Gys J. de Jongh

Psoriasis is associated with increased beta-defensin genomic copy number

Psoriasis is a common inflammatory skin disease with a strong genetic component. We analyzed the genomic copy number polymorphism of the β-defensin region on human chromosome 8 in 179 Dutch individuals with psoriasis and 272 controls and in 319 German individuals with psoriasis and 305 controls. Comparisons in both cohorts showed a significant association between higher genomic copy number for β-defensin genes and risk of psoriasis.

β-Defensin-2 Protein Is a Serum Biomarker for Disease Activity in Psoriasis and Reaches Biologically Relevant Concentrations in Lesional Skin

Background Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2) is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis. Methodology/Principal Findings We found that systemic levels of hBD-2 showed a weak but significant correlation with beta defensin copy number in healthy controls but not in psoriasis patients with active disease. In psoriasis patients but not in atopic dermatitis patients, we found high systemic hBD-2 levels that strongly correlated with disease activity as assessed by the PASI score. Our findings suggest that systemic levels in psoriasis are largely determined by secretion from involved skin and not by genomic copy number. Modelling of the in vivo epidermal hBD-2 concentration based on the secretion rate in a reconstructed skin model for psoriatic epidermis provides evidence that epidermal hBD-2 levels in vivo are probably well above the concentrations required for in vitro antimicrobial and chemokine-like effects. Conclusions/Significance Serum hBD-2 appears to be a useful surrogate marker for disease activity in psoriasis. The discrepancy between hBD-2 levels in psoriasis and atopic dermatitis could explain the well known differences in infection rate between these two diseases.

Cystatin M/E Is a High Affinity Inhibitor of Cathepsin V and Cathepsin L by a Reactive Site That Is Distinct from the Legumain-binding Site A NOVEL CLUE FOR THE ROLE OF CYSTATIN M/E IN EPIDERMAL CORNIFICATION

Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, and we have previously reported that both proteins are likely to be involved in the regulation of stratum corneum formation in skin. Although cystatin M/E contains a predicted binding site for papain-like cysteine proteases, no high affinity binding for any member of this family has been demonstrated so far. We report that human cathepsin V (CTSV) and human cathepsin L (CTSL) are strongly inhibited by human cystatin M/E. Kinetic studies show that Ki values of cystatin M/E for the interaction with CTSV and CTSL are 0.47 and 1.78 nm, respectively. On the basis of the analogous sites in cystatin C, we used site-directed mutagenesis to identify the binding sites of these proteases in cystatin M/E. We found that the W135A mutant was rendered inactive against CTSV and CTSL but retained legumain-inhibiting activity. Conversely, the N64A mutant lost legumain-inhibiting activity but remained active against the papain-like cysteine proteases. We conclude that legumain and papain-like cysteine proteases are inhibited by two distinct non-overlapping sites. Using immunohistochemistry on normal human skin, we found that cystatin M/E co-localizes with CTSV and CTSL. In addition, we show that CTSL is the elusive enzyme that processes and activates epidermal transglutaminase 3. The identification of CTSV and CTSL as novel targets for cystatin M/E, their (co)-expression in the stratum granulosum of human skin, and the activity of CTSL toward transglutaminase 3 strongly imply an important role for these enzymes in the differentiation process of human epidermis.

Expression of the Vanin Gene Family in Normal and Inflamed Human Skin: Induction by Proinflammatory Cytokines

The vanin gene family encodes secreted and membrane-bound ectoenzymes that convert pantetheine into pantothenic acid and cysteamine. Recent studies in a mouse colitis model indicated that vanin-1 has proinflammatory activity and suggest that pantetheinases are potential therapeutic targets in inflammatory diseases. In a microarray analysis of epidermal gene expression of psoriasis and atopic dermatitis lesions, we identified vanin-3 as the gene showing the highest differential expression of all annotated genes that we studied (19-fold upregulation in psoriasis). Quantitative real-time PCR analysis confirmed the microarray data on vanin-3 and showed similar induction of vanin-1, but not of vanin-2, in psoriatic epidermis. Immunohistochemistry showed that vanin-3 is expressed in the differentiated epidermal layers. Using submerged and organotypic keratinocyte cultures, we found that vanin-1 and vanin-3 are induced at the mRNA and protein level by psoriasis-associated proinflammatory cytokines (Th17/Th1) but not by Th2 cytokines. We hypothesize that increased levels of pantetheinase activity are part of the inflammatory-regenerative epidermal differentiation program, and may contribute to the phenotype observed in psoriasis.

Genetically Programmed Differences in Epidermal Host Defense between Psoriasis and Atopic Dermatitis Patients

In the past decades, chronic inflammatory diseases such as psoriasis, atopic dermatitis, asthma, Crohn’s disease and celiac disease were generally regarded as immune-mediated conditions involving activated T-cells and proinflammatory cytokines produced by these cells. This paradigm has recently been challenged by the finding that mutations and polymorphisms in epithelium-expressed genes involved in physical barrier function or innate immunity, are risk factors of these conditions. We used a functional genomics approach to analyze cultured keratinocytes from patients with psoriasis or atopic dermatitis and healthy controls. First passage primary cells derived from non-lesional skin were stimulated with pro-inflammatory cytokines, and expression of a panel of 55 genes associated with epidermal differentiation and cutaneous inflammation was measured by quantitative PCR. A subset of these genes was analyzed at the protein level. Using cluster analysis and multivariate analysis of variance we identified groups of genes that were differentially expressed, and could, depending on the stimulus, provide a disease-specific gene expression signature. We found particularly large differences in expression levels of innate immunity genes between keratinocytes from psoriasis patients and atopic dermatitis patients. Our findings indicate that cell-autonomous differences exist between cultured keratinocytes of psoriasis and atopic dermatitis patients, which we interpret to be genetically determined. We hypothesize that polymorphisms of innate immunity genes both with signaling and effector functions are coadapted, each with balancing advantages and disadvantages. In the case of psoriasis, high expression levels of antimicrobial proteins genes putatively confer increased protection against microbial infection, but the biological cost could be a beneficial system gone awry, leading to overt inflammatory disease.

Differential gene expression in premalignant human epidermis revealed by cluster analysis of serial analysis of gene expression (SAGE) libraries

Serial analysis of gene expression (SAGE) has been used for quantitative analysis of gene expression. We applied cluster analysis on multiple SAGE libraries derived from premalignant epidermal tissue (actinic keratosis), normal human epidermis, and cultured keratinocytes. The samples were obtained from skin biopsies without contamination by dermal tissue or blood. A total of 60,000 transcripts (tags) were analyzed. Two‐way cluster analysis was applied to both the transcripts and the tissues, resulting in separation of the cultured cells from the epidermal samples, and clustering of many, presumably coregulated, genes. Two clusters of genes, strongly up‐regulated in the tumor tissue compared with normal epidermis, were investigated in more detail. The differential expression of genes could be confirmed in actinic keratosis from four patients. Several of these genes have been previously associated with carcinogenesis or are likely to be important on the basis of their presumed function. Automated literature search tools show that a subgroup of these genes is coexpressed in other tissues and is part of an epidermal differentiation gene cluster on chromosome 1q 21. We conclude that cluster analysis on large data sets uncovers clear partitions and correlations that could be confirmed by independent methods. We predict that these partitions will lead to biological interpretations that can be relevant for understanding the processes of carcinogenesis and tumor progression.