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714 Rhodomyrtone suppresses TNF-α and IL-17A-induced keratinocyte inflammatory responses: A potential therapeutic for psoriasis
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Rhodomyrtone suppresses TNF-a and IL-17A-induced keratinocyte inflammatory responses: A potential therapeutic for psoriasis J Chorachoo1, S Lambert2, LW Roberts3, S Voravuthikunchai1 and A Johnston3 1 Prince of Songkhla University, Hay Yai, Songkhla, Thailand, 2 Univ. Mich, Ann Arbor, MI and 3 University of Michigan, Ann Arbor, MI Psoriasis is a chronic inflammatory skin disease characterized by cytokine-driven epidermal hyperproliferation and inflammation. There is an ongoing need for discovery of new effective therapeutics for psoriasis and medicinal plants are potential sources. Rhodomyrtone is a bioactive plant extract from Rhodomyrtus tomentosa leaves, which has been shown to have anti-proliferative effects. To determine whether rhodomyrtone has potential for countering psoriatic skin inflammation we examined its effects on imiquimod (IMQ)-induced skin inflammation in mice. Rhodomyrtone significantly decreased epidermal hyperplasia (IMQ+vehicle 74.2611.38mm vs. IMQ+2.35mg/in2 rhodomyrtone 45.816.87mm, n¼9 mice/group, day 15, p < 0.001). Using normal human skin organ cultures treated with TNFa+IL-17A to recapitulate the cytokine-driven skin inflammation in psoriasis, we found that 400ng/ml rhodomyrtone significantly decreased DEFB4, IL36G, LCN2, S100A7, and S100A8 mRNA expression (p < 0.0001) as well as HBD-2, IL-36g, and S100A7 protein expression as assessed immunohistochemically, and significantly decreased HBD-2, IL-36g and S100A7 secretion (p < 0.0001, all). Using RNA-seq of monolayer keratinocytes (KC), rhodomyrtone inhibited 194 out of 665 KC genes >2-fold induced by TNF-a, a number of which were confirmed by RT-PCR and ELISA of conditioned media (CXCL1, IL6 and IL8 secretion, p < 0.0001, all). Western blotting revealed that rhodomyrtone inhibited TNF-a-induced ERK, JNK, p38 and NF-kBp65 phosphorylation, suggesting that rhodomyrtone acts by modulating MAP kinase and NF-kB signaling pathways. These results suggest that rhodomyrtone may be useful in preventing or slowing the progression of psoriasis and other inflammatory diseases.
Sorafenib is a new stimulator for human skin-type mast cell degranulation and maturation Y Mizukami1, K Sugawara1 and D Tsuruta2 1 Osaka City University Graduate School of Medicine, Osaka, Japan and 2 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan Sorafenib is known as a multi-kinase inhibitor for the treatment of advanced hepatocellular carcinoma, renal cell carcinoma and thyroid carcinoma by targeting various types of receptors and signaling molecules, including vascular endothelial growth factor receptors, platelet-derived growth factor receptor, c-Kit and Raf-1. Although sorafenib treatment is reported as only approved systemic therapy for advanced hepatocellular cancer, it has also been known to cause diverse cutaneous adverse reactions including hand-foot reaction, facial and scalp eruptions, alopecia and pruritus. However, the mechanism of these adverse effects has not been well-investigated. Mast cells (MC)s are reported to be associated with various types of skin diseases including drug eruption. Here we focused on skin type MC biology by using human skin organ culture system with sorafenib. As a result, sorafenib significantly activated MC degranulation and increased both Kit and tryptase positive skin type MC number in situ. However, sorafenib did not affect MC proliferation and apoptosis. This suggests that, just as neuropeptides including corticotropin-releasing hormone or endocannabinoid, anandamide which we have previously reported, sorafenib stimulated MC maturation from resident precursor cells. Furthermore, sorafenib significantly increased the expression of Akt but not Erk within Kit positive MCs. Importantly, a PI3K inhibitor, wortmannin abrogated the increased number of MCs by sorafenib. This suggests that the compensatory upregulation of PI3Ksignaling was induced by the inhibition of MAP kinase signaling by sorafenib.Sorafenib also significantly upregulated stem cell factor, a well-known MC growth factor expression within the epidermis of organ cultured skin samples. Our results indicate that sorafenib is a new activator for human skin type MCs degranulation and maturation, and MCs could be a new target for treating adverse effects by sorafenib.
