Vet Dermatol 2020

DOI: 10.1111/vde.12894

Serum canine thymus and activation-regulated
chemokine (TARC/CCL17) concentrations correlate withdisease severity and therapeutic responses in dogs withatopic dermatitis
Ryota Asahina* , Kazunori Ueda†, Yuri Oshima‡, Toshitaka Kanei*, Masahiro Kato§,
Masutaka Furue¶, Toshihiro Tsukui§, Masahiko Nagata**
and Sadatoshi Maeda**Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan†Yokohama Yamate Dog and Cat Medical Center, 27-4 Kashiwaba Naka-ku Yokohama, Kanagawa, 231-0866, Japan‡Dermatology Services for Dogs and Cats, TANDEM Hirano Bld. 1F, 2-11-14 Hirano Koto-ku, Tokyo, 135-0023, Japan§Animal Life Science Laboratories, Nippon Zenyaku Kogyo Co., Ltd., 1-1 Tairanoue Sasagawa Asaka-machi Koriyama, Fukushima, 963-0196, Japan¶Department of Dermatology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi Higashi-ku, Fukuoka, 812-8582, Japan**Dermatology Service, Veterinary Specialists Emergency Center, 815 Ishigami Kawaguchi, Saitama, 333-0823, Japan
Correspondence: Sadatoshi Maeda, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido,
Gifu 501-1193, Japan. E-mail: sadat@gifu-u.ac.jp

Background – Thymus and activation-regulated chemokine (TARC/CCL17) has been implicated in the pathogenesis of canine atopic dermatitis (cAD). Serum TARC concentrations are a reliable biomarker for human atopic dermatitis; however, their potential as a biomarker for cAD has not been investigated.
Hypothesis/Objectives – To investigate whether serum TARC concentrations correlate with disease severity
and therapeutic responses for cAD.
Animals – Thirty-nine dogs with cAD and 42 healthy dogs were recruited.
Methods and materials – Serum TARC concentrations in dogs with cAD and healthy dogs were measured bysandwich ELISA with anti-canine TARC antibodies. The clinical severity of cAD was scored using the validated
Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04). Serum TARC concentrations werecompared between dogs with cAD and healthy controls, and their relationship with CADESI-04 was examined.
Serum TARC concentrations also were measured in 20 dogs with cAD treated with prednisolone or oclacitinib for
four weeks.
Results – Serum TARC concentrations were significantly higher in dogs with cAD than in healthy dogs
(P < 0.001). In dogs with cAD, serum TARC concentrations correlated with CADESI-04 scores (q = 0.457,
P < 0.01). Furthermore, serum TARC concentrations significantly decreased in treated dogs with the attenuationof clinical signs (P < 0.001). Changes in serum TARC concentrations before and after treatment correlated withthose in CADESI-04 scores (q = 0.746, P < 0.001).
Conclusions and clinical relevance – Serum TARC concentrations have potential as a clinical and research tool
for the objective evaluation of disease severity and therapeutic responses for cAD.

Introduction
Canine atopic dermatitis (cAD) is a chronic inflammatory
and pruritic skin disease. It shares many clinical characteristics with its counterpart in humans, such as a genetic
predisposition, early age of onset, predilection sites ofaffected skin, a relationship with epidermal barrier
defects and frequent colonization by Staphylococcus.1
Furthermore, dogs with cAD show characteristic laboratory findings, with elevated allergen-specific immunoglobulin (Ig)E concentrations and increased eosinophil counts

Accepted 9 July 2020
Ryota Asahina and Kazunori Ueda are joint first authors.
Sources of Funding: This study was self-funded.
Conflict of Interest: No conflicts of interest have been declared.

in peripheral blood that may be attributed to excessive
amounts of Type 2 helper T (Th2) cytokines.2–4 Previous
studies reported that concentrations of Th2 cytokines,
including interleukin (IL)-4, IL-5 and IL-13, in serum,5
peripheral blood mononuclear cells3 and lesional skin,6–8
were higher in atopic dogs than in healthy dogs. One
study also found elevated numbers of IL-4-expressing
helper T cells in the peripheral blood of dogs with cAD.9
Therefore, cAD is a Th2-associated inflammatory disease,similar to human atopic dermatitis (AD).
Thymus and activation-regulated chemokine (TARC/
CCL17) is a functional ligand for CC chemokine receptor 4(CCR4), which is selectively expressed on Th2 cells.10,11
The number of CCR4+ helper T cells was reported to beelevated in the peripheral blood of dogs with cAD.12 Furthermore, previous studies demonstrated the preferential
transcription of CCR4 and its ligand TARC in the lesional© 2020 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the European Society of Veterinary Dermatology and the American College of Veterinary Dermatology
1This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License,
which permits use, distribution and reproduction in any medium, provided the original work is properly cited andis not used for commercial purposes.

Asahina et al.

skin of dogs with cAD, and not in nonlesional and healthyskin.13,14 Immunohistochemical analyses revealed that
TARC was expressed in the lesional keratinocytes ofcAD.11 In an in vitro study using canine keratinocytes,
TARC was induced by an agonist of protease-activatedreceptor-2 that recognized proteases derived from house
dust mites (HDM), a major allergen in cAD.15 Previous
studies showed that the transcription of TARC was
upregulated, with the greatest increases being observed
among the genes examined in the lesional skin of experimentally sensitized dogs with HDM antigens.16,17 In
another study using an experimental cAD model, a CCR4
antagonist reduced the infiltration of CCR4+ mononuclear
cells into skin.18 Therefore, TARC likely plays a crucial rolein the pathogenesis of cAD by facilitating the migration ofTh2 cells to lesional skin.
Clinical studies of human AD demonstrated that serumor plasma TARC concentrations correlated with disease
severity.19–23 Additionally, among several biomarkers,
such as serum lactate dehydrogenase (LDH), total IgE
concentrations and peripheral eosinophil counts, serum
TARC concentrations more strongly correlated with clinical scores.24,25 Serum TARC concentrations are currently
the most sensitive biomarker for assessing the disease
severity of human AD. Furthermore, serum TARC concentrations in patients with human AD decreased withcorresponding improvements in clinical signs after treatments with topical agents, including corticosteroids and/or tacrolimus19,22,26 and oral ciclosporin.20,25 Accordingly,
serum TARC concentrations also have been used as an
objective marker to evaluate therapeutic responses inhuman AD.24
Although increasing evidence implicates TARC in cAD,
it currently remains unclear whether serum TARC concentrations have potential as a biomarker for cAD. In thepresent study, we investigated whether serum TARC
concentrations correlate with disease severity and therapeutic responses for cAD using a canine TARC-specific
ELISA system.

Methods and materials
Animals
Thirty-nine client-owned dogs with spontaneous cAD in 17 veterinary
hospitals were recruited consecutively for the present study. The
age of animals ranged between one and 15 years (median age eight
years); there were 22 females (16 neutered) and 17 males (nine neutered). Breeds included Shiba inus (n = 19), mongrels (n = 8), French
bulldogs (n = 4) and one each of the following breeds: beagle, Bordercollie, Labrador retriever, Maltese, miniature dachshund, Samoyed,
toy poodle and West Highland white terrier. The diagnosis of cAD
was based on the combination of compatible clinical features27 andthe fulfilment of at least five of eight criteria.28 Other pruritic skin diseases, such as flea allergy dermatitis, scabies, demodicosis, and bacterial or Malassezia dermatitis, were excluded based on routinedermatological examinations and therapeutic trials. In cases exhibiting year-round clinical signs, an elimination diet trial for a minimum ofeight weeks was performed to rule out a cutaneous adverse foodreaction. All affected dogs had serological evidence of allergen-specific IgE. Tests for Dermatophagoides farinae (Der f) HDM or Der f 2
were performed on all dogs (Nippon Zenyaku Kogyo, Zenoaq;
Fukushima, Japan). In cases in which these tests were negative,

other serum allergen-specific IgE tests for environmental allergens,such as Der f HDM, Der f 2, grasses and pollens, were performed (atAnimal Allergy Clinical Laboratories; Kanagawa, Japan).29
Forty-two healthy dogs were used as control samples. Dogs wereconsidered to be healthy based on their medical history and a physical examination. The age of animals ranged between one and 13
years (median age 4.5 years); there were 24 females (15 neutered)and 18 males (seven neutered). They originated from a research beagle colony (n = 12) or were privately owned (n = 30). The privately
owned dog breeds included Shiba inu (n = 21), golden retriever(n = 5) and one each of the following breeds: cavalier King Charles
spaniel, French bulldog, Shetland sheepdog and toy poodle.
Research beagles were kept for experimental purposes under a protocol approved by the Animal Care and Use Committee of our institute (#17014). In Japan, ethics committees are not available forprivate-practice animal hospitals. Even so, this study was conductedin accordance with the ethical codes of the Japan Veterinary Medical
Association.30

Clinical score assessment and treatment for cAD
The validated Canine Atopic Dermatitis Extent and Severity Index,
4th iteration (CADESI-04) was used to evaluate the severity oflesions.31 Twenty of 39 affected dogs had been treated with oral
prednisolone (Predonin, Shionogi & Co., Ltd.; Osaka, Japan; 0.5–
1.0 mg/kg once daily for six days, then every other day; n = 11) ororal oclacitinib (Apoquel, Zoetis Inc.; Florham Park, NJ, USA; 0.4–
0.6 mg/kg twice daily for 14 days, then once daily; n = 9) for 28 days,
according to a previous study.32 CADESI-04 scores were evaluatedbefore and after treatment.

Blood samples
Blood samples were collected from dogs with cAD and client-owned
healthy dogs that visited the clinic for routine check-ups or vaccinations; the owners of all dogs consented to sample collection for the
purpose of this study. One millilitre blood samples were obtained
from each dog and centrifuged to separate serum. For all recruiteddogs, any anti-inflammatory agents, including glucocorticoids, ciclosporin, oclacitinib or antihistamines were withdrawn for at least two
weeks.33 Blood collection was performed on all dogs with cAD.
Twenty of 39 atopic dogs also underwent blood sampling after fourweeks of treatment with oral prednisolone or oclacitinib. Serum samples were stored at !30°C until analysed.

Recombinant canine TARC
The recombinant canine TARC protein was produced using an
Escherichia coli expression system. The cDNA sequence, excluding
the signal sequence, was inserted into an E. coli expression vector(pGEX-4T-1, 28954549, GE Healthcare Life Sciences; Little Chalfont,
UK). The recombinant canine TARC protein was purified as a fusion
protein with glutathione S-transferase (GST); the mature protein and
GST protein were cleaved using thrombin (GE Healthcare Life
Sciences; 27-0846-01) and only the mature protein was purified.

