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622 Skin barrier, innate immunity and staphylococci homeostasis
Innate Immunity, Microbiology, Inflammation | ABSTRACTS 618
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Isolation of antifungal compounds from Ou-gon which is widely supplemented in Kampo medicines X Da and E Morita Department of Dermatology, Shimane University Faculty of Medicine, Shimane, Japan Background: Development of safe antifungal drug is beneficial for clinical use because immunosuppressive drugs including anticancer drugs are more frequently used and fungal infections will be a limitation of their clinical application.Kampo medicine mainly contain crude extracts of natural products such as plants, animals, and minerals. Since plants synthesize numerous antimicrobial components such as plant defensins, Kampo medicines likely contain potent antimicrobial constituents. We have tested antifungal activity of 61 commercially available Kampo medicines by using micro-broth dilution assay with T. rubrum, and found that 7 of them had antifungal activity. Among these 7 Kampo medicines 6 contained Ou-gon which derived from the roots of Scutellaria baicalensis Georgi, and a crude extract of Ou-gon exhibited significant antifungal activity. This study aims to identify antifungal components contained in Ou-gon against dermatophytes, and determine their antifungal mechanism. Method: T. rubrum was used for antifungal activity assay. The antifungal activity assay was performed by measuring 595 nm absorbance in micro-broth dilution assay. Active components of Ou-gon were analyzed by high performance liquid chromatography (HPLC), and identified by UV absorption spectrum and liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS/MS). TUNEL, SYTOX-Green Uptake analyses, and electron microscopy studies were used to clarify the antifungal mechanism of active components. Result: Upon HPLC analysis, two low molecular weight-compounds were isolated having potent antifungal activity. The two compounds were identified as Baicalein and Wogonin by UVabsorption spectrum and LC-ESIMS/MS. Baicalein showed significant antifungal activity for T. rubrum, T. mentagrophytes, A. fumigatus and C. albicans. Wogonin showed antifungal activity for all except C. albicans. Detection of antifungal mechanism of Baicalein and Wogonin suggested that their mode of action is apoptosislike programmed cell death. Conclusion: Baicalein and Wogonin are major compounds to have antifungal activity in Kampo medicine. This study may contribute to the development of new and safe antifungal drugs, especially for the clinical treatment of pathogenic fungal infections.
The C5a-C5aR1 axis suppresses the Aldara-induced immune activation R Shibuya1, A Kitoh1 and K Kabashima2 1 Department of Dermatology, Kyoto Univ., Kyoto, Japan and 2 Department of Dermatology, Kyoto Univ., Kyoto, Japan Aldara, a cream containing the toll-like receptor (TLR) 7 agonist imiquimod, is used for anogenital human papillomavirus infection and non-melanoma skin cancers. Growing evidence suggests that its therapeutic effects depend on the immune activation. In particular, the infiltration of plasmacytoid dendritic cells (pDCs) in the treated skin is considered to be essential for the effects of Aldara. However, the precise mechanism underlying how Aldara induces therapeutic immune activation remains unclear. Here we investigated the possible role of C5a, an active component of complement system, in the Aldara-induced immune activation. C5a receptor 1-deficient (C5ar1-/-) mice exhibited more severe ear swelling with enhanced infiltration of inflammatory cells following topical application of Aldara compared to wild-type (WT) mice. Flow cytometric analysis revealed that Aldara-induced pDC accumulation was significantly enhanced in C5ar1-/- mice, indicating that C5aR1 signaling suppresses pDC infiltration in the treated skin. Among genes encoding pDC-attracting chemokines such as Ccl2, Ccl5, Cxcl10, Cxcl12, and Rarres2, only Ccl2 expression was upregulated in the Aldara-treated skin. Aldara-induced Ccl2 expression was significantly higher in C5ar1-/- mice than WT mice. Given the previous report that pDCs infiltrate to the Aldara-treated skin in a CCL2-dependent manner, enhanced pDC accumulation in the Aldara-treated skin of C5ar1-/- mice is likely due to increased CCL2 expression at least in part. Because TLR7-independent inflammasome activation in keratinocytes has been implicated for the Aldara-induced immune activation, we assessed Il1b expression by keratinocytes after Aldara treatment. The mRNA expression of Il1b in the epidermis of Aldara-treated skin was significantly higher in C5ar1-/- mice than WT mice. Collectively, our results indicate that the C5a-C5aR1 axis suppresses Aldara-induced pDC accumulation and keratinocyte activation in the treated skin.
