Franziska Rademacher

The Pattern Recognition Receptor NOD2 Mediates Staphylococcus aureus-Induced IL-17C Expression in Keratinocytes

IL-17C is an important epithelial cell-derived cytokine activating innate immunity by the induction of antimicrobial peptides and cytokines. Here, we investigated the role of the cytosolic pattern recognition receptor nucleotide-binding oligomerization domain-containing protein 2 (NOD2) for the Staphylococcus aureus-mediated induction of IL-17C. Activation of NOD2 in HEK293 cells overexpressing NOD2 induced the IL-17C promoter, an activity that was significantly reduced in cells overexpressing the Crohns disease-associated NOD2 mutation 3020insC (1007fs) or the Crohns disease- and atopic dermatitis-associated NOD2-R702W variant. The first NF-κB-binding site in the IL-17C promoter was critical for NOD2-mediated IL-17C induction. Infection of human primary keratinocytes with S. aureus induced NOD2 and IL-17C gene expression. Overexpression of NOD2 in keratinocytes augmented S. aureus-mediated IL-17C gene expression as compared with NOD2-R702W overexpression. S. aureus-induced IL-17C expression was diminished in NOD2 small interfering RNA (siRNA)-treated keratinocytes. Moreover, significantly less S. aureus bacteria survived in keratinocytes overexpressing NOD2 but not in cells overexpressing the NOD2-R702W variant. Finally, S. aureus showed an increased survival in keratinocytes treated with NOD2 or IL-17C siRNA. In summary, our study provides evidence that S. aureus activates NOD2 in keratinocytes, resulting in an increased expression of IL-17C, a mechanism that may be dysregulated in atopic dermatitis.

IL-17A and IFN-γ Synergistically Induce RNase 7 Expression via STAT3 in Primary Keratinocytes

Human keratinocytes produce several antimicrobial peptides and proteins (AMP) which contribute to the protection of human skin against infection. RNase 7 is a major AMP involved in cutaneous defense with a high expression in keratinocytes and a broad spectrum of antimicrobial activity. The cytokine IL-17A has been recently identified as a potent inducer of several AMP in keratinocytes. Since the role of IL-17A to induce RNase 7 expression is unknown we analyzed IL-17A alone and in combination with other cytokines to induce RNase 7 expression in keratinocytes. Whereas IL-17A alone only weakly induced RNase 7 expression, the synergistic combination of IL-17A and IFN-γ (IL-17A/IFN-γ) was identified as a potent inducer of RNase 7 expression. This combination was more effective in inducing RNase 7 than the combination of IL-17A/TNF-α, a combination previously identified as a strong inducer of psoriasis-related immune response genes including several AMP. IFN-γ and IL-17A both have been reported to activate the transcription factor STAT3 (Signal transducer and activator of transcription 3). Therefore we investigated the influence of STAT3 on the IL-17A/IFN-γ -mediated RNase 7 induction. The use of a STAT3 inhibitor as well as siRNA-mediated downregulation of STAT3 resulted in a diminished IL-17A/IFN-γ -mediated RNase 7 induction in keratinocytes indicating that STAT3 is involved in this process. Similarly as seen with RNase 7, treatment of keratinocytes with IL-17A/IFN-γ revealed also a synergistic induction of gene expression of the AMP human beta-defensin (hBD)-2 and -3 as well as the S100 protein psoriasin (S100A7) indicating that the combination of IL-17A/IFN-γ is a potent inducer of various AMP classes in general. This was also reflected by an increase of the Staphylococcus aureus-killing activity of IL-17A/IFN-γ -treated keratinocytes.

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.

Infection of keratinocytes with Trichophytum rubrum induces epidermal growth factor-dependent RNase 7 and human beta-defensin-3 expression.

Human keratinocytes are able to express various antimicrobial peptides (AMP) to protect the skin from exaggerated microbial colonization and infection. Recently, in vitro growth-inhibiting activity of the skin-derived AMP psoriasin, RNase 7 and human beta-defensin (hBD)-2 against dermatophytes such as Trichophyton (T.) rubrum have been reported. To evaluate whether keratinocytes are able to respond to T. rubrum infection by an induced expression of AMP we exposed primary keratinocytes to living conidia of T. rubrum. This led to conidia germination and mycelial growth which was paralleled by a strong gene induction of the skin-derived AMP RNase 7 and hBD-3. Gene expression of the AMP psoriasin (S100A7) and hBD-2 were only slightly induced. The T. rubrum-mediated RNase 7 gene induction was accompanied by increased secretion of RNase 7. Parallel treatment of the keratinocytes with T. rubrum and the cytokine combination IL-17A/IFN-γ resulted in synergistic induction of RNase 7 and hBD-3 expression. Since patients receiving therapy by inhibition of the epidermal growth factor receptor (EGFR) more often suffer from dermatophytoses we investigated whether EGFR may be involved in the T. rubrum-mediated RNase 7 and hBD-3 induction. Primary keratinocytes incubated with an EGFR blocking antibody as well as with the EGFR antagonist AG1478 showed a significantly diminished RNase 7 and hBD-3 induction upon exposure of the keratinocytes to T. rubrum indicating that EGFR is involved in the T. rubrum-mediated induction of RNase 7 and hBD-3. The growth of T. rubrum in vitro was inhibited by hBD-3 in a dose-dependent manner suggesting that hBD-3 may contribute to cutaneous innate defense against T. rubrum. Taken together our data indicate that keratinocytes are able to initiate a fast defense response towards T. rubrum by the increased expression of AMP active against T. rubrum. A dysregulation of AMP may contribute to chronic and recurring dermatophytoses.