Anti-canine TARC monoclonal and polyclonal
antibodies
The cDNA of canine TARC was inserted into a mammalian expression vector (pcDNA3.1, V79020, Thermo Fisher Scientific; Waltham,
MA, USA). Mice (BALB/c) were immunized with plasmid DNA toestablish a monoclonal antibody. The recombinant protein was used
for monoclonal antibody screening. Rabbits were immunized withthe same plasmid DNA that was used to develop the monoclonal
antibody in order to generate a polyclonal antibody. After DNA immunization, an increase in the antibody titre was confirmed using a
recombinant protein and blood was collected to obtain a polyclonal
antibody. The production of anti-canine TARC antibodies using miceand rabbits was approved by the Animal Care and Use Committee of
our institute (#Z120720, #Z130413).

© 2020 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the European Society of Veterinary Derma2
tology and the American College of Veterinary Dermatology

Serum TARC in canine atopic dermatitis

Table 1. Breed, age, sex, weight, Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04) scores and serum thymus andactivation-regulated chemokine (TARC) concentrations at baseline and after treatment in dogs with atopic dermatitis
Baseline

After treatment

Case

Breed

Age
(years)Sex

Weight(kg)

CADESI-04
score

Serum TARC
concentration(pg/mL)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39

Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
French bulldog
French bulldog
French bulldog
Mixed breed
Mixed breed
BeagleMalteseMiniature dachshund
Toy poodle
West Highland white terrier
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Mixed breed
Mixed breed
Mixed breed
Mixed breed
Mixed breed
Mixed breed
Border collie
French bulldog
Labrador retriever
Samoyed

6
6
7
7
8
8
11
13
13
13
9
7
11
5
11
12
11
10
2
9
1
3
8
8
10
11
13
14
15
3
3
5
6
6
8
10
1
11
7

SF
CM
F
CM
SF
SF
SF
SF
SF
CM
SF
SF
F
M
M
SF
SF
SF
CM
M
M
M
F
M
M
F
F
SF
SF
SF
CM
F
CM
CM
M
CM
SF
CM
SF

8
11
8
11
8
10
14
8
7.2
10
11
10.6
8.2
13
23
9
6.5
3
4.2
8
8.1
11
10.5
24
9
9
8
15.2
7.4
5.7
19
9
6.8
11
8.6
17.4
8
33
23

41
50
40
7
54
37
25
105
32
41
65
22
139
43
48
19
53
13
14
46
4
65
34
103
46
15
28
68
69
10
18
42
20
44
59
36
4
24
15

3,143
2,415
1,591
663
2,113
2,447
4,563
5,469
2,135
1,753
5,006
3,275
3,173
6,502
4,406
953
1,558
4,060
1,338
2,195
2,540
7,152
2,040
6,372
3,626
1,553
1,443
2,153
6,816
3,261
1,300
769
2,057
5,078
1,287
3,760
945
1,535
2,227

CADESI-04
score

Serum
TARC
concentration(pg/mL)

Treatment

19
25
10
4
10
9
17
53
2
12
35
2
117
7
15
5
32
0
4
17

1,693
346
310
309
189
1,532
3,937
564
195
183
1,825
251
1,376
3,564
2,052
150
884
2,708
632
177

Prednisolone
Prednisolone
Prednisolone
Prednisolone
Oclacitinib
Prednisolone
Prednisolone
Prednisolone
Oclacitinib
Prednisolone
Oclacitinib
Oclacitinib
Oclacitinib
Prednisolone
Oclacitinib
Prednisolone
Oclacitinib
Oclacitinib
Prednisolone
Oclacitinib

CM, castrated male; F, female; M, male; SF, spayed female.

ELISA
The anti-TARC monoclonal antibody with carbonate buffer (0.25 lg/mL) was immobilized on ELISA plates (Thermo Scientific Nunc,
439454, Thermo Fisher Scientific) in 100 lL, and incubated at 4°C
overnight. ELISA plates were washed using PBST (Phosphate buffered saline containing 0.05% Tween-20), 100 lL of sera diluted
two-fold with a blocking buffer (four-fold Block Ace in PBST, UK-B80,
Yukijirushi; Tokyo, Japan) was added and then plates were kept atroom temperature for 2 h. After washing with washing buffer, theanti-TARC polyclonal antibody, 400-fold diluted with blocking buffer,
was added in 100 lL increments and the plates were kept at room
temperature for 2 h. After washing, horseradish peroxidase (HRP)conjugated goat anti-rabbit IgG (656120, Invitrogen; Carlsbad, CA,USA), 2,000-fold diluted with blocking buffer, was added to theplates, which were then kept at room temperature for 2 h. Afterwashing, 3,3-,5,5-tetramethylbenzidine (3405-100TAB, SigmaAldrich, St Louis, MO, USA) solution was added to the plates in
100 lL increments and plates were then kept at room temperaturefor 10 min. To stop the reaction, 50 lL of 2N H2SO4 was added tothe plates. Plates were read at a dual wavelength of 450–570 nmwith a micro plate leader (model 680, 168-1000, Bio-Rad; Hercules,
CA, USA). The assay was performed in duplicate on the same day.
Diluted recombinant TARC protein from 98 to 50,000 pg/mL, insteadof sera, was added to the plate in duplicate to prepare a calibration
curve. Optical density values were increased proportionally with concentrations of the recombinant canine TARC (R2 = 0.9961,
P < 0.001; Figure S1). The workflow of ELISA is shown in Figure S2.

Statistical analysis
Serum TARC concentrations were measured as continuous variables. The results of serum TARC concentrations were represented
as medians with interquartile ranges (IQR: 25–75th percentiles)
owing to the non-normal data distribution. Differences in serum
TARC concentrations as the dependent variables were analysedusing the Wilcoxon–Mann–Whitney U-test for comparisons between
the independent variables of group – dogs with cAD and healthy
dogs. The Wilcoxon signed rank test was used to compare paired
serum TARC concentrations before and after treatment. Similarly,© 2020 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the European Society of Veterinary Dermatology and the American College of Veterinary Dermatology
3Asahina et al.

Table 2. Breed, age, sex, weight and serum thymus and activation-regulated chemokine (TARC) concentrations in healthy dogs
Case

Breed

Age (years)

Sex

Weight (kg)Serum TARC
concentration (pg/mL)

1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42

Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
Shiba inu
BeagleBeagleBeagleBeagleBeagleBeagleBeagleBeagleBeagleBeagleBeagleBeagleGolden retriever
Golden retriever
Golden retriever
Golden retriever
Golden retriever
Cavalier King Charles spaniel
French bulldog
Shetland sheepdog
Toy poodle

0.6
1
1
2
2
2
3
4
6
6
6
6
7
7
7
8
8
9
11
12
13
2
3
3
3
3
3
3
4
4
4
5
9
3
4
8
9
10
5
1
8
5

M
CM
CM
SF
SF
SF
SF
M
F
SF
M
M
F
SF
SF
SF
M
SF
CM
SF
CM
M
F
F
F
M
M
M
F
F
M
M
F
CM
SF
SF
SF
SF
SF
F
CM
CM

6.4
7.9
13
11.2
13.2
11.5
8.7
8
9.2
8.5
10.5
10.2
6.6
12.9
9.9
12.3
8.7
10.3
10.4
9.7
11
10
10
10
10
10
10
10
10
10
10
10
10
33.6
28
24.5
29
28.7
8.5
12.8
13.5
3.4

1,330
180
205
183
416
117
283
249
0
150
2,243
123
0
409
192
164
343
201
157
614
146
164
245
178
194
132
149
124
189
229
281
322
146
346
205
250
172
127
0
391
122
82

CM, castrated male; F, female; M, male; SF, spayed female.

CADESI-04 scores were collected as continuous variables. The relationship between serum TARC concentrations and CADESI-04
scores was analysed by the Spearman rank correlation test. A valueof P < 0.05 was considered to be significant. Statistical analyses
were performed using PRISM 8 software (GraphPad; San Diego, CA,
USA).

Results
Animals and clinical data
Forty-two healthy dogs (male:female ratio 18:24; medianage 4.5 years; median weight 10.0 kg) and 39 dogs withcAD (17:22; 8 years; 9.0 kg) were recruited. No significant differences were observed in age, sex or weight
between healthy and atopic dogs (data not shown). In
both groups, Shiba inus accounted for approximately halfof the cases (21 of 42 healthy dogs, 19 of 39 atopic dogs).
Index scores in 39 atopic dogs ranged between 4 and 139(median 40) on the CADESI-04 scale, which has amaximum score of 180.31 The severity of cAD included
mild (CADESI-04 index 0–34, 17 dogs), moderate (35–59,
15 dogs) or severe (≥60, seven dogs) according to the
CADESI-04 severity categories.31 CADESI-04 scores of allhealthy dogs were 0. Twenty dogs with cAD were treatedwith prednisolone (11 dogs) or oclacitinib (nine dogs). The
median CADESI-04 score was 41 (range 7–139) before
treatment and decreased to 11 (range 0–117) after treatment. Breed, age, sex, weight, treatment and CADESI-04
scores at baseline and after treatment in dogs with cAD
are listed in Table 1. The breed, age, sex and weight ofhealthy dogs are shown in Table 2.
Serum TARC concentrations in dogs with cAD and
healthy dogs
Serum TARC concentrations were >10-fold higher in dogs
with cAD than in healthy dogs. The median serum TARC
concentration of dogs with cAD was 2,227 pg/mL (IQR

© 2020 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the European Society of Veterinary Derma4
tology and the American College of Veterinary Dermatology

Serum TARC in canine atopic dermatitis

Figure 2. Relationship between serum thymus and activation-regulated chemokine (TARC) concentrations and Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04) scores indogs with atopic dermatitis (n = 39).
Serum TARC concentrations in dogs positively correlated withCADESI-04 scores (q = 0.457, P < 0.01). **P < 0.01 by the Spearman rank correlation test.

Figure 1. Serum thymus and activation-regulated chemokine (TARC)
concentrations in dogs with atopic dermatitis (cAD) (n = 39) andhealthy dogs (n = 42).
Serum TARC concentrations were significantly higher in dogs withatopic dermatitis than in healthy controls (P < 0.001). The lines within
the boxes indicate the median serum TARC concentrations and theupper and lower boundaries of the boxes represent the 25th and75th percentiles, respectively. ***P < 0.001 by the Wilcoxon–Mann–
Whitney U-test.

1,556–3,910), whereas that of healthy dogs was 183 pg/
mL (IQR 146–273). Serum TARC concentrations were significantly higher in dogs with cAD than in healthy dogs
(P < 0.001; Figure 1).
Relationship between serum TARC concentrations
and CADESI-04 scores in dogs with cAD
In order to assess the utility of serum TARC concentrations as a marker for clinical severity, we examined therelationship between serum TARC concentrations and
CADESI-04 scores. Serum TARC concentrations in dogs
with cAD correlated with CADESI-04 scores (q = 0.457,
P < 0.01; Figure 2).
Serum TARC concentrations before and after
treatment in dogs with cAD
We measured serum TARC concentrations in 20 dogs
with cAD before and after systemic treatment with oral
prednisolone or oclacitinib. In all 20 dogs, serum TARC
concentrations significantly decreased after four weeks
of treatment in accordance with the amelioration of skinlesions (P < 0.001). Serum TARC concentrations
decreased from 2,431 pg/mL (IQR 1,713–4,147) before
treatment to 598 pg/mL (IQR 237–1,726) after treatment(Figure 3).

