- Email: [email protected]
135 Holographic microscopy as a new tool for imaging the effect of linear and cyclic siloxanes (silicones) after dermal application
Epidermal Structure and Function | ABSTRACTS 131
132
Epidermal autophagy and nucleophagy O Akinduro1, CA Harwood1, R O’Shaughnessy2 and D Bergamaschi1 1 Centre for Cell Biology and Cutaneous Research, Blizard Institute, Barts and The London SMD, QMUL, London, United Kingdom and 2 Livingstone Skin Research Centre for Children, Institute of Child Health, UCL, London, United Kingdom Epidermal keratinocytes migrate through the epidermis to the granular layer where, upon terminal differentiation they progressively lose organelles and convert into anucleate cells or corneocytes. Autophagy is a conserved lysosomal degradation pathway which eliminates protein aggregates and damaged organelles in order to maintain cytoplasmic homeostasis. In keratinocytes autophagy has been described as a mechanism of senescent cell death, a stress response leading to induction of differentiation, and a pro-survival mechanism protecting from UV-induced damage. However, the role of autophagy in epidermis is poorly understood. We provide a comprehensive profile of autophagy marker expression in developing epidermis. We reinforce the importance of mTORC1 in the regulation of autophagy which is constitutively active in epidermal granular layer where, by electron microscopy, we identified double membrane-autophagosomes. Moreover we demonstrate that differentiating keratinocytes undergo a selective form of nucleophagy characterized by accumulation of LC3/ LAMP2/p62 positive autolysosomes. By immunofluorescence we provide evidence that these perinuclear vesicles display positivity for histone interacting protein HP1a and localize in proximity with lamin A and B1 accumulation. We also confirmed these finding in epidermal tissues, including newborn mice and adult human skin, where we report LC3 puncta coincident with misshapen nuclei within the granular layer. This process of nuclear degradation relies on autophagy integrity as confirmed by lack of nucleophagy upon depletion of WIPI1 or ULK1. Finally, we report deregulated expression or location of most of the autophagic markers and lack of LC3 in parakeratotic psoriatic skin, suggesting that impaired autophagy contributes to the pathogenesis of psoriasis. Our findings provide the first evidence for the existence of epidermal nucleophagy and may ultimately improve treatment options for patients with epidermal barrier defects.
Identification of novel pathways linked to the pathogenesis of Recessive X-Linked Ichthyosis F McGeoghan1, M Menon1, P Dewan1, M Caley1, M Donaldson2 and EA O’Toole1 1 Centre for Cell Biology and Cutaneous Research, Barts and the London School of Medicine and Dentistry, London, United Kingdom and 2 Dermatology TA, GSK, London, United Kingdom Recessive X-Linked Ichthyosis (RXLI) results from a deficiency of steroid sulfatase (STS) and is characterized by an accumulation of cholesterol sulfate in the upper layers of the epidermis, resulting in a scaling phenotype and barrier dysfunction. To further understand the pathomechanisms of RXLI, we have generated an RNA-Seq data set of the transcriptome of keratinocytes with siRNA-induced loss of STS. To validate these data, we generated a 3D XLRI model using 2 telomerase-immortalized keratinocyte cell lines with stable knockdown of STS using different lentiviral shRNA clones. Knockdown of STS was confirmed by western blotting (P < 0.001). We found the 3D model representative of RXLI in patients with acanthosis of the epidermis on Haematoxylin and Eosin staining and altered Nile Red staining. We also saw an increase in expression of the late differentiation marker, involucrin, in this model, which has been observed in patient samples. Transglutaminase 1 expression and activity were also decreased. RNA-Seq analysis showed significant dysregulation of genes involved in differentiation (ACER1) and lipid metabolism (CYP4F22, UGCG). Other altered genes correlated with extracutaneous features of RXLI, such as ALDH1A1 (corneal opacity) and OXTR (prolonged labour, ADHD). We validated our RNA-Seq data, using our model of RXLI and patient samples, showing significant decreases of these genes via Western blotting and immunostaining. We also profiled the lipid content of our RXLI model providing novel data on lipid changes in RXLI compared to control. In summary, we have a 3D organotypic model which simulates the expected phenotype in vitro and this was used to further explore the molecular pathogenesis of RXLI, providing novel pathways which can be linked to the clinical features of the disease and may provide therapeutic targets.
