Mesenchymal Stem Cells and Extracellular Vesicles Derived from Canine Adipose Tissue Ameliorates Inflammation, Skin Barrier Function and Pruritus by Reducing JAK/STAT Signaling in Atopic Dermatitis

Canine atopic dermatitis (AD) is a common chronic inflammatory skin disorder resulting from imbalance between T lymphocytes. Current canine AD treatments use immunomodulatory drugs, but some of the dogs have limitations that do not respond to standard treatment, or relapse after a period of time. Thus, the purpose of this study was to evaluate the immunomodulatory effect of mesenchymal stem cells derived from canine adipose tissue (cASCs) and cASCs-derived extracellular vesicles (cASC-EVs) on AD. First, we isolated and characterized cASCs and cASCs-EVs to use for the improvement of canine atopic dermatitis. Here, we investigated the effect of cASCs or cASC-EVs on DNCB-induced AD in mice, before using for canine AD. Interestingly, we found that cASCs and cASC-EVs improved AD-like dermatitis, and markedly decreased levels of serum IgE, (49.6%, p = 0.002 and 32.1%, p = 0.016 respectively) epidermal inflammatory cytokines and chemokines, such as IL-4 (32%, p = 0.197 and 44%, p = 0.094 respectively), IL-13 (47.4%, p = 0.163, and 50.0%, p = 0.039 respectively), IL-31 (64.3%, p = 0.030 and 76.2%, p = 0.016 respectively), RANTES (66.7%, p = 0.002 and 55.6%, p = 0.007) and TARC (64%, p = 0.016 and 86%, p = 0.010 respectively). In addition, cASCs or cASC-EVs promoted skin barrier repair by restoring transepidermal water loss, enhancing stratum corneum hydration and upregulating the expression levels of epidermal differentiation proteins. Moreover, cASCs or cASC-EVs reduced IL-31/TRPA1-mediated pruritus and activation of JAK/STAT signaling pathway. Taken together, these results suggest the potential of cASCs or cASC-EVs for the treatment of chronic inflammation and damaged skin barrier in AD or canine AD.


Characterization of cASCs and cASC-EVs
The profiles of cASCs revealed a homogeneous population of cells, positive for mesenchymal markers, such as CD29, CD44, and CD90 ( Figure 1A). As expected, the isolated cASCs were successfully differentiated into adipocytes, chondrocytes or osteocytes with characterization of stem cells that can differentiate into each cell phenotype ( Figure 1B). Next, we investigated the size distribution and concentration of cASC-EV particles using NTA. The expected exosomal size of cASCs is around 182 nm ( Figure 1C). To determine the characterization of cASC-EVs, the exosomal specific markers were identified by western blot analysis. As shown Figure 1D, Alix, flotillin and CD9, as the exosomal specific markers, were clearly detected in the isolated cASC-EVs. In addition, we confirmed that several surface CD molecules, including CD105, CD49e, CD9, CD41b, CD81, CD44, and CD29, were markedly expressed in cASC-EV, but not in human major histocompatibility complex, such as human leukocyte antigen (HLA-DRDPDQ and HLA-ABC) and co-stimulatory molecules, such as CD80 and CD86 [33] ( Figure 1E). These results suggest that the low immunogenicity of cASCs and cASC-EVs allow their allogeneic use for autoimmune diseases. addition, we confirmed that several surface CD molecules, including CD105, CD49e, CD9, CD41b, CD81, CD44, and CD29, were markedly expressed in cASC-EV, but not in human major histocompatibility complex, such as human leukocyte antigen (HLA-DRDPDQ and HLA-ABC) and co-stimulatory molecules, such as CD80 and CD86 [33] (Figure 1E). These results suggest that the low immunogenicity of cASCs and cASC-EVs allow their allogeneic use for autoimmune diseases.

cASCs and cASC-EVs Alleviate Atopic Dermatitis Induced by DNCB Treatment
To confirm the therapeutic potential of cASCs and cASC-EVs on an in vivo model, we established an atopic dermatitis (AD) mouse model induced by topical DNCB treatment ( Figure 2A). AD-like skin lesions by DNCB showed skin severity with excoriation, scaling, edema, and erythema. Whereas, cASCs and cASC-EVs treatment

cASCs and cASC-EVs Alleviate Atopic Dermatitis Induced by DNCB Treatment
To confirm the therapeutic potential of cASCs and cASC-EVs on an in vivo model, we established an atopic dermatitis (AD) mouse model induced by topical DNCB treatment ( Figure 2A). AD-like skin lesions by DNCB showed skin severity with excoriation, scaling, edema, and erythema. Whereas, cASCs and cASC-EVs treatment alleviated DNCB-induced dermatitis compared to vehicle treatment ( Figure 2B,D, Tables 1 and 2). In addition, cASCs and cASC-EVs treatment resulted in suppression of epidermis hyperplasia, parakeratosis, and dermal edema induced by DNCB ( Figure 2C,E and Table 1). Repeated treatment of topical DNCB remarkably increased serum IgE production, whereas administration of cASCs and cASC-EVs reduced levels of serum IgE as a major characteristic of AD ( Figure 2F and Table 1). alleviated DNCB-induced dermatitis compared to vehicle treatment ( Figure 2B,D, Tables  1 and 2). In addition, cASCs and cASC-EVs treatment resulted in suppression of epidermis hyperplasia, parakeratosis, and dermal edema induced by DNCB ( Figure 2C,E and Table  1). Repeated treatment of topical DNCB remarkably increased serum IgE production, whereas administration of cASCs and cASC-EVs reduced levels of serum IgE as a major characteristic of AD ( Figure 2F and Table 1).

cASCs and cASC-EVs Ameliorated Skin Inflammation in DNCB-Induced Atopic Dermatitis Mice
We further focused on determining local infiltration by mast cells. It is well known that histamine released by activated mast cells in a region of atopic dermatitis increases the permeability of blood vessels, causing immune cells to penetrate into tissues [34]. Thus, it is necessary to reduce activated mast cells to decrease allergic reactions like AD. cASC and cASC-EV administration markedly decreased the number of mast cells compared to the vehicle group on the dorsal skin and ears of mice treated with DNCB ( Figure 3A,B and Table 1). In addition, we examined cytokine and chemokine productions in dorsal skin on day 28. The levels of the Th2 cytokines and chemokines, including IL-4, IL-13, IL-31, TSLP, RANTES and TARC, were enhanced by DNCB, while administration of cASC and cASC-EV reduced the levels of IL-4, IL-13, IL-31, TSLP, RANTES, and TARC in dorsal skin treated with DNCB ( Figure 3C, Tables 1 and 2). These results demonstrate that cASC and cASC-EV suppress the immune response of AD by regulating mast cell infiltration and the production of Th2-related cytokines and chemokines.   in dorsal skin treated with DNCB ( Figure 3C, Tables 1 and 2). These results demonstrate that cASC and cASC-EV suppress the immune response of AD by regulating mast cell infiltration and the production of Th2-related cytokines and chemokines.  (min-max) and the others represent mean ± SD. Significant value was * p < 0.05, ** p < 0.01 vs. DNCB + Vehicle.

cASCs and cASC-EVs Promote Skin Barrier Repair in DNCB-Induced Atopic Dermatitis Mice
To evaluate skin barrier function, transepidermal water loss (TEWL) and hydration were measured by using tewameter and corneometer in DNCB-induced AD-like skin lesions. As shown in Figure 4A, multiple DNCB-exposures markedly resulted in the increment of TEWL and decrement of hydration on dorsal skin. However, increased TEWL of AD-like skin lesion markedly decreased in cASCs and cASC-EVs groups on day 28 compared to the vehicle group ( Figure 4A and Table 1). Moreover, cASCs and cASC-EVs significantly recovered reduced hydration compared to the vehicle group ( Figure 4B and Table 1). We also confirmed whether cASCs and cASC-EVs affect the expression levels of epidermal differentiation proteins, such as keratin1 (K1), filaggrin (FLG), loricrin (LOR), and involucrin (INV), to improve AD in dorsal skin lesions treated with DNCB. The expression of keratin1, filaggrin, loricrin, and involucrin significantly decreased in DNCB-induced AD-like skin lesions, which markedly improved following administration of cASCs and cASC-EVs ( Figure 4C,E and Table 1). Collectively, these results indicate that cASCs and cASC-EVs might be effective in accelerating skin barrier repair.

