Skin-Derived TSLP Triggers Progression from EpidermalBarrier Defects to Asthma
Shadmehr Demehri1, Mitsuru Morimoto1, Michael J. Holtzman2, Raphael Kopan1*
1 Department of Developmental Biology and Division of Dermatology, Washington University School of Medicine, Saint Louis, Missouri, United States of America,
2 Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri, United States of America

Abstract
Asthma is a common allergic lung disease frequently affecting individuals with a prior history of eczema/atopic dermatitis(AD); however, the mechanism underlying the progression from AD to asthma (the so-called ‘‘atopic march’’) is unclear. Here
we show that, like humans with AD, mice with skin-barrier defects develop AD-like skin inflammation and are susceptible toallergic asthma. Furthermore, we show that thymic stromal lymphopoietin (TSLP), overexpressed by skin keratinocytes, isthe systemic driver of this bronchial hyper-responsiveness. As an AD-like model, we used mice with keratinocyte-specificdeletion of RBP-j that sustained high systemic levels of TSLP. Antigen-induced allergic challenge to the lung airways of RBPj–deficient animals resulted in a severe asthmatic phenotype not seen in similarly treated wild-type littermates. Eliminationof TSLP signaling in these animals blocked the atopic march, demonstrating that high serum TSLP levels were required tosensitize the lung to allergic inflammation. Furthermore, we analyzed outbred K14-TSLPtg mice that maintained highsystemic levels of TSLP without developing any skin pathology. Importantly, epidermal-derived TSLP was sufficient totrigger the atopic march, sensitizing the lung airways to inhaled allergens in the absence of epicutaneous sensitization.
Based on these findings, we propose that in addition to early treatment of the primary skin-barrier defects, selective
inhibition of systemic TSLP may be the key to blocking the development of asthma in AD patients.
Citation: Demehri S, Morimoto M, Holtzman MJ, Kopan R (2009) Skin-Derived TSLP Triggers Progression from Epidermal-Barrier Defects to Asthma. PLoS Biol 7(5):e1000067. doi:10.1371/journal.pbio.1000067
Academic Editor: Yong-Jun Liu, MD Anderson Cancer Center, United States of America
Received September 12, 2008; Accepted February 9, 2009; Published May 19, 2009
Copyright: ß 2009 Demehri et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: SD and RK were supported by a grant from the National Institutes of Health-National Institute of General Medical Sciences (GM55479-10). MM was
supported in part by Washington University and by the Toyobo Biotechnology Foundation Long-Term Research Grant and the Japanese Society for the
Promotion of Science. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Abbreviations: AD, atopic dermatitis; BAL, bronchoalveolar lavage; IL, interleukin; OVA, ovalbumin; Th2, T-helper2; TSLP, thymic stromal lymphopoietin.
* E-mail: Kopan@wustl.edu

tends to be the first manifestation of the atopic march [3]. Anothersupportive observation relates to filaggrin, a skin cornified
envelope protein that is absent from the lung epithelia [7,8].
Although controversial [9], it seems that AD patients with filaggrin
loss-of-function mutations exhibit increased incidence of asthma[10–12]. This is consistent with the third hypothesis, that loss of an
epidermal-specific barrier protein can trigger systemic atopy inhumans. Mechanistically, it is suspected that epicutaneous
sensitization with allergens underlies the development of airway
hyperreactivity in mice [13] and in humans with filaggrin
mutations [10,11]. However, it is unclear whether intrinsic skinbarrier defects can trigger asthma in the absence of any
epicutaneous sensitization. If epicutaneous exposure is not
required, it will imply that systemic factor(s) produced by ADskin may be involved in sensitizing the bronchial epithelia. Suchfactors will be important as therapeutic targets in preventing
asthma.
To address this question, we studied mice lacking Notch
signaling in the skin. Skin keratinocytes are organized in highlyinterconnected basal, spinous, granular, and cornified layers,
forming an elaborate barrier protecting the organism from theoutside environment. One of the major molecular regulators of
this structure is Notch signaling [14–16]. Notch is a transmembrane receptor interacting with ligands expressed on the surface ofneighboring keratinocytes [16,17]. Upon activation, sequential

Introduction
Allergic asthma is a chronic lung disease characterized by Thelper2 (Th2)–mediated inflammation and airway obstruction in
response to allergen exposure [1]. Asthma is an increasingly
common disorder affecting more than 300 million individuals
around the world [2]. Atopic dermatitis (AD) is another prevalentallergic disease, and 17% of children in the United States sufferfrom this disorder [3]. In comparison to its 4–8% prevalence in thegeneral population, asthma develops in up to 70% of patients withhistory of severe AD, a phenomenon referred to as ‘‘atopic march’’[3]. It may be possible to prevent asthma in these at-risk
individuals by early diagnosis of AD and blockage of the atopic
march. To achieve this, it is critical that we understand themechanism triggering the development of asthma in AD patients.
A survey of the literature has identified several possible
mechanisms underlying the clinical link between AD and asthma.
These include: (a) a systemic immune system disorder leading toexcessive Th2 response at epithelial surfaces exposed to allergens[4], (b) a barrier defect shared by both skin and lung epithelia that
leads to overstimulation of the immune cells by invading allergens[5], or (c) systemic consequences of a skin-specific barrier defectcausing immune cells to mount an allergic inflammation at any
allergen-exposed epithelial surface [6]. Epidemiological data
supporting the third hypothesis include the observation that AD
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Skin-Derived TSLP Triggers Asthma

manipulation blocked the development of asthma in animals withpersistent AD-like pathology and inflammation. To ask if TSLP
overexpression by skin keratinocytes is sufficient, we used outbred
transgenic mice overexpressing TSLP in epidermal keratinocytes
and showed that epidermal-derived TSLP was sufficient to confera severe asthmatic phenotype even in the absence of any skindefect. These findings establish that high systemic availability of
TSLP [15] can sensitize the lung to allergens, and provide a novel
molecular mechanism for the atopic march. Serum TSLP is thus
an important potential therapeutic target in preventing asthma in
AD patients.

Author Summary
Eczema (atopic dermatitis) is a common allergic skininflammation that has a particularly high prevalence
among children. Importantly, a large proportion of people
suffering from eczema go on to develop asthma later inlife. Although the susceptibility of eczema patients toasthma is well documented, the mechanism that mediates‘‘atopic march’’—the progression from eczema to asthma—is unclear. We used genetic engineering to generate
mice with chronic skin-barrier defects and a subsequenteczema-like disorder. With these mice, we were able toinvestigate how skin-specific defects predisposed thelungs to allergic asthma. We identified thymic stromal
lymphopoietin (TSLP), a cytokine that is secreted bybarrier-defective skin into the systemic circulation, as theagent sensitizing the lung to allergens. We demonstrated
that high systemic levels of skin-derived TSLP were both
required and sufficient to render lung airways hypersensitive to allergens. Thus, these data suggest that early
treatment of skin-barrier defects to prevent TSLP overexpression, and systemic inhibition of TSLP, may be crucial inpreventing the progression from eczema to asthma.

Results
RBP-jCKO Mice Develop an AD-Like Skin Phenotype
The ablation of Notch signaling in skin keratinocytes byremoving RBP-j severely impairs the differentiation of basal layer
keratinocytes and maintenance of upper spinous and granular cell
layers [15,16]. Such a defect in epidermal stratification leads to an
aberrant skin-barrier function signified by transepidermal water
loss and penetration of dye through the defective barrier in RBP-j–
deficient mice at birth [15,16]. After birth, the persistence ofbarrier defects in RBP-j–deficient skin is evident by the presence ofreactive epidermal hyperplasia, TSLP overexpression, and upregulation of antimicrobial peptides (Figure S2 and Table S1)[15,16,24,25]. Defective skin-barrier function has been suggested
to be a hallmark of AD [6,7]. In agreement with this notion, theimpaired skin-barrier function in RBP-jCKO mice initiated an
inflammatory cascade culminating in the development of an ADlike skin phenotype. The hyperplastic epidermis, acanthosis,
hyperkeratosis, parakeratosis, and mast cell infiltration were
evident in RBP-jCKO skin as early as 1 wk after birth, followedby dramatic dermal mast cell accumulation, serum IgE elevation,and systemic Th2 cell expansion in adult RBP-j–deficient animals(Figure 1A–1C). Therefore, adult RBP-jCKO mice resemble
humans with AD to a degree that allows us to examine thesystemic consequences of an allergic inflammation in the skin.

proteolysis of Notch releases its intracellular domain, which thentranslocates into the nucleus, binds to RBP-j (the DNA-binding
partner of Notch), and activates downstream targets [17].
Keratinocyte-specific deletion of Notch signaling pathway components impairs epidermal differentiation, resulting in skin-barrier
defects [15,16]. We have recently shown that Notch signaling loss
in the skin also triggers a severe neonatal B-lymphoproliferative
disorder (B-LPD; [15]). This systemic disease is directly caused byelevated levels of thymic stromal lymphopoietin (TSLP) released
into the circulation by Notch-deficient keratinocytes failing todifferentiate [15]. TSLP, a general biomarker for skin-barrier
defects [15], is an interleukin-7 (IL-7)–like cytokine produced byepithelial cells and is implicated in the pathogenesis of both AD
and asthma [18–22]. TSLP expression is sustained as long as
barrier defects persist [15]. Thus, the potentially high systemic
availability of skin-derived TSLP and its central role in promoting
asthma bring up the possibility that TSLP may be the factor
predisposing AD patients to asthma.
Identifying a clear mechanism for the atopic march in thecomplex network of genetic, immunological, and environmental
factors that contribute to AD has been a challenge to the field andhas resulted in much debate as to the right therapeutic approachfor preventing asthma [6]. To determine the mechanisms
underlying the progression from AD to asthma, we first generated
animals that lacked Notch signaling in a portion of their skinsurfaces by embryonic removal of RBP-j from keratinocytes using
the Msx2-Cre transgene (Msx2-Cre/+;RBP-jflox/flox or RBP-jCKO).
Msx2-Cre is ectopically expressed at embryonic day 9.5 (E9.5) inclusters of ectodermal cells, resulting in a chimeric pattern of RBP-jdeletion in the skin [23]. The presence of unaffected skin surfaces
allowed RBP-jCKO animals to live for approximately 100 days on
average with a few surviving up to one year (Figure S1) [15,16],
presenting a model in which to determine whether a defectiveskin-barrier could cause AD-like symptoms and render susceptibility to asthma in the absence of epicutaneous sensitization. We
found that defective skin-barrier function in adult RBP-j–deficientanimals caused the development of an AD-like allergic inflammation and a subsequent susceptibility to asthma. To determine
whether TSLP is required for this susceptibility, we deleted the
TSLP receptor in RBP-jCKO mice and showed that this genetic
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AD-Like Skin Disease Predisposes RBP-jCKO Animals toAllergic Asthma
The lungs of 10-wk-old mice with RBP-j–deficient skins werenormal and did not show any sign of allergic inflammation understandard housing conditions; however, the longest-living mutant
animals (52 wk) did develop spontaneous lung inflammation(Figure 1D). To determine whether this phenotype was a trueindicator of increased susceptibility to an asthmatic phenotype, weused an ovalbumin (OVA)-induced model of allergic inflammation
with 5- to 7-wk-old RBP-jCKO mice. This protocol faithfully
mimics the development of asthma in humans [18]. OVA-treatedRBP-jCKO animals developed a more severe lung inflammationcompared with OVA-treated wild-type littermates (Figure 2).
Although the wild-type animals tolerated the intranasal OVA
challenge well, two out of ten OVA-treated RBP-jCKO mice diedduring this procedure following a period of severe laboredbreathing. In the surviving mutants, the number of bronchoalveolar lavage (BAL) leukocytes and the percentage of BAL
eosinophils were significantly higher compared with those of thewild-type littermates (Figure 2A and 2B). The absolute number of
eosinophils was approximately 7-fold higher in the mutant mice.
In addition, IgE was detectable only in the BAL fluid of RBP-j–
deficient animals (Figure 2C). Histology of the lungs from OVAchallenged RBP-jCKO and wild-type controls clearly confirmed
the existence of severe airway inflammation in RBP-jCKO mice,
including significant leukocyte infiltration around the airways and2

