Impairment of skin barrier function in NC/Nga Tnd mice as a possible model for atopic dermatitis

BACKGROUND: The pathogenesis and aetiology of atopic dermatitis (AD) remain unclear. Establishment of suitable animal models should aid elucidation of the pathogenesis and development of therapy. OBJECTIVES: We focused on biophysical and biochemical parameters in the skin of NC/Nga Tnd mice to evaluate similarities to and differences from AD. METHODS: Biophysical (transepidermal water loss and skin surface conductance) and biochemical parameters (ceramide contents and activity of ceramide-metabolizing enzymes) were measured in NC/Nga Tnd mice in which spontaneous dermatitis appeared under ambient laboratory conditions (ALC). RESULTS: Biophysical parameters suggested impairment of water retention properties and barrier function. The amount of ceramide in NC/Nga Tnd mice under ALC decreased significantly. These dermatological features resembled those of AD, as did the clinical signs and histological changes. CONCLUSIONS: The results described here and previous immunological studies on AD suggest that the NC/Nga Tnd mouse may be a suitable model for certain aspects of AD.     

Effect of pedunculagin investigated by non‐invasive evaluation on atopic‐like dermatitis in NC/Nga mice

Background/purpose: Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disorder that is becoming increasingly prevalent. Experimental animal models have been an indispensable tool for studying its pathological mechanisms and for in vivo testing of novel therapeutic approaches. AD‐like lesions can be induced experimentally in NC/Nga mice. Pedunculagin, an ellagitannin purified from the Manchurian alder, Alnus hirsuta var. microphylla, Betulaceae, is a novel immunomodulator. To evaluate the effect of pedunculagin for AD‐like lesions in NC/Nga mice, using clinical and non‐invasive methods. Methods: AD‐like lesions were induced in NC/Nga mice using 2,4,6‐trinitrochlorobenzene (TNCB). A cream containing 0.1% or 0.5% pedunculagin was applied to the positive treatment group, and the base cream without pedunculagin was applied to the negative treatment group. The control group did not receive any kind of topical agents. We evaluated the therapeutic efficacy of pedunculagin for AD by statistical evaluation of the clinical severity score using non‐invasive biomedical engineering tools before treatment, and 1 day, 3 days, 1 week, 2 weeks and 4 weeks afterwards. Results: An AD‐like skin rash was successfully induced using TNCB in NC/Nga mice. The group receiving higher concentrations of pedunculagin showed faster and greater improvement. Conclusion: Our results suggest that remedies made from natural materials like pedunculagin are now showing promise for medical applications, and many new studies are expected to explore this potential.     

Adipose‐derived stem cells attenuate atopic dermatitis‐like skin lesions in NC/Nga mice

There is an unmet need in novel therapeutics for atopic dermatitis (AD). We examined the effects of autologous adipose‐derived stem cells (ADSCs) on AD‐like skin lesions induced by the application of 2,4‐dinitrochlorobenzene (DNCB) in NC/Nga mice. Autologous ADSCs and ADSC‐conditioned medium (ADSC‐CM) were injected intralesionally three times. Clinical severity and histopathologic findings were compared in sham naïve control, saline‐treated, ADSC‐treated, ADSC‐CM‐treated and 2.5% cortisone lotion‐applied animals. The severity index, skin thickness, mast cell number, as well as expression levels of thymic stromal lymphopoietin, CD45, chemoattractant receptor‐homologous molecule, chemokine ligand 9 and chemokine ligand 20 were significantly lower in mice treated with ADSC, ADSC‐CM, or 2.5% cortisone lotion. Tissue levels of interferon‐γ as well as serum levels of interleukin‐33 and immunoglobulin E levels were also decreased in those groups. We conclude that autologous ADSCs improved DNCB‐induced AD‐like skin lesions in NC/Nga mice by reducing inflammation associated with Th2 immune response and interferon‐γ.     

Time‐dependent progression from the acute to chronic phases in atopic dermatitis induced by epicutaneous allergen stimulation in NC/Nga mice

Atopic dermatitis (AD) is a complicated skin condition influenced by genetic background and environmental factors. In this study, we applied Dermatophagoides farinae body extract (DfE) to the barrier‐disrupted skin of NC/Nga mice twice a week for 8 weeks to identify the clinical and immunological factors in AD progression. Repeated application of the DfE to the skin of NC/Nga mice showed the similar consequences for the natural course of progression in human AD, histologically and immunologically. We confirmed that the AD‐like skin lesions in NC/Nga mice did not last for the whole period of our experiment in spite of repeated topical applications of DfE twice a week. Topical DfE stimulation increased the skin mRNA expressions of Th1‐, Th2‐ and Th17‐related cytokines in the acute phase. The expression patterns of IL‐4 and IL‐13 in splenic T cells and skin lesions were consistent with the time course alterations of clinical features of AD‐like skin symptoms. We also showed that there was a remission phase either just before or right after the chronic phase in this experimental model. Interestingly, splenic T‐cell‐derived IL‐5 expression began to increase in the chronic phase, while skin‐derived IL‐5 mRNA expression increased in the acute phase. In conclusion, our results suggest that we should pay attention to the characteristics of each stage of AD progression and choose a suitable corresponding stage of animal model not only to elucidate the pathogenesis of AD but also to develop and evaluate therapeutic drugs for AD.     

Aggravation of atopic dermatitis‐like symptoms by consecutive low concentration of formaldehyde exposure in NC/Nga mice

Formaldehyde (FA) has been known to be associated with development of asthma (AS) and atopic dermatitis (AD). In this study, we investigated whether FA inhalation would affect the provocation or exacerbation of AD‐like symptoms. Atopic‐prone NC/Nga mice were exposed to low (0.2 ppm) and high (1.0 ppm) concentration of FA by inhalation. Combined exposure to low concentration of FA inhalation and topical house dust mite (HDM) stimulation significantly upregulated HDM‐induced total plasma IgE and IgG2a production, Th1‐, Th2‐, Th17‐related cytokine as well as COX‐2 mRNA expressions in the skin. Interestingly, independent FA inhalation, especially at low concentration (0.2 ppm), increased the skin mRNA expressions of IL‐13, IL‐17E/IL‐25 and COX‐2, even though it failed to induce AD‐like skin inflammation. In conclusion, we suggest that increased skin mRNA expressions of IL‐13, IL‐25/IL‐17E and COX‐2 by independent low concentration of FA exposure might be a key factor to exacerbate HDM‐mediated AD‐like skin inflammation.     

Suppressive effect of mangosteen rind extract on the spontaneous development of atopic dermatitis in NC/Tnd mice

Atopic dermatitis (AD) is a chronic and relapsing skin disorder characterized by pruritic and dry skin lesions with allergic inflammation. Recent studies have revealed anti‐inflammatory and anti‐allergic effects of xanthones in mangosteen through regulation of the nuclear factor (NF)‐κB signaling pathway. Activation of NF‐κB signals is responsible for allergic inflammation in AD. To develop a new preventive therapy for AD, we examined the effects of the natural medicine, mangosteen rind extract (ME), on AD in a murine model. ME (250 mg/kg per day) was administrated to NC/Tnd mice, a model for human AD, for 6 weeks to evaluate its preventive effects on AD. We also confirmed the effects of ME on various immune cell functions. Oral administration of ME prevented the increase of clinical skin severity scores, plasma total immunoglobulin E levels, scratching behavior, transepidermal water loss and epidermal hyperplasia in NC/Tnd mice; moreover, no adverse effects were noted. We demonstrated that ME suppressed thymic stromal lymphopoietin and interferon‐γ mRNA expression both in vitro and in vivo. Not only immunoglobulin E production from splenic B cells but also immunoglobulin E‐mediated degranulation of bone marrow‐derived cultured mast cells was significantly reduced by the addition of ME to the culture. In addition, mRNA expression levels of nerve growth factor were decreased in ME‐administrated NC/Tnd mice compared with those of controls. Keratinocyte proliferation was well‐controlled by ME application. Oral administration of ME exhibited its suppressive potential on the early development of AD by controlling inflammation, itch and epidermal barrier function.     

Oral administration of β‐carotene or lycopene prevents atopic dermatitis‐like dermatitis in HR‐1 mice

Atopic dermatitis (AD) is a chronic relapsing eczematous skin disease. Certain populations of patients are resistant to standard therapies with topical steroids and/or calcineurin inhibitors, and require systemic medication, such as immunosuppressants. Recently, several reports have shed light on the anti‐allergic effects of carotenoids. Therefore, we investigated the effect of p.o. administration of β‐carotene or lycopene on AD‐like symptoms of HR‐1 hairless mice fed with a low zinc/magnesium diet. Mice were divided into four groups: (i) fed with a standard diet (Co group); (ii) low zinc/magnesium diet (HR group); (iii) low zinc/magnesium and β‐carotene diet (HR‐C group); and (iv) low zinc/magnesium and lycopene diet (HR‐L group). They were then fed these diets for 8 weeks. Severities of dermatitis were assessed by their appearance, and histopathological and hematological observations. Mice in the HR group developed AD‐like dermatitis both clinically and histologically. HR‐C and HR‐L group mice also developed xerosis and wrinkle‐like skin changes, but they were milder than those of HR group mice. Histological analysis revealed that epidermis thickening and inflammatory cell infiltration in the skin of the HR‐C and HR‐L groups were both statistically less than those of the HR group. The concentration of thymus and activation regulated chemokine in the skin of the HR‐L group and the concentration of CCL27 in the skin of the HR‐C group were significantly lower than those of the HR group, respectively. In conclusion, p.o. administration of β‐carotene or lycopene prevents AD‐like symptoms in association with a suppression of T‐helper 2 chemokines in a murine model. Ingestion of carotenoids may be beneficial for patients with AD.     

Therapeutic potential of topically administered γ‐AlOOH on 2,4‐dinitrochlorobenzene‐induced atopic dermatitis‐like lesions in Balb/c mice

Boehmite (γ‐AlOOH) has a wide range of applications in a variety of industrial and biological fields. However, little is known about its potential roles in skin diseases. The current study investigated its effect on atopic dermatitis (AD). Following characterization, cytotoxicity, pro‐inflammatory response and oxidative stress associated with boehmite were assessed, using TNF‐α‐induced keratinocytes and mast cells. In addition, therapeutic effects of boehmite, topically administered to Balb/c mice induced by 2,4‐dinitrochlorobenzene (DNCB), were evaluated. Expression of cytokines (TLSP, IL‐25 and IL‐33) and the generation of ROS from keratinocytes induced by TNF‐α were significantly inhibited by boehmite without affecting cell viability. MAPKs (ERK, JNK and p38) required for cytokine expression were suppressed by boehmite treatment. Up‐regulation of cytokines (TSLP, IL‐4, IL‐5, IL‐13, RANTES) in human mast cells treated with phorbol 12‐myristate 13‐acetate and calcium ionophore was also suppressed by boehmite. Boehmite improved the AD severity score, epidermal hyperplasia and transepidermal water loss in DNCB‐induced AD‐like lesions. Moreover, Th2‐mediated cytokine expression, mast cell hyperplasia and destruction of the skin barrier were improved by boehmite treatment. Overall, we demonstrated that boehmite may potentially protect against AD.     

Inhibition of atopic dermatitis-like skin lesions by topical application of a novel ceramide derivative, K6PC-9p, in NC/Nga mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease that commonly begins in childhood. K6PC-9p (N-(Ethyl dihydrogenphosphate)-2-hexyl-3-oxo-decanamide) is a synthetic ceramide derivative of PC-9S (N-Ethanol-2-mirystyl-3-oxo-staramide), which was known to be effective in atopic patients. In this study, we examined the effect of topical application of K6PC-9p on skin inflammation and AD-like skin lesions in mouse models. K6PC-9p dose-dependently inhibited phorbol ester-induced increase in ear thickness in BALB/c mice. Moreover, topical application of K6PC-9p suppressed dust mite extract-induced AD-like skin lesions in NC/Nga mice. Histopathological analysis revealed that both ear swelling and leucocyte infiltration were suppressed by K6PC-9p treatment. K6PC-9p also suppressed IL-4 and TNF-alpha expression in the ears and mast cell infiltration into the ears in NC/Nga mice. Further study demonstrated that K6PC-9p inhibited ConA-induced IL-4 secretion and LPS-induced macrophage activation. Taken together, our results showed that topical application of K6PC-9p exerts beneficial effects in animal model of skin inflammation and AD, suggesting that K6PC-9p might be a promising topical agent for the treatment of inflammatory skin diseases.     

Extracellular superoxide dismutase ameliorates house dust mite‐induced atopic dermatitis‐like skin inflammation and inhibits mast cell activation in mice

Extracellular superoxide dismutase (EC‐SOD) is an enzyme that catalyses the dismutation of superoxide anions. It has multiple functions, such as reactive oxygen species scavenging, anti‐angiogenic, anti‐inflammatory, antichemotatic and antitumor activities. Recently, we demonstrated that EC‐SOD inhibits ovalbumin‐induced allergic airway inflammation in mice. However, the anti‐allergic effect of EC‐SOD on skin tissue and the role of EC‐SOD in mast cells, which are important for allergic responses, have not been well studied. In this study, we investigated whether EC‐SOD can alleviate atopic dermatitis in mice and inhibit mast cell activation. Treatment with human recombinant EC‐SOD ameliorated house dust mite‐induced atopic dermatitis in mice. Furthermore, the levels of pro‐allergic cytokine gene expression and histamine release increased in EC‐SOD KO mast cells and decreased in EC‐SOD overexpressing mast cells, suggesting that EC‐SOD inhibits mast cell activation. Consistently, a passive cutaneous anaphylaxis experiment showed more blood leakage from EC‐SOD KO mouse ear skin, implying that the lack of EC‐SOD increases allergic responses. These results suggest that EC‐SOD inhibits mast cell activation and atopic dermatitis and that the loss of EC‐SOD causes more severe allergic responses, implying that EC‐SOD might be a good drug candidate for treatment of allergic disorders, such as atopic dermatitis.     

Evidence for a regulatory loop between IFN‐γ and IL‐33 in skin inflammation

Interleukin‐33 has recently gained much attention due to its role in allergic responses. It has been shown to amplify Th2 responses and to act as a damage‐associated molecular pattern. IL‐33 acts on a broad range of cells and has been proposed to link innate and adaptive features of allergic responses. It was the aim of this study to investigate this property of IL‐33 in the inflammatory response characterising atopic dermatitis (AD). We have analysed the response of skin‐resident cells derived from patients with AD and healthy donors with regard to the expression of IL‐33 and its receptor ST2. The functional impact of IL‐33 on CD4+ T cells was investigated. Keratinocytes and dermal fibroblasts clearly differ in their regulation of IL‐33. In fibroblasts, the concerted action of TNF‐α and IL‐1β was the strongest inducer, whereas IFN‐γ is clearly the key molecule that upregulates IL‐33 in keratinocytes with a more pronounced response of cells derived from patients with AD. Keratinocytes from patients with AD showed a markedly higher constitutive expression level of surface ST2. CD4+ T cells respond to IL‐33. Unexpectedly, IL‐33 failed to induce a significant secretion of IL‐5 or IL‐13. By contrast, high amounts of IFN‐γ were detectable if IL‐33 was added to the T‐cell receptor‐stimulated cells or in combination with IL‐12. These results suggest that IL‐33 and IFN‐γ are closely interlinked in epidermal AD inflammation. IFN‐γ induces IL‐33 in keratinocytes and IL‐33 acts on activated T cells to further increase the release of IFN‐γ, therefore contributing to drive skin inflammation towards chronic responses.     

