IL-23 from Langerhans Cells Is Required for the Development of Imiquimod-Induced Psoriasis-Like Dermatitis by Induction of IL-17A-Producing cd T Cells

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1 ORIGINAL ARTICLE IL-23 from Langerhans Cells Is Required for the Development of Imiquimod-Induced Psoriasis-Like Dermatitis by Induction of -Producing cd T Cells Ryutaro Yoshiki 1, Kenji Kabashima 2, Tetsuya Honda 2, Satoshi Nakamizo 2, Yu Sawada 2, Kazunari Sugita 1, Haruna Yoshioka 1, Shun Ohmori 1, Bernard Malissen 3, Yoshiki Tokura 4 and Motonobu Nakamura 1 Psoriasis is a common chronic inflammatory skin disease that involves dysregulated interplay between immune cells and keratinocytes. Recently, it has been reported that IL-23 induces CCR6 þ gd T cells, which have the pivotal role in psoriasis-like skin inflammation in mice of producing and IL-22. Langerhans cells (LCs) are a subset of dendritic cells that reside in the epidermis and regulate immune responses. The role of LCs has been extensively investigated in contact hypersensitivity, but their role in psoriasis remains to be clarified. In this study, we focused on Th17-related factors and assessed the role of LCs and gd T cells in the development of psoriasis using a mouse psoriasis model triggered by topical application of imiquimod (). LC depletion by means of diphtheria toxin (DT) in Langerin DT receptor knocked-in mice suppressed hyperkeratosis, parakeratosis, and ear swelling in the -treated regions. In addition, LC-depleted mice showed decreased levels of Th17-related cytokines in -treated skin lesions. Moreover, the -treated skin of LC-depleted mice showed a decreased number of -producing CCR6 þ gd T cells. These results suggest that LCs are required for the development of psoriasis-like lesions induced by in mice. Journal of Investigative Dermatology (214) 134, ; doi:1.138/jid ; published online 3 April 214 INTRODUCTION Psoriasis is an inflammatory epidermal hyperproliferative skin disease that affects 2 3% of the population in Caucasians. In this disease, patients develop erythematous papules and scaly plaques over the skin surface. Several lines of evidence, such as the presence of activated CD4 þ and CD8 þ T cells in psoriatic plaques (Ferenczi et al., 2), the results of studies on human skin xenografts in mice (Boyman et al., 24), and the therapeutic efficacy of T cell targeted drugs (Di Cesare et al., 29), have suggested the involvement of T cell 1 Department of Dermatology, University of Occupational and Environmental Health, Kitakyushu, Japan; 2 Department of Dermatology, Kyoto University, Kyoto, Japan; 3 Centre d Immunologie de Marseille-Luminy, Institut National de la Santé et de la Recherche Médicale U631, Centre National de la Recherche Scientifique UMR612, Université de la Méditerrannée, Marseille, France and 4 Department of Dermatology, Hamamatsu University School of Medicine, Hamamatsu, Japan Correspondence: Ryutaro Yoshiki, Department of Dermatology, University of Occupational and Environmental Health, 1-1 Iseigaoka, Kitakyushu , Japan. yockey@med.uoeh-u.ac.jp or Kenji Kabashima, Department of Dermatology, Kyoto University Graduate School of Medicine, 54 Shogoin- Kawara, Kyoto , Japan. kaba@kuhp.kyoto-u.ac.jp Abbreviations: ddcs, dermal dendritic cells; DT, diphtheria toxin;, imiquimod; LCs, Langerhans cells; TLR, Toll-like receptor; TNF, tumor necrosis factor Received 12 December 212; revised 26 December 213; accepted 7 January 214; accepted article preview online 25 February 214; published online 3 April 214 mediated immune responses in this disease. In addition, the expression levels of IL-1, tumor necrosis factor (TNF), IL-12, IL-17, IL-22, and IL-23 are elevated in psoriatic skin in humans (Di Cesare et al., 29), and IL-23R gene polymorphisms are associated with psoriasis (Capon et al., 27). These findings suggest the involvement of these cytokines in the pathogenesis of this skin disease. Recent studies have demonstrated that the IL-23/Th17 pathway is linked to a number of inflammatory diseases, including psoriasis and animal models of multiple sclerosis, arthritis, and inflammatory bowel disease (Cua et al., 23). The most persuasive evidence for the role of IL-23/Th17 in psoriasis comes from clinical studies. Treatment with an anti- IL-12/IL-23p4 Ab (ustekinumab) is effective against psoriasis (Krueger et al., 27). TNF-a-neutralizing antibodies are also widely used to treat psoriasis and significantly improve psoriasis area severity index scores. Notably, inhibition of TNF-a by a soluble TNF-a receptor neutralizing antibody (etanercept) was associated with reduced Th17 responses (Zaba et al., 27). As Th1 responses were not affected by the administration of etanercept, this finding suggests that Th17 cells are particularly important in driving psoriasis. Consistently, amelioration of psoriasis has been associated with reduced Th17 responses (Zaba et al., 27). Th17 cytokines, principally and IL-22, exert profound effects on keratinocytes (Nograles et al., 28). In psoriasis lesions, keratinocytes are activated and proliferate at 1912 Journal of Investigative Dermatology (214), Volume 134 & 214 The Society for Investigative Dermatology

2 substantially faster rates than normal keratinocytes (Iizuka, 1994). has been shown to induce cytokine and chemokine production by keratinocytes, whereas IL-22 induces antimicrobial peptide production by keratinocytes and is a direct potent stimulator of keratinocyte growth (Nograles et al., 28). More recently, topical treatment with imiquimod (), a ligand for Toll-like receptor (TLR) 7 and TLR8, was reported as a novel mouse model for psoriasis-like skin inflammation inducing acanthosis, parakeratosis, and a mixed inflammatory infiltrate (van der Fits et al., 29). is used in humans for topical treatment of genital and perianal warts caused by human papillomavirus, actinic keratosis, and superficial basal cell carcinoma. Clinical application of can induce psoriasis or exacerbate the disease in patients with wellcontrolled psoriasis (Fanti et al., 26). In mice, induced dermatitis development is critically dependent on the IL-23/IL-17 axis (van der Fits et al., 29). Moreover, administration of IL-23 into mouse skin results in the epidermal recruitment of CCR6 þ cells, a subtype of gd T cells that produces (Mabuchi et al., 211), and treated mice show increased numbers of IL-17-producing gd T cells (Roller et al., 212). However, it remains unclear how these IL-17-producing gd T cells are induced in the -treated skin, specifically in regard to the involvement of skin resident dendritic cells (DCs). Recent immunological studies have demonstrated that there are Langerhans cells (LCs), Langerin dermal DCs (ddcs), and Langerin þ ddcs in murine skin (Ginhoux et al., 27). LCs are a subset of skin-resident DCs that form a dense network in the epidermis (Romani et al., 21; Egawa and Kabashima, 211). The role of LCs has been well studied in contact hypersensitivity, the induction of which is a classical technique permitting the examination of cutaneous adaptive immune responses (Honda et al., 212; Kaplan et al., 212). Transgenic mice with an inducible or constitutive ablation of LCs showed enhanced contact hypersensitivity (Kaplan et al., 25), whereas other LC ablation mouse models developed reduced or unaffected contact hypersensitivity (Bennett et al., 27). In this study, we explored the role of LCs in a mouse model in which psoriasis-like skin inflammation is triggered by topical application of. LC depleted mice showed reduced ear swelling and decreased levels of Th17-related cytokines in -treated skin regions. LC-depleted -treated skin showed decreased production and expression of CCR6 from skin gd T cells. We also found that LCs produce IL-23 in -applied skin regions and that IL-23-producing cells were reduced at draining lymph nodes after application. These results suggest that LCs are required for generation of psoriasislike lesions in response to in mouse models. RESULTS LC depleted mice show reduced -induced psoriasis-like skin inflammation Consistent with a previous report (van der Fits et al., 29), topical application of on the skin of wild-type mice for 5 days induced psoriasis-like lesions featuring redness, scales, and crust formation. To evaluate the function of LCs in the pathogenesis of psoriasis, LCs were depleted with diphtheria toxin (DT) in Langerin-DT receptor (DTR) knocked-in mice. LCs were completely ablated from the epidermis within 24 h after injection of DT. We also investigated whether Langerin þ ddcs are involved in -induced psoriasis-like skin inflammation. It has been reported that Langerin þ ddcs recolonize in the dermis in 5 days or less after DT injection (Bursch et al., 27). Ten days after DT injection, LCs are still absent in the epidermis but ddcs are present (Supplementary Figure 1a online). cream was applied on both ears of LC/ Langerin þ ddc depleted mice or on ears of LC-depleted mice for 5 consecutive days. When the absolute number of each LC/DC subset per skin specimen was calculated, the subset of Langerin þ ddcs absolutely disappeared by application, but that of LCs and Langerin ddcs was not affected (Supplementary Figure 1a, b, d, e, f online). In addition, the application increased the number of Gr1 þ cells regardless of the depletion of LCs or Langerin þ ddcs (Supplementary Figure 1c, g online). Ear thickness was measured on day 6 after the start of application. Compared with -treated non-dt control mice, LCdepleted and -treated mice showed milder increases in ear thickness (Figure 1a and b). To exclude the involvement of newly differentiated LCs, we applied a daily DT treatment and application for 5 consecutive days to ensure efficient ablation of LC throughout the course of disease. A daily DT treatment and application did not affect the result of the ear thickness change (Supplementary Figure 2 online). Microscopic evaluation of skin sections from mice treated with revealed changes characteristic of psoriasis-like lesions, such as acanthosis, parakeratosis, desquamation, and dermal infiltration of immune cells. In contrast, administration had qualitatively and quantitatively fewer effects on Langerin þ cell (including LCs and Langerin þ ddcs) depleted mice ( application 1 day after DT treatment; Day 1) or LC-depleted mice ( application 1 days after DT treatment; Day 1) (Figure 1a). Hyperproliferation of keratinocytes in -induced psoriatic regions was also monitored by immunohistochemical staining of Stat3 and Ki67. -treated skin keratinocytes of control C57BL/6 mice were highly positive for Stat3 and Ki67 as compared with -treated Langerin þ cell depleted (Day 1) or LC-depleted mice (Day 1) (Figure 1a and c). These findings indicate that LCs are critical for the induction of -induced psoriasis-skin inflammation. LC depleted skin shows decreased IL-12/23p19, IL-12/23p4,, IL-22, and TNF-a mrna induction by Recently, the roles of Th1 and Th17 cytokines in the pathogenesis of -induced psoriasis-like skin inflammation have been demonstrated (van der Fits et al., 29). To characterize immune responses in -treated skin of LCdepleted mice, the expression of cytokines was measured by real-time PCR. cream was applied on both ears of LC/ Langerin þ ddc depleted mice or only LC-depleted mice for 5 consecutive days. On day 6, the mice were killed and mrna from both ears was extracted. In control C57BL/6J mice,

3 DT: DT: + DT: day 1 DT: day 1 H&E (x1) H&E (x4) Stat3 (x1,) Ki-67 (x1,) μm 45 3 Cells per 3 μm Stat3 Ki67 Ear thickness change 3 15 Stat3/Ki67-positive cells 2 1 Figure 1. Langerhans cell depleted mice show decreased -induced psoriasis-like skin inflammation. Langerin þ cells were depleted by DT in Langerin-DTR knocked-in mice. Langerin þ ddcs, but not Langerhans cells, repopulated in the skin 1 days later. C57BL/6J mice or Langerin-DTR knocked-in mice were treated daily with cream or control cream application on both ears for 5 consecutive days. (a) H&E-stained and immunohistochemical staining of Stat3, Ki-67 sections. (b) The ear swelling at day 6. (c) The numbers of Stat3- or Ki67-positive keratinocytes. Data are representative of four independent experiments and each group consisted of more than four mice. Po.5 between indicated groups. Normal bar ¼ 2 mm, dotted bar ¼ 1 mm. ddcs, dermal dendritic cells; DT, diphtheria toxin; DTR, DT receptor; H&E, hematoxylin and eosin;, imiquimod. application of induced IL-12/23p19, IL-12/23p4,, IL-22, and TNF-a mrna expression (Figure 2a e), whereas expression of IL-12p35 mrna was not detected by real-time PCR (data not shown). In LC-depleted and -treated mice, the induction of IL-12/23p19, IL-12/ 23p4,, IL-22, and TNF-a mrnas was suppressed on both day 1 and day 1 (Figure 2a e). As IL-23 maintains IL-17-producing cells, the above findings suggest that stimulates the production of IL-23, but not of IL-12, in LCs, which in turn promotes Th17 in -treated skin lesions. induces IL-23 production from LCs after application of Recently, it was postulated that IL-23, a cytokine driving the development of IL-17- and IL-22-producing Th17 cells, is functionally involved in the pathogenesis of psoriasis. Expression of IL-23 is increased in psoriasis lesional skin (Lee et al., 24), and increased numbers of Th17 cells are present (Lowes et al., 28). Application of leads to hyperplasia of the epidermis. In the same fashion, intradermal injection of IL-23 in mouse skin results in erythema, a mixed inflammatory infiltrate, and epidermal hyperplasia (Chan et al., 26). However, the source of IL-23 in psoriasis is still unclear. To investigate the source of IL-23 in -treated psoriasis-like skin, we first examined the expression of TLR7, a receptor for, in the skin-composing cells. In the steady state, LCs and Langerin þ and Langerin ddcs expressed TLR7, whereas gd T cells, Gr1 þ cells, and keratinocytes were TLR7 negative irrespective of application (Figure 3a and b). Recently, it has been reported that the -containing 1914 Journal of Investigative Dermatology (214), Volume 134

4 IL-12p19 mrna IL-12/23p4 mrna mrna IL-22 mrna TNF-α mrna Figure 2. application induces cytokine mrna production in skin. cream was applied on both ears of LC/Langerin þ d DC depleted mice or LC-depleted mice for 5 consecutive days. Real-time PCR analysis revealed that LC-depleted skin showed less expression of (a) IL-12p19, (b) IL-12/23p4, (c) IL- 17A, (d) IL-22, and (e) TNF-a mrna after application. Error bars indicate s.d. based on a single, pooled treatment group that was divided into three wells for RT-PCR measurement. Similar results were obtained in two independent experiments and each group consisted of more than four mice. Po.5 between indicated groups. DC, dendritic cell; DT, diphtheria toxin;, imiquimod; LC, Langerhans cell; TNF, tumor necrosis factor. cream Aldara induces inflammation largely independently of TLR7 and vehicle cream also promotes inflammasome activation in cultured keratinocytes (Walter et al., 213). To examine whether affects the expression of TLR7 to skin containing cells, we measured the TLR7-mRNA expression by real-time PCR. LCs, gd T cells, and KCs showed no remarkable change of TLR7-mRNA; however, only Langerin ddcs showed high expression of TLR7-mRNA (Figure 3c). As shown in Supplementary Figure 1b online, Langerin þ ddcs disappeared from -applied skin and expression of TLR7 from Langerin þ ddcs could not be measured. We next examined the IL-23-producing cells in the -applied skin by means of flow cytometry. -applied skin contained more IL-23-producing cells than did control mice (Figure 3d and e). To identify which cells produced IL-23, applied skins were analyzed by flow cytometry stained by EpCAM, Langerin, gdtcr, and Gr1. Only both EpCAM- and Langerin-positive cells or LCs produced IL-23 after application to the skin (Figure 3f). In control skin, B1% or less of LCs produce IL-23. However, B8% of LCs produced IL-23 in -applied skin (Figure 3g). These results suggest that administration induces IL-23 production in LCs. Decreased infiltration of -producing CCR6 þ cd Tcellsin LC depleted skin after application It has been reported that IL-23-responsive dermal gd T cells are the major IL-17 producers in the skin (Cai et al., 211), and were revealed to express CCR6 in a murine model of psoriasis (Mabuchi et al., 211). We next investigated the production of and CCR6 expression from gd T cells in -treated skin. cream was applied on both ears of LC/Langerin þ ddc-depleted mice or on ears of only LC-depleted mice for 5 consecutive days. On day 6, epidermal-cell and dermal-cell suspensions were prepared from -treated skin. When was applied to non-dt-treated mice, increased numbers of -producing gdtcr mid þ T cells were detected in the epidermis, which were reduced in LC-depleted mice (Figure 4a and b). On the other hand, similar numbers of -producing gdtcr mid þ cells were observed in the dermis of -treated skin regardless of the presence of LCs (Figure 4c and d). These producing gdtcr mid þ cells were Vg4 and CCR6 positive (Supplementary Figure 3 online). We also analyzed the positive cells in the CD4 þ T-cell subset. The number of IL- 17A-producing CD4 þ T cells increased in the -treated epidermis and dermis. The number of -producing CD4 þ cells was decreased in the dermis of LC- or Langerin þ ddc-depleted skin (Figure 4e h). These data indicate that LCs induce infiltration of IL 17A-producing CCR6 þ gd TCR mid þ Vg4 þ cells into epidermis and psoriasis-like inflammation after application. LC-depleted and -treated mice show decreased numbers of IL-12/23p4-positive cells and -producing cd Tcellsat regional lymph nodes To analyze IL-23-producing cells and -producing cells in the draining lymph nodes, we next performed flow cytometric analysis of regional lymph node cells at the end of a 5-day course of treatment. Compared with control mice, mice that had just received 5 days of treatment exhibited increased numbers of IL-23-producing major histocompatibility complex (MHC) class II þ cells in the draining lymph nodes. On the other hand, Langerin þ cell depleted or LC-depleted mice did not show any significant change in the number of IL-23-producing cells (Figure 5a and c). These IL-23-producing MHC class II þ cells were positive for EpCAM and Langerin, whereas IL-23-non-producing MHC class II þ cells were negative for EpCAM and Langerin (Figure 5b). Surprisingly, almost all LCs in draining lymph nodes produce IL-23. These results indicate that IL-23-producing MHC class

