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The Journal of Immunology, 2007, 178: 584-591.
Copyright © 2007 by The American Association of Immunologists, Inc.

Suppression of Skin Lesions by Transdermal Application of CpG-Oligodeoxynucleotides in NC/Nga Mice, a Model of Human Atopic Dermatitis1

Joe Inoue and Yukihiko Aramaki2

School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Atopic dermatitis (AD) is a pruritic inflammatory skin disease characterized by an elevation of the total IgE level in plasma, the infiltration of mast cells and eosinophils, and the expression of cytokines by Th2 cells. NC/Nga mice kept in conventional conditions are known to develop skin lesions resembling human AD. We examined in this study the alterations of immune response in NC/Nga mice kept in conventional conditions, following transdermal application of CpG-oligodeoxynucleotides (ODN), which plays a critical role in immunity via the augmentation of Th1-type and suppression of Th2-type responses. CpG-ODN remarkably changed the immune response from type Th2 to Th1 as determined from cytokine mRNA and Ab levels. The serum IgE level was decreased and the expression of IgG2a was up-regulated. The application of CpG-ODN to the skin also decreased inflammatory infiltration of mast cells, and suppression in the skin lesions was observed. Furthermore, the generation of regulatory T cells, which are considered immune suppressive T cells, was observed in the skin on treatment with CpG-ODN. These results suggested CpG-ODN is effective for immunotherapy in patients with AD, which is characterized by Th2-dominated inflammation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Atopic disorders have a complex and chronic pathogenesis that provides many potential cellular and molecular targets for therapeutic intervention, but may also include redundant pathways mediating disease (1, 2). In atopic dermatitis (AD),3 the skin lesions show a chronically relapsing inflammatory disorder with prurience and eczema usually associated with elevated serum IgE levels (3, 4). The skin lesions of AD patients are characterized by the presence of inflammatory infiltrates consisting of T lymphocytes, monocytes/macrophages, eosinophils, and mast cells (5, 6).

Dysregulated Th1 or Th2 responses are thought to be central to the pathology of diseases such as AD and asthma, which are characterized by Th2-dominated allergic inflammation (7, 8, 9). Th2-like immune responses mediated by IL-4, IL-5, and IL-13 are key to the pathogenesis of atopic disorders (8, 9) because the up-regulation of IgE production, one of the major causes of atopic inflammation, has been extensively regulated with Th2 cytokines, IL-4 and IL-13. Th2 cell numbers are increased in lesional tissue of patients who suffer from atopic diseases and patients with AD frequently show elevated IgE levels in response to many kinds of allergens such as mite Ag and house dust (10, 11). Recently, many experiments have shown that the T cells constitutively expressing CD25, regulatory T cells (Tregs), are stably anergic and suppressive, and regulate various autoimmune diseases and allergic diseases (12, 13, 14). Therefore, it is very important to consider the balance of Th1, Th2, and Tregs in patients with immune diseases especially in allergic diseases.

Both bacterial dinucleotides flanked by certain bases (CpG-motif) and synthetic oligodeoxynucleotides (ODN) containing a CpG-motif (CpG-ODN) activate cells such as B cells, macrophages, and dendritic cells through TLR9 (15, 16, 17). Signaling through TLR9 leads to the secretion of proinflammatory cytokines such as IL-1, IL-6, TNF-{alpha}, and IL-12 (15, 16, 17). IL-12 acts on T cells and NK cells inducing the production of cytokines, primarily IFN-{gamma}. Consequently, CpG-ODN could be useful as an adjuvant for cellular and humoral immunity (18). It is also important that CpG-ODN-activated dendritic cells induced the generation of Tregs with a strong immune-suppressive function (19). So we expect that the ability of CpG-ODN to shift the immune response from type Th2 to Th1 with the generation of Tregs can be exploited to develop an immunotherapy for patients with diseases like AD that are associated with a Th2-predominant immune response.

NC/Nga mice are an inbred strain established from Japanese fancy mice in 1957 by Kondo et al. (20). When kept under conventional conditions, they started to scratch themselves at ~8 wk, and their skin became dry and scaly. Within the next several weeks, the mice developed lesions on the ears, back, neck, and face. Immunohistochemical examination of the lesions in conventional NC/Nga mice reveal hyperkeratosis, acanthosis, and parakeratosis, all of which resemble the typical features of the skin observed in patients with AD (9, 21). The lesions show lymphocyte infiltration, macrophage infiltration, and mast cell and eosinophil degranulation. In addition, the level of IgE in the serum gradually increases (22). All these immunological, histological, and biochemical changes in conventional NC/Nga mice resemble human AD.

We have reported that administration of CpG-ODN through the barrier-disrupted skin may shift the immune response from type Th2 to Th1 and drastically attenuated the production of IgE in mice undergoing an IgE-type immune response (23, 24). In this study, we examined the alterations in immune response in conventional NC/Nga mice, which spontaneously develop AD-like symptoms and high Th2-immune responses following a application of CpG-ODN to the skin. We also investigated the generation of Tregs in the skin lesions following the application of CpG-ODN. CpG-ODN remarkably changed the immune response from type Th2 to Th1 as determined from cytokine mRNA and Ab levels. CpG-ODN induced Tregs to appear in the skin lesions. These results suggested that CpG-ODN could change cytokine production and generate Tregs in conventional NC/Nga mice, and may be effective for immunotherapy in patients with AD.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and reagents

NC/Nga mice (male, 6-wk-old) were purchased from Nihon SLC Co. The sequence for control ODN (non-CpG-ODN) was 5'-TCCATGAGCTTCCTGAGTCT-3' and CpG-ODN was 5'-TCCATGACGTTCCTGATGCT-3', and HPLC-purified phosphorothioate ODN was obtained from Sigma-Genosys. Rhodamine (Rho)-labeled CpG-ODN was also obtained from Sigma-Genosys.

Animal experiment

Animal use and relevant experimental procedures were approved by the Tokyo University of Pharmacy and Life Science Committee on the Care and Use of Laboratory Animals (permission no. 2004-003). Mice housed under conventional conditions were anesthetized with an i.p. injection of Nembutal (1.5 mg/mouse) and their back was shaved. After the application of CpG-ODN or Rho-labeled CpG-ODN onto the shaved skin, the skin was excised at specific points in time, embedded in Tissue-Tek OTC compound (Miles), and snap-frozen in liquid nitrogen. The skin was then cut with a cryostat into 10-µm vertical sections. To analyze the distribution of Rho-CpG-ODN, for the observation of skin hypertrophy, and for immune staining for CD4+CD25+ cells, the dermal skin was fixed with 10% formalin at 4°C for 16 h, embedded, snap-frozen, and cut as described. The sections were examined by confocal laser scanning microscopy (MicroRadiance; Bio-Rad) or with an optical microscope following H&E staining and toluidine blue staining. The number of infiltrated cells was discussed per microscope field, and two skin sections from each mouse were used. Anti-CD4 mAb, anti-c-kit mAb, anti-foxp3 mAb, and anti-CD25 mAb, using Alexa Fluor 488- and Alexa Fluor 594-conjugated secondary Ab, and nuclear staining with 4',6'-diamidino-2-phenylindole were used for immune staining and the sections were examined by fluorescent microscope.

Cytokine determination

Mice housed under conventional conditions were anesthetized with an i.p. injection of Nembutal (1.5 mg/mouse) and their back was shaved. After the application of CpG-ODN (50 µg) or non-CpG-ODN (50 µg) onto the shaved skin, the skin was excised and homogenated with the Polytron homogenizer in 1 ml of saline containing protease inhibitors (0.17 mg/ml PMSF, 0.02 mg/ml leupeptin, and 0.01 mg/ml aprotinin). The homogenate was centrifuged at 2200 x g for 5 min and the supernatant was collected. The level of IL-12p70 in the skin was determined with a sandwich ELISA using pairs of purified capture and biotinylated detection mAbs (all obtained from BD Pharmingen) recognizing murine IL-12p70, according to the manufacturer’s directions.

Ab determination

Blood samples from mice treated with CpG-ODN (50 µg) or non-CpG-ODN (50 µg) every week from 11 to 18 wk were collected on specific days from the retro-orbital plexus and sera were pooled. The levels of IgG1, IgG2a, and IgE in sera were determined with a sandwich ELISA using pairs of purified capture and biotinylated detection mAbs (all obtained from BD Pharmingen) recognizing murine IgG1, IgG2a, and IgE, according to the manufacturer’s instructions.

