1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders.

1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders.

1,25-dihydroxyvitamin D3 (Vitamin D) is a potent immunomodulator capable of generating regulatory T cells (Tregs) and contributing to immune tolerance. Additionally, vitamin D has been shown to promote mucosal immunity when used as a vaccine adjuvant. We show here that pretreatment of an area of skin with the synthetic vitamin D analog calcipotriol combined with transcutaneous immunization results in the induction of CD4⁺CD25⁺ Tregs capable of inhibiting the elicitation of a contact hypersensitivity response. We also demonstrate that topical calcipotriol has significant effects on the immune response to subcutaneously injected vaccines, and compare it with another common topical immunosuppressant, the corticosteroid betamethasone-17-valerate (BMV). Functionally, calcipotriol and BMV treatment both result in the suppression of CD8⁺ T cell priming in response to subcutaneous vaccination, despite the topical co-administration of the potent Th1 inducing TLR9 agonist unmethylated CpG DNA. The effects of calcipotriol on the humoral response are subtler as we observe marginally increased production of antigen-specific IgG1 immunoglobulins along with a strong suppression of the IgG2a isotype. This is in contrast to pretreatment with BMV, which instead suppresses the production of IgG1 and IgA antibodies. In the draining lymph nodes of calcipotriol treated animals, we see no change in the percentage of Foxp3⁺ CD4⁺ T cells post-immunization, but show that tolerance is transferable with the adoptive transfer of CD4⁺CD25⁺ cells. Despite a decrease in the percentage of antigen-bearing APCs in the DLN of calcipotriol treated animals, the DCs maintain high expression of co-stimulatory markers and can induce CD4⁺ T cell proliferation ex vivo. Our data indicate that calcipotriol has distinct effects on immune responses to subcutaneous vaccines consistent with its role as an immunomodulator, although the mechanism(s) through which it is acting remain unclear. We believe that further research is warranted into its potential use as part of a treatment modality for allergy and autoimmune disorders. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22000号 / 医博第4514号 / 新制||医||1038(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 生田 宏一, 教授 竹内 理, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Abstract Glucocorticoids are the most important and widely used treatment modality in dermatology. A large variety of topical as well as systemic preparations is available. Most patients treated with glucocorticoids respond quickly to the treatment, but some are considered insensitive or even resistant to glucocorticoid therapy. Currently, there is no known measurable variable, through which the response can be predicted. Glucocorticoids mediate their actions through glucocorticoid receptors (GR). Several isoforms of GR exist, but the α (GRα) and β (GRβ) isoforms are clinically the most important. Based on previous studies, it has been proposed that the abundance of GR isoforms or the GRβ: GRα –ratio could affect individual responsiveness to corticosteroid treatment. In particular, up-regulation of GRβ expression has been shown to be linked to resistance to corticosteroid treatment. This thesis comprises three sub-studies. Firstly, we wanted to determine whether GRα and GRβ are expressed in inflammatory skin diseases. Secondly, we examined if the expression is altered by corticosteroid treatment in eczema atopicum, bullous pemphigoid and psoriasis. Finally, we measured the effects of a topical vitamin D3 analogue (calcipotriol) combined with betamethasone compared with betamethasone monotherapy on inflammatory biomarkers of psoriasis. Our studies provide detailed novel data about the expression of GRα and GRβ. GRα and GRβ were shown to be expressed in the blood lymphocytes and lesional skin of patients with eczema atopicum, bullous pemphigoid and psoriasis, as well as in the skin of patients with eczema nummulare, lichen simplex chronicus and lichen ruber planus. Systemic corticosteroid treatment was shown to affect the expression of GRα and GRβ in eczema atopicum and bullous pemphigoid, but the inconsistent variation in their expression between patients prevented us from drawing firm conclusions. Neither GRα nor GRβ as a single marker were found to be a suitable predictor of corticosteroid responsiveness. Clinical and laboratory analyses showed that topical treatment of psoriasis with calcipotriol/betamethasone combination ointment is more beneficial measured by both than betamethasone monotherapy. / Tiivistelmä Glukokortikoideja (”kortisoni”) käytetään tulehduksellisten ihotautien hoidossa paikallisesti tai systeemisenä lääkkeenä. Suurin osa potilaista reagoi hoitoon nopeasti, mutta osalla hoitovaste on heikompi tai ilmenee hitaasti. Tällä hetkellä ei tunneta keinoja ennustaa luotettavasti kortisonihoidon vastetta. Glukokortikoidit vaikuttavat elimistössä glukokortikoidireseptorien (GR) kautta. Glukokortikoidireseptorista tunnetaan useita alatyyppejä, joista tärkeimmät ovat α (GRα) ja β (GRβ). Aiemman tiedon pohjalta on pidetty mahdollisena, että GR-alatyyppien suhteella tai määrällä on merkitystä kortisonivasteen syntymisessä. Erityisesti on arveltu, että ylimäärä GRβ:aa voisi estää kortisonihoidon vaikutusta. Tässä väitöskirjassa tavoitteena on ollut selvittää, tapahtuuko GR-alatyyppien ilmenemisessä muutoksia tulehduksellisia ihosairauksia sairastavilla potilailla sekä tutkia, miten kortisonihoito vaikuttaa GR-tasoihin atooppista ihottumaa, pemfigoidia ja psoriaasia sairastavilla potilailla. Lisäksi olemme verranneet paikallishoitoa pelkällä kortisonivoiteella D-vitamiinijohdos kalsipotriolin ja kortisonin yhdistelmähoitoon psoriaatikoilla. Tutkimus on antanut uutta yksityiskohtaista tietoa GRα:n ja GRβ:n esiintymisestä ihossa ja tulehdussoluissa ihosairauksia sairastavilla potilailla. Tutkimustulosten perusteella voidaan todeta, että GRα ja GRβ esiintyvät atooppista ihottumaa, pemfigoidia ja psoriaasia sairastavien potilaiden ihossa ja veren tulehdussoluissa sekä nummulaari-ihottumaa, neurodermatiittia ja punajäkälää sairastavien potilaiden ihossa. Suun kautta annettu kortisonihoito vaikuttaa GRα- ja GRβ–lähetti-RNA:n ilmenemiseen, mutta potilaskohtaiset erot ovat suuret, eikä kumpikaan, GRα tai GRβ, sovellu yksinään ennustamaan kortisonihoidon vastetta. Paikallisella kortisonihoidolla D-vitamiinijohdos kalsipotrioliin yhdistettynä on suotuisampi vaikutus psoriaasin tulehduksellisiin välittäjäaineisiin ja tulehdussoluihin kuin pelkällä paikallisella kortisonihoidolla.