slightly lower than the cortisone-treated group (P=0.0081) (Figure 2a, c). As shown in Figure 2b and Figure 2d, the number of in ltrated mast cell was signi cantly reduced in mice treated with ADSCs (45.11 ± 2.232 cells, P < 0.0001) and cortisone (43.09 ± 1.810 cells, P < 0.0001) when compared to the PBS-treated group (66.60 ± 3.069 cells). Similarly, the level of serum IgE was signi cantly increased in PBS-treated mice on day 50, whereas treatment with ADSCs and cortisone markedly reduced the level of IgE, which parallels the changes observed in the in ltration of mast cells (Figure 2e).

ADSCs decrease the expression of IL-4R and IL-17
We next determined whether ADSCs could in uence the pro-in ammatory cytokine that produced in OVAinduced AD mice. Since the pathogenesis of AD involves the dysfunction of multiple helper T cell types, we performed qRT-PCR to detected the pro-in ammatory cytokines associated with Th1 (TNF-α, IFN-γ), Th2 (IL-13, IL-4, IL-4R), and Th17 (IL-17A) cells. As shown in Figure 3

ADSCs normalize the alteration of IL-17 signaling pathway in OVA-induced skin lesions
To decipher the mechanisms that ADSCs improve the skin lesion, skin lesions from normal control, PBS-, ADSCs-, and cortisone-treated mice were implemented high-throughput RNA sequencing. The result of principal component analysis (PCA) showed that ADSCs group was clearly separated from the normal control, PBS, or cortisone groups (Figure 4a), suggesting that ADSCs regulate a different gene expression program during AD recovery.
By a threshold minimum of |log2FC| ≥ 1 (Padj ≤ 0.05), a total of 4771 DEGs was identi ed in the skin specimens of the four groups. All DEGs were organized into a heatmap ( Figure 4b). As shown in Figure   4c, there are 2965 DEGs between the PBS and normal control groups, 2641 DEGs between the ADSCs and PBS groups, 933 DEGs between the cortisone and PBS groups, and 1204 DEGs between the ADSCs and cortisone groups.
We then performed GO and KEGG analyses on up-regulated DEGs between the PBS-treated and normal control groups. GO analysis showed that the top 10 terms associated with biological process (BP), cellular component (CC), and molecular function (MF) were mostly related to epidermis hyperplasia, leukocyte migration, and cytokine related activities (Figure 5a). These results correlate with epidermal thickening and an elevated in ammatory response after OVA sensitization, which is consistent with the histological analyses (Figure 2). In comparison with the normal control group, the result of KEGG analysis showed a signi cant change of the IL-17 signaling pathway in PBS group, illustrating that Th17 cells were remarkably activated after OVA sensitization (Figure 5b).
Subsequently, GO and KEGG analyses were conducted on the down-regulated DEGs identi ed between the of ADSCs and PBS groups, as well as the cortisone and PBS groups. The result of GO analysis revealed that the top 10 BP, CC, and MF terms were primarily related to leukocyte activities, bacterium response, and cytokine related activities (Figure 5c), which are correlated with alleviation of in ammatory responses after ADSCs and cortisone treatment. As for KEGG analysis, a signi cant change in the IL-17 signaling pathway was found between the ADSCs and PBS group (ranked ninth) as well as the cortisone and PBS groups (ranked second) (Figure 5d), which suggests that ADSCs and cortisone effectively suppress Th17 cell activation similarly.

ADSCs suppress the proliferation and activation of Th17 cell in vitro
To further identify the mechanism associated with ADSCs-derived inhibition of Th17 cells activation, a direct co-culture of ADSCs and mature Th17 cells was performed. The formation of T-cell receptor (TCR)activated T-cell clusters were observed to evaluate the Th17 cell suppression capability of ADSCs. As shown in Figure 6a, after 72 hours of culturing, less TCR-activated T-cell clusters were formed in the coculture group compared to the monocultured Th17 cells. In addition, the monocultured Th17 cells have proliferated to an amount of 3.774 ± 0.05802 ×10 6 in 72 hours, whereas only 0.6310 ± 0.06552 ×10 6 Th17 cells were counted in the co-culture group, even less than the initial cell number (2×10 6 ) ( Figure 6b).
Correspondingly, the expression levels of IL-17A (Figure 6c), as well as RAR-related orphan receptor γT (RORγT) (Figure 6e), the master transcription factor of Th17 cell differentiation, were reduced under the co-culture condition. The expression of IL-17F was also reduced in the co-culture group, but there was no statistical signi cance ( Figure 6d). Figure 7a, there was no obvious morphological difference between the co-cultured and monocultured ADSCs. Whereas, in comparison to monocultured ADSCs, the expression of programed death ligand 1 (PD-L1), transforming growth factor-β (TGF-β) and prostaglandin E2 (PGE2) were upregulated in co-cultured ADSCs (Figure 7b to 7d), indicating that these ADSCs derived protein and factors might have roles in the suppression of Th17 cells.

Discussion
AD is an allergic in ammatory skin disease characterized by (1) disrupted epidermal barrier function [15], (2) dysregulated immune responses, (3) pruritic skin lesions. Stem cells possess immunomodulatory properties and may serve as a promising therapeutic candidate for AD. In the present study, we demonstrate that subcutaneous administration of ADSCs can ameliorate OVA-induced murine AD, probably through down regulating the IL-17 secretion by Th17 Cells.
The delivery methods of MSCs in AD models mainly include subcutaneous and intravenous injections [16,17]. Although previous study has reported that ADSCs have homing effects to the injured tissue after intravenous injection [18], other study indicates that this administration route would lead to low viability of stem cells because of pulmonary interception [19]. In addition, some studies demonstrated that local injection of ADSCs have higher viability than intravenous injection [19] [20]. Therefore, we administrated ADSCs directly to the lesion sites utilizing subcutaneous injection. Our results indicated that subcutaneous administration of ADSCs successfully relieved the symptoms, and reduced thickening of the skin.
Mast cells, which are mainly recruited by IgE [21], are considered to play an signi cant role in the persistence of AD symptoms [22]. Mashiko et al reported that the inhibition of mast cell activities leads to the amelioration of AD, whereas increased mast cell activities aggravated AD symptoms [23]. To evaluate whether ADSCs in uence the recruitment of mast cells to AD lesion site, we determined the amount of skin mast cells and serum IgE was present in our mouse model. Our data showed that the number of in ltrated mast cells and serum IgE levels were signi cantly decreased after ADSCs treatment, suggesting an inhibitory effect on mast cell recruitment and local allergic responses.
Although AD is generally considered as a Th2 dominant disease, recent studies reported that other Th cell subsets might be involved in the pathogenesis of AD, such as Th1 and Th17 [24][25][26]. Therefore, we performed qRT-PCR to assess whether ADSCs could in uence the expression of Th1-, Th2-and Th17de ning cytokines. Our data showed that IL-4, IL-4R, IL-13, IFN-γ, TNF-α, and IL-17 were signi cantly increased in lesion sites after OVA induction. In vitro and in vivo studies have shown that the immunosuppressive ability of MSCs depends on preliminary activation by immune cells through the secretion of Th1 associated cytokine IFNγ [27,28] alone, or together with TNFα [29]. The increase of IFN-γ and TNF-α in our present study may attribute to the activation of ADSCs. Type 2 in ammation mediated by IL-4 and IL-13 is known to play a central role in AD [30]. Moreover, the activation of IL-4/IL-13/IL-4R axis promotes the differentiation of Th2 cells, which subsequently mediates the pro-allergic adaptive immune response [31,32]. As a hub for IL-4 and IL-13 acting on downstream in ammatory cells, the IL-4R complex activates multiple signaling pathways that regulate allergic responses, which promote multiple processes in AD immune dysregulation [33]. Interestingly, ADSCs treatment downregulated IL-4R rather than IL-4 and IL-13, indicating that ADSCs may inhibit Th2 in ammation by suppressing the IL-4R expression in various downstream in ammatory cells, but not by decreasing the production of Th2-related cytokines directly.
However, the underlying mechanisms remain to be clari ed.
Recent studies have indicated that Th17 cells, and the associated cytokine IL-17, are profoundly involved in the pathogenesis of certain skin disorders, such as psoriasis [34,35]. In addition, IL-17 has been found to participate in allergen-speci c immune responses [36,37]and exhibits increased expression in skin trauma and skin barrier dysfunction-associated diseases [38]. IL-17 has been reported to regulate epidermal cell proliferation conditions and skin thickening [39]. Considering the proin ammatory properties of IL-17, it is interesting to study if, and how Th17 cells are involved in the pathogenesis of AD. Our qRT-PCR results revealed that the expression of IL-17A in skin samples was effectively inhibited in ADSC-treated mice. Moreover, RNA-seq analyses identi ed a remarkable transcriptomic down-regulation in IL-17 signaling pathways in ADSC-treated mice, indicating that the therapeutic effect of ADSCs on AD may be attributed to down-regulating IL-17 secretion by Th17 Cells.
Although the mechanisms behind the immunosuppressive effect of MSCs on Th17 cells have not been fully elucidated, several processes were recently reported, including a dependence on cell-cell contactbased inhibition and the production of soluble factors [40]. In this study, co-culture of ADSCs and Th17 cells was performed to further investigate the immunoregulatory effect of MSCs on Th17 cells. The suppression of IL-17A and IL-17F expression were highly comparable to the T-cell cluster results, suggested that ADSCs exert a stronger immunosuppressive effect on Th17 cells. PD-L1 is an expressed on the cytomembrane of MSCs and plays an important role in the negative regulation of immune responses [41] [42,43]. Our data revealed that PD-L1 accumulated signi cantly in the co-culture system, indicating that the inhibition of Th17 cells by ADSCs may be partially attributed to the cellular contact of MSCS through PD-L1. Cytokines secreted by MSCs were also reported to have an immunosuppressive effect on immune cell populations. As MSC-derived PGE2 and TGF-β have been reported to play an signi cant role in regulating multiple immune suppressive effects [44][45], they were quanti ed in our coculture assay. The results revealed that the co-culture group contained higher levels of PGE2 and TGF-β, in comparison with cells cultured alone, which may explain the immunosuppressive effect of ADSCs on Th17 cells. Collectively, ADSCs exerted an immunosuppressive effect on Th17 cells, which may be attributed to both cell-cell contact inhibition and immunosuppressive cytokines secretion, but further studies are required to clarify the exact mechanisms.
Recent researchs indicated that AD is a highly heterogenous in ammatory skin disease characterized by activation of multiple immune reaction [46][47][48]. Therapeutic options for AD still remain limited, in part because currently available models do not adequately capture all immune and barrier features of the human AD skin [49][50][51], being the OVA-induced and NC/Nga mice that best represent it [52]. There remains diversity in immune types between the experimental AD studies and these differences may have resulted from the use of different animal models, model methods and examination methods, etc [53][54][55]. Although studies haved highlighted a critical role for the Th2 signals in AD[56-58], activation of Th17 and Th22 pathway was also reported to play an important role in the development of AD [59]. Especially, recent studies suggested an increased Th17 and Th22 activation in Asian AD and the percentage of Th17 signal level was signi cantly correlated with the severity of AD[60-62]. Therefore, our study may provide some new insights into the understanding and therapeutic approach of Th17 signalling of AD.
This study had some limitations. First, this study applied an OVA-induced AD mice model. Use of reliable animal models (i.e. xenograft or bioengineered skin-humanized mouse) that can capture more AD features would be desirable. Second, recent study demonstrated that the different MSC lines had different immumodulatory properties e cacy[63], therefore, a comparision of therapeutic effect between different MSC lines on the AD mice skin would deepen our understanding regarding the therapeutic mechanisms of AD based on MSCs. Third, the therapeutic effects of stem cell are mainly attributed to their tissue regenerative capacity and immunomodulatory ability, but this study only investigated the immunomodulatory ability of ADSCs on AD. Further studies to clarify the tissue regenerative capacity of ADSCs on AD are required.

Conclusion
MSC-based therapy is a promising and potent approach for human allergic diseases, especially for AD. In this study, subcutaneous injection of ADSCs ameliorated AD by down-regulating IL-17 secretion of Th17 cells in ovalbumin-induced mouse model. Our results demonstrated that ADSCs might become a promising therapeutic medication for AD. Moreover, our ndings might further expand the knowledge about the mechanisms of MSC-based therapy for AD.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.