Immunity in the Developing Lung Foxa2 Programs Th2 Cell-Mediated

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Foxa2 Programs Th2 Cell-Mediated Innate Immunity in the Developing Lung Gang Chen, Huajing Wan, Fengming Luo, Liqian Zhang, Yan Xu, Ian Lewkowich, Marsha Wills-Karp and Jeffrey A. This information is current as Whitsett of September 27, 2021. J Immunol 2010; 184:6133-6141; Prepublished online 5 May 2010; doi: 10.4049/jimmunol.1000223

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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*

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 Downloaded from 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: 6133–6141. http://www.jimmunol.org/ fter birth, the respiratory tract is recurrently exposed to inflammation is dominated by Th2 cell activation and production pathogens, allergens, and toxicants. A multilayered in- of IL-13, IL-4, IL-5, eotaxins, and other mediators that influence A nate and acquired host defense system develops to innate immune responses, Ab production, goblet cell metaplasia, maintain pulmonary homeostasis in the face of these challenges. airway remodeling, and hyperreactivity (5). IL-4 binds to two Th2-dominated inflammation and mucus hyperproduction are distinct receptor complexes, whereas IL-13 only binds to one of common features of acute and chronic pulmonary disorders, in- these complexes. Specifically, IL-4 binds to the IL-4Ra–chain, the cluding asthma. Normally, inflammatory responses are precisely functional receptor subunit of both the type I receptor, which is balanced to achieve mucociliary clearance and removal of a heterodimer of IL-4Ra and the gc-chain, and the type II re- pathogens. Various signaling and transcriptional programs in- ceptor, which is a heterodimer of IL-4Ra and IL-13Ra1. IL-13 by guest on September 27, 2021 fluence allergic lung inflammation and mucus hyperproduction, does not bind to IL-4Ra directly but binds to IL-13Ra1 and can including the IL-4R/STAT6 (1), TLR/NF-kB (2), and epidermal only activate the type II receptor. As the common subunit of re- growth factor receptor/MAPK (3, 4) pathways. Allergen-induced ceptor 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 *Section of Neonatology, Perinatal and Pulmonary Biology, Perinatal Institute, Cin- antisense oligonucleotides suppressed airway Th2 inflammation, cinnati Children’s Hospital Medical Center, Cincinnati, OH 45229; xDivision of hyperresponsiveness, and mucus production after allergen expo- Immunobiology, University of Cincinnati College of Medicine, Cincinnati, OH 45220; †West China Developmental and Stem Cell Institute, Sichuan University sure in vivo (8, 9), indicating the pivotal role of IL-4Ra in me- Second University Hospital, Chengdu; and ‡West China Hospital of Sichuan Univer- diating Th2 response. There is increasing evidence that respiratory sity, Chengdu, People’s Republic of China epithelial cells lining conducting airways play important roles in Received for publication January 29, 2010. Accepted for publication March 25, 2010. modulation of inflammatory responses to allergens, pathogens, This work was supported by the American Lung Association (to H.W.), National and injurious agents (10, 11). Innate immune responses induced Institutes of Health Grants HL095580 and HL090156 (to J.A.W. and Y.X.), and by epithelial cells in response to pathogen are crucial for dendritic National Heart, Lung, and Blood Institute Grant AR47363, which supported the flow cytometry core at Cincinnati Children’s Hospital Medical Center. cells (DCs) to initiate and maintain allergic Th2 cell responses in The sequences presented in this article have been submitted to the National Center experimental asthma (12, 13). Recent studies, from this laboratory for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.gov/ and others, demonstrated that expression of Foxa2, a member of geo/query/) under accession number GSE19204. the forkhead box family of transcription factors expressed in Address correspondence and reprint requests to Dr. Jeffrey A. Whitsett at the current the respiratory epithelium, is inhibited during allergen or IL-13– address: Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, MLC7029, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail address: induced goblet cell metaplasia and Th2 inflammation (14, 15). [email protected] Deletion of Foxa2 in respiratory epithelial cells in the mouse The online version of this article contains supplemental material. impaired surfactant production and lung maturation before birth Abbreviations used in this paper: AHR, airway hyperresponsiveness; a.u., arbitrary and caused spontaneous inflammation and goblet cell metaplasia units; BALF, bronchoalveolar lavage fluid; Chia, acidic chitinase; DC, dendritic cell; after birth, indicating a requirement for Foxa2 in normal postnatal E, embryonic day; EGFP, enhanced GFP; Eos, eosinophil; GO, gene ontology; Lym, lymphocyte; Mac, macrophage; mDC, myeloid dendritic cell; Neu, neutrophil; pDC, pulmonary homeostasis (16). Goblet cell metaplasia induced by plasmacytoid dendritic cell; PN, postnatal day; qRT-PCR, quantitative RT-PCR; allergens or IL-13 was dependent on Stat6 in a process associated Spdef, SAM pointed domain-containing Ets transcription factor; Tarc, thymus and with the induction of SAM pointed domain-containing Ets tran- activation-regulated chemokine. scription factor (Spdef) and Foxa3, indicating that these tran- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 scription factors interact in a network influencing airway epithelial www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000223 6134 Foxa2 AND Th2-MEDIATED LUNG INFLAMMATION differentiation (14, 17, 18). Recent studies demonstrated that induced at 6 wk of age by provision of doxycycline 24 h before mice were goblet cells induced by pulmonary allergens are derived from the intranasally sensitized with OVA (Supplemental Fig. 8). The control mice differentiation of resident Clara cells that serve as progenitor cells were the single transgenic littermates (Scgb1a1-rtTA) that received the same doxycycline and OVA treatments. Doxycycline was continued until in the bronchiolar epithelium (18, 19). In initial studies, in which the time of sacrifice. Foxa2 was deleted in the respiratory epithelium prior to birth, mucous cell metaplasia, alveolar remodeling, and inflammation mRNA microarray analysis were observed (14). The characteristics and mechanisms un- Lung cRNA was hybridized to the murine genome MOE430 chips (con- derlying lung inflammation caused by loss of Foxa2 in airway sisting of 45,000 gene entries, Affymetrix, Santa Clara, CA) according to epithelial cells are unclear at present. the manufacturer’s protocol. The RNA quality and quantity, probe prep- In this study, we demonstrate that respiratory epithelial cell- aration, labeling, hybridization, and image scans were carried out in the CCHMC Affymetrix Core using standard procedures. Affymetrix Micro- specific deletion of Foxa2 caused spontaneous Th2 cytokine/che- array Suite 5.0 (Affymetrix) was used to scan and quantitate the gene chips mokine-mediated inflammation and goblet cell metaplasia. The under default scan settings. Normalization was performed using the Robust hypothesis, that the inflammatory response was mediated by the Multichip Average model. Data were further analyzed using affylmGUI spontaneous activation of IL-4R signaling, was tested using a mAb (http://bioinf.wehi.edu.au/affylmGUI) from R/Bioconductor package to block IL-4R signaling in neonatal mice. Deletion of Foxa2 in- (www.bioconductor.org). Differentially expressed genes were selected with the threshold of p value of ,0.01, fold change $1.5, and a minimum duced expression of a network of genes influencing or associated of two present calls in three samples with relatively higher expression. with Th2-cell mediated inflammation and DC recruitment and ac- Gene ontology (GO) analysis was performed using the web-based tool tivation, indicating the role of respiratory epithelial cell in pro- David (Database for annotation, visualization, and integrated discovery). gramming inflammatory responses in the developing lung in Overrepresented pathways were identified by comparison of the overlap of Downloaded from differentially expressed genes identified postdeletion of FOXA2 and all a process regulated by Foxa2 and requiring IL-4Ra signaling. genes in MOE430 mouse genome. Gene sets associated with known pathways and disease states were identified from the Kyoto Encyclopedia Materials and Methods of Genes and Genomes (www.genome.ad.jp/kegg/), GenMAPP (www. Transgenic mice and animal husbandry genmapp.org/), and GEArrays (www.sobiosciences.com/microarrays.php/). A pathway was considered to be overrepresented when a probability p Animals were maintained according to protocols approved by the In- value was #0.01 and gene hits $5. Potential protein/protein or protein/ stitutional Animal Care and Use Committee at Cincinnati Children’s DNA interactions were identified using Ingenuity Pathway Analysis (In- http://www.jimmunol.org/ Hospital Research Foundation (Cincinnati, OH). Mice were housed in genuity Systems, Redwood City, CA). Ingenuity Pathway Analysis soft- a pathogen-free barrier facility in humidity- and temperature-controlled ware maps the differentially expressed genes identified from the rooms on a 12:12 h light/dark cycle and were allowed food and water ad microarray experiment onto the interactome according to Ingenuity Path- LoxP/LoxP libitum. SFTPC-rtTA/tetO7CMV-Cre/Foxa2 compound transgenic way Knowledge Base, a large curated database of published literature mice were generated as previously described (14) and used to permanently findings related to mammalian biology. Genetic networks preferentially delete Foxa2 in the fetal lung. Pregnant dams were maintained on doxy- enriched in the sets of mRNAs were generated based on their connectivity. cycline food from embryonic day (E) 6.5 to E12.5 to delete Foxa2 from Statistical scores were calculated to rank the resulting networks and respiratory epithelial cells during fetal development, producing SFTPC/ pathways using Fisher’s right-tailed exact test. The score indicates the Foxa2Δ/Δ mice. degree of relevance of a network to the input gene set, taking into account Conditional expression of Foxa2 in respiratory epithelial cells was the number of network-eligible genes and the size of the network. by guest on September 27, 2021 achieved by producing tetO7-Foxa2-IRES-EGFP transgenic mice that were then mated to Scgb1a1-rtta (line II) mice (20). Full-length rat Foxa2 Cytokine and quantitative RT-PCR assays coding sequence together with the 59 untranslated region and 39 untranslated region was isolated from pRc/CMV-rFoxa2 (14) at EcoRI sites To determine cytokine levels, BALF was collected as previously described (22). BALF from each adult mouse was concentrated using 0.5 ml (VIVA and cloning into the BamHI site of the pOtet7-IRES-EGFP vector (21) (the latter provided by Dr. Kenneth Campbell, Cincinnati Children’s Hospital SPIN4, catalog number VS0413, Sartorius, Bohemia, NY) and subjected to Medical Center, Cincinnati, OH) via blunt-end ligation. Transgenes were ELISA to determine the concentrations cytokines using the mouse IL-4, IL-5, identified by PCR using the primer set: 59-AGC AAA GAC CCC AAC IL-13, and IFN-g ELISA kits from eBioscience, San Diego, CA (catalog GAG AAG C-39 and 59-CAA ACA ACA GAT GGC TGG CAA C-39. numbers 88-7044, 88-7054, 88-7137, 88-7104, and 88-7384, respectively). The Ccl17 ELISA kit was purchased from R&D Systems (MCC170; Min- Histology and immunohistochemistry neapolis, 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 Immunohistochemistry was performed on 5-mm lung sections using rabbit total RNAwas purified by RNeasy Mini Kit (catalog number 74104, Qiagen, anti-Foxa2 (1:2000–1:8000), guinea pig anti-Spdef (1:4000) generated in Valencia, CA) and reverse transcribed into cDNA by Verso cDNA kit (catalog this laboratory, goat anti-Foxa3 (1:100; SC-5361, Santa Cruz Biotechnology, number AB-1453/A, Thermo Fisher Scientific, Waltham, MA). Quantitative Santa Cruz, CA), mouse mAb against Muc5ac (1:500, ab3649, Abcam, RT-PCR (qRT-PCR) was performed on a 7300 real-time PCR system Cambridge, MA) (18). Sections were processed with Ag retrieval using (Applied Biosystems, Foster City, CA) with the TaqMan probes Foxa2 heating in citrate buffer. Anti-mouse IL-4Ra mAb (lot number 9382-25B, 11/ (Mm00839704_mH), Il4 (Mm00445259_m1), Il5 (Mm99999063_m1), Il13 12/03, AS/CH) was kindly provided by Amgen (Thousand Oaks, CA). (Mm99999190_m1), Ccl11 (Mm00441239_g1), Ccl24 (Mm00444701_m1), Ccl17 (Mm01244826_g1), Il33 (Mm01195784_m1), Il6 (Mm99999064_m1), Anti–IL-4R treatment Ccl20 (Mm01268753_g1), and Ifn-g (Mm00801778_m1) and normalized to Neonatal SFTPC/Foxa2Δ/Δ pups and control littermates were injected i.p. endogenous 18s rRNA for control (probe part number 4352930E). qRT-PCR on postnatal day (PN) 2 and PN9 with either anti–IL-4Ra Ab (50 mg/g was performed with an n of 3 mice for each genotype at PN11 and PN15. body weight) or equal volume of sterile saline. On PN15, mice were an- esthetized and the lungs lavaged five times with 0.3 ml saline, and the cells Flow cytometry were counted from bronchoalveolar lavage fluid (BALF). Cytospins of 3 4 Lung cell suspensions were incubated with anti-CD16/32 (clone 2.4G2) for BALF cells (5 10 ) were stained with a Diff-Quik kit (catalog number 30 min and then staining reactions were performed at 4˚C. Myeloid DCs 24606, Polysciences, Warrington, PA). Cells (400–500) were counted to (mDCs; CD11c+,CD11b+,Gr1neg, and CD317neg) and plasmacytoid DCs determine numbers and percentage of macrophages, eosinophils, neu- (pDCs; CD11clow,CD11bneg,Gr1+, and CD317+) were quantified using trophils, and lymphocytes. Lung tissue from a second group of mice was AF647-conjugated CD11c (HL3), PE-Cy7–conjugated CD11b (M1/70), fixed with 4% paraformaldehyde and processed into paraffin blocks for APC-eFluor780–conjugated anti-Gr1 (RB6-8C5), and AF488-conjugated immunohistochemistry. anti-317 (eBio927, eBioscience). To measure costimulatory molecule Pulmonary OVA sensitization expression, cells were stained with PE-conjugated anti-CD80 (16-10A1), anti-CD86 (GL1), or B7-DC (TY25). For intracellular T cell cytokine Mice were sensitized to OVA by systemic and pulmonary administration staining, lung cells were cultured overnight in the presence of PMA using protocols previously described (18). Expression of Foxa2 in the (100 ng/ml) and ionomycin (1 mg/ml). Cytokine secretion was blocked Scgb1a1-rtTA/tetO7-Foxa2-IRES-EGFP (Scgb1a1/Foxa2-IE) mice was with a combination of brefeldin A and monensin (eBioscience) for 4 h. The Journal of Immunology 6135

Cells were stained with PE-Cy7–conjugated anti-CD4 (RM4-5) and PE- test. Difference between two groups was considered significant when the p conjugated anti–IL-13 (eBio13A). All mAbs were purchased from value was ,0.05 for all tests. eBioscience. Data were acquired with an LSRII flow cytometer (BD Bio- sciences, San Jose, CA) equipped with lasers tuned to 488 nm, 633 nm, and 405 nm. Spectral overlap was compensated using the FACSDiVa Results software (BD Biosciences) and analyzed using FlowJo software (Tree Conditional deletion of Foxa2 caused Th2 cell-mediated Star, Ashland, OR). pulmonary inflammation Plasmid and luciferase reporter assay To delete Foxa2 expression in respiratory epithelium, dams of SFTPC-rtTA/tetO7-CMV-Cre/Foxa2LoxP/LoxP transgenic em- The control plasmid used in luciferase reporter assay expressing enhanced GFP (EGFP) was made by isolation of the IRES-EGFP fragment from bryos were treated with doxycycline from E6.5 to E12.5. Ap- pIRES2-EGFP (catalog number 6029-1, Clontech, Mountain View, CA) by proximately 30% of the mutant mice died in the postnatal period BamHI and NotI sites and subcloned into the BamHI site of the as previously reported (14, 16). Surviving mice develop sponta- p3XFLAG-Myc-CMV plasmid (catalog number E6401, Sigma-Aldrich, St. neous pulmonary eosinophilic inflammation, goblet cell meta- Louis, MO) via blunt-end ligation. The Foxa2 expression plasmid was made by cloning rat Foxa2 cDNA from pRC/CMV-rFoxa2 using PCR plasia, and airspace enlargement in the first 2 wk of life (Fig. 1A). primers: 59-ATG AAT TCA ATG CTG GGA GCC GTG AAG-39 and 59- On PN15, pulmonary inflammation was seen histologically and ATG AAT TCG CGG ACG AGT TCA TAA TAGG-39 and inserting into supported by quantitation of inflammatory cells in BALF. Num- the EcoRI site of the control vector. AccuPrime Taq DNA Polymerase bers of eosinophils, lymphocytes, and macrophages were increased (catalog number 12339-016, Invitrogen, Carlsbad, CA) was used for am- in BALF from Foxa2Δ/Δ mice (Fig. 1B). Deletion of Foxa2 was plification of DNA fragments. The mouse Ccl17-pGL3 plasmid was cloned by amplification of the 2-kb