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532 A “multi-omic” investigation of the effects of long wavelength ultraviolet light on primary human keratinocytes
Photobiology and Pigmentation | ABSTRACTS 530
531
Soy isoflavones and nicotinamide down-regulate pro-inflammatory mediators in vitro R Di Caprio1, S Lembo2, A Balato3, F Gasparri4 and G Monfrecola1 1 Deparment of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy, 2 Department of Medicine and Surgery, University of Salerno, Salerno, Italy, 3 Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy and 4 Department of Pharmacy (DIFARMA), University of Salerno, Salerno, Italy The aim of this study was to evaluate, in vitro, the anti-inflammatory and antioxidant properties of soy isoflavones and nicotinamide (NCT), singularly or in combination, in UVBirradiated keratinocytes. Soy isoflavones were used in two different forms: ISOSOY and Lo ISO-3s. The first, is a standardized glycine soy extract titrated to 90% in isoflavone aglycones (with genistein:daidzein¼1:4), soluble in dimethylsulfoxide. The second, consists in microencapsulated soy extracts, enriched in daidzein and genistein, obtained by spray-drying technique using a sodium-carboxymethylcellulose based matrix, soluble in water. Immortalized human keratinocytes were pre-incubated for 2h with ISOSOY and NCT or with Lo ISO3s and NCT, singularly or in combination, and irradiated with UVB 60 mJ/cm2. Substances toxicity was assessed through cell viability and their possible anti-inflammatory effect was explored analyzing IL-6, TNF-a and COX-2 gene expression (qRT-PCR) 24h after UVB irradiation. None of the tested substances resulted toxic to cells. All of them showed anti-inflammatory properties alone or in combination with NCT. In detail, Lo ISO-3s was more effective than ISOSOY in down-regulating IL-6 and COX-2 increases (p<0.01), while ISOSOY was more effective than Lo ISO-3s in down-regulating TNF-a increases (p<0.01). A part from TNF-a, Lo ISO-3s was more effective than NCT in reducing UVB-induced inflammation, while ISOSOY, generally, resulted less effective. In conclusion, soy isoflavones and NCT, used alone or in combination, have demonstrated a potential immuno-modulatory and cytoprotective effect representing an interesting option to improve or prevent UV-induced/aggravated clinical conditions.
Human skin mast cells express photoreceptors H Siiskonen2, S Buscone1, I Castellano Pellicena1, A Smorodchenko3, NE Uzunbajakava4, NV Botchkareva1, M Maurer3 and J Scheffel3 1 Faculty of Life Sciences, Centre for Skin Sciences, Bradford, United Kingdom, 2 Dermatology, Kuopio University Hospital, Kuopio, Finland, 3 Department of Dermatology and Allergy, Charite´ - Universita¨tsmedizin Berlin, Berlin, Germany and 4 Philips Research Eindhoven, Eindhoven, Netherlands Circadian clocks critically influence mast cell (MC) functions and drive the daily rhythms in IgE/MCemediated allergic reactions. In the skin, these clocks may be regulated by photoreceptors (PRs) such as cryptochromes (CRY) and opsins (OPN), which have recently been shown to be expressed in keratinocytes. Whether skin MCs express PRs has not been investigated yet. Therefore, we studied the expression of selected PRs in human MCs using qRT-PCR. To this end, we used freshly isolated cutaneous MCs from breast (n¼3), eyelid (n¼1) and abdominal skin (n¼1); cultured MCs from breast (n¼2) and foreskin (n¼2); cultured CD34-positive peripheral blood stem cell-derived MCs (PSCMCs); and LAD2-MCs. We identified that freshly isolated MCs from breast skin, cultured MCs from breast- and foreskin and LAD2-MCs express CRY1, OPN1 medium-wave-length, OPN2 and OPN3. Expression of CRY1, OPN1 medium-wave-length and OPN3 was also detected in freshly isolated MCs from eyelid skin. Of the PRs assessed, CRY1 was the most prominently expressed, and we also found CRY1 expression at the protein level in cultured MCs from breast skin and foreskin and from freshly isolated MCs from breast skin. The expression levels of PRs varied between different donors and skin areas. MCs freshly isolated from abdominal skin and PSCMCs were negative for the investigated PRs, suggesting that expression of CRYs and OPNs might be connected to MC differentiation and functionality. Our results show, for the first time, that human skin MCs express several PRs. We will next investigate a potential role of these PRs in MCs by studying their involvement in circadian oscillation of MC function and in MCmediated skin responses.
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A “multi-omic” investigation of the effects of long wavelength ultraviolet light on primary human keratinocytes M Narzt1, IM Nagelreiter1, V Bochkov3, J Latreille4, M Fedorova5, Z Ni5, J Grillari2, E Tschachler1 and F Gruber1 1 Dermatology, MUW, Vienna, Austria, 2 VIBT-BOKU, Vienna, Austria, 3 University of Graz, Graz, Austria, 4 ChanelPB, Paris, France and 5 University of Leipzig, Leipzig, Germany Ultraviolet A light is the dominant environmental oxidative stressor for the skin and a major factor for skin aging. In dermal fibroblasts UVA oxidizes phospholipids which in turn induce Nrf2 activation and autophagy as stress responses. The role of keratinocytes (NHEK), in the initial events of UVA - photoaging is not well understood. We studied which lipid mediators UVA would generate in KC and how they are correlated to UVA induced changes in mRNA, microRNA and protein expression. The oxidized phospholipidome of cultured NHEK immediately or after 24h stress recovery showed, using HPLC-MS/MS, that 173 distinct lipid species were significantly induced immediately after UVA exposure, of which 120 lipid species had declined to their baseline values after 24h. In parallel, gene ontology and pathway analysis of global mRNA expression 7h after the stress showed that the 81 mRNAs induced both by UVA and in vitro oxidized lipids which partially mimic the UVA response could be attributed to the action of upstream regulators Nrf2, the UPR, and PPAR signalling. Among the upregulated genes we found mitochondrial phospholipases, peroxiredoxins and other enzymes capable of metabolizing isoprostanoid-PL and other lipid mediators recently correlated to (skin) aging. Together our data suggest that UV-induced phospholipids initiate a transcriptional response that not only induces synthesis of antioxidant stress response genes but also enzymes to specifically metabolize and detoxify bioactive oxidized membrane lipids.
Distinct photoprotective effects on UVA irradiated-keratinocytes N Bechetoille1, E Metral2, F Demarne1, O Damour2 and W Rachidi3 1 GATTEFOSSE, R&D, Saint-Priest, France, 2 Laboratoire des Substituts Cutane´s, Edouard Herriot Hospital, Lyon, France and 3 CEA, Laboratoire Le´sions des Acides Nucle´iques, Grenoble, France Skin is exposed every days to sun radiations. Among them, UVA radiations penetrate through the epidermis and induce oxidative damages (DNA, proteins, lipids) in germinative cells of the epidermal basal layer. Moreover, UVA are now known for inducing pyrimidine dimers (CPD) as well. While basal cells allow epidermis renewal and homeostasis, it is essential to protect them and maintain their integrity. For this, several UV filters can be used as physical shield. The aim of the study was to test the photoprotective effects of plant extracts on keratinocytes exposed to UVA. In order to be sure that the photoprotective effects were not due to a physical filter action, keratinocytes were treated with plant extracts before being exposed to UVA (50 J/cm2). Cytotoxicity, clonogenicity, DNA damages (8-oxoG by the alkaline comet assay and pyrimidine dimers (CPD) by HPLC-MS/MS) and oxidative stress (ROS production) were assessed. All plant extracts displayed a potent photoprotective effects against UVA as demonstrated by cytotoxicity and clonogenicity studies. Interestingly, two-thirds of plant extracts decreased single strand break and alkali-labile sites as well as oxidative DNA damages such as 8oxoG and CPD. Surprisingly, a third of plant extracts did not show any influence on DNA damages although they showed a photoprotective effect. All plant extracts will be soon assessed on the DNA repair activities by using our new biochip developed and patented in our laboratory. All together, our results emphasize the importance of using different techniques to explore cellular responses against UVA stress and demonstrate the photoprotective effects of products against UV irradiation.
