Tsutomu Hirasawa

Characterization of dermatitis arising spontaneously in DS-Nh mice maintained under conventional conditions: another possible model for atopic dermatitis

DS-Nh (DS Nh/+) mice spontaneously develop dermatitis when they are housed in a conventional environment. In this study, we analyzed the clinical and histopathological features of dermatitis in DS-Nh mice, which is characterized by erythema, edema, and erosion on the face, neck, chest and flexor surfaces of their forelegs with marked scratching behavior. Histopathological examination, including immunohistochemistry, revealed that inflammatory cells consisting of mast cells, eosinophils, CD4-positive T cell-dominant lymphocytes and CD11b-positive macrophages infiltrated the skin lesions. The cytokine production pattern of inflammatory cells in a lesional skin tissue was shifted to the Th2-type (IL-4) rather than the Th1 type (IFN-gamma). Serum IgE levels were elevated and correlated with the severity of the clinical skin conditions. These skin symptoms were observed in association with a colonization of Staphylococcus aureus. Similar clinical and histopathological symptoms were inducible with repeated percutaneous immunization of heat-killed S. aureus on the back of SPF DS-Nh mice. These results suggest that the spontaneous dermatitis that occurs in conventionally raised DS-Nh mice is comparable to a certain type of human atopic dermatitis (AD), which is associated with S. aureus, a recognized environmental factor. Thus, we consider that DS-Nh mice offer a useful model for investigating the pathogenesis of AD and for developing new therapeutic approaches or drugs for treating AD.

TRPV3 as a Therapeutic Target for Itch

Abbreviations: AD, atopic dermatitis; AEW, acetone ether water; PGP9.5, protein gene product 9.5; TRPV3, transient receptor potential vanilloid subfamily 3

Heme oxygenase 1 attenuates the development of atopic dermatitis–like lesions in mice: Implications for human disease

BACKGROUND Oxidative stress has been implicated in the exacerbation of atopic dermatitis (AD). OBJECTIVE We sought to investigate the pathophysiologic roles of inducible antioxidant heme oxygenase (HO) 1 in the development of AD. METHODS Serum HO-1 levels of patients with AD (n = 100) and age-matched healthy control subjects (n = 72) were determined by means of ELISA. The relationships between serum HO-1 levels and clinical severities, laboratory parameters, and cytokines/chemokines were assessed. Skin lesions of patients with AD and psoriasis were analyzed by means of immunohistochemistry. A murine AD model, DS-Nh, was used to further investigate localization and function of HO-1. Evaluation of symptoms, serum IgE and IL-18 levels, immunoblotting results, and histologic analyses of skin were performed. The effect of intraperitoneally administered hemin, a potent HO-1 inducer, or zinc protoporphyrin IX, an inhibitor of HO, was monitored. RESULTS Serum HO-1 levels were significantly increased in patients with AD compared with those seen in healthy control subjects and were associated with AD disease severity. Serum HO-1 levels correlated with serum IgE, lactate dehydrogenase, IL-18, and thymus and activation-regulated chemokine levels. HO-1-expressing cells were accumulated in skin lesions of patients with AD and DS-Nh mice. Immunofluorescence of mouse skin lesions revealed that HO-1-positive cells were macrophages and dendritic cells. Treatment with hemin, but not with zinc protoporphyrin IX, attenuated the development of the skin lesions in DS-Nh mice and reduced serum IL-18 levels. CONCLUSION HO-1 levels were increased in sera and skin lesions of patients with AD. Enhancement of HO-1 attenuated the development of skin lesions in mice, suggesting that HO-1 induction offers a promising therapeutic strategy for AD.

Spontaneous scratching behaviour in DS-Nh mice as a possible model for pruritus in atopic dermatitis.

Itching is one of the major clinical symptoms in atopic dermatitis (AD) and complicates the management of this pathological condition. An animal model of AD‐like pruritus would contribute to a better understanding of AD and could lead to the development of safe and effective antipruritic agents. DS non‐hair (DS‐Nh) mice raised under conventional conditions spontaneously develop pruritus, which is associated with a dermatitis similar to human AD. There is a significant positive correlation between disease severity and the period of scratching behaviour in DS‐Nh mice. In the present study, we found that levels of histamine and nerve growth factor (NGF) in serum and/or skin tissue were higher in DS‐Nh mice with AD‐like dermatitis than in age‐matched mice without dermatitis. The histopathological data indicated that nerve fibres extend into and mast cells infiltrate the surrounding area of the skin lesion. NGF production by XB‐2 cells, which was derived from mouse keratinocytes, was enhanced by histamine via the H1 receptor. We also found that prolonged treatment with an H1‐antagonist was effective against pruritus through depression of the production of NGF, which is thought to be generated by keratinocytes. We conclude that DS‐Nh mice can serve as a suitable model for gaining a better understanding of pruritus in AD, and that prolonged treatment with an H1‐antagonist may be beneficial in patients with AD‐associated pruritus.

DS-Nh as an experimental model of atopic dermatitis induced by Staphylococcus aureus producing staphylococcal enterotoxin C.

DS–Nh mice raised under conventional conditions spontaneously develop dermatitis similar to human atopic dermatitis (AD), which is associated with staphylococcal infection. In the present study, we show that Staphylococcus aureus producing staphylococcus exotoxin C (SEC) was recovered from the culture of the skin lesions of DS–Nh mice with AD‐like dermatitis and that the serum levels of anti‐SEC antibodies from these mice were elevated. We describe here how to promote experimental AD by epicutaneous injection with SEC‐producing S. aureus to DS–Nh mice. In order to assess the role of SEC in the pathogenesis of AD, the mitogenic activity, TCRBV repertoire analysis and the production of IL‐4 and IFN‐γ from spleen mononuclear cells (MNC) from DS–Nh stimulated by SEC were compared with those due to SEA, SEB and TSST. The weakest was the mitogenic activity of SEC, and higher IL‐4 responses and lower IFN‐γ responses to SEC showed correlation with TCRBV8S2‐positive T cells, which were selectively stimulated by SEC. We also demonstrate that SEC‐producing S. aureus was able to survive in DS–Nh after intradermal injection. These results suggest a possible role for SEC in the pathogenesis of AD through host–S. aureus relationships.

Role of TRPV3 in immune response to development of dermatitis

BackgroundRecently, it has been reported that the Gly573Ser substitution of transient receptor potential V3 (TRPV3) leads to increased ion-channel activity in keratinocytes. Our previous studies have indicated that the spontaneous hairless and dermatitis phenotypes of DS-Nh mice, which were newly established as an animal model of atopic dermatitis (AD), are caused by TRPV3Gly573Ser. Although this substitution causes hairlessness in several kinds of rodents, in our investigations, dermatitis developed in only a few animals. Here, we generated NC/Nga-Nh mice to elucidate the role of TRPV3Gly573Ser in NC/Nga mice, which is one of the most studied animal models of AD.MethodsTo establish and validate the new AD animal model, NC/Nga-Nh mice were generated using NC/Nga and DS-Nh mice, and their clinical features were compared. Next, T-cell receptor (TCR) Vβ usage in splenocytes, evaluation of bacterial colonization, and serological and histological analyses were carried out. Finally, repeated-hapten-application dermatitis was induced in these mice.ResultsNC/Nga-Nh mice did not develop spontaneous dermatitis, whereas DS-Nh mice displayed this phenotype when maintained under the same conditions. Serological analysis indicated that there really was a phenotypic difference between these mice, and TCR repertoire analysis indicated that TCRVβ haplotypes played an important role in the development of dermatitis. Artificial dermatitis developed in DS and NC/Nga-Nh mice, but not in DS-Nh and NC/Nga mice. Histological and serological analyses indicated that mouse strains were listed in descending order of number of skin mast cells: DS-Nh > DS ≈ NC/Nga-Nh > NC/Nga, and serum IgE levels were increased after 2,4,6 trinitrochlorobenzene application in these mice. Serum IgE level in DS-Nh mice was lower than that mesured in other strains.ConclusionOur results confirm the contribution of the TRPV3Gly573Ser gene to the development of repeated hapten dermatitis, but not spontaneous dermatitis in NC/Nga mice.

Effects of TNCB sensitization in DS-Nh mice, serving as a model of atopic dermatitis, in comparison with NC/Nga mice.

Background: It has been predicted that a type-1 and type-2 helper T cell (Th1/Th2) imbalance exists in atopic dermatitis (AD). In DS-Nh mice, an AD mouse model, Staphylococcus aureus increases on the skin surface. Objective: To investigate whether the Th1-dominant response has an influence on the development of AD, we induced chronic allergic hypersensitivity with 2,4,6-trinitrochlorobenzene (TNCB ) in two AD mouse models: NC/Nga mice and DS-Nh mice. Th1 and Th2 cytokine production of splenocytes was assessed under stimulation with staphylococcal enterotoxin B (SEB) which induces a Th1 response in DS-Nh mice with or without TNCB sensitization. Methods: We examined clinical skin changes, transepidermal water loss (TEWL), the number of S. aureus on the skin and the serum IgE levels in these mice treated repeatedly with TNCB under conventional conditions (free of fur mites). The splenocytes of DS-Nh mice were cultured with SEB and the cytokine levels in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Results: Significant skin changes were observed on the skin even where TNCB was not applied in both mice treated with TNCB. Increases in S. aureus on the skin and serum IgE levels were detected in DS-Nh mice, but not in NC/Nga mice. In DS-Nh mice, IFN-γ and IL-13 production of splenocytes increased in the mice treated with TNCB. Conclusion: These results suggest that there might be a different mechanism of dermatitis induction between NC/Nga and DS-Nh mice. Th1 responses might play an important role in the development of dermatitis and increase in serum IgE levels in DS-Nh mice through an increase in IL-13 production.

Effects of zinc deficient diet on development of atopic dermatitis-like eruptions in DS-Nh mice

BACKGROUND Zinc is one of the essential dietary factors and zinc deficiency diminishes the immune system. However, the mechanisms by which zinc deficiency affects the immune system are not fully understood. OBJECTIVE We analyzed the mechanisms of zinc deficiency affecting the allergic response using a DS-Nh mouse model of atopic dermatitis. METHODS Male DS-Nh mice were fed a zinc deficient diet for 4 weeks. We measured transepidermal water loss (TEWL) and epidermal moisture level, assessed the skin eruption score, and examined the frequency of lymphocyte subpopulation in spleen and thymus by flow cytometry. The suppressive effect of CD25+CD4+ T cells was analyzed in vitro. The amount of cytokines produced by the spleen cells and the serum IgE levels were measured by ELISA. RESULTS In DS-Nh mice fed the zinc deficient diet, skin eruptions were exacerbated and serum IgE levels and number of S. aureus on the skin surface was increased. IFN-gamma and IL-13 production by spleen cells was increased. The number of CD25+CD4+ T cells in spleen was significantly decreased, while the percentage of Foxp3 positive cells in the CD25+CD4+ T cells was comparable to those of the controls. CD25+CD4+ T cells from mice fed the zinc deficient diet maintained a suppressive function compared with those from the controls. CONCLUSION These findings indicate that zinc deficiency influences the skin barrier system and immune system, and suggests that zinc deficiency acts as an exacerbation factor of atopic dermatitis.

DNA vaccination against macrophage migration inhibitory factor improves atopic dermatitis in murine models

BACKGROUND Atopic dermatitis (AD) is a common chronic inflammatory skin disease. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that has been implicated in the pathogenesis of AD. Recently, we developed a novel DNA vaccine that generates neutralizing endogenous anti-MIF antibodies. OBJECTIVE This study explores the preventive and therapeutic effects of this MIF-DNA vaccine in mouse models of AD. METHODS Two different AD model mice (DS-Nh and NC/Nga) received MIF-DNA vaccination to analyze preventive and therapeutic effects, as assessed by clinical skin scores, histologic findings, and serum IgE levels. RESULTS In murine models of AD, MIF-DNA vaccination prevented the occurrence of the AD skin phenotype. Furthermore, administration of MIF-DNA vaccine to mice that had already developed AD produced a rapid improvement in AD skin manifestation. There were reduced histologic signs of inflammation and lower serum IgE levels in treated mice compared with those seen in control animals. Finally, passive transfer of IgG from MIF-DNA vaccinated mice to AD mice also produced a significant therapeutic effect. These results demonstrate that MIF-DNA vaccination not only prevents the development of AD but also improves the symptoms of pre-existing AD. CONCLUSION Taken together, the induction of an anti-MIF autoantibody response using MIF-DNA vaccination appears to be a useful approach in the treatment of AD.

Impact of T‐cell receptor Vβ haplotypes on the development of dermatitis in DS‐Nh mice: synergistic production of interleukin‐13 caused by staphylococcal enterotoxin C and peptide glycans from Staphylococcus aureus

Although the pathogenic role of interleukin‐13 (IL‐13) is a key for atopic dermatitis (AD), the mechanism of IL‐13 production in AD remains unclear. To investigate the role of the T‐cell receptor Vβ (TCR Vβ) haplotype in the development of dermatitis and the production of IL‐13 in the naturally occurring dermatitis model by staphylococcal enterotoxin C (SEC)‐producing Staphylococcus aureus, we raised DS‐Nh mice harbouring the TCR Vβa haplotype with a central deletion in the TCRBV gene segments, including TCR Vβ8S2. Observation and histopathological analysis of the two mouse substrains with spontaneous dermatitis indicated that later onset and weaker severity of AD‐like dermatitis were identified in mice with TCR Vβa compared to those with TCR Vβb. Immunohistochemical examination revealed the infiltration of a large number of CD4‐bearing T cells in the skin lesions in mice with TCR Vβb but not in those with TCR Vβa. Interestingly, much lower levels of serum IL‐13 were detected in mice with the TCR Vβa than in those with the TCR Vβb haplotype. In vitro, synthetic ligands (Pam2CSK4) of toll‐like receptor 2 (TLR2) synergistically produced IL‐13 with SEC in splenocytes of mice with TCR Vβb but not of those with TCR Vβa, and natural killer T cells were essential for this synergism. Our findings suggested that this TCR Vβ‐haplotype‐dependent synergism with TLR2 plays an important role in the development of AD‐like dermatitis in DS‐Nh mice.