Epicutaneous sensitization with ovalbumin, staphylococcal enterotoxin B and vitamin D analogue induces atopic dermatitis in mice

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1 中南大学学报 ( 医学版 ) J Cent South Univ (Med Sci) 17, (9) 13 DOI: /j.issn Epicutaneous sensitization with ovalbumin, staphylococcal enterotoxin B and vitamin D analogue induces atopic dermatitis in mice TAN Lina 1, LU Jianyun 1, CHEN Meilin 1, XIANG Yaping 1, CHENG Qingmei, LIANG Yunsheng 3, HUANG Jinhua 1, HUANG Jian 1, CHEN Jing 1, GAO Lihua 1 ( 1. Department of Dermatology, Third Xiangya Hospital, Central South University, Changsha 113;. Department of Physiology, School of Basic Medical Sciences, Central South University, Changsha 113; 3. Department of Dermatology, Second Xiangya Hospital, Central South University, Changsha 111, China ) ABSTRACT KEY WORDS Objective: To illuminate a method for establishment of a cost-efficient atopic dermatitis (AD) mouse model by topical application of ovalbumin (OVA), super-antigen staphylococcal enterotoxin B (SEB), and calcipotriene ointment (CO) on the back of BALB/c mice. Methods: Experimental mice were topically treated with or /CO every other day during 15 days of induction. Clinical alterations on the skin area were monitored every other day. Epidermal thickness were measured by reflectance confocal microscope (RCM) before harvest. Inflammatory cells in skin biopsies were marked by hematoxylin-eosin (HE) staining. Blood sample and skin biopsies were measured by ELISA and quantitative real-time PCR to detect the expression of IL-, IL-, IL-31, interferon (IFN)-γ, tumor necrosis factor (TNF)-α pruritus-associated nerve growth factor (NGF), and serum IgE. Results: Human AD-like cutaneous local inflammatory reaction was characterized by the accumulation of inflammatory cells, increased epidermal thickness and serum IgE levels as well as Th1 cell-associated cytokines (IFN-γ, TNF-α), Th cell-associated cytokines (IL-, IL-31), and NGF in the /CO group compared with that in the normal control group or the OVA/ SEB group. Conclusion: /CO can induce an AD-like mouse model with lower economic and time consumption. atopic dermatitis; vitamin D analogue; BALB/c mice; inflammatory cytokines; IgE; epidermal thickness Date of reception: First author: TAN Lina, @qq.com Corresponding author: GAO Lihua, @qq.com Foundation item: This work was supported by the Natural Science Foundation of Hunan Province (15JJ61), the Health and Family Planning Research Foundation of Hunan Province (B15-3), and Development and Reform Commission Project of Hunan Province ([1]658), China.

2 1 中南大学学报 ( 医学版 ), 17, (9) 卵清蛋白 金黄色葡萄球菌肠毒素 B 和维生素 D 类似物诱导小鼠特应性皮炎模型 谭丽娜 1, 鲁建云 1, 陈美琳 1, 向亚平 1, 程庆梅, 梁云生 3, 黄进华 1, 黄健 1, 陈静 1 1, 高丽华 ( 中南大学 1. 湘雅三医院皮肤科, 长沙 113;. 基础医学院生理学系, 长沙 113;3. 湘雅二医院皮肤科, 长沙 111) [ 摘要 ] 目的 : 阐明利用卵清蛋白 (ovalbumin,ova) 金黄色葡萄球菌肠毒素 B(staphylococcal enterotoxin B,SEB) 和维生素 D 类似物 (calcipotriene ointment,co) 局部刺激小鼠背部建立特应性皮炎 (atopic dermatitis,ad) 动物模型的方法 方法 : 将 BALB/c 小鼠背部去毛随机分为 3 组 : 对照组,,/CO, 分别给予相应的处理 隔天给药并拍照记录皮损状态, 于第 15 天处死小鼠, 处死前用皮肤共聚焦显微镜测量表皮厚度 用 ELISA 方法检测血清 IL-, IL-,IL-31, 干扰素 (interferon,ifn)-γ, 肿瘤坏死因子 (tumor necrosis factor,tnf)-α, 神经生长因子 (nerve growth factor,ngf) 和 IgE 水平,HE 染色检测皮损处炎症情况, 实时荧光定量 PCR 检测皮损处上述因子的 mrna 表达水平 结果 :/CO 组皮损处炎症细胞浸润和表皮增厚明显, 血清 IgE 水平显著增高, 血清和皮损处 IL-,IL-31, IFN-γ,TNF-α 和 NGF 表达水平均显著高于对照组或 组 结论 :/CO 局部刺激可短时间 低成本地诱导 BALB/c 小鼠皮肤 AD 样改变 [ 关键词 ] 特应性皮炎 ; 维生素 D 类似物 ;BALB/c 小鼠 ; 炎症因子 ; 免疫球蛋白 E; 表皮厚度 Atopic dermatitis (AD) is one of the most common chronic skin inflammatory diseases that affect 1% % of infants and 1% 3% of adults worldwide. The main feature of AD is the presence of eczematoid, dry and pruritic skin lesions [1-]. The pathogenesis of AD is multifactorial and involves interactions between environmental and genetic factors, thus making it difficult to be effectively treated and resulting in a huge economic burden [3]. Patients with AD usually have a thicker epidermis and the presence of abnormal inflammatory cells in skin lesion, as well as high levels of IgE and IL- in the serum compared to healthy individuals. To investigate the etiology of AD, different AD mouse models have been developed, including 1) application of epicutaneous sensitizers such as house dust mite, super-antigen, haptens, trinitrochlorobenzene (TNCB), etc; ) transgenic mice with manipulated selected genes, such as staphylococcal enterotoxin B (SEB); 3) spontaneous AD-like skin lesions developed in mice. Considering the cost and time to establish various models, application of epicutaneous (EC) sensitizers, such as ovalbumin (OVA), and super-antigens, such as SEB, to induce AD-like skin lesions in mice is one of the most common approaches []. Spergel et al [5] described the presence of a cellular infiltration rich in T cells and eosinophils, as well as increased levels of IL-, IL-5, and interferon (IFN)-γ mrna in the local AD-like skin lesion area of mice after the application of protein antigen OVA. Laouini et al [6] also found that EC exposure to SEB induces acutely elevated Th cytokine IL-, but not the Th1 cytokine IFN-γ in an AD-like skin lesion area. New insights into the recent findings of Savinko et al [7] suggested that exposure to both SEB and elicites a mixed Th1/Th-associated cytokine and chemokine expression profile in lesion skin locally. Huang et al [8] also reported that BALB/c mice subjected to EC application of the recombinant mite induce localized dermatitis characterized by pronounced epidermal hyperplasia and spongiosis, skin infiltration with CD + /CD8 + T cells, and a locally and systematically skewed Th response. Calcipotriene is a vitamin D analogue approved as an effective drug for the treatment of psoriasis [9]. Surprisingly, Li et al [1] found that topical application of vitamin D analogue induces the expression of thymic stromal lymphopoietin (TSLP) in epidermal keratinocytes, by which to lead to an atopic dermatitis-like skin lesion. Furthermore, application of calcipotriene induces an AD-like phenotype in mice as defined by clinical and histological parameters and increases expression of IL- transcripts locally [11]. Although these mouse models are effective to study AD-like lesion, they are costly and time consuming. It is well known that topical OVA and SEB exposure induces AD-like skin lesions, and recently vitamin analogues, like calcipotriene ointment (CO), have been proven to exacerbate AD. To develop more effective AD-like models, we reported an AD-like mouse model by sensitization of mouse skin with SEB, OVA, and CO.

3 Epicutaneous sensitization with ovalbumin, staphylococcal enterotoxin B and vitamin D analogue induces atopic dermatitis in mice, TAN Lina, et al 15 1 Materials and methods 1.1 Materials Animals: 6 8 weeks old female BALB/c mice were obtained from the Laboratory Animal Center of Xiangya School of Medicine at Central South University, which supplied a pathogen-free and humidity controlled environment for mouse housing. Animal related studies were conducted in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals adopted and promulgated by the United National Institutes of Health. All experimental mouse protocols were approved by the Review Committee for the Use of Human or Animal Subjects of Xiangya School of Medicine, Central South University. SEB (Sigma, USA) stock was prepared at.5 μg/ml in sterile phosphate-buffered saline (). Aliquots were storage at until use. OVA (Sigma, USA) stock was prepared at 1 mg/ml in sterile. Aliquots were storage at until use. CO was produced by LEO Laboratories Limited (Approval number: H183). Other materials included chloral hydrate (liq.), test chambers (used for patch-test),, medical infusion paste, and Veet (depilatory cream, France). 1. Contact sensitization and elicitation procedure The skin of the dorsal back (1 cm ) of 8 weeks old female mice were marked, shaved, and depilated under anesthesia with 1% chloral hydrate. Twenty-four hours later, the hairless area was sensitized according to 3 approaches:, /CO, and. Treated skin patch would be then sensitized about hours. The dose of SEB and OVA was 1.5 and 5 μg/patch, respectively. The amount of CO solution was 1 mg/ patch. The marked skin patch was consecutively treated every other day for a total of 8 times during a 15-day experimental period (Figure 1). Results from 8 mice per treatment group were collected and analyzed. 1. Skin thickness analysis The thickness of the skin in each group was measured by reflectance confocal microscope (RCM, VivaScope 15, LUCID NIC) before the mice were killed. 1.5 Quantitative real-time PCR analysis Skin biopsies obtained from the treated patch areas were snap frozen in liquid nitrogen and stored at 8 until use. RNA extraction was performed after skin tissus had been grounded into a powder [1]. RNA quantity was determined by measuring optical density at 6/8 nm. cdna was synthesized using.5 μg of total RNA in total volume of μl reaction mixture with revert aid first strand cdna synthesis kit (Thermo Scientific, USA). The cdna was then used for quantitative real-time PCR (qrt-pcr, ABI Prism 77; Applied Biosystems, USA) as previously described [13]. The expression of specific genes was analyzed by delta-delta Ct method, with β-actin as an internal control. Three mice from each group were analyzed. The primers used are listed in Table 1. Table 1 Primers sequences for the quantitative real-time PCR Primers IL- IL-31 IL- TNF-α IFN-γ NGF β-actin Sequence F: 5'-GCCATATCCACGGATGCGACAA-3' R: 5'-GGTGTTCTTCGTTGCTGTGAGG-3' F: 5'-CATCTCGGTCATCATAGCACATC-3' R: 5'-TTCATCATATTTCCAGGCACAG-3' F: 5'-AGATGAACTTGGACCTCTGCG-3' R: 5'-ACTCATCATCGAATTGGCACTC-3' F: 5'-GGAACTGGCAGAAGAGGCACTC-3' R: 5'-GTAGACAGAAGAGCGTGGTGGC-3' F: 5'-CTCAAGTGGCATAGATGTGGAAG-3' R: 5'-TGCTGATGGCCTGATTGTCT-3' F: 5'-ATAAAGGTTTTGCCAAGGACG-3' R: 5'-AGTGGGCTTCAGGGACAGAG-3' F: 5'-TTGCAGCTCCTTCGTTGCC-3' R: 5'-GACCCATTCCCACCATCACA-3' TNF-: Tumor necrosis factor; NGF: Nerve growth factor 1.3 Skin histology After the 8th sensitization, the treated skin patches were harvested, fixed in 1% buffered formalin, and embedded in paraffin for routine histopathological examination using μm sections. Images were obtained under the microscope (The Product Mode: OLYMPUS CX1). 1.6 Plasma cytokine analysis On the 16th day after induction, mice were killed and plasma was collected under centrifugation at r/min for 1 minutes, and immediately frozen at 8. The samples were complied with Bio-Plex Pro kit (BIO-RAD, USA) to examine the levels of cytokines (IL-, IL-, IL- 31, IFN-γ, TNF-α, and NGF). Results were analyzed by Bio-Plex system.