Effects of β‐carotene on oxazolone‐induced atopic dermatitis in hairless mice

Skin acts as a barrier, which protects internal tissues and promotes moisture retention. Atopic dermatitis (AD) is an inflammatory skin disease associated with a variety of genetic and environmental factors that involve helper T cells. β‐Carotene (provitamin A) exhibits antioxidant activity and activates the immune system. However, it is not clear whether inflammation in AD skin is improved by posttreatment with β‐carotene. In the current study, we investigated the effects of β‐carotene on the skin of hairless mice with oxazolone‐induced inflammation/oedema (Ox‐AD mice). We found that skin inflammation was significantly reduced by oral administration of β‐carotene. In addition, treatment with β‐carotene suppressed protein levels of TNF‐α, IL‐1β and MCP‐1, as well as mRNA expression associated with IL‐1β, IL‐6, IL‐4 and Par‐2 in skin tissues. Furthermore, the mRNA and protein levels of filaggrin, a structural protein in the epidermal stratum corneum, were elevated by β‐carotene administration as compared with Ox‐AD mice. β‐Carotene significantly reduced the activity of proMMP‐9, but not proMMP‐2. These results suggest that in Ox‐AD mice, β‐carotene improves skin inflammation by suppressing the expression of inflammatory factors, promoting filaggrin expression and reducing MMP‐9 activity. β‐Carotene is a potent anti‐inflammatory agent that improves the barrier functions of AD skin.     

Effect of topical application and intraperitoneal injection of oregonin on atopic dermatitis in NC/Nga mice

Please cite this paper as: Effect of topical application and intraperitoneal injection of oregonin on atopic dermatitis in NC/Nga mice. Experimental Dermatology 2010; 19 : e37–e43. Abstract: The diarylheptanoid, oregonin (ORE), which was isolated from the bark of Alnus japonica Steudel that grows natively in Korea, has been known to exert antioxidative, anti‐inflammatory, anti‐cancer and immune response inhibitory effects. The antioxidative effect of ORE was observed on the superoxide and 1,1‐diphenyl‐2‐picrylhydrazyl radical, as well as on the expression of inducible nitric oxide synthase and cyclooxygenase‐2 in lipopolysaccharide‐treated RAW264.7 macrophages. The statistically significant inhibitory action of ORE against production of cytokines induced by bacterial products or by interleukin (IL)‐1β, free radicals and nitrogen species, and a corresponding increase in cellular calcium concentration because of ORE were confirmed in bone marrow and spleen dendritic cells that are known to play important functions in the development and advancement of atopic dermatitis (AD). It was thus expected that ORE would exert a beneficial effect in the treatment of AD. A study on the pharmaceutical benefits of ORE against AD has not yet been conducted in vivo. We therefore used an in vivo AD animal model, namely the NC/Nga mice, and by applying ORE onto the animals through skin application as well as intraperitoneal injection, we attempted to evaluate the benefits of ORE in this system. Evaluation of ORE was conducted by following the SCORE method to score the effect, as well as by measuring the Th2 cytokines IL‐4, IL‐5 and IL‐13 levels from serum and lymphocytes, and IgE and eosinophil levels from serum. Additionally, the expression of mRNA and protein levels was estimated using real‐time polymerase chain reaction and Western blotting analysis. The following categories of clinical evaluation, Th2 cytokines IL‐4, IL‐5 and IL‐13 values, serum IgE levels, serum eosinophil levels, and mRNA and protein expression levels of iNOS and COX‐2, were evaluated from topical application and intraperitoneal injection groups of ORE. The effects of ORE on AD in NC/Nga mice were confirmed as being similar to the positive control group, while a significant difference with the negative control group was observed. The results presented in this report suggest that ORE might be beneficial in the treatment of AD.     

Oral administration of poly‐γ‐glutamic acid prevents the development of atopic dermatitis in NC/Nga mice

Bacillus subtilis‐derived poly‐γ‐glutamic acid (γPGA) has demonstrated adjuvant activity in promoting Th1/Th17 cell differentiation. Here, the NC/Nga (NC) mouse model was used to determine whether γPGA modulates the outcome of atopic dermatitis (AD), which is known to be a Th2‐biased immune disease. We found that oral administration of γPGA dramatically reduced the development of AD in NC mice. Antigen‐presenting cells activated with γPGA produced pro‐inflammatory cytokines, such as IL12/23 and IFNγ, which, in turn, induced the differentiation of Th1 and Th17 cells. Concomitantly, Th2 responses, such as high levels of serum IgE, were dramatically decreased. Furthermore, in vivo γPGA treatment altered several cellular components of allergic reactions, such as mast cells and eosinophils. Taken together, our results strongly demonstrate that in vivo treatment with γPGA at early time points can prevent the development of AD in NC mice and suggest that γPGA may have therapeutic applications for human AD.     

Heparinoid suppresses Der p‐induced IL‐1β production by inhibiting ERK and p38 MAPK pathways in keratinocytes

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro‐inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen‐induced interleukin (IL)‐1β release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase‐1 release, suggesting that heparinoid did not affect HDM allergen‐induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro‐IL‐1β, but also suppressed IL‐1β messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal‐regulated kinase and p38 pathways, which are required for IL‐1β expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL‐1β mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte‐mediated skin inflammation.     

REGγ accelerates melanoma formation by regulating Wnt/β‐catenin signalling pathway

It has been reported that the proteasome activator REGγ is associated with multiple oncogenic pathways in human cancers. However, the role of REGγ in the development of melanoma and the underlying mechanisms remain unclear. In this study, we attempted to investigate the effects of REGγ on human melanoma cell proliferation in vitro and in vivo. We demonstrated that knockdown of REGγ inhibited melanoma cell growth and arrested melanoma cell at G1 phase. Furthermore, depletion of REGγ also inhibited the xenograft growth of human melanoma. Mechanistically, REGγ activates Wnt/β‐catenin signal pathway by degrading GSK‐3β in melanoma cell lines and mouse models. Transient knockdown of β‐catenin effectively blocked cell proliferation in REGγ wild‐type melanoma cells. In human melanoma samples, REGγ was overexpressed and positively correlated with β‐catenin levels. This study demonstrates that REGγ is a central molecule in the development of melanoma by regulating Wnt/β‐catenin pathway. This suggests that targeting REGγ could be an alternative therapeutic approach for melanoma.     

Induction of scratching behaviour and dermatitis in various strains of mice cohabiting with NC/Nga mice with chronic dermatitis

BACKGROUND: NC/Nga (NC) mice with similar pathological and behavioural features as seen in human atopic dermatitis are used as a model of the disease. Under normal circumstances, spontaneous and persistent scratching occurs in NC mice and this can lead to the onset of skin inflammation. OBJECTIVES: We examined the induction of scratching behaviour in NC, BALB/c, ICR and C3H/HeN mice, and of dermatitis in NC and BALB/c mice, by cohabitation with mice with dermatitis. METHODS: NC, BALB/c, ICR and C3H/HeN mice were kept together with NC mice with chronic dermatitis (CNV-NC) for 2 weeks, and the numbers of scratching episodes were counted. NC and BALB/c mice were also kept together with CNV-NC mice for 24 weeks and the skin severity score was assessed. The score was assessed for a further 8 weeks after separation of these mice. RESULTS: The number of scratching episodes in NC, BALB/c, ICR and C3H/HeN mice was increased by cohabitation with CNV-NC mice. Cohabitation with CNV-NC mice led to dermatitis in both NC and BALB/c mice. The number of scratching episodes and the skin severity score in BALB/c mice were about half of those in NC mice. When cohabitation with CNV-NC mice stopped, the number of scratching episodes and the skin severity score decreased in BALB/c mice, but not in NC mice. Changes in the histopathological data of BALB/c mice supported the severity of skin inflammation. CONCLUSIONS: Our study demonstrates that scratching behaviour and dermatitis can be induced in various strains of mice by cohabitation with CNV-NC mice, and that cessation of cohabitation leads to a recovery in BALB/c mice but not in NC mice.