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Dihomo-γ-linolenic acid prevents the development of atopic dermatitis through prostaglandin D1 production in NC/Tnd mice

  • Yosuke Amagai
    Affiliations
    Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan
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  • Kumiko Oida
    Affiliations
    Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan
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  • Akira Matsuda
    Affiliations
    Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of Animal Life Science, Institute of Agriculture, Tokyo, Japan
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  • Kyungsook Jung
    Affiliations
    Laboratory of Comparative Animal Medicine, Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan
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  • Saki Kakutani
    Affiliations
    Institute for Health Care Science, Suntory Wellness Ltd., Osaka, Japan
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  • Takao Tanaka
    Affiliations
    Institute for Health Care Science, Suntory Wellness Ltd., Osaka, Japan
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  • Kenshiro Matsuda
    Affiliations
    Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan
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  • Hyosun Jang
    Affiliations
    Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan
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  • Ginae Ahn
    Affiliations
    Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan
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  • Yan Xia
    Affiliations
    Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan
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  • Hiroshi Kawashima
    Affiliations
    Institute for Health Care Science, Suntory Wellness Ltd., Osaka, Japan
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  • Hiroshi Shibata
    Affiliations
    Institute for Health Care Science, Suntory Wellness Ltd., Osaka, Japan
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  • Hiroshi Matsuda
    Correspondence
    Corresponding authors at: Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan. Tel.: +81 42 367 5925; fax: +81 42 367 5916.
    Affiliations
    Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan

    Laboratory of Veterinary Molecular Pathology and Therapeutics, Division of Animal Life Science, Institute of Agriculture, Tokyo, Japan
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  • Akane Tanaka
    Correspondence
    Corresponding authors at: Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan. Tel.: +81 42 367 5925; fax: +81 42 367 5916.
    Affiliations
    Cooperative Major in Advanced Health Science, Graduate School of Bio-Applications and System Engineering, Tokyo University of Agriculture and Technology, Tokyo, Japan

    Laboratory of Comparative Animal Medicine, Division of Animal Life Science, Institute of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan
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      Highlights

      • DGLA containing diet suppressed the development of dermatitis in NC/Tnd mice.
      • Oral DGLA increased both PGD1 and PGD2 levels in the skin of the mice.
      • PGD1 levels were negatively correlated with the duration of scratching behavior.
      • Topical application of PGD1 significantly reduced itching behavior.
      • Mast cells may be one of the important sources of PGD1 after DGLA application.

      Abstract

      Background

      Atopic dermatitis (AD) is a chronic and relapsing skin disorder with pruritic skin symptoms. We previously reported that dihomo-γ-linolenic acid (DGLA) prevented the development of AD in NC/Tnd mice, though the mechanism remained unclear.

      Objective

      We attempted to investigate the mechanism of preventive effect of DGLA on AD development in NC/Tnd mice.

      Methods

      The clinical outcomes of NC/Tnd mice that were given diets containing DGLA, arachidonic acid, or eicosapentaenoic acid were compared. Lipid mediator contents in the skin in each group were also quantified. In addition, release of lipid mediators from RBL-2H3 mast cells treated with either DGLA or prostaglandin D1 (PGD1) was measured. Furthermore, effect of PGD1 on gene expression of thymic stromal lymphopoietin (TSLP) in PAM212 keratinocyte cells was determined.

      Results

      Only DGLA containing diet suppressed the development of dermatitis in vivo. By quantifying the 20-carbon fatty acid-derived eicosanoids in the skin, the application of DGLA was found to upregulate PGD1, which correlated with a better outcome in NC/Tnd mice. Moreover, we confirmed that mast cells produced PGD1 after DGLA exposure, thereby exerting a suppressive effect on immunoglobulin E-mediated degranulation. PGD1 also suppressed gene expression of TSLP in keratinocytes.

      Conclusion

      These results suggest that oral administration of DGLA causes preventive effects on AD development in NC/Tnd mice by regulating the PGD1 supply.

      Abbreviations:

      AA (arachidonic acid), AD (atopic dermatitis), ALA (α-linolenic acid), β-HEX (β-hexosaminidase), BrdU (5-bromo-2′-deoxy-uridine), BSA (bovine serum albumin), DGLA (dihomo-γ-linolenic acid), DNP (dinitrophenyl), EPA (eicosapentaenoic acid), FBS (fetal bovine serum), GLA (γ-linolenic acid), HETE (hydroxyeicosatetraenoic acid), HEPE (hydroxyeicosapentaenoic acid), HETrE (hydroxyeicosatrienoic), IgE (immunoglobulin E), LA (linoleic acid), PG (prostaglandin), PMA (phorbol 12-myristate 13-acetate), PUFA (polyunsaturated fatty acids), RT-PCR (reverse transcription and polymerase chain reaction), SRM (selected reaction monitoring), TSLP (thymic stromal lymphopoietin)

      Keywords

      1. Introduction

      Atopic dermatitis (AD) is a common, chronic inflammatory skin disease associated with dryness and itching of the skin [
      • Ring J.
      • Alomar A.
      • Bieber T.
      • Deleuran M.
      • Fink-Wagner A.
      • Gelmetti C.
      • et al.
      Guidelines for treatment of atopic eczema (atopic dermatitis) part I.
      ]. The etiology of the disease is multifactorial; genetic, environmental, immunological, psychological, and physical factors are considered to be involved in the onset and exaggeration of AD [
      • Ring J.
      • Alomar A.
      • Bieber T.
      • Deleuran M.
      • Fink-Wagner A.
      • Gelmetti C.
      • et al.
      Guidelines for treatment of atopic eczema (atopic dermatitis) part I.
      ,
      • Schmid-Ott G.
      • Jaeger B.
      • Kapp A.
      • Werfel T.
      Different expression of cytokine and membrane molecules by circulating lymphocytes on acute mental stress in patients with atopic dermatitis in comparison with healthy controls.
      ,
      • Palmer C.N.
      • Irvine A.D.
      • Terron-Kwiatkowski A.
      • Zhao Y.
      • Liao H.
      • Lee S.P.
      • et al.
      Common loss-of-function variants of the epidermal barrier protein filaggrin are a major predisposing factor for atopic dermatitis.
      ,
      • Cork M.J.
      • Danby S.G.
      • Vasilopoulos Y.
      • Hadgraft J.
      • Lane M.E.
      • Moustafa M.
      • et al.
      Epidermal barrier dysfunction in atopic dermatitis.
      ]. Among them, food intake is an important factor affecting AD symptoms. In particular, fatty acids have been considered to play a critical role in inflammatory responses because they are a source of various kinds of lipid mediators [
      • Schäfer L.
      • Kragballe K.
      Abnormalities in epidermal lipid metabolism in patients with atopic dermatitis.
      ,
      • Trak-Fellermeier M.A.
      • Brasche S.
      • Winkler G.
      • Koletzko B.
      • Heinrich J.
      Food and fatty acid intake and atopic disease in adults.
      ,
      • Sala-Vila A.
      • Miles E.
      • Calder P.
      Fatty acid composition abnormalities in atopic disease: evidence explored and role in the disease process examined.
      ]. Therefore, many attempts to investigate the efficacy of a diet containing essential fatty acids in the treatment of AD have been made, though most of them were unsuccessful [
      • Berth-Jones J.
      • Graham-Brown R.
      Placebo-controlled trial of essential fatty acid supplementation in atopic dermatitis.
      ,
      • Van Gool C.
      • Zeegers M.
      • Thijs C.
      Oral essential fatty acid supplementation in atopic dermatitis-a meta-analysis of placebo-controlled trials.
      ]. In addition, the efficacy of eicosanoids has been investigated using a spontaneous AD mouse model [
      • Arai I.
      • Takano N.
      • Hashimoto Y.
      • Futaki N.
      • Sugimoto M.
      • Takahashi N.
      • et al.
      Prostanoid DP1 receptor agonist inhibits the pruritic activity in NC/Nga mice with atopic dermatitis.
      ,
      • Honma Y.
      • Arai I.
      • Hashimoto Y.
      • Futaki N.
      • Sugimoto M.
      • Tanaka M.
      • et al.
      Prostaglandin D2 and prostaglandin E2 accelerate the recovery of cutaneous barrier disruption induced by mechanical scratching in mice.
      ,
      • Sugimoto M.
      • Arai I.
      • Futaki N.
      • Hashimoto Y.
      • Honma Y.
      • Nakaike S.
      Increased scratching counts depend on a decrease in ability of cutaneous prostaglandin D2 biosynthesis in NC/Nga mice with atopic dermatitis.
      ]. The group of Nakaie et al. [
      • Arai I.
      • Takano N.
      • Hashimoto Y.
      • Futaki N.
      • Sugimoto M.
      • Takahashi N.
      • et al.
      Prostanoid DP1 receptor agonist inhibits the pruritic activity in NC/Nga mice with atopic dermatitis.
      ,
      • Honma Y.
      • Arai I.
      • Hashimoto Y.
      • Futaki N.
      • Sugimoto M.
      • Tanaka M.
      • et al.
      Prostaglandin D2 and prostaglandin E2 accelerate the recovery of cutaneous barrier disruption induced by mechanical scratching in mice.
      ,
      • Sugimoto M.
      • Arai I.
      • Futaki N.
      • Hashimoto Y.
      • Honma Y.
      • Nakaike S.
      Increased scratching counts depend on a decrease in ability of cutaneous prostaglandin D2 biosynthesis in NC/Nga mice with atopic dermatitis.
      ] focused on the role of prostaglandin D2 (PGD2) and showed a negative correlation between PGD2 and AD progression. After mechanical scratching, PGD2 levels in the skin were increased in BALB/c mice but not in NC/Nga AD model mice [
      • Takaoka A.
      • Arai I.
      • Sugimoto M.
      • Futaki N.
      • Sakurai T.
      • Honma Y.
      • et al.
      Role of scratch-induced cutaneous prostaglandin D2 production on atopic-like scratching behaviour in mice.
      ]. These observations indicated that PGD2 might have suppressive effect on itch sensation and the subsequent development of AD in the mice. However, topical application of a PGD2 receptor agonist did not exert a potent inhibitory effect on AD symptoms [
      • Sugimoto M.
      • Arai I.
      • Futaki N.
      • Hashimoto Y.
      • Sakurai T.
      • Honma Y.
      • et al.
      The anti-pruritic efficacy of TS-022, a prostanoid DP1 receptor agonist, is dependent on the endogenous prostaglandin D2 level in the skin of NC/Nga mice.
      ].
      We recently reported that oral administration of dihomo-γ-linolenic acid (DGLA) prevented the development of AD in NC/Tnd mice [
      • Kawashima H.
      • Tateishi N.
      • Shiraishi A.
      • Teraoka N.
      • Tanaka T.
      • Tanaka A.
      • et al.
      Oral administration of dihomo-γ-linolenic acid prevents development of atopic dermatitis in NC/Nga mice.
      ]. DGLA is metabolized to either the series 1 PGs (PGD1, PGE1 or PGF) as well as arachidonic acid (AA) which is a source of various kinds of leukotrienes and prostanoids such as PGD2 and PGE2 (Fig. 2A) [
      • Shimizu T.
      Lipid mediators in health and disease: enzymes and receptors as therapeutic targets for the regulation of immunity and inflammation.
      ]. Inhibitory effects of PGD1 on the PGD2/E1/E2-mediated increase in vascular permeability were reported [
      • Flower R.J.
      • Kingston W.P.
      Proceedings Prostaglandin D1 inhibits the increase in vascular permeability in rat skin produced by prostaglandin E1, E2 and D2.
      ]; however, there has been little information about the effects of the series 1 PGs on allergic inflammation, including AD. In this study, we compared the clinical outcomes of several diets containing different fatty acids in NC/Tnd mice to clarify the lipid mediators that mainly contribute to the better outcome of mice following DGLA supplementation. Our results revealed that PGD1 upregulation is involved in the prevention of AD by suppressing both mast cells and keratinocytes activations.

      2. Materials and methods

      2.1 Reagents

      PGD1, PGE1, PGF, PGD2, PGE2, PGF, PGD3, PGE3, PGF, PGD2-d4, PGE2-d4, 8(S)-hydroxyeicosatrienoic (HETrE), 15(S)-HETrE, 5(S)-hydroxyeicosatetraenoic acid (HETE), 8(S)-HETE, 12(S)-HETE, 15(S)-HETE, 5(S)-hydroxyeicosapentaenoic acid (HEPE), 8(S)-HEPE, 12(S)-HEPE, 5(S)-HETE-d8, and 15(S)-HETE-d8 were obtained from Cayman Chemical (Ann Arbor, MI).

      2.2 Mice

      NC/Tnd mice were maintained in conventional circumstances, as previously described [
      • Matsuda H.
      • Watanabe N.
      • Geba G.P.
      • Sperl J.
      • Tsudzuki M.
      • Hiroi J.
      • et al.
      Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice.
      ]. All experiments with animals complied with the standards specified in the guidelines of the University Animal Care and Use Committee of the Tokyo University of Agriculture and Technology, as well as with the guidelines for the use of laboratory animals provided by Science Council of Japan.

      2.3 Diet

      Mice were fed a defined diet containing several types of fatty acid (Table 1) from 8 to 13 weeks of age. The mice were given a pellet diet and water ad libitum. The diet was a modified AIN-76A containing 5% (w/w) lipids, which consisted of a mixture of corn oil, lard, olive oil, and either DGLA oil, AA oil, or eicosapentaenoic acid (EPA) ethyl ester (Table 1). DGLA oil was obtained as described previously [
      • Kawashima H.
      • Tateishi N.
      • Shiraishi A.
      • Teraoka N.
      • Tanaka T.
      • Tanaka A.
      • et al.
      Oral administration of dihomo-γ-linolenic acid prevents development of atopic dermatitis in NC/Nga mice.
      ]. AA oil and EPA ethyl ester was obtained from Suntory Ltd. (Osaka, Japan) and Bizen Chemical (Okayama, Japan), respectively. The fatty acid compositions of the diets were adjusted to be similar to each other and for the amounts of total n-6 fatty acids (linoleic acid [LA] + γ-linolenic acid [GLA] + DGLA) to be approximately 40%.
      Table 1Fatty acid composition of lipids in each experimental diet.
      DietProportion of fatty acid (%)
      DGLAAAEPA
      Control000
      20:3 DGLA11.40.90
      20:4 AA012.10
      20:5 EPA0012.8

      2.4 Clinical skin severity score and scratching analysis

      The clinical features of dermatitis were scored twice a week according to criteria described previously [
      • Matsuda H.
      • Watanabe N.
      • Geba G.P.
      • Sperl J.
      • Tsudzuki M.
      • Hiroi J.
      • et al.
      Development of atopic dermatitis-like skin lesion with IgE hyperproduction in NC/Nga mice.
      ], which consisted of five major clinical signs and symptoms of AD: itching, erythema/hemorrhage, edema, excoriation/erosion, and scaling/dryness. The scratching behavior of the mice was analyzed using a SCLABA-Real® system (Noveltec Inc., Kobe, Japan) [
      • Orito K.
      • Chida Y.
      • Fujisawa C.
      • Arkwright P.
      • Matsuda H.
      A new analytical system for quantification scratching behaviour in mice.
      ]. Scratching behavior was quantified as the frequency and duration of scratching per 20 min in the double-blind evaluation.

      2.5 Analysis of fatty acids of skin homogenates

      The quantification of each fatty acid in the skin was conducted according to a previously described method [
      • Kawashima H.
      • Tateishi N.
      • Shiraishi A.
      • Teraoka N.
      • Tanaka T.
      • Tanaka A.
      • et al.
      Oral administration of dihomo-γ-linolenic acid prevents development of atopic dermatitis in NC/Nga mice.
      ].

      2.6 Analysis of lipid mediators of skin homogenates and RBL-2H3 cells

      The extraction and analysis of lipid mediators were conducted based on previous methods with some modification [
      • Kakutani S.
      • Kawashima H.
      • Tanaka T.
      • Shiraishi-Tateishi A.
      • Kiso Y.
      Uptake of dihomo-γ-linolenic acid by murine macrophages increases series-1 prostaglandin release following lipopolysaccharide treatment.
      ,
      • Tateishi N.
      • Kakutani S.
      • Kawashima H.
      • Shibata H.
      • Morita I.
      Dietary supplementation of arachidonic acid increases arachidonic acid and lipoxin A4 contents in colon, but does not affect severity or prostaglandin E2 content in murine colitis model.
      ]. Briefly, skin tissue frozen in liquid nitrogen was ground using a Multi-Beads Shocker MB701(S) (Yasui Kikai, Osaka, Japan) and homogenized with ice-cold ethanol. Fixed amounts of PGD2-d4, PGE2-d4, 5(S)-HETE-d8, and 15(S)-HETE-d8 were added as an internal standard. After centrifugation, each supernatant was dried by centrifugal evaporation, and the residues were dissolved in methanol and diluted with water/acetic acid (1000/5, v/v) for clean-up with solid phase extraction (SPE) cartridges (Empore disk cartridge C18-SD; 3 M, St. Paul, MN). An Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA) equipped with a Cadenza CD-C18 column (3 μm, 2 mm i.d. × 150 mm; Imtakt, Kyoto, Japan) and a quadruple linear ion trap hybrid mass spectrometer, 4000 Q TRAP, with an electrospray interface (Applied Biosystems/MDS SCIEX, Concord, Canada) was used for quantification. The flow rate was 0.2 mL/min and the column temperature was set at 30 °C. Solvent A was acetonitrile/water (50/50, v/v) and solvent B was methanol/formic acid (10,000/1, v/v). The analytes were separated using the following gradient: 0–2.5 min, 0–100% solvent B; 2.5–5 min, 100% solvent B; 5–5.75 min, 100–0% solvent B; and 5.75–14.8 min, 0% solvent B. The mass spectrometer was operated in negative ion mode with selected reaction monitoring (SRM) as summarized in Table 2. Lipid mediators in the culture medium of RBL-2H3 cells [
      • Morita Y.
      • Siraganian R.P.
      Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation.
      ] were analyzed using the same method as the above though grinding and homogenization steps were skipped.
      Table 2SRM transitions and collision energy conditions.
      AnalyteQ1 (m/z)Q3 (m/z)Collision energy (V)
      PGD1/PGE1353317−20
      PGD2/PGE2351271−26
      PGD3/PGE3349269−21
      PGF355311−30
      PGF353309−28
      PGF351307−26
      5-HETE319115−20
      5-HEPE317115−20
      8-HETrE321157−23
      8-HETE319155−22
      8-HEPE317155−20
      12-HETE319179−21
      12-HEPE317179−20
      15-HETrE321221−23
      15-HETE319219−19
      PGD2-d4/PGE2-d4355275−26
      5-HETE-d8327116−22
      15-HETE-d8327226−20

      2.7 Cell culture and determination of cell activation

      RBL-2H3 mast cells [
      • Morita Y.
      • Siraganian R.P.
      Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation.
      ] were maintained in α-minimum essential medium (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (FBS; Filtron, Brooklyn, Australia) and antibiotics. A β-hexosaminidase (β-HEX) assay was performed according to the method described by Ortega et al. [
      • Ortega E.
      • Hazan B.
      • Zor U.
      • Pecht I.
      Mast cell stimulation by monoclonal antibodies specific for the Fc epsilon receptor yields distinct responses of arachidonic acid and leukotriene C4 secretion.
      ]. For immunoglobulin E (IgE) cross-linkage, cells were first sensitized with 5 μg/ml monoclonal anti-dinitrophenyl (DNP) IgE (clone SPE7; Sigma Aldrich, Taufkirchen, Germany) for 4 h and stimulated by DNP-bovine serum albumin (DNP-BSA; Sigma Aldrich) for 3 h. The absorbance was measured with an ImmunoMini NJ-2300 (Nalge Nunc International K.K., Tokyo, Japan).

      2.8 Growth activity of RBL-2H3 cells

      A 5-bromo-2′-deoxy-uridine (BrdU) incorporation assay was conducted according to the method described previously [
      • Amagai Y.
      • Tanaka A.
      • Ohmori K.
      • Matsuda H.
      Establishment of a novel high-affinity IgE receptor-positive canine mast cell line with wild-type c-kit receptors.
      ].

      2.9 Determination of thymic stromal lymphopoietin (TSLP) mRNA expression

      PAM212 keratinocyte cells [
      • Terranova V.P.
      • Rohrbach D.H.
      • Martin G.R.
      Role of laminin in the attachment of PAM 212 (epithelial) cells to basement membrane collagen.
      ] were maintained in Dulbecco's modified Eagle medium (Life Technologies) supplemented with 10% FBS and antibiotics. Cells were incubated with DGLA and PGD1 for 48 h, and then stimulated with 50 nM phorbol 12-myristate 13-acetate (PMA; Sigma Aldrich) or dimethyl sulfoxide for 6 h. The real-time reverse transcription and polymerase chain reaction (RT-PCR) was performed using an ABI 7000 system (Life Technologies) according to the manufacturer's instructions. The following primers were used; a TSLP-specific forward primer (5′-cga gca aat cga gga ctg tga g-3′), a TSLP-specific reverse primer (5′-ctt cgg gag tta ctg gtg acg-3′), an ACTB-specific forward primer (5′-cat ccg taa aga cct cta tgc caa c-3′) and an ACTB-specific reverse primer (5′-atg gag cca ccg atc cac a-3′).

      2.10 Topical application of PGD1 or PGD2

      Conventional NC/Tnd mice aged 14–17 weeks were topically applied PGD1 or PGD2, which are dissolved in ethanol at 0.1 mg/mL, respectively, habituated in a cage for 30 min, and the number of scratching on either the nape or rostral part of the back was measured for 20 min every hour. As a sham control, ethanol alone was applied. In order to compare alterations in the number of scratching behaviors in the same mice, they were measured in each mouse following application of either PGD1 or PGD2 a week after the same measurement following ethanol treatment.

      2.11 Statistical analyses

      Dunnett's test, Tukey's test, Steel's test and Spearman's rank correlation test were performed for statistical analysis, and a p value of <0.05 was considered statistically significant. The Spearman's coefficients are denoted by rs.

      3. Results

      To investigate whether the preventive effect of DGLA on AD was obtained directly from DGLA-derived PGs or mediated through AA, diets containing either AA or DGLA (Table 1) were fed to NC/Tnd mice and clinical skin scores, as well as the scratching frequency of the mice, were compared. As controls, a diet containing another 20-carbon fatty acid, EPA, and a normal diet that contained neither of these fatty acids were administered (Table 1). As shown in Fig. 1A , DGLA abrogated the exaggeration of AD development, while both AA and EPA did not suppress the increase of clinical scores in the mice. In addition, compared to the control, only DGLA decreased the number and duration of scratching behaviors (Fig. 1B and C).
      Figure thumbnail gr1
      Fig. 1The effects of various diets on the development of AD and scratching behaviors in NC/Tnd mice. (A) Clinical skin severity scores of the mice that were fed each fatty acid-containing diet. Data represent the mean and SE of results from 7 mice in each group. * p < 0.05 compared to control by using Steel's test. Scratching frequency (B) and total scratching duration (C) of NC/Tnd mice are shown. Both sets of data were based on a 20-min observation in mice that were given each diet for 2 or 4 weeks. * p < 0.05 compared to the control by using Dunnett's test.
      Polyunsaturated fatty acids (PUFAs) are converted to various kinds of eicosanoids as shown in Fig. 2A . EPA, which is originated from α-linolenic acid (ALA; n-3 PUFA), is converted to the series 3 PGs. On the other hand, LA (n-6 PUFA) is converted to DGLA resulting in direct conversion to the series 1 PGs as well as in indirect conversion to the series 2 PGs through AA. Because skin absorption of fatty acids derived from each diet was confirmed (Fig. 2B for DGLA, and data not shown for other fatty acids), we assumed that amounts of each eicosanoid produced in NC/Tnd mice were different and some of them may be highly correlated with the exaggeration of AD symptoms. Therefore, we measured the amount of eicosanoids in the skin of NC/Tnd mice fed each diet for 5 weeks. PGs, especially the series 1 PGs, were significantly upregulated only in the DGLA diet-treated group, while there were no significant differences in most mediators that are converted by lipoxygenase (Fig. 2C). The series 3 PGs, produced from EPA, were detected only in the EPA diet-treated group. On the other hand, PGD2 and PGE2, which are converted from AA, were significantly increased following both DGLA and AA treatment, although there were no significant differences between those two groups (Fig. 2C). To further explore the correlation between the production of lipid mediators and AD-related symptoms, the amount of lipid mediators and duration of scratching behavior in the mice fed each diet for 5 weeks were compared (Fig. 2D). Corresponding to the results shown in Fig. 2C, a significant negative correlation was observed between AD symptoms and PGD1 (rs = −0.52) and a positive correlation was observed with PGE2 (rs = 0.44). Regarding the correlation between clinical scores and lipid mediators, weak negative correlation with PGD1 (rs = −0.21) and weak positive correlation with PGE2 (rs = 0.26) was observed, though it was not statistically significant (Fig. 2E).
      Figure thumbnail gr2
      Fig. 2Analysis of lipid mediators produced in NC/Tnd mice that were fed diets containing different fatty acids. (A) Outline of the conversion pathway of eicosanoids from PUFAs. (B) Absorption of each fatty acid in NC/Tnd mice which were fed each fatty acid-containing diet. The mice were fed the each diet for 5 weeks, and the amount of DGLA was quantified. Data represent the mean and SE of the results from 7 mice in each group. ** p < 0.01 compared to the control by using Tukey's test. (C) Lipid mediators in the skin of NC/Tnd mice. Mice were fed each diet for 5 weeks, and the content of each mediator in the skin was quantified. Data represent the mean and SE of the results from 7 mice in each group. *, ** p < 0.05, 0.01 compared to the control by using Tukey's test, respectively. (D) Relationships between the number of scratching behaviors and the amount of lipid mediators in the skin. The data are based on the results obtained in mice after receiving each diet for 5 weeks. The amount of lipid mediators is indicated as the ratio to total PGs in the skin. The lines were calculated using the method of least squares. * and ** denote p < 0.05 and p < 0.01, respectively, for Spearman's rank correlation. (E) Relationships between the clinical scores and the amount of lipid mediators in the skin. The data are based on the results obtained in mice after receiving each diet for 5 weeks. The amount of lipid mediators is indicated as the ratio to total PGs in the skin. The lines were calculated using the method of least squares.
      These results indicate that series 1 PGs, especially PGD1 play an important role in the prevention of AD development in NC/Tnd mice. However, few studies have determined the role and source of these PGDs. Because mast cells are a primary source of PGD2 in the skin [
      • Benyon R.C.
      • Robinson C.
      • Holgate S.T.
      • Church M.K.
      Prostaglandin D2 release from human skin mast cells in response to ionophore A23187.
      ,
      • Benyon R.C.
      • Robinson C.
      • Church M.K.
      Differential release of histamine and eicosanoids from human skin mast cells activated by IgE-dependent and non-immunological stimuli.
      ], we hypothesized that PGD1 was also produced from mast cells. To test this possibility, DGLA, AA or EPA was supplemented to the culture medium of an RBL-2H3 mast cell line [
      • Morita Y.
      • Siraganian R.P.
      Inhibition of IgE-mediated histamine release from rat basophilic leukemia cells and rat mast cells by inhibitors of transmethylation.
      ]. As shown in Fig. 3A , DGLA supplementation significantly upregulated PGD1 production in RBL-2H3 cells in a dose-dependent manner. In contrast, other series 1 PGs as well as series 2/3 PGs were not increased by DGLA treatment in these cells (Fig. 3B and C). Moreover, neither AA nor EPA increased the production of any PGs in the mast cells (Fig. 3A–C). We next evaluated the effect of each mediator on mast cell function. As shown in Fig. 3D, DGLA, but neither AA nor EPA, inhibited mast cell degranulation in a dose-dependent manner. In contrast, no mediators affected the growth of the cells (Fig. 3E). To further explore whether the inhibitory effect of DGLA on mast degranulation was mediated by PGD1, β-HEX assay was carried out with RBL-2H3 cells incubated with PGD1. Corresponding to the results obtained in DGLA-treated cells, PGD1 suppressed the degranulation of the cells (Fig. 3F).
      Figure thumbnail gr3
      Fig. 3Involvement of mast cells in PG production and activation. (A–C) Production of PG mediators in RBL-2H3 cells. Cells were treated with DGLA, AA or EPA for 48 h, and the prostanoids produced within the cells were quantified. Data represent the mean and SE of 3 independent experiments. (D) Degranulation responses of RBL-2H3 cells. Cells were treated with anti-DNP IgE and DNP-BSA after the treatment of DGLA, AA, or EPA for 48 h. Data represent the mean and SE of 3 independent experiments. * p < 0.05 compared to the control by using Dunnett's test. (E) Effects of each fatty acid on the growth activity of RBL-2H3 cells. Cells were incubated with each fatty acid for 48 h in the presence of BrdU, and the incorporation of BrdU was quantified. Data represent the mean and SE of 3 independent experiments. (F) Degranulation responses of RBL-2H3 cells. Cells were treated with anti-DNP IgE and DNP-BSA after the treatment of PGD1 for 48 h. Data represent the mean and SE of 3 independent experiments. * p < 0.05 compared to the control by using Dunnett's test.
      TSLP is a cytokine released from epithelial cells and well-known to trigger allergic inflammation at the onset of AD [
      • Soumelis V.
      • Reche P.A.
      • Kanzler H.
      • Yuan W.
      • Edward G.
      • Homey B.
      • et al.
      Human epithelial cells trigger dendritic cell-mediated allergic inflammation by producing TSLP.
      ,
      • Wang Y.
      • Ito T.
      • Wang Y.
      • Homey B.
      • Watanabe N.
      • Martin R.
      • et al.
      Maintenance and polarization of human TH2 central memory t cells by thymic stromal lymphopoietin-activated dendritic cells.
      ]. Next we examined effects of DGLA on TSLP expression in keratinocyte. PAM212 keratinocytes were treated with either DGLA or PGD1, activated with PMA and real-time RT-PCR was carried out to quantify the expression of TSLP mRNA. As shown in Fig. 4, PGD1 suppressed the TSLP mRNA expression in PAM212 cells. Although DGLA also tended to suppress, there was not statistical difference (Fig. 4).
      Figure thumbnail gr4
      Fig. 4Inhibitory effects of DGLA and PGD1 on keratinocyte activations. Cells were treated with DGLA or PGD1 for 48 h, stimulated with 50 nM PMA for 6 h, and then real-time RT-PCR was conducted. Data represent the mean and SE of 4 independent experiments. * p < 0.05 compared to the PMA treatment group by using Dunnett's test.
      Finally, PGD1 was administered pericutaneously to the established AD in NC/Tnd mice. As shown in Fig. 5, topical application of PGD1 at 0.1 mg/ml significantly decreased the number of scratching behaviors in NC/Tnd mice especially for the first 12 h, while PGD2 showed limited effect. However, 0.01 mg/ml or less concentration of PGD1 application did not show any inhibitory effect (data not shown). We also applied PGD1 daily for 8 days to evaluate effects on clinical severity, though little inhibitory effect was identified (data not shown).
      Figure thumbnail gr5
      Fig. 5The effects of PGD1 and PGD2 application on the development of AD and scratching behavior in NC/Tnd mice. Either PGD1 or PGD2 at 0.1 mg/ml dissolved in ethanol was applied to the skin of NC/Tnd mice, and the numbers of scratching behavior was counted. Each point represents the mean and SE of the accumulated number of scratching behaviors of 6 mice in each group.

      4. Discussion

      In this study, we revealed that DGLA prevents AD via PGD1 production for the first time. DGLA solely prevented AD development in NC/Tnd mice despite the fact that AA is converted from DGLA, indicating the significance of series 1 PGs in the inhibition of dermatitis progression (Fig. 6). The notion is also supported by the results that PGD1 suppressed TSLP mRNA expression and mast cell activation in vitro as well as the number of scratching behavior in vivo. In addition, systemic alteration of the composition of lipid mediators may be beneficial in the prevention of AD, considering our result that the serum levels of PGE2 were negatively correlated with the reduction in the frequency of scratching behaviors in NC/Tnd mice. Compared to healthy donors, patients with AD have been reported to have lower DGLA levels as well as normal LA levels in the serum [
      • Sala-Vila A.
      • Miles E.
      • Calder P.
      Fatty acid composition abnormalities in atopic disease: evidence explored and role in the disease process examined.
      ], suggesting that AD patients may possess abnormalities in lipid metabolism, especially in the process related to the conversion of LA to DGLA. In fact, lower transcriptional levels of both Δ6-desaturase and elongase 5, which are the enzymes necessary for the conversion of LA to DGLA, have been noted in pediatric patients with AD [
      • Chisaguano A.M.
      • Montes R.
      • Pérez-Berezo T.
      • Castellote A.I.
      • Guerendiain M.
      • Bustamante M.
      • et al.
      Gene expression of desaturase (FADS1 and FADS2) and elongase (ELOVL5) enzymes in peripheral blood: association with polyunsaturated fatty acid levels and atopic eczema in 4-year-old children.
      ]. Thus, supplements or agents that modify the enzymatic activity of those proteins or other molecules that target the metabolism of these lipids may help to prevent or ameliorate AD symptoms.
      Figure thumbnail gr6
      Fig. 6Schematic representation of the relationship between the amount of lipid mediators and the development of AD. Under normal conditions, the amount of most mediators is low, though the sum effect of those mediators results in the exaggeration of AD in NC/Tnd mice. DGLA supplementation results in the upregulation of PGs, in particular those that inhibit AD exaggeration, thus providing a preventive effect on AD.
      PGD2 receptor 1 (DP1) and DP2 are well-known receptors for PGD2 and PGJ2 [
      • Kim N.
      • Luster A.D.
      Regulation of immune cells by eicosanoid receptors.
      ]. DP1 activation shows suppressive effects on allergic reactions, while DP2 activation promotes allergic inflammation [
      • Monneret G.
      • Gravel S.
      • Diamond M.
      • Rokach J.
      • Powell W.S.
      Prostaglandin D2 is a potent chemoattractant for human eosinophils that acts via a novel DP receptor.
      ,
      • Mandal A.K.
      • Zhang Z.
      • Ray R.
      • Choi M.S.
      • Chowdhury B.
      • Pattabiraman N.
      • et al.
      Uteroglobin represses allergen-induced inflammatory response by blocking PGD2 receptor-mediated functions.
      ,
      • Fujitani Y.
      • Kanaoka Y.
      • Aritake K.
      • Uodome N.
      • Okazaki-Hatake K.
      • Urade Y.
      Pronounced eosinophilic lung inflammation and Th2 cytokine release in human lipocalin-type prostaglandin D synthase transgenic mice.
      ]. Limited effects of PGD2 on AD symptoms in this study as well as the ones reported by Nakaie et al. [
      • Sugimoto M.
      • Arai I.
      • Futaki N.
      • Hashimoto Y.
      • Sakurai T.
      • Honma Y.
      • et al.
      The anti-pruritic efficacy of TS-022, a prostanoid DP1 receptor agonist, is dependent on the endogenous prostaglandin D2 level in the skin of NC/Nga mice.
      ] was probably due to DP2 activation mediated by PGD2 and its metabolites [
      • Kim N.
      • Luster A.D.
      Regulation of immune cells by eicosanoid receptors.
      ,
      • Ulven T.
      • Kostenis E.
      Targeting the prostaglandin D2 receptors DP and CRTH2 for treatment of inflammation.
      ]. Though there is little information regarding the PGD1 and its receptors, several cellular responses to PGD1 are similar to those induced by PGD2. For example, both PGD1 and PGD2 activate peroxisome proliferator-activated receptors [
      • Yu K.
      • Bayona W.
      • Kallen C.B.
      • Harding H.P.
      • Ravera C.P.
      • McMahon G.
      • et al.
      Differential activation of peroxisome proliferator-activated receptors by eicosanoids.
      ] and inhibit growth of glioma cells [
      • Sugino H.
      • Miwa T.
      • Ueno R.
      • Hayaishi O.
      Alteration in morphology and induction of glutamine synthetase in rat glioma C6BU-1 cells cultured with prostaglandin D2-supplemented media.
      ]. It suggests that DP1 and DP2 may also be provided as receptors for PGD1. Since PGD1 suppressed activation of keratinocyte and mast cell in vitro partially, there is a possibility that the binding affinity of PGD1 to DP1 was stronger than that to DP2 on those cells. Different outcomes of DP1 and DP2 activation are characterized by the opposing effects on cyclic AMP (cAMP) production; while DP1 activates cAMP and downstream protein kinase A (PKA), DP2 downregulates both of them [
      • Hata A.N.
      • Zent R.
      • Breyer M.D.
      • Breyer R.M.
      Expression and molecular pharmacology of the mouse CRTH2 receptor.
      ,
      • Hirai H.
      • Tanaka K.
      • Yoshie O.
      • Ogawa K.
      • Kenmotsu K.
      • Takamori Y.
      • et al.
      Prostaglandin D2 selectively induces chemotaxis in T helper type 2 cells, eosinophils, and basophils via seven-transmembrane receptor CRTH2.
      ]. Interestingly, IgE-mediated mast cell degranulation requires cAMP downregulation [
      • Serra-Pages M.
      • Olivera A.
      • Torres R.
      • Picado C.
      • de Mora F.
      • Rivera J.
      E-prostanoid 2 receptors dampen mast cell degranulation via cAMP/PKA-mediated suppression of IgE-dependent signaling.
      ], supporting the premise that PGD1 activated DP1 signaling and resulted in functional suppression of mast cells via cAMP upregulation. Because PMA strongly increases intracellular cAMP and following TSLP expression [
      • Yoshimura M.
      • Cooper D.M.
      Type-specific stimulation of adenylylcyclase by protein kinase C.
      ], inhibitory effects of PGD1 in vitro may be a consequence of cAMP consumption by DGLA pretreatment. It raises the possibility that DGLA supplementation can alter the cellular responsiveness to external stimuli and prevent excessive inflammatory reactions. Though PGD1 may be a novel candidate for controlling AD symptoms, topical application of PGD1 on the skin of NC/Tnd mice exerted little beneficial effect on their clinical symptoms, probably due to the instability and short half-life of PGD1 [
      • Vergroesen A.J.
      Physiological effects of dietary linoleic acid.
      ]. Constant supplementation of PGD1 through oral administration of DGLA may be superior to direct PGD1 application. In addition, our in vivo experiments showed that DGLA uptake enhanced the production of several lipid mediators other than PGD1 including PGE1 and 15-HETrE, which may also be involved in the control of AD symptoms directly or indirectly and providing the benefit of DGLA supplementation.
      In conclusion, our results reveal that production of PGD1 is one of the mechanisms of the preventive effects of DGLA on AD progression. Therefore, supplementation of DGLA or the modification of these metabolic pathways may provide a novel therapeutic strategy for AD.

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