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Published March 4, 2016, doi:10.4049/jimmunol.1402943 The Journal of Immunology

Control of the Physical and Antimicrobial Skin Barrier by an IL-31–IL-1 Signaling Network

Kai H. Ha¨nel,*,†,1,2 Carolina M. Pfaff,*,†,1 Christian Cornelissen,*,†,3 Philipp M. Amann,*,4 Yvonne Marquardt,* Katharina Czaja,* Arianna Kim,‡ Bernhard Luscher,€ †,5 and Jens M. Baron*,5

Atopic dermatitis, a chronic inflammatory skin disease with increasing prevalence, is closely associated with skin barrier defects. A cy- tokine related to disease severity and inhibition of differentiation is IL-31. To identify its molecular targets, IL-31–dependent expression was determined in three-dimensional organotypic skin models. IL-31–regulated are involved in the formation of an intact physical skin barrier. Many of these genes were poorly induced during differentiation as a consequence of IL-31 treatment, resulting in increased penetrability to allergens and irritants. Furthermore, studies employing cell-sorted skin equivalents in SCID/NOD mice demonstrated enhanced transepidermal water loss following s.c. administration of IL-31. We identified the IL-1 network as a downstream effector of IL-31 signaling. Anakinra, an IL-1R antagonist, blocked the IL-31 effects on skin differentiation. In addition to the effects on the physical barrier, IL-31 stimulated the expression of antimicrobial peptides, thereby inhibiting bacterial growth on the three-dimensional organotypic skin models. This was evident already at low doses of IL-31, insufficient to interfere with the physical barrier. Together, these findings demonstrate that IL-31 affects keratinocyte differentiation in multiple ways and that the IL-1 cytokine network is a major downstream effector of IL-31 signaling in deregulating the physical skin barrier. Moreover, by interfering with IL- 31, a currently evaluated drug target, we will have to consider that low doses of IL-31 promote the antimicrobial barrier, and thus a complete inhibition of IL-31 signaling may be undesirable. The Journal of Immunology, 2016, 196: 000–000.

he skin is the largest organ of the body and forms It is extensively modified and processed and fulfills essential func- an indispensable barrier to protect against penetration by tions in the formation of the skin barrier. In addition, processed T environmental pathogens, allergens, or irritants. Another profilaggrin enhances moisturization and contributes to the acidity of major function of the skin is to inhibit transepidermal water loss the , both important for sustaining the integrity of the skin (TEWL) and thus minimize dehydration (1, 2). Inflammatory skin barrier (1, 9, 10). Profilaggrin is processed to filaggrins, which are diseases like (AD), a highly pruritic skin disor- cross-linked to and induce bundling of filaments. This to- der, are characterized by impairment of skin barrier function, in- gether with other and enzymatic activities substantially creased skin surface pH, and allergen priming, as well as augments the mechanical stability of keratin filaments (2, 9, 11). decreased hydration of the (3–5). In addition, Thus, filaggrin is central to the development of an efficient skin enhanced susceptibility to infections (e.g., by Staphylococcus barrier in combination with other processes that result in the gen- aureus) has been observed in patients with AD with an overall eration of the cornified envelope (2, 3, 5, 12, 13), including the altered skin microbiota (6–8). Mechanisms underlying this atten- strengthening of cell–cell contacts by the maturation of uation of skin barrier functions and the contribution of inflam- to corneodesmosomes (3, 5, 8, 9, 14, 15), the formation of an matory to this phenotype are incompletely understood. envelope (8, 9, 16, 17), and the production of an antimicrobial At the genetic level, impaired skin barrier function can be linked barrier by synthesizing antimicrobial peptides (AMPs) and fatty to reduction or loss of expression of the structural profilaggrin. acids (9, 16, 18–21).

*Department of Dermatology and Allergology, Medical School, RWTH Aachen Uni- The sequences presented in this article have been submitted to the National Center versity, 52074 Aachen, Germany; †Institute of and Molecular Biol- for Biotechnology Information’s Omnibus (http://www.ncbi.nlm. ogy, Medical School, RWTH Aachen University, 52074 Aachen, Germany; and nih.gov/geo/) under accession number GSE76880. ‡Department of Dermatology, College of Physicians and Surgeons, Columbia Uni- Address correspondence and reprint requests to Prof. Bernhard Luscher€ or Prof. Jens versity, New York, NY 10032 M. Baron, Institute of Biochemistry and Molecular Biology, RWTH Aachen Univer- 1K.H.H. and C.M.P. are cofirst authors. sity, Pauwelsstraße 30, 52074 Aachen, Germany (B.L.) or Department of Dermatol- ogy and Allergology, RWTH Aachen University, Pauwelsstraße 30, 52074 Aachen, 2Current address: Chiltern International, Bad Homburg, Germany. Germany (J.M.B.). E-mail addresses: [email protected] (B.L.) or jensmalte. 3Current address: Novartis Pharma, Nurnberg,€ Germany. [email protected] (J.M.B.) 4Current address: Department of Dermatology, Stadt- und Landkreis Hospital Heil- The online version of this article contains supplemental material. bronn, Heilbronn, Germany. Abbreviations used in this article: AD, atopic dermatitis; AMP, antimicrobial peptide; 5B.L. and J.M.B. are cosenior authors. CsSSE, cell-sorted skin equivalent; 3D, three-dimensional; hBD, human b-defensin; HDF, human dermal fibroblast; HPRT, phosphoribosyl trans- ORCIDs: 0000-0001-7983-657X (C.C.); 0000-0002-1824-1318 (K.C.); 0000-0003- ferase; NHEK, normal human epithelial keratinocyte; qRT-PCR, quantitative RT- 4609-1182 (A.K.); 0000-0002-1174-6946 (J.M.B.). PCR; rh, recombinant human; TEWL, transepidermal water loss. Received for publication November 21, 2014. Accepted for publication February 8, 2016. Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 This work was supported by Deutsche Forschungsgemeinschaft (BA 1803/7-1) and the START program of the Medical School of RWTH Aachen University (to C.C., B.L., and J.M.B.).

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402943 2 IL-31–IL-1 SIGNALING CONTROLS SKIN BARRIER

AD and other common dermatologic and allergologic diseases study will likely be relevant when considering interfering with IL- are associated with loss-of-function in FLG, which 31 function. encodes profilaggrin, further supporting the key role of this protein in skin barrier formation (22–24). FLG mutations are strongly Materials and Methods associated with AD, although only about half of the heterozygotes Primary cell culture and skin equivalents develop clinical disease (10, 25). Interestingly, the acute lesional Normal human epithelial (NHEKs) and human dermal fi- skin of these patients with AD carrying FLG mutations exhibits broblasts (HDFs) were prepared from sterile human skin samples (approved lower levels of filaggrin expression as compared with the clini- by the ethic committee of the Medical School of the RWTH Aachen cally unaffected skin of the same patients. These findings suggest University) and cultivated under regular cell-culture conditions. Organo- that additional factors may contribute to the regulation of filaggrin typic skin equivalents of NHEKs and HaCaT-IL31RA cells were con- structed as described previously (1, 41), cultured over a period of up to 10 d expression and the AD phenotype (1, 26, 27). Indeed, AD is as- at the air–liquid interphase, and treated with recombinant human (rh)IL-31 sociated with the deregulated expression of various cytokines, (PeproTech, Hamburg, Germany) or rhIL-1a (PeproTech). The recon- including IL-4, IL-13, IL-22, IL-25, and IL-31, which are known structs were harvested, cut into pieces, and either fixed according to a to modulate the expression of structural proteins implicated in the standardized protocol for routine histology or embedded in Tissue Tec (O. formation of the skin barrier (9, 28–30). IL-31 belongs to the IL-6 C.T.) compound (Sakura Finetek, Zoeterwoude, the Netherlands) for cryo- sectioning. Parts of the cultures were stored in RNA later (Ambion/Applied family of proinflammatory cytokines and signals through hetero- Biosystems, Darmstadt, Germany) for RNA isolation. dimeric receptors composed of the oncostatin M and the IL-31–specific receptor IL-31RA (30–32). IL-31 expression is HaCaT cells with inducible IL-31RA increased in lesions and serum of patients with AD and correlates HaCaT cells were obtained from N. Fusenig (German Research with disease severity (30, 33–36). Furthermore, distinct haplo- Center, Heidelberg, Germany) (50). IL-31RA was expressed stably in types of the IL31 gene are associated with AD (27, 37, 38). HaCaT cells using a tet-inducible lentiviral expression vector as described previously (1). In morphological studies employing human three-dimensional (3D) skin equivalents, we recently demonstrated that IL-31 dis- Cell-sorted skin equivalents turbs the differentiation of keratinocytes, interferes with filaggrin Cell-sorted skin equivalents (CsSSEs) were based on protocols as described expression, and weakens the lipid envelope formation (1, 9, 24, 39, previously (12). Briefly, 48 h before implantation, 4–6-wk-old SCID/NOD 40). In this study, we report on our studies to clarify further the mice were fed with doxycycline (2 mg/ml) provided in the drinking water supplemented with 5% sucrose. On day 21, two silicon chambers were molecular and functional consequences of IL-31 signaling in implanted onto the muscle fascia of the mouse back. Twenty-four hours keratinocytes. Primary normal keratinocytes express very low later, a cell slurry of 106 cells of each HDFs and HaCaT cells (chamber 1) levels of IL-31RA, which can be induced by IFN-g (33–36, 41). or HDFs and HaCaT-IL31RA cells (chamber 2) were seeded into the However, such treatment has additional consequences, including chambers. From days 4 to 7, 20 mg rhIL-31 in 50 ml PBS or 50 ml PBS for cell arrest, making it difficult to distinguish between IFN-g–and control were applied s.c. under the skin equivalents. On day 6, the chambers were removed followed by TEWL measurements on day 7. Mice IL-31–specific effects. To avoid interference by IFN-g stimula- were sacrificed and skin equivalents were either embedded in paraffin or tion, we used a donor-independent reproducible system applying cryoconserved for further investigation. HaCaT cells expressing the IL-31RA receptor (1). We demon- Skin barrier analysis strate that IL-31 influences the formation of the skin barrier in multiple ways. In addition to deregulating the expression of For skin barrier analysis, fluorescently labeled recombinant timothy grass structural proteins, including filaggrin, IL-31 represses pollen major allergen phl p1 (Biomay, Vienna, Austria) was applied topically on 7-d-old HaCaT-IL31RA organotypic skin equivalents for 45 and proteins involved in filaggrin processing and the formation of min and the luminescence measured. To analyze the vulnerability of an desmosomes, whereas genes encoding tight junction proteins were organotypic skin equivalent to the penetration by irritating agents, 7-d-old not affected. The net result is a weakening of the physical barrier. HaCaT-IL31RA organotypic skin equivalents were treated topically with As downstream effector of IL-31 signaling, we identified the IL-1 0.2% SDS for 40 min, and 24 h later, the expression of IL1A was analyzed cytokine network. Through this network, IL-31 also enhances the by quantitative RT-PCR (qRT-PCR), and the IL-1a release was measured by ELISA (IL-1a: DY200; R&D Systems, Wiesbaden, Germany). antimicrobial barrier by inducing the expression of AMPs, in- cluding human b-defensin (hBD)-2 and -3 and members of the Penetration assay 2+ S100 Ca -binding (S100A8, S100A9, and HaCaT models were treated with 10 mmol Lucifer Yellow/Biocytin (L6950; S100A12). Invitrogen) from the basolateral side at room temperature for 1 h and These findings suggest that IL-31 is a key player in the path- subsequently processed for cryoconservation and sectioning. ogenesis of AD. Furthermore, we uncovered a positive effect of TEWL analysis in cell-sorted skin equivalents IL-31 on the antimicrobial barrier function of the skin. IL-31 stimulates the expression of AMPs with a broad spectrum of an- TEWL was measured using a Tewameter TM210 (Courage+Khazaka, Cologne, Germany) according to the protocol of the manufacturer. timicrobial activities against skin pathogens, including S. aureus. Of note is that these latter effects are measurable in response to Ex vivo explants low concentrations of IL-31, which are insufficient to affect the Ex vivo skin explants were prepared from sterile human skin samples physical barrier. Thus, IL-31 appears to exert bifunctional effects, (approved by the ethics committee of the Medical School of the RWTH with low doses promoting the antimicrobial barrier but high doses Aachen University). Subcutaneous fat was carefully removed, and skin additionally impairing the physical barrier. Low levels of IL-31 samples were rinsed three times in sterile PBS containing penicillin, may be produced by different skin-resident immune cells, whereas streptomycin, and amphotericin B (Biochrom, Berlin, Germany). Eight- millimeter punch biopsies were taken from these skin samples and high levels are associated with different inflammatory skin dis- placed on the bottom of a polycarbonate membrane insert (3-mm pore size; eases, including AD, allergic contact dermatitis, and prurigo Nunc, Rochester, NY). Then inserts were placed in six-well plates, and nodularis (30), but also with tumors such as mastocytosis and skin grafts were cultured at the air–liquid interphase and kept in the cell cutaneous T cell lymphoma (42–45), and with pruritus in many culture incubator at 37˚C with 5% CO2. The medium consisting of equal volumes of DMEM (Life Technologies, Carlsbad, CA) and KGM (Lonza) pathological conditions (46–49). Thus, these findings suggest that supplemented with 5% FCS, 50 mg/ml L-ascorbic acid (Sigma-Aldrich), IL-31 is an emerging therapeutic target. The differential effects of and a concentration of 1.2 mmol with and without 100, 500, or low and high doses of IL-31 on the skin barrier described in this 1000 ng/ml rhIL-31 (PeproTech) was changed daily for 4 d. The Journal of Immunology 3

Analysis of gene expression using expression arrays all measurements were performed in triplicates in separate reaction wells. Statistical significance was evaluated by using the two-sided Student t test For gene expression analysis, HaCaT-IL31RA organotypic skin equivalents on all of the experiments as indicated in the figure legends. were stimulated with rhIL-31 (100 ng/ml; PeproTech) for 2, 8, 24, or 48 h. mRNA was then purified and analyzed on GeneChip Human Exon 1.0 ST Light microscopy and immunofluorescence arrays as reported previously (1, 51). Data visualization and analysis were performed with GeneSpring GX software (Agilent Technologies, Bo¨blingen, For microscopy and immunofluorescence analyses of the 3D skin Germany). The primary data have been assigned the Gene Expression models, 4-mm cryosections were processed as described previously (1, 54). Omnibus accession number GSE76880 (http://www.ncbi.nlm.nih.gov/geo/). The following Abs specific for the indicated proteins were used: filaggrin (sc-66192; Santa Cruz Biotechnology), S100A7 (MCA5253Z; AbD Western blotting Serotec, Dusseldorf,€ Germany), hBD-2 (ab63982; Abcam, Cambridge, U.K.), and IL-1a (ab9614; Abcam); and the DNA was stained with DAPI Cells were lysed in RIPA buffer (10 mmol Tris/HCl [pH 7.4], 150 mmol (Applichem, Darmstadt, Germany). NaCl, 1% Nonidet P-40, 1% deoxycholic acid, 0.1% SDS, and 0.5% Trasylol) containing a inhibitor mixture (Proteobloc; Fermentas, Flow cytometry Waltham, MA), on ice (52). The lysates were sonicated twice for 30 s on ice and cleared by centrifugation. Proteins were separated using 10–12% Cultured HaCaT-IL31RA cells were washed with PBS/EDTA and disso- SDS-PAGE and then blotted on nitrocellulose membranes for the detection ciated by addition of trypsin/EDTA. To inactivate the trypsin, culture of selected proteins using specific Abs. Abs were purchased from either medium containing 10% FCS was added, and then the cells were washed Technology (p-STAT3, p-p38, p65, p-p65, and IkBa)or with PBS. For analysis of the surface expression of IL-1R1 and IL-1R2, the Santa Cruz Biotechnology (STAT3 and p38; Santa Cruz, CA). Abs specific cells were fixed in 3.7% paraformaldehyde for 20 min, washed, blocked for and were purchased from Sigma-Aldrich and MP Bio- with PBS/1% BSA, and incubated with receptor-specific Abs IL-1R1 (ab medicals, respectively. 40774; Abcam) and IL-1R2 (ab89159; Abcam) at room temperature for 30 min. The cell-surface fluorescence intensity was measured by adding Analysis of antimicrobial activity fluorescently labeled secondary Abs on an FACS Canto flow cytometry system (BD Biosciences, Franklin Lakes, NJ). For the analysis of antimicrobial activity, HaCaT-IL31RA organotypic skin equivalents were stimulated with or without rhIL-31 for 10 d. Cultures of S. ELISA aureus stably expressing GFP (ATCC29213) (53) were grown in Luria- Bertani medium containing 20 mg/ml chloramphenicol overnight and then Cells were treated as specified in the figure legends and lysed in RIPA buffer diluted 1:10 for an additional 2 h until an OD of 1 was reached. The as described before or the supernatants were collected (55). The samples bacteria were diluted 1:100 in Luria-Bertani and applied topically onto the were either directly applied to the ELISA or diluted in the provided di- 3D skin equivalents. The models were harvested directly or incubated at lution buffer and measured according to the manufacturer’s recommen- 37˚C and 5% CO2 for 8 or 16 h. The models were cut into three parts. One dations (IL-1a: DY200, R&D Systems; IL-1b: DY201, R&D Systems; part was used for immunohistochemistry, the other two parts were lysed, hBD-2: 900-K172, PeproTech; hBD-3, S100A7, S100A9, and S100A12: and genomic DNA or RNA was isolated. RNA was isolated using the Cloud-Clone, Houston, TX). NucleoSpin RNA II Kit (Macherey-Nagel, Dren, Germany) according to the manufacturer’s instructions for Gram-positive bacteria. cDNA was synthesized with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Genomic DNA was prepared using the peqGOLD Bacterial DNA Kit (Macherey-Nagel). GFP DNA and cDNA was quantified by real-time PCR using the TaqMan system and Assay-on-Demand gene expression products for GFP (Mr03989638_mr; Applied Biosystems). The real-time PCR re- actions were performed with the TaqMan Gene Expression Master Mix (Applied Biosystems). Genomic DNA measurements were performed in duplicates, and cDNA was analyzed in triplicates. For determining the relative bacterial growth, the changes of threshold cycle values for GFP from 0 to 8 or 16 h were calculated. The values given in Fig. 9 are 2DCT (i.e., fold change compared with the 0-h values). The experiments were performed in triplicates. RNA preparation, reverse transcription, and qRT-PCR The RNeasy Mini Kit (Qiagen, Hilden, Germany) was used for total RNA extraction, according to the manufacturer’s instruction, and residual ge- nomic DNA was removed by DNaseI (Qiagen) digestion. A total of 1 mg RNA was reverse transcribed into cDNA by using the QuantiTect reverse transcription (Qiagen) and analyzed by quantitative real-time PCR by using the Corbett RotorGene system (Qiagen). The real-time PCR reac- tions were performed with the SensiFAST SYBR Kit (Bioline, Luckenwalde, Germany). All primer pairs used were QuantiTect primer assays (Qiagen) except the primer pairs for hypoxanthine guanine phosphoribosyl trans- ferase (HPRT) (forward, 59-TGACACTGGCAAAACAATGCA-39 and reverse, 59-GGTCCTTTTCACCAGCAAGCT-39) and OSMRb (forward, 59-GTGTGGGTGCTTCTCCTGCTTC-39 and reverse, 59-TCTGTGCTAAT- GACTGTGCTTG-39). All measurements were performed in duplicates. The relative quantification was calculated by using the comparative cycle threshold method and normalized to HPRT. In case of organotypic 3D models, the tissues were mechanically disrupted and homogenized by using tissue lyzer (Qiagen). Total RNA was extracted with Nucleo Spin RNA II (Macherey-Nagel) according to the manufacturer’s protocol. Purified RNA was reverse transcribed by using High Capacity RNA-to- cDNA Master Mix (Applied Biosystems, Foster City, CA). TaqMan ex- periments were carried out on an ABI PRISM 7300 sequence detection FIGURE 1. IL-31 deregulates the expression of genes associated with the system (Applied Biosystems) using Assay-on-Demand gene expression A products (Applied Biosystems) for FLG (Hs00418578_m1), S100A7 physical skin barrier in organotypic HaCaT-IL31RA 3D models. ( )qRT-PCR (Hs00161488_m1), and DEFB4A (Hs00823638_m1) according to the analysis of the indicated genes in HaCaT-IL31RA cells stimulated with rhIL-31; manufacturer’s recommendations. An Assay-on-Demand product for mean values 6 SD; n =3.(B and C) qRT-PCR analysis of a 10-d HaCaT- HPRT mRNA (Hs99999909_m1) was used as an internal reference to IL31RA 3D model; mean values 6 SD; n =3.Thep values were calculated normalize the target transcripts. For FLG, S100A7, DEFB4A,andHPRT, using Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. 4 IL-31–IL-1 SIGNALING CONTROLS SKIN BARRIER

Sera from patients suffering from AD were collected (approved by the kallikrein-like peptidase 7, two important involved in the ethics committee of the Medical School of the RWTH Aachen University) processing of filaggrin during keratinocyte differentiation, were 2 and stored at 80˚C. IL-31 serum concentrations were determined by repressed (Supplemental Fig. 1B) (56, 60, 61). These effects were ELISA (Human IL-31 DuoSet DY2824E; R&D Systems). ELISA plates were precoated with anti-human IL-31 Ab, blocked with PBS/1% BSA, verified in HaCaT-IL31RA monolayer cultures (Fig. 1A) and in and incubated for 2 h with serum samples. The samples were either di- HaCaT-IL31RA organotypic skin equivalents stimulated with IL-31 rectly applied to the ELISA or diluted in PBS/1% BSA. Detection was (Fig. 1B). Although the findings were reproduced in the 3D models, performed according to the manufacturer’s recommendations. the gene expression pattern was more complex in the monolayer cultures, suggesting that observations made in the latter have to be Results interpreted with caution. This may relate to the lack of appropriate IL-31 controls the expression of genes associated with the cellular interactions and differentiation-associated effects. Unlike physical skin barrier genes expressing desmosomal proteins, tight junction genes were IL-31 disturbs keratinocyte differentiation in organotypic 3D skin not deregulated (Fig. 1C). Together, these findings are consistent models with either NHEKs or HaCaT-IL31RA cells (HaCaT cells with the IL-31–induced defect in skin differentiation observed expressing the IL-31RA under the control of doxycycline) (1). We previously (1, 30) and suggest defects in the physical barrier due to analyzed gene expression in organotypic 3D models of HaCaT- reduced expression of structural proteins and corneodesmosomes. IL31RA cells in response to IL-31 after 2, 8, 24, or 48 h. IL-31 IL-31 impairs skin barrier function treatment resulted in altered expression of a broad range of genes (Supplemental Table I), including many that encode differentiation- We assessed the integrity of the epidermal barrier upon IL-31 associated structural proteins (Supplemental Fig. 1A). Moreover, treatment. We incubated a 3D model with fluorescently labeled genes encoding desmosomal proteins and filaggrin-processing en- recombinant timothy grass pollen major allergen (phl p1) (4). The zymes were repressed (Supplemental Fig. 1B). Most notably, the allergen did not penetrate the control organotypic epidermis, genes encoding desmoglein 1 and 4 but also 1 and 2 and whereas it was enriched in deeper layers upon IL-31 treatment were downregulated. Desmoglein 1 and desmocollin (Fig. 2A). Moreover, biotin applied basolaterally accumulated in 1 are essential components of corneodesmosomes and targets of the stratum corneum only when the model was pretreated with IL- proteases important for skin desquamation (1, 56–58). Corneo- 31 (Fig. 2B) (6). The former and latter findings are consistent with desmosin is covalently linked to the cornified envelope (6, 59). Moreover, the expression of the genes encoding -14 and