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Possible involvement of interleukin-33 and basophils in the pathophysiology of human atopic dermatitis via ST2 receptor Y Nonomura1, A Otsuka1, JA Seidel1, C Nakashima1 and K Kabashima2 1 Department of Dermatology, Kyoto University, Kyoto, Japan and 2 Department of Dermatology, Kyoto Univ., Kyoto, Japan Atopic dermatitis (AD) is an inflammatory, relapsing and itchy skin disorder mainly due to Th2 immune responses. We previously reported that basophils are required in Th2 induction in a murine AD model. However, their role in human AD remains largely unknown. To address this issue, we evaluated the expression levels of activation markers on basophils in AD patients; CD63, CD69 and CD203c. But no significant difference was observed between AD patients and healthy controls. Interestingly, the IL-33 receotpr (ST2) expression on basophils was significantly elevated in AD patients. In addition, the serum IL-33 level was significantly higher in AD patients. We also found that the ST2 expression level on basophils was correlated with the serum IgE level, but not with the serum IL-33 level. Consistently, antigenspecific anti-IgE stimulation upregulated the expression levels of ST2 on basophils in vitro. By means of immunohistochemistry, IL-33 expression was observed only in the nucleus of keratinocytes in the normal skin, but it was upregulated even in the cytoplasm of keratinocytes in the AD skin lesion. IL-33 induction in the cytoplasm of keratinocytes was also observed in skin from healthy controls after scratching. Consistently, IL-33 protein production in the supernatant using an organ culture system was increased by the scratched skin. These results indicated that high levels of serum IL-33 in AD patients are derived from the damaged epidermis, such as by scratching. In summary, our findings suggested that IL-33 is induced from the damaged epidermis, such as by scratching, in AD, and that IL-33 acts on basophils via upregulated ST2 for activation, which might contribute to the exacerbation of allergic inflammation of AD patients.
Skin barrier, innate immunity and staphylococci homeostasis S Gauthier1, L Costes1, F Legendre1, S Leoty-okombi2, D Rival2, V Andre-Frei3 and M Chavan4 1 INVIVOGEN, Toulouse, France, 2 BASF BEAUTY CARE SOLUTIONS, Lyon, France, 3 BASF BCS, Lyon, France and 4 BASF CORP, Tarrytown, NY Skin colonization by microorganism is driven by the ecology of the skin surface. This ecology depends on topographical location of microorganisms, endogenous host and environmental factors. Dry skin conditions are correlated with skin and microbiota dysbiosis. The epidermis is the skin physical barrier, limiting penetration by microorganisms and toxins while retaining moisture. Epidermal lipids, such as ceramides, also play a fundamental role in establishing this barrier. To strengthen it, keratinocytes also stimulate the skin innate immunity by expressing Toll Like Receptors (TLRs) and Dectins which recognize pathogens and trigger skin defense cascade in order to invade them. TLRs can detect bacteria (such as S. aureus), viruses and some fungus while Dectins sense mainly fungus. In this study, we trained keratinocytes to recognize microorganisms by stimulating of TLR-2 (x8) and Dectin-1a (x10) with a yeast extract in HEK Blue cell line. After 10 days of reconstructed epidermis treatment, the yeast extract significantly increased the total and main classes of skin lipids synthesis, namely, ceramides, and cholesterol/ cholesterol sulfate ratio. Moreover, the yeast extract speed up skin biopsies barrier recovery after solvent disruption and strengthened ZO-1 tight junctions in differentiated keratinocytes. In clinical trials, the skin barrier function was confirmed to be increased after 14 days (TEWL -6.9% vs placebo). Regarding microbiome, there was no direct change in vitro in the aerobic growth of S. epidermidis and S. aureus in presence of the yeast extract but it significantly decreases IL8 synthesis induced by S. aureus. Clinical trials after 14 days with the yeast extract also demonstrated an increase proportion of S. epidermidis versus S. aureus positively correlated with dry skin condition improvement.
Novel diagnostic approach in detecting skin infection: Identification of bacterial-specific volatile organic compounds in bacterial biofilms on human cutaneous wound models M Ashrafi1, L Novak-Frazer1, M Bates2, M Baguneid3, T Alonso-Rasgado1, R Rautemaa-Richardson1 and A Bayat1 1 University of Manchester, Manchester, United Kingdom, 2 MCBA Consulting, Cardiff, Wales, United Kingdom and 3 University Hospital of South Manchester, Manchester, United Kingdom Microorganisms release volatile organic compounds (VOCs) and the ability to identify these could lead to non-invasive diagnosis. The aims were to assess bacterial biofilm formation and identify VOC profiles in human ex-vivo cutaneous wound models. Staphylococcus aureus (MSSA), Pseudomonas aeruginosa (PA) and Streptococcus pyogenes (SP) biofilms were formed on incisional and excisional cutaneous wound substrates. Biofilm development was determined using histological assessment, XTT assay, fluorescence and scanning electron microscopy. The headspace of 240 cultures and controls were sampled at days 0, 1, 3 and 5 and VOCs separated by gas chromatography and detected by mass spectrometry with relative abundances compared. 3-methylbutanal (P0.033) and pentanal (P < 0.001) were unique to MSSA; octamethyl-cyclotetrasiloxane (P0.018), hydrogen cyanide (P0.042), 5-methyl-1heptanol (P0.046), 6-methyl-1-heptanol (P0.033), 2-nonanone (P0.029) and 2-undecanone (P0.04) to PA; 1,2,4-benzenetricarboxylic acid (P0.036) and ethanol (P0.021) to SP, compared to controls. 2-methyl-1-propanol (P0.043), 1-butanol (P0.017) and 3methyl-1-butanol (P0.026) were unique to two or more bacteria. We present for the first time bacterial biofilm formation on novel human ex-vivo cutaneous wound models and their bacterial-specific VOC profiles. These models have a role in experimental biofilm studies and VOC detection has potential clinical translatability in the non-invasive diagnosis of cutaneous infections.
Cutaneous acquisition of Staphylococcus quorum-sensing agr mutations protects against atopic dermatitis development Y Nakamura1, H Takahashi1, A Takaya1, Y Katayama2, F Yamade1, Y Kusuya1, N Shimojo1, G Nunez3 and H Matsue1 1 Chiba University, Chiba, Japan, 2 Department of Dermatology, Chiba University, Chiba, Japan and 3 University of Michigan Medical School, Ann Arbor, MI Atopic dermatitis (AD) is commonly associated with colonization by Staphylococcus aureus in the affected skin. To understand pathogen-host interactions in the development of AD, we performed whole genome sequencing (WGS) of S. aureus strains isolated from the cheek skin of 268 Japanese infants 1 month (M) and 6 Ms after birth. 6-M-old infants with S. aureus colonization raised the risk of developing AD compared to infants with no colonization (odds ratio 5.433, p < 0.001). Based on WGS analyses, higher ratios of nonsynonymous to synonymous mutations (>1) were detected in S. aureus strains at age 6 M continuously colonized from age 1M, regardless of AD development, indicating the adaptive genomic changes by the positive selection in the infant skin. Surprisingly, accumulation of dysfunctional mutations in the agr quorum-sensing system was observed only in strains from 6-M-old infants who did not develop AD, which correlated with reduced infant skin colonization. Reconstitution of mutant strains with wild-type agr restored the expression of the agr-controlled regulatory RNA, RNAIII. Consistently, the wild-type S. aureus triggered robust inflammatory disease in the skin, whereas agr-deleted S. aureus had an impaired ability to colonize the skin and induced significantly less inflammation in mice. Thus, cutaneous acquisition of loss-offunction mutations in the quorum-sensing agr system of S. aureus in infant skin protects against the development of AD.
www.jidonline.org S107
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Isolation of antifungal compounds from Ou-gon which is widely supplemented in Kampo medicines X Da and E Morita Department of Dermatology, Shimane University Faculty of Medicine, Shimane, Japan Background: Development of safe antifungal drug is beneficial for clinical use because immunosuppressive drugs including anticancer drugs are more frequently used and fungal infections will be a limitation of their clinical application.Kampo medicine mainly contain crude extracts of natural products such as plants, animals, and minerals. Since plants synthesize numerous antimicrobial components such as plant defensins, Kampo medicines likely contain potent antimicrobial constituents. We have tested antifungal activity of 61 commercially available Kampo medicines by using micro-broth dilution assay with T. rubrum, and found that 7 of them had antifungal activity. Among these 7 Kampo medicines 6 contained Ou-gon which derived from the roots of Scutellaria baicalensis Georgi, and a crude extract of Ou-gon exhibited significant antifungal activity. This study aims to identify antifungal components contained in Ou-gon against dermatophytes, and determine their antifungal mechanism. Method: T. rubrum was used for antifungal activity assay. The antifungal activity assay was performed by measuring 595 nm absorbance in micro-broth dilution assay. Active components of Ou-gon were analyzed by high performance liquid chromatography (HPLC), and identified by UV absorption spectrum and liquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS/MS). TUNEL, SYTOX-Green Uptake analyses, and electron microscopy studies were used to clarify the antifungal mechanism of active components. Result: Upon HPLC analysis, two low molecular weight-compounds were isolated having potent antifungal activity. The two compounds were identified as Baicalein and Wogonin by UVabsorption spectrum and LC-ESIMS/MS. Baicalein showed significant antifungal activity for T. rubrum, T. mentagrophytes, A. fumigatus and C. albicans. Wogonin showed antifungal activity for all except C. albicans. Detection of antifungal mechanism of Baicalein and Wogonin suggested that their mode of action is apoptosislike programmed cell death. Conclusion: Baicalein and Wogonin are major compounds to have antifungal activity in Kampo medicine. This study may contribute to the development of new and safe antifungal drugs, especially for the clinical treatment of pathogenic fungal infections.
The C5a-C5aR1 axis suppresses the Aldara-induced immune activation R Shibuya1, A Kitoh1 and K Kabashima2 1 Department of Dermatology, Kyoto Univ., Kyoto, Japan and 2 Department of Dermatology, Kyoto Univ., Kyoto, Japan Aldara, a cream containing the toll-like receptor (TLR) 7 agonist imiquimod, is used for anogenital human papillomavirus infection and non-melanoma skin cancers. Growing evidence suggests that its therapeutic effects depend on the immune activation. In particular, the infiltration of plasmacytoid dendritic cells (pDCs) in the treated skin is considered to be essential for the effects of Aldara. However, the precise mechanism underlying how Aldara induces therapeutic immune activation remains unclear. Here we investigated the possible role of C5a, an active component of complement system, in the Aldara-induced immune activation. C5a receptor 1-deficient (C5ar1-/-) mice exhibited more severe ear swelling with enhanced infiltration of inflammatory cells following topical application of Aldara compared to wild-type (WT) mice. Flow cytometric analysis revealed that Aldara-induced pDC accumulation was significantly enhanced in C5ar1-/- mice, indicating that C5aR1 signaling suppresses pDC infiltration in the treated skin. Among genes encoding pDC-attracting chemokines such as Ccl2, Ccl5, Cxcl10, Cxcl12, and Rarres2, only Ccl2 expression was upregulated in the Aldara-treated skin. Aldara-induced Ccl2 expression was significantly higher in C5ar1-/- mice than WT mice. Given the previous report that pDCs infiltrate to the Aldara-treated skin in a CCL2-dependent manner, enhanced pDC accumulation in the Aldara-treated skin of C5ar1-/- mice is likely due to increased CCL2 expression at least in part. Because TLR7-independent inflammasome activation in keratinocytes has been implicated for the Aldara-induced immune activation, we assessed Il1b expression by keratinocytes after Aldara treatment. The mRNA expression of Il1b in the epidermis of Aldara-treated skin was significantly higher in C5ar1-/- mice than WT mice. Collectively, our results indicate that the C5a-C5aR1 axis suppresses Aldara-induced pDC accumulation and keratinocyte activation in the treated skin.
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Possible involvement of interleukin-33 and basophils in the pathophysiology of human atopic dermatitis via ST2 receptor Y Nonomura1, A Otsuka1, JA Seidel1, C Nakashima1 and K Kabashima2 1 Department of Dermatology, Kyoto University, Kyoto, Japan and 2 Department of Dermatology, Kyoto Univ., Kyoto, Japan Atopic dermatitis (AD) is an inflammatory, relapsing and itchy skin disorder mainly due to Th2 immune responses. We previously reported that basophils are required in Th2 induction in a murine AD model. However, their role in human AD remains largely unknown. To address this issue, we evaluated the expression levels of activation markers on basophils in AD patients; CD63, CD69 and CD203c. But no significant difference was observed between AD patients and healthy controls. Interestingly, the IL-33 receotpr (ST2) expression on basophils was significantly elevated in AD patients. In addition, the serum IL-33 level was significantly higher in AD patients. We also found that the ST2 expression level on basophils was correlated with the serum IgE level, but not with the serum IL-33 level. Consistently, antigenspecific anti-IgE stimulation upregulated the expression levels of ST2 on basophils in vitro. By means of immunohistochemistry, IL-33 expression was observed only in the nucleus of keratinocytes in the normal skin, but it was upregulated even in the cytoplasm of keratinocytes in the AD skin lesion. IL-33 induction in the cytoplasm of keratinocytes was also observed in skin from healthy controls after scratching. Consistently, IL-33 protein production in the supernatant using an organ culture system was increased by the scratched skin. These results indicated that high levels of serum IL-33 in AD patients are derived from the damaged epidermis, such as by scratching. In summary, our findings suggested that IL-33 is induced from the damaged epidermis, such as by scratching, in AD, and that IL-33 acts on basophils via upregulated ST2 for activation, which might contribute to the exacerbation of allergic inflammation of AD patients.
Skin barrier, innate immunity and staphylococci homeostasis S Gauthier1, L Costes1, F Legendre1, S Leoty-okombi2, D Rival2, V Andre-Frei3 and M Chavan4 1 INVIVOGEN, Toulouse, France, 2 BASF BEAUTY CARE SOLUTIONS, Lyon, France, 3 BASF BCS, Lyon, France and 4 BASF CORP, Tarrytown, NY Skin colonization by microorganism is driven by the ecology of the skin surface. This ecology depends on topographical location of microorganisms, endogenous host and environmental factors. Dry skin conditions are correlated with skin and microbiota dysbiosis. The epidermis is the skin physical barrier, limiting penetration by microorganisms and toxins while retaining moisture. Epidermal lipids, such as ceramides, also play a fundamental role in establishing this barrier. To strengthen it, keratinocytes also stimulate the skin innate immunity by expressing Toll Like Receptors (TLRs) and Dectins which recognize pathogens and trigger skin defense cascade in order to invade them. TLRs can detect bacteria (such as S. aureus), viruses and some fungus while Dectins sense mainly fungus. In this study, we trained keratinocytes to recognize microorganisms by stimulating of TLR-2 (x8) and Dectin-1a (x10) with a yeast extract in HEK Blue cell line. After 10 days of reconstructed epidermis treatment, the yeast extract significantly increased the total and main classes of skin lipids synthesis, namely, ceramides, and cholesterol/ cholesterol sulfate ratio. Moreover, the yeast extract speed up skin biopsies barrier recovery after solvent disruption and strengthened ZO-1 tight junctions in differentiated keratinocytes. In clinical trials, the skin barrier function was confirmed to be increased after 14 days (TEWL -6.9% vs placebo). Regarding microbiome, there was no direct change in vitro in the aerobic growth of S. epidermidis and S. aureus in presence of the yeast extract but it significantly decreases IL8 synthesis induced by S. aureus. Clinical trials after 14 days with the yeast extract also demonstrated an increase proportion of S. epidermidis versus S. aureus positively correlated with dry skin condition improvement.
Novel diagnostic approach in detecting skin infection: Identification of bacterial-specific volatile organic compounds in bacterial biofilms on human cutaneous wound models M Ashrafi1, L Novak-Frazer1, M Bates2, M Baguneid3, T Alonso-Rasgado1, R Rautemaa-Richardson1 and A Bayat1 1 University of Manchester, Manchester, United Kingdom, 2 MCBA Consulting, Cardiff, Wales, United Kingdom and 3 University Hospital of South Manchester, Manchester, United Kingdom Microorganisms release volatile organic compounds (VOCs) and the ability to identify these could lead to non-invasive diagnosis. The aims were to assess bacterial biofilm formation and identify VOC profiles in human ex-vivo cutaneous wound models. Staphylococcus aureus (MSSA), Pseudomonas aeruginosa (PA) and Streptococcus pyogenes (SP) biofilms were formed on incisional and excisional cutaneous wound substrates. Biofilm development was determined using histological assessment, XTT assay, fluorescence and scanning electron microscopy. The headspace of 240 cultures and controls were sampled at days 0, 1, 3 and 5 and VOCs separated by gas chromatography and detected by mass spectrometry with relative abundances compared. 3-methylbutanal (P0.033) and pentanal (P < 0.001) were unique to MSSA; octamethyl-cyclotetrasiloxane (P0.018), hydrogen cyanide (P0.042), 5-methyl-1heptanol (P0.046), 6-methyl-1-heptanol (P0.033), 2-nonanone (P0.029) and 2-undecanone (P0.04) to PA; 1,2,4-benzenetricarboxylic acid (P0.036) and ethanol (P0.021) to SP, compared to controls. 2-methyl-1-propanol (P0.043), 1-butanol (P0.017) and 3methyl-1-butanol (P0.026) were unique to two or more bacteria. We present for the first time bacterial biofilm formation on novel human ex-vivo cutaneous wound models and their bacterial-specific VOC profiles. These models have a role in experimental biofilm studies and VOC detection has potential clinical translatability in the non-invasive diagnosis of cutaneous infections.
Cutaneous acquisition of Staphylococcus quorum-sensing agr mutations protects against atopic dermatitis development Y Nakamura1, H Takahashi1, A Takaya1, Y Katayama2, F Yamade1, Y Kusuya1, N Shimojo1, G Nunez3 and H Matsue1 1 Chiba University, Chiba, Japan, 2 Department of Dermatology, Chiba University, Chiba, Japan and 3 University of Michigan Medical School, Ann Arbor, MI Atopic dermatitis (AD) is commonly associated with colonization by Staphylococcus aureus in the affected skin. To understand pathogen-host interactions in the development of AD, we performed whole genome sequencing (WGS) of S. aureus strains isolated from the cheek skin of 268 Japanese infants 1 month (M) and 6 Ms after birth. 6-M-old infants with S. aureus colonization raised the risk of developing AD compared to infants with no colonization (odds ratio 5.433, p < 0.001). Based on WGS analyses, higher ratios of nonsynonymous to synonymous mutations (>1) were detected in S. aureus strains at age 6 M continuously colonized from age 1M, regardless of AD development, indicating the adaptive genomic changes by the positive selection in the infant skin. Surprisingly, accumulation of dysfunctional mutations in the agr quorum-sensing system was observed only in strains from 6-M-old infants who did not develop AD, which correlated with reduced infant skin colonization. Reconstitution of mutant strains with wild-type agr restored the expression of the agr-controlled regulatory RNA, RNAIII. Consistently, the wild-type S. aureus triggered robust inflammatory disease in the skin, whereas agr-deleted S. aureus had an impaired ability to colonize the skin and induced significantly less inflammation in mice. Thus, cutaneous acquisition of loss-offunction mutations in the quorum-sensing agr system of S. aureus in infant skin protects against the development of AD.
www.jidonline.org S107