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632 Photoprotection by constitutive melanin: Correlating deoxyribonucleic acid damage in vivo and skin cancer incidence
ABSTRACTS | Photobiology and Pigmentation 627
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A single sunburn influences the immune component of human skin for up to 14 days SM Pilkington, N Al-Gazaq, S Murphy, A Nicolaou and LE Rhodes Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom Sunburn is prevalent and while the clinical effects disappear quickly, repeated episodes have long term consequences such as skin cancer. Pro-inflammatory eicosanoid expression and leukocyte infiltration characterise the peak of the sunburn response, however, little is known of these processes during the resolution phase. We examined changes in eicosanoid expression and leukocyte infiltration up to 14 days post acute sunburn. Photoprotected buttock skin was exposed to 3xMED of broadband UVR and skin erythema measured over 14 days (n¼13; 20-58yrs, 8F, phototype I-III). Biopsies and suction blister fluid were sampled from unexposed and UVR-exposed skin over the 14 day time-course and analysed by immunohistochemistry (neutrophil elastase, CD4, CD8, CD68, CD1a, COX-2, EP4) and LCMS/MS (eicosanoids). Erythema, COX-2, eicosanoid production and infiltration of innate immune cells (neutrophils and macrophage), peaked at D1 post UVR. By D7 erythema showed 60% resolution, while expression of the prostaglandin E2 receptor EP4 remained elevated at D14. Infiltration of adaptive immune cells (CD4+, CD8+ T cells) peaked at D4-D7 and remained above baseline at D14 (p<0.01, p<0.001 respectively). Moreover, the CD4+:CD8+ ratio rose post-UVR, peaking at 4.3:1 at D1, compared with 2:1 at D0. In contrast, epidermal Langerhans cell numbers showed maximum reduction (w50%) at D4-D7 (p<0.01), only fully returning to baseline at D14. Acute UVR exposure has profoundly impacts epidermal antigen presenting cell number persisting for up to 2 weeks following the initial insult. Thus a single sunburn may trigger a prolonged immunosuppressive environment compromising ability to respond to neoplastic change and exogeneous challenge.
Dual vibration resonance frequency CARS microscopy imaging of basal cell carcinoma to achieve stain free histopathology ´ Krolopp2, K Lorincz3, R Szipocs1,2 and NM Wikonkal3 1 Wigner RCP, Institute for N Kiss3,1, A Solid State Physics and Optics, Budapest, Hungary, 2 R&D Ultrafast Lasers Ltd, Budapest, Hungary and 3 Dept. of Dermatology, Semmelweis University, Budapest, Hungary Basal cell carcinoma (BCC) is the most common malignancy in Caucasians. Nonlinear microscopy has been previously utilized for the imaging of BCC. To date, experiments were carried out solely on frozen sections, and the captured images do not correlate with the standard H&E staining. Recently, Freudiger et al. introduced a novel method to visualize tissue morphology analogous to H&E staining, using coherent anti-Stokes Raman scattering (CARS) technique. In our present work, we introduce a novel algorithm to post-process images obtained from dual vibration resonance frequency (DVRF) CARS measurements to increase the chemical selectivity of the imaging and to acquire high-quality pseudo H&E images of ex vivo healthy and BCC skin samples. We adapted our CARS setup to utilize the distinct vibrational properties of CH3 (found mainly in proteins) and CH2 bonds (primarily in lipids). In a narrowband setup, the central wavelength of the pump laser is set to 791 nm and 796 nm to obtain optimal excitation of the CH3 and CH2 bonds. Due to the partial overlap of the excitation spectra and the 5-10 nm FWHM spectral bandwidth of our lasers, we set the wavelengths to 790 nm (for excitation of proteins) and 800 nm (for lipids). In addition to the spectral overlap, nonresonant background from water molecules also reduces the chemical selectivity which can be significantly improved if we subtract the DVRF images from each other. As a result, we acquired two images: one for “lipids” and one for ”proteins” when we properly set a multiplication factor to minimize the non-specific CARS and autofluorescence background. By merging these images, we obtained high contrast H&E “stained” ex vivo microscope images of BBC’s. Nonlinear microscope systems upgraded for real time DVRF CARS measurements, providing pseudo H&E images can be suitable for in vivo assessment of BCC in the future.
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Suppression of autophagy by Tyrosinase-Creemediated deletion of Atg7 leads to accumulation of p62/sequestosome 1 in pigment cells and neurons S Sukseree1, S Bergmann1, F Gruber1, M Narzt1, IM Nagelreiter1, L LARUE2, E Tschachler1 and L Eckhart1 1 Department of Dermatology, Medical University of Vienna, Vienna, Austria and 2 Institut Curie, PSL Research University, INSERM U1021, CNRS UMR3347, Orsay, France Autophagy is a lysosomal digestion mechanism that plays ubiquitous and cell type-specific roles. We have previously generated Tyrosinase (Tyr)-Cre Atg7f/f mice in which the tyrosinase promoter directs the expression of the Cre recombinase to pigment cells. Cre-mediated deletion of the floxed Atg7 gene suppresses autophagy and dysregulates the antioxidant response of epidermal melanocytes in these mice. As the tyrosinase promoter was also reported to be active during the development other neuroectoderm-derived cell types, we investigated whether Tyr-Cre Atg7f/f mice have a brain phenotype. Indeed, the autophagy adaptor and substrate p62/sequestosome 1 accumulated in a subset of brain neurons of TyrCre Atg7f/f mice but not in control mice. The number and size of p62-positive aggregates increased with the age of Tyr-Cre Atg7f/f mice. In contrast to mouse models lacking autophagy in all neurons, the alterations of Tyr-Cre Atg7f/f neurons were not associated with overt abnormalities in health and behavior of mice up to an age of 2 years. These results suggest that Tyr-Cre Atg7f/f mice represent a new model for the study of autophagy-related processes in the aging brain.
Multi-omics identify nuclear protein 1 (Nupr1/p8) as central regulator of redox stress mediated by ultraviolet A light M Narzt1,2, IM Nagelreiter1,2 , V Bochkov3 , J Latreille4, M Fedorova5, Z Ni5 , F Sialana6 , G Lubec6, M Bilban7, E Tschachler1, J Grillari8,2 and F Gruber1,2 1 Dermatology, Medical University of Vienna, Vienna, Austria, 2 Christian Doppler Laboratory Skin Aging, MUW, Vienna, Austria, 3 University of Graz, Graz, Austria, 4 CHANEL PB, Pantin, France, 5 University of Leipzig, Leipzig, Germany, 6 Pharmaceutical Chemistry, University of Vienna, Vienna, Austria, 7 KILM, Medical University of Vienna, Vienna, Austria and 8 VIBT, BOKU, Vienna, Austria Ultraviolet A light is the dominant environmental oxidative stressor for the skin and a major factor for skin aging. We studied which oxidized phospholipid (PL) mediators UVA would generate in primary epidermal keratinocytes (NHEK) and how oxidized lipids contribute to UVA- induced changes in the transcriptome and the proteome. HPLC-MS/MS analysis of the oxidized phospholipidome of cultured NHEK immediately or after 24h stress recovery showed dynamic changes in carbonyl containing lipid mediators, which mostly declined to baseline values after 24 hours whereas PL-hydroperoxides and other oxygenated PL species remained elevated. We then studied which effects the exposure to either UVA or to in-vitro UVA-oxidized phospholipids would have on the transcriptome and proteome of NHEK. Using pathway analysis we identified that both stressors induced the antioxidant response dependent on Nrf2, whereas UVA additionally induced the unfolded protein response (UPR). Both stressors also induced a battery of lipid metabolizing enzymes, some of which are capable of reducing and detoxifying reactive carbonyls on lipids. A bioinformatic analysis of upstream regulatory elements in both types of stress suggested Nupr1 as upstream transcription regulatory factor. Indeed, inactivation of Nupr1 dysregulated the cell cycle, the expression of antioxidant response genes and lipid metabolizing enzymes. This suggests that Nupr1 is a novel and central player in the response of keratinocytes to aging promoting redox stress.
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Photoprotective effect of Skin-derived Precursors on skin photodamage in Balb/c hairless mice J Zhong1, D Xian2 and X Xiong1 1 Department of Dermatology, the Affiliated Hospital of Southwest Medical University, Luzhou, China and 2 Department of Neurobiology, Southwest Medical University, Luzhou, China Skin photodamage a special damage in skin, is associated with UV-induced overproduction of reactive oxygen species (ROS) and the inactivation of NF-E2-related factor 2 (Nrf2). Skinderived precursor cells (SKPs), a population of dermal stem cells, are considered to be involved in wound repair and skin regeneration through the activation of Nrf2. However, no reports concentrate on the treatment of skin photodamage with SKPs. To investigate the photoprotective role of SKPs in UV-induced photodamaged mice, 50 Balb/c hairless mice were divided into five groups (n ¼ 10): normal group, model group, prevention group, treatment group, and control group. The latter four groups were dorsally exposed to a twoweek UVA+UVB irradiation, whereas no any intervention was carried out in normal group. Mice in the prevention group received weekly SKP injections for two weeks the day before irradiation. Mice in the treatment and Hanks groups respectively received a two-time injection of SKPs and Hanks after irradiation. One week after final intervention, skin appearance, pathological characteristics, and oxidative indicators were evaluated by ELISA, immunohistochemical analysis, and western blotting. After irradiation, lesions appeared as erythema, edema, scales and wrinkles on mice dorsal skin; however, these were significantly ameliorated by subcutaneous SKP injection. Hyperkeratosis, acanthosis, and spongiosis in the epidermis, as well as dermal papillae edema and inflammatory cell infiltration, were observed in both model and control groups; however, these conditions resolved with SKPs. In addition, SKPs increased Nrf2, HO-1, GPX, SOD, CAT and GSH expression, while decreasing levels of ROS, MDA, and H2O2. Together with these findings, it is suggested that SKPs have a photoprotective role against UV-induced damage in mice, which may be associated with their ability to scavenge photo-oxidative insults and activate Nrf2.
S300 Journal of Investigative Dermatology (2017), Volume 137
Photoprotection by constitutive melanin: Correlating deoxyribonucleic acid damage in vivo and skin cancer incidence D Fajuyigbe1, SM Lwin1, B Diffey2, RP Sarkany1 and AR Young1 1 St John’s Institute of Dermatology, King’s College London, London, United Kingdom and 2 Dermatological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom Ultraviolet radiation (UVR) causes DNA damage, which initiates skin carcinogenesis. Epidemiological studies show a lower skin cancer incidence in people with dark skin compared to fair skin. This is attributed to photoprotection by epidermal melanin. Some authors have shown that the degree of UVR-induced DNA damage is influenced by constitutive pigmentation with greater protection (maximum of 7 fold) afforded by dark skin. We have studied how a skin cancer biomarker differs in Caucasian (Skin phototype (SPT) I/II) and West African (SPT VI) subjects. Upper buttocks of subjects (6 SPT I/II, 6 SPT VI) were exposed to a dose series of solar simulated radiation (SSR). Exposures were based on standard erythema doses (SED). The SED range for a given SPT was based on minimal erythema dose (MED) because the MED of SPT VI is w10 times higher than SPT I/II. Biopsies were taken immediately after exposure, sectioned and stained with a monoclonal antibody against cyclobutane pyrimidine dimers (CPD). We found a comparable amount of CPD in the overall epidermis, in each SPT group, when the SSR dose in SPT VI is 10 fold greater than in SPT I/II, thus indicating an overall melanin DNA protection of 10. These results suggest that UVRinduced CPD in the whole epidermis is linked to the erythema response. Comparisons at different epidermal zones (upper, mid, basal) showed that unlike SPTI/II, SPT VI showed a clear effect of epidermal zone with fewer CPD with increasing epidermal depth. The basal layer contains proliferative stem cells thus lesions here may be relevant to greater susceptibility to skin cancer. The protective effect of melanin in the basal layer was about 40 fold; a figure close to the 32 fold difference in melanoma incidence in dark and fair individuals. Our data stress the importance of the localisation of epidermal biomarkers for skin cancer that has been largely ignored in previous studies.
628
A single sunburn influences the immune component of human skin for up to 14 days SM Pilkington, N Al-Gazaq, S Murphy, A Nicolaou and LE Rhodes Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom Sunburn is prevalent and while the clinical effects disappear quickly, repeated episodes have long term consequences such as skin cancer. Pro-inflammatory eicosanoid expression and leukocyte infiltration characterise the peak of the sunburn response, however, little is known of these processes during the resolution phase. We examined changes in eicosanoid expression and leukocyte infiltration up to 14 days post acute sunburn. Photoprotected buttock skin was exposed to 3xMED of broadband UVR and skin erythema measured over 14 days (n¼13; 20-58yrs, 8F, phototype I-III). Biopsies and suction blister fluid were sampled from unexposed and UVR-exposed skin over the 14 day time-course and analysed by immunohistochemistry (neutrophil elastase, CD4, CD8, CD68, CD1a, COX-2, EP4) and LCMS/MS (eicosanoids). Erythema, COX-2, eicosanoid production and infiltration of innate immune cells (neutrophils and macrophage), peaked at D1 post UVR. By D7 erythema showed 60% resolution, while expression of the prostaglandin E2 receptor EP4 remained elevated at D14. Infiltration of adaptive immune cells (CD4+, CD8+ T cells) peaked at D4-D7 and remained above baseline at D14 (p<0.01, p<0.001 respectively). Moreover, the CD4+:CD8+ ratio rose post-UVR, peaking at 4.3:1 at D1, compared with 2:1 at D0. In contrast, epidermal Langerhans cell numbers showed maximum reduction (w50%) at D4-D7 (p<0.01), only fully returning to baseline at D14. Acute UVR exposure has profoundly impacts epidermal antigen presenting cell number persisting for up to 2 weeks following the initial insult. Thus a single sunburn may trigger a prolonged immunosuppressive environment compromising ability to respond to neoplastic change and exogeneous challenge.
Dual vibration resonance frequency CARS microscopy imaging of basal cell carcinoma to achieve stain free histopathology ´ Krolopp2, K Lorincz3, R Szipocs1,2 and NM Wikonkal3 1 Wigner RCP, Institute for N Kiss3,1, A Solid State Physics and Optics, Budapest, Hungary, 2 R&D Ultrafast Lasers Ltd, Budapest, Hungary and 3 Dept. of Dermatology, Semmelweis University, Budapest, Hungary Basal cell carcinoma (BCC) is the most common malignancy in Caucasians. Nonlinear microscopy has been previously utilized for the imaging of BCC. To date, experiments were carried out solely on frozen sections, and the captured images do not correlate with the standard H&E staining. Recently, Freudiger et al. introduced a novel method to visualize tissue morphology analogous to H&E staining, using coherent anti-Stokes Raman scattering (CARS) technique. In our present work, we introduce a novel algorithm to post-process images obtained from dual vibration resonance frequency (DVRF) CARS measurements to increase the chemical selectivity of the imaging and to acquire high-quality pseudo H&E images of ex vivo healthy and BCC skin samples. We adapted our CARS setup to utilize the distinct vibrational properties of CH3 (found mainly in proteins) and CH2 bonds (primarily in lipids). In a narrowband setup, the central wavelength of the pump laser is set to 791 nm and 796 nm to obtain optimal excitation of the CH3 and CH2 bonds. Due to the partial overlap of the excitation spectra and the 5-10 nm FWHM spectral bandwidth of our lasers, we set the wavelengths to 790 nm (for excitation of proteins) and 800 nm (for lipids). In addition to the spectral overlap, nonresonant background from water molecules also reduces the chemical selectivity which can be significantly improved if we subtract the DVRF images from each other. As a result, we acquired two images: one for “lipids” and one for ”proteins” when we properly set a multiplication factor to minimize the non-specific CARS and autofluorescence background. By merging these images, we obtained high contrast H&E “stained” ex vivo microscope images of BBC’s. Nonlinear microscope systems upgraded for real time DVRF CARS measurements, providing pseudo H&E images can be suitable for in vivo assessment of BCC in the future.
