Combination Therapy with Betamethasone and Josamycin Demonstrates Superior Therapeutic Efficacy in an NC/Nga Mouse Model of Atopic Dermatitis

Abstract

We have previously demonstrated the excellent bactericidal activity of josamycin against Staphylococcus aureus isolated from patients with atopic dermatitis (AD), with therapeutic efficacy equal to that of betamethasone. The present study was designed to evaluate the effectiveness of combination therapy with betamethasone and josamycin for AD. Betametasone (0.1%) and josamycin (0.1%) were topically administered to NC/Nga mice with severe AD-like skin lesions. Skin severity scores, histological changes in skin lesions, and serum immunoglobulin E (IgE) levels were assessed as indicators of therapeutic efficacy. Topical treatment with both drugs suppressed the skin severity score to a greater degree than betamethasone alone. This was associated with a reduction of epidermal thickening, a reduced density of dermal cellular infiltration, a decreased mast cell count in the dermis, and a reduced serum IgE level. In addition, both drugs in combination markedly reduced the expression of interferon (IFN)-γ and interleukin (IL)-4 in auricular lymph node cells, as well as the S. aureus count on the lesioned skin. These results show that simultaneous topical application of both drugs can ameliorate severe AD-like skin lesions in NC/Nga mice. It is suggested that combination therapy with betamethasone and josamycin would be beneficial for control of severe AD lesions colonized by S. aureus by inhibiting the development of both T helper (Th) type 1 (Th1) and Th2 cells and also through elimination of superficially located S. aureus.

INTRODUCTION

Atopic dermatitis (AD) is a chronic inflammatory skin disease with immunopathologic features that vary depending on the duration of the lesions.¹⁾ AD significantly impacts the QOL of patients, and its worldwide prevalence is considered to be about 10–30% in children and about 2–10% in adults.^2,3) AD develops early in childhood, and its onset is thought to be related to multiple genetic abnormalities, decreased skin barrier function, immune response abnormality, and environmental factors.⁴⁾ In particular, the resulting defective skin barrier function disturbs the normal immune system through stimulation of the microbiome and plays major roles in the pathogenesis of AD.⁵⁾ The majority of AD patients show superficial skin colonization by Staphylococcus aureus and increased expression of T helper (Th) type 2 (Th2) cytokines such as interleukin (IL)-4, IL-5, and IL-13 in peripheral blood mononuclear cells or skin lesions.^6,7) S. aureus can be isolated from 96–100% of AD skin lesions, whereas only 0–10% of healthy individuals show skin colonization by this organism.^8,9) Furthermore, the incidence of S. aureus detected in the lesioned skin of AD patients is higher than that in non-lesioned skin, and the S. aureus bacterial cell count is also significantly higher in lesioned than in non-lesioned skin.⁹⁾ We have also demonstrated that chronic skin colonization with S. aureus may augment Th2 cell development in AD patients.¹⁰⁾ Besides, we have clarified that cell wall components of S. aureus induce an increase in the numbers of eosinophils, mast cells and Th2 cells, which are closely associated with exacerbation of skin inflammation.^11–14) Therefore, treatment of AD with antibiotics has a beneficial effect, not only in patients with impetiginized AD but also those without clinical signs of superinfection.

We have shown in previous studies that a macrolide antibiotic, josamycin, has excellent bactericidal activity against S. aureus strains isolated from AD skin lesions, and suppresses the exacerbation of AD-like skin inflammation in NC/Nga mice as affectively as betamethasone upon topical application to the lesional skin.^15,16) Therefore, in the present study using NC/Nga mice, we evaluated the effect of combination therapy with betamethasone and josamycin for severe AD lesions, which could not be fully controlled by betamethasone alone.

MATERIALS AND METHODS

Mice

The mice used for the study were 6-week-old female specific pathogen-free NC/Nga mice obtained from Japan SLC (Hamamatsu, Japan). The animals were housed in plastic cages with sterilized paper bedding in a clean, air-conditioned room at 24 °C and allowed access to a standard laboratory diet and water ad libitum. All procedures performed on the mice were in accordance with the guidelines and approval of the Animal Care and Use Committee of Meiji Pharmaceutical University.

Reagents

A mite antigen, Dermatophagoides farinae extract (Biostir AD), was purchased from Biostir Inc. (Osaka, Japan). Betamethasone and josamycin were obtained from Tokyo Chemical Industry (Tokyo, Japan) and Adipogen (Liestal, Switzerland), respectively. The vehicle employed was white petrolatum including 5% (w/w) liquid paraffin, and used to prepare 0.1% (w/w) betamethasone ointment and 0.1% (w/w) josamycin ointment.

Induction of AD-Like Skin Lesions

The dorsal hair of NC/Nga mice was shaved with a hair clipper and finally removed with a depilatory cream. Skin barrier disruption was then achieved by topical application of 150 μL of 4% sodium dodecyl sulfate to the dorsal skin (120 μL/8 cm²) and auricle skin of both ears (15 μL/1 cm²). After 3 h, 100 mg Biostir AD was applied topically to the dorsal skin (80 mg/8 cm²) and auricle skin of both ears (10 mg/1 cm²). These procedures were repeated every 3 or 4 d. Figure 1 shows the design of the experimental schedule.

Fig. 1. Experimental Schedule for Induction of AD-Like Dermatitis in NC/Nga Mice and Treatment with Betamethasone and Josamycin Ointment

Measurement of Skin Severity Score

Dermatitis severity was assessed macroscopically using the scoring system described previously.¹⁶⁾ Briefly, one skin lesion (1 cm²) on each ear and one skin lesion (8 cm²) on the back were scored on the basis of the following criteria. The dermatitis score (minimum 0; maximum 30 [= 3 regions × 2 points × 5 symptoms]) was calculated as the sum of the individual scores for the three regions, and graded as 0 (no symptoms), 1 (less than 1/3 of the skin area) or 2 (1/3 and more of the skin area), for each of the following 5 symptoms: redness/scratch marks, edema/lichenification/thickening, hemorrhage/scabbing, erosion, and desquamation.

Topical Application of Betamethasone and Josamycin to NC/Nga Mice

After the skin severity score had reached approximately 18–20, which corresponds to severe AD-like skin inflammation, vehicle, 0.1% betamethasone ointment or 0.1% josamycin ointment was applied topically to the dorsal skin and the auricle skin of both ears (50 mg/body [= 50 mg/10 cm² skin]) in each group once per day, except Sunday. Figure 1 shows the design of the experimental schedule.

Histopathological Observations

The dorsal skin was removed 24 d after assessment of skin severity, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 5 μm. Tissue sections were then stained with hematoxylin–eosin (H&E) stain, toluidine blue (TB) stain (for staining mast cells) and Luna stain (for staining eosinophils), respectively, and observed microscopically. The number of lymphocytes, mast cells and eosinophils, respectively, was counted in 16 high-power fields (×400) of each skin section and expressed as cells/mm². The epidermal thickness was measured at 10 places per skin section, and the mean value was taken as the epidermal thickness for that specimen.

Measurement of Serum Total Immunoglobulin E (IgE)

Blood samples were collected from the heart 24 d after assessment of skin lesion severity. The total serum IgE concentration was measured by enzyme-linked immunosorbent assay (ELISA) using a mouse IgE ELISA kit (Cedarlane, ON, Canada).

Quantification of Th1 and Th2 Cytokine Production from T Lymphocytes in Lymph Nodes

Auricular lymph node cells were harvested on the 24th day of assessment of skin severity, and adjusted to 1 × 10⁶ cells/mL in RPMI 1640 medium with L-glutamine (Sigma-Aldrich, St. Louis, MO, U.S.A.) containing 10% fetal bovine serum (Sigma-Aldrich), 25 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (Hepes) (Sigma-Aldrich), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco RBL, Grand Island, NY, U.S.A.). The cultures (0.2 mL/well) were incubated in 96-well culture plates (Nunc, Roskilde, Denmark) in the presence of Dynabeads Mouse T-Activator CD3/CD28 (Life Technologies, Oslo, Norway) at 37 °C in a humidified atmosphere with 5% CO₂. The culture supernatants were collected after incubation for 48 h, and the interferon (IFN)-γ and IL-4 concentrations were measured using ELISA kits for quantification of murine IFN-γ and IL-4, respectively (R & D Systems, Minneapolis, MN, U.S.A.).

Detection of S. aureus on Skin

Bacterial isolates were obtained from each skin lesion by applying a “Film Stamp” for 10 s to the affected dorsal skin (8 cm² area [= 2 cm × 4 cm]).⁹⁾ After incubation overnight on tryptic soy agar plates at 37 °C, colonies were characterized by color and diameter, and colony numbers were expressed as colony-forming units (CFU) per 8 cm² skin area. Microscopic examination of Gram-stained colonies and the PS Latex (Eiken Chemical, Tokyo, Japan) slide agglutination test was also carried out for organism identification. S. aureus was finally identified from the reaction profile based on the 20 biochemical tests included in the API STPH system (Biomérieux, Marcy-I’Etoile, France).

Statistical Analysis

The data were expressed as means (± standard error of the mean (S.E.M.)), and differences between means were analyzed using the Tukey–Kramer multiple comparison test or Steel–Dwass multiple comparison test (for comparison of skin severity scores). Differences at p < 0.05 were considered to be statistically significant. All statistical analyses were conducted using BellCurve for Excel (Social Survey Research Information Co., Ltd., Tokyo, Japan).

RESULTS

Therapeutic Effects of Topically Applied Betamethasone and Josamycin on AD-Like Skin Lesions in NC/Nga Mice

Assessment of skin lesion severity in NC/Nga mice and medication with ointment were started 25 d after initial sensitization with the mite antigen (Fig. 1). Before treatment, the sensitized mice had severe AD-like skin lesions, and the clinically evident skin lesion severity decreased gradually with the medication (Fig. 2). After 14 d of assessment, the combined therapeutic efficacy of josamycin ointment and betamethasone ointment became apparent and persisted throughout the experimental period, the efficacy far exceeding that of betamethasone ointment alone (Figs. 2, 3). Topical application of vehicle alone had no effect on the dermatitis. All of the positive control (untreated) mice exhibited AD-like skin lesions characterized by redness/scratch marks, edema/lichenification/thickening, hemorrhage/scabbing, erosion and desquamation (Fig. 3). In the negative control (normal skin) mice, no superficial lesions were observed throughout the experimental period.

Fig. 2. Effects of Topically Applied Betamethasone and Josamycin on Skin Severity Score

The results for each experimental group are expressed as means ± S.E.M. (n = 6). **  p < 0.05 versus untreated, ^§ p < 0.01 versus untreated, ^† p < 0.05 versus betamethasone (Steel–Dwass test).

Fig. 3. Macroscopic Features of AD-Like Skin Lesions in NC/Nga Mice

The photograph shows skin lesions on the 24th day of assessment.

Effects of Topical Application of Betamethasone and Josamycin on Histopathological Changes in Dorsal Skin

As shown in Fig. 4a, no abnormal histopathological changes were evident in the dorsal skin of negative control (normal skin) mice throughout the experimental period. However, positive control (untreated) mice on day 24 of assessment showed epidermal hyperplasia and dense infiltration of inflammatory cells such as lymphocytes, mast cells and eosinophils, similar to the skin lesions of AD patients (Figs. 4a–c). Topical application of vehicle alone had no influence on these histopathological changes, and topical application of betamethasone ointment alone inhibited the dermis infiltration of inflammatory cells to a slight degree (Figs. 4a–c, 5). Furthermore, the degree of this inhibition elicited by the combined topical application of betamethasone ointment and josamycin ointment was superior to that elicited by betamethasone alone. Notably, among the above inflammatory cells, the number of mast cells increased to the greatest extent in positive control mice, and the number was markedly reduced by application of betamethasone alone and to an even greater extent by application of josamycin and betamethasone together, but not by vehicle alone (Figs. 4b, 5). Similarly, the combination of the two ointments also ameliorated the degree of epidermal hyperplasia significantly (Figs. 4a, 6).

Fig. 4. Histopathological Analysis of AD-Like Skin Lesions in NC/Nga Mice

Skin sections were stained with hematoxylin-eosin stain (a), toluidine blue stain (b) and Luna stain (c), and observed at ×100. Arrowheads in Luna stain (c) point to eosinophils. The photograph shows sections of skin lesions on the 24th day of assessment.

Fig. 5. Quantification of Lymphocytes, Mast Cells and Eosinophils in the Skin of NC/Nga Mice

Skin sections stained with hematoxylin-eosin stain, toluidine blue stain or Luna stain were observed at ×400 and the numbers of lymphocytes, mast cells and eosinophils were counted. The results for each experimental group are expressed as means ± S.E.M. (n = 6). * p < 0.01 versus normal mouse, ** p < 0.05 versus untreated, ^§ p < 0.01 versus untreated, ^† p < 0.05 versus betamethasone.

Fig. 6. Quantification of Epidermal Hyperplasia in the Skin of NC/Nga Mice

Epidermal thickness was measured in hematoxylin–eosin-stained skin sections. The results for each experimental group are expressed as means ± S.E.M. (n = 6). * p < 0.01 versus normal mouse, ^§ p < 0.01 versus untreated, ^† p < 0.05 versus betamethasone.

Effects of Topical Application of Betamethasone and Josamycin on Serum Total IgE Levels

Serum total IgE levels on the 24th day of assessment of skin lesion severity were significantly elevated in positive control mice (untreated) (Fig. 7). Although the increased concentration of IgE in serum was not reduced by topical application of vehicle, it was reduced significantly by application of betamethasone alone and the combined application of betamethasone and josamycin. However, the level of reduction in the serum concentration of IgE resulting from application of betamethasone alone was inferior to that obtained by combined application of betamethasone and josamycin.

Fig. 7. Effects of Topically Applied Betamethasone and Josamycin on IgE Concentration in the Serum of NC/Nga Mice with AD-Like Skin Lesions

The serum total IgE levels on the 24th day of assessment were measured by ELISA. The results for each experimental group are expressed as means ± S.E.M. (n = 6). * p < 0.01 versus normal mouse, ** p < 0.01 versus untreated, ^§ p < 0.01 versus untreated, ^† p < 0.05 versus betamethasone.

Effects of Topical Application of Betamethasone and Josamycin on Th1 and Th2 Cell Development

To examine Th1 and Th2 cell development in the auricular lymph nodes on day 24 of skin severity assessment, ELISA for IFN-γ and IL-4 was carried out by using culture supernatants of lymph node cells stimulated through cell surface CD3/CD28 molecules for 48 h. Figure 8 shows that production of the Th1 cytokine IFN-γ and the Th2 cytokine IL-4 from lymph node cells was significantly increased by mite antigen sensitization (untreated) and that this was suppressed by topical treatment with betamethasone alone and combined application of betamethasone and josamycin, but not by vehicle. In addition, the levels of inhibition of Th1 and Th2 cell development induced by the combined application of betamethasone and josamycin markedly exceeded those achieved by topical application of betamethasone alone.

Fig. 8. Effects of Topically Applied Betamethasone and Josamycin on Development of Th1 and Th2 Cells in Auricular Lymph Nodes of NC/Nga Mice with AD-Like Skin Lesions

Auricular lymph node cells on the 24th day of skin severity assessment were stimulated through their surface CD3/CD28 molecules, and the concentrations of IFN-γ and IL-4 in the culture supernatants were determined by ELISA. Each culture was prepared in triplicate, and the mean value was obtained as a representative result for one experiment. The results are expressed as means ± S.E.M. (n = 6). * p < 0.01 versus normal mouse, ** p < 0.05 versus untreated, ^§ p < 0.01 versus untreated, ^† p < 0.05 versus betamethasone.

Effects of Topical Application of Betamethasone and Josamycin on Skin Colonization by S. aureus

Figure 9 shows the CFU counts of S. aureus per 8 cm² area of dorsal skin in the NC/Nga mice on day 24 of skin severity assessment. The lesioned skin of untreated mice was colonized by large numbers of S. aureus. Whereas topical application of vehicle alone had no influence on the S. aureus skin CFU values, that of betamethasone ointment decreased the CFU values slightly. Furthermore, when betamethasone was used topically in combination with josamycin, skin colonization by S. aureus was suppressed very strongly compared with the use of betamethasone alone.

Fig. 9. Effects of Topically Applied Betamethasone and Josamycin on Numbers of S. aureus Cells on AD-Like Skin Lesions in NC/Nga Mice

The results for each experimental group are expressed as means ± S.E.M. (n = 6). * p < 0.01 versus normal mouse, ** p < 0.05 versus untreated, ^§ p < 0.01 versus untreated, ^† p < 0.01 versus betamethasone.

DISCUSSION

Th1/Th2 immune balance is fundamentally involved in a range of immunological diseases, including allergy. It is well known that Th2 immunity is responsible for allergic immune responses and the subsequent pathogenesis of allergic inflammation.^17–19) AD is one such allergy-related condition, characterized by a marked increase in the count of Th2 cells in both peripheral blood and acute skin lesions.⁶⁾ On this basis, it has been proposed that the Th2 immune response plays a key pathogenetic role in AD, and this is supported by the presence of blood eosinophilia and enhanced serum IgE levels in most AD patients.^20,21) Our previous study has also suggested that the Th1 immune response also plays an instrumental role in AD-related chronic inflammation.²²⁾ Recently, therefore, we have been focusing on an immunoregulatory method for prevention of Th1 and Th2 cell development in AD patients.