Relationship between changes in serum TARC
concentrations and CADESI-04 scores
In order to assess the utility of serum TARC concentrations as a marker for treatment responses, we examined
the relationship between changes in TARC concentrations and CADESI-04 scores after treatment. Changes inserum TARC concentrations correlated with those in
CADESI-04 scores (q = 0.746, P < 0.001; Figure 4).

Discussion
To the best of the authors’ knowledge, this is the firststudy to investigate the relationship between serum
TARC concentrations and the disease severity of cAD. To
evaluate the usefulness of serum TARC concentrations
as a biomarker for cAD, we established a sandwich ELISA
system for quantifying canine TARC. Previous studies
demonstrated that plasma and serum TARC concentrations in human patients with AD correlated with disease
severity.19–23 Serum TARC concentrations were found to
be markedly higher than plasma TARC concentrations inpatients with AD because platelets in serum released
large amounts of TARC.19 Furthermore, serum TARC concentrations were stable independent of the freeze–thaw
process, whereas those in plasma contaminated with platelets were elevated after freeze–thaw due to the releaseof TARC.34 A meta-analysis proposed serum TARC concentrations as the most reliable biomarker for the disease
severity of human AD.35 Therefore, herein we used
serum samples to quantify canine TARC.
The results obtained showed that serum TARC concentrations were >10-fold higher in dogs with cAD than in
healthy controls, which is consistent with previous findings on human AD.19,22 The median serum TARC concentrations in dogs with cAD and healthy controls were
2,227 pg/mL (IQR 1,556–3,910) and 183 pg/mL (IQR
146–273), respectively. These concentrations closely corresponded to previously reported values from human© 2020 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the European Society of Veterinary Dermatology and the American College of Veterinary Dermatology
5Asahina et al.

Figure 4. Relationship between changes in serum thymus and activation-regulated chemokine (TARC) concentrations and Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04)
scores (n = 20).
Changes in serum TARC concentrations before and after treatment
with either oral prednisolone (n = 11) or oclacitinib (n = 9) positively
correlated with the CADESI-04 scores (q = 0.746, P < 0.001).
***P < 0.001 by the Spearman rank correlation test.

Figure 3. Serum thymus and activation-regulated chemokine (TARC)
concentrations before and after treatment in dogs with atopic dermatitis (cAD) (n = 20).
Serum TARC concentrations showed a significant decrease after fourweeks of treatment with either oral prednisolone (n = 11) or oclacitinib (n = 9) (P < 0.001). ***P < 0.001 by the Wilcoxon–Mann–Whitney U-test.

counterparts in the literature with an average serum
TARC concentration of 1,480 pg/mL (IQR 640–3,540) inpatients with AD and 250 pg/mL (IQR 240–260) in healthy
controls.25 However, a previous study also showed that ahigh serum TARC concentration was not specific tohuman AD. An elevated serum TARC concentration also
has been reported in human patients with other skin diseases, such as bullous pemphigoid, scabies, polymorphic
prurigo, cutaneous T-cell lymphoma, drug eruption, pustular dermatosis and other internal disorders.24 Based onthese findings, serum TARC concentrations have notbeen solely used to establish a diagnosis of human AD.
Thus, additional studies that evaluate TARC concentrations in dogs with other diseases, besides cAD, areneeded to assess the effect of other inflammatory or neoplastic skin conditions on TARC concentrations.
In the present study, serum TARC concentrations in
two healthy dogs were >1,000 pg/mL (cases 1 and 11;
Table 2). This anomaly may be partially due to an age-related difference in TARC concentrations. One of the twodogs with a high serum TARC concentration (Case 1) was
only 7 months old; the mean age of the healthy controls
with lower serum TARC concentrations was 4.5 years. A
previous study on healthy human subjects reported that
serum TARC concentrations were higher in infants (<1year old) than in children (>2–5 and 6–16 years old) andadults (>16 years old).36 The same study also found thatto achieve the same diagnostic accuracy of human AD,
different cut-off values were required for different age
groups.36 Because serum TARC concentrations in healthy
human subjects differ among infants, children and adults,
healthy thresholds also vary according to age, based on
current evidence.24 Therefore, further studies are needed
to investigate age-related differences in serum TARC concentrations in healthy and affected dogs in order to establish appropriate reference ranges. Additionally, it isimportant to note that one (Case 1) of these two outliers
subsequently developed the typical clinical signs of cAD,suggesting that TARC concentrations became elevatedbefore the onset of cAD. This hypothesis is supported byprevious findings indicating the potential of serum TARC
concentrations to detect subclinical dermal inflammationor the early stages of human AD.37,38 Because theremaining outlier (Case 11) was lost to follow-up, wewere unable to confirm whether this subject eventuallydeveloped cAD or another disease that may have caused
an elevated TARC concentration. Although it may be ofclinical significance, it was not possible to confirm the utility of TARC concentrations in predicting the onset of cAD
in the present study. Thus, further studies are necessary
to evaluate that serum TARC concentrations would be agood marker to predict onset of cAD, which may lead toprophylactic administration of anti-inflammatory agents.
The present results showed that serum TARC concentrations correlated with CADESI-04 scores, suggesting
the potential of TARC as a useful objective measure forthe disease severity of cAD, similar to human AD.20–22 In
previous studies on human AD, among available markers,
including total IgE concentrations, peripheral eosinophil
counts and serum LDH concentrations, serum TARC
more strongly correlated with clinical scores.24,25 In© 2020 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the European Society of Veterinary Derma6
tology and the American College of Veterinary Dermatology