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Figure 1. RBP-jCKO mice lacking RBP-j (and thus, notch signaling) in skin keratinocytes develop a progressive, AD-like disease
culminating in aged animals with lung inflammation. (A) H&E and Toluidine blue staining of the skin documents severe AD-like changes in
RBP-j–deficient mice, including epidermal hyperkeratosis, parakeratosis, acanthosis, skin inflammation, and increased number of dermal mast cells(insets) detectable as early as 1 wk after birth, which worsen as RBP-jCKO animals age (6 wk and 10 wk). Note that the keratin cysts formed in thedermis of RBP-jCKO mice are due to the destruction of RBP-j–deficient hair follicles [42]. (B) The progression of skin inflammation is reflected in RBPjCKO serum IgE levels, which are dramatically elevated by 10 wk of age (10 wk; n = 4 for each group; *p,0.01). (C) Intracellular cytokine staining of
CD4+ T cells isolated from lymph nodes (mixture of inguinal and axillary) or spleen shows a robust interleukin (IL)-4-positive and interferon (IFN)-cnegative Th2 cell presence in 10-wk-old RBP-jCKO animals, indicating the development of a full-blown AD-like allergic inflammation. Percentage ofcells in each quadrant is included (representative data are presented). (D) H&E and periodic acid-Schiff (PAS) staining of the lung do not show any
signs of inflammation in 10-wk-old RBP-jCKO animals; however, at 52 wk (1 y), the mutant mice develop lung inflammation. The pathology of RBPjCKO lung includes immune cell infiltration around the airways and blood vessels (arrowheads), goblet cell hyperplasia in the airways (arrow; insets),
and airway remodeling (representative pictures are presented; scale bar: 50 mm).
doi:10.1371/journal.pbio.1000067.g001

localized TSLP overexpression in lung epithelium is capable ofinducing asthma [18,19,21], we have articulated an alternative
hypothesis: TSLP may be a systemic signal that sensitizes theanimals to allergen exposure in the lung. In that case, highsystemic levels of epidermal-derived TSLP should render RBPjCKO animals susceptible to the asthmatic phenotype upon
exposure to allergen.
To test this hypothesis we deleted the IL7Ra subunit of TSLP
receptor in RBP-jCKO animals (Msx2-Cre/+;RBP-jflox/flox;IL7ra-/-;or RBP-jCKO;IL7ra-/- [27]). This strategy was chosen because
the TSLPR subunit of the TSLP receptor is linked to the RBP-jlocus. The inhibition of TSLP reception reduced the severity of
local AD-like inflammation in the skin of RBP-jCKO animals as
judged by the reduction of dermal mast cells in RBPjCKO;IL7ra-/- mice relative to their RBP-jCKO littermates[28]. However, a significant elevation in mast cell number was stilldetectable in RBP-jCKO;IL7ra-/- skin compared to wild type
(Figure 3A and 3B). In addition, epidermal hyperplasia and TSLP
overexpression that mark the presence of postnatal skin-barrier
defects persisted in aged RBP-jCKO;IL7ra-/- mice (Figure 3A
and Figure S4). Importantly, deletion of IL7Ra did not affect theintensity of systemic Th2 response to allergic skin inflammation

blood vessels, extensive goblet cell hyperplasia in the large airways,
and distinct appearance of goblet cells in medium-sized airways(Figure 2D). Considering that RBP-j is not deleted in the lung
(Figures S3 and S7), these data clearly show that the skin-barrier
defect can serve as a primary risk factor for development of asthma
in a normal lung.

TSLP Signaling Is Required for the Atopic March in RBP-j–
Deficient Animals
It is possible that atopic skin lesions are the essential components
downstream of skin-barrier defects that initiate systemic atopy in
RBP-jCKO mice [3]. If this hypothesis were true, Th2 cells
generated at the site of inflamed skin would migrate to other sites,
including lung mucosa, and release high levels of Th2-derived
cytokines, which would sensitize the lung airways to allergic
inflammation. This model, however, is challenged by findings thatshow that Th2 cells generated in mouse models of AD specificallyhome to skin [26]. In addition, it is unclear whether the presenceof AD lesions is required for initiation of the atopic march [11].
Skin-barrier defects led to systemic TSLP elevation [15], which
remained elevated in the serum of RBP-jCKO animals throughout
life (Figure S2). On the basis of these observations and that
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Figure 2. Five- to seven-wk-old RBP-jCKO mice develop a severe allergic lung inflammation in an OVA-induced murine model ofasthma. (A and B) There are more leukocytes (A) and a higher percentage of eosinophils (B) present in BAL fluid collected from OVA-treated RBPjCKO mice in comparison to their wild-type littermates (Msx2-Cre/+;RBP-jflox/+, RBP-jflox/flox, RBP-jflox/+), indicating a severe airway inflammation in thesemutant animals (M, macrophages; E, eosinophils; L, lymphocytes; N, neutrophils). (C) IgE reaches detectable levels only in BAL fluid from RBP-j–
deficient mice, further indicating the high intensity of OVA-induced allergic inflammation in the RBP-jCKO lung. (D) Comparing lung histology of RBPjCKO with wild-type littermates sensitized and challenged with OVA shows a significantly more intense lung inflammation in RBP-jCKO mice. H&E andPAS staining shows greater immune cell infiltration around the airways and blood vessels (arrowheads), increased airway remodeling, goblet cell
hyperplasia (insets), and mucus overproduction in the mutant lung (representative pictures are presented; scale bar: 50 mm). ‘‘Controls’’ refers to RBPjCKO and wild-type animals treated with PBS instead of OVA (n = 4 for each group; *p,0.05, comparing the adjacent groups). These data are
confirmed in additional independent experiments.
doi:10.1371/journal.pbio.1000067.g002

its consequences (e.g., elevated IgE) could confer susceptibility toasthma in the absence of TSLP signaling.
We challenged the lung airways of age- and sex-matched RBPjCKO;IL7ra-/-, RBP-jCKO;IL7ra+/- (RBP-jCKO), IL7ra-/-,and wild-type littermates with OVA. To prevent RBP-jCKO
lethality, all the animals in this experiment received intranasal
OVA challenges only twice. Despite this reduced exposure, RBPjCKO animals developed a severe asthmatic response, which wasabsent in RBP-jCKO;IL7ra-/- mice (Figure 3E–3H). Histologicaland serum IgE levels remained elevated in RBP-jCKO;IL7ra-/mice (Figure 3C and 3D). This could be due to other cytokines/
chemokines made by defective skin barrier in RBPjCKO;IL7ra-/- animals [15], which together with exposure toallergens/pathogens through the compromised skin, sustain arobust systemic Th2 response even in the absence of TSLP
reception [28]. Therefore, RBP-jCKO;IL7ra-/- animals provide asuitable system in which to determine if skin-barrier defects andAD-like skin inflammation including a systemic Th2 response with

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Figure 3. TSLP signaling blockade rescues asthmatic phenotype of RBP-jCKO mice. (A) H&E and Toluidine blue staining of the skin shows
the persistence of epidermal hyperplasia, hyperkeratosis, parakeratosis, and acanthosis in 10-wk-old RBP-jCKO;IL7ra-/- skin. However, the local skininflammation and number of dermal mast cells (insets) are markedly reduced in RBP-jCKO;IL7ra-/- skin (representative pictures are presented; scalebar: 50 mm [28]). (B) The quantitative analysis of mast cell infiltration in the dermis of RBP-jCKO;IL7ra-/- (R;I) shows that there is still more mast cell
accumulation in RBP-jCKO;IL7ra-/- compared to wild-type (Wt) and IL7ra-/- (I) skin. The bar graphs show the average number of mast cells in ten
random 1006 microscope fields (*p,0.001). (C) Serum IgE levels of 10-wk-old RBP-jCKO;IL7ra-/- mice are highly elevated and are comparable tothose of RBP-jCKO (Msx2-Cre/+;RBP-jflox/flox;IL7ra+/-, R) littermates (*p,0.01). (D) Intracellular cytokine staining shows a significant population of IL-4producing Th2 cells in inguinal/axillary lymph nodes and spleen of 10-wk-old RBP-jCKO;IL7ra-/- animals (representative data are presented). (E)Comparing lung histology of 5- to 7-wk-old RBP-jCKO;IL7ra-/-, RBP-jCKO, IL7ra-/-, and wild-type littermates sensitized and challenged with OVA
shows a complete reversal of the intense RBP-jCKO lung inflammation in RBP-jCKO;IL7ra-/- mice. H&E and PAS staining shows the muted
inflammatory response around the airways and blood vessels (arrowheads), and lack of airway remodeling or goblet cell hyperplasia (insets) in RBPjCKO;IL7ra-/- lung (representative pictures are presented; scale bar: 50 mm). (F and G) The average number of leukocytes (F) and percentage of
eosinophils (G) in BAL fluid from OVA-treated RBP-jCKO;IL7ra-/- lung is lower than that of wild-type lung treated similarly, indicating that deletion of
IL7Ra rescues the allergic lung inflammation in RBP-jCKO mice (M, macrophages; E, eosinophils; L, lymphocytes; N, neutrophils). (H) IgE remains below
detection levels in BAL fluid from OVA-treated RBP-jCKO;IL7ra-/- mice, further confirming that the intense OVA-induced allergic inflammation in RBPjCKO lung is absent in RBP-jCKO;IL7ra-/- lung. To avoid death among RBP-jCKO cohort, all the animals in this study are challenged intranasally with
OVA twice. Although the asthmatic response among RBP-jCKO;IL7ra-/-, IL7ra-/-, and wild-type groups is minimal, a severe response is seen in RBPjCKO mice. ‘‘Controls’’ refers to RBP-jCKO;IL7ra-/-, RBP-jCKO, IL7ra-/-, and wild-type animals treated with PBS instead of OVA (n = 4 for each group;*p,0.01, compared to wild-type cohort). These data are confirmed in additional independent experiments.
doi:10.1371/journal.pbio.1000067.g003

that in this outbred background and in the absence of skin-barrier
defects, TSLP overproduction was insufficient to attract mast cells
to the skin (Figure 5B). In addition, serum IgE levels and Th2 cellnumbers in the peripheral lymph nodes of K14-TSLPtg mice wereindistinguishable from the wild type, confirming the absence ofallergic skin inflammation in K14-TSLPtg animals that were notexposed to any external stimulus (e.g., allergen exposure; Figure 5C
and 5D). Therefore, outbred K14-TSLPtg mice constituted aanalysis could not detect goblet cell hyperplasia or significant
inflammation around the airways and vasculature in RBPjCKO;IL7ra-/- lungs (Figure 3E). Total white blood cell countand eosinophilia in BAL fluid from OVA-treated RBPjCKO;IL7ra-/- mice was indistinguishable from IL7ra-/- littermates (Figure 3F and 3G). In addition, IgE was undetectable in
BAL fluid from OVA-treated RBP-jCKO;IL7ra-/- mice
(Figure 3H). These findings show that without a TSLP signal,no progression from allergic skin inflammation to asthma isobserved, indicating that atopic march requires TSLP signals.