4 16 中南大学学报 ( 医学版 ), 17, (9) Statistical analysis Non-parametric Mann-Whitney test and ANOVA were used to compare difference among groups, and a twotailed P<.5 was considered statistically significant for all comparisons. Results.1 Topical exposure to /CO induced skin inflammation and exacerbated allergic dermatitis Topical exposure to /CO induced local erythema and epidermal thickening. With continuous stimulation, remarkable local skin changes, such as erythema, oozing, bleeding and crusting were further developed. sensitized mice only showed mild redness and scaling. The appearance of the -treated control mice were normal (Figure 1A 1C). Histologic analysis was consistent with the skin clinical changes. Topical /CO exposure significantly induced skin thickening and increased accumulation of inflammatory cells in the dermis compared with mice treated with OVA/ SEB, which mainly induced hyperplasia (increased collagen in the dermis) with a mild inflammatory response (a small amount of inflammatory cells under tested patches), while vehicle ()-sensitized skin patch was normal (Figure 1E 1G, 1I 1; 1H, 1J, ). Additionally, under the RCM, the epidermal thickness was greatly increased in /CO-treated skin compared with either or groups (Figure 1D). A Epidermal thickness/μm OVA OVA B C SEB SEB D CO E F G H I J Figure 1 Skin surface feature, epidermal thickness and H&E staining showing skin lesions after treatment Skin surface features after treatment with (A), (B), and /CO(C). Epidermal thickness of mice (D) measured by RCM. HE staining showing skin lesions after treatment (E-J; 1E 1G, 1I 1; 1H, 1J, ). E and H represent the control () group. F and I represent the group. G and J represent the /CO group. Bar size is μm. /CO group showed that marked hyperplasia of the epidermis, infiltration of numerous inflammatory cells, hyperkeratosis and parakeratosis coexist. /CO stimulated the expression of TH1/Th-type cytokines and pruritus-related cytokines Th1-type cytokines, including IFN-γ and TNF-α were increased in the /CO-treated skin in comparison with the control group. The expression level of IL- was higher in the group than that in the /CO group (Figure ). The expressions of Th-

5 Epicutaneous sensitization with ovalbumin, staphylococcal enterotoxin B and vitamin D analogue induces atopic dermatitis in mice, TAN Lina, et al 17 type cytokines (IL-, IL-31) were locally elevated in the /CO group compared with the control group, while IL- was only slightly increased in the group (Figure ). Additionally, pruritus-related cytokine NGF was significantly increased in the /CO group compared with the and control groups. The expression of NGF in the and control groups was similar (Figure ). Relative IL- expression 8 6 Relative IL-31 expression 3 1 /CO /CO P<.5 Relative TNF-α expression 8 6 Relative NGF expression P<.5 /CO /CO Relative INF-γ expression /CO Relative IL- expression /CO Figure mrna expression of IL-, IL-, IL-31, TNF-α, IFN-γ, and NGF in skin lesions of mice.3 Effect of /CO on serum IgE levels, Th1/Th-type cytokines, and pruritus-related NGF As shown in Figure 3, serum IgE levels were increased in the /CO group compared with the control group. Levels of Th1-type cytokines (TNF-α, IL- ) and Th-type cytokines (IL-, IL-31) were increased in both and /CO groups. The efficiency of /CO for AD induction was stronger than. Surprisingly, plasma IFN-γ levels had no significant difference among the three groups. Pruritusrelated cytokine NGF levels were significantly increased in the /CO group compared with the and control groups, and the NGF levels did not change in the group (Figure 3).

