The role of IL-31 in skin barrier formation

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der RWTH Aachen University zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigte Dissertation vorgelegt von

Master of Science Biotechnologie Kai Herbert Hänel aus Neuss

Berichter: Universitätsprofessor Dr. rer. nat. Bernd Lüscher apl. Prof. Dr. rer. nat. Dirk Ostareck

Tag der mündlichen Prüfung: 26.05.2015

Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar. II

No man should escape our universities without knowing how little he knows.

J. Robert Oppenheimer

Parts of this thesis were prepublished

Hänel, K.H., Cornelissen, C., Lüscher, B., and Baron, J.M. (2013). and the Skin Barrier. International Journal of Molecular Sciences, 14(4), 6720–6745.

Hänel, K.H ., Cornelissen, C., Amann, P.M., Marquardt, Y., Czaja, K., Kim, A., Bickers, D.R., Lüscher, B. and Baron, J.M. Control of the mechanical and antimicrobial skin barrier by an IL-31 – IL-1 axis (manuscript submitted) III

Abstract

The skin is the largest organ in the human body and its major function is to protect from harmful environmental influences and to prevent dehydration. It forms the first barrier against biological, chemical and physical stress. (AD) is a relapsing inflammatory skin disease. Hallmarks of AD are pruritic, eczematous erythematous plaques resulting in reduced skin barrier function, increasing transepidermal water loss and, affected by this, dry skin. AD commonly begins in early childhood and affects 10-20% of children and 1-3% of adults in industrialized countries. AD patients show significant higher levels of IL-31 in serum and increased IL31 mRNA levels in lesional skin samples. These higher levels are correlated with serum IgE, disease severity, and subjective intensity. In previous work it was observed that IL-31 interferes with keratinocyte differentiation and proliferation. The differentiation defect was associated with a profound repression of terminal differentiation markers, including filaggrin, an essential factor for skin barrier formation, and a reduced lipid envelope. To identify its molecular targets, IL-31-dependent expression was determined in 3-dimensional organotypic skin models in this work. IL- 31 regulated were 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 subcutaneous administration of IL-31. The IL-1 network was identified as a downstream effector of IL-31 signaling. , an IL-1 receptor 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. This was evident already at low doses of IL-31, which were insufficient to interfere with the physical barrier. Moreover IL-31 induced the expression and release of the itch mediator TSLP in human 3- dimensional organotypic skin models.

Together these findings demonstrated 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. Therefore when targeting IL-31 for therapeutic purposes it needs to be considered that low doses of IL-31 promote the antimicrobial barrier and thus a complete inhibition of IL-31 signaling may be undesirable. IV

Zusammenfassung

Die Haut ist das größte Organ des menschlichen Körpers und seine Hauptfunktion ist es, vor schädlichen Umwelteinflüssen zu schützen und Austrocknung zu verhindern. Die Haut bildet die erste Barriere des Körpers gegen biologische, chemische und physikalische Belastungen. Atopische Dermatitis (AD) ist eine rezidivierende, entzündliche Hauterkrankung. Kennzeichen der AD sind juckende, gerötete Plaques, erhöhter transepidermaler Wasserverlust, eine verringerte Hautbarriere und dadurch verursacht, trockene Haut. AD beginnt häufig in der frühen Kindheit und betrifft 10-20% der Kinder und 1-3% der Erwachsenen in den Industrieländern. AD-Patienten zeigen deutlich erhöhte IL-31 Serum Werte und ebenfalls erhöhte IL31 -mRNA-Level in Proben lesionaler Haut. Diese erhöhten IL-31 Level korrelieren mit dem Serum-IgE, der Schwere der Erkrankung und der Intensität des Juckreizes. In früheren Arbeiten wurde beobachtet, dass IL-31 die Keratinozyten-Differenzierung und Proliferation stört. Der Defekt in der Differenzierung ist mit einer Unterdrückung der terminalen Differenzierungsmarker und einer reduzierten Lipidhülle verbunden. Um neue molekulare Ziele zu identifizieren, wurde in dieser Arbeit die IL-31-abhängige Genexpression in 3-dimensionalen organotypischen Hautmodellen untersucht. IL-31 regulierte Gene, welche an der Bildung einer intakten physikalischen Hautbarriere beteiligt sind. Viele dieser Proteine waren als Folge der IL-31-Behandlung während der Differenzierung geringer exprimiert als ohne IL-31, was zu einer erhöhten Penetrierung von Allergenen und Reizstoffen führte. Darüber hinaus zeigten Experimente mit Zellsortierten Hautäquivalenten in SCID / NOD-Mäusen einen erhöhten transepidermalen Wasserverlust nach subkutaner Verabreichung von IL-31. Das IL-1- Zytokin-Netzwerk wurde als ein Downstream-Effektor des IL-31-Signalwegs identifiziert. Anakinra, ein IL-1-Rezeptor-Antagonist, blockierte effektiv den oben beschriebenen Einfluss von IL-31 auf die Differenzierung. Zusätzlich zu der Wirkung auf die physische Barriere stimulierte IL-31 die Expression von antimikrobiellen Peptiden. Dies zeigte sich bereits bei niedrigen Dosen von IL-31, welche nicht ausreichten, um die physische Barriere zu beeinträchtigen. Außerdem induzierte IL-31 die Expression und die Freisetzung des Zytokins TSLP in 3-dimensionale organotypischen Hautmodellen. Dieses Zytokin wurde durch andere Arbeitsgruppen als ein Vermittler von Juckreiz identifiziert.

Zusammengefasst zeigen diese Ergebnisse, dass IL-31 die Differenzierung der Keratinozyten in vielfältiger Weise beeinflusst und dass das IL-1-Zytokin-Netzwerk ein Downstream -Effektor des IL-31-Signalwegs in der Deregulierung der physikalischen V

Hautbarriere ist. Bei der Inhibierung von IL-31 als ein neu identifizierter Angriffspunkt zur Wirkstoffentwicklung in atopischen Hautkrankheiten wird zu berücksichtigen sein, dass niedrige Dosen von IL-31 die antimikrobielle Barriere fördern und deswegen eine vollständige Hemmung der IL-31 Signalwegs unerwünscht sein kann. VI

Table of contents

1 Introduction ...... 8 The human skin ...... 9 The cornified envelope ...... 10 Atopic Dermatitis...... 12 The human Skin microbiome ...... 13 The skin microbiome in atopic dermatitis ...... 15 Antimicrobial peptides in human skin ...... 15 Human defensins ...... 16 S100 Family ...... 20 Cytokines and the skin barrier ...... 23 Cytokine Families ...... 23 Concluding Remarks ...... 34 Aim of this work ...... 37

2 Results ...... 38 IL-31 controls the expression of genes associated with the mechanical barrier ...... 39 IL-31 weakens the skin barrier ...... 41 IL-31 promotes transepidermal water loss in mouse model ...... 43 IL-31 stimulates the IL-1 network ...... 45 IL-1 network is not affected by IL-20 and IL-24...... 49 Role of IL-1α in IL-31 signaling ...... 50 IL-1α affects the skin barrier ...... 50 Regulation of antimicrobial peptides by IL-31 is dependent on IL-1α induction ...... 53 IL-1α regulates the expression of AMPs ...... 53 IL-31 influences the expression of AMPs ...... 54 Regulation of antimicrobial peptides by IL-31 is dependent on IL-1α induction ...... 57 Role of low IL-31 doses in human skin barrier formation...... 58 IL-31 promotes the expression and release of TSLP ...... 60

3 Discussion ...... 63 Role of IL-31 in Atopic Dermatitis ...... 64 IL-31 weakens the skin barrier ...... 65 IL-31 stimulates the IL-1 network ...... 69 Regulation of antimicrobial peptides by IL-31 is dependent on IL-1α induction ...... 73 Bi-functional role of IL-31 in human skin barrier formation ...... 75 IL-31 promotes the expression and release of TSLP ...... 76 VII

4 Conclusion and Perspectives ...... 79

5 Material and Methods ...... 82 Experimental procedures ...... 83 Consumables and Reagents ...... 83 Inhibitors and cytokines ...... 88 Cell lines ...... 88 Methods ...... 89 Methods in molecular biology ...... 89 Methods in cell biology ...... 91 Methods in protein biochemistry ...... 94

6 References ...... 98

7 Appendix ...... 122 List of figures ...... 123 List of tables ...... 123 Abbreviations...... 124 CV ...... 127 Scientific Contribution ...... 128 Eidesstatliche Versicherung ...... 129 Danksagung ...... 130 INTRODUCTION 8

1 INTRODUCTION INTRODUCTION 9

THE HUMAN SKIN

The skin is the largest organ in the human body and its major function is to protect from harmful environmental influences and to prevent dehydration. The skin barrier consists mainly of differentiating epidermal keratinocytes. It forms the first barrier against, biological, chemical and physical stress. The skin is composed of three different layers, epidermis, dermis and hypodermis.

The epidermis is the outermost layer and first defense organ of the human body to the environment. To maintain this function keratinocytes undergo different developmental stages from cycling keratinocytes in the stratum basale (SB), through differentiating cells in the stratum spinosum (SS) and the stratum granulosum (SG) to finally culminate into anucleated and flat corneocytes surrounded by cross-linked and extruded lipids in the stratum corneum (SC). These are highly differentiated, nucleus-free cells that form an insoluble and rigid structure referred to as the cornified envelope (CE). In Figure 1 the epidermal barrier and the processes, which are needed to develop an appropriate barrier, are summarized. The correct formation and desquamation of the CE is essential for the barrier function of the skin (Boniface et al., 2007; Proksch et al., 2008). Thus, all components, which are involved, need to be highly regulated in a differentiation-associated manner, i.e. their timely and quantitative expression, to ensure the proper development of the epidermal barrier.

INTRODUCTION 10

THE CORNIFIED ENVELOPE

Proteins of the mammalian epidermis consists to 80-90% of keratins and filaggrin (Roop, 1995; Nemes and Steinert, 1999). The large profilaggrin (>400kDa) is processed to monomeric filaggrin that aggregates with keratin filaments into bundles during differentiation. Subsequently filaggrin is completely proteolyzed into single amino acids by a variety of proteases, including caspase 14 (Denecker et al., 2008). These single amino acids are responsible for the hydration of the stratum corneum and thus inhibit transepidermal water loss (TEWL). During cornification other proteins including involucrin, loricrin, trichohyalin and small proline-rich proteins are crosslinked by either disulphide bonds or lysine-isopeptide bonds, which are formed by various transglutaminases (Nemes and Steinert, 1999). These cross links provide a rigid structure and build an insoluble barrier. Tight junctions (TJ) and corneodesmosomes connect the anucleated corneocytes, these side-by-side connections are important for the cohesion of the CE. TJ are cell–cell junctions that connect neighboring cells. They control diffusion of molecules through the epithelium and thus build a barrier. Furthermore TJ separate the apical from the basolateral part of a cell membrane, which prevent diffusion of membrane proteins. Due to this TJ allow the specialized functions of each cell surface. In human epidermis, various TJ proteins have been identified, including occludin, members of the claudin family (1, 4, and 7), JAM-1 (junctional adhesion molecule-1), ZO-1 (zonula occludens protein 1) and MUPP-1 (multi-PDZ protein-1) (Pummi et al., 2001; Brandner et al., 2002; Langbein et al., 2002; Brandner et al., 2003; Wilke et al., 2006. 2006).

INTRODUCTION 11

TEWL Allergen Irritant UV Pathogens

Lipid Envelope Cornified Envelope

Desquamation Cornified Keratinocytes (Corneocytes)

Stratum Cornified Corneum Envelope

Lipid Extrusion Filaggrin/ Late Corneo- and Cross- Keratin Differentiating desmosomes linking Network Keratinocytes Stratum

Granulosum Loricrin Synthesis

Lipid Early Profilaggrin Desmosome Synthesis/ Differentiating Synthesis Maturation Processing Keratinocytes

Stratum Involucrin Spinosum Synthesis

Keratin Proliferating Desmosomes Synthesis Keratinocytes Basale Stratum

Figure 1: Schematic representation of the differentiation process undergoing in keratinocytes. Keratinocytes differentiate from a proliferating state in the basal layer (Stratum basale) to dead corneocytes in the outermost layer (Stratum corneum). During this differentiation process the lipid envelope, the filaggrin / keratin network, and the cornified envelope is formed and desmosomes mature to corneodesmosomes. Together these components form a compact barrier against the outside preventing entry of harmful components, for example allergens, pathogens, irradiation and other irritants, into the skin and body. Furthermore the barrier inhibits the trans-epidermal water loss (TEWL) and associated loss of solutes.

Within the CE, the strong cohesion of corneocytes is provided by corneodesmosomes. These are cellular junctions derived from desmosomes. Desmogleins (DSG) 1-4, desmocollins (DSC) 1-3 and corneodesmosin (CDSN) are components of the extracellular part of corneodesmosomes in the human epidermis and are responsible for cell-cell connection. Intracellular parts of DSC and DSG are connected to a plaque formed by two different families of desmosomal proteins, the armadillo repeat protein family, with plakoglobin and plakophillin, and the plakin family with desmoplakin, envoplakin, periplakin and plectin. These plaques are connected to the intermediate filament cytoskeleton of the cell and by these build a rigid structure. During differentiation the composition of desmogleins and desmocollins changes within the epidermal layer. From DSG 2 and 3 in the stratum basale to DSG 1 and DSG 4 in the upper stratum granulosum. Moreover DCS 1 replaces DSC 3 during the differentiation and their number and size rise significantly in the spinous layers and decreases in the granular compartment (Niessen, 2007). INTRODUCTION 12

The intercellular space between the tightly bound corneocytes is filled with a mixture of lipids, which have originated from within the golgi apparatus and are transported by lamellar granules. These bodies fuse during the late differentiation process with the plasma membrane and subsequently secrete their cargo into the intercellular space. Lipids secreted from the lamellar bodies are modified and arranged. The final lipid composition consist of a mixture of ceramides, cholesterol and free fatty acids besides several other lipids (Madison, 2003). This lipid composition serves as a water barrier and is required for an effective skin barrier function.

ATOPIC DERMATITIS

Atopic dermatitis (AD, ‘‘eczema,’’ ICD-10: L20, OMIM ®: 603165) is a relapsing inflammatory skin disease. Hallmarks of AD are pruritic, eczematous erythematous plaques resulting in reduced skin barrier function, increasing TEWL and, caused by this, dry skin (Boniface et al., 2007; Guttman-Yassky et al., 2011; Proksch et al., 2008). AD commonly begins in early childhood and affects 10-20% of children and 1-3% of adults in industrialized countries (Gazel et al., 2006; Bieber, 2010). 85% of the affected individuals develop AD in the first five years of life (Kay et al., 1994) and nearly 80% of children suffering from AD develop rhinitis or asthma during lifetime (Eichenfield et al., 2003). This aggravation of atopic manifestations is also known as the “atopic / allergic march”. This disease development suggest that epicutaneous allergen sensitization may predispose people to atopic respiratory diseases (Leung and Bieber, 2003; Palmer et al., 2006; Sandilands et al., 2007). Histological analysis of acute and chronic atopic dermatitis skin shows decreased cornification and decreased expression of terminal differentiation proteins as well as decreased lipids. On a cellular basis AD is also characterized by an increased number of eosinophils and mast cells in the dermis and a lack of neutrophils in the epidermis (Leung and Bieber, 2003).

INTRODUCTION 13

The reasons for the development of AD are not yet fully understood. AD is a genetically complex disease with many chromosomal regions containing pathophysiology relevant genes. The gene encoding filaggrin has been identified as a major locus causing skin barrier deficiency (Palmer et al., 2006), along with eleven other risk loci in European individuals (Ellinghaus et al., 2013). Interestingly not all patients suffering from AD show a genetic defect within one of the known risk loci. This led some researchers to the conclusion that besides genetic factors AD is triggered by immunological factors. This implication can be strengthened by the fact that nearly 40% of children with AD have skin rashes after the uptake of food allergens (Sampson, 1999) and furthermore skin lesions can develop after challenge with aeroallergens in sensitized patients with AD (Tupker et al., 1996).

THE HUMAN SKIN MICROBIOME

The human skin as interface to the environment is permanently in contact with microorganisms but usually healthy human skin does not show sign of bacteria infections. The human skin houses a plenteous and diverse population of bacteria, fungi, and viruses. By 16S RNA gene phylotyping, 19 phyla are known to be part of the bacterial skin microbiome. The four phyla, Actinobacteria (51.8%), Firmicutes (24.4%), Proteobacteria (16.5%), and Bacteroidetes assigned together 99% of the identified sequences. Moreover the majority (62%) of the 205 identified genera were part of the Corynebacterium , Propionibacterium and Staphylococcus genera (Grice et al., 2009). The skin flora varies between skin sites, which provide many different niches for large populations of microbes. These niches differ in humidity, temperature, pH, and the composition of antimicrobial peptides and lipids. Furthermore discrete niches are formed by skin structures like hair follicles and eccrine glands (Grice et al., 2008). Moreover the skin flora also differs between individuals (Schommer and Gallo, 2013), shown by a genomic approach in which 129 males and 113 females were tested at different body sites. The approach also revealed that the diversity within samples from one subject (alpha-diversity) is different from the INTRODUCTION 14 diversity between samples from the same sample site among other subjects (beta diversity) (Human Microbiome Project Consortium, 2012). The composition of resident microorganisms varies qualitatively and quantitatively but is stable over time. However, the mechanism responsible for this balance remain largely unknown (Costello et al., 2009; Human Microbiome Project Consortium, 2012).

The interaction of microbes with a host can be divided into three different categories. The interaction can either have a positive, a negative or no effect on involved subjects. A lot of environmental factors including community, diet, hygiene products, lifestyle, stress and the uptake of drugs influence the composition of the skin microbiome. Besides these, also age and genetic predisposition play an important role for the microbiome (Schommer and Gallo, 2013). All these factors can influence the host-microbe and also the microbe- microbe interaction on the skin. A shift of microbial population that alter host–microbiome interactions has been associated with disease manifestations, regardless which factor has affected the change (Srinivas et al., 2013; Kong et al., 2012; Fry et al., 2013). One step further, a potential dysfunction of the capacity to establish the normal microbiome, may promote the development to a chronic disease (Schommer and Gallo, 2013).

INTRODUCTION 15

THE SKIN MICROBIOME IN ATOPIC DERMATITIS

As discussed above the reduced skin barrier in AD patients lead to an effortless penetration of bacteria and viruses. Indeed patients suffering from AD have an increased bacterial colonization and are particularly prone to infections with Staphylococcus aureus and viruses such as herpes and vaccinia (Leung, 2013; Kim et al., 2013). The alteration of the skin microbiome may be caused by the dysfunction of the skin barrier. Findings in patients with AD demonstrate that a drastic change in the microbiome occurs during AD development compared to healthy control patients (Kong et al., 2012; Hata and Gallo, 2008). Interestingly, AD therapy is associated with changes in bacterial diversity. In particular the pathogen S. aureus ’ proportion is increased during disease flares compared to baseline or post-therapy. Yet another member of the Staphylococcus family, S. epidermidis , spreads on the skin during flares. Still increases in Streptococcus , Propionibacterium , and Corynebacterium species could also be observed following therapy (Kong et al., 2012).

ANTIMICROBIAL PEPTIDES IN HUMAN SKIN

Antimicrobial peptides (AMPs) are small peptides with a size between 12 and 50 amino acids with broad activity against bacteria, viruses and fungi. The group of AMPs is very heterogeneous with different modes of action in killing pathogens. The characteristics of AMPs mentioned in the following including expression pattern, antimicrobial targets and their chemo-attractant properties are summarized in Table 1. The first AMP was isolated from the skin of the African claw frog which was called “magainin” in 1987 (Zasloff, 1987). Keratinocytes of the skin produces a variety of antimicrobial peptides. Structures of the groups mentioned in the following are schematically represented in Figure 2, namely the S100 family, members of the human beta defensins. Besides their differences in structure, the expression patterns of AMPs also vary. Some of the antimicrobial peptides are constitutively expressed in the epidermis and not induced in inflammatory conditions; a INTRODUCTION 16 second group is constitutively expressed in the skin but additionally induced upon . The third group of AMPs is only present in inflammatory skin but not present in healthy human skin.

A SP Pro region AMP

B N -helix -helix -helix -helix C

Figure 2: Representation of structural elements of human antimicrobial peptides in human skin. A. Human defensins are synthesized as pre‐pro peptides that are cleaved to release their antimicrobial peptides (AMPs) that interact with negatively charged bacterial surface moieties. Dotted lines indicate disulfide bonds. SP, Signaling peptide; B. S100 protein have two helix-loop-helix motifs which are responsible for the binding of Ca2+. S100 proteins can form homo- or heterodimers as well as oligomers that change their conformation upon calcium binding.

HUMAN DEFENSINS

The family of defensins contains small cationic peptides with six to eight cysteine residues. These residues form disulfide bonds, which are responsible for the division into subfamilies: α-defensins, β-defensins and θ-defensins. The six members of the α-defensin subfamily are primarily expressed in neutrophils, natural killer cells and certain T-cell subsets (Zhao and Lu, 2014). β-defensins, a subfamily with 26 members, are expressed and secreted by many kinds of epithelial cells as well as by leukocytes (Weinberg et al., 2012). The last group is not present in humans, chimpanzees and gorillas but was found to occur in old world monkeys such as rhesus macaques (Li et al., 2014). Due to the fact that only the human β-defensins are expressed by keratinocytes only these defensins are described in the following.

INTRODUCTION 17

Human β-defensin-1

The human β-defensin 1 (hBD-1), a 36 amino acid basic peptide, was the first discovered human β-defensin originally isolated from human blood filtrate in 1995 (Bensch et al., 1995). The protein is encoded by the DEFB1 gene (Bensch et al., 1995). Later it was found in suprabasal keratinocytes, sweat ducts, and sebaceous glands of human skin (Fulton et al., 1997; Ali et al., 2001). Either overexpression of DEFB1 lead to increased expression of keratinocyte differentiation marker proteins (Frye et al., 2001) or differentiation of keratinocytes by Ca 2+ lead to upregulation of DEFB1 gene expression (Abiko et al., 2003; Frye et al., 2001; Harder et al., 2004). Proinflammatory cytokines such as IL-1α, or bacteria e.g. Pseudomonas aeruginosa do not induce hBD-1 expression in keratinocytes (Harder et al., 2004). HBD-1 shows activity against E. coli and P. aeruginosa (Singh et al., 1998). Micromolar concentrations of recombinant hBD-1 are needed to kill various clinical strains of E. coli (Singh et al., 1998) and a concentration of 1 μg/ml recombinant hBD-1 is required to kill 50% of P. aeruginosa . In comparison native hBD-1 shows only minor antimicrobial activity (Zucht et al., 1998). Generally no activity against gram-positive bacteria such as the pathogen S. aureus has been reported yet. Besides that, hBD-1 is also able to recruit immune cells such as immature DC and memory T-cells to sites of invasion via CCR6 receptor signaling and thus modulate the adaptive immune response (Yang et al., 1999).

Human β-defensin-2

In 1997, the second member of the human β-defensin family was isolated from lesional psoriatic scale extracts using an E. coli affinity column and is encoded by the DEFB4A gene (Harder et al., 1997). In contrast to hDB-1 expression, human β-defensin-2 (hBD-2) expression is induced in inflamed regions of the skin and is not detected in non-inflamed regions. Proinflammatory cytokines such as IL-1α, IL-1β and, TNF-α are effective DEFB4A inducers (Huh et al., 2002; Sørensen et al., 2003; Liu et al., 2002; Harder et al., 1997). Furthermore IL-22 has been detected as a potent stimulus for DEFB4A expression (Wolk et al., 2004). In addition, bacteria such as P. aeruginosa or INTRODUCTION 18

Fusobacterium nucleatum stimulate the expression of hBD-2 (Schroder and Harder, 1999). In IL-1α stimulated keratinocytes hBD-2 could be detected in lamellar bodies and the intercellular space. By the release into the intracellular space within the epidermis hBD-2 can accumulate to high local concentrations (Oren et al., 2003). The concentration of hBD-2 in IL-1α stimulated keratinocytes were estimated in a range of 3.5-16 µM (Liu et al., 2002). Consistent with that, in psoriatic scales of human skin hBD-2 concentrations of 2-10 µM were determined (Harder and Schröder, 2005).

Like hBD-1, hBD-2 shows activity against gram-negative bacteria such as E. coli and P. aeruginosa . The concentration needed to kill 90% of all bacteria (LD 90) was estimated with 10 µg/ml. For killing of 90% of the yeast Candida albicans 25 µg/ml were needed and against the gram-positive pathogen S. aureus hBD-2 has only bacteriostatic effects at concentration greater than 100 µg/ml (Singh et al., 1998; Tomita et al., 2000; Liu et al., 2002; Harder et al., 1997). Besides its antimicrobial activity, hBD-2 is a also chemoattractant for TNF-α-induced neutrophils and it can also recruit mast cells via a phospholipase C-dependent pathway (Niyonsaba et al., 2002).

Human β-defensin-3

The third member, hBD-3, was also isolated from psoriatic scales and the DEFB103A gene was cloned in 2001 from keratinocytes and lung epithelial cells (Harder et al., 2001; García et al., 2001; Jia et al., 2001). The highly basic AMP (pI 10.08) with 67 amino acids and a 22 amino acid precursor is 43% identical to hBD-2 (García et al., 2001; Jia et al., 2001; Harder et al., 2001). DEFB103A is expressed in a variety of tissues, such as keratinocytes (García et al., 2001; Jia et al., 2001; Harder et al., 2001). TNF-α induces hDB-3 at a low level compared to the induction that IFN-γ achieves (Jia et al., 2001; García et al., 2001; Nomura et al., 2003). Interestingly, IL-1 and IL-6 lead to a further increased expression of DEFB103A during inflammation by modulating the transactivation of the EGF-receptor, even though these cytokines do not induce DEFB103A expression directly (Sørensen et al., INTRODUCTION 19

2005). DEFB103A gene expression is also induced by bacterial components (García et al., 2001; Harder et al., 2001).

Table 1: Properties of skin derived AMPs Properties of skin derived AMPs

Name Antimicrobial activity Chemotactic Expression Expression Gram Gram Fungi properties pattern Stimuli + - Human beta defensin family

constitutively Ca 2+ hBD-1 + + ? T-cells, DC expressed in differentiation skin

IL-1α, IL-1β, T-cells, DC, induced upon TNF-α, IL- hBD-2 + + + MC inflammation 22, bacterial components

TNF-α, IFNγ, T-cells, DC, induced upon hBD-3 + + + bacterial inflammation components

Ca 2+ binding S100 family

TNF-α, IL- T-cells, induced upon S100A7 + + ? 1β, bacterial neutrophils inflammation components

UVB, IL-1α, IL-6, IL-8, S100A8 neutrophils induced upon + + + TNF-α, inflammation S100A9 bacterial components

monocytes induced upon TNF-α, LPS S100A12 ? ? ? neutrophils inflammation

Compared to hBD-1 and hBD-2, hBD-3 exhibits antimicrobial activity against S. aureus , including multiresistant strains (MRSA). Moreover, hBD-3 exhibits a broad spectrum of antimicrobial activity against many potentially pathogenic gram-negative and gram- positive bacteria and fungi (García et al., 2001; Harder et al., 2001; Hoover et al., 2003; Maisetta et al., 2003; Sahly et al., 2003). HBD-3 kills S. aureus by perforation of the cell INTRODUCTION 20 wall. The plasma membrane is resolved within 30 minutes and bacteria are totaly lysed after 2h of contact. These effects remind of those seen when S. aureus is treated with penicillin (Giesbrecht et al., 1998; Harder et al., 2001). As shown by other members of the hBD family hBD-3 induces chemotaxis of immune cells. HBD-3 recruits monocytes and HEK 293 cells transfected with CCR6 (Wu et al., 2003).

S100 PROTEIN FAMILY

The S100 protein family is characterized by two calcium-binding sites that have helix- loop-helix conformation. They have more functions than to act as antimicrobial peptide, for example they are involved in the regulation of Ca 2+ homeostasis, cell growth, differentiation, and inflammatory response. This low molecular weight protein family is widely expressed in cells and tissues (Heizmann et al., 2002; Donato, 2003). Many of the 21 members of the S100 family are encoded on 1q21 where also the epidermal differentiation complex (EDC) is located (Hardas et al., 1996; Wicki et al., 1996; Volz et al., 1993). All S100A family members from S100A2 to S100A12 and S100A15 are expressed in the human epidermis or in cultured keratinocytes (Böni et al., 1997; Xia et al., 1997; Broome et al., 2003; Wolf et al., 2003).

S100A7 (Psoriasin)

The 11 kDa protein psoriasin was originally discovered in psoriatic skin lesions as a new Ca 2+ -binding S100 protein of unknown biological function (Madsen et al., 1991). Psoriasin is distributed in the cytoplasm of keratinocytes. During differentiation it accumulates and localizes to the cell periphery of terminally differentiated keratinocytes (Broome et al., 2003). Furthermore, sebocytes, the lipid-secreting cells of sebaceous glands, are also a source of psoriasin (Gläser et al., 2005). Psoriasin is secreted in vivo by the human body with different concentrations at different sites. The highest concentrations can be found at the forehead, armpit, palm, and at the sole of feet (Gläser et al., 2005). These are all areas INTRODUCTION 21 where high bacterial colonization is present. Further data indicates that the highly hydrophobic psoriasin is also stored in the lipid layer of healthy skin (Gläser et al., 2005). The proinflammatory cytokines TNF-α and IL-1β trigger the expression of psoriasin in human keratinocytes. In addition the contact of keratinocytes with E. coli induces the expression and secretion of psoriasin. Psoriasin shows antimicrobial activity against the gram-negative bacteria E. coli with LD 90 of 0.5 µM but against P. aeruginosa considerably less active. Similarly psoriasin has only a low activity with an LD 90 >30 µM against the gram-positive S. aureus (Gläser et al., 2005). This is in line with a minor activity against the commensal Staphylococcus epidermidis (Gläser et al., 2005). No morphological changes of E. coli's cell walls could be observed by electron microscopy after the treatment with psoriasin. This indicates that psoriasin acts differently from many cationic antimicrobial peptides (Zasloff, 2002). It is suggested that psoriasin kills E. coli by the sequestration of zinc (Gläser et al., 2005).

S100A8 and S100A9

S100A8 and S100A9 were found in the synovial fluid of patients with rheumatoid arthritis in 1987 (Odink et al., 1987). S100A8 and S100A9 form homo- as well as heterodimers and are expressed at very low levels in normal human epidermis (Teigelkamp et al., 1991). Both proteins are overexpressed in psoriasis and are present in the basal, granular and spinous layers (Broome et al., 2003). Additionally they are induced during inflammatory bowel disease (Lügering et al., 1995; Schmid et al., 1995). S100A8 and S100A9 expression is induced upon epidermal injury and UVB irradiation (Thorey et al., 2001). Keratinocytes overexpressing S100A8 and S100A9 show a reduction in cell division and enhanced expression of the differentiation markers filaggrin and involucrin. E. coli and P. aeruginosa as well as the cytokines IL-1α, IL-6, IL-8 and TNF-α trigger the expression of S100A8 and S100A9 in human keratinocytes (Abtin et al., 2010; Mørk et al., 2003; Nukui et al., 2008). The complex formed by S100A8 and S100A9 exhibits antimicrobial activity against gram-negative bacteria such as E. coli and also against gram-positive bacteria such as S. aureus and the commensal S. epidermidis (Brandtzaeg et al., 1995). INTRODUCTION 22

Furthermore activity against the fungus C. albicans was shown (Murthy et al., 1993). The sequestration of essential trace elements, as mentioned earlier for S100A7, such as zinc or manganese have been proposed as a potential mechanism of antimicrobial action (Murthy et al., 1993; Sohnle et al., 2000; Corbin et al., 2008). It has been demonstrated that S100A8 and S100A9 also can act as an intracellular antimicrobial peptide complex and by this can increase epithelial resistance against bacterial pathogens (Champaiboon et al., 2009; Nisapakultorn et al., 2001b; a). When injected into mice, S100A8 stimulates the local accumulation of neutrophils and macrophages (Lau et al., 1995), whereas S100A9 stimulates neutrophil adhesion (Newton and Hogg, 1998).

S100A12

S100A12 (calgranulin C, EN-RAGE) is a RAGE (Receptor for Advanced Glycation Endproducts) receptor ligand (Hofmann et al., 1999) and was first characterized in 1996 (Wicki et al., 1996). S100A12 is overexpressed in many inflammatory diseases (Yang et al., 2001; Rouleau et al., 2003; Foell et al., 2003a; b), for example in psoriasis (Mirmohammadsadegh et al., 2000; Semprini et al., 2002), where it accumulates in the suprabasal epidermal layers (Mirmohammadsadegh et al., 2000; Semprini et al., 2002). LPS as well as TNF-α induce the expression of S100A12 mRNA in monocytes (Yang et al., 2001). It was shown that S100A12 has activity against the parasite Brugia malayi. S100A12 inhibits the movement of the worms and affects microfilariae (an early stage of nematode development) motility (Gottsch et al., 1999). Furthermore S100A12, like S100A8 and S100A9, induces neutrophil adhesion and also migration (Rouleau et al., 2003).

INTRODUCTION 23

CYTOKINES AND THE SKIN BARRIER

Cytokines are small proteins, which are important in and in regulating cell growth and differentiation. Cells release them and they affect other cells by acting through receptors on the cell surface. The large group of cytokines includes , , , and tumor necrosis factors but not hormones and growth factors. Keratinocytes and other skin resident cells produce cytokines that are responsible for the control of cellular communication. For example cytokines influence keratinocyte proliferation and differentiation, at least in part by modulating the gene expression program in these cells. One consequence is the expressional control of other cytokines resulting in a feedback loop and complex network of signaling molecules that affect the physiology of keratinocytes and the quality of the skin barrier. Deregulated cytokine expression can thus contribute to dysfunctions of the epidermal barrier as it is observed in many diseases, including atopic dermatitis (AD) and psoriasis.

CYTOKINE FAMILIES

Cytokines and their receptors can be classified based on the three-dimensional structure of the receptors. This classification results in six groups: 1. type I cytokine receptors with a conserved WSXWS amino acid motif in the extracellular domain. 2. Interleukin type II cytokine receptors, which are similar to type I receptors but lack this conserved motif. 3. The Immunoglobulin (Ig) superfamily sharing structural homology with immunoglobulin domains in their extracellular part. 4. The receptor (TNFR) family sharing a cysteine-rich extracellular cytokine-binding domain. 5. The IL-17 family of cytokine receptors sharing only little homology with the receptors of the other cytokine families. Finally within group six there are receptors that couple to G proteins. In Figure 3 the in the following mentioned cytokine receptor families are summarized together with their signaling pathways. Cytokines belonging to the above- INTRODUCTION 24 mentioned cytokine families, which are reported to have an impact on skin barrier function, will be described.

Cytokine Receptor Families

Interleukin Type I Interleukin Type II TNFR Ig Family tumor necrosis factor receptor JAKs Family Family STATs Cytokine IL-2 IL-6 IL-12 IFN IL-10 TNF IL-1 Signal Transducer and Families Activator of TRAFs TNF receptor associated factors IRAK Interleukin-1 receptor-associated kinase

WSXWS Fibronectin like domain motif

Cysteine rich motifs

Ig like domain immunoglobulin Intracellular domain

Transmembrane domain

JAK/STAT PI3K/AKT MAPK NFκB Janus kinase/ Phosphoinositide Mitogen-activated nuclear factor Signal Transducer and 3-kinase protein kinases kappa-light- Activator of Transcription chain-enhancer of activated B cells

Figure 3: Cytokine receptor complexes, their ligand families and corresponding signaling pathways involved in skin barrier regulation. The cytokine receptor families are grouped according to the three-dimensional structure of their receptors. The receptors of the interleukin type I and interleukin type II families possess extracellular fibronectin like domains. These two families differ only in the WSXWS motif present in the type I but not type II receptors. The tumor necrosis factor receptor (TNFR) family has cysteine-rich motifs in their extracellular regions able to bind ligands. The immunoglobulin (Ig) superfamily shares homology with immunoglobolin domains in their extracellular regions structural.

Interleukin Type I

The interleukin type I cytokine receptors can be further subdivided into five sub-groups of which three are relevant. The cytokines mentioned in the following text can be divided into the following sub-groups. The γ-chain containing IL-2 family receptor for the cytokine thymic stromal lymphopoietin (TSLP) (Liao et al., 2011); the IL-6 or gp130 family, a sub- group that includes gp130 in their receptor complexes with the members IL-31 and (OSM) (Garbers et al., 2012). The extracellular part of the interleukin type I receptor family consist of fibronectin like domains with a WSXWS domain specific for this type of receptor family. Signaling of these receptors through their intracellular parts activates the JAK / STAT, MAP kinase and PI3K / AKT pathways (Figure 3). INTRODUCTION 25

The IL-2 Family of Cytokines The IL-2 family contains six members, IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21. The members of this family share the IL-2 receptor common γ-chain (CD132). In the following briefly the IL-7-like cytokine thymic stromal lymphopoietin (TSLP) is discussed (Ziegler and Artis, 2010). TSLP binds to a heterodimeric receptor complex consisting of the IL-7 receptor alpha and the thymic stromal lymphopoietin receptor (TSLPR). The TSLPR is most closely related to the common γ-chain, which binds TSLP only with low affinity (Ziegler and Artis, 2010).

TSLP

Thymic stromal lymphopoietin (TSLP) is increased in AD skin lesions compared to healthy controls (Alysandratos et al., 2010). Serum levels of TSLP were also elevated in children suffering from AD compared to controls (Lee et al., 2010). Furthermore an association between genetic variants in TSLP and AD was found (Gao et al., 2010). A broad variety of cytokines including, IL-1α IL-4, IL-13, IL-31, and TNF-α, were shown to induce the expression and release of TSLP from keratinocytes (Bogiatzi et al., 2007; Danso et al., 2014). TSLP release from epithelia cells was also shown upon stimulation with microbial products, such as flagellin and physical injury (Le et al., 2011; Allakhverdi et al., 2007). Furthermore infections with S.aureus triggers Th2 inflammation in keratinocytes via TSLP (Vu et al., 2010). The released TSLP activates dendritic cells leading to production of proallergic cytokines (Soumelis et al., 2002). In addition TSLP activated dendritic cells prime naïve Th-cells to produce IL-5, IL-13 and TNF-α. Furthermore they also inhibit the expression of IL-10 and IFN-γ (Watanabe et al., 2004; Soumelis et al., 2002). Beside all these proinflammatory properties TSLP also induces itch by acting directly on a subset of sensory neurons (Wilson et al., 2013b).

INTRODUCTION 26

The IL-6 Family of Cytokines In the following the IL-6 family cytokines IL-31 and OSM are briefly discussed. Beyond these two the IL-6 family contains more members and all activate the signal transducing receptor protein, (gp130), except for IL-31. The receptor heterodimer for IL-31 consists of a unique gp130-like receptor chain IL-31RA, and the receptor subunit beta (OSMR β).

IL-31 and OSM

AD patients show significant higher levels of IL-31 in serum and increased IL31 mRNA levels in lesional skin samples in contrast to healthy individuals (Kim et al., 2011). These higher levels are correlated with serum IgE, disease severity, and subjective itch intensity (Schmuth et al., 2007; Kim et al., 2011; Nobbe et al., 2012; Raap et al., 2008; Neis et al., 2006). As described above, IL-31 signals via its heterodimeric receptor complex composed of the IL-31RA and the OSMR β (Dillon et al., 2004). IL-31 interferes with keratinocyte differentiation in organotypic 3D cell culture models, where the keratinocytes at the air- liquid interphase are able to differentiate into a skin-like structure. Treatment of these models with IL-31 leads to a reduced epidermal thickness, disturbed epidermal constitution, altered alignment of the SB and poor development of the SG. The observed differentiation defect is associated with a profound suppression of terminal differentiation markers, including filaggrin (FLG ), an essential factor for skin barrier formation, and a reduced lipid envelope (Cornelissen et al., 2012b). Moreover transgenic mice overexpressing IL-31 develop severe pruritus, alopecia (hair loss) and skin lesions (Dillon et al., 2004). IL-31 may also play a role in the sensation of itch because several itch- associated skin diseases like prurigo nodularis (Sonkoly et al., 2006), chronic spontaneous urticaria (Raap et al., 2010), and allergic contact dermatitis (Neis et al., 2006) exhibit high protein levels in blood serum and skin mRNA levels of IL-31. Treatment of Nc / Nga mice (a model for AD) with anti-IL-31 antibodies resulted in a reduction in scratching behavior (Takaoka et al., 2006; Grimstad et al., 2009) Dorsal root ganglia that are meant to be responsible for the sensation of itch express the IL-31 receptor and their unmyelinated INTRODUCTION 27 nerve fibers might be directly stimulated by IL-31. It might also be possible that IL-31 triggers the expression of pruritic factors, which subsequently activate the nerve fibers leading to the sensation of itch (Cornelissen et al., 2012a). As this is mediated by neurons, the regulation of pruritus by IL-31 might be completely unrelated to its suppression of keratinocyte differentiation (Cornelissen et al., 2012a).

Both OSM production and OSM receptor expression are enhanced in inflammatory skin diseases. OSM is a potent inducer of keratinocyte migration and triggers hyperplasia of reconstituted human epidermis (Boniface et al., 2007). OSM suppresses the expression of the ‘‘classical’’ epidermal differentiation markers (e.g filaggrin) causing a reduction of cornified cells in reconstituted human epidermis (Gazel et al., 2006). Cells as well as skin models show similar phenotypes when treated with either IL-31 or OSM. This could be due to the similarity of both receptor complexes. So far IL-31 is the only cytokine of the IL-6 family that does not signal through a receptor complex that contains gp130, which makes it a unique member within this family. Thus to understand IL-31 signaling the expression pattern of the OSMR β needs to be considered.

High serum levels of IL-31 in AD patients, impaired differentiation in organotypic models (that do not include inflammatory cells or factors besides the ones that may be produced by keratinocytes), and the increased scratching behavior in AD mice provoked the conclusion that high IL-31 expression is one of the causes rather than only a symptom of AD. This suggests that anti-IL-31 treatment, either by blocking the IL-31 receptor complex or by reducing IL-31 cytokine levels, may be beneficial for patients (Hanel et al., 2013). INTRODUCTION 28

Interleukin Type II

Type II cytokine receptors are divided into three sub-groups, i.e. the IL-10 family and the type I and type II families. All three sub-groups have been found to affect the proliferation and differentiation of keratinocytes. In particular the IL-10 family has various effects on the skin (Zdanov, 2010). Such as the Interleukin type I cytokine receptors, the type II receptors use the same signaling pathways ((MAP kinase, JAK / STAT, and PI3K / AKT) (Figure 3)).

IL-10 Family

IL-20

The expression of the IL20 gene is also increased in inflammatory skin diseases like AD or psoriasis (Kunz et al., 2006) and IL20 mRNA is upregulated in lesional compared to non- lesional psoriatic skin (Otkjaer et al., 2005). IL-20 induces proliferation of keratinocytes in monolayer culture, even if a neutralizing antibody against EGFR blocks autocrine growth. In line with these results, IL-20 triggers acanthosis or hyperplasia in stratified epidermal culture systems and epidermal thickening in the early cell layers in a dose dependent manner (Sa et al., 2007). In organotypic 3D co-cultures of keratinocytes with fibroblasts IL-31 is able to promote the expression of IL20 and IL24 , suggesting that these two cytokines are downstream effectors of IL-31. Indeed, IL-20 and IL-24 possess a small inhibitory effect on the differentiation within the 3D model. This phenotype is also characterized by a reduced expression of filaggrin and keratin 10. It is worth mentioning that these effects are relatively minor compared to IL-31, indicating that IL-31 induces additional mediators that control keratinocyte differentiation (Cornelissen et al., 2012b).

Overexpression of IL-20 in transgenic mice causes neonatal lethality with skin abnormalities including aberrant epidermal expression of several keratins indicating a prominent role of IL-20 in epidermal development during embryogenesis (Blumberg et al., 2001). Histological analyses of the skin of different IL-20 transgenic mice display a INTRODUCTION 29 thickened epidermis, hyperkeratosis, a compact SC and the cells of this layer express differentiation and proliferation markers that are normally confined to the basal and suprabasal layers of the skin. Overexpressing IL-20 in other tissues also causes these changes in the skin, indicating that circulating and epidermis infiltrating IL-20 is most likely responsible for the observed phenotype (Rich and Kupper, 2001).

IL-24

Similar to IL-20, IL-24 expression is increased in inflammatory skin diseases like AD or psoriasis (Kunz et al., 2006) and IL-24 induces, as well as the other IL-10 family members IL-19 and IL-20, the proliferation of keratinocytes in monolayer cultures. Like IL-20, IL- 24 promotes acanthosis / hyperplasia in a reconstituted human epidermis culture system and epidermal thickening in the early cell layers in a dose dependent manner. IL-24 decreases keratin 10 (KRT10 ) mRNA expression (Boniface et al., 2005) while it increases keratin 16 (KRT16 ) mRNA (Sa et al., 2007). Keratin 16 is upregulated in the suprabasal layers of interfollicular epidermis showing hyperproliferation and abnormal differentiation and thus also serves as a psoriasis marker protein (Bhawan et al., 2004). IL-24 is able to inhibit TGF α-induced migration of keratinocytes in an in vitro model of wound repair and interferes with TGF α-mediated proliferation of keratinocytes (Poindexter et al., 2010). In organotypic co-cultures with fibroblasts, IL-24 together with IL-20 impairs differentiation and reduces the expression of FLG and KRT10 (Cornelissen et al., 2012b). IL-24 leads to an increase of epidermal thickness in transgenic mice (He and Liang, 2010). Together these findings document that IL-24 function in normal skin is distinct from skin with an inflammatory response. In the former, IL-24 increases the proliferation and impairs normal differentiation whereas in inflamed skin both proliferation and migration of keratinocytes is inhibited. This suggests that the consequences of IL-24 are modulated or negated by other cytokines / signals present during inflammation. It would be interesting to define the nature of these factors that crosstalk with IL-24 signaling.

INTRODUCTION 30

These observations indicate a number of different roles for the cytokines of the IL-10 family (with the exception of IL-10 itself, whose function is rather distinct in comparison to the other family members) in regulating keratinocyte proliferation and differentiation in the epidermal layer. IL19 , IL20 and IL24 are clustered together with IL10 in the IL10 gene cluster on chromosome 1q32 whereas IL22 is located on chromosome 12q14 (Blumberg et al., 2001; Pestka et al., 2004). This clustering of the genes and the fact that IL-19, IL-20 and IL-24 signal via the same receptor components (IL-20R1, IL-20R2, IL-22R1) and thus are able to activate the same signaling pathways may be one reason for these overlapping but not identical phenotypes in response to these cytokines. It seems that all these cytokines have a role in regulating the skin barrier by controlling keratinocyte proliferation and migration as well as abrogating the expression of genes that encode components of the skin barrier (Hanel et al., 2013).

Interferons The family of interferons can be subdivided into two groups. Group I interferons, binding to IFNA receptors (IFNAR), include IFN-α, IFN-β and IFN-ω. IFN-γ is the only member of the group II interferons, which interact with the IFNG receptors (IFNGR).

IFN-α and IFN-γ

IFNG expression is increased in inflammatory skin disease like AD or psoriasis (Wolk et al., 2004; Kunz et al., 2006). In these diseases higher levels of IFN-α and IFN-γ can alter the response to different cytokines by regulating the expression of their corresponding receptors. It was shown that IFN-γ enhances the expression of the IL-31 receptor (Feld et al., 2010; Heise et al., 2009) and of the OSM receptor (Dambacher et al., 2007), while reducing IL-4 receptor expression (Albanesi et al., 2000), whereas IFN-α enhances the expression of the IL-22 receptor (Tohyama et al., 2012). IFN-α as well as IFN-γ have a modulatory effect on the expression of the integrins α3, α6 and β1. IFN-α treatment has an inhibitory effect on integrin α6 expression in basal cells of reconstructed skin (Tenaud et INTRODUCTION 31 al., 2002). The integrins α6 and β4 are main components of the hemidesmosomes, which are essential protein complexes that mediate the firm attachment of epithelial cells to the underlying basement membranes (Litjens et al., 2006). Mice with a conditional ablation of integrin α6 in the epidermis present skin fragility and inflammation (Niculescu et al., 2011), the latter probably as a consequence of impaired barrier formation. Moreover α3β1 integrins play crucial roles in the organization of epithelial and endothelial tissues by exerting functions related to cell adhesion and migration (Tsuji, 2004). In contrast to the decreased epidermis-dermis interaction, the levels of transcripts for acid sphingomyelinase (SMase) and glucocerebrosidase (GCase), both enzymes play important roles in ceramide synthesis, are stimulated in response to IFN-γ (Sawada et al., 2012). SMase hydrolyses sphingomyelin thereby producing ceramides 2 and 5 (Uchida et al., 2000), whereas GCase degrades glucosylceramide to produce the ceramides 1 to 7 (Hamanaka et al., 2002). Ceramides are an integral part of the lipid envelope accounting for 30% - 40% of stratum corneum lipids by weight. In combination with cholesterol and free fatty acids (FA), ceramides form the extracellular lamellar membrane structures. This subserves the permeability barrier and is important to prevent water loss due to evaporation (Madison,

2003). Thus through activation of the expression of SMase and GCase, IFN-γ enhances ceramide synthesis and consequently strengthens the lipid envelope. Consistent with these findings is that IFN-γ suppresses TEWL (Hatano et al., 2005).

In conclusion, IFNs seem to play a role in maintaining the barrier function of the skin by regulating receptors important for cytokine signaling, including the IL-4 and IL-31 receptors. Cytokines belonging to these receptors have a tremendous impact on the formation of the skin barrier (see above). Furthermore IFNs seem to directly influence the detachment of keratinocytes from the basal membrane, an essential step in the differentiation process from basal to primary differentiating suprabasal cells. Additionally IFNs are involved in ceramide synthesis and therefore impact the formation of the lipid envelope and thus are able to regulate TEWL.

INTRODUCTION 32

Ig superfamily (receptors with extracellular immunoglobulin (Ig)-like domains)

The IL-1 family of cytokines includes a growing number of members that play important roles in immune regulation and inflammatory processes (summarized in (Barksby et al., 2007)). In the following the functions of those family members, i.e. IL-1α and IL-1β, which have been analyzed in combination with epidermal differentiation and skin diseases are addressed.

IL-1α and IL-1β The cutaneous expression of IL1A transcripts is reduced in inflammatory skin diseases like psoriasis or AD whereas no significant changes are seen for IL1B (Wolk et al., 2004). In contrast an increase of secreted IL-1α in lesional skin of psoriasis patients was observed (Portugal-Cohen et al., 2012). After acute permeability barrier disruption, an increase in the epidermal expression of IL-1α and IL-1β occurs, which might have a protective function (Wood et al., 1992). Indeed stimulating keratinocytes with IL-1α enhances the synthesis of barrier lipids in monolayer cell culture models (Barland et al., 2004). In contrast O’Shaughnessy and colleagues found that IL-1α leads to hyperkeratosis without increased lipid synthesis and causes a reduction of keratin 10 and involucrin expression.

Moreover IL-1α promotes the expression of the fatty acid desaturase 2 in organotypic 3D cultures (O'Shaughnessy et al., 2010). These contrasting findings regarding the influence of IL-1α on lipid synthesis in particular could be due to differences in the experimental setup. Barland and colleagues used calcium differentiated keratinocytes in a monolayer culture (Barland et al., 2004), while OShaughnessy et al. treated organotypic 3D cultures with IL- 1α (O'Shaughnessy et al., 2010). 3D co-cultures with fibroblasts serve as a more physiological model for epidermal development and homeostasis, in which the Ca 2+ gradient develops in the epidermal layer comparably to normal skin. Moreover the fibroblasts provide physiologically relevant cytokines and growth factors. In contrast Ca 2+ - induced differentiation of a monolayer culture results in a highly simplified version of the differentiation program. This might explain the differences observed in the two models in response to IL1-α. Although this seems an obvious explanation, it should be noted that INTRODUCTION 33 explicit studies addressing the differences of keratinocyte differentiation between the 3D co-culture model and a monolayer culture treated with Ca 2+ are missing. In other words it remains open to what extent these different models are comparable. Nevertheless it will be important to define the role of IL-1α in more detail, as this important cytokine is likely to be involved in keratinocyte differentiation.

The intra-cutaneous administration of IL-1α was able to improve barrier function in mice by enhancing the lipid synthesis and the lamellar body formation (Barland et al., 2004). In another study, IL-1α injected into mice led to an increased barrier recovery after perturbation and IL-1α stimulation restored epidermal permeability and the antimicrobial barrier that were compromised by topical application of tacrolimus, an immunosuppressive drug (Jung et al., 2011). IL-1α upregulates the expression of genes associated with cell adhesion, proliferation, and epidermal differentiation (Yano et al., 2008). Conversely, transgenic mice that overexpress IL-1α in basal keratinocytes under the control of the K14 promoter develop spontaneous inflammatory skin lesions, as well as dermal neutrophil infiltration even in non-lesional skin (Groves et al., 1995). In all epithelial cells the IL-1α precursor is constitutively expressed, translocates to the nucleus and can modulate gene transcription and thus modifies cell behavior and cellular differentiation (Dinarello, 2009). Overexpressing this biological active precursor in keratinocytes has distinct consequences to the skin barrier in comparison to injecting a biologically active IL-1α that binds to its cell surface receptor and induces intracellular signaling. IL-1α signaling attracts lymphocytes and neutrophils via the expression of CCL20 in keratinocytes (Yano et al., 2008). Mice with a knockout of the IL-1 receptor type 1 develop a more profound barrier deficit than age-matched wild-types and an increase in IL-1α protein expression in the skin after acute permeability barrier perturbation. IL-1β treatment leads to an increased expression of the main thigh junction proteins occludin and claudin-1 at early time points after treatment. Furthermore ZO-1, which connects tight junctions with the cytoskeleton, was also up regulated in organotypic 3D models at early time points after treatment. In contrast to this early response to the IL-1β treatment, after 96 h of stimulation the expression of all these proteins is decreased. In line with the altered expression of these INTRODUCTION 34 structural proteins also a functional effect could be observed. The treatment with IL-1β leads to an increased transepithelial resistance, indicating an increased barrier of calcium differentiated keratinocytes as a consequence of the early response and a decreased resistance at later stages (Kirschner et al., 2009).

It appears that IL-1α and IL-1β have profound effects on epidermal differentiation that need to be tightly controlled under physiological conditions. They are able to strengthen the epidermal barrier via influencing the mechanical attachment of cells and the formation of the lipid envelope. This might be highly relevant in a response to e.g. mechanical injury of the skin and wound repair. Nevertheless a deregulated expression of IL-1α seems to promote an inflammatory skin phenotype e.g. manifested by attraction of inflammatory cells. Together it seems worth proposing that the role of IL-1α and β in skin physiology and pathology needs to be studied in greater detail, for example to answer the question whether deregulated IL-1 expression occurs in skin diseases and if this might contribute to disease symptoms like barrier disruption.

CONCLUDING REMARKS

The various cytokines act at multiple levels to control barrier formation, like the cornification process (e.g. IL-31), the composition of the lipid envelope (e.g. IFN-γ) and cell-cell adhesion (e.g. IL-1α). Cytokines and their targets in the formation of the skin barrier, even those that were not mentioned in the text before, are summarized in Figure 4. Many discussed cytokines alter the expression of structurally important proteins involved in the formation of the cornified envelope, including loricrin and profilaggrin. Some also prevent the development or maintenance of tight junctions, corneodesmosomes and hemidesmosomes, thereby affecting the interaction of terminally differentiated cells in the cornified envelope or the basal cell layer. Moreover cytokines control the expression of genes that encode enzymes key to process the proteins and lipids associated with terminal INTRODUCTION 35 differentiation. Because these processes are highly balanced it is not surprising that many cytokines provoke a weakening of the barrier, albeit their effects are mediated by different components. These contribute to inefficient barrier formation, beginning with effects on early differentiation processes to the disturbance of terminal events that define the barrier proper. In addition to these “antagonistic” cytokines for barrier formation, others cytokines appear to be important for the efficient repair of barrier activity. This indicates that the interaction of different cytokines will need to be addressed to understand how cytokine balance is relevant for the protective function of the skin.

IL-4; IL-13

Epidermal Barrier Desquamation

Stratum Cornified Corneum Envelope

Lipid Extrusion Filaggrin/ Late Corneo- and Cross- Keratin Differentiating desmosomes IL-4; IL-13; linking Network Keratinocytes IL-22; TNF Stratum IL-4; IL-13; OSM; Granulosum IL-31; IL-20; IL-24; Loricrin IL-22; IL-1 ; TNF; Synthesis IFN ; TNF IL-17; IL-25 IL-22 Early Lipid Profilaggrin Desmosome Differentiating Processing Synthesis Maturation IL-4; IL-13; Keratinocytes IL-22 IL-6; IL19; IL-20;

Stratum IL-4; OSM; IL-31; IL-21; IL-22; IL-24; IL-1 ; Involucrin Spinosum IL-20; IL-24; IL-22; IL-1 IL-22; IFN ; Synthesis IFN ; IL-1 IL-1 ; TNF IL-31; IFN ; IL-1 ; IL-17

Keratin Proliferating Lipid Synthesis Desmosomes Synthesis Keratinocytes Basale Stratum

Figure 4: Regulatory effects of the cytokines on the barrier formation. This figure illustrates the relevant effects of cytokines on the formation of the skin barrier and selected targets in the differentiation process documented either in cell culture or in animal studies. Arrows indicate the consequences on these processes. (é) Indicates that the cytokine enhances or promotes the target process; (ê) indicates that the cytokine downregulates or inhibits the target process.

It should be noted that in most cases it is still unclear what the mechanistic base is for these effects. This may be at least in part due to the difficulties in analyzing individual steps during the differentiation process, as this is only efficiently occurring in vivo or in 3D organotypic cultures. Moreover the terminal differentiation events, which result in the cornified envelope, are challenging to study at the cellular level beyond documenting protein expression by for example immuno-histochemical analyses because the cells do no INTRODUCTION 36 longer proliferate and cannot easily be separated from earlier differentiation stages. As a consequence, in most situations it is not clear how a specific cytokine controls the expression of genes relevant for the differentiation process. Together the studies that are presently available do not allow precise conclusions about how certain cytokines affect barrier formation. In particular it is typically not known whether a cytokine controls the expression of a relevant gene directly or whether these are secondary effects.

The mechanistic studies are further complicated, by many additional cells beyond keratinocytes that infiltrate the epidermis of animals. Many of these are immune cells that respond to different cytokines and also produce cytokine cocktails making the analysis of individual cytokines complex. Nevertheless it is striking that the majority of the cytokines, which disrupt the barrier function of the epidermis in model systems, are also deregulated in skin diseases, including AD and psoriasis. Moreover the overexpression of these cytokines typically correlates with disease severity. An additional important level of complexity is given by the fact that the disruption of the skin barrier facilitates the invasion of pathogens and allergens. In turn this contributes to local inflammatory immune responses. In other words, deregulated cytokine expression in the skin can weaken barrier function and thus aggravate the inflammatory response, thereby establishing a viscous circle. Thus it is quite obvious that the management of cytokine expression and activity is important to control and ameliorate disease development in the skin. INTRODUCTION 37

AIM OF THIS WORK

Patients suffering from inflammatory skin diseases such as AD have profound barrier defects (Bouwstra and Ponec, 2006). These defects in the barrier formation lead to increased penetration of the skin by allergens and pathogens, thereby aggravating disease symptoms. In line with these observations, around 90% of all AD patients suffer from bacterial super infections (Schittek, 2011). In contrast to the pathological appearance, the relevant pathogenic mechanism leading to AD remains largely unclear. One hint might be, that AD patients show significantly higher levels of IL-31 in serum and increased IL31 mRNA levels in lesional skin samples in contrast to healthy individuals (Kim et al., 2011). These higher levels correlate with disease severity and subjective itch intensity (Schmuth et al., 2007; Kim et al., 2011; Nobbe et al., 2012; Raap et al., 2008; Neis et al., 2006). Previous work has shown that IL-31 is an important regulator of keratinocyte differentiation (Cornelissen et al., 2012a).

Therefore the aim of this thesis was to investigate the role of IL-31 on skin barrier function in in vitro and in vivo models. To study this, expression arrays were used to identify genes, which are differentially expressed during the organotypic differentiation of keratinocytes of untreated and IL-31 treated cells. In vitro models including monolayer cultures, 3- dimensional organotypic skin equivalents and, a cell-sorted skin equivalent (CeSSE) in the mouse were used. Moreover a second part of this thesis was to identify IL-31 signaling pathways as well as factors induced by IL-31 and their functional consequences. Furthermore a part of this work was to identify the potential role of IL-31 in healthy human skin. RESULTS 38

2 RESULTS RESULTS 39

IL-31 CONTROLS THE EXPRESSION OF GENES ASSOCIATED

WITH THE MECHANICAL BARRIER

Previous studies of our group in cooperation with the Institute of Dermatology and Allergology revealed that IL-31 is an important regulator of keratinocyte differentiation (Cornelissen et al., 2012b). However there are no reports describing the influence of IL-31 on the skin barrier formation. Due to this fact it was of general interest to identify this influence and furthermore to recognize downstream signaling pathways. To understand the role of IL-31 for the formation of the skin barrier, human recombinant (rh) IL-31 was applied to organotypic 3D models of HaCaT-IL31RA cells and gene expression was analyzed using Affymetrix gene arrays after 2, 8, 24 or 48 h. These organotypic 3D models consist of two different cell types. The basal, dermal part of these skin models is constructed with human dermal fibroblasts suspended in bovine collagen. On top of this, the epidermal layer is seeded by HaCaT keratinocytes expressing the IL-31 receptor alpha (RA) under the control of a doxycycline inducible expression vector (Cornelissen et al., 2012b; Neis et al., 2010). These layers are lifted to and cultivated at the air-liquid interphase to induce keratinocyte differentiation. (For a better understanding, the models are lifted to the air-liquid interphase two days after seeding the keratinocytes and for all following experiments day 0 is defined as the day when the models were lifted to the air- liquid interphase.) IL-31 treatment resulted in altered expression of approximately 570 genes including a broad range of genes encoding for example differentiation-associated structural proteins (Figure 5 A). Genes encoding desmosomal proteins and filaggrin processing enzymes were repressed (Figure 5 B). Most notably the genes encoding desmoglein 1 and 4 ( DSG1 and 4), but also desmocollin 1 and 2 ( DSC1 and 2) and corneodesmosin ( CDSN ) were down regulated. DSG1 and DSC1 are essential components of corneodesmosome and are targets of proteases important for skin desquamation (Hoste et al., 2011; Amagai and Stanley, 2012; Ishida-Yamamoto et al., 2011). CDSN is covalently linked to the cornified envelope (CE) (Jonca et al., 2002). Also the expression of the genes encoding caspase-14 ( CASP14 ) and kalikrein-like peptidase 7 ( KLK7 ), two important proteases involved in the processing of filaggrin during keratinocyte RESULTS 40 differentiation were repressed (Hoste et al., 2011; Hvid et al., 2011; Yamamoto et al., 2011) (Figure 5 B).

A 5 4 3 2

2 [h] IL-31 1 8 0 -1 24

foldchange -2 -3 48 -4 -5 FLG KRT10 KRT4 KRT6A KRT7 KRT13 KRT16 KRT18 KRT23 KRTDAP IVL KRT1 KRT6B KRT6C KRT8 KRT15 KRT17 KRT19 KRT77

DSC1 DSG4 FLG CASP14 KLK7 B DSG1 DSC2 0 0 2 2

-1 IL-31 -4 [h] IL-31 8 8 -8 -2 [h] -12 24 24

foldchange -3 fold fold change -16 48 48 -20 -4 HaCaT-IL31RA mono HaCaT-IL31RA 3D 10 days HaCaT-IL31RA 3D 10 days C 4 D E IL-31 * 3 0 h 1 1 * *** ** *** ** ** ** *** +IL-31 +IL-31 24 h 2 0.5 -IL-31 48 h 0.5 -IL-31

1 * ** * * *** ***

** *** * 72 h rel.expression *** rel. expression

rel. expression 0 0 0 2 4 1 4 G1 G4 N 1 1 1 S S SC1 SP D D D DSCCDSN KLK7 JUP CG D SG4 SC SC2 KLK7 ASP1 LDN GNL OCLN DSG1 D D D ASP C C C C

Figure 5: IL-31 deregulates the expression of genes associated with the mechanical skin barrier. A and B. HaCaT-IL31RA organotypic 3D models were stimulated with or without 100 ng/ml rhIL-31 for 2, 8, 24 or 48 h. Gene expression was analyzed using Affymetrix exon arrays. Indicated are the fold changes relative to the untreated control. C. qRT-PCR analysis of the indicated genes in HaCaT-IL31RA monolayer cells stimulated with 100 ng/ml rhIL-31 for the indicated time, mean values ± SD, n = 3. D and E. qRT-PCR analysis of a 10 day HaCaT-IL31RA 3D organotypic skin model, models were treated every second day with 100 ng/ml rhIL-31, mean values ± SD, n = 3. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001).

A first confirmation of the array data was done in HaCaT-IL31RA monolayer cultures treated with IL-31 for up to 72 h using qRT-PCR (Figure 5 C). In this experimental setting downregulation of the genes encoding DSG1 , DSG4 , DSC1 , CASP14 and KLK7 could be observed. However DSC2 gene expression was upregulated upon IL-31 stimulation (Figure 5 C) and DSG1 gene expression returned to initial levels after 72 h IL-31 treatment (Figure 5 C), which was not seen in the array data (Figure 5 B). Furthermore, the expressions of these genes was analyzed by qRT-PCR in HaCaT-IL31RA organotypic skin equivalents, differentiated and stimulated every second day with IL-31 for 10 days. In these skin equivalents DSG1 , DSG4 , DSC1 , DSC2 , CDSN , CASP14 and KLK7 were significantly downregulated upon IL-31 treatment (Figure 5 D). In a similar experimental setting it was tested if the gene expression of tight junction proteins was also affected by IL-31. Unlike genes expressing desmosomal proteins, tight junction genes were not deregulated or to a RESULTS 41 minor extend such as both cingulin family members CGN (cingulin) and CGNL (cingulin-like 1) (Figure 5 E).

IL-31 WEAKENS THE SKIN BARRIER

The reduced expression of genes encoding proteins relevant for the mechanical barrier in response to IL-31, as shown before, prompted to assess the integrity of the epidermal barrier. In order to determine the impact of IL-31 on desmosomal maturation and the formation of the cornified envelope, 10-day-old HaCaT-IL31RA organotypic skin models either treated with IL-31 every second day or untreated, were incubated with biotin, either applied from the apical or the basolateral side of the model (Figure 6 A). The application from the apical side should test the formation of the CE. Application from the basolateral side was for testing desmosome maturation. In the IL-31 treated organotypic model apically applied biotin (arrows) penetrated into the upper layer of the CE (Figure 6 A, upper panel). When biotin was applied from the basolateral side, biotin accumulated between the keratinocytes and fibroblasts in the untreated control. In the IL-31 treated model biotin did not accumulate at the basal membrane but it was also found in the CE (Figure 6 A, lower panel). Moreover fluorescently labeled biotin added to the dermal side of a 10-day-old organotypic HaCaT-IL31RA model penetrated through all nucleated epidermal layers and accumulated in the cornified envelope only when the model was stimulated with IL-31 (Figure 6 B). To further evaluate these findings and to mimic a more in vivo situation, a HaCaT-IL31RA organotypic 3D model was incubated with fluorescently labeled recombinant timothy grass pollen major allergen (phl p1) (Suphioglu, 2000). Immunofluorescence imaging showed that the allergen stayed on top of the model and did not penetrate the control organotypic epidermis, whereas it was enriched in deeper layers upon IL-31 treatment (Figure 6 C).

RESULTS 42

Figure 6: IL-31 weakens the skin barrier. A. HaCaT-IL31RA organotypic skin equivalents were treated with or without 100 ng/ml rhIL-31 every second day. The models were incubated with biotin applied from the apical (upper panel) or basolateral side (lower panel) for 60 min at day 10. Sections were analyzed by immunohistochemistry. Scale bar lines 1 and 3, 100 µm, scale bar lines 2 and 4, 200 µm. B. HaCaT-IL31RA organotypic skin equivalents (stimulated and cultivated as in A) were incubated at day 10 basolaterally with labeled biotin (green) for 60 min and histological sections were stained for DNA with DAPI (blue). Scale bar 100 µm C. 7- day-old HaCaT-IL31RA 3D models (stimulated and cultivated as in A). Fluorescently labeled recombinant timothy grass pollen major allergen (phl p1) was applied topically for 45 min. Histological sections were HE stained (line 2 and 4) and the location of phl p1 (green) was determined by fluorescence microscopy and DNA was stained with DAPI (blue, line 1 and 3). Scale bar HE stain 200 µm, scale bar fluorescence microscopy 100 µm. D. 7-day-old HaCaT-IL31RA 3D models were treated with 0.2% SDS and/or 100 ng/ ml rhIL-31 for 40 min. IL-1α release was measured by ELISA and IL1A mRNA expression was measured by qRT-PCR after 24 h. Mean values of 2 experiments are displayed. n.d., not determinable. The data is part of Hänel et al., manuscript submitted. Panel C and D were performed in collaboration with and by Philipp M. Amann (Department of Dermatology and Allergology, University Hospital Aachen).

Besides allergens and pathogens several irritants also affect the human skin. Upon treatment with irritants human keratinocytes are able to release inflammatory mediators in vitro and in vivo (Perkins et al., 1999; Roguet, 1999). It was also shown that the cytokine IL-1α is produced and released in a concentration and time-dependent manner after keratinocytes were treated with irritants such as sodium dodecyl sulfate (SDS) or UVB light, even at levels which are not cell cytotoxic (Cohen et al., 1991). Further publications have elaborated that IL-1α is a good marker for induced cell damage (Bernhofer et al., 1999b; a; Perkins et al., 1999; Mühlen et al., 2004). It was tested if IL-31 alone or in combination with SDS triggers an irritating feature. For this purpose IL-31, SDS or both together were applied apically to fully developed 7-day-old HaCaT-IL31RA organotypic skin models, the applied substances were washed away after 40 min, and IL1A gene expression and IL-1α release were measured after 24 h. Topical treatment with the irritant SDS promoted expression and release of IL-1α in an organotypic model pretreated with IL- 31 compared to untreated controls (Figure 6 D). Furthermore the combined treatment of RESULTS 43

IL-31 and SDS resulted in a higher IL-1α secretion and IL1A gene expression than the sum of the individual treatments. Moreover, IL-31 alone was sufficient to promote IL1A mRNA expression and, in contrast to SDS alone, able to trigger IL-1α release into the cell culture medium (Figure 6 D). Together these findings demonstrate, that IL-31 alters the skin barrier and promotes the uptake of allergens and pathogens in the skin. Moreover IL- 31 treatment induces IL1A gene expression and IL-1α release from an organotypic skin model.

IL-31 PROMOTES TRANSEPIDERMAL WATER LOSS IN MOUSE MODEL

To further determine the impact of IL-31 on skin differentiation in vivo , an epidermal mouse model based on a cell-sorted skin equivalent (CsSE) was refined (Wang et al., 2000; Skazik et al., 2014). Plastic chambers were transplanted on the back of SCID mice. Two days before transplanting the chamber, drinking water was supplemented with doxycycline to induce the IL-31 receptor expression in keratinocytes, which would be seeded later into the plastic chambers (Figure 7 A). One day after transplanting the chambers, HaCaT keratinocytes either expressing the IL-31 receptor (HaCaT-IL31RA) or not (HaCaT) were seeded together with human dermal fibroblasts (HDF) into these chambers. In Figure 7 B macroscopic pictures of CsSE on the back of a SCID mouse are shown. The mice were injected subcutaneously below the chambers with IL-31 daily till the end of the experiment, starting four days after seeding the cell slurry (Figure 7 A). At day six the chambers were removed and one-day later trans-epidermal water loss (TEWL) was measured in the different models. Measurement of TEWL is a non-invasive method for determining the barrier function of skin (Gioia and Celleno, 2002). Temperature and relative humidity were measured at two different heights above the skin in a hollow cylinder and by this TEWL is indirectly calculated. If the skin barrier is somehow damaged an increase of TEWL can be observed. HaCaT-IL31RA cells exhibited a profound defect in epidermal development with a reduction in filaggrin expression as observed in immunofluorescence imaging. It also showed a reduced lipid barrier formation visualized RESULTS 44 by reduced nile red staining (Figure 7 C), resulting in disturbed barrier formation as shown by significant increased TEWL (Figure 7 D).

A

Day-3 Day-1 Day 0 Day 4 Day 6 Day 7 Day 8 DOX Chamber Adding Cells IL-31 Chamber TEWL Sacrifice Application Transplantation Removal Measuring IF-Staining

B D HaCaT- 100 HaCaT IL31RA * 80 60 x hour)

2 40 TEWL TEWL 20 (g/m 0 - T a C C aCaT HE filaggrin nile red H Ha 31RA IL HaCaT IL31RA HaCaT-

Figure 7: IL-31 promotes transepidermal water loss. A. Treatment Procedure for generation of Cell-sorted Skin Equivalent (CsSE). Mice were fed with doxycycline and subcutaneously treated with 20 µg rhIL-31 at day 4 to 7. HaCaT or HaCaT-IL31RA cells were seeded together with human dermal fibroblast at day 0. B. Macroscopic pictures of a 6-day-old CsSE. C. Histological sections of CsSE were stained with HE, for filaggrin (green), the DNA with DAPI (blue), and lipids with nile red. Scale bar HE 200 µm, scale bar immuno fluorescence 100 µm. D. Transepidermal water loss (TEWL) was measured on 7 day old CsSEs, with a Tewameter, mean values ± SD, n = 3. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001). The data is part of Hänel et al., manuscript submitted. Animal experiments were performed in collaboration with and by Philipp M. Amann and Jens M. Baron (Department of Dermatology and Allergology, University Hospital Aachen). RESULTS 45

IL-31 STIMULATES THE IL-1 NETWORK

In the above-described experiments it was seen that IL-31 alone stimulates IL1A gene expression in differentiated organotypic skin models (Figure 6). To assess if the IL1 gene is a downstream target of IL-31 treatment in keratinocytes, qRT-PCR was used to analyze the expression of the IL1 gene family. IL-31 stimulated in a time-dependent manner the expression of IL1A and IL1B as well as the gene encoding the IL-1 receptor subunit IL1R2, in monolayer HaCaT-IL31RA cells (Figure 8 A). The IL-1 receptor subunit IL1R1 and the natural IL1 receptor antagonist IL1RN were not significantly regulated upon IL-31 treatment (Figure 8 A). To further investigate if the IL-1 receptor subunits were also presented at the cell surface, FACS analysis of HaCaT-IL31RA monolayer cells was performed. This analysis revealed an increase of IL-1R2 at the cell surface upon 72 h IL-31 treatment (Figure 8 B). It was of particular interest to determine whether the amount of intracellular IL-1α and IL-1β is affected by IL-31 stimulation and IL-1α and IL-1β is also released into the culture medium. To address this question, ELISA technique was used to monitor the intra- and extracellular amount of IL-1α and IL-1β upon IL-31 stimulation in HaCaT-IL31RA monolayer cells. An increase of both IL-1α and IL-1β concentration within the cells could be observed, whereas only the IL-1α concentration raised in the cell culture medium and no additional release of IL-1β was seen (Figure 8 C).

This observation was further investigated in a slightly different experimental ELISA setting. The proinflammatory cytokine IL-1β is produced as an inactive precursor pro-IL- 1β in the cytosol after different stimuli. The inactive precursor is cleaved by caspase-1, which can be activated by cytosolic multi-protein-complexes called 'inflammasomes' (Franchi et al., 2012). Nigericin, a microbial toxin derived from Streptomyces hygroscopicus, was used to activate the NALP3 inflammasome. Upon treatment with either nigericin or IL-31, no additional release of IL-1β was seen but when HaCaT- IL31RA monolayer cells were prestimulated with IL-31 and later challenged with nigericin, an increase of both IL-1α and IL-1β in the cell culture medium was observed (Figure 8 D). As mentioned before, there are large differences in the physiology and RESULTS 46 reactions to substances and cytokines between keratinocyte monolayer cultures and organotypic skin models. This issue was addressed by determining the reactions regarding the IL-1 network also in 3D organotypic skin models. By immuno-histological staining an increase of IL-1α expression could be detected within a HaCaT-IL31RA organotypic skin model (Figure 8 E). Furthermore also the release of IL-1α from these models could be detected using ELISA technique (Figure 8 F). However release of IL-1β was not seen (data not shown), which was consistent with the findings in HaCaT-IL31RA monolayer cells (Figure 8 C).

Two studies from 2011 showed that i) IL31 mRNA levels in skin lesions of AD patients correlate with their IL-31 serum levels (Kim et al., 2011) and ii) serum levels obtained from children during AD, during a flare, but also when quiescence, are significantly higher than those in healthy controls (Ezzat et al., 2011). Considering these studies, blood sera of 26 AD patients were analyzed with ELISA technique to determine the concentration of IL-

31 and IL-1α. First the IL-31 levels were detected and sera were correspondingly grouped in a high level (above 4.1 ng/ml) and a low level (below 4.1 ng/ml) cohort. The measurement of IL-1α revealed that high IL-31 levels correlated with higher IL-1α levels

(Figure 8 G). RESULTS 47

HaCaT-IL31RA mono A 25

* IL-31 20 0 h C HaCaT-IL31RA mono 24 h 15 cell lysates cell lysates 48 h 16 16 *** *** *** *** 10 72 h 12 12 ** *** *** (pg/ml) (pg/ml) rel. expression 5 8 8 α β 4 4 IL-1 0 IL-1 IL1A IL1B IL1R1IL1R2 IL1RN 0 0 - IL-31 - IL-31 HaCaT-IL31RA mono supernatant supernatant B 16 100 20

IL1R1 IL1R2 * 15 12 80 +IL31

10 (pg/ml)

(pg/ml) 8 β 60 - IL31 α Ab Control 5 4 IL-1 40 Cells only IL-1 0 0 - IL-31 - IL-31 % of maximum of % 20 0 100 101 102 103 104 101 102 103 104 FITC-A

HaCaT-IL31RA mono E IL-1α D 200 40 *** *** 160 32 - 120 24 (pg/ml) (pg/ml) β α 80 16

40 * 8 IL-31 IL-1 IL-1 0 0 - in - in in 31 c cin 31 c - ri - ri IL eri IL e g ge igeric Ni N -31/Ni IL IL-31/Nig HaCaT-IL31RA 3D HaCaT-IL31RA mono F 10 days G 500 H 5

80 ** 400 *** IL1A

4 * 60 ** IL20 300 3 40 (pg/ml) (pg/ml) α 200 α 2 20 100 IL-1 * ** ** ** *** *** ***

IL-1 1 0 0 rel. expression - IL-31 low IL-31 high IL-31 0 DMSO PI3K ERK p38 JAK JNK -

IL-31 Figure 8: IL-31 stimulates the IL-1 signaling network. A. qRT-PCR analysis of the indicated genes in monolayer HaCaT-IL31RA cells stimulated with 100 ng/ml rhIL-31 for the indicated times, mean values ± SD, n = 3. B. FACS analysis of IL-1R1 and IL-1R2 expression on monolayer HaCaT-IL31RA cells in response to 100ng/ml rhIL-31 (72 h), one representative experiment shown. C. IL-1α and IL-1β proteins were analyzed by ELISA from cell lysates and supernatants of monolayer HaCaT-IL31RA cells treated with 100 ng/ml rhIL-31, mean values SD, ± n = 3. D. Same setting as in C but cells were challenged with 20µM for 24 h of nigericin after 24 rhIL-31 treatment, proteins were measured in the supernatant. mean values SD, ± n = 3. E. Histological sections of a 10-day-old HaCaT-IL31RA 3D organotypic skin model treated every second day with 100 ng/ml rhIL-31. The model was stained for IL- 1α (green) and for DNA (blue). Scale bar 200 µm. F. IL-1α was measured by ELISA in the supernatant of 3D organotypic skin models as in E, mean values ± SD, n = 3. G. IL-1α levels in 26 human sera were compared, 14 sera with an IL-31 concentration below and 12 sera with an IL-31 concentration above 4.1 ng/ml. IL-1α and IL-31 levels were measured by ELISA. H. Monolayer HaCaT-IL31RA cells were treated with 100 ng/ml rhIL-31 for 4h. In addition, the indicated kinase inhibitors (JAK inhibitor I [100 nM]; c-Jun N-terminal kinase inhibitor II [20 µM]; SB202190, selective for p38 MAP kinases [20 µM]; U0126, selective for extracellular signal-regulated kinases [20 µM]; Wortmannin, selective for PI3K kinases [500 nM]) were added 1 h prior to stimulation with rhIL-31. The expression of the indicated genes was analyzed using qRT-PCR and normalized to the rhIL-31 / DMSO control, mean values ± SD, n = 3. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001). RESULTS 48

In addition, our research group has shown before that IL-31 induces the expression of IL20 and IL24 genes in HaCaT-IL31RA keratinocytes and that IL-20 and IL-24 may contribute to the IL-31 phenotype (Cornelissen et al., 2012b). To analyze if the induction of the genes encoding IL1A and IL1B was regulated similarly to the IL20 and IL24 expression, monolayer HaCaT-IL31RA cells were treated with specific inhibitors for different signaling pathways downstream of the IL-31 receptor complex. Inhibitors targeting PI3K kinases, p38 and extracellular signal-regulated kinases (ERK) interfered with the induction of IL1A and IL1B by IL-31 (Figure 8 H; IL1B not shown but similar to IL1A data). However, a pan JAK inhibitor against JAK 1, JAK 2, JAK 3 and Tyk 2 used at a concentration more than 6-fold higher than the highest IC 50, did not inhibit the expression of both IL1A and IL1B in HaCaT-IL31RA cells (Figure 8 H). In contrast to these findings the induction of the expression of IL20 and IL24 was dependent on the JAK kinase, p38 and ERK pathways (Figure 8 H and (Cornelissen et al., 2012b)). In conclusion IL-31 induced the IL1A and IL1B expression via the PI3K, p38 and ERK pathways and induces the release of IL-1α. The release of IL-1β could only be detected after additional nigericin stimulation. Moreover high IL-31 serum levels correlated with higher IL-1α levels in AD patients. RESULTS 49

IL-1 NETWORK IS NOT AFFECTED BY IL-20 AND IL-24

As it was identified before that IL-31 affects the IL-1 signaling pathway, it should subsequently be tested, if this effect is direct or indirect, i.e. whether or not the genes of the IL-1 signaling network are directly controlled by IL-31 activated signaling pathways. IL- 31 effect. As already mentioned, IL-31 induces the gene expression of IL20 and IL24 immediately after treatment (Cornelissen et al., 2012b). Furthermore IL-20 and IL-24 were shown to mediate parts of the IL-31 effect in organotypic skin modes (Cornelissen et al., 2012b). Partially the same signaling pathways such as p38 and ERK were necessary to induce either IL1A or IL20 and IL24 gene expression (Figure 8 H and Cornelissen et al., 2012b). Therefore it should be tested in the following experiments if IL1A or IL1B gene expression was triggered by IL-20 or IL-24. For this purpose 3D organotypic HaCaT- IL31RA models were used and treated with IL-20, IL-24 or with both. Gene expression was analyzed after seven days of treatment using qRT-PCR. Neither IL-20 nor IL-24 nor a combination of both induced IL1A and IL1B gene expression (Figure 9). Interestingly the gene expression of both IL1 genes was slightly reduced upon treatment.

1.5 1.5 n IL1A IL1B o i 1.2 1.2 s s

e 0.9 0.9 r * p *

x 0.6 0.6 e

.

l 0.3 0.3 e rel. expression r 0 0 Ctrl. IL-20 IL-24 IL-20 Ctrl. IL-20 IL-24 IL-20 IL-24 IL-24

Figure 9: IL-20 and IL-24 do not induce IL-1 gene expression. qRT-PCR analysis of the indicated genes in 10-day-old HaCaT-IL31RA organotypic skin models stimulated with 20 ng/ml of rhIL-20 or rhIL-24, mean values ± SD, n = 3. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001).

RESULTS 50

ROLE OF IL-1α IN IL-31 SIGNALING

IL-1α AFFECTS THE SKIN BARRIER

It has been shown by various research groups that IL-1α can influence the formation of the skin barrier (Wood et al., 1992; Barland et al., 2004; O'Shaughnessy et al., 2010). To investigate in which way IL-1α affects the expression of the essential skin barrier protein filaggrin, 10-day-old HaCaT-IL31RA and 5-day-old primary organotypic skin models with NHEK cells were used. After lifting the models to the air liquid interphase they were treated every second day with IL-31 or IL-1α. It could be observed that IL-1α inhibited FLG mRNA expression to a comparable extent as IL-31 (Figure 10 A). Moreover in monolayer cell culture experiments with HaCaT-IL31RA cells treated up to 72 h with IL- 1α, gene expression of desmosomal proteins and filaggrin processing enzymes was analyzed with qRT-PCR (Figure 5 C). IL-1α also altered the expression of desmosomal proteins, as shown before for IL-31 (Figure 5 C). In HaCaT-IL31RA cells treated with IL- 1α for up to 72 h the genes encoding desmoglein 1 and 4 ( DSG1 and 4) but also desmocollin 1 and 2 ( DSC1 and 2) and the genes encoding caspase-14 ( CASP14 ) and kalikrein-like peptidase 7 ( KLK7 ) were down regulated (Figure 10 B).

HaCaT-IL31RA A NHEK 5 days 10 days B 1.6 1.2 1.2 IL-1 α 1.0 1.0 1.2 0 h exp. exp.