Here we demonstrated that combined therapy with betamethasone and josamycin markedly ameliorated severe AD-like skin lesions in an NC/Nga mouse model, with a therapeutic efficacy far superior to that of betamethasone alone. In comparison to the vehicle control group, skin lesion severity assessed macroscopically in terms of redness/scratch marks, edema/lichenification/thickening, hemorrhage/scabbing, erosion and desquamation was significantly decreased by simultaneous application of 0.1% betamethasone ointment and 0.1% josamycin ointment. These findings were also supported by histopathologic analysis. Combined topical application of betamethasone and josamycin inhibited epidermal hyperplasia and dense infiltration of inflammatory cells such as mast cells, eosinophils and lymphocytes more effectively than betamethasone alone. We had shown previously that Langerhans cells (LCs) treated with betamethasone and josamycin, respectively, inhibited both Th1 cell and Th2 cell development in lymph nodes.^15,23) This suggested that combined topical application of betamethasone and josamycin would target LCs in the epidermis, which would then move to lymph nodes, where Th1 cell development and subsequent IFN-γ production, and Th2 cell development and subsequent IL-4 production, would be additively down-regulated. This was properly reflected in the levels of Th1/Th2 cytokines in auricular lymph nodes on day 24. It is known that the Th2 cytokine response is dominant in the acute phase of AD, and that in the late phase, the Th1 cytokine response is increased in addition to the Th2 cytokine response, contributing to chronic inflammation.^6,20,24) These facts indicate that combined topical application of betamethasone and josamycin would exert an additive effect on both acute and chronic inflammation, thus contributing to improved amelioration of severe AD-like skin lesions in NC/Ng mice.

About 70–80% of patients with AD show increased serum levels of IgE, which are associated with disease severity.⁶⁾ Elevation of serum IgE levels was also observed in mite antigen-treated NC/Nga mice, and topical application of betamethasone alone significantly reduced the serum IgE concentration. IL-4 receptor-mediated signaling in B cells is essential for induction of IgE synthesis.²⁵⁾ Therefore, elevation of serum IgE levels in mite antigen-treated NC/Nga mice and its inhibition by topical application of betamethasone could be explained by the degree of IL-4 expression in lymph nodes. Furthermore, since topical application of betamethasone and josamycin together additively reduced the serum IgE concentration and IL-4 expression level in NC/Nga mice, the difference in therapeutic efficacy between topical application of betamethasone alone and that induced by combined topical application of betamethasone and josamycin could be partly explained by the serum IgE concentration, which precisely reflects the degree of the Th2 immune response.

On the other hand, we showed previously that S. aureus isolated from the skin lesions of AD patients was susceptible to josamycin.¹⁵⁾ Since in most AD patients the skin shows superficial S. aureus colonization and barrier disruption due to a reduction of filaggrin,²⁶⁾ bacterial products such as staphylococcal enterotoxins, lipoteichoic acid and peptidoglycan would likely penetrate the skin and induce the production of Th2 cells, Th1 cells, and their related chemokines. This in turn would induce a Th2 immune response and a subsequent Th1 immune response, thus augmenting skin inflammation.^{6,10,11,14,27)} Therefore, the marked effect of topical josamycin on the skin lesions of AD patients likely involves bactericidal action against S. aureus and inhibition of the Th1 and Th2 immune response through inhibition of LC-mediated production of allergen-specific Th1 and Th2 cells, as well as mast cell-mediated production of allergen-specific Th1 cells, unlike immunosuppressants such as tacrolimus or steroids.^15,27) We believe that this LC-mediated inhibitory effect of jocamycin on Th1/Th2 cell differentiation, along with its excellent bactericidal activity against S. aureus from AD patients, is an important factor in its therapeutic effect. Up to the present, we have been focusing on macrolides, tetracyclines, and fluoroquinolones as antibiotics with immunomodulatory effects, and after repeated screening we have found that josamycin best satisfies the above conditions.^15,16,22,28)

