Oral Administration of Poly-γ-Glutamate Ameliorates Atopic Dermatitis in Nc/Nga Mice by Suppressing Th2-Biased Immune Response and Production of IL-17A

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Oral Administration of Poly-γ-Glutamate Ameliorates Atopic Dermatitis in Nc/Nga Mice by Suppressing Th2-Biased Immune Response and Production of IL-17A Tae-Young Lee, Doo-Jin Kim, Ji-Na Won, Il-Han Lee, Moon-Hee Sung, Haryoung Poo Journal of Investigative Dermatology Volume 134, Issue 3, Pages 704-711 (March 2014) DOI: 10.1038/jid.2013.389 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Poly-γ-glutamate (γ-PGA) activates dendritic cells (DCs) to produce IL-12 in an Nc/Nga mouse model. DCs derived from bone marrow (BM) cells were cultured in the presence of γ-PGA or lipopolysaccharide (LPS). (a) DCs were harvested and the surface expression of CD40, CD80, CD86, and class II major histocompatibility complex (MHC) was analyzed by flow cytometry. (b) The concentration of IL-12 in the culture media was determined by ELISA. Data are presented as the mean±standard error of the mean (SEM), and representative of four independent experiments with similar results. Dex, dexamethasone; PBS, phosphate-buffered saline; PMB, polymyxin B; w/o, without. Journal of Investigative Dermatology 2014 134, 704-711DOI: (10.1038/jid.2013.389) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Oral administration of poly-γ-glutamate (γ-PGA) alleviates atopic dermatitis (AD) symptoms in a dose-dependent manner. (a) Dermatitis index scores were determined every 2 weeks as described in the Materials and Methods section, and are presented as the mean±SEM. Photographs showing representative clinical characteristics were taken at week 15. (b) Scratching behavior was video recorded for 20 min and the episodes were manually counted in a blinded manner. Data are presented as the mean±SEM. (c) Mice (n=10 per group) were given different doses of γ-PGA for 6 weeks, and their clinical symptoms were monitored. Similar results were obtained from three independent experiments. Dex, dexamethasone; PBS, phosphate-buffered saline. *P<0.05, **P<0.01. Journal of Investigative Dermatology 2014 134, 704-711DOI: (10.1038/jid.2013.389) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Serum levels of IgE and IgG1 are negatively regulated by poly-γ-glutamate (γ-PGA) treatment. Nc/Nga mice (n=10 per group) were treated with γ-PGA (400 μg per day) for 6 weeks, and serum samples were taken every 2 weeks for analysis. The levels of (a) serum IgE, (b) IgG1, and (c) IgG2a were measured by ELISA. (d) After the 6-week-long treatments with different doses of γ-PGA, serum levels of IgE were evaluated by ELISA. All data are presented as the mean±SEM and representative of three independent experiments. Dex, dexamethasone; PBS, phosphate-buffered saline. *P<0.05, **P<0.01. Journal of Investigative Dermatology 2014 134, 704-711DOI: (10.1038/jid.2013.389) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Poly-γ-glutamate (γ-PGA) treatment reduces T helper type 2 (Th2) cytokines and IL-17A in atopic dermatitis (AD) skin lesions. At week 15, dorsal skin biopsies were taken from each group (n=10 per group), homogenized, and analyzed for the levels of (a) IL-4, (b) IL-5, and (c) IL-17A. Data are expressed as the mean±SEM and results from three independent experiments. Dex, dexamethasone; PBS, phosphate-buffered saline. *P<0.05. Journal of Investigative Dermatology 2014 134, 704-711DOI: (10.1038/jid.2013.389) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Poly-γ-glutamate (γ-PGA) suppresses tissue inflammation and the accumulation of mast cells in atopic dermatitis (AD) skin lesions. Dorsal skin samples were stained with (a) hematoxylin and eosin (H&E) or (b) toluidine blue and examined under an optical microscope (upper panels, respectively). (b, lower panel) The number of mast cells was counted from 10 randomly selected low-power fields. Data are presented as the mean±SEM, and similar results were obtained in two independent experiments. Scale bar=100 μm. Dex, dexamethasone; PBS, phosphate-buffered saline. *P<0.05. Journal of Investigative Dermatology 2014 134, 704-711DOI: (10.1038/jid.2013.389) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Oral administration of poly-γ-glutamate (γ-PGA) has therapeutic effects on established, higher severity atopic dermatitis (AD) in Nc/Nga mice. Nc/Nga mice (n=5 per group) were housed under conventional conditions for 18 weeks and then orally treated with phosphate-buffered saline (PBS), dexamethasone (Dex) (50 μg per day), or γ-PGA (400 μg per day) for an additional 12 weeks. (a–c) The levels of total IgE, IgG1, and IgG2a were determined from serum samples, respectively. (d–i) Total splenocytes were stimulated with concanavalin A for 48 hours and the concentrations of IL-4, IL-5, IL-10, IL-12, IFN-γ, and IL-17A were measured from the culture media, respectively. All data are expressed as the mean±SEM. Similar results were obtained from two independent experiments. SPF, specific pathogen free. *P<0.05, **P<0.01. Journal of Investigative Dermatology 2014 134, 704-711DOI: (10.1038/jid.2013.389) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions