See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/342699816

Establishment of a Mouse Model for Post‐Inflammatory Hyperpigmentation

Article  in  Pigment Cell & Melanoma Research · July 2020

DOI: 10.1111/pcmr.12911

CITATIONS READS

2 55

9 authors, including:

Kimiko Nakajima Kazumasa Wakamatsu

Kochi University Fujita Health University
98 PUBLICATIONS   1,352 CITATIONS    516 PUBLICATIONS   17,714 CITATIONS   

SEE PROFILE SEE PROFILE

Shosuke Ito Masahiro Hayashi

Fujita Health University University of Colorado
535 PUBLICATIONS   18,744 CITATIONS    74 PUBLICATIONS   658 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Free ElectronLaser View project

Genetic architecture of plumage colour variation in the barn owl. View project

All content following this page was uploaded by Kazumasa Wakamatsu on 19 September 2020.

The user has requested enhancement of the downloaded file.

The official journal of
INTERNATIONAL FEDERATION OF PIGMENT CELL SOCIETIES · SOCIETY FOR MELANOMA RESEARCH

PIGMENT CELL & MELANOMA

Research
Establishment of a mouse model for
post-inflammatory hyperpigmentation
Shoko Nakano | Yuko Abe | Kimiko Nakajima |
Shigetoshi Sano | Osamu Yamamoto | Kazumasa Wakamatsu |
Shosuke Ito | Masahiro Hayashi | Tamio Suzuki

DOI: 10.1111/pcmr.12911

If you wish to order reprints of this article,

please see the guidelines here

Supporting Information for this article is freely available here

EMAIL ALERTS
Receive free email alerts and stay up-to-date on what is published
in Pigment Cell & Melanoma Research – click here

Submit your next paper to PCMR online at http://mc.manuscriptcentral.com/pcmr

Subscribe to PCMR and stay up-to-date with the only journal committed to publishing
basic research in melanoma and pigment cell biology

As a member of the IFPCS or the SMR you automatically get online access to PCMR. Sign up as
a member today at www.ifpcs.org or at www.societymelanomaresarch.org

To take out a personal subscription, please click here

More information about Pigment Cell & Melanoma Research at www.pigment.org
 |
Received: 21 May 2020    Accepted: 23 June 2020

DOI: 10.1111/pcmr.12911

ORIGINAL ARTICLE

Establishment of a mouse model for post-inflammatory

hyperpigmentation

Shoko Nakano1  | Yuko Abe1  | Kimiko Nakajima2  | Shigetoshi Sano2  |

Osamu Yamamoto3  | Kazumasa Wakamatsu4  | Shosuke Ito4  |
Masahiro Hayashi1  | Tamio Suzuki1

1
Department of Dermatology, Faculty of
Medicine, Yamagata University, Yamagata, Abstract
Japan Post-inflammatory hyperpigmentation (PIH) is a common cutaneous condition that
2
Department of Dermatology, Kochi Medical
can cause a disfigured appearance. However, the pathophysiology of PIH remains
School, Kochi University, Nankoku, Japan
3
Division of Dermatology, Department of
poorly understood, at least in part, because an appropriate animal model for research
Medicine of Sensory and Motor Organs, has not been established. In order to analyze the pathomechanism of PIH, we suc-
Faculty of Medicine, Tottori University,
Yonago, Japan
cessfully induced PIH in a hairless version of transgenic mice (hk14-SCF Tg/HRM)
4
Department of Chemistry, Fujita Health that have a human-type epidermis containing melanin by repeated hapten application
University School of Medical Sciences, of 2,4-dinitrofluorobenzene. Histopathologic observation showed epidermal hyper-
Toyoake, Japan
plasia, predominant infiltrations of inflammatory cells, and melanin-containing cells
Correspondence in the dermis just after elicitation of the atopic dermatitis-like condition. At week 2,
Tamio Suzuki, Department of Dermatology,
Faculty of Medicine, Yamagata University, the findings were similar to the characteristics of PIH, that is, an increase of melanin
2-2-2, Iida-Nishi, Yamagata 990-9585, Japan. without spongiosis or liquid degeneration in the epidermis and an increase in dermal
Email: tamsuz@med.id.yamagata-u.ac.jp
melanophages. Dynamic analysis of melanin showed that the melanin in the dermis
Funding information remained for a longer duration than in the epidermis. Furthermore, immunohisto-
Eli Lilly Japan K.K.; Japan Society for
the Promotion of Science, Grant/Award chemical staining revealed that the majority of cells containing melanin were positive
Number: JP19K08742 for the anti-CD68 antibody, but negative for the anti-F4/80 antibody. These data
suggest that novel treatments of PIH should be targeted against macrophages and
should eventually lead to the development of new treatment modalities.

KEYWORDS

macrophage, melanin, melanocyte, mouse model, pigmentation

1 |  I NTRO D U C TI O N
Post-inflammatory hyperpigmentation (PIH) is an acquired pigmentary
disorder that results from a preceding inflammatory skin disorder, injury,
or similar cause (Callender, St Surin-Lord, Davis, & Maclin, 2011). PIH is
more common in darker-skinned individuals of Fitzpatrick skin photo-
types III–VI than in individuals with lighter skin (Davis & Callender, 2010)
and frequently occurs in east Asians. The causes of PIH are various,
including inflammatory conditions, such as atopic dermatitis, contact
dermatitis, and psoriasis, infections, treatments such as laser irradiation
and chemical peeling, sunburn, injury, and other forms of skin damage
(Silpa-Archa, Kohli, Chaowattanapanit, Lim, & Hamzavi, 2017). Among
those causes, PIH after atopic dermatitis often covers wide areas of skin
and can be a significant concern for many patients. PIH also causes cos-
metic issues that markedly diminish the quality of life (QOL) and restrict
social activities (Seghers et al., 2014). However, the pathophysiology of
PIH remains poorly understood, one reason being that an appropriate
animal model for research has not been established.

© 2020 John Wiley & Sons A/S . Published by John Wiley & Sons Ltd

Pigment Cell Melanoma Res. 2020;00:1–10.  |

wileyonlinelibrary.com/journal/pcmr     1
 |
2       NAKANO et al.
Melanin, the cause of PIH, is a pigment that is synthesized in
melanosomes, organelles produced in melanocytes (Bonaventure,
Domingues, & Larue,  2013). Melanosomes are transferred to sur-
rounding keratinocytes, where they move to the perinuclear area
and form a melanin cap that protects the nuclear DNA from ultra-
violet radiation (D'Mello, Finlay, Baguley, & Askarian-Amiri, 2016).
Under normal circumstances, melanin is mostly found in the basal
layer of the epidermis and is not present in the dermis. In the pro-
cess of the development of PIH, damage and inflammation, such as
dermatitis, occurs in the basal layer of the epidermis, which results in
melanin moving down from the epidermis to the dermis in the skin.
That melanin is then phagocytosed by macrophages and sometimes
fibroblasts in the dermis (Silpa-Archa et al., 2017). Therefore, an an-
imal model for PIH must have melanin in the epidermis; however, in
contrast to human skin, the epidermis of wild-type mice does not
contain melanocytes and melanin is not transferred to epidermal
keratinocytes. Previous attempts to induce PIH in murine skin by
repeatedly generating conditions that cause PIH in humans, such as
contact dermatitis, have thus far proved unsuccessful.
Our laboratory has successfully created a hairless version of
transgenic (Tg) mice (hk14-SCF Tg/HRM) that have a human-type
epidermis that contains a proportion of melanocytes similar to
human epidermis and in which melanin is also transferred to kera-
tinocytes (Kunisada et al., 1998). That Tg mouse has been used as
a murine model of Japanese human skin (Abe et al., 2016) and has
enabled extremely accurate studies of acquired pigmentation disor-
ders. In this study, we successfully induced PIH in that Tg mouse by
the induction of repeated contact dermatitis and have established a
reproducible mouse model of PIH.
2 |  M ATE R I A L S A N D M E TH O DS
2.1 | Production of a mouse model for PIH
Hk14-SCF Tg/HRM mice were previously developed and have mel-
anocytes in their epidermis, which mimics human skin (Kunisada
et al., 1998). In this study, male 16- to 20-week-old mice were used
unless otherwise noted. 2,4-dinitrofluorobenzene (DNFB; Wako) was
used as a hapten to induce chronic contact dermatitis as shown sche-
matically in Figure 1a. This experiment complied with the principles of
the Yamagata University Regulations for Animal Experiments and was
approved by the Animal Experiment Ethics Committee (R2070).
2.2 | Measurement of mouse dorsal skin color
The melanin content (melanin index) of the dorsal skin of 5 mice was
measured three times by evaluating skin color using a Mexameter®
(MX18; Courage + Khazaka Electronic GmbH), and the mean value
was calculated. This device quantifies the melanin content of the
skin in terms of its melanin index (Masuda, Yamashita, Hirao, &
Takahashi, 2009).
Significance
Studies of post-inflammatory hyperpigmentation (PIH),
which have rarely been investigated to date, can be ad-
vanced by using the mouse model established in this study.
In particular, in addition to the analysis of macrophages
that play an important role in the pathology of PIH, the de-
velopment of effective and useful treatments will advance.
This paper will be good news for many patients who suffer
from PIH.
2.3 | Histological observation of mouse dorsal skin
After mice were anesthetized using isoflurane, skin tissues were
excised and fixed in 10% formalin and then embedded in paraffin,
after which sections approximately 4 μm thick were prepared. These
sections were subjected to hematoxylin and eosin staining, Fontana-
Masson staining, and immunohistochemical staining with an anti-
Melan-A antibody (NBP1-30151, 1:100; Novus Biologicals).
2.4 | Changes in mRNA expression
levels of inflammatory cytokines after the
induction of repeated contact dermatitis
The mRNA expression levels of inflammatory cytokines in epider-
mal tissue were measured using real-time polymerase chain reaction
(PCR). The induction group contained 20 hk14-SCF Tg/HRM mice
(male:female ratio 1:1), and biopsies were taken from their dorsal skin
24 hr after the final application of DNFB. The no-treatment control
group contained 10 untreated hk14-SCF Tg/HRM mice (male:female
ratio 1:1). Each skin biopsy tissue was homogenized, and total RNA
was extracted using an RNA extraction kit (RNeasy Fibrous Tissue
Mini Kit; QIAGEN), after which cDNA was synthesized (RNA PCR
Kit Version 3.0; TaKaRa). Real-time PCR was performed on each
cDNA using SYBR Green detection by a Mx3000P system (Agilent),
following the Brilliant III Ultra-Fast SYBR® Green QRT PCR Master
Mix protocol (Agilent) using specific primers for IFNγ, TNFα, IL-2,
IL-4, IL-5, and IL-13 at the conditions reported previously (Nakajima
et al., 2011). The expression levels of mRNAs were analyzed by rela-
tive quantification using hypoxanthine phosphoribosyl transferase
(HPRT) as the internal control gene.
2.5 | Measurement of areas of epidermal and
dermal melanin distribution by image analysis
Fontana-Masson-stained tissue specimens were photographed with
an all-in-one optical microscope (BZ-X700; KEYENCE), and areas
of melanin distribution were measured using BZ-analyzer software
(KEYENCE). The epidermis was observed with a 44× objective and
NAKANO et al. |
      3

F I G U R E 1   Production of a mouse (a)

model for PIH and change in skin color.
(a) Tg mice were sensitized by the Sensitization Elicitation Follow-up time
application of 100 µl 0.5% DNFB to the
day 0, 1 (before) 7 14 21 28 35
abdominal region of each mouse twice on
DNFB (days)
successive days. At a frequency of twice
1 week
a week, 100 µl 0.15% DNFB was then
applied repeatedly on the dorsal skin of DNFB
0 2 4 8 12
each mouse a total of nine times. DNFB; (weeks)
2, 4-dinitrofluorobenzene. (b) Melanin Contact dermatitis
content (melanin index) of the dorsal skin repeatedly
was measured by evaluating skin color (b)
with a Mexameter®. The dorsal skin of
500
**

The mean of melanin index

each mouse was measured three times,
and mean values were calculated (n = 5).
400
** ** **
⁑p < .001. *p < .05. (c) The mice were
Error bars, means ± SEM. Welch's t-test.

300 * *
photographed once a week *
200
* *
Elicitation
repeatedly
100
before 0 1 2 3 4 5 6 7 8 9 11 12
Follow-up time (weeks)
(c)

the dermis with a 20× objective. Three random visual fields were 2.7 | Electron microscopic observation of dermal
measured in each specimen, and the means of five mice (five speci- melanin-containing cells
mens) were calculated.
Dorsal skin biopsies were taken 2 weeks after the induction of in-
flammation from two mice. After fixation, they were embedded
2.6 | Quantitative analysis of melanin using a in epoxy resin and ultra-thin sections were prepared and then ob-
specific degradation product served using a JEM-1400 transmission electron microscope (JEOL).

Dorsal skin biopsies were taken at 0, 2, 4, and 8 weeks after

the induction of inflammation from three mice. Pieces of biopsy
tissues were excised with a 5-mm punch scalpel and immersed
in 1 M sodium chloride aqueous solution for 48 hr, after which
the epidermis and the dermis were separated and homogenized
separately in 400 µl water with a Ten-Broeck glass homogenizer.
Aliquots of 100 µl were subjected to alkaline hydrogen per-
oxide oxidation (Ito et al., 2011) and hydroiodic acid hydrolysis
(Wakamatsu et al., 2002). Eumelanin was analyzed as a specific
degradation product, PTCA, while pheomelanin was analyzed as
4-amino-3-hydroxyphenylalanine (4-AHP) produced by the hy-
droiodic acid hydrolysis.
2.8 | Immunohistochemical staining of dermal
melanin-containing cells
Immunohistochemical staining was conducted on specimens from
five mice. After the specimens had been deparaffinized, antigen
retrieval was carried out by adding ethylenediamine tetraacetic
acid (EDTA; Nichirei) at pH 9 and autoclaving at 120°C for 20 min.
Blocking was carried out by placing the specimens in 3% skim milk/
phosphate-buffered saline (PBS) for 30  min. The primary antibody
(F4/80, ab6640, 1:100; Abcam) was incubated overnight at 4°C. The
specimens were then incubated at room temperature for 60 min
 |
4       NAKANO et al.
with anti-rabbit FITC (ab150061, 1:100; Abcam) as the secondary
antibody and were then counterstained with hematoxylin (Merck)
before slides were prepared. Another primary antibody (CD68,
ab125212, 1:100; Abcam) was stained using the same procedure.
The sections were photographed with an all-in-one optical micro-
scope (BZ-X700; KEYENCE), and the numbers of melanin-containing
cells and F4/80-positive cells in the dermis were counted. Counts
were performed independently by two or more investigators. The
dermis was observed using a 20× objective, three random visual
fields were photographed for each specimen, and the mean values
for five animals (five specimens) were calculated.
3 |   R E S U LT S
3.1 | Induction and histopathologic analysis of PIH
In order to analyze the pathomechanisms of PIH, we firstly induced
chronic contact dermatitis by repeated hapten application of 2,4-di-
nitrofluorobenzene (DNFB) on the dorsal skin of hk14-SCF Tg/HRM
mice according to previous studies (Inagaki et al., 2006; Shiohara,
Hayakawa, & Mizukawa, 2004). The experimental design is shown
in Figure 1a. Briefly, the mice were sensitized by the application of
100 µl 0.5% DNFB in acetone/olive oil (4:1) to the abdominal region
of each mouse twice on successive days. After one week, 100 µl
0.15% DNFB was applied to the dorsal skin of each mouse at a fre-
quency of twice a week for a total of 9 applications, after which the
color (melanin index) of the dorsal skin of each mouse was measured.
The untreated skin is denoted as “before.” As shown in Figure 1b, the
results revealed that the melanin index peaked at week 1 compared
to untreated skin and then gradually decreased until week 12. From
week 0 to week 8, statistically significant increases in the melanin
index were observed between the DNFB-treated and untreated
mice. On the backs of the PIH mice, lichenified lesions, scales, and
crusting were apparent at weeks 0 and 1 (Figure 1c). At week 2, hy-
perpigmentation was the predominantly observed phenotype, after
which the PIH slowly disappeared.
Skin biopsies were taken for evaluation at “before” and at weeks
0, 1, 2, and 8. Hematoxylin–eosin staining revealed epidermal hy-
perplasia and predominant infiltrations of inflammatory cells in the
dermis at weeks 0 and 1 (Figure 2a, upper row). Fontana-Masson
staining revealed that melanin and melanin-containing cells in the
epidermis and the dermis apparently increased at weeks 0 and
1 and subsequently decreased to week 8 (Figure 2a, middle row).
Immunostaining with the anti-melan-A antibody, which is a specific
marker of melanocytes, revealed a large increase in melanocytes at
weeks 0 and 1 (Figure 2a, lower row), and the ascent of melanocytes
from the epidermis was also detectable at week 1 (Figure 2a, ar-
rowheads in lower row). From week 2, the number of melanocytes
was similar to that of the untreated group. Highly magnified images
showed prominent epidermal hyperplasia, granular layer thickening,
and mild spongiotic changes in the epidermis at week 0 (Figure 2b),
which was similar to the changes of chronic eczema in atopic
dermatitis and chronic contact dermatitis. At week 2, the epidermal
changes seen at week 0 were no longer apparent (Figure 2b).
3.2 | Changes in inflammatory cytokine mRNA
expression levels after chronic contact dermatitis
We also confirmed that PIH model mice had skin inflammation simi-
lar to atopic dermatitis. The cytokine profile at 24 hr after the final
application of DNFB revealed that expression levels of both Th1 cy-
tokines (IFN-γ, TNF-α, and IL-2) and Th2 cytokines (IL-4, IL-5, and IL-
13) were increased significantly in the induced inflammation group
compared with the no-treatment control (Figure 3), which indicates
that the inflammation caused in the dorsal skin of the mice resem-
bles atopic dermatitis in humans. Both male and female mice were
used in this experiment, but no difference in cytokine expressions
was observed between male and female mice.
3.3 | Kinetic analysis of epidermal and dermal
melanin in PIH model mice
To evaluate changes of melanin content in the epidermis and dermis
of PIH model mice, we used image analysis to measure the areas of
melanin distribution. The results showed that the area of distribu-
tion of epidermal melanin peaked at week 0 and decreased quickly
by week 2 (Figure 4a). On the other hand, in the dermis, the area
of distribution of melanin also peaked at week 0; however, the sig-
nificantly increased melanin area in the dermis continued until week
4 and then slowly declined at week 12 (Figure 4b). Those changes
were clearly seen in the dermis-to-epidermis ratio of melanin area
levels (Figure 4c). The amount of melanin area in the dermis peaked
at 4 weeks relative to the epidermis. These observations were sup-
ported by the results of quantitative chemical measurement of
melanin in the epidermis and dermis. We analyzed the content of
eumelanin as pyrrole-2,3,5-tricarboxylic acid (PTCA), an eumelanin-
specific degradation product, because more than 95% of melanin
in this mouse skin was reported to be eumelanin (Abe et al., 2016).
At weeks 2 and 4, a statistically significant increase in the dermis/
epidermis ratio was observed and that ratio returned to the original
level after 8 weeks (Figure 4d). Pheomelanin levels, present at much
lower levels than eumelanin, showed a similar pattern of distribution
between the epidermis and the dermis, at much lower levels (data
not shown).
3.4 | Observations by electron microscopy
We observed melanin-containing cells in the dermis using trans-
mission electron microscopy (Figure 5). The dorsal skin at week 2
showed the disappearance of histopathologic changes character-
istic of acute dermatitis, namely epidermal hyperplasia, spongio-
tic changes, and inflammatory cell infiltrates in the upper dermis,
NAKANO et al. |
      5

Follow-up time (weeks)

(a)
before 0 1 2 8
Hematoxylin-Eosin
Fontana-Masson
Melan-A

(b)
Follow-up time
0 2 (weeks)
a b

F I G U R E 2   Histological observations of mouse dorsal skin. (a) Middle-power fields: Hematoxylin–eosin staining (upper row), Fontana-
Masson staining (middle row), and anti-melan-A antibody staining (lower row). The elimination of melanocytes from the epidermis is
indicated in the middle panel in the bottom row (arrowheads). Scale bar = 100 µm. (b) high-power fields: At week 0, obvious epidermal
hyperplasia, granular layer thickening, and mild spongiotic changes in the epidermis were observed. At week 2, the epidermal changes seen
at week 0 were no longer apparent. Scale bars = 50 µm

and those histological findings were similar to PIH in human skin.
Melanin-containing cells were distributed from the papillary dermis
to a moderately deep area of the reticular dermis. Most of these
melanin-containing cells were macrophages, and a few melanin-
containing fibroblasts were detectable, which remained only in the
papillary dermis (Figure 5a–c). Macrophages were found to contain
more melanin granules than fibroblasts. Further, in some areas, mast
cells containing large amounts of histamine granules were observed.
These mast cells appeared to be attached and have interactions with
macrophages and fibroblasts (Figure 5d), indicating that mast cells
might have a role in PIH.
3.5 | Immunohistochemical staining of dermal
melanin-containing cells
In order to investigate the characteristics of macrophages contain-
ing melanin in the dermis, we carried out immunohistochemical
staining with antibodies to several macrophage markers. Only two
of them, anti-CD68 antibody and anti-F4/80 antibody, reacted with
the melanin-containing cells after the start of DNFB treatment, but
not before the treatment. The majority of cells containing melanin
were positive for anti-CD68 antibody (Figure 6a), but in contrast,
only 17%-30% of macrophages containing melanin were positive for
 6       | NAKANO et al.

F I G U R E 3   Changes in mRNA
expression levels of inflammatory
cytokines after the induction of repeated
contact dermatitis. In the induction group,
skin biopsies were taken 24 hr after the
ninth application of DNFB to the dorsal
skin. The mRNA expression levels of
Th1 and Th2 cytokines of the induction
group (n = 20) increased compared to the
no-treatment group (n = 10). Error bars,
means ± SEM. Welch's t-test. *p < .05

F I G U R E 4   Analysis of melanin
Melanin distribution area per field of

Melanin distribution area per field of

500 600 distribution. (a, b) Measurements of

view at a magnification of 440x (µ

view at a magnification of 200x (µ

(a) Epidermis (b) ** Dermis areas of epidermal and dermal melanin

450
400
* 500
** distribution using image analysis. Three
350 400 fields of views were taken at random
300
per sample, and the average values of
250 300 ** five animals were calculated (n = 5). (c)
200
150 200 Ratio of the melanin are of the dermis
100 100 to that of the epidermis, calculated from
50 (a) and (b) (n = 5). (d) Ratio of melanin
0 0
before 0 2 4 12 before 0 2 4 12
distribution in the dermis to that in
Follow-up time (weeks) Follow-up time (weeks) the epidermis by chemical analysis.
The amounts of epidermal and dermal
5 melanin were characterized by PTCA, an
4.5 ** 0.6
* *
Dermis/Epidermis Ratio

eumelanin-specific degradation product,

Dermis/Epidermis Ratio

(c) (d)
by chemical analysis

means ± SEM. Welch's t-test. ⁑p < .001.

0.5 were measured (n = 3). Error bars,
3.5
3 0.4
*p < .05
2.5
2
** 0.3
1.5
** 0.2
1
0.5 0.1
0 0.0
before 2 4 12 before 2 4 8
Follow-up time (weeks) Follow-up time (weeks)

the anti-F4/80 antibody (Figure 6b). The pattern of changes in the

melanin index of the skin and the number of macrophages found in 4 | D I S CU S S I O N
the dermis is similar, but there was no correlation with the change in
the number of F4/80 positive cells (Figures 2 and 6). In this study, a model mouse mimicking PIH after atopic dermatitis
Finally, we examined whether melanin was synthesized within was created for the purpose of analyzing the pathogenesis of PIH,
melanosomes taken up by macrophages and fibroblasts in the upper which has been rarely studied in the past. We used hk14-SCF Tg/
dermis. Since tyrosinase activity is essential for melanogenesis, the HRM mice, in which melanocytes are localized in the basal layer of
presence or absence of tyrosinase activity in melanosomes was con- the epidermis and in which the epidermal keratinocytes have mela-
firmed by DOPA staining. As a result, melanosomes in dermal macro- nin similar to human skin.
phages and fibroblasts were negative for tyrosinase (Figure S1). This Histopathologically, PIH is defined as normal or increased amounts
result reveals that melanin synthesis does not occur in the phagocy- of melanin in the epidermis and dermal melanophage proliferation that
tosed melanosomes. does not accompany the vacuolization of basal cells (Park et al., 2017;
NAKANO et al. |
      7

F I G U R E 5   Observation of dermal
cells that contain melanin by electron (a) (b)
microscopy. Electron micrographs of
follow-up at 2 weeks. (a, b) Most of these
melanin-containing cells are macrophages.
(c) A few melanin-containing fibroblasts
are detectable. Enlarged images of
fibroblasts and macrophages are shown.
(d) Mast cells containing a large amount
of histamine granules are observed. F,
fibroblast; M, macrophage; MC, mast cell;
L, lymphocyte

(c) (d)

Silpa-Archa et al., 2017). In our PIH model, repeated application of the
hapten DNFB led to the infiltration of inflammatory cells with spongi-
osis in the epidermis, but those changes disappeared after two weeks
(Figure 2b). At week 2, the histological findings were similar to the
characteristic findings of PIH, that is, an increase in epidermal melanin
content and an increase in dermal melanophages (Figure 2a). From the
above findings, the skin conditions at week 2 to week 8 were very
similar to those of PIH in human skin, especially after atopic dermatitis,
and this model mouse well reflected the characteristics of human PIH.
In order to investigate the pathophysiology of PIH and to de-
velop novel treatments for PIH, several studies have been reported,
in which some used human skin, for example, the use of 35% tri-
chloroacetic acid to induce pigmentation (Isedeh et al., 2016) and
suction blisters to observe pigmentation (Passeron et al., 2018).
Those methods involve human subjects and have risks of scarring
and thus are ethically challenging methods. Our study successfully
established a mouse model using hk14SCF Tg-HRM mice, in which
induction of an atopic dermatitis-like response triggers reproducible

(a) (b)
16 16
14 14
Number of cells per field

Number of cells per field

12 12
10 10
8 8
F I G U R E 6   Immunohistochemical 6 6
observation of dermal cells that contain 4 4
melanin. The numbers of all cells
2 2
containing melanin, and CD68 (a)- or
F4/80 (b)-positive cells were measured 0 0
before 0 2 4 12 before 0 2 4 12
in the skins at before, and at weeks 0,
2, 4, and 12. Three visual fields were Follow-up me (weeks) Follow-up me (weeks)
selected at random per specimen (n = 5),
All cells containing melanin All cells containing melanin
and average values were calculated. Error
CD68+ cells containing melanin F4/80+ cells containing melanin
bars, means ± SEM
 |
8       NAKANO et al.
PIH, thus constituting a highly useful model for future research of
PIH.
Atopic dermatitis is a disease process characterized by a pruritic
rash with repeated flares and remission (Brunner, Guttman-Yassky, &
Leung, 2017). Histopathology of the skin of many patients with atopic
dermatitis reveals an environment dominated by Th2 cytokines, such
as IL-4 and IL-13, which is thought to decrease filaggrin expression
and upregulate IgE (Kubo, 2017). Common animal models of atopic
dermatitis include the NC/Nga mouse (Matsuda et al., 1997), a hapten
induction model (Nagai, Matsuo, Hiyama, Inagaki, & Kawada, 1997)
and the IL-18 Tg mouse (Konishi et al., 2002). In each of those mod-
els, immunological analysis showed an environment with increased
levels of Th2 cytokines and serum IgE (Shiohara et al., 2004). In our
PIH mouse model, visual inspection (Figure 1c) and histopathologic
inspection (Figure 2a) were identical to those of atopic dermatitis
models. Regarding cytokines, both Th1 and Th2 cytokine levels were
higher in our mice compared with controls (Figure 3). Recent studies
suggested the involvement of Th17 cells, which produce IL-17A and
IL-22 (Brunner et al., 2017).
Dynamic analysis of melanin in the PIH mouse model showed
that the melanin area in the epidermis at week 2 rapidly decreased
and was not significantly different from the non-treatment group,
but melanin distribution in the dermis was still higher at week 4
(Figure 4a–c). Moreover, dermal melanin remained for a longer du-
ration than epidermal melanin, suggesting that melanin metabolism
in the dermis is slower than in the epidermis by 4 weeks or more.
Chemical quantitative analysis of a eumelanin-specific degradation
product (PTCA) showed increasing levels of the dermis/epidermis
ratio at weeks 2 and 4 (Figure 4d). Regarding melanin in hk14SCF
Tg-HRM skin, the quantity of eumelanin was found to be >20 times
greater than that of pheomelanin (Abe et al., 2016), and we spec-
ulated that the effect of pheomelanin on pigmentation (melanin
index) was limited.
Removal of melanin following pigmentation is accomplished via
either the epidermis or the dermis. In the epidermis, the turnover
of melanosomes in keratinocytes is accompanied by the spread of
melanin toward the surface of the epidermis, allowing visualization
of pigmentation in the skin. Melanin within keratinocytes is removed
through the desquamation process. However, how proliferating me-
lanocytes are removed is still unknown. This mouse model would
be a good tool to study the turnover of epithelial melanocytes, as
the removal of epithelial melanocytes can be observed (Figure 2a,
arrowheads in lower row). Regarding the dermal route of melanin
removal, it has been reported that, in Kitl-Tg mice that have an ex-
cessive proliferation of melanocytes in the epidermis, there was an
increase in melanin levels in dermal lymphatics proportional with
the number of weeks (Yoshino, Yamazaki, & Hayashi, 2008; Yoshino,
Yamazaki, Shultz, & Hayashi, 2006), showing that melanin is trans-
ported to the lymphatic system. However, it is still unknown whether
melanin breakdown occurs within phagocytes.
Our investigation by electron microscopy of the skin of PIH
model mice revealed that the majority of melanin in the dermis was
inside macrophages, and some was in fibroblasts (Figure 5). Dermal
macrophages can be divided into those that remain fixed in the
tissue and those that wander through the tissue. The wandering
macrophages are those that infiltrate the tissue during inflamma-
tion, moving through the dermis and migrating to the lymphatics
(Ginhoux, Guilliams, & Naik, 2016). In mice, tissue macrophages were
reported to be CD11b+, CD64hi, MerTK+, and CCR2lo, whereas wan-
dering macrophages were CD11b+, CD64lo, MerTK− to lo, and CCR2+
(Tamoutounour et al., 2013), but those are not distinguished by the
expression of F4/80. We observed many melanin-containing cells
that were positive for CD68, suggesting that those cells were mac-
rophages. On the other hand, another macrophage surface marker
F4/80 (Lin et al., 2005) was negative in the majority of melanin-con-
taining cells (Figure 6), indicating that there are some populations of
macrophages in the dermis and that F4/80-negative macrophages
might play an important role in PIH.
Conventional treatments for PIH include laser therapy, chemical
peeling, and topical application of retinoids, hydroquinone, nicotin-
amide, and kojic acid (Chaowattanapanit, Silpa-Archa, Kohli, Lim, &
Hamzavi, 2017); however, the effects on dermal melanin-containing
cells have been unclear. Our PIH model mouse can be an excellent
tool for the scientific evaluation of treatments for PIH. According to
our study, over-produced melanin by inflammation in the epidermis is
degraded and excreted faster than melanin in the dermis, and the epi-
dermis quickly returns to the state before the inflammation. On the
other hand, melanin phagocytosed in the dermis was clinically recog-
nized as PIH due to its slow degradation rate, and it was confirmed
that it becomes a major problem. Therefore, for PIH treatment, drugs
that stimulate the activity of dermal macrophages instead of inhib-
iting melanin synthesis and that activate macrophage migration and
melanin degradation may be good candidates. None of the drugs cur-
rently in use have demonstrated such effects, and the activity of der-
mal macrophages appears to be a future therapeutic target.
In conclusion, we established an excellent animal model of PIH in
mice, and studies using this model demonstrated that the majority of
melanin remaining in the dermis is phagocytosed by macrophages,
particularly F4/80-negative macrophages, suggesting that novel
treatments of PIH should be targeted against macrophages. Further
investigations using this PIH mouse model will further illuminate the
challenging pathophysiology of PIH and should eventually lead to
the development of new treatment modalities.
AC K N OW L E D G M E N T S
We thank Dr. Hitomi Aoki, and Professor Takahiro Kunisada, Gifu
University Graduate School of Medicine, for their excellent advice
about the mice. This work was supported by JSPS KAKENHI Grant
Number JP19K08742, with funding from Eli Lilly Japan K.K.
C O N FL I C T O F I N T E R E S T
This study was performed through joint research agreement be-
tween Yamagata University and Eli Lilly Japan K.K., with funding
from Eli Lilly Japan K.K. to TS.
NAKANO et al.
AU T H O R C O N T R I B U T I O N
Conceptualization, project administration, and funding acquisition:
TS; methodology: YA, KN, SS, TS; validation: SN, KW, SI; formal anal-
ysis: SN, SI; investigation: SN, YA, KN, OY, KW, SI; resources: SN, YA,
TS; data curation: SN, OY, SI; writing - original draft preparation: SN,
TS; writing -review and editing: AY, KN, SS, OY, KW, SI, MH; visuali-
zation: SN, OY, KW, SI, TS; supervision:SS, TS.
DATA AVA I L A B I L I T Y S TAT E M E N T
No datasets were generated or analyzed during the current study.
ORCID
Shoko Nakano  https://orcid.org/0000-0002-8465-6108
Yuko Abe  https://orcid.org/0000-0002-0903-873X
Kimiko Nakajima  https://orcid.org/0000-0002-0341-3252
Shigetoshi Sano  https://orcid.org/0000-0002-9812-0216
Osamu Yamamoto  https://orcid.org/0000-0002-9992-5294
Kazumasa Wakamatsu  https://orcid.org/0000-0003-1748-9001
Shosuke Ito  https://orcid.org/0000-0001-9182-5144
Masahiro Hayashi  https://orcid.org/0000-0002-1674-4093
Tamio Suzuki  https://orcid.org/0000-0002-4669-9721
REFERENCES
Abe, Y., Okamura, K., Kawaguchi, M., Hozumi, Y., Aoki, H., Kunisada, T., …
Suzuki, T. (2016). Rhododenol-induced leukoderma in a mouse model
mimicking Japanese skin. Journal of Dermatological Science, 81(1),
35–43. https://doi.org/10.1016/j.jderm​sci.2015.10.011
Bonaventure, J., Domingues, M. J., & Larue, L. (2013). Cellular and mo-
lecular mechanisms controlling the migration of melanocytes and
melanoma cells. Pigment Cell & Melanoma Research, 26(3), 316–325.
https://doi.org/10.1111/pcmr.12080
Brunner, P. M., Guttman-Yassky, E., & Leung, D. Y. (2017). The immu-
nology of atopic dermatitis and its reversibility with broad-spec-
trum and targeted therapies. The Journal of Allergy and Clinical
Immunology, 139(4S), S65–S76. https://doi.org/10.1016/j.
jaci.2017.01.011
Callender, V. D., St Surin-Lord, S., Davis, E. C., & Maclin, M. (2011).
Postinflammatory hyperpigmentation: Etiologic and therapeutic
considerations. American Journal of Clinical Dermatology, 12(2), 87–
99. https://doi.org/10.2165/11536​930-00000​0 000-00000
Chaowattanapanit, S., Silpa-Archa, N., Kohli, I., Lim, H. W., & Hamzavi,
I. (2017). Postinflammatory hyperpigmentation: A comprehensive
overview: Treatment options and prevention. Journal of the American
Academy of Dermatology, 77(4), 607–621. https://doi.org/10.1016/j.
jaad.2017.01.036
Davis, E. C., & Callender, V. D. (2010). Postinflammatory hyperpigmen-
tation: A review of the epidemiology, clinical features, and treat-
ment options in skin of color. The Journal of Clinical and Aesthetic
Dermatology, 3(7), 20–31.
D'Mello, S. A., Finlay, G. J., Baguley, B. C., & Askarian-Amiri, M. E.
(2016). Signaling pathways in melanogenesis. International Journal
of Molecular Sciences, 17(7), 1144. https://doi.org/10.3390/ijms1​
7071144
Ginhoux, F., Guilliams, M., & Naik, S. H. (2016). Editorial: Dendritic
Cell and macrophage nomenclature and classification. Frontiers in
Immunology, 7, 168. https://doi.org/10.3389/fimmu.2016.00168
Inagaki, N., Shiraishi, N., Igeta, K., Itoh, T., Chikumoto, T., Nagao, M., …
Nagai, H. (2006). Inhibition of scratching behavior associated with
allergic dermatitis in mice by tacrolimus, but not by dexamethasone.
|
      9
European Journal of Pharmacology, 546(1–3), 189–196. https://doi.
org/10.1016/j.ejphar.2006.07.019
Isedeh, P., Kohli, I., Al-Jamal, M., Agbai, O. N., Chaffins, M., Devpura, S., …
Hamzavi, I. H. (2016). An in vivo model for postinflammatory hyper-
pigmentation: An analysis of histological, spectroscopic, colorimetric
and clinical traits. British Journal of Dermatology, 174(4), 862–868.
https://doi.org/10.1111/bjd.14184
Ito, S., Nakanishi, Y., Valenzuela, R. K., Brilliant, M. H., Kolbe, L., &
Wakamatsu, K. (2011). Usefulness of alkaline hydrogen peroxide
oxidation to analyze eumelanin and pheomelanin in various tissue
samples: Application to chemical analysis of human hair melanins.
Pigment Cell & Melanoma Research, 24(4), 605–613. https://doi.
org/10.1111/j.1755-148X.2011.00864.x
Konishi, H., Tsutsui, H., Murakami, T., Yumikura-Futatsugi, S., Yamanaka,
K.-I., Tanaka, M., … Mizutani, H. (2002). IL-18 contributes to the spon-
taneous development of atopic dermatitis-like inflammatory skin le-
sion independently of IgE/stat6 under specific pathogen-free con-
ditions. Proceedings of the National Academy of Sciences of the United
States of America, 99(17), 11340–11345. https://doi.org/10.1073/
pnas.15233​7799
Kubo, M. (2017). Innate and adaptive type 2 immunity in lung allergic
inflammation. Immunological Reviews, 278(1), 162–172. https://doi.
org/10.1111/imr.12557
Kunisada, T., Yoshida, H., Yamazaki, H., Miyamoto, A., Hemmi, H.,
Nishimura, E., … Hayashi, S. (1998). Transgene expression of steel
factor in the basal layer of epidermis promotes survival, prolif-
eration, differentiation and migration of melanocyte precursors.
Development, 125(15), 2915–2923.
Lin, H.-H., Faunce, D. E., Stacey, M., Terajewicz, A., Nakamura, T., Zhang-
Hoover, J., … Stein-Streilein, J. (2005). The macrophage F4/80 recep-
tor is required for the induction of antigen-specific efferent regula-
tory T cells in peripheral tolerance. Journal of Experimental Medicine,
201(10), 1615–1625. https://doi.org/10.1084/jem.20042307
Masuda, Y., Yamashita, T., Hirao, T., & Takahashi, M. (2009). An innovative
method to measure skin pigmentation. Skin Research and Technology,
15(2), 224–229. https://doi.org/10.1111/j.1600-0846.2009.00359.x
Matsuda, H., Watanabe, N., Geba, G. P., Sperl, J., Tsudzuki, M., Hiroi, J., …
Ra, C. (1997). Development of atopic dermatitis-like skin lesion with
IgE hyperproduction in NC/Nga mice. International Immunology, 9(3),
461–466. https://doi.org/10.1093/intim​m/9.3.461
Nagai, H., Matsuo, A., Hiyama, H., Inagaki, N., & Kawada, K. (1997).
Immunoglobulin E production in mice by means of contact sensi-
tization with a simple chemical, hapten. The Journal of Allergy and
Clinical Immunology, 100(6 Pt 2), S39–S44. https://doi.org/10.1016/
s0091-6749(97)70003-4
Nakajima, K., Kanda, T., Takaishi, M., Shiga, T., Miyoshi, K., Nakajima, H.,
… Sano, S. (2011). Distinct roles of IL-23 and IL-17 in the development
of psoriasis-like lesions in a mouse model. The Journal of Immunology,
186(7), 4481–4489. https://doi.org/10.4049/jimmu​nol.1000148
Park, J. Y., Park, J. H., Kim, S. J., Kwon, J. E., Kang, H. Y., Lee, E. S., &
Kim, Y. C. (2017). Two histopathological patterns of postinflamma-
tory hyperpigmentation: Epidermal and dermal. Journal of Cutaneous
Pathology, 44(2), 118–124. https://doi.org/10.1111/cup.12849
Passeron, T., Nouveau, S., Duval, C., Cardot-Leccia, N., Piffaut, V.,
Bourreau, E., … Bernerd, F. (2018). Development and validation of
a reproducible model for studying post-inflammatory hyperpig-
mentation. Pigment Cell Melanoma Res, 31(5), 649–652. https://doi.
org/10.1111/pcmr.12692
Seghers, A. C., Lee, J. S. S., Tan, C. S., Koh, Y. P., Ho, M. S. L., Lim, Y. L., …
Tang, M. B. Y. (2014). Atopic dirty neck or acquired atopic hyperpig-
mentation? An epidemiological and clinical study from the National
Skin Centre in Singapore. Dermatology, 229(3), 174–182. https://doi.
org/10.1159/00036​2596
Shiohara, T., Hayakawa, J., & Mizukawa, Y. (2004). Animal models for
atopic dermatitis: Are they relevant to human disease? Journal of
 |
10       NAKANO et al.