FIGURE 2. IL-31 weakens the skin barrier. (A) HaCaT-IL31RA 3D FIGURE 3. IL-31 promotes transepidermal water loss. (A) Macroscopic models were stimulated with or without rhIL-31. Fluorescently labeled phl pictures of a 6-d-old CsSSE. Mice were fed with doxycycline and s.c. p1 was applied topically for 45 min. Histological sections were H&E treated with 20 mg rhIL-31 at days 4 to 7. (B) Histological sections of stained, and the location of phl p1 (green) was determined by fluorescence CsSSE were stained with H&E, for filaggrin (green), the DNA with DAPI microscopy. Scale bars, 200 mm (H&E) and 100 mm (fluorescence). (B) (blue), and with nile red. Scale bars, 200 mm (H&E) and 100 mm Biotin (green) was applied basolaterally for 60 min. DNA was labeled (immunofluorescence). (C) TEWL was measured on 7-d-old CsSSEs; with DAPI (blue). Scale bars, 100 mm. (C) 3D models were treated mean values 6 SD; n = 3. The p values were calculated using Student t with 0.2% SDS and/or 100 ng/ml rhIL-31 for 40 min. IL-1a release and test. (D) Human ex vivo skin explants were cultivated with and without IL1A mRNA expression were measured after 24 h. Mean values of two 100, 500, or 1000 ng/ml rhIL-31 and harvested at day 4. Histological experiments. n.d., not detected. sections were stained for filaggrin (green). *p , 0.05. The Journal of Immunology 5 defects in the stratum corneum and in cellular adhesion, respec- NOD mice do not have T and B cells, we stained the sections for tively, as expected from the reduced expression of relevant genes F4/80, a marker found on macrophages and other myeloid cells. (Fig. 1) (1). The IL-31–treated models were also more vulnerable Indeed, this revealed some infiltrating myeloid cells; however, no to the application of irritants. Topical treatment with the irritant differences between the HaCaT and HaCaT-IL31RA models were SDS increased expression and release of IL-1a in a fully developed observed (data not shown). To expand on these findings, we used 3D model pretreated with IL-31 compared with untreated controls human ex vivo skin explants that were treated with increasing (Fig. 2C) (11, 16, 25). Moreover, IL-31 was sufficient to induce doses of IL-31 in culture (Fig. 3D). expression was re- IL1A mRNA expression and release of the cytokine (Fig. 2C). duced when 1 mg/ml IL-31 was applied for 4 d. Together, these To determine the impact of IL-31 on skin differentiation in vivo, findings suggest strongly that the profound effect of IL-31 on the we developed an epidermal mouse model based on a CsSSE as gene expression program of keratinocytes results in an impairment described (12). In this in vivo skin reconstitution assay, we com- of the physical skin barrier. pared the morphology, filaggrin expression, lipid layer, and TEWL of HaCaT keratinocytes with HaCaT-IL31RA cells seeded to- IL-31 activates the IL-1 signaling network gether with HDFs in chambers placed on the back of SCID/NOD IL-1a is a proinflammatory cytokine secreted by inflammatory mice (Fig. 3A). Both models were injected s.c. with rhIL-31 daily. cells upon activation of the inflammasome (similar to IL-1b) (62, HaCaT-IL-31RA cells exhibited a profound defect in epidermal 63). Physiological levels of IL-1a enhance barrier formation and development with a reduction in filaggrin expression and a re- wound healing following mechanical injury (16, 25, 64–67), duced nile red staining (Fig. 3B), resulting in disturbed barrier promote the synthesis of epidermal lipids and the formation of formation as shown by increased TEWL (Fig. 3C). The TEWL lamellar bodies, and induce the upregulation of genes and proteins values in the control models were higher compared with normal associated with , proliferation, and epidermal dif- skin of healthy individuals (typically 20–22 g/m2 3 h), most ferentiation in keratinocytes (16, 19, 25). In contrast, dysregulat- likely due to incomplete barrier formation at the time of analysis. ion of the IL-1 signaling network has been associated with We noted that some immune cells infiltrated the models. As SCID/ different skin diseases, including AD (22). Enhanced expression

