Airway and Lung Pathology Due to Mucosal Surface
Dehydration in ?-Epithelial Na?Channel-Overexpressing
Mice: Role of TNF-? and IL-4R? Signaling, Influence of
Neonatal Development, and Limited Efficacy of Glucocorticoid
Alessandra Livraghi,2* Barbara R. Grubb,* Elizabeth J. Hudson,* Kristen J. Wilkinson,*
John K. Sheehan,* Marcus A. Mall,†Wanda K. O’Neal,* Richard C. Boucher,* and
Scott H. Randell*
Overexpression of the epithelial Na?channel ? subunit (Scnn1b gene, ?ENaC protein) in transgenic (Tg) mouse airways dehy-
drates mucosal surfaces, producing mucus obstruction, inflammation, and neonatal mortality. Airway inflammation includes
macrophage activation, neutrophil and eosinophil recruitment, and elevated KC, TNF-?, and chitinase levels. These changes
recapitulate aspects of complex human obstructive airway diseases, but their molecular mechanisms are poorly understood. We
used genetic and pharmacologic approaches to identify pathways relevant to the development of Scnn1b-Tg mouse lung pathology.
Genetic deletion of TNF-? or its receptor, TNFR1, had no measurable effect on the phenotype. Deletion of IL-4R? abolished
transient mucous secretory cell (MuSC) abundance and eosinophilia normally observed in neonatal wild-type mice. Similarly,
IL-4R? deficiency decreased MuSC and eosinophils in neonatal Scnn1b-Tg mice, which correlated with improved neonatal sur-
vival. However, chronic lung pathology in adult Scnn1b-Tg mice was not affected by IL-4R? status. Prednisolone treatment
ablated eosinophilia and MuSC in adult Scnn1b-Tg mice, but did not decrease mucus plugging or neutrophilia. These studies
demonstrate that: 1) normal neonatal mouse airway development entails an IL-4R?-dependent, transient abundance of MuSC and
eosinophils; 2) absence of IL-4R? improved neonatal survival of Scnn1b-Tg mice, likely reflecting decreased formation of as-
phyxiating mucus plugs; and 3) in Scnn1b-Tg mice, neutrophilia, mucus obstruction, and airspace enlargement are IL-4R?- and
TNF-?-independent, and only MuSC and eosinophilia are sensitive to glucocorticoids. Thus, manipulation of multiple pathways
will likely be required to treat the complex pathogenesis caused by airway surface dehydration. The Journal of Immunology,
2009, 182: 4357–4367.
moter in transgenic (Tg) mice, results in epithelial Na?hyperab-
irway epithelial overexpression of the epithelial Na?
channel ? subunit (?ENaC protein, Scnn1b gene),
driven by the Clara cell secretory protein (CCSP)3pro-
sorption, airway surface liquid (ASL) dehydration, impaired mu-
cus clearance, airway inflammation, and early postnatal mortality
(1). The Scnn1b-Tg mouse model recapitulates many features of
cystic fibrosis (CF) and other human airway diseases associated
with relative dehydration of airway surfaces (2), including chronic
bronchitis (CB) and chronic obstructive pulmonary disease
(COPD). At birth, the lungs of Scnn1b-Tg mice are morphologi-
cally normal, but they rapidly develop time-dependent abnormal-
ities (3). Tracheal mucus obstruction is associated with neonatal
mortality, and, in surviving mice, mucus plugging and mucous
secretory cell (MuSC) metaplasia progressively extend into the
intrapulmonary bronchi. The inflammatory infiltrate is character-
ized by enlarged/highly vacuolated macrophages, persistent neu-
trophilia associated with elevated KC, MIP-2, and TNF-?, and
transient eosinophilia with increased levels of IL-13 and eotaxin-1
(from 2 to 6 wk). YM1, YM2, and acidic mammalian chitinase, all
associated with Th2-type inflammation in asthma and helmintic
infection (4–7), are also elevated in Scnn1b-Tg mice. Moreover,
Scnn1b-Tg mice exhibit transient and spotty necrotic degener-
ation of Clara cells in the intrapulmonary airways, peaking at
day 3 and being completely resolved by day 10, and early neo-
natal air-trapping that later results in emphysematous changes
(3). As surviving Scnn1b-Tg mice age, lymphocytic aggregates
similar to those described in the lungs of COPD patients (8)
become more frequent, suggesting progressive development of
*Cystic Fibrosis/Pulmonary Research and Treatment Center, The University of North
Carolina at Chapel Hill, Chapel Hill, NC, 27599; and†Department of Pediatrics III,
Pediatric Pulmonology and Cystic Fibrosis Center, University of Heidelberg, Heidel-
Received for publication August 5, 2008. Accepted for publication January 21, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was funded by North American Cystic Fibrosis Foundation (CFF) Grant
LIVRAG04I0 (to A.L.), Deutsche Forschungsgemeinschaft MA2081/2-1 grant (to
M.A.M.), National Institutes of Health (NIH) Grant P30 DK065988 and CFF grant R026-
CR02 (to W.K.O.), NIH Grants P50 HL060280, P01 HL034322, P30 DK065988, and
P50 HL084934 (to R.C.B.), and CFF Grant RANDEL07P0 (to S.H.R.).
2Address correspondence and reprint requests to Dr. Alessandra Livraghi, Cystic
Fibrosis/Pulmonary Research and Treatment Center, University of North Carolina at
Chapel Hill, CB 7248 Thurston Bowles Building, Room 6029, Chapel Hill, NC
27599. E-mail address: email@example.com
3Abbreviations used in this paper: CCSP, Clara cell secretory protein; Tg, transgenic;
ASL, airway surface liquid; CF, cystic fibrosis; CB, chronic bronchitis; COPD, chronic
obstructive pulmonary disease; MuSC, mucous secretory cells; BAL, bronchoalveolar
lavage; WT, wild type; KO, knockout; BALF, bronchoalveolar lavage fluid; AB/PAS, Al-
cian blue/periodic acid-Schiff; Scnn1b, sodium channel nonvoltage gated 1, ? subunit.
Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00
The Journal of Immunology
adaptive immune responses (9). Collectively, the presence of
MuSC metaplasia, airspace enlargement, and inflammatory
markers of both Th1- and Th2-type responses suggests that the
mucosal immune response in Scnn1b-Tg mice is multifactorial
and shares features of both aerotoxin- and allergen-mediated
lung pathologies, such as COPD and asthma.