Dermatological Science, 36(1), 1–9. https://doi.org/10.1016/j.jderm​ Yoshino, M., Yamazaki, H., Shultz, L. D., & Hayashi, S. (2006). Constant
sci.2004.02.013 rate of steady-state self-antigen trafficking from skin to regional
Silpa-Archa, N., Kohli, I., Chaowattanapanit, S., Lim, H. W., & Hamzavi, lymph nodes. International Immunology, 18(11), 1541–1548. https://
I. (2017). Postinflammatory hyperpigmentation: A comprehen- doi.org/10.1093/intim​m/dxl087
sive overview: Epidemiology, pathogenesis, clinical presentation,
and noninvasive assessment technique. Journal of the American
Academy of Dermatology, 77(4), 591–605. https://doi.org/10.1016/j. S U P P O R T I N G I N FO R M AT I O N
jaad.2017.01.035
Additional supporting information may be found online in the
Tamoutounour, S., Guilliams, M., Montanana Sanchis, F., Liu, H., Terhorst,
D., Malosse, C., … Henri, S. (2013). Origins and functional special- Supporting Information section.
ization of macrophages and of conventional and monocyte-derived
dendritic cells in mouse skin. Immunity, 39(5), 925–938. https://doi.
org/10.1016/j.immuni.2013.10.004 How to cite this article: Nakano S, Abe Y, Nakajima K, et al.
Wakamatsu, K., Ito, S., & Rees, J.L. (2002). The usefulness of 4-amino-3-hy-
Establishment of a mouse model for post-inflammatory
droxyphenylalanine as a specific marker of pheomelanin. Pigment Cell
Res, 15(3), 225–232. https://doi:10.1034/j.1600-0749.2002.02009.x hyperpigmentation. Pigment Cell Melanoma Res. 2020;00:1–10.
Yoshino, M., Yamazaki, H., & Hayashi, S. (2008). Analysis of capturing https://doi.org/10.1111/pcmr.12911
skin antigens in the steady state using milk fat globule EGF factor
8-deficient skin-hyperpigmented mice. Immunology Letters, 115(2),
131–137. https://doi.org/10.1016/j.imlet.2007.10.017

View publication stats