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.     

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.     

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.     

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.     

Heat-killed bacillus Calmette-Guérin and Mycobacterium kansasii antigen 85B combined vaccination ameliorates dermatitis in a mouse model of atopic dermatitis by inducing regulatory T cells

Background Atopic dermatitis (AD) is a recurrent inflammatory skin disease characterized by dominant T‐helper (Th) 2 cytokine response. Bacillus Calmette–Guérin (BCG) has been used for preventing tuberculosis, and is regarded as a strong Th1 cytokine inducer. Antigen (Ag) 85B is a secretory protein present in Mycobacterium species that induces Th1 cytokine production. Objectives We investigated the effects of combined vaccination of heat‐killed BCG (hkBCG) and Mycobacterium kansasii Ag85B in an AD mouse model. Methods For the AD model, keratin 14 promoter‐derived caspase‐1 overexpressing mice (KCASP1Tg) were used. The mice received a combination therapy of hkBCG at age 3 weeks and Ag85B twice weekly for 11 weeks from the 4th week; Ag85B monotherapy from the 4th week; hkBCG monotherapy at the 3rd week; or control saline. Areas of skin lesions, cytokine mRNA expression and serum interleukin (IL)‐18 and immunoglobulin (Ig) E levels were analysed. Inducible Foxp3+ regulatory T cells (iTreg), IL‐10‐producing T cells (Tr1), and interferon (IFN)‐γ/IL‐4/IL‐17‐producing T cells were evaluated in the spleen. Results Saline‐treated mice and hkBCG monotherapy mice spontaneously developed severe dermatitis. However, combined therapy with hkBCG and Ag85B significantly suppressed the development of skin lesions and mast cell infiltrations. Elevations of the serum IgE and IL‐18 levels were significantly suppressed with combined therapy. Mice treated with hkBCG and Ag85B had a normal number of iTreg in the spleen, and decreased number of both IL‐4‐ and IL‐17‐producing CD4+ T cells. The effect of Ag85B monotherapy was limited. Conclusions Combined vaccination with hkBCG and Ag85B decreases AD skin lesions by inducing regulatory T cells, suggesting that this vaccination is a potent and novel therapeutic strategy for AD.     

Hyperproduction of IFN-gamma by CpG oligodeoxynucleotide-induced exacerbation of atopic dermatitis-like skin lesion in some NC/Nga mice

Under conventional conditions, NC/Nga mice spontaneously develop an atopic dermatitis (AD)-like skin lesion accompanied by immunoglobulin E (IgE) hyperproduction and the expression of T helper 2 (Th2) cytokines. CpG DNA activates a strong interferon-gamma (IFN-gamma)-dominated T helper 1 (Th1) response, while inhibiting Th2-dependent allergies. In this study, we examined whether CpG oligodeoxynucleotide (ODN) could prevent the development of the skin lesions in NC/Nga mice. Sixteen of 26 NC/Nga mice did not exhibit dermatitis after CpG ODN was administered intraperitoneally every 2 wk for a total of five times. CpG ODN administration induced IFN-gamma production, which inhibited the production of Th2 cytokines (interleukin (IL)-4, IL-5, and IL-13) in both spleen and lymph node cells and culminated in a decrease in the serum IgE level. These data suggest that the CpG ODN has a therapeutic effect against AD; however, some mice (10 of 26) treated with CpG ODN exhibited an exacerbation of dermatitis accompanied by the hyperproduction of IFN-gamma, although Th2 cytokines were suppressed. These results suggest that the suppression of Th2 cytokines may not completely prevent dermatitis and that IFN-gamma may play a role in developing dermatitis in some NC/Nga mice.     

