α-MSH-Stimulated Tolerogenic Dendritic Cells Induce Functional Regulatory T Cells and Ameliorate Ongoing Skin Inflammation

ORIGINAL ARTICLE

a-MSH-Stimulated Tolerogenic Dendritic Cells Induce Functional Regulatory T Cells and Ameliorate Ongoing Skin Inflammation Matteo Auriemma1,4,5, Thomas Brzoska1,5, Lars Klenner1,2, Verena Kupas1, Tobias Goerge1,2, Maik Voskort1, Zuotao Zhao1, Tim Sparwasser3, Thomas A. Luger1,2,5 and Karin Loser1,2,5 The neuropeptide a-melanocyte-stimulating hormone (a-MSH) is a well-known mediator of skin pigmentation. More recently, it has been shown that a-MSH also exerts a strong anti-inflammatory and immunosuppressive activity. To elucidate the mechanisms underlying a-MSH-induced immunosuppression, we investigated whether a-MSH affects dendritic cell/T cell communication, as especially this interaction has an important role in the regulation of immune responses. Here, we show that a-MSH, by binding to MC-1R, induced tolerogenic dendritic cells, which were capable of expanding CD4 þ CD25 þ Foxp3 þ regulatory T cells (Tregs) in vitro, as well as in vivo. Notably, those a-MSH-induced Tregs were functional as they efficiently inhibited cutaneous contact allergy and ongoing psoriasis-like skin inflammation in mice. Furthermore, a-MSH induced tolerogenic dendritic cells capable of generating functional Tregs in human blood. Interestingly, human Tregs expanded via a-MSH-stimulated dendritic cells suppressed the proliferation and cytokine secretion of pathogenic T-helper-17 (Th17) cells from individuals with psoriasis. Taken together, these data indicate that a-MSH induced immunosuppressive Tregs in vitro and in vivo, which inhibited disease progression in a mouse model of psoriasis-like skin inflammation and suppressed the activation and proliferation of effector T cells from subjects with psoriasis. Journal of Investigative Dermatology (2012) 132, 1814–1824; doi:10.1038/jid.2012.59; published online 15 March 2012

INTRODUCTION a-MSH is a 13-amino-acid peptide hormone derived by posttranslational processing from the precursor protein proopiomelanocortin, which also encodes for other bioactive peptide hormones, including ACTH, b- and g-MSH, or endogenous endorphins such as b-endorphin (Brzoska et al., 2008). a-MSH can be detected in numerous cell types such as melanocytes, keratinocytes, epithelial cells, B cells, natural killer cells, and subsets of T cells (Botchkarev et al., 1999; Luger et al., 2000; Loser et al., 2010). Although originally discovered in the pituitary gland and named for its effects on

1

Department of Dermatology, University of Mu¨nster, Mu¨nster, Germany; Interdisciplinary Center of Clinical Research (IZKF), University of Mu¨nster, Mu¨nster, Germany and 3Institute of Infection Immunology, TWINCORE, Center for Experimental and Clinical Infection Research, Hannover, Germany

2

4

Current address: Department of Dermatology, G. d’Annunzio University of Chieti-Pescara, Chieti, Italy.

5

These authors contributed equally to this work.

Correspondence: Karin Loser, Department of Dermatology, University of Mu¨nster, von-Esmarch-Strasse 58, Mu¨nster 48149, Germany. E-mail: [email protected] Abbreviations: a-MSH, a-melanocyte-stimulating hormone; bmDC, bone marrow–derived DC; CHS, contact hypersensitivity; DC, dendritic cell; PBS, phosphate-buffered saline; Th17, T-helper-17; TNF-a, tumor necrosis factor-a; Treg, regulatory T cell Received 11 September 2011; revised 7 January 2012; accepted 2 February 2012; published online 15 March 2012

1814 Journal of Investigative Dermatology (2012), Volume 132

pigmentation, more recent studies revealed that a-MSH interacts with immune cells, thereby exerting antimicrobial, anti-inflammatory, and immunomodulatory activities (Luger and Brzoska, 2007; Brzoska et al., 2008; Catania et al., 2010). The biological effects of a-MSH are mediated by binding to one of five melanocortin receptors, termed MC-1R to MC-5R, which belong to the superfamily of G-protein coupled receptors with seven-transmembrane domains (Cone et al., 1996). Melanocortin receptor ligation by a-MSH leads to the activation of adenylate cyclase and the subsequent elevation of intracellular cyclic cAMP levels (Elliott et al., 2004). MC-1R was the first melanocortin receptor that has been cloned from melanocytes and was shown to have a crucial role during pigmentation, as functional mutations of this receptor greatly influenced fur color in mammals, as well as hair and skin color in humans (Chhajlani and Wikberg, 1992; Mountjoy et al., 1992). However, MC-1R is not only expressed in melanocytes but is also detectable in the majority of nonmelanocytic cell types such as mucosal cells of the gastrointestinal tract or immune cells, including macrophages, monocytes, dendritic cells (DCs), mast cells, and neutrophils (Andersen et al., 2005; Cooper et al., 2005; Brzoska et al., 2008). More recently, MC-1R was also found in CD8 þ T cells (Loser et al., 2010), suggesting a widespread function of this receptor. At least the anti-inflammatory effects of a-MSH have been linked to MC-1R engagement as mice with a nonfunctional MC-1R showed a significantly increased & 2012 The Society for Investigative Dermatology

M Auriemma et al. a-MSH-Induced Tregs Ameliorate Inflammation

gastrointestinal inflammation upon a-MSH stimulation in a model of experimental colitis, whereas a-MSH considerably improved inflammation in the gut of wild-type controls (Maaser et al., 2006). Besides ameliorating gastrointestinal inflammation, a-MSH has been demonstrated to reduce delayed-type hypersensitivity responses in the skin, and to inhibit the development of experimental autoimmune encephalomyelitis or allergic airway inflammation (Grabbe et al., 1996; Raap et al., 2003; Han et al., 2007), indicating a rather general role for a-MSH in the regulation of inflammatory immune responses. However, the molecular mechanisms underlying the anti-inflammatory and immunomodulatory activities of a-MSH are not completely understood. It has

8.6%

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been shown that the inhibition of NF-kB activation and translocation, as well as the protection against IkBa phosphorylation, finally leading to reduced production of pro-inflammatory cytokines, seems to be critical for the a-MSH-mediated effects on immune cells (Bo¨hm et al., 1999; Ichiyama et al., 1999; Hill et al., 2006). Here, we show that a-MSH induced tolerogenic DCs, as well as an enhanced capacity to expand functional CD4 þ CD25 þ Foxp3 þ regulatory T cells (Tregs). These Tregs were able to efficiently suppress the proliferation and cytokine secretion of pathogenic Th17 effector cells in ongoing psoriasis-like skin inflammation of mice and in Th17 cells from individuals with psoriasis.

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Figure 1. a-Melanocyte-stimulating hormone (a-MSH) induces tolerogenic dendritic cells (DCs). (a) Animals were injected with a-MSH or phosphate-buffered saline (PBS), and 48 hours after elicitation of contact hypersensitivity (CHS) ear swelling was assessed. Data are shown as mean ear swelling±SD and are representative of 12 mice in each group (*Po0.05 vs. PBS-treated mice). (b) Flow cytometry of T cells from mice injected with PBS or a-MSH (n ¼ 8 mice in each group) 48 hours after elicitation of CHS. (Left) Representative dot blots and (right) statistical analyses of Foxp3 þ cells in regional lymph nodes are shown; cells are gated for CD4. Foxp3 staining was performed after cell permeabilization (*Po0.05 vs. PBS-treated mice). (c, d) Bone marrow–derived DCs (bmDCs) were stimulated with a-MSH or PBS, and the expression of (c) CD80, CD86, PD-L1, PD-L2, and (d) CD205 was quantified by flow cytometry or real-time PCR. Histogram overlays are gated for CD11c, and one representative out of three independent experiments is shown. (e) The IL-10 concentration in culture supernatants from a-MSH- or PBS-stimulated DCs was quantified using FlowCytomix kits, and IL-10 mRNA levels were analyzed by real-time quantitative PCR (*Po0.05 vs. PBS-stimulated DCs). (f) DCs from C57BL/6Je/e mice were stimulated with a-MSH, and the expression of CD80, CD86, and CD205 was analyzed by flow cytometry. Histogram overlays are gated for CD11c, and one representative experiment is shown.

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a-MSH induces tolerogenic DCs by binding to MC-1R

To investigate the mechanism underlying the anti-inflammatory effects of a-MSH, we performed contact hypersensitivity (CHS) experiments. As shown in Figure 1, mice injected with a-MSH before sensitization to DNFB developed a signifi-

CD4+ T cells from CD4+ T cells from cocultures with cocultures with α-MSH-stim. DC PBS-stim. DC 6.0% 12.8%

cantly reduced ear swelling compared with phosphatebuffered saline (PBS)-treated controls (Figure 1a), which was associated with increased levels of Tregs in regional lymph nodes (Figure 1b). To dissect direct effects of a-MSH on Tregs, CD4 þ T cells from wild-type mice were stimulated with different concentrations of a-MSH (105 to 1015 M) for 1–7

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Figure 2. a-Melanocyte-stimulating hormone (a-MSH)-stimulated DCs expand functional regulatory T cells (Tregs). (a) CD4 þ T cells from cocultures with a-MSH-stimulated or control DCs were analyzed for the expression of Foxp3, and (left) one representative dot-plot and (right) statistical analyses of four independent experiments are shown (*Po0.05 vs. CD4 þ T cells from cocultures with phosphate-buffered saline (PBS)-stimulated DCs). Foxp3 staining was performed after cell permeabilization and cells are gated for CD4. (b) CD4 þ T cells from cocultures with a-MSH-stimulated or control DCs were analyzed for the expression of Foxp3, Nrp-1, and CTLA-4 by flow cytometry. CTLA-4 and Foxp3 staining was performed after cell permeabilization, and (top) representative histogram overlays and (bottom) statistical analyses of four independent experiments are shown (*Po0.05 vs. CD4 þ T cells from cocultures with PBS-stimulated DCs). Cells are gated for CD4 þ Foxp3 þ . (c) The relative mRNA levels of Foxp3, IL-10, Nrp-1, CTLA-4, and TGF-b were quantified in CD4 þ T cells from cocultures with a-MSH- or PBS-stimulated DCs. Data are shown as mean±SD (*Po0.05 vs. CD4 þ T cells from cocultures with PBS-stimulated DCs). (d) CD4 þ T cells were purified from cocultures with a-MSH- or PBS-treated DCs, and proliferation assays were performed by stimulating freshly isolated CD4 þ CD25 T cells with anti-CD3 and anti-CD28 in the absence or presence of CD4 þ T cells from cocultures. Mean values of 3H-thymidine incorporation±SD are shown (*Po0.05 vs. effector T cells þ CD4 þ T cells from cocultures with PBS-stimulated DCs). (e) Wild-type (wt) mice were injected with CD4 þ T cells from cocultures with a-MSH- or PBS-stimulated DCs 24 hours before induction of contact allergy. Data are shown as mean ear swelling assessed 48 hours after elicitation of contact hypersensitivity (CHS) and are representative of 15 mice in each group (*Po0.05 vs. transfer of CD4 þ T cells from cocultures with PBS-stimulated DCs). (f) CD4 þ T cells were cocultured with a-MSH- or PBS-stimulated DCs. Subsequently, the numbers of Foxp3 þ Helios þ and Foxp3 þ Helios Tregs were quantified by flow cytometry. Cells are gated for CD4; Foxp3 and Helios staining was performed after cell permeabilization; and one representative experiment is shown. c.p.m., counts per minute.

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days. However, none of these assays resulted in the upregulation of Treg numbers or the induction of suppressive activity (Supplementary Figure S1a and b online), indicating that a-MSH had no direct effect on CD4 þ T cells and suggesting that the expansion of Tregs during CHS might rather be an indirect effect of a-MSH. As tolerogenic DCs have been described to induce functional Tregs (Mahnke et al., 2003; Loser et al., 2006), we stimulated bone marrow–derived DCs (bmDCs) with a-MSH and assessed the DC phenotype. Compared with PBS-treated controls, aMSH-stimulated DCs showed a reduced expression of CD80 and CD86, but increased levels of the negative co-stimulatory B7-family molecules PD-L1 and PD-L2 (Figure 1c). Moreover, a-MSH-stimulated DCs expressed elevated levels of CD205 and IL-10 (Figure 1d and e), markers characteristic for tolerogenic DCs (Adler and Steinbrink, 2007; Mahnke et al., 2007). As not only the induction of a tolerogenic phenotype but also the decreased expression of IL-12 or IL-23 might contribute to immunosuppressive DC functions (Gee et al., 2009), we quantified these cytokines in PBS- and a-MSHstimulated DCs. However, a-MSH did not affect the secretion of IL-12 or IL-23 (Supplementary Figure S2 online), suggesting a minor role of IL-12 and IL-23 deprivation for the immunosuppressive effects of a-MSH. As signaling via MC-1R has been implicated in the immunosuppressive activity of a-MSH (Brzoska et al., 2008), we analyzed the a-MSH-mediated induction of tolerogenic DCs in C57BL/6Je/e mice characterized by a point mutation in the MC-1R gene, leading to a nonfunctional receptor (Robbins et al., 1993). However, a-MSH neither induced the downregulation of maturation markers nor the upregulation of CD205 and IL-10 in bmDCs from C57BL/6Je/e mice (Figure 1f and data not shown), indicating that binding of a-MSH to a functional MC-1R seems to be crucial for the induction of tolerogenic DCs. a-MSH-stimulated DCs generate functional Tregs

Next, CD4 þ T cells were cocultured with a-MSH-stimulated DCs or PBS-treated controls and, subsequently, Treg numbers were quantified. Interestingly, in cocultures with a-MSHstimulated DCs, we identified 2-fold increased levels of Foxp3 þ cells expressing high levels of markers associated with Treg function, such as CTLA-4, IL-10, TGF-b, or Nrp-1 (Figure 2a–c). Furthermore, Tregs expanded by a-MSHstimulated DCs were immunosuppressive in vitro and in vivo, as they inhibited the proliferation of activated CD4 þ CD25 effector T cells (Figure 2d) and reduced the development of CHS upon adoptive transfer into sensitized mice (Figure 2e). Using a transwell system or neutralizing soluble factors such as IL-10 or TGF-b revealed that both direct cell contact and immunosuppressive cytokines were responsible for the inhibitory effect of Tregs induced by aMSH-stimulated DCs (Supplementary Figure S3 online). To scrutinize whether a-MSH-treated DCs mediated the induction of Tregs from conventional CD4 þ CD25 T cells rather than the expansion of pre-existing Tregs, thymicand peripherally induced derived Foxp3 þ Helios þ Foxp3 þ Helios Tregs (Thornton et al., 2010) were quantified

