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414 Influence of filaggrin genotype on regulatory T cells in atopic dermatitis
Inflammation, Immunity and Infection | ABSTRACTS 412
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Atopic dermatitis and filaggrin deficiency lead to characteristic shifts of skin microbiome H Baurecht, E Rodrı´guez, F Thielking and S Weidinger Department of Dermatology, Allergology and Venereology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Genomic approaches to characterize skin bacteria have revealed characteristic site-specificities. In addition, in children with AD, shifts of microbial community structures were described at creases and in particular during flares, which have been postulated to mirror the disturbed skin barrier function and/or cutaneous inflammation at these sites. But it is yet unclear whether they are restricted to predilection sites or a general feature of the skin of AD patients. Further, the impact of distinct abnormalities of epidermal barrier integrity and function such as an inherited filaggrin deficiency and of the acuteness of local inflammation on cutaneous microbial community structures have not been examined so far. The skin microbiome was determined by bacterial 16S rRNA sequencing at 4 different body sites of 10 AD patients and 10 healthy controls matched for age, sex and FLG mutation status. In addition, in AD patients acute and chronic eczema lesions were analysed. Independly from the presence of AD, filaggrin deficiency was associated with decrease of diversity and Proteobacteria abundance. Irrespective of the body site, patients with AD showed an increase of Staphilococci with a predominance of S. epidermidis, which was particularly marked in predilection sites. The cutaneous microbiome at acute and chronic lesions is highly similar independently from the body site. Compared to nonlesional skin, there were gradual shifts in diversity from acute to chronic lesions. S. aureus showed a higher abundance on chronic than on acute lesions. Distinct disturbances of epidermal barrier function such as filaggrin deficiency are associated with shifts of the microbial community composition characterized by a reduced diversity and Proteobacteria abundance. In AD, there is a generalized increase of Staphilococci also on non-predilection sites. The impact of inflammation, i.e. affection by eczema, appears to overlay locoregional influences on microbiome composition.
Epigenetics modification and autophagy associated to UV radiations on skin models E Caviola VITROSCREEN, Milan, Italy Human aging is characterized by a low-grade systemic inflammation, a condition that has been designated as “inflamm-aging”. Mediators of the inflammatory UV-induced response can further be associated and induce genetic and epigenetic changes. UV radiations induce autophagy, a catabolic process that regulate cellular response to UV and directly contribute to the process of photoaging. An experimental model on a human 3D reconstructed skin models has been developed in order to reproduce at molecular and morphological level mechanisms associated to photoaging and inflamm-aging process. The protocol is based on the stress induced by UVA radiation (exposure to 12 J/cm2) and UVA +UVB (2MED) delivered by Oriel 1KW Xenon, Spectra Physics Lamp (Mod 6271-Xenon 1000 W). The following miR-21, miR -22 and miR -126 belonging to NF-kB signaling and FOXO, SIRT-1, ATR, AMK, TSC1/2, P62, SESN2, UVRAG genes relevant for autophagy process have been quantified by qRT-PCR. The ratio or amount of methylated DNA has been measured through an ELISA-like reaction. The phosphorylated/not phosphorylated P53 has been investigated by IF and LC3 immunostaining has been performed to identify the morphology of auto phagosome. UVA induced NF-kB activation through miR-21 activation leading to inflammatory response has been observed at early timepoint (6h) and after 24h an increased DNA methylation has been observed. In skin tissues exposed to 2 MED an increased miR-22 expression has been quantified providing a window in time for cells to repair the DNA damage and mitigate the detrimental impact of UV radiation on skin. The activation of autophagy process has been observed and more pronounced after repeated UVA+UVB radiation at gene and protein level. Reference molecules and topical formulations have been tested for their efficacy in protecting from UVA induced damages showing anti-inflammatory activity.
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Influence of filaggrin genotype on regulatory T cells in atopic dermatitis V Moosbrugger-Martinz2, M Bellutti1, K Ladsta¨tter1, M Schmuth1, S Dubrac1 and R Gruber1,3 1 Department of Dermatology, Venereology and Allergology, Medical University of Innsbruck, Innsbruck, Austria, 2 Department of Dermatology, Venereology and Allergology, Medical University of Innsbruck, Innsbruck, Austria and 3 Department of Human Genetics, Medical University of Innsbruck, Innsbruck, Austria Atopic dermatitis (AD) is considered to result from skin barrier impairment coupled with environmental factors and abnormal immune reactivity. Although previous studies describe an important role of regulatory T cells (Tregs) in the pathogenesis of AD, the interaction between Tregs and epidermal barrier dysfunction in AD patients remains unclear. We here report a heterogeneous AD cohort, which has been characterized for filaggrin (FLG) mutation status by next generation sequencing and for phenotypical grading by assessment of EASI score, grade of erythema, transepidermal water loss (TEWL) and total serum IgE. AD patients revealed an increased EASI score, grade of erythema and TEWL, regardless of filaggrin genotype, when compared to healthy controls. Total serum IgE was increased in AD patients without filaggrin mutations when compared to healthy controls and AD patients carrying filaggrin null mutations. For characterization of circulating Tregs we identified demethylation of CpG dinucleotides in a conserved region of FoxP3 intron 1, representing stable Tregs with suppressive function, and percentages of CD4+ CD25+ CD127-/low Tregs in peripheral blood of AD patients and healthy controls. Although our cohort presented no significant differences in percentages of Tregs with demethylated FoxP3i1, we observed an increased proportion of CD4+ CD25+ CD127-/low Tregs within the CD4+ cell population only in FLG wt/wt AD patients. Within the Treg population, proportions of effector CCR4+CD45RAlow Tregs were enhanced, exhibiting increased proportions of Th2 Tregs, whereas proportions of Th1 Tregs were decreased in FLG wt/wt AD patients. Thus, in AD patients neither disease severity nor filaggrin genotype influences levels of suppressive Tregs, while Tregs with plasticity are increased in AD patients without filaggrin mutations.
No difference in UVB induced changes in antigen presenting cells and cytokines between subjects with and without FLG null-mutation S Simonsen1, JP Thyssen1, A Christiansen2, C Bonefeld3, C Geisler3 and L Skov1 1 Dept. of Dermatology and Allergy, Univ. of CPH, CPH, Denmark, 2 Dept. of Ophthalmology, Univ. of CPH, CPH, Denmark and 3 Inst. of International Health, Immunology and Microbiology, Univ. of CPH, CPH, Denmark Epidermal filaggrin deficiency may affect UVB induced immune responses. We examined if common filaggrin gene (FLG) null-mutations were associated with altered responses in antigen presenting cells and cytokines following UVB irradiation of human skin in vivo. Atopic heterozygotic FLG null-mutation carriers and healthy wildtype subjects(WT) were included. Skin on the volar forearm was irradiated with 1.5 minimal erythema dose UVB. Suction blisters were raised on skin irradiated 24 and 72 hours earlier and on non-irradiated control skin(C). Antigen presenting cell numbers and activation markers were examined in epidermal single cell suspensions by flow cytometry. Concentrations of IL-1b, IL-6, IL-10, IL-12p70, TNF-a, TGF-b1, and CCL20 were measured in blister fluid. As expected, UVB irradiation caused a significant decrease in Langerhans cell (LC) numbers, a significant increase in monocyte numbers, and a significant upregulation of activation markers CD86 and OX40 ligand on LCs. These changes did not differ between heterozygotic and WT subjects: deltaLC 24hrs - C; P¼0.44, deltaLC 72hrs - C; P¼0.20, deltaMonocytes 24hrs - C; P¼0.19, deltaMonocytes 72hrs - C; P¼0.38, deltaCD86 24hrs - C; P¼0.38, deltaCD86 72hrs - C; P¼0.10, deltaOX40L 24hrs - C; P¼0.28, deltaOX40L 72hrs - C; P¼0.51(N¼8+8). UVB irradiation upregulated IL-1b, IL-6, and TGF-b1, but these changes were not influenced by FLG mutation status either: deltaIL-1b 24hrs - C; P¼0.84(N¼5+6), deltaIL-1b 72hrs - C; P>0.99(N¼5+6), deltaIL-6 24hrs - C; P¼0.91(N¼5+6), deltaIL-6 72hrs - C; P¼0.91(N¼5+6), deltaTGF-b1 24hrs - C; P¼0.96(N¼5+7), deltaTGF-b1 72hrs - C; P¼0.43(N¼5+7). Taken together, we found no difference in changes of antigen presenting cells and cytokines in UVB irradiated skin from subjects with and without a FLG null-mutation.
