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567 Optimal ultraviolet wavelength to induce natural regulatory T cells
Photobiology | ABSTRACTS 567
568
Optimal ultraviolet wavelength to induce natural regulatory T cells S Muramatsu and A Morita Geriatrick and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Narrowband ultraviolet B (NB-UVB) phototherapy (311-nm) is widely used to treat refractory skin disorders, such as psoriasis and atopic dermatitis. Recent studies reported that the mechanisms underlying the effects of phototherapy include inhibition of cell proliferation in keratinocytes, induction of apoptosis in pathogenetically relevant cells, and expansion of regulatory T (Treg) cells. Treg cells suppress inflammatory skin disorders. Although broadband UVB (290-320 nm) induces Treg cells, the optimal UV wavelength for inducing Treg is not known. Here we investigated which wavelength effectively induces Treg cells in a mouse model. Shaved C57BL6J mice were irradiated with several UV wavelengths (290 nm, 300 nm, 310 nm, and 320 nm) using a monochromator. After a single dose of 100 mJ/cm2 UV exposure, the lymph nodes and spleen were collected and analyzed with a flow cytometer. After 300 nm and 310 nm UV exposure, CD4+CD25+Foxp3+neuropilin-1+ (Natural Treg: nTreg) cells were increased by 3% to 4% in lymph nodes compared with those in sham irradiated mice (300 nm: from 9.17% to 13.5%. 310 nm: from 7.37% to 10.3%). The optimal UV wavelength range for inducing nTreg was 300 nm to 310 nm. This finding suggests that NB-UVB therapy effectively induces nTreg cells and mediates therapeutic effects in skin diseases.
Neutrophil NETosis is involved in UVB induced-skin inflammation M Liu1,2, M Sharma1,2 and VP Werth1,2 1 University of Pennsylvania, Philadelphia, PA and 2 Philadelphia VAMC, Philadelphia, PA Excessive exposure to UVB in sunlight causes photodamage that triggers autoimmune skin inflammation. Skin inflammation can exacerbate photodamage and, if recurring or longlasting, can induce pathogenic alterations. The responses to UVB exposure include the release of inflammatory cytokines, including TNF-a, from keratinocytes, and the recruitment of inflammatory cells to the skin. Neutrophils are the first group of immune cells to be recruited to the site of photodamage, prior to the infiltration of monocyte/macrophages and lymphocytes. Accumulating evidence indicates the important roles of neutrophils in sterile inflammation, however, little is known about their involvement in UVB-induced skin inflammation. Neutrophil NETosis is a newly characterized neutrophil cell death that releases neutrophil extracellular traps (NETs). NETs have been detected in the lupus skin. NETs exhibit IL-17, and drive type I interferon (IFN) release from pDCs. These two important cytokines interact together to sustain and amplify autoimmune and inflammatory responses in lupus pathogenesis. However, the role of neutrophil NETosis in UVB-induced skin inflammation has not been explored. In the current study, we found that exposure of female C57/BL6 wild-type (WT) mice to UVB (250 mJ/cm2/day) for 5 consecutive days can induce skin inflammation with increased NETotic neutrophils. Our preliminary studies showed that these NETotic structures in the UVB-irradiated skin also exhibit proinflammatory cytokine IL-17. In searching for a relevant stimulus that mediates neutrophil NETosis in UVB-exposed WT mice, we found that TNFa was upregulated in UVB-irradiated skin. In addition, treatment of human primary neutrophils with TNFa in vitro can induce neutrophil NETosis. Therefore, our studies indicate that UVB exposure of mice can induce skin inflammation with increased NETotic neutrophils, which exhibit proinflammatory cytokine IL-17. UVB-induced TNF-a may mediate neutrophil NETosis in the inflamed skin.
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YB-1 interacts with DNp63a in UVB exposed human skin DN Syed, RK Lall, J Chamcheu, Y Soon and H Mukhtar Dermatology, University of Wisconsin, Madison, WI Exposure to ultraviolet (UV) radiation induces skin cancer through dysregulated cell proliferation causing neoplastic transformation. The Y-box binding protein1 (YB-1) performs a variety of cellular functions, including regulation of transcription and translation, DNA repair, drug resistance, and stress responses to extracellular signals. Reduction of YB-1 expression causes growth inhibition or apoptosis in cancer cells indicating its critical role in cell proliferation. The role of YB-1 in UV exposed human skin has not been studied. We observed a significant increase in YB-1 phosphorylation at Ser102 in monolayer cultures of epidermal keratinocytes exposed to UVB (40mJ/cm2:4h) radiation. This data was reciprocated in a 3-D epidermal model comprised of multiple layers of well differentiated keratinocytes where increased phosphorylation of YB-1 was observed at 4 and 24 h post UVB. The epidermal protein DNp63a involved in keratinocyte proliferation and differentiation showed a similar pattern of expression with an initial increase at 4 h followed by downregulation at extended time points. Interestingly the decrease in YB-1/DNp63a expression correlated with apoptosis of UVB-exposed keratinocytes. YB-1-overexpressing keratinocytes exhibited increased DNp63a levels while a significant decrease was noted in YB-1-depleted keratinocytes. Immunoprecipitation studies confirmed functional interaction of YB-1 with DNp63a in UVB exposed keratinocytes. In a chronic UV exposure model, where keratinocytes were subjected to repetitive sub-apoptotic UVB doses (10mJ/cm2), increased expression of YB-1 was associated with upregulation of DNp63a levels. Our studies suggest that YB-1 interaction with DNp63a may be an important regulatory mechanism that modulates the UV response of epidermal keratinocytes. The increase in YB-1/DNp63a expression is likely associated with an initial protective response mounted by keratinocytes following UVB exposure. Our ongoing studies target the precise functionality of this pathway in UV exposed human skin.
The AHR-p27 axis modulates DNA damage responses in UV-irradiated keratinocytes in vitro and in vivo S Shaik, M Pollet, J Krutmann and T Haarmann-Stemmann Leibniz-Research Institute for Environmental Medicine, Du¨sseldorf, Germany The aryl hydrocarbon receptor (AHR) is a ligand-activated and UVB-sensitive transcription factor. Previously, we have demonstrated that the AHR represses apoptosis in UVB-exposed epidermal keratinocytes (KC), indicating that this transcription factor may be critically involved in photocarcinogenesis. In fact, we observed that, in contrast to AHR+/+ mice, AHR-/- animals developed 50% less skin tumors in a chronic UVB irradiation study and thus were largely protected against the carcinogenicity of UVB radiation. In vitro studies further indicated that the AHR down-regulates the tumor suppressor p27 (KIP1) in KC to suppress apoptosis, which may explain the ‘pro-carcinogenic’ action of cutaneous AHR signaling. Now we asked if p27 has a direct impact on UVB-induced DNA damage responses in KC. We found that overexpression of human p27 in HaCaT KC resulted in an enhanced occurrence of UVB-induced apoptosis 24h after UVB-exposure, thus mirroring the effects of chemical and genetic AHR inhibition. Interestingly, overexpression of human p27 also affected the clearance of UVB-induced cyclobutane pyrimidine dimers (CPDs) early (4h) after irradiation, indicating that the AHR may influence both apoptosis and DNA repair by modulating p27. Studies using AHR antagonists and RNAi further demonstrated that the AHR inhibits CPD removal and thus probably NER in HaCaT cells. By silencing xeroderma pigmentosum complementation group A expression and excluding an involvement of early apoptosis, we demonstrated that the acceleration of CPD clearance through AHR inhibition was in fact due to a more pronounced activity of the NER machinery. Importantly, we also observed a higher level of the p27 protein in the skin of AHR-/- mice, which was accompanied by a faster removal of mutagenic CPDs 48h after a single exposure to UVB radiation. These studies further support the concept that the AHR serves as a negative regulator of DNA damage responses in UVB-irradiated KC and thus might be a molecular target for skin cancer prevention.
571
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FOXO3a-dependent expression of Trail and Bim is regulated by mTORC2 in UVB-induced apoptosis RP Feehan and LM Shantz Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA Ultraviolet B (UVB) radiation, the primary cause of non-melanoma skin cancer (NMSC), activates the rapamycin-sensitive mammalian target of rapamycin complex 1 (mTORC1) and the rapamycin-resistant mTORC2 in keratinocytes. Our lab has shown that mTORC2 mediates the survival of UVB-damaged cells. FOXO3a is an important regulator of apoptosis and acts as a tumor suppressor in several types of cancer. We also demonstrated that the UVBinduced cell survival response results from an attenuation of FOXO3a activity by AKT activation, which is reversed by mTORC2 inhibition. FOXO3a is known to promote apoptosis through the coordinated expression of a variety of target genes. To test the direct UVBinduced transcriptional activity of FOXO3a, we used spontaneously immortalized human keratinocytes (HaCaT cells). mTORC2 was inhibited pharmacologically or by shRNA targeting mSIN1, an essential structural protein in mTORC2. FOXO3a activity was modulated by shRNA knockdown. Cells were exposed to an apoptotic dose of UVB in all experiments. Using a luciferase reporter construct to test transcription of the Forkhead Response Element (FHRE), we observed a significant drop in FHRE activity 3h post UVB in control cells compared to their No UVB counterparts (P<0.001). In contrast, FHRE activity was not significantly different with or without UVB exposure when cells were treated with either the TOR kinase inhibitor torin 2 or mSIN1 knockdown, suggesting TORC2 inhibition preserves FOXO3a-dependent transcription. RT-qPCR measured the direct FOXO3a transcriptional targets Trail and Bim, both of which induce apoptosis. Torin 2 treatment increased Trail and Bim expression significantly compared to controls both prior to and 6h after UVB exposure (P<0.0001). This effect was reversed by knockdown of FOXO3a (P<0.0001). These results suggest that Trail and Bim transcription is increased in a FOXO3a-dependent manner when mTORC2 is disrupted. Thus, these gene products may play a role in the increased sensitivity to UVB-induced apoptosis seen with inhibition of mTORC2.
Protein kinase C delta is a central regulator of DNA damage responses G Patil, A Wautlet, J Scheck, C Negro and M Denning Loyola University Chicago, Maywood, IL DNA damage signaling is important to both cancer etiology and therapy since many carcinogens (UV radiation, ionizing radiation) and cancer therapeutics (5-fluorouracil) function by eliciting DNA damage. Protein kinase C delta (PKCd) is a serine/threonine protein kinase activated in response to diverse DNA damaging agents, including UV radiation and ionizing radiation (IR), where it participates in apoptosis and cell cycle checkpoint activation. Furthermore, PKCd expression is lost in squamous carcinomas and functions as a tumor suppressor gene. Since cell cycle checkpoints and apoptosis are coupled to DNA damage repair, we examined the role of PKCd in UVB radiation and IR DNA damage repair, survival and mutagenesis. Wild type mouse embryo fibroblasts (MEFs) exposed to a non-apoptotic dose of UVB (5 mJ/cm2) were competent at repairing cyclopyrimidine dimers (CPDs) as determined by quantitative immunofluorescence microscopy. In contrast, PKCd null MEFs had significantly reduced repair of UVB-induced CPD adducts (p<0.001). Despite this, PKCd null MEFs had significantly improved survival following UVB exposure compared with wild type MEFs, as measured by clonogenic survival assays (p<0.01 at 2 mJ/cm2; p<0.05 at 5, 10 mJ/cm2). Consistent with the lack of CPD repair, PKCd null MEFs had elevated UV-induced mutagenesis frequency. PKCd null MEFs also had a defect in their repair of IR-induced DNA double strand breaks from 5 Gy of X-rays, as determined by neutral comet assays, but also had improved clonogenic survival compared with wild type MEFs (p<0.005 at 2 Gy; p<0.01 at 5 Gy). The reduced repair of both the UVB-induced CPDs and IR-induced DNA double strand breaks could be improved by transduction of full-length PKCd into the PKCd null MEFs. Finally, PKCd null MEFs had reduced p53 serine 15 phosphorylation in response to both UVB and IR, suggesting that PKCd is required for full p53 activation. These results indicate that PKCd is involved in core DNA damage signaling and its loss enhances survival and mutagenesis following DNA damage, potentially contributing to its tumor suppressive functions.