0.8 0.8 *** *** *** ** 24 h

0.8 *** *** ** 0.6 **

0.6 ** FLG FLG ** 48 h *** *** *** *** 0.4 0.4 0.4 *** rel. 0.2 rel. 0.2 *** 72 h *** 0 0 rel. expression 0 -IL-31 IL-1α -IL-31 IL-1α 4 4 7 1 K G1 G C2 L S S S SP K D D DSC1 D A C

Figure 10: IL-1α deregulates the expression of genes associated with the mechanical skin barrier. A. NHEK 3D organotypic skin model (one representative experiment) and HaCaT-IL31RA 3D organotypic skin model were stimulated with 100 ng/ml rhIL-31 and 10 ng/ml rhIL-1α, FLG expression measured by qRT-PCR mean values ± SD, n = 3. B. qRT-PCR analysis of the indicated genes in HaCaT-IL31RA monolayer cells stimulated with 10 ng/ml rhIL-1α, mean values ± SD, n = 3. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001). RESULTS 51

These findings demonstrate that in our cell culture models IL-1α regulates the expression of filaggrin and desmosomal proteins in a comparable way to IL-31 (Figure 5 C and Figure 10 B). All genes analyzed with qRT-PCR after treating the cells either with IL- 31 or IL-1α were regulated in the same direction except for DSC2 . DSC2 was up regulated upon IL-31 treatment (Figure 5 C) but repressed upon IL-1α treatment in monolayer cell culture experiments (Figure 10 B). Genes associated with skin differentiation i.e. FLG (Figure 10) and genes associated with the skin barrier i.e. DSG1 , DSG4 , DSC1 , CASP14 and KLK7 (Figure 5 and Figure 10) were regulated the same way upon either IL-31 or IL- 1α treatment. Moreover IL-31 induces directly or indirectly the IL-1 network with promoting the release of IL-1α into the extracellular space (Figure 8). These facts raised the question, if the effects regarding the skin barrier, which could be observed in organotypic models treated with IL-31, were indeed mediated via the IL-1 network. To evaluate the downstream effect of IL-1α after IL-31 treatment, the IL-1 receptor antagonist anakinra, which inhibits the binding of IL-1 to IL-1R1 was used (Dinarello et al., 2012). Anakinra (brand name Kineret®) is a potent IL-1 signaling inhibitor effectively used in patients suffering from rheumatoid arthritis (RA) and neonatal-onset multisystem inflammatory disease (NOMID) (Jesus and Goldbach-Mansky, 2014). Primary organotypic skin models with NHEK cells were cultivated for two day to examine mRNA expression and for five days to analyze protein expression. These models were either treated with IL- 31 or IL-1α every second day. Moreover anakinra was added to block the IL-1α signaling one-day prior to the cytokine treatment. Both IL-31 and IL-1α were able to suppress filaggrin gene and protein expression to a comparable extent (Figure 11 A and B). These downregulating effects of both IL-31 and IL-1α on filaggrin gene and protein expression were reverted by anakinra. This is indicated by the decreased green staining for filaggrin in the IL-31 and IL-1α treated samples and an increased staining intensity in the anakinra pretreated models (Figure 11 B). This implies that IL-1α may be at least partially necessary for the response to IL-31 (Figure 11 A and B). Anakinra alone had even a slightly increasing effect on filaggrin expression (Figure 11 B).

RESULTS 52

A NHEK 2 days 2.0 1.6 exp. 1.2 * FLG

0.8 * 0.4 rel. 0 - IL-1α IL-31 Ana IL-1α IL-31 /Ana /Ana

HaCaT-IL31RA mono α α B NHEK 5 days C IL-31 IL-31/Ana IL-1 IL-1 /Ana Ana HE Filaggrin -1530153015 30 15 30 1530 m STAT3 phospho- STAT3 p38 IL-31 - phospho- α p38 IL-1 p65

phospho -p65

IκBα Ana Ana IL-31/ tubulin / α Ana IL-1 actin Figure 11: Role of IL-1α in IL-31 signaling. A. FLG expression in NHEK 3D organotypic skin model treated with 10 ng/ml rhIL-1α, 100 ng/ml rhIL-31 and one day before with 3 µg/ml anakinra (Ana) was analyzed by qRT-PCR, mean values ± SD, n = 3. B. NHEK 3D organotypic skin models were treated with cytokines and anakinra as described in A. Histological sections were stained with HE, for filaggrin (green) andthe DNA was stained with DAPI (blue). Scale bar HE 200 µm, scale bar immunofluorescence 100 µm. C. The indicated signaling molecules were analyzed by Western blot technique. HaCaT-IL31RA monolayer cells were pretreated with 3 µg/ml anakinra (Ana) one day before and then stimulated with 10 ng/ml rhIL-1α, 100 ng/ml rhIL-31 for the indicated time periods. Cells were pretreated with 3 µg/ml anakinra for 30 min as indicated. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001).

As another step to investigate if the observed IL-31 effects in 3D models are a result of the IL-1 network, the activation of signaling kinases downstream of IL-1α were analyzed. The phosphorylation status of STAT3, p38 and p65, known downstream effectors of the IL-31 and IL-1 signaling, were analyzed in HaCaT-IL31RA monolayer cells (Cornelissen et al., 2012a; Barksby et al., 2007). Moreover degradation of the inhibitor protein I kappa B alpha (IκBα) was analyzed (Barksby et al., 2007). Cells were pretreated for 30 minutes with anakinra and the cytokines were added for the indicated time (Figure 11 C). Presence of IL-31 but not IL-1α promoted the phosphorylation of STAT3 and this effect was not inhibited by anakinra pretreatment. However, the phosphorylation of p65 and the degradation of IκBα was influenced by the presence of IL-1α but not IL-31 and this effect was blocked by anakinra pretreatment. On the other hand, addition of anakinra efficiently prevented the phosphorylation of the MAP-Kinase p38, which was enhanced in the RESULTS 53 presence of each, IL-31 or IL-1α. Summarizing, anakinra pretreatment blocks IL-31 and IL-1α mediated filaggrin reduction. Moreover both cytokines IL-31 and IL-1α induce p38 phosphorylation but only IL-1α induces p65 phosphorylation and the degradation of IκBα.

IL-31 induces STAT3 phosphorylation.

REGULATION OF ANTIMICROBIAL PEPTIDES BY IL-31 IS

DEPENDENT ON IL-1α INDUCTION

IL-1α REGULATES THE EXPRESSION OF AMP S

In addition to effects on various genes encoding proteins associated with barrier function, literature data supports that several genes encoding antimicrobial peptides (AMPs) are induced in response to IL-1α including genes encoding S100 Ca 2+ -binding proteins, human beta defensin 2 (hBD2/DEFB4A ) and human beta defensin 3 (hBD3/DEFB103A ) (Yano et al., 2008; Bando et al., 2007). To check if the genes encoding for S100A7, S100A8, S100A9, S100A12, DEFB103A and DEFB4A respond to IL-1α in HaCaT-IL31RA monolayer cultures, their expression was analyzed by qRT-PCR. The expression of DEFB4A was significantly increased within 24 h of IL-1α treatment (Figure 12). However DEFB103A gene expression was not significantly induced upon IL-1α treatment. The analyzed S100 genes, S100A7 , S100A8 , S100A9 and S100A12 were induced upon IL-1α treatment. In contrast to S100A7 and S100A12 , gene expression of S100A8 and S100A9 was increased within the first 48 h but the induction was reduced to a lower level after 72 h (Figure 12).

RESULTS 54

60 20 ** n IL-1 α IL-1 α o i s

45 *** 0 h 15 0 h s e *** r 24 h 10 24 h p 30 x 48 h * * e 48 h

* 15 5 *** . l 72 h 72 h e rel. expression r 0 0 DEFB4A DEFB103 S100A7 S100A9 S100A8 S100A12

Figure 12: IL-1α regulates the expression of antimicrobial peptides. HaCaT-IL31RA cells were stimulated with 10 ng/ml rhIL-1α for the indicated time periods and gene expression was analyzed with qRT-PCR, mean values ± SD, n = 3. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001).

In summary IL-1α inhibited the FLG expression in HaCaT and NHEK organotypic skin models as well as the expression of desmosomal proteins (Figure 10 and Figure 12). Furthermore it also induced the expression of AMPs (Figure 12), which are a known defense mechanism of the skin (Schröder and Harder, 2006).

IL-31 INFLUENCES THE EXPRESSION OF AMP S

As seen before, IL-1α mediates downstream effects of IL-31 on the filaggrin protein and gene expression level (Figure 11). Furthermore it was shown that IL-1α induced the expression of AMPs in HaCaT-IL31RA cells. These findings lead to the question whether IL-31 can induce the expression of antimicrobial peptide genes in a similar manner as IL- 1α. The gene expression of S100A7, S100A8, S100A9, S100A12 , DEFB103A and DEFB4A was analyzed in HaCaT-IL31RA monolayer cultures stimulated with IL-31 for up to 72 h using qRT-PCR (Figure 13 A). The expression of all genes was significantly increased upon IL-31 stimulation. The AMP encoding genes were also induced by IL-1α in HaCaT-

IL31RA cells, as indicated previously, albeit the effect was less strong in comparison to the response to IL-31 (Figure 12). Moreover when organotypic HaCaT-IL31RA models were challenged with IL-31, the concentration of human beta defensin proteins in the cell culture medium was increased (Figure 13 B). Additionally staining intensity of S100A7 and S100A9 proteins was enhanced in 10-day-old 3D organotypic models after IL-31 stimulation (Figure 13 C).

RESULTS 55

A HaCaT-IL31RA mono 1600 300 IL-31 1200 225 0 h

800 * 150 24 h *

** 48 h

400 ** 75 * ** ** * 72 h

rel. expression 0 rel. expression 0 DEFB4A DEFB103 S100A7 S100A9 S100A8 S100A12 B HaCaT-IL31RA 3D 10 days

3 3 * 6 * * 2.25 2.25 4.5 1.5 1.5 3 0.75 0.75 1.5 hBD2 (ng/ml) hBD3 (ng/ml)

0 0 S100A7 (ng/ml) 0 - IL-31 - IL-31 - IL-31

800 800 600 600 400 400 200 200 S100A9 (ng/ml) 0 (ng/ml)S100A12 0 - IL-31 - IL-31 C S100A7 hBD-2 IL-31 -

Figure 13: IL-31 regulates the expression of antimicrobial peptides. A. HaCaT-IL31RA cells were stimulated with 100 ng/ml rhIL-31 for the indicated time periods and gene expression was analyzed by qRT-PCR, mean values ± SD, n = 3 B. The indicated proteins were measured by ELISA in the supernatants of 10-day-old HaCaT-IL31RA 3D organotypic skin models stimulated every second day with 100 ng/ml rhIL-31, mean values ± SD n = 3. C. Histological sections of 10-day-old HaCaT-IL31RA 3D organotypic skin models stimulated every second day with 100 ng/ml rhIL-31were stained with HE, for the indicated proteins (green), and the DNA was stained with DAPI (blue). Scale bar 100 µm. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001). RESULTS 56

To further analyze the IL-31 mediated AMP gene expression, pulse experiments were performed. Our research group had shown that only a relative short IL-31 pulse was sufficient to de-regulate the differentiation process in keratinocytes, with filaggrin as a sensitive marker. A reduction of FLG expression in 3D organotypic skin models measured after 7 days could be observed after an IL-31 pulse of 2 h at the first day, while other genes responded less sensitive (Cornelissen et al., 2012b). Inspired by these effects on differentiation, the pulse experiment was used to analyze AMP gene expression. 7-day-old organotypic HaCaT-IL31RA models were treated at the first day of differentiation with IL- 31 for 2, 4, 6, 8 h or were treated permanently for the 7 days (Figure 14). Afterwards the IL-31 pulse medium was changed and the models were cultivated in the absence of IL-31. DEFB103A was already induced 20-fold after a 2 h IL-31 stimulation whereas induction of DEFB4A and S100A7, S100A8 , S100A9 and S100A12 required a permanent IL-31 presence.

1500 500 10 DEFB4A DEFB103A S100A7 * 1200 400 8 900 300 6 600 200 4 1 21 23 38 43 1 0.9 0.7 0.8 0.8 300 1 2.6 3.7 5.3 6.2 100 2 * rel.expression 0 rel.expression 0 rel.expression 0 0 2 4 6 8 perm. 0 2 4 6 8 perm. 0 2 4 6 8 perm. IL-31 [h] IL-31 [h] IL-31 [h]

50 150 25 ** S100A8 S100A9 S100A12 *** 40 *** 120 20 30 90 15 20 60 10 1 2.3 1.5 2.1 1.2 1 3.4 2.3 4.4 5 1 1.6 1.5 2.7 2.4 10 30 5

rel. expression 0 rel. expression 0 rel. expression 0 0 2 4 6 8 perm. 0 2 4 6 8 perm. 0 2 4 6 8 perm. IL-31 [h] IL-31 [h] IL-31 [h]

Figure 14: Permanent IL-31 treatment induces AMP expression. qRT-PCR analysis of the indicated genes in HaCaT-IL31RA organotypic 3D models stimulated with 100 ng/ml rhIL-31 for the indicated times at day 0 or permanent every second day. Skin equivalents were harvested after 7 days, mean values ± SD, n = 3. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001).

Overall the analyzed data indicated that both cytokines IL-1α and IL-31 promote the expression of antimicrobial peptides in HaCaT-IL31RA cells, irrespective of monolayer or 3D cultured. Furthermore the release of certain AMPs, i.e. hBD-2 hBD-3, S100A9 and S100A12, into the cell culture medium upon IL-31 treatment could be shown. RESULTS 57

REGULATION OF ANTIMICROBIAL PEPTIDES BY IL-31 IS

DEPENDENT ON IL-1α INDUCTION

In former experiments it was seen that the presence of either IL-1 (Figure 12) or IL-31 (Figure 13) triggered the expression of AMPs in keratinocytes. As a next step it should be tested, if IL-1 is required for the IL-31 effect on AMPs by the use of the IL-1 receptor antagonist anakinra. Therefore HaCaT-IL31RA monolayer cells were stimulated for 72 h with IL-31, IL-1α and anakinra, or a given combination. All genes analyzed were not at all or to a lesser extent induced by IL-31 when the IL-1 signaling was blocked by anakinra. This also applied to the induction with IL-1α (Figure 15).

HaCaT -IL31RA mono 1000 DEFB4A 375 ** DEFB103A 200 S100A7

800 * 300 160 600 225 120 400 150 80

200 *** 75 40 ** ** *** *** rel. expression 0 - IL-1α IL-31 Ana IL-1α IL-31 0 - IL-1α IL-31 Ana IL-1α IL-31 0 - IL-1α IL-31 Ana IL-1α IL-31 Ana Ana Ana Ana Ana Ana

100 S100A8 100 S100A9 37.5 S100A12

80 80 30 *** 60 60 22.5 40 40 15 *

20 20 ** 7.5 ** *** *** rel. expression *** 0 - IL-1α IL-31 Ana IL-1α IL-31 0 - IL-1α IL-31 Ana IL-1α IL-31 0 - IL-1α IL-31 Ana IL-1α IL-31 Ana Ana Ana Ana Ana Ana Figure 15:Regulation of antimicrobial peptides by IL-31 is dependent on IL-1α induction. The indicated genes were analyzed by qRT-PCR. HaCaT-IL31RA monolayer cells were treated for 72 hours with either 10 ng/ml rhIL-1α, 100 ng/ml rhIL-31 and 3 µg/ml anakinra (Ana), mean values ± SD, n = 3. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001).

In summary all these results show that the presence of IL-1 is necessary for the induction of AMPs by IL-31, as seen before for the regulation of differentiation markers i.e. FLG and for the regulation of different barrier proteins. RESULTS 58

ROLE OF LOW IL-31 DOSES IN HUMAN SKIN BARRIER

FORMATION

IL-31 prevents the formation of the mechanical barrier but stimulates the antimicrobial barrier. To evaluate whether these two effects can be separated and furthermore to identify a potential role of IL-31 in healthy human skin, IL-31 was titrated in HaCaT-IL31RA 3D organotypic skin models and skin differentiation was analyzed after ten days. As shown before, 100 ng/ml of IL-31 were sufficient to prevent filaggrin expression and terminal differentiation (Figure 5, Figure 7 and Figure 11 and (Cornelissen et al., 2012b)). Even a dose of 10 ng/ml of IL-31 could prevent filaggrin expression in organotypic HaCaT- IL31RA 3D models (Figure 16 A). Lower doses had no significant effects on the gene expression of the differentiation marker FLG and INV and on the expression of desmosomal proteins (Figure 16 B), and were also insufficient to prevent filaggrin protein expression (Figure 16 A).

However at a concentration of 1 ng/ml IL-31 enhanced expression of IL1A and IL1B and of the genes encoding several antimicrobial peptides (S100A8, S100A9, S100A12, DEFB103A and DEFB4A ) was observed in HaCaT-IL31RA monolayer cells after 72 h (Figure 16 C).

RESULTS 59

HaCaT-IL31RA 3D 10 days HaCaT-IL31RA ABHE Filaggrin 3D 10 days 2.5 FLG - 2 1.5 1 0.5

0.5 ** *** 0 00.5 1 10 100 1 1.5 INV 1.2 IL-31ng/ml 0.9 10 0.6

0.3 *** 100 0 00.5 1 10 100 IL-31 ng/ml HaCaT-IL31RA mono 5 C 12 150 4 10 120 3 0 ng/ml 8 1ng/ml 90 2 6 100 ng/ml 60 4 1 rel.expression rel. expressionexpression rel. 2 30 0

rel.expression DSG1 DSC1 0 0 IL1A IL1B DEFB4A DEFB103A DSG4 DSC2 200 150 0 ng/ml 100 1ng/ml 50 100 ng/ml rel.expression 0 S100A7 S100A8 S100A9 S100A12

Figure 16: Role of low IL-31 doses in human skin barrier formation. A. HaCaT-IL31RA 3D organotypic skin models were stimulated with rhIL-31 in the indicated concentration every second day for 10 days and histological sections were stained with HE or for filaggrin (green) and DNA with DAPI (blue). Scale bar HE 200 µm, scale bar IF 100 µm. B. qRT-PCR analysis of the indicated genes in 10-day-old HaCaT-IL31RA 3D organotypic models stimulated as in A, mean values ± SD, n = 3. C. qRT-PCR analysis of the indicated genes in HaCaT-IL31RA cells treated for 72 h with the indicated concentration of rhIL-31, mean values ± SD, n = 3. P-values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001).

Taken together these findings demonstrate that low doses of IL-31 enhance the expression of genes encoding AMPs (Figure 13 and Figure 16), whereas higher doses have a profound effect on the mechanical barrier (Figure 6 and Figure 7).

RESULTS 60

IL-31 PROMOTES THE EXPRESSION AND RELEASE OF TSLP

Various studies either in mice or in dogs have shown that IL-31 has an important role in inducing itch (Takaoka et al., 2006; Dillon et al., 2004; Gonzales et al., 2013; Arai et al., 2013). In further support of this hypothesis IL-31 expression is increased in the lesional skin of AD patients and the IL-31 receptors are localized in dorsal root ganglia. Recent studies have shown that repeated administration of IL-31 in mice also increased the expression of IL-31 receptors in dorsal root ganglia (Arai et al., 2014).

A recent clinical study addressed the role of IL-31 in human skin in vivo . Ten patients with AD and ten healthy volunteers were challenged with IL-31 and NaCl (negative control) by skin prick testing (Hawro et al., 2014). They concluded that IL-31 does not induce immediate itch responses in humans. Furthermore the late onset of IL-31-induced itch supports the notion that IL-31 exerts its pruritic effect indirectly via keratinocytes and secondary mediators, rather than through its receptors on cutaneous nerves (Hawro et al., 2014). Sooner afterwards it was demonstrated that IL-31 treated epidermal models were a source of TSLP (Danso et al., 2014). Moreover two papers revealed that the receptor complexes IL-31 and TSLP mediate itch via the same subset of neurons. TSLP acts directly on a subset of TRPA1 positive sensory neurons to trigger itch behavior (Wilson et al., 2013b). IL-31 mediated itch was significantly reduced in TRPA1 and TRPV1-deficient mice (Cevikbas et al., 2013). Altogether these findings let to the idea that IL-31 treatment on keratinocytes may promote the secretion of TSLP and by this mediate itch behavior. RESULTS 61

Figure 17: IL-31 promotes the expression and release of TSLP. A. TSLP gene expression was analyzed in monolayer HaCaT-IL31RA cells upon treatment with 100 ng/ml rhIL-31 using qRT-PCR. B. TSLP in the supernatant of a 10-day-old HaCaT-IL31RA 3D model treated every second day with 100 ng/ml rhIL-31, mean values ± SD, n = 3. C. TSLP in the supernatant of a 5-day-old NHEK 3D organotypic skin model treated with 100 ng/ml rhIL-31 for the indicated time periods, mean values ± SD, n = 3. D. TSLP gene expression was analyzed in HaCaT-IL31RA skin models upon treatment with 10 ng/ml rhIL-1α or rhIL-1β using qRT-PCR, mean values ± SD, n = 3. P- values were calculated using Student’s t-test (* p<0.05; ** p<0.01; *** p<0.001).

To test if IL-31 affects TSLP gene expression and TSLP release from keratinocytes, qRT- PCR were performed on samples from monolayer HaCaT-IL31RA cells treated with IL-31 for up to 72 h. A significant increase expression of the TSLP gene was observed (Figure 17 A). To test if TSLP is also released from HaCaT-IL31RA 3D organotypic skin models into the cell culture medium, ELISA was performed after ten days of stimulation with IL- 31. A significant increase of TSLP concentration in the cell culture medium was measured (Figure 17 B). To identify if TSLP could be a possible secondary mediator upon IL-31 treatment. A five-day-old organotypic skin equivalent was apically challenged with IL-31 and a sample of the surrounding cell culture medium was taken every 30 min. Release of TSLP into the medium was measured by ELISA. TSLP was measured first 30 min after IL- 31 application, TSLP concentration peaked at 1.5 h (20 pg/ml) and after 2.5 h no TSLP could be detected in the medium anymore (Figure 17 C). As shown before, several genes, which were induced by IL-31, were indirect IL-31 target genes and were induced by either IL-1α or IL-1β. As a consequence HaCaT-IL31RA monolayer cells were treated either with IL-1α or with IL -1β for up to 72 h and TSLP gene expression was analyzed. No significant change in TSLP gene expression could be observed by treatments with both RESULTS 62 cytokines (Figure 17 D). In summary IL-31 promotes TSLP gene expression and release from HaCaT-IL31RA keratinocytes an effect that appeared independent of IL-1α or IL -1β. DISCUSSION 63

3 DISCUSSION DISCUSSION 64

ROLE OF IL-31 IN ATOPIC DERMATITIS

AD is a common chronic inflammatory disease characterized by a defect in keratinocyte differentiation and skin barrier formation (Leung and Bieber, 2003). Consequences of the impaired skin barrier are the increased percutaneous penetration of allergens and pathogens, thereby promoting inflammation that can be further aggravated by secondary infections. Furthermore the impaired skin barrier is characterized by an increased TEWL. Alterations in genes encoding key structural components for proper keratinocyte differentiation and skin barrier formation are associated with barrier dysfunction. A seminal finding was the identification of FLG mutations in patients with ichthyosis vulgaris and AD (Smith et al., 2006; Palmer et al., 2006; Sandilands et al., 2007; Brown and McLean, 2012). Also cytokines are deregulated in AD patients and were postulated to participate in disease progression (Leung and Bieber, 2003; Hanel et al., 2013). One of these deregulated cytokines is IL-31. Serum IL-31 expression is increased and correlates with disease severity in these patients (Kim et al., 2011; Neis et al., 2006; Raap et al., 2008; Nobbe et al., 2012). Furthermore IL31 mRNA levels of lesional AD skin are elevated compared to non-lesional and healthy skin (Sonkoly et al., 2006). IL31 transcripts could be further induced after topical exposure of the non-lesional skin of AD patients to either dust mite allergens (from D. farinae and D. pteronyssimus ) or staphylococcal super antigens (SEB and TSST-1) (Sonkoly et al., 2006). Source of IL-31 production in the skin are either resident or infiltrating immune cells (Cornelissen et al., 2012a). One major population that produce IL-31 are activated T-cells. Upon treatment with various inflammatory stimuli such as UV radiation, allergens, pathogens and histamines, T-cells promote IL31 gene expression and the release of IL-31 (Bilsborough et al., 2006; Cornelissen et al., 2011; Dillon et al., 2004; Gutzmer et al., 2009; Sonkoly et al., 2006). Recently it was shown that IL-4, a prominent cytokine in AD, induces IL-31 expression in TH1 cells (Stott et al., 2013) and downregulates FLG expression (Howell et al., 2009). This subset of T-cells was also shown to have upregulated IL31 mRNA expression upon activation with either anit-CD3 or anti-CD28 (Zhang et al., 2008). Moreover circulating T- cells positive for the skin-homing cutaneous lymphocyte antigen (CLA) produce IL-31. Furthermore, cells isolated from patients with AD produce higher levels of IL-31 DISCUSSION 65 compared to either psoriasis patients or healthy individuals (Bilsborough et al., 2006). By recognizing skin related allergens these CLA positive cells accumulate in acute lesions of AD and release higher amounts of IL-4 and IL-13 (Akdis et al., 1997). Furthermore mast cells induce IL31 mRNA expression when treated with the antimicrobial peptides, such as hBDs (hBD-1 to hBD-4) or LL-37, an effect that could be further enhanced by pretreating the cells with IgE (Niyonsaba et al., 2010). AD lesions providing an inflammatory environment, with elevated IgE levels. Only 10% of skin lesions are not colonized with S. aureus resulting in an increased amount of bacteria antigens and an increased expression of antimicrobial peptides. All these products promote the expression and release of IL-31 from various cell (Leung, 1997; Guttman-Yassky et al., 2011; Ott et al., 2009). Due to this IL-31 seems to have a major contribution to the pathomechanism of AD.

IL-31 WEAKENS THE SKIN BARRIER

Previously our research group demonstrated that IL-31 inhibits keratinocyte proliferation and differentiation without affecting apoptosis with FLG being a repressed target of IL-31 signaling (Cornelissen et al., 2012b). 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 and barrier formation. Moreover it was unclear whether IL-31 signaling was directly or indirectly responsible for affecting FLG expression.

In this work it was observed that IL-31 interferes with the expression of many genes associated with skin barrier formation in 3D organotypic skin models of HaCaT-IL31RA cells, in contrast to monolayer cultures where only a few genes were deregulated (Cornelissen et al., 2012b). HaCaT-IL31RA cells were preferably used because primary NHEK cells often show donor dependent variability, which was noted before by studying the response to rhIL-31. HaCaT cells, as a donor independent cell system, are spontaneously immortalized primary keratinocytes that are able to differentiate in DISCUSSION 66 organotypic skin models (Boukamp et al., 1988). HaCaT cells do not express the IL- 31RA but still express the OSM receptor, which is one of the subunits of the heterodimeric receptor complex. That is why a cell line was established in our group that expresses the IL-31RA under the control of doxycycline (Cornelissen et al., 2012b).

Altogether Affymetrix exon array data identified approximately 570 genes that were altered more than two-fold in response to IL-31. Indeed many genes encoding proteins associated with corneodesmosomes, such as desmogleins or desmocolleins, with the formation of terminally differentiated corneocytes and with the production of natural moisturizing factors, including filaggrin and its processing enzymes caspase-14 and KLK7, were deregulated in response to IL-31 (Figure 5). The findings made by the Affymetrix arrays were only partially reproduced in the monolayer culture but completely consistent with findings in the organotypic skin models (Figure 5). This suggests that observations made in monolayer cultures have to be carefully interpreted. This may be related to the lack of appropriate cellular interactions. 3D organotypic skin cultures with fibroblasts serve as a more physiological model for epidermal development and homeostasis, in which a calcium gradient develops in the epidermal layer, which is comparable to normal skin. As a result a complete skin-like epidermal barrier is formed. Moreover fibroblasts provide physiologically relevant cytokines and growth factors that lack in monolayer cultures. In contrast monolayer cultures are a highly simplified and rather rudimental model and results generated in this model should be interpreted with caution and always compared with results from either organotypic or in vivo models or ideally should be confirmed in these settings.

The observations made in monolayer and organotypic 3D skin models (Figure 5) implicate that IL-31 treatment does not only modulate keratinocyte differentiation and proliferation as shown before (Cornelissen et al., 2012b) but also affects the gene expression pattern of desmosomal and filaggrin processing enzymes such as CASP14 and KLK7 (Figure 5). Interestingly IL-31 did not affect the expression pattern of genes expressing tight junction DISCUSSION 67 proteins (Figure 5). In contrast to this finding treatment of keratinocytes with high amounts of histamine results in a similar expression pattern of structural proteins, including filaggrin and keratin 10 (Gschwandtner et al., 2013; Cornelissen et al., 2012b), but it also affects the expression of tight junction proteins beyond the expression of desmosomal structures (Gschwandtner et al., 2013). Thus IL-31 showed a more selective effect in the experiments displayed in this work (Figure 5). This argues for different signaling pathways controlling different aspects of the differentiation process in keratinocytes. Consistent with these findings bronchial epithelia cells also show no alteration of the tight junctions determined by transepithelial electrical resistance when they were treated with IL-31 (Parker et al., 2012).

The functional consequences of suppressed desmosomal gene expression were confirmed by assessing the integrity of the skin barrier in an organotypic skin model (Figure 6) and in a CsSE model (Figure 7). Within these models poor differentiation and reduced expression of late differentiation markers, e.g. filaggrin and lipids, could be observed (Figure 6 and Figure 7). Moreover an increased penetration of biotin from the dermal site, and the phl p1 allergen from the apical site into 3D models was seen (Figure 6). Additionally high vulnerability to skin irritants and enhanced TEWL were measured (Figure 6 and Figure 7). All these observations point to a severe disturbance of the mechanical and functional properties of the skin barrier regarding formation of the cornified envelope. Additionally a defect in cellular adhesion could be implicated by the enhanced penetration of biotin from the dermal site (Figure 6). These findings are consistent with the change in desmosomal gene expression upon IL-31 treatment (Figure 5) and the reduced expression of keratinocyte differentiation markers such as filaggrin and keratin 10 as shown before (Cornelissen et al., 2012b). The impaired formation of the cornified envelope upon IL-31 treatment may be a cooperative effect between the reduction of filaggrin, filaggrin processing enzymes, keratin 10, and desmosomal proteins. This idea is supported by the finding that filaggrin is essential for the formation of the stratum corneum and for skin barrier function (Mildner et al., 2010; Sandilands et al., 2009). The enhanced penetration of biotin also implies that IL-31 treated keratinocytes did not form a basal membrane DISCUSSION 68 structure (Figure 6). These might also be explained by the disorganized β4-integrin, which in untreated organotypic models is located at the basal membrane and in IL-31 treated models spreads through all epidermal layers (Cornelissen et al., 2012b). However disorganization of β4-integrin might not be the only cause but the disturbance of the basal membrane structure might be further mediated or supported by the reduced expression of desmosomal proteins (Figure 5).

In summary different in vitro and in vivo approaches were used, namely monolayer cell culture experiments, organotypic 3D models, and a mouse model, to further define the role of IL-31 for the skin barrier integrity. Together these findings are consistent with the IL- 31-induced defect in skin differentiation observed previously (Cornelissen et al., 2012b) and suggest defects in the mechanical barrier due to reduced expression of structural proteins and corneodesmosomes. Furthermore IL-31 exhibits the potential to increase the irritating properties of different external stimuli as shown for SDS (Figure 6). These findings indicate that IL-31 may play an important role in altering the skin barrier. All these features shown here to be triggered by IL-31 treatment were also found in the skin of AD patients (Neis et al., 2006), strengthening the idea that elevated IL-31 concentrations in the skin of AD patients not only correlate with the disease sights and symptoms but IL-31 also seems to be a driver of the pathogenic mechanism leading to the AD skin phenotype.

Recently an ‘outside-inside,’ then back to ‘outside’ pathogenic mechanism in AD was postulated, expanding on the previous view that AD is largely a disease of immunologic etiology (Cornelissen et al., 2012b; Elias and Schmuth, 2009). The authors suggested that primary inherited barrier abnormalities in AD ultimately stimulate downstream paracrine mechanisms that could further compromise skin barrier function, driving an ‘outside- inside-outside’ pathogenic loop in AD. This model suggests a cyclic interplay between inherited barrier abnormalities and exogenous and endogenous stressors with key functions of different secreted signaling molecules. More precisely, inherited barrier abnormalities allow enhanced uptake of allergens and antigens, which trigger increased IL-31 expression, DISCUSSION 69 as well as other factors, in immune cells, including Th2 cells, and enhanced IL-31RA expression on keratinocytes (Cornelissen et al., 2012a). The elevated IL-31 expression can then result in a negative feedback loop in which the released IL-31 affects again the skin barrier. The findings made in this thesis endorse this model.

IL-31 STIMULATES THE IL-1 NETWORK

Of particular interest was the observation that not only expression of many genes encoding components of the IL-1 signaling network was affected by IL-31 but also an increased IL- 1α and IL-1β release was stimulated by IL-31 (Figure 8 A-C, E, F), supporting a concept in which IL-1 signaling is downstream of IL-31. Interestingly, a combination of IL-31 and the inflammasome activator nigericin resulted in a synergistic effect concerning IL-1α and IL- 1β release (Figure 8 D). Considering the increase of the intracellular concentrations of IL- 1α and IL-1β (Figure 8 C) the synergistic effect can be explained by the cleavage of pro- IL-1β by caspase-1, which is activated upon inflammasome activation (Barksby et al., 2007). The cleaved and activated IL-1β is then released from the cell either by small plasma membrane microvesicles (MacKenzie et al., 2001) or endocytotic vesicles (Andrei et al., 2004). This synergistic effect is in line with other observations in monocytes. In these cells a second stimulus is required to release active IL-1 cytokines. An initial stimulus, e.g. LPS, causes accumulation of pro-IL-1β in the cytosol and only a modest IL- 1β secretion (Dinarello, 1998). IL-1β release is then strongly induced by extracellular adenosine triphosphate (ATP) that activates procaspase-1 and subsequently pro-IL-1β (Ferrari et al., 2006). Together these results suggest that IL-1 signaling is an important mediator of the effects of IL-31 (Figure 11 and Figure 15). The importance of these findings is supported by published findings of known consequences of this network on skin diseases. The proinflammatory IL-1 signaling network is known to be an important mediator of innate immunity (Dinarello, 2009). Physiological levels of IL-1α enhance barrier formation and wound healing following mechanical injury (Barland et al., 2004; Jung et al., 2010), promote the synthesis of epidermal lipids and the formation of lamellar bodies, and induce the upregulation of genes and proteins associated with cell adhesion, DISCUSSION 70 proliferation and epidermal differentiation in keratinocytes (Barland et al., 2004; Jung et al., 2010; Yano et al., 2008). In contrast, deregulation of the IL-1 signaling network has been associated with different skin diseases, including AD (Jensen, 2010). Thus over- activation of the network, for example due to mutations in inflammasome components or as a result of IL-1RA deficiency, can lead to autoinflammatory diseases with severe skin involvement (Contassot et al., 2012; Schmitz et al., 2011). Indeed increased secretion of IL-1α has been observed in lesional skin of patients with psoriasis (Barksby et al., 2007). Exposure of keratinocytes to IL-1α provokes hyperkeratosis (O'Shaughnessy et al., 2010) and enhanced expression of IL-1α, e.g. by intradermal injection, promotes an inflammatory skin phenotype (Jung et al., 2011). Additionally, keratinocyte-specific overexpression of IL-1α leads to the development of spontaneous inflammatory skin lesions in mice (Groves et al., 1995). The findings of this work, e.g. the reduction in filaggrin expression on gene and protein level after IL-1α or IL-31 treatment (Figure 10 A and Figure 11 A and B), support the inhibitory character of IL-1α for keratinocyte differentiation with IL-31 playing an important upstream role (Figure 8), suggesting that this cascade of cytokines controls skin differentiation. This is also supported by the observation that the effect of either IL-31 or IL-1α to repress filaggrin expression was abolished when the IL-1 signaling cascade was inhibited by anakinra (Figure 11 A and B). Moreover hyperkeratosis and other effects suggest that IL-31 controls through additional pathways other aspects that modify IL-1α signaling. The putatively involved signaling pathways and kinases will be discussed in more detail below.

Furthermore by regulating the NF-κB signaling, IL-1α also regulates immune-reactive properties of IFN-γ (Hurgin et al., 2007), which is the mayor stimulus to induce the expression of the IL-31 receptor on keratinocytes (Heise et al., 2009). Moreover these signaling pathways offer entry sites for crosstalk with other signals that might influence differentiation. It is also of interest that FLG loss-of-function mutations are associated with enhanced IL-1α and IL-1β expression (Kezic et al., 2012). Besides the stimulation with IL-

31 keratinocytes produce IL-1α upon mechanical injury by UVB. Furthermore the proinflammatory cytokine TNF-α induces the expression of IL-1α (Kutsch et al., 1993) and DISCUSSION 71 vice versa IL-1α induces TNF-α expression and release from endothelia cells (Imaizumi et al., 2000). Studies with knockout mice show that IL ‐1α is important for T-cell priming during contact hypersensitivity and for the induction of high levels of serum IgE following immunization with ovalbumin and furthermore IL-1 induce the proliferation and survival of T-cells (Sims and Smith, 2010). To sum up IL-1α and IL-1β induce gene expression in different immune cell types, such as monocytes or macrophages, epithelial and endothelial cells, and chondrocytes or fibroblasts (Jeong et al., 2004; Wolter et al., 2008; Holzberg et al., 2003; Vincenti and Brinckerhoff, 2001; Allantaz et al., 2007). By elevating the levels of intracellular IL-1α and IL-1β without releasing IL-1β as seen in this work (Figure 8 A- D), IL-31 primes the cells for a second stimulus leading to an aggravated immune reaction in the skin. Interestingly IL-1β is required for neutrophil recruitment and an effective innate immune response against cutaneous challenges with the pathogen S. aureus in an in vivo mouse model (Miller et al., 2007). Upon contact with staphylococcal exotoxin T-cell produce and release IL-31 (Sonkoly et al., 2006). Moreover IL-31 increases the intracellular levels of IL-1β in keratinocytes (Figure 8 C), which can be released upon an additional stimulus such as nigericin (Figure 8 D). This potential cascade indicates a role for IL-31 immune response against the pathogen S. aureus .

However both cytokines, IL-1α and IL-1β, are required for full development of asthma. They promote eosinophil infiltration into the lung, goblet cell hyperplasia, adhesion molecule expression, production of IL-4, IL-5 and allergen-specific IgG and IgE antibodies, and airway hyper responsiveness (Sims and Smith, 2010). As mentioned in the introduction 80% of all children suffering from AD develop rhinitis or asthma during their lifetime (Eichenfield et al., 2003). The increased gene expression of IL1A and IL1B as well as the boosted release of IL-1α and the elevated intracellular amount of IL-1β upon IL-31 treatment could promote the progression of the atopic march.

As mentioned before, by elevating the levels of intracellular IL-1α and IL-1β, without releasing IL-1β, IL-31 primes the cells for a second stimulus leading to an aggravated DISCUSSION 72 immune reaction in the skin, which would not appear if the IL-1 network would not be active. The patients' sera analysis has also shown that a correlation between IL-31 and IL- 1α is not only present in the in vitro models, but there is also a correlation between elevated IL-31 serum levels and increased IL-1α serum levels in patients. Due to the indirect modulation of the IFN-γ signaling as mentioned above, the elevated levels of IL-1α might have an effect on IL-31RA signaling, Moreover by inducing IL-1α gene expression and release IL-31 seems to induce a feed-forward loop which enhances signaling. By this IL-31 might be an important cytokine in an inflammatory environment as provided in acute AD lesions. It could be that elevated IL-31 levels over a longer time period lead to a formation of a chronic inflammatory environment and by this to disease progression.

Having set these observations in a larger disease-related context, it is now interesting to evaluate the relationship between IL-31 and the IL-1 network more profoundly on a molecular level. Analysis of different IL-31 downstream signaling pathways showed that IL1A and IL20/IL24 expression was affected by different pathways, such as p38 MAP kinase and ERK pathway (Figure 8 H). Interestingly the IL-31 targets IL-20 and IL-24 in combination showed an effect on keratinocyte differentiation (Cornelissen et al., 2012b) but neither IL-20 nor IL-24 induced the expression of IL1A or IL1B . This implies that IL- 31 affects the skin barrier via a minimum of two different effector networks, which must be regulated by different signaling pathways downstream of the IL-31 receptor (Figure 8 H). These networks were also compared in monolayer cell culture. Although it was discussed above that this experimental setting should be interpreted with caution, it can provide first indications of pathways for future studies. It could be observed that the addition of anakinra efficiently prevented the phosphorylation of the MAP kinase p38, which was enhanced in response to IL-31 or IL-1α. However, the phosphorylation of p65 and the degradation of IκBα was influenced by the presence of IL-1α but not IL-31 and this effect was also blocked by anakinra. These differences in phosphorylation and degradation of downstream signaling molecules might be due to the fact that after IL-31 stimulation IL-1α must be efficiently released from the cells before it can activate the DISCUSSION 73 signaling pathway. Moreover the induced phosphorylation of p38 upon IL-31, which is inhibited by the addition of anakinra, again supports a possible downstream role of IL-1α in IL-31 signaling.

Altogether IL-31 induces many of different cytokines such as IL-1α, IL-1β, IL-20 and IL- 24 when acting on keratinocytes. Through these targets IL-31 is able to influence a variety of different processes in the human skin and possibly in other organs when these mediators are released into the serum.

REGULATION OF ANTIMICROBIAL PEPTIDES BY IL-31 IS

DEPENDENT ON IL-1α INDUCTION

IL-31 also modulated the expression of genes encoding different AMPs in monolayer cell cultures as well as in 3D skin models (Figure 13 and Figure 14). These effects were at least in part dependent on the activation of the IL-1 signaling network as they were inhibited by anakinra (Figure 15). These observations are in line with literature data as IL-1α has been known to regulate AMP expression in several cell types, including keratinocytes ((Figure 12) (Bando et al., 2007; Jung et al., 2011; Hayashi et al., 2007; Krisanaprakornkit et al., 2003; Liu et al., 2002; Moon et al., 2002)). Still it is striking that IL-31 seems to have a much higher gene induction potential as IL-1α for several AMPs (Figure 15). This may be an indicator for synergistic effects between the IL-31 and additional downstream signaling pathways in inducing AMP expression. Moreover the expression pattern of the different AMPs upon IL-31 treatment varies. Observations comparable to those of the structural proteins were made, where FLG was identified as a sensitive marker for IL-31 treatment (Cornelissen et al., 2012b). The same experimental setup was used to analyze the AMP gene expression. In this setting DEFB103A was already induced 20-fold after a 2 h IL-31 stimulation whereas induction of DEFB4A and the S100A , S100A8 , S100A9 and S100A12 DISCUSSION 74 required a permanent IL-31 presence (Figure 14). Thus this defines DEFB103A like FLG as a sensitive marker. Keratinocyte expression and secretion of AMPs represent the first line of defense against cutaneous pathogens (Harder et al., 2007; Wiesner and Vilcinskas, 2010). To promote their antimicrobial properties AMPs are as well as corneocytes embedded in an acidic lipid envelope. This lipid envelope together with the AMPs largely inhibits the growth of various microbes (Drake et al., 2008; Elias and Choi, 2005). Increased amounts of the AMPs psoriasin, RNase-7, hBD-2 and hBD-3 were found in lesional and non-lesional skin and fluids obtained in washings from the skin of AD patients compared to healthy controls (Harder et al., 2010; Gambichler et al., 2008; Gläser et al., 2009).

