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Calcipotriol modulates IL-22 receptor expression and keratinocyte proliferation in IL-22-induced epidermal hyperplasia
Journal of Dermatological Science 71 (2013) 76–77
Contents lists available at SciVerse ScienceDirect
Journal of Dermatological Science journal homepage: www.jdsjournal.com
Letter to the Editor Calcipotriol modulates IL-22 receptor expression and keratinocyte proliferation in IL-22induced epidermal hyperplasia Interleukin-22 (IL-22) is a member of the IL-10 cytokine family which has proinflammatory activities [1]. In the skin, IL-22 is mainly produced by activated CD4+ helper T cells (Th17 and Th22 cells) and regulates the proliferation and differentiation of epidermal keratinocytes (KCs) [2,3]. Previous reports have demonstrated that IL-22 induced an acanthosis of reconstituted human epidermis, an organotypic culture of multilayered human KCs [4,5]. In addition, high IL-22 expression was observed in the skin and serum of patients with psoriasis [6]. Neutralization of IL22 prevented the development of skin inflammations in murine psoriasis models [7]. These observations suggest that IL-22 plays an important role in the development of psoriasis skin lesions. Calcipotriol, also known as MC-903, is a synthetic analog of vitamin D3. It exerts its effect through an intranuclear vitamin D receptor that is expressed in various cell types, including KCs [8]. It has been used for the treatment of hyperproliferative epidermal disorders such as psoriasis. Calcipotriol is known to suppress proliferation and induce the maturation of KCs [9]; however, it remains unclear whether calcipotriol modulates the responsiveness of KCs to IL-22. Therefore, analyzing the effect of calcipotriol on IL-22-induced epidermal hyperplasia in the context of the pathogenesis of psoriasis is important. To achieve this, we cultured reconstituted human epidermis (TMLSE-013A; Toyobo, Osaka, Japan) at 37 8C with 5% CO2 at the air-medium interface for three days. Next, we added 20 ng/ml IL22 (R&D Systems, Minneapolis, USA) and/or 107 M calcipotriol (LEO Pharma, Ballerup, Denmark) into the culture medium and incubated for additional four days. The medium was changed every other day. In order to evaluate epidermal thickness, reconstituted human epidermis sections were stained with hematoxylin and eosin (H&E) and three images were taken for each sample using a digital microscope (BIOREVO BZ-9000; Keyence, Osaka, Japan). Treatment of IL-22 resulted in acanthosis with hypogranulosis compared to the control (Fig. 1A, B, and E). The enhanced acanthosis induced by IL-22 was abrogated by calcipotriol (Fig. 1D), although treatment of calcipotriol alone did not show any significant effect (Fig. 1C). In order to explain the inhibitory effect of calcipotriol on epidermal hyperplasia in human KCs, we evaluated whether calcipotriol inhibits keratinocyte proliferation induced by IL-22 and how calcipotriol decreases the responsiveness of KCs to IL-22. To this end, we performed a keratinocyte proliferation assay. 5 103 normal human epidermal KCs (NHEKs; Kurabo, Osaka, Japan) per well were cultured with complete human KC growth supplement (Humedia-KB2, Kurabo) in a 96-well plate for 24 h. Over the next 24 h, growth supplements (bovine pituitary extract and human epidermal growth factor) were depleted for starvation. NHEKs were then cultured with or without 20 ng/ml IL-22 and
107 M calcipotriol, pulsed with 0.5 mCi 3H-thymidine for the next 24 h, and subjected to liquid scintillation counting. Similar to the results obtained in reconstituted human epidermis (Fig. 1), IL-22 enhanced 3H-thymidine uptake and this effect was attenuated by the addition of calcipotriol (Fig. 2A). Similar inhibitory effects by calcipotriol was observed in keratinocyte proliferation induced by IL-21 (Moniaga et al., unpublished data) which is another psoriasis-associated cytokine [10].
Fig. 1. Calcipotriol inhibited IL-22-induced acanthosis on reconstituted human epidermis. (A–E) Epidermal thickness was measured using H&E-stained sections. Reconstituted human epidermis was cultured for four days in basal medium (A), with 20 ng/ml IL-22 (B), with 107 M calcipotriol (C), and with a combination of IL22 and calcipotriol (D). Scale bars, 60 mm. (E) Columns show the mean standard deviation (SD) of epidermis thickness. * p < 0.05. Data are representative of three independent experiments with similar results.
0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.03.011
Letter to the Editor / Journal of Dermatological Science 71 (2013) 76–77
77
psoriasis. Therefore, it is intriguing to examine whether vitamin D3 derivatives modulate the responsiveness of KCs to IL-22 in the context of the pathogenesis of psoriasis. In this study, we found that calcipotriol inhibits the IL-22-induced epidermal hyperplasia by decreasing KC proliferation and down-modulating the IL-22R expression on KCs. Our data provide evidence that calcipotriol inhibits IL-22 dependent epidermal hyperplasia, such as psoriasis. These new findings may expand our knowledge of the pathogenesis of psoriasis and the therapeutic strategy to control its development. Funding This study was partly supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Ministry of Health, Labor, and Welfare of Japan. Acknowledgments We are grateful to Ms. Hiromi Doi and Ms. Kaori Tomari for their technical assistance. References
Fig. 2. Calcipotriol inhibited IL-22-induced proliferation and IL-22 receptor expression of NHEKs. (A) NHEKs were incubated with or without 20 ng/ml IL-22 and 107 M calcipotriol in growth supplement-depleted medium for 24 h. The proliferation was assessed using 3H-thymidine incorporation. (B) The mRNA (upper panel) and protein expression (lower panel) levels of IL22R in NHEKs treated with or without IL-22 and/or calcipotriol. Columns show the mean SD. * p < 0.05. Data are representative of three independent experiments with similar results.
To explain how calcipotriol decreases the responsiveness of KCs to IL-22, we hypothesized that calcipotriol may decrease the expression level of IL-22 receptor (IL-22R) on KCs. To address this issue, we cultured NHEKs with or without 20 ng/ml IL-22 and 107 M calcipotriol for three days in Humedia-KB2 medium. Cells were then collected for quantitative polymerase chain reaction (qPCR) and western blot analysis for IL-22R. qPCR analysis was performed using 50 -TGT GCC GAG TGA AGA CAC-30 (forward) and 50 -TGG TGA CAT ATC TGT AGC TCA-30 (reverse) primers, and western blot analysis was performed with rabbit anti-human IL22RA1 antibody (R&D Systems, Minneapolis, USA) and rabbit antihuman beta actin antibody (Abcam, Osaka, Japan). The calcipotriol treatment was down-modulated the expression level of IL-22R both in mRNA (Fig. 2B, upper panel) and protein (Fig. 2B, lower panel) levels. Although we cannot exclude a possibility that the relationship between IL-22R down regulation and impair keratinocyte proliferation by calcipotriol is just correlative, our results suggest that calcipotriol suppressed the expression level of IL-22R on KCs, thereby impairing the proliferative-response to IL-22. It was proposed that calcipotriol inhibits KC proliferation by deactivating epidermal growth factor receptor-related genes, such as early growth response-1 and polo-like kinase-2 [8], since all of these parameters are altered in an identical manner in the KCs of patients suffering from psoriasis [5]. Recently, it was demonstrated that epidermal hyperplasia in psoriasis depends on IL-22 [1]. It is well known that vitamin D3 derivatives control the skin lesions of
[1] Boniface K, Guignouard E, Pedretti N, Garcia M, Delwail A, Bernard FX, et al. A role for T cell-derived interleukin 22 in psoriatic skin inflammation. Clin Exp Immunol 2007;150:407–15. [2] Zheng Y, Danilenko DM, Valdez P, Kasman I, Eastham-Anderson J, Wu J, et al. Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 2007;445:648–51. [3] Eyerich S, Eyerich K, Pennino D, Carbone T, Nasorri F, Pallotta S, et al. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest 2009;119:3573–85. [4] Boniface K, Bernard FX, Garcia M, Gurney AL, Lecron JC, Morel F. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. J Immunol 2005;174:3695–702. [5] Wolk K, Haugen HS, Xu W, Witte E, Waggie K, Anderson M, et al. IL-22 and IL20 are key mediators of the epidermal alterations in psoriasis while IL-17 and IFN-gamma are not. J Mol Med (Berl) 2009;87:523–36. [6] Wolk K, Kunz S, Witte E, Friedrich M, Asadullah K, Sabat R. IL-22 increases the innate immunity of tissues. Immunity 2004;21:241–54. [7] Ma HL, Liang S, Li J, Napierata L, Brown T, Benoit S, et al. IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation. J Clin Invest 2008;118:597–607. [8] Kristl J, Slanc P, Krasna M, Berlec A, Jeras M, Strukelj B. Calcipotriol affects keratinocyte proliferation by decreasing expression of early growth response1 and polo-like kinase-2. Pharm Res 2008;25:521–9. [9] Takahashi H, Ibe M, Kinouchi M, Ishida-Yamamoto A, Hashimoto Y, Iizuka H. Similarly potent action of 1,25-dihydroxyvitamin D3 and its analogues, tacalcitol, calcipotriol, and maxacalcitol on normal human keratinocyte proliferation and differentiation. J Dermatol Sci 2003;31:21–8. [10] Caruso R, Botti E, Sarra M, Esposito M, Stolfi C, Diluvio L, et al. Involvement of interleukin-21 in the epidermal hyperplasia of psoriasis. Nat Med 2009;15:1013–5.
Catharina Sagita Moniaga, Gyohei Egawa, Yoshiki Miyachi, Kenji Kabashima* Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan *Corresponding author at: Department of Dermatology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawara, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: +81 75 751 3310; fax: +81 75 761 3002 E-mail address: [email protected] (C.S. Moniaga)
6 September 2012 12 March 2013 Accepted 26 March 2013
Contents lists available at SciVerse ScienceDirect
Journal of Dermatological Science journal homepage: www.jdsjournal.com
Letter to the Editor Calcipotriol modulates IL-22 receptor expression and keratinocyte proliferation in IL-22induced epidermal hyperplasia Interleukin-22 (IL-22) is a member of the IL-10 cytokine family which has proinflammatory activities [1]. In the skin, IL-22 is mainly produced by activated CD4+ helper T cells (Th17 and Th22 cells) and regulates the proliferation and differentiation of epidermal keratinocytes (KCs) [2,3]. Previous reports have demonstrated that IL-22 induced an acanthosis of reconstituted human epidermis, an organotypic culture of multilayered human KCs [4,5]. In addition, high IL-22 expression was observed in the skin and serum of patients with psoriasis [6]. Neutralization of IL22 prevented the development of skin inflammations in murine psoriasis models [7]. These observations suggest that IL-22 plays an important role in the development of psoriasis skin lesions. Calcipotriol, also known as MC-903, is a synthetic analog of vitamin D3. It exerts its effect through an intranuclear vitamin D receptor that is expressed in various cell types, including KCs [8]. It has been used for the treatment of hyperproliferative epidermal disorders such as psoriasis. Calcipotriol is known to suppress proliferation and induce the maturation of KCs [9]; however, it remains unclear whether calcipotriol modulates the responsiveness of KCs to IL-22. Therefore, analyzing the effect of calcipotriol on IL-22-induced epidermal hyperplasia in the context of the pathogenesis of psoriasis is important. To achieve this, we cultured reconstituted human epidermis (TMLSE-013A; Toyobo, Osaka, Japan) at 37 8C with 5% CO2 at the air-medium interface for three days. Next, we added 20 ng/ml IL22 (R&D Systems, Minneapolis, USA) and/or 107 M calcipotriol (LEO Pharma, Ballerup, Denmark) into the culture medium and incubated for additional four days. The medium was changed every other day. In order to evaluate epidermal thickness, reconstituted human epidermis sections were stained with hematoxylin and eosin (H&E) and three images were taken for each sample using a digital microscope (BIOREVO BZ-9000; Keyence, Osaka, Japan). Treatment of IL-22 resulted in acanthosis with hypogranulosis compared to the control (Fig. 1A, B, and E). The enhanced acanthosis induced by IL-22 was abrogated by calcipotriol (Fig. 1D), although treatment of calcipotriol alone did not show any significant effect (Fig. 1C). In order to explain the inhibitory effect of calcipotriol on epidermal hyperplasia in human KCs, we evaluated whether calcipotriol inhibits keratinocyte proliferation induced by IL-22 and how calcipotriol decreases the responsiveness of KCs to IL-22. To this end, we performed a keratinocyte proliferation assay. 5 103 normal human epidermal KCs (NHEKs; Kurabo, Osaka, Japan) per well were cultured with complete human KC growth supplement (Humedia-KB2, Kurabo) in a 96-well plate for 24 h. Over the next 24 h, growth supplements (bovine pituitary extract and human epidermal growth factor) were depleted for starvation. NHEKs were then cultured with or without 20 ng/ml IL-22 and
107 M calcipotriol, pulsed with 0.5 mCi 3H-thymidine for the next 24 h, and subjected to liquid scintillation counting. Similar to the results obtained in reconstituted human epidermis (Fig. 1), IL-22 enhanced 3H-thymidine uptake and this effect was attenuated by the addition of calcipotriol (Fig. 2A). Similar inhibitory effects by calcipotriol was observed in keratinocyte proliferation induced by IL-21 (Moniaga et al., unpublished data) which is another psoriasis-associated cytokine [10].
Fig. 1. Calcipotriol inhibited IL-22-induced acanthosis on reconstituted human epidermis. (A–E) Epidermal thickness was measured using H&E-stained sections. Reconstituted human epidermis was cultured for four days in basal medium (A), with 20 ng/ml IL-22 (B), with 107 M calcipotriol (C), and with a combination of IL22 and calcipotriol (D). Scale bars, 60 mm. (E) Columns show the mean standard deviation (SD) of epidermis thickness. * p < 0.05. Data are representative of three independent experiments with similar results.
0923-1811/$36.00 ß 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jdermsci.2013.03.011
Letter to the Editor / Journal of Dermatological Science 71 (2013) 76–77
77
psoriasis. Therefore, it is intriguing to examine whether vitamin D3 derivatives modulate the responsiveness of KCs to IL-22 in the context of the pathogenesis of psoriasis. In this study, we found that calcipotriol inhibits the IL-22-induced epidermal hyperplasia by decreasing KC proliferation and down-modulating the IL-22R expression on KCs. Our data provide evidence that calcipotriol inhibits IL-22 dependent epidermal hyperplasia, such as psoriasis. These new findings may expand our knowledge of the pathogenesis of psoriasis and the therapeutic strategy to control its development. Funding This study was partly supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Ministry of Health, Labor, and Welfare of Japan. Acknowledgments We are grateful to Ms. Hiromi Doi and Ms. Kaori Tomari for their technical assistance. References
Fig. 2. Calcipotriol inhibited IL-22-induced proliferation and IL-22 receptor expression of NHEKs. (A) NHEKs were incubated with or without 20 ng/ml IL-22 and 107 M calcipotriol in growth supplement-depleted medium for 24 h. The proliferation was assessed using 3H-thymidine incorporation. (B) The mRNA (upper panel) and protein expression (lower panel) levels of IL22R in NHEKs treated with or without IL-22 and/or calcipotriol. Columns show the mean SD. * p < 0.05. Data are representative of three independent experiments with similar results.
To explain how calcipotriol decreases the responsiveness of KCs to IL-22, we hypothesized that calcipotriol may decrease the expression level of IL-22 receptor (IL-22R) on KCs. To address this issue, we cultured NHEKs with or without 20 ng/ml IL-22 and 107 M calcipotriol for three days in Humedia-KB2 medium. Cells were then collected for quantitative polymerase chain reaction (qPCR) and western blot analysis for IL-22R. qPCR analysis was performed using 50 -TGT GCC GAG TGA AGA CAC-30 (forward) and 50 -TGG TGA CAT ATC TGT AGC TCA-30 (reverse) primers, and western blot analysis was performed with rabbit anti-human IL22RA1 antibody (R&D Systems, Minneapolis, USA) and rabbit antihuman beta actin antibody (Abcam, Osaka, Japan). The calcipotriol treatment was down-modulated the expression level of IL-22R both in mRNA (Fig. 2B, upper panel) and protein (Fig. 2B, lower panel) levels. Although we cannot exclude a possibility that the relationship between IL-22R down regulation and impair keratinocyte proliferation by calcipotriol is just correlative, our results suggest that calcipotriol suppressed the expression level of IL-22R on KCs, thereby impairing the proliferative-response to IL-22. It was proposed that calcipotriol inhibits KC proliferation by deactivating epidermal growth factor receptor-related genes, such as early growth response-1 and polo-like kinase-2 [8], since all of these parameters are altered in an identical manner in the KCs of patients suffering from psoriasis [5]. Recently, it was demonstrated that epidermal hyperplasia in psoriasis depends on IL-22 [1]. It is well known that vitamin D3 derivatives control the skin lesions of
[1] Boniface K, Guignouard E, Pedretti N, Garcia M, Delwail A, Bernard FX, et al. A role for T cell-derived interleukin 22 in psoriatic skin inflammation. Clin Exp Immunol 2007;150:407–15. [2] Zheng Y, Danilenko DM, Valdez P, Kasman I, Eastham-Anderson J, Wu J, et al. Interleukin-22, a T(H)17 cytokine, mediates IL-23-induced dermal inflammation and acanthosis. Nature 2007;445:648–51. [3] Eyerich S, Eyerich K, Pennino D, Carbone T, Nasorri F, Pallotta S, et al. Th22 cells represent a distinct human T cell subset involved in epidermal immunity and remodeling. J Clin Invest 2009;119:3573–85. [4] Boniface K, Bernard FX, Garcia M, Gurney AL, Lecron JC, Morel F. IL-22 inhibits epidermal differentiation and induces proinflammatory gene expression and migration of human keratinocytes. J Immunol 2005;174:3695–702. [5] Wolk K, Haugen HS, Xu W, Witte E, Waggie K, Anderson M, et al. IL-22 and IL20 are key mediators of the epidermal alterations in psoriasis while IL-17 and IFN-gamma are not. J Mol Med (Berl) 2009;87:523–36. [6] Wolk K, Kunz S, Witte E, Friedrich M, Asadullah K, Sabat R. IL-22 increases the innate immunity of tissues. Immunity 2004;21:241–54. [7] Ma HL, Liang S, Li J, Napierata L, Brown T, Benoit S, et al. IL-22 is required for Th17 cell-mediated pathology in a mouse model of psoriasis-like skin inflammation. J Clin Invest 2008;118:597–607. [8] Kristl J, Slanc P, Krasna M, Berlec A, Jeras M, Strukelj B. Calcipotriol affects keratinocyte proliferation by decreasing expression of early growth response1 and polo-like kinase-2. Pharm Res 2008;25:521–9. [9] Takahashi H, Ibe M, Kinouchi M, Ishida-Yamamoto A, Hashimoto Y, Iizuka H. Similarly potent action of 1,25-dihydroxyvitamin D3 and its analogues, tacalcitol, calcipotriol, and maxacalcitol on normal human keratinocyte proliferation and differentiation. J Dermatol Sci 2003;31:21–8. [10] Caruso R, Botti E, Sarra M, Esposito M, Stolfi C, Diluvio L, et al. Involvement of interleukin-21 in the epidermal hyperplasia of psoriasis. Nat Med 2009;15:1013–5.
Catharina Sagita Moniaga, Gyohei Egawa, Yoshiki Miyachi, Kenji Kabashima* Department of Dermatology, Kyoto University Graduate School of Medicine, Kyoto 606-8507, Japan *Corresponding author at: Department of Dermatology, Kyoto University Graduate School of Medicine, 54 Shogoin-Kawara, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: +81 75 751 3310; fax: +81 75 761 3002 E-mail address: [email protected] (C.S. Moniaga)
6 September 2012 12 March 2013 Accepted 26 March 2013