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.

The Inflammasome and the Epidermal Growth Factor Receptor (EGFR) Are Involved in the Staphylococcus aureus-Mediated Induction of IL-1alpha and IL-1beta in Human Keratinocytes.

Staphylococcus (S.) aureus is an important pathogen causing various infections including those of the skin. Keratinocytes are able to sense invading S. aureus and to initiate a fast defense reaction by the rapid release of innate defense mediators such as antimicrobial peptides and cytokines. There is increasing evidence that the cytokines IL-1alpha and IL-1beta, which both signal through the IL-1 receptor, play an important role in cutaneous defense against S. aureus. The aim of this study was to gain more insight into the underlying mechanisms leading to the S. aureus-induced IL-1alpha and IL-1beta expression in keratinocytes. Infection of human primary keratinocytes with S. aureus led to the induction of gene expression and protein secretion of IL-1alpha and IL-1beta. Full S. aureus-induced IL-1 protein release required the inflammasome components caspase-1 and ASC (apoptosis-associated speck-like protein containing a CARD) whereas gene induction of IL-1alpha and IL-beta by S. aureus was not dependent on caspase-1 and ASC. Since patients receiving anti-cancer therapy by inhibition of the epidermal growth factor receptor (EGFR) often suffer from skin infections caused by S. aureus we additionally evaluated whether the EGFR pathway may be involved in the IL-1alpha and IL-1beta induction by S. aureus. Inactivation of the EGFR with a blocking antibody decreased the S. aureus-mediated IL-1alpha and IL-1beta induction in primary keratinocytes. Moreover, the use of siRNA experiments revealed that ADAM17 (A Disintegrin and A Metalloprotease 17), a metalloproteinase known to mediate the shedding and release of EGFR ligands, was required for full induction of IL-1alpha and IL-1beta in keratinocytes infected with S. aureus. A failure of keratinocytes to adequately upregulate IL-1alpha and IL-1beta may promote S. aureus skin infections.

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.

RNase 7 participates in cutaneous innate control of Corynebacterium amycolatum

Nondiphtheria corynebacteria are typical members of the skin microbiota. However, in addition to being harmless inhabitants of healthy skin commensal skin-derived corynebacteria such as C. amycolatum occasionally also cause infections. This suggests that human skin must harbor adequate mechanisms to control the growth of corynebacteria on the skin surface. Here we show that keratinocytes are able to detect the presence of C. amycolatum leading to the epidermal growth factor receptor (EGFR)-dependent induction of the antimicrobial protein RNase 7. C. amycolatum-mediated induction of RNase 7 was also confirmed in a human 3D skin equivalent. The functional relevance of these findings was demonstrated by potent antimicrobial activity of RNase 7 against C. amycolatum and C. xerosis. In addition, the capacity of human stratum corneum to restrict the growth of C. amycolatum was significantly attenuated when RNase 7 was inactivated by a specific RNase 7-neutralizing antibody. Taken together, the interaction of RNase 7 with C. amycolatum indicates that RNase 7 may function as important effector molecule to control the growth of corynebacteria on human skin.

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 psoriasin in keratinocytes: Implications for the cutaneous barrier

Millions of patients around the world suffer minor or major extremity amputation due to progressive wound healing complications of chronic or infected wounds, the therapy of which remains a challenge. One emerging therapeutic option for the treatment of these complicated wounds is the local application of an autologous thrombocytes concentrate lysate (e.g. platelet-released growth factors ((PRGF)) or Vivostat PRF®) that contains a multitude of chemokines, cytokines and growth factors and is therefore supposed to stimulate the complex wound healing process. Although PRGF and Vivostat PRF® are already used successfully to support healing of chronic, hard-to-heal and infected wounds the underlying molecular mechanisms are not well understood. Psoriasin, also termed S100A7, is a multifunctional antimicrobial protein expressed in keratinocytes and is involved in various processes such as wound-healing, angiogenesis, innate immunity and immune-modulation. In this study, we investigated the influence of PRGF on psoriasin expression in human primary keratinocytes in vitro and the influence of Vivostat PRF® on psoriasin expression in experimentally generated skin wounds in vivo. PRGF treatment of primary keratinocytes caused a significant concentration- and time-dependent increase of psoriasin gene and protein expression in vitro that were partially mediated by the epidermal growth factor receptor (EGFR) and the interleukin-6 receptor (IL-6R). In accordance with these cell culture data, Vivostat PRF® induced a significant psoriasin gene and protein expression when applied to artificially generated skin wounds in vivo. The observed psoriasin induction in keratinocytes may contribute to the wound healing-promoting effects of therapeutically used thrombocyte concentrate lysates.