Resident epidermal LCs, which are gradually lost from the epidermis in active disease phase psoriasis, are responsible for maintaining a suppressive skin environment by balancing the anti‐inflammatory IL‐10 and pro‐inflammatory IL‐23 axis.

Several subtypes of APCs are found in psoriasis patients, but their involvement in disease pathogenesis is poorly understood. Here, we investigated the contribution of Langerhans cells (LCs) and plasmacytoid DCs (pDCs) in psoriasis. In human psoriatic lesions and in a psoriasis mouse model (DKO* mice), LCs are severely reduced, whereas pDCs are increased. Depletion of pDCs in DKO* mice prior to psoriasis induction resulted in a milder phenotype, whereas depletion during active disease had no effect. In contrast, while depletion of Langerin‐expressing APCs before disease onset had no effect, depletion from diseased mice aggravated psoriasis symptoms. Disease aggravation was due to the absence of LCs, but not other Langerin‐expressing APCs. LCs derived from DKO* mice produced increased IL‐10 levels, suggesting an immunosuppressive function. Moreover, IL‐23 production was high in psoriatic mice and further increased in the absence of LCs. Conversely, pDC depletion resulted in reduced IL‐23 production, and therapeutic inhibition of IL‐23R signaling ameliorated disease symptoms. Therefore, LCs have an anti‐inflammatory role during active psoriatic disease, while pDCs exert an instigatory function during disease initiation.

Introduction Psoriasis is a frequent pathology of the skin affecting about 2% of the total Western population. It is characterized by inflamed lesions that display abnormal keratinocyte proliferation and differentiation as well as prominent immune cell infiltration. Both the innate and the adaptive immune system play a role in the pathomechanism of psoriasis (Nestle et al, 2009), and several cues point to a role of keratinocytes in psoriasis etiology (Nickoloff, 2006). In human psoriatic skin, an overall increase of dendritic cells (DCs) has been found both in the epidermis and in the dermis (Lowes et al, 2005; Wagner et al, 2010). DC types that are normally absent in healthy skin, such as TNF and iNOS‐producing DCs (Tip‐DCs) (Lowes et al, 2005), slanDCs (Schakel et al, 2006), and plasmacytoid DCs (pDCs) (Nestle et al, 2005), have been shown to infiltrate predominantly the dermal compartment of psoriatic skin. Whereas little is known about the roles of the different DC subsets in psoriasis, recent reports indicate that DCs are an important source of IL‐23, a cytokine that seems to have, along with TNF‐α and IL‐17, a central role in psoriasis pathology (Brunner et al, 2013; Di Cesare et al, 2009; Gunther et al, 2013; Wohn et al, 2013). Likewise, polymorphisms in the IL‐23 receptor (IL‐23R) have been associated with psoriasis (Di Meglio et al, 2013), and blocking IL‐23 is successful in the treatment of psoriasis (Crow, 2012). Recent findings indicate that inhibitors of TNF‐α signaling, which are similarly useful in therapy, seem to function via blockage of DC‐derived IL‐23 (Brunner et al, 2013; Gunther et al, 2013). IL‐23 promotes the maintenance of T cells producing IL‐17 and IL‐22, which are abundant in and contribute to many of the hallmarks seen in psoriasis. In psoriatic skin, these are constituted by both CD4+ and CD8+ TCRαβ+ T cells, as well as γδ T cells, and the recently discovered innate lymphoid cells (ILCs) (Dyring‐Andersen et al, 2014; Lowes et al, 2014). pDCs have been detected in low numbers even within uninvolved skin of psoriatic patients and have therefore been implicated in the conversion of healthy into lesional skin (Nestle et al, 2005). In mice engrafted with human psoriatic skin, the formation of lesions could be inhibited by pre‐treatment of mice with antibodies that blocked pDC‐specific type I IFN secretion (Nestle et al, 2005). Therefore, targeting pDCs as a therapeutic measure against clinically manifest psoriasis has been discussed. Another DC subset that has been suspected to be involved in psoriasis are Langerhans cells (LCs), which are constitutively resident within the epidermis. In contrast to most other immune cells that recycle from the bone marrow, the LC compartment renews under steady‐state conditions from an epidermis‐resident precursor population that is maintained from an early embryonic age throughout life (Chorro et al, 2009; Hoeffel et al, 2012; Merad et al, 2008). In addition, severe inflammation may provoke additional recruitment of a developmentally unrelated LC precursor from the bone marrow (Merad et al, 2008; Nagao et al, 2012). While LCs are the only DCs present within healthy epidermis, at least four different types of DCs are present in murine dermis (Tamoutounour et al, 2013), among them a subset of DCs that expresses Langerin, termed Langerin‐positive dermal DCs (Lan+ DDCs). In humans, a counterpart for Lan+ DDCs exists, but lacks Langerin expression, and is identified by expression of CD141 (Haniffa et al, 2012). In mice, Lan+ DDCs can be discriminated from LCs by their additional expression of the αE integrin (CD103) (Merad et al, 2008). The role of LCs and Lan+ DDCs could be studied using diphtheria‐toxin (DT)‐based mouse models that express either the DT receptor (DTR) or DT under the control of the Langerin promoter, thus allowing inducible or constitutive depletion of LCs and Lan+ DDCs, which are herein mentioned as Lan+ (Lan+) APCs. These studies demonstrated that dependent on the context, LCs could act either pro‐ or anti‐inflammatory (Bobr et al, 2010; Igyarto et al, 2011; Ouchi et al, 2011; Romani et al, 2012; Shklovskaya et al, 2011), while Lan+ DDCs have proinflammatory roles in most settings (Bedoui et al, 2009; Romani et al, 2012; Seneschal et al, 2014). Psoriasis etiology is linked with an array of predisposing genes located within several psoriasis susceptibility regions (PSORS). Jun and JunB are members of the activator protein‐1 (AP‐1) family and act in a heterodimeric fashion together with other AP‐1 members. They are located within the susceptibility regions PSORS7 (Jun) and PSORS2 (Junb) (Schonthaler et al, 2013; Zenz et al, 2005). Interestingly, a regional loss of JunB expression is observed in human psoriatic epidermis (Guinea‐Viniegra et al, 2014). A similar observation has been made for systemic lupus erythematosus (SLE) with cutaneous involvement (Pflegerl et al, 2009). Embryonic deletion of both Jun and JunB within the epidermis leads to fatal cachexia of neonatal mice (Guinea‐Viniegra et al, 2009; Zenz et al, 2005). Their deletion in adult mice via a tamoxifen (Tx)‐inducible cre recombinase in keratin 5 expressing cells (Junf/f JunBf/f K5creER = DKO* mice) leads within 14 days after Tx treatment to a skin phenotype that is strongly reminiscent of human psoriasis (Zenz et al, 2005). DKO* mice present many psoriatic hallmarks, ranging from epidermal changes such as keratinocyte hyperproliferation, parakeratosis, and prominent rete ridge formation to epidermal and dermal immune infiltrates, excess of proinflammatory cytokines (Zenz et al, 2005) and hypervascularization (Schonthaler et al, 2009). Additionally, DKO* mice exhibit molecular parallels to human psoriasis, specifically a similar global protein expression pattern (Schonthaler et al, 2013), complement activation (Schonthaler et al, 2013), and increased TNF‐α shedding (Zenz et al, 2005). In this study, we employed patient biopsies, an Imiquimod (Imi)‐induced skin inflammation mouse model, and the DKO* mice to investigate the function of LCs and pDCs in psoriasis. We show that LC numbers were severely diminished within human psoriatic plaques, while pDC numbers were increased. In order to investigate the consequences of LC and pDC absence during defined phases of psoriatic inflammation, we employed DKO* mice bred to either Langerin‐DTR (LanDTR) mice (Kissenpfennig et al, 2005), or to BDCA2‐DTR mice (Swiecki et al, 2010), in which LCs or pDCs could be inducibly depleted by injection of DT, respectively. We found that depletion of pDCs prior to disease initiation attenuated disease development in the DKO* model, whereas their depletion during fully developed psoriasis‐like inflammation had no effect. Conversely, LCs were not essential during the initiation of the phenotype, but their depletion during ongoing disease exacerbated skin inflammation. Our findings demonstrate that pDCs, which infiltrate during the early disease phase, are important instigators of psoriasis‐like disease, while LCs serve to protect immune homeostasis in established inflammation.

Discussion The contribution of the distinct DC subsets that are present in the skin of psoriatic patients is only poorly understood. Recently, two studies suggested that a dermal DC subset may be involved in psoriasis initiation via the production of IL‐23 (Wohn et al, 2013), a key cytokine that mediates expansion of IL‐17‐ and IL‐22‐producing cells, promoting important events in psoriasis pathology, including neutrophil infiltration and epidermal thickening (Di Cesare et al, 2009). In the present study, we report that two other skin DC subtypes, pDCs and LCs, contribute selectively to distinct stages, initiation and propagation, of the inflammatory process in the skin according the model shown in Supplementary Fig S6. pDCs were shown previously to be abundantly present within both lesional and non‐lesional skin of psoriatic patients (Nestle et al, 2005). In contrast, we found significant numbers of pDCs only in psoriatic skin, with low numbers in non‐lesional skin, at 2 cm distant from the adjacent lesion. Different to our investigation, the previous study analyzed skin 0.5 cm distant from the lesion, which might still represent an activated skin area in psoriasis (Nestle et al, 2005). In a mouse model of xenotransplanted human non‐lesional skin of psoriatic patients, injection of anti‐BDCA2 antibodies, which block pDC‐specific IFN‐α secretion, prevented the development of psoriatic lesions (Nestle et al, 2005). We also found that the presence of pDCs was necessary only for the initiation of psoriatic disease in DKO* mice since their depletion attenuated the psoriatic phenotype. pDCs were dispensable for maintenance of chronic inflammation, which might explain the inefficiency of anti‐IFNα‐based therapies in psoriatic patients (Bissonnette et al, 2010). In a second mouse model of skin inflammation that is based on the topical application of the synthetic TLR7 agonist Imiquimod, we and others (Wohn et al, 2013) have found that the development of skin inflammation was independent of pDCs. This discrepancy to the DKO* model might be due to the fact that Imiquimod induces only an acute and transient skin inflammation, thus mimicking only very early steps in psoriasis inflammation. In contrast, the DKO* psoriasis model exhibits chronic inflammation, which remains constant over a longer period of time, thereby likely modeling the human disease. We found that LCs were reduced in lesions of psoriatic patients as well as in the DKO* psoriatic mouse model. In DKO* mice, the disappearance of LCs was independent of the presence of pDCs, since pDC depletion in DKO* mice did not affect epidermal LC frequencies. Other groups that reported a reduction of LC numbers in psoriatic skin found that LC numbers reverted back to normal levels when patients had successfully been treated (Romani et al, 2012). In another study, following skin trauma, a portion of LCs underwent mass emigration directly after the insult, while the majority of LCs did not emigrate upon further stimulation (Dearman et al, 2004). Similarly, in patients with early‐onset psoriasis, LCs from non‐lesional skin were unable to migrate in response to cytokine stimulation (Cumberbatch et al, 2006). Therefore, the differential migration capacities of LCs from psoriatic skin and the fact that only about 30% of LCs can be induced to emigrate, might reflect the existence of two types of LCs in humans. In support of this hypothesis are the observations that during inflammatory conditions, LCs can originate either from bone marrow‐derived precursors or from preexisting epidermal LC precursors (Chorro et al, 2009; Elnekave et al, 2014; Ginhoux et al, 2006; Nagao et al, 2012). In DKO* mice, but not in Imi‐treated mice, we found that depleted LCs were replaced from bone marrow rather than from skin‐resident precursors, suggesting that two different types of LCs exist which might differently react to inflammatory conditions. We found that elimination of LCs aggravated psoriasis‐like inflammation. Lan+ DDCs, which have been shown to prime cutaneous adaptive immune responses in several instances (Romani et al, 2012), did not influence the chronic disease phase. LC depletion was associated with an increase in the frequency of epidermal immune cells known to be key mediators in psoriasis such as neutrophils, or Lanneg APCs, that are efficient producers of TNF‐α in psoriasis (Lowes et al, 2005). Multiple lines of evidence argue for a local or systemic tolerogenic role of LCs during inflammatory conditions such as UV irradiation (Yoshiki et al, 2010), allergic contact dermatitis (Gomez de Aguero et al, 2012), or infections (Kautz‐Neu et al, 2011). In contrast, LCs are required for the efficient generation of immune responses in other situations, such as for antigen‐specific T helper 17 (TH17) cells during fungal skin infections (Igyarto et al, 2011), and vaginal immunization (Hervouet et al, 2010). In DKO* mice, LCs within the epidermis as well as in lymph nodes exhibited higher levels of CD205, PD‐L1, and CD86, which have been associated with DC‐mediated generation of regulatory T cells (Bonifaz et al, 2002), peripheral T‐cell tolerance (Salomon et al, 2001), and protection from spontaneous autoimmunity (Probst et al, 2005). PD‐L1 can be upregulated in response to IL‐10, which has also been implicated in the induction of peripheral tolerance. We detected increased IL‐10 expression in LCs isolated from DKO* mice. IL‐10 has been shown to negatively regulate the production of proinflammatory cytokines (D'Andrea et al, 1993; de Waal Malefyt et al, 1991). IL‐10 production by DCs seems to be crucial for the establishment of tolerance after UV irradiation in the skin (Ghoreishi & Dutz, 2006). Psoriatic patient skin lacks IL‐10 compared to healthy individuals, which is likely due to the severe reduction of LCs in psoriatic lesions (Nickoloff et al, 1994). Therefore, LCs may directly prevent psoriasis aggravation via IL‐10 and PD‐L1. An increasing body of evidence points to a critical role for IL‐23 signaling in the pathogenesis of psoriasis. We found that IL‐23 was increased in the skin of DKO* mice and epidermal IL‐23 levels seem to be antagonistically regulated by both types of DCs investigated. In the absence of LCs during the chronic phase of psoriatic inflammation, epidermal IL‐23 levels increased, while absence of pDCs during psoriasis initiation led to a reduction of dermal IL‐23 levels (Supplementary Fig S6). These changes in IL‐23 did not affect the levels of IL‐17 and IL‐22. This is surprising, given the fact that IL‐23 has been reported to mediate its effects by supporting a robust IL‐17 response. However, two recent papers indicate that IL‐23‐driven pathology in both an asthma and a colitis model were independent of the presence of IL‐17 (Izcue et al, 2008; Peng et al, 2010). Likewise, IL‐23 stimulated the secretion of antimicrobial peptides in keratinocytes (Kanda & Watanabe, 2008), molecules that have been implicated in the instigation of psoriasis (Lowes et al, 2014). Recently, in two studies, IL‐23 production following stimulation with Imi was attributed to myeloid DCs such as conventional DCs (Wohn et al, 2013), CD103+ DC, and macrophages of the dermis (Riol‐Blanco et al, 2014). The latter two populations are also present in high abundance in the early stage of psoriatic inflammation of DKO* mice. However, other groups have reported that IL‐23 is produced by keratinocytes in psoriasis (Piskin et al, 2006). Therefore, it is possible that DCs and macrophages are involved in the early steps of psoriasis etiology, with keratinocytes taking over the production of IL‐23 once the inflammatory cascade is fully pronounced. We found that in DKO* mice that were treated with an antibody directed against the murine IL‐23R, chronic psoriasis‐like inflammation was significantly ameliorated. Successful therapy of psoriatic patients with antibodies targeting molecules within the IL‐23 axis, such as Ustekinumab, an antibody against the p40 subunit shared by IL‐12 and IL‐23, has been established in clinical trials (Rustin, 2012). Furthermore, promising results in clinical trials were also obtained with an antibody targeting the specific p19 subunit of IL‐23 (Alexander, 2013). However, antibody‐based therapies are costly and come with a certain risk of side effects owing to systemic immunosuppression (Crow, 2012). Thus, strategies aimed at modulating the local composition of DC subtypes in psoriatic lesions might represent a novel approach for the treatment of psoriasis in the future.

Materials and Methods Mice Mice harboring loxP‐flanked alleles of Jun and JunB and expressing K5cre‐ERT have been previously described (Zenz et al, 2005). Junf/fJunBf/f K5cre‐ERT mice (mixed background) were bred to LanDTR (Kissenpfennig et al, 2005) and BDCA2‐DTR mice (Swiecki et al, 2010) (both of C57BL/6J background). To delete Jun and JunB and induce psoriasis‐like disease, K5‐creER positive (DKO*) or negative (Jun/JunBf/f) mice were injected with 1 mg tamoxifen (Tx, Sigma‐Aldrich) in an emulsion with sunflower seed oil (Sigma)/ethanol mixture (10:1) intraperitoneally on 5 consecutive days. Deletion of Jun and JunB was verified by PCR. Similarly, 300 ng of Diphtheria toxin (DT, List Biological Laboratories, in PBS) was injected intraperitoneally into experimental mice according to the schemes indicated in the figures. For LC depletion, DT was applied every third day, and for pDC depletion, every other day. LC and pDC depletion was > 90% as determined in the epidermis or the spleen, respectively. Mice were kept in the animal facility of the Medical University of Vienna in accordance with institutional policies and federal guidelines. Animal experiments were approved by the Animal Experimental Ethics Committee of the Medical University of Vienna and the Austrian Federal Ministry of Science and Research. (Animal license numbers: GZ 66.009/124‐BrGT/2003; GZ 66.009/109‐BrGT/2003; BMWF‐66.009/0073‐II/10b/2010 BMWF‐66.009/0074‐II/10b/2010; BMWFW‐66.009/0200‐WF/II/3b/2014; and BMWFW‐66.009/0199‐WF/II/3b/2014). Patient material, histology, and histomorphometry Skin was obtained under an approved protocol (EK700/2009, Ethics Committee of the Medical University of Vienna), according to the Declaration of Helsinki. Patients suffering from chronic plaque‐type psoriasis with a PASI > 10 that had undergone no systemic or topical treatment for at least 4 weeks, and age‐matched healthy volunteers were enrolled in the study after providing written informed consent. 6 mm punch biopsies were taken from the abdomen under local anesthesia, embedded in optimal cutting temperature compound O.C.T.™ (Tissue‐Tek®, Sakura Finetek, Zoeterwoude, Netherlands), and stored at −80°C until further processing. Non‐lesional biopsies were taken 2 cm distant from the margin of a chronic psoriasis plaque. 7‐μm cryosections were fixed in acetone and incubated with a mouse anti‐Langerin or an anti‐BDCA‐2 antibody in PBS with 2% BSA overnight at 4°C. After incubation with 1% H 2 O 2 for 10 min, antibody binding was visualized using conventional immunohistochemical staining (Dako REAL™ Detection Systems HRP/AEC, Dako AutostainerLink 48, Dako, Glostrup, Denmark). For LC and pDC quantification, immunohistological images were acquired using a Zeiss Observer.Z1 microscope (Carl Zeiss, Oberkochen, Germany) equipped with TissueFAXS® and 2 sites per sample were analyzed using HistoQuest® software (both Tissue Gnostics, Vienna, Austria). Scoring of the psoriatic phenotype To monitor psoriasis severity of individual mice, a psoriasis severity scoring system modified from Singh et al (2010) was used rating the degree of erythema, swelling, and scaling of the skin separately for five dermatomes (ears, tail, paws, snout, back skin). We attributed a score of 0–4 to each of the dermatomes, defining a score of 0 as absence of pathological symptoms, 1 as isolated, sparse lesions or visible rubor, 2 as several lesions accompanied by low‐grade swelling, 3 as moderate inflammation of most parts of the dermatome, and a score of 4 as intense swelling, redness and scaling of the complete dermatome and the absence of healthy skin. The phenotype score was attributed to each dermatome of each mouse in a blinded fashion and summarized as a cumulative score. Bone marrow chimeric mice Host CD45.2 mice were exposed to whole body gamma irradiation, applying the lethal dose of 10 Gray. Subsequently, CD45.1 donor bone marrow cells were isolated, and T cells were depleted either via biotinylated antibodies against CD3 (Biolegend) and CD90 (Biolegend), followed by negative magnetic sorting with IMag™ streptavidin‐coated magnetic beads (BD Biosciences), or using MACS CD3 microbeads (Miltenyi) according to the manufacturer's protocol. Of 3.5 × 106 bone marrow cells (depleted of T cells) were injected into the tail vein of each host animal, and mice were maintained for 6 weeks on acidified