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Angustias Page, Manuel Navarro, Marina Garín, Paloma Pérez, M

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Presentation on theme: "Angustias Page, Manuel Navarro, Marina Garín, Paloma Pérez, M"— Presentation transcript:

1 IKKβ Leads to an Inflammatory Skin Disease Resembling Interface Dermatitis 
Angustias Page, Manuel Navarro, Marina Garín, Paloma Pérez, M. Llanos Casanova, Rodolfo Moreno, José L. Jorcano, José L. Cascallana, Ana Bravo, Angel Ramírez  Journal of Investigative Dermatology  Volume 130, Issue 6, Pages (June 2010) DOI: /jid Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 IκB kinase (IKK) and NF-κB activities are increased in K5-IKKβ transgenic (Tg) mice. (a) Diagrammatic representation of the transgene. (b) Western blot analysis of total protein extracts from the back skin of 8-week-old male wild-type (WT) and Tg mice. The membranes were sequentially probed with antibodies for the indicated proteins. β-Actin was used as a loading control. (c) Immunohistochemical analysis of K5-IKKβ transgene expression in back skin of 9-month-old L1 mice. Antibodies against both vesicular stomatitis virus (VSV)-tag and IKKβ gave positive signals in hair follicles (arrowhead) and basal epidermal cells (arrows), in a similar pattern to that obtained for K5; in these staining conditions, endogenous IKKβ is not detected. (d) In vitro kinase assay. Western blots for IKKβ and IKKα are also shown. Protein extracts proceed from female mice. (e) Electrophoretic mobility shift assay (EMSA) analysis of the DNA-binding activity of total protein extracts from the back skin of male mice of the indicated genotypes using a radiolabeled NF-κB oligonucleotide (left panel). WT and L2 protein extracts were subjected to a supershift assay using antibodies against p65 or p50, as indicated (right panel). The components present in each band, as judged by the supershifts obtained, are indicated on the left margin. Numbers below the left panel indicate the mean intensity of the p50/p50 and p50/p65 bands in relation to the WT sample. (f) NF-κB activity estimated by ELISA analysis of p50 binding to NF-κB consensus sequences using back skin extracts of 32-day-old mice. Scale bar=100μm in the photographs of panel c. (*P<0.05, Mann–Whitney (Wilcoxon) W-test.) Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Changes associated with IKKβ overexpression in keratinocytes. (a) The K5-IKKβ transgene is functional in keratinocytes. Keratinocytes (K) and fibroblasts (F) from L1 and wild-type (WT) mice were separated and cultured in vitro, and protein extracts were blotted against the indicated antibodies. (b) WT and transgenic (Tg) keratinocytes respond to tumor necrosis factor-α (TNF-α) treatment with a transient increase in P-p65 and P-IκBα, and a reduction in IκBα, although the baseline (t=0) concentrations of these proteins differ greatly between the Tg and WT mice. (c–d) IKKβ overexpression protects keratinocytes against apoptosis when treated with TNF-α and cycloheximide (CHX). Representative images of cultured cells are shown in (c), and the percentage of surviving cells in (d). Scale bar=500μm. (*P<0.05, Mann–Whitney (Wilcoxon) W-test.) Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Skin phenotype in transgenic (Tg) mice. (a,b) Phenotypic manifestations in Tg mice are shown: erythema in young mice (a) and hair loss and skin lichenification in older mice from L2 line (b). (c) Severe skin phenotype in aged L2 Tg mice. Extended areas of the epidermis are bald, hyperplastic, and show altered hair follicles and severe pigment incontinence (arrows). Insert: higher magnification, showing necrotic basal keratinocytes (arrow) and a region with epidermal clefting by severe vacuolar degeneration of keratinocytes in the basal layer (arrowheads). (d) Lichenoid infiltration associated with vacuolar alteration of the basal epidermal cells in skin from other locations. Note in the Tg tissues the presence of inflammatory cells affecting the basal cell layer and the upper dermis (arrows). The epidermis (E) and dermis (D) are indicated. (e) Ultrastructure of epidermal keratinocytes showing severe vacuolar degeneration of the cytoplasm and nucleus. (f) Increased expression of differentiation markers in altered Tg skin of L2 males. K5 is expressed in all the layers of the epidermis and hair follicles; K6 expression is limited to hyperplastic epidermal layers. Scale bar=500μm in panel c, =50μm in inset of panel c, =5μm in panel e, and =100μm in panels d and f. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Skin inflammation in K5-IKKβ transgenic (Tg) mice. (a–e) Back skin sections of wild-type (WT) and L2 Tg males were stained with antibodies specific for CD45, F4/80, and CD3 (characteristic of infiltrating cells, macrophages, and T lymphocytes, respectively) at different ages (a: P2; b: P13; c: P16; d: P32; e: 4 months for CD45 and F4/80 and 2 months for CD3). Note the increased infiltration of macrophages and other inflammatory cells in the skin of Tg mice from 13 days onwards. Scale bar=200μm in panels a–c and =100μm in panels d and e. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 The phenotype of skin is independent of T and B cells. Gross (a, b) and microscopic appearance (c, d) of skin transplanted onto NOD/SCID mice at 15 weeks after transplantation. IKKβ immunohistochemistry (e, f) and the presence of positive cells for CD45 (g, h) and F4/80 (i, j) antigens are shown. (a, c, e, g, i) K5-IKKβ transgenic (Tg) L2 male transplants. (b, d, f, h, j) Transplants from a wild-type (WT) littermate. In c and d, arrows indicate hair follicles from transplanted skin (pigmented and anagenic) and arrowheads indicate unpigmented and telogenic follicles from receptor origin. In i–j, arrows indicate some positive cells for the indicated staining; arrowheads indicate pigmented hair follicles. Scale bar=500μm in panels c and d and =200μm in panels e–j. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Cytokine and chemokine levels in the serum and in cultured keratinocytes from transgenic mice. (a) Levels of cytokines and chemokines in the serum of 32-day-old transgenic (Tg) and wild-type (WT) male mice. For IL-1α, IL-5, and CCL-2, the actual values have been multiplied by 10 (indicated by × 10), to see more clearly the differences between WT and Tg samples (i.e., the actual average value for IL-1α WT samples is 40pgml–1). (b) Concentration of cytokines and chemokines accumulated in the supernatants after 48hours of culture of the same number of keratinocytes from WT, L1, or L2 mice. The values for GM-CSF and CCL-2 have been divided by 10 and 100, respectively (*P<0.05, Mann–Whitney (Wilcoxon) W-test). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

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