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Suppression of autophagy by Tyrosinase-Creemediated deletion of Atg7 leads to accumulation of p62/sequestosome 1 in pigment cells and neurons S Sukseree1, S Bergmann1, F Gruber1, M Narzt1, IM Nagelreiter1, L LARUE2, E Tschachler1 and L Eckhart1 1 Department of Dermatology, Medical University of Vienna, Vienna, Austria and 2 Institut Curie, PSL Research University, INSERM U1021, CNRS UMR3347, Orsay, France Autophagy is a lysosomal digestion mechanism that plays ubiquitous and cell type-specific roles. We have previously generated Tyrosinase (Tyr)-Cre Atg7f/f mice in which the tyrosinase promoter directs the expression of the Cre recombinase to pigment cells. Cre-mediated deletion of the floxed Atg7 gene suppresses autophagy and dysregulates the antioxidant response of epidermal melanocytes in these mice. As the tyrosinase promoter was also reported to be active during the development other neuroectoderm-derived cell types, we investigated whether Tyr-Cre Atg7f/f mice have a brain phenotype. Indeed, the autophagy adaptor and substrate p62/sequestosome 1 accumulated in a subset of brain neurons of TyrCre Atg7f/f mice but not in control mice. The number and size of p62-positive aggregates increased with the age of Tyr-Cre Atg7f/f mice. In contrast to mouse models lacking autophagy in all neurons, the alterations of Tyr-Cre Atg7f/f neurons were not associated with overt abnormalities in health and behavior of mice up to an age of 2 years. These results suggest that Tyr-Cre Atg7f/f mice represent a new model for the study of autophagy-related processes in the aging brain.
Multi-omics identify nuclear protein 1 (Nupr1/p8) as central regulator of redox stress mediated by ultraviolet A light M Narzt1,2, IM Nagelreiter1,2 , V Bochkov3 , J Latreille4, M Fedorova5, Z Ni5 , F Sialana6 , G Lubec6, M Bilban7, E Tschachler1, J Grillari8,2 and F Gruber1,2 1 Dermatology, Medical University of Vienna, Vienna, Austria, 2 Christian Doppler Laboratory Skin Aging, MUW, Vienna, Austria, 3 University of Graz, Graz, Austria, 4 CHANEL PB, Pantin, France, 5 University of Leipzig, Leipzig, Germany, 6 Pharmaceutical Chemistry, University of Vienna, Vienna, Austria, 7 KILM, Medical University of Vienna, Vienna, Austria and 8 VIBT, BOKU, Vienna, Austria Ultraviolet A light is the dominant environmental oxidative stressor for the skin and a major factor for skin aging. We studied which oxidized phospholipid (PL) mediators UVA would generate in primary epidermal keratinocytes (NHEK) and how oxidized lipids contribute to UVA- induced changes in the transcriptome and the proteome. HPLC-MS/MS analysis of the oxidized phospholipidome of cultured NHEK immediately or after 24h stress recovery showed dynamic changes in carbonyl containing lipid mediators, which mostly declined to baseline values after 24 hours whereas PL-hydroperoxides and other oxygenated PL species remained elevated. We then studied which effects the exposure to either UVA or to in-vitro UVA-oxidized phospholipids would have on the transcriptome and proteome of NHEK. Using pathway analysis we identified that both stressors induced the antioxidant response dependent on Nrf2, whereas UVA additionally induced the unfolded protein response (UPR). Both stressors also induced a battery of lipid metabolizing enzymes, some of which are capable of reducing and detoxifying reactive carbonyls on lipids. A bioinformatic analysis of upstream regulatory elements in both types of stress suggested Nupr1 as upstream transcription regulatory factor. Indeed, inactivation of Nupr1 dysregulated the cell cycle, the expression of antioxidant response genes and lipid metabolizing enzymes. This suggests that Nupr1 is a novel and central player in the response of keratinocytes to aging promoting redox stress.
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Photoprotective effect of Skin-derived Precursors on skin photodamage in Balb/c hairless mice J Zhong1, D Xian2 and X Xiong1 1 Department of Dermatology, the Affiliated Hospital of Southwest Medical University, Luzhou, China and 2 Department of Neurobiology, Southwest Medical University, Luzhou, China Skin photodamage a special damage in skin, is associated with UV-induced overproduction of reactive oxygen species (ROS) and the inactivation of NF-E2-related factor 2 (Nrf2). Skinderived precursor cells (SKPs), a population of dermal stem cells, are considered to be involved in wound repair and skin regeneration through the activation of Nrf2. However, no reports concentrate on the treatment of skin photodamage with SKPs. To investigate the photoprotective role of SKPs in UV-induced photodamaged mice, 50 Balb/c hairless mice were divided into five groups (n ¼ 10): normal group, model group, prevention group, treatment group, and control group. The latter four groups were dorsally exposed to a twoweek UVA+UVB irradiation, whereas no any intervention was carried out in normal group. Mice in the prevention group received weekly SKP injections for two weeks the day before irradiation. Mice in the treatment and Hanks groups respectively received a two-time injection of SKPs and Hanks after irradiation. One week after final intervention, skin appearance, pathological characteristics, and oxidative indicators were evaluated by ELISA, immunohistochemical analysis, and western blotting. After irradiation, lesions appeared as erythema, edema, scales and wrinkles on mice dorsal skin; however, these were significantly ameliorated by subcutaneous SKP injection. Hyperkeratosis, acanthosis, and spongiosis in the epidermis, as well as dermal papillae edema and inflammatory cell infiltration, were observed in both model and control groups; however, these conditions resolved with SKPs. In addition, SKPs increased Nrf2, HO-1, GPX, SOD, CAT and GSH expression, while decreasing levels of ROS, MDA, and H2O2. Together with these findings, it is suggested that SKPs have a photoprotective role against UV-induced damage in mice, which may be associated with their ability to scavenge photo-oxidative insults and activate Nrf2.
S300 Journal of Investigative Dermatology (2017), Volume 137
Photoprotection by constitutive melanin: Correlating deoxyribonucleic acid damage in vivo and skin cancer incidence D Fajuyigbe1, SM Lwin1, B Diffey2, RP Sarkany1 and AR Young1 1 St John’s Institute of Dermatology, King’s College London, London, United Kingdom and 2 Dermatological Sciences, Institute of Cellular Medicine, Newcastle University, Newcastle, United Kingdom Ultraviolet radiation (UVR) causes DNA damage, which initiates skin carcinogenesis. Epidemiological studies show a lower skin cancer incidence in people with dark skin compared to fair skin. This is attributed to photoprotection by epidermal melanin. Some authors have shown that the degree of UVR-induced DNA damage is influenced by constitutive pigmentation with greater protection (maximum of 7 fold) afforded by dark skin. We have studied how a skin cancer biomarker differs in Caucasian (Skin phototype (SPT) I/II) and West African (SPT VI) subjects. Upper buttocks of subjects (6 SPT I/II, 6 SPT VI) were exposed to a dose series of solar simulated radiation (SSR). Exposures were based on standard erythema doses (SED). The SED range for a given SPT was based on minimal erythema dose (MED) because the MED of SPT VI is w10 times higher than SPT I/II. Biopsies were taken immediately after exposure, sectioned and stained with a monoclonal antibody against cyclobutane pyrimidine dimers (CPD). We found a comparable amount of CPD in the overall epidermis, in each SPT group, when the SSR dose in SPT VI is 10 fold greater than in SPT I/II, thus indicating an overall melanin DNA protection of 10. These results suggest that UVRinduced CPD in the whole epidermis is linked to the erythema response. Comparisons at different epidermal zones (upper, mid, basal) showed that unlike SPTI/II, SPT VI showed a clear effect of epidermal zone with fewer CPD with increasing epidermal depth. The basal layer contains proliferative stem cells thus lesions here may be relevant to greater susceptibility to skin cancer. The protective effect of melanin in the basal layer was about 40 fold; a figure close to the 32 fold difference in melanoma incidence in dark and fair individuals. Our data stress the importance of the localisation of epidermal biomarkers for skin cancer that has been largely ignored in previous studies.