RT-PCR

The changes in cytokine and chemokine mRNA expression in lymph node (LN) and skin were determined by RT-PCR. Total RNA (1 µg) was isolated with Isogen solution (Nippon Gene) as reported previously (25). cDNA was synthesized using SuperScript III. Then, cDNAs were amplified with primers specific for each cytokine. The primers are: IL-4 (forward) 5'-AGTTGTCATCCTGCTCTTCTTT-3', (reverse) 5'-GACTGGGACTCATTCATGGTGC-3'; IL-5 (forward) 5'-CAAAAAGAGAAGTGTGGCGAGG-3', (reverse) 5'-TAGATAGGAGCAGGAAGCCC-CG-3'; IL-10 (forward) 5'-G-GACAACATACTGCTAACCGACTC-3', (reverse) 5'-AAAATCACTCTTCACCTGCTC-CAC-3'; IL-12 p40 (forward) 5'-CAGAAGCTAACCATCTCCTGGTTTG-3', (reverse) 5'-TCCGGAGTAATTTGGTGGTTCACAC-3'; IL-13 (forward) 5'-GCAACGGCAGCATGGTATGGAG-3', (reverse) 5'-TGGTATAGGGGAGGCTGGAGAC-3'; IFN-{gamma} (forward) 5'-CTCAAGTGGCATAGATGT-3', (reverse) 5'-GAGATAATCTGGCTCTGCAGGATT-3'; MDC (forward) 5'-TCTGATGCAGGTCCCTATGGT-3', (reverse) 5'-TTATGGAGTAGCTTCTTCAC-3'; TARC, (forward) 5'-CAGGAAGTTGGTGAGCTGGTATA-3', (reverse) 5'-TTGTGTTCGCCTGTAGTGCATA-3'; TNF-{alpha} (forward) 5'-CACCACGCTCTTCTGTCTACTGAAC-3', (reverse) 5'-CCGGACTCCGTGATGTCTAAGTACT-3'; COX-2 (forward) 5'-CAGAACCGCATTGCCTCTG-3', (reverse) 5'-TTGAAGGTGTCGGGCAGC-3'; TGF-beta1 (forward) 5'-CTTTAGGAAGGACCTGG-GTT-3', (reverse) 5'-CAGGAGGCGACAATCATGTT-3'; foxp3 (forward) 5'-CAGCTGCCTACAGTGCCCCTAG-3', (reverse) 5'-CATTTGCCAGCAGTGGGTAG-3'; and beta-actin (forward) 5'-GCACCACACCTTCTACAATGAG-3', (reverse) 5'-TTGGCATAGAGGTCTTTACGGA-3'. beta-Actin primers were used as an internal control. Primers designed based on the mouse sequences were obtained from Sigma-Genosys. beta-Actin primers were used as an internal control.

Statistical analysis

The paired Student’s t test was used to compare paired groups. ANOVA was used for multigroup analysis. Values of p > 0.05 were considered to indicate lack of significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Th2-predominant immune response in conventional NC/Nga mice

When NC/Nga mice were kept under conventional conditions, they started to scratch themselves at ~8 wk, and their skin became dry and scaly (21). Within the next several weeks, the mice developed lesions on the ears, back, neck, and face (Fig. 1a). In AD patients (3, 4), skin lesions show a chronically relapsing inflammatory disorder with prurience and eczema usually associated with elevated serum IgE levels and a Th2-predominant immune response. In conventional NC/Nga mice, serum IgE levels were extremely high (Fig. 1b). mRNA levels of cytokines and chemokines in the draining LN were evaluated by RT-PCR, and Th2-type cytokines and chemokines were expressed but not IFN-{gamma}, which is classified as a Th1-type cytokine as shown in Fig. 1c. From these findings, we confirmed that the conventional NC/Nga mice used in this experiment show high Th2-like immune responses, and the up-regulation of IgE production regulated by Th2 cytokines IL-4 and IL-13 is one of the major causes of atopic inflammation.


Figure 1
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  		FIGURE 1. Th2-predominant immune responses in conventional (CONV) NC/Nga mice. a, The macroscopic appearance in NC/Nga mice kept under conventional conditions. Blood samples from conventional NC/Nga mice were collected from the retro-orbital plexus, and sera were pooled. b, Serum IgE levels in conventional NC/Nga mice were determined with ELISA. c, The mRNA levels of cytokines in the draining LN in conventional NC/Nga mice were evaluated by RT-PCR. Each value represents the mean ± SD for five mice. **, p < 0.01 compared with mice kept under specific pathogen-free conditions.

 
Localization of CpG-ODN in the skin of conventional NC/Nga mice

In AD patients, a deficiency in the barrier function of the skin at the lesional site was reported (26). Therefore environmental allergens, such as mites and house dusts, easily penetrate such susceptible skin. So we first examined the penetration of CpG-ODN by the barrier-disrupted AD skin in conventional NC/Nga mice. Rhodamine-labeled CpG-ODN (Rho-CpG-ODN) was applied to the shaved back and its localization was examined by confocal laser scanning microscopy. In the skin of NC/Nga mice kept under specific pathogen-free conditions, fluorescent signals for rhodamine were observed faintly in the stratum corneum, suggesting that CpG-ODN did not penetrate the s.c. layer (Fig. 2). In the conventional NC/Nga mice, however, red fluorescence generated from rhodamine was observed around the corium at 12 h after the application, indicating that CpG-ODN, having a m.w. of >6000 and a negative charge, easily penetrated the skin. Interestingly, the high density of fluorescence in the skin was observed (Fig. 2, open arrows). From these findings, the barrier function of conventional NC/Nga mouse skin was disrupted as in AD patients.


Figure 2
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  		FIGURE 2. Localization of CpG-ODN to the skin lesions of conventional (CONV) NC/Nga mice. Rhodamine-labeled CpG-ODN (50 µg) was applied to the skin and the localization of Rho-labeled CpG-ODN in the skin was determined at 12 h by confocal laser scanning microscopy. High-density fluorescence of the skin is indicated (open arrows).

 
Effect of CpG-ODN on production of IL-12 and Abs in conventional NC/Nga mice

We next examine the effect of CpG-ODN on the production of IL-12 at 12 h. CpG-ODN significantly enhanced the production of IL-12 in the skin, which is known to induce Th1-predominant immune responses as shown in Fig. 3a. To examine the changes in levels of Abs, 50 µg of CpG-ODN or non-CpG-ODN was applied to the skin lesions every week eight times from week 11, which is when the lesions first develop. Total IgE levels decreased from 24 wk and a significant difference from the control and non-CpG-ODN was observed at 28–33 wk (Fig. 3b). In contrast, IgG2a levels increased significantly at 20–24 wk in mice applied CpG-ODN (Fig. 3c). The total amount of IgG1 that is characterized as a Th2-type Ab decreased drastically as did the profile of IgE production (Fig. 3d). These results suggest that the application of CpG-ODN changes immune responses in conventional NC/Nga mice, which show a Th2-predominant reaction.


Figure 3
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  		FIGURE 3. Effect of CpG-ODN on IL-12 and Ab production in conventional NC/Nga mice. At 12 h after the application of CpG-ODN (50 µg) or non-CpG-ODN (50 µg) onto the shaved skin of conventional NC/Nga mice, the skin was excised. a, The level of IL-12p70 in the skin was determined with a sandwich ELISA. Blood samples from mice treated with saline ({blacksquare}), CpG-ODN (•), or non-CpG-ODN ({circ}) every week from week 11–18 were collected on specific days from the retro-orbital plexus, and sera were pooled. The levels of IgE (b), IgG2a (c), and IgG1 (d) were determined with a sandwich ELISA. Each value represents the mean ± SD for nine mice. **, p < 0.01, and *, p < 0.05 compared with control.

 
Changes in immune responses in CpG-ODN-treated conventional NC/Nga mice

Next we examined the changes in mRNA levels of cytokines and chemokines in the draining lymph node (LN) at 33 wk because production of IgE decreased significantly compared with the control at that time (Fig. 3). As shown in Fig. 4, mRNA levels of Th2-type cytokines and chemokines were decreased in CpG-ODN-treated NC/Nga mice. Conversely, mRNA levels of Th1-type cytokines, IFN-{gamma} and IL-12, increased slightly in CpG-ODN-treated NC/Nga mice. Interestingly, the expression of IFN-{gamma} mRNA in LN was also observed in untreated mice (control). mRNA levels of TNF-{alpha} and COX-2, inflammatory markers, decreased in CpG-ODN-treated mice. These findings suggest that transdermal application of CpG-ODN changes the immune response of NC/Nga, which is predominantly of type Th2. The decrease in IL-4 and IL-13 mRNA expressions in CpG-ODN-treated mice is suppose to be a cause of suppression in IgE production. Interestingly, CpG-ODN did not induce strong Th1 immune responses and inflammatory marker expression in 33 wk.


Figure 4
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  		FIGURE 4. Effect of CpG-ODN on cytokine mRNA expression in conventional NC/Nga mice. LN from mice treated with saline or CpG-ODN (50 µg) every week from week 11 to 18 were collected at 33 wk, and the cytokine and chemokine mRNA expression in LN was determined by RT-PCR analysis (a), and normalized by beta-actin levels (b). beta-Actin primers were used as an internal control.

 
Improvement of skin lesions in CpG-ODN-treated NC/Nga mice

In AD patients, the skin lesions show a chronically relapsing inflammatory disorder with prurience and eczema (3, 4). We thus examined the effect of CpG-ODN on the appearance of skin lesions in conventional NC/Nga mice. In untreated NC/Nga mice (control) and non-CpG-ODN-treated mice, eczematous injury was observed on the back, which resembles typical features of AD. Compared with control mice and non-CpG-ODN-treated mice, a drastic decrease in these injuries was observed in CpG-ODN-treated mice (Fig. 5). The number of these eczematous lesions was significantly decreased and 18.0 ± 6.23 for control, 18.25 ± 4.62 for non-CpG-ODN-treated mice, and 6.13 ± 4.19 for CpG-ODN-treated mice (p < 0.05 compared with control and non-CpG-ODN-treated mice), respectively.


Figure 5
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  		FIGURE 5. Macroscopic observations of skin lesions in CpG-ODN-treated NC/Nga mice. Conventional NC/Nga mice treated with saline, CpG-ODN (50 µg) or non-CpG-ODN (50 µg) every week from week 11 to 18 were examined at 33 wk. The number of eczematous lesions was counted. Each value represents the mean ± SD for nine mice.

 
Improvements in epidermal skin lesions and mast cells infiltration to the skin in CpG-ODN-treated NC/Nga mice

Histochemical examination of the lesions in conventional NC/Nga mice reveals hyperkeratosis, acanthosis, and parakeratosis, all of which are typical features of the skin of patients with AD. The infiltration of lymphocytes, macrophages, mast cells and eosinophils was observed in the skin lesions. To analyze the effect of CpG-ODN on skin hypertrophy and granulocyte infiltration in conventional NC/Nga mice, CpG-ODN-treated skin was stained with H&E and truidin blue, and then examined with an optical microscope. As shown in Fig. 6a, the acanthosis was clearly suppressed in CpG-ODN-treated mice compared with control mice. Also, granulocyte infiltration, stained as red dots in Fig. 6b, was decreased in the CpG-ODN-treated mice. The number of infiltrated cells was 36.33 ± 15.86 for the control mice and 8.72 ± 3.59 for the CpG-ODN-treated mice (p < 0.05 compared with control). Skin sections were also stained with anti-c-kit mAb (green) and the localization of fluorescence was examined with a fluorescence microscope. As shown in Fig. 6c, mast cell infiltration was also decreased in the CpG-ODN-treated mice. These results indicated that CpG-ODN changes not only the Th2-predominant immune response in conventional NC/Nga mice but also in skin conditions, skin hypertrophy, and mast cell infiltration.


Figure 6
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  		FIGURE 6. Histochemical examination of the lesions in CpG-ODN-treated NC/Nga mice. Skin sections from mice treated with saline or CpG-ODN (50 µg) every week from week 11 to 18 were collected at 33 wk. Skin sections were stained with H&E (a), and truidin blue (b), a hypertrophy and mast cell infiltration were examined with an optical microscope. c, Skin sections were also stained with anti-c-kit mAb (green) and then the localization of fluorescence was examined with a fluorescence microscope.