Serum TARC in canine atopic dermatitis

studies on cAD, although total IgE concentrations weresignificantly higher in dogs with cAD than in healthy controls, they did not correlate with clinical severity according
to the Canine Atopic Dermatitis Lesion Index.2 Furthermore, the lesional severity scores of dogs with cAD did
not correlate with the serum concentrations of othercytokines, including IL-17, IL-31 and macrophage migration inhibitory factor.39 Therefore, serum TARC concentrations appear to be a more accurate marker thanpreviously studied biomarkers and correspond well withthe lesional severity of cAD.
In the present study, we also investigated whether
serum TARC concentrations reflected therapeutic
responses. To the best of our knowledge, a serum biomarker has not yet been used to assess spontaneouscAD before and after treatment in individual subjects. We
demonstrated that serum TARC concentrations
decreased with corresponding improvements in clinicalsigns in all dogs treated with prednisolone or oclacitinib,
both of which are first-line treatments in the managementof cAD exacerbations. We also found a strong correlation
between changes in TARC concentrations and CADESI04 scores after treatment. The present results show thepotential of serum TARC concentrations as an objective
marker for evaluating therapeutic responses in cAD treated with prednisolone or oclacitinib.
The comparison of TARC concentrations before andafter treatment with prednisolone and oclacitinib would
have been clinically interesting. However, because dogs
were not randomized into each group, there was a risk ofallocation bias leading to inaccurate interpretation. A previous study using a mouse model of allergic dermatitis
reported that oclacitinib reduced the TARC concentrationin skin.40 In this model, oclacitinib also suppressed IL-31,
TNF-a and TSLP, although abrupt withdrawal led to a
rapid increase of these cytokines.40,41 TNF-a has beenshown to be the mediator inducing TARC release fromcanine keratinocytes.42,43 Thus, oclacitinib may reduce
serum TARC concentrations by inhibiting TNF-a in keratinocytes. Common treatment regimens for cAD also
may include the use of topical glucocorticoids, oral ciclosporin or lokivetmab injections, which were not evaluatedin the present study. In human patients with AD, serum
TARC concentrations decreased after the treatmentswith topical glucocorticoids19,22,26 or oral ciclosporin.20,25
Additionally, an in vitro study revealed that IL-31 induced
TARC in human keratinocytes.44 These studies suggestthat serum TARC has the potential to act as a marker forevaluating therapeutic responses in cAD treated withother drugs, although the generalizability of our resultscannot be assumed for these drugs. Therefore, specificinteractions between serum TARC concentrations andother treatment modalities require further investigation.
In conclusion, serum TARC concentrations correlatedwith the lesional severity scores of cAD, indicating that
serum TARC concentrations are an objective marker forassessing disease severity and therapeutic responses incAD. The assessment of disease severity and efficacy oftreatment for cAD has relied on traditional markers susceptible to various confounders. The use of TARC as abiomarker will allow standardized evaluations not only inclinical settings, but also importantly in research settings,

thereby facilitating the cross-comparison of trials and its
use as a surrogate end-point. Further studies are needed
to confirm the potential of serum TARC concentrations as
an objective and predictive marker in the treatment monitoring of cAD.

Acknowledgements
The authors would like to thank Takahiro Akita (Akita Animal Hospital), Daisuke Itsukaichi (Poplar Animal Hospital),
Chiaki Kitanaka (SENA Animal Hospital Rakuhoku Animal
Wellness Center), Jin Kozakai (Ai Animal Hospital
Kashima), Yuko Machida (AZ Animal Hospital), Hisanori
Mutoh (Miyaki Animal Clinic), Hiromi Oboso (Cookie Animal Hospital), Taichi Oshima (Taichi Animal Hospital) andAkihiro Sugeno (Sugeno Animal Hospital) for collecting
blood samples.

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Supporting Information
Additional Supporting Information may be found in the
online version of this article.
Figure S1. A standard curve of the sandwich ELISA with
serial dilutions of purified recombinant canine TARC ranging from 98 to 50,000 pg/mL.
Figure S2. The workflow of the sandwich ELISA assay.

!sume
!
Re
! te
! implique!e dans laContexte – La TARC/CCL17 (thymus and activation-regulated chemokine) a e!nie de la dermatite atopique canine (cAD). Les concentrations se!riques de TARC sont un biomarpathoge
queur fiable chez l’homme atopique ; cependant, leur potentiel en tant que biomarqueur pour cAD n’a pas! te!e!tudie!.
e© 2020 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the European Society of Veterinary Derma8
tology and the American College of Veterinary Dermatology

Serum TARC in canine atopic dermatitis#ses/Objectifs – Etudier si les concentrations se!riques de TARC corre#lent avec la se
!ve
!rite
! de laHypothe!ponses the!rapeutiques pour cAD.
maladie et les re! te! inclus.
Sujets – Trente – neuf chiens avec cAD et 42 chiens sains ont e!riels et me!thodes – Les concentrations se!riques en TARC chez les chiens avec cAD et les chiens
Mate
! te
! mesure!es par ELISA avec anticorps anti-TARC canin. La se
!ve
!rite
! clinique de cAD a e! te! note!esains ont e
par CADESI-04 (Canine Atopic Dermatitis Extent and Severity Index, 4th iteration). Les concentrations de!riques ont e! te! compare!es entre les chiens avec cAD et les chiens sains contro^les, et leur relation
TARC se! te! e!tudie!e. Les concentrations se!riques de TARC ont aussi e! te! mesure!es chez 20
avec CADESI-04 a e!s par prednisolone ou oclacitinib pendant quatre semaines.
chiens avec cAD traite!sultats – Les concentrations se!riques de TARC e!taient significativement plus e!leve!es chez les chiensRe!riques deavec cAD que chez les chiens sains (P < 0.001). Chez les chiens avec cAD, les concentrations se!laient avec les scores de CADESI-04 (q = 0.457, P < 0.01). En outre, les concentrations se!riques
TARC corre!s avec atte
!nuation des signes cliniques (P <de TARC diminuaient significativement chez les chiens traite!riques de TARC avant et apre#s traitement corre!laient avec
0.001). Les changements de concentrations seceux des scores CADESI-04 (q = 0.746, P < 0.001).
!riques de TARC ont le potentiel d’e^tre unConclusions et importance clinique – Les concentrations se!valuation objective de l’intensite! de la maladie et de la re!ponse the!raoutil clinique et de recherche pour l’epeutique de la CAD.
RESUMEN! n – el timo y la quimiocina regulada por activacio!n (TARC/CCL17) se han implicado en la
Introduccio!nesis de la dermatitis ato!pica canina (cAD). Las concentraciones se!ricas de TARC son un biomarcapatoge!pica humana; sin embargo, no se ha investigado su potencial como biomarcador fiable en la dermatitis ato
dor en cAD.
! tesis/Objetivos – investigar si las concentraciones se!ricas de TARC se correlacionan con la gravedad
Hipo!uticas en cAD.
de la enfermedad y las respuestas terape
Animales – se incluyeron treinta y nueve perros con cAD y 42 perros sanos.
!todos y materiales – Las concentraciones se!ricas de TARC en perros con cAD y perros sanos seMemidieron mediante ELISA s!andwich con anticuerpos anti-TARC caninos. La gravedad cl!ınica de la cAD se! utilizando el !ındice validado de extensio!n y gravedad de la dermatitis ato!pica canina, cuarta versio!nvaloro(CADESI-04). Se compararon las concentraciones de TARC en suero entre perros con cAD y controles! su relacio!n con CADESI-04. Tambie!n se midieron las concentraciones se!ricas desanos, y se examino
TARC en 20 perros con cAD tratados con prednisolona u oclacitinib durante cuatro semanas.
!ricas de TARC fueron significativamente m!
Resultados – las concentraciones se
as altas en perros con cAD!ricas de TARC se correlacionaque en perros sanos (P <0,001). En perros con cAD, las concentraciones se!ricas deron con las puntuaciones de CADESI-04 (q = 0,457, P <0,01). Adem!
as, las concentraciones se!n de los signos cl!ınicos (PTARC disminuyeron significativamente en los perros tratados con la disminucio!ricas de TARC antes y despue!s del tratamiento se correla<0,001). Los cambios en las concentraciones se
cionaron con los de los valores de CADESI-04 (q = 0,746, P <0,001).
!ricas de TARC tienen potencial como herraConclusiones y relevancia cl!ınica – las concentraciones se!n para la evaluacio!n objetiva de la gravedad de la enfermedad y las respuesmienta cl!ınica y de investigacio!uticas en cAD.
tas terape
Zusammenfassung
Hintergrund – Dem Thymus und Activation-regulated Chemokine (TARC/CCL17) wird in der Pathogenese
der caninen atopischen Dermatitis (cAD) eine Rolle zugeschrieben. Serum TARC Konzentrationen sind ein€r dieverl€asslicher Biomarker bei der atopischen Dermatitis des Menschen; ihr Potential als Biomarker fu
cAD wurde jedoch noch nicht untersucht.
Tiere – Neununddreißig Hunde mit cAD und 42 gesunde Hunde wurden rekrutiert.
Methoden und Materialien – Serum TARC Konzentrationen bei Hunden mit cAD und bei gesunden Hun€rpern gemessen. Der klinische Schweden wurden mittels Sandwich ELISA mit anti-caninen TARC Antikoregrad der cAD wurde mittels validiertem Canine Atopic Dermatitis Extent and Severity Index, 4te
Ausgabe (CADESI-04) beurteilt. Die Serum TARC Konzentrationen wurden zwischen den Hunden mit cAD
und den gesunden Kontrollen verglichen und ihre Beziehung mittels CADESI-04 untersucht. Es wurden
auch die TARC Konzentrationen bei 20 Hunden mit cAD, die mit Prednisolon oder Oclacitinib vier Wochenlang behandelt worden waren, gemessen.
€her als bei den
Ergebnisse – Die Serum TARC Konzentrationen waren bei Hunden mit cAD signifikant ho
gesunden Hunden (P < 0,001). Bei Hunden mit cAD korrelierten die Serum TARC Konzentrationen mit den
CADESI-04 Werten (q = 0.746, P < 0.001). Weiters nahmen die Serum TARC Konzentrationen bei behandelten Hunden mit Abnahme der klinischen Zeichen ab (P < 0,001). Ver€
anderungen bei den Serum TARC
Konzentrationen vor und nach der Behandlung korrelierten mit denen der CADESI-04 Werte (q = 0.746, P <0.001).
© 2020 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the European Society of Veterinary Dermatology and the American College of Veterinary Dermatology
9Asahina et al.