One strategy to identify key signaling pathways in the develop-
ment of lung pathology is to crossbreed Scnn1b-Tg mice to mice
deficient in putatively relevant inflammatory mediators. Based on
the Scnn1b-Tg mouse phenotype, we focused our studies on two
pathways, namely TNF-? and IL-4R?. Exogenous administration
or transgenic overexpression of TNF-? in murine airways pro-
motes mucus secretion, lymphoid hyperplasia, and emphysema
(10–13). Conversely, genetic ablation of TNF-?-mediated signal-
ing prevents cigarette smoke-induced matrix breakdown, macro-
phage and neutrophil influx, and late-onset emphysema (14). IL-
4R? is a shared component of the receptors for IL-4 and IL-13,
two cytokines that trigger Th2-type airway inflammation and re-
modeling (15, 16) and have been highly implicated in the patho-
genesis of allergy and asthma (17, 18). Ablation of IL-4R? sig-
naling has been shown to suppress IL-4- and IL-13-induced airway
MuSC metaplasia and eosinophilic inflammation (19) and inhibit
accumulation of chitinases YM1/2 in bronchoalveolar lavage
(BAL) of allergic mice (20). In particular, Clara cell-targeted de-
letion of IL-4R? was sufficient to prevent allergen-induced MuSC
metaplasia (21). Finally, in vitro studies showed that both TNF-?
(22–24) and IL-4/IL-13 (25) inhibited ENaC-mediated Na?ab-
sorption and could thus modify the Scnn1b-Tg mouse phenotype.
To study the contribution of TNF-? and IL-4R? signaling to the
development of Scnn1b-Tg mouse lung pathology, we crossbred
Scnn1b-Tg mice with mice deficient in TNF-?, TNF-?R1, or IL-
4R? and studied survival, lung pathology, BAL cell, cytokine/
chemokine, and mucin content, and airway ion transport proper-
ties. As a complementary approach to our genetic studies, we
tested whether established Scnn1b-Tg mouse lung disease was
modulated by pharmacologic treatment with prednisolone, a broad
spectrum antiinflammatory glucocorticoid (26) shown to reduce
eosinophilia and MuSC metaplasia in murine models of atopic
Materials and Methods
All mice were housed in individually ventilated microisolator cages, in a
specific pathogen-free facility maintained at the University of North Caro-
lina at Chapel Hill, on a 12-h day/night cycle. They were fed a regular
chow diet and given water ad libitum. Hemizygous Scnn1b-Tg mice
(Scnn1b-Tg?/?, or Scnn1b-Tg) and littermate controls (Scnn1b-Tg nega-
tive, or wild type (WT)) were obtained by breeding Scnn1b-Tg mice with
C3H/HeN:C57BL6/N (C3:B6) F1mice (Taconic) and genotyped for
Scnn1b-Tg expression by PCR of genomic DNA, as originally described
(1). To generate Scnn1b-Tg/IL-4R?-deficient mice and appropriate litter-
mate controls, we first bred Scnn1b-Tg mice with IL-4R? knockout (KO)
mice (IL-4R??/?, strain BALB/c-Il4ratm1Sz/J (32), kindly provided to us
by Dr. Beverly Koller, University of North Carolina at Chapel Hill; IL-
4R?-deficient mice exhibit loss of IL-4- and IL-13-mediated responses
upon challenge, but they do not exhibit phenotypic abnormalities at base-
line (32)) and generated IL-4R? heterozygous (IL-4R??/?) Scnn1b-Tg
mice. IL-4R??/?Scnn1b-Tg mice were then bred with IL-4R? KO mice
to obtain experimental animals of four predicted genotypes: IL-4R??/?,
WT; IL-4R??/?, WT; IL-4R??/?, Scnn1b-Tg; and IL-4R??/?, Scnn1b-
Tg. We note that this breeding strategy generated control mice heterozy-
gous for the deleted gene of interest, which were not expected to be dif-
ferent from homozygous WT mice. However, we gained the advantage that
experimental animals of all four genotypes, with the expected Mendelian
distribution of 25% each, shared the identical environment (i.e., littermate
controls). We used multiple breeders and multiple litters per breeder to
minimize founder and litter order effects. Although nonrandom strain ef-
fects can never be ruled out, the use of littermate controls is the best
possible approach for reducing the chance of misinterpreting transgene or
knockout effects (33). The same breeding strategy was used to generate
experimental animals for the TNF-? KO ? Scnn1b-Tg and TNF-?R1
KO ? Scnn1b-Tg crosses, with the exception that we used inbred
C57BL6/N Scnn1b-Tg mice, recently generated by backcrossing the orig-
inal C3:B6 Scnn1b-Tg mouse (line 6608) with C57BL/6N inbred mice for
12 generations (9). Both TNF-? KO (B6;129-Tnftm1Gkl/J (34), mixed 129S/
SvEv:C57BL/6J background, stock 003008) and TNF-?R1 KO (B6.129-
Tnfrsf1atm1Mak/J (35), C57BL/6J background, stock 002818) mice were
from The Jackson Laboratory. WT inbred C57BL/6N, C3H/HeN, and
BALB/cJ mice for studies of normal neonatal development were obtained
from The Jackson Laboratory and Taconic. For prednisolone treatment
experiments, we used inbred C57BL6/N Scnn1b-Tg mice and their WT
littermates. All animal studies were approved by the Institutional Animal
Care and Use Committee of the University of North Carolina at Chapel Hill
and performed according to the principles outlined by the Animal Welfare
and the National Institutes of Health guidelines for the care and use of
animals in biomedical research.
Weaned 5- to 6-wk-old C57BL/6N Scnn1b-Tg mice and WT littermates
were administered prednisolone (20 mg/kg/day) by i.p. injection, for 2 wk.
Prednisolone (provided by Pfizer) was administered twice a day, that is,
two injections of 10 mg/kg in 100 ?l of sterile vehicle (0.1% Pluronic P105
in sterile, pyrogen-free 150 mM NaCl plus 10% DMSO), ?10 h apart.
Control mice (not receiving prednisolone) were injected with 100 ?l of
vehicle on the same schedule. Mice were weighed every day before each
injection. At the end of the 2 wk treatment, BAL was performed and lung
tissue harvested for histological evaluation of lung pathology, as described
For 10-day-old or older mice, we used a standardized procedure to obtain
both BAL cell counts and lung histology from each animal. Mice were
euthanized by exsanguination under deep avertin (2,2,2-tribromoethanol)
anesthesia and the chest cavity was opened to ligate the left main bronchus.
A blunt needle (20-gauge for adults, 22-gauge for 5- to 10-day-old pups)
was inserted through a small incision in the upper trachea and tied in place
with 3.0 silk. After ligation of the left main stem bronchus, BAL was
performed on the right lobes by instilling a volume of room temperature,
sterile PBS determined by the formula: (mouse weight (g) ? 0.0175 ml ?
ml PBS instilled) (36). Due to their small size, 5-day-old pups were subject
to either whole lung lavage (mouse weight (g) ? 0.035 ml ? ml PBS
instilled) or fixation for histology, but not both. BAL was performed by
gently injecting and retrieving the PBS volume three times. This procedure
was conducted a second time with an equal volume of PBS, and fractions
were pooled. Return volume was consistently ?80% of the instilled vol-
ume. BAL cells were pelleted by centrifugation at 1000 ? g for 5 min at
4°C and the cell-free supernatant (BAL fluid, or BALF) was collected and
stored at ?80°C for further analysis. BAL cells were resuspended in 100
?l of PBS and total cells were counted with a hemocytometer. Cytospin
slides of 30,000–60,000 cells/slide were obtained (StatSpin CytoFuge 2),
air dried, and stained with modified Giemsa for differential cell counts
(Newcomer Supply) of at least 200 cells per slide. After BAL, the left
bronchial ligature was removed and the left lung was immersion-fixed in
10% neutral-buffered formalin to prevent dislodging of airway luminal
Fixed lungs were embedded in paraffin oriented to maximize longitudinal
sectioning of primary bronchi, sectioned to a thickness of 4–6 ?m, and
stained with H&E for assessment of lung morphology and Alcian blue/
periodic acid-Schiff staining (AB/PAS) for mucopolysaccharides. The se-
verity of lung pathology was graded semiquantitatively on a scale ranging
from 0 to 3 for the following features: 1) airway obstruction, that is, air-
ways obstructed by AB/PAS-positive mucus: 0, no obstruction; 1, one
airway partially or totally obstructed; 2, two airways partially or totally
obstructed; 3, three or more airways partially or totally obstructed; 2)
MuSC abundance, that is, estimated percentage of AB/PAS positive cells
in airway epithelium: 0, none; 1, 0–5% MuSC; 2, 5–20% MuSC; 3, ?20%
MuSC; 3) airspace enlargement, that is, enlargement of the alveoli in the
parenchymal space: 0, none; 1, spotty; 2, 50% of parenchyma; 3, ?50% of
parenchyma; 4) lymphoid hyperplasia, that is, perivascular, peribronchial,
or parenchymal lymphoid aggregates: 0, none; 1, one nodule per lung sec-
tion; 2, two nodules per lung section; 3, three or more nodules per lung
section; and 5) airway inflammation, that is, interstitial thickening and
inflammatory cell infiltrate: 1, one airway; 2, two airways; 3, three or more
4358 MECHANISMS OF LUNG PATHOLOGY CAUSED BY ASL DEHYDRATION
airways. To confirm the results of the semiquantitative score for MuSC
abundance, we used MetaMorph image analysis software (MDS Analytical
Technologies) and determined the percentage of airway epithelial area pos-
itive for AB/PAS staining. Briefly, three random fields within the left lung
proximal main stem bronchus were photographed with an upright Nikon
Microphot-SA microscope interfaced with a DXM 1200 color camera (Ni-
kon Instruments) at ?20 magnification. The AB/PAS-positive area was
measured by thresholding and was divided by the total epithelial area, to
give the volume density of stored mucosubstances. Tissue blocks received
a numerical code at time of embedding, and scoring of the slides was
performed by an investigator blinded to specimen genotype.
Agarose gel mucin Western blot
This method was used to measure secreted mucins, as described in detail
(37). BAL samples were centrifuged at low speed (1000 ? g for 5 min).
Total protein concentration of BALF was determined using the Microplate
BCA protein assay, according to the manufacturer’s instructions (Thermo
Scientific), and was used to control for equivalent loading, since the large
molecular sieve of agarose gels does not allow retention of globular pro-
teins conventionally used for normalization. An equal volume of 8 M gua-
nidine hydrochloride (GuHCl) was added to the BALF. GuHCl-dispersed
samples were dialyzed against 6 M urea, reduced with 10 mM DTT, and
alkylated with 25 mM iodoacetamide. Alternatively, BALF was directly
diluted 1/5 in 6 M urea plus 0.1% SDS, reduced, and alkylated. Equal
volumes of reduced samples (20–25 ?l) were run on 1% agarose gel using
a submerged gel electrophoresis apparatus with Tris acetate-EDTA/SDS
buffer, at 80 V for 90–120 min. Gels were vacuum-blotted onto nitrocel-
lulose membranes, blocked with Odyssey blocking buffer (OBB; LI-COR
Biosciences), and probed with a rabbit polyclonal Ab raised against puri-
fied cervical mucins (“reduced subunit antibody”, described in Refs. 38,
39). This Ab recognizes the cysteine-rich domain of almost all mucins and
is thus a “pan-mucin” detection reagent. Alternatively, blots were probed
with a rabbit polyclonal Ab against murine Muc-5b (described in Refs. 40,
41). Pan-mucin and Muc5b Abs were diluted 1/2000 and 1/1000 in OBB
plus 0.1% Tween 20 (OBBT), respectively. The secondary Ab was Alexa
Fluor 680 goat anti-rabbit IgG, diluted 1/15,000 in OBBT. Detection and
analysis of specific signals were performed using the Odyssey infrared
imaging system (LI-COR Biosciences).
Measurement of cytokines and chemokines in BALF
Aliquots of cell-free BALF, stored at ?80°C, were used to measure mouse
TNF-?, KC, IL-4, IL-5, IL-13, IL-17, MCP-1, IL-6, MIP-2, and INF?
using a bead-based assay (Upstate (Millipore) Beadlyte multiplex assays/
Luminex), according to the manufacturer’s instructions.
Ussing chamber measurements of airway bioelectric properties
Freshly excised tracheas were mounted in Ussing chambers and equili-
brated as described (42). After recording the basal short-circuit current
(Isc), the following drugs were added sequentially to the chambers: amilo-
ride (10?4M, apical), forskolin (10?5M, apical), UTP (10?4M, apical),
and bumetanide (10?4M, basolateral), and changes in Iscwere recorded.
Statistical analyses were performed using SigmaStat 3.1 or GraphPad
Prism 4.0. Survival curves were compared using Kaplan-Meier log rank
analysis and Holm-Sidak multiple comparison. One-way ANOVA fol-
lowed by Tukey’s post hoc test for multiple comparisons were used to
determine significant differences among groups. A p value of ?0.05 was
considered statistically significant.
means ? SEM.
Alldataare expressed as
TNF-? signaling is not essential for airway inflammation and
remodeling in Scnn1b-Tg mice
To investigate the role of TNF-? in airway inflammation and re-
modeling in Scnn1b-Tg mice, we crossbred Scnn1b-Tg mice (in-
bred line C57BL/6N) with mice lacking either TNF-? ligand
(TNF-??/?mouse, C57:129Sv mixed background) or TNF-?R1
(TNF-?R1?/?mouse, C57BL/6J background), and produced four
possible genotypes as described in Materials and Methods. Both
crosses gave similar results, and data for the TNF-? KO cross are
shown in Fig. 1 and in supplemental Fig. 1, D and E,4while data
for the TNF-?R1 KO cross are provided in supplemental Fig.
Lack of TNF-? or TNF-?R1 did not alter survival of Scnn1b-Tg
mice, and all mice had comparable, high survival, ranging between
80 and 95% (Fig. 1A and supplemental Fig. 1A). Survival of
Scnn1b-Tg mice differed from previously published studies (1),
likely due to strain differences. In fact, ongoing backcross studies
in our laboratory, aimed at obtaining inbred strains of Scnn1b-Tg
mice, have shown that survival is significantly increased in the
C57BL/6N and 129S1/SvImJ backgrounds (9) compared with the
original mixed C3:B6 background (1).
TNF-? was significantly elevated in BAL from TNF-??/?
Scnn1b-Tg mice in comparison to TNF-??/?WT littermates (Fig.
1B, left panel), consistent with previous reports (3). As expected
due to TNF-? gene deletion, TNF-? was undetectable in BAL
samples from TNF-??/?Scnn1b-Tg mice. KC was significantly
elevated in both TNF-??/?Scnn1b-Tg mice and TNF-??/?
Scnn1b-Tg mice (Fig. 1B, right panel), indicating that absence of
TNF-? did not impact the production of this neutrophil
Histological lesions typically observed in Scnn1b-Tg mice are
illustrated in Fig. 1C. Semiquantitative scoring of these lesions in
5-wk-old mice from the TNF-? KO ? Scnn1b-Tg cross revealed
significant mucus plugging, airspace enlargement, lymphoid hy-
perplasia, and airway inflammation in Scnn1b-Tg mice compared
with WT littermates, irrespective of TNF-? or TNF-?R1 status
(Fig. 1D and supplemental Fig. 1B). TNF-? or TNF-?R1 defi-
ciency did not alter lung histology in WT mice. Absence of TNF-?
or TNF-?R1 did not prevent neutrophil and eosinophil infiltration in
Scnn1b-Tg mouse lung, as assessed by BAL differential cell counts
(Fig. 1E and supplemental Fig. 1C). Large, foamy alveolar macro-
phages (see Fig. 1C10) were present in both TNF-?-sufficient and
-deficient Scnn1b-Tg mice. Furthermore, TNF-? ablation did not af-
fect lung histopathology or BAL differential cell counts in 10-day-old
Scnn1b-Tg or WT mice (supplemental Fig. 1, D and E).
TNF-? has been shown to down-regulate ENaC activity in air-
way epithelial cells in vitro (22–24). To test whether lack of
TNF-? altered Na?transport, we studied freshly excised tracheas
from 5-wk-old mice in Ussing chambers (Fig. 1F). Amiloride-
sensitive short-circuit current (Isc) was significantly greater in
Scnn1b-Tg mice than in WT mice, consistent with increased ENaC
activity and as previously reported (1), but it was not affected by
TNF-? status. Collectively, these data indicate that TNF-? or
TNFR1 are not essential for development of airway and lung pa-
thology in Scnn1b-Tg mice.
Absence of IL-4R? enhances neonatal survival in Scnn1b-Tg
To determine the role of IL-4R? signaling in the development of
Scnn1b-Tg mouse lung pathology, we crossed Scnn1b-Tg (mixed
C3:B6 background) and IL-4R? KO mice (IL-4R? KO, BALB/cJ
background), as described in Materials and Methods. As expected,
the survival of WT mice was high (100%) and was unaffected by
the absence of IL-4R?. In contrast, Scnn1b-Tg mice exhibited
characteristic early postnatal mortality (Fig. 2A). In agreement
with our backcross studies, introduction of the BALB/cJ back-
ground reduced Scnn1b-Tg mouse survival in comparison to the
mixed C3:B6 background (32% vs 50% for C3:B6:BALB and
C3:B6 (1), respectively). However, genetic ablation of IL-4R? sig-
nificantly increased survival of Scnn1b-Tg mice (54% vs 32% for
4The online version of this article contains supplemental material.
4359The Journal of Immunology
IL-4R??/?Scnn1b-Tg vs IL-4R??/?Scnn1b-Tg mice; Fig. 2A).
By genotyping pups at days 1–3, we verified that all four geno-
types were present in the expected Mendelian proportions at birth
(supplemental Fig. 2A). The improvement in survival occurred be-
tween 5 and 12 days, the window of peak mortality for Scnn1b-Tg
mice, and stabilized thereafter (Fig. 2A).
Absence of IL-4R? decreases neonatal MuSC abundance and
eosinophilia in WT and Scnn1b-Tg mice
To elucidate the reason(s) for improved survival in IL-4R?-defi-
cient Scnn1b-Tg mice, we examined lung histology and BAL cell
counts in 10-day-old pups. Although MuSC are rare in adult WT
unchallenged mice, we detected luminal mucus and rather abun-
dant MuSC in the bronchi of 10-day-old IL-4R??/?WT mice
(Fig. 2, B3 and C). However, both mucus and MuSC were virtually
absent in IL-4R??/?WT mice (Fig. 2, B4 and C). Although bron-
chial mucus plugging, as detected histologically, was similar in
both IL-4R?-sufficient and -deficient Scnn1b-Tg mice (Fig. 2C),
MuSC were significantly reduced in IL-4R?-deficient Scnn1b-Tg
mice (Fig. 2, B7, B8, and C), which was confirmed by computer
image analysis of AB/PAS-positive stored mucosubstances (sup-
plemental Fig. 2B). However, absence of IL-4R? did not amelio-
rate the parenchymal air space enlargement and airway inflamma-
tory lesions already evident in young Scnn1b-Tg mice (Fig. 2C).
Lymphoid hyperplasia was never observed in 10-day-old animals.
Absence of IL-4R? decreased BAL eosinophils in 10-day-old
Scnn1b-Tg mice in comparison to IL-4R??/?Scnn1b-Tg mice
(Fig. 2D), but failed to reduce the pronounced macrophage and
background (TNF-? KO) decreases mortality of Scnn1b-Tg mice compared with the original C3H/HeN:C57BL/6N strain, irrespective of TNF-? status. See
text for details. B, BAL cytokine/chemokine profile in 5-wk-old mice. ?, p ? 0.05 vs TNF-??/?WT mice. The dotted line represents the assay lower
detection limit. C, Representative photomicrographs illustrating characteristic lesions in 5-wk-old TNF-??/?Scnn1b-Tg mice. H&E stain unless indicated.
1, Mucus plugs, AB/PAS stain, scale bar ? 100 ?m. 2, Mucous secretory cells, AB/PAS stain, scale bar ? 50 ?m. 3 and 4, Distal airspace enlargement,
comparing WT (3) and Scnn1b-Tg mice (4), scale bar ? 200 ?m. 5 and 6, Lymphoid hyperplasia, at low (5, scale bar ? 200 ?m) and high (6, scale bar ?
50 ?m) magnification. 7 and 8, Airway inflammation, at low (7, scale bar ? 50 ?m) and high (8, scale bar ? 20 ?m) magnification. Cytospins from WT
(9) and Scnn1b-Tg mice (10), illustrating large/foamy macrophages, neutrophils (arrows), and eosinophils (arrowhead), Giemsa stain, scale bar ? 20 ?m.
D, Semiquantitative histopathology scores indicating similar lesions in TNF-??/?and TNF-??/?Scnn1b-Tg mice. ?, p ? 0.05 vs TNF-??/?WT mice.
E, Differential BAL cell counts. ?, p ? 0.05 vs TNF-??/?WT mice. F, Ion transport properties of freshly excised tracheal tissues. “Basal” indicates the
Iscbefore drug application. The change in Isc(?) after sequential drug addition is shown. “Residual” Iscis the Iscremaining after amiloride application.
?, p ? 0.05 vs TNF-??/?WT mice.
Minimal effect of TNF-? deletion on the Scnn1b-Tg mouse phenotype. A, Survival curves. Introduction of the 129S/SvEv:C57BL/6J
4360 MECHANISMS OF LUNG PATHOLOGY CAUSED BY ASL DEHYDRATION
neutrophil infiltrate. Low, but readily detectable numbers of eo-
sinophils were also present in BAL from 10-day-old IL-4R??/?
WT mice (4890 ? 100 cells/ml), which was reduced in IL-4R??/?
WT mice (900 ? 260 cells/ml).
Similar to the results we obtained for genetic deletion of TNF-?,
we did not detect changes in airway ion transport properties of
either WT or Scnn1b-Tg mice due to the absence of IL-4R? (sup-
plemental Fig. 2C).
Absence of IL-4R? does not mitigate chronic lung pathology in
surviving adult Scnn1b-Tg mice
We next tested whether absence of IL-4R? modified chronic lung
pathology or inflammation in surviving 5-wk-old Scnn1b-Tg mice.
As expected, no differences were detected in adult WT mice due
to the presence or absence of IL-4R? (Fig. 3, A1 and A2). Ab-
sence of IL-4R? minimally altered lung histology scores and
BAL parameters in adult Scnn1b-Tg mice. Specifically, airway
mucus plugging, MuSC abundance, air space enlargement, lym-
phoid hyperplasia, and airway inflammation were equivalent in
IL-4R?-deficient and -sufficient Scnn1b-Tg mice (Fig. 3, A3, A4,
and B). As seen in 10-day-old animals, BAL eosinophils were
reduced in IL-4R??/?Scnn1b-Tg mice in comparison to IL-4R?-
sufficient mice, but marked neutrophilia persisted in Scnn1b-Tg
mice regardless of IL-4R? status (Fig. 3C).
Absence of IL-4R? does not modify the BAL inflammatory
mediator profile of neonatal or adult Scnn1b-Tg mice
Genetic deletion of IL-4R? reduced neonatal MuSC and improved
survival in Scnn1b-Tg mice, but it did not eliminate chronic neu-
trophilic inflammation. To elucidate the IL-4R?-independent
mechanisms driving the development of chronic lung pathology,
we analyzed BALF cytokines in 10-day-old and 5-wk-old
Scnn1b-Tg mice. INF?, MCP-1, and IL-17 were below the detec-
tion limit in all samples. KC and TNF-? levels were elevated in
Scnn1b-Tg mice compared with WT littermates at both early and
late time points and were not altered by IL-4R? status (Fig. 4, A
and B). A trend toward higher IL-4, IL-5, and IL-13 levels was
observed in 10-day-old Scnn1b-Tg mice in comparison to WT lit-
termates, which normalized by 5 wk of age (Fig. 4C–E; IL-4,
2.0 ? 0.7 vs 0.02 ? 0.02; IL-5, 127 ? 37 vs 15.2 ? 7; IL-13,
17.6 ? 6 vs 4.3 ? 1 pg/ml for 10-day-old Scnn1b-Tg and WT
mice, respectively; n ? 10). Absence of IL-4R? caused a small but
significant decrease in IL-5 in 5-wk-old Scnn1b-Tg mice. In agree-
ment with a previous study (43), transient eosinophilia in 10-day-
old WT mice was not associated with increased BALF levels of
IL-4, IL-5, and IL-13 in comparison to adult mice.
Elements of Scnn1b-Tg airway and lung pathology are
In view of the minimal impact of TNF-?, TNFR1, or IL-4R? ge-
netic removal on adult Scnn1b-Tg mouse lung pathology/inflam-
mation, we investigated the effects of glucocorticoid treatment.
Daily systemic administration of 20 mg/kg prednisolone for 2 wk,
starting at 5–6 wk of age, inhibited the body weight gain (2.5
g/week) typical of both Scnn1b-Tg and WT littermates (supple-
mental Fig. 3, A and B), likely reflecting catabolic effects of high-
dose systemic glucocorticoids (48) and indicating effective drug
delivery. Prednisolone administration markedly diminished MuSC
abundance and eosinophilia and significantly reduced the inci-
dence of lymphoid aggregates in Scnn1b-Tg mice, but it failed to
ameliorate mucus plugging, airway inflammation, and neutrophilia
(Fig. 5A–C). BAL lymphocyte counts were decreased by pred-
nisolone, but not significantly (2620 ? 867 and 993 ? 580 lym-
phocytes/ml BAL for vehicle and prednisolone, respectively). In
Scnn1b-Tg mice, prednisolone administration increased the BALF
total protein content (Fig. 5D), but it did not affect the levels of
KC, MIP-2, TNF-?, and IL-6, which were significantly elevated in
comparison to WT littermates (data not shown). Interestingly,
IL-4R? improves neonatal survival
cells and eosinophils in 10-day-old
Scnn1b-Tg mice. A, Survival curves.
Introduction of the BALB/c/J back-
ground (IL-4R? KO) increases mor-
tality of Scnn1b-Tg mice compared
C57BL/6N strain, and absence of IL-
4R? improved survival. See text for
details. #, p ? 0.014 vs IL-4R??/?
Scnn1b-Tg mice. B, Representative
photomicrographs of trachea (1 and 2
for WT and 5 and 6 for Scnn1b-Tg
mice) and lung (3 and 4 for WT and
7 and 8 for Scnn1b-Tg mice) of IL-
4R??/?and IL-4R??/?mice, AB/
PAS stain. Scale bars: low magnifi-
cation ? 200 ?m, high magnification
insets ? 20 ?m. C, Semiquantitative
histopathology scores. ?, p ? 0.05 vs
IL-4R??/?WT mice. #, p ? 0.05 vs
IL-4R??/?Scnn1b-Tg mice. D, Dif-
ferential BAL cell counts. ?, p ?
0.05 vs IL-4R??/?WT mice. #, p ?
0.05 vs IL-4R??/?Scnn1b-Tg mice.
Genetic deletion of
4361The Journal of Immunology
prednisolone also reduced the normal abundance of bronchial
MuSC in WT mice (Fig. 5, A and B2). We assessed whether the
prednisolone-induced reduction in MuSC correlated with de-
creased BAL mucin protein content, as assessed by agarose gel
Western blots. As shown in Fig. 5E, BAL mucin content was sig-
nificantly higher in Scnn1b-Tg mice compared with WT mice, and
it was not reduced as a function of prednisolone administration.
Normal postnatal development of mouse airways entails a
spatially and temporally confined abundance of MuSC and
The presence of AB/PAS-positive MuSC and eosinophils in the
airways of 10-day-old IL-4R?-sufficient WT mice and their ab-
sence in IL-4R?-deficient mice prompted us to investigate whether
the appearance of MuSC and eosinophils was a normal develop-
mental feature of the mouse respiratory system. We analyzed tra-
cheal and lung histology in 5-day-old, 10-day-old, and 5-wk-old
mice from C57BL/6N, C3H/HeN, and BALB/cJ inbred strains and
found a distinct developmental pattern. At day 5, when submuco-
sal glands are rudimentary (44, 45), AB/PAS-positive cells were
abundant in the trachea (illustrated for the C57BL/6N strain in Fig.
6A1) and absent in the bronchi (Fig. 6A4), whereas by day 10 their
frequency diminished in the trachea (Fig. 6A2) and increased in the
proximal portion of the main stem bronchi (Fig. 6A5). At 5 wk,
MuSC were virtually absent in the trachea (Fig. 6A3), and the
number of bronchial MuSC stabilized at the low levels found in
adult mice (Fig. 6A6). This pattern was conserved among inbred
strains (Fig. 6B for C57BL/6N and supplemental Fig. 4, A and B,
for C3H/HeN and BALB/cJ).
BAL differential cell counts from 5-day-old, 10-day-old, and
5-wk-old C57BL/6N, C3H/HeN, and BALB/cJ mice revealed
mainly macrophages, although we detected rare neutrophils in 5-
to 10-day-old mice, which were absent in adult animals (Fig. 6C
for C57BL/6N and supplemental Fig. 4, C and D, for C3H/HeN
and BALB/cJ). Notably, we also found a subtle but consistent
degree of eosinophilia in 10 day-old C57BL/6N and C3H/HeN mice.
Eosinophil counts were significantly lower in 10 day-old BALB/cJ
mice (supplemental Fig. 4D) compared with C57BL/6N and C3H/
HeN mice, which is consistent with previous studies comparing al-
lergic BALB/cJ and C57BL/6N mice and the reported lower affinity
of the BALB/c IL-4R? variant for IL-4 (46, 47).
BAL secreted mucin content is increased in Scnn1b-Tg mice
To test for a biochemical correlate of the time-dependent changes
in MuSC observed in WT mice, and to assess how BALF mucin
content may be affected by airway surface dehydration, we per-
formed a time-course analysis in WT mice and their Scnn1b-Tg
mouse phenotype. A, Representative photomicrographs of lung histology in
WT (1 and 2) and Scnn1b-Tg (3 and 4) mice, H&E stain, scale bar ? 200
?m. B, Semiquantitative histopathology scores. ?, p ? 0.05 vs IL-4R??/?
WT mice. C, Differential BAL cell counts. ?, p ? 0.05 vs IL-4R??/?WT
mice. #, p ? 0.05 vs IL-4R??/?Scnn1b-Tg mice.
Minimal effect of IL-4R? deletion on the adult Scnn1b-Tg
Scnn1b-Tg mice has minimal effect
on sustained BAL TNF-? and KC and
transient Th-2 cytokines. BAL cyto-
kines in 10-day-old and 5-wk-old
mice are shown. The dotted line rep-
resents the assay lower detection
limit. ?, p ? 0.05 vs age-matched IL-
4362MECHANISMS OF LUNG PATHOLOGY CAUSED BY ASL DEHYDRATION
littermates (C57BL/6N line). At all ages, the BALF mucin content,
assessed with Abs that detect either all mucins (Fig. 7A) or murine
Muc5b (supplemental Fig. 5A), were greater in Scnn1b-Tg mice
than in WT mice, whereas the BALF total protein content for
Scnn1b-Tg mice was only slightly increased in comparison to WT
mice at 5 days and 8 wk of age (Fig. 7B). In both WT and
Scnn1b-Tg littermates, BALF mucins were greatest in 5- and 10-
day-old mice and declined in older animals. This temporal pattern
of mucin glycoprotein expression is consistent with prior Muc5ac,
Muc5b, Muc4, and Gob5 mRNA expression studies (3) and sug-
gests that relative mucin abundance is a consistent feature of
neonatal airway development, which is further augmented by
impaired mucus clearance in Scnn1b-Tg mice. Analysis of
BALF mucin content in 5-day-old, 10-day-old, and 5-wk-old
ment of Scnn1b-Tg mice reduces
MuSC and eosinophils but not neutro-
phils or BAL mucin content. A, Semi-
quantitative histopathology scores. ?,
p ? 0.05 vs vehicle-treated WT mice.
Scnn1b-Tg mice. B, Representative
photomicrographs of lung sections
from WT (1 and 2) and Scnn1b-Tg
mice (3 and 4) treated with vehicle (1
and 3) or prednisolone (2 and 4), AB/
PAS stain, scale bars: low magnifica-
tion ? 100 ?m, high magnification
insets ? 20 ?m. C, BAL differential
cell counts. ?, p ? 0.05 vs vehicle-
treated WT mice. #, p ? 0.05 vs ve-
hicle-treated Scnn1b-Tg mice. D,
BALF total protein content. ?, p ?
0.05 vs vehicle-treated WT mice;
Scnn1b-Tg mice. E, Agarose gel
Western blots and corresponding den-
sitometry of BAL samples from vehi-
cle- and prednisolone-treated mice.
Blots were probed with Muc5b Ab. ?,
p ? 0.05 vs WT mice.
are transiently abundant during nor-
mal mouse airway development. A,
Representative photomicrographs of
trachea and bronchi from C57BL/6N
mice at age 5 days (1 and 4), 10 days
(2 and 5), and 5 wk (3 and 6), AB/
PAS stain, scale bars: low magnifica-
tion ? 200 ?m, high magnification
insets ? 20 ?m. B, Semiquantitative
MuSC in tracheas and bronchi of
C57BL/6N mice at age 5 days (5 dd),
10 days (10 dd), and 5 wk. C, Differ-
ential BAL cell counts in C57BL/6N
mice at 5 days, 10 days, and 5 wk of
age. ?, p ? 0.05 vs 5-day-old and
MuSC and eosinophils
4363 The Journal of Immunology
WT mice of diverse genetic backgrounds (inbred C57BL/6N,
C3H/HeN, and BALB/cJ) revealed modest age- and strain-de-
pendent changes in BALF total and Muc5b mucin content (sup-
plemental Fig. 5B–D).
Adequate airway mucosal surface hydration is essential for effec-
tive mucus clearance and lung health. The dynamic progression of
lung disease following disruption of mucus clearance in
Scnn1b-Tg mice suggests a complex host response to airway sur-
face dehydration (3). We investigated the role of TNF-? and IL-
4R? signaling during postnatal lung development and identified
aspects of Scnn1b-Tg lung pathology that are uniquely susceptible
to modification of these pathways and to corticosteroid treatment.
Our studies indicated that TNF-? signaling is not required for
triggering or sustaining inflammation, airway remodeling, and dis-
tal lung pathology (air space enlargement) in Scnn1b-Tg mice. The
earlyappearance ofair trapping/airspace
Scnn1b-Tg mice suggests that neonatal airway inflammation might
shift the protease/antiprotease balance, impairing alveolarization
and generating proinflammatory signals via extracellular matrix
degradation, for example, Pro-Gly-Pro peptide (49) and hyaluro-
nan (50). Development of pulmonary lymphoid aggregates in
Scnn1b-Tg mice was also TNF-?-independent. Since similar nod-
ules are found in lungs of mice repetitively challenged with aero-
solized allergen (51, 52) or Haemophilus influenzae lysate (53), we
speculate that lymphoid hyperplasia is caused by greater and/or
more sustained exposure to environmental Ags due to mucus sta-
sis, which in turn enhances adaptive immune responses. Finally,
TNF-? inhibits ENaC expression and activity in vitro (22–24) and
can down-regulate the rat CCSP promoter (54), which in
Scnn1b-Tg mice drives ?ENaC overexpression. However, we
found that absence of TNF-? did not alter airway epithelial ion
transport properties in excised tracheal tissue from WT or
Scnn1b-Tg mice, suggesting that TNF-? does not affect either
ENaC activity or the CCSP promoter in vivo.
Breeding Scnn1b-Tg mice with IL-4R? gene-deleted mice re-
vealed the existence of IL-4R?-dependent and -independent ele-
ments of Scnn1b-Tg mouse lung pathology and provided new in-
sights regarding the role of IL-4R? signaling during normal
neonatal airway development.
In murine models of allergic asthma, eosinophils are involved in
collagen deposition and airway smooth muscle hyperplasia, but
they are not required for airway hyperreactivity or MuSC meta-
plasia (55, 56). Our data suggest that eosinophils are not a major
determinant of lung pathology in Scnn1b-Tg mice. Decreased eo-
sinophils correlated with fewer MuSC in neonatal but not adult
IL-4R??/?Scnn1b-Tg mice, and lack of eosinophils did not ame-
liorate other aspects of lung pathology in Scnn1b-Tg mice. More-
over, although a time-dependent role for eosinophils in promoting
MuSC abundance is conceivable based on correlations observed
between neonatal MuSC and eosinophilia (see Figs. 2, C and D, 6,
B and C, and supplemental Fig. 4, A and C), the abundant MuSC
found in normal 10-day-old BALB/c/J mice (supplemental Fig.
4B), despite the virtual absence of eosinophils (supplemental Fig.
4D), suggests that a causal association is unlikely.
The transient and spatially restricted appearance of MuSC in the
airways of neonatal WT mice was completely ablated in the ab-
sence of IL-4R? (Fig. 2C). We speculate that neonatal MuSC ex-
pansion is driven by local, basal levels of Th2 cytokines (IL-4,
IL-13, and IL-5; Fig. 4C–E), which are difficult to detect once
diluted in BAL. In comparison to WT littermates, neonatal
Scnn1b-Tg mice had higher levels of Th2 cytokines (Fig. 4C–E),
which likely increased MuSC (Fig. 2C). However, absence of IL-
4R? mitigates this response. The residual MuSC in neonatal IL-
4R??/?Scnn1b-Tg mice as compared with IL-4R??/?WT mice
(Fig. 2C) indicates the existence of an IL-4R?-independent path-
way that triggers MuSC in neonatal Scnn1b-Tg mice. Indeed, this
pathway could also be active in adult Scnn1b-Tg mice, in which
MuSC are abundant (Fig. 3B) but Th2 cytokine levels return to-
ward baseline (Fig. 4C–E).
The similarity in the MuSC distribution pattern among neonatal
WT mice (Figs. 2B3 and 6A5), Scnn1b-Tg mice (Fig. 2B7), and
OVA-challenged mice (57), namely abundance in the proximal
main stem bronchi and a gradual decrease distally, suggests that
specific populations of airway epithelial cells are primed to differ-
entiate into MuSC in response to external stimuli. Reports of tem-
poral and spatial expression of transcription factors involved in
promoting or suppressing MuSC, such as SPDEF (58) and FOXA2
(59), support this hypothesis.
Scnn1b-Tg mice exhibited mucus plugging equivalent to IL-4R?-
sufficient Scnn1b-Tg mice, despite decreased MuSC (Fig. 2C).
Similar results obtained upon prednisolone treatment suggest that
airway surface dehydration and defective mucus clearance are ma-
jor determinants of mucus accumulation over a wide range of
MuSC abundance. Moreover, even in WT mice, the high degree of
variation in MuSC abundance detected histologically at 5 days, 10
days, and 5 wk of age (Fig. 6, A and B, and supplemental Fig. 4,
A and B) in comparison to the modest changes observed in BALF
total and Muc5b mucins (supplemental Fig. 5, B and C) suggests
that BAL mucin content depends on factors other than the intra-
cellular content of stored mucosubstances alone. We hypothesize
that constitutively secreted mucins (40), including Muc5b as de-
tected in BAL (supplemental Fig. 5, A and C) and shed cell surface
mucins (Muc1, Muc4, and Muc16), are an integral part of secreted
mucus in WT mice and contribute to mucus obstruction in
Scnn1b-Tg mouse airways. However, the survival advantage of
elevated in neonatal and adult Scnn1b-Tg mice. A, Agarose gel Western
blots and corresponding densitometry of BAL samples from C57BL/6N
Scnn1b-Tg mice and WT littermates at age 5 days (5 dd), 10 days (10 dd),
4 wk, and 8 wk. Membranes were probed with pan-mucin Ab. ?, p ? 0.05
vs WT mice of corresponding age; #, p ? 0.05 vs 10-day-old mice of
corresponding genotype. B, BALF total protein content. ?, p ? 0.05 vs WT
mice of corresponding age.
