Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2010 Feb 18;5(2):e9258.
doi: 10.1371/journal.pone.0009258.

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin

Alexis Dumortier  1 , André-Dante Durham, Matteo Di Piazza, Sophie Vauclair, Ute Koch, Gisèle Ferrand, Isabel Ferrero, Shadmehr Demehri, Lynda Li Song, Andrew G Farr, Warren J Leonard, Raphael Kopan, Lucio Miele, Daniel Hohl, Daniela Finke, Freddy Radtke
Affiliations

Atopic dermatitis-like disease and associated lethal myeloproliferative disorder arise from loss of Notch signaling in the murine skin

Alexis Dumortier et al. PLoS One. .

Abstract

Background: The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth.

Methodology and principal findings: To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia.

Significance: Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors declare no competing financial interests, except that WJL is an inventor on patent applications related to TSLP.

Figures

Figure 1
Figure 1. Loss of Notch signaling in post-natal epidermis leads to a severe form of atopic dermatitis and lethality.
(A) Representative photograph of Control (Ctrl) and N1N2K5 mice 38 days post first injection of tamoxifen showing loss of hair, thick, dry, and scaly skin. (B) Survival curve of control (Ctrl, n = 20) and N1N2K5 (n = 24) mice after tamoxifen injection. The survival curve is the combined result of 3 individual experiments. (C) Southern blot analysis of genomic DNA from scraped epidermis from control (Ctrl, n = 2) and N1N2K5 mice (n = 3) showing the floxed and the recombined (Recomb) alleles of Notch1 and Notch2 respectively. The recombination efficiency is >70% for both genes. Three individual experiments were performed. (D) Representative HE staining on control (Ctrl) and N1N2K5 dorsal skin sections showing a thickened epidermal layer (e) a massively infiltrated dermis (d) with large epidermoid cysts (c) from degenerated hair follicles (hf) and absence of subcutis (sc) above the muscles (m). Asterisks indicate enlarged regions of the skin showing acanthosis, hyperkeratosis and spongiosis of the epidermis as well as eosinophil infiltrates (arrows) around dilated blood vessels (bv) in the dermis (n = 8, 4 individual experiments were performed). (E) Goldner's Trichrome (GT) readily shows the spongiosis and hyperkeratosis (n = 7, 4 individual experiments were performed). (F) Toluidine blue (TB) staining on control (Ctrl) and N1N2K5 skin sections showing massive infiltration of mast cells (dark blue) (n = 7, 4 individual experiments were performed). (G) Quantitative RT-PCR on dermis-derived RNA for the T helper specific cytokine IL-4 from Ctrl and N1N2K5 mice. The experiment was performed in triplicates (n = 3 per sample group, two individual experiments were performed). (H) A 16-fold increase in serum IgE levels is observed in N1N2K5 compared to Ctrl mice. The experiment was performed in triplicates (n = 3 per sample group, three individual experiments). (* p<0.01; ** p<0.001). [Scale bars: 50 µm].
Figure 2
Figure 2. Notch signaling deficient epidermis massively produces TSLP.
(A) qRT-PCR analysis of inflammatory cytokines on scraped epidermis of control (Ctrl, n = 3), N1N2K5 (n = 3) and RBP-JK5 (n = 3) mice (* p<0.01; ** p<0.001) showing relative increased expression of a wide panel of cytokines in mutant mice. TSLP shows the highest relative increase (125 fold) in mRNA among the tested cytokines. Three individual experiments were performed. (B) Serum TSLP levels in control (Ctrl, n = 4), N1N2K5 (n = 4) and RBP-JK5 (n = 4) mice revealing a 400 fold increase of this cytokine in mutant mice. The experiment was performed in triplicates. (C-D) Control (Ctrl) and N1N2K5 new born skin was grafted onto Athymic nu/nu mice and allowed to grow for 2 months (-Tamoxifen, n = 3). After induction of Cre-mediated recombination (+Tamoxifen, n = 3), the graft develops a similar phenotype to N1N2K5 mice. H/E and Toluidin blue staining shows acanthosis, hyperkeratosis, spongiosis, epidermoid cysts and massive infiltration of mast cells in the dermis of the N1N2K5 derived graft. Three individual experiments were performed. (E) TSLP serum levels of athymic nu/nu mice after grafting the skin of control (Ctrl) or N1N2K5 mice and subsequent gene inactivation. Serum from 3 Ctrl and 3 grafted Athymic nu/nu mice were pooled for the analysis. Bars represent the mean of two individual experiments.
Figure 3
Figure 3. Notch receptor expression is down regulated in skin samples of AD patients.
H/E staining of representative skin sections derived from (A) human control skin (n = 9) and lesional sites from the following human skin disorders: (D) atopic dermatitis (n = 9), (G) psoriasis (n = 4), and (J) lichen planus (n = 4). (B, E, F, H, I, K and L) show panNotch staining using a DL4-IgG fusion protein while (C) shows control staining with a hIgG isotype control antibody. Nuclei are counterstained with DAPI. (E-F) shows down regulation of Notch receptor expression in skin sections from two different AD patients, while (H-I) reveals the presence of Notch expression on sections from two psoriasis patients and (K-L) from two patients suffering from lichen planus. [Scale bars: 50 µm].
Figure 4
Figure 4. N1N2K5 mice develop a myeloproliferative disorder (MPD).
