Inhibition of RNase 7 by RNase inhibitor promotes inflammation and Staphylococcus aureus growth: Implications for atopic dermatitis.

BACKGROUND: The antimicrobial ribonuclease RNase 7 is abundantly expressed in the epidermis of lesional skin of atopic dermatitis (AD). Host RNase inhibitor (RI) binds to RNase 7 and blocks its ribonuclease activity. This study aimed to evaluate the impact of RNase 7-RI interactions on AD. METHODS: Cultured human primary keratinocytes, with siRNA-mediated downregulation of RNase 7 and RI, were stimulated with the synthetic RNA polyinosinic-polycytidylic acid (poly I:C). Induction of proinflammatory mediators was analyzed by real-time PCR and ELISA. RI expression in AD non-lesional and lesional skin biopsies and healthy controls was analyzed by real-time PCR and immunostaining. RI protein release in vivo on the AD skin surface was determined by western blot. Antimicrobial and ribonuclease assays were used to investigate the functional role of RI. RESULTS: RNase 7 inhibited the RNA-induced expression of proinflammatory mediators in keratinocytes. Accordingly, downregulation of RNase 7 in keratinocytes enhanced RNA-mediated induction of proinflammatory mediators, whereas downregulation of RI had the opposite effect. RI was released by damaged keratinocytes and epidermis. In vivo expression and release of RI on the skin surface were enhanced in lesional AD skin. Rinsing solution from the surface of lesional AD skin blocked the ribonuclease activity of RNase 7. The anti-Staphylococcus aureus activity of RNase 7 was abrogated by RI. CONCLUSIONS: Our data suggest a novel role of RI as a trigger factor of inflammation in AD by blocking the ribonuclease and antimicrobial activity of RNase 7, thereby enhancing RNA-mediated inflammation and S. aureus growth.

Antimicrobial Peptides (AMPs) and the Microbiome in Preterm Infants: Consequences and Opportunities for Future Therapeutics.

Antimicrobial peptides (AMPs) are crucial components of the innate immune system in various organisms, including humans. Beyond their direct antimicrobial effects, AMPs play essential roles in various physiological processes. They induce angiogenesis, promote wound healing, modulate immune responses, and serve as chemoattractants for immune cells. AMPs regulate the microbiome and combat microbial infections on the skin, lungs, and gastrointestinal tract. Produced in response to microbial signals, AMPs help maintain a balanced microbial community and provide a first line of defense against infection. In preterm infants, alterations in microbiome composition have been linked to various health outcomes, including sepsis, necrotizing enterocolitis, atopic dermatitis, and respiratory infections. Dysbiosis, or an imbalance in the microbiome, can alter AMP profiles and potentially lead to inflammation-mediated diseases such as chronic lung disease and obesity. In the following review, we summarize what is known about the vital role of AMPs as multifunctional peptides in protecting newborn infants against infections and modulating the microbiome and immune response. Understanding their roles in preterm infants and high-risk populations offers the potential for innovative approaches to disease prevention and treatment.

The influence of the commensal skin bacterium Staphylococcus epidermidis on the epidermal barrier and inflammation: Implications for atopic dermatitis.

The skin microbiota is a crucial component in maintaining cutaneous barrier function. Staphylococcus epidermidis is considered as a beneficial commensal member of the cutaneous microbiota promoting skin health. However, S. epidermidis is also frequently detectable in the skin of patients with the inflammatory skin disease atopic dermatitis (AD) and some studies reported a significantly higher presence of S. epidermidis in severe AD as compared to mild AD. Therefore, this study aimed to analyse the impact of S. epidermidis on the expression of cutaneous inflammatory mediators and skin barrier molecules. Various S. epidermidis skin-derived isolates activated the proinflammatory transcription factor NF-kappaB and induced expression of AD-associated proinflammatory cytokines in human primary keratinocytes and 3D skin equivalents. Skin barrier molecules such as filaggrin were downregulated by S. epidermidis. In general, AD-derived S. epidermidis strains elicited a higher response than strains derived from the skin of healthy individuals. Taken together, our results provide further evidence that the abundance of S. epidermidis in AD may trigger the inflammatory scenario associated with this disease.

Platelet-Released Growth Factors Influence Wound Healing-Associated Genes in Human Keratinocytes and Ex Vivo Skin Explants.

Platelet-released growth factors (PRGFs) or other thrombocyte concentrate products, e.g., Platelet-Rich Fibrin (PRF), have become efficient tools of regenerative medicine in many medical disciplines. In the context of wound healing, it has been demonstrated that treatment of chronic or complicated wounds with PRGF or PRF improves wound healing in the majority of treated patients. Nevertheless, the underlying cellular and molecular mechanism are still poorly understood. Therefore, we aimed to analyze if PRGF-treatment of human keratinocytes caused the induction of genes encoding paracrine factors associated with successful wound healing. The investigated genes were Semaphorin 7A (SEMA7A), Angiopoietin-like 4 (ANGPLT4), Fibroblast Growth Factor-2 (FGF-2), Interleukin-32 (IL-32), the CC-chemokine-ligand 20 (CCL20), the matrix-metalloproteinase-2 (MMP-2), the chemokine C-X-C motif chemokine ligand 10 (CXCL10) and the subunit B of the Platelet-Derived Growth Factor (PDGFB). We observed a significant gene induction of SEMA7A, ANGPLT4, FGF-2, IL-32, MMP-2 and PDGFB in human keratinocytes after PRGF treatment. The CCL20- and CXCL10 gene expressions were significantly inhibited by PRGF therapy. Signal transduction analyses revealed that the PRGF-mediated gene induction of SEMA7A, ANGPLT4, IL-32 and MMP-2 in human keratinocytes was transduced via the IL-6 receptor pathway. In contrast, EGF receptor signaling was not involved in the PRGF-mediated gene expression of analyzed genes in human keratinocytes. Additionally, treatment of ex vivo skin explants with PRGF confirmed a significant gene induction of SEMA7A, ANGPLT4, MMP-2 and PDGFB. Taken together, these results describe a new mechanism that could be responsible for the beneficial wound healing properties of PRGF or related thrombocytes concentrate products such as PRF.

Antimicrobial peptides and proteins: Interaction with the skin microbiota.

The cutaneous microbiota comprises all living skin microorganisms. There is increasing evidence that the microbiota plays a crucial role in skin homeostasis. Accordingly, a dysbiosis of the microbiota may trigger cutaneous inflammation. The need for a balanced microbiota requires specific regulatory mechanisms that control and shape the microbiota. In this review, we highlight the present knowledge suggesting that antimicrobial peptides (AMPs) may exert a substantial influence on the microbiota by controlling their growth. This is supported by own data showing the differential influence of principal skin-derived AMPs on commensal staphylococci. Vice versa, we also illuminate how the cutaneous microbiota interacts with skin-derived AMPs by modulating AMP expression and how microbiota members protect themselves from the antimicrobial activity of AMPs. Taken together, the current picture suggests that a fine-tuned and well-balanced AMP-microbiota interplay on the skin surface may be crucial for skin health.

Free human DNA attenuates the activity of antimicrobial peptides in atopic dermatitis.

BACKGROUND: The high susceptibility of AD patients to microbial skin infections has been attributed to a deficient antimicrobial peptide (AMP) expression, which is contradicted by a growing amount of recent studies clearly demonstrating that AMP expression is not impaired in lesional skin of AD patients. The reasons for the high susceptibility of AD patients to microbial infections are still unknown. METHODS: The influence of self-DNA on the antimicrobial activity of RNase 7, LL-37, and hBD2 has been investigated using antibacterial and antiviral assays. The amount of self-DNA on skin has been analyzed by skin rinsings and subsequent quantification using dsDNA assays. DNA source was identified by qPCR. RESULTS: Complex formation of the AMPs with self-DNA significantly impaired their antibacterial activity against Staphylococcus aureus and their antiviral activity against HSV-1. The inhibition of the antibacterial activity was dependent on the DNA concentration but not on the length of the DNA molecules. Of note, we detected significant higher amounts of cell-free self-DNA in skin rinses taken from lesional AD skin compared to skin rinses from non-lesional skin and from normal skin of healthy donors. Consequently, rinse solution from AD lesional skin prevented antibacterial activity of LL-37. CONCLUSION: Our study indicates that extracellular self-DNA is released in considerable amounts in AD skin lesions and AMP-self-DNA-complex formation leads to a significant loss of antibacterial and antiviral activity in atopic dermatitis. Studies on strategies to reduce the amount of extracellular DNA in AD are needed to identify possible methods relevant in clinical settings.

Expression of epidermal antimicrobial peptides is increased in tinea pedis.

