WO2007073151A1

WO2007073151A1 - Psoriatic skin equivalents

Info

Publication number: WO2007073151A1
Application number: PCT/NL2005/050090
Authority: WO
Inventors: Josephus Schalkwijk; Evert Nicolaas Lamme; Anna Maria Gertruda Bergers; Patrick Laurentius Johannes Maria Zeeuwen; Gerardus Johannes De Jongh; Geuranne Sandra Tjabringa
Original assignee: Stichting Katholieke Universiteit
Priority date: 2005-12-21
Filing date: 2005-12-21
Publication date: 2007-06-28

Abstract

The present invention relates to the field of psoriasis. Psoriatic skin equivalents are provided in vitro, as well as methods for making and using these.

Description

Psoriatic skin equivalents

FIELD OF THE INVENTION

The present invention relates to the field of inflammatory skin diseases and in vitro skin equivalents. Especially skin equivalents resembling lesional psoriatic skin (herein called "psoriatic skin equivalents") are provided, as well as methods (and compositions) for making such skin equivalents. The psoriatic skin equivalent may be used for identifying new compounds and compositions for the treatment or prophylaxis of psoriasis and for the analysis of the effect of compounds or compositions on psoriatic skin, whereby potentially adverse effects of the compounds or compositions can be identified.

BACKGROUND OF THE INVENTION

The skin consists of two layers, the dermis and epidermis. At the start of a physiological inflammatory skin response, immune cells are attracted from the bloodstream in the dermis by mediators secreted by the cells in the epidermis. While in the normal situation the inflammation resolves spontaneously, in some cases the immune cells that have infiltrated in the epidermis initiate an ongoing cycle of cell activation between the immune cells and epidermal cells (keratinocytes). This can lead to a chronic inflammatory skin disease of which psoriasis and atopic dermatitis are the most prominent examples. Although these inflammatory skin diseases resemble each other to a certain extent, the causes and phenotypic results on the molecular level are not identical. The medications used in these diseases can be similar (e.g. corticosteroids, UV-B light, cyclosporin) but can also be quite distinct.

It is a challenge for experimental drug research in dermatology to develop in vitro models by creating tissue engineered skin equivalents, especially skin equivalents which resemble a specific skin disease.

Some mammalian skin equivalents have been described. For example WO99/45770 describes mammalian skin equivalents. However, in order to generate skin equivalents which resemble disease states, it is suggested therein to use the cells of an individual with a particular skin disorder, as the skin equivalent will apparently maintain the characteristics of that disorder. There are a number of disadvantages in having to use cells of such individuals, such as availability and costs. Further no information is provided as to how the skin equivalent needs to be treated in order to maintain the disorder phenotype.

It is an object of the present invention to provide methods for making human skin equivalents that resemble particular skin disorders (especially psoriasis), starting from keratinocytes obtained from healthy volunteers. It is also an embodiment to provide methods for maintaining the skin equivalent in the specific disorder state. Further, a general method for identifying alternative compounds suitable for generating and maintaining a psoriatic skin equivalent is provided.

GENERAL DEFINITIONS

A "psoriatic skin equivalent" or a "skin equivalent resembling psoriasis" refers to an in vitro cell culture comprising, or consisting of, an epidermis which comprises both morphological and molecular features of psoriatic skin.

"Morphological features of psoriatic skin" or "morphological features resembling psoriatic skin" refer to the presence of at least one, but preferably several, of the following morphological characteristics: 1. an enlarged suprabasal compartment (acanthosis); 2. the presence of nuclei in the stratum corneum (parakeratosis); 3. elongation of rete ridges with hyperproliferative basal cells

"Molecular features of psoriatic skin" or "molecular features resembling psoriatic skin" refer herein to the upregulation of gene expression (mRNA transcription and corresponding protein levels) of at least two of the following psoriasis specific genes compared to the expression level in control skin equivalents: HBD2 (human beta- defensin 2), CXCL8 (interleukin-8), SPRR2c (Small proline-rich protein 2c), skin- derived antileukoproteinase (SKALP). At the same time, the expression level of at least one, or preferably both, of the following genes is unchanged or downregulated compared to the expression level in control skin equivalents: human Calmodulin Like Skin Protein (CLSP) and Carbonic Anhydrase 2 (C A2). mRNA sequences for each of these genes are depicted (as corresponding DNA) in the sequences listing as SEQ ID NO: 1-6. In addition, PCR primer pairs for amplification are provided in SEQ ID NO: 9-18. The primer pair depicted in SEQ ID NO: 7 and 8 amplifies the reference gene (beta-actin).

"IL-I" refers herein to IL-I alpha and/or IL-I beta.

A "control skin equivalent" is, for example, a skin equivalent obtainable using the same method, but without psoriasis inducing/maintaining proteins (e.g. IL-I, IL-6, TNF-α and KGF) added to the medium. Alternatively, a control skin equivalent may also be a normal human epidermis or a skin equivalent resembling a normal (healthy) human epidermis. Similarly, an atopic dermatitis (AD) skin, or skin equivalent resembling an atopic dermatitis, may be used as control. "Upregulation" of gene expression refers to an amount of mRNA transcript levels of at least about 2 times the level of the control sample (obtained from a skin equivalent not treated with cytokines), preferably at least about 3x, 4x, 5x, 10x, 2Ox, 30x or more. "Downregulation" of gene expression refers to an amount of mRNA transcript levels of at least about 2 times lower than the level of the control sample (obtained from a skin equivalent not treated with cytokines), preferably at least about 3x, 4x, 5x lower.

The term "nucleic acid sequence" (or nucleic acid molecule) refers to a DNA or RNA molecule in single or double stranded form. An "isolated nucleic acid sequence" refers to a nucleic acid sequence which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a bacterial host cell or in the plant nuclear or plastid genome.

The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3 -dimensional structure or origin. The term "gene" means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3 'non-translated sequence comprising e.g. transcription termination sites.

"Expression of a gene" refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA. The coding sequence encodes a biologically active protein or peptide In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. Thus, the indefinite article "a" or "an" thus usually means "at least one", e.g. "a cell" refers also to several cells in the form of cell cultures, tissues, etc. It is further understood that, when referring to "sequences" herein, generally the actual physical molecules with a certain sequence of subunits (e.g. amino acids) are referred to.

"Sequence identity" and "sequence similarity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms. Sequences may then be referred to as "substantially identical" or "essentially similar" when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length, maximizing the number of matches and minimises the number of gaps. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA. Also, EmbossWin version 2.10.0 can be used, using the program 'needle' (which corresponds to GAP) with the same parameters as for GAP above. Alternatively percent similarity or identity may be determined by searching against databases, using algorithms such as FASTA, BLAST, etc.

"Stringent hybridisation conditions" can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequences at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridisations (Northern blots using a probe of e.g. lOOnt) are for example those which include at least one wash in 0.2X SSC at 63 °C for 20min, or equivalent conditions. Stringent conditions for DNA-DNA hybridisation (Southern blots using a probe of e.g. lOOnt) are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

DETAILED DESCRIPTION OF THE INVENTION In one embodiment of the invention a method is provided for the in vitro reconstruction of an epidermis having the histological characteristics of a psoriatic epidermis, and the molecular signature (altered gene expression) found in psoriasis.

The method relates to seeding normal keratinocytes on an a-cellular dermal matrix and to allow normal epidermal differentiation to occur. At the moment that a fully differentiated epidermis, resembling normal skin, has been (re)generated, an inflammatory stimulus is given by adding a cocktail of specific proteins (cytokines and/or growth factors) to the culture medium. This stimulus replaces the immune cells normally found in psoriatic epidermis as a source of inflammatory mediators. Hereafter, e.g. within 4-8 days, an epidermis is generated resembling a psoriatic epidermis, which can be used to screen potential anti-inflammatory/anti-psoriatic drugs by applying them topically onto the epidermis or adding them to the culture medium.

Thus, the invention relates to a method for making a psoriatic skin equivalent in vitro, comprising the steps of:

(a) seeding normal human keratinocytes onto a dermal matrix,

(b) allowing the keratinocytes to differentiate into an epidermis, (c) adding a composition comprising a suitable amount of at least two proteins which induce, in the epidermal cells, the transcription of at least two genes selected from the group consisting of: β-defensin (HBD2), skin-derived antileukoproteinase (SKALP), Small Proline Rich Protein 2c (SPRR2c) and Interleukin 8 (CXCL8), and optionally

(d) harvesting said psoriatic epidermis.

Normal, healthy human keratinocytes are used. In steps (a) and (b) known methods for keratinocyte seeding, proliferation and differentiation may be used. For example the methods of Ponec et al. (1997, J Invest Dermatology 109: 348-355) and Gibbs et al. (2000, Wound Repair 8: 192-203), incorporated herein by reference, may be used. See also the Examples.

In step (c) suitable amounts of at least two proteins are added to the culture medium (of cultures which are about 10 days old). The proteins added are proteins which induce the transcription of at least two psoriasis specific genes in the epidermal cells. Thus any protein may be added which induces mRNA transcription of at least two of the following genes in the epidermal cells: the endogenous β-defensin (HBD2) gene, SKALP gene, Small Proline Rich Protein 2c (SPRR2c) gene and Interleukin 8 (CXCL8) gene. Preferably transcription of at least three, more preferably of at least all four genes is induced by addition of the proteins. In addition, preferably the expression level of human Calmodulin Like Skin Protein (CLSP) and/or Carbonic Anhydrase 2 (CA2) is not altered or is downregulated following addition of the proteins.

Upregulation and/or downregulation of the expression levels can be determined by various methods known in the art and as shown in the Examples. For example quantitative PCR (e.g. quantitative RT-PCR) can be carried out, using mRNA (or corresponding cDNA) extracted from the protein-treated cells and control epidermal cells in order to quantify the mRNA (cDNA) levels of the above transcripts. Also, Northern blot analysis or (quantitative) RT-PCR, using for example probes or primer pairs, respectively, specific for the transcripts may be carried out. Alternatively, the protein level may be determined, e.g. using antibody based detection of the proteins (e.g. SDS-PAGE followed by Western blot analysis; ELISA assays, immunohistochemical assays, etc). See also the Examples.

Thus, for example the levels of one or more of the mRNA transcripts provided in SEQ ID NO: 1-6, or sequences essentially similar thereto, can be detected. As reference levels, preferably the transcript levels of a housekeeping gene, such as the actin gene, are determined. Sequences essentially similar to SEQ ID NO: 1-6 include sequences comprising at least 70, 75, 85, 95, 90, 95, 98, 99%, or more, sequence identity to those of SEQ ID NO: 1-6, whereby sequence identity is determined by pairwise alignment (over the entire length) using the GAP parameters as defined above. PCR primers for use in quantitative PCR detection methods are provided herein (SEQ ID NO: 7-18), but off course any stretch of at least about 10, 11, 12, 13, 14, 15, 17, 18, 20, 22, 24, 25, or more, consecutive nucleotides of the transcripts, e.g. of SEQ ID NO: 1-6 or of the sequences essentially similar to SEQ ID NO: 1-6, may be used. Also degenerate primer pairs may be used. The same applies to probes for nucleic acid hybridization, which may be generated as known in the art. For example, the whole sequence, or any fragment of the sequences provided herein, or of an essentially similar sequence, may be used to make probes for (e.g. stringent) nucleic acid hybridization.

The proteins added in step (c) also induce morphological changes, whereby the skin equivalent resembles human psoriatic skin in morphology. Especially, an enlarged suprabasal compartment and parakeratosis is seen in the protein treated epidermis. The presence of morpho logical features resembling psoriasis can be detected using known histological methods. For example, the skin equivalent may be harvested and paraffin sections of the protein treated samples and control samples may be analyzed for their morphology.

In one embodiment suitable amounts of at least two proteins selected from the group consisting of human Interleukin-6 (IL-6), Interleukin-1 (IL-I), Tumor Necrosis Factor alpha (TNF-α) and Keratinocyte Growth Factor (KGF), are added, as these are suitable for induction of both morphological and molecular features of psoriasis. In another embodiment at least three or even more preferably all four of these proteins are added in suitable amounts. Substantially pure proteins (e.g. purified from natural sources or produced using recombinant methods) are commercially available, e.g. from Sigma- Aldrich and other supplies.

What a suitable amount is for each protein can be determined by the skilled person. The amount should be high enough to induce morphological and molecular features of psoriasis, but not too high to have a negative effect on these features. For example, a final concentration of 10, 20, 30, 40, 50, 100 ng of one protein / ml medium can be tested in combination with a final concentration of 10, 20, 30, 40, 50, 100 ng of another protein / ml medium and the most suitable concentration can be chosen.

In particular, at least IL-I and TNF-α are added during step (c) to the medium, preferably in synergistically effective amounts. Synergistically effective amounts of IL- 1 and TNF-α are protein amounts of at least about 10ng, or more, of IL-I per ml medium and at least about 2 ng, 5 ng, 10 ng, or more, of TNF-α. Preferably the amount of TNF-α does not exceed about 30 ng protein/ml medium, as this may have a negative effect on morphology. In another embodiment, besides IL-I and TNF-α, IL-6 or KGF are added during step (c). In yet a further embodiment, besides IL-I and TNF-α, IL-6 and KGF are added during step (c).

In another embodiment of the invention a psoriatic skin equivalent obtainable by the above method is provided. Such a skin equivalent is characterized by having both morphological and molecular features of psoriasis. Thus, the transcription profile of the above genes is indicative of the psoriatic state (upregulation of certain psoriasis specific genes and downregulation or no change in transcription of others) and the morphology is indicative of psoriasis. The psoriatic skin equivalent may be present on a dermal matrix, or it may be isolated therefrom, or it may be present on a synthetic filter. In particular it is present on a de-epidermized dermis (DED) dermal substrate or tissue- engineered bio-matrices or (semi)-synthetic biocompatible matrices or on coated or uncoated polycarbonate-based membrane filter inserts, e.g. for 24-well or 6-well plates (e.g. commercially available from Costar "Trans-Wells^®" or from Millipore "Millicell Culture Plate Inserts^®"), and is used for screening test substances for an anti-psoriatic effect. Thus, a method for using a psoriatic skin equivalent is provided, whereby the method comprises contacting the skin equivalent with one or more test compounds or test compositions and determining whether said contact alters one or more of the morphological and/or molecular features of psoriasis. See also the Examples, where it is shown that contact with retinoic acid results in downregulation of HBD2 and SPRR2C.

The "contact" may be brought about by adding purified test substances, or test compositions, to the medium on which the skin equivalent is maintained, and/or by addition to the upper surface of the skin equivalent. Thereafter, any change of the molecular and/or morphological characteristics is analyzed. Test substances or compositions which significantly alter one or more of the molecular and/or morphological features of psoriasis may then be identified for further use. Especially, substances or compositions which significantly reduce at least one, but preferably several (most preferably essentially all) of the morpho logical and/or molecular features of psoriasis are identified and selected. These may be suitable for the manufacture of anti-psoriasis medicaments (both for therapy and for prophylaxis), that can be used for systemical or topical treatment. The method may be repeated several times, for example with different purity levels or different concentrations of the compound.

The method may also be used to analyze any adverse effect a compound or composition may have in vitro. For example, it was found that the anti-psoriasis compound retinoic acid, when contacted with the psoriatic skin equivalent, showed as undesired side effect a downregulation of CKlO. Thus, such unwanted side effects can be analyzed or monitored using the present method.

In a further embodiment compositions (or "cocktails") for use in the above methods are provided. Such compositions comprise suitable amounts of at least two proteins which, when contacted with differentiated healthy keratinocytes, induce molecular features of psoriasis (i.e. increase transcription of at least two psoriasis specific genes) and which induce morphological features characteristic of psoriasis, as described above. The composition preferably comprises at least two proteins selected from IL-6, IL-I, TNF-α and KGF. Most preferably at least IL-I and TNF-α are present in suitable amounts. In a further embodiment, besides IL-I and TNF-α, IL-6 or KGF is present in the cocktail in a suitable amount. In yet a further embodiment, besides IL-I and TNF-α, IL-6 and KGF are present in the cocktail in suitable amounts. The absolute amounts of each protein in the composition may vary. It may be highly concentrated (1Ox or 10Ox), so that addition to the medium dilutes the protein concentration to the suitable amount described above.

The compositions of the invention may also be used for maintaining a psoriatic epidermis in a psoriatic state. It may, thus, be brought into contact with psoriatic tissue in vitro. The psoriatic epidermis may be isolated from a human subject or it may be a psoriatic skin equivalent.

Thus, it is also an embodiment of the invention to provide a method for maintaining psoriatic epidermis in a psoriatic state in vitro. Because the present inventors identified the growth factors and cytokines necessary for inducing and maintaining psoriasis, also the receptors and signaling pathways which need to be activated in the keratinocytes are known. It was found that the induction of at least two, or preferably three, of the following three signaling pathways leads to the development or maintainance of the morpho logical and molecular features of psoriasis: 1. The IRAK-NFKB and TRAF-NFKB signaling pathway;

2. The Jak-STAT signalling pathway; and

3. The MAPK signaling pathway.

For example, IL-6 binds to the IL-6 receptor on the keratinocytes and stimulates the Jak-STAT pathway. Similarly, IL-I (alpha or beta) binds to the IL-I receptor and stimulates the IRAK- NFKB and the MAPK signaling pathway; TNF-α binds to the TNF receptor and stimulates the TRAF- NFKB and MAPK signaling pathway. KGF binds to the Receptor Tyrosine Kinase KGF receptor and stimulates the MAPK signaling pathway.

Thus, any two proteins may be used in the above methods and compositions which stimulate at least two of these signaling pathways in healthy (or psoriatic, in case of maintenance) keratinocytes. Various other proteins (such as ligands of the TLR family, or chemokines) may be tested for their ability to stimulate these pathways indirectly and may then be used to generate and/or maintain a psoriatic skin equivalent, as described.

Inhibitors of various signaling intermediates can be tested for their ability to inhibit the psoriatic markers in the skin equivalent model. Experimental compounds of the inhibitor classes comprise kinase inhibitors, phosphatase inhibitors, molecules that interfere with protein-protein or protein-DNA interaction, protease inhibitors, inhibitors of G-protein coupled receptors. Also protein-based drugs (biologicals) such as antibodies, cytokines or receptor antagonists can be tested in this model system.

SEQUENCES

SEQ ID NO 1 : mRNA of human β-defensin (HBD2) SEQ ID NO 2: mRNA of human SKALP

SEQ ID NO 3: mRNA of human Small Proline Rich Protein 2c (SPRR2c)

SEQ ID NO 4: mRNA of human Interleukin 8 (CXCL8)

SEQ ID NO 5: mRNA of Calmodulin Like Skin Protein (CLSP)

SEQ ID NO 6: mRNA of Carbonic Anhydrase 2 (CA2) SEQ ID NO 7 and 8: PCR primer pair (forward and reverse primer) for beta-actin transcript detection and quantification

SEQ ID NO 9 and 10: PCR primer pair (forward and reverse primer) for HBD2 transcript detection and quantification

SEQ ID NO 11 and 12: PCR primer pair (forward and reverse primer) for CXCL8 transcript detection and quantification

SEQ ID NO 13 and 14: PCR primer pair (forward and reverse primer) for SPRR2c transcript detection and quantification

SEQ ID NO 15 and 16: PCR primer pair (forward and reverse primer) for CLSP transcript detection and quantification SEQ ID NO 17 and 18: PCR primer pair (forward and reverse primer) for CA2 transcript detection and quantification FIGURES Figure 1

Hematoxylin and eosin-stained paraffin sections of a skin equivalent on a DED dermal substrate. Control equivalents (A) were left untreated and the others were stimulated after 7 days of exposure to air with epidermal growth factor 5 ng/ml (EGF)(B), keratinocyte growth factor 10ng/ml (KGF)(C), and interleukin-6 25ng/ml (IL-6)(D) for 4 days. Figure 2 Beta-defensin 2 staining of paraffin sections of skin equivalents on a DED dermal substrate. After 7 days of exposure to air, control equivalents (A) were left untreated and the others were stimulated for 4 days with TNF-α 30ng/ml (B), IL- lα lOng/ml (C), and a combination of IL- lα 10ng/ml and TNF-α 10ng/ml (D). Note that positive staining in the dermis is not cell-associated but indicates staining of HBD2 secreted by keratinocytes, that associates with dermal structures. Figure 3

Hematoxylin and eosin staining of paraffin sections of skin equivalents on a DED dermal substrate. After 7 days of exposure to air, control equivalents (A) were not stimulated and the others were stimulated for 4 days with TNF-α 30ng/ml (B), IL- lα 10ng/ml (C), and a combination of IL-I α 10ng/ml and TNF-α 30ng/ml (D). Figure 4

Hematoxylin and eosin staining of paraffin sections of a skin equivalent on a DED dermal substrate. After 7 days of exposure to air, control equivalents (A) were left untreated and the others were stimulated for 4 days with cocktail of IL-6, KGF, IL- lα and TNF-α all at 10ng/ml (B).

The following non-limiting Examples illustrate the different embodiments of the invention. Unless stated otherwise in the Examples, all techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, and Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. See also Dieffenbach and Dveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratory Press, and McPherson at al. (2000) PCR-Basics: From Background to Bench, First Edition, Springer Verlag, Germany).

EXAMPLES

1. Validation of discriminative read-out markers for psoriasis and atopic dermatitis

1.1 Material and Method

The following markers have been described in the scientific literature (Nomura et al.,

2003, Allergy Clin Immunol. 112(6): 1195-202; see Table 1) as being highly upregulated in psoriasis or atopic dermatitis (AD). The inventors validated these markers and used them for characterization of their skin equivalent models by qPCR.

Table 1

Pure epidermis was obtained from biopsies of involved skin from psoriasis and AD patients (n=3). The epidermis was isolated by an incubation for 4h in a solution containing 12 mg/ml dispase in PBS, followed by mechanical removal of the purified epidermis. Total RNA was isolated using Trizol (Invitrogen) according to manufacturer's instructions. RNA concentrations were determined and integrity verified with agarose gel electrophoresis. Two μg of DNAse-treated RNA from each sample was used to generate cDNA using the Superscript™ II Reverse Transcriptase (Invitrogen) according to manufacturers instructions using per reaction 250 ng Oligo-dT(15) primers (Promega). Generated cDNA samples were diluted 50 times and stored frozen in aliquots. 1/200 of initial sample was used as template cDNA in the amplication RT- PCR mixture (25 μl) containing 2x SYBR Green Supermix (Biorad), and 300 nM forward and reverse primers. The iCycler of BioRad was used for detecting real-time PCR products. Primers for genes and house-keeping genes were almost all designed to span intron-exon boundaries to distinguish PCR products generated from genomic versus cDNA template. Each PCR reaction was optimized to ensure reaction efficiencies in between 90-110%, that a single band of the appropriate length (66-187 bp) was amplified and that no bands corresponding to genomic DNA amplification or primer-dimer pairs were present. The PCR cycling conditions were performed for all of the samples as follows: 2 min at 50°C; 10 min at 95°C for polymerase activation; and 40 cycles for the melting (95°C, 15 s) and annealing/extension (60°C for 1 min) steps. Each sample was run on separate 96-well plates with all tested primers pairs including the house keeping genes in duplo. The ΔΔCT method was used to determine relative expression for each gene compared with the house keeping gene beta-actin (see Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001 Dec;25(4):402- 8). In addition to the qPCR analysis the inventors examined a number of proteins for increased expression in lesional psoriatic skin by immunohistochemistry. Skin biopsies were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Sections (5 μm) were cut, deparaffinized, and rehydrated for immunohistochemical analysis. After incubation with primary antibodies, sections were stained with avidin-biotin-peroxidase complex system (streptABcomplex/HRP; Dako), as described by the suppliers. All sections were counterstained with hematoxylin.

1.2 Results

The difference in mRNA expression level between psoriasis and AD epidermis for the genes indicated above is presented in Table 2. Table 2

Real-time PCR analysis of lesional psoriatic and AD skin. Figures indicate fold difference between the two diseases. Positive values mean a higher level in psoriasis, whereas negative values mean a higher level in AD

The selected genes clearly show large differences in mRNA expression between psoriasis and AD. The inventors confirmed this difference at the protein level, by semi- quantiative analysis of HBD2 expression in biopsies of psoriasis and AD patients.

