Efficacy of Allogeneic and Xenogeneic Exosomes for the Treatment of Canine Atopic Dermatitis: A Pilot Study

Simple Summary Canine atopic dermatitis is a multifactorial allergic skin disease that lacks permanent curative treatment, and a single treatment strategy does not show efficacy in all cases. Therefore, novel and innovative therapeutic options are urgently needed. Exosomes, one of the major types of extracellular vesicles, have been investigated as an alternative treatment for various diseases in humans; however, the efficacy or side effects of exosomes in the treatment of canine atopic dermatitis are elusive. In this study, we investigated the therapeutic potential of canine- and human-derived exosomes against canine atopic dermatitis using six experimental models randomly assigned to control, canine exosome, or human exosome groups. Our findings revealed that canine- and human-derived exosomes alleviated canine atopic dermatitis in clinical, immunological, and microbiological aspects and that the treatment with exosomes was well tolerated. Abstract Canine atopic dermatitis (CAD) is a genetically predisposed inflammatory pruritic skin disease. The available treatments for CAD have several adverse effects and vary in efficacy, indicating the need for the development of improved treatments. In this study, we aimed to elucidate the therapeutic effects of allogeneic and xenogeneic exosomes on CAD. Six laboratory beagle dogs with CAD were randomly assigned to three treatment groups: control, canine exosome (cExos), or human exosome (hExos) groups. Dogs in the cExos and hExos groups were intravenously administered 1.5 mL of cExos (5 × 1010) and hExos (7.5 × 1011) solutions, respectively, while those in the control group were administered 1.5 mL of normal saline three times per week for 4 weeks. Skin lesion score and transepidermal water loss decreased in cExos and hExos groups compared with those in the control group. The exosome treatments decreased the serum levels of inflammatory cytokines (interferon-γ, interleukin-2, interleukin-4, interleukin-12, interleukin-13, and interleukin-31) but increased those of anti-inflammatory cytokines (interleukin-10 and transforming growth factor-β), indicating the immunomodulatory effect of exosomes. Skin microbiome analysis revealed that the exosome treatments alleviated skin bacterial dysbiosis. These results suggest that allogeneic and xenogeneic exosome therapy may alleviate CAD in dogs.

CAD poses a significant challenge and negatively affects the quality of life of affected dogs and their owners because of the lack of a permanent curative treatment and poor knowledge of its pathogenesis [4,14,15].Pruritus is often the most common symptom in dogs; therefore, immediate and sustained control of pruritus is the primary therapeutic goal for managing CAD in veterinary practice [16,17].However, the management of CAD is multimodal, and no single treatment strategy is likely to be successful in all cases [13,18].Therefore, novel and innovative therapeutic options are urgently needed.
Mesenchymal stem cell (MSC) therapy has been extensively investigated as an alternative treatment for various diseases in humans; however, its safety and efficacy are controversial [19].Additionally, the low viability of transplanted cells due to anoikis caused by the loss of cell adhesion is a primary limitation of MSC-based therapies [20][21][22].However, recent studies suggest that the therapeutic effects of MSCs are attributable to the biological molecules they secrete in the form of microscopic particles called extracellular vesicles (EVs) [20].
Most eukaryotic and prokaryotic cells release EVs, which are important mediators of intercellular communication [23].EVs are released by all cell types under physiological and pathological conditions and are present in all body fluids [24,25].Exosomes are one of the major types of EVs, with diameters ranging from 40 to 100 nm [25][26][27], and they contain cell type-specific combinations of proteins, including enzymes, cytokines, and growth factors, as well as DNA, coding and noncoding RNAs, metabolites, and lipids [24,28].Exosomes can travel through the bloodstream to cells in distant organs and easily fuse with recipient cells [20,29].Additionally, exosomal contents are resistant to degradation via both endogenous and exogenous enzymes or RNases [29].Exosomes do not require cell culture facilities and have easier storage and distribution than stem cells.These properties render them an attractive alternative to stem cells in human medicine.However, studies investigating the efficacy or side effects of exosomes derived from canines (cExos) or humans (hExos) in CAD treatment have not been reported.
Therefore, this study aimed to investigate the therapeutic potential of cExos and hExos in CAD models.We hypothesized that intravenous injection of allogeneic and xenogeneic exosomes would be tolerable and alleviate CAD.Additionally, we speculated that allogeneic exosomes would be more effective than xenogeneic exosomes in CAD treatment.