cASCs and cASC-EVs Suppressed IL-31-Mediated Pruritus and Phosphorylated STATs in DNCB-Induced Atopic Dermatitis Mice
We then evaluated whether cASCs and cASC-EVs could decrease pruritus in DNCBinduced AD-like skin lesions. To understand the potential role of cASCs and cASC-EVs, we performed fluorescent IHC of TRPA1, activated under inflammatory conditions, on AD dorsal skin [35]. The expression of TRPA1 significantly increased in the epidermal and dermal regions of DNCB-induced AD ( Figure 5A and Table 1). Conversely, the expression of TRPA1 was rare in cASC and cASC-EV-administered skin lesions ( Figure 5A and Table 1). Our previous data showed that the expression of IL-13 induced by DNCB was decreased by the administration of cASCs and cASC-EVs ( Figure 3C and Table 1). It has been reported that IL-13 highly stimulates TRPA1 expression in mast cells [36] and IL-31-induced pruritus decreased in TRPA1-deficient mice [37]. Additionally, cASC and cASC-EVs markedly inhibited the expression of IL-31 receptor A (IL-31RA) and the Oncostatin M Receptor (OSMR) known as IL-31 receptors ( Figure 5B and Table 1) as well as IL-31 ( Figure 3C and Table 1). Several reports have shown that IL-4, IL-13, and IL-31 induces the expression of specific genes by activating STATs following activation of Janus-kinses (JAKs) [38,39]. To evaluate the regulation of JAK-STATs signaling by cASCs and cASC-EVs in DNCB-induced AD like lesions, we investigated the activation of STAT1 and STAT3 in dorsal skin. cASC and cASC-EVs markedly decreased levels of phosphorylated STAT1 and STAT3 induced by DNCB. These results suggest that increase of Th2-related cytokines and IL-31-induced TRPA1 expression are induced by STAT1 and STAT3 signaling, and cASC and cASC-EVs improve AD by suppressing STAT1 and STAT3 signaling. levels of epidermal differentiation proteins, such as keratin1 (K1), filaggrin (FLG), loricrin (LOR), and involucrin (INV), to improve AD in dorsal skin lesions treated with DNCB. The expression of keratin1, filaggrin, loricrin, and involucrin significantly decreased in DNCB-induced AD-like skin lesions, which markedly improved following administration of cASCs and cASC-EVs ( Figure 4C,E and Table 1). Collectively, these results indicate that cASCs and cASC-EVs might be effective in accelerating skin barrier repair. (D) Transcripts of keratin 1, filaggrin, loricrin, and involucrin were quantified using real-time PCR (n = 6/group). (E) Immunoblots with anti-filaggrin, anti-loricrin, anti-involucrin, and anti-β-actin antibodies using lysates from skin. All data represent mean ± SD. Significant value was * p < 0.05, ** p < 0.01 vs. DNCB + Vehicle. (D) Transcripts of keratin 1, filaggrin, loricrin, and involucrin were quantified using real-time PCR (n = 6/group). (E) Immunoblots with anti-filaggrin, anti-loricrin, anti-involucrin, and anti-β-actin antibodies using lysates from skin. All data represent mean ± SD. Significant value was * p < 0.05, ** p < 0.01 vs. DNCB + Vehicle. STAT3 in dorsal skin. cASC and cASC-EVs markedly decreased levels of phosphorylated STAT1 and STAT3 induced by DNCB. These results suggest that increase of Th2-related cytokines and IL-31-induced TRPA1 expression are induced by STAT1 and STAT3 signaling, and cASC and cASC-EVs improve AD by suppressing STAT1 and STAT3 signaling. (C) Immunoblots with anti-p-STAT1, anti-STAT1, anti-p-STAT3, anti-STAT3 or anti-β-actin antibodies using lysates from skin. Western blots were analyzed quantitatively. All data represent mean ± SD. Significant value was * p < 0.05, ** p < 0.01 vs. DNCB + Vehicle.

Discussion
Mesenchymal stem cells have inflammatory therapeutic functions through the interaction of paracrine factors, such as extracellular vesicles, cytokines, chemokines, and growth factors, with immune cells. EVs secreted from MSCs contain various active molecules, such as bioactive proteins, lipids, mRNAs, and microRNAs. Numerous recent studies have reported that the immunomodulatory ability of hMSCs can be useful in the treatment of allergic diseases, such as asthma and dermatitis [40][41][42][43][44]. In addition, studies have been conducted on the immunosuppressive effects of EVs secreted by MSCs. MSC-EVs are considered a new alternative to reducing potential side effects, such as the uncontrolled differentiation and proliferation potential of MSCs [18,19,45,46]. Thus, many researchers are trying to research the immunological therapeutic application of MSCs and MSC-EVs to cure AD, which is a common inflammatory skin disease, resulting from skin barrier defects and immunological hyperactivity. However, there are few therapeutic studies of canine AD on cMSCs [47,48], and not yet any studies on the efficacy of cMSC-EVs for the treatment of canine AD. Here, MSCs were isolated from canine adipose tissue, and EVs were further isolated and characterized as shown in Figure 1. In addition, cASCs and cASC-EVs were subcutaneously injected into DNCB-induced AD in mice to analyze their immunomodulatory ability, before using them for canine AD. Subcutaneous administration of cASC led to a high level of therapeutic efficacy. Interestingly, the application of cASC-EVs showed a similar therapeutic efficacy compared to cASC administration. In this study, we demonstrated that cASCs and cASC-EVs suppress DNCB-induced AD in mice, probably through the inhibition of Th2 cytokine and chemokines, causing improvement of skin barrier function and pruritus.
Canine AD results in various factors, including a large amount of IgE, T lymphocyte polarization, and mast cell hyperplasia [49]. Furthermore, canine AD disease typically begins with Th2 cells, and progresses to a chronic disease with a mixed Th2/Th1 profiles [50,51]. Schlotter et al. reported that Th2 cells prominently distributed in the acute skin lesions of canine AD produce cytokines, such as interleukin (IL)-4, IL-13 and IL-31 [23]. In chronic AD, IL-2, IFN-γ and IL-18 secreted from Th1 cells are detected [24,50]. IL-4 and IL-13 are involved in the proliferation and differentiation of B cells, and promote the production of IgE antibodies, by activating the infiltration of eosinophils to the site of inflammation [24,50]. In addition, IL-31 is actively involved in pruritus [51,52]. It seems that the cytokine pathway of canine AD is very similar to that of human AD [53][54][55]. According to a previous study, 22 of 26 AD dogs administered allogeneic canine adipose MSCs (cAd-MSC) were treated without systemic immunosuppressant medication at follow-up of 6 months [48]. None of the dogs showed systemic local adverse reactions after administration of allogeneic cAd-MSC [48]. Moreover, several studies have proven that injected MSCs migrate to damaged and inflammatory tissues, resulting in a therapeutic effect on the diseases [32,[56][57][58]. In AD, MSCs migrate to skin lesions through draining lymph nodes and reduce cell infiltration, such as T cells in skin lesions [32]. In the present study, we also observed infiltration of mast cells, and higher expression of IL-4, IL-13, IL-31, TSLP, RANTES and TARC in AD-like region of mice treated with DNCB. On the other hand, the administration of cASC and cASC-EVs reduced mast cell infiltration and the production of Th2-related cytokines and chemokines ( Figure 3). Moreover, the enhanced levels of serum IgE in DNCB-induced AD mice was markedly decreased by cASC and cASC-Evs ( Figure 2). These results indicated that cASCs and cASC-EVs inhibited skin inflammation in DNCB-induced AD mice through modulation of inflammatory circumstance, mediated by activation of T cells and mast cells.
AD is a pruritic skin disorder with barrier dysfunction and elevated expression of IL-4 and IL-13. In addition, the barrier dysfunction is correlated with the downregulation of barrier-related molecules, including K1, FLG, LOR and IVL. IL-4 and IL-13 potently suppress the expression of barrier-related molecules which play crucial roles in maintaining the structural integrity and functions of stratum corneum, and help in maintaining the homeostasis of stratum corneum [59,60]. The expression and release of IL-31 induced by IL-4 in Th2 cells are a recently identified cytokine with a well-defined role in the pathogenesis of pruritus. Interleukin-31 activates the JAK1/JAK2 and STAT3 (also to less extent STAT1 and STAT5) pathways via IL-31R which is a heterodimer composed of IL-31RA and OSMR [61,62]. In this study, we confirmed the downregulation of K1, FLG, LOR and IVL in AD-like regions of mice treated with DNCB ( Figure 4). Furthermore, IL-31/IL-31R was upregulated in AD-like regions. However, cASCs and cASC-EVs clearly recovered the downregulated barrier-related genes and proteins as well as the upregulated IL-31/IL-31R expression. In addition, STAT1 and 3 activations in AD-like regions were significantly suppressed by cASCs and cASC-EVs ( Figure 5). Taken together, for improvement of AD, cASCs and cASC-EVs regulated histamine or IgE release by prohibiting infiltration of mast cells, and skin barrier dysfunction induced by IL-4 and IL-13 by blocking infiltration of T cells, and pruritus skin stimulated by the phosphorylation of STAT1, and 3 through the increment of IL-31/IL-31R expression in T cell by IL-4. However, using 6 mice per group in this study has shown limitation in the significance of some data in statistical analysis, although there is tendency for positive effects of cASCs and cASC-EVs on AD-skin.
Similar immunomodulatory effects of cASC-EVs, compared to cASCs, on AD were demonstrated in this study. MSC-EVs have been studied in various disease models [63,64]. Although clinical trial results for human-derived MSCs confirmed the therapeutic efficacy of human-MSCs [65], so far, there is no direct evidence of the clinical relevance of canines to exosomes derived from canines. The current study provides a basis for the treatment of AD in canines as well as humans, as it has been known that the paracrine factors, containing EVs, are one of the therapeutic effects of MSCs [18,19].

Isolation, Culture, and Characterization of cASC
The adipose tissue was obtained from healthy beagle dogs with the approval of the Ethics Committee for Experimental Animal Research at Kyungpook National University (IACUCSGU2019_15). ASCs were isolated from tissues and cultures as previous described [66,67]. Primary cultures were carried out with Dulbecco's modified Eagle's medium /F12 (cytiva, Logan, UT, USA) containing 10% fetal bovine serum (cytiva, Logan, UT, USA), 10 ng/mL epidermal growth factor (EGF, ProSpec, Rehovot, Israel), 2 ng/mL basic fibroblast growth factor (bFGF, PeproTech, Rocky Hill, NJ, USA). 10 ng/mL insulinlike growth factor (IGF, ProSpec, Rehovot, Israel), 2.5 mM L-glutamine (Gibco, Grand island, NY, USA) 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Grand island, NY, USA). Cells were detached when confluence was over 80%, and sub-cultured at a concentration of 10 4 cells/cm 2 for continued passaging. The remaining cells were cryopreserved, and stored in liquid nitrogen. All experiments and in vivo implantation were conducted at passage 2. The cells were characterized for the expression of several stem cell markers by flow cytometry (Beckman Coulter, Brea, CA, USA) before they were used in the experiments.

Isolation and Characterization of cASC-EV
ASC-EV were isolated from the ASCs CM by the tangential flow filtration (TFF)-based method [46,68]. Briefly, the CM was filtrated through a 0.22-µm polyethersulfone membrane filter (Merck Millipore, Billerica, MA, USA) to remove non-ev particles, such as cells, cell debris, micro-vesicles and apoptotic bodies. The CM was then concentrated by tangentialflow filtration with a 500 kDa molecular weight cut-off filter membrane cartridge (Pall, Port Washington, NY, USA), and then buffer exchange was performed by diafiltration with PBS (WELGENE, Gyeongsan, Korea). Isolated ASC-EV were aliquoted into polypropylene disposable tubes, and stored at −80 • C until use. Before using, frozen ASC-EV were left at 4 • C until completely thawed and were not frozen again. The characterization and profile analysis of ASC-EV were conducted following the Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV2018) recommended by the International Society of Extracellular Vesicles (ISEV) [15].
To determine size distribution and particle concentration, cASC-EV diluted with PBS were analyzed by nanoparticle tracking analysis (NTA) using a NanoSight LM10 (Malvern Panalytical, Worcestershire, UK) equipped with a 642-nm laser. cASC-EV, diluted with PBS to between 100 and 200 particles per frame, were scattered and illuminated by the laser beam and their movements under Brownian motion were captured for 60 s each at a camera level of 16. Videos were then analyzed by the NTA 3.2 software using constant settings. To provide a representative result, at least 5 videos were captured and >2000 validated tracks were analyzed for each individual sample. The NTA instrument was regularly checked with 100 nm-sized standard beads (Malvern Panalytical, Worcestershire, UK). To provide representative size distribution of particle, size distribution profiles from each video replicates were averaged.