Allergen-loaded microneedle patches efficiently induce Treg cells in atopic dermatitis C Park1, S Kim2, JU Shin3, J Kim1, Z Sun1 and K Lee1 1 Yonsei University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 2 Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea and 3 Columbia University Medical Center, New York, NY Regulatory T (Treg) cells are a key player in inducing immune tolerance in atopic dermatitis. There is no efficient method to develop Treg cells rather than subcutaneous route with allergen delivery. Recently, new drug delivery system like microneedle patches is used more conveniently and effectively to deliver drugs into the skin, directly dermis. Therefore, we developed Dermatophagoides farinae (DF)-loaded hyaluronic acid (HA) microneedle patches and confirmed the stability and allergenicity. First, we found that 10 mg of DF-loaded HA microneedle patches successfully delivered allergen into the dermis where MHCII+ antigen presenting cells captured DF within 1 hour after patch application. In DFesensitized atopic dermatitis mouse model, 5-week application of 10 mg DF-loaded microneedle patches reduced Th2 cytokines such as IL-4, IL-13 and significantly induced Foxp3 and IL-10 comparable to 100 ug DF subcutaneous delivery although 10 ug DF subcutaneous delivery did not have any effect on Treg. We also found that 10 mg DF-loaded microneedle patches induced DF-specific IgG4 significantly compared to 10 ug DF subcutaneous delivery. Taken together, we found that DF-loaded HA microneedle patches had 10 times more effective allergen-specific immune tolerance or Treg development than subcutaneous route or epicutaneous patch, through the bypass of 500 Da rule interfering allergen delivery.
Nrf2 pathway is implicated in the antioxidant effect of baicalein on melanocytes of vitiligo patients S Guo1, J Ma2, B Liu2, T Gao3 and C Li3 1 Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China, 2 Department of Dermatology, Xijing Hospital, Fourth Military Medical University, xi’an, Shaanxi, China and 3 Department of Dermatology, Xiijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China Oxidative stress caused by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), which accumulates in the epidermis of vitiligo patients, plays a major role in the loss of melanocytes. Baicalein, as a flavonoid extracted from the Scutellaria baicalensis, has been showed to have anti-oxidant effect. Therefore, we inferred that baicalein could protect melanocytes of vitiligo patients (PIG3V) from oxidative stress. In the present study, we investigated the protective effect and mechanisms of baicalein against H2O2-induced damage of PIG3V. Initially, baicalein significantly inhibited H2O2-induced cytotoxicity and apoptosis in PIG3V. Furthermore, nuclear factor E2-related factor 2 (Nrf2), a major antioxidant pathway, was activated by baicalein. We then knocked down the expression of Nrf2 in PIG3V by lentivirus infection, and found that the protective effect of baicalein on PIG3V against H2O2 was insignificant after Nrf2 suppression. Additionally, the expression of heme oxygenase-1(HO-1), one of the target genes of Nrf2, was decreased in Nrf2 knock-down cells. Furthermore, we found that cells with Nrf2 knock-down were more sensitive to H2O2 and the cell proliferation was also suppressed. In conclusion, baicalein can protect melanocytes of vitiligo patients from oxidative stress via Nrf2/HO-1 pathway.
Mechanistic physiological modeling as a tool for enhancing dermatology research C Friedrich1, K Kudrycki1, M Weis1, M Reed2, M Pryor1 and R Baillie1 1 Rosa & Co. LLC, San Carlos, CA and 2 Rosa & Co. LLC, San Carlos, CA Skin diseases range from benign to life threatening and affect most people at some life stage. Despite recent advances, many mechanistic details of etiology and pathogenesis remain to be elucidated and there is a need to increase research efficiency for advancement of new treatments. Mechanistic physiological modeling can help to meet these needs. Rosa’s PhysioPDÔ Platforms are graphical, mathematical models that incorporate engineering approaches and scientific data analysis to clarify physiology and drug interactions. Simulated experiments can be used to test hypotheses, elucidate the connections between drug action and physiological outcomes, explore the efficacy, toxicity, and therapeutic dose range, select the correct drug targets, and the patient target population. When used in conjunction with preclinical and clinical research, this can provide a rigorous method for advancing dermatologic research. With participation from industry clients, we have studied acne, atopic dermatitis (AD), psoriasis, skin aging, and erythema in over a dozen research projects. For example, in AD, PhysioPD research was used to clarify mechanisms of action and help prioritize acquired assets for development. In acne, in sillico research identified sebogenesis and inflammation as top drivers for acne pathophysiology and demonstrated that only one compound out of three was likely to be superior to the standard of care. In skin aging, a sensitivity analysis highlighted hyaluronic acid and other key drivers for improvements in skin appearance as promising treatment targets. Each research project has elucidated interactions between skin structure/function and immunology and enabled investigation of hypotheses linking pathologies to clinical outcome scores, e.g., SCORAD. In the context of dermatology R&D, mechanistic modeling is an efficient way to increase confidence in a candidate therapy and enables a focused use of resources.