www.jidonline.org S101
568
Optimal ultraviolet wavelength to induce natural regulatory T cells S Muramatsu and A Morita Geriatrick and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan Narrowband ultraviolet B (NB-UVB) phototherapy (311-nm) is widely used to treat refractory skin disorders, such as psoriasis and atopic dermatitis. Recent studies reported that the mechanisms underlying the effects of phototherapy include inhibition of cell proliferation in keratinocytes, induction of apoptosis in pathogenetically relevant cells, and expansion of regulatory T (Treg) cells. Treg cells suppress inflammatory skin disorders. Although broadband UVB (290-320 nm) induces Treg cells, the optimal UV wavelength for inducing Treg is not known. Here we investigated which wavelength effectively induces Treg cells in a mouse model. Shaved C57BL6J mice were irradiated with several UV wavelengths (290 nm, 300 nm, 310 nm, and 320 nm) using a monochromator. After a single dose of 100 mJ/cm2 UV exposure, the lymph nodes and spleen were collected and analyzed with a flow cytometer. After 300 nm and 310 nm UV exposure, CD4+CD25+Foxp3+neuropilin-1+ (Natural Treg: nTreg) cells were increased by 3% to 4% in lymph nodes compared with those in sham irradiated mice (300 nm: from 9.17% to 13.5%. 310 nm: from 7.37% to 10.3%). The optimal UV wavelength range for inducing nTreg was 300 nm to 310 nm. This finding suggests that NB-UVB therapy effectively induces nTreg cells and mediates therapeutic effects in skin diseases.
Neutrophil NETosis is involved in UVB induced-skin inflammation M Liu1,2, M Sharma1,2 and VP Werth1,2 1 University of Pennsylvania, Philadelphia, PA and 2 Philadelphia VAMC, Philadelphia, PA Excessive exposure to UVB in sunlight causes photodamage that triggers autoimmune skin inflammation. Skin inflammation can exacerbate photodamage and, if recurring or longlasting, can induce pathogenic alterations. The responses to UVB exposure include the release of inflammatory cytokines, including TNF-a, from keratinocytes, and the recruitment of inflammatory cells to the skin. Neutrophils are the first group of immune cells to be recruited to the site of photodamage, prior to the infiltration of monocyte/macrophages and lymphocytes. Accumulating evidence indicates the important roles of neutrophils in sterile inflammation, however, little is known about their involvement in UVB-induced skin inflammation. Neutrophil NETosis is a newly characterized neutrophil cell death that releases neutrophil extracellular traps (NETs). NETs have been detected in the lupus skin. NETs exhibit IL-17, and drive type I interferon (IFN) release from pDCs. These two important cytokines interact together to sustain and amplify autoimmune and inflammatory responses in lupus pathogenesis. However, the role of neutrophil NETosis in UVB-induced skin inflammation has not been explored. In the current study, we found that exposure of female C57/BL6 wild-type (WT) mice to UVB (250 mJ/cm2/day) for 5 consecutive days can induce skin inflammation with increased NETotic neutrophils. Our preliminary studies showed that these NETotic structures in the UVB-irradiated skin also exhibit proinflammatory cytokine IL-17. In searching for a relevant stimulus that mediates neutrophil NETosis in UVB-exposed WT mice, we found that TNFa was upregulated in UVB-irradiated skin. In addition, treatment of human primary neutrophils with TNFa in vitro can induce neutrophil NETosis. Therefore, our studies indicate that UVB exposure of mice can induce skin inflammation with increased NETotic neutrophils, which exhibit proinflammatory cytokine IL-17. UVB-induced TNF-a may mediate neutrophil NETosis in the inflamed skin.