in cocultures. Whereas the numbers of Foxp3 þ Helios þ Tregs were similar in both groups, we observed upregulated levels of Foxp3 þ Helios Tregs when CD4 þ T cells were cocultured with a-MSH-stimulated DCs compared with PBS-stimulated DCs (3.2±0.5% in cocultures with PBSstimulated DCs vs. 9.7±0.3% in cocultures with a-MSHstimulated DCs; Figure 2f). Moreover, Ki-67 staining revealed an increased proliferation index in Foxp3 þ Helios T cells from cocultures with a-MSH-treated DCs versus PBS-treated DCs (Supplementary Figure S4a online), suggesting that a-MSH-treated DCs mediate the peripheral induction of Tregs. To analyze whether a-MSH-stimulated DCs induced Tregs from conventional CD4 þ T cells in vivo, DEREG mice (Lahl et al., 2007) were treated with diphtheria toxin to deplete Tregs and inoculated with PBS- or a-MSH-stimulated DCs. Interestingly, Treg numbers were increased in recipients of a-MSH-treated DCs compared with controls and, furthermore, induced Helios Tregs showed an enhanced proliferation as evidenced by BrdU incorporation (Supplementary Figure S4b and c online), thus supporting that a-MSHstimulated DCs favor the de novo induction of Tregs. a-MSH ameliorates ongoing psoriasis by inducing functional Tregs in vivo

As an impaired suppressor function of Tregs has been associated with the progression of psoriasis (Sugiyama et al., 2005; Yun et al., 2010), we investigated whether the a-MSH-mediated induction of functional Tregs might ameliorate ongoing psoriasis-like skin inflammation. Therefore, mice were topically treated with imiquimod (van der Fits et al., 2009) and injected with a-MSH or an equal amount of anti-tumor necrosis factor-a (TNF-a) because TNF-a blockade represents an approved treatment of psoriasis (Sabat et al., 2007; Nestle et al., 2009). Notably, compared with controls, recipients of a-MSH showed a significant reduction in skin inflammation, which was similar to mice that received antiTNF-a (Figure 3a). This was paralleled by the in vivo induction of tolerogenic DCs in a-MSH-treated mice as evidenced by the decreased expression of CD80 and CD86, as well as the upregulation of IL-10 in major histocompatibility complex class II þ cells (Figure 3b and data not shown). Moreover, treatment of psoriatic mice with a-MSH resulted in 2-fold increased numbers of Foxp3 þ Tregs in regional lymph nodes (placebo-treated mice: 9.9±2.1%; imiquimod-treated mice þ PBS: 11.1±3.0%; imiquimodtreated mice þ a-MSH: 26.8±2.4%; imiquimod-treated mice þ anti-TNF-a: 17.2±1.8%; Figure 3c), which secreted high levels of IL-10 (Figure 3c), suggesting that a-MSH indeed generated functional Tregs in vivo by the induction of tolerogenic DCs. In psoriasis, pathogenic Th17 cells are crucial for disease progression (van der Fits et al., 2009; Clark, 2010). Thus, we investigated whether a-MSH-induced Tregs might inhibit the proliferation or cytokine secretion of Th17 cells. As shown in Figure 3d, psoriatic mice that received a-MSH exhibited decreased numbers of CD4 þ T cells expressing IL-17 compared with recipients of PBS. Furthermore, CD4 þ T cells from a-MSH-treated mice showed reduced mRNA levels www.jidonline.org 1817

M Auriemma et al. a-MSH-Induced Tregs Ameliorate Inflammation

of IL-17, IL-22, or the Th17-specific transcription factor RORgt (Figure 3e), indicating that a-MSH-induced Tregs might indeed have inhibited the proliferation and cytokine

secretion of pathogenic Th17 cells. It is worth mentioning that the effect of a-MSH on Th17 cell numbers and cytokine secretion was comparable to that of systemic anti-TNF-a

Imiquimod + PBS

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M Auriemma et al. a-MSH-Induced Tregs Ameliorate Inflammation

treatment (Figure 3d and e). Taken together, these data indicate that a-MSH, via binding to MC-1R, generated tolerogenic DCs, which induced functional Tregs able to suppress the activity of pathogenic Th17 cells in vivo, thus resulting in the amelioration of ongoing imiquimod-induced psoriasis. Ex vivo–generated a-MSH-stimulated DCs ameliorate psoriasis-like skin inflammation

Next, we analyzed whether ex vivo–generated a-MSHstimulated DCs were able to ameliorate ongoing psoriasis by inducing functional Tregs in vivo. Therefore, a-MSHstimulated bmDCs from Thy1.1 þ donors were injected into imiquimod-treated Thy1.2 þ recipients. It is worth noting that the tolerogenic phenotype of adoptively transferred DCs was conserved in vivo as demonstrated by flow cytometry and real-time quantitative PCR of re-isolated Thy1.1 þ DCs at day 9 after the start of imiquimod treatment. Moreover, injected Thy1.1 þ DCs migrated to regional lymph nodes but were not detectable in cutaneous lesions (data not shown). In contrast to controls, injection of a-MSH-stimulated DCs significantly reduced erythema and epidermal thickness in imiquimodtreated recipients and induced Tregs in regional lymph nodes (Figure 4a and b). These Tregs inhibited the IL-17 secretion in CD4 þ T cells and potentially also the migration of pathogenic Th17 cells from draining lymph nodes to lesional skin (Figure 4b), indicating that ex vivo-generated a-MSH-treated DCs were able to reduce disease severity by expanding functional Tregs in vivo. a-MSH-stimulated human DCs induce immunosuppressive Tregs