5 ( ) % Max LCs TLR7 Langerin + ddcs Langerin ddcs γδ T cells Gr1 + cells KCs Relative amount of TLR7 mrna LCs NS Relative amount of TLR7 mrna Langerin ddcs ( ) (+) ( ) (+) (+) % Max LCs TLR7 Langerin + ddcs Not detected Langerin ddcs γδ T cells Gr1 + cells KCs Relative amount of TLR7 mrna γδ T cells NS Relative amount of TLR7 mrna 1.5 ( ) (+) ( ) (+) 1.5 KCs NS MHC-class II.1 % 2.1 %.2 %.5 % IL-23 IL-23-producing MHC- class II-positive cells 8, 6, 4, 2, Cells per ear Langerin Gated in IL-23-producing MHC-class II-positive fraction EpCAM Percentage of IL-23- producing Langerhans cells % Figure 3. LCs produce IL-23 after application of. cream was applied to mice ears for 5 consecutive days and whole-skin suspension was analyzed by real-time PCR. (a) TLR7 was expressed on Langerin ddcs by application of. (b, c) Whole-skin suspension was stained with PE-conjugated IL-23 for flow cytometry. LC-depleted skin did not show an increase in the number of IL-23-producing cells. Data points represent individual frequencies. (d, e) CD45 þ MHC class II positive cells were gated by EpCAM and Langerin. Other CD45-positive cells were gated by gdtcr and Ly-6G (Gr1). Only LCs showed IL-23 production by application (f, g). Data are representative of four independent experiments and each group consisted of more than five mice. ddcs, dermal dendritic cells; DT, diphtheria toxin;, imiquimod; KCs, keratinocytes; LCs, Langerhans cells; MHC, major histocompatibility complex; NS, not significant; TLR, Toll-like receptor. II þ cells are LCs. Similarly, we found that the number of -producing gd T cells was significantly increased in the -treated mice, and decreased in the Langerin þ cell depleted or LC-depleted mice (Figure 5d and e). We also studied -producing CD4 þ T cells. The number of IL- 17A-producing CD4 þ T cells was increased by treatment with, which was decreased by depletion of Langerin þ cells or only LCs (Figure 5f and g). Of note, the total number of IL- 17A-producing CD4 þ T cells was less than a tenth of producing gd T cells. Thus, gd T cells were the main source of in the draining lymph nodes of -treated mice, and LC abrasion led to reduced numbers of -producing gd T cells. These results suggest that LCs are required for the IL-23 pathways that lead to IL-17 production by gd T cells in the draining lymph nodes following treatment. IL-23 from skin-derived LCs induces psoriatic skin inflammation On the basis of the indication that IL-23 from LCs may increase IL-17 production by gd T cells in the draining lymph nodes, we further explored a role of IL-23 from LCs by transplanting bone marrow (BM) cells of IL-23-deficient (IL23KO) mice into X-ray-irradiated wild-type (WT) mice or vice versa. As LCs are resistant to X-ray irradiation (Merad et al., 24), IL-23-producing LCs existed in the skin of X-rayirradiated WT mice. Two months after transplanting BM cells of WT or IL23KO mice into X-ray-irradiated WT or IL23KO mice, mice received topical applications of commercially available cream on both ears for 5 consecutive days. Ear thickness was measured on day 6 after the start of application. Compared with -treated WT mice in which both donors and recipients were WT, IL23KO mice, in which both donors and recipients were IL23 KO mice, showed a decrease in ear thickness (Figure 6). When transplanting BM cells of WT mice into X-ray-irradiated IL23KO mice ear thickness significantly decreased, whereas ear thickness did not show any change compared with WT mice when transplanting IL23KO BM cells into irradiated WT mice (Figure 6). In Figure 6, WT control showed less swelling than 1916 Journal of Investigative Dermatology (214), Volume 134

6 Epidermis 3. % 5.8 % 3.4 % 2.2 % γδ TCR -producing γδ TCR mid + cells 15, 1, 5, Dermis 3. % 2.6 % 1.9 % 1.3 % γδ TCR -producing γδ TCR mid + cells 5, 4, 3, 2, 1, N.S. Epidermis 8, NS.1 %.3 %.3 %.2 % -producing CD4 + cells 6, 4, 2, CD4 Dermis.5 % 1.8 %.2 %.1 % -producing CD4 + cells 2, 15, 1, 5, CD4 Figure 4. -producing CCR6 þ cd T cells are reduced in a Langerhans cell (LC)-depleted and imiquimod ()-applied epidermis. gd TCR-positive cells in the epidermis and dermis. Expression of gdtcr, CD4, and intracellular was examined in diphtheria toxin (DT)± and -applied skin by flow cytometry. (a d) application induced the -producing gdtcr mid þ cells in the epidermis and LC depletion suppressed the induction of gdtcr mid þ cells. There was no change in the recruitment of -producing gdtcr mid þ cells in the dermis. (e h) There was no significant increase in the number of producing CD4 þ cells in the epidermis, but -producing CD4 þ cells increased in the dermis. Data are representative of four independent experiments and each group consisted of more than five mice. NS, not significant. Po.5 between indicated groups. mice shown in Figure 1b. This difference was possibly due to the procedures of X-ray irradiation and BM transplantation. WT mice in Figure 6 were X-ray irradiated once and then transplanted with WT BM cells. Hence, total numbers and distribution of lymphocytes in BM-transplanted WT mice in Figure 6 are supposed to be different from those in normal WT mice shown in Figure 1. Taken together, these data indicated that IL-23 from LCs has a critical role in the -induced skin inflammation, possibly that of modulating IL-17-producing gd T cells in the draining lymph nodes. DISCUSSION In this report, we analyzed the pathomechanisms of psoriasiform dermatitis caused by in Langerin-DTR knocked-in mice. The -treated mouse skin resembles human plaquetype psoriasis with respect to erythema, skin thickening, scaling, and epidermal alterations (acanthosis, parakeratosis), but these findings were not observed in a LC-depleted state. In addition, LC-depleted mice showed decreased levels of Th17- related cytokines in -treated skin lesions. Moreover, the -treated skin of LC-depleted mice showed a decreased

7 .28 % 1.4 %.11%.19 % IL % 2. % 1.4 % 1.2 % MHC-class II Langerin IL-23-producing MHC-class II + cells 2. % 94.1 % 1.4 % 2.5 % EpCAM Langerin IL-23-non-producing MHC-class II + cells.3 %.2 % 88.5 % 11 % EpCAM IL-23-producing MHC-class II + cells Cells per ear 5, 4, 3, 2, 1,.7 %.46 %.11 %.2 % 1 γδ TCR -producing γδ T cells Cells per ear 15, 1, 5,.2 %.4 %.2 %.1 % CD4 -producing CD4 + T cells Cells per ear 2, 1,5 1, 5 Figure 5. IL-23-producing LCs migrate to the draining lymph nodes and LCs are indispensable for induction of -producing cd T cells. Regional lymph nodes from -applied mice were analyzed by flow cytometry. (a, c) application induces IL-23-producing MHC class II þ cells in the draining lymph nodes, but LC-depleted mice did not show induction of IL-23-producing MHC class II þ cells. (b) IL-23-producing cells in the draining lymph node express EpCAM and Langerin, the markers for LCs. (d g) Increase in the number of -producing gd T cells and CD4 þ T cells in the draining lymph node was suppressed by the depletion of LCs. Data are representative of four independent experiments and each group consisted of more than five mice. DT, diphtheria toxin;, imiquimod; LCs, Langerhans cells; MHC, major histocompatibility complex. Po.5. number of -producing CCR6 þ Vg4 þ gdtcr þ mid cells. These results suggest that LCs are required for the development of murine psoriasis-like lesions induced by. The importance of T cells in the pathogenesis of psoriasis is supported by the response of patients to treatment with agents that affect T-cell functions, such as cyclosporine. Several cytokines have been implicated in the pathogenesis of psoriasis, including TNF-a, IFN-g, IL-12, IL-17, IL-22, and IL-23 (van der Fits et al., 29). It has recently been reported that IL-23 expression is elevated in psoriatic skin lesions (Lee et al., 24), IL-23 induces psoriasis-like dermatitis (Chan et al., 26), polymorphisms in IL-23p19, IL-12/23p4, and IL-23R are associated with the development of psoriasis (Capon et al., 27), and antibody against IL-12/23p4 can ameliorate psoriasis in human skin (Krueger et al., 27). IL-23 is essential for the differentiation and survival of IL-17-producing cells (Diveu et al., 28), and IL-17 expression is augmented in psoriatic skin. In addition, anti-il-17 antibody administration ameliorates psoriasis, indicating that IL-17 has an important role in the pathogenesis of psoriasis (Di Cesare et al., 29) Journal of Investigative Dermatology (214), Volume 134

8 Bone marrow from WT mouse Bone marrow from IL-23 KO mouse Recipient Donor μm WT (X-ray-irradiated) WT WT IL-23 KO (X-ray-irradiated) IL-23 KO IL-23 KO IL-23 KO WT WT IL-23 KO application has been shown to induce -producing gd T cells in the regional lymph nodes (Roller et al., 212) and IL-23p19 mrna in mice (van der Fits et al., 29). However, the source of IL-23 in -treated areas remained unclear. Here, we clearly demonstrated that LCs were the major DC subset to produce IL-23 in the -treated skin, and that IL-23 induced the infiltration of IL-17-producing Vg4 þ gdtcr mid þ cells to the epidermis. These findings are consistent with the recent studies, which demonstrated that LCs are required for induction of IL-17-producing cells in Candida albicans-infected skin lesions (Igyarto et al., 211). Recently, Wohn et al. (213) reported that Langerin-negative ddcs, rather than LCs, are responsible for -induced psoriatic plaque formation Their conclusion was seemingly contrary to ours in which LCs are indispensable for induced psoriatic plaque formation. These two apparently contradictory experimental results using Langerin-DTR mice may be caused by the different -induction methods used by the two laboratories. Wohn et al. (213) applied on NS LC IL-23 (+) LN IL-23 (+) LC IL-23 (+) LN IL-23 ( ) LC IL-23 ( ) LN IL-23 ( ) LC IL-23 ( ) LN IL-23 (+) Figure 6. Skin-derived IL-23-producing LCs have a critical role in induced skin inflammation. (a) Recipient WT C57BL/6J mice or IL-23 KO mice (C57BL/6J background) underwent 9 Gy total body irradiation on day 1, and received bone marrow single-cell suspensions from WT C57BL/6J mice or IL-23 KO mice on day. LCs remained in the skin even after the irradiation, whereas LN cells of the recipients were derived from donors. (b) Two months after transplantation, cream was applied on each chimera mouse and increase in ear thickness was measured on day 6. Ear thickness decreased when recipients were IL23KO mice. Data are representative of four independent experiments and each group consisted of more than five mice., imiquimod; KO, knockout; LCs, Langerhans cells; LN, lymph node; NS, not significant; WT, wild type. Po.5. the back of mice, whereas we painted on the ears of mice. In human beings, psoriasis is predisposed to take place on knees and elbows. This predisposition may reflect the difference in nature and the number of LCs and Langerinnegative ddcs depending on the body sites. Moreover, time course of DT application is different between the two laboratories. Wohn et al. applied DT on day 3 and painted, whereas we applied DT on day 1 and applied. The time course of the DT application affects the number of LCs as shown in Supplementary Figure 1 online. These differences in the -applied areas and the timing of DT application possibly gave rise to the different results. It remains unclear whether directly acts on LCs in vivo. Recently, it has been reported that Aldara cream, which contains, induces inflammation largely independently of TLR7, and keratinocyte death and IL-1 release also occur in response to the vehicle cream in the absence of (Walter et al., 213). However, the role of for psoriatic skin inflammation is still unclear. Intriguingly, the expression level of TLR7 is rather high in dermal DCs compared with LCs. We examined the production of IL-23 from LCs or dermal DCs by stimulating them with in vitro. However, we could detect no increase in IL-23 production from LCs or dermal DCs (data not shown). Therefore, there may be other pathways besides TLR7 that are involved in upregulating IL-23 production from LCs. Therefore, it should be clarified whether directly acts on LCs to produce IL-23 through TLR7 or indirectly induces LC activation possibly through other immune cells, such as plasmacytoid DCs or keratinocytes in the future. Recently, CCR6 was shown to be a marker of peripheral IL- 17A-expressing gd T cells (Cua and Tato, 21). These gd T cells are observed following IL-23 intracutaneous injections (Mabuchi et al., 211). We now demonstrate that the accumulation of -producing CCR6 þ gd T cells in the epidermis and the draining lymph nodes is decreased in the LC-depleted mice. This finding supports the hypothesis that LCs are the main source of IL-23 for the induction of -producing CCR6 þ gd T cells in the -induced dermatitis model. It was reported that topical application of induces migration of LCs from treated skin into the draining lymph nodes (Suzuki et al., 2). In line with this, we demonstrated the accumulation of IL-23- producing LCs in the draining lymph nodes after application and the induction of -producing CCR6 þ gd T cells in the lymph node cell suspensions by IL-23. Therefore, LCs may function in two ways. First, treatment induces the accumulation of IL-23-producing LCs in the draining lymph nodes for the induction of -producing gd T cells. These gd T cells migrate to the epidermal area of the -treated skin, possibly via CCR6, where LCs may enhance the functions or survival of -producing gd T cells for the development of psoriatic skin lesions. Although -induced psoriasis-like skin lesions in mice share some clinical and histological characteristics with human psoriasis (Chan et al., 26), there are several differences between them. Most notably, the human psoriatic skin lesion contains predominantly at cells and not many gd T cells. However, it has recently been reported that dermal -producing gd T cells were significantly

9 increased in human psoriatic skin (Cai et al., 211), and that production of the local chemokine CCL2, a ligand for CCR6, is also increased in human psoriatic skin (Kryczek et al., 28). The increased frequency of gd T cells in psoriatic skin suggests that gd T cells may be crucial in the development of psoriasis not only in mice but also in humans. In conclusion, here we demonstrate that treatment induces IL-23 production by LCs in mice, and that LC depletion resulted in attenuated -induced psoriasis-like dermatitis with decreased numbers of infiltrating producing gd T cells. In addition, these -producing gd T cells were induced by IL-23 in the draining lymph nodes. These findings suggest that LCs seem to be the main cutaneous DC subset in the development of -induced psoriasis. MATERIALS AND METHODS Mice and treatments Seven 1-week-old C57BL/6J mice were obtained from SLC (Shizuoka, Japan). Mice were maintained on a 12-hour light/dark cycle under specific pathogen-free conditions. Langerin DTR knocked-in mice were generated (Kissenpfennig et al., 25). To deplete Langerin þ cells, mice were injected intraperitoneally with DT (2 ng each; Sigma-Aldrich, St Louis, MO). IL23KO mice were obtained from Keio University (Hasegawa et al., 213). Protocols were approved by the Institutional Animal Care and Use Committee of the University of Occupational and Environmental Health. Mice at 8 11 weeks of age received a daily topical dose of 62.5 mg of commercially available cream (5%) (Mochida Pharmaceutical, Tokyo, Japan) on both ears for 5 consecutive days. Generation of bone marrow chimera mice To generate bone marrow chimera mice, recipient WT C57BL/6J mice or IL-23 KO mice (C57BL/6J background) underwent 9 Gy total body irradiation on day 1, and received bone marrow single-cell suspensions from WT C57BL/6J mice or IL-23 KO mice on day intravenously through the tail vein. Recipient mice were treated with oral antibiotics for 2 months after transplantation. Culture medium RPMI-164 (Gibco BRL Life Technology, Grand Island, NY) was supplemented with 1% heat-inactivated fetal calf serum, 2 mm L-glutamine, M 2-mercaptoethanol, 1 5 Msodiumpyruvate, 25 mm HEPES, 1% nonessential amino acids, 1 units per ml penicillin, and 1 mg per ml streptomycin (all from Gibco). Preparation of skin suspensions Skin sheets from mouse ears were floated in.2% of trypsin in PBS (ph 7.4; Sigma-Aldrich) for 3 min at 37 1C as described previously (Tokura et al., 1994). The epidermis was separated from the dermis with forceps in PBS supplemented with 1% fetal calf serum. Both epidermis and dermis were minced and incubated for 1 h at 37 1C in PBS with collagenase II (Sigma-Aldrich). The obtained cells were filtered through a 4-mm filter. Flow cytometry Cells were immunostained with various combinations of fluorescence-conjugated mabs and analyzed with a FACSCanto flow cytometer (BD Biosciences, San Diego, CA) and FlowJo software (Tree star, Ashland, OR). The expression levels of cell surface or intracellular molecules and intracytoplasmic cytokines were analyzed using the following antibodies: Alexa Fluor 488-conjugated anti-gdtcr (clone; ebiogl3), PE-conjugated anti-il-12/23p4 (clone; C17.8), (clone; ebio17b7), IL-22 (clone; 1H8PWSR), TNF-a (clone; MP6-XT22); PerCP-conjugated CD45 antibody (clone; 3-F11); biotin-conjugated anti-mouse CD27 (Langerin) (clone; ebiol31); and PE-Cy7-conjugated anti-ly-6g (clone; RB6-8C5) (ebioscience, San Diego, CA) and anti-epithelial cell adhesion molecule (EpCAM, clone; G8.8), Vg4 (clone; UC3-1A6), Vg5 (clone; 536) (Biolegend, San Diego, CA), and anti-tlr7 antibody (clone; IMG4G6) (IMGENEX, San Diego, CA); APC-conjugated CCR6 antibody (clone; 1476) (BD Biosciences). All mabs were used at a concentration of 1 5 mg per 1 6 cells, and each incubation was performed for 3 min at 4 1C, followed by two washes in PBS supplemented with 5% fetal calf serum and.2% sodium azide. Viable cells were identified by 7-AAD uptake. For intracellular cytokine staining, cells were incubated in the presence of Goldi Stop (BD Biosciences) for 2 hours. Intracytoplasmic cytokines were detected in permeabilized cell suspensions using a BD cytofix/ cytoperm Plus Kit (BD Biosciences). Histology and immunohistochemistry Paraffin-embedded skin specimens were prepared using routine methods. The sections were stained with H&E. For immunohistochemistry, sections were deparaffinized and hydrated by washing sections in xylene followed by graded alcohol series. To unmask antigens, sections were incubated in 1 mm citric acid (ph 6) at 95 1C for 3 min. Sections were blocked with normal serum for 6 min at room temperature followed by incubation with primary antibodies (Stat3, Ki67; DAKO, Glostrup, Denmark) overnight at 4 1C. Samples were washed and incubated for 6 min with secondary antibodies. Real-time PCR Total RNA was extracted from skin with the SVTotal RNA isolation system (Promega, Madison, WI) according to the manufacturer s protocol. Murine IL-23p19, IL-12/23p4,, IL-22, and TNF-a gene expression levels were quantified in a two-step RT-PCR. cdna was reverse transcribed from total RNA samples using the TaqMan RT reagents (Applied Biosystems, Foster City, CA). Target gene expression was quantified using TaqMan Gene Expression Assay (Applied Biosystems) in the ABI PRISM 7 sequence detection system (Applied Biosystems). The probe was synthesized with VIC as a reporter dye and Tamra as the quencher dye. The forward primer, reverse primer, and Taqman probe were purchased from Applied Biosystems (IL-23p19; Mm11611_g1, IL-12/23p4; Mm434174_m1, ; Mm439619_m1, IL-22; Mm444241_m1, TNF-a; Mm443258_m1, TLR7; Mm44659_m1). As an endogenous control for these PCR quantification studies, GAPDH gene expression was measured using the TaqMan rodent GAPDH control reagents (Applied Biosystems). Gene expression was calculated using the DDCt method. CONFLICT OF INTEREST The authors state no conflict of interest. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at Journal of Investigative Dermatology (214), Volume 134

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