promoter region from C57/B6 mouse genomic associated with increased expression of Th2 cytokines and che- Downloaded from DNA using primers: 59-GGA CCT GAA ATA GTC AGC ATCC-39 and 59- mokines, including IL-13, IL-4, IL-5, and Ccl17 (thymus and CTG AGG TGA AGG TCT TCA TGGG-39 and cloned into the pGL3- activation-regulated chemokine [Tarc]) as measured in BALF basic plasmid (catalog number E1751, Promega, Madison, WI). The lu- (Fig. 1C). CD4+ T cells that were recruited into the lung were ciferase assay was performed by transfection of MLE-15 cells with the mCcl17-pGL3 plasmid along with either control plasmid-expressing EGFP further analyzed by flow cytometry, revealing a significant in- or Foxa2-EGFP at a 1:1 ratio using the transfection reagent Lipofectamine crease of the median fluorescent intensity of IL-13 and IL-17a and 2000 (catalog number 11668-019, Invitrogen). CMV-bGal plasmid was abundance of Th2 and Th17 cell populations in the lungs of Δ Δ cotransfected to serve as an internal control, and the luciferase activity was Foxa2 / mice. Expression mRNA of IL-6, a cytokine required for http://www.jimmunol.org/ normalized to b-galactosidase activity after 24 h of transfection. Th17 cell differentiation (23, 24), was significantly induced. In Statistics contrast, Ifn-g mRNA was not altered (Supplemental Fig. 1). Δ/Δ Statistical differences in cell counts and concentrations of proteins assessed Taken together, Foxa2 mice develop spontaneous pulmonary by ELISA were determined using Student t test (two-tailed and unpaired). inflammation that is typical of enhanced Th2 cytokine/chemokine The statistic method applied to analyze qRT-PCR was the Mood’s Median activity during the postnatal period of development. by guest on September 27, 2021

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. 6136 Foxa2 AND Th2-MEDIATED LUNG INFLAMMATION

Inhibition of IL-4R–mediated signaling inhibited eosinophilic inflammation, goblet cell metaplasia and mucus hyperproduction inflammation and goblet cell differentiation induced by Foxa2 in Foxa2Δ/Δ mice were inhibited by the IL-4Ra Ab. Goblet cell deletion markers, including Alcian blue, and Spdef, Foxa3, and Muc5ac A key pathway that regulates allergic inflammation and tissue staining were also inhibited by neutralizing Ab against IL-4Ra remodeling in the airways involves the Th2 cytokines IL-4 and IL- (Fig. 2B), demonstrating that the goblet cell metaplasia was de- 13 and the activation of the IL-4Ra subunit (IL-4Ra) (25). IL-4 is pendent upon activated IL-4Ra signaling caused by deletion of primarily involved in promoting the differentiation and pro- Foxa2. liferation of Th2 cells and the synthesis of IgE, whereas IL-13 has a critical role in mediating airway hyperresponsiveness (AHR), Loss of Foxa2 in respiratory epithelium enhances mDC goblet cell metaplasia, and mucus hypersecretion, the elements recruitment and activation that are most closely linked to clinical manifestations of asthma. Th2-cell mediated inflammation is dependent on activation and Blocking IL-4R signaling pathway with neutralizing Ab against migration of pulmonary DCs to the respiratory epithelium (31). IL-4Ra or mutagenesis of IL-4Ra was sufficient to inhibit re- As professional APCs, DCs are uniquely capable of fully activating sponses to both IL-4 and IL-13 cytokines in vivo and in vitro (26– naive T cells. In the lung, two distinct phenotypes of DCs have been 28). Specifically, loss of IL-4Ra in Clara cells inhibited mucus identified that have markedly different effects on T cell function. production after OVA intrapulmonary exposure in adult mice (29), mDCs (defined as CD11c+, CD11b+, Gr1neg, and CD317neg cells) and loss of IL-4Ra significantly decreased spontaneously induced promote robust T cell activation and efficiently promote allergen- mucous cells and eosinophilia in neonatal mice (PN10) (30). To induced AHR (32). In contrast, pDCs (defined as CD11clow, test whether pulmonary inflammation and goblet cell differentia- CD11bneg, Gr1+, and CD317+ cells) promote the development of Downloaded from tion caused by conditional deletion of Foxa2 in the airway epi- Foxp3+ regulatory T cells, preventing allergen-induced AHR (33). thelium was dependent upon IL-4R–mediated signaling, Foxa2Δ/Δ Thus, to test the hypothesis that excessive Th2 cell activation ob- mice were treated with IL-4Ra mAb at PN2 and PN9. Eosino- served in mice lacking Foxa2 in the respiratory epithelium philic inflammation was significantly inhibited by anti–IL-4Ra was preceded by dysregulation in pulmonary DC recruitment or Ab, as shown by the reduction of inflammatory cells and eosi- activity, we assessed mDC and pDC recruitment and activation nophils in BALF (Fig. 2A). Consistent with inhibition of Th2 at PN8, PN11, and PN15 by flow cytometry. Comparing the http://www.jimmunol.org/ by guest on September 27, 2021

