Reversible Imiquimod Effects on Skin Tissue of Psoriasis Mice

Model: An Experimental Study

Thianti Sylviningrum

, Afifah

, Dody Novrial

, Brian Wasita

, Bambang Purwanto

Harijono K. Sentono

Doctoral Degree of Medical Science Programme, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia

Department of Pharmacology, Faculty of Medicine, Universitas Jenderal Soedirman, Purwokerto, Indonesia

Department of Anatomical Pathology, Faculty of Medicine, Universitas Jenderal Soedirman, Purwokerto, Indonesia

Department of Anatomical Pathology, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia

Department of Internal Medicine, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia

Department of Dermatology and Venereology, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia

Keywords: Imiquimod, skin, mice model, psoriasis.

Abstract: Psoriasis is an incurable inflammatory disease with erythematous, scaly, and thick skin. Imiquimod (IMQ)-

induced psoriasis mice have been widely used since it may resemble psoriasis human. Imiquimod effects on

development, peak, and resolution of mice skin are crucial. Thus, this study aims at evaluating IMQ effects

on psoriasis mice model skin tissue changes. Twenty-seven female Balb/c mice 8-11 weeks, 20-25 grams

body weight (BW) were randomize equally into control (A) and experimental groups (B,C). Over 25 grams

BW mice after acclimatization were excluded. The mice had shaved their back then topically applied

emollient (A) and IMQ (B,C) for 7 consecutive days. On day 8 (A,B) and 15 (C), mice were terminated and

back skin harvested for histopathological examination. Psoriasis Area Severity Index (PASI) and Baker’s

scores were used to measure macroscopic and microscopic skin changes. All groups score differences were

assessed using Kruskal Wallis then Mann Whitney tests. Skin changes gradually appeared from day 2 to 7 of

IMQ applications and faded after 2 days IMQ discontinuation. On day 7, all mice showed means of PASI

scores 0(A);9,00±2,69(B);8,11±1,62(C) with significant differences between experimental to control groups

(p = 0.00). Means of PASI scores from mice group C on day 15 showed similar to group A (p = 0.10). Means

of Baker’s scores from all groups were 1,16±0,25(A); 3,33±1,25(B);1,39±0,22(C) and group B mice showed

different significant scores from others (p = 0.00). As conclusion, the IMQ showed a reversible effect on skin

tissue changes in psoriasis mice model.The abstract should summarize the contents of the paper and should

contain at least 70 and at most 200 words. It should be set in 9-point font size, justified and should have a

hanging indent of 2-centimenter. There should be a space before of 12-point and after of 30-point.

1 INTRODUCTION

Psoriasis is an autoimmune disease characterized by

the erythematous plaque covered by the thick, silvery,

and scaly skin lesions with the chronic inflammatory

background (Boehncke & Schön, 2015). The disease

has complex pathophysiological pathways involving

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https://orcid.org/0000-0001-5338-6005

the interactions of multi-genetic and environmental

factors. Psoriatic patients who have skin and/or

systemic manifestations may decrease their quality of

life, and need long-term treatment possibly associated

with higher side effects and lower compliance

(Belinchón et al., 2016).World Health Organization

(WHO) reported that there were approximately 2% of

psoriasis patients throughout the world and tended

260

Sylviningrum, T., Aﬁfah, ., Novrial, D., Wasita, B., Purwanto, B. and Sentono, H.

Reversible Imiquimod Effects on Skin Tissue of Psoriasis Mice Model: An Experimental Study.

DOI: 10.5220/0010491002600266

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 260-266

ISBN: 978-989-758-499-2

increasing in the last 20 years (WHO, 2016). This

condition indicates that more studies are required to

investigate the pathways involved in the development

of psoriasis and treatment method associated with a

good response and minimum side effects.

Studies on pathophysiological and drug

development in human psoriasis patients may face the

ethical problems. Thus, psoriasis animal model is

preferable. Imiquimod (IMQ)-induced psoriasis

mouse model is the most widely used research

instrument because it is inexpensive, easy to perform,

and immediately induce the acute inflammatory

process resembles human psoriasis. This procedure

may also trigger the psoriasis skin manifestations,

such as erythematous, skin hyperplasia, and scaling.

The pathognomonic histopathological changes

of human psoriasis were also found, including munro

abscess, acanthosis, keratinocyte hyperplasia,

parakeratosis (Hawkes et al., 2017). The application

of 62.5mg imiquimod daily may induce the psoriasis

skin lesions on the back-shaved Balb/c mice through

their activation of Interleukin (IL)-23/IL-17 axis

(Bocheńska et al., 2017). The previous studies

showed that IMQ might affect a rapid, temporary, and

reversible inflammation on the human skin (Van der

Kolk et al., 2018), yet the information on the duration

of inflammation development process during the

application of IMQ on mice was various and limited.

The duration of inflammation process in IMQ-

induced psoriasis mouse model is essential for the

momentum to do the treatment procedures for new

drug development.

2 MATERIALS AND METHODS

This study is an experiment study with posttest-only

control design that held in February to April 2020 in

Pharmacology Department, Medical Faculty of

Universitas Jenderal Soedirman, Central Java,

Indonesia. Twenty-seven female Balb/c mice aged 8-

11 weeks and weighed 20-25 grams were divided into

three groups: control group (A), first treatment group

(B), and second treatment group (C) consisting of 9

mice per group. The female Balb/c mice were

obtained from the Department of Pharmacology,

Faculty of Medicine, Universitas Gadjah Mada,

Indonesia.

All mice underwent 7-day acclimatization

period and were under-maintained in 12-12 hour light

and dark cycle with ad libitum food and drink. After

the acclimatization period, all mice were then

anesthetized with 0.1 ml intramuscular injection per

10 gram BW, 80mg/kgBW with ketamine cocktail;

12.5 mg/kg BW with xylazine cocktail; 3mg/kg BW

with acepromazine cocktail (Vertebrate Animal

Research, 2020). After anesthetized, 2x2 cm2 of all

mice’s back was shaved using an animal shaver and

applied with the depilatory cream Veet®. For the next

consecutive 7 days, each mouse in group A was

applied with 62.5 mg Noroid cream® (Soho Global

Health, Indonesia) on the shaved back skin, and

62.5mg of Aldara cream® (3M Pharmacy, United

Kingdom) that contains 5% imiquimod was applied

on the shaved back skin of each mouse in group B and

C. On day 8, the PASI scores of mice in all groups

were evaluated and terminated using the cervical

dislocation for those mice in group A and B followed

by back skin tissue harvesting. This step was repeated

on day 15 for mice in group C.

All skin tissues underwent a histopathological

examination using the hematoxylin and eosin

staining. The components of PASI scores for mice

were interpreted as erythematous (0-4), scaling (0-4),

thickness (0-4) with the total score of 0-12 by 2

certified dermatologists (Luo et al., 2016). To

minimize the subjectivity in evaluating the erythema,

the erythema level was scored based on color, 0 = no

color changes; 1 = light pink; 2 = pink; 3 = red; and 4

= dark red/violet. The scaling was scored based on the

observance, while the skin thickness was evaluated

by the histopathological examination. Two

pathologists evaluated and scored the

histopathological examination results using Baker’s

scoring system as presented in Table 1 (Baker et al.,

1992).

Table 1. Baker’s scoring system

Items Score

Keratin Munro Abscess 2.0

Hyperkeratosis 0.5

Parakeratosis 1.0

Epidermis Thinning above papillae 0.5

Lengthening and

clubbin

of rete rid

1.5

Acanthosis 0.5

Lack of

ranular la

1.0

Dermis L

mphoc

tic infiltrate

Mil

0.5

Moderate 1.0

Marked 2.0

Papillary congestion 0.5

Kruskal Wallis test and Mann Whitney test were

used to measure the difference between PASI and

Baker’s scores of the groups. The p value of < 0.05

Reversible Imiquimod Effects on Skin Tissue of Psoriasis Mice Model: An Experimental Study

261

was considered having a significant difference

between variables. This research was approved by the

Health Research Committee, Faculty of Medicine,

Universitas Jenderal Soedirman Number: Ref:

03IIIGPMII/2020.

3 RESULTS

All mice included in this study have completed with

the acclimatization period. The mice were treated in

accordance with the procedure mentioned in the

method section. By the end of day 7, all mice in group

B and C had psoriasis-like skin inflammation lesions,

but those in group A did not. The mice in group C had

the normal skin after 7-day application

discontinuation of IMQ (Figure 1).

During the application of IMQ on the mice in

group B and C, there was an increasing erythema

score from day 2, that is, the preceding scaling and

skin thickness manifested starting from day 3. On day

7 as the last application of IMQ on the mice in groups

B and C resulted in the highest erythema, scaling, and

thickness scores. In group C mice, there was a gradual

decrease in skin scaling and thickness 1 day after

IMQ exposure discontinuation. Meanwhile the

erythema score began to subsided on day 2 after the

last application of IMQ. All PASI score components

had returned to 0 score on day 13 (Figure 2). The mice

in group A did not show any changes in their skin

erythema, scaling, and thickness during the study.

In this study, the PASI mean scores in all

groups were respectively 0 (group A); 9.00 ± 2.69

(group B); 8.11 ± 1.62 (group C on day 7); and 0 for

group C on day 14. The Kruskal Wallis test result

showed the significant difference of PASI scores on

day 7 among all groups (p = 0.000). Post hoc study

showed the significant difference of PASI scores

between groups A and B, groups A and C on day 7,

group B and C on day 14, also groups C on day 7 and

day 14 (p = 0.000). Meanwhile, no significant

difference in PASI scores was found between those in

group B and C on day 7 (p = 0.465), also those in

group A and C on day 14 (p = 1.000).

The skin tissue was stained using hematoxylin

and eosin further scored using Baker’s system. Mice

in group A and C showed normal epidermis and

dermis layers. Psoriasis-like histopathological

changes were found in the mice in group B, such as

parakeratosis, Munro microabscess, acanthosis,

thinning above papillae, neutrophils and lymphocytes

infiltrate, and papillary congestions (Figure 3). The

following data were mean of Baker’s scores ±

standard deviation in group A, B, and C; 1.16 ± 0.25;

3.33 ± 1.25; and 1.39 ± 0.22. Baker’s scoring system

used in this study found significant differences among

all groups (p = 0.000). Further tests showed the

statistically significant differences of the mice in

group A and B (p = 0.000); group B and C (p = 0.001),

yet not between mice in group A and C ( p = 0.065).

Figure 1. Psoriasis-skin lesion like developed by the mice

in group B and C after the application 5% imiquimod cream

(Aldara®), while mice in group A had no changes in their

skin appearances. This psoriasis-like skin manifestation

was gradually decreased after the application

discontinuation of 5% imiquimod cream in group C.

4 DISCUSSIONS

Psoriasis is an inflammatory disease that may be

induced by multifactor, including drugs. Imiquimod

is known as one of the drugs that may induce psoriasis

in humans (Balak et al., 2017), meanwhile, IMQ

application in mice could trigger psoriasis-like skin

inflammation (Bocheńska et al., 2017). The use of

IMQ to induce psoriasis mice may provide the

benefits of investigating the pathogenesis and

developing new drugs for psoriasis.

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262

Figure 2. Progressively increasing mean of PASI scores components in groups B and C in the 7-day application of IMQ were

as the culmination point. In the mice of group C, these PASI score components also showed gradually after discontinuation

of IMQ applications.

In this study, psoriasis-like skin inflammation

induced by IMQ in mice was successful, evidenced

by the mean of PASI scores of mice in treatment

groups significantly higher than control one. The

results of this study showed similar findings from Luo

et al. (2016) that stated IMQ was a strong inducer of

psoriasiform changes in the mice skin tissue.8 The

IMQ exposures will bond to the TLR7 on the surface

of keratinocytes (Li et al., 2013), macrophages, and

plasmacytoid dendritic cells5 followed by NF-κβ,

mitogen-activated kinase protein, and activations of

the inflammatory signaling pathways (Ma et al.,

2020).

The IMQ exposure may cause the maturation of

dendritic cells and the mature ones will release IL-23.

The IL-23 may induce the differentiation and

activation of Th17 cells (Ueyama et al., 2014). The

activated Th17 cells will continue releasing

inflammatory cytokines and the chemo-attractants

that possibly causing the neutrophil migrations from

the dermal vascular to the epidermal layers, and

induce the keratinocyte cells to produce antimicrobial

peptides (AMPs) including cathelicidin. In psoriasis,

cathelicidin may induce inflammation by activating

immune cells via multiple pathways (Takahashi et al.,

2020). All of these processes may produce gradual

and prolonged inflammation in psoriasis (Kelhälä et

al., 2014). The skin tissue inflammation in mice

induced by the IMQ has gradually appeared. In this

study, the inflammation signs were initiated by

erythematous from day 2, followed by skin scaling

and thickness that visible from day 3. These skin

inflammation changes also showed disappeared

sequentially after discontinuation of IMQ exposures.

Unlike erythematous score that showed reduced later,

skin scaling and thickness disappeared earlier started

from day 1 after IMQ exposure cessation. These

gradual changes could be explained by

immunological circuits involving immune cells as

described above. Therefore, it is important to

determine the peak occurring inflammation to

become the point where tre Previous studies showed

that psoriasis histopathological features may have

similarities with chronic dermatitis (Ghasemi Basir et

al., 2018) and other psoriasiform dermatitis

(Chanadanwale et al., 2015). Previous study

mentioned that different mice strains that induced

with IMQ may give distinct responses that correspond

to other human skin disorders such as wounds or

infections (Swindell et al., 2017). Therefore, a

DAY

PASI COMPONENTS' MEAN SCORES IN GROUP C

Erythema Scaling Thickness

Reversible Imiquimod Effects on Skin Tissue of Psoriasis Mice Model: An Experimental Study

263

histopathological examination should be done to

determine psoriasiform changes apart from clinical

features evaluation in psoriasis-like inflammation

mice. Based on human psoriasis and psoriasiform

dermatitis skin biopsies, a prior study mentioned that

acanthosis, parakeratosis, hyperkeratosis, Munro

microabscess, dilated blood vessels, and

inflammatory infiltrates in the upper dermis including

neutrophils and lymphocytes consistent with

psoriasis (Chanadanwale et al., 2015).

Figure 3. Histopathological changes was not found in the mice in group A and C. Mice in group B showed the Munro abscess

(blue arrow), acanthosis (red arrow), parakeratosis (green arrow), thinning above papillae (black arrow), mild-moderate

lymphocyte infiltration (brown arrow), and papillary congestion (yellow arrow) which was pathognomonic to the psoriasis.

In this present study, these histopathological

features are also found in the skin tissue of group B

mice. The mice which were not applied with IMQ

(group A) or 7-day application discontinuation of

IMQ (group C) showed that the histopathological

images mimicking psoriasis were not found. The

mean of Baker's score 3.33 ± 1.25 in the group B mice

significantly differed from other groups. These

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

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264

outcomes proved that imiquimod induction in mice

for 7 days succeeded in providing the same

psoriasiform histopathological features as those

found in human psoriasis. The results of this study are

consistent with those conducted by Luo et al. (2016),

that the repeated applications of IMQ could cause

changes in the histopathological features resembling

psoriasis.8 These histopathological changes could be

initiated by skin irritation after application of IMQ

following the mice back shave that leads to the

increase of infiltrating lymphocytes, monocytes, and

dermal dendritic cells including plasmacytoid

dendritic cells (Luo et al., 2016; Ueyama et al., 2014;

Chiricozzi et al., 2018).

Furthermore, imiquimod induced maturation of

plasmacytoid dendritic cells into activated myeloid

dendritic cells which acted as the main source of IL-

23 (Girolomoni et al., 2017). The increase of IL-23

will direct Th17 to release IL-17 and IL-22 that

trigger epidermal hyperplasia, acanthosis, and

parakeratosis.21 Hereafter, IL-17, and IL-22 also

induce the keratinocytes to produce IL-8 and AMPs

as chemoattractants for neutrophils migration from

dermal vascular to epidermis layers which may

appear as the Munro/Kogoj microabscess (Luo et al.,

2016; Kelhälä et al., 2014; Chiricozzi et al., 2018;

Moos et al., 2019). These steps of the inflammation

process in psoriasis may elucidate the

histopathological changes induced by IMQ in the skin

of mice.

This study also indicated that the mice skin tissues

exposed to IMQ may cause the temporary induction

of psoriasiform histopathological changes. The

limitation of this study is we cannot identify the

stages of psoriasiform histopathological changes

during IMQ exposure or discontinuation, unlike the

clinical manifestation of gradual inflammation

changes that can be observed and scored.

Nevertheless, to the best of our knowledge, this is the

first study that shows the inflammatory effects of

IMQ in Balb/C female mice are temporary and the

period for these inflammatory signs subsided. The

duration of IMQ-induced inflammation in psoriasis

mice model is useful for further research to identify

pathogenesis and drug development for psoriasis.

5 CONCLUSIONS

The IMQ may induce reversible acute inflammation

with clinical and histopathological changes

resembling psoriasis in humans as treated on the

female Balb/c mice.

ACKNOWLEDGMENTS

We thank the following individuals, namely Lantip

Rujito who help us to write this article, Shinta Prima

Ardinas and Nirwan for their proficiencies,

experiences, and assistance throughout laboratory

aspects of our study.

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