Pericarpium Zanthoxylum extracts alleviate UVB-induced photoaging through activating AMPK/ mTOR signaling in HaCaT Cells D Hao, X Jiang and D Du Department of Dermatology, West China Hospital, SiChuan University, Chengdu, Sichuan, China The Pericarpium Zanthoxylum (PZs) is a plant mainly grows in Chinese Sichuan region, are used in traditional Chinese medicine. The main pathways involved in skin aging, mTOR (mammalian target of rapamycin), which is controlled by the AMPK (adenosine monophosphate activated protein kinase) cascade.The aim of this study was to evaluate the photoprotective effects and antioxidant properties of PZs, and explore its molecular mechanisms on UVB irradiated HaCaT keratinocytes. The cells were exposed to UVB (40mJ/cm2), after irradiation these cells incubated with PZs for 6, 24 and 48 hours, after which supernants, mRNA, DNA and protein samples were taken for analysis. Cell viability was determined by CCK8 assay; the antioxidant potential was assessed the production of ROS by DCFH-DA assay. TNF-alpha, IL-6 and IL-8 in supernatants were quantified by ELISA. The mRNA expressions of inflammation-related genes were evaluated by real time q-PCR. Western blotting was used to identify functions of AMPK signaling for the expression of AMPK and mTOR.The results demonstrate that PZs promote cell proliferation in the range of 5w20 ug/ml , and no significant cytotoxicity until 200 ug/ml (treated for 72h). PZs has a significant antiinflammatory activity, while ROS production shown no significant different in each groups. Furthermore, PZs can stimulate AMPK pathways, as well as suppress mTOR activity, thus presented the protective capacities against UVB irradiation on HaCaT cells. In addition, AMPK inhibitor can restrain the anti-inflammatory capabilities of PZs on UVB-induced HaCaT cells.Taken together, these data suggest that PZs may be useful in the treatment or prevention of photodamaging, probably acting via lowering the secretion of inflammatory cytokines and activing of AMPK /mTOR pathway.
www.jidonline.org S123
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Rhodomyrtone suppresses TNF-a and IL-17A-induced keratinocyte inflammatory responses: A potential therapeutic for psoriasis J Chorachoo1, S Lambert2, LW Roberts3, S Voravuthikunchai1 and A Johnston3 1 Prince of Songkhla University, Hay Yai, Songkhla, Thailand, 2 Univ. Mich, Ann Arbor, MI and 3 University of Michigan, Ann Arbor, MI Psoriasis is a chronic inflammatory skin disease characterized by cytokine-driven epidermal hyperproliferation and inflammation. There is an ongoing need for discovery of new effective therapeutics for psoriasis and medicinal plants are potential sources. Rhodomyrtone is a bioactive plant extract from Rhodomyrtus tomentosa leaves, which has been shown to have anti-proliferative effects. To determine whether rhodomyrtone has potential for countering psoriatic skin inflammation we examined its effects on imiquimod (IMQ)-induced skin inflammation in mice. Rhodomyrtone significantly decreased epidermal hyperplasia (IMQ+vehicle 74.2611.38mm vs. IMQ+2.35mg/in2 rhodomyrtone 45.816.87mm, n¼9 mice/group, day 15, p < 0.001). Using normal human skin organ cultures treated with TNFa+IL-17A to recapitulate the cytokine-driven skin inflammation in psoriasis, we found that 400ng/ml rhodomyrtone significantly decreased DEFB4, IL36G, LCN2, S100A7, and S100A8 mRNA expression (p < 0.0001) as well as HBD-2, IL-36g, and S100A7 protein expression as assessed immunohistochemically, and significantly decreased HBD-2, IL-36g and S100A7 secretion (p < 0.0001, all). Using RNA-seq of monolayer keratinocytes (KC), rhodomyrtone inhibited 194 out of 665 KC genes >2-fold induced by TNF-a, a number of which were confirmed by RT-PCR and ELISA of conditioned media (CXCL1, IL6 and IL8 secretion, p < 0.0001, all). Western blotting revealed that rhodomyrtone inhibited TNF-a-induced ERK, JNK, p38 and NF-kBp65 phosphorylation, suggesting that rhodomyrtone acts by modulating MAP kinase and NF-kB signaling pathways. These results suggest that rhodomyrtone may be useful in preventing or slowing the progression of psoriasis and other inflammatory diseases.