569
570
YB-1 interacts with DNp63a in UVB exposed human skin DN Syed, RK Lall, J Chamcheu, Y Soon and H Mukhtar Dermatology, University of Wisconsin, Madison, WI Exposure to ultraviolet (UV) radiation induces skin cancer through dysregulated cell proliferation causing neoplastic transformation. The Y-box binding protein1 (YB-1) performs a variety of cellular functions, including regulation of transcription and translation, DNA repair, drug resistance, and stress responses to extracellular signals. Reduction of YB-1 expression causes growth inhibition or apoptosis in cancer cells indicating its critical role in cell proliferation. The role of YB-1 in UV exposed human skin has not been studied. We observed a significant increase in YB-1 phosphorylation at Ser102 in monolayer cultures of epidermal keratinocytes exposed to UVB (40mJ/cm2:4h) radiation. This data was reciprocated in a 3-D epidermal model comprised of multiple layers of well differentiated keratinocytes where increased phosphorylation of YB-1 was observed at 4 and 24 h post UVB. The epidermal protein DNp63a involved in keratinocyte proliferation and differentiation showed a similar pattern of expression with an initial increase at 4 h followed by downregulation at extended time points. Interestingly the decrease in YB-1/DNp63a expression correlated with apoptosis of UVB-exposed keratinocytes. YB-1-overexpressing keratinocytes exhibited increased DNp63a levels while a significant decrease was noted in YB-1-depleted keratinocytes. Immunoprecipitation studies confirmed functional interaction of YB-1 with DNp63a in UVB exposed keratinocytes. In a chronic UV exposure model, where keratinocytes were subjected to repetitive sub-apoptotic UVB doses (10mJ/cm2), increased expression of YB-1 was associated with upregulation of DNp63a levels. Our studies suggest that YB-1 interaction with DNp63a may be an important regulatory mechanism that modulates the UV response of epidermal keratinocytes. The increase in YB-1/DNp63a expression is likely associated with an initial protective response mounted by keratinocytes following UVB exposure. Our ongoing studies target the precise functionality of this pathway in UV exposed human skin.
The AHR-p27 axis modulates DNA damage responses in UV-irradiated keratinocytes in vitro and in vivo S Shaik, M Pollet, J Krutmann and T Haarmann-Stemmann Leibniz-Research Institute for Environmental Medicine, Du¨sseldorf, Germany The aryl hydrocarbon receptor (AHR) is a ligand-activated and UVB-sensitive transcription factor. Previously, we have demonstrated that the AHR represses apoptosis in UVB-exposed epidermal keratinocytes (KC), indicating that this transcription factor may be critically involved in photocarcinogenesis. In fact, we observed that, in contrast to AHR+/+ mice, AHR-/- animals developed 50% less skin tumors in a chronic UVB irradiation study and thus were largely protected against the carcinogenicity of UVB radiation. In vitro studies further indicated that the AHR down-regulates the tumor suppressor p27 (KIP1) in KC to suppress apoptosis, which may explain the ‘pro-carcinogenic’ action of cutaneous AHR signaling. Now we asked if p27 has a direct impact on UVB-induced DNA damage responses in KC. We found that overexpression of human p27 in HaCaT KC resulted in an enhanced occurrence of UVB-induced apoptosis 24h after UVB-exposure, thus mirroring the effects of chemical and genetic AHR inhibition. Interestingly, overexpression of human p27 also affected the clearance of UVB-induced cyclobutane pyrimidine dimers (CPDs) early (4h) after irradiation, indicating that the AHR may influence both apoptosis and DNA repair by modulating p27. Studies using AHR antagonists and RNAi further demonstrated that the AHR inhibits CPD removal and thus probably NER in HaCaT cells. By silencing xeroderma pigmentosum complementation group A expression and excluding an involvement of early apoptosis, we demonstrated that the acceleration of CPD clearance through AHR inhibition was in fact due to a more pronounced activity of the NER machinery. Importantly, we also observed a higher level of the p27 protein in the skin of AHR-/- mice, which was accompanied by a faster removal of mutagenic CPDs 48h after a single exposure to UVB radiation. These studies further support the concept that the AHR serves as a negative regulator of DNA damage responses in UVB-irradiated KC and thus might be a molecular target for skin cancer prevention.
