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Nga mice, an atopic dermatitis model
ABSTRACTS | Inflammation, Immunity and Infection 418
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Protection against AhR-induced oxidative stress and premature skin aging H Chajra2, M Kim1, M Frechet2 and E Jung1 1 Biospectrum, Seoul, Korea (the Republic of) and 2 Clariant Production France, Toulouse, France Pollution is a major concern in our urbanized and industrialized countries. As a result, the skin is in contact with particulate matter (PM), which are tiny pollution particles associated to polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HM). Deleterious effects of these pollutants on the skin are now well described. Key players induced by pollutants following activation of Aryl hydrocarbon receptor (AhR) are the noxious Reactive Oxygen Species (ROS). We showed in keratinocytes that AhR was activated following application of calibrated HM or PAHs and in the mean time we measured an increase of ROS production. A more close-to-reality model, ex vivo skin explant, was then exposed to a mix of HM and PAHs to check for pollutants damaging effects. After HM/PAHs treatment, we measured an increase of malonaldehyde (MDA), a lipid peroxidation product. This oxidative stress causes dramatic biological consequences occurring during skin aging: a deterioration of dermoepidermal junction (DEJ), an overexpression of MMP-1 protein, and a decrease of collagen type I expression. AhR activation regulates the expression of the NADPH oxidases (NOX) that are major contributors of ROS production. Intracellular ROS activate AP-1 signalling which in turn stimulates inflammatory pathways evidenced here by COX2 and MMP1 expression. Camellia Japonica flower extract exhibits an interesting antioxidant activity measured by DPPH assay (radical scavenging), thus constituting a good candidate for skin protection from harmful pollutants. We showed in keratinocytes, that this extract limits AhR activation and abrogates HM and PAHs dependent ROS induction. In addition, we showed in ex vivo model that this extract limits NOX related inflammatory pathways activation by inhibiting MMP1 and COX2 induction but stimulates collagen type I expression and preserves the global tissue morphology. Moreover, clinical results confirmed this anti-aging effect. In conclusion, we propose Camellia japonica flower extract as a powerful active ingredient fighting against pollutants-induced premature skin aging.
Inhibition of Phosphodiesterase 4 (PDE 4) by East Indian Sandalwood Oil (EISO): A new therapeutic option for inflammatory skin diseases M Sharma Dept. of Urologic Sciences, UBC, Vancouver Prostate Centre, Vancouver, BC, Canada Phosphodiesterase 4 (PDE4) is overactive in patients with chronic relapsing inflammatory skin diseases such as psoriasis (PS) and atopic dermatitis (AD)/eczema. PDE4 regulates pro-inflammatory cytokines/chemokine production by degrading cyclic adenosine monophosphate (cAMP) that activates protein kinase A, which in turn represses NFkB-driven proinflammatory cytokine/chemokine expression. Here we report results from a single center clinical trial of pediatric patients suffering from mild to severe AD/eczema demonstrating that a topical East Indian Sandalwood oil (EISO) formulation is well-tolerated and significantly improved symptoms. EISO has anti-inflammatory and anti-proliferative activities in skin diseases, but the mechanism for these activities has yet to be elucidated. We hypothesized that the ability of EISO to alleviate chronic inflammatory skin conditions might be linked to PDE4 inhibition. To test this possibility, PDE4 expression was evaluated in organotypic psoriatic and normal human skin models. EISO treatment for 2 days reduced expression of PDE4 and reversed psoriasis pathology and cytokines/chemokine (TNF-a, MCP-1, IL-6 and IL-8) production. EISO treatment inhibited NFkB activation and downregulation of total cellular cAMP-PDE activity in lipopolysaccharides (LPS) stimulated human dermal fibroblasts. These effects correlated with decreased PDE4 isoforms A, B and D at messenger RNA level and reduced levels of pro-inflammatory cytokines/chemokines in LPS stimulated dermal fibroblasts treated with EISO. Additionally, EISO inhibited enzymatic activity of the three PDE4 isoforms in vitro. These results suggest that the ability of EISO to alleviate AD symptoms is in part due to suppressed expression and activity of PDE4 isomers that allows for cAMP-regulated inhibition of NFkB. EISO may therefore be an attractive new natural therapeutic for the chronic skin diseases like PS and AD.
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Staphylococcus aureus from atopic dermatitis skin alters cytokine production triggered by monocyte-derived Langerhans cell K Iwamoto1, M Moriwaki1, Y Niitsu1,2, M Saino1, J Hisatsune2, M Sugai2 and M Hide1 1 Department of Dermatology, Graduate school of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan and 2 Department of Bacteriology, Graduate school of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases. The skin of patients with AD presents as a disbalance of the microbiome with a strong colonization by Staphylococcus aureus, which positively correlates with the severity of the disease. In addition, S. aureus isolated from patients with AD was reported to be more likely to produce superantigens. However, the effect of colonized S. aureus on the skin immune system has not been fully elucidated. The aim of this study was to explore whether S. aureus strains isolated from AD skin (S. aureus - AD) were able to skew T cell responses via Langerhans cells (LC) as compared to a standard strain of S. aureus NCTC8325 or S. epidermidis. Monocyte-derived LC (MoLC) were prepared from peripheral blood of healthy controls and patients with AD. MoLC were stimulated with heat-inactivated S. aureus NCTC8325, S. aureus - AD, or S. epidermidis, respectively. After stimulation, CD1a-sorted MoLC were cocultured with autologous CD4+ T cells and then T cell responses were analyzed. Compared to S. aureus NCTC8325 and S. epidermidis, MoLC stimulated by S. aureus - AD induced significantly high and rapid proliferation of T cells with significantly high amounts of IL-2 and less IFN-g production. The mRNA from T cells after co-culture showed imbalanced Th1/Th2 (decreased TBX21/GATA3 ratio) expression. No significant differences were found on T cell responses against S. aureus between healthy controls and patients with AD. These data illustrate S. aureus - AD can skew T cell responses via LC toward imbalanced Th1 / Th2 skin immunity.
Dipotassium glycyrrhizate attenuates the development of atopic dermatitis-like lesions in staphylococcus aureus treated human skin equivalent H Choi, M Bahng, N Park and Y Kim Amorepacific co., ltd, Yongin-si, Korea (the Republic of) Atopic dermatitis is characterized by intense itching and recurrent eczematous lesions. The pathogenesis of atopic dermatitis is not fully understood. The triggers of atopic dermatitis include diet, allergen exposure, stress, and irritants. Environmental factors that could trigger atopic dermatitis include colonization or infection of the skin with microbes, such as Staphylococcus aureus (S.aureus). The microbe infection irritates the skin and results in inflammation. Licorice (Glycyrrhiza glabra) is a traditional soothing herb growing in several regions of the world. It is known that licorice has anti-inflammatory, anti-bacterial, antioxidative, anti-viral and expectorant properties and is effective in the detoxification. Dipotassium glycyrrhizate (GR-K) is one of natural biological active materials isolated from the licorice plant. The aims of this study are to evaluate the anti-inflammatory effect of GR-K in S. aureus treated normal human keratinocytes (NHKs) and to investigate the barrier recovery effect of GR-K in S.aureus treated human skin equivalent (SE). We found that S.aureus treated NHKs secreted pro-inflammatory cytokines IL-1, IL-6 and TNFa. We then evaluated whether the pretreatment of GR-K suppresses pro-inflammatory cytokines. As results, GR-K inhibited secretion of IL-6 and TNFa but not IL-1. We then made atopic dermatitis-like skin equivalent (SE) model to examine skin barrier recovery effect of GR-K. Epidermis of S.aureus treated SE showed hyperplasia which is one of phenotypes of atopic dermatitis. Histological analysis revealed that GR-K pretreatment suppressed a considerable amount of epidermal hyperplasia. Moreover, tight junction permeability assay revealed that impaired tight junctions in S.aureus treated SE was recovered by GR-K pretreatment. Taken together, these results mean GR-K has skin barrier recovery effect that may be due to suppressing the secretion of pro-inflammatory cytokines in epidermal cells.
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Galectin-7 suppresses the erythema and cytokine productions in Nc/Nga mice, an atopic dermatitis model M Nakamura, Y Sawada, E Mashima, R Kubo, T Yamaguchi, S Haruyama, M Yoshioka and E Okada Department of Dermatology, University of Occupational and Environmental Health, Kitakyushu, Japan Atopic dermatitis is a chronic inflammatory skin disease. Urocanic acid (UCA) is an epidermal chromophore that undergoes trans to cis isomerization after UVB irradiation. cisUCA is a potent inhibitor of cutaneous acquired immunity. To search a new therapeutic approach for atopic dermatitis, we first explored the genes which are up-regulated by cis-UCA in normal human epidermal keratinocytes (NHEK) by DNA microarray analysis and investigated its role in vitro and in vivo. DNA microarray experiment revealed that cis-UCA increased the expression of a gene encoding a beta-galactoside-binding lectin, galectin-7, LGALS7B. To elucidate a role of galectin-7 in the control of cytokine production by T lymphocytes, we cultured Jurkat cells with galetin-7. Galectin-7 administration inhibited the expression of interleukin-2 (IL2) and interferon-gamma (IFNG) mRNA by Jurkat cells. Enzyme linked immunosorbent assay revealed that the IL-2 concentration of culture sup of Jurkat cells was down-regulated by galectin-7. FACS analysis revealed the down-modulation of the MHC molecules in bone marrow derived dendritic cells by the addition of galectin-7. Atopic dermatitis-like skin lesion was induced in the dorsal skin of NC/Nga mice by repeated topical application of Dermatophagoides farniae body ointment. Local injections of galectin-7 into erythematous regions in NC/Nga mice ameliorated erythema. Histological examination revealed a decrease in the number of infiltrating lymphocytes into the dermis by galectin-7. Quantitative real-time RT-PCR analysis showed that expression of Il4 mRNA, Il17 mRNA, Ifng mRNA in the skin was suppressed by the injections of galectin-7. Taken together, these data suggested that galectin-7 may play important roles in down-regulation of cutaneous immune system both in vitro and in vivo and can be developed into novel immunosuppressive therapies for atopic dermatitis.
S264 Journal of Investigative Dermatology (2017), Volume 137
STAT 3 and its role in the cutaneous defense H Mu¨ller, M Simanski, F Rademacher, L Schro¨der and J Harder Department of Dermatology and Allergology, University Hospital Schleswig-Holstein, Kiel, Germany Our study is based on the Hyper-IgE syndrome (HIES) which is a primary immunodeficiency due to a mutation in the STAT3-gene (“signal transducer and activator of transcription 3”). Patients suffer from severe skin infections mainly caused by Staphylococcus aureus (SA). It has been shown that the STAT3-mutation decreases the differentiation of T-cells into TH17-cells which are especially important for the activation of the skin’s defense system. This provides an explanation why HIES-patients are highly susceptible to skin infections. Beyond that we questioned if the STAT3-mutation also affects keratinocytes themselves in their innate defense system. Therefore we examined keratinocytes regarding both the gene expression of antimicrobial peptides (AMPs) which are physiologically involved in the killing of SA (hBD-3 and RNase 7) and the gene expression of IL-17C, an important cytokine produced by keratinocytes. Primary keratinocytes were cultured with a STAT3 Inhibitor or with a STAT3 siRNA to mimic the STAT3-dysregulation of HIES-patients in vitro. Subsequently the keratinocytes were stimulated with living SA overnight. Afterwards the RNA was isolated and reverse transcribed into cDNA which served as a target for the “real-time” PCR to quantify the gene expression of AMPs and IL-17C. Our results show a SA-mediated gene induction of hBD-3, RNase 7 and IL17C. This induction was decreased by blocking STAT3 with a STAT3 inhibitor or a STAT3 siRNA which indicates that the SA-induced expression of those genes is mediated by STAT3. Based on our data we assume that IL-17C, hBD-3 and RNase 7 are less induced during the infection with SA in keratinocytes of HIES-patients. A decreased induction of IL-17C may lead to a reduced activation of the skin’s defense system. Since hBD-3 and RNase 7 have the capacity to kill SA, one may hypothesize that the failure to adequately induce these genes leads to increased infections with this pathogen. Our findings could initiate further research in this field and may lead to novel treatment options for HIES-patients by the application of AMPs.
