Control of the Physical and Antimicrobial Skin Barrier by an IL-31–IL-1 Signaling Network
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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 keratinocyte differentiation is IL-31. To identify its molecular targets, IL-31–dependent gene expression was determined in three-dimensional organotypic skin models. IL-31–regulated genes 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 cytokine 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: 3233–3244. he skin is the largest organ of the human 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 epidermis, 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 atopic dermatitis (AD), a highly pruritic skin disor- cross-linked to and induce bundling of keratin filaments. This to- der, are characterized by impairment of skin barrier function, in- gether with other proteins 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 stratum corneum (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 desmosomes uation of skin barrier functions and the contribution of inflam- to corneodesmosomes (3, 5, 8, 9, 14, 15), the formation of an lipid matory cytokines 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 protein 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 Biochemistry and Molecular Biol- for Biotechnology Information’s Gene Expression 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, hypoxanthine guanine 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 3234 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 mutations 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 keratinocytes (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 receptor 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 Cancer 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