571
572
FOXO3a-dependent expression of Trail and Bim is regulated by mTORC2 in UVB-induced apoptosis RP Feehan and LM Shantz Cellular and Molecular Physiology, Penn State College of Medicine, Hershey, PA Ultraviolet B (UVB) radiation, the primary cause of non-melanoma skin cancer (NMSC), activates the rapamycin-sensitive mammalian target of rapamycin complex 1 (mTORC1) and the rapamycin-resistant mTORC2 in keratinocytes. Our lab has shown that mTORC2 mediates the survival of UVB-damaged cells. FOXO3a is an important regulator of apoptosis and acts as a tumor suppressor in several types of cancer. We also demonstrated that the UVBinduced cell survival response results from an attenuation of FOXO3a activity by AKT activation, which is reversed by mTORC2 inhibition. FOXO3a is known to promote apoptosis through the coordinated expression of a variety of target genes. To test the direct UVBinduced transcriptional activity of FOXO3a, we used spontaneously immortalized human keratinocytes (HaCaT cells). mTORC2 was inhibited pharmacologically or by shRNA targeting mSIN1, an essential structural protein in mTORC2. FOXO3a activity was modulated by shRNA knockdown. Cells were exposed to an apoptotic dose of UVB in all experiments. Using a luciferase reporter construct to test transcription of the Forkhead Response Element (FHRE), we observed a significant drop in FHRE activity 3h post UVB in control cells compared to their No UVB counterparts (P<0.001). In contrast, FHRE activity was not significantly different with or without UVB exposure when cells were treated with either the TOR kinase inhibitor torin 2 or mSIN1 knockdown, suggesting TORC2 inhibition preserves FOXO3a-dependent transcription. RT-qPCR measured the direct FOXO3a transcriptional targets Trail and Bim, both of which induce apoptosis. Torin 2 treatment increased Trail and Bim expression significantly compared to controls both prior to and 6h after UVB exposure (P<0.0001). This effect was reversed by knockdown of FOXO3a (P<0.0001). These results suggest that Trail and Bim transcription is increased in a FOXO3a-dependent manner when mTORC2 is disrupted. Thus, these gene products may play a role in the increased sensitivity to UVB-induced apoptosis seen with inhibition of mTORC2.
Protein kinase C delta is a central regulator of DNA damage responses G Patil, A Wautlet, J Scheck, C Negro and M Denning Loyola University Chicago, Maywood, IL DNA damage signaling is important to both cancer etiology and therapy since many carcinogens (UV radiation, ionizing radiation) and cancer therapeutics (5-fluorouracil) function by eliciting DNA damage. Protein kinase C delta (PKCd) is a serine/threonine protein kinase activated in response to diverse DNA damaging agents, including UV radiation and ionizing radiation (IR), where it participates in apoptosis and cell cycle checkpoint activation. Furthermore, PKCd expression is lost in squamous carcinomas and functions as a tumor suppressor gene. Since cell cycle checkpoints and apoptosis are coupled to DNA damage repair, we examined the role of PKCd in UVB radiation and IR DNA damage repair, survival and mutagenesis. Wild type mouse embryo fibroblasts (MEFs) exposed to a non-apoptotic dose of UVB (5 mJ/cm2) were competent at repairing cyclopyrimidine dimers (CPDs) as determined by quantitative immunofluorescence microscopy. In contrast, PKCd null MEFs had significantly reduced repair of UVB-induced CPD adducts (p<0.001). Despite this, PKCd null MEFs had significantly improved survival following UVB exposure compared with wild type MEFs, as measured by clonogenic survival assays (p<0.01 at 2 mJ/cm2; p<0.05 at 5, 10 mJ/cm2). Consistent with the lack of CPD repair, PKCd null MEFs had elevated UV-induced mutagenesis frequency. PKCd null MEFs also had a defect in their repair of IR-induced DNA double strand breaks from 5 Gy of X-rays, as determined by neutral comet assays, but also had improved clonogenic survival compared with wild type MEFs (p<0.005 at 2 Gy; p<0.01 at 5 Gy). The reduced repair of both the UVB-induced CPDs and IR-induced DNA double strand breaks could be improved by transduction of full-length PKCd into the PKCd null MEFs. Finally, PKCd null MEFs had reduced p53 serine 15 phosphorylation in response to both UVB and IR, suggesting that PKCd is required for full p53 activation. These results indicate that PKCd is involved in core DNA damage signaling and its loss enhances survival and mutagenesis following DNA damage, potentially contributing to its tumor suppressive functions.
www.jidonline.org S101