This finding seems to be at odds with the observations of increased superinfection with bacteria in patients suffering from AD (Arikawa et al., 2002; Guzik et al., 2005). However the increased availability of AMPs in these washings may not reflect the total amounts. More likely the reduced or the absence of a lipid barrier in these patients prevents the attachment of AMPs to the epidermal layer and thus allows more efficient extraction. Furthermore the reduction of desmosomal proteins and the reduction of filaggrin and keratins 1 and 10 weaken the skin barrier leading to an increased penetration of bacteria into the skin. This is further complicated by the fact that persistent, relapsing and difficult to treat S. aureus infections are associated with the formation of a slow growing small colony variant phenotype (SCV) (Proctor et al., 2006; Vaudaux et al., 2006). Bacteria associated with SCV show reduced susceptibility toward AMPs (Gläser et al., 2014). Of note is also that different cytokines associated with the complex cytokine milieu of AD lesions may have antagonistic effects on the expression of AMPs by keratinocytes. For example the Th2 cytokines IL-4, IL-13 and IL-33 have been shown to inhibit the induction of AMPs by TNF-α and IFN-γ (Albanesi et al., 2007). Thus the positive consequence of IL-31 on the microbial barrier, as reported here, is most certainly modified by other factors, with many potentially reverting or antagonizing the IL-31 effect. Well-designed experimental setups will be required in the future to investigate the cytokine interplay the AD lesions. DISCUSSION 75

BI-FUNCTIONAL ROLE OF IL-31 IN HUMAN SKIN BARRIER

FORMATION

Concentration-dependent differences in the effects of IL-31 on the barrier formation and on the induction of AMPs were observed (Figure 16). Although low levels of IL-31 did not significantly induce AMP gene expression (Figure 16 C) a first positive trend on gene induction was indicated. Thus low doses of IL-31 might promote the antibacterial barrier (Figure 16 C) without compromising the mechanical barrier, indicated by no significant influence on the differentiation markers filaggrin and involucrin, as well as no effect on desmosomal gene expression (Figure 16 A and B). This might indicate a potentially important physiological role of IL-31. This is supported by the fact that the IL-31RA is upregulated by S. aureus α-toxin and staphylococcal enterotoxin B in monocytes (Kasraie et al., 2009), suggesting a role in innate immunity. Furthermore PBMCs of AD patients secrete significantly more IL-31 compared to those of healthy patients upon stimulation with α-toxin and staphylococcal enterotoxin B (Niebuhr et al., 2011). This regulation seems to be specific as for example IL31 mRNA is not upregulated in response to herpes simplex or influenza virus infection (Sonkoly et al., 2006). The expression of IL-31RA in keratinocytes is also stimulated by the IFN-γ and by activation of the TLR2 receptor (Kasraie et al., 2011). The activation of the IL-31 signaling network by different bacteria and the subsequent production and release of different AMPs by keratinocytes is of clinical interest. It is known that eccrine sweat, a complex mixture of minerals, proteins and proteolytic enzymes, contains IL-31. While sweat is not harmful to healthy skin, it can aggravate the lesions of AD patients. Sweat stimulates IL-1β and IL-

1RA expression in keratinocytes, possibly due to the presence of IL-31, and also contains

IL-1α and IL-1β (Dai et al., 2013). The presence of these cytokines in human sweat is consistent with their anti-microbial function as described here. These findings suggest that the induction of IL-1α by IL-31 and the subsequent upregulation of AMPs are independent of the IL-31-induced barrier disruption that includes repression of many factors associated with the mechanical barrier (Figure 5, Figure 6 and Figure 7). The different immune cells that reside in the skin or are recruited into the skin by diverse signaling mechanisms are DISCUSSION 76 integrated into a complex network of cells that crosstalk at multiple levels. For example IL31 gene expression in mast cells is induced upon treatment with hBDs (hBD-1 to hBD-4) and LL-37 (Niyonsaba et al., 2010). By revealing the fact that upon IL-31 stimulation keratinocytes release increased amounts of hBD-2 and hBD-3 (Figure 13), a potential feed- forwardloop could be identified. In this feed-forward loop mast cells are a potential source of IL-31 and may on one hand further alter the differentiation of keratinocytes and thus promote the pathogenesis of diseases such as AD and on the other hand promote the antimicrobial barrier.

In summary, this study reveals bilateral effects of IL-31 on the mechanical and the antimicrobial skin barrier. The reduction of the mechanical barrier mediated by genetic factors or a deregulated cytokine milieu, these two are likely to cooperate, results in the increased penetration with allergens and pathogens. It is likely that in individuals genetically susceptible to AD, deregulation of the IL-31 and the IL-1 signaling networks promote a feedback loop that can aggravate a proinflammatory environment. The results also indicate that pharmacological modulation of IL-31 and IL-1α activity in AD and possibly other skin diseases might be therapeutically beneficial.

IL-31 PROMOTES THE EXPRESSION AND RELEASE OF TSLP

It was of interest that IL-31 promoted the gene expression in monolayer cell cultures as well as the release of TSLP in organotypic 3D models (Figure 17). Beside the effects on different immune cells TSLP was shown to induce directly itch on a defined subset of neurons (Wilson et al., 2013b). Of note a positive correlation was shown between itch intensity and either IL31 mRNA or IL-31 serum levels (Ezzat et al., 2011; Kim et al., 2011). In many animal models the role of IL-31 in promoting itch was addressed and in all models an increase in scratching behavior was observed (Dillon et al., 2004; Gonzales et al., 2013; Arai et al., 2013; 2014). As mentioned in the introduction, dorsal root ganglia that are meant to be responsible for the sensation of itch express the IL-31 receptor and their unmyelinated nerve fibers might be directly stimulated by IL-31 (Cornelissen et al., DISCUSSION 77

2012a). Interestingly repeated administration of IL-31 promotes IL-31RA expression in dorsal root ganglia (Arai et al., 2014), indicating a further role in chronic itch mediated by IL-31. However IL-31–induced itch was significantly reduced in transient receptor potential cation channel vanilloid subtype 1 (TRPV1)-deficient and transient receptor channel potential cation channel ankyrin subtype 1 (TRPA1)–deficient mice but not in c- kit or proteinase-activated receptor 2 deficient mice (Cevikbas et al., 2013). The TRPA1 channel is also necessary to promote TSLP mediated itch behavior (Wilson et al., 2013b). Furthermore TRPA1 is necessary to promote chronic itch in a AD mouse model (Wilson et al., 2013a). Taken together this indicates a possible link between IL-31 and TSLP in itch mediation.

However subcutaneous injection of IL-31 does not induce itch immediately but only with a delay of more than 30 min (Hawro et al., 2014). Consistently treatment of keratinocytes with IL-31 resulted in TSLP secretion after 30 min (Figure 17) and permanent treatment over 10 days with IL-31 triggered also TSLP secretion (Figure 17). This strengthens the hypothesis of a link between both cytokines and a possible mechanism for the mediation of itch. So far the molecular mechanisms of this potential interaction has not been studied. Itch may be even mediated directly via the IL-31RA, which is expressed on a subset of neurons which (Bando et al., 2006) or as suggested by the findings above indirectly via the release of TSLP. Further studies are necessary to clarify the mechanism. However if it was mediated via triggering the TSLP expression it is well possible that the IL-31 – IL-1 axis is not involved in itch promotion. This can be concluded from the observation that neither IL- 1α nor IL-1β stimulated TSLP expression (Figure 17 D).

Itch and associated scratching behavior is a central mechanism to aggravate AD symptoms. Patients suffering from AD scratch their skin and thereby promote the development of lesions and the uptake allergens and pathogens. Beside these physiological implications itch is one of the most bothersome symptoms, strongly affecting the quality of life (Raap et al., 2011). The prevalence of mental health disorders such as depression, anxiety, conduct DISCUSSION 78 disorder, and autism was significant higher in children and adults suffering from AD than compared to healthy individuals (Yaghmaie et al., 2013; Schmitt et al., 2009; Yang et al., 2010; Hashizume and Takigawa, 2006). Moreover dose-dependent relationship was observed between the prevalence of a mental health disorder and the severity of the skin disease (Yaghmaie et al., 2013).

In summary the correlation between IL-31 and the mediation of itch makes IL-31 a potential therapeutical target in AD. CONCLUSION AND PERSPECTIVES 79

4 CONCLUSION AND PERSPECTIVES CONCLUSION AND PERSPECTIVES 80

The findings in this thesis that IL-31 on the one hand weakens the skin barrier and on the other hand regulates the expression of AMPs dependent on induction of the IL-1 signaling network leads to a more comprehensive understanding of the pathomechanism underlying the decreased barrier function and aggravated AD disease, affected by elevated IL-31 levels. Moreover these findings indicate a novel role of IL-31 in the human host defense by promoting the expression of AMPs. It is from outstanding interest that effects mediated by IL-31 were dose dependent and only high doses of IL-31 affected the skin barrier, whereas low doses of IL-31 strengthened the antimicrobial barrier. Interestingly IL-31 stimulated the secretion and release of TSLP in an in vitro organotypic 3D model 30 min after treatment. This finding is chronological in line with the induced itch by IL-31 in healthy and AD volunteers (Hawro et al., 2014). All these findings together suggest that IL-31 may not directly interact with either the formation of the skin barrier and mediating itch but rather indirectly orchestrates secretion of a plenty of cytokines (IL-1, IL-20, IL24, TSLP) from keratinocytes. Through these cytokines IL-31 a variety of effects within the skin, and contributes to the pathogenesis of AD. Thus in conclusion this thesis supports the hypothesis that high IL-31 levels in AD skin are a cause of AD rather than a symptom. Findings made should be concerned when IL-31 is targeted, a complete inhibition of IL-31 seems to be undesirable. Moreover a potential vicious cycle has been identified, in which IL-31 secreted from immune cells upon contact with AMPs leads to the promotion of AMP release from keratinocytes (Figure 18). CONCLUSION AND PERSPECTIVES 81

Direct Itch (IL-31-mediated) Sensory neuron

Itch TSLP

Mechanical stress Mast keratinocyte cell IL-31

AMPs

Differentiation and barrier defect (IL-31-induced) Skin barrier defect

Increased penetration by allergens and pathogens

Figure 18: Schematic overview of the interaction of mast cells with keratinocytes. Signalling resulting in reduced keratinocyte differentiation, e.g. by IL-31 with mast cells being one source, provokes skin barrier defects that allow increased penetration by allergens and pathogens. The skin barrier is also disrupted as a consequence of itch-induced scratching. In response to IL-31 keratinocytes produce AMPs and TSLP. As a consequence different cells in the skin are stimulated, including mast cells. Together this suggests that a feed-forward loop is activated that enhances skin barrier defects and associated symptoms (vicious cycle). AMP, antimicrobial peptide; TSLP, thymic stromal lymphopoietin.

By identifying IL-1 signaling as a downstream effector of IL-31 signaling, it might be of interest to identify IL-31 effects on keratinocyte behavior with excluding secondary signaling effects of IL-1. In line with this it might also be interesting to identify the role of IL-1α as a transcription factor in IL-31 signaling. It has been shown that the N-terminal IL- 1α acts as a nuclear oncoprotein and by this can modulate inflammation (Stevenson et al., 1997; Werman et al., 2004). And moreover the correlation of IL-31, IL-1α and AMPs within the skin of healthy individuals and AD patients has to be analyzed carefully as well as the role of the elevated intracellular IL-1β levels in the development of AD. To learn more about itch mediating properties of IL-31 it will be helpful to have a closer look on TSLP secretion and its contribution to IL-31 mediated itch. MATERIAL AND METHODS 82

5 MATERIAL AND METHODS

MATERIAL AND METHODS 83

EXPERIMENTAL PROCEDURES

Materials and methods are described according to standard protocols used in the Institute of Biochemistry and Molecular Biology, RWTH Aachen University, and modified regarding individual differences in experimental procedures.

CONSUMABLES AND REAGENTS

Consumables

Consumables were purchased from:

Amersham Biosciences, Ansell, Becton Dickinson, Biometra, Bio-Rad, Brand, Braun, Corning, Costar, Eppendorf, Falcon, Fisher Scientific, Fuji, Greiner, Kimberlex-Clark, Merck, Millipore, Nalgene, Nerbe Plus, Nunc, Roth, Sarstedt, Sartorius, Schleicher&Schuell, Stratagene, TPP, VWR

Reagents

Reagents, with a purity of at least analytical grade, were obtained from:

Abcam, AbD Serotec, Applichem, BD Biosciences, Biozym, Calbiochem, Cellsignaling, Clontech, Difco, Eurogentec, Fermentas, Fluka, GE Healthcare, Hoechst, Gibco, Invitrogen, InvivoGen, Jackson Immuno Research, Macherey&Nagel, MBL, Merck, MP Biomedicals, New England Biolabs, Novagen, Perkin Elmer, Pierce, Promega, Qiagen, Rockland, Roche, Roth, Sigma Aldrich, Stratagene, Tulip Biolabs, Zymo Research

MATERIAL AND METHODS 84

Solutions

All solutions were prepared and stored according to standard protocols used in the Institute of Biochemistry and Molecular Biology.

PBS buffer

140 mM NaCl | 2.6 mM KCl | 2 mM Na 2HPO 4 | 1.45 mM KH 2PO 4

RIPA lysis buffer 10 mM Tris, pH 7.4 | 150 mM NaCl | 1% NP-40 | 0.1% SDS | 1% Deoxycholate (DOC) | 0.5% Aprotinin

Running buffer (Laemmli), pH 8.3 25 mM Tris | 250 mM glycine pH 8.3 | 0.1% (w/v) SDS

4x Sample buffer 320 mM Tris, pH 6.8 | 40% (v/v) glycerol | 20% (w/v) SDS | 0.5% (w/v) bromophenol blue | 200 mM β-mercaptoethanol

Protein standard PageRuler prestained protein ladder 10-170 kDa (Fermentas) or Protein marker VI (10- 245) prestained (AppliChem)

Semi-dry transfer buffer 25 mM Tris | 192 mM glycine | 20% (v/v) methanol

PBS-T PBS | 0.05% (v/v) Tween® 20

Blocking buffer 5% (w/v) skim milk powder (AppliChem) in PBS-T

Ponceau S solution 0.2% (w/v) Ponceau S in 3% (v/v) trichloroacetic acid (TCA) MATERIAL AND METHODS 85

Oligonucleotides

Table 2: qPCR primers qPCR primers and Taq Man Probes

Gene Sequence / catalogue number hypoxanthine guanine forward: 5’-TGACACTGGCAAAACAATGCA-3’; reverse: phosphoribosyl transferase 5’-GGTCCTTTTCACCAGCAAGCT-3’ (HPRT)

forward: 5’-GTGTGGGTGCTTCTCCTGCTTC-3’; reverse: OSMRb 5’-TCTGTGCTAATGACTGTGCTTG-3’

DEFB4A QT01852277

DEFB103A QT01529535

S100A7 QT00018746

S100A8 QT12503907

S100A9 QT00018739

S100A12 QT01860873

FLG QT02448138

KLK7 QT00028343

CASP14 QT00046011

DSC1 QT00047971

DSC2 QT00016128

DSG1 QT00001617

DSG4 QT00064687

CDSN QT01192625

JUP QT01003170

CGN QT00005446

DSP QT00085218

CLDN1 QT00225764

CGNL1 QT00024808

OCLN QT00081844

IL1A QT00001127

IL1B QT00021385 MATERIAL AND METHODS 86

IL1R1 QT00081263

IL1R2 QT00006972

IL1RN QT00014238

IL20 QT00044905

IVL QT00082586

FLG Hs00418578_m1

S100A7 Hs00161488_m1

DEFB4A Hs00823638_m1

HPRT Hs99999909_m1

MATERIAL AND METHODS 87

Antibodies

Table 3: Antibodies

Primary antibodies

Antigen Species Company / cat.no Information

Filaggrin Mouse Santa Cruz/sc-66192 IL1a Rabbit Santa Cruz/sc-1253

IL1R1 Rabbit Abcam/ab40774

IL1R2 Mouse Abcam/ab89159

S100A7 Mouse ABDSerotec/MCA5253Z

HBD-2 Rabbit Abcam/Ab63982 STAT3 Rabbit Santa Cruz/sc-8019

(p-)STAT3 Rabbit Cell signaling/9131S Tyr705 p38 Rabbit Santa Cruz/sc-7149

(p-)p38 Mouse Cell signaling/9219 Thr180/Tyr182 p65 Rabbit Cell signaling/3987 (p-)p65 Rabbit Cell signaling/3031 IkBa Rabbit Cell signaling/9242 Tubulin Mouse Sigma/T-6557 Actin Rabbit Sigma/A2066 Secondary antibodies

Antigen Species Company/cat.no Information α-rabbit IgG+IgM goat Jackson Immuno Research HRP-conjugated α-mouse IgG+IgM goat Jackson Immuno Research HRP-conjugated α-rabbit IgG+IgM goat Invitrogen Alexa Fluor 488-conjugated α-mouse IgG+IgM goat Invitrogen Alexa Fluor 488-conjugated α-rabbit IgG+IgM goat Invitrogen Alexa Fluor 555-conjugated α-mouse IgG+IgM goat Invitrogen Alexa Fluor 555-conjugated MATERIAL AND METHODS 88

INHIBITORS AND CYTOKINES

Table 4: Inhibitors

Inhibitors

Name Target Company / cat.no Final concentration UO126 MEK1 Calbiochem/662005 20 µM SB202190 p38 Calbiochem/559388 20 µM Jak Inh I JAK kinase Calbiochem/420099 100 nM JNK Inh I JNK kinase Calbiochem/420119 20 µM Wortmannin PI3-kinase Calbiochem/69675 500 nM Anakinra IL1 receptor Swedish Orphan Biovitrum 3 µg/ml

Table 5: Recombinant human Cytokines

Recombinant human Cytokines

Antigen Company / cat.no Final concentration rhIL-31 Peprotech/200-31 100 ng/ml rhIL-1 alpha Peprotech/200-01A 10 ng/ml rhIL-1 beta Peprotech/200-01B 50 ng/ml rhIL-20 R&D/1102IL 20 ng/ml rhIL-24 R&D/1965IL 20 ng/ml

CELL LINES

HaCaT HaCaT cells are a spontaneously transformed cell line derived from keratinocytes of histological normal skin of a 63 years old male (Boukamp et al., 1988) and were a generous gift of N. Fusenig.

HaCaT-IL31RA HaCaT derived cell line containing a stably integrated cassette that leads to the expression of a HA-tagged IL-31RA upon doxycycline addition to the cell culture medium. The cell line was generated by lentiviral infection of wt HaCaT cells with the vector pSLIK- IL31RA-HA. MATERIAL AND METHODS 89

METHODS

METHODS IN MOLECULAR BIOLOGY

RNA isolation and cDNA synthesis

RNA was isolated from cells using Qiagen QIAshredder colums and RNeasy Kit with the optional on-column DNase digestion (RNase-free DNase Set, Qiagen) according to manufacturer’s protocol. In case of organotypic 3D models, tissues were primarily mechanically disrupted and homogenized by using a tissue lyzer (Qiagen). RNA was eluted from the column in 20 µl TE buffer. RNA purity and concentration were determined with the NanoDrop TM device. 1 µg of RNA was reverse transcribed into complementary (c)DNA using QuantiTect Reverse Transcription Kit TM (Qiagen) following company’s manual. cDNA was diluted 1:10 with TE and analyzed by quantitative Real-Time PCR or stored at -20 °C. qPCR

To determine the gene expression quantitative PCR (qPCR) was used. The process of qPCR is based on the principle of conventional PCR with the fluorescent dye SYBR Green providing the quantification of amplified DNA. The dye incorporates into double-stranded DNA and emits a light signal after excitation at a specific wavelength. The emitted fluorescence signal is detected after each elongation step within a PCR cycle in Real Time . Thereby the fluorescence increases proportionally to the amount of PCR product and thus allows calculated conclusions on the amount of PCR product. Experiments were either run on a Rotor-Gene 6000/Q (Corbett/Qiagen) in 10 µl reaction volume using SensiFast™ SYBR No-ROX Kit (Bioline). TaqMan ® experiments were carried out in 25 µl on an ABI PRISM 7300 sequence detection system (Applied Biosystems) using Assay-on-Demand gene expression products (Applied Biosystems). MATERIAL AND METHODS 90

Table 6: PCR pipetting scheme PCR pipetting scheme 2x SensiFast™ 5 µl QuantiTect primer assays 1:4 diluted in TE 1 µl individually ordered oligonucleotides, 100nM, 1:40 diluted DNA template cDNA (1:20) 2 µl

RNase-free H 2O ad 10 µl

Table 7: PCR program PCR program Step Temperature Time Cycles initial denaturation 95 °C 10 min 1 denaturation 95 °C 15 sec 40 cycles annealing 60 °C 15 sec elongation 72 °C 15 sec Melting curve 60-95 °C in 0.5 °C steps 5 sec each step

Table 8: TaqMan ® pipetting scheme PCR pipetting scheme TaqMan® Gene Expression Master Mix 12.5 µl Taqman probe 1.25 µl

DNA template cDNA 5 µl

RNase-free H 2O ad 25 µl

Table 9: TaqMan ® program PCR program Step Temperature Time Cycles initiation 50°C 2 min 1 initial denaturation 95 °C 10 min 1 denaturation 95 °C 15 sec annealing and 60 °C 60 sec 40 cycles elongation

Relative mRNA expression was calculated by the comparative C t method and normalized to the housekeeping gene HPRT (hypoxanthine guanine phosphoribosyl transferase) using Rotor-Gene Q software. In the text the whole procedure from isolating RNA over cDNA synthesis and qPCR processes were combined and named as qRT-PCR. MATERIAL AND METHODS 91

METHODS IN CELL BIOLOGY

Human cell cultivation

All cell lines were incubated in humidified atmosphere at 37 °C with 5% CO 2. HaCaT, HaCaT-IL31RA and Human dermal fibroblasts cells were cultivated under adherent conditions in DMEM-GlutaMAX I (Gibco), supplemented with 10% heat-inactivated fetal calf serum (FCS, Gibco). NHEK (Normal human epidermal keratinocytes) were cultivated in KGM supplemented with BPM, hEGF, insulin and hydrocortisone. To sustain stability of the integrated vector the medium of HaCaT-IL31RA were supplemented with 800 µg/ml geniticin (G418).

Seeding and passaging of cells For passaging the cells medium was exhausted and the cells were once carefully washed with PBS. All cell lines were incubated in Trypsin / EDTA (0,5%/0,02%, NHEKs: 0,25%/0,01%) until they detached from the plastic surface, medium was added to stop the process of trypsinization. Subsequent cells were counted either using the Casy Cell Counter system (Innovatis) or a chamber slide and the cells were seeded in the appropriate dilutions in new culture plates.

Cryoconservation For cryoconservation, 80% confluent adherent cells were removed from dishes by trypsinization. Cells were pelleted by centrifugation. The cell pellet was re-suspended in 1 ml FCS containing 10% DMSO (v/v) and transferred into a cryotube. Tubes were frozen down to -80°C using a Mr. Frosty TM (Sigma Aldrich) freezing container. For long-term storage, tubes were transferred to -150 °C. For cell thawing, tubes were quickly warmed in a 37 °C water bath and directly transferred into 9 ml prewarmed cell culture medium. Cells were centrifuged and again re-suspended MATERIAL AND METHODS 92 in 10 ml prewarmed cell culture medium. Finally cells were seeded in the appropriate dilutions in new culture plates.

Isolation of HDFs and NHEKs Normal human epithelial keratinocytes (NHEKs) and human dermal fibroblasts (HDFs) were prepared from sterile human skin samples (approved by the ethic committee of the Medical School of the RWTH Aachen University). Yvonne Marquard (Dermatology Department, Medical School, RWTH Aachen University) isolated HDFs and NHEKs from human skin samples.

Normal human keratinocytes were isolated from skin biopsies of adult skin and foreskin. The biopsies were rinsed with 70% ethanol, washed 3 times in PBS with antibiotics / antimycotics and cut into pieces of 1cm 2. To separate dermis and epidermis these pieces were incubated in 50 units/ml Caseinolytic Dispase (BD Bioscience) for a minimum of 2h at 37°C. Single cell suspension of keratinocytes was released by gentle pipetting after incubation for 30 min at 37°C in 0.25 mg/ml trypsin/EDTA (Lonza). The Trypsin Neutralisation Solution TNS (Lonza) inactivated the remained trypsin. Human dermal fibroblasts (HDF) were isolated by incubating the remaining dermis in dispase. Afterwards collagenase 1A 100U/ml (Sigma) digestion was performed. The generated single cell suspensions was seeded in the appropriate dilutions as described before.

Keratinocytes were used in their first to second passage for organotypic skin equivalents or monolayer cultures.

NHEK and HaCaT skin equivalents NHEK skin equivalents were constructed as described in (Neis et al., 2010). To construct either HaCaT or HaCaT-IL31RA skin equivalents bovine collagen type I was mixed in a ratio 8:1 with HBSS (Gibco) w/o Ca 2+ /Mg - and 1 M NaOH was added to neutralize the pH. 1 ml collagen gel was added on top of a 6-well insert with 3.0 µm pore size and incubated for 1 h at 37°C. HDFs with a cell number of 4x10 6 cells/ml were suspended in FCS. In a ratio of 8:1:1 bovine collagen, HBSS and HDFs were mixed. The pH of the collagen, MATERIAL AND METHODS 93

HBSS solution was neutralized with 1 M NaOH before adding the cells. 3 ml of this suspension were plated onto each gel made before. The new formed gels were incubated for 3 h at 37°C. For the next day HaCaT 3D medium, normal HaCaT culture medium as described before supplemented with 50 µg/ml ascorbic acid was added below and above the gel. At the next day 4x10 6 HaCaT cells in 2 ml medium were added on top of the model. After 2-3 h of incubation at 37°C and 5% CO 2 2 ml additional medium was given to the cells. At least the model was lifted to the air liquid interphase after 2 days of co- cultivating HDFs and HaCaT.

Cell-sorted skin equivalents (CsSE) CsSE were based on protocols as described previously (Wang et al., 2000; Neis et al., 2010). Briefly, 48 h before implantation, 4-6 weeks old SCID / NOD mice were fed with doxycycline (2 mg/ml) provided in the drinking water supplemented with 5% sucrose. On day -1 two silicon chambers were implanted onto the muscle fascia of the mouse back. 24 h later a cell slurry of 1-2 million HDFs and HaCaT cells (chamber 1) or HDFs and HaCaT-IL31RA cells (chamber 2) were seeded into the chambers. From day 4 to 7, 20 µg/ml rhIL-31 (or PBS as control) were applied subcutaneously under the skin equivalents. On day 6 the chambers were removed followed by TEWL measurements on day 7. Mice were sacrificed and skin equivalents were either embedded in paraffin or cryoconserved for further investigation. Philipp M. Amann, Yvonne Marquard and Jens M. Baron (Dermatology Department, Medical School, RWTH Aachen University) planned and conducted the animal experiments.

Skin barrier analysis For skin barrier analysis fluorescently labeled recombinant timothy grass pollen major allergen, phl p1 (Biomay, Vienna/Austria) was applied topically on a 7 day-old HaCaT- IL31RA organotypic skin equivalents for 45 min and analyzed by immunofluorescence. To analyze the vulnerability of an organotypic skin equivalent to the penetration by irritating agents, 7 day-old HaCaT-IL31RA organotypic skin equivalents were treated topically with MATERIAL AND METHODS 94

0.2% SDS for 40 min and 24 h later the expression of IL1A was analyzed by qRT-PCR and the IL-1α release was measured by ELISA.

Penetration assay HaCaT-IL31RA models were treated with 10 mM Lucifer Yellow / Biocytin (Invitrogen, L6950) at room temperature for 1 h. Models were subsequently processed for cryoconservation.

Transepidermal water loss analysis in cell-sorted skin equivalents TEWL was measured using a Tewameter TM210 (Courage+Khazaka, Cologne, Germany) according to the protocol of the manufacturer.

METHODS IN PROTEIN BIOCHEMISTRY

Protein sample preparation

From cells To prepare whole cell protein lysates, adherent cells were washed with ice-cold PBS. Subsequently, 300 µl RIPA lysis buffer plus freshly added Protease Inhibitor cocktail was added per 10 cm dish. Adherent cells were collected by scraping and transferred to an Eppendorf tube. RIPA lysates were sonicated in a water-bath sonicator (Bioruptor®, Diagenode) for 15 min with 30-second cycles at maximum intensity and were subsequently centrifuged at 13,000 x g, for 30 min at 4 °C. Supernatants were transferred to a new Eppendorf tube and stored at -80 °C or directly analyzed by SDS-PAGE or ELISA.

Lowry Assay

Lowry assay was performed using the RC/DC Protein Assay Kit from Bio-Rad according to the manufacturers protocol. In brief 20 µl of reagent S was mixed with 1 ml of reagent A to obtain A’.Subsequently 25 µl reagent A’ were mixed with 200 µl B and added to the MATERIAL AND METHODS 95

RIPA cell-lysate in a 96 well plate. The lysate and reagent were incubated for 15 min at RT and wavelength at 750 nm was read. For determination of the protein amounts in the unknown samples a BSA standard curve was used.

SDS-PAGE

Proteins were electrophoretically separated under denaturing conditions in discontinuous sodium dodecyl sulfate polyacrylamide gels (SDS-PAGE) according to the method described by Laemmli (Laemmli, 1970). SDS running gels were prepared with 10-15% acrylamide, depending on the size of the analyzed protein. Independent from the running gel, stacking gels contained 5% acrylamide. Prior to loading, protein samples were mixed with 4x sample buffer and boiled for approx. 3 min. Separation of gels was performed in a BioRad electrophoresis cell filled with Laemmli running buffer. In order to estimate protein sizes, a protein standard was also applied to each SDS-PAGE.

Western blot and immunodetection

Separated proteins were transferred from acrylamide gel onto a nitrocellulose membrane (Millipore) using the semi-dry Western blotting system. Gel, nitrocellulose membrane and Whatman filter paper soaked with semi-dry transfer buffer were placed between the two electrode plates of a blotting chamber. Transfer was performed for 70 min with

2 1.5 mA/cm of nitrocellulose membrane. To confirm successful protein transfer, membrane was stained with Ponceau S solution for 2 min.

For immunological detection, the membrane was blocked with 5% non fat dry milk in PBS-T for a minimum of 30 min and subsequently incubated at 4 °C overnight with primary antibody in PBS-T. After removing residual antibodie wiht three washing steps in PBS-T, membrane was incubated for 1 h at RT with HRP-coupled secondary antibody in PBS-T. The detection was performed by using horseradish peroxidase–labeled secondary antibodies with either ‘‘Chemiluminescence ECL kit’’ (Pierce/Thermo Scientific, Rockford, Ill) or ‘‘SuperSignal West Femto Maximum Sensitivity Substrate’’ (Pierce). This substrates for HRP resulted in chemiluminescence signals, and were immediately detected by LAS-3000 Imager (Fujifilm). MATERIAL AND METHODS 96

Flow cytometry

Cells were detached from the surface as described before. 1x10 6 cells per sample were washed twice with PBS, and 1 ml 3.7% PFA was added under vortexing conditions. Cells were incubated for 20 min at RT, wash twice with PBS/1%BSA and blocked for 30 min in the same solution. After blocking the cells, 200 µl primary antibody was added for 1 h at RT. Samples were washed twice with PBS/1%BSA and incubated with 200 µl secondary antibody for 20 min at RT in the dark. Finally the cells were washed three times with PBS/1%BSA and resuspend in 500 µl PBS/1%BSA. The cell surface fluorescence intensity was measured on a FACS- Canto flow cytometry system (BD, Franklin Lakes, NJ, USA).

ELISA

Cells were treated as described in the figure legends and lysed in RIPA buffer as described before (Pandithage et al., 2008) or the supernatants were collected. The samples were either directly applied to the ELISA or diluted in the provided dilution buffer, and measured according to the manufacture’s recommendations.

Table 10: ELISA ELISAs

Gene Company / cat.no

IL1A R&D/DY200

IL1B R&D/DY201

S100A7 Cloud-Clone Corp/SEC035HU

S100A9 Cloud-Clone Corp/SEB793HU

S100A12 Cloud-Clone Corp/SEB080HU

HBD-2 Peprotech/900-K172

HBD-3 Cloud-Clone Corp/SEE132Hu

MATERIAL AND METHODS 97

HE staining of organotypic skin models

4 µm thin slices were cut from cryo-conserved three dimensional skin equivalents or CsSE and transferred onto microscope slides. 5 min staining with hemalum followed by 10 min incubation in water that increases the pH of the samples, colors acidic structures like nucleus and ER (and a few other objects) blue. A basic counterstaining with an aqueous solution of 0.1% eosin, which colors other, basic structures in various shades of red, follows the nuclear staining. Eosin is removed by washing two times for 3 min in water. The water within the samples is removed by 70% ethanol followed by washing two times for 3 min in 90% ethanol and two times for 3 min in 100% ethanol. Finally the slides were washed two times for 3 min in Xylol and embedded with Vitro Clud® (Langenbrinck) under a cover slip.

Immunostaining of organotypic skin models

For light microscopy and immunofluorescence analyses of the 3D skin models, 4 µm slices were cut from cryo-conserved three dimensional skin equivalents and transferred onto microscope slides. The slices were air dried and washed once with PBS w/o Ca 2+ /Mg +. Primary Antibody was diluted in Antibody dilutent (Dako) and incubated for 1 h at RT. The slides were washed three times with PBS and then secondary fluorescently labeled antibody was diluted in Antibody dilutent (Dako) and incubated for 1 h at RT in the dark. The slices were washed again with PBS and nuclear staining was performed for 5 min with

DAPI 1:5000 in ddH 2O. Finally the slices were washed three times with ddH 2O and mounted under a cover slip with mouning medium (Dako).

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7 APPENDIX

APPENDIX 123

LIST OF FIGURES

Figure 1: Schematic representation of the differentiation process undergoing in keratinocytes...... 11 Figure 2: Representation of structural elements of human antimicrobial peptides in human skin...... 16 Figure 3: Cytokine receptor complexes, their ligand families and corresponding signaling pathways involved in skin barrier regulation...... 24 Figure 4: Regulatory effects of the cytokines on the barrier formation...... 35 Figure 5: IL-31 deregulates the expression of genes associated with the mechanical skin barrier...... 40 Figure 6: IL-31 weakens the skin barrier...... 42 Figure 7: IL-31 promotes transepidermal water loss...... 44 Figure 8: IL-31 stimulates the IL-1 signaling network...... 47 Figure 9: IL-20 and IL-24 do not induce IL-1 gene expression...... 49 Figure 10: IL-1α deregulates the expression of genes associated with the mechanical skin barrier...... 50 Figure 11: Role of IL-1α in IL-31 signaling...... 52 Figure 12: IL-1α regulates the expression of antimicrobial peptides...... 54 Figure 13: IL-31 regulates the expression of antimicrobial peptides...... 55 Figure 14: Permanent IL-31 treatment induces AMP expression...... 56 Figure 15:Regulation of antimicrobial peptides by IL-31 is dependent on IL-1α induction...... 57 Figure 17: IL-31 promotes the expression and release of TSLP...... 61 Figure 18: Schematic overview of the interaction of mast cells with keratinocytes...... 81

LIST OF TABLES

Table 1: Properties of skin derived AMPs ...... 19 Table 2: qPCR primers ...... 85 Table 3: Antibodies ...... 87 Table 4: Inhibitors ...... 88 Table 5: Recombinant human Cytokines ...... 88 Table 6: PCR pipetting scheme ...... 90 Table 7: PCR program ...... 90 Table 8: TaqMan® pipetting scheme ...... 90 Table 9: TaqMan® program ...... 90 Table 10: ELISA...... 96 APPENDIX 124

ABBREVIATIONS

Abbreviations Abbreviated form full text form °C degree Celsius 3D three dimensional A ampere A. dest destilled water aa amino acid AAM alternatively activated macrophage Ab antibody ACD allergic contact dermatitis AD atopic dermatitis AE atopic eczema AKT v-akt murine thymoma viral oncogene homolog 1 Amp ampicillin ATCC American Type Culture Collection bio biotin Bp BSA bovine serum albumin ca. circa CBD cytokine binding domain CCL CC chemokine receptor ligand CD cluster of differentiation CD Crohn’s disease Cdc2 CDK1, cyclin-dependent kinase 1 CDK cyclin dependent kinase cDNA copy DNA CE cornified envelope HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HGF hepatocyte growth factor HPRT hypoxanthine guanine phosphoribosyl transferase hr(s) hour(s) HRP horse radish peroxidase hyg hygromycine i.e. that is IF immunofluorescence IFN interferon Ig immune globulin IGF insulin like growth factor IL Interleukin IL-31RA IL-31 receptor alpha INV Involucrin IRF interferon regulatory factor ITGB4 β 4-integrin JAK janus tyrosine kinase JNK Jun N-terminal kinase K keratin k kilo APPENDIX 125

KGF keratinocyte growth factor Ki67 antigen identified by Ki 67 KO knock out l liter LCE late cornified envelope LDH lactate dehydrogenáse LPS lipopolysaccharide m milli M molar (Mol/l) MAPK mitogen activated protein kinase MAPKAPK2 mitogenic activated protein kinase activated kinase 2 max maximum MB mega bases MC MEK MAP2K, mitogen-activated protein kinase kinase min minimum min minutes MMP matrix metallo- proteases mRNA messenger RNA n nano NCID notch intracellular domain Neo neomycin NF κ -B nuclear factor kappa B NHEK normal human epidermal keratinocyte NP-40 Nonidet P-40 OSM oncostatin M OSMR oncostatin M receptor OVA ovalbumin PAGE polyacrylamide gel electrophoresis PBMC peripheral blood mononuclear cell PBS phosphate buffered saline PCR polymerase chain reaction PEG poly-ethylene glycol PI3-K Phosphoinositide-3-kinase PKB/C protein kinase B/C PKC protein kinase C rh recombinant human RNA ribonucleic acid ROS reactive oxygen species rpm rounds per minute qRT-PCR quantitative reverse transcriptase polymerase chain SB reaction stratum basale SC stratum corneum SDS sodium dodecyl sulfate SEB superantigen staphylococcal enterotoxin B SEMF Human colonic subepithelial myofibroblast SG stratum granulosum shRNA small hairpin RNA SNP single nucleotide polymorphisms SOCS suppressor of cytokine signaling APPENDIX 126

SPF specific pathogen free SPR small proline rich SS stratum spinosum STAT signal transducers and activators of transcription TE Tris-EDTA TG transglutaminase TGF transforming growth factor TIMP-1 TIMP metallopeptidase inhibitor 1 TLR toll like receptor TNF tumor necrosis factor TRE tetracycline responsive element TSB tryptic soy broth Tyr, Y tyrosine U unit UV ultra violet light v/v volume per volume VEGF vascular epithelial growth factor w/v weight per volume WB western blot WT wild type x times/-fold β -ME β -mercaptoethanol λ wavelength of light μ micro OMIM ® Online Mendelian Inheritance in Man ® ICD-10 International Classification of Diseases

APPENDIX 127

CV

KAI HERBERT HÄNEL

Geburtstag und -ort:: 21. Februar 1985, Neuss

Berufserfahrung

01/2012-12/2014 Wissenschaftlicher Angestellter Institut für Biochemie und Molekularbiologie, Uniklinik RWTH Aachen

Ausbildung

Seit 01/2012 Promotionsstudent Institut für Biochemie und Molekular Biologie, Uniklinik RWTH Aachen

10/2009-12/2011 Master of Science in Biotechnologie/Molekularer Biotechnologie RWTH Aachen

09/2006-09/2009 Bachelor of Science in Chemie und Biotechnologie Hochschule Niederrhein, Krefeld

08/2005-06/2006 Ausbildung zum staatlich geprüften chemisch-technischen Assistenten Heinrich Hertz Berufskolleg, Düsseldorf

06/2005 Abitur Marie-Curie-Gymnasium, Neuss APPENDIX 128

SCIENTIFIC CONTRIBUTION

PUBLICATIONS

2014 Hänel, K.H ., Cornelissen, C., Amann, P.M., Marquardt, Y., Czaja, K., Kim, A., Bickers, D.R., Lüscher, B. and Baron, J.M. Control of the mechanical and antimicrobial skin barrier by an IL-31 – IL-1 axis (Manuscript submitted)

11/2014 Altrichter, S., Hawro, T., Hänel, K ., Czaja, K., Lüscher, B., Maurer, M., Church, M.K. and Baron, J.M. (2014), Successful omalizumab treatment in chronic spontaneous urticaria is associated with lowering of serum IL-31 levels. Journal of the European Academy of Dermatology and Venereology. doi: 10.1111/jdv.12831

03/2013 Hänel, K.H., Cornelissen, C., Lüscher, B., and Baron, J.M. (2013). Cytokines and the Skin Barrier. International Journal of Molecular Sciences, 14(4), 6720–6745.

AWARDS

03/2014 Mainzer Abstract-Preis der DGAKI 26. Mainzer Allergieworkshop der Deutschen Gesellschaft für Allergologie und klinische Immunologie, Mainz

03/2013 ADF/ECARF-Award for European Allergy Research “IL-31 affects the formation of the mechanical skin barrier and strengthens the antimicrobial defense of the skin partially by upregulating IL-1αlpha expression” APPENDIX 129

EIDESSTATLICHE VERSICHERUNG

Ich versichere hiermit an Eides statt. dass ich die vorliegende Dissertation selbstständig und ohne unzulässige fremde Hilfe erbracht habe. Ich habe keine anderen als die angegebenen Quellen und Hilfsmittel benutzt.

______

Ort, Datum Unterschrift

APPENDIX 130

DANKSAGUNG

Vielen Dank an Herrn Prof. Dr. Lüscher für die Übertragung des Projektes, seinen Enthusiasmus, sowie seine ständige Unterstützung und Diskussionsbereitschaft.

Herrn Prof. Dr. Dirk Ostareck danke ich für die Übernahme des Referates.

Mein Dank gilt ebenfalls Herrn Prof. Dr. Jens Malte Baron. Ihm möchte ich für die anregenden Diskussionen und die Möglichkeiten, meine Forschung auf mehreren Konferenzen vorzutragen danken.

Der gesamten Arbeitsgruppe "Experimentelle Dermatologie und Allergologie" danke ich sehr, sie hat in ganz wesentlichen Teilen zu dieser Arbeit beigetragen. Mein besonderer Dank hierbei gilt Yvonne Marquardt und Katharina Czaja, ohne Euch wäre diese Arbeit nicht in dieser Form möglich gewesen.

Danke an Dr. Christian Cornelissen für die Einarbeitung und Betreuung in der ersten Zeit dieses Projektes.

Allen aktuellen und ehemaligen Mitarbeitern der AG Lüscher danke ich für das hervorragende Arbeitsklima, die wissenschaftlichen Diskussionen und die Aktivitäten auch abseits der Arbeit. Mareike Bütepage, Laura Eckei, Dr. Karla Fejis, Dr. Franzi Flick, Annika Gross, Particia Hans, Dr. Jörg Hartkamp, Lora Hefele, Dr. Nico Herzog, Dr. Ferdinant Kappes, Max Kaufmann, Eva Lausberg, Babara Lippok, Dr. Juliane Lüscher-Firtzlaf, Gabi Lützeler, Dr. Daniala Otten, Dr. Christian Preisinger, Jürgen Stahl, Marieke Sternkopf, Nathalie Tomanek, Dr. Jörg Vervoorts-Weber.

Danke an das #1 Labor der AG Lüscher, es war jeden Tag aufs Neue super mit euch: Dr. Alexandra Forst, Dr. Andrea Ullius, Marc Dohmen, Carolina Pfaff, Elena Buerova, Alex Stephan und Elena Meuser, Danke.

Ein genau so großer, wenn nicht größerer Dank geht an die besten Büro Kollegen Dr. Andrea Ullius, Dr. Georgios Agalarides und Dr. Patricia Verheugd! Für alle APPENDIX 131 zukünftigen Büro-Gemeinschaften wird es eine Herausforderung sein, auf euer Niveau zu kommen.

Danke auch an Marc und Patrice für dass montägliche "Aufarbeiten und Verarbeiten" der Handball-Erlebnisse vom Wochenende. Es wird mir fehlen, mit euch zu fachsimpeln.

Claudia Schubert, vielen Dank für die schöne Kaffee-Klatsch Zeit.

Bei Stefan Reimig möchte ich mich für den Soccer-Tuesday und die FIFA-Abende bedanken. Dein Arbeitseifer hat mich das ein oder andere Mal motiviert und angespornt, fertig zu werden.

Bei meinen Eltern möchte ich dafür bedanken, dass sie mir mein Studium überhaupt durch ihre Unterstützung ermöglicht haben und für Ihr großes Interesse während der gesamten Zeit.

Meinen Geschwistern, Ute, Saskia und Dominik danke ich für das Interesse und die Unterstützung, die Sie mir die gesamte Zeit entgegengebracht haben.

Mein letzter und Größter dank gilt Dir, liebe Alex. Ich könnte hier Seiten füllen mit Beispielen wie und wo Du überall zum gelingen dieser Arbeit beigetragen hast und dich mit Lob überschütten. Ich fasse mich jedoch kurz und sage: DANKE für einfach alles!