TSLP Induces Mast Cell Development and Aggravates Allergic Reactions through the Activation of MDM2 and STAT6  Na-Ra Han, Hyun-A Oh, Sun-Young Nam, Phil-Dong Moon, Do-Won Kim, Hyung-Min Kim, Hyun-Ja Jeong  Journal of Investigative Dermatology  Volume 134, Issue 10, Pages 2521-2530 (October 2014) DOI: 10.1038/jid.2014.198 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Population and maturation of mast cells are reduced in thymic stromal lymphopoietin (TSLP)-/-. (a) Several organs were stained with alcian blue and safranine O. Mast cells were counted in five tissue sections randomly selected per mouse (mm2). *P<0.05; significantly different from mast cells of wild-type mice (WT) (C57BL/6). (b) c-Kit+ (phycoerythrin) cells of dorsal skin were examined with a confocal laser-scanning microscope. Bar=10μm. (c) Scanning electron microscopy (SEM) shows morphological differences between cell surface microvilli (arrow) and size in dorsal skin from WT and TSLP-/- (original magnification × 15,000). Bar=3μm. (d) Transmission electron microscopy (TEM) shows numerous secretory granules that have released their contents (arrowheads). Tissues from both WT and TSLP-/- have numerous intact granules (arrow). G, granules; N, nucleus (original magnification × 10,000). Bar=2μm. Journal of Investigative Dermatology 2014 134, 2521-2530DOI: (10.1038/jid.2014.198) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Thymic stromal lymphopoietin (TSLP) induces mast cell development. (a) Bone marrow cells were cultured with IL-3 for 2 weeks and treated with TSLP for 3 days. (b) IC-2 cells were cultured with TSLP or IL-3 for 3 days. Bar=30μm. (c) HMC-1 cells were stimulated with TSLP or neutralizing antibody (Ab) for 2 days. (d) Viability was measured with an 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. (e) Bone marrow cells were gated on live mast cells by flow cytometry. (f) Bone marrow cells were cultured with TSLP or neutralizing Ab for 3 weeks. (g) Bone marrow cells were cultured with TSLP for 2 weeks and treated with IL-3, TSLP, or neutralizing Ab for 3 days. (h) Sorted mast cells were stained with alcian blue and safranine O (A & S) or toluidine blue. Bar=40μm. Journal of Investigative Dermatology 2014 134, 2521-2530DOI: (10.1038/jid.2014.198) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 MDM2 is required for mast cell proliferation. Cells were stimulated with thymic stromal lymphopoietin (TSLP) for (a) 8hours or (b) 48hours and western blot analysis was performed. (c) Caspase-3 activity was measured 48hours after stimulation with TSLP in HMC-1 cells. (d, e) Production was measured 8hours after stimulation with TSLP in HMC-1 cells. PMACI, phorbol 12-myristate 13-acetate (PMA), and calcium ionophore–stimulated cells. MDM2 expression was analyzed in wild-type mice (WT) and TSLP-/- using (f) real-time PCR and (g) western blot analysis. Proliferation was measured with BrdU incorporation assay in transfected (h) bone marrow–derived mast cells (BMMCs) and (i) HMC-1 cells. (j, k) Production was measured 8hours after stimulation with TSLP in transfected HMC-1 cells. B, unstimulated cells; 20+antibody (Ab), neutralizing Ab (20ngml−1) against TSLP was added in the presence of TSLP (20ngml−1). GM-CSF, granulocyte-macrophage colony–stimulating factor. We have adjusted some blot figures. Because of incubating membrane with antibodies in blocking buffer, blots can appear with or without background depending on the quality of antibodies. Journal of Investigative Dermatology 2014 134, 2521-2530DOI: (10.1038/jid.2014.198) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Signal transducers and activators of transcription 6 (STAT6) is required for mast cell development. (a) Thymic stromal lymphopoietin (TSLP) or IL-3 was treated in bone marrow–derived mast cells (BMMCs), IC-2 cells, or HMC-1 cells. (b) pSTAT6+ (FITC) c-Kit+ (phycoerythrin) cells were examined with a confocal laser-scanning microscope. Bar=10μm. (c) BMMCs cultured with TSLP for 2 weeks were treated with neutralizing antibody (Ab) for 3 days. (d, e) Live cells gated by flow cytometry were numbered at 3 weeks. (f) Immunoprecipitation (IP) was performed from nuclear extracts. (g) Cells were treated with leflunomide for 2hours and stimulated with TSLP for 8hours. (h) mRNA and (i) protein expression was analyzed in wild-type mice (WT) (BALB/c) and STAT6-/-. (j) mRNA expression was analyzed in cells cultured with TSLP for a week. We have adjusted some blot figures. Because of incubating membrane with antibodies in blocking buffer, blots can appear with or without background depending on the quality of antibodies. Journal of Investigative Dermatology 2014 134, 2521-2530DOI: (10.1038/jid.2014.198) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Thymic stromal lymphopoietin (TSLP) and signal transducers and activators of transcription 6 (STAT6) promote mast cell–mediated allergic reactions. (a) Intraperitoneal injections were given with compound 48/80 to wild-type mice (WT) or TSLP-/- (n=5). (b) The amount of dye based on passive cutaneous anaphylaxis (PCA) reaction was calculated with a spectrophotometer (n=5). (c) mRNA expression in skin tissues under PCA reaction was analyzed using reverse transcription-PCR analysis. (d) Serum cytokines were measured from mice under PCA reaction by ELISA. (e) Intraperitoneal injections were given with compound 48/80 to WT or STAT6-/- (n=6). (f) Serum histamine was assayed from mice under systemic anaphylactic reaction. (g) The amount of dye based on PCA reaction was calculated with a spectrophotometer (n=6). (h) Skin tissues under PCA reaction were stained by alcian blue–nuclear fast red. (i) Serum cytokines were measured from mice under PCA reaction by ELISA. Journal of Investigative Dermatology 2014 134, 2521-2530DOI: (10.1038/jid.2014.198) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 MDM2 by thymic stromal lymphopoietin (TSLP) increases atopic dermatitis (AD) reactions. (a) MDM2 expression was measured from AD patients with an antibody (Ab) microarray. (b) Levels of MDM2 and p53 were analyzed using western blot analysis. AD, AD patients; NOR, normal controls. (c) mRNA expression was analyzed in DNFB-induced ear lesions. NON, normal dorsal skin (n=4). (d) mRNA (upper) and protein (lower) expression was analyzed in DNFB-induced dorsal lesions (n=4). (e) mRNA and protein expression was analyzed in wild-type mice (WT) and TSLP-/- (n=3). (f) MDM2+ (FITC) c-Kit+ (TRITC) cells were examined with a confocal laser-scanning microscope. Bar=2μm. (g) mRNA expression was analyzed in dorsal lesions of small interfering RNA (siRNA)-injected groups (n=2). (h) TSLP levels in dorsal lesions were analyzed using ELISA. (i–k) Caspase activities and (l) tryptase activity were measured in dorsal lesions. We have adjusted some blot figures. Because of incubating membrane with antibodies in blocking buffer, blots can appear with or without background depending on the quality of antibodies. Journal of Investigative Dermatology 2014 134, 2521-2530DOI: (10.1038/jid.2014.198) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions