Encapsulated Activated-Grape Seed Extract (ACTIVITIS (TM)) inhibits demethylation of PP2A promoting anti-aging benefits and barrier repair for human skin

S | Epidermal Structure and Barrier Function 108 Antimicrobial peptide hBD-3 improves Th2 cytokine-mediated impairment of tight junction barrier through autophagy activation G Peng, Y Umehara, M Komatsu, K Okumura, H Ogawa, S Ikeda and F Niyonsaba 1 Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, Bunkyo, Tokyo, Japan, 2 Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Bunkyo, Tokyo, Japan, 3 Department of Physiology, Juntendo University Graduate School of Medicine, Bunkyo, Tokyo, Japan and 4 Faculty of International Liberal Arts, Juntendo University Graduate School of Medicine, Bunkyo, Tokyo, Japan Th2 cytokines such as IL-4 and IL-13 attenuate the skin barrier function and modulate autophagy. We previously showed that the antimicrobial peptide human b-defensin (hBD)-3 improves the tight junction (TJ) barrier function in keratinocytes; however, its effect on Th2 cytokine-mediated impairment of TJ barrier function remains unclear. We aimed to evaluate the effects of hBD-3 on IL-4and IL-13-mediated impairment of TJ barrier in human keratinocytes and explore the underlying mechanism. We assessed the expression of autophagy marker LC3, and TJ-related proteins and the signaling pathways in keratinocytes by western blot, autophagosome/autolysosome formation by immunofluorescence and electron microscopy, expression and distribution of TJ-related proteins by real-time PCR and immunofluorescence, respectively. We found that hBD-3 increased the expression of LC3, and enhanced the formation of autophagosomes/autolysosomes in keratinocytes. Besides, hBD-3 induced activation of mTOR and MAPK signaling pathways, which were required for the hBD-3mediated activation of autophagy, as evidenced by the inhibitory effects of their specific inhibitors. hBD-3 also rescued the downregulation of TJ proteins, including claudin-1 and -4 in IL-4+IL-13-treated keratinocytes. Interestingly, autophagy deficiency following infection of keratinocytes with Atg3 mutant adenovirus abolished hBD-3-mediated TJ barrier improvement, suggesting that hBD-3 regulates TJ barrier function through autophagy activation. Our findings provide novel evidence that hBD-3 might be a therapeutic target for the treatment of skin diseases that are characterized by dysfunction of autophagy and skin barrier. S20 Journal of Investigative Dermatology (2021), Volume 141 109 IL-4 and IL-13 cytokines drive sex steroid hormone synthesis and lipid abnormalities in sebocyte during atopic dermatitis pathogenesis C Zhang, M Chinnappan, M Artami, K Eckert, G Vale, J McDonald and T Harris-Tryon 1 Department of Dermatology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States, 2 Department of Molecular Genetics; Center for Human Nutrition, The University of Texas Southwestern Medical Center, Dallas, Texas, United States and 3 Department of Immunology, The University of Texas Southwestern Medical Center, Dallas, Texas, United States One of the hallmark features of atopic dermatitis (AD) is an elevation of the type 2 cytokines, IL-4 and IL-13. Thus, the development of a monoclonal antibody against the IL-4 receptor, Dupilumab, has revolutionized therapy for AD patients. Though the central role of IL-4 and IL-13 in AD is clear, we still have an incomplete understanding of how these immune cytokines drive changes in the skin epithelium. Sebaceous glands are specialized sebum-producing epithelial cells that release a mixture of lipids, free fatty acid, and antimicrobial proteins to the skin surface. Little is known about how sebocyte biology changes in AD. Here we show the impact of type 2 cytokines on sebocytes and find that IL-4 and IL-13 stimulate the expression of 3-beta hydroxysteroid dehydrogenase (HSD3B1). HSD3B1 is a rate-limiting enzyme in sex steroid hormone synthesis. Using liquid chromatography-tandem mass spectrometry, we demonstrate that IL-4 and IL-13 can enhance HSD3B1 dependent androgen production. Further, in an HSD3B1 dependent manner, IL-4 and IL-13 drive lipid abnormalities in human sebocyte cells through regulation of INSIG1 expression. Consistent with our findings in sebocytes, the expression of HSD3B1 is highly elevated in the skin of AD patients and can be restored by Dupilumab treatment. Taken together, these data suggest that targeting sex steroid hormone synthesis pathway could be a potential therapeutic avenue to restore normal skin barrier function in AD patients. 