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 Crohn's disease-associated NOD2 mutation 3020insC (1007fs) or the Crohn's 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.
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.
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.
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 human ribonuclease RNase 7 has been originally isolated from human skin and is a member of the human RNase A superfamily. RNase 7 is constantly released by keratinocytes and accumulates on the skin surface. The expression of RNase 7 in keratinocytes can be induced by diverse stimuli such as cytokines, growth factors, and microbial factors. RNase 7 exhibits a potent broad spectrum of antimicrobial activity against various microorganisms and contributes to control bacterial growth on the skin surface. The ribonuclease and antimicrobial activity of RNase 7 can be blocked by the endogenous ribonuclease inhibitor. There is also increasing evidence that RNase 7 exerts immunomodulatory activities and may participate in antiviral defense. In this review, we discuss how these characteristics of RNase 7 contribute to innate cutaneous defense and highlight its role in skin infection and inflammation. We also speculate how a potential dysregulation of RNase 7 promotes inflammatory skin diseases and if RNase 7 may have therapeutic potential.
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