(A) Representative images of spleen and lymph node of control (Ctrl) and N1N2K5 mice showing splenomegaly and lymphadenopathy (n = 12, three individual experiments). (B) HE staining, B220 and CD11b immunofluorescence on spleen sections showing a loss of normal splenic architecture with fibrosis, loss of follicular structures (B220+ B cells) and increase in CD11b+ myeloid cells in N1N2K5 mice (n = 12, three individual experiments). (C) Representative HE staining on liver sections from control and N1N2K5 mice. The liver structure with terminal hepatic venules (v) and portal tracts (T) is changed due to periportal invasion of inflammatory cells and fibrotic reactions in Notch mutant mice (arrows and insert, n = 8, two individual experiments). (D) Representative flow cytometric analysis of splenic myeloid and B cells showing a massive increase in myeloid cells (CD11b+Gr-1int) and loss of follicular (B220+CD23+CD21int) and marginal zone B cells (B220+CD23lo/-CD21+) in N1N2K5 mice. (E) Representative cytometric analysis of bone marrow myeloid and B cells showing an increase in myeloid (CD11b+Gr-1int) and a block of B cell development at the pre-pro B stage (B220+CD43+). Numbers indicate the percentage of cells in each gate. Results are representative of n = 12 per sample group of three individual experiments. [Scale bars: 50 µm].
Figure 5
Figure 5. TSLP is causative of both AD and MPD in N1N2K5 mice.
TSLPR−/− mice were indistinguishable from wild type mice and therefore only the results of TSLPR−/− mice are shown. (A) Representative skin sections of TSLPR−/−, N1N2K5 and N1N2K5 TSLPR−/− mice stained for HE (upper panels), Ki67 (middle panels) and Toluidin blue (lower panels). Notch mutant mice lacking TSLPR have a markedly less proliferative epidermis, do not develop spongiosis and have large a reduction in dermal inflammatory cells (arrows, n = 6 per sample group, three individual experiments). (B) Myeloid cell counts in peripheral blood, and (C) spleen and lymph node (LN) macroscopy from TSLPR−/−, N1N2K5 and N1N2K5TSLPR−/− mice showing a rescue of the MPD phenotype. The bar diagrams represent mean values ± SD (n = 3 for each genotype of mice, three individual experiments). Representative flow cytometric analysis of myeloid and B cells of the spleen (D) and bone marrow (E) from TSLPR−/−, N1N2K5 and N1N2K5 TSLPR−/− mice stained for CD11b and Gr1, CD21 and CD23 (gated on B220+ splenic B cells), or B220 and CD43 (n = 6 per sample group, three individual experiments). [Scale bars: 50 µm].
Figure 6
Figure 6. G-CSF is responsible for the cell non-autonomous development of the TSLP-induced MPD.
(A) EPLM cultures on ST-2 cells in presence of IL-7, or increasing concentrations of rmTSLP showing an increase in B cell numbers, but no increase of myeloid cells. The experiment was performed twice, and each cytokine concentration was analyzed as triplicates. (B) Southern blot analysis of genomic DNA from scraped epidermis from control (Ctrl), RBP-JK5, and RBP-JK5G-CSF+/− mice showing the floxed and the recombined (Recomb) alleles of the RBP-J gene. Recombination efficiency is similar for the two mutant mouse strains. Representative blot for n = 3 mice of each genotype, three individual experiments. (C) HE staining on skin (two upper panels) and spleen (bottom panels) sections from control (Ctrl), RBP-JK5 and RBP-JK5/G-CSF+/− mice. Macroscopy of the spleens in inserts (n = 5 mice per sample group from two individual experiments). (D) Serum G-CSF and TSLP levels in control (Ctrl), RBP-JK5 and RBP-JK5/G-CSF+/− mice (* = p value<0.02). The bar diagrams represent mean values ± SD (n = 5 per sample group of mice from two individual experiments). Representative flow cytometric analysis of myeloid and B cells of the spleen (E) and the bone marrow (F) from control (Ctrl), RBP-JK5 and RBP-JK5/G-CSF+/− mice stained for CD11b and Gr1, or CD21 and CD23 (gated on B220+ splenic B cells), or B220 and CD43 within the BM compartment (n = 5 per sample group from two individual experiments). [Scale bars: 50 µm].
Figure 7
Figure 7. Model for the role of Notch signaling in adult skin and how its loss results in the development of AD, MPD and oteopenia.
Notch receptors are expressed in the suprabasal cell layer of the skin. Skin specific loss of Notch signaling leads to pronounced secretion of TSLP by epithelial cells. High TSLP serum levels in the embryo or neonates cause a cell non-autonomous B-LPD. In contrast, the presence of TSLP in adult mice results in the recruitment of mast cells and eosinophiles within the dermis of Notch mutant mice, thereby contributing to massive inflammation and the development of an AD-like disease. At very high TSLP serum levels, G-CSF is produced by a currently unknown cell type, causing the cell non-autonomous development of MPD and osteopenia.
See this image and copyright information in PMC

References

    1. Fuchs E, Raghavan S. Getting under the skin of epidermal morphogenesis. Nat Rev Genet. 2002;3:199–209. - PubMed
    1. Artavanis-Tsakonas S, Rand MD, Lake RJ. Notch signaling: cell fate control and signal integration in development. Science. 1999;284:770–776. - PubMed
    1. Wilson A, Radtke F. Multiple functions of Notch signaling in self-renewing organs and cancer. FEBS Lett. 2006;580:2860–2868. - PubMed
    1. Ilagan MX, Kopan R. SnapShot: notch signaling pathway. Cell. 2007;128:1246. - PubMed
    1. Rangarajan A, Talora C, Okuyama R, Nicolas M, Mammucari C, et al. Notch signaling is a direct determinant of keratinocyte growth arrest and entry into differentiation. Embo J. 2001;20:3427–3436. - PMC - PubMed

Publication types

MeSH terms