Our recent study demonstrated that the lesional skin of NC/Nga mice was colonized by a large number of S. aureus cells, and that the number of such cells present on the lesioned skin was related to the severity of the skin lesions.²⁹⁾ As a result, topical administration of josamycin to those mice would control both lesion severity and S. aureus skin colonization. Furthermore, we have previously observed that josamycin, when topically administered to the lesional skin of NC/Nga mice, suppressed the severity of skin lesions, the associated histological changes, the serum level of IgE, and Th1/Th2 cell development to the same degree as topical application of betamethasone.¹⁶⁾ Similar findings were also obtained in the present study (data not shown). Although treatment with betamethasone alone also decreased the number of S. aureus cells on the lesioned skin of NC/Nga mice, this reflected the steroidal effect of betamethasone, and not any intrinsic antibacterial activity. However, we confirmed that S. aureus colonization was eliminated more effectively by using an ointment containing both betamethasone and josamycin, resulting in a more marked decrease of the skin severity score than that achieved with betamethasone alone. This additive effect would have been due to not only the antibacterial activity, but also the immunosuppressive effects of josamycin. Thus, it can be said that the additive effect of betamethasone and josamycin on the lesional skin of NC/Nga mice is associated with an antibacterial action against S. aureus in addition to suppression of Th1/Th2 cell differentiation. Most of the S. aureus strains isolated from the lesioned skin of AD patients are susceptible to josamycin.¹⁵⁾ Furthermore, since josamycin has a relatively large molecular size, it cannot penetrate normal skin. Therefore, it is thought that topical application of josamycin to the skin would have few side effects and is highly safe. However, once the skin inflammation has healed and the skin barrier function has been restored, it should be confirmed that S. aureus colonization has been completely eliminated. If no S. aureus is detected on the skin, withdrawal of topical josamycin would allow the skin flora to normalize. Then, if necessary, proactive therapy with betamethasone alone would lead to a complete cure of AD.³⁰⁾

CONCLUSION

The results of the present study using NC/Nga mice have demonstrated that combined topical application of betamethasone and josamycin can additively inhibit the development of severe AD-like skin lesions, as well the Th1/Th2 immune response, through both immunomodulatory and bactericidal actions. This new therapeutic strategy appears to show promise for AD lesions with superficial S. aureus colonization.

Acknowledgments

This work was supported by JSPS KAKENHI Grant No. JP22K06779.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Weidinger S, Beck LA, Bieber T, Kabashima K, Irvine AD. Atopic dermatitis. Nat. Rev. Dis. Primers, 4, 1 (2018).
• 2) Chamlin SL, Chren MM. Quality-of-life outcomes and measurement in childhood atopic dermatitis. Immunol. Allergy Clin. North Am., 30, 281–288 (2010).
• 3) Deckers IA, McLean S, Linssen S, Mommers M, van Schayck CP, Sheikh A. Investigating international time trends in the incidence and prevalence of atopic eczema 1990–2010: a systematic review of epidemiological studies. PLOS ONE, 7, e39803 (2012).
• 4) Bieber T. Atopic dermatitis. N. Engl. J. Med., 358, 1483–1494 (2008).
• 5) Koh LF, Ong RY, Common JE. Skin microbiome of atopic dermatitis. Allergol. Int., 71, 31–39 (2022).
• 6) Leung DY, Boguniewicz M, Howell MD, Nomura I, Hamid QA. New insights into atopic dermatitis. J. Clin. Invest., 113, 651–657 (2004).
• 7) Ong PY, Leung DY. Immune dysregulation in atopic dermatitis. Curr. Allergy Asthma Rep., 6, 384–389 (2006).
• 8) Guzik TJ, Bzowska M, Kasprowicz A, Czerniawska-Mysik G, Wójcik K, Szmyd D, Adamek-Guzik T, Pryjma J. Persistent skin colonization with Staphylococcus aureus in atopic dermatitis: relationship to clinical and immunological parameters. Clin. Exp. Allergy, 35, 448–455 (2005).
• 9) Matsui K, Nishikawa A, Suto H, Tsuboi R, Ogawa H. Comparative study of Staphylococcus aureus isolated from lesional and non-lesional skin of atopic dermatitis. Microbiol. Immunol., 44, 945–947 (2000).
• 10) Matsui K, Nishikawa A. Peptidoglycan from Staphylococcus aureus induces Th2 immune response in mice. J. Investig. Allergol. Clin. Immunol., 22, 80–86 (2012).
• 11) Matsui K, Nishikawa A. Lipoteichoic acid from Staphylococcus aureus induces Th2-prone dermatitis in mice sensitized percutaneously with an allergen. Clin. Exp. Allergy, 32, 783–788 (2002).
• 12) Matsui K, Nishikawa A. Percutaneous application of peptidoglycan from Staphylococcus aureus induces an increase in mast cell numbers in the dermis of mice. Clin. Exp. Allergy, 35, 382–387 (2005).
• 13) Matsui K, Wirotesangthong M, Nishikawa A. Percutaneous application of peptidoglycan from Staphylococcus aureus induces eosinophil infiltration in mouse skin. Clin. Exp. Allergy, 37, 615–622 (2007).
• 14) Matsui K, Nishikawa A. Percutaneous application of peptidoglycan from Staphylococcus aureus induces infiltration of CCR4⁺ cells into mouse skin. J. Investig. Allergol. Clin. Immunol., 21, 354–362 (2011).
• 15) Matsui K, Tamai S, Ikeda R. Effects of macrolide antibiotics on Th1 cell and Th2 cell development mediated by Langerhans cells. J. Pharm. Pharm. Sci., 19, 357–366 (2016).
• 16) Matsui K, Tachioka K, Onodera K, Ikeda R. Topical application of josamycin inhibits development of atopic dermatitis-like skin lesions in NC/Nga mice. J. Pharm. Pharm. Sci., 20, 38–47 (2017).
• 17) Kay AB. Allergy and allergic diseases: first of two parts. N. Engl. J. Med., 344, 30–37 (2001).
• 18) Herrick CA, Bottomly K. To respond or not to respond: T cells in allergic asthma. Nat. Rev. Immunol., 3, 405–412 (2003).
• 19) Nguyen TH, Casale TB. Immune modulation for treatment of allergic disease. Immunol. Rev., 242, 258–271 (2011).
• 20) Grewe M, Bruijnzeel-koomen CAFM, Schöpf E, Thepen T, Langeveld-Wildschut AG, Ruzicka T, Krutmann J. A role of Th1 and Th2 cells in the immunopathogenesis of atopic dermatitis. Immunol. Today, 19, 359–361 (1998).
• 21) Leung DY, Bieber T. Atopic dermatitis. Lancet, 361, 151–160 (2003).
• 22) Matsui K, Nojima Y, Kajiwara Y, Busujima K, Mori Y. Topical application of doxycycline inhibits Th2 cell development mediated by Langerhans cells and exerts a therapeutic effect on atopic dermatitis. J. Pharm. Pharm. Sci., 23, 86–99 (2020).
• 23) Matsui K, Tamai S, Ikeda R. Betamethasone, but not tacrolimus, suppresses the development of Th2 cells mediated by Langerhans cell-like dendritic cells. Biol. Pharm. Bull., 39, 1220–1223 (2016).
• 24) Sabin BR, Peters N, Peters AT. Chapter 20: atopic dermatitis. Allergy Asthma Proc., 33 (Suppl. 1), S67–S69 (2012).
• 25) Wu LC, Scheerens H. Targeting IgE production in mice and humans. Curr. Opin. Immunol., 31, 8–15 (2014).
• 26) Brown SJ, McLean WHI. One remarkable molecule: filaggrin. J. Invest. Dermatol., 132, 751–762 (2012).
• 27) Matsui K, Kanai S, Ikuta M, Horikawa S. Mast cells stimulated with peptidoglycan from Staphylococcus aureus augment the development of Th1 cells. J. Pharm. Pharm. Sci., 21, 296–304 (2018).
• 28) Matsui K, Kashima A, Motegi A. Norfloxacin, a fluoroquinolone antibiotic, inhibits Langerhans cell-mediated Th1 and Th2 cell development. J. Pharm. Pharm. Sci., 22, 122–130 (2019).
• 29) Matsui K, Nakamura M, Obana N. Effects of josamycin on scratching behavior in NC/Nga mice with atopic dermatitis-like skin lesions. Biol. Pharm. Bull., 44, 798–803 (2021).
• 30) Schmitt J, von Kobyletzki L, Svensson Å, Apfelbacher C. Efficacy and tolerability of proactive treatment with topical corticosteroids and calcineurin inhibitors for atopic eczema: systematic review and meta-analysis of randomized controlled trials. Br. J. Dermatol., 164, 415–428 (2011).