IL-4 and IL-12 Polymorphisms are Associated with Response to Suplatast Tosilate , a Th 2 Cytokine Inhibitor , in Patients with Atopic Dermatitis

Th2-related immune and inflammatory responses have been implicated in the pathogenesis of atopic dermatitis (AD), but few clinical lines of evidence have been reported regarding how and whether Th2-related responses are associated with other risk factors in the treatment of AD patients. In this study, the associations between the polymorphisms of genes related to the pathophysiology of AD and the efficacy of suplatast tosilate, an oral immunemodulator known to downregulate Th2-related allergic responses, were analyzed in adult patients with chronic AD. Patients were recruited from our previous study, where suplatast tosilate was evaluated for its efficacy when used in combination with topical steroids. The genotypes of 35 single nucleotide polymorphisms (SNPs) of 27 genes related to AD pathogenesis were then determined in 17 responders and 18 non-responders, as defined by the improvement rate in their AD skin scores. While no significant difference in the patient background was observed between responders and non-responders, significant associations were noted between the response to treatment with suplatast tosilate and three SNPs of IL-4 (-590C/T: P=0.04, -33C/T: P=0.04) and IL-12B (1188A/C: P=0.03), but not for the other SNPs. Of note, ethnic differences in the genotype frequencies of IL-4 -590C/T and IL-12B 1188A/C SNPs were found. In conclusion, the present results raise the possibility that AD patients who tend to produce more IL-4 and IL-12 may be susceptible to suplatast tosilate treatment and that ethnic variations should be considered to further understand the role of Th2-related responses.


INTRODUCTION
AD is one of the most common chronic inflammatory diseases of the skin.While its causes and mechanisms have not yet been fully elucidated, its pathogenesis has been *Address correspondence to this author at the Tokushima Research Center, Taiho Pharmaceutical Co., Ltd., 224-2, Ebisuno Hiraishi, Kawauchi-cho, Tokushima, 771-0194, Japan; Tel: +81-88-665-6014; Fax: +81-88-665-6554; E-mail: makiniwa@taiho.co.jp characterized by altered skin barrier function and immune dysregulation.Recent genome-wide association studies or polymorphism analyses of candidate genes have further extended the view that patients with AD have genetically determined risk factors that affect the skin barrier and immune responses [1][2][3][4][5][6].It should be noted, however, that few clinical lines of evidence have been reported regarding how these factors interact with each other and, more importantly, whether the down-or up-regulation of any gene or factor may affect the treatment of AD patients.
In the present study, we focused on suplatast tosilate, which has been approved in Japan as an oral immunemodulator for AD, allergic rhinitis, and asthma [7][8][9][10].This agent has been used as an adjunct to or in combination with topical anti-inflammatory and immunosuppressive agents and has been shown to reduce the requirements for these agents [11,12].Suplatast tosilate has been referred to as a Th2 cytokine inhibitor because 1) it was discovered based on its capacity to suppress murine IgE formation without affecting cellular immune responses [13], 2) it suppressed IL-4 and IL-5 production by Th2 cells [14], 3) it suppressed allergic inflammation via indirect actions on Th2 cells as well as direct actions on eosinophils [15][16][17][18][19], and 4) it affected certain biomarkers such as Th1/Th2 ratio during the treatment of allergic asthmatic patients [20].Although its mechanism of action remains to be identified at a molecular level, it is reasonable to assume that its efficacy in allergic patients may be ascribed to the modulation of the Th2related immune and inflammatory responses.
We previously performed a clinical study investigating the efficacy of suplatast tosilate in adult chronic AD patients who also received topical steroids but had responded poorly to adjunct therapy such as antihistamines [21,22].This study revealed that suplatast tosilate markedly improved the AD skin symptom scores in a certain group of patients, but not in all patients, enabling us to recruit suplatast tosilate responders and non-responders and to analyze the polymorphism of genes related to the pathophysiology of AD retrospectively.The results implicated the efficacy of suplatast tosilate in the modulation of both Th1 and Th2 responses in AD therapy and suggested ethnic variations in the pathogenesis of AD.

Study Subjects
One hundred and sixty-three patients with atopic dermatitis whose conditions had been poorly controlled by other anti-allergic drugs at 11 hospitals between 2004 and 2006 were enrolled in our previous study [21,22].The Th2 cytokine inhibitor suplatast tosilate (300 mg/day) was administered to these patients.After 4 weeks or more of treatment, the skin symptom score was determined to evaluate the usefulness of suplatast tosilate.The effectiveness of suplatast tosilate was evaluated by comparing the improvement rate relative to the baseline skin symptom score [21,22].To classify the patients into a highly effective group and a poorly effective group, we divided all the cases into three groups based on the improvement rate of the skin symptom scores.The one-third of cases with the highest improvement rates were defined as "responders", and the one-third of cases with the lowest improvement rates were defined as "non-responders".As a result, 54 "responder" patients had an improvement rate of more than 58%, and 53 "non-responder" patients had an improvement rate of less than 25%.From these patients, a total of 35 patients (17 responders and 18 non-responders) provided informed consent to undergo SNP genotyping using DNA from a blood sample.The improvement rate for the 17 highresponders with an improvement rate of more than 58% for their skin symptom scores was 68.8 ± 12.2%.On the other hand, that of the 18 non-responders with an improvement rate of less than 25% was 1. 4

Statistical Analysis
The associations between the clinical parameters and the response to treatment with suplatast tosilate were evaluated using a likelihood ratio chi-square test for categorical data or the Student t-test for continuous data.The effect of suplatast tosilate on the clinical parameters was evaluated using a paired t-test.Hardy-Weinberg equilibrium of the genotyping results was evaluated using a Pearson's chi-square test.A likelihood ratio chi-square test was used to analyze the associations between the genotype of each SNP and the response to treatment with suplatast tosilate.All the analyses were performed using the statistical software JMP 7.0.1 and the SAS statistical package, version 9.1.3(SAS Institute Inc., Cary, NC, USA).Differences were considered significant when P<0.05.Because this was the study with a small sample size, a correction for multiple comparisons was not made.

Patient Characteristics and Effect of Suplatast Tosilate on Th2-Related Parameters
No significant differences between the responders and the non-responders were observed with regard to sex, age, and the pre-treatment values for the skin symptom scores, IgE levels or eosinophil counts (Table 2).Note that suplatast tosilate treatment did not affect the serum IgE level or the eosinophil count in either the responders or the nonresponders (Table 3).

Genotype and Allele Frequencies
The genotyping results and the allele frequencies of the 35 SNPs are shown in Table 4.Among 34 SNPs (excluding the Filaggrin Tyr3105Asp SNP), the genotype frequencies were the values expected under Hardy-Weinberg equilibrium, and no significant difference was observed between the minor allele frequencies reported in this study and those shown in the SNPper (http://snpper.chip.org/bio/snpperenter)or NCBI databases (http://www.ncbi.nlm.nih.gov/snp/) (data not shown), indicating that the genotyping analysis was performed accurately.Regarding the genotyping results for the Filaggrin Tyr3105Asp SNP, the genotype frequency deviated from the values expected from Hardy-Weinberg equilibrium, and the minor allele frequency was significantly higher than that shown in the SNPper database (data not shown).This analysis may not have been performed accurately because the primer design region for the Filaggrin Tyr3105Asp SNP was highly homologous with other genes.

Associations between Genotype and Response to Treatment with Suplatast Tosilate
Significant associations between the response to treatment with suplatast tosilate and three SNPs of IL-4 (-590C/T: P=0.04, -33C/T: P=0.04) and IL-12B (1188A/C: P=0.03) were observed among the 35 SNPs genotyped in this study (Table 4).The genotyping results of two SNPs (-590C/T and -33C/T) within the IL-4 promoter region were perfectly identical among the 35 patients in this study.Therefore, the IL-4 -590C/T SNP, as a representative of the two SNPs of IL-4 that showed a significant association with the response, was used in further analyses.For the IL-4 -590C/T SNP, patients with a C/C genotype had a significantly lower response rate to suplatast tosilate than those with a T/T or T/C genotype (0.0% vs 53.1%, P=0.04) (Table 5).On the other hand, for the IL-12B 1188A/C SNP, patients with an A/A genotype had a significantly lower response rate to suplatast tosilate than those with a C/C or C/A genotype (12.5% vs 59.3%, P=0.02, odds ratio=10.2) (Table 5).In a combination analysis of the two SNPs (IL-4 -590C/T and IL-12B 1188A/C), patients with either IL-4 -590C/T C/C or IL-12B 1188A/C A/A had a significantly lower response rate than those with other genotype combinations (10.0%vs 64.0%, P=0.002, odds ratio=16.0)(Table 5).

Ethnic Differences in Genotype Frequency of IL-4 -590C/T and IL-12B 1188A/C SNPs
The genotype frequencies of IL-4 -590C/T T/T and IL-12B 1188A/C C/C, which were associated with a strong response to suplatast tosilate, were significantly higher among Asians than among Europeans based on the results of the HapMap project (Table 6, http://www.ncbi.nlm.nih.gov/snp/).SNPs of 27 allergy-related genes.Of note, no significant difference was seen in the minor allele frequencies of each SNP, with the exception of one SNP (Filaggrin Tyr3105Asp) with a low reliability, based on a comparison with an SNP control database for the Japanese population (SNPper or NCBI).These findings suggest that no genetic polymorphisms associated with susceptibility to AD were observed among the SNPs investigated in this study.
The results of the present SNP genotyping analysis clearly indicated that suplatast tosilate efficacy was associated with IL-4 and IL-12B polymorphisms, but not with any of the others, indicating that patients with a T/T or T/C genotype for the IL-4 -590C/T SNP as well as a C/C or C/A genotype for the IL-12B 1188A/C SNP responded to suplatast tosilate treatment at a significantly higher rate.Moreover, patients with both the IL-4 -590 T/T or T/C genotype and the IL-12B 1188 C/C or C/A genotype had a significantly higher response rate than those with other genotype combinations.Of note, SNPs in the regulatory sequences of genes are associated with the varying production of relevant cytokines.In fact, the promoter sequence of IL-4 -590T was reported to show a greater binding to nuclear transcription factors than that of -590C [23,24].Nakashima et al., reported that PBMC with a -590T/T or -590T/C genotype produced higher levels of IL-4 than those with a -590C/C genotype [25].In general, IL-4 is a major Th2 cytokine that plays an essential role in the class-switching of B cells to IgEproducing cells, Th2 cell differentiation, and the initial phase of tissue inflammation during the Th2-dominant phase of atopic diseases.Recently, IL-4 has been also reported to play an important role in regulating skin homeostasis and innate barrier function in AD lesions [26].In fact, Burchard et al., has reported that the sequence variant in the IL-4 promoter region is associated with the asthma FEV1 (Forced expiratory volume in one second) [27].It is reasonable, therefore, to assume that patients with the IL-4 -590T allele tend to develop a Th2dominated immune response, leading to the susceptibility of these patients to suplatast tosilate which down-regulates Th2related responses including skin manifestations.
Suplatast tosilate has been reported to suppress IgE formation and eosinophil counts presumably through inhibition of Th2 cytokine in basic experiments and in several clinical studies in patients with atopic asthma [13][14][15][16][17][18][19][20].However, no significant decreases in Th2-related parameters, such as the total IgE level and the eosinophil count, were observed after suplatast tosilate treatment even among the responders in this study.The reason for this contradiction is clearly unknown, but, as a possibility, a Th2-dependency may be decreased during the chronic phase of AD by the effect such as infections.In this sense, of great interest is the observation that patients with the IL-12B -1188 C/C genotype responded to suplatast tosilate treatment at a high rate.The IL-12B -1188 C allele was reported to result in IL-12B mRNA with a lower transcriptional activity and stability than that for the A allele [28].Although IL-12 p40 encoded by IL-12B is a component of IL-12 (a p40 and p35 heterodimer) [29], the IL-12 p40 homodimer was reported to function as an antagonist of IL-12 action [30,31].In fact, PBMC with a -1188IL-12B C/C genotype having a high response rate to suplatast tosilate reportedly produced significantly higher levels of biologically active IL-12 upon stimulation with LPS or PPD than those of other genotypes with the A allele [32].IL-12 is known to induce IFN-production [33], to suppress IgE synthesis [34], and to promote Th1 cell maturation [33].The results, therefore, suggest that the efficacy of suplatast tosilate is associated with the Th1-immune response that also underlies the AD symptoms.This finding is partly in accordance with an observation by Matsui et al., [35], who reported that PBMC from suplatast tosilate responders with childhood asthma produced higher amounts of IFN-than those of suplatast tosilate non-responders.The findings are also more directly in accordance with those by Murakami et al., [36], who found that suplatast tosilate treatment significantly suppressed the elevated expression of IL-4, IL-5, and IFN-mRNA in caspase-1 transgenic mice that spontaneously developed ADlike dermatitis.They also observed that suplatast tosilate treatment significantly decreased the expression of IL-18, which induced Th1 responses in synergy with IL-12, possibly explaining the mechanism of suplatast tosilate efficacy for Th1related responses.
The present results suggest that both Th2-and Th1-related responses may play more important roles in AD patients who responded to suplatast tosilate treatment than other factors, and ethnic differences may exist in the mechanism of chronic AD, since the allele frequencies of the IL-4 and IL-12B polymorphisms associated with the efficacy of suplatast tosilate differ significantly in the Japanese population compared with the European population.It should be noted, however, that these data were based on a retrospective analysis of a small sample size.Therefore, these results have to be confirmed in a largescale prospective study.Moreover, further large-scale investigations taking ethnic differences into account are also needed to clarify the exact mechanism of chronic AD.

Table 2 . Clinical Characteristics of the Patients
*The P-values for categorical data and for continuous data were obtained using the likelihood ratio chi-square test and Student t-test, respectively.

Table 3 . Effect of Suplatast Tosilate on Skin Symptom Score, Serum IgE Level, and Eosinophil Count in Atopic Dermatitis Patients
*P-values were obtained using a paired t-test.