Gene expression of enzymes for tryptophan degradation pathway is upregulated in the skin lesions of patients with atopic dermatitis or psoriasis

BACKGROUND: Atopic dermatitis (AD) and psoriasis are common inflammatory skin diseases. Although many reports implicate Th2 cytokines in the pathophysiology of AD and Th1 cytokines in psoriasis, the precise etiology of these diseases remains elusive. OBJECTIVE: We investigated novel AD- or psoriasis-related genes to further understand the pathogenesis of these diseases. METHODS: We performed a comprehensive analysis of mRNA expression in skin biopsies from AD or psoriasis patients using DNA microarrays. Quantitative PCR was then used to monitor the expression of novel disease-related genes in human keratinocytes or pinnae from NC/Nga mice. RESULTS: Levels of mRNA for IDO (indoleamine 2,3-dioxygenase) and kynureninase, enzymes constituting the tryptophan degradation pathway, were found to be upregulated in the skin lesions as compared to the uninvolved skin of patients with AD or psoriasis. Expression of these two genes was induced in human epidermal keratinocytes stimulated with IFN-gamma in vitro. Moreover, in NC/Nga mice, the expression of kynureninase mRNA in the ear skin was induced following development of AD-like skin lesions. CONCLUSION: The tryptophan degradation pathway may play an important role in the pathophysiology of AD and psoriasis.     

A single dose of interleukin‐31 (IL‐31) causes continuous itch‐associated scratching behaviour in mice

We investigated the effects of a single dose of mouse interleukin‐31 (IL‐31) on scratching behaviour in comparison with spontaneous skin‐lesion‐ or serotonin (5‐HT)‐ induced scratching behaviour in NC/Nga and BALB/c mice. Intradermal (i.d.) injection of IL‐31 caused a gradual increase in long‐lasting scratching (LLS, over 1.5 s) about 3 h after administration followed by a gradual decrease for over 24 h after administration. I.d. injection of IL‐31 significantly increased the total LLS counts/24 h but not short‐lasting scratching (SLS, 0.3–1.5 s). In skin‐lesioned NC/Nga mice, the LLS but not SLS counts were significantly higher than those in non‐skin‐lesioned NC/Nga mice. We also investigated 5‐HT‐induced scratching in BALB/c mice, SLS but not LLS increased immediately after the injection and then decreased to baseline after at 20 min. These results suggest that IL‐31 may participate in the sensation of itching and promote scratching behaviour in skin‐lesioned NC/Nga mice, an animal model of atopic dermatitis (AD).     

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).     

Modulation of HMGB1 translocation and RAGE/NFκB cascade by quercetin treatment mitigates atopic dermatitis in NC/Nga transgenic mice

Quercetin, glycosylated form of flavonoid compound, has potent antioxidant and anti‐inflammatory properties. In this study, we have investigated the effects of quercetin on skin lesion, high‐mobility group box (HMGB)1 cascade signalling and inflammation in atopic dermatitis (AD) mouse model. AD‐like lesion was induced by the application of house dust mite extract to the dorsal skin of NC/Nga transgenic mouse. After AD induction, quercetin (50 mg/kg, p.o) was administered daily for 2 weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for HMGB1, receptor for advanced glycation end products (RAGE), toll‐like receptor (TLR)4, nuclear factor (NF)κB, nuclear factor erythroid‐2‐related factor (Nrf)2, kelch‐like ECH‐associated protein (Keap)1, extracellular signal‐regulated kinase (ERK)1/2, cyclooxygenase (COX)2, tumor necrosis factor (TNF)α, interleukin (IL)‐1β, IL‐2Rα and other inflammatory markers in the skin of AD mice. In addition, serum levels of T helper (Th) cytokines (interferon (IFN)γ, IL‐4) were measured by enzyme‐linked immunosorbent assay. Quercetin treatment attenuated the development of AD‐like skin lesions. Histological analysis showed that quercetin inhibited hyperkeratosis, parakeratosis, acanthosis, mast cells and infiltration of inflammatory cells. Furthermore, quercetin treatment downregulated cytoplasmic HMGB1, RAGE, nuclear p‐NFκB, p‐ERK1/2, COX2, TNFα, IL‐1β, IL‐2Rα, IFNγ and IL‐4 and upregulated nuclear Nrf2. Our data demonstrated that the HMGB1/RAGE/NFκB signalling might play an important role in skin inflammation, and quercetin treatment could be a promising agent for AD by modulating the HMGB1/RAGE/NFκB signalling and induction of Nrf2 protein.     

Therapeutic effects of streptococcal preparation OK-432 on atopic dermatitis-like lesions in NC/Nga mice: possible shift from a Th2- to Th1-predominance

BACKGROUND: The inducement of Th1 cell-mediated immune response, possibly brought about through bacterial stimulation, may serve to control atopic disorders such as atopic dermatitis (AD). The streptococcal preparation, OK-432, has been shown a potent Th1 inducer through the action of IL-12. NC/Nga mice under ordinary conditions have been found to contract dermatitis similar to human AD. OBJECTIVE: Examination was made of the therapeutic effects of OK-432 local intra- and/or subcutaneous injections on AD-like lesions in NC/Nga mice. METHODS: Immunohistochemical staining with IL-4/IL-12p40 and CD80/86 and phosphorylated STAT4/p-STAT6 and RT-PCR for IL-4/IL-12p40 and STAT6/STAT4 mRNA was conducted for the evaluation of OK-432 treatment of spontaneous AD-like lesions in NC/Nga mice. RESULTS: At 5 weeks following injection of OK-432, for treating head and back lesions in NC/Nga mice, 10 of 12 OK-432 treated NC mice were found to have clinically improved quite considerably. On the head and back skin of OK-432-treated mice, IL-12p40/CD80 positive cellular infiltration was conspicuous, in contrast to non-treated mice. IL-4/CD86 positive cellular infiltrates in OK-432-treated mice had decreased significantly more than in non-treated mice and IL-4 mRNA expression was virtually absent in OK-432-treated mice. P-STAT4 positive cells could be seen abundantly present in OK-432-treated mice, and p-STAT6 positive cells were much fewer than in non-treated mice. CONCLUSIONS: OK-432-treatment appears to induce Th1 cellular response and to down-regulate that of the Th2 pathway in AD-like lesions of NC/Nga mice. The present results demonstrate bacterial components from such Streptococcus to likely constitute an effective new therapeutic approach in the treatment of AD.     

Topical application of rapamycin ointment ameliorates Dermatophagoides farina body extract‐induced atopic dermatitis in NC/Nga mice

Atopic dermatitis (AD), a chronic inflammatory skin disease characterized by relapsing eczema and intense prurigo, requires effective and safe pharmacological therapy. Recently, rapamycin, an mTOR (mammalian target of rapamycin) inhibitor, has been reported to play a critical role in immune responses and has emerged as an effective immunosuppressive drug. In this study, we assessed whether inhibition of mTOR signalling could suppress dermatitis in mice. Rapamycin was topically applied to inflamed skin in a murine AD model that was developed by repeated topical application of Dermatophagoides farina body (Dfb) extract antigen twice weekly for 7 weeks in NC/Nga mice. The efficacy of topical rapamycin treatment was evaluated immunologically and serologically. Topical application of rapamycin reduced inflammatory cell infiltration in the dermis, alleviated the increase of serum IgE levels and resulted in a significant reduction in clinical skin condition score and marked improvement of histological findings. In addition, increased mTOR phosphorylation in the lesional skin was observed in our murine AD model. Topical application of rapamycin ointment inhibited Dfb antigen‐induced dermatitis in NC/Nga mice, promising a new therapy for atopic dermatitis.     

DAMP molecules S100A9 and S100A8 activated by IL‐17A and house‐dust mites are increased in atopic dermatitis

S100A9 and S100A8 are called damage‐associated molecular pattern (DAMP) molecules because of their pro‐inflammatory properties. Few studies have evaluated S100A9 and S100A8 function as DAMP molecules in atopic dermatitis (AD). We investigated how house‐dust mites affect S100A9 and S100A8 expression in Th2 cytokine‐ and Th17 cytokine‐treated keratinocytes, and how secretion of these molecules affects keratinocyte‐derived cytokines. Finally, we evaluated expression of these DAMP molecules in AD patients. S100A9 expression and S100A8 expression were strongly induced in IL‐17A‐ and Dermatophagoides (D.) farinae‐treated keratinocytes, respectively. Furthermore, co‐treatment with D. farinae and IL‐17A strongly increased expression of S100A9 and S100A8 compared with D. farinae‐Th2 cytokine co‐treatment. The IL‐33 mRNA level increased in a dose‐dependent manner in S100A9‐treated keratinocytes, but TSLP expression did not change. S100A8/A9 levels were also higher in the lesional skin and serum of AD patients, and correlated with disease severity. Taken together, S100A9 and S100A8 may be involved in inducing DAMP‐mediated inflammation in AD triggered by IL‐17A and house‐dust mites.     