Animal Care and Induction of AD Model
Six-week-old male BALB/c mice were supplied by DBL, Inc. (Incheon, Korea). Solid feed (no antibiotics added) and water were supplied until the day of the experiment, and the animals were used for experiments following a 1-week adaptation period at temperatures of 23 ± 2 • C, humidity of 55 ± 10% and a 12-12 h light-dark cycle. All animal experiments were conducted in accordance with the principles of laboratory animal care of the National Institutes of Health (NIH, Bethesda, MD, USA) and with the approval of the Ethics Committee for Experimental Animal Research at Sogang University (IACUCSGU2019_15).
Repeated application of 2,4-dinitrochlorobenzene (DNCB; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) to a local site causes AD-like skin lesions. In the first inflammation induction process, 100 µL of 1% DNCB solution (acetone:olive oil = 4:1) was applied to the same dorsal area twice for 3 days in all groups, except in the normal group (NOR). The vehicle (acetone:olive oil = 4:1) was used to treat the NOR group. Following a latent period of 4 days, 100 µL of 0.4% DNCB was used for a second sensitization. 8 h after every DNCB exposure, cASCs (2 × 10 6 cells/head) or cASC-EVs (2 × 10 10 particles/head) were subcutaneously applied twice or 5 times for 2 weeks, respectively. Vehicle (saline) served as a negative control.
Basal transepidermal water loss (TEWL) was measured with a TM300 connected to MPA5 (Courage + Khazaka, Cologne, Germany) and stratum corneum (SC) hydration was assessed with a Corneometer CM820 (Courage + Khazaka, Cologne, Germany). 2 days after the last treatment of DNCB, skin and blood samples were collected to evaluate the allergic inflammation and skin barrier function.

Measurement of Dermatitis Score and Epidermal Thickness
Atopic dermatitis severity and epidermal thickness were measured as described previously [69]. After mice were anesthetized with 2% isoflurane, skin lesions were photographed. Scores of 0 (none), 1 (mild), 2 (moderate), and 3 (severe) were given for each of the four symptoms: (i) erythema/hemorrhage, (ii) edema, (iii) excoriation/erosion and (iv) scaling/dryness. Maximum score is 12. Epidermal thickness of 10 randomly selected areas in three sections of each mouse was measured using a microscope (DM750; Leica, Wetzlar, Germany) aided by haematoxylin and eosin (H&E) staining and ImageJ version 1.51 (NIH, Bethesda, MD, USA). Assessments were performed in a blinded manner.

Measurement of Total Serum IgE and Inflammatory Cytokines
Blood collected from mice was placed in an e-tube and centrifuged at 4 • C and 850× g for 30 min. Following centrifugation, supernatant was stored at −70 • C until measurement. Serum IgE was measured using an enzyme-linked immunosorbent assay (ELISA). Each sample was added onto a 96-well plate treated with the primary antibody using a mouse IgE ELISA Kit (Invitrogen, Waltham, MA, USA), and washed four times with the working solution. After washing, the samples were treated with the secondary antibody, HRPconjugated goat anti-mouse IgE (1:10,000), for 1 h after which a color development reagent was used. Following color development, the reaction was stopped using a stop solution, and absorbance was measured at 450 nm using a microplate reader (Spectra Max 250, Molecular Devices, Sunnyvale, CA, USA).

Statistical Analysis
Continuous variables were tested for normal distribution by using the Shapiro-Wilk test. Data are presented as mean ± SD or as median [IQR] (min-max). Statistical analysis was performed by independent samples t-test for normally distributed data, or Mann-Whitney-U test was used. A value of p < 0.05 was considered statistically significant. All calculations were carried out by using SPSS Statistics 17 (SPSS, Chicago, IL, USA).

Conclusions
In conclusion, this study demonstrated the effect of cASCs and cASC-EVs in improvement of impaired skin in AD. cASCs and cASC-EVs positively regulated inflammatory responses and repair of defective epidermal permeability barrier functions by modulating gene expression, including epidermis differentiation and immune response. Furthermore, cASCs and cASC-EVs ameliorated pruritus by reducing IL-31/TRPA1 through the inhibition of JAK-STATs signaling activation. We further need to study the downregulation of STATs to clarify a more accurate relationship between IL-31/TRPA1 and STATs. These findings indicate that cASCs or cASC-EVs could be a novel therapeutic strategy to treat canine AD.