Characterization of Spink6 in mouse skin: the conserved inhibitor of kallikrein-related peptidases is reduced by barrier injury.

The proteolytic regulation of the desquamation process by kallikrein-related peptidases (KLKs) is crucial for epidermal barrier function, and elevated KLK levels have been reported in atopic dermatitis. KLKs are controlled by specific inhibitors of the serine protease inhibitor of Kazal-type (Spink) family. Recently, SPINK6 was shown to be present in human stratum corneum. In order to investigate its role in epidermal barrier function, we studied mouse Spink6. Sequence alignment revealed that the Kazal domain of Spink6 is highly conserved in animals. Recombinant Spink6 efficiently inhibited mouse Klk5 and human KLK2, KLK4, KLK5, KLK6, KLK7, KLK12, KLK13, and KLK14, whereas human KLK1 and KLK8 were not inhibited. Spink6 was expressed in mouse epidermis mainly in the stratum granulosum, and the inner root sheath of hair follicles. Stimulation with flagellin, EGF, and IL-1ß did not alter Spink6 expression, whereas stimulation with tumor necrosis factor-α (TNFα)/IFNγ and all-trans retinoic acid resulted in a significant downregulation of Spink6 expression in cultured primary mouse keratinocytes. Mechanically and metabolically induced skin barrier dysfunction resulted both in a downregulation of Spink6 expression. Our study indicates that Spink6 is a potent inhibitor of KLKs and involved in skin barrier function.

Murine filaggrin-2 is involved in epithelial barrier function and down-regulated in metabolically induced skin barrier dysfunction.

The S100 fused-type proteins (SFTPs) are thought to be involved in the barrier formation and function of the skin. Mutations in the profilaggrin gene, one of the best investigated members of this family, are known to be the major risk factors for ichthyosis vulgaris and atopic dermatitis. Recently, we identified human filaggrin-2 as a new member of the SFTP family. To achieve further insight into its function, here the murine filaggrin-2 was analysed as a possible orthologue. The 5' and 3' ends of the mouse filaggrin-2 cDNA of the BALB/c strain were sequenced and confirmed an organization typical for SFTPs. Murine filaggrin-2 showed an expression pattern mainly in keratinizing epithelia in the upper cell layers on both mRNA and protein levels. The expression in cultured mouse keratinocytes was increased upon elevated Ca(2+) levels. Immunoblotting experiments indicated an intraepidermal processing of the 250-kDa full-length protein. In metabolically (essential fatty acid-deficient diet) induced skin barrier dysfunction, filaggrin-2 expression was significantly reduced, whereas filaggrin expression was up-regulated. In contrast, mechanical barrier disruption with acetone treatment did not affect filaggrin-2 mRNA expression. These results suggest that filaggrin-2 may contribute to epidermal barrier function and its regulation differs, at least in parts, from that of filaggrin.

Induction of regulatory T cells by a murine ß-defensin.

ß-Defensins are antimicrobial peptides of the innate immune system produced in the skin by various stimuli, including proinflammatory cytokines, bacterial infection, and exposure to UV radiation (UVR). In this study we demonstrate that the UVR-inducible antimicrobial peptide murine ß-defensin-14 (mBD-14) switches CD4(+)CD25(-) T cells into a regulatory phenotype by inducing the expression of specific markers like Foxp3 and CTLA-4. This is functionally relevant because mBD-14-treated T cells inhibit sensitization upon adoptive transfer into naive C57BL/6 mice. Accordingly, injection of mBD-14, comparable to UVR, suppresses the induction of contact hypersensitivity and induces Ag-specific regulatory T cells (Tregs). Further evidence for the ability of mBD-14 to induce Foxp3(+) T cells is provided using DEREG (depletion of Tregs) mice in which Foxp3-expressing cells can be depleted by injecting diphtheria toxin. mBD-14 does not suppress sensitization in IL-10 knockout mice, suggesting involvement of IL-10 in mBD-14-mediated immunosuppression. However, unlike UVR, mBD-14 does not appear to mediate its immunosuppressive effects by affecting dendritic cells. Accordingly, UVR-induced immunosuppression is not abrogated in mBD-14 knockout mice. Together, these data suggest that mBD-14, like UVR, has the capacity to induce Tregs but does not appear to play a major role in UVR-induced immunosuppression. Through this capacity, mBD-14 may protect the host from microbial attacks on the one hand, but tame T cell-driven reactions on the other hand, thereby enabling an antimicrobial defense without collateral damage by the adaptive immune system.

Differential suppression of epidermal antimicrobial protein expression in atopic dermatitis and in EFAD mice by pimecrolimus compared to corticosteroids.

It has been suggested that the increased rate of bacterial infection in atopic dermatitis (AD) may be caused by reduced antimicrobial protein (AMP) expression. We were interested whether common treatments in AD affect antimicrobial defense. We investigated the effects of topically applied corticosteroids betamethasone valerate (BV) and triamacinolone acetonide (TA) and those of the calcineurin inhibitor pimecrolimus for 3 weeks on AMP expression in AD. BV and TA treatment in AD led to a significant reduction in AMP expression; protein expression of human beta-defensins (hBD)-2 and hBD-3, psoriasin, RNase 7 and cathelicidin LL-37 was below the level in skin of healthy controls. After pimecrolimus treatment, AMP expression was also reduced but less compared to BV and TA; the expression levels of hBD-2, psoriasin and RNase 7 still remained above the control levels. In essential fatty acid-deficient (EFAD) mice, a model of chronic skin barrier disease with inflammation, expression of the mouse beta-defensins mBD-1, mBD-3 and mBD-14 (orthologues for hBD-1, hBD-2 and hBD-3, respectively), was reduced by both treatments, again more pronounced by BV compared to pimecrolimus. In summary, we found that treatment for AD with corticosteroids in human skin and EFAD mice caused a strong reduction in AMPs; reduction was less with pimecrolimus. This result may explain the clinical observation that prolonged treatment with topical corticosteroids sometimes leads to bacterial infection.

Mechanical and metabolic injury to the skin barrier leads to increased expression of murine ß-defensin-1, -3, and -14.

Protection of the skin against microbiological infection is provided by the permeability barrier and by antimicrobial proteins. We asked whether the expression of murine ß-defensins (mBDs)-1, -3, and -14-orthologs of human ß-defensins hBD-1, -2, and -3, respectively--is stimulated by mechanically/physicochemically (tape stripping or acetone treatment) or metabolically (essential fatty acid-deficient (EFAD) diet) induced skin barrier dysfunction. Both methods led to a moderate induction of mBD-1 and mBD-14 and a pronounced induction of mBD-3 mRNA. Protein expression of the mBDs was increased as shown by immunohistology and by western blotting. Artificial barrier repair by occlusion significantly reduced the increased expression of mBD-14 after mechanical injury and of all three mBDs in EFAD mice, supporting an interrelationship between permeability and the antimicrobial barrier. mBD-3 expression was stimulated in vitro by tumor necrosis factor-α (TNF-α), and a neutralizing anti-TNF-α antibody significantly reduced increased mBD-3 expression after barrier injury in mouse skin, indicating that induction of mBD-3 expression is mediated by cytokines. The expression of mBD-14 was stimulated by transforming growth factor-α and not by TNF-α. In summary, we demonstrated upregulation of mBD1, -3, and -14 after mechanically and metabolically induced skin barrier disruption, which may be an attempt to increase defense in the case of permeability barrier dysfunction.