water. Subsequently, chimerism was verified in peripheral blood collected from the tail via flow cytometric analysis of CD45.1 and CD45.2. Chimerism was routinely > 90%. Isolation of cells from epidermal, dermal, lymph node, and splenic suspensions Mice were euthanized by cervical dislocation, and skin cells were isolated from ears and tails. Dorsal and ventral ear halves were separated, and tail skin was peeled from residual tissue. Skin sheets were then placed on 0.8% trypsin for 45 min (Fisher Scientific) at 37°C. Epidermis and dermis were separated, and epidermal pieces were incubated for 30 min at 37°C in PBS containing 8% FCS (PAA) and 100 μg/ml DNase I (Sigma‐Aldrich). Dermal pieces were incubated in PBS with 1% FCS, 100 μg/ml DNase I, and 100 μg/ml Liberase TM (Roche) for 30 min at 37°C. Epidermal and dermal cell suspensions for flow cytometric analysis shown in Fig 2B were isolated as previously described (Tamoutounour et al, 2013). Auricular lymph nodes and spleen were isolated and incubated for 30 min at 37°C in PBS supplemented with 1% FCS, 100 μg/ml DNase I and 50 μg/ml Liberase TM. After red blood cell lysis, suspensions were filtered through a 70‐μm cell strainer (BD Biosciences). Spleens were flushed with PBS containing 1% FCS, 100 μg/ml DNaseI, and 50 μg/ml Liberase TM and incubated in this enzyme mix for 30 min at 37°C. Flow cytometry Single cell suspensions were stained with fluorescent antibodies for 30 min on ice after blocking Fc‐receptors with anti‐CD16/CD32 antibody. For intracellular IL‐17 staining of DKO* skin, dermal and epidermal cell suspensions were pooled and stimulated for 4.5 h with 500 ng/ml PMA (Sigma) and 500 ng/ml ionomycin (Sigma) in the presence of GolgiPlug (BD Biosciences) for the last 4 h. For a list of monoclonal antibodies used, see Supplementary Table S1. Gating for flow cytometric analysis in Fig 2B was performed as previously described (Tamoutounour et al, 2013). In brief, subsets were gated as: CD11b+DCs (CD11b+CD64−CCR2+Ly‐6C−MHC‐II+ CD24lo) MHC‐IIlomoDCs (CD11b+CD64loCCR2+Ly‐6ChiMHC‐IIlo CD24−) MHC‐II+moDCs (CD11b+CD64loCCR2+Ly‐6CloMHC‐II+ CD24−) MHC‐II+ dermal macrophages (CD11b+CD64hiCCR2loLy‐6CloMHC‐II+ CD24−) Dead cells were excluded by fixable dead cell stainings (Fisher Scientific, ebioscience). For intracellular stainings, cells were fixed in 2% PFA (Roth) and subsequently permeabilized using PermWash buffer (BD Biosciences). For Ki67, IL‐17, and FoxP3 stainings, a FoxP3 Fix/Perm buffer set (Biolegend) was used. Flow cytometry was performed on a LSRFortessa cell analyzer (BD Biosciences), and data were analyzed with FlowJo 7.6.4 software (Treestar). All flow cytometric gatings were performed on live cells following exclusion of doublets with FSC‐A/FSC‐H. Gates for activation markers and intracellular FoxP3, BrdU, Ki‐67, and IL‐17 stainings were set according to a corresponding isotype control. Numbers in flow cytometric plots and within graphs depicting quantifications of flow cytometric stainings indicate the percentage of a population of live single cells. In vivo BrdU labeling For proliferation studies of LCs, mice received one intraperitoneal injection of 1.5 mg 5‐Bromo‐2′deoxyuridine (BrdU, Calbiochem) followed by 1 week of BrdU application via drinking water (0.8 mg/ml). BrdU content was analyzed by flow cytometry using a BrdU Flow Kit (BD Biosciences). Imiquimod treatment Ears and/or shaved back skin of 8‐ to 12‐week‐old C57BL/6 mice were treated topically with Aldara, a 5% Imiquimod cream formulation every other day for 14 days, as previously described by our group (Drobits et al, 2012), resulting in a total of 7 imiquimod applications. Alternatively, for the data shown in Supplementary Figs S2K–M and S4D–I, Imi was applied daily on 6 consecutive days according to the treatment regimen described by van der Fits (van der Fits et al, 2009) and mice were analyzed on day 7. Skin thickness measurement Ears were cut off at the base and split in half, and the lower ear half was embedded in paraffin. 4‐μm sections were stained with hematoxylin and eosin (Sigma‐Aldrich). Images were obtained with a Nikon eclipse 80i microscope; histomorphometric analysis was performed using the Lucia system. Epidermal and dermal thickness were measured on 10 random fields on 3–4 independent pictures per sample, magnification 4×, using Adobe Photoshop CS4 (Adobe). Immunofluorescence stainings Tissues were embedded in O.C.T.™ (Sakura), and 5‐μm cryosections were generated and fixed in acetone before processing. Epidermal ear sheets were generated by separating epidermis from dermis with 3.8% ammoniumthiocyanate (VWR) and fixed in 4% PFA (Roth). Samples were blocked for 30 min at room temperature in 1% bovine serum albumin (Sigma‐Aldrich) in PBS containing 5% goat serum (PAA) and 0.1% Triton (Sigma‐Aldrich) and were incubated with the indicated antibodies overnight at 4°C in the same buffer. Apoptosis of epidermal LCs was assessed by co‐staining with antibodies against Langerin and active caspase‐3 followed by a secondary staining with the DyLight 594 goat anti‐rabbit IgG (Vector Laboratories). Total RNA isolation and RT–PCR analysis of murine cells and tissues Total RNA from epidermal cells was isolated with TRIzol Reagent (Invitrogen). Complementary DNA (cDNA) synthesis was performed with SuperScript First‐Strand Synthesis System (Invitrogen) according to the manufacturer's instructions. qRT–PCRs were carried out using SYBR Green Mix (Applied Biosystems), according to the manufacturer's instructions. For a list of primer sequences employed, see Supplementary Table S2. PCRs were performed on a 7500 Fast Real‐Time PCR System (Applied Biosystems, California USA) under the following conditions: an initial incubation at 50°C for 20 s and 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 54°C for 1 min. Relative quantification of RNA was calculated by ΔΔC t method. Omission of cDNA or reverse transcriptase enzyme was used as negative controls. Isolation of LCs and epidermal cells LCs were collected as previously described (Holcmann et al, 2009). In vivo inhibition of IL‐23R signaling The monoclonal antibody to mouse IL‐23R (21A4) was generated at Merck Research Laboratories (Palo Alto). To inhibit IL‐23 signaling, mice with established disease were treated by either intraperitoneal injection with 300 μg anti‐IL23R antibody or an isotype mouse IgG1 antibody (27F11) every other day. Mice were grouped randomly, and phenotype score was assessed weekly. Ears were analyzed by histology, and tail skin was used for flow cytometry. Microscopy Confocal microscopic pictures were acquired on a Zeiss LSM700 and evaluated using the ZEN2010 software. Graphs and statistics Experiments were performed at least two times, and data are represented as mean ± standard error of the mean (SEM). All graphs and statistical analyses were generated GraphPad Prism4 and Adobe illustrator software. Unpaired two‐tailed student's t‐test, Mann–Whitney U‐test, and Wilcoxon signed‐rank test were used to assess statistical significance (*P < 0.05, **P < 0.01, ***P < 0.001), as indicated in the figure legends.

Author contribution EG designed the experiments and performed most of them. AK performed the experiments related to pDCs. PMB performed the analysis in human patient samples and together with GS participated in the interpretation of the data. BD, NA, TK, and MH performed experiments. HBS and EFW provided the DKO* mice and helped with the interpretation of data. MH participated in the design, analysis, and interpretation of data. MS conceived and supervised the whole project and provided the requested funding.

Acknowledgements We are grateful to Martina Hammer for maintaining our mouse colonies. We thank Alexandra Bogusch, Sarah Bardakji, Elena Schmidt, and Lisa Bierbaumer for excellent technical assistance. Ly5.1 mice were kindly provided by Wilfried Ellmeier. LanDTR mice and BDCA2‐DTR mice were gifts of Bernard Malissen and Marco Colonna, respectively. The monoclonal anti‐IL‐23R antibody was a generous gift of Schering‐Plough Biopharma. We thank Juan Guinea‐Viniegra, Özge Uluçkan, Karin Komposch, and Rainer Zenz for critical reading of the manuscript. We express our thanks to Thomas Bauer for designing of the graphical abstract. This work was supported by the Austrian Science Fund (FWF) grants DK W1212, P18782, and SFB‐23‐B13 and the Austrian Federal Government's GEN‐AU program ‘Austromouse’ (GZ 200.147/1‐VI/1a/2006 and 820966). B.D. was a recipient of a DocForte fellowship from the Austrian Academy of Sciences (ÖAW). E.F.W. is funded by the Banco Bilbao Vizcaya Argentaria (BBVA) Foundation and a European Research Council Advanced Grant (ERC FCK/2008/37).

Conflict of interest The authors declare that they have no conflict of interest.