Foxa2 Programs Th2 Cell-Mediated Innate
Immunity in the Developing Lung
This information is current as
of June 16, 2017.
Gang Chen, Huajing Wan, Fengming Luo, Liqian Zhang,
Yan Xu, Ian Lewkowich, Marsha Wills-Karp and Jeffrey A.
Whitsett
J Immunol published online 5 May 2010
http://www.jimmunol.org/content/early/2010/05/05/jimmun
ol.1000223
http://www.jimmunol.org/content/suppl/2010/05/05/jimmunol.100022
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Supplementary
Material
Published May 5, 2010, doi:10.4049/jimmunol.1000223
The Journal of Immunology
Foxa2 Programs Th2 Cell-Mediated Innate Immunity in the
Developing Lung
Gang Chen,* Huajing Wan,† Fengming Luo,‡ Liqian Zhang,* Yan Xu,*
Ian Lewkowich,x Marsha Wills-Karp,x and Jeffrey A. Whitsett*
A
fter birth, the respiratory tract is recurrently exposed to
pathogens, allergens, and toxicants. A multilayered innate and acquired host defense system develops to
maintain pulmonary homeostasis in the face of these challenges.
Th2-dominated inflammation and mucus hyperproduction are
common features of acute and chronic pulmonary disorders, including asthma. Normally, inflammatory responses are precisely
balanced to achieve mucociliary clearance and removal of
pathogens. Various signaling and transcriptional programs influence allergic lung inflammation and mucus hyperproduction,
including the IL-4R/STAT6 (1), TLR/NF-kB (2), and epidermal
growth factor receptor/MAPK (3, 4) pathways. Allergen-induced
*Section of Neonatology, Perinatal and Pulmonary Biology, Perinatal Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; xDivision of
Immunobiology, University of Cincinnati College of Medicine, Cincinnati, OH
45220; †West China Developmental and Stem Cell Institute, Sichuan University
Second University Hospital, Chengdu; and ‡West China Hospital of Sichuan University, Chengdu, People’s Republic of China
Received for publication January 29, 2010. Accepted for publication March 25, 2010.
This work was supported by the American Lung Association (to H.W.), National
Institutes of Health Grants HL095580 and HL090156 (to J.A.W. and Y.X.), and
National Heart, Lung, and Blood Institute Grant AR47363, which supported the flow
cytometry core at Cincinnati Children’s Hospital Medical Center.
The sequences presented in this article have been submitted to the National Center
for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.gov/
geo/query/) under accession number GSE19204.
Address correspondence and reprint requests to Dr. Jeffrey A. Whitsett at the current
address: Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical
Center, MLC7029, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address:
[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this paper: AHR, airway hyperresponsiveness; a.u., arbitrary
units; BALF, bronchoalveolar lavage fluid; Chia, acidic chitinase; DC, dendritic cell;
E, embryonic day; EGFP, enhanced GFP; Eos, eosinophil; GO, gene ontology; Lym,
lymphocyte; Mac, macrophage; mDC, myeloid dendritic cell; Neu, neutrophil; pDC,
plasmacytoid dendritic cell; PN, postnatal day; qRT-PCR, quantitative RT-PCR;
Spdef, SAM pointed domain-containing Ets transcription factor; Tarc, thymus and
activation-regulated chemokine.
Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000223
inflammation is dominated by Th2 cell activation and production
of IL-13, IL-4, IL-5, eotaxins, and other mediators that influence
innate immune responses, Ab production, goblet cell metaplasia,
airway remodeling, and hyperreactivity (5). IL-4 binds to two
distinct receptor complexes, whereas IL-13 only binds to one of
these complexes. Specifically, IL-4 binds to the IL-4Ra–chain, the
functional receptor subunit of both the type I receptor, which is
a heterodimer of IL-4Ra and the gc-chain, and the type II receptor, which is a heterodimer of IL-4Ra and IL-13Ra1. IL-13
does not bind to IL-4Ra directly but binds to IL-13Ra1 and can
only activate the type II receptor. As the common subunit of receptor complexes for both IL-4 and IL-13, IL-4Ra is required for
mediating signal transduction of these two cytokines (1, 6, 7).
Targeting IL-4Ra function by antagonists or its expression by
antisense oligonucleotides suppressed airway Th2 inflammation,
hyperresponsiveness, and mucus production after allergen exposure in vivo (8, 9), indicating the pivotal role of IL-4Ra in mediating Th2 response. There is increasing evidence that respiratory
epithelial cells lining conducting airways play important roles in
modulation of inflammatory responses to allergens, pathogens,
and injurious agents (10, 11). Innate immune responses induced
by epithelial cells in response to pathogen are crucial for dendritic
cells (DCs) to initiate and maintain allergic Th2 cell responses in
experimental asthma (12, 13). Recent studies, from this laboratory
and others, demonstrated that expression of Foxa2, a member of
the forkhead box family of transcription factors expressed in
the respiratory epithelium, is inhibited during allergen or IL-13–
induced goblet cell metaplasia and Th2 inflammation (14, 15).
