Cross-Linking of SPINK6 by Transglutaminases Protects from Epidermal Proteases

Extracellular kallikrein-related peptidases (KLKs) are involved in the desquamation process and the initiation of epidermal inflammation by different mechanisms. Their action is tightly controlled by specific protease inhibitors. Recently, we have identified the serine protease inhibitor of Kazal-type (SPINK) 6 as a selective inhibitor of KLKs in human stratum corneum extracts. As SPINK6 is expressed in the same localization as transglutaminases (TGM) and contains TGM substrate motifs, SPINK6 was tested to be cross-linked in the epidermis. Recombinant SPINK6 was shown to be cross-linked to fibronectin (FN) by TGM1 by western blot analyses. Moreover, SPINK6 was cross-linked in epidermal extracts and cultured keratinocytes by immunoblotting analyses. The use of TGM1 and TGM3 resulted in different immunoreactivities in western blot analyses of SPINK6 and epidermal extracts, suggesting substrate specifities of different TGMs for SPINK6 cross-linking in the epidermis. Conjugated SPINK6 exhibited protease inhibitory activity in keratinocytes and stratum corneum extracts; cross-linked SPINK6 protected FN from KLK5-mediated cleavage, whereas a lower KLK-inhibiting SPINK6-GM mutation did not. In conclusion, we demonstrated that SPINK6 is cross-linked in keratinocytes and human epidermis and remains inhibitory active. Thus, cross-linked SPINK6 might protect specific substrates such as FN from KLK cleavage and contributes to the regulation of proteases in the epidermis

Decreased Susceptibility of Staphylococcus aureus Small-Colony Variants toward Human Antimicrobial Peptides

Staphylococcus aureus is a frequent resident of human nose and skin in many individuals, but it is also able to cause a variety of serious infections including those of the skin and soft tissue. There is increasing evidence that particularly persistent, relapsing, and difficult-to-treat infections caused by S. aureus are associated with the formation of the small-colony variant (SCV) phenotype. The aim of this study was to investigate the hypothesis that (i) skin-derived antimicrobial peptides (AMPs) exhibit a reduced activity against SCVs and (ii) that switching into the SCV phenotype may endow S. aureus with a decreased susceptibility toward the killing activity of human stratum corneum. Here, we show that clinically derived S. aureus SCVs are less susceptible to the bactericidal activity of different human skin-derived AMPs as compared with their isogenic corresponding wild-type strains. Similarly, a S. aureus hemB mutant displaying the SCV phenotype was less susceptible to the antimicrobial activity of AMPs than its hemB-complemented mutant. These findings were accompanied by a higher resistance of SCVs to the killing activity of human stratum corneum. Switching into the SCV phenotype may help S. aureus to subvert cutaneous innate defense, thus contributing to the establishment and persistence of infection

Flagellin Delivery by Pseudomonas aeruginosa Rhamnolipids Induces the Antimicrobial Protein Psoriasin in Human Skin

The opportunistic pathogen Pseudomonas aeruginosa can cause severe infections in patients suffering from disruption or disorder of the skin barrier as in burns, chronic wounds, and after surgery. On healthy skin P. aeruginosa causes rarely infections. To gain insight into the interaction of the ubiquitous bacterium P. aeruginosa and healthy human skin, the induction of the antimicrobial protein psoriasin by P. aeruginosa grown on an ex vivo skin model was analyzed. We show that presence of the P. aeruginosa derived biosurfactant rhamnolipid was indispensable for flagellin-induced psoriasin expression in human skin, contrary to in vitro conditions. The importance of the bacterial virulence factor flagellin as the major inducing factor of psoriasin expression in skin was demonstrated by use of a flagellin-deficient mutant. Rhamnolipid mediated shuttle across the outer skin barrier was not restricted to flagellin since rhamnolipids enable psoriasin expression by the cytokines IL-17 and IL-22 after topical application on human skin. Rhamnolipid production was detected for several clinical strains and the formation of vesicles was observed under skin physiological conditions. In conclusion we demonstrate herein that rhamnolipids enable the induction of the antimicrobial protein psoriasin by flagellin in human skin without direct contact of bacteria and responding cells. Hereby, human skin might control the microflora to prevent colonization of unwanted microbes in the earliest steps before potential pathogens can develop strategies to subvert the immune response

Enhanced Expression and Secretion of Antimicrobial Peptides in Atopic Dermatitis and after Superficial Skin Injury

Human skin can defend itself against potentially invading microorganisms by production of antimicrobial peptides (AMPs). The expression of AMPs in atopic dermatitis (AD) is still emerging. To gain more insight into the role of AMPs in AD, we systematically analyzed the expression of ribonuclease 7 (RNase 7), psoriasin, and human β-defensins (hBD)-2 and -3 in AD compared with psoriatic and healthy control skin as well as after experimental barrier disruption. Immunostaining revealed enhanced expression of all AMPs in the lesional skin of untreated AD and psoriasis when compared with non-lesional skin and controls. Accordingly, induced in vivo secretion of RNase 7, psoriasin, and hBD-2 was detected using ELISA on lesional skin in AD and in even higher concentrations in psoriasis. The secretion of AMPs did not correlate with severity of AD and Staphylococcus aureus colonization. Skin barrier disruption caused enhanced immunoreactivity of hBD-2 and hBD-3 after 24hours. Strong secretion of RNase 7 was already detected after 1hour, whereas hBD-2 secretion was significantly enhanced after 24hours only under occlusion. Thus, a disturbed skin barrier may trigger AMP induction in AD and psoriasis. The functional role of AMP in AD, especially with regard to the control of S. aureus colonization, needs further analysis