110 Deletion of TNFAIP6 gene in human keratinocytes by CRISPR/Cas9 edition demonstrates a role for TSG-6 to retain hyaluronan inside epidermis C Evrard, E Faway, E De Vuyst, O Svensek, O De Backer, B Flamion, C Lambert de Rouvroit and Y Poumay Universite de Namur, Namur, Belgium TNFa-stimulated gene 6 (TSG-6) protein is found in human skin, where it exerts anti-inflammatory properties and contributes to wound healing. In human epidermis, TSG-6 is mainly secreted in the extracellular matrix between keratinocytes where it interacts with HA. This work aims at better understanding TSG-6 and HA functions in epidermal physiology. Reconstructed human epidermis (RHE) incubated with Th2 interleukins to mimic atopic dermatitis (AD) or RHE infected with Trichophyton rubrum dermatophytes were compared with RHE cultured in normal conditions. In both pathological conditions, mRNA expression levels and protein release of TSG-6 were strongly upregulated in parallel to HA production, suggesting that they might play a role together in challenged epidermal tissues. Potential role was investigated by creating TSG-6 cells using the CRISPR/Cas9 system to edit TNFAIP6 gene in N/TERT keratinocytes, an immortalized human cell line which produces keratinized layers in tissue reconstruction. Two TSG-6 clones harboring major deletions in both alleles of the target gene were used to reconstruct RHE. TSG-6 RHE exhibit normal epidermal morphology with efficient barrier and typical localization of HA and differentiation markers. Their phenotype was further analyzed through RNA sequencing. Despite no alteration in the expression of genes involved in HA metabolism, an increased amount of HA was detected in medium underneath TSG-6 RHE in concomitance with a reduced epidermal HA content, especially in conditions that mimic AD and dermatophytosis. This enhanced HA leakage from either challenged and untreated TSG-6 epidermis is reversed when TSG-6 expression is reintroduced in the tissue, suggesting TSG-6 critical involvement to cross-link and thus retain HA in epidermal extracellular matrix. In addition to other organs and tissues, this work demonstrates overexpression and function of TSG-6 in challenged epidermis. 111 Type XVII collagen modulates epidermal patterning 6 1 6 6,2 6 6 Y Wang , H Kitahata , H Kosumi , M Watanabe , Y Fujimura , S Takashima , S Osada, T Hirose, W Nishie, M Nagayama, H Shimizu and K Natsuga 1 Department of Physics, Chiba University, Chiba, Japan, 2 Department of Life Sciences and Systems Biology, University of Turin, Turin, Italy, 3 Department of Dermatology, Nippon Medical School, Tokyo, Japan, 4 Molecular Biology, Yokohama City University School of Medicine Graduate School of Medicine, Yokohama, Japan, 5 Research Institute for Electronic Science, Hokkaido University, Hokkaido, Japan and 6 Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Many mammals show epidermal patterning, as seen in alternating mouse tail scales and in the microtopography of the human skin surface. However, the mechanisms underlying the development of such patterning are poorly understood. Here, we show that type XVII collagen (COL17), a niche for epidermal stem cells, is a factor in determining epidermal patterning. Col17-null mice exhibit slender epidermal tail scales, a condition that is alleviated by human COL17 overexpression. Aberrant cell polarity, a feature of Col17-null mouse epidermis, does not account for the slender-scale phenotype. Skin regeneration in mice after wounding is accompanied by slender-scale epidermis, a condition that is rescued by human COL17 overexpression. The revertant mosaicism and diseased skin seen in human junctional epidermolysis bullosa demonstrate that COL17-positive and negative skin harbor mutually distinct epidermal patterns. These results demonstrate that COL17 defines mouse tail scale morphology and human skin microtopography. Our study sheds light on the role of the stem cell niche in tissue pattern formation. 112 Identification of a desmoglein-1 reducing component of human stratum corneum contained in wild thyme (Thymus serpyllum) extract A Tada and C Nakahara POLA Chemical Industries, Inc., Yokohama, Japan The outermost layer of the skin, the stratum corneum (SC) has the very important function of preventing excessive transpiration via the epidermis (inner-to-outer barrier) and preventing invasion by foreign substances (outer-to-inner barrier). The SC also contributes to the softness and mechanical strength of the skin. In the histological examination of normal human skin, when observing thin skin sections with hematoxylin/eosin staining, the SC consists of two layers, an outer layer with a basket-weave (BW) structure, and an inner layer with a compact structure. Reportedly, the layer with the BW structure contributes to the barrier function and to SC flexibility. We hypothesized that by identifying the relevant component that develops the BW structure, the barrier function and flexibility of the SC might be restored, leading to healthy skin. Since it is known that a major component of corneodesmosomes is desmoglein 1 (Dsg1), the degradation of which is a necessary process for generating the BW structure of the SC, we performed a quantitative and distribution pattern analysis of Dsg1 as a marker of the completeness of BW structure formation in the SC. Dsg1 is involved in adhesion between SC cells, so we investigated its components after trea