534
535
Vitiligo: Studying the dermal compartment D Kovacs, E Bastonini, M Ottaviani, C Cota, E Migliano, M Dell’Anna and M Picardo San Gallicano Dermatologic Institute, Rome, Italy Homeostasis of pigmentary system is regulated by strictly balanced interactions at both molecular and cellular level and a multitude of messengers and growth factors released by epidermal and dermal cells regulate melanocyte activities and consequently skin pigmentation. Vitiligo is characterized by the progressive disappearance of melanocytes. Increasing evidences are currently highlighting an intrinsic metabolic impairment of melanocytes, favouring the increase of intracellular oxidative stress, which, in turn, may promote the onset of a premature senescent-like phenotype. Despite the important role of the dermis in regulating melanocyte physiology, few data have evaluated its involvement in vitiligo. The aim of our study was to investigate the dermal compartment of non lesional vitiligo skin, focusing on the features of extracellular matrix proteins and fibroblasts. We employed primary cultures of non lesional vitiligo fibroblasts and performed a parallel ex vivo evaluation on skin biopsies collected from the same patients from whom the cells were derived. Our results demonstrate that vitiligo fibroblasts display increased basal ROS levels associated with the up-modulation of stress-induced markers, e.g. p53 and its target gene GADD45. The higher intrinsic oxidative stress favours the transition of fibroblasts into a myofibroblasts-like phenotype, as assessed by the positive expression of markers related to myofibroblasts including alphasmooth muscle actin, the glycoprotein fibronectin, the intermediate filament associated protein vimentin and the secretory proteins interleukin IL-6 and hepatocyte growth factor. The results point to the involvement of the entire skin in vitiligo, showing the presence of modifications not restricted to melanocytes, which constitute the long-established major player of the disease, but extended to the dermis. The focus on normal appearing skin may therefore allow to recognize the appearance of cellular phenomena before the clear clinical onset of the disease and possibly restrict the spread of the lesions.
Keratinocytes Stem cells are more resistant to UVA radiation than their direct progeny W Rachidi1, E Metral2, F Demarne2, N Bechetoille2 and O Damour3 1 INAC/SyMMES/LAN, CEA, Grenoble, France, 2 GATTEFOSSE, Saint Priest, France and 3 Banque de Tissus et Cellules, Hospices Civils de Lyon, Lyon, France Epidermis is in constant renewal during all lifelong. This is possible thank to a special keratinocyte population located at the basal layer, the Keratinocyte Stem Cells (KSC) which are surrounded by their direct progeny, called keratinocyte progenitors or transient amplifying cells (TA), that arise from their division. In another hand, skin is exposed every day to sun radiations. Among them, UVA radiations penetrate through the epidermis and induce damages to KSC and TA. Although keratinocyte precursor cells are the most likely cells of origin of skin carcinomas and/or photoaging, surprisingly few studies addressed the specific responses of these cells to UV radiation. In this study, the response to UVA irradiation of human KSC and TA, was investigated immediately after cell sorting and during the primary culture, in order to investigate cell phenotypes close to those found in vivo in the basal layer of epidermis. Cell populations enriched in KSC were sorted by flow cytometry from normal human skin samples using the phenotype defined by a6hi and low transferrin receptor expression (a6bri/CD71dim), whereas the TA were sorted on the a6bri/CD71bri phenotype. These two populations were irradiated with different UVA doses (from 0 to 50 J/cm2) and proliferative and clonogenic capacities were performed. Both the MTT assay and the clonogenic assay showed that the KSC were photo-resistant whereas TA were photo-sensitive. Investigations of DNA repair by comet test showed that both single DNA strand breaks and oxidative DNA damages were repaired quicker and more efficiently in KSC than TA. Our results show for the first time that keratinocyte populations enriched for stem cells from human epidermis are more resistant to UVA radiation by its high endogenous DNA repair capacity. Our future work will try to identify factors or pathways responsible for this differential photo-sensivity and DNA repair capacity between KSC and TA.