Molecular Identification and Expression Analysis of Filaggrin-2, a Member of the S100 Fused-Type Protein Family

Genes of the S100 fused-type protein (SFTP) family are clustered within the epidermal differentiation complex and encode essential components that maintain epithelial homeostasis and barrier functions. Recent genetic studies have shown that mutations within the gene encoding the SFTP filaggrin cause ichthyosis vulgaris and are major predisposing factors for atopic dermatitis. As a vital component of healthy skin, filaggrin is also a precursor of natural moisturizing factors. Here we present the discovery of a member of this family, designated as filaggrin-2 (FLG2) that is expressed in human skin. The FLG2 gene encodes a histidine- and glutamine-rich protein of approximately 248 kDa, which shares common structural features with other SFTP members, in particular filaggrin. We found that FLG2 transcripts are present in skin, thymus, tonsils, stomach, testis and placenta. In cultured primary keratinocytes, FLG2 mRNA expression displayed almost the same kinetics as that of filaggrin following Ca2+ stimulation, suggesting an important role in molecular regulation of epidermal terminal differentiation. We provide evidences that like filaggrin, FLG2 is initially expressed by upper granular cells, proteolytically processed and deposited in the stratum granulosum and stratum corneum (SC) layers of normal epidermis. Thus, FLG2 and filaggrin may have overlapping and perhaps synergistic roles in the formation of the epidermal barrier, protecting the skin from environmental insults and the escape of moisture by offering precursors of natural moisturizing factors

Antimicrobial peptides as defense molecules at the interface of the host and bacteria

Microbes surround us. However, most of the time we are unaware of them. Our epithelial surfaces, which provide the physical barrier and separate us from the environment, are usually free of signs of a battle taking place. Over the past several years, it has been realized that the epithelium is capable of mounting its own battery of defensive chemicals. Besides the constituents of fluids, such as tears, saliva, and sweat that are secreted onto the surface of epithelia, antimicrobial peptides defend this barrier. The aim of this project was to investigate antimicrobial components at different body surfaces like lung, skin and intestine. In the first study the human beta defensin-2 was discovered as the major antimicrobial peptide from lung epithelial cells against Pseudomonas aeruginosa, a bacterium often affecting the lungs of patients suffering from cystic fibrosis. It was demonstrated that mucoid forms of P. aeruginosa as well as the proinflammatory cytokines IL-1beta and TNF-alpha are able to induce the expression human beta defensin-2 in respiratory epithelial cells. In the second study the four human beta defensins hBD1-4 were examined in keratinocytes. The expression of three defensins, namely hBD2, -3 and -4 were inhibited by co-cultivation with retinoic acid. These defensins exhibited upregulation during keratinocyte differentiation and stimulation by proinflammatory cytokines and bacterial contact. Co-incubation with retinoic acid exhibited the induction of hBD2, -3 and -4. Since retinoic acid is used topically in certain skin diseases and leads sometimes to bacterial superinfection, the here described inhibitory effect of retinoic acid on the regulation of defensins could explain this phenomenon. In the third study it was demonstrated that most of the antimicrobial components in the small intestine of mice localize to the mucus layer building an effective shield between the bacteria in the intestinal lumen and the epithelial cells. Mucus preparation exhibited broad antimicrobial activity. Besides alpha defensins, other antimicrobial active factors like lysozyme, phospholipase A2 and ribosomal proteins could be identified. The results of this study imply that the mucus is not only a physical barrier but serves as a structure to keep antimicrobial peptides and proteins in high concentration. Recent studies have suggested an important role of antimicrobial peptides and the mucus in inflammatory bowl diseases. In the fourth study the heparin/heparan sulfate interacting protein (HIP), which is similar to the ribosomal protein L29, was identified as one of the major antimicrobial active substance in lung extracts by HPLC-fractionation, N-terminal sequencing and mass-spectrometry analysis. HIP/RPL29 was also detected in extracts of the small intestine. HIP/RPL29 exhibited broad antimicrobial activity and notably against P. aeruginosa strains. The HIP/RPL29 protein was found to be localized specifically to the epithelial surface of the lungs and intestines by immunohistochemistry. We suggest that HIP/RPL29 fulfils a function as an abundant antimicrobial factor of the epithelial innate defense shield against invading bacteria both in lungs and in the small intestine

Identification of Lympho-Epithelial Kazal-Type Inhibitor 2 in Human Skin as a Kallikrein-Related Peptidase 5- Specific Protease Inhibitor

Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5