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467 Immunological signature of amyopathic dermatomyositis
ABSTRACTS | Inflammation, Immunity and Infection 466
467
The tryptophan photoproduct FICZ improves atopic dermatitis by enhancement of fillagrin production H Uchi, M Kiyomatsu-Oda and M Furue Dermatology, Kyushu University, Fukuoka, Japan 6-formylindolo[3,2-b]carbazole (FICZ) is a potent endogenous ligand for aryl hydrocarbon receptor (AHR), however, the biological role of FICZ in atopic dermatitis (AD) is unknown. In this study, we demonstrated that FICZ augmented the expression of filaggrin (FLG) in cultured keratinocytes in an AHR-dependent manner, and that topical application of FICZ ameliorated eczema in a mouse model of AD-like dermatitis induced by mite antigen together with improvement of transepidermal water loss (TEWL). Histologically, FICZ significantly reduced the epidermal and dermal thickness as well as the number of mast cells. These findings provide a new strategic basis for developing new drugs for atopic dermatitis.
Immunological signature of amyopathic dermatomyositis C Cassius1,2,3, R Amode1,2, M Jachier1,3, M Branchtein2, M Bagot1,2,3, M Battistella4,3, H Le Buanec2,3 and J Bouaziz1,2,3 1 Dermatology, Hoˆpital Saint-Louis, Paris, France, 2 Skin research institute, Hoˆpital Saint-Louis, INSERM UMR-976, Paris, France, 3 Universite´ Paris Diderot, Paris, France and 4 Pathology, Hoˆpital Saint-Louis, Paris, France Classical dermatomyositis (cDM) is an auto-immune mediated inflammatory myopathy associated with specific skin symptoms. Amyopathic DM (aDM) is characterized by the lack of muscle involvement. cDM is associated with autoantibodies and lymphocyte infiltration in the skin and the muscle. No specific immunological investigation has been performed to date in aDM patients. Eight aDM patients (skin samples, serum and blood mononuclear cells before any suppressive treatment), 19 healthy blood donors (blood mononuclear cells and serum) and 8 normal skin samples (for skin microarray) were analyzed. T cell population phenotyping was performed using mutiparameter flow cytometry (18 colors). Cytokine level in the serum was measured using Luminex technology. RNA was extracted on frozen skin sections for transcriptome analysis (HTA 2.0, Affymetrix). Microarray data were analyzed by Linear Model for Microarray data suite for “R” using student t-test and cytometer data with Mann-Withney U-test. Comparisons between patients and healthy donors revealed: i/ blood modified CD8+ T-cells profile (increased frequency of CD3+CD8+CD28null), decreased frequency of innate cells (iNKT, MAIT cells); ii/ conserved regulatory T-cell blood frequency; iii/ increased serum level of inflammatory cytokines (IL-6, IL-10, TNFa), iv/ skin upregulation of type I interferon response genes and cytotoxicity genes. Our study highlights a systemic inflammation observed in aDM patients, confirms some immunological abnormalities observed in cDM patients and paves the way for the use of new targeted anti-inflammatory systemic and topical drugs in aDM patients.
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Methodological improvement of imiquimod-induced psoriasiform dermatitis model S Horva´th, A´ Keme´ny, R Komlo´di, A Perkecz, C Gyo¨mo¨rei, E Pinte´r and R Gyulai University of Pe´cs, Pe´cs, Hungary While imiquimod (IMQ)-induced skin inflammation is currently the most widely accepted animal model for psoriasis, it has several practical limitations. Especially, unintended systemic consequences (e.g., splenomegaly, weight loss, or animal death) are of major concern, as they may directly or indirectly affect investigational parameters. Therefore, we modified the IMQ-induced psoriasiform dermatitis protocol in order to minimize the systemic effects while maintaining similar skin reactions. In this study we compared the two experimental paradigms e the original (van der Fits, 2009) and our modified method - for the induction of psoriasiform dermatitis. In the original protocol (OP) group, 62.5 mg Aldara cream (5% IMQ) was applied on the shaved back skin of C57BL/6 mice on each day of the 5-day experiment. In control animals vaseline was used. In the modified protocol (MP) group, 20 mg Aldara or vaseline was applied simultaneously in Finn chambers on the dorsal skin of C57BL/6 mice. Skin thickness, blood perfusion, body weight and skin scaling were daily measured. Spleen and skin samples were collected and weighted at the end of the experiment and histopathological alterations were evaluated on H&E stained sections. In the OP group, skin thickness and skin blood perfusion increased by 30-40% and 40%, respectively, on day 3 and 4. However, significant weight loss, spleen enlargement and untimely death was observed in some of the animals. Edema formation and blood perfusion values were similar in the MP group, but with significantly decreased systemic effects and no animal loss. Histology of skin sections clearly showed equivocal hallmarks of psoriasis in both experimental groups. Our new method using Finn chambers for IMQ application leads to no or considerably reduced systemic inflammatory reactions in the animals, while fully reproducing the psoriatic alterations of the skin. Having the psoriasiform and the control skin areas are on the same mice decreases the likelihood of inter-animal differences, and allows for the normalization of systemic IMQ effects.
Xenobiotic metabolism is triggered in atopic dermatitis K Schwabenbauer1, A Elentner1, R Gruber1, M Hermann2, M Schmuth1 and S Dubrac1 1 Department of Dermatology, Venereology and Allergology, Medical University of Innsbruck, Innsbruck, Austria and 2 KMT Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Center for Operative Medicine, Medical University of Innsbruck, Innsbruck, Austria Pollution is known to exacerbate asthma and atopic dermatitis (AD). Moreover, mice overexpressing xenobiotic receptors such as Aryl hydrocarbon receptor (AHR) or pregnane x receptor (PXR), in the epidermis develop AD-like symptoms. Furthermore, we have found increased expression of genes involved in xenobiotic metabolism in the skin of patients with AD but not with ichthyosis vulgaris (IV) by using RNA sequencing technology. To further investigate the possible link between enhanced metabolism of noxious molecules in AD and to validate data from our RNA sequencing analysis, we performed immunohistochemistry and real-time PCR on skin biopsies from AD patients with wild type or mutated fillaggrin or from patients with IV or from healthy controls. We found increased expression of Phase 1 (CYPs) and Phase II (UGTs) enzymes in the skin of AD patients when compared to patients with IV or to healthy controls. Increased xenobiotic metabolism in the skin, resulting from systemic or topical exposure, might trigger cellular processes such as oxidative stress, and cell apoptosis, ultimately leading to or sustaining skin inflammation. Furthermore, data from this work should encourage campaigns to drastically reduce exposure of at-risk populations such as pregnant women and children to xenobiotics. However, the mechanisms by which increased glucuronidation of drugs, pesticides, chemicals and endocrine disruptors trigger skin inflammation remain to be elucidated.
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Cell type-specific SOCS3 expression abnormalities in keratinocytes and macrophages from patients suffering from atopic dermatitis or psoriasis as compared to healthy controls J Zeitvogel, I Klug and T Werfel Hannover Medical School, Hannover, Germany In chronic inflammatory skin diseases a de-regulated immune regulation occurs with a prolonged and increased expression of pro-inflammatory cytokines. Less is known about the impact of regulatory molecules in such diseases. Therefore, it was the aim of this study to investigate the expression profile of Suppressor of Cytokine Signalling (SOCS)3 in inflammatory skin diseases. Skin biopsies from healthy donors and patients suffering from atopic dermatitis or psoriasis were analysed by immunohistological staining for the expression pattern of SOCS3. Moreover, keratinocytes and macrophages derived from patients and healthy controls were checked by qRT-PCR for their expression of SOCS3 mRNA under basal and under inflammatory conditions. We found a reduced expression profile of SOCS3 in the epidermis from lesional atopic dermatitis and psoriasis skin as compared to skin from healthy controls. Cell culture experiments confirmed these findings. A diminished SOCS3 expression was found in keratinocytes from atopic dermatitis patients as compared to cells from healthy donors. Interestingly, macrophages from patients suffering from psoriasis or atopic dermatitis showed an opposed behaviour with increased SOCS3 mRNA expression levels. A promoter reporter assay revealed that in macrophages SOCS3 is mainly regulated on the level of transcription whereas in keratinocytes other mechanisms seemed to be involved. A demethylation assay with the drug 5-Aza-2’-deoxycytidine significantly increased SOCS3 expression in keratinocytes. We concluded that SOCS3 expression is altered in a cell type-specific manner in atopic dermatitis and psoriasis patients which may contribute to the dysregulated immune response seen in inflammatory skin diseases.