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). The Journal of Immunology 6137 frequencies of DC subsets in control and Foxa2Δ/Δ mice revealed pendent or focal changes in its expression would not be detected in a significant increase in the frequency of both mDCs and pDCs the current study design. 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 Foxa2 regulates a genetic network associated with pulmonary mice, there was a substantial increase in the mDC/pDC ratio in Th2 inflammation and goblet cell differentiation Foxa2Δ/Δ mice, particularly at PN15, suggesting an environment To investigate the role of Foxa2 and its downstream targets asso- better suited to T cell activation in Foxa2Δ/Δ mice. Comparing the ciated with the Th2 inflammation and goblet cell hyperplasia, RNAs expression of DC activation-associated costimulatory molecule were isolated from the lungs of Foxa2Δ/Δ and control littermates at expression on mDCs from control and Foxa2Δ/Δ mice demonstrated PN15. Lung cRNA was hybridized to the murine genome MOE430 significantly elevated frequencies of mDCs expressing B7-DC chips and differential gene expression and GO analyzed. Deletion (Fig. 3B), B7-H1 (Fig. 3C), and CD86 (Fig. 3D). mDCs expressing of Foxa2 significantly influenced the expression of 665 mRNAs. CD80 were also increased in Foxa2Δ/Δ mice, although changes Among these, 516 out of 665 mRNAs were induced, and 148 were were not statistically significant. MHC class II was not different reduced in response to deletion of Foxa2 in the respiratory epi- from control and Foxa2Δ/Δ mice (Supplemental Fig. 2). In- thelium (Fig. 4A). The complete microarray dataset has been terestingly, although pDCs were also generally more activated in submitted to Gene Expression Omnibus under accession number Foxa2Δ/Δ mice than in control mice, by PN15, a time when there GSE19204 (www.ncbi.nlm.nih.gov/geo/query/). GO analysis sug- was robust activation of T cell cytokine production, the levels of gested that the induced genes are primarily enriched in “Immune/ pDC activation had decreased and were indistinguishable from inflammatory response” (p value: 3.8E-13), indicating that Foxa2 controls (Fig. 3B–D). Collectively, these data demonstrate that plays a suppressive role in immune/inflammatory responses and Downloaded from concomitant with the increased T cell activation, the recruitment mucus production in the postnatal lung; nearly 40% of induced and activation of pulmonary mDCs was increased in Foxa2Δ/Δ genes encode cytokines, chemokines, and their receptors, and mice. Expression of Th2 cytokine mRNAs, including Il4 and Il13, many are known to be associated with asthma or experimental was significantly increased in the lungs of Foxa2Δ/Δ mice at PN11 allergen sensitization (Supplemental Table I). The overlap between and PN15 (Fig. 4B, Supplemental Fig. 3). Mechanisms underlying the known asthma-related genes (genes causing, predisposing, or

the recruitment and activation of DCs in the lung are not well es- protecting from asthma) and those responding to the deletion of http://www.jimmunol.org/ tablished at present. The IL-7–like cytokine thymic stromal lym- Foxa2 was highly statistically significant. Pathway analysis further phopoietin, produced by epithelial cells, is known to influence DCs indicated the “dendritic and Ag presenting,” “T cell and B cell expressing OX40 ligand, causing differentiation of naive CD4+ activation,” “cytokine–cytokine receptor interaction,” “eicosanoid T cells to Th2 cells that produce IL-4, IL-13, and TNF but not IL-10 signaling,” and “JAK-STAT signaling pathway” were