FIGURE 4. IL-31 stimulates the IL-1 signaling network. (A) qRT-PCR analysis of the indicated genes in HaCaT-IL31RA cells stimulated with rhIL-31; mean values 6 SD; n =3.(B and C) IL-1a and IL-1b proteins were analyzed from cell lysates and supernatants by ELISA; mean values 6 SD; n =3.(D) Histological sections of HaCaT-IL31RA cells stained for IL-1a (green) and for DNA (blue). Scale bar, 200 mm. (E)IL-1a in the supernatant of 10-d HaCaT- IL31RA 3D models; mean values 6 SD; n =3.(F)IL-1a levels in 26 human sera: 14 with IL-31 below and 12 with IL-31 .4.1 ng/ml were compared. (G) HaCaT-IL31RA cells were treated with IL-31 for 4 h. In addition, the indicated kinases were blocked with the following inhibitors: JAK inhibitor I (100 nmol); JNK inhibitor II (20 mmol); SB202190, selective for p38 MAPKs (20 mmol); U0126, selective for ERKs (20 mmol); and wortmannin, selective for PI3K kinases (500 nmol). The inhibitors were added 1 h prior to stimulation with IL-31. The expression of the indicated genes were analyzed using qRT-PCR; mean values 6 SD; n =3.Thep values were calculated using Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. 6 IL-31–IL-1 SIGNALING CONTROLS SKIN BARRIER of IL-1a (e.g., artificially by intradermal injection) promotes an tivities of the IL-1 network (Fig. 5B, 5C). We verified the func- inflammatory skin phenotype (25). We observed that IL-31 stim- tionality of anakinra by monitoring the induction of IL-31/IL-1a– ulated the expression of IL1A and IL1B, the genes encoding the induced signaling pathways (Fig. 5D). Anakinra efficiently IL-1R subunits IL1R1 and IL1R2, and the natural IL-1R antagonist blocked the activation of the MAPK p38 by both cytokines and the IL1RN, as well as several genes of IL-1R–associated proteins phosphorylation of p65–NF-kB by IL-1a, but had no effect on IL- (Fig. 4A, Supplemental Fig. 1D). Consistent with the mRNA data, 31–induced STAT3 activation. This supports the concept of an FACS analysis of HaCaT-IL31RA cells revealed an increase of IL- important role of IL-1a downstream of IL-31 for keratinocyte 1R1 and IL-1R2 cell-surface expression (Supplemental Fig. 2), differentiation. enhanced expression of IL-1a and IL-1b upon IL-31 stimulation IL-31 regulates the antimicrobial barrier dependent on IL-1a (Fig. 4B), and their release when treated with nigericin (Fig. 4C). activation In the 3D model, IL-31 increased the expression (Fig. 4D) and the release of IL-1a (Fig. 4E). In contrast, IL-1b could not be detected In addition to effects on various genes encoding proteins associated in the supernatant of the 3D model (not shown), consistent with with barrier function, our array data indicated that several genes the findings in HaCaT-IL31RA monolayer cells (Fig. 4B). Addi- encoding AMPs were deregulated in response to IL-31 (Supplemental 2+ tional signals might be required for IL-1b release. An interaction Fig. 1E), including genes encoding S100 Ca -binding proteins and between IL-31 and IL-1a was also apparent when these cytokines human b-defensin-2 (hBD2/DEFB4A)and-3(hBD3/DEFB103A). were compared in sera of 26 patients with AD. High IL-31 levels These genes are known IL-1a targets in keratinocytes grown in (.4.1 ng/ml) correlated with high IL-1a levels (Fig. 4F). The monolayers (19). We verified the induction of S100A7, S100A8, specificity of induction of the IL1A gene was addressed by using S100A9, S100A12, DEFB103A,andDEFB4A in HaCaT-IL31RA different kinase inhibitors. PI3K kinases, p38, and ERKs were monolayer cultures (Fig. 6A). The expression of all genes increased essential to induce the IL1A gene (Fig. 4G). In contrast to these strongly over time. Moreover, IL-31 stimulated the release of these findings, the induction of the expression of IL20 was dependent on AMPs and the expression of S100A7 and S100A9 in 3D organotypic the JAK kinase, p38, and ERK pathways (Fig. 4G), comparable to models of HaCaT-IL31RA cells (Fig. 6B, 6C). The AMP encoding previous findings (1). This suggested that IL-1 signaling pathways genes were also induced by IL-1a in HaCaT-IL31RA cells, as in- are potential downstream mediators of IL-31 and that the regu- dicated previously for S100A7 and DEFB4A (19), albeit less ef- lation of the IL-1 network is distinct from how IL20 and IL24 are ficiently when compared with the response to IL-31 (Fig. 7). controlled. Anakinra reduced or inhibited the effects of both IL-1a and IL-31, providing further support for IL-1a being a downstream effector of IL-1a interferes with keratinocyte differentiation IL-31. Treatment of HaCaT-IL31RA and NHEK organotypic skin models with IL-1a inhibited filaggrin mRNA and protein expression Bifunctional effects of IL-31 on the epidermal barrier comparable to IL-31 and interfered with keratinocyte differenti- IL-31 prevents the formation of the physical barrier but stimulates ation (Fig. 5A, 5B). The IL-31 effects on filaggrin expression and the antimicrobial barrier, at least in part by activating IL-1 sig- keratinocyte differentiation were blocked by anakinra, a potent IL- naling. To evaluate whether the two effects can be separated, we 1R antagonist that inhibits the binding of IL-1 to IL-1R1 (28), titrated IL-31 and analyzed skin differentiation. A dose of 10 ng/ml indicating that IL-1a mediates at least part of the response to IL- IL-31 was enough to prevent filaggrin expression in organotypic 31 (Fig. 5B, 5C). Anakinra alone was also slightly increasing HaCaT-IL31RA models (Fig. 8A). Lower doses had minor or no filaggrin expression, possibly as a result of repressing basal ac- effects on the expression of FLG and INV and genes encoding

FIGURE 5. IL-1a is a down- stream effector of IL-31 signaling. (A) NHEK (one representative ex- periment) and HaCaT-IL31RA 3D models were stimulated with rhIL- 31 and rhIL-1a, and FLG expression was measured by qRT-PCR. (B) NHEK 3D models were treated with rhIL-1a, rhIL-31, and anakinra (Ana). Histological sections were stained with H&E for filaggrin (green) and the DNA with DAPI (blue). Scale bars, 200 mm(H&E) and 100 mm (immunofluorescence). (C) FLG expression in NHEK 3D models was analyzed by qRT-PCR; mean values 6 SD; n =3.(D) The indicated signaling molecules were analyzed on Western blots. The p values were calculated using Stu- dent t test. *p , 0.05, ***p , 0.001. The Journal of Immunology 7

doses have a profound effect on the physical barrier. The latter likely antagonizes enhancement of the antimicrobial barrier.