S272 Journal of Investigative Dermatology (2017), Volume 137
Isolation and characterization of T cells from human prenatal skin R Reitermaier1, T Ayub1, P Kienzl1, A Scharrer2, M Mildner1, C Worda3, W Eppel3, C Schuster1 and A Elbe-Buerger1 1 Dermatology, Medical University of Vienna, Vienna, Austria, 2 Pathology, Medical University of Vienna, Vienna, Austria and 3 Obstetrics &Gynecology, Medical University of Vienna, Vienna, Austria Adult human skin is home to huge numbers of T cells that as pivotal part of the immune system fulfill important roles in immune surveillance and play a key role in cutaneous immunity. T cells are already present in fetal skin, yet their function remains elusive. The aim of this study is to delineate the nature and significance of fetal skin T cells and to compare it with T cells in adult skin. As T cells are extremely rare in fetal skin we compared established and novel isolation techniques and succeeded to enrich them in sufficient numbers to better characterize them. The highest T cell yield was achieved with an automatic tissue dissociator in combination with an appropriate dissociation kit. This methodology preserved surface markers such as CD4, CD8, CD45RA and CD45RO upon single cell isolation. Flow cytometry analysis showed that prenatal CD3+ T cells were predominantly positive for CD4 and that higher CD8+ T cell percentages were found in fetal compared to adult skin. Further examination showed that most of the fetal skin T cells had a naive phenotype, while adult T cells largely expressed a memory phenotype. Moreover, we were able to expand sizable numbers of fetal T cells in vitro, using different culture conditions, which principally preserved the phenotype before culture. These studies will expand our understanding of the formation of important prerequisites for a functional skin immune system and consequently help to better understand many human skin diseases characterized by T cell activation and proliferation.
467
The tryptophan photoproduct FICZ improves atopic dermatitis by enhancement of fillagrin production H Uchi, M Kiyomatsu-Oda and M Furue Dermatology, Kyushu University, Fukuoka, Japan 6-formylindolo[3,2-b]carbazole (FICZ) is a potent endogenous ligand for aryl hydrocarbon receptor (AHR), however, the biological role of FICZ in atopic dermatitis (AD) is unknown. In this study, we demonstrated that FICZ augmented the expression of filaggrin (FLG) in cultured keratinocytes in an AHR-dependent manner, and that topical application of FICZ ameliorated eczema in a mouse model of AD-like dermatitis induced by mite antigen together with improvement of transepidermal water loss (TEWL). Histologically, FICZ significantly reduced the epidermal and dermal thickness as well as the number of mast cells. These findings provide a new strategic basis for developing new drugs for atopic dermatitis.
Immunological signature of amyopathic dermatomyositis C Cassius1,2,3, R Amode1,2, M Jachier1,3, M Branchtein2, M Bagot1,2,3, M Battistella4,3, H Le Buanec2,3 and J Bouaziz1,2,3 1 Dermatology, Hoˆpital Saint-Louis, Paris, France, 2 Skin research institute, Hoˆpital Saint-Louis, INSERM UMR-976, Paris, France, 3 Universite´ Paris Diderot, Paris, France and 4 Pathology, Hoˆpital Saint-Louis, Paris, France Classical dermatomyositis (cDM) is an auto-immune mediated inflammatory myopathy associated with specific skin symptoms. Amyopathic DM (aDM) is characterized by the lack of muscle involvement. cDM is associated with autoantibodies and lymphocyte infiltration in the skin and the muscle. No specific immunological investigation has been performed to date in aDM patients. Eight aDM patients (skin samples, serum and blood mononuclear cells before any suppressive treatment), 19 healthy blood donors (blood mononuclear cells and serum) and 8 normal skin samples (for skin microarray) were analyzed. T cell population phenotyping was performed using mutiparameter flow cytometry (18 colors). Cytokine level in the serum was measured using Luminex technology. RNA was extracted on frozen skin sections for transcriptome analysis (HTA 2.0, Affymetrix). Microarray data were analyzed by Linear Model for Microarray data suite for “R” using student t-test and cytometer data with Mann-Withney U-test. Comparisons between patients and healthy donors revealed: i/ blood modified CD8+ T-cells profile (increased frequency of CD3+CD8+CD28null), decreased frequency of innate cells (iNKT, MAIT cells); ii/ conserved regulatory T-cell blood frequency; iii/ increased serum level of inflammatory cytokines (IL-6, IL-10, TNFa), iv/ skin upregulation of type I interferon response genes and cytotoxicity genes. Our study highlights a systemic inflammation observed in aDM patients, confirms some immunological abnormalities observed in cDM patients and paves the way for the use of new targeted anti-inflammatory systemic and topical drugs in aDM patients.
468
469
Methodological improvement of imiquimod-induced psoriasiform dermatitis model S Horva´th, A´ Keme´ny, R Komlo´di, A Perkecz, C Gyo¨mo¨rei, E Pinte´r and R Gyulai University of Pe´cs, Pe´cs, Hungary While imiquimod (IMQ)-induced skin inflammation is currently the most widely accepted animal model for psoriasis, it has several practical limitations. Especially, unintended systemic consequences (e.g., splenomegaly, weight loss, or animal death) are of major concern, as they may directly or indirectly affect investigational parameters. Therefore, we modified the IMQ-induced psoriasiform dermatitis protocol in order to minimize the systemic effects while maintaining similar skin reactions. In this study we compared the two experimental paradigms e the original (van der Fits, 2009) and our modified method - for the induction of psoriasiform dermatitis. In the original protocol (OP) group, 62.5 mg Aldara cream (5% IMQ) was applied on the shaved back skin of C57BL/6 mice on each day of the 5-day experiment. In control animals vaseline was used. In the modified protocol (MP) group, 20 mg Aldara or vaseline was applied simultaneously in Finn chambers on the dorsal skin of C57BL/6 mice. Skin thickness, blood perfusion, body weight and skin scaling were daily measured. Spleen and skin samples were collected and weighted at the end of the experiment and histopathological alterations were evaluated on H&E stained sections. In the OP group, skin thickness and skin blood perfusion increased by 30-40% and 40%, respectively, on day 3 and 4. However, significant weight loss, spleen enlargement and untimely death was observed in some of the animals. Edema formation and blood perfusion values were similar in the MP group, but with significantly decreased systemic effects and no animal loss. Histology of skin sections clearly showed equivocal hallmarks of psoriasis in both experimental groups. Our new method using Finn chambers for IMQ application leads to no or considerably reduced systemic inflammatory reactions in the animals, while fully reproducing the psoriatic alterations of the skin. Having the psoriasiform and the control skin areas are on the same mice decreases the likelihood of inter-animal differences, and allows for the normalization of systemic IMQ effects.
Xenobiotic metabolism is triggered in atopic dermatitis K Schwabenbauer1, A Elentner1, R Gruber1, M Hermann2, M Schmuth1 and S Dubrac1 1 Department of Dermatology, Venereology and Allergology, Medical University of Innsbruck, Innsbruck, Austria and 2 KMT Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Center for Operative Medicine, Medical University of Innsbruck, Innsbruck, Austria Pollution is known to exacerbate asthma and atopic dermatitis (AD). Moreover, mice overexpressing xenobiotic receptors such as Aryl hydrocarbon receptor (AHR) or pregnane x receptor (PXR), in the epidermis develop AD-like symptoms. Furthermore, we have found increased expression of genes involved in xenobiotic metabolism in the skin of patients with AD but not with ichthyosis vulgaris (IV) by using RNA sequencing technology. To further investigate the possible link between enhanced metabolism of noxious molecules in AD and to validate data from our RNA sequencing analysis, we performed immunohistochemistry and real-time PCR on skin biopsies from AD patients with wild type or mutated fillaggrin or from patients with IV or from healthy controls. We found increased expression of Phase 1 (CYPs) and Phase II (UGTs) enzymes in the skin of AD patients when compared to patients with IV or to healthy controls. Increased xenobiotic metabolism in the skin, resulting from systemic or topical exposure, might trigger cellular processes such as oxidative stress, and cell apoptosis, ultimately leading to or sustaining skin inflammation. Furthermore, data from this work should encourage campaigns to drastically reduce exposure of at-risk populations such as pregnant women and children to xenobiotics. However, the mechanisms by which increased glucuronidation of drugs, pesticides, chemicals and endocrine disruptors trigger skin inflammation remain to be elucidated.
470
471
Cell type-specific SOCS3 expression abnormalities in keratinocytes and macrophages from patients suffering from atopic dermatitis or psoriasis as compared to healthy controls J Zeitvogel, I Klug and T Werfel Hannover Medical School, Hannover, Germany In chronic inflammatory skin diseases a de-regulated immune regulation occurs with a prolonged and increased expression of pro-inflammatory cytokines. Less is known about the impact of regulatory molecules in such diseases. Therefore, it was the aim of this study to investigate the expression profile of Suppressor of Cytokine Signalling (SOCS)3 in inflammatory skin diseases. Skin biopsies from healthy donors and patients suffering from atopic dermatitis or psoriasis were analysed by immunohistological staining for the expression pattern of SOCS3. Moreover, keratinocytes and macrophages derived from patients and healthy controls were checked by qRT-PCR for their expression of SOCS3 mRNA under basal and under inflammatory conditions. We found a reduced expression profile of SOCS3 in the epidermis from lesional atopic dermatitis and psoriasis skin as compared to skin from healthy controls. Cell culture experiments confirmed these findings. A diminished SOCS3 expression was found in keratinocytes from atopic dermatitis patients as compared to cells from healthy donors. Interestingly, macrophages from patients suffering from psoriasis or atopic dermatitis showed an opposed behaviour with increased SOCS3 mRNA expression levels. A promoter reporter assay revealed that in macrophages SOCS3 is mainly regulated on the level of transcription whereas in keratinocytes other mechanisms seemed to be involved. A demethylation assay with the drug 5-Aza-2’-deoxycytidine significantly increased SOCS3 expression in keratinocytes. We concluded that SOCS3 expression is altered in a cell type-specific manner in atopic dermatitis and psoriasis patients which may contribute to the dysregulated immune response seen in inflammatory skin diseases.
S272 Journal of Investigative Dermatology (2017), Volume 137
Isolation and characterization of T cells from human prenatal skin R Reitermaier1, T Ayub1, P Kienzl1, A Scharrer2, M Mildner1, C Worda3, W Eppel3, C Schuster1 and A Elbe-Buerger1 1 Dermatology, Medical University of Vienna, Vienna, Austria, 2 Pathology, Medical University of Vienna, Vienna, Austria and 3 Obstetrics &Gynecology, Medical University of Vienna, Vienna, Austria Adult human skin is home to huge numbers of T cells that as pivotal part of the immune system fulfill important roles in immune surveillance and play a key role in cutaneous immunity. T cells are already present in fetal skin, yet their function remains elusive. The aim of this study is to delineate the nature and significance of fetal skin T cells and to compare it with T cells in adult skin. As T cells are extremely rare in fetal skin we compared established and novel isolation techniques and succeeded to enrich them in sufficient numbers to better characterize them. The highest T cell yield was achieved with an automatic tissue dissociator in combination with an appropriate dissociation kit. This methodology preserved surface markers such as CD4, CD8, CD45RA and CD45RO upon single cell isolation. Flow cytometry analysis showed that prenatal CD3+ T cells were predominantly positive for CD4 and that higher CD8+ T cell percentages were found in fetal compared to adult skin. Further examination showed that most of the fetal skin T cells had a naive phenotype, while adult T cells largely expressed a memory phenotype. Moreover, we were able to expand sizable numbers of fetal T cells in vitro, using different culture conditions, which principally preserved the phenotype before culture. These studies will expand our understanding of the formation of important prerequisites for a functional skin immune system and consequently help to better understand many human skin diseases characterized by T cell activation and proliferation.