Timed NF-κB Inhibition in Skin Reveals Dual Independent Effects on Development of HED/EDA and Chronic Inflammation  Maria H. Ulvmar, Inderpreet Sur, Sylvie Mémet, Rune Toftgård  Journal of Investigative Dermatology  Volume 129, Issue 11, Pages 2584-2593 (November 2009) DOI: 10.1038/jid.2009.126 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Inhibition of NF-κB during embryogenesis leads to a reversible skin inflammation within the first week after birth. (a) Skin from 4-day-old K5-tTA/TRE-IκBα mice. Skin sections were stained with hematoxylin and eosin or with antibodies F4/80 (macrophages), CalA (neutrophils), and (P-JNK) phosphorylated activated JNK. (b) Skin from 25-day old-mice, in which transgene expression was suppressed by doxycycline (dox) in the drinking water from day 4 after birth. Staining for F4/80 (macrophages) and CalA (neutrophils) show normalization of the skin. Scale bars 50μm. Journal of Investigative Dermatology 2009 129, 2584-2593DOI: (10.1038/jid.2009.126) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 K5-tTA/TRE-IκBα mice show a partial phenocopy of the dl/cr/ta mouse mutants, mimicking human HED/EDA. Hematoxylin and eosin staining of tail skin, paws, eyelid, and tongue. K5-tTA/TRE-IκBα mice lack hair follicles in the tail, lack or have hypoplastic sweat glands in the paws, and lack meibomian glands in the eyelids. Structures indicated by arrows. The mucus (black arrow) and the serous glands (gray arrow) of the tongue are missing or hypoplastic, respectively. Full body pictures show absence of hair behind the ears and on the back in K5-tTA/TRE-IκBα mice. Scale bars 50μm. Journal of Investigative Dermatology 2009 129, 2584-2593DOI: (10.1038/jid.2009.126) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Induction of IκBα after birth leads to the development of inflammation days 20–23 PN. Hematoxylin and eosin staining of dorsal skin from wild-type and K5-tTA/TRE-IκBα mice on days 22, 24, 28, and 36 after birth. Dox was removed on days 2–3 PN. The transgenic skin is inflamed and hyperplastic, with concomitant hyperkeratinization from days 20–23 (here illustrated by samples from day 22), which progress with time. Scale bars 50μm. Journal of Investigative Dermatology 2009 129, 2584-2593DOI: (10.1038/jid.2009.126) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Nuclear exclusion of p50 is associated with the onset of transgene expression and the development of inflammation and hyperplasia. Dorsal skin from 21-day-old wild-type and K5-tTA/TRE-IκBα mice, stained for the p50 and p65 NF-κB subunits. Dox was removed on days 2–3 PN. Scale bars 25μm. Journal of Investigative Dermatology 2009 129, 2584-2593DOI: (10.1038/jid.2009.126) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 F4/80-positive macrophages are the first immune cells to invade K5-tTA/TRE-IκBα transgenic skin after the onset of transgene expression at day 20. Panel showing wild-type and transgenic skin at days 21, 28, and 36 PN. Dox was removed on days 2–3 PN. K6 (keratin 6) marks hyperplastic foci at day 21, which extend to the entire epidermis at days 28 and 37. F4/80 (macrophages) infiltrate at day 21 followed by CalA (neutrophils)-positive cells, here shown at day 28. Scale bars 50μm. Journal of Investigative Dermatology 2009 129, 2584-2593DOI: (10.1038/jid.2009.126) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Upregulation of macrophage-derived VEGFA contribute to blood vessel formation in K5-tTA/TRE-IκBα mice. (a) Frozen sections of skin on day 32 PN, double-stained for F4/80 (macrophages) and VEGFA. The overlay reveals that a large portion of the macrophages express VEGFA. (b) Staining for blood vessels (CD31) and lymphatic vessels (Lyve-1) in transgenic and wild-type skin on day 36 after birth (dox is removed on days 2–3 PN). Representative pictures are shown. Arrows indicate stained vessels. Scale bars 50μm. (c) Histogram showing decrease in the number of vessels in inflamed skin from K5-tTA/TRE-IκBα mice treated with VEGF-Trap. Results from two pairs of littermates: L1 and L2, treated and untreated mice are shown. L1: P=0.009, L2: P=0.001. Journal of Investigative Dermatology 2009 129, 2584-2593DOI: (10.1038/jid.2009.126) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Early upregulation of MCP-1, MCP-2, and MCP-3 in the epidermal compartment of K5-tTA/TRE-IκBα mice. (a) Real-time PCR analysis of TNFα mRNA expression in K5-tTA/TRE-IκBα compared with wild-type mice. (Dox was removed on days 2–3 after birth). No upregulation of TNFα can be seen until late, that is, day 36. (b) Real-time PCR analysis of MCP-1, MCP-2, and MCP-3 in K5-tTA/TRE-IκBα compared with wild-type mice. Upregulation can be seen from day 24 onward. (c) Expression of MCP-1, MCP-2, and MCP-3 in K5-tTA/TRE-IκBα epidermis and dermis relative to wild-type epidermis or dermis, respectively, at day 4 after birth. (The transgene is on throughout development). All the data depicted represent the mean±SD of duplicate samples. Journal of Investigative Dermatology 2009 129, 2584-2593DOI: (10.1038/jid.2009.126) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions