Conceived and designed the experiments: FR. Performed the experiments: AD ADD MDP SV UK GF IF SD LLS DF. Analyzed the data: RK LM DH. Contributed reagents/materials/analysis tools: AGF WJL RK LM DH. Wrote the paper: FR.
The authors declare no competing financial interests, except that WJL is an inventor on patent applications related to TSLP.
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.
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.
Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.
The skin epidermis and its appendages represent a constantly renewing physical barrier that protects against mechanical injuries, infective organisms and excessive loss of water
Most of our current knowledge regarding Notch signaling in skin and hair follicles is derived from both gain and loss of function studies in primary keratinocytes, or from genetic studies inactivating different Notch signaling components during embryonic development
The role of Notch signaling in adult skin is less clear and has only been partially investigated
Here, we show that simultaneous ablation of Notch1 and Notch2 signaling in the adult skin results in a severe form of atopic dermatitis-like disease as a result of highly elevated levels of TSLP. The AD-like disease is accompanied by a cell non-autonomous G-CSF induced myeloproliferative disorder and osteopenia, all of which is caused by TSLPR mediated signaling.
The function of Notch signaling during skin homeostasis was characterized using mice bearing homozygously floxed alleles for
(
N1N2K5 mice start to lose hair as soon as 1 week post tamoxifen injection and show complete and irreversible hair loss after 1 month (
Histological analysis of Notch mutant mice revealed a massive dermal hypercellularity, a complete loss of subcutaneous fat, the presence of numerous hyperproliferative epidermoid cysts in the dermis and a thickened and hyperkeratinized epidermis (acanthosis and hyperkeratosis) (
Consequently, we investigated more specifically the strong inflammatory response within the Notch deficient epidermis. For this purpose, we selectively isolated RNA from the epidermis of control, N1N2K5 and RBP-JK5 mice and conducted quantitative real-time PCR analysis for a panel of inflammatory cytokines, many of which were significantly increased (
(
It seemed essential to exclude the possibility that the observed phenotypes may be caused by ectopic expression of the Cre recombinase in an organ other than the skin. Thus, we transplanted the skin of control and N1N2K5 newborn pups onto athymic
To investigate whether TSLP expression in the skin is sufficient to cause AD, transgenic mice expressing TSLP under the
Since loss of Notch signaling and TSLP expression are linked in mouse skin, we assessed a possible role for Notch in the etiology of AD in humans. Therefore, we analyzed the presence of Notch receptors with a tagged ligand (DL4-FC)
H/E staining of representative skin sections derived from (
Despite the fact that decreased Notch receptor expression is found in both AD and squamous cell carcinoma (SCC) patients
(
As MPD may occur due to neoplastic alterations intrinsic to hematopoietic cells, we ensured that the phenotype observed was not due to aberrant Cre-recombinase activity in hematopoietic progenitors. To exclude this possibility we generated BM chimeras by transplanting CD45.1+ wild-type BM into CD45.2+ control, N1N2K5 or RBP-JK5 mice. The reconstitution efficiency for control, N1N2K5 and RBP-JK5 chimeras was >80% (data not shown). The hematopoietic system of N1N2K5 (data not shown) and RBP-JK5, but not control, BM chimeras (which are reconstituted from WT cells) developed an identical hematopoietic phenotype to the
We assessed whether excessive TSLP-mediated signaling is indeed responsible for the development of both the AD-like and MPD phenotypes. For this purpose TSLPR−/− mice
TSLPR−/− mice were indistinguishable from wild type mice and therefore only the results of TSLPR−/− mice are shown. (
As TSLP is mostly known to influence and/or promote B cell development
(
The increase of immature granulocytes in the spleen and BM (
However, the median survival of RBP-JK5/G-CSF+/− compared to RBP-JK5 mice doubled (105 versus 52 days, n = 14), suggesting that the primary cause of death of Notch mutant mice is the G-CSF-induced MPD and not the skin disorder.
N1N2K5 and RBP-JK5, (but not N1K5 or N2K5) mice show clinical, histological and cellular features that are typical of human AD. AD is the most common childhood skin disorder
TSLP was the cytokine found to be most dramatically increased in both N1N2K5 and RBP-JK5 mutant mice. TSLP is an IL-7-like cytokine and is able to support B cell differentiation and T cell proliferation
Postnatal inactivation of Notch signaling in murine skin leads to hyperkeratinization of the epidermis, formation of epidermoid cysts in the dermis, and loss of subcutaneous fat. Hyperkeratosis could be the result of deregulated Notch-dependent control of terminal differentiation
Loss of Notch signaling in the developing epidermis of the embryo or neonates results in a loss of epithelial barrier function
However, additional cell intrinsic mechanisms within skin epithelial cells have recently been shown to contribute to TSLP expression in the absence of barrier defects or microbial products
We show that human AD patients have reduced Notch receptor levels on the cell surface of the suprabasal epithelium and they are known to have an increased risk to develop hematological malignancies
TSLP being responsible for the development of the MPD was unexpected. Most reports involve TSLP in B cell homeostasis and/or B cell development, together with its implication in the development of allergic diseases. TSLP was originally described as a cytokine, which leads to increased B cell differentiation when added to BM B cell progenitors
In conclusion, our studies reveal novel aspects of Notch signaling in adult versus embryonic skin; it is essential to control local and systemic inflammatory responses. Loss of Notch signaling in the embryonic skin leads to a cell non-autonomous B-cell lymphoproliferative disease as previously reported
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.
All animal work was conducted according to Swiss national guidelines. All mice were kept in the animal facility under EPFL animal care regulations. They were housed in individual cages at 23±1°C with a 12-h light /dark cycle. All animals were supplied with food and water ad libitum. This study has been reviewed and approved by the Service Vétérinaire Cantonal of Etat de Vaud.
Single-cell suspensions of BM were prepared and stained following standard protocols for fluorescence-activated cell-sorter scanner (FACS) analysis using the following monoclonal antibody conjugates: CD43-FITC (clone S7, BD Pharmingen), Gr-1-PE-Cy7 (clone RB5-8C5, ebiosciences); B220 (RA3.6B2)–Alexa Fluor 647; CD11b (M1/70)-Alexa Fluor 647 and CD45.2 (104)–PE. All antibodies were purified from hybridoma supernatants and conjugated in our laboratory according to standard protocols. Alexa Fluor 647 conjugates were prepared using the appropriate Alexa Fluor protein labeling kits (Invitrogen). PE conjugates were prepared using kits purchased from Prozyme. Single-cell suspensions were stained with the respective antibodies and analyzed using a FACSCalibur, FACSCanto (Becton Dickinson) or CyAn flow cytometer (Dako). Dead cells and debris were eliminated by appropriate gating on forward and side scatter. The data were analyzed using FlowJo (TreeStar, Inc.) software. EPLMs were stained and sorted on a FACSAria flow cytometer (Becton Dickinson) as previously described (Balciunaite et al., 2005). Sorted EPLMs were culture on irradiated (3000 rads) ST2 stromal cells in the presence of recombinant murine TSLP (555-TS-010, R&S Systems).
Keratinocytes were scraped from frozen skin of control or N1N2K5 and RBP-JK5 mice and total RNA was isolated using TRIZOL reagent (Invitrogen). RNA was quantified using a ND-100 NanoDrop spectrophotometer (NanoDrop Technologies). 1 µg of total RNA was reverse-transcribed using the Quantitect reverse transcription kit (Qiagen). 18S was used to control for equal cDNA inputs. Real-time PCR was conducted with a LightCycler system (Roche Diagnostics). Reactions were performed using primers and template mixed with the LightCycle DNA master SYBR Green kit and run for 45 cycles. Specificity of the reactions was determined by subsequent melting curve analysis. LightCycler analysis software was used for quantifications, and background fluorescence was removed using the noise band. The number of cycles needed to reach the crossing point for each sample was used to calculate the amount of each product using the 2-ΔΔCP method. Relative levels of expression were normalized to 18S or HPRT expression.
Paraffin embedded skin samples were sectioned at 4 µm. The sections were dewaxed and antigen retrieval was performed in trisodium citrate solution at 95°C for 20′. Sections were blocked in 1% BSA in TBS-Tween for 45 minutes. 20 µg/ml of Delta4-IgG fusion protein
Additional experimental procedures can be found online as supporting information (
Supplementary Experimental Procedures
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Loss of both Notch1 and Notch2, or RBP-J, but not Notch1 or Notch2 alone, leads to the development of a severe skin phenotype. Representative HE staining, Keratin14 (K14), Keratin1 (K1), Loricrin (Lori) and Ki67 immunohistochemistry and Toluidine Blue (TB) staining on dorsal skin sections from control (Ctrl), N1K5, N2K5, N1N2K5 and RBP-JK5 mice (all within 30-40 days of tamoxifen injection) (n = 8, from 4 individual experiments). Mice lacking Notch2 in the epidermis are indistinguishable from controls, whereas Notch1 deficient mice show K14 expression throughout the epidermis (whereas it is confined to the basal layer in controls), have a hyperproliferative epidermis and a mild increase in toluidine blue positive mast cells. Loss of Notch signaling in N1N2K5 or RBP-JK5 mice leads to an even more hyperproliferative phenotype with Ki67 positive cells throughout the entire epidermis, which is also K14 positive. However, K1 and Loricrin are still expressed in the upper layers of the epidermis. Loss of Notch signaling also leads to a massive dermal infiltration of mast cells [Scale bars: 50 µm].
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Analysis of DCs and other bone marrow derived cells in the skin. (A). Histograms show the staining of CD45 in total dermal cell suspensions from control (Ctrl) or N1N2K5 mice. The percentage of CD45+ cells is indicated. The bar diagram indicates the percentage of T cells (CD3+ cells), B cells (CD19+ cells), dendritic cells (DCs, CD11b+ CD11c+ cells), neutrophils (Ly6G+ cells) and other myeloid cells (CD11b+ CD11c-) within the CD45+ gate in control (Ctrl) or N1N2K5 dermis. Dot plots show analysis of DCs (gated as CD45+ CD11c+) populations in the dermis. CD11b versus CD103 staining displays an increase of dermal Langerin+ DCs (CD11b+ CD103+) in N1N2K5 mice (41.2%) compared to control (Ctrl) mice (21.3%). CD11b versus Ly6C staining shows an increase of inflammatory DCs (CD11b+ LyC+) in N1N2K5 dermis (53.5%) compared to Ctrl dermis (7.5%). Plasmacytoid DCs (CD11b- CD11c+ PDCA1+) were not detected either in control (Ctrl) or N1N2K5 dermis (data not shown). Prior to flow cytometric analysis the CD45 population as well as the populations of DCs were enriched using magnetic MACS cell separation. (B) Histograms show CD45 staining in total epidermal cell suspensions from control (Ctrl) and N1N2K5 mice. The percentage of CD45+ cells is indicated. The bar diagram indicates the relative percentage of murine dendritic epidermal T cells (DETCs, CD3+ Vγ3+), Langerhans cells (LCs, CD11b+ CD11c+), and CD11b+ CD11c- cells within the CD45+ gate in control and N1N2K5 epidermis. Flow cytometric analysis was performed after magnetic MACS cell enrichment. Cells analyzed were a pooled sample size of n = 8 for control or n = 8 for N1N2K5 from two individual experiments.
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Quantitative RT-PCR on dermis-derived RNA for the T helper specific cytokines IFNγ, IL-12, IL-13, IL-17a, IL-21, IL-22 from Ctrl and N1N2K5 mice. Two to four individual experiments were performed (n = 3 per sample group); each experiment was run in triplicates (* p<0.01).
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Skin-specific transgenic TSLP expression is sufficient to induce a similar AD phenotype as the Notch signaling deficient mice. Representative HE staining, Ki67 immunohistochemistry and Toluidine Blue (TB) staining on skin sections from control (Ctrl, n = 4), N1N2K5 (n = 8) and K14-TSLP mice (n = 4). The results shown are representative of 2 individual experiments. Both N1N2K5 and K14-TSLP mice show acanthosis, hyperkeratosis as well as dermal infiltration by mast cells [Scale bars: 50 µm].
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Loss of TSLP signaling or reduction of GCSF levels rescues the MPD. (A) Absolute cell numbers in the spleen of control, N1N2K5, N1N2K5 TSLPR−/−, RPB-JK5 and RBP-JK5/GCSF+/− mice. (B) Splenic absolute cell numbers of immature (CD11b+Gr.1lo/int) and mature (CD11b+Gr.1lo/int) myeloid populations, as well as (C) marginal zone (B220+CD21+CD23lo) and follicular (B220+CD21intCD23+) B cells. (n = 5 per sample group from two individual experiments).