Airway Hyper-Responsiveness in RBP-jCKO Mice Is
Blocked by Inhibiting TSLP Signaling
Airway hyper-responsiveness is a hallmark of allergic asthma[18]. To determine whether skin-derived TSLP could cause
airway hyper-responsiveness in RBP-jCKO mice, we used the
OVA-induced model of allergic inflammation and challenged thelung airways of age- and sex-matched RBP-jCKO;IL7ra-/-, RBPjCKO, IL7ra-/-, and wild-type littermates twice with OVA.
Thereafter, we measured the total lung resistance of OVA-treatedmice at basal state (vehicle alone) or in response to increasing dosesof nebulized methacholine. As expected, OVA-treated RBPjCKO mice mounted a more severe airway resistance comparedwith their wild-type littermates when exposed to low doses ofmethacholine, and could not tolerate the higher doses ofmethacholine (Figure 4). RBP-jCKO;IL7ra-/-, however, did not
develop any significant airway resistance even when exposed tohigh doses of methacholine (Figure 4). These results confirm thatskin-barrier defects predispose animals to asthmatic phenotypes
and that TSLP is required for this predisposition.

Figure 4. Severe airway hyper-responsiveness among OVAtreated RBP-jCKO mice is dependent on TSLP signaling. Ageand sex-matched RBP-jCKO;IL7ra-/-, RBP-jCKO, IL7ra-/-, and wild-type
littermates are sensitized and challenged with OVA as in Figure 3.
Airway reactivity at the basal state (vehicle alone; V) and in response toincreasing doses of methacholine (2.5, 5, 10, 20, 40, and 80 mg/ml) is
monitored in OVA-treated animals using total lung resistance (RL; n = 4
for each group). OVA-treated RBP-jCKO animals mount a severe airway
hyper-responsiveness at 5 mg/ml of methacholine and do not toleratemethacholine doses less than or equal to 10 mg/ml, developing fatal
labored breathing. OVA-treated RBP-jCKO;IL7ra-/-, IL7ra-/-, and wildtype mice, on the other hand, show moderate response only to thehighest dose of methacholine (*p,0.01). Data are presented as mean +standard deviation of percent increase in total lung resistancecompared to basal state (vehicle alone; V) in each group. These
measurements are confirmed in another independent set of experiments.
doi:10.1371/journal.pbio.1000067.g004

TSLP Overexpression by the Skin Is Sufficient to Trigger
the Atopic March
Although we showed that TSLP signaling is necessary for thedevelopment of asthma in RBP-j–deficient animals with chronic
allergic skin inflammation, it remains unclear whether otherfactors associated with AD-like skin lesions contribute to this atopic
march. TSLP overexpression in mouse skin led to an AD-like
pathology on an inbred background [26]. However, we discoveredthat on an outbred background, the same K14-TSLPtg mice [15]maintained high serum TSLP levels under normal conditions(approximately 450 pg/ml) without any skin or lung inflammation(Figure 5 and Figure S5) [29]. Normal skin morphology and thelack of mast cell hyperplasia evident in K14-TSLPtg mice confirmed
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Figure 5. K14-TSLPtg mice exhibit no sign of skin or lung allergic inflammation under normal conditions. (A) Skin-derived TSLP
overexpression leads to high systemic TSLP levels in the serum of K14-TSLPtg mice (n = 10 for each group; *p,0.01). (B–D) Under normal conditions,skin TSLP overproduction on an outbred C57BL/6-CD1 background does not elicit any allergic inflammation in K14-TSLPtg mice. Note the normal skinhistology (B), normal serum IgE levels (n = 10 in each group)(C), and normal number of peripheral IL-4-producing Th2 cells (D) in K14-TSLPtg mice atpostnatal day 120 (P120). (E) H&E and PAS stained lung sections of K14-TSLPtg animals confirm that no inflammation or airway remodeling occurred at
P120 (scale bar: 50 mm).
doi:10.1371/journal.pbio.1000067.g005

for TSLP expression prior to OVA treatment (Figures S6 and S7).
Total leukocyte count was less than 5-fold higher in BAL fluid
from K14-TSLPtg mice compared with similarly treated wild-type
animals (Figure 6B), which was mainly due to a severe BAL fluid
eosinophilia in the transgenic animals (Figure 6C). In addition, IgEreached detectable levels only in BAL fluid from OVA-treated
K14-TSLPtg mice (Figure 6D), which also experienced significantly
higher serum IgE levels (Figure 6E) (1.6 mg/ml versus 0.18 mg/ml
in OVA-treated wild-type serum, p,0.05). Thus, K14-TSLPtg micedeveloped an intense Th2 response upon allergen exposure. In the
airway reactivity test, OVA-treated K14-TSLPtg mice showed asevere airway hyper-responsiveness to low doses of nebulized
methacholine, which was not seen among similarly treated wildtype littermates (Figure 6F).

suitable model in which to examine whether epidermal TSLP
overexpression alone would increase susceptibility to asthma uponallergen exposure.
Next, we sensitized and challenged the lung airways of 5- to 7wk-old K14-TSLPtg mice and their age- and sex-matched wild-type
littermates three times with allergen. As seen in OVA-treatedRBP-jCKO animals exposed to three doses of intranasal OVA,
K14-TSLPtg mice developed a severe asthmatic response andsignificant lethality, which were not seen in the control group(Figure 6). Two out of eight OVA-treated transgenic mice diedduring the intranasal challenge. Histological examination of lungs
from surviving K14-TSLPtg animals revealed pronounced airway
remodeling with goblet cell hyperplasia and inflammation aroundthe lung airways and vasculature (Figure 6A), which were negative
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Figure 6. OVA treatment reveals that K14-TSLPtg mice are prone to asthmatic phenotype. (A) Histological analysis shows severe
inflammatory cell infiltrates (arrowheads), airway remodeling, and goblet cell hyperplasia (arrow; insets) in the lung airways of 5- to 7-wk-old K14TSLPtg animals that are sensitized and challenged with OVA, but not in similarly treated wild-type littermates (scale bar: 50 mm). (B–D) BAL fluid
analysis shows more leukocytes (B), a higher percentage of eosinophils (C), and specific appearance of IgE in lung airways of OVA-treated K14-TSLPtg
mice (D), confirming the higher intensity of the asthmatic phenotype conferred by the TSLP overexpression in the morphologically normal skin (M,macrophages; E, eosinophils; L, lymphocytes; N, neutrophils). (E) The severe allergic inflammation in OVA-treated K14-TSLPtg lungs leads to drastic
elevation of serum IgE in the animals. ‘‘Controls’’ refers to K14-TSLPtg and wild-type mice treated with PBS alone (n = 4 for each group; *p,0.05,
comparing the adjacent groups). (F) Total lung resistance (RL) measurements show that OVA-treated K14-TSLPtg lung airways are hyper-responsive toincreasing doses of nebulized methacholine as compared to OVA-treated wild types (V: vehicle-only basal measurement; n = 4 for each group;*p,0.05). These findings are confirmed in additional independent experiments.
doi:10.1371/journal.pbio.1000067.g006

Even though outbred K14-TSLPtg animals developed a severe
asthmatic phenotype, their skin retained its normal appearance
after OVA treatment, confirming that the lung inflammation did
not depend on a concurrent skin lesion (Figure S8). Although lowlevels of TSLP were expressed in the trachea, the K14-TSLP
transgene was not expressed in the lung of K14-TSLPtg animals and
TSLP protein was not detectable in their BAL fluid (Figures S6and S7). Therefore, these observations are most consistent with amodel in which systemic TSLP is sufficient to predispose mice toallergic lung inflammation.

of TSLP-mediated activation remains a subject for future
investigation. Of note, elevated TSLP expression is also reported
in psoriatic skin [34]; however, there is no evidence linking psoriasis
to predisposition to asthma. This phenomenon could be explainedby the dominance of Th1 response in patients with psoriasis and theunresponsiveness of their immune cells to TSLP [34].
The findings outlined in this report have a potentially important
implication for human health. They provide a plausible explanation of why a large number of AD patients develop asthma andother allergic disorders later in life [35,36]. Because TSLP
overproduction is triggered by skin-barrier defects and faithfully
mirrors the severity of these defects [15], our data emphasize thatan early and aggressive treatment of the underlying skin-barrier
defects in AD-prone patients [10,11] may be more beneficial inpreventing asthma than treating the outbreaks of AD lesions [6].
Although serum TSLP levels in AD patients are yet to be
determined, high TSLP expression levels in human AD lesions[37] suggest that this diffusible cytokine could also reach systemiclevels sufficient to trigger atopic march in AD patients. If indeed
serum TSLP levels are elevated in AD patients, our findings
suggest that an aggressive management of TSLP levels in thesepatients will lower the incidence of asthma later in their lives.

Discussion
This report describes the mechanism that mediates the
progression of a local allergic skin disease to a disseminatedallergic inflammation downstream of skin-barrier defects in mice.
We show that creating an intrinsic skin-barrier defect (by removing
Notch signaling specifically from some skin keratinocytes) results inthe sensitization of the lung airways to allergens. Similar to theclinical cases, barrier-defective mice require a secondary insult byallergens to expose their predisposition to asthma. Importantly, weshow that epicutaneous sensitization with a common allergen [3] is
not required to achieve this pronounced lung hyper-responsiveness. Therefore, RBP-jCKO animals demonstrate that skinbarrier impairment provides a signal that promotes developmentof a distant atopy in the lung, most likely by a systemic, diffusible
cytokine. We identified this signal to be TSLP, a central player inthe early stages of allergic inflammation [18–20] and a barrierdefect–sensitive product of skin keratinocytes that is released prior
to development of an AD-like skin lesion [15]. In contrast to TSLP
overexpression by lung epithelia, which does not result in systemic
accumulation of this cytokine [18], overexpression of TSLP in theskin results in high systemic availability [15]. Therefore, skin iscapable of acting as a signaling organ, driving susceptibility toallergic inflammation in another barrier organ (i.e., lung) byreleasing TSLP.
Importantly, we were able to show that TSLP was necessary to
predispose RBP-j–deficient animals to the asthmatic phenotype.
Concomitant removal of IL7Ra subunit of the TSLP receptor in
RBP-jCKO mice prevented the atopic march despite persistentAD-like pathology and elevated serum IgE levels. Complementing
this experiment is the observation that overexpression of TSLP byskin keratinocytes leads to high serum levels of TSLP that are
sufficient to sensitize the lung airways to allergic inflammation in theabsence of any skin pathology. If the low levels of TSLP in the
trachea of K14-TSLPtg mice contribute to the phenotype, they do soby adding to the already high levels of skin-derived serum TSLP.
Because TSLP can activate dendritic cells [30], T cells [31,32], andmyeloid cells [33], we speculate that its high systemic levels directlyprime these immune cells at distant sites (e.g., the lung) to mount an
intense allergic inflammation in response to a second stimulatory
signal (e.g., allergens in the lung airways); however, the specificcontribution of these immune cells to the atopic march downstream
PLoS Biology | www.plosbiology.org

Materials and MethodsMice
Msx2-Cre/+;RBP-jflox/flox (RBP-jCKO), Msx2-Cre/+;RBP-jflox/flox;
IL7ra-/- (RBP-jCKO;IL7ra-/-), and K14-TSLP tg mice weregenerated as previously described [15]. All animals were kept inmixed C57BL/6 and CD1 outbred genetic background, which areresistant to Th2-mediated inflammation [29]. Age- and sexmatched mutant and wild-type littermates were used in eachanalysis. All mice were maintained in the animal facility underWashington University animal care regulations.

OVA Treatment
The allergic sensitization and lung airways challenge with OVA
was carried out as previously outlined [18]. In brief, wild-type andmutant mice at 5–7 wk of age were sensitized on days 0 and 14 by
250 ml intraperitoneal injection of antigen solution containing
50 mg OVA (Sigma) dissolved in 1.3 mg aluminum hydroxide gel(Sigma) and PBS. On days 21, 22, and 23, mice were intranasallychallenged with 150 mg OVA dissolved in 40 ml of PBS. Micecohorts designated as ‘‘controls’’ included the mutant and wildtype animals that underwent the same regimen without OVA
antigen. On day 24, all the animals were humanely euthanized forthe analysis. To avoid mortality among RBP-jCKO animals, all
the age- and sex-matched littermates used in the rescue
experiments presented in Figures 3 and 4 were challenged only
twice with OVA. Note that RBP-jCKO;IL7ra-/- mice could
tolerate the repeated OVA intranasal challenge, but RBP-jCKO
mice developed severe responses after even two exposures. The
OVA experiments were conducted on male and female animals;
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Skin-Derived TSLP Triggers Asthma

however, within-sex analyses were performed to avoid any sex
effect on phenotypes observed.

PCR
Conventional PCR for the RBP-j allele was performed on
genomic DNA isolated from skin and lung of adult RBP-jCKO
mice using KlenTaq10 (DNA Polymerase Technology) supplemented with 1.3 M final concentration of betaine (amplification
cycles = 32). The following primers were used to distinguishbetween deleted (RBP-jD) and floxed (RBP-jflox) alleles of RBP-j:
Deleted allele: 59-TGTTTGCCACCAGAATCTGTTTGTTATTTGC-39 and 59-ATTTGCTTGAGGCTTGATGTTCTGTATTGC-39.
Floxed allele: 59-TGTTTGCCACCAGAATCTGTTTGTTATTTGC-39 and 59-AGGTACCTGGTACTAACTGTCTGGGACCG-39.

Airway Reactivity Test
The airway responsiveness to aerosolized methacholine wasdetermined in OVA-treated animals by measuring total lung
resistance and dynamic compliance as previously outlined [38]. In
brief, mice were treated with OVA as described in the preceding
section (two intranasal OVA challenges for all mice in rescue
experiments (Figure 4) and three intranasal OVA challenges for all
mice in sufficiency experiments (K14-TSLPtg and wild type;Figure 6)). The animals were then anesthetized for airway reactivity
test on day 24 post OVA sensitization. They were ventilated
through a tracheotomy and monitored for intrapleural pressureusing an oroesophageal tube. PBS (vehicle) or methacholine (Sigma)
in PBS were delivered at 3-min intervals using an in-line nebulizer.
The respiratory flow signals were collected between deliveries usinga pneumotach (SenSym SCXL004, Buxco Electronics). For detailed
description of the device, refer to [39].

qRT-PCR
mRNA isolated from P4 epidermis and lung of RBP-jCKO andwild-type littermates was used to perform qRT-PCR analysis aspreviously described [41]. The primers used to amplify TSLP were:59-CCAGGCTACCCTGAAACTGA-39 and 59-TCTGGAGATTGCATGAAGGA-39.

Histology
Dorsal skin samples were harvested from the mice and fixed in
4% PFA at 4 uC overnight. Lungs were inflated through thetrachea to 25-cm water pressure with 4% PFA prior to excisionfrom the chest and fixation. These lungs were PFA-fixed for 24 hr
at 4 uC and embedded in paraffin. The paraffin-embedded tissues
were sectioned at 5–6 mm and stained with hematoxylin and eosin(H&E), toluidine blue, or periodic acid-Schiff (PAS). For RBP-j
immunohistochemical staining, anti–RBP-j antibody (clone
T6709, Institute of Immunology) and biotinylated anti–rat
secondary antibody were used. HRP-conjugated streptavidin and
DAB substrate kit (Pierce) were used to visualize the signal.
Hematoxylin was used to counterstain the sections. For TSLP
immunostaining, paraffin-embedded tissue samples and biotinylated anti-TSLP antibody (R&D Systems) were used. Sections
were counterstained with DAPI nuclear stain.

Statistical Analysis
The quantitative measurements were assessed using Student ttest as the test of significance and presented as mean 6 standard
deviation in bar graph format. Our studies were conducted inoutbred cohorts of animals; therefore, we used nested ANOVA to
exclude any effect of between-family differences and make certain
the significant differences observed were solely attributable to thegene removed (RBP-jCKO and RBP-jCKO;IL7ra-/-) or overexpressed (K14-TSLPtg).

Accession Numbers
The Genbank (http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for proteins discussed in this article are as follows:
RBP-j, NM_009035; TSLP, NM_021367.

Supporting Information

BAL Analyses

Figure S1 RBP-jCKO animals die prematurely due to theirsevere skin phenotype (p,0.001 compared to wild-type life span,
log-rank test). A few mutant mice that survive up to one year (redcircles), however, develop spontaneous lung inflammation.
Found at: doi:10.1371/journal.pbio.1000067.s001 (78 KB TIF)BAL fluid was collected by infusing the lungs of the anesthetizedmouse with 1-ml PBS through tracheal insertion of a Surflo catheter(Terumo Medical). Leukocyte count in BAL fluid was determinedusing a Hemavet 950 analyzer (Drew Scientific). BAL fluid was spun
down and the supernatant was stored for cytokine/immunoglobulin
analysis. The cell pellet was resuspended in PBS and used for
differential cell count after Giemsa staining on the slide.

Figure S2 Serum TSLP levels in RBP-jCKO mice are highly

elevated. TSLP overproduction is evident in RBP-jCKO serum at
1 wk after birth, reaching extreme levels in the adult animals (n = 4
for each group; *p,0.01, comparing the mutants to the wild-type
littermates).
Found at: doi:10.1371/journal.pbio.1000067.s002 (55 KB TIF)

ELISA
Serum TSLP levels were determined using Quantikine mouse
TSLP kit according to manufacturer’s instructions (R&D Systems).
Serum and BAL fluid IgE levels were measured using Mouse IgE
ELISA kit (Immunology Consultants Laboratory).

Figure S3 Lung epithelium is normal in RBP-jCKO animals.
(A) PCR analysis of DNA isolated from adult RBP-jCKO (Msx2Cre/+;RBP-jflox/flox) and wild-type (RBP-jflox/flox) skin and lung showsthat RBP-j locus is intact (i.e., RBP-j is not deleted) in the lung (D:deleted allele; M: molecular marker; S: skin; L: lung). (B)Immunohistochemical analysis for RBP-j protein confirms that
RBP-j is present in the lung airway epithelium. Skin sections
stained under the same condition are presented as controls (scalebar: 50 mm).
Found at: doi:10.1371/journal.pbio.1000067.s003 (7.35 MB TIF)

Flow Cytometry
Intracellular cytokine staining was conducted to estimate Th2cell census as previously described [26]. Single cell suspensions
were prepared from spleen and lymph node samples and cultured
in presence of PMA (50 ng/ml), ionomycin (1 mg/ml) andmonensin (10 mg/ml) for 4 h. Cells were then stained withphycoerythrin (PE)-cy7 conjugated anti-CD4 antibody (552775,
BD Bioscience Pharmingen). After fixation in 2% PFA andpermeabilization with 0.5% saponin, cells were stained with PE
conjugated anti-IL4 (554389) and APC conjugated anti-IFN-c(554412) antibodies from BD Bioscience Pharmingen [40].
PLoS Biology | www.plosbiology.org

Figure S4 Serum TSLP levels of 10-wk-old RBPjCKO;IL7ra-/- mice are highly elevated. This indicates thatskin-barrier defects caused by the loss of RBP-j in epidermal
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Skin-Derived TSLP Triggers Asthma

keratinocytes persist in the absence of IL7Ra (n = 5 for each group;* p,0.01). Note that serum TSLP levels in RBP-jCKO;IL7ra-/mice are consistently and significantly higher than in RBP-jCKO
animals. We are currently investigating the underlying reason forthis surge.
Found at: doi:10.1371/journal.pbio.1000067.s004 (50 KB TIF)

Found at: doi:10.1371/journal.pbio.1000067.s008 (1.3 MB TIF)
Table S1 Major antimicrobial peptides are overexpressed in

postnatal epidermis of RBP-jCKO animals. This overexpression
indicates the persistence of the skin-barrier defect in RBP-jCKO
mice after birth [25]. Data are extracted from a microarray study
on P9 epidermis of RBP-jCKO and wild-type littermates aspreviously described [15]. The fold increase of antimicrobial
peptide mRNA in RBP-j–deficient epidermis relative to wild typeis presented.
Found at: doi:10.1371/journal.pbio.1000067.s009 (37 KB PDF)

Figure S5 The ear and skin of K14-TSLPtg mice appear normal

at P120. This emphasizes that in an outbred genetic background(C57BL/6 and CD1 mix) these transgenic animals do not develop
any skin inflammation under normal conditions.
Found at: doi:10.1371/journal.pbio.1000067.s005 (1.3 MB TIF)

Acknowledgments

Figure S6 There is no TSLP overexpression detectable in K14TSLPtg lung airways or parenchyma. Because the K14 gene isexpressed in basal cells located in the trachea, we analyzed mRNAlevels by qRT-PCR on samples isolated from epidermis, trachea
and lung of K14-TSLPtg and wild-type mice. This analysis showsthat the K14-TSLP transgene is active in basal cells within the
trachea, but the overall levels are 200-fold lower than those madeby epidermal keratinocytes (***p,0.0000001 and *p,0.01
compared to wild type).
Found at: doi:10.1371/journal.pbio.1000067.s006 (145 KB TIF)

The authors would like to thank Ms. Ahu Turkoz and Suzanne Swanson
for their excellent technical assistance; Drs. Michael Walter and Adrian
Shifren for numerous discussions and encouragement during the executionof these studies; and Drs. David Ornitz, Jeffery Miner, and Anne Bowcock
and the members of the Kopan laboratory for careful reading of themanuscript. We thank Dr. Andrew Farr for IL7ra-/- and K14-TSLPtg, Dr.
Gail Martin for Msx2-Cre, and Dr. Tasuku Honjo for RBP-jflox/flox mice.

Author Contributions
The author(s) have made the following declarations about theircontributions: Conceived and designed the experiments: SD RK.
Performed the experiments: SD MM MJH. Analyzed the data: SD MM.
Contributed reagents/materials/analysis tools: MJH. Wrote the paper: SD
RK.

Figure S7 Lung epithelium does not overexpress TSLP in adult
K14-TSLPtg, RBP-jCKO, or RBP-jCKO;IL7ra-/- animals. Immunofluorescence staining for TSLP protein (red) confirms that
TSLP is overexpressed only in the epidermal keratinocytes of themutant mice. All sections are stained under the same conditions.
Dotted lines outline the basement membrane and asterisks
highlight the epidermal keratin cysts present in RBP-jCKO skin(scale bar: 50 mm).
Found at: doi:10.1371/journal.pbio.1000067.s007 (5.3 MB TIF)

Note Added in Proof
After this manuscript was accepted for publication, similar observations
were reported by Zhang et al: Zhang Z, Hener P, Frossard N, Kato S,
Metzger D, et al. (2009). Thymic stromal lymphopoietin overproduced bykeratinocytes in mouse skin aggravates experimental asthma. Proc Natl
Acad Sci U S A 106: 1536–1541.

The skin of OVA-treated K14-TSLPtg mice remains
normal. H&E and toluidine blue staining of K14-TSLPtg and wildtype skin show no significant signs of cutaneous inflammation in
the transgenic mice (scale bar: 50 mm).
Figure S8References
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