Biphasic increase in scratching behaviour induced by topical application of Dermatophagoides farinae extract in NC/Nga mice

Atopic dermatitis (AD) is a chronic inflammatory skin disease accompanied by severe itching and eczematous lesion. In this study, we applied an ointment containing Dermatophagoides farinae body (Dfb) extract repeatedly on the dorsal skin of NC/Nga mice with barrier disruption to investigate the characteristics of this murine model of human AD. Following repeated topical application of Dfb ointment twice weekly for 2 weeks, the dermatitis score increased gradually, accompanied by an elevation of total immunoglobulin E level in plasma. Topical application of Dfb ointment also caused epidermal hyperplasia and accumulation of inflammatory cells in the lesional skin and increased expression of T‐helper (Th) 1/Th2/Th17 cytokines in axillary lymph node cells. Furthermore, increased sprouting of intraepidermal nerve fibres was observed with an increase in the content of nerve growth factor and decrease in that of semaphorin 3A in the lesional skin. These findings suggest that the characteristics in this model were similar to those observed in patients with AD. Interestingly, it was observed for the first time that scratching behaviour increased in a biphasic fashion by topical application of Dfb ointment in addition to an increase in spontaneous scratching behaviour in this model. It is also suggested that further clarifying the underlying mechanisms of scratching behaviour in this model leads not only to elucidating the pathogenesis of AD but also to discovering novel therapeutic drugs for AD.     

Lipoteichoic acid-related molecule derived from the streptococcal preparation, OK-432, which suppresses atopic dermatitis-like lesions in NC/Nga mice

Bacterial stimulation may serve to control atopic disorders such as atopic dermatitis (AD) through inducement of Th1 cell-mediated immune response. The lipoteichoic acid (LTA)-related molecule (okLTA) from streptococcal preparation, OK-432, has been shown to be a potent Th1 inducer through the action of IL-12. Examination was made of the therapeutic effects of this okLTA injected intra- and/or subcutaneously into AD-like lesions in NC/Nga mice, particularly in the vicinity of the suppressor of cytokine signaling (SOCS) regulatory pathways. Using immunohistochemical staining with IL-4/IL-12p40 and phosphorylated STAT6/p-STAT4 and RT-PCR for IL-4/IL-12p40, STAT6/STAT4 and mRNA expression and in situ hybridization of SOCS3 and 5, evaluation was made of the immunoregulatory effects of this okLTA in the treatment of spontaneous AD-like lesions in NC/Nga mice. Following the injection of okLTA, remarkable improvement in the lesions of NC/Nga mice was noted. In okLTA-treated skin, IL-12p40/p-STAT4 positive cellular infiltration was extensive while IL-4/p-STAT6 positive cell infiltration was seen to diminish considerably, compared to untreated NC mice. SOCS3 in situ expression in okLTA-treated mice was noted to be significantly less compared to untreated NC mice, in which the expression was prominent. SOCS5 in situ expression was rather, though not significantly, strong in okLTA-treated mice. okLTA treatment is clearly shown to induce Th1 cellular response and down-regulate immune response in the Th2 pathway through SOCS3 reduction in AD-like lesions of NC/Nga mice. The present results demonstrate that bacterial wall components such as okLTA should serve as an effective new therapeutic approach for treating AD.     

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.     

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.     

Repeated topical challenge with chemical antigen elicits sustained dermatitis in NC/Nga mice in specific-pathogen-free condition

NC/Nga mice are known to develop skin lesions resembling to atopic dermatitis (AD) in conventional but not in specific-pathogen-free (SPF) condition. An epicutaneous application of 2,4-dinitrofluorobenzene (DNFB) increased skin thickness in C3H as well as NC/Nga mice in SPF environment, and the response was enlarged by repeating the challenge at weekly intervals. Although the skin reaction in C3H mice was ameliorated when the challenge was discontinued after the fifth application, the reaction in NC/Nga mice was sustained at least for 3 wk. Analyses of cytokine production by CD4+ cells from the draining lymph node proximal to the lesions revealed that, unlike C3H mice, NC/Nga mice fail to induce T helper 2 (Th2) cytokine interleukin-4 (IL-4), whereas the level of Th1 cytokine interferon-gamma in NC/Nga mice is equivalent to that of C3H mice. In addition, NC/Nga mice highly expressed IL-12, a cytokine-preventing formation of Th2 response, whereas C3H mice did not. Administration of anti-IL-12 antibody reduced duration of dermatitis in DNFB-treated NC/Nga mice. Taken together, our data suggest that IL-12 plays a role in the persistent skin reaction in NC/Nga mice. The action of IL-12 might be mediated by the decrease in IL-4 production.     

