Constitutive expression of the antimicrobial peptide RNase 7 is associated with Staphylococcus aureus infection of the skin.

BACKGROUND: Staphylococcus aureus infections of the skin are a public health problem of growing importance. Antimicrobial peptides in human skin are believed to play an important role in innate defense against intruding pathogens. This study aimed to clarify whether their baseline expression influences the propensity of healthy individuals to develop S. aureus-positive skin infections. METHODS: Using real-time polymerase chain reaction technique and a prospective case-control design, we determined the expression of messenger RNA coding for human beta-defensin 2 and 3 as well as RNase 7 in unaffected skin of 20 travelers returning with Staphylococcus aureus-positive skin infection (case patients) relative to levels in 40 matched control subjects. RESULTS: Expression of RNase 7 was found to be 64% higher in unaffected skin of control subjects, compared with unaffected skin of case patients (95% confidence interval, 17%-131%; P = .007). This association remained stable after controlling for S. aureus nasal carriage, smoking, level of accommodation, and history of allergy. No such association was present for human beta-defensin 2 or 3. CONCLUSIONS: In conjunction with the existing evidence from in vitro studies, these findings suggest that antimicrobial peptides found at high baseline levels in healthy skin, such as RNase 7, confer protection against S. aureus infection of the skin.

Generation of multiple stable dermcidin-derived antimicrobial peptides in sweat of different body sites.

Antimicrobial peptides (AMPs) are effector molecules of innate immunity. Dermcidin (DCD), a recently discovered AMP with broad-spectrum activity, is produced constitutively by the eccrine sweat glands and secreted into sweat. In this study, we investigated the proteolytic processing, site-specific expression, and stability of DCD peptides in eccrine sweat. Using surface-enhanced laser desorption ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and reversed-phase high-pressure liquid chromatography analysis, we identified in eccrine sweat 14 proteolytically processed DCD peptides. Semiquantitative SELDI-TOF-MS analysis indicated that processing of DCD-1L is individually different, but generates a few dominant peptides. At body sites with a high probability for contact with pathogenic microorganisms, a high amount of antimicrobial active DCD peptides was detected in sweat. Furthermore, we show that the secretion rate of DCD is constant during a period of prolonged sweating and that DCD peptides are stable in sweat over several hours. Other known AMPs like the human cathelicidin LL-37 and alpha- or beta-defensins were not detected in significant quantity in eccrine sweat. Owing to the durable and abundant presence, DCD-derived peptides contribute to the first line of defense by building a constant barrier that overlies the epithelial skin.

Skin commensals amplify the innate immune response to pathogens by activation of distinct signaling pathways.

Little is known about the impact of different microbial signals on skin barrier organ function and the interdependency between resident microflora and pathogenic microorganisms. This study shows that commensal and pathogenic staphylococci differ in their ability to induce expression of antimicrobial peptides/proteins (AMPs) and activate different signaling pathways in human primary keratinocytes. Whereas secreted factors of skin commensals induce expression of the AMPs HBD-3 and RNase7 in primary human keratinocytes via Toll-like receptor (TLR)-2, EGFR, and NF-κB activation, those of pathogenic staphylococci activate the mitogen-activated protein kinase and phosphatidylinositol 3-kinase/AKT signaling pathways and suppress NF-κB activation. Interestingly, commensal bacteria are able to amplify the innate immune response of human keratinocytes to pathogens by increased induction of AMP expression and abrogation of NF-κB suppression, suggesting that the two activation pathways can act in a synergistic way. These data indicate that commensal and pathogenic microorganisms evolved specific mechanisms to modulate innate immunity of the skin.

The role of antimicrobial peptides in human skin and in skin infectious diseases.

Antimicrobial peptides or proteins (AMPs) represent an ancient and efficient innate defense mechanism which protects interfaces from infection with pathogenic microorganisms. In human skin AMPs are produced mainly by keratinocytes, neutrophils, sebocytes or sweat glands and are either expressed constitutively or after an inflammatory stimulus. In several human skin diseases there is an inverse correlation between severity of the disease and the level of AMP production. Skin lesions of patients with atopic dermatitis show a diminished expression of the beta-defensins and the cathelicidin LL-37. Furthermore, these patients have a reduced amount of the AMP dermcidin in their sweat which correlates with an impaired innate defense of human skin in vivo. In addition, decreased levels of AMPs are associated with burns and chronic wounds. In contrast, overexpression of AMPs can lead to increased protection against skin infections as seen in patients with psoriasis and rosacea, inflammatory skin-diseases which rarely result in superinfection. In other skin diseases, e.g. in patients with acne vulgaris, increased levels of AMPs are often found in inflamed or infected skin areas indicating a role of these peptides in the protection from infection. These data indicate that AMPs have a therapeutical potential as topical anti-infectives in several skin diseases. The broad spectrum of antimicrobial activity, the low incidence of bacterial resistance and their function as immunomodulatory agents are attractive features of AMPs for their clinical use.

Cathepsin D is present in human eccrine sweat and involved in the postsecretory processing of the antimicrobial peptide DCD-1L.

The protein pattern of healthy human eccrine sweat was investigated and 10 major proteins were detected from which apolipoprotein D, lipophilin B, and cathepsin D (CatD) were identified for the first time in human eccrine sweat. We focused our studies on the function of the aspartate protease CatD in sweat. In vitro digestion experiments using a specific fluorescent CatD substrate showed that CatD is enzymatically active in human sweat. To identify potential substrates of CatD in human eccrine sweat LL-37 and DCD-1L, two antimicrobial peptides present in sweat, were digested in vitro with purified CatD. LL-37 was not significantly digested by CatD, whereas DCD-1L was cleaved between Leu(44) and Asp(45) and between Leu(29) and Glu(30) almost completely. The DCD-1L-derived peptides generated in vitro by CatD were also found in vivo in human sweat as determined by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. Furthermore, besides the CatD-processed peptides we identified additionally DCD-1L-derived peptides that are generated upon cleavage with a 1,10-phenanthroline-sensitive carboxypeptidase and an endoprotease. Taken together, proteolytic processing generates 12 DCD-1L-derived peptides. To elucidate the functional significance of postsecretory processing the antimicrobial activity of three CatD-processed DCD-1L peptides was tested. Whereas two of these peptides showed no activity against Gram-positive and Gram-negative bacteria, one DCD-1L-derived peptide showed an even higher activity against Escherichia coli than DCD-1L. Functional analysis indicated that proteolytic processing of DCD-1L by CatD in human sweat modulates the innate immune defense of human skin.

Deficiency of dermcidin-derived antimicrobial peptides in sweat of patients with atopic dermatitis correlates with an impaired innate defense of human skin in vivo.

Antimicrobial peptides are an integral part of the epithelial innate defense system. Dermcidin (DCD) is a recently discovered antimicrobial peptide with a broad spectrum of activity. It is constitutively expressed in human eccrine sweat glands and secreted into sweat. Patients with atopic dermatitis (AD) have recurrent bacterial or viral skin infections and pronounced colonization with Staphylococcus aureus. We hypothesized that patients with AD have a reduced amount of DCD peptides in sweat contributing to the compromised constitutive innate skin defense. Therefore, we performed semiquantitative and quantitative analyses of DCD peptides in sweat of AD patients and healthy subjects using surface-enhanced laser desorption ionization time-of-flight mass spectrometry and ELISA. The data indicate that the amount of several DCD-derived peptides in sweat of patients with AD is significantly reduced. Furthermore, compared with atopic patients without previous infectious complications, AD patients with a history of bacterial and viral skin infections were found to have significantly less DCD-1 and DCD-1L in their sweat. To analyze whether the reduced amount of DCD in sweat of AD patients correlates with a decreased innate defense, we determined the antimicrobial activity of sweat in vivo. We showed that in healthy subjects, sweating leads to a reduction of viable bacteria on the skin surface, but this does not occur in patients with AD. These data indicate that reduced expression of DCD in sweat of patients with AD may contribute to the high susceptibility of these patients to skin infections and altered skin colonization.