Study Subjects
Six laboratory beagle dogs with previously developed experimental CAD were used for the study [30].The dogs had been sensitized epicutaneously twice a week for 12 weeks to house dust mite (HDM), Dermatophagoides farinae (Der f ) (18M03, CITEQ biologics, Groningen, Netherlands, mixed with mineral oil).Successful sensitization was confirmed with Der f -specific IgE assay and intradermal test.After the end of the sensitization, six dogs were challenged twice a week for four weeks with the same HDM paste to induce clinical signs of atopic dermatitis.Immediately after the completion of the mite challenge, six CAD model dogs were included in this study.The animals were raised in individual kennels made of stainless steel in a room in the Konkuk Laboratory Animal Research Center.The kennels were cleaned once daily using high-pressure water, and the temperature and humidity of the room were maintained at 22-24 • C and 45-55%, respectively.During the experimental period, the animals were fed a conventional diet based on daily energy requirements and had ad libitum access to clean tap water.To avoid contamination, other dogs were not allowed to enter the room, and access was restricted to individuals directly involved in this study.Additionally, items such as bedding, clothes, toys, and bathing were prohibited from the animal house.The six CAD models were randomly assigned to allogeneic exosomes, xenogeneic exosomes, and control treatment groups.Every group had two dogs, and they were maintained throughout the entire study period.No concurrent medications were administered during the study period.This study was approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University (IACUC approval no.KU21075, approval date 2 June 2021).

Production of cExos and hExos
Allogeneic exosomes (cExos) used in this study were isolated from canine adipose tissue-derived MSCs.The xenogeneic exosomes (hExos) were Expi293F cell-derived exosomes loaded with super-repressor IκB (srIκB).cExos and hExos were obtained following the methods reported in previous studies [31][32][33].Briefly, both upstream and downstream processes were used in this study.In the upstream process, the cells were cultivated for 4 days using the WAVE method under blue-light exposure.Afterward, the conditioned media was subjected to ultrafiltration and purification to obtain the purified exosomes in the downstream process.

Study Design
On day 0 (D0), skin lesion scoring was performed using a scale that was a modified form of the canine atopic dermatitis extent and severity index (CADESI-04), and transepidermal water loss (TEWL) was measured.Additionally, a skin swab was obtained from the same site for skin microbiome analysis.Blood samples were collected for circulating cytokine analysis.
Prior to the treatments, cExos and hExos were thawed and diluted with Dulbecco's phosphate buffer saline.Thereafter, the animals were intravenously injected with 1.5 mL of cExos (5 × 10 10 EVs) or hExos (7.5 × 10 11 EVs) solution via the cephalic vein.The exosome dosage in this study was determined based on the effective dose and half-life of exosomes identified in our previous studies [32,34], which involved a different disease model.Dogs in the control group were intravenously administered 1.5 mL of normal saline (0.9% sodium chloride).The injection was repeated three times a week at two-, two-, and three-day intervals for four weeks.Overall, each experimental animal received a total of 12 doses during the 4-week period.
To assess the safety of cExos and hExos, the dogs were monitored for signs of allergic reaction or anaphylaxis for 30 min following every injection, and physical examinations were performed daily during the study period.Lesion scoring was performed after four and eight doses of the treatments and at the end of the experiment using the modified CADESI-04 scale [35].Additionally, at baseline and the end of the study period (on day 28; D28), TEWL measurement, skin microbiome analysis, and circulating cytokine analysis were performed (Figure 1).
dosage in this study was determined based on the effective dose and half-life of exosomes identified in our previous studies [32,34], which involved a different disease model.Dogs in the control group were intravenously administered 1.5 mL of normal saline (0.9% sodium chloride).The injection was repeated three times a week at two-, two-, and threeday intervals for four weeks.Overall, each experimental animal received a total of 12 doses during the 4-week period.
To assess the safety of cExos and hExos, the dogs were monitored for signs of allergic reaction or anaphylaxis for 30 min following every injection, and physical examinations were performed daily during the study period.Lesion scoring was performed after four and eight doses of the treatments and at the end of the experiment using the modified CADESI-04 scale [35].Additionally, at baseline and the end of the study period (on day 28; D28), TEWL measurement, skin microbiome analysis, and circulating cytokine analysis were performed (Figure 1).

Evaluation of Skin Lesions
The severity of skin lesions was scored on days 0, 10, 19, and 28 using a modified CADESI-04 scale [35] by a veterinarian who was blinded to the study details.Erythema, lichenification, and excoriation lesions of the right groin were scored from 0 to 3, with a maximum score of 9.

Evaluation of Skin Barrier Function
The water barrier integrity of the stratum corneum was evaluated based on the TEWL measured in the right groin region on D0 and D28 using a VapoMeter ® (Delfin Technologies, Kuopio, Finland), according to the manufacturer's instructions [36].All measurements were performed in the same room in which the dogs were raised.Mean values were calculated after obtaining five consecutive values.

Evaluation of Skin Lesions
The severity of skin lesions was scored on days 0, 10, 19, and 28 using a modified CADESI-04 scale [35] by a veterinarian who was blinded to the study details.Erythema, lichenification, and excoriation lesions of the right groin were scored from 0 to 3, with a maximum score of 9.

Evaluation of Skin Barrier Function
The water barrier integrity of the stratum corneum was evaluated based on the TEWL measured in the right groin region on D0 and D28 using a VapoMeter ® (Delfin Technologies, Kuopio, Finland), according to the manufacturer's instructions [36].All measurements were performed in the same room in which the dogs were raised.Mean values were calculated after obtaining five consecutive values.

Skin Microbiome Analysis
Skin swab samples were collected from the right groin before and after the treatment (D0 and D28).The swab samples were stored at −80 °C until bacterial DNA extraction, PCR amplification, and 16S rRNA sequencing.PCR amplification of the extracted DNA was performed using fusion primers targeting the V3-V4 regions of the 16S rRNA gene.Sequencing was performed at CJ Bioscience, Inc. (Seoul, Republic of Korea) using MiSeq (Illumina, San Diego, CA, USA), an integrated next-generation sequencing instrument.The primer sequences used for the PCR and data analysis after sequencing were the same as those in a previous study [30].

Statistical Analysis
The sample size was too small to perform statistical analysis, and only a descriptive assessment was performed.For comparison between groups, the change in baseline was calculated because the pretreatment values were different in all dogs.The change from baseline value was defined as the difference between post-treatment and pretreatment values.All values are expressed as the mean ± standard error.

Evaluation of Skin Barrier Function
TEWL was measured in each group before treatment (D0) and after all treatment doses (D28).The TEWL score was reduced in the cExos and hExos groups compared to that in the control group, indicating that cExos and hExos treatment improved skin barrier integrity in the dogs.Specifically, the change in TEWL value (post-treatment value-baseline value) was 1.9 ± 2.5, −34.65 ± 1.25, and −20.0 ± 13.0 g/m 2 /h in the control, cExos, and hExos groups, respectively (Figure 4).

Evaluation of Skin Barrier Function
TEWL was measured in each group before treatment (D0) and after all treatment doses (D28).The TEWL score was reduced in the cExos and hExos groups compared to that in the control group, indicating that cExos and hExos treatment improved skin barrier integrity in the dogs.Specifically, the change in TEWL value (post-treatment value-baseline value) was 1.9 ± 2.5, −34.65 ± 1.25, and −20.0 ± 13.0 g/m 2 /h in the control, cExos, and hExos groups, respectively (Figure 4).