Skin allergy caused by air-oxidized terpenes: Mechanism of action and role of transcription factor Nrf2 C Raffalli1,2 1 Universite´ Paris-Saclay, Saint Pierre le`s Nemours, France and 2 Qualiwell SAS, Boulogne Billancourt, France Many fragrance terpenes are considered as low skin sensitizers in allergic contact dermatitis (ACD), a skin pathology in which dendritic cells play a crucial role. On air exposure, those prehaptens autoxidize to form different oxidation products such as epoxides, diols and allylic hydroperoxides. Allylic hydroperoxides are haptens which bind through radical reactions to proteins in the skin to start the immunotoxic process. They are considered as strong skin sensitizers. In this work, we studied how two terpenes, linalool and limonene, and their allylic hydroperoxides could induce the activation of dendritic cells. We tested those molecules on the THP-1 model cell line, as a surrogate of dendritic cells, used in the h-CLAT in vitro test. Using a molecular fluorescent probe (alexa 488 c5 maleimide), we quantified the oxidation of cell-surface thiols by flow cytometry. Our results show that linalool and limonene hydroperoxides decrease cell surface thiols on THP-1 cells after 30 minutes and 1 hour of treatment. Those thiols are then recycled 2 hours post exposure. This oxidation leads to a decrease of GSH/GSSG ratio after 1 hour of treatment, measured by luminescence and an activation of MAPKs (P38 MAPK, JNK, ERK) and NF-KB pathways, measured by western blot. In response to limonene and linalool hydroperoxides, the expression of cell-surface markers CD54 and CD86, measured by flow cytometry, is induced. Mixture of linalool hydroperoxides induces the transcription of il-1b measured by RT-qPCR. Thus, stimulation of THP-1 cells by hydroperoxides leads to a pro-inflammatory phenotype and maturation. Furthermore, the accumulation of Nrf2, 6 hours post stimulation, is directly correlated with a higher Antioxidant Response Element (ARE) transcriptional activity and an induction of Nrf2 target genes (ho-1 and nqo-1) in response to linalool and limonene hydroperoxides. To conclude, our results show that thiols oxidation in response to allylic hydroperoxides trigger different events leading to Nrf2 activation and in fine to the maturation of THP-1 cells.
Inflammatory cytokine mediated induction of serine racemase in atopic dermatitis Y Yoshihisa1, M Nakagawa2, M Rehman3, S Matsukuma2, T Makino1, H Mori4 and T Shimizu1 1 Dermatology, University of Toyama, Toyama, Japan, 2 Advanced Technology Research Center, Fancl Research Institute, Yokohama, Japan, 3 Radiological Sciences, University of Toyama, Toyama, Japan and 4 Molecular Neuroscience, University of Toyama, Toyama, Japan Serine racemase (SR) is an enzyme that catalyzes the conversion of L-serine to pyruvate or Dserine, an endogenous agonist for N-methyl-D-aspartate (NMDA) receptors. Our previous study showed the presence of SR protein in the epidermis of wild type (WT) mice but not in SR knockout (KO) mice. Moreover, SR immune-reactivity was only found in the granular and cornified layers of epidermis in WT mice. It was suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of skin barrier. However, its role in skin barrier dysfunction like atopic dermatitis (AD) remains to be elusive. Therefore, this study was aimed to determine the content of D-serine in stratum corneum in AD patients and healthy controls, using tape stripping method. Skin samples were collected from cheek and upper arm of AD patient’s lesion and healthy persons. Significant difference was found in Dbody ratio between AD involved, uninvolved and healthy control in cheek (p<0.005, p<0.001) and upper-arms (p<0.001, p<0.001), respectively. Furthermore, immune-histochemical analysis also revealed increased SR expression in the epidermis of AD patients compared to healthy control. In addition, keratinocytes stimulation with TNF-a or MIF, results in the increased expression of SR. Taken together, these findings suggest that D-serine particularly express in AD lesional skin and SR expression in the keratinocytes is linked to inflammatory cytokines.
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Atopic dermatitis and filaggrin deficiency lead to characteristic shifts of skin microbiome H Baurecht, E Rodrı´guez, F Thielking and S Weidinger Department of Dermatology, Allergology and Venereology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany Genomic approaches to characterize skin bacteria have revealed characteristic site-specificities. In addition, in children with AD, shifts of microbial community structures were described at creases and in particular during flares, which have been postulated to mirror the disturbed skin barrier function and/or cutaneous inflammation at these sites. But it is yet unclear whether they are restricted to predilection sites or a general feature of the skin of AD patients. Further, the impact of distinct abnormalities of epidermal barrier integrity and function such as an inherited filaggrin deficiency and of the acuteness of local inflammation on cutaneous microbial community structures have not been examined so far. The skin microbiome was determined by bacterial 16S rRNA sequencing at 4 different body sites of 10 AD patients and 10 healthy controls matched for age, sex and FLG mutation status. In addition, in AD patients acute and chronic eczema lesions were analysed. Independly from the presence of AD, filaggrin deficiency was associated with decrease of diversity and Proteobacteria abundance. Irrespective of the body site, patients with AD showed an increase of Staphilococci with a predominance of S. epidermidis, which was particularly marked in predilection sites. The cutaneous microbiome at acute and chronic lesions is highly similar independently from the body site. Compared to nonlesional skin, there were gradual shifts in diversity from acute to chronic lesions. S. aureus showed a higher abundance on chronic than on acute lesions. Distinct disturbances of epidermal barrier function such as filaggrin deficiency are associated with shifts of the microbial community composition characterized by a reduced diversity and Proteobacteria abundance. In AD, there is a generalized increase of Staphilococci also on non-predilection sites. The impact of inflammation, i.e. affection by eczema, appears to overlay locoregional influences on microbiome composition.
Epigenetics modification and autophagy associated to UV radiations on skin models E Caviola VITROSCREEN, Milan, Italy Human aging is characterized by a low-grade systemic inflammation, a condition that has been designated as “inflamm-aging”. Mediators of the inflammatory UV-induced response can further be associated and induce genetic and epigenetic changes. UV radiations induce autophagy, a catabolic process that regulate cellular response to UV and directly contribute to the process of photoaging. An experimental model on a human 3D reconstructed skin models has been developed in order to reproduce at molecular and morphological level mechanisms associated to photoaging and inflamm-aging process. The protocol is based on the stress induced by UVA radiation (exposure to 12 J/cm2) and UVA +UVB (2MED) delivered by Oriel 1KW Xenon, Spectra Physics Lamp (Mod 6271-Xenon 1000 W). The following miR-21, miR -22 and miR -126 belonging to NF-kB signaling and FOXO, SIRT-1, ATR, AMK, TSC1/2, P62, SESN2, UVRAG genes relevant for autophagy process have been quantified by qRT-PCR. The ratio or amount of methylated DNA has been measured through an ELISA-like reaction. The phosphorylated/not phosphorylated P53 has been investigated by IF and LC3 immunostaining has been performed to identify the morphology of auto phagosome. UVA induced NF-kB activation through miR-21 activation leading to inflammatory response has been observed at early timepoint (6h) and after 24h an increased DNA methylation has been observed. In skin tissues exposed to 2 MED an increased miR-22 expression has been quantified providing a window in time for cells to repair the DNA damage and mitigate the detrimental impact of UV radiation on skin. The activation of autophagy process has been observed and more pronounced after repeated UVA+UVB radiation at gene and protein level. Reference molecules and topical formulations have been tested for their efficacy in protecting from UVA induced damages showing anti-inflammatory activity.
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Influence of filaggrin genotype on regulatory T cells in atopic dermatitis V Moosbrugger-Martinz2, M Bellutti1, K Ladsta¨tter1, M Schmuth1, S Dubrac1 and R Gruber1,3 1 Department of Dermatology, Venereology and Allergology, Medical University of Innsbruck, Innsbruck, Austria, 2 Department of Dermatology, Venereology and Allergology, Medical University of Innsbruck, Innsbruck, Austria and 3 Department of Human Genetics, Medical University of Innsbruck, Innsbruck, Austria Atopic dermatitis (AD) is considered to result from skin barrier impairment coupled with environmental factors and abnormal immune reactivity. Although previous studies describe an important role of regulatory T cells (Tregs) in the pathogenesis of AD, the interaction between Tregs and epidermal barrier dysfunction in AD patients remains unclear. We here report a heterogeneous AD cohort, which has been characterized for filaggrin (FLG) mutation status by next generation sequencing and for phenotypical grading by assessment of EASI score, grade of erythema, transepidermal water loss (TEWL) and total serum IgE. AD patients revealed an increased EASI score, grade of erythema and TEWL, regardless of filaggrin genotype, when compared to healthy controls. Total serum IgE was increased in AD patients without filaggrin mutations when compared to healthy controls and AD patients carrying filaggrin null mutations. For characterization of circulating Tregs we identified demethylation of CpG dinucleotides in a conserved region of FoxP3 intron 1, representing stable Tregs with suppressive function, and percentages of CD4+ CD25+ CD127-/low Tregs in peripheral blood of AD patients and healthy controls. Although our cohort presented no significant differences in percentages of Tregs with demethylated FoxP3i1, we observed an increased proportion of CD4+ CD25+ CD127-/low Tregs within the CD4+ cell population only in FLG wt/wt AD patients. Within the Treg population, proportions of effector CCR4+CD45RAlow Tregs were enhanced, exhibiting increased proportions of Th2 Tregs, whereas proportions of Th1 Tregs were decreased in FLG wt/wt AD patients. Thus, in AD patients neither disease severity nor filaggrin genotype influences levels of suppressive Tregs, while Tregs with plasticity are increased in AD patients without filaggrin mutations.
No difference in UVB induced changes in antigen presenting cells and cytokines between subjects with and without FLG null-mutation S Simonsen1, JP Thyssen1, A Christiansen2, C Bonefeld3, C Geisler3 and L Skov1 1 Dept. of Dermatology and Allergy, Univ. of CPH, CPH, Denmark, 2 Dept. of Ophthalmology, Univ. of CPH, CPH, Denmark and 3 Inst. of International Health, Immunology and Microbiology, Univ. of CPH, CPH, Denmark Epidermal filaggrin deficiency may affect UVB induced immune responses. We examined if common filaggrin gene (FLG) null-mutations were associated with altered responses in antigen presenting cells and cytokines following UVB irradiation of human skin in vivo. Atopic heterozygotic FLG null-mutation carriers and healthy wildtype subjects(WT) were included. Skin on the volar forearm was irradiated with 1.5 minimal erythema dose UVB. Suction blisters were raised on skin irradiated 24 and 72 hours earlier and on non-irradiated control skin(C). Antigen presenting cell numbers and activation markers were examined in epidermal single cell suspensions by flow cytometry. Concentrations of IL-1b, IL-6, IL-10, IL-12p70, TNF-a, TGF-b1, and CCL20 were measured in blister fluid. As expected, UVB irradiation caused a significant decrease in Langerhans cell (LC) numbers, a significant increase in monocyte numbers, and a significant upregulation of activation markers CD86 and OX40 ligand on LCs. These changes did not differ between heterozygotic and WT subjects: deltaLC 24hrs - C; P¼0.44, deltaLC 72hrs - C; P¼0.20, deltaMonocytes 24hrs - C; P¼0.19, deltaMonocytes 72hrs - C; P¼0.38, deltaCD86 24hrs - C; P¼0.38, deltaCD86 72hrs - C; P¼0.10, deltaOX40L 24hrs - C; P¼0.28, deltaOX40L 72hrs - C; P¼0.51(N¼8+8). UVB irradiation upregulated IL-1b, IL-6, and TGF-b1, but these changes were not influenced by FLG mutation status either: deltaIL-1b 24hrs - C; P¼0.84(N¼5+6), deltaIL-1b 72hrs - C; P>0.99(N¼5+6), deltaIL-6 24hrs - C; P¼0.91(N¼5+6), deltaIL-6 72hrs - C; P¼0.91(N¼5+6), deltaTGF-b1 24hrs - C; P¼0.96(N¼5+7), deltaTGF-b1 72hrs - C; P¼0.43(N¼5+7). Taken together, we found no difference in changes of antigen presenting cells and cytokines in UVB irradiated skin from subjects with and without a FLG null-mutation.
Skin allergy caused by air-oxidized terpenes: Mechanism of action and role of transcription factor Nrf2 C Raffalli1,2 1 Universite´ Paris-Saclay, Saint Pierre le`s Nemours, France and 2 Qualiwell SAS, Boulogne Billancourt, France Many fragrance terpenes are considered as low skin sensitizers in allergic contact dermatitis (ACD), a skin pathology in which dendritic cells play a crucial role. On air exposure, those prehaptens autoxidize to form different oxidation products such as epoxides, diols and allylic hydroperoxides. Allylic hydroperoxides are haptens which bind through radical reactions to proteins in the skin to start the immunotoxic process. They are considered as strong skin sensitizers. In this work, we studied how two terpenes, linalool and limonene, and their allylic hydroperoxides could induce the activation of dendritic cells. We tested those molecules on the THP-1 model cell line, as a surrogate of dendritic cells, used in the h-CLAT in vitro test. Using a molecular fluorescent probe (alexa 488 c5 maleimide), we quantified the oxidation of cell-surface thiols by flow cytometry. Our results show that linalool and limonene hydroperoxides decrease cell surface thiols on THP-1 cells after 30 minutes and 1 hour of treatment. Those thiols are then recycled 2 hours post exposure. This oxidation leads to a decrease of GSH/GSSG ratio after 1 hour of treatment, measured by luminescence and an activation of MAPKs (P38 MAPK, JNK, ERK) and NF-KB pathways, measured by western blot. In response to limonene and linalool hydroperoxides, the expression of cell-surface markers CD54 and CD86, measured by flow cytometry, is induced. Mixture of linalool hydroperoxides induces the transcription of il-1b measured by RT-qPCR. Thus, stimulation of THP-1 cells by hydroperoxides leads to a pro-inflammatory phenotype and maturation. Furthermore, the accumulation of Nrf2, 6 hours post stimulation, is directly correlated with a higher Antioxidant Response Element (ARE) transcriptional activity and an induction of Nrf2 target genes (ho-1 and nqo-1) in response to linalool and limonene hydroperoxides. To conclude, our results show that thiols oxidation in response to allylic hydroperoxides trigger different events leading to Nrf2 activation and in fine to the maturation of THP-1 cells.
Inflammatory cytokine mediated induction of serine racemase in atopic dermatitis Y Yoshihisa1, M Nakagawa2, M Rehman3, S Matsukuma2, T Makino1, H Mori4 and T Shimizu1 1 Dermatology, University of Toyama, Toyama, Japan, 2 Advanced Technology Research Center, Fancl Research Institute, Yokohama, Japan, 3 Radiological Sciences, University of Toyama, Toyama, Japan and 4 Molecular Neuroscience, University of Toyama, Toyama, Japan Serine racemase (SR) is an enzyme that catalyzes the conversion of L-serine to pyruvate or Dserine, an endogenous agonist for N-methyl-D-aspartate (NMDA) receptors. Our previous study showed the presence of SR protein in the epidermis of wild type (WT) mice but not in SR knockout (KO) mice. Moreover, SR immune-reactivity was only found in the granular and cornified layers of epidermis in WT mice. It was suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of skin barrier. However, its role in skin barrier dysfunction like atopic dermatitis (AD) remains to be elusive. Therefore, this study was aimed to determine the content of D-serine in stratum corneum in AD patients and healthy controls, using tape stripping method. Skin samples were collected from cheek and upper arm of AD patient’s lesion and healthy persons. Significant difference was found in Dbody ratio between AD involved, uninvolved and healthy control in cheek (p<0.005, p<0.001) and upper-arms (p<0.001, p<0.001), respectively. Furthermore, immune-histochemical analysis also revealed increased SR expression in the epidermis of AD patients compared to healthy control. In addition, keratinocytes stimulation with TNF-a or MIF, results in the increased expression of SR. Taken together, these findings suggest that D-serine particularly express in AD lesional skin and SR expression in the keratinocytes is linked to inflammatory cytokines.
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