BALF total mucin content varies as a function of age and is
4364MECHANISMS OF LUNG PATHOLOGY CAUSED BY ASL DEHYDRATION
IL-4R?-deficient Scnn1b-Tg mice suggests that, especially during
the neonatal period, modifications of the mucus secretory system
can be beneficial to prevent fatal airway obstruction when mucus
clearance is impaired. The absence of significant differences in
tracheal epithelial ion transport properties due to the absence of
IL-4R? in Scnn1b-Tg mice (supplemental Fig. 2C) supports our
conclusion that the increased survival of IL-4R?-deficient
Scnn1b-Tg mice reflects modifications in MuSC and mucus secre-
tion, not Na?transport.
Although IL-4R? removal had an impact on the neonatal phe-
notype, lung lesions in adult IL-4R?-sufficient and -deficient
Scnn1b-Tg mice were indistinguishable. In contrast to allergen
challenge (21) or the late response to viral infection (60), which
require the IL-13/IL-4R? signaling axis to elicit lung pathology,
our findings strongly suggest that airway surface dehydration/mu-
cus stasis is a unique stimulus that activates multiple IL-4R?-in-
dependent effector pathways leading to inflammation and remod-
eling in Scnn1b-Tg mice.
Given the complexity of the inflammatory responses in
Scnn1b-Tg mice, we tested whether a broad-spectrum antiinflam-
matory agent could ameliorate or reverse adult lung lesions. Glu-
cocorticoids are widely used both clinically and experimentally to
blunt inflammation and mucus hypersecretion (27–31, 61, 62), and
they can effectively reduce eosinophils, mast cells, CD4?T lym-
phocytes, dendritic cells, and proinflammatory cytokines in asthma
(63, 64). However, in obstructive lung diseases characterized by
neutrophilic inflammation, for example, chronic bronchitis and
COPD, inflammation appears to be corticosteroid-resistant (65,
66). In Scnn1b-Tg mice, prednisolone treatment blunted eosino-
philia and MuSC (Fig. 5A–C), similar to other mouse models (62,
67, 68) but did not reduce neutrophil influx, the appearance of
large foamy macrophages, or mucus accumulation in the airway
lumen. In COPD, unresponsiveness to glucocorticoids has been
attributed to inhibition of histone deacetylase 2 activity by oxida-
tive and nitrosative stress (26), a condition that may also occur in
Scnn1b-Tg mice. Alternatively, glucocorticoids have been shown
to spare or enhance innate immunity while repressing adaptive
responses (69, 70). We suggest that chronic neutrophil recruitment
and macrophage activation are due to a glucocorticoid-insensitive,
innate immune response triggered by accumulation of environ-
mental and endogenous stimuli in stagnant mucus, which likely
involves signaling through pattern recognition receptors, such
Our studies with neonatal Scnn1b-Tg mice and WT littermates
led us to discover a temporal and spatial pattern of MuSC and
inflammatory cell abundance in the airways of unchallenged WT
mice suggestive of an active phase of maturation and adaptation of
innate immune responses (Fig. 6). Perinatal changes in MuSC/
mucus composition and BAL leukocytes have been described in
several species (71–76), including humans (77–81). Surprisingly,
we found little information regarding these normal developmental
changes in mouse airways. In agreement with our studies, one
study that focused on mouse submucosal gland development inci-
dentally showed abundant MuSC in the superficial epithelium at
postnatal day 4 (44), and there is one report of eosinophilia in
10-day-old C3HeB/FeJ mice as compared with adult animals (43).
Our histological sectioning protocol and the multiple time points
studied enabled us to visualize the dynamic changes in MuSC and
BAL cells. We hypothesize that immediately after birth, when the
acquired immune system is still immature (82, 83), airway muco-
sal immunity relies on secreted mucus as a primary barrier to pro-
tect the host from inhaled, potentially pathogenic particles. A
growing body of evidence indicates that neonatal events, for ex-
ample, bacterial colonization (84, 85), viral infection (86, 87), or
allergen exposure (88), have a profound impact on later immune
responses. In this context, effective mucus clearance is critical be-
cause it determines the concentration and dwell time of particles/
pathogens/chemical mediators, the extent of inflammatory and pa-
renchymal cell activation, and the propensity for airway
obstruction. While mucus abundance may offer broad airway pro-
tection (89), it may become life-threatening when coupled with
defective clearance, as in Scnn1b-Tg mice. Indeed, different sur-
vival rates observed in Scnn1b-Tg mice of different genetic back-
grounds (Wanda O’Neal, unpublished observation and Ref. 9)
likely reflect diversity in the amount/physical properties of se-
creted mucus or in the architecture of the proximal airways.
In summary, we propose that in neonatal Scnn1b-Tg mice, im-
paired mucus clearance strongly interacts with the normal, IL-
4R?-dependent abundance of MuSC and eosinophils. This inter-
action produces both potentially lethal mucus obstruction and
delays the normal regression of MuSC and eosinophils. In addition
to exogenous aerotoxins and endogenous mediators trapped in
static mucus, epithelial cell necrosis, transiently present in the
lower airways of newborn Scnn1b-Tg mice (3), contributes to pro-
mote local inflammation (90) and disease onset. Although unre-
solved/lingering MuSC abundance and eosinophilia can be blunted
by corticosteroid treatment, neutrophilia and mucus obstruction
persist and are likely responsible for maintaining airway and lung
disease in Scnn1b-Tg mice. Future studies elucidating the interac-
tion of mucus clearance with other components of airway mucosal
immune system during postnatal development, and identifying the
molecular mechanisms that sustain airway inflammation and re-
modeling in adults, will suggest therapeutic approaches for com-
mon human obstructive airway diseases.
The authors thank Kimberly Burns, Donald Joyner and Tracy Eldred for
outstanding technical assistance with histology; Troy Rogers for assistance
with Ussing chamber studies; the University of North Carolina Michael
Hooker Microscopy Facility, funded by an anonymous private donor, for
assistance with imaging; the Thurston Arthritis Research Center, Clinical
Proteomics Laboratory (directed by Dr. R. Roubey) for Luminex assays;
Dr. Camille Ehre, Dr. Mehmet Kesimer, and Genevieve DeMaria for pro-
viding the Muc5b Ab and assistance with the mucin Western blots; Dr. J.
Schwabe and Athena Jin for assistance with histology scoring; and Dr.
S. L. Tilley for reading the manuscript.
The authors have no financial conflicts of interest.
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