Serum Cytokine Levels
Next, we evaluated the changes in the serum levels of IFNγ, IL-2, IL-4, IL-10, IL-12, IL-13, IL-31, and TGFβ (Figure 5; Table 1).Treatment with the exosomes decreased the levels of inflammatory cytokines and increased the serum levels of anti-inflammatory cytokines compared to that in the control group.

Serum Cytokine Levels
Next, we evaluated the changes in the serum levels of IFNγ, IL-2, IL-4, IL-10, IL-12, IL-13, IL-31, and TGFβ (Figure 5; Table 1).Treatment with the exosomes decreased the levels of inflammatory cytokines and increased the serum levels of anti-inflammatory cytokines compared to that in the control group.

Skin Microbiome Analysis
A total of 420,237 and 658,452 reads were obtained from the skin swab samples of all the subjects before and after treatment, respectively.Compared with before treatment, the number of operational taxonomic units (OTUs) increased in cExos and hExos groups after treatment (Table 2).The numbers of normalized reads were 61,927 and 30,956 in all groups before and after the treatments, respectively.The alpha diversity indices were calculated after normalizing the reads before and after the treatment in each sample (Table 2).Changes in OTUs and Chao1 and Shannon indices after the treatments were calculated (Table 3) and are presented in Figure 6.Also, principal coordinate analysis (PCoA) plots from the generalized UniFrac metric were constructed (Figure 7).

Discussion
Currently, available treatment strategies for CAD include symptomatic treatments and specific interventional therapies [37,38].Symptomatic therapy includes glucocorticoids; calcineurin inhibitors such as cyclosporine and tacrolimus; the Janus kinase inhibitor oclacitinib; and the monoclonal interleukin-31 antibody, lokivetmab [18,39].Glucocorticoids are inexpensive and show rapid action; however, they can cause side effects, such as polyuria/polydipsia, polyphagia, muscle atrophy, skin atrophy, and iatrogenic hyperadrenocorticism [10,38,40].Additionally, the side effects of cyclosporine were reported in 55% of the 15 clinical trials of CAD, especially gastrointestinal side effects such as vomiting and diarrhea [41,42].Although long-term administration of oclacitinib seems to be well tolerated, new or exacerbated existing neoplasia has been observed in dogs treated with oclacitinib [43,44].Lokivetmab showed more pronounced antipruritic efficacy than cyclosporine but had a lower anti-inflammatory effect [45,46].Interventional therapies include allergen-specific immunotherapy, which is the only etiological treatment for CAD [47].Various routes have been used to administer allergen-specific immunotherapy; however, the risk of anaphylaxis must be considered [48,49].
Treatment with cExos and hExos ameliorated skin lesions in dogs with CAD compared with that in the control group, as evidenced by a decrease in modified CADESI-04 scores in the cExos and hExos groups.Additionally, cExos and hExos treatments reduced TEWL, indicating that exosome therapy improved skin barrier function.Moreover, exosome treatments decreased the serum levels of inflammatory cytokines, including IFNγ, IL-2, IL-4, IL-12, IL-13, and IL-31, and increased the levels of anti-inflammatory cytokines, including IL-10 and TGFβ.This trend was more pronounced in the exosome treatment

Discussion
Currently, available treatment strategies for CAD include symptomatic treatments and specific interventional therapies [37,38].Symptomatic therapy includes glucocorticoids; calcineurin inhibitors such as cyclosporine and tacrolimus; the Janus kinase inhibitor oclacitinib; and the monoclonal interleukin-31 antibody, lokivetmab [18,39].Glucocorticoids are inexpensive and show rapid action; however, they can cause side effects, such as polyuria/polydipsia, polyphagia, muscle atrophy, skin atrophy, and iatrogenic hyperadrenocorticism [10,38,40].Additionally, the side effects of cyclosporine were reported in 55% of the 15 clinical trials of CAD, especially gastrointestinal side effects such as vomiting and diarrhea [41,42].Although long-term administration of oclacitinib seems to be well tolerated, new or exacerbated existing neoplasia has been observed in dogs treated with oclacitinib [43,44].Lokivetmab showed more pronounced antipruritic efficacy than cyclosporine but had a lower anti-inflammatory effect [45,46].Interventional therapies include allergen-specific immunotherapy, which is the only etiological treatment for CAD [47].Various routes have been used to administer allergen-specific immunotherapy; however, the risk of anaphylaxis must be considered [48,49].
Treatment with cExos and hExos ameliorated skin lesions in dogs with CAD compared with that in the control group, as evidenced by a decrease in modified CADESI-04 scores in the cExos and hExos groups.Additionally, cExos and hExos treatments reduced TEWL, indicating that exosome therapy improved skin barrier function.Moreover, exosome treatments decreased the serum levels of inflammatory cytokines, including IFNγ, IL-2, IL-4, IL-12, IL-13, and IL-31, and increased the levels of anti-inflammatory cytokines, including IL-10 and TGFβ.This trend was more pronounced in the exosome treatment groups than in the control group, indicating that exosome therapy has an immunomodulatory effect.
Given that IL-31 is a potent pruritogenic cytokine, the decrease in IL-31 levels emphasizes the antipruritic effect of exosomes [50].
The alpha diversity indices represent the species richness or evenness of the microbiome.The findings of this study showed an increase in OTUs and Chao1 and Shannon indices after exosome treatment.Consistent with our findings, a previous study has shown that allergic dogs have lower species diversity in the skin microbiome than healthy dogs [51].Therefore, the results of this study suggest that the exosome treatments improved skin microbiome dysbiosis in the CAD model.
MSC-derived EVs exhibit the same functions as stem cells, including anti-inflammatory and proregenerative activities [52].Additionally, MSC-derived EVs are smaller and less immunogenic than MSCs, making them safer alternatives to cell injections [52].In human medicine, the therapeutic effects of MSCs have been largely attributed to the EVs they secrete, supporting the regenerative and immunomodulatory capabilities of MSCs [53].However, this approach is still in the early stages of research, and there has only been one study on the therapeutic use of MSC-derived exosomes in veterinary medicine [24].Canine MSC-derived microvesicles or exosomes significantly promote the healing of cutaneous wounds, collagen synthesis, and vascularization at wound sites in dogs [54].Although the therapeutic effects of autogenous or allogeneic adipose-derived MSCs on CAD [55,56] or experimental CAD models [57] have been examined, studies have yet to examine the effects of exosome therapy on CAD.Additionally, the therapeutic effect of xenogeneic exosomes in dogs with CAD is yet to be reported.
In this study, we investigated the therapeutic effects of human-derived exosomes isolated from the Expi293F cell line, which was derived from the HEK293 cell line.These are human embryonic kidney cell lines that are easy to manipulate and show high growth capacity; therefore, they are commonly used to produce therapeutic proteins or exosomes in human medicine [33,58].We used the engineered exosome loaded with srIκB, a nondegradable form of IκB that prevents the nuclear translocation of NFκB [59].NFκB is one of the most important regulators of proinflammatory gene expression and is highly activated at sites of inflammation in diverse diseases [60].The effects of srIκB loaded exosomes for sepsis, acute kidney injury, and preterm birth have been confirmed in several studies [32,61,62].
Nevertheless, the immunogenicity of xenogeneic transplantation is a serious issue that should be considered in therapeutic research.The intravenous administration of Expi293F-derived EVs to mice did not induce significant toxicity [63].Similarly, cExos and hExos treatments (allogeneic and xenogeneic exosomes) were well tolerated in the present study, as evidenced by the absence of any allergic reaction, anaphylaxis, and abnormal findings.However, the duration of this study was only 4 weeks; therefore, further studies are necessary to verify the long-term safety of xenogeneic exosomes.
In the present study, it was difficult to compare the therapeutic efficacy of canine-and human-derived exosomes due to differences in their administration dosage, the origin of cells, and the small number of study subjects.A comparative study of the regenerative effects of allogeneic and xenogeneic adipose tissue-derived EVs on full-thickness skin wounds in rats showed that both exosomes had almost the same effects [64].Collectively, these results indicate that EVs from the same tissue carry similar contents and that the species might not be the main factor for EV function.However, further studies are necessary to verify the efficacy of allogeneic and xenogeneic exosomes in CAD.
The xenogeneic exosomes used in this study were not derived from human MSC but from an embryonic cell line that was previously stored.However, the xenogeneic exosomes had a similar immunomodulatory effect as the allogeneic MSC-derived exosomes in CAD.Cell line-derived exosomes are widely used to produce human industrial bioproducts due to the ease of large-scale production and extraction from existing cells [58].The largescale production of therapeutic exosomes is an unexplored field in veterinary medicine, indicating the need for further studies.
Although promising results were obtained, this study has some limitations.For example, statistical analysis and comparisons could not be performed due to the small number of study subjects; therefore, a definite conclusion on the efficacy of exosomes for CAD cannot be inferred.However, the positive findings of this study support the need for larger placebo-controlled trials involving patients with CAD.Additionally, we did not compare the histopathology and immunohistochemistry at the lesion sites before and after treatment in this study to minimize invasive tests.However, we evaluated changes in TEWL, circulating cytokines, and the skin microbiome.Furthermore, an experimental CAD model was used in this study; therefore, the effects of exosomes on patients with spontaneous CAD cannot be predicted.Further studies are required to verify the efficacy of exosomes in patients with CAD.