Sorafenib is a new stimulator for human skin-type mast cell degranulation and maturation Y Mizukami1, K Sugawara1 and D Tsuruta2 1 Osaka City University Graduate School of Medicine, Osaka, Japan and 2 Department of Dermatology, Osaka City University Graduate School of Medicine, Osaka, Japan Sorafenib is known as a multi-kinase inhibitor for the treatment of advanced hepatocellular carcinoma, renal cell carcinoma and thyroid carcinoma by targeting various types of receptors and signaling molecules, including vascular endothelial growth factor receptors, platelet-derived growth factor receptor, c-Kit and Raf-1. Although sorafenib treatment is reported as only approved systemic therapy for advanced hepatocellular cancer, it has also been known to cause diverse cutaneous adverse reactions including hand-foot reaction, facial and scalp eruptions, alopecia and pruritus. However, the mechanism of these adverse effects has not been well-investigated. Mast cells (MC)s are reported to be associated with various types of skin diseases including drug eruption. Here we focused on skin type MC biology by using human skin organ culture system with sorafenib. As a result, sorafenib significantly activated MC degranulation and increased both Kit and tryptase positive skin type MC number in situ. However, sorafenib did not affect MC proliferation and apoptosis. This suggests that, just as neuropeptides including corticotropin-releasing hormone or endocannabinoid, anandamide which we have previously reported, sorafenib stimulated MC maturation from resident precursor cells. Furthermore, sorafenib significantly increased the expression of Akt but not Erk within Kit positive MCs. Importantly, a PI3K inhibitor, wortmannin abrogated the increased number of MCs by sorafenib. This suggests that the compensatory upregulation of PI3Ksignaling was induced by the inhibition of MAP kinase signaling by sorafenib.Sorafenib also significantly upregulated stem cell factor, a well-known MC growth factor expression within the epidermis of organ cultured skin samples. Our results indicate that sorafenib is a new activator for human skin type MCs degranulation and maturation, and MCs could be a new target for treating adverse effects by sorafenib.
Allergen-loaded microneedle patches efficiently induce Treg cells in atopic dermatitis C Park1, S Kim2, JU Shin3, J Kim1, Z Sun1 and K Lee1 1 Yonsei University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea, 2 Brain Korea 21 PLUS Project for Medical Science, Yonsei University College of Medicine, Seoul, Seoul-t’ukpyolsi, Republic of Korea and 3 Columbia University Medical Center, New York, NY Regulatory T (Treg) cells are a key player in inducing immune tolerance in atopic dermatitis. There is no efficient method to develop Treg cells rather than subcutaneous route with allergen delivery. Recently, new drug delivery system like microneedle patches is used more conveniently and effectively to deliver drugs into the skin, directly dermis. Therefore, we developed Dermatophagoides farinae (DF)-loaded hyaluronic acid (HA) microneedle patches and confirmed the stability and allergenicity. First, we found that 10 mg of DF-loaded HA microneedle patches successfully delivered allergen into the dermis where MHCII+ antigen presenting cells captured DF within 1 hour after patch application. In DFesensitized atopic dermatitis mouse model, 5-week application of 10 mg DF-loaded microneedle patches reduced Th2 cytokines such as IL-4, IL-13 and significantly induced Foxp3 and IL-10 comparable to 100 ug DF subcutaneous delivery although 10 ug DF subcutaneous delivery did not have any effect on Treg. We also found that 10 mg DF-loaded microneedle patches induced DF-specific IgG4 significantly compared to 10 ug DF subcutaneous delivery. Taken together, we found that DF-loaded HA microneedle patches had 10 times more effective allergen-specific immune tolerance or Treg development than subcutaneous route or epicutaneous patch, through the bypass of 500 Da rule interfering allergen delivery.
Nrf2 pathway is implicated in the antioxidant effect of baicalein on melanocytes of vitiligo patients S Guo1, J Ma2, B Liu2, T Gao3 and C Li3 1 Department of Dermatology, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China, 2 Department of Dermatology, Xijing Hospital, Fourth Military Medical University, xi’an, Shaanxi, China and 3 Department of Dermatology, Xiijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi, China Oxidative stress caused by reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), which accumulates in the epidermis of vitiligo patients, plays a major role in the loss of melanocytes. Baicalein, as a flavonoid extracted from the Scutellaria baicalensis, has been showed to have anti-oxidant effect. Therefore, we inferred that baicalein could protect melanocytes of vitiligo patients (PIG3V) from oxidative stress. In the present study, we investigated the protective effect and mechanisms of baicalein against H2O2-induced damage of PIG3V. Initially, baicalein significantly inhibited H2O2-induced cytotoxicity and apoptosis in PIG3V. Furthermore, nuclear factor E2-related factor 2 (Nrf2), a major antioxidant pathway, was activated by baicalein. We then knocked down the expression of Nrf2 in PIG3V by lentivirus infection, and found that the protective effect of baicalein on PIG3V against H2O2 was insignificant after Nrf2 suppression. Additionally, the expression of heme oxygenase-1(HO-1), one of the target genes of Nrf2, was decreased in Nrf2 knock-down cells. Furthermore, we found that cells with Nrf2 knock-down were more sensitive to H2O2 and the cell proliferation was also suppressed. In conclusion, baicalein can protect melanocytes of vitiligo patients from oxidative stress via Nrf2/HO-1 pathway.
Mechanistic physiological modeling as a tool for enhancing dermatology research C Friedrich1, K Kudrycki1, M Weis1, M Reed2, M Pryor1 and R Baillie1 1 Rosa & Co. LLC, San Carlos, CA and 2 Rosa & Co. LLC, San Carlos, CA Skin diseases range from benign to life threatening and affect most people at some life stage. Despite recent advances, many mechanistic details of etiology and pathogenesis remain to be elucidated and there is a need to increase research efficiency for advancement of new treatments. Mechanistic physiological modeling can help to meet these needs. Rosa’s PhysioPDÔ Platforms are graphical, mathematical models that incorporate engineering approaches and scientific data analysis to clarify physiology and drug interactions. Simulated experiments can be used to test hypotheses, elucidate the connections between drug action and physiological outcomes, explore the efficacy, toxicity, and therapeutic dose range, select the correct drug targets, and the patient target population. When used in conjunction with preclinical and clinical research, this can provide a rigorous method for advancing dermatologic research. With participation from industry clients, we have studied acne, atopic dermatitis (AD), psoriasis, skin aging, and erythema in over a dozen research projects. For example, in AD, PhysioPD research was used to clarify mechanisms of action and help prioritize acquired assets for development. In acne, in sillico research identified sebogenesis and inflammation as top drivers for acne pathophysiology and demonstrated that only one compound out of three was likely to be superior to the standard of care. In skin aging, a sensitivity analysis highlighted hyaluronic acid and other key drivers for improvements in skin appearance as promising treatment targets. Each research project has elucidated interactions between skin structure/function and immunology and enabled investigation of hypotheses linking pathologies to clinical outcome scores, e.g., SCORAD. In the context of dermatology R&D, mechanistic modeling is an efficient way to increase confidence in a candidate therapy and enables a focused use of resources.
Pericarpium Zanthoxylum extracts alleviate UVB-induced photoaging through activating AMPK/ mTOR signaling in HaCaT Cells D Hao, X Jiang and D Du Department of Dermatology, West China Hospital, SiChuan University, Chengdu, Sichuan, China The Pericarpium Zanthoxylum (PZs) is a plant mainly grows in Chinese Sichuan region, are used in traditional Chinese medicine. The main pathways involved in skin aging, mTOR (mammalian target of rapamycin), which is controlled by the AMPK (adenosine monophosphate activated protein kinase) cascade.The aim of this study was to evaluate the photoprotective effects and antioxidant properties of PZs, and explore its molecular mechanisms on UVB irradiated HaCaT keratinocytes. The cells were exposed to UVB (40mJ/cm2), after irradiation these cells incubated with PZs for 6, 24 and 48 hours, after which supernants, mRNA, DNA and protein samples were taken for analysis. Cell viability was determined by CCK8 assay; the antioxidant potential was assessed the production of ROS by DCFH-DA assay. TNF-alpha, IL-6 and IL-8 in supernatants were quantified by ELISA. The mRNA expressions of inflammation-related genes were evaluated by real time q-PCR. Western blotting was used to identify functions of AMPK signaling for the expression of AMPK and mTOR.The results demonstrate that PZs promote cell proliferation in the range of 5w20 ug/ml , and no significant cytotoxicity until 200 ug/ml (treated for 72h). PZs has a significant antiinflammatory activity, while ROS production shown no significant different in each groups. Furthermore, PZs can stimulate AMPK pathways, as well as suppress mTOR activity, thus presented the protective capacities against UVB irradiation on HaCaT cells. In addition, AMPK inhibitor can restrain the anti-inflammatory capabilities of PZs on UVB-induced HaCaT cells.Taken together, these data suggest that PZs may be useful in the treatment or prevention of photodamaging, probably acting via lowering the secretion of inflammatory cytokines and activing of AMPK /mTOR pathway.
www.jidonline.org S123