Discussion AD is a common chronic inflammatory disease characterized by a defect in keratinocyte differentiation and skin barrier formation (27). Consequences of the impaired skin barrier are TEWL and increased percutaneous penetration of allergens and pathogens, thereby promoting inflammation that can be further aggravated by secondary infections. Alterations in genes encoding key structural components for proper keratinocyte differentiation and skin bar- rier formation are associated with barrier dysfunction. A seminal finding was the identification of FLG mutations in patients with vulgaris and AD (9, 24, 39, 40). Also, cytokines, in- cluding IL-31, are dysregulated in AD and postulated to partici- pate in disease progression (29). Serum IL-31 expression is increased and correlates with disease severity in these patients (33–36). IL-31 is produced by immune cells that can either in- filtrate the skin or are resident in skin (30). Recently, it was shown that IL-4, a prominent cytokine in AD, induces IL-31 expression in Th1 cells (42) and downregulates FLG expression (46). It is possible that IL-31 is a downstream effector of IL-4 in controlling keratinocyte differentiation. Previously, we demonstrated that IL- 31 inhibits keratinocyte proliferation and differentiation without effects on , with FLG being a repressed target of IL-31 signaling (1). However, it remained open whether FLG is one of few genes that are deregulated in response to IL-31 or whether this cytokine has a broader effect on skin differentiation. Moreover, it was unclear whether IL-31 signaling was directly responsible for affecting FLG expression. We observed that IL-31 interferes with the expression of many genes associated with skin barrier formation in 3D organotypic A FIGURE 6. IL-31 regulates the expression of AMPs. ( ) qRT-PCR models of HaCaT-IL31RA cells (Supplemental Table I), whereas 6 analysis of the indicated genes stimulated with rhIL-31; mean values only a few genes were deregulated in monolayers (1). Indeed, SD; n =3.(B) The indicated proteins were measured in the supernatants of many genes encoding proteins associated with corneodesmosomes, 3D models by ELISA; mean values 6 SD; n =3.(C) Histological sections of HaCaT-IL-31RA were stained with H&E for the indicated proteins such as desmogleins or desmocolleins, with the formation of (green) and the DNA with DAPI (blue). Scale bar, 100 mm. The p values terminally differentiated and with the production of were calculated using Student t test. *p , 0.05, **p , 0.01. natural moisturizing factors, including filaggrin and its processing enzymes caspase-14 and kallikrein-like peptidase 7, were deregulated in response to IL-31 (Fig. 1). Altogether, our array desmosomal proteins (Fig. 8B, 8C), and were insufficient to pre- data identified ∼570 genes that were altered .2-fold in response vent differentiation (Fig. 8A). However, 1 ng/ml IL-31 was suf- to IL-31. Our results indicate that IL-31 has a profound effect on ficient to enhance the expression of IL1A and IL1B and of the the gene expression program that defines keratinocyte differenti- genes encoding several antimicrobial peptides (S100A8, S100A9, ation. The functional consequences of this reprogramming were S100A12, DEFB103A, and DEFB4A) in HaCaT-IL31RA cells confirmed by measuring keratinocyte differentiation in 3D organo- (Fig. 8D). To address the functional relevance of induced AMP typic and CsSSE models and by assessing the integrity of the expression, we cocultivated bacteria with HaCaT-IL31RA 3D skin barrier. The poor differentiation, reduced expression of late models. Antimicrobial activity against the pathogen S. aureus was differentiation markers, increased penetration of biotin and phl p1 evident both at low (1 ng/ml) and high (100 ng/ml) concentrations allergen into 3D models, high vulnerability to skin irritants, and of IL-31 compared with untreated control (Fig. 9). In organotypic enhanced TEWL all point to a severe disturbance of the physical HaCaT-IL31RA models, the addition of S. aureus inflicted severe and functional properties of the skin barrier (Figs. 2, 3). Thus, IL- damage to the skin model, resulting in the destruction of the 31 has multidimensional effects on keratinocyte behavior. In epidermis and infiltration of bacteria into the dermis (Fig. 9A–C). contrast to recent findings in which histamine treatment of 3D This was efficiently prevented when the models were treated with organotypic skin models results in a disturbed barrier formation IL-31. Quantification of the S. aureus–associated GFP fluores- and a downregulation of many differentiation-associated factors, cence demonstrated a significant reduction of infiltrating bacteria including desmosomal and tight junction proteins (6), IL-31 (Fig. 9D). In agreement with these observations, the analysis of showed a more selective effect. In particular, no altered expres- genomic GFP sequences as well as GFP mRNA indicated that sion of genes associated with tight junctions was measured. This bacterial growth was substantially inhibited (Fig. 9E, 9F). Thus, argues for different signaling pathways controlling different as- these findings demonstrate that even low doses of IL-31 have a pects of the differentiation process in keratinocytes. bacteriostatic effect on S. aureus, most likely due to the induction Of particular interest to us was the observation that many genes of several AMPs. Taken together, these findings demonstrate that encoding components of the IL-1 signaling network were affected low doses of IL-31 enhance the expression of genes encoding by IL-31, including increased IL-1a release (Fig. 4), supporting AMPs, thus promoting the antimicrobial barrier, whereas higher the concept that IL-1 signaling is downstream of IL-31. The 8 IL-31–IL-1 SIGNALING CONTROLS SKIN BARRIER

FIGURE 7. Regulation of AMPs by IL-31 is de- pendent on IL-1a induction. The indicated genes were analyzed by qRT-PCR. HaCaT-IL31RA cells were treated for 72 h; mean values 6 SD; n = 3. The p values were calculated using Student t test. *p , 0.05, **p , 0.01, ***p , 0.001. Ana, anakinra.

proinflammatory IL-1 signaling network is known to be an im- expression of IL-1a leads to the development of spontaneous in- portant mediator of innate immunity (62). Overactivation of the flammatory skin lesions in mice (72). In contrast to these findings, network, for example, due to mutations in inflammasome com- the intracutaneous administration of IL-1a was shown to improve ponents or as a result of IL-1RA deficiency can lead to auto- epidermal barrier function (16, 25) and results in the upregulation inflammatory diseases with severe skin involvement (68, 69). of genes associated with cell adhesion, proliferation, and differ- Indeed, increased secretion of IL-1a has been observed in lesional entiation (19). Our findings support the inhibitory role of IL-1a on skin of patients with psoriasis (70). Exposure of keratinocytes to keratinocyte differentiation and suggest that IL-1 signaling is an IL-1a provokes hyperkeratosis (71). Keratinocyte-specific over- important mediator of the effects of IL-31 (Fig. 5), suggesting that

FIGURE 8. Bifunctional role of IL-31 in human skin barrier formation. (A) HaCaT-IL31RA 3D models were stimulated with rhIL-31 for 10 d, and histological sections were stained with H&E or for filaggrin (green) and DNA with DAPI (blue). Scale bars, 200 mm (H&E) and 100 mm (immunofluorescence). (B and C)qRT- PCR analysis of the indicated genes in 10-d 3D HaCaT- IL31RA models stimulated with rhIL-31; mean values 6 SD; n =3.(D) qRT-PCR analysis of the indicated genes in HaCaT-IL31RA cells stimulated with rhIL-31; mean val- ues 6 SD; n =3.Thep values were calculated using Student t test. **p , 0.01, ***p , 0.001. The Journal of Immunology 9

FIGURE 9. IL-31 promotes the antimicrobial defense. HaCaT- IL31RA 3D models (10 d) were treated with or without IL-31 as in- dicated. Subsequently, S. aureus– expressing GFP were applied to the apical surface and incubated for the times displayed. The models were harvested and prepared for stainings and DNA or RNA extraction. (A) Sections were stained with H&E. (B) Gram staining of histological sections. (C) S. aureus was visual- ized in sections using an S. aureus– specific Ab (red). The DNA was stained with DAPI (blue). Scale bars, 100 mm(A–C). (D) Quantifi- cation of S. aureus fluorescence, which was measured from three sections using the ImageJ software (National Institutes of Health). (E) DNA was prepared and GFP mea- sured using quantitative PCR, - tive bacterial growth was calculated, and mean values 6 SD of three ex- periments are shown. (F) GFP mRNA was measured using qRT- PCR. The relative bacterial growth was calculated, and mean values 6 SD of three experiments measured in triplicates are displayed. The p values were calculated using Stu- dent t test. *p , 0.05, **p , 0.01, ***p , 0.001.

this cascade of cytokines controls skin differentiation. Moreover, the skin of AD patients compared with healthy controls (2, 5, 80– these signaling pathways offer entry sites for crosstalk with other 84). This finding seems to be at odds with the observations of signals that might influence differentiation. It is also of interest increased superinfection with bacteria in patients suffering from that FLG loss-of-function mutations are associated with enhanced AD (5, 8, 9, 85, 86). However, the increased availability of AMPs IL-1a and IL-1b expression (63). This finding suggests that the in these washings may not reflect the total amounts. More likely, lack of filaggrin stimulates IL-1 signaling that in turn may ag- the reduced or the absence of a lipid barrier in these patients gravate the consequences of the genetic defect. prevents the attachment of AMPs to the epidermal layer and thus IL-31 also modulated the expression of genes encoding different allows more efficient extraction. Furthermore, the reduction of AMPs (Fig. 5). These effects were at least in part dependent on the desmosomal proteins and the reduction of filaggrin and 1 activation of the IL-1 signaling network as they were inhibited by and 10 weaken the skin barrier, leading to an increased penetration anakinra (Figs. 5, 7). IL-1a has been known to regulate AMP of bacteria into the skin. This is further complicated by the fact expression in several cell types, including keratinocytes (25, 64– that persistent, relapsing, and difficult-to-treat S. aureus infections 67, 73). Expression and secretion of AMPs by keratinocytes are associated with the formation of a slow-growing small colony represent the first line of defense against cutaneous pathogens (18, variant phenotype (87, 88). Bacteria associated with small colony 20). Corneocytes are embedded in an acidic lipid envelope that variant phenotype show reduced susceptibility toward AMPs (9, largely inhibits the growth of various microbes (3, 5, 74, 75). 18, 20, 21, 89). Of note also is that different cytokines associated There are at least three groups of AMPs expressed in the skin. In with the complex cytokine milieu of AD lesions may have antago- the first group, AMPs like hBD-1 are constitutively expressed (7, nistic effects on the expression of AMPs by keratinocytes. For ex- 8, 18). Members of the second group that includes psoriasin/ ample, the Th2 cytokines IL-4, IL-13, and IL-33 have been shown to S100A7 are present in normal skin and further induced in re- inhibit the induction of AMPs by TNF-a and IFN-g (90). Thus, the sponse to skin inflammation and wounding (2, 76). Members of positive consequence of IL-31 on the antimicrobial barrier, as the third group, for example, hBD-2, hBD-3, and LL-37, are only reported in this study, is most certainly modified by other factors, detectable in inflamed skin (2, 77–79). Increased amounts of the with many potentially reverting or antagonizing the IL-31 effect. AMPs psoriasin, RNase-7, hBD-2, and hBD-3 were found in We have observed concentration-dependent differences in the lesional and nonlesional skin and fluids obtained in washings from effects of IL-31 on the barrier formation and the induction of AMPs 10 IL-31–IL-1 SIGNALING CONTROLS SKIN BARRIER

(Figs. 6–8). Low doses of IL-31 promote the antibacterial barrier the IL-31 and the IL-1 signaling networks promote a feedback loop without compromising the physical barrier. We suggest that this is that can aggravate a proinflammatory environment. Our results also a potentially important physiological role of IL-31. This is sup- indicate that pharmacological modulation of IL-31 and IL-1a ported by the fact that the IL-31RA is upregulated by S. aureus activity in AD and possibly other skin diseases might be thera- a-toxin and staphylococcal enterotoxin B in (91), peutically beneficial. suggesting a role in innate immunity. Furthermore, PBMCs of patients with AD secrete significantly more IL-31 compared with Acknowledgments those of healthy controls upon stimulation with a-toxin and We thank Juliane Luscher-Firzlaff€ for helpful discussions during the early staphylococcal enterotoxin B (26, 92). This regulation seems to be phase of the project, Bernd Denecke and Sebastian Huth for help with specific, and IL31 RNA is not upregulated in response to herpes bioinformatics, and Oleg Krut and Lothar Rink for the staphylococcus simplex or influenza infection (30). The expression of IL- aureus strain stably expressing GFP. 31RA in keratinocytes is also stimulated by the inflammatory cytokine IFN-g and activation of the TLR2 receptor (30, 93). The Disclosures activation of the IL-31 signaling network by different bacteria and The authors have no financial conflicts of interest. the subsequent production and release of different AMPs by keratinocytes is of clinical interest. It is known that eccrine sweat, References a complex mixture of minerals, proteins, and proteolytic enzymes, 1. Cornelissen, C., Y. Marquardt, K. Czaja, J. Wenzel, J. Frank, J. Luscher-Firzlaff,€ contains IL-31. Although sweat is not harmful to healthy skin, it B. Luscher,€ and J. M. Baron. 2012. 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