www.jidonline.org S251
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Soy isoflavones and nicotinamide down-regulate pro-inflammatory mediators in vitro R Di Caprio1, S Lembo2, A Balato3, F Gasparri4 and G Monfrecola1 1 Deparment of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy, 2 Department of Medicine and Surgery, University of Salerno, Salerno, Italy, 3 Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy and 4 Department of Pharmacy (DIFARMA), University of Salerno, Salerno, Italy The aim of this study was to evaluate, in vitro, the anti-inflammatory and antioxidant properties of soy isoflavones and nicotinamide (NCT), singularly or in combination, in UVBirradiated keratinocytes. Soy isoflavones were used in two different forms: ISOSOY and Lo ISO-3s. The first, is a standardized glycine soy extract titrated to 90% in isoflavone aglycones (with genistein:daidzein¼1:4), soluble in dimethylsulfoxide. The second, consists in microencapsulated soy extracts, enriched in daidzein and genistein, obtained by spray-drying technique using a sodium-carboxymethylcellulose based matrix, soluble in water. Immortalized human keratinocytes were pre-incubated for 2h with ISOSOY and NCT or with Lo ISO3s and NCT, singularly or in combination, and irradiated with UVB 60 mJ/cm2. Substances toxicity was assessed through cell viability and their possible anti-inflammatory effect was explored analyzing IL-6, TNF-a and COX-2 gene expression (qRT-PCR) 24h after UVB irradiation. None of the tested substances resulted toxic to cells. All of them showed anti-inflammatory properties alone or in combination with NCT. In detail, Lo ISO-3s was more effective than ISOSOY in down-regulating IL-6 and COX-2 increases (p<0.01), while ISOSOY was more effective than Lo ISO-3s in down-regulating TNF-a increases (p<0.01). A part from TNF-a, Lo ISO-3s was more effective than NCT in reducing UVB-induced inflammation, while ISOSOY, generally, resulted less effective. In conclusion, soy isoflavones and NCT, used alone or in combination, have demonstrated a potential immuno-modulatory and cytoprotective effect representing an interesting option to improve or prevent UV-induced/aggravated clinical conditions.
Human skin mast cells express photoreceptors H Siiskonen2, S Buscone1, I Castellano Pellicena1, A Smorodchenko3, NE Uzunbajakava4, NV Botchkareva1, M Maurer3 and J Scheffel3 1 Faculty of Life Sciences, Centre for Skin Sciences, Bradford, United Kingdom, 2 Dermatology, Kuopio University Hospital, Kuopio, Finland, 3 Department of Dermatology and Allergy, Charite´ - Universita¨tsmedizin Berlin, Berlin, Germany and 4 Philips Research Eindhoven, Eindhoven, Netherlands Circadian clocks critically influence mast cell (MC) functions and drive the daily rhythms in IgE/MCemediated allergic reactions. In the skin, these clocks may be regulated by photoreceptors (PRs) such as cryptochromes (CRY) and opsins (OPN), which have recently been shown to be expressed in keratinocytes. Whether skin MCs express PRs has not been investigated yet. Therefore, we studied the expression of selected PRs in human MCs using qRT-PCR. To this end, we used freshly isolated cutaneous MCs from breast (n¼3), eyelid (n¼1) and abdominal skin (n¼1); cultured MCs from breast (n¼2) and foreskin (n¼2); cultured CD34-positive peripheral blood stem cell-derived MCs (PSCMCs); and LAD2-MCs. We identified that freshly isolated MCs from breast skin, cultured MCs from breast- and foreskin and LAD2-MCs express CRY1, OPN1 medium-wave-length, OPN2 and OPN3. Expression of CRY1, OPN1 medium-wave-length and OPN3 was also detected in freshly isolated MCs from eyelid skin. Of the PRs assessed, CRY1 was the most prominently expressed, and we also found CRY1 expression at the protein level in cultured MCs from breast skin and foreskin and from freshly isolated MCs from breast skin. The expression levels of PRs varied between different donors and skin areas. MCs freshly isolated from abdominal skin and PSCMCs were negative for the investigated PRs, suggesting that expression of CRYs and OPNs might be connected to MC differentiation and functionality. Our results show, for the first time, that human skin MCs express several PRs. We will next investigate a potential role of these PRs in MCs by studying their involvement in circadian oscillation of MC function and in MCmediated skin responses.