Involvement of IL-31 on scratching behavior in NC/Nga mice with atopic-like dermatitis

Pruritus is an important symptom in atopic dermatitis (AD), but the major pruritogen has not been identified. NC/Nga mice, spontaneously develop an eczematous AD-like skin lesion when kept under conventional conditions, but not under specific pathogen-free (SPF) conditions, have been thought to be an animal model for AD. In this study, to determine whether newly identified cytokine, IL-31, may be involved in pruritus of AD, we examined the IL-31 expression in spontaneous dermatitis model which showed itch-associated long-lasting (over 1.5 s duration) scratching behavior and compared with that of hapten-induced contact dermatitis model without itch-associated long-lasting scratching behavior, using NC/Nga mice. In NC/Nga mice cohabited with NC/Nga mice which developed severe dermatitis for 2 weeks (conventional NC/Nga mice), the numbers of long-lasting scratching counts were significantly increased. Yet in 2,4,6-trinitrochlorobenzene (TNCB)-sensitized and challenged mice (TNCB-applied NC/Nga mice), no significant increase in long-lasting scratching counts was observed. In conventional NC/Nga mice with long-lasting scratching behavior, expression of IL-31 mRNA was increased, while in TNCB-applied NC/Nga mice without long-lasting scratching behavior, the expression of IL-31 mRNA were unchanged. There was a good correlation between the scratching counts and expression of IL-31 mRNA in conventional NC/Nga mice, but not so in TNCB-applied NC/Nga mice. These results suggest that IL-31 causes the itch-associated scratching behavior in conventional NC/Nga mice, an experimental animal model for AD.     

Mechanism of pathogenesis of imiquimod‐induced skin inflammation in the mouse: A role for interferon‐alpha in dendritic cell activation by imiquimod

Topical application of imiquimod (IMQ), a Toll‐like receptor (TLR)7 ligand, can induce and exacerbate psoriasis, a chronic inflammatory skin disorder. In a mouse model of IMQ‐induced psoriasis‐like skin inflammation, T‐helper (Th)17 cells and interleukin (IL)‐17/IL‐22‐producing γδ‐T cells have been shown to play a pivotal role. However, the mechanisms of induction of the Th17 pathway and development of psoriasis‐like skin inflammation by IMQ treatment remain unclear. In this study, we investigated pathogenic mechanisms of IMQ‐induced psoriasis‐like skin inflammation in mice. We first confirmed that, together with an increase in IL‐17 and IL‐22 production, application of IMQ to mouse skin induced the expression of cytokines required for activation of the Th17 pathway, and pro‐inflammatory mediators involved in the pathology of psoriasis. Analysis of Tlr7^?/? mice demonstrated that most of the in vivo effects of IMQ were mediated via TLR7. In an in vitro study using plasmacytoid dendritic cells (DCs), IMQ induced production of interferon (IFN)‐α, IL‐23, IL‐6 and tumor necrosis factor (TNF)‐α. Furthermore, when we analyzed in vitro‐generated bone marrow‐derived DCs with features similar to TNF‐α and inducible nitric oxide synthase (iNOS)‐producing DCs, IL‐23, IL‐6, IL‐1β, TNF‐α and iNOS/NO production was weakly induced by IMQ alone and further enhanced after co‐stimulation with IMQ and IFN‐α. These in vitro effects of IMQ were also mediated via TLR7 and the synergistic effect of IMQ, and IFN‐α was suggested to be caused by upregulation of TLR7 expression by IFN‐α. These results demonstrate part of the mechanism by which the Th17 pathway and psoriasis‐like skin inflammation are induced by IMQ and IFN‐α in a mouse model.