Deletion of Foxa2 in respiratory epithelial cells in the mouse
impaired surfactant production and lung maturation before birth
and caused spontaneous inflammation and goblet cell metaplasia
after birth, indicating a requirement for Foxa2 in normal postnatal
pulmonary homeostasis (16). Goblet cell metaplasia induced by
allergens or IL-13 was dependent on Stat6 in a process associated
with the induction of SAM pointed domain-containing Ets transcription factor (Spdef) and Foxa3, indicating that these transcription factors interact in a network influencing airway epithelial
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After birth, the respiratory tract adapts to recurrent exposures to pathogens, allergens, and toxicants by inducing the complex
innate and acquired immune systems required for pulmonary homeostasis. In this study, we show that Foxa2, expressed selectively
in the respiratory epithelium, plays a critical role in regulating genetic programs influencing Th2 cell-mediated pulmonary
inflammation. Deletion of the Foxa2 gene, encoding a winged helix/forkhead box transcription factor that is selectively expressed
in respiratory epithelial cells, caused spontaneous pulmonary eosinophilic inflammation and goblet cell metaplasia. Loss of Foxa2
induced the recruitment and activation of myeloid dendritic cells and Th2 cells in the lung, causing increased production of Th2
cytokines and chemokines. Loss of Foxa2-induced expression of genes regulating Th2 cell-mediated inflammation and goblet cell
differentiation, including IL-13, IL-4, eotaxins, thymus and activation-regulated chemokine, Il33, Ccl20, and SAM pointed
domain-containing Ets transcription factor. Pulmonary inflammation and goblet cell differentiation were abrogated by treatment
of neonatal Foxa2Δ/Δ mice with mAb against IL-4Ra subunit. The respiratory epithelium plays a central role in the regulation of
Th2-mediated inflammation and innate immunity in the developing lung in a process regulated by Foxa2. The Journal of
Immunology, 2010, 184: 000–000.
2
Materials and Methods
Transgenic mice and animal husbandry
Animals were maintained according to protocols approved by the Institutional Animal Care and Use Committee at Cincinnati Children’s
Hospital Research Foundation (Cincinnati, OH). Mice were housed in
a pathogen-free barrier facility in humidity- and temperature-controlled
rooms on a 12:12 h light/dark cycle and were allowed food and water ad
libitum. SFTPC-rtTA/tetO7CMV-Cre/Foxa2LoxP/LoxP compound transgenic
mice were generated as previously described (14) and used to permanently
delete Foxa2 in the fetal lung. Pregnant dams were maintained on doxycycline food from embryonic day (E) 6.5 to E12.5 to delete Foxa2 from
respiratory epithelial cells during fetal development, producing SFTPC/
Foxa2Δ/Δ mice.
Conditional expression of Foxa2 in respiratory epithelial cells was
achieved by producing tetO7-Foxa2-IRES-EGFP transgenic mice that were
then mated to Scgb1a1-rtta (line II) mice (20). Full-length rat Foxa2
coding sequence together with the 59 untranslated region and 39 untranslated region was isolated from pRc/CMV-rFoxa2 (14) at EcoRI sites
and cloning into the BamHI site of the pOtet7-IRES-EGFP vector (21) (the
latter provided by Dr. Kenneth Campbell, Cincinnati Children’s Hospital
Medical Center, Cincinnati, OH) via blunt-end ligation. Transgenes were
identified by PCR using the primer set: 59-AGC AAA GAC CCC AAC
GAG AAG C-39 and 59-CAA ACA ACA GAT GGC TGG CAA C-39.
Histology and immunohistochemistry
Immunohistochemistry was performed on 5-mm lung sections using rabbit
anti-Foxa2 (1:2000–1:8000), guinea pig anti-Spdef (1:4000) generated in
this laboratory, goat anti-Foxa3 (1:100; SC-5361, Santa Cruz Biotechnology,
Santa Cruz, CA), mouse mAb against Muc5ac (1:500, ab3649, Abcam,
Cambridge, MA) (18). Sections were processed with Ag retrieval using
heating in citrate buffer. Anti-mouse IL-4Ra mAb (lot number 9382-25B, 11/
12/03, AS/CH) was kindly provided by Amgen (Thousand Oaks, CA).
Anti–IL-4R treatment
Neonatal SFTPC/Foxa2Δ/Δ pups and control littermates were injected i.p.
on postnatal day (PN) 2 and PN9 with either anti–IL-4Ra Ab (50 mg/g
body weight) or equal volume of sterile saline. On PN15, mice were anesthetized and the lungs lavaged five times with 0.3 ml saline, and the cells
were counted from bronchoalveolar lavage fluid (BALF). Cytospins of
BALF cells (5 3 104) were stained with a Diff-Quik kit (catalog number
24606, Polysciences, Warrington, PA). Cells (400–500) were counted to
determine numbers and percentage of macrophages, eosinophils, neutrophils, and lymphocytes. Lung tissue from a second group of mice was
fixed with 4% paraformaldehyde and processed into paraffin blocks for
immunohistochemistry.
Pulmonary OVA sensitization
Mice were sensitized to OVA by systemic and pulmonary administration
using protocols previously described (18). Expression of Foxa2 in the
Scgb1a1-rtTA/tetO7-Foxa2-IRES-EGFP (Scgb1a1/Foxa2-IE) mice was
induced at 6 wk of age by provision of doxycycline 24 h before mice were
intranasally sensitized with OVA (Supplemental Fig. 8). The control mice
were the single transgenic littermates (Scgb1a1-rtTA) that received the
same doxycycline and OVA treatments. Doxycycline was continued until
the time of sacrifice.
mRNA microarray analysis
Lung cRNA was hybridized to the murine genome MOE430 chips (consisting of 45,000 gene entries, Affymetrix, Santa Clara, CA) according to
the manufacturer’s protocol. The RNA quality and quantity, probe preparation, labeling, hybridization, and image scans were carried out in the
CCHMC Affymetrix Core using standard procedures. Affymetrix Microarray Suite 5.0 (Affymetrix) was used to scan and quantitate the gene chips
under default scan settings. Normalization was performed using the Robust
Multichip Average model. Data were further analyzed using affylmGUI
(http://bioinf.wehi.edu.au/affylmGUI) from R/Bioconductor package
(www.bioconductor.org). Differentially expressed genes were selected
with the threshold of p value of ,0.01, fold change $1.5, and a minimum
of two present calls in three samples with relatively higher expression.
Gene ontology (GO) analysis was performed using the web-based tool
David (Database for annotation, visualization, and integrated discovery).
Overrepresented pathways were identified by comparison of the overlap of
differentially expressed genes identified postdeletion of FOXA2 and all
genes in MOE430 mouse genome. Gene sets associated with known
pathways and disease states were identified from the Kyoto Encyclopedia
of Genes and Genomes (www.genome.ad.jp/kegg/), GenMAPP (www.
genmapp.org/), and GEArrays (www.sobiosciences.com/microarrays.php/).
A pathway was considered to be overrepresented when a probability p
value was #0.01 and gene hits $5. Potential protein/protein or protein/
DNA interactions were identified using Ingenuity Pathway Analysis (Ingenuity Systems, Redwood City, CA). Ingenuity Pathway Analysis software maps the differentially expressed genes identified from the
microarray experiment onto the interactome according to Ingenuity Pathway Knowledge Base, a large curated database of published literature
findings related to mammalian biology. Genetic networks preferentially
enriched in the sets of mRNAs were generated based on their connectivity.
Statistical scores were calculated to rank the resulting networks and
pathways using Fisher’s right-tailed exact test. The score indicates the
degree of relevance of a network to the input gene set, taking into account
the number of network-eligible genes and the size of the network.
Cytokine and quantitative RT-PCR assays
To determine cytokine levels, BALF was collected as previously described
(22). BALF from each adult mouse was concentrated using 0.5 ml (VIVA
SPIN4, catalog number VS0413, Sartorius, Bohemia, NY) and subjected to
ELISA to determine the concentrations cytokines using the mouse IL-4, IL-5,
IL-13, and IFN-g ELISA kits from eBioscience, San Diego, CA (catalog
numbers 88-7044, 88-7054, 88-7137, 88-7104, and 88-7384, respectively).
The Ccl17 ELISA kit was purchased from R&D Systems (MCC170; Minneapolis, MN). The ELISA was performed with an n of 6–8 mice of each
genotype (Foxa2Δ/Δ mice and their littermate controls) at PN15. Whole-lung
total RNA was purified by RNeasy Mini Kit (catalog number 74104, Qiagen,
Valencia, CA) and reverse transcribed into cDNA by Verso cDNA kit (catalog
number AB-1453/A, Thermo Fisher Scientific, Waltham, MA). Quantitative
RT-PCR (qRT-PCR) was performed on a 7300 real-time PCR system
(Applied Biosystems, Foster City, CA) with the TaqMan probes Foxa2
(Mm00839704_mH), Il4 (Mm00445259_m1), Il5 (Mm99999063_m1), Il13
(Mm99999190_m1), Ccl11 (Mm00441239_g1), Ccl24 (Mm00444701_m1),
Ccl17 (Mm01244826_g1), Il33 (Mm01195784_m1), Il6 (Mm99999064_m1),
Ccl20 (Mm01268753_g1), and Ifn-g (Mm00801778_m1) and normalized to
endogenous 18s rRNA for control (probe part number 4352930E). qRT-PCR
was performed with an n of 3 mice for each genotype at PN11 and PN15.
Flow cytometry
Lung cell suspensions were incubated with anti-CD16/32 (clone 2.4G2) for
30 min and then staining reactions were performed at 4˚C. Myeloid DCs
(mDCs; CD11c+, CD11b+, Gr1neg, and CD317neg) and plasmacytoid DCs
(pDCs; CD11clow, CD11bneg, Gr1+, and CD317+) were quantified using
AF647-conjugated CD11c (HL3), PE-Cy7–conjugated CD11b (M1/70),
APC-eFluor780–conjugated anti-Gr1 (RB6-8C5), and AF488-conjugated
anti-317 (eBio927, eBioscience). To measure costimulatory molecule
expression, cells were stained with PE-conjugated anti-CD80 (16-10A1),
anti-CD86 (GL1), or B7-DC (TY25). For intracellular T cell cytokine
staining, lung cells were cultured overnight in the presence of PMA
(100 ng/ml) and ionomycin (1 mg/ml). Cytokine secretion was blocked
with a combination of brefeldin A and monensin (eBioscience) for 4 h.
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differentiation (14, 17, 18). Recent studies demonstrated that
goblet cells induced by pulmonary allergens are derived from the
differentiation of resident Clara cells that serve as progenitor cells
in the bronchiolar epithelium (18, 19). In initial studies, in which
Foxa2 was deleted in the respiratory epithelium prior to birth,
mucous cell metaplasia, alveolar remodeling, and inflammation
were observed (14). The characteristics and mechanisms underlying lung inflammation caused by loss of Foxa2 in airway
epithelial cells are unclear at present.