significantly (34, 35). Similarly, CCL20 is a chemokine produced in epithelial induced. In contrast, “glutathione metabolism” (1.2E-04) was the cells and capable of inducing DC migration via interaction with most significantly overrepresented canonical pathway in mRNAs CCR6 expressed on the immature DCs (36, 37). Ccl20, but not Tslp decreased postdeletion of Foxa2 (Supplemental Table II). Expres- mRNA expression, was induced in the lungs of Foxa2Δ/Δ mice at sion of mRNAs of major Th2 cytokines and chemokines in the PN15 (Supplemental Fig. 4), suggesting a potential mechanism by whole lung was confirmed by qRT-PCR. Consistent with the severe by guest on September 27, 2021 which DCs are recruited and activated in the lungs of Foxa2Δ/Δ eosinophilic inflammation and Th2 lymphocytes infiltration seen in mice. Although Tslp mRNA was not increased in whole lung at Foxa2Δ/Δ mice, Il4, Il5, and Il13 mRNAs in the whole lung were PN11 or PN15 (data not shown), it remains possible that time de- significantly increased, whereas expression of the Th1 cytokine Ifn-

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 Foxa2 Δ/Δ mice at PN11 to PN15 (A). Al- though both subsets of DCs were found with greater frequency in the lungs of Foxa2 Δ/Δ mice, there was a substantial increase in the mDC/pDC ratio in Foxa2 Δ/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. Collec- tively, these data demonstrate that, concomitant with the increased T cell activation observed in Foxa2 Δ/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). 6138 Foxa2 AND Th2-MEDIATED LUNG INFLAMMATION Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

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. g was not changed (Fig. 4B, Supplemental Fig. 1). Epithelial cells Conditional expression of Foxa2 in the respiratory epithelium lining the respiratory and gastrointestinal tract play a pivotal role in inhibited allergen-induced goblet cell differentiation initiation, regulation, and resolution of innate and adaptive immune A transgenic mouse in which Foxa2 was conditionally expressed in response by expressing a wide range of immune response genes the respiratory epithelium was produced to test whether increased including costimulatory molecules, chemokines, cytokines, and expression of Foxa2 was sufficient to inhibit allergen-induced PGs (38). Of particular interest, respiratory epithelium expresses inflammation or goblet cell differentiation (Supplemental Fig. 6). IL-33, a cytokine that promotes Th2 cytokine production (39) and Double-transgenic mice Scgb1a1-rtTA/tetO -Foxa2-IRES-EGFP CCL17 (40, 41) that chemoattracts Th2 cells via interactions with 7 (Scgb1a1/Foxa2-IE) were treated with doxycycline, and the in- CCR4 that is selectively expressed on the Th2 cells (42). Expres- sion of Il33 and Ccl17 mRNAs was significantly induced in the duction of Foxa2 and EGFP expression was confirmed by im- Foxa2Δ/Δ mice at PN15 (Fig. 4B). CCL17 (Tarc), a potent T cell munohistochemistry and fluorescence microscopy (Fig. 5A, chemoattractant induced in bronchiolar epithelial cells of asth- Supplemental Fig. 6). Goblet cells, although normally rare in the matics (40, 41), likely plays an important role in the pulmonary surface epithelium lining the conducting airways of adult mice, inflammation seen in the Foxa2Δ/Δ mice. The effects of Foxa2 on are usually present in young mice (30, 43). Expression of Foxa2 Tarc gene expression were assessed in MLE-15 cells in vitro. from PN4-PN18 by doxycycline treatment blocked the normal Foxa2 inhibited the Tarc promoter by ∼40% (Supplemental Fig. 5). postnatal goblet cell differentiation (Supplemental Fig. 7). Con- A schematic summarizing the effects of Foxa2 gene deletion on ditional expression of Foxa2 in the respiratory epithelium in adult Th2-mediated processes in is provided by Fig. 6. mice inhibited goblet cell differentiation (Fig. 5B, Supplemental The Journal of Immunology 6139

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 Downloaded from 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 http://www.jimmunol.org/ and goblet cell metaplasia. The proposed network indicates regulatory relationships but does not imply direct transcriptional control of each gene by Foxa2.