Conclusions
Conclusively, the findings of the study showed that allogeneic and xenogeneic exosome treatment was feasible and well tolerated in the experimental CAD model, with no evidence of adverse events.Additionally, this study provides a basis for future large-scale studies to confirm the long-term safety and efficacy of allogeneic and xenogeneic exosomes in CAD.

Figure 3 .
Figure 3. Changes in lesion scores at different time points compared with the baseline values.Dose 0, D0; Dose 4, D10; Dose 8, D19; Dose 12, D28.All values are expressed as the mean ± standard error.

Figure 3 .
Figure 3. Changes in lesion scores at different time points compared with the baseline values.Dose 0, D0; Dose 4, D10; Dose 8, D19; Dose 12, D28.All values are expressed as the mean ± standard error.

Figure 4 .
Figure 4. Changes in TEWL values at specific time points compared with the baseline values.Dose 0, D0; Dose 12, D28.All values are expressed as the mean ± standard error.TEWL, transepidermal water loss.

Figure 4 .
Figure 4. Changes in TEWL values at specific time points compared with the baseline values.Dose 0, D0; Dose 12, D28.All values are expressed as the mean ± standard error.TEWL, transepidermal water loss.

16 Figure 6 .
Figure 6.Analysis of alpha diversity of the skin microbiome.Changes in (A) observed OTUs, (B) Chao1, and (C) Shannon indices compared with the baseline values.Dose 0, D0; Dose 12, D28.All values are expressed as the mean ± standard error.

Figure 6 .
Figure 6.Analysis of alpha diversity of the skin microbiome.Changes in (A) observed OTUs, (B) Chao1, and (C) Shannon indices compared with the baseline values.Dose 0, D0; Dose 12, D28.All values are expressed as the mean ± standard error.

Figure 7 .
Figure 7. Principal coordinates analysis (PCoA) plot using the generalized UniFrac metric before (A) and after treatment (B), respectively.PCoA plot of all twelve samples (C).

Figure 7 .
Figure 7. Principal coordinates analysis (PCoA) plot using the generalized UniFrac metric before (A) and after treatment (B), respectively.PCoA plot of all twelve samples (C).

Table 2 .
Summary of the alpha diversity indices of the skin microbiome pre and post-treatment.

Table 3 .
Changes in observed OTUs and Chao1 and Shannon indices in the control, cExos, and hExos groups compared with the baseline values.