In addition, the inventors could, at the protein level, confirm previous markers that are known to be increased in psoriasis compared to normal skin and/or AD skin, such as SKALP, CKl 6, CKl 7 and SLPI. Using immunohistochemistry the inventors found increased expression of these genes in psoriasis compared to normal skin (data not shown). The increased disease-specific expression of the indicated genes is for most genes also found when compared to AD skin, with the exception of CKl 6 and CKl 7. In conclusion, skin equivalents resembling a psoriatic epidermis should have an upregulated expression level of markers such as HBD2, CXCL8, SKALP, SLPI and SPRR2c when compared to AD or normal skin or skin equivalents resembling these conditions. The expression of CLSP and CA2 should be comparable to (or lower than) the level found in control or AD skin or models resembling these conditions. 2. - Effect of cytokines and growth factors on epidermal morphology 2.1 Material and Methods

Human epidermal skin equivalents were generated according to a previously described method (Ponec et al. J Invest Dermatol. 1997; 109:348-55; Gibbs S et al. Wound Repair Regen. 2000; 8: 192-203).

Briefly, primary keratinocytes passage 1 were seeded on de-epidermized dermis (DED) at a density of 150.000-200.000 cells/cm² and cultured submerged for 3 days in the medium previously described containing 5% Fetal Bovine Serum (FBS) and 5 ng/ml KGF. After 3 days, the equivalents were raised to the air- liquid interphase and medium was changed as previously described. At this moment serum was omitted, but the medium continued to contain 5ng/ml KGF. Medium was refreshed after 3 days. After 7 days (10 day old cultures), medium was refreshed again and the appropriate active factors were added (EGF, KGF and IL-6 (Sigma)). This medium was changed after 48h, and after 96h the skin equivalents were harvested, routinely processed for paraffin embedded histological analysis or used to isolate the mRNA from the epidermis as described above. Note that the reconstructed skin that was obtained is only composed of an epidermal layer that is similar to human epidermis, whereas dermal cells are absent. For convenience these reconstructed epidermal equivalents are referred to as 'skin equivalents'.

Histology and immunohistochemistry:

Harvested skin equivalents were washed in phosphate-buffered saline (PBS), a part was fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin whereas the other part was snap-frozen. Sections (5 μm) were cut, deparaffinized in ethanol, and rehydrated in preparation for morphological (hematoxylin/eosin stain, H&E) or immunohistochemical analysis of cytokeratin 10 and 17, SKALP, SLPI and Ki67. After incubation with primary antibodies, sections were stained with avidin-biotin-peroxidase complex system (streptABcomplex/HRP; Dako), as described by the suppliers with the following minor modifications: PBS was used instead of TRIS buffered saline and for Ki67 staining antigen retrieval was performed by immersing slides in 0.1 M citrate buffer (pH 6.0) for 15 min at 100°C followed by slow cooling to room temperature prior to staining of the sections. All sections were counterstained with hematoxylin. 2.2 Results

In Figure 1, the control epidermal equivalent (A) without active factors in the medium, can be compared to equivalents treated for 4 days with various growth factors. The addition of EGF (B) induced elongation of the rete ridges into the dermis and some parakeratosis as found in the psoriatic skin. The addition of KGF (C) showed no morphological differences compared the control, whereas the addition of IL-6 did result in epidermis with an enlarged suprabasal compartment and some parakeratosis as found in psoriatic epidermis. Interestingly, only in the IL-6 stimulated culture, the immunohistochemical analysis showed (mild) expression of HDB2 and some focally expressed cytokeratin 17 in the epidermis (not shown). SKALP protein was present in IL-6 and highly expressed in EGF stimulated skin equivalent, whereas it was absent in the control and KGF treated equivalents. These data indicate that IL-6 could be used to partly induce the psoriatic phenotype in the in vitro reconstructed epidermis.

3. - Upregulation of psoriasis specific markers requires both IL- lα and TNF-α 3.1 Material and Method

In the literature, it has been demonstrated that IL- lα is able to induce the expression of HBD2 in skin equivalents (Liu et al. J Invest Dermatol 202; 118:275-281). TNF-α was found to induce HBD2 in keratinocytes under submerged conditions (Harder et al. Nature. 1997; 387:861). The effect of these cytokines on the expression of a comprehensive array of psoriasis- specific or AD-specific markers has not been investigated in skin equivalents. The issue whether cytokines and/or growth factors act in synergy to induce a disease-specific phenotype has never been addressed before.

In the following example, skin equivalents were generated as described above. After 7 days exposure to air, the equivalents were stimulated with IL- lα, TNF-α or a combination of both factors. The concentrations used are indicated in the figure below. The expression of the different molecular markers HBD2, CXCL8, SPRR2c, CLSP, CA2 and the control HBD3 was investigated using RT-PCR. At the protein level, HBD2 expression was evaluated histologically. 3.2 Results

Figure 2 shows skin equivalents stimulated with IL- lα and TNF-α or a combination of both cytokines, stained for HBD2.

The control and TNF-α 30ng/ml alone did not induce significant expression of HBD2 (A and B). IL- lα showed some induction of HBD2 expression mainly in some upper suprabasal cells (C), but the combination of IL- lα and TNF-α showed the strongest staining for HBD2 (D). Most cells in the suprabasal layer stained positive.