BACKGROUND: Tinea pedis is often chronic or recurrent, but not all individuals are equally susceptible to this infection. Dermatophytes are able to induce the expression of antimicrobial peptides and proteins (AMPs) in human keratinocytes and certain AMPs can inhibit the growth of dermatophytes. OBJECTIVE: The focus of this study was to analyse the secretion of relevant AMPs, especially RNase 7, human beta-defensin-2 (hBD-2) and the S-100 protein psoriasin (S100A7), in patients with confirmed tinea pedis. METHODS: To verify the diagnosis, skin scales were obtained from all patients (n = 13) and the dermatophytes were identified by potassium hydroxide mount, culture and molecular analysis. To determine the AMP concentrations, the lesional skin area of the foot was rinsed with a buffer that was subsequently analysed by ELISA. The corresponding area of the other unaffected foot as well as defined healthy skin areas of the forearm and forehead and samples from age and gender-matched healthy volunteers served as controls. RESULTS: In tinea pedis patients the AMP concentrations were higher in lesional skin than in non-lesional skin and in healthy skin of controls. In particular, concentrations of hBD-2 and psoriasin were significantly elevated. CONCLUSIONS: The induction of AMPs in tinea pedis might be triggered directly by the dermatophytes; furthermore, attendant inflammation and/or differentiation processes may play a role. Our results indicate that there is no defect in the constitutive expression and induction of the analysed AMPs by dermatophytes in the epidermis of affected patients. However, it is not known why the elevated AMP concentrations fail to efficiently combat dermatophyte growth.

Platelet-Released Growth Factors Induce Genes Involved in Extracellular Matrix Formation in Human Fibroblasts.

Platelet concentrate products are increasingly used in many medical disciplines due to their regenerative properties. As they contain a variety of chemokines, cytokines, and growth factors, they are used to support the healing of chronic or complicated wounds. To date, underlying cellular mechanisms have been insufficiently investigated. Therefore, we analyzed the influence of Platelet-Released Growth Factors (PRGF) on human dermal fibroblasts. Whole transcriptome sequencing and gene ontology (GO) enrichment analysis of PRGF-treated fibroblasts revealed an induction of several genes involved in the formation of the extracellular matrix (ECM). Real-time PCR analyses of PRGF-treated fibroblasts and skin explants confirmed the induction of ECM-related genes, in particular transforming growth factor beta-induced protein (TGFBI), fibronectin 1 (FN1), matrix metalloproteinase-9 (MMP-9), transglutaminase 2 (TGM2), fermitin family member 1 (FERMT1), collagen type I alpha 1 (COL1A1), a disintegrin and metalloproteinase 19 (ADAM19), serpin family E member 1 (SERPINE1) and lysyl oxidase-like 3 (LOXL3). The induction of these genes was time-dependent and in part influenced by the epidermal growth factor receptor (EGFR). Moreover, PRGF induced migration and proliferation of the fibroblasts. Taken together, the observed effects of PRGF on human fibroblasts may contribute to the underlying mechanisms that support the beneficial wound-healing effects of thrombocyte concentrate products.

Skin microbiota analysis in human 3D skin models-"Free your mice".

In the May issue of Experimental Dermatology 2018, we published a review article focusing on human 3D skin models in the context of microbiota research. The principal intention was to provide an overview of present and future concepts to use skin models in microbiota analyses. With the present viewpoint, we would like to draw the reader's attention again to the use of human skin models in microbiota research with the aim to highlight the benefits and necessity of human skin models to analyse the human skin-microbiota interaction. This is accompanied by a critical view on mice models that often are not suitable to analyse the functional impact of the human skin microbiota. In addition, we present novel and future concepts highlighting the benefits of human 3D skin models in microbiota research.

Dysregulated Expression of Antimicrobial Peptides in Skin Lesions of Patients with Cutaneous T-cell Lymphoma.

Mycosis fungoides and Sézary syndrome belong to the group of primary cutaneous T-cell lymphomas. Because of the inflammatory appearance of the skin lesions, we hypothesized that antimicrobial peptides might be dysregulated in these conditions, similar to in inflammatory skin conditions. Samples from 20 patients with cutaneous T-cell lymphoma were analysed using immunohistochemistry and enzyme-linked immunoassay (ELISA) of skin washing fluids of hBD-2, hBD-3, RNase 7 and psoriasin. Immunohistochemistry results were compared with previous analyses of healthy and psoriatic skin. ELISA and immunohistochemistry revealed a higher expression of psoriasin in lesional cutaneous T-cell lymphoma compared with non-lesional and healthy samples. Immunohistochemistry showed an increase in hBD-2 in lesional cutaneous T-cell lymphoma skin compared with healthy skin. The expression profile of antimicrobial peptides in cutaneous T-cell lymphoma appears to be dysregulated, indicating a potential role of antimicrobial peptides in cutaneous T-cell lymphoma. A larger prospective study and functional studies are needed to improve our understanding of the role of antimicrobial peptides in cutaneous T-cell lymphoma.

Platelet-Released Growth Factors and Platelet-Rich Fibrin Induce Expression of Factors Involved in Extracellular Matrix Organization in Human Keratinocytes

Platelet-released growth factor (PRGF) is a thrombocyte concentrate lysate which, like its clinically equivalent variations (e.g., Vivostat PRF® (platelet-rich fibrin)), is known to support the healing of chronic and hard-to-heal wounds. However, studies on the effect of PRGF on keratinocytes remain scarce. This study aims to identify genes in keratinocytes that are significantly influenced by PRGF. Therefore, we performed a whole transcriptome and gene ontology (GO) enrichment analysis of PRGF-stimulated human primary keratinocytes. This revealed an increased expression of genes involved in extracellular matrix (ECM) organization. Real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) analysis confirmed the PRGF-mediated induction of selected ECM-related factors such as transforming growth factor beta-induced protein, fibronectin 1, matrix metalloproteinase-9, transglutaminase 2, fermitin family member 1, collagen type I alpha 1 and collagen type XXII alpha 1. PRGF-induced expression of the above factors was influenced by blockade of the epidermal growth factor receptor (EGFR), a receptor playing a crucial role in wound healing. A differential induction of the investigated factors was also detected in skin explants exposed to PRGF and in experimentally generated in vivo wounds treated with Vivostat PRF®. Together, our study indicates that the induction of ECM-related factors may contribute to the beneficial wound-healing effects of PRGF-based formulations.

Staphylococcus epidermidis-induced Interleukin-1 Beta and Human Beta-defensin-2 Expression in Human Keratinocytes is Regulated by the Host Molecule A20 (TNFAIP3).

Staphylococcus epidermidis is an abundant skin commensal capable of activating cutaneous defense responses, such as induction of cytokines and antimicrobial peptides. To permanently colonize human skin and prevent inflammation S. epidermidis needs to control the induction of host defense mediators. We report here that S. epidermidis induces expression of the host regulator protein A20 in human keratinocytes, thereby controlling expression and release of interleukin-1 beta. siRNA-mediated knockdown of A20 expression strongly enhanced the induction of interleukin-1 beta gene expression and protein release in keratinocytes stimulated with S. epidermidis. Furthermore, siRNA-mediated knockdown of A20 resulted in enhanced gene expression and secretion of the antimicrobial peptide human beta-defensin-2 in keratinocytes facing S. epidermidis. Mechanistically, A20 negatively controlled S. epidermidis-induced activation of the transcription factor NF-kappaB. Together, these data indicate that S. epidermidis exploits A20 to attenuate cutaneous defense responses, which may help S. epidermidis to persist on human skin.

Skin microbiota and human 3D skin models.

Although the role of the microbiota in skin homeostasis is still emerging, there is growing evidence that an intact microbiota supports the skin barrier. The increasing number of research efforts that are trying to shed more light on the human skin-microbiota interaction requires the use of suitable experimental models. Three-dimensional (3D) skin equivalents have been established as a valuable tool in dermatological research because they contain a fully differentiated epidermal barrier that reflects the morphological and molecular characteristics of normal human epidermis. In this review, we provide an overview of current 3D skin models and illustrate the potential of 3D skin models to study the human skin-microbiota interplay.

The role of RNase 7 in innate cutaneous defense against Pseudomonas aeruginosa.

The ribonuclease RNase 7 is a major skin-derived human antimicrobial protein expressed in keratinocytes. Here we show that the gram-negative pathogen Pseudomonas aeruginosa secretes factor(s) that induced RNase 7 gene and protein expression in human primary keratinocytes. The metalloprotease inhibitor marimastat, the epidermal growth factor receptor (EGFR) inhibitor AG1478 and the EGFR blocking antibody cetuximab significantly attenuated this induction, indicating an important role of the EGFR for the P. aeruginosa-mediated RNase 7 induction. In line with this, siRNA-mediated downregulation of ADAM17, a metalloprotease known to proteolytically mediate the release of soluble EGFR ligands, decreased the P. aeruginosa-mediated RNase 7 induction in keratinocytes. The impact of the EGFR was also demonstrated in a human 3D skin equivalent where blockade of the EGFR diminished induction of RNase 7 by P. aeruginosa. Blockade of Toll-like receptor 5 (TLR5), a pattern recognition receptor (PRR) known to be activated by P. aeruginosa, only moderately reduced the P. aeruginosa-mediated RNase 7 induction in keratinocytes. The functional relevance of RNase 7 to participate in cutaneous defense against P. aeruginosa was demonstrated by antibodies that neutralized the antimicrobial activity of RNase 7. These antibodies significantly inhibited the capacity of human stratum corneum skin extracts to control growth of P. aeruginosa. Taken together, our results indicate that P. aeruginosa induces the expression of RNase 7 in keratinocytes in an EGFR-dependent manner. Enhanced release of RNase 7 contributes to control cutaneous growth of P. aeruginosa.

Basal cells contribute to innate immunity of the airway epithelium through production of the antimicrobial protein RNase 7.

Basal cells play a critical role in the response of the airway epithelium to injury and are recently recognized to also contribute to epithelial immunity. Antimicrobial proteins and peptides are essential effector molecules in this airway epithelial innate immunity. However, little is known about the specific role of basal cells in antimicrobial protein and peptide production and about the regulation of the ubiquitous antimicrobial protein RNase 7. In this study, we report that basal cells are the principal cell type producing RNase 7 in cultured primary bronchial epithelial cells (PBEC). Exposure of submerged cultured PBEC (primarily consisting of basal cells) to the respiratory pathogen nontypeable Haemophilus influenzae resulted in a marked increase in expression of RNase 7, although this was not observed in differentiated air-liquid interface cultured PBEC. However, transient epithelial injury in air-liquid interface-cultured PBEC induced by cigarette smoke exposure led to epidermal growth factor receptor-mediated expression of RNase 7 in remaining basal cells. The selective induction of RNase 7 in basal cells by cigarette smoke was demonstrated using confocal microscopy and by examining isolated luminal and basal cell fractions. Taken together, these findings demonstrate a phenotype-specific innate immune activity of airway epithelial basal cells, which serves as a second line of airway epithelial defense that is induced by airway epithelial injury.

The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes.

Platelet-released growth factors (PRGF) and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF®)) contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3) is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR). In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds.

Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes.

Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF®) came recently into the physicians' focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10) and late (transglutaminase-1 and involucrin) differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR-) dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo.

Platelet-released growth factors induce the antimicrobial peptide human beta-defensin-2 in primary keratinocytes.

Platelet-released growth factors (PRGF) and its related clinically used formulations [e.g. Vivostat platelet-rich fibrin (PRF(®) )] are thrombocyte concentrate lysates that support healing of chronic, hard-to-heal and infected wounds. Human beta-defensin-2 (hBD-2) is an antimicrobial peptide expressed in human keratinocytes exhibiting potent antimicrobial activity against wound-related bacteria. In this study, we analysed the influence of PRGF on hBD-2 expression in human primary keratinocytes and the influence of Vivostat PRF(®) on hBD-2 expression in experimentally generated skin wounds in vivo. Treatment of primary keratinocytes with PRGF caused a significant increase in hBD-2 gene and protein expressions in a concentration- and time-dependent manner. The use of blocking antibodies revealed that the PRGF-mediated hBD-2 induction was partially mediated by the epidermal growth factor receptor and the interleukin-6 receptor (IL-6R). Luciferase gene reporter assays indicated that the hBD-2 induction through PRGF required activation of the transcription factor activator protein 1 (AP-1), but not of NF-kappaB. In concordance with these cell culture data, Vivostat PRF(®) induced hBD-2 expression when applied to experimentally generated skin wounds. Together, our results indicate that the induction of hBD-2 by thrombocyte concentrate lysates can contribute to the observed beneficial effects in the treatment of chronic and infected wounds.

RNase 7 in Cutaneous Defense.

RNase 7 belongs to the RNase A superfamily and exhibits a broad spectrum of antimicrobial activity against various microorganisms. RNase 7 is expressed in human skin, and expression in keratinocytes can be induced by cytokines and microbes. These properties suggest that RNase 7 participates in innate cutaneous defense. In this review, we provide an overview about the role of RNase 7 in cutaneous defense with focus on the molecular mechanism of the antimicrobial activity of RNase 7, the regulation of RNase 7 expression, and the role of RNase 7 in skin diseases.

Human skin engages different epidermal layers to provide distinct innate defense mechanisms.

Keratinocytes are able to sense bacteria or bacterial products leading to a rapid defense reaction by the increased expression of antimicrobial peptides and cytokines. Recent data by Percoco and collaborators published in Experimental Dermatology indicate that bacteria colonizing the skin surface induce a differential spatial expression pattern of antimicrobial peptides and cytokines. Using laser capture microdissection followed by real-time PCR as well as immunohistochemistry and transmission electron microscopy, the authors provide evidence that antimicrobial peptides such as human beta-defensin (hBD)-2 and -3 were more strongly induced in the uppermost epidermal layers, whereas the main induction of cytokines such as IL-1beta and IL-6 occurred in the lower parts of the epidermis.