To investigate the relevance of a-MSH/MC-1R signaling for the induction of tolerogenic human DCs, we characterized the phenotype of a-MSH- versus PBS-stimulated HLA-DR þ DCs. Notably, a-MSH treatment resulted in a reduced expression of CD80 and CD86, as well as an upregulation of IL-10 (Figure 5a). Similar to mouse bmDCs, a-MSH-stimulated human DCs expanded functional Tregs, as 2-fold increased numbers of Foxp3 þ T cells, which efficiently suppressed the proliferation of activated CD4 þ CD25 effector T cells, were detectable in cocultures with a-MSH-stimulated DCs compared with PBS-treated DCs (Figure 5b and c). It is worth mentioning that the a-MSH-mediated induction of tolerogenic DCs and expansion of immunosuppressive Tregs was dependent on MC-1R ligation, as stimulation of HLA-DR þ DCs from

individuals with variants in the MC-1R gene, known to compromise receptor function (Loser et al., 2010), neither resulted in the downregulation of maturation markers nor the generation of Tregs upon coculture with CD4 þ T cells (Figure 5d and data not shown). Tregs expanded by a-MSH-stimulated DCs suppressed the proliferation of pathogenic Th17 cells from individuals with psoriasis

Next, we investigated whether Tregs induced by a-MSHstimulated DCs are able to suppress the activity of Th17 cells from individuals with psoriasis. It is worth noting that, also in peripheral blood from individuals with psoriasis, a-MSH stimulation generated tolerogenic DCs as shown by the induction of IL-10 and the downregulation of maturation markers (Supplementary Figure S5 online and data not shown). Interestingly, upon coculture with CD4 þ T cells from the same individuals, a-MSH-stimulated DCs induced functional, immunosuppressive Tregs (Figure 6a and b). To analyze whether Tregs generated by a-MSH-stimulated DCs inhibited the proliferation and cytokine secretion in Th17 cells from individuals with psoriasis, we cocultured CD4 þ T cells with a-MSH-stimulated DCs and quantified IL17 þ cells. As shown in Figure 6c, substantial amounts of CD4 þ IL-17 þ T cells were detectable in cocultures with control DCs, whereas the numbers of Th17 cells decreased significantly when CD4 þ T cells were cocultured with aMSH-stimulated DCs, thus suggesting that Tregs induced by a-MSH-treated DCs might suppress Th17 activity. This was further strengthened by the decreased expression of IL-17, IL22, and RORc (markers that are implicated in the pathogenicity of Th17 cells; Ma et al., 2008) in CD4 þ T cells from individuals with psoriasis that have been cocultured with a-MSH-stimulated DCs (Figure 6d). To confirm that indeed Tregs induced by a-MSH-stimulated DCs inhibited Th17 activity, we purified CD4 þ T cells from cocultures with a-MSH- or PBS-treated DCs and mixed them with freshly isolated CD4 þ T cells from peripheral blood of individuals with psoriasis. Interestingly, in contrast to T cells from cocultures with control DCs, CD4 þ T cells from cocultures with a-MSH-stimulated DCs significantly decreased the numbers of Th17 cells (Figure 6e), indicating that a-MSH reduced the activity of Th17 cells from individuals with psoriasis by the induction of tolerogenic DCs and functional Tregs. Furthermore, these data suggest that a-MSH might represent a potential, to our knowledge previously unreported, therapeutic alternative for the treatment of psoriasis.

Figure 3. a-Melanocyte-stimulating hormone (a-MSH) ameliorates ongoing psoriasis by inducing regulatory T cells (Tregs). (a) Typical skin pathologies of BALB/c mice topically treated with imiquimod for 8 days and injected intravenously (i.v.) with a-MSH or anti-tumor necrosis factor-a (TNF-a) (n ¼ 8 mice in each group). Hematoxylin and eosin (H&E) staining of skin tissue: original magnification  200, bar ¼ 25 mm. (b) DCs from regional lymph nodes of mice injected with a-MSH expressed elevated levels of IL-10. Cells are gated for major histocompatibility complex (MHC-II þ ), and IL-10 staining was performed after cell permeabilization. One representative histogram overlay is depicted. (c, left) Increased numbers of Tregs and (right) upregulated IL-10 mRNA expression in CD4 þ T cells from lymph nodes draining cutaneous lesions of a-MSH-treated mice (*Po0.05 vs. injection of phosphate-buffered saline (PBS)). (d) Reduced Th17 numbers in lymph nodes from a-MSH-injected mice. Cells are gated for CD4, and IL-17 staining was performed after cell permeabilization. (Left) One representative dot-plot for each group and (right) statistical analyses of % IL-17-expressing CD4 þ T cells from regional lymph nodes are shown (*Po0.05 vs. injection of PBS). (e) a-MSH decreased the expression of Th17-associated genes in CD4 þ T cells from lymph nodes draining cutaneous lesions. Relative mRNA levels of RORgt, IL-17, and IL-22 are shown from n ¼ 6 mice in each group (*Po0.05 vs. injection of PBS).

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CD4/IL-17/DAPI Figure 4. a-Melanocyte-stimulating hormone (a-MSH)-stimulated DCs ameliorate imiquimod-induced psoriasis. (a) Typical skin pathologies of BALB/c mice treated with imiquimod and injected with Thy1.1 þ phosphate-buffered saline (PBS)- or a-MSH-stimulated DCs (n ¼ 8 mice in each group). Hematoxylin and eosin (H&E) staining of skin tissue: original magnification  200, bar ¼ 25 mm. (b) Immunofluorescence staining of regional lymph nodes and lesional skin from mice treated with imiquimod and injected with PBS- or a-MSH-stimulated DCs using antibodies against CD4, Foxp3, and IL-17. One representative image is shown, and CD4 þ Foxp3 þ regulatory T cells (Tregs) and CD4 þ IL-17 þ Th17 cells are marked by arrows. Original magnification  400, bar ¼ 25 mm. DAPI, 40 ,6-diamidino-2-phenylindole.

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Counts

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CD4+ T cells cocultured with α-MSH-stimulated DC 24.2%

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PBS-stim. DC from individuals with MC-1R variants α-MSH-stim. DC from individuals with MC-1R variants

60 40

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80

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Figure 5. a-Melanocyte-stimulating hormone (a-MSH) induces tolerogenic human dendritic cells (DCs) capable of generating functional regulatory T cells (Tregs). (a) DCs from peripheral blood were stimulated with a-MSH or phosphate-buffered saline (PBS), and the expression of CD80, CD86, and IL-10 was quantified by flow cytometry. Histogram overlays are gated for HLA-DR, and one representative experiment is shown. IL-10 staining was performed after cell permeabilization. (b) CD4 þ T cells from cocultures with a-MSH-stimulated DCs were analyzed for the expression of Foxp3 and CD25 by flow cytometry. Cells are gated for CD4; Foxp3 staining was performed after cell permeabilization; and (left) one representative experiment and (right) statistical analyses of n ¼ 7 donors are shown (*Po0.05 vs. CD4 þ T cells from cocultures with PBS-stimulated DCs). (c) Freshly isolated CD4 þ CD25 effector T cells were activated with anti-CD3 and anti-CD28 in the absence or presence of CD4 þ T cells from cocultures with PBS- or a-MSH-stimulated DCs. Mean values of 3H-thymidine incorporation±SD are shown from one out of three independent experiments (*Po0.05 vs. effector T cells þ CD4 þ T cells from cocultures with PBS-stimulated DCs). (d) Signaling via MC-1R is critical for the a-MSH-mediated induction of tolerogenic DCs. HLA-DR þ DCs were purified from peripheral blood of individuals with loss-of-function variants in the MC-1R gene, stimulated with a-MSH, and the expression of CD86 was analyzed by flow cytometry. Cells are gated for HLA-DR, and one representative histogram overlay is shown. c.p.m., counts per minute.

DISCUSSION Here, we show that a-MSH induced tolerogenic DCs by decreasing the expression of co-stimulatory molecules and upregulating the immunosuppressive cytokine IL-10. Notably, a-MSH-stimulated tolerogenic DCs of murine and human origin expanded functional Tregs, which efficiently inhibited the proliferation of effector T cells in vitro, as well as in vivo. Moreover, a-MSH, by generating Tregs, reduced the proliferation and cytokine secretion of pathogenic Th17 cells, and thereby ameliorated ongoing psoriasis in a mouse model. As in mice, a-MSH inhibited the activity of Th17 cells from individuals with psoriasis, suggesting a-MSH as a

therapeutic option for the treatment of psoriasis, which to our knowledge is previously unreported. Upon inflammation, DCs receive maturation signals by a variety of stimuli, resulting in effector T-cell activation (Banchereau et al., 2000; Barton and Medzhitov, 2003). However, immunomodulatory agents have been shown to induce tolerogenic DCs characterized by a reduced expression of co-stimulatory receptors and an upregulated secretion of anti-inflammatory cytokines (Guilliams et al., 2010; Maldonado and von Andrian, 2010). Accordingly, the immunomodulatory neuropeptide vasoactive intestinal peptide (VIP) inhibits the capacity of DCs to stimulate effector T cell proliferation by www.jidonline.org 1821

M Auriemma et al. a-MSH-Induced Tregs Ameliorate Inflammation

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Figure 6. a-Melanocyte-stimulating hormone (a-MSH)-stimulated DCs induce functional regulatory T cells (Tregs) capable of inhibiting the activity of Th17 cells from individuals with psoriasis. (a) CD4 þ T cells were purified from peripheral blood of healthy donors or individuals with psoriasis and cocultured with a-MSH-stimulated DCs. The numbers of Foxp3 þ Tregs were quantified by flow cytometry from n ¼ 8 individuals in each group, and Foxp3 staining was performed after cell permeabilization. (b) Coculture with a-MSH-stimulated DCs restored the suppressor function in CD4 þ T cells from individuals with psoriasis. Proliferation assays were performed by mixing CD4 þ T cells from cocultures with phosphate-buffered saline (PBS)- or a-MSH-stimulated DCs and freshly isolated, anti-CD3/anti-CD28-activated CD4 þ CD25 effector T cells from the same individuals. Mean values of 3H-thymidine incorporation±SD are shown from one out of three independent experiments (*Po0.05 vs. proliferation of effector T cells alone). (c) Reduced IL-17 expression in CD4 þ T cells from individuals with psoriasis after coculture with a-MSH-stimulated DCs. CD4 þ T cells were purified from cocultures, and the IL-17 expression was quantified by flow cytometry in n ¼ 7 individuals in each group. IL-17 staining was performed after cell permeabilization (*Po0.05 vs. coculture with PBS-stimulated DCs). (d) CD4 þ T cells from individuals with psoriasis that have been cocultured with a-MSH-stimulated DCs show decreased relative mRNA levels of IL-17, IL-22, and RORc (n ¼ 9 individuals in each group; *Po0.05 vs. coculture with PBS-stimulated DCs). (e) CD4 þ T cells from cocultures with a-MSH-stimulated DCs suppress the proliferation of Th17 cells. CD4 þ T cells were purified from cocultures with a-MSH-stimulated or control DCs and mixed with freshly isolated CD4 þ T cells from individuals with psoriasis. Subsequently, the numbers of IL-17-expressing cells were quantified by flow cytometry in n ¼ 5 individuals in each group (*Po0.05 vs. coculture with PBS-stimulated DCs). c.p.m., counts per minute.

decreasing the expression of CD80 and CD86 and by reducing the production of pro-inflammatory cytokines (Delgado et al., 2005b). Similarly, treatment of DCs with NDP-MSH, an analog of a-MSH and closely related to VIP, downregulates CD86 and thereby impairs the allostimulatory capacity of DCs (Rennalls et al., 2010). Recently, a-MSH has been shown to decrease the expression of CD83 and CD86, thus preventing the maturation of monocyte-derived DCs (Min et al., 2011). Similar to the observations made by Rennalls et al. (2010) and Min et al. (2011), we detected reduced levels of the maturation markers CD80 and CD86 in a-MSH-stimulated murine and human DCs, which in combination with the increased IL-10 secretion points to an immunosuppressive phenotype. Interestingly, in monocyte-derived DCs the a-MSH-mediated induction of a tolerogenic phenotype was mediated by the upregulation of Annexin A1 (Min et al., 2011). Hence, it would be worthwhile to analyze whether also in murine bmDCs or HLA-DR þ DCs a-MSH upregulates Annexin A1, thereby providing a possible explanation why the tolerogenic phenotype of adoptively transferred DCs was conserved in vivo. However, the induction of tolerogenic DCs was dependent on a functional MC-1R as a-MSH neither downregulated co-stimulatory 1822 Journal of Investigative Dermatology (2012), Volume 132

receptors nor upregulated IL-10 in DCs from C57BL/6Je/e mice or individuals with variants in the MC-1R gene associated with a compromised receptor function (Figures 1f and 5c). In contrast to our observations, Cooper et al. (2005) have shown that MC-1R variants did not alter a-MSH-induced immunosuppression. These inconsistent data might be explained by the different variant alleles present in the individuals that have been analyzed. Whereas alleles analyzed by Cooper et al. (2005) are mainly associated with a red hair/fair skin phenotype, the variant alleles in our subjects (R151C, R160W, R142H, and D294H) have been implicated in an impaired downstream signaling of MC-1R and incomplete ability to bind a-MSH (Loser et al., 2010). Immunosuppressive DCs are critically involved in the induction and maintenance of peripheral tolerance. Although the detailed mechanism is still unclear, it has been shown that by repetitive stimulation with tolerogenic DCs CD4 þ T cells differentiate into functional Tregs. Accordingly, tolerogenic DCs generated by VIP induced the peripheral expansion of immunosuppressive Tregs, which inhibited contact allergy (Delgado et al., 2005a). Besides VIP, a-MSH has been suggested to expand functional Tregs, as CD4 þ T

M Auriemma et al. a-MSH-Induced Tregs Ameliorate Inflammation

cells cocultured in the presence of a-MSH and antigenpresenting cells inhibited the development of experimental autoimmune uveoretinitis upon adoptive transfer into immunized mice (Taylor and Namba, 2001). However, the cellular and molecular mechanism underlying a-MSH-mediated Treg induction has not been elucidated. Here we show that a-MSH generates tolerogenic DCs most likely by upregulating IL-10. The DC-derived IL-10 might directly contribute to Treg induction, as signaling via the IL-10 receptor has been shown to over-ride the dephosphorylation of CD28, thereby inhibiting the secretion of pro-inflammatory cytokines (Maldonado and von Andrian, 2010). As the IL-10 expression was significantly increased in mouse and human DCs after stimulation with aMSH (Figures 1e and 5a), and as blocking IL-10 signaling prevented the generation of Tregs (unpublished observation), it is conceivable that IL-10 might have an important role during Treg induction by a-MSH-stimulated DCs. Recently, GirardMadoux et al. (2012) demonstrated that murine IL-10 receptor–deficient DCs expressed high levels of IL-10 during CHS, which surprisingly resulted in prolonged and enhanced contact allergy, suggesting that IL-10 signaling in DCs and T cells is critical to modulate inflammatory immune responses. On the basis of these data, it would be interesting to investigate whether IL-10 secreted by a-MSH-stimulated DCs primarily modulates T cells or might also be crucial to maintain the tolerogenic DC phenotype, which could possibly explain the long-lasting immunosuppressive effect of a-MSH, although the half-life of this peptide is relatively short (Rudman et al., 1983). However, it remained to be clarified whether a-MSHstimulated DCs mediated the induction of Tregs from conventional CD4 þ CD25 T cells or the expansion of thymic-derived Tregs. Notably, in cocultures of CD4 þ T cells with a-MSH-stimulated DCs, we observed increased numbers of Helios induced compared with Helios þ thymicderived Tregs, and, moreover, upon adoptive transfer a-MSHstimulated DCs generated Tregs in Treg-depleted mice (Figure 2f and Supplementary Figure S4 online), suggesting that a-MSH-treated tolerogenic DCs mediated the de novo induction rather than the expansion of existing Tregs. In accordance with our data, Gonzalez-Rey et al. (2006) demonstrated that VIP administration to CD25-depleted mice led to the induction of functional CD4 þ CD25 þ Tregs, indicating that both neuropeptides, VIP and a-MSH, might promote the peripheral induction of Treg from conventional T cells. Induced Tregs are a heterogeneous population, but a common hallmark is the expression of anti-inflammatory cytokines such as IL-10 and the inhibitory surface receptor CTLA-4 (Vignali et al., 2008). Similarly, CD4 þ T cells from cocultures with a-MSH-stimulated DCs upregulated CTLA-4 and IL-10 (Figure 2b and c). It is worth noting that direct stimulation of CD4 þ T cells with a-MSH neither resulted in the upregulation of CTLA-4 and IL-10 nor the induction of suppressive activity most likely because murine, in contrast to human, CD4 þ T cells did not express MC-1R (Andersen et al., 2005; Loser et al., 2010). The anti-inflammatory effects in vivo and the capacity to induce Tregs suggest melanocortins as potential drugs for the

treatment of inflammatory disorders. Hence, systemic administration of a-MSH inhibited the development of contact allergy or reduced gastrointestinal inflammation (Grabbe et al., 1996; Maaser et al., 2006). Here, we demonstrated that a-MSH ameliorated ongoing psoriasis-like skin inflammation in mice and decreased the activity of Th17 cells from individuals with psoriasis (Figures 3 and 6). The pathogenesis of psoriasis has been associated with an impaired suppressor function of Tregs, resulting in the overproduction of proinflammatory cytokines such as TNF-a, IL-17, or IL-22 mainly by Th1 and Th17 cells (Sugiyama et al., 2005; Nestle et al., 2009). In this study, we could show that the beneficial effect of a-MSH was mediated by the induction of tolerogenic DCs and functional Tregs, which downregulated the proliferation and cytokine secretion of human and murine Th17 cells and thereby ameliorated disease, thus suggesting a-MSH as a potential therapeutic alternative for the treatment of individuals with psoriasis. Long-term TNF-a inhibitor treatment of individuals with psoriasis exacerbated disease in some cases (Ko et al., 2009), and analyses in a mouse model revealed that this might be mediated by a decrease in Treg numbers (Ma et al., 2010). On the basis of our data, it is conceivable that in psoriasis treatment a-MSH could shift the immune responses from a Th17-dominated inflammation back to a tolerance-controlled status by expanding functional Tregs through the induction of tolerogenic DCs. MATERIALS AND METHODS Materials and Methods can be found in the Supplementary Information online. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank Kerstin Vischedyk for excellent technical assistance. This work was supported by the Interdisciplinary Center of Clinical Research (IZKF) grants Lo2/017/07 and Lo2/004/11 to KL, and by the German Research Association (DFG) grants Lo817/2-1 to Karin Loser and GO1360/4-1 to Tobias Goerge.

Author contributions MA performed most of the experiments, TB provided reagents, as well as advice in parts of the study, and helped to write the paper; VK, LK, MV, and ZZ assisted with some experiments; TG collected blood samples from healthy donors and individuals with psoriasis, and TS generated DEREG mouse mutants. TAL provided expertise. KL conceived the study, analyzed data, and wrote the paper. SUPPLEMENTARY MATERIAL Supplementary material is linked to the online version of the paper at http:// www.nature.com/jid

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