and IL-13) and Ccl17 (Tarc) production and Foxa2 inhibiting Tarc gene promoter activity in vitro. The inflammatory effects of Foxa2 FIGURE 5. Foxa2 inhibited allergen-induced goblet cell differentiation. deletion were inhibited by blocking IL-4R signaling in the de- Scgb1a1/Foxa2-IE transgenic mice were treated with doxycycline for 3 d, veloping mouse lung. Taken together, the respiratory epithelium, increasing Foxa2 expression as detected by immunohistochemistry using via Foxa2, plays a critical role in programming the innate immune by guest on September 27, 2021 a high dilution of Foxa2 Ab at 4 wk of age (A, original magnification system during postnatal development of the lung and serves to 3 100). Foxa2 was not prominent at this Ab dilution in single transgenic inhibit the development of Th2-dominated innate immunity. Scgb1a1-rtTA or Scgb1a1/Foxa2-IE (without doxycycline) transgenic mice. Doxycycline was administered to Scgb1a1-rtTA and Scgb1a1/Foxa2-IE Th2 cytokine-dominated inflammation postdeletion of Foxa2 in transgenic mice 1 d before intranasal OVA exposure. Foxa2 markedly in- the lung hibited goblet cell differentiation as shown by decreased Alcian blue, Allergic airway inflammation is characterized by recruitment and Muc5ac, Spdef, and Foxa3 staining (B, original magnification 3100). In- serts are the higher magnification (34) of the regions indicated by arrows. activation of Th2 lymphocytes and eosinophils, goblet cell meta- Scale bar, 50 mm. Figures are representative of n = 3 for each genotype. plasia, and mucus hyperproduction in a process regulated by a number of cytokines and chemokines. A number of the com- ponents of the IL-4R signaling pathway (e.g., IL-4, IL-13, and Fig. 8). However, expression of Foxa2 in respiratory epithelial IL-4Ra) were increased in Foxa2Δ/Δ mice. IL-13 is produced cells of the mature lung prior to OVA sensitization did not alter primarily in Th2 cells and regulates many asthma-related pro- Th2 cytokine production or inflammation. Inflammatory cell cesses, including mucus hyperproduction, eosinophil recruitment counts (total or differential), as well as IL-4, IL-5, IL-13, IL-10, and survival, and airway hyperreactivity (44). IL-13 blockade and IFN-g concentrations, were similar in BALF from Foxa2- abrogated many of the features of asthma (45), demonstrating that expressing and control mice after OVA exposure (Supplemental IL-13 is an important mediator in Th2 responses and asthma Fig. 9A,9B). These finding are consistent with previous ob- pathogenesis. The present observation, that inhibition of IL-4Ra servations supporting the role of Foxa2 in regulating epithelial cell signaling substantially inhibited pulmonary eosinophilic inflam- differentiation (14, 16). The present findings demonstrate that mation and goblet cell metaplasia postdeletion of Foxa2, supports Foxa2 also plays an important role in modulating Th2 immunity in the important role of Foxa2 in inhibiting development of Th2- postnatal development, but does not directly control Th2-mediated mediated innate immunity that is