Schlussfolgerungen und klinische Bedeutung – Die Serum TARC Konzentrationen haben Potential als
klinisches und wissenschaftliches Werkzeug zur objektiven Evaluierung der Schwere der Erkrankung und
der therapeutischen Resonanz bei cAD.
要約
背景 – 胸腺および活性化調節ケモカイン（TARC / CCL17）は、犬アトピー性皮膚炎（cAD）の病因に関与している。血清TARC濃度は、ヒトアトピー性皮膚炎の信頼できるバイオマーカーである。ただし、cADに対するバイオマーカーとしての可能性は調査されていない。
仮説/目的 – 本研究の目的は、血清TARC濃度が疾患の重症度およびcADの治療反応と相関するかどうかを調査することであった。
供試動物 – cAD犬39頭と健常犬42頭を募った。
材料と方法 – cAD犬および健常犬の血清TARC濃度を、抗犬TARC抗体を用いたサンドイッチELISAで測定した。 cADの臨床的重症度は、検証済みの犬アトピー性皮膚炎の程度および重症度指数、4thインテレーション（CADESI-04）を使用してスコア化された。cAD犬および健常犬間の血清TARC濃度を比較し、
CADESI-04との関係を調査した。血清TARC濃度も、プレドニゾロンまたはオクラシチニブで4週間治療されたcADの犬20頭で測定した。
結果 – 血清TARC濃度は、健常犬よりもcAD犬で有意に高かった（P <0.001）。 cAD犬では、血清TARC濃度がCADESI-04スコアと相関していた（q= 0.457、P <0.01）。さらに、治療された犬の血清TARC濃度
は、臨床徴候の減衰とともに有意に減少した（P <0.001）。治療前後の血清TARC濃度の変化は、CADESI-04スコアの変化と相関していた（q= 0.746、P <0.001）。
結論と臨床的関連性 – 血清TARC濃度は、cADの疾患の重症度および治療反応を客観的に評価する臨床および研究ツールとしての可能性を秘めている。
摘要
背景 – 胸腺和活化调节趋化因子(TARC/CCL17)与犬异位性皮炎(cAD)的发病机制有关。血清TARC浓度是人类特应性皮炎的可靠生物标志物；然而，尚未有研究报道其作为cAD生物标志物的潜力。
假设/目的 – 研究血清TARC浓度是否与cAD的疾病严重程度和治疗反应相关。
动物 – 招募了39 只cAD犬和42只健康犬。
方法和材料 – 通过夹心ELISA和抗犬TARC抗体测定cAD犬和健康犬的血清TARC浓度。使用犬异位性皮炎
程度和严重指数第4版(CADESI-04)对cAD的临床严重程度进行评分。比较cAD犬和健康对照犬的血清TARC
浓度，并检查其与CADESI-04的关系。还测定了泼尼松龙或奥拉替尼治疗4周的20只cAD犬，测定了血清
TARC浓度。
结果 – cAD犬血清TARC浓度显著高于健康犬(P<0.001)。对于cAD犬，血清TARC浓度与CADESI-04评分相关(q=0.457，P<0.01)。此外，治疗犬的血清TARC浓度显著降低，临床症状减轻(P<0.001)。治疗前后血清
TARC浓度变化与CADESI-04评分相关(q=0.746 ，P<0.001)。
结论和临床相关性 – 检测血清TARC浓度，有可能作为客观评价疾病严重程度和cAD治疗反应的临床和研
究工具。
Resumo^nese
Contexto – A quimiocina regulada pelo timo e ativac!~ao (TARC / CCL17) tem sido implicada na patoge!pica canina (DAC). As concentrac!o~es se!ricas de TARC s~da dermatite ato
ao um biomarcador confi!
avel para!pica humana; no entanto, seu potencial como biomarcador para CAD n~dermatite ato
ao foi investigado.
! tese/Objetivos – Investigar se as concentrac!o~es se!ricas de TARC se correlacionam com a gravidade
Hipo^uticas para DAC.
da doenc!a e as respostas terape
Animais – Trinta e nove c~aes com DAC e 42 c~
aes saud!
aveis foram recrutados.
!todos e materiais – As concentrac!o~es se!ricas de TARC em c~Me
aes com DAC e em c~aes saud!
aveis foram
medidas por ELISA sandu!ıche com anticorpos anti-TARC caninos. A gravidade cl!ınica da DAC foi avaliada!pica canina validado, 4ª iterac!~
usando o !ındice de extens~ao e gravidade da dermatite atoao (CADESI-04). As~es se!ricas de TARC foram comparadas entre c~concentrac!oaes com DAC e controles saud!
aveis, e sua~es se!ricas de TARC tambe!m foram mensuradas em
relac!~ao com CADESI-04 foi avaliada. As concentrac!o20 c~aes com DAC tratados com prednisolona ou oclacitinib por quatro semanas.
~es se!ricas de TARC foram significativamente maiores em c~
Resultados – As concentrac!oaes com DAC do~es se!ricas de TARC se correlacionaque em c~aes saud!aveis (P <0,001). Em c~aes com DAC, as concentrac!o~es de CADESI-04 (q = 0,457, P <0,01). Ale!m disso, as concentrac!o~es se!ricas de TARC
ram com as pontuac!odiminu!ıram significativamente nos c~aes tratados, com a atenuac!~
ao dos sinais cl!ınicos (P <0,001).
~es se!ricas de TARC antes e depois do tratamento correlacionaram-se com as
Mudanc!as nas concentrac!odos escores CADESI-04 (q = 0,746, P <0,001).
~ es e releva^ncia cl!ınica – As concentrac!o~es se!ricas de TARC te^m potencial como uma ferraConcluso^uticasmenta cl!ınica e de pesquisa para a avaliac!~ao objetiva da gravidade da doenc!a e das respostas terapepara DAC.

© 2020 The Authors. Veterinary Dermatology published by John Wiley & Sons Ltd on behalf of the European Society of Veterinary Derma10
tology and the American College of Veterinary Dermatology