419
Protection against AhR-induced oxidative stress and premature skin aging H Chajra2, M Kim1, M Frechet2 and E Jung1 1 Biospectrum, Seoul, Korea (the Republic of) and 2 Clariant Production France, Toulouse, France Pollution is a major concern in our urbanized and industrialized countries. As a result, the skin is in contact with particulate matter (PM), which are tiny pollution particles associated to polycyclic aromatic hydrocarbons (PAHs) and heavy metals (HM). Deleterious effects of these pollutants on the skin are now well described. Key players induced by pollutants following activation of Aryl hydrocarbon receptor (AhR) are the noxious Reactive Oxygen Species (ROS). We showed in keratinocytes that AhR was activated following application of calibrated HM or PAHs and in the mean time we measured an increase of ROS production. A more close-to-reality model, ex vivo skin explant, was then exposed to a mix of HM and PAHs to check for pollutants damaging effects. After HM/PAHs treatment, we measured an increase of malonaldehyde (MDA), a lipid peroxidation product. This oxidative stress causes dramatic biological consequences occurring during skin aging: a deterioration of dermoepidermal junction (DEJ), an overexpression of MMP-1 protein, and a decrease of collagen type I expression. AhR activation regulates the expression of the NADPH oxidases (NOX) that are major contributors of ROS production. Intracellular ROS activate AP-1 signalling which in turn stimulates inflammatory pathways evidenced here by COX2 and MMP1 expression. Camellia Japonica flower extract exhibits an interesting antioxidant activity measured by DPPH assay (radical scavenging), thus constituting a good candidate for skin protection from harmful pollutants. We showed in keratinocytes, that this extract limits AhR activation and abrogates HM and PAHs dependent ROS induction. In addition, we showed in ex vivo model that this extract limits NOX related inflammatory pathways activation by inhibiting MMP1 and COX2 induction but stimulates collagen type I expression and preserves the global tissue morphology. Moreover, clinical results confirmed this anti-aging effect. In conclusion, we propose Camellia japonica flower extract as a powerful active ingredient fighting against pollutants-induced premature skin aging.
Inhibition of Phosphodiesterase 4 (PDE 4) by East Indian Sandalwood Oil (EISO): A new therapeutic option for inflammatory skin diseases M Sharma Dept. of Urologic Sciences, UBC, Vancouver Prostate Centre, Vancouver, BC, Canada Phosphodiesterase 4 (PDE4) is overactive in patients with chronic relapsing inflammatory skin diseases such as psoriasis (PS) and atopic dermatitis (AD)/eczema. PDE4 regulates pro-inflammatory cytokines/chemokine production by degrading cyclic adenosine monophosphate (cAMP) that activates protein kinase A, which in turn represses NFkB-driven proinflammatory cytokine/chemokine expression. Here we report results from a single center clinical trial of pediatric patients suffering from mild to severe AD/eczema demonstrating that a topical East Indian Sandalwood oil (EISO) formulation is well-tolerated and significantly improved symptoms. EISO has anti-inflammatory and anti-proliferative activities in skin diseases, but the mechanism for these activities has yet to be elucidated. We hypothesized that the ability of EISO to alleviate chronic inflammatory skin conditions might be linked to PDE4 inhibition. To test this possibility, PDE4 expression was evaluated in organotypic psoriatic and normal human skin models. EISO treatment for 2 days reduced expression of PDE4 and reversed psoriasis pathology and cytokines/chemokine (TNF-a, MCP-1, IL-6 and IL-8) production. EISO treatment inhibited NFkB activation and downregulation of total cellular cAMP-PDE activity in lipopolysaccharides (LPS) stimulated human dermal fibroblasts. These effects correlated with decreased PDE4 isoforms A, B and D at messenger RNA level and reduced levels of pro-inflammatory cytokines/chemokines in LPS stimulated dermal fibroblasts treated with EISO. Additionally, EISO inhibited enzymatic activity of the three PDE4 isoforms in vitro. These results suggest that the ability of EISO to alleviate AD symptoms is in part due to suppressed expression and activity of PDE4 isomers that allows for cAMP-regulated inhibition of NFkB. EISO may therefore be an attractive new natural therapeutic for the chronic skin diseases like PS and AD.
420
421
Staphylococcus aureus from atopic dermatitis skin alters cytokine production triggered by monocyte-derived Langerhans cell K Iwamoto1, M Moriwaki1, Y Niitsu1,2, M Saino1, J Hisatsune2, M Sugai2 and M Hide1 1 Department of Dermatology, Graduate school of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan and 2 Department of Bacteriology, Graduate school of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan Atopic dermatitis (AD) is one of the most common chronic inflammatory skin diseases. The skin of patients with AD presents as a disbalance of the microbiome with a strong colonization by Staphylococcus aureus, which positively correlates with the severity of the disease. In addition, S. aureus isolated from patients with AD was reported to be more likely to produce superantigens. However, the effect of colonized S. aureus on the skin immune system has not been fully elucidated. The aim of this study was to explore whether S. aureus strains isolated from AD skin (S. aureus - AD) were able to skew T cell responses via Langerhans cells (LC) as compared to a standard strain of S. aureus NCTC8325 or S. epidermidis. Monocyte-derived LC (MoLC) were prepared from peripheral blood of healthy controls and patients with AD. MoLC were stimulated with heat-inactivated S. aureus NCTC8325, S. aureus - AD, or S. epidermidis, respectively. After stimulation, CD1a-sorted MoLC were cocultured with autologous CD4+ T cells and then T cell responses were analyzed. Compared to S. aureus NCTC8325 and S. epidermidis, MoLC stimulated by S. aureus - AD induced significantly high and rapid proliferation of T cells with significantly high amounts of IL-2 and less IFN-g production. The mRNA from T cells after co-culture showed imbalanced Th1/Th2 (decreased TBX21/GATA3 ratio) expression. No significant differences were found on T cell responses against S. aureus between healthy controls and patients with AD. These data illustrate S. aureus - AD can skew T cell responses via LC toward imbalanced Th1 / Th2 skin immunity.
Dipotassium glycyrrhizate attenuates the development of atopic dermatitis-like lesions in staphylococcus aureus treated human skin equivalent H Choi, M Bahng, N Park and Y Kim Amorepacific co., ltd, Yongin-si, Korea (the Republic of) Atopic dermatitis is characterized by intense itching and recurrent eczematous lesions. The pathogenesis of atopic dermatitis is not fully understood. The triggers of atopic dermatitis include diet, allergen exposure, stress, and irritants. Environmental factors that could trigger atopic dermatitis include colonization or infection of the skin with microbes, such as Staphylococcus aureus (S.aureus). The microbe infection irritates the skin and results in inflammation. Licorice (Glycyrrhiza glabra) is a traditional soothing herb growing in several regions of the world. It is known that licorice has anti-inflammatory, anti-bacterial, antioxidative, anti-viral and expectorant properties and is effective in the detoxification. Dipotassium glycyrrhizate (GR-K) is one of natural biological active materials isolated from the licorice plant. The aims of this study are to evaluate the anti-inflammatory effect of GR-K in S. aureus treated normal human keratinocytes (NHKs) and to investigate the barrier recovery effect of GR-K in S.aureus treated human skin equivalent (SE). We found that S.aureus treated NHKs secreted pro-inflammatory cytokines IL-1, IL-6 and TNFa. We then evaluated whether the pretreatment of GR-K suppresses pro-inflammatory cytokines. As results, GR-K inhibited secretion of IL-6 and TNFa but not IL-1. We then made atopic dermatitis-like skin equivalent (SE) model to examine skin barrier recovery effect of GR-K. Epidermis of S.aureus treated SE showed hyperplasia which is one of phenotypes of atopic dermatitis. Histological analysis revealed that GR-K pretreatment suppressed a considerable amount of epidermal hyperplasia. Moreover, tight junction permeability assay revealed that impaired tight junctions in S.aureus treated SE was recovered by GR-K pretreatment. Taken together, these results mean GR-K has skin barrier recovery effect that may be due to suppressing the secretion of pro-inflammatory cytokines in epidermal cells.
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Galectin-7 suppresses the erythema and cytokine productions in Nc/Nga mice, an atopic dermatitis model M Nakamura, Y Sawada, E Mashima, R Kubo, T Yamaguchi, S Haruyama, M Yoshioka and E Okada Department of Dermatology, University of Occupational and Environmental Health, Kitakyushu, Japan Atopic dermatitis is a chronic inflammatory skin disease. Urocanic acid (UCA) is an epidermal chromophore that undergoes trans to cis isomerization after UVB irradiation. cisUCA is a potent inhibitor of cutaneous acquired immunity. To search a new therapeutic approach for atopic dermatitis, we first explored the genes which are up-regulated by cis-UCA in normal human epidermal keratinocytes (NHEK) by DNA microarray analysis and investigated its role in vitro and in vivo. DNA microarray experiment revealed that cis-UCA increased the expression of a gene encoding a beta-galactoside-binding lectin, galectin-7, LGALS7B. To elucidate a role of galectin-7 in the control of cytokine production by T lymphocytes, we cultured Jurkat cells with galetin-7. Galectin-7 administration inhibited the expression of interleukin-2 (IL2) and interferon-gamma (IFNG) mRNA by Jurkat cells. Enzyme linked immunosorbent assay revealed that the IL-2 concentration of culture sup of Jurkat cells was down-regulated by galectin-7. FACS analysis revealed the down-modulation of the MHC molecules in bone marrow derived dendritic cells by the addition of galectin-7. Atopic dermatitis-like skin lesion was induced in the dorsal skin of NC/Nga mice by repeated topical application of Dermatophagoides farniae body ointment. Local injections of galectin-7 into erythematous regions in NC/Nga mice ameliorated erythema. Histological examination revealed a decrease in the number of infiltrating lymphocytes into the dermis by galectin-7. Quantitative real-time RT-PCR analysis showed that expression of Il4 mRNA, Il17 mRNA, Ifng mRNA in the skin was suppressed by the injections of galectin-7. Taken together, these data suggested that galectin-7 may play important roles in down-regulation of cutaneous immune system both in vitro and in vivo and can be developed into novel immunosuppressive therapies for atopic dermatitis.
S264 Journal of Investigative Dermatology (2017), Volume 137
STAT 3 and its role in the cutaneous defense H Mu¨ller, M Simanski, F Rademacher, L Schro¨der and J Harder Department of Dermatology and Allergology, University Hospital Schleswig-Holstein, Kiel, Germany Our study is based on the Hyper-IgE syndrome (HIES) which is a primary immunodeficiency due to a mutation in the STAT3-gene (“signal transducer and activator of transcription 3”). Patients suffer from severe skin infections mainly caused by Staphylococcus aureus (SA). It has been shown that the STAT3-mutation decreases the differentiation of T-cells into TH17-cells which are especially important for the activation of the skin’s defense system. This provides an explanation why HIES-patients are highly susceptible to skin infections. Beyond that we questioned if the STAT3-mutation also affects keratinocytes themselves in their innate defense system. Therefore we examined keratinocytes regarding both the gene expression of antimicrobial peptides (AMPs) which are physiologically involved in the killing of SA (hBD-3 and RNase 7) and the gene expression of IL-17C, an important cytokine produced by keratinocytes. Primary keratinocytes were cultured with a STAT3 Inhibitor or with a STAT3 siRNA to mimic the STAT3-dysregulation of HIES-patients in vitro. Subsequently the keratinocytes were stimulated with living SA overnight. Afterwards the RNA was isolated and reverse transcribed into cDNA which served as a target for the “real-time” PCR to quantify the gene expression of AMPs and IL-17C. Our results show a SA-mediated gene induction of hBD-3, RNase 7 and IL17C. This induction was decreased by blocking STAT3 with a STAT3 inhibitor or a STAT3 siRNA which indicates that the SA-induced expression of those genes is mediated by STAT3. Based on our data we assume that IL-17C, hBD-3 and RNase 7 are less induced during the infection with SA in keratinocytes of HIES-patients. A decreased induction of IL-17C may lead to a reduced activation of the skin’s defense system. Since hBD-3 and RNase 7 have the capacity to kill SA, one may hypothesize that the failure to adequately induce these genes leads to increased infections with this pathogen. Our findings could initiate further research in this field and may lead to novel treatment options for HIES-patients by the application of AMPs.