 
Generation of Tregs in CpG-ODN-treated skin

To clarify the mechanisms of this effect of CpG-ODN, we first focused on the change in cytokine levels of the skin. mRNA levels of TGF-beta and IL-10, which are cytokines considered to suppress inflammation especially in immune diseases, increased following transdermal application of CpG-ODN (Fig. 7a). In contrast, the mRNA expression of COX-2, a marker of inflammation, decreased. Furthermore, we examined the mRNA expression of foxp3, a marker of Tregs, because Tregs produce immune suppressive cytokines TGF-beta and IL-10. As shown in Fig. 7a, foxp3 was expressed in the CpG-ODN-treated skin, which also expressed TGF-beta and IL-10. These findings suggest that the up-regulation of TGF-beta and IL-10 mRNA expression results in Tregs being generated in the skin. Second, we analyzed the induction and migration of Tregs into the skin. Skin sections were stained with anti-CD4 mAb (green) and anti-CD25 mAb (red), a marker of Treg, and then examined with a fluorescent microscope. As shown in Fig. 7b, CD4+ cells were detected in both control skin and CpG-ODN-treated skin as green fluorescence. In contrast, CD4+CD25+ double-positive cells were observed only in the CpG-ODN-treated skin (Fig. 7b, white arrows). Skin sections were also stained with anti-foxp3 mAb (green) and foxp3+ cells were detected only in the CpG-ODN-treated skin (Fig. 7c, white arrows). These results indicated that CpG-ODN induces the induction and migration of Tregs in the skin of conventional NC/Nga mice.


Figure 7
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  		FIGURE 7. Generation of Tregs in the skin of CpG-ODN-treated conventional NC/Nga mice. Skin from mice treated with saline or CpG-ODN (50 µg) every week from week 11 to 18 were collected at 33 wk. a, The TGF-beta, IL-10, foxp3, and COX-2 mRNA expression in the skin was determined by RT-PCR analysis. b and c, Skin sections were also stained with anti-CD4 mAb (green), anti-foxp3 mAb (green), anti-CD25 mAb (red), and 4',6'-diamidino-2-phenylindole (blue), and the localization of fluorescence was examined with a fluorescent microscope. White arrows in b show CD4+CD25+ double-positive cells, white arrows in c show foxp3+ cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The ability of CpG-ODN to shift the immune response from type Th2 to Th1 is expected to be used in immunotherapy for patients with diseases like AD and asthma that are associated with a Th2-predominant immune response (27, 28, 29). Youn et al. (30) reported that CpG-ODN reverses established allergen-induced airway remodeling. It is also reported that CpG-ODN inhibits established Th2-type immune responses through production of IFN-{gamma} and IL-10 (31). In a model of AD, Kootiratrakarn et al. (32) showed that CpG-ODN could improve an established eosinophilic inflammation in the skin. These studies suggested that CpG-ODN is a potent inducer of Th1 and improves allergic responses even in established Th2-type response models. Recently, it has been reported that CpG-ODN-activated dendritic cells induce the generation of Tregs with a strong immune suppressive function (19). In another study, blocking the suppressor activity of Tregs and increasing Th2 cell frequency enhanced allergen-specific Th2 cell activation ex vivo (14). We thus investigated whether the topical application of CpG-ODN to conventional NC/Nga mice, a frequently used model of human AD with Th2-predominant immune responses, changes the immune response from type-Th2 to Th1 and induces the generation of Tregs.

First we examined the penetration of CpG-ODN from the barrier-disrupted AD skin in conventional NC/Nga mice, and the penetration of CpG-ODN and the high density of fluorescence in the skin was observed (Fig. 2). We have reported that administration of CpG-ODN through the barrier-disrupted skin also showed the localization of CpG-ODN in the skin, and the time-dependent migration into the lower part of the skin was observed (24). We expected that dendritic cells may be the one of the reasons of this localization and may be connected with the immune responses. In fact, the application of CpG-ODN through the barrier-disrupted skin up-regulates the migration of CD11c+ cell to LNs (data not shown). Thus, the activation of epidermal and dermal dendritic cells by CpG-ODN may be the one of the mechanisms of the changing in the immune responses.

Our examination showed that the application of CpG-ODN changed the immune response from type Th2 to type Th1 on the basis of the Ig isotype in the sera (Fig. 3, b–d) and expression of cytokine mRNA in LN (Fig. 4). Total IgE and IgG1 levels were decreased from 24 wk and a significant difference was observed at 28–33 wk (Fig. 3, b and d). In contrast, production of IgG2a was significantly increased at 20–24 wk (Fig. 3c). Interestingly, the increase in IgG2a production (20 wk) was slightly faster than the decrease in IgE and IgG1 production (24 wk) (Fig. 3, b–d) and the level of IgG2a returned to the control value from 24 wk. These findings indicated that the Th1-type immune response was up-regulated by CpG-ODN until 24 wk ("early phase"), and also the balance of Th1/Th2 was changed at this time. After 24 wk, these immune responses may be all suppressed with this balance maintained ("late phase"). Recently, it was reported that Tregs act as suppressor T cells that down-regulate other effecter T cells and inflammation. This down-regulation in "late phase" may be the result in the induction of Tregs.

However, the Th1 cytokine IFN-{gamma} is found in the skin lesions at the chronic stage of the disease and Th1 cells cause a deterioration of disease at this stage (2). In our study, control animals will be in the chronic stage at 33 wk because IFN-{gamma} was expressed in the mice (Fig. 4). It is still unclear whether CpG-ODN heals the skin at chronic stages of the disease. Peng et al. (33) reported that CpG-ODN vaccinations may fail to down-regulate ongoing IgE responses and the application induces a transient skin delayed-type hypersensitivity with production of IFN-{gamma}. Moreover, STAT-6-deficient NC/Nga mice, which fail to produce IgE and Th2 cytokines and highly express IFN-{gamma} in skin lesions, developed AD-like skin lesions at an equivalent frequency to normal NC/Nga littermates (34). But another report suggested that CpG-ODN could reserve the established eosinophilic inflammation in the skin with IFN-{gamma} production (32). The mechanisms of the effect of CpG-ODN on established Th2-immune responses are still unclear; therefore, further investigation is needed in chronically affected NC/Nga mice.

Macroscopic observation and histochemical examination revealed an improvement of the AD-like skin lesions in CpG-ODN-treated NC/Nga mice (Figs. 5 and 6). The clear expression of suppressive cytokines, TGF-beta and IL-10, and generation of Tregs in the CpG-ODN-treated skin were also observed (Fig. 7). It is reported that Tregs develop mainly in the presence of IL-10 and TGF-beta (35). We have examined IL-10 production in the skin and LN, and observed an up-regulation in CpG-ODN-treated skin and LN (data not shown). It is also reported in NC/Nga mice that the s.c. injection of recombinant TGF-beta suppressed eczematous skin lesions associated with reduced serum IgE levels, and histological analysis showed that TGF-beta inhibited the infiltration of inflammatory cells such as mast cells and eosinophils into the skin (36). Furthermore, many studies have shown that CCR2, CCR4, CCR5, CCR6, E-selectin and P-selectin ligands, and beta1 integrin were important for the homing of Tregs to various peripheral sites (37). CpG-ODN induces the production of various chemokines, ligands of these receptors, such as MCP-1, eotaxin, RANTES, TARC, and MIP-1 and MIP-3, from APCs and keratinocytes (15, 16, 38, 39). These findings strongly support our results and suggest that topically applied CpG-ODN is a potent generator of Tregs in the skin.

The current findings are the first demonstration of treating AD with CpG-ODN as the skin lesions first develop in NC/Nga mice. In summary, CpG-ODN remarkably changed the immune responses. The serum IgE level was decreased and the production of IgG2a was up-regulated. CpG-ODN also improved the skin lesions and the inflammatory infiltration of mast cells decreased with the generation of Tregs in the skin. These results suggested that CpG-ODN is effective for immunotherapy in patients with AD, which is characterized by Th2-dominated inflammation. Additional studies are required to determine whether clinical trials that use CpG-ODN reduce serum IgE levels and improve skin lesions with the generation of Tregs.


   Acknowledgments
 
We are grateful to T. Yoshida, H. Kumamoto, M. Ishida, K. Kunii, and Y. Saigo for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by Grant-in-aid 14657594 for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to Y.A.). Back

2 Address correspondence and reprint requests to Dr. Yukihiko Aramaki, School of Pharmacy, Tokyo University of Pharmacy and Life Science 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. E-mail address: aramaki{at}ps.toyaku.ac.jp Back

3 Abbreviations used in this paper: ODN, oligodeoxynucleotide; AD, atopic dermatitis; Treg, regulatory T cell; LN, lymph node. Back

Received for publication December 7, 2005. Accepted for publication October 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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