133
134
Understanding the role of ABCA12 in the pathogenesis of Harlequin Ichthyosis F Enjalbert1, P Dewan1, M Caley1, B Fell1, M Donaldson2, DP Kelsell1 and EA O’Toole1 1 Centre for Cell Biology and Cutaneous Research, Barts and the London School of Medicine and Dentistry, London, United Kingdom and 2 Dermatology TA, GSK, Uxbridge, United Kingdom ATP-binding cassette transporter A12 (ABCA12), a lipid transporter, is known to be critical for skin barrier integrity. Mutations in this gene cause the most severe form of Autosomal Recessive Congenital Ichthyosis (ARCI): Harlequin Ichthyosis (HI). HI patients have marked hyperkeratosis at birth with fissuring, leading to life-threatening complications due to increased risk of infection, trans-epidermal water and heat loss. To understand the pathomechanisms of HI, we used siRNA knockdown of ABCA12 in primary keratinocytes with subsequent calcium-induced differentiation to model HI as well as a HPV- immortalised HI patient-derived cell line. RNA-sequencing was performed on the siABCA12 primary keratinocytes and siC controls. 136 genes were significantly down-regulated and 87 genes were significantly up-regulated (FDR < 0.01). Functional annotation clustering analysis performed using DAVID showed changes in the following biological processes: inflammatory response, endomembrane system, cytoplasmic bound vesicle/secretary granule, cell cycle, apoptosis and epithelial development. Early expression of differentiation markers was observed in the 3D organotypic model recapitulating the HI epidermis phenotype. Further experiments were performed to validate changes seen in nitric oxide pathway signaling genes. We found that Arginase-1, a regulator of nitric oxide synthase activity, was down regulated in the HI organotypic epidermis. Also, the master regulator of apoptosis and inflammation, STAT1 and STAT1-Tyr701, were strongly upregulated in the HI patient cell line compared to control. STAT1 activation is required for the production of nitric oxide from L-arginine. These data suggest that nitric oxide signaling may be dysregulated in HI skin.
In vitro biological activities of a cream designed for sensitive skin treatment D Pinto, A Benedusi, F Rinaldi and B Marzani Giuliani S.p.A., Milano, Italy Sensitive skin syndrome is a condition of subjective and self-reported cutaneous hyperreactivity to environmental stimuli. This condition is characterized by a decline in epidermal barrier function linked to an imbalance in stratum corneum intracellular lipids, while signs such as flushing, blushing erythema are due to sub-chronic inflammation, vascular reactivity and a hypersensitive reaction. TRPV1 was identified to contribute to this skin condition, facilitating neurogenic inflammation leading to skin sensitivity resulting in pain or itch with a burning sensation. TRPV1 up-regulation due to TNF-alpha stimulation highlights the correlation between inflammation process and hypersensitivity skin reactions. Due to multifactorial pathogenesis of this skin disorders, a protective cream should be formulated to include different actives able to improve the variety of aspects of sensitive skin. Lichtena MedÒ (Giuliani, Milan, Italy) contains moisturizers (betaine, inositol, trehalose, glycerin), emollients (allantoin and Butyrospermum parkii butter), antioxidant (tocotrienols, Rosmarinus officinalis, Olea europea extract, Calendula officinalis extracts, bisabolol, beta-sitosterol) and anti-inflammatory compounds (sorbityl furfural palmitate, glicyrrhetinic acid). To evaluate Lichtena MedÒ preclinical activity, we performed an in vitro test with a topical application of cream (2 mg/cm2) on PhenionÒ Full-Thickness Skin Model treated with SDS as inflammatory stimulus. Inflammation-related genes (TNF-alpha; IL-1beta; IL-8; PTGE2) and TRPV1 gene were evaluated by qRT-PCR, while the DCFH-DA antioxidant assay were used for the epidermal and dermal evaluations. The results show that Lichtena MedÒ has a statistically significant antiinflammatory activity by a down-regulation of TNF-alpha, IL-1beta, IL-8 and PTGE2 genes associated with a significant antioxidant activity in counteracting ROS production during inflammation process both in epidermidis and derma layers. This in vitro study suggest the usefulness of Lichtena MedÒ cream in the treatment of signs associated with sensitive skin syndrome.
135
136
Holographic microscopy as a new tool for imaging the effect of linear and cyclic siloxanes (silicones) after dermal application D Krenczkowska1, K Szymkowska1, K Mojsiewicz- Pienkowska1 and E Stachowska2 1 Department of Physical Chemistry, Medical University of Gdansk, Gdansk, Poland and 2 Department of Metrology and Measurement System, Poznan University of Technology, Poznan, Poland Siloxanes, due to beneficial properties, are common ingredients in cosmetic and pharmaceutical formulations. Particularly volatile methyl siloxanes are usually used as excipients. They should give desired properties of formulation, but not destructively interact with structures of the skin. Generally, siloxanes are regarded as biologically inert compounds. However, after analyzing chemical structure and physicochemical properties (e.g. MW< 500 Da, logP< 5) it can be considered they can penetrate stratum corneum(SC). The aim of the study was to assess the possibility of low molecular weight siloxanes: linear octamethyltrisiloxane L3 and cyclic decamethylocyclopentasiloxane D5 to penetrate SC and observation of possible interaction with SC structures. In studies of human skin digital transmission holographic microscope was used for the first time. Based on comparison differences between images of test (siloxane application) and control samples (water application) were observed. In control samples the structure of SC is uniform. Whereas, images of test samples presented that in the SC structure there are dark areas. It indicates a disorder of SC. Siloxanes due to lipophilic properties penetrate by transepidermal transport through the lipid matrix causing lipid extraction. This provides to collapse of corneocytes and formation of specific cavity, visible as dark area. 3D images and quantitative data from measurements provide information about the magnitude of this phenomenon. In control samples regularly corneocytes with average size of width 20 mm, and thickness of about 1 mm were observed. However, in test samples it was not possible to observe clearly distinguishable structures, possible to measure. It has been found that the use of siloxanes influence on the human skin barrier loss. Holographic microscopy is useful method in the evaluation of SC alteration after low molecular siloxanes application.
MiR-10a controls the proliferation and inflammatory responses of human primary keratinocytes. MiR-10a controls the proliferation and inflammatory responses of human primary keratinocytes T Runnel1, H Hermann1, E Urgard1, K Kingo1, L Sahmatova1, CA Akdis2, T Maimets1 and A Rebane1 1 University of Tartu, Tartu, Estonia and 2 University of Zu¨rich, Zu¨rich, Switzerland MiR-10 family genes are located in the Hox gene clusters and have been shown to be involved in the regulation of several processes, such as cell proliferation, migration and inflammatory response modulation. Abnormal miR-10a/b expression has been linked to increased invasiveness of various cancers. In immune system, miR-10a helps to control the stability and plasticity of helper T cells. The aim of the study was to investigate how miR-10a affects keratinocytes (KCs) and regulates their immune responses. The methods used include miRNA profiling of KCs from atopic dermatitis (AD) patients, overexpression of miR-10a to study changes in KC gene expression and cell cycle, KC differentiation, and luciferase assays to verify direct targets of miR-10a. Our results show miR-10a to be upregulated in cultured human primary KCs as well as skin biopsies from AD patients compared to healthy individuals. In human KCs, miR-10a expression was higher in proliferating conditions and decreased in differentiation-inducing growth conditions, namely during in vitro reconstruction of the skin and high calcium environment. Overexpression of miR-10a in KCs downregulated the previously characterized miR-10a direct target MAP3K7 (TAK1) from the NF-kB signalling pathway and hyaluronan synthase 3 (HAS3), a putative novel miR-10a direct target, which is known to be dysregulated in AD. Subsequent luciferase assays confirmed HAS3 as a novel direct target of miR-10a. In addition, miR-10a overexpression down-regulated many genes linked to cell cycle regulation, epithelial development or genes in the NF-kB pathway. Accordingly, cell cycle analysis of miR-10a-transfected KCs revealed less cells to be in the Sphase compared to controls. We conclude that miR-10a downregulates the proliferation of KCs through targeting multiple factors involved in the regulation of the cell cycle and has a regulatory role for immune responses in both healthy and diseased skin.
www.jidonline.org S183
132
Epidermal autophagy and nucleophagy O Akinduro1, CA Harwood1, R O’Shaughnessy2 and D Bergamaschi1 1 Centre for Cell Biology and Cutaneous Research, Blizard Institute, Barts and The London SMD, QMUL, London, United Kingdom and 2 Livingstone Skin Research Centre for Children, Institute of Child Health, UCL, London, United Kingdom Epidermal keratinocytes migrate through the epidermis to the granular layer where, upon terminal differentiation they progressively lose organelles and convert into anucleate cells or corneocytes. Autophagy is a conserved lysosomal degradation pathway which eliminates protein aggregates and damaged organelles in order to maintain cytoplasmic homeostasis. In keratinocytes autophagy has been described as a mechanism of senescent cell death, a stress response leading to induction of differentiation, and a pro-survival mechanism protecting from UV-induced damage. However, the role of autophagy in epidermis is poorly understood. We provide a comprehensive profile of autophagy marker expression in developing epidermis. We reinforce the importance of mTORC1 in the regulation of autophagy which is constitutively active in epidermal granular layer where, by electron microscopy, we identified double membrane-autophagosomes. Moreover we demonstrate that differentiating keratinocytes undergo a selective form of nucleophagy characterized by accumulation of LC3/ LAMP2/p62 positive autolysosomes. By immunofluorescence we provide evidence that these perinuclear vesicles display positivity for histone interacting protein HP1a and localize in proximity with lamin A and B1 accumulation. We also confirmed these finding in epidermal tissues, including newborn mice and adult human skin, where we report LC3 puncta coincident with misshapen nuclei within the granular layer. This process of nuclear degradation relies on autophagy integrity as confirmed by lack of nucleophagy upon depletion of WIPI1 or ULK1. Finally, we report deregulated expression or location of most of the autophagic markers and lack of LC3 in parakeratotic psoriatic skin, suggesting that impaired autophagy contributes to the pathogenesis of psoriasis. Our findings provide the first evidence for the existence of epidermal nucleophagy and may ultimately improve treatment options for patients with epidermal barrier defects.
Identification of novel pathways linked to the pathogenesis of Recessive X-Linked Ichthyosis F McGeoghan1, M Menon1, P Dewan1, M Caley1, M Donaldson2 and EA O’Toole1 1 Centre for Cell Biology and Cutaneous Research, Barts and the London School of Medicine and Dentistry, London, United Kingdom and 2 Dermatology TA, GSK, London, United Kingdom Recessive X-Linked Ichthyosis (RXLI) results from a deficiency of steroid sulfatase (STS) and is characterized by an accumulation of cholesterol sulfate in the upper layers of the epidermis, resulting in a scaling phenotype and barrier dysfunction. To further understand the pathomechanisms of RXLI, we have generated an RNA-Seq data set of the transcriptome of keratinocytes with siRNA-induced loss of STS. To validate these data, we generated a 3D XLRI model using 2 telomerase-immortalized keratinocyte cell lines with stable knockdown of STS using different lentiviral shRNA clones. Knockdown of STS was confirmed by western blotting (P < 0.001). We found the 3D model representative of RXLI in patients with acanthosis of the epidermis on Haematoxylin and Eosin staining and altered Nile Red staining. We also saw an increase in expression of the late differentiation marker, involucrin, in this model, which has been observed in patient samples. Transglutaminase 1 expression and activity were also decreased. RNA-Seq analysis showed significant dysregulation of genes involved in differentiation (ACER1) and lipid metabolism (CYP4F22, UGCG). Other altered genes correlated with extracutaneous features of RXLI, such as ALDH1A1 (corneal opacity) and OXTR (prolonged labour, ADHD). We validated our RNA-Seq data, using our model of RXLI and patient samples, showing significant decreases of these genes via Western blotting and immunostaining. We also profiled the lipid content of our RXLI model providing novel data on lipid changes in RXLI compared to control. In summary, we have a 3D organotypic model which simulates the expected phenotype in vitro and this was used to further explore the molecular pathogenesis of RXLI, providing novel pathways which can be linked to the clinical features of the disease and may provide therapeutic targets.
133
134
Understanding the role of ABCA12 in the pathogenesis of Harlequin Ichthyosis F Enjalbert1, P Dewan1, M Caley1, B Fell1, M Donaldson2, DP Kelsell1 and EA O’Toole1 1 Centre for Cell Biology and Cutaneous Research, Barts and the London School of Medicine and Dentistry, London, United Kingdom and 2 Dermatology TA, GSK, Uxbridge, United Kingdom ATP-binding cassette transporter A12 (ABCA12), a lipid transporter, is known to be critical for skin barrier integrity. Mutations in this gene cause the most severe form of Autosomal Recessive Congenital Ichthyosis (ARCI): Harlequin Ichthyosis (HI). HI patients have marked hyperkeratosis at birth with fissuring, leading to life-threatening complications due to increased risk of infection, trans-epidermal water and heat loss. To understand the pathomechanisms of HI, we used siRNA knockdown of ABCA12 in primary keratinocytes with subsequent calcium-induced differentiation to model HI as well as a HPV- immortalised HI patient-derived cell line. RNA-sequencing was performed on the siABCA12 primary keratinocytes and siC controls. 136 genes were significantly down-regulated and 87 genes were significantly up-regulated (FDR < 0.01). Functional annotation clustering analysis performed using DAVID showed changes in the following biological processes: inflammatory response, endomembrane system, cytoplasmic bound vesicle/secretary granule, cell cycle, apoptosis and epithelial development. Early expression of differentiation markers was observed in the 3D organotypic model recapitulating the HI epidermis phenotype. Further experiments were performed to validate changes seen in nitric oxide pathway signaling genes. We found that Arginase-1, a regulator of nitric oxide synthase activity, was down regulated in the HI organotypic epidermis. Also, the master regulator of apoptosis and inflammation, STAT1 and STAT1-Tyr701, were strongly upregulated in the HI patient cell line compared to control. STAT1 activation is required for the production of nitric oxide from L-arginine. These data suggest that nitric oxide signaling may be dysregulated in HI skin.
In vitro biological activities of a cream designed for sensitive skin treatment D Pinto, A Benedusi, F Rinaldi and B Marzani Giuliani S.p.A., Milano, Italy Sensitive skin syndrome is a condition of subjective and self-reported cutaneous hyperreactivity to environmental stimuli. This condition is characterized by a decline in epidermal barrier function linked to an imbalance in stratum corneum intracellular lipids, while signs such as flushing, blushing erythema are due to sub-chronic inflammation, vascular reactivity and a hypersensitive reaction. TRPV1 was identified to contribute to this skin condition, facilitating neurogenic inflammation leading to skin sensitivity resulting in pain or itch with a burning sensation. TRPV1 up-regulation due to TNF-alpha stimulation highlights the correlation between inflammation process and hypersensitivity skin reactions. Due to multifactorial pathogenesis of this skin disorders, a protective cream should be formulated to include different actives able to improve the variety of aspects of sensitive skin. Lichtena MedÒ (Giuliani, Milan, Italy) contains moisturizers (betaine, inositol, trehalose, glycerin), emollients (allantoin and Butyrospermum parkii butter), antioxidant (tocotrienols, Rosmarinus officinalis, Olea europea extract, Calendula officinalis extracts, bisabolol, beta-sitosterol) and anti-inflammatory compounds (sorbityl furfural palmitate, glicyrrhetinic acid). To evaluate Lichtena MedÒ preclinical activity, we performed an in vitro test with a topical application of cream (2 mg/cm2) on PhenionÒ Full-Thickness Skin Model treated with SDS as inflammatory stimulus. Inflammation-related genes (TNF-alpha; IL-1beta; IL-8; PTGE2) and TRPV1 gene were evaluated by qRT-PCR, while the DCFH-DA antioxidant assay were used for the epidermal and dermal evaluations. The results show that Lichtena MedÒ has a statistically significant antiinflammatory activity by a down-regulation of TNF-alpha, IL-1beta, IL-8 and PTGE2 genes associated with a significant antioxidant activity in counteracting ROS production during inflammation process both in epidermidis and derma layers. This in vitro study suggest the usefulness of Lichtena MedÒ cream in the treatment of signs associated with sensitive skin syndrome.
135
136
Holographic microscopy as a new tool for imaging the effect of linear and cyclic siloxanes (silicones) after dermal application D Krenczkowska1, K Szymkowska1, K Mojsiewicz- Pienkowska1 and E Stachowska2 1 Department of Physical Chemistry, Medical University of Gdansk, Gdansk, Poland and 2 Department of Metrology and Measurement System, Poznan University of Technology, Poznan, Poland Siloxanes, due to beneficial properties, are common ingredients in cosmetic and pharmaceutical formulations. Particularly volatile methyl siloxanes are usually used as excipients. They should give desired properties of formulation, but not destructively interact with structures of the skin. Generally, siloxanes are regarded as biologically inert compounds. However, after analyzing chemical structure and physicochemical properties (e.g. MW< 500 Da, logP< 5) it can be considered they can penetrate stratum corneum(SC). The aim of the study was to assess the possibility of low molecular weight siloxanes: linear octamethyltrisiloxane L3 and cyclic decamethylocyclopentasiloxane D5 to penetrate SC and observation of possible interaction with SC structures. In studies of human skin digital transmission holographic microscope was used for the first time. Based on comparison differences between images of test (siloxane application) and control samples (water application) were observed. In control samples the structure of SC is uniform. Whereas, images of test samples presented that in the SC structure there are dark areas. It indicates a disorder of SC. Siloxanes due to lipophilic properties penetrate by transepidermal transport through the lipid matrix causing lipid extraction. This provides to collapse of corneocytes and formation of specific cavity, visible as dark area. 3D images and quantitative data from measurements provide information about the magnitude of this phenomenon. In control samples regularly corneocytes with average size of width 20 mm, and thickness of about 1 mm were observed. However, in test samples it was not possible to observe clearly distinguishable structures, possible to measure. It has been found that the use of siloxanes influence on the human skin barrier loss. Holographic microscopy is useful method in the evaluation of SC alteration after low molecular siloxanes application.
MiR-10a controls the proliferation and inflammatory responses of human primary keratinocytes. MiR-10a controls the proliferation and inflammatory responses of human primary keratinocytes T Runnel1, H Hermann1, E Urgard1, K Kingo1, L Sahmatova1, CA Akdis2, T Maimets1 and A Rebane1 1 University of Tartu, Tartu, Estonia and 2 University of Zu¨rich, Zu¨rich, Switzerland MiR-10 family genes are located in the Hox gene clusters and have been shown to be involved in the regulation of several processes, such as cell proliferation, migration and inflammatory response modulation. Abnormal miR-10a/b expression has been linked to increased invasiveness of various cancers. In immune system, miR-10a helps to control the stability and plasticity of helper T cells. The aim of the study was to investigate how miR-10a affects keratinocytes (KCs) and regulates their immune responses. The methods used include miRNA profiling of KCs from atopic dermatitis (AD) patients, overexpression of miR-10a to study changes in KC gene expression and cell cycle, KC differentiation, and luciferase assays to verify direct targets of miR-10a. Our results show miR-10a to be upregulated in cultured human primary KCs as well as skin biopsies from AD patients compared to healthy individuals. In human KCs, miR-10a expression was higher in proliferating conditions and decreased in differentiation-inducing growth conditions, namely during in vitro reconstruction of the skin and high calcium environment. Overexpression of miR-10a in KCs downregulated the previously characterized miR-10a direct target MAP3K7 (TAK1) from the NF-kB signalling pathway and hyaluronan synthase 3 (HAS3), a putative novel miR-10a direct target, which is known to be dysregulated in AD. Subsequent luciferase assays confirmed HAS3 as a novel direct target of miR-10a. In addition, miR-10a overexpression down-regulated many genes linked to cell cycle regulation, epithelial development or genes in the NF-kB pathway. Accordingly, cell cycle analysis of miR-10a-transfected KCs revealed less cells to be in the Sphase compared to controls. We conclude that miR-10a downregulates the proliferation of KCs through targeting multiple factors involved in the regulation of the cell cycle and has a regulatory role for immune responses in both healthy and diseased skin.
www.jidonline.org S183