In Table 3, qPCR analysis of some of these equivalents is shown. From table 3 it is clear that the IL- lα stimulus alone did not result in large changes of mRNA levels for most genes.

Table 3 Real-time PCR analysis of epidermal skin equivalents that were treated with IL- lα 10ng/ml or IL-lα+TNF-α lOng/ml. The expression of level of the selected genes was compared to control skin equivalent treated without cytokines. The relative increase or decrease was measured using the 2^to(deltaCt) formula and given as the fold difference compared to control. Positive values indicate a higher level than in control, whereas negative values mean a lower value than in controls.

Gene IL-Ia IL-I α + TNF-α

HBD2 3.5 36.4

CXCL8 -1.5 3.1

SPRR2C 1.2 3.5

CLSP -3.1 -3.5

CA2 -1.7 -2.4

The combination IL- lα and TNF-α, however, clearly showed synergistic effects on gene expression. The strongest upregulation was found for HDB2 confirming the histological results. Interestingly, all psoriasis specific genes HBD2, CXCL8 and SPRR2c showed to be upregulated and the AD genes CLSP and CA2 to be downregulated. These results clearly demonstrate that both inflammatory cytokines IL- lα and TNF-α are required to induce the psoriatic epidermal phenotype in the skin equivalent.

4 - Titration of inflammatory cytokine concentrations to assess the lower and upper limits for induction of the required phenotype

4.1 Material and Methods

The experiment described in Example 3 was repeated, but with different concentrations for IL- lα and TNF-α alone and a combination of IL-I α 10, 20 or 30 ng/ml with 2, 10 or 30 ng/ml TNF-α. Analysis was performed as described above.

4.2 Results

In Figure 3, the control skin equivalent is shown (A) and an equivalent stimulated with 30ng/ml TNF-α (B). The morphology of the basal keratinocytes is clearly altered compared to control. Only a TNF-α concentration of 10 ng/ml showed a normal epidermal morphology. Similar results were obtained when TNF-α 30ng/ml was combined with IL- lα 10, 20 or 30ng/ml. In the figure, this is shown for the combination IL- lα 10 ng/ml and TNF-α 30 ng/ml (D). A dramatic negative effect on the morphology of the basal keratinocytes was observed, with areas where the epidermis detached from the underlying dermis. The combination IL- lα 10 ng/ml and TNF-α 10 ng/ml (C) showed normal epidermal morphology. The lowest IL- lα concentration that induced HBD2 expression was 10 ng/ml. IL- lα concentrations up to 100 ng/ml did not dramatically increase HBD2 staining at the histological protein level nor did they negatively affect the epidermal morphology. TNF-α did not induce the HBD2 protein expression in skin equivalents not even at a concentration as high as 100ng/ml. The combination of a concentration range of IL-I α with 2 ng of TNF-α did not show a synergistic upregulation of HBD2. IL- lα in combination with 10ng/ml TNF-α induced a strong upregulation of HBD2 expression compared to IL- lα alone, indicating that the lowest TNF-α concentration required for a synergistic effect is between 2 and lOng/ml. In conclusion, the lowest effective IL- lα concentration is 10 ng/ml. The lowest TNF-α concentration to achieve a synergistic effect is between 2 and 10ng/ml. Suitable TNF-α concentrations to be used are between 10 and 30ng/ml. Concentrations of 30 ng/ml and higher are likely to be deleterious to the epidermal morphology.

5. - Evaluation of different cvtokines/growth factors in combination with IL- lα and TNF-α

5.1 Material and Methods

In subsequent experiments, combinations of EGF, KGF, IL-6 with a cocktail of IL-I α and TNF-α (both at 10ng/ml) were evaluated.

5.2 Results

KGF + IL- lα + TNF-α: In combination with KGF, the skin equivalent showed a morphology comparable to control skin equivalent. Molecular markers for psoriasis were moderately to strongly upregulated, whereas the AD markers CLSP and CA2 gave a slight downregulation as determined by qPCR.

EGF + IL-lα + TNF-α: the epidermal morphology was strongly negatively affected in combination with EGF. The epidermis was only a few layers thick and the expression profile of analyzed molecular markers did not meet the requirements indicating that EGF is not a good candidate to be used to induce a psoriatic phenotype in the skin equivalents.

IL-6 + IL-lα + TNF-α: The morphology of IL-6 treated epidermis previously showed an enlarged suprabasal compartment and parakeratosis. Here, in combination with IL- lα and TNF-α parakeratosis was still observed, but enlargement of the suprabasal compartment did not occur. The expression of the psoriasis markers HBD2 (128x) and SPRR2c (12x) was strongly up regulated and CXCL8 moderately (3.2x). The AD markers CLSP (4.3x) and CA2 (6.5x) were down regulated.

In conclusion, skin equivalents stimulated with IL-6 in combination with IL-lα and TNF-α strongly mimic the psoriatic phenotype, being positive for the histological marker parakeratosis and showing the upregulated or downregulated expression of the psoriasis and AD molecular markers, respectively.

Ideally, the skin equivalent should also have an enlarged suprabasal compartment (acanthosis) indicative for a hyperproliferative epidermis. It was reasoned that the IL-6, II- lα and TNF-α cocktail used above, most likely required the presence of an additional strong epithelial mitogen, e.g. KGF. This was evaluated in the following experiment. The skin equivalents were produced as described previously and cultured for 4 days with or without IL-6+KGF+IL-l+TNFα to investigate the effect on the epidermal morphology (see Figure 4).

The skin equivalent stimulated with the IL-6+KGF+IL-l+TNFα cocktail showed an enlarged suprabasal compartment and parakeratosis (B). Moreover when compared to control cultures the expression of HBD2, SPRR2C, CXCL8 was strongly upregulated, whereas CLSP and CA2 were downregulated (not shown).

In conclusion, reconstituted skin equivalents stimulated for 96h with a cocktail consisting of KGF, IL-6, IL- lα and TNF-α (all at 10ng/ml) induces an epidermal phenotype that resembles psoriatic epidermis both at the morphological and molecular level.

6. - Use of a psoriasis-like skin equivalent to evaluate the efficacy of the anti-psoriatic drug retinoic acid

A skin equivalent resembling a psoriatic epidermis was obtained by stimulation in KGF, IL-6, IL- lα and TNF-α, and subsequently cultured with or without retinoic acid at 10^"6 M. The control skin equivalents showed an enlarged suprabasal compartment and parakeratosis. The epidermal skin equivalents treated with retinoic acid still showed an enlarged suprabasal compartment, but no parakeratosis. This is a remarkable effect in a short culture period, because in vivo retinoic acid works rather slow. In table 3 the expression of molecular markers is compared for control skin equivalents with and without retinoic acid. Table 3: Real-time qPCR analysis of skin equivalents treated with or without retinoic acid (10^~6 M). The relative increase or decrease was measured using the 2^in(deitac^t) f_ormui_a g^ indicated as the fold increase (+) or decrease (-) caused by retinoic acid.

Fold up (+) or down (-)

Gene regulation by retinoic acid

HBD2 - 6

SPRR2c - 22

CLSP + 1.3

CA2 - 2.5

CKlO - 40

From Table 3 it is evident that treatment with retinoic acid resulted in diminished expression levels of the psoriasis markers HBD2 and SPRR2c. The expression of the AD markers CA2 and CLSP was hardly changed. CKlO was included as a positive control because it is known to respond to retinoic acid. Downregulation of this gene can actually be regarded as an unwanted side effect of retinoic acid in anti-psoriatic therapy i.e. suppression of normal differentiation. As such, the model can also be used to monitor adverse drug effects in vitro.

In conclusion, the optimal skin equivalent resembling psoriatic epidermis as described in Example 5 can be used to evaluate the effect of an anti-psoriatic drug.

Claims

1. A method for making a psoriatic skin equivalent in vitro, comprising the steps of: (a) seeding normal human keratinocytes onto a dermal matrix,

(b) allowing the keratinocytes to differentiate into an epidermis,

(c) adding a composition comprising a suitable amount of at least two proteins which induce, in the epidermal cells, the transcription of at least two genes selected from the group consisting of: β-defensin (HBD2), skin-derived antileukoproteinase (SKALP), Small Proline Rich Protein

2c (SPRR2c) and Interleukin 8 (CXCL8).

2. The method according to claim 1, wherein said proteins are selected from the group consisting of: Interleukin-6 (IL-6), Interleukin- 1 (IL-I), Tumor Necrosis Factor alpha (TNF-α) and Keratinocyte Growth Factor (KGF).

3. The method according to claim 2, wherein said proteins are added in an amount of at least IOng protein per milliliter medium and, preferably, not more than 30ng TNF- α protein per milliliter medium.

4. The method according to any one of claim 1-3, wherein the dermal matrix is selected from a de-epidermized dermis (DED) dermal substrate, a tissue- engineered bio-matrix, a (semisynthetic biocompatible matrix and a coated or uncoated polycarbonate-based membrane filter insert.

5. A psoriatic skin equivalent, obtainable by the method according to any one of claims 1-4.

6. A method for using a psoriatic skin equivalent according to claim 5, comprising contacting said skin equivalent with one or more test compounds or compositions and determining whether said contact alters the morphological and/or molecular features of psoriasis.

7. The method according to claim 6, further comprising identifying of a compound or composition which reduces one or more of the morphological and/or molecular features of psoriasis.

8. A composition for stimulating the development of a psoriatic skin equivalent or for maintaining a psoriatic skin equivalent, comprising a suitable amount of at least two proteins selected from: IL-6, IL-I, TNF-α and KGF.

9. The composition according to claim 8 comprising a synergistic amount of IL-I and TNF-α.

10. Use of a synergistically effective amount of IL-I and TNF-α for the induction and/or maintenance of a human psoriatic skin equivalent in vitro.

11. Use of a composition according to claim 8 or 98 in a method according to any one of claims 1-4, 7 and 7.

12. Use of IL-6 for the induction and/or maintainance of a human psoriatic skin equivalent in vitro, especially for the induction of epidermal parakeratosis in vitro. .