In this study, we demonstrate that respiratory epithelial cellspecific deletion of Foxa2 caused spontaneous Th2 cytokine/chemokine-mediated inflammation and goblet cell metaplasia. The
hypothesis, that the inflammatory response was mediated by the
spontaneous activation of IL-4R signaling, was tested using a mAb
to block IL-4R signaling in neonatal mice. Deletion of Foxa2 induced expression of a network of genes influencing or associated
with Th2-cell mediated inflammation and DC recruitment and activation, indicating the role of respiratory epithelial cell in programming inflammatory responses in the developing lung in
a process regulated by Foxa2 and requiring IL-4Ra signaling.
Foxa2 AND Th2-MEDIATED LUNG INFLAMMATION
The Journal of Immunology
Cells were stained with PE-Cy7–conjugated anti-CD4 (RM4-5) and PEconjugated anti–IL-13 (eBio13A). All mAbs were purchased from
eBioscience. Data were acquired with an LSRII flow cytometer (BD Biosciences, San Jose, CA) equipped with lasers tuned to 488 nm, 633 nm,
and 405 nm. Spectral overlap was compensated using the FACSDiVa
software (BD Biosciences) and analyzed using FlowJo software (Tree
Star, Ashland, OR).
Plasmid and luciferase reporter assay
Statistics
Statistical differences in cell counts and concentrations of proteins assessed
by ELISA were determined using Student t test (two-tailed and unpaired).
The statistic method applied to analyze qRT-PCR was the Mood’s Median
test. Difference between two groups was considered significant when the p
value was ,0.05 for all tests.
Results
Conditional deletion of Foxa2 caused Th2 cell-mediated
pulmonary inflammation
To delete Foxa2 expression in respiratory epithelium, dams of
SFTPC-rtTA/tetO7-CMV-Cre/Foxa2LoxP/LoxP
transgenic
embryos were treated with doxycycline from E6.5 to E12.5. Approximately 30% of the mutant mice died in the postnatal period
as previously reported (14, 16). Surviving mice develop spontaneous pulmonary eosinophilic inflammation, goblet cell metaplasia, and airspace enlargement in the first 2 wk of life (Fig. 1A).
On PN15, pulmonary inflammation was seen histologically and
supported by quantitation of inflammatory cells in BALF. Numbers of eosinophils, lymphocytes, and macrophages were increased
in BALF from Foxa2Δ/Δ mice (Fig. 1B). Deletion of Foxa2 was
associated with increased expression of Th2 cytokines and chemokines, including IL-13, IL-4, IL-5, and Ccl17 (thymus and
activation-regulated chemokine [Tarc]) as measured in BALF
(Fig. 1C). CD4+ T cells that were recruited into the lung were
further analyzed by flow cytometry, revealing a significant increase of the median fluorescent intensity of IL-13 and IL-17a and
abundance of Th2 and Th17 cell populations in the lungs of
Foxa2Δ/Δ mice. Expression mRNA of IL-6, a cytokine required for
Th17 cell differentiation (23, 24), was significantly induced. In
contrast, Ifn-g mRNA was not altered (Supplemental Fig. 1).
Taken together, Foxa2Δ/Δ mice develop spontaneous pulmonary
inflammation that is typical of enhanced Th2 cytokine/chemokine
activity during the postnatal period of development.
FIGURE 1. Deletion of Foxa2 in respiratory epithelium caused pulmonary eosinophilic inflammation and goblet cell differentiation. The Foxa2D/D mice
spontaneously developed airway inflammation, eosinophilic infiltration, and goblet cell metaplasia at PN15. Loss of Foxa2 expression was confirmed by
immunohistochemical staining. Increased eosinophils in BALF were revealed by Diff-Quik staining (A, original magnification 3400). Numbers of total cells,
eosinophils, macrophages, lymphocytes, and neutrophils in BALF were assayed. Increased numbers of total cells and eosinophils were found in BALF from
Foxa2D/D mice (B). Expression of Th2 cytokines (IL-4, IL-5, and IL-13) and chemokine Ccl17 (Tarc) was increased in BALF of Foxa2D/D mice as determined
by ELISA (C). Inserts in A are the higher magnification of the regions indicated by arrows. The graphs represent means 6 SE; n = 6 for each genotype. Scale
bar, 50 mm. pp , 0.05, versus controls using Student t test (two-tailed, unpaired). Eos, eosinophil; Lym, lymphocyte; Mac, macrophage; Neu, neutrophil.
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The control plasmid used in luciferase reporter assay expressing enhanced
GFP (EGFP) was made by isolation of the IRES-EGFP fragment from
pIRES2-EGFP (catalog number 6029-1, Clontech, Mountain View, CA) by
BamHI and NotI sites and subcloned into the BamHI site of the
p3XFLAG-Myc-CMV plasmid (catalog number E6401, Sigma-Aldrich, St.
Louis, MO) via blunt-end ligation. The Foxa2 expression plasmid was
made by cloning rat Foxa2 cDNA from pRC/CMV-rFoxa2 using PCR
primers: 59-ATG AAT TCA ATG CTG GGA GCC GTG AAG-39 and 59ATG AAT TCG CGG ACG AGT TCA TAA TAGG-39 and inserting into
the EcoRI site of the control vector. AccuPrime Taq DNA Polymerase
(catalog number 12339-016, Invitrogen, Carlsbad, CA) was used for amplification of DNA fragments. The mouse Ccl17-pGL3 plasmid was cloned
by amplification of the 2-kb promoter region from C57/B6 mouse genomic
DNA using primers: 59-GGA CCT GAA ATA GTC AGC ATCC-39 and 59CTG AGG TGA AGG TCT TCA TGGG-39 and cloned into the pGL3basic plasmid (catalog number E1751, Promega, Madison, WI). The luciferase assay was performed by transfection of MLE-15 cells with the
mCcl17-pGL3 plasmid along with either control plasmid-expressing EGFP
or Foxa2-EGFP at a 1:1 ratio using the transfection reagent Lipofectamine
2000 (catalog number 11668-019, Invitrogen). CMV-bGal plasmid was
cotransfected to serve as an internal control, and the luciferase activity was
normalized to b-galactosidase activity after 24 h of transfection.
3
4
Inhibition of IL-4R–mediated signaling inhibited eosinophilic
inflammation and goblet cell differentiation induced by Foxa2
deletion
inflammation, goblet cell metaplasia and mucus hyperproduction
in Foxa2Δ/Δ mice were inhibited by the IL-4Ra Ab. Goblet cell
markers, including Alcian blue, and Spdef, Foxa3, and Muc5ac
staining were also inhibited by neutralizing Ab against IL-4Ra
(Fig. 2B), demonstrating that the goblet cell metaplasia was dependent upon activated IL-4Ra signaling caused by deletion of
Foxa2.
Loss of Foxa2 in respiratory epithelium enhances mDC
recruitment and activation
Th2-cell mediated inflammation is dependent on activation and
migration of pulmonary DCs to the respiratory epithelium (31).
As professional APCs, DCs are uniquely capable of fully activating
naive T cells. In the lung, two distinct phenotypes of DCs have been
identified that have markedly different effects on T cell function.
mDCs (defined as CD11c+, CD11b+, Gr1neg, and CD317neg cells)
promote robust T cell activation and efficiently promote allergeninduced AHR (32). In contrast, pDCs (defined as CD11clow,
CD11bneg, Gr1+, and CD317+ cells) promote the development of
Foxp3+ regulatory T cells, preventing allergen-induced AHR (33).
Thus, to test the hypothesis that excessive Th2 cell activation observed in mice lacking Foxa2 in the respiratory epithelium
was preceded by dysregulation in pulmonary DC recruitment or
activity, we assessed mDC and pDC recruitment and activation
at PN8, PN11, and PN15 by flow cytometry. Comparing the
FIGURE 2. Inhibition of IL-4R–suppressed goblet cell differentiation and pulmonary inflammation in Foxa2Δ/Δ mice. IL-4Ra mAb was administered i.p. at
PN2 and PN9. Eosinophilic inflammation was inhibited, shown by reduced total cell and eosinophil numbers (A). Goblet cell differentiation was inhibited by
Ab treatment, indicated by lack of Spdef, Muc5ac, and Foxa3 staining in Foxa2Δ/Δ mice (B). Inserts show the higher magnification (346) of eosinophils in
perivascular regions pointed by arrows (left panel insets). B, H&E, Spdef and Muc5ac panels were taken at 3100 and Foxa3 panels were taken at 3400. Scale
bar, 50 mm. Graphs represent means 6 SD. n = 6 for each genotype. pp , 0.01, versus controls using Student t test (two-tailed, unpaired).
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A key pathway that regulates allergic inflammation and tissue
remodeling in the airways involves the Th2 cytokines IL-4 and IL13 and the activation of the IL-4Ra subunit (IL-4Ra) (25). IL-4 is
primarily involved in promoting the differentiation and proliferation of Th2 cells and the synthesis of IgE, whereas IL-13 has
a critical role in mediating airway hyperresponsiveness (AHR),
goblet cell metaplasia, and mucus hypersecretion, the elements
that are most closely linked to clinical manifestations of asthma.
Blocking IL-4R signaling pathway with neutralizing Ab against
IL-4Ra or mutagenesis of IL-4Ra was sufficient to inhibit responses to both IL-4 and IL-13 cytokines in vivo and in vitro (26–
28). Specifically, loss of IL-4Ra in Clara cells inhibited mucus
production after OVA intrapulmonary exposure in adult mice (29),
and loss of IL-4Ra significantly decreased spontaneously induced
mucous cells and eosinophilia in neonatal mice (PN10) (30). To
test whether pulmonary inflammation and goblet cell differentiation caused by conditional deletion of Foxa2 in the airway epithelium was dependent upon IL-4R–mediated signaling, Foxa2Δ/Δ
mice were treated with IL-4Ra mAb at PN2 and PN9. Eosinophilic inflammation was significantly inhibited by anti–IL-4Ra
Ab, as shown by the reduction of inflammatory cells and eosinophils in BALF (Fig. 2A). Consistent with inhibition of Th2
Foxa2 AND Th2-MEDIATED LUNG INFLAMMATION
The Journal of Immunology
FIGURE 3. Loss of Foxa2 in respiratory epithelium
enhanced recruitment and activation of mDCs. The
percentages of both mDCs and pDCs were increased in
the lung of Foxa2Δ/Δ mice at PN11 to PN15 (A). Although both subsets of DCs were found with greater
frequency in the lungs of Foxa2Δ/Δ mice, there was
a substantial increase in the mDC/pDC ratio in Foxa2Δ/Δ
mice. To compare the activation of pulmonary mDCs
and pDCs in control and Foxa2Δ/Δ mice, expression of
costimulatory molecules was assessed in gated populations by flow cytometry. Frequencies of mDCs expressing B7-DC (B), B7-H1 (C), and CD86 (D) were
increased in Foxa2Δ/Δ mice at PN11 to PN15. Collectively, these data demonstrate that, concomitant with the
increased T cell activation observed in Foxa2Δ/Δ mice,
there was enhanced recruitment and activation of pulmonary mDCs compared with control mice. n = 8
at PN8; n = 5 at PN11; and n = 4 at PN15 of each
genotype. Graphs represent means 6 SE. pp , 0.05;
ppp , 0.01; pppp , 0.001, versus controls as determined by Student t test (two-tailed, unpaired).
pendent or focal changes in its expression would not be detected in
the current study design.
Foxa2 regulates a genetic network associated with pulmonary
Th2 inflammation and goblet cell differentiation
To investigate the role of Foxa2 and its downstream targets associated with the Th2 inflammation and goblet cell hyperplasia, RNAs
were isolated from the lungs of Foxa2Δ/Δ and control littermates at
PN15. Lung cRNA was hybridized to the murine genome MOE430
chips and differential gene expression and GO analyzed. Deletion
of Foxa2 significantly influenced the expression of 665 mRNAs.
Among these, 516 out of 665 mRNAs were induced, and 148 were
reduced in response to deletion of Foxa2 in the respiratory epithelium (Fig. 4A). The complete microarray dataset has been
submitted to Gene Expression Omnibus under accession number
GSE19204 (www.ncbi.nlm.nih.gov/geo/query/). GO analysis suggested that the induced genes are primarily enriched in “Immune/
inflammatory response” (p value: 3.8E-13), indicating that Foxa2
plays a suppressive role in immune/inflammatory responses and
mucus production in the postnatal lung; nearly 40% of induced
genes encode cytokines, chemokines, and their receptors, and
many are known to be associated with asthma or experimental
allergen sensitization (Supplemental Table I). The overlap between
the known asthma-related genes (genes causing, predisposing, or
protecting from asthma) and those responding to the deletion of
Foxa2 was highly statistically significant. Pathway analysis further
indicated the “dendritic and Ag presenting,” “T cell and B cell
activation,” “cytokine–cytokine receptor interaction,” “eicosanoid
signaling,” and “JAK-STAT signaling pathway” were significantly
induced. In contrast, “glutathione metabolism” (1.2E-04) was the
most significantly overrepresented canonical pathway in mRNAs
decreased postdeletion of Foxa2 (Supplemental Table II). Expression of mRNAs of major Th2 cytokines and chemokines in the
whole lung was confirmed by qRT-PCR. Consistent with the severe
eosinophilic inflammation and Th2 lymphocytes infiltration seen in
Foxa2Δ/Δ mice, Il4, Il5, and Il13 mRNAs in the whole lung were
significantly increased, whereas expression of the Th1 cytokine Ifn-
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frequencies of DC subsets in control and Foxa2Δ/Δ mice revealed
a significant increase in the frequency of both mDCs and pDCs
found in the lung of Foxa2Δ/Δ mice (Fig. 3A). Although both subsets
of DCs were found with greater frequency in the lungs of Foxa2Δ/Δ
mice, there was a substantial increase in the mDC/pDC ratio in
Foxa2Δ/Δ mice, particularly at PN15, suggesting an environment
better suited to T cell activation in Foxa2Δ/Δ mice. Comparing the
expression of DC activation-associated costimulatory molecule
expression on mDCs from control and Foxa2Δ/Δ mice demonstrated
significantly elevated frequencies of mDCs expressing B7-DC
(Fig. 3B), B7-H1 (Fig. 3C), and CD86 (Fig. 3D). mDCs expressing
CD80 were also increased in Foxa2Δ/Δ mice, although changes
were not statistically significant. MHC class II was not different
from control and Foxa2Δ/Δ mice (Supplemental Fig. 2). Interestingly, although pDCs were also generally more activated in
Foxa2Δ/Δ mice than in control mice, by PN15, a time when there
was robust activation of T cell cytokine production, the levels of
pDC activation had decreased and were indistinguishable from
controls (Fig. 3B–D). Collectively, these data demonstrate that
concomitant with the increased T cell activation, the recruitment
and activation of pulmonary mDCs was increased in Foxa2Δ/Δ
mice. Expression of Th2 cytokine mRNAs, including Il4 and Il13,
was significantly increased in the lungs of Foxa2Δ/Δ mice at PN11
and PN15 (Fig. 4B, Supplemental Fig. 3). Mechanisms underlying
the recruitment and activation of DCs in the lung are not well established at present. The IL-7–like cytokine thymic stromal lymphopoietin, produced by epithelial cells, is known to influence DCs
expressing OX40 ligand, causing differentiation of naive CD4+
T cells to Th2 cells that produce IL-4, IL-13, and TNF but not IL-10
(34, 35). Similarly, CCL20 is a chemokine produced in epithelial
cells and capable of inducing DC migration via interaction with
CCR6 expressed on the immature DCs (36, 37). Ccl20, but not Tslp
mRNA expression, was induced in the lungs of Foxa2Δ/Δ mice at
PN15 (Supplemental Fig. 4), suggesting a potential mechanism by
which DCs are recruited and activated in the lungs of Foxa2Δ/Δ
mice. Although Tslp mRNA was not increased in whole lung at
PN11 or PN15 (data not shown), it remains possible that time de-
5
6
Foxa2 AND Th2-MEDIATED LUNG INFLAMMATION
g was not changed (Fig. 4B, Supplemental Fig. 1). Epithelial cells
lining the respiratory and gastrointestinal tract play a pivotal role in
initiation, regulation, and resolution of innate and adaptive immune
response by expressing a wide range of immune response genes
including costimulatory molecules, chemokines, cytokines, and
PGs (38). Of particular interest, respiratory epithelium expresses
IL-33, a cytokine that promotes Th2 cytokine production (39) and
CCL17 (40, 41) that chemoattracts Th2 cells via interactions with
CCR4 that is selectively expressed on the Th2 cells (42). Expression of Il33 and Ccl17 mRNAs was significantly induced in the
Foxa2Δ/Δ mice at PN15 (Fig. 4B). CCL17 (Tarc), a potent T cell
chemoattractant induced in bronchiolar epithelial cells of asthmatics (40, 41), likely plays an important role in the pulmonary
inflammation seen in the Foxa2Δ/Δ mice. The effects of Foxa2 on
Tarc gene expression were assessed in MLE-15 cells in vitro.
Foxa2 inhibited the Tarc promoter by ∼40% (Supplemental Fig. 5).
A schematic summarizing the effects of Foxa2 gene deletion on
Th2-mediated processes in is provided by Fig. 6.
Conditional expression of Foxa2 in the respiratory epithelium
inhibited allergen-induced goblet cell differentiation
A transgenic mouse in which Foxa2 was conditionally expressed in
the respiratory epithelium was produced to test whether increased
expression of Foxa2 was sufficient to inhibit allergen-induced
inflammation or goblet cell differentiation (Supplemental Fig. 6).
Double-transgenic mice Scgb1a1-rtTA/tetO7-Foxa2-IRES-EGFP
(Scgb1a1/Foxa2-IE) were treated with doxycycline, and the induction of Foxa2 and EGFP expression was confirmed by immunohistochemistry and fluorescence microscopy (Fig. 5A,
Supplemental Fig. 6). Goblet cells, although normally rare in the
surface epithelium lining the conducting airways of adult mice,
are usually present in young mice (30, 43). Expression of Foxa2
from PN4-PN18 by doxycycline treatment blocked the normal
postnatal goblet cell differentiation (Supplemental Fig. 7). Conditional expression of Foxa2 in the respiratory epithelium in adult
mice inhibited goblet cell differentiation (Fig. 5B, Supplemental
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 4. Deletion of Foxa2 in respiratory epithelial cells influenced expression of mRNAs mediating Th2-like inflammation and goblet cell differentiation. A, Lung cRNAs were produced from Foxa2Δ/Δ and control littermate at PN15 (n = 3 of each genotype) and hybridized to murine genome MOE430
chips. Affymetrix microarray analysis (Affymetrix) revealed increased mRNAs delete expression of Th2 cytokines, chemokines including Il4, Il13, Ccl17,
Ccl22, Ccl11, and Ccl24, as well as goblet cell-associated genes Clca3 and Agr2 postdeletion of Foxa2. B, Expression of mRNAs of Th2 cytokines (Il4, Il5,
Il13, and Il33) and chemokines (Ccl11, Ccl24, and Ccl17), were increased in Foxa2Δ/Δ mice at PN15 as detected by qRT-PCR. Graphs represent mean 6 SEM
at a.u.s. n = 3 for each genotype for qRT-PCR assay. pp , 0.05, versus control determined by Mood’s Median test. a.u., arbitrary unit.
The Journal of Immunology
7
FIGURE 5. Foxa2 inhibited allergen-induced goblet cell differentiation.
Scgb1a1/Foxa2-IE transgenic mice were treated with doxycycline for 3 d,
increasing Foxa2 expression as detected by immunohistochemistry using
a high dilution of Foxa2 Ab at 4 wk of age (A, original magnification
3100). Foxa2 was not prominent at this Ab dilution in single transgenic
Scgb1a1-rtTA or Scgb1a1/Foxa2-IE (without doxycycline) transgenic mice.
Doxycycline was administered to Scgb1a1-rtTA and Scgb1a1/Foxa2-IE
transgenic mice 1 d before intranasal OVA exposure. Foxa2 markedly inhibited goblet cell differentiation as shown by decreased Alcian blue,
Muc5ac, Spdef, and Foxa3 staining (B, original magnification 3100). Inserts are the higher magnification (34) of the regions indicated by arrows.
Scale bar, 50 mm. Figures are representative of n = 3 for each genotype.
Fig. 8). However, expression of Foxa2 in respiratory epithelial
cells of the mature lung prior to OVA sensitization did not alter
Th2 cytokine production or inflammation. Inflammatory cell
counts (total or differential), as well as IL-4, IL-5, IL-13, IL-10,
and IFN-g concentrations, were similar in BALF from Foxa2expressing and control mice after OVA exposure (Supplemental
Fig. 9A, 9B). These finding are consistent with previous observations supporting the role of Foxa2 in regulating epithelial cell
differentiation (14, 16). The present findings demonstrate that
Foxa2 also plays an important role in modulating Th2 immunity in
postnatal development, but does not directly control Th2-mediated
inflammatory responses in the mature lung.
Discussion
Deletion of Foxa2 in respiratory epithelial cells caused spontaneous pulmonary inflammation associated with goblet cell metaplasia and enhanced expression of Th2 cell-associated cytokines,
chemokines, and their receptors. Recruitment and activation of
mDCs and Th2 lymphocytes were increased in Foxa2Δ/Δ mice
lungs and associated with increased Th2 cytokines (IL-4, IL-5,
and IL-13) and Ccl17 (Tarc) production and Foxa2 inhibiting Tarc
gene promoter activity in vitro. The inflammatory effects of Foxa2
deletion were inhibited by blocking IL-4R signaling in the developing mouse lung. Taken together, the respiratory epithelium,
via Foxa2, plays a critical role in programming the innate immune
system during postnatal development of the lung and serves to
inhibit the development of Th2-dominated innate immunity.
Th2 cytokine-dominated inflammation postdeletion of Foxa2 in
the lung
Allergic airway inflammation is characterized by recruitment and
activation of Th2 lymphocytes and eosinophils, goblet cell metaplasia, and mucus hyperproduction in a process regulated by
a number of cytokines and chemokines. A number of the components of the IL-4R signaling pathway (e.g., IL-4, IL-13, and
IL-4Ra) were increased in Foxa2Δ/Δ mice. IL-13 is produced
primarily in Th2 cells and regulates many asthma-related processes, including mucus hyperproduction, eosinophil recruitment
and survival, and airway hyperreactivity (44). IL-13 blockade
abrogated many of the features of asthma (45), demonstrating that
IL-13 is an important mediator in Th2 responses and asthma
pathogenesis. The present observation, that inhibition of IL-4Ra
signaling substantially inhibited pulmonary eosinophilic inflammation and goblet cell metaplasia postdeletion of Foxa2, supports
the important role of Foxa2 in inhibiting development of Th2mediated innate immunity that is dependent upon IL-4R signaling.
Consistent with the observed Th2-mediated inflammation in
Foxa2Δ/Δ mice, a number of factors involved in T cell and eosinophil recruitment, including colony-stimulating factor receptors
Csf2ra and Csf2rb (2- and 4-fold), Il1b (2-fold), tumor necrosis
signaling factor-associated genes Tnfrsf1b, -4, -9, -13, -14, and
-18, were induced (2–10-fold). Likewise, mRNAs encoding lymphocyte chemoattractants, Ccl13 (3.4-fold), Ccl22 (4.6-fold), Ccr9
(4-fold), Ccr5 (4-fold), and Ccr2 (2.6-fold), were induced, the
latter being required for pulmonary DC accumulation in asthma
Downloaded from http://www.jimmunol.org/ by guest on June 16, 2017
FIGURE 6. Deletion of Foxa2 influences Th2 cell-mediated inflammation in the developing lung. Deletion of the epithelial-specific
transcription factor Foxa2 caused spontaneous Th2 cell-mediated lung
inflammation and goblet cell metaplasia. Loss of Foxa2 increased the
expression of epithelial-derived cytokines (IL-33) and chemokines
(CCL17, CCL20, and eotaxins) that mediate the chemoattraction of mDCs,
Th2 cells, and eosinophils in the lung. In association with activation of
mDCs, naive T cells were polarized to Th2/Th17 cells producing IL-17,
IL-4, and IL-13, in turn inducing Spdef to cause goblet cell metaplasia,
features common to asthma. Lung inflammation caused by deletion of
Foxa2 was inhibited by a mAb against IL-4Ra that inhibited eosinophilia
and goblet cell metaplasia. The proposed network indicates regulatory
relationships but does not imply direct transcriptional control of each gene
by Foxa2.
8
Role of Foxa2 in the regulation of pulmonary DCs
A network of DCs is closely associated with the respiratory epithelium (56). Both mDCs and pDCs are present in the lung, but
mDC subsets generally dominate the airway mucosa (57). In the
current study, both mDCs and pDCs were detected in the lungs of
normal neonatal mice, although mDCs were typically more
abundant than pDCs. We previously demonstrated that the numbers of mDCs present typically exceed pDCs by 10–25-fold in the
adult mouse lung (58). Thus, in neonatal mice, the lung appears to
be a more tolerogenic environment, capable of promoting the
development of T regulatory rather than activated T effector cells.
In further support of this concept, we observed that although the
level of mDC activation increased from PN8 to PN15 in control
mice, the activation status of pulmonary pDCs peaked at PN11
and decreased thereafter, suggesting that between PN11 and
PN15, there is a shift toward the activation of a population of
pulmonary DCs better suited to promote T cell activation. Thus, in
the absence of overt immunization, the neonatal lung may represent a generally suppressive environment, favoring the development of tolerance rather than overt immune responses.
Collectively, these data demonstrate that, concomitant with the
increased T cell activation observed in Foxa2Δ/Δ mice, there is also
greater recruitment and activation of pulmonary mDCs in the
absence of Foxa2. Fig. 6 summarizes the present findings regarding mechanisms by which Foxa2 influences lung inflammation in the developing mouse.
Goblet cell differentiation caused by deletion of Foxa2 in the
developing lung seen in the current study was associated with Th2
cell activation and the induction of Spdef and Foxa3 that appear to
function in a transcriptional network in the respiratory epithelium
(18). Consistent with recently published studies (59), we demonstrated that forced expression of Foxa2 in the adult mouse inhibited goblet cell differentiation, blocking Spdef and Foxa3, but
did not influence allergen-induced inflammation or eosinophilic
infiltration. These findings support the role of Foxa2 in the instruction of innate immunity during development rather than
a direct role for Foxa2 in suppressing inflammation during OVA
sensitization in the mature mouse.
Acknowledgments
We thank Ann Maher for preparation of the manuscript, Amgen for providing the IL-4Ra mAb, and Dr. Fred Finkelman for scientific input.
Disclosures
The authors have no financial conflicts of interest.
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