6 18 中南大学学报 ( 医学版 ), 17, (9) IL-31 level in lge serum/(ng. ml 1 ) IL- level in P<.1 P<.5 /CO P<.1 /CO /CO A D G IL- level in IFN-γ level in /CO /CO Figure 3 Serum levels of IgE (A) and cytokine [IL-(B), TNF-α(C), IL-31(D), IFN-γ(E), NGF(F) and IL-(G)] in mice B E TNF-α level in NGF level in P<.5 /CO /CO C F 3 Discussion We report that topical application of sensitizers, /CO, on the dorsal skin of mouse back induces a human AD-like animal model with features including skin inflammation, severe allergic dermatitis, thickened epidermis and the presence of inflammatory cells in affected areas as well as increased serum levels of IgE and IL-. This human AD-like mouse model is easy to establish and convenient to use, thus provides an effective tool to study AD etiology and pathogenesis, as well as develops future therapeutic alternatives for human AD. Worth noting that EC exposure to /CO elicited an intense local cutaneous inflammatory response compared with the and control groups. This inflammatory reaction was characterized by erythema, oozing, bleeding and crusting, increased epidermal thickness with infiltration of numerous inflammatory cells. Without the CO, induced a lower epidermal thickness and needed longer time (about 8 weeks) to establish the AD-like mouse model. Results were verified by RCM, as shown in Figure 1D. RCM, adopted by confocal laser scanning microscopy (CLSM), is a novel tool that provides skin images in horizontal plane at a level of resolution that allows the view of microanatomics tructures [1]. Compared with pathologic examination, RCM is a noninvasive, real-time, dynamic skin diagnostic imaging technique that is used to track the normal morphology and physiological functions of live cells, and provides information of the tissue microstructure and metabolic process without the need to kill the mice. RCM was initially used for the diagnosis of skin tumors, hemangiomas, and infectious skin diseases and is useful in the skin examination of patients with AD. During the acute stage of AD, Th cytokines IL-, IL-5, and IL-13 are predominant, whereas Th1 cytokine IFN-γ is expressed at late stages of the disease [15]. Our study shows a higher expression of IL-, IL-31, IFN-γ, IL-, and TNF-α in the group exposed to /CO sensitization. These results are consistent with previous studies from Laouini et al [6-7] who suggested that SEB and exposure elicites a mixed Th1/Th-associated cytokine expression profile within the skin. Additionally, IL-31 and NGF were highly expressed in the /CO group. IL-31 is overexpressed in pruritic skin diseases like AD compared with non-pruritic skin inflammation like psoriasis. In patients with AD, activated leukocytes express significantly higher levels of IL-31 compared with the control group [16]. NGF, a neurotrophin that is closely link to atopic dermatitis,

7 Epicutaneous sensitization with ovalbumin, staphylococcal enterotoxin B and vitamin D analogue induces atopic dermatitis in mice, TAN Lina, et al 19 is released by proliferating keratinocytes. NGF levels in patients with AD were also higher than those in a healthy group, and were related to the severity of pruritus, erythema, and inflammatory cells [17]. The relationship between NGF and pruritic diseases (such as AD) is still not well understood. Current results suggest that both IL- 31 and NGF may serve as novel targets for developing antipruritic drugs in the future. In conclusion, our study indicates that the combination of clinical features, histopathology and cytokine expression could be obtained from human AD mouse model. Considering that topically application of OVA, SEB, or both to establish an AD mouse model usually requires 7 or more weeks, and CO-treated models require transgenic mice. Our current model has a good effect on the lower cost and time saving, which are crucial for efficient study for this challenging skin disorder, where time, costs and quality of life of patient are key to seek a relief in the cardinal symptom of this pruritus condition. Although the relationship between CO and AD has not been completely understood, data from multiple clinical studies indicate that CO could induce or worsen the condition. To our knowledge, this is the first study that demonstrates the add of CO to a basic treated mice could elicit a perfect AD model on normal skin of mice, moreover, this model has a very similar skin lesions seen in human patients with AD. References [1] Lifschitz C. The impact of atopic dermatitis on quality of life[ J]. Ann Nutr Metab, 15, 66 (Suppl 1): 3-. [] Leung DY, Jain N, Leo HL. New concepts in the pathogenesis of atopic dermatitis[ J]. Curr Opin Immunol, 3, 15(6): [3] Boguniewicz M, Leung DYM. 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Clin Rev Allergy Immunol, 16, 51(3): (Edited by CHEN Liwen) 本文引用 : 谭丽娜, 鲁建云, 陈美琳, 向亚平, 程庆梅, 梁云生, 黄进华, 黄健, 陈静, 高丽华. 卵清蛋白 金黄色葡萄球菌肠毒素 B 和维生素 D 类似物诱导小鼠特应性皮炎模型 [ J]. 中南大学学报 ( 医学版 ), 17, (9): DOI: /j.issn Cite this article as: TAN Lina, LU Jianyun, CHEN Meilin, XIANG Yaping, CHENG Qingmei, LIANG Yunsheng, HUANG Jinhua, HUANG Jian, CHEN Jing, GAO Lihua. Epicutaneous sensitization with ovalbumin, staphylococcal enterotoxin B and vitamin D analogue induces atopic dermatitis in mice[ J]. Journal of Central South University. Medical Science, 17, (9): DOI: / j.issn