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.     

Deficiency of n‐6 polyunsaturated fatty acids is mainly responsible for atopic dermatitis‐like pruritic skin inflammation in special diet‐fed hairless mice

Hairless mice fed a special diet, HR‐AD, develop atopic dermatitis (AD)‐like skin inflammation with skin barrier defects and itch‐related scratching; however, the ingredient(s) causing the dermatitis remains unclear. In this study, we examined whether deficiency of certain polyunsaturated fatty acids (PUFAs) is involved in HR‐AD‐induced AD. High‐purity PUFAs were given to HR‐AD‐fed mice by dietary supplementation or gavage. Fatty acid levels in the serum and skin were determined by using gas chromatography–mass spectrometry. In serum from HR‐AD‐fed mice, linoleic acid (LA, 18:2n‐6) and α‐linolenic acid (ALA, 18:3n‐3), as well as their metabolites, were markedly decreased. When mice were fed HR‐AD supplemented with LA or ALA in an amount equal to that contained in a normal diet, the development of AD‐like symptoms was completely prevented by supplementation with LA but not with ALA. Relatively high dose of ALA slightly alleviated skin barrier defects, but did neither itch‐related scratching nor skin inflammation. On the other hand, gavage administration of LA metabolites, such as γ‐linolenic acid and arachidonic acid (AA), significantly ameliorated established dermatitis without increasing LA in the serum and skin. Moreover, AA‐induced amelioration of dermatitis was not affected by pharmacological blockade of 5‐lipoxygenase (5‐LOX) and cyclooxygenase (COX), suggesting no involvement of 5‐LOX‐ or COX‐mediated AA metabolites in the amelioration. In conclusion, our results indicate that deficiency of n‐6 PUFAs is mainly responsible for AD‐like symptoms by HR‐AD feeding. Thus, this model could be useful for studying the pathomechanisms associated with deficiency of n‐6 PUFAs in AD.     

Dietary deficiencies of unsaturated fatty acids and starch cause atopic dermatitis‐like pruritus in hairless mice

Hairless mice fed with a special diet (named HR‐AD) show atopic dermatitis (AD)‐like pruritic skin inflammation that is almost completely resolved with the supplementation of an unsaturated fatty acid (UFA), the linoleic acid (LA). This suggests that the dietary deficiency of LA is the key cause of this dermatitis. However, because there is no appropriate control diet for HR‐AD, the involvement of other dietary ingredients cannot be ruled out. Furthermore, it has not yet been tested whether only UFA deficiency can cause such AD‐like pruritus. In this study, using semi‐purified custom diets, we attempted to reproduce this syndrome. Four‐week‐old hairless mice were maintained on a widely used standard diet American Institute of Nutrition‐76A (AIN‐76A), its modifications, or HR‐AD. Several modifications of fat and carbohydrate components revealed that dietary deficiency of both UFAs and cornstarch was required to induce severe skin barrier dysfunction as typically occurred in HR‐AD‐fed mice. An UFA‐ and cornstarch‐deficient diet caused severe AD‐like pruritus comparable to HR‐AD, despite weak Th2 immune responses and absence of immunoglobulin E production. On the other hand, a diet lacking UFAs but containing cornstarch significantly alleviated the development of pruritic dermatitis. Furthermore, the supplementation of wheat starch similarly improved skin barrier function. In conclusion, this study showed that a lack of certain starches might also be the cause of diet‐induced AD. Our findings could help to reproduce the diet‐induced AD itch model and also provide evidence that certain starches can have protective and ameliorative effects on AD‐like pruritus.     

Amelioration of atopic‐like skin conditions in NC/Tnd mice by topical application with distilled Alpinia intermedia Gagnep extracts

Alpinia intermedia, a perennial plant that belongs to the Zingiberaceae family, has been used in folk medicine for a long time in the southern region of Japan. Because skin care is an effective approach that enables patients to manage their atopic dermatitis (AD), various herbal ingredients with few adverse effects have been evaluated for use in AD patients in recent years. In this study, we examined whether distilled extracts obtained from A. intermedia were beneficial for AD‐like skin conditions in NC/Tnd mice. Topical application with the A. intermedia extracts significantly reduced the severity of AD, transepidermal water loss and scratching behavior in the mice. Supplementation of the extracts to cell cultures suppressed the expression of Tslp mRNA in PAM212 keratinocytes, degranulation in bone marrow‐derived cultured mast cells (BMCMC), and neurite outgrowth in PC12 cells and dorsal root ganglia. In addition, the component analysis revealed that β‐pinene was a major constituent of the A. intermedia extracts. The inhibitory effects of β‐pinene both in vivo and in vitro were also demonstrated. These results indicate that topical application with the A. intermedia extract to the skin of NC/Tnd mice improved the condition of the skin by suppressing multiple inflammatory responses. The extracts may become novel skin‐care remedies for AD patients.     

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.     

Topical acidic cream prevents the development of atopic dermatitis‐ and asthma‐like lesions in murine model

Long‐standing or repeated skin barrier damage followed by atopic dermatitis (AD) is the initial step of the atopic march that eventually progresses to respiratory allergies. Maintenance of an acidic pH in the stratum corneum (SC) is an important factor for normal skin barrier function. We performed this study to determine whether an oxazolone (Ox)‐induced AD murine model can develop airway inflammation by topical application and nasal inhalation of a house dust mite, Dermatofagoides pteronyssinus (Dp), which is a novel ‘atopic march animal model’, and whether an acidic SC environment, made by repeated application of acidic cream, can interrupt this atopic march. During repeated treatment with Ox and Dp to make an atopic march murine model, acidic cream (pH 2.8) and neutral cream (pH 7.4) adjusted by citric acid and sodium hydroxide mixed with vehicle were applied twice daily. Repeated treatment with Ox and Dp to hairless mice induced AD‐like skin lesions followed by respiratory allergy, defining it as an atopic march model. Acidic cream inhibited the occurrence of respiratory allergic inflammation as well as AD‐like skin lesions. These results indicate that a novel atopic march animal model can be developed by repeated topical and nasal treatments with house dust mite on Ox‐induced AD mice and that the acidification of SC could be a novel intervention method to block the atopic march.     

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.     

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.     

Increased alpha‐melanocyte‐stimulating hormone (α‐MSH) levels and melanocortin receptors expression associated with pigmentation in an NC/Nga mouse model of atopic dermatitis

Please cite this paper as: Increased alpha‐melanocyte‐stimulating hormone (α‐MSH) levels and melanocortin receptors expression associated with pigmentation in an NC/Nga mouse model of atopic dermatitis. Experimental Dermatology 2010; 19: 132–136. Abstract: Patients with a specific subtype of atopic dermatitis (AD) display particular patterns of pigmentation, such as ripple pattern pigmentation on the neck, pigmented macules on the lip and diffuse pigmentation. However, the mechanism underlying these patterns has not been determined. The purpose of our research is to investigate the factors influencing this type of pigmentation in AD. We observed that AD model mice (NC/Nga mice) displayed an increase in the number of 3, 4‐dihydroxyphenylalanine (Dopa)‐positive melanocytes in the epidermis and intestine (jejunum and colon) while in the inflammatory state. The plasma levels of alpha‐melanocyte‐stimulating hormone (α‐MSH) and adrenocoticotropin (ACTH) also increased in NC/Nga mice with dermatitis. Furthermore, the expression of melanocortin receptor 5 and melanocortin receptor 1 (MC1R) increased in the skin, and melanocortin receptor 3 (MC3R) expression increased in the intestine. However, the changes in the Dopa‐positive cells of conventional NC/Nga mice were not induced by treatment with either agouti (an MC1R antagonist) or agouti‐related protein (an MC3R antagonist). These results indicate that the pigmentation of AD is related to increased levels of α‐MSH, MC1R (in the skin) and MC3R (in the intestines).     

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.     

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.     

A putative antipruritic mechanism of the phosphodiesterase‐4 inhibitor E6005 by attenuating capsaicin‐induced depolarization of C‐fibre nerves

E6005, a potent, selective phosphodiesterase (PDE) 4 inhibitor, has been developed as a novel topical agent of atopic dermatitis (AD). It has been shown to inhibit itching in patients with AD as well in mouse models. To study the mechanism underlying the anti‐pruritic effect of E6005, we examined its effect on the activation of dorsal root ganglion (DRG) neurons associated with the itch sensation. Depolarization of DRG neurons by a transient receptor potential vanilloid 1 (TRPV 1) activator, capsaicin was attenuated by E6005 as well as by a 3′,5′‐cyclic adenosine monophosphate (cAMP) elevator, forskolin. E6005 elevated intracellular levels of cAMP in DRG cells. Taken together, these results suggest that E6005 suppresses TRPV1‐mediated C‐fibre depolarization through elevation of cAMP levels, thereby exerting an anti‐pruritic effect. Thus, E6005 shows the potential to be a new agent for managing pruritus in various skin disorders, including AD.     

Malassezia yeasts activate the NLRP3 inflammasome in antigen‐presenting cells via Syk‐kinase signalling

Although being a normal part of the skin flora, yeasts of the genus Malassezia are associated with several common dermatologic conditions including pityriasis versicolour, seborrhoeic dermatitis (SD), folliculitis, atopic eczema/dermatitis (AE/AD) and dandruff. While Malassezia spp. are aetiological agents of pityriasis versicolour, a causal role of Malassezia spp. in AE/AD and SD remains to be established. Previous reports have shown that fungi such as Candida albicans and Aspergillus fumigatus are able to efficiently activate the NLRP3 inflammasome leading to robust secretion of the pro‐inflammatory cytokine IL‐1β. To date, innate immune responses to Malassezia spp. are not well characterized. Here, we show that different Malassezia species could induce NLRP3 inflammasome activation and subsequent IL‐1β secretion in human antigen‐presenting cells. In contrast, keratinocytes were not able to secrete IL‐1β when exposed to Malassezia spp. Moreover, we demonstrate that IL‐1β secretion in antigen‐presenting cells was dependent on Syk‐kinase signalling. Our results identify Malassezia spp. as potential strong inducers of pro‐inflammatory responses when taken up by antigen‐presenting cells and identify C‐type lectin receptors and the NLRP3 inflammasome as crucial actors in this process.     

The hypomagnesic rat model: dermatitis-prone hairless rats with mild magnesium depletion fed a diet low in lipids did not develop pruritic dermatitis

BACKGROUND/PURPOSE: In the hypomagnesic dermatosis of rats, low-dietary magnesium leads to lowered serum Mg2+, universal dermatitis and scratching. The model is postulated to work better if the diet also has a low content of lipids. Pruritus is seen in patients suffering from anorexia nervosa and resolves on weight restoration. Lipid depletion of the diet and/or lowered body weight may therefore be important in developing pruritus. The purpose of the present study was to investigate whether a diet low in lipids can induce pruritus in dermatitis prone rats because of mild magnesium depletion thereby focusing on the role of lipids in the hypomagnesic dermatosis rat model. METHODS: Fourteen male hairless rats, 3 weeks of age, were fed a diet deficient in lipids and with a lower content of magnesium for a 4-week period and compared with 11 controls fed a normal diet. The skin was inspected daily and transepidermal water loss (TEWL), serum Mg2+ and scratching activity were measured. At the end, skin biopsies were taken from the flank and ears. RESULTS: Serum Mg2+ was significantly lowered in the diet group (P<0.001) but the rats developed no dermatitis or increased TEWL compared with 11 rats in a control group. Biopsies were normal with no sign of inflammation. Rats in the diet group had inferior weight gain, were less active and spent more time asleep than control rats (P<0.01). Furthermore, scratching activity monitored in the last week of the study as the number of scratch-sequences per awake minute was reduced in the diet group (P<0.001). CONCLUSION: The lipid-deficient diet was not able to induce pruritic dermatitis despite the fact that the rats were dermatitis prone because of mild magnesium depletion. It is not confirmed that dietary lipid depletion plays any significant role in the hypomagnesic dermatitis rat model. The dermatitis and the model appear to be strictly dependent on efficient magnesium depletion manufactured with very low magnesium levels.     

Characterization of skin lesions induced by skin‐tropic α‐ and β‐papillomaviruses in a patient with epidermodysplasia verruciformis

Epidermodysplasia verruciformis (EV) is a rare, lifelong, autosomal recessive skin disease associated with an unusual susceptibility to infections with ubiquitous β‐human papillomaviruses (β‐HPVs), and in some cases also skin‐tropic α genotypes. In this case report, HPV infection patterns were correlated with pathology and clinical manifestations of skin lesions from a patient with EV, without loss‐of‐function mutations in the EVER genes. HPV infection was investigated by both polymerase chain reaction (PCR) and laser capture microdissection (LCM) PCR, alongside immunofluorescence for the viral proteins E4 and L1. Analysis of eyebrow hair bulbs revealed multiple β‐genus HPV infections, including HPV20 and HPV24, which were consistently found in all 11 skin lesions on the patient. Six lesions were also positive for the skin tropic α‐genotype, HPV27. Clear‐cut differences between two wart‐like lesions, one caused by a skin‐tropic α‐genotype and the other by β‐genotypes (as detected by LCM PCR) are shown, including the high cellular proliferation rate in β‐HPV‐induced lesions. Widespread expression of the early protein E4 was also evident in skin lesions positive for HPV20 by LCM PCR in both tumours and nearby intraepidermal proliferative areas. L1 expression was restricted to areas of intraepidermal proliferation showing productive infection. The patient's inability to control HPV infections is conclusive to the uncontrolled replication of few genotypes from both α and β genera, which cause proliferative lesions with clear‐cut clinical and histological features.     

Oral supplementation with Lactobacillus rhamnosus CGMCC 1.3724 prevents development of atopic dermatitis in NC/NgaTnd mice possibly by modulating local production of IFN‐γ

Abstract: Prevalence of allergies has increased during the last two decades. Alteration of the gut microbiota composition is thought to play a crucial role in development of atopic diseases. Oral administration of probiotics has been reported to treat and/or prevent symptoms of atopic diseases in infants, but the results are still controversial. We investigated the potential efficacy of dietary interventions by a probiotic strain on prevention and treatment of atopic dermatitis (AD) in a human‐like AD model, NC/NgaTnd mice by perinatal administration. Pregnant NC/NgaTnd mice were orally treated with the probiotic strain Lactobacillus rhamnosus CGMCC 1.3724 (LPR), which was followed by treatment of pups until 12 weeks of age. LPR‐treated mice exhibited significant lower clinical symptoms of dermatitis, reduced scratching frequency, lower levels of plasma total Immunoglobulin E and higher levels of interferon‐γ in skin biopsies, compared with untreated mice. The protective effect was also observed when mice started to be treated at weaning time (5 weeks of age) even with limited supplementation period of 2 weeks. However, treatment of mice with the probiotic starting 1 week after the onset of the disease (8 weeks of age) had limited effects. The usefulness of LPR for primary prevention of AD was supported.     

Increased interleukin‐36γ expression in skin and sera of patients with atopic dermatitis and mycosis fungoides/Sézary syndrome

Interleukin (IL)‐36γ is expressed by keratinocytes and functions as a key initiator of inflammation in the skin. IL‐36γ expression is enhanced by tumor necrosis factor‐α and IL‐17A, having a strong association with psoriasis. In this study, we examined the role of IL‐36γ in atopic dermatitis (AD) and mycosis fungoides (MF)/Sézary syndrome (SS). Serum levels of IL‐36γ in AD patients and MF/SS patients were elevated compared with those of healthy controls. Importantly, serum IL‐36γ levels in AD patients positively correlated with Eczema Area and Severity Index and those of MF/SS patients positively correlated with serum soluble IL‐2 receptor levels. IL‐36γ mRNA levels in AD skin and MF/SS skin were significantly higher than those of normal skin. IL‐36γ mRNA levels in MF/SS skin positively correlated with IL‐17A mRNA levels. Immunohistochemical staining revealed that IL‐36γ was highly expressed in keratinocytes in lesional skin of AD and MF/SS. Taken together, our study demonstrated that IL‐36γ expression was increased in sera and skin of patients with AD and MF/SS as was reported in psoriatic patients.