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The MPD in mice lacking Notch signaling in the epidermis is cell non-autonomous. (A) Representative flow cytometric analysis of chimeras (WT CD45.1+ BM cells derived from n = 3 mice were transplanted into 9 lethally irradiated RBP-JK5 mice). WT cells adopt a similar myeloproliferative phenotype in the BM and spleen as in the RBP-JK5 mice. (B) Representative flow cytometric analysis of reverse chimeras (CD45.2+ BM cells derived from 3 sick RBP-JK5 mice were intravenously transplanted into 9 lethally irradiated WT CD45.1+ recipients). RBP-JK5 derived BM cells show normal hematopoiesis in a WT environment. The results in A and B are derived from 3 individual experiments.
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Loss of Notch signaling in the epidermis leads to osteopenia and is due to high G-CSF levels. (A) Representative X-ray analysis of femurs from control (Ctrl) and N1N2K5 mice showing a marked decrease in bone density in the mutant bone. (B) Femur length of control (Ctrl) and N1N2K5 mice (* = p value<0.05, n = 10, three individual experiments). (C) Femur cortical thickness of control (Ctrl) and N1N2K5 mice (* = p value<0.001, n = 10, three individual experiments). (D) Representative HE staining on femoral bone sections from control (Ctrl), N1N2K5, RBP-JK5 and RBP-JK5/G-CSF+/− mice showing a loss of endosteal cells in N1N2K5 and RBP JK5 mice and rescue of this phenotype in the RBP-JK5/G-CSF+/− mice (n = 5 per sample group, two individual experiments). [Scale bars: 50 µm].
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Intact stratum corneum in N1N2K5 mice. Down regulation or loss of LEKTI is frequently observed in skin with altered desquamation, impaired keratinization and skin barrier defects. LEKTI deficiency causes abnormal desmosome cleavage in the upper granular layer through degradation of desmoglein 1. This leads to defective stratum corneum adhesion and thus to the loss of barrier function. Positive staining for (A) the multi-domain serine protease inhibitor LEKTI (lympho epithelial kazal-type inhibitor) and (B) Desmoglein indicates the presence of an intact epidermis in both control (Ctrl) and N1N2K5 mice 5 weeks after gene inactivation (n = 3 mice per sample group, two individual experiments). [Scale bars: 50 µm].
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TSLP is required for B-LPD occurrence in Notch signaling-deficient animals. TSLP binds to a heterodimeric receptor that shares one of its subunits (IL-7rα) with IL-7 receptor. Inhibiting TSLP effects by removing either TSLPR (TSLPR−/−) or IL-7rα (IL7rα−/−) subunit of TSLP receptor leads to disappearance of B-LPD in the mutant mice. (A) WBC counts of RBP-j-deficient animals lacking IL-7rα (Msx2-Cre/+; RBP-jflox/flox; IL-7rα−/− or RBP-jCKO;IL7rα−/−) are within the normal range at P14. (B) Flow cytometric analysis on peripheral blood shows no signs of B-LPD in RBP-jCKO;IL7rα−/− mice compared to their Msx2-Cre/+; RBP-jflox/flox; IL-7Rα+/− (RBP-jCKO) littermates at P14. Representative results are shown with B220+ B cell percentage (red) in the upper right corner of each dot plot. IL-7rα−/− and wild type littermates are also analyzed as controls. Note that WBC counts and B cell percentage of RBP-jCKO;IL7rα−/− and IL-7rα−/− mice are lower than wild-type (IL-7rα+/−) littermates. This may be due to a simultaneous inhibition of IL-7 reception and reduction in baseline B and T cells especially in adulthood. (C) WBC counts of N1N2CKO animals lacking IL-7rα (Msx2-Cre/+; Notch1flox/flox; Notch2flox/flox; IL-7rα−/− or N1N2CKO;IL7rα−/−) are normal at P14. (D) Similar normalization of WBC counts is seen with PSDCKO animals that have lost the TSLPR arm of TSLP receptor (Msx2-Cre/+; PS1flox/flox; PS2flox/flox; TSLPR−/− or PSDCKO;TSLPR−/−). (E, F) B-LPD prevention leads to increased life span among (E) N1N2CKO;IL7rα−/− and (F) PSDCKO;TSLPR−/− animals compared to their N1N2CKO and PSDCKO littermates respectively (p<0.001, log rank test). In each panel, data are compiled from 4 animals in each group from two individual experiments.
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We thank Daniel Metzger and Pierre Chambon for the TSLP-luciferase reporter construct and the