532
533
A “multi-omic” investigation of the effects of long wavelength ultraviolet light on primary human keratinocytes M Narzt1, IM Nagelreiter1, V Bochkov3, J Latreille4, M Fedorova5, Z Ni5, J Grillari2, E Tschachler1 and F Gruber1 1 Dermatology, MUW, Vienna, Austria, 2 VIBT-BOKU, Vienna, Austria, 3 University of Graz, Graz, Austria, 4 ChanelPB, Paris, France and 5 University of Leipzig, Leipzig, Germany Ultraviolet A light is the dominant environmental oxidative stressor for the skin and a major factor for skin aging. In dermal fibroblasts UVA oxidizes phospholipids which in turn induce Nrf2 activation and autophagy as stress responses. The role of keratinocytes (NHEK), in the initial events of UVA - photoaging is not well understood. We studied which lipid mediators UVA would generate in KC and how they are correlated to UVA induced changes in mRNA, microRNA and protein expression. The oxidized phospholipidome of cultured NHEK immediately or after 24h stress recovery showed, using HPLC-MS/MS, that 173 distinct lipid species were significantly induced immediately after UVA exposure, of which 120 lipid species had declined to their baseline values after 24h. In parallel, gene ontology and pathway analysis of global mRNA expression 7h after the stress showed that the 81 mRNAs induced both by UVA and in vitro oxidized lipids which partially mimic the UVA response could be attributed to the action of upstream regulators Nrf2, the UPR, and PPAR signalling. Among the upregulated genes we found mitochondrial phospholipases, peroxiredoxins and other enzymes capable of metabolizing isoprostanoid-PL and other lipid mediators recently correlated to (skin) aging. Together our data suggest that UV-induced phospholipids initiate a transcriptional response that not only induces synthesis of antioxidant stress response genes but also enzymes to specifically metabolize and detoxify bioactive oxidized membrane lipids.
Distinct photoprotective effects on UVA irradiated-keratinocytes N Bechetoille1, E Metral2, F Demarne1, O Damour2 and W Rachidi3 1 GATTEFOSSE, R&D, Saint-Priest, France, 2 Laboratoire des Substituts Cutane´s, Edouard Herriot Hospital, Lyon, France and 3 CEA, Laboratoire Le´sions des Acides Nucle´iques, Grenoble, France Skin is exposed every days to sun radiations. Among them, UVA radiations penetrate through the epidermis and induce oxidative damages (DNA, proteins, lipids) in germinative cells of the epidermal basal layer. Moreover, UVA are now known for inducing pyrimidine dimers (CPD) as well. While basal cells allow epidermis renewal and homeostasis, it is essential to protect them and maintain their integrity. For this, several UV filters can be used as physical shield. The aim of the study was to test the photoprotective effects of plant extracts on keratinocytes exposed to UVA. In order to be sure that the photoprotective effects were not due to a physical filter action, keratinocytes were treated with plant extracts before being exposed to UVA (50 J/cm2). Cytotoxicity, clonogenicity, DNA damages (8-oxoG by the alkaline comet assay and pyrimidine dimers (CPD) by HPLC-MS/MS) and oxidative stress (ROS production) were assessed. All plant extracts displayed a potent photoprotective effects against UVA as demonstrated by cytotoxicity and clonogenicity studies. Interestingly, two-thirds of plant extracts decreased single strand break and alkali-labile sites as well as oxidative DNA damages such as 8oxoG and CPD. Surprisingly, a third of plant extracts did not show any influence on DNA damages although they showed a photoprotective effect. All plant extracts will be soon assessed on the DNA repair activities by using our new biochip developed and patented in our laboratory. All together, our results emphasize the importance of using different techniques to explore cellular responses against UVA stress and demonstrate the photoprotective effects of products against UV irradiation.
534
535
Vitiligo: Studying the dermal compartment D Kovacs, E Bastonini, M Ottaviani, C Cota, E Migliano, M Dell’Anna and M Picardo San Gallicano Dermatologic Institute, Rome, Italy Homeostasis of pigmentary system is regulated by strictly balanced interactions at both molecular and cellular level and a multitude of messengers and growth factors released by epidermal and dermal cells regulate melanocyte activities and consequently skin pigmentation. Vitiligo is characterized by the progressive disappearance of melanocytes. Increasing evidences are currently highlighting an intrinsic metabolic impairment of melanocytes, favouring the increase of intracellular oxidative stress, which, in turn, may promote the onset of a premature senescent-like phenotype. Despite the important role of the dermis in regulating melanocyte physiology, few data have evaluated its involvement in vitiligo. The aim of our study was to investigate the dermal compartment of non lesional vitiligo skin, focusing on the features of extracellular matrix proteins and fibroblasts. We employed primary cultures of non lesional vitiligo fibroblasts and performed a parallel ex vivo evaluation on skin biopsies collected from the same patients from whom the cells were derived. Our results demonstrate that vitiligo fibroblasts display increased basal ROS levels associated with the up-modulation of stress-induced markers, e.g. p53 and its target gene GADD45. The higher intrinsic oxidative stress favours the transition of fibroblasts into a myofibroblasts-like phenotype, as assessed by the positive expression of markers related to myofibroblasts including alphasmooth muscle actin, the glycoprotein fibronectin, the intermediate filament associated protein vimentin and the secretory proteins interleukin IL-6 and hepatocyte growth factor. The results point to the involvement of the entire skin in vitiligo, showing the presence of modifications not restricted to melanocytes, which constitute the long-established major player of the disease, but extended to the dermis. The focus on normal appearing skin may therefore allow to recognize the appearance of cellular phenomena before the clear clinical onset of the disease and possibly restrict the spread of the lesions.
Keratinocytes Stem cells are more resistant to UVA radiation than their direct progeny W Rachidi1, E Metral2, F Demarne2, N Bechetoille2 and O Damour3 1 INAC/SyMMES/LAN, CEA, Grenoble, France, 2 GATTEFOSSE, Saint Priest, France and 3 Banque de Tissus et Cellules, Hospices Civils de Lyon, Lyon, France Epidermis is in constant renewal during all lifelong. This is possible thank to a special keratinocyte population located at the basal layer, the Keratinocyte Stem Cells (KSC) which are surrounded by their direct progeny, called keratinocyte progenitors or transient amplifying cells (TA), that arise from their division. In another hand, skin is exposed every day to sun radiations. Among them, UVA radiations penetrate through the epidermis and induce damages to KSC and TA. Although keratinocyte precursor cells are the most likely cells of origin of skin carcinomas and/or photoaging, surprisingly few studies addressed the specific responses of these cells to UV radiation. In this study, the response to UVA irradiation of human KSC and TA, was investigated immediately after cell sorting and during the primary culture, in order to investigate cell phenotypes close to those found in vivo in the basal layer of epidermis. Cell populations enriched in KSC were sorted by flow cytometry from normal human skin samples using the phenotype defined by a6hi and low transferrin receptor expression (a6bri/CD71dim), whereas the TA were sorted on the a6bri/CD71bri phenotype. These two populations were irradiated with different UVA doses (from 0 to 50 J/cm2) and proliferative and clonogenic capacities were performed. Both the MTT assay and the clonogenic assay showed that the KSC were photo-resistant whereas TA were photo-sensitive. Investigations of DNA repair by comet test showed that both single DNA strand breaks and oxidative DNA damages were repaired quicker and more efficiently in KSC than TA. Our results show for the first time that keratinocyte populations enriched for stem cells from human epidermis are more resistant to UVA radiation by its high endogenous DNA repair capacity. Our future work will try to identify factors or pathways responsible for this differential photo-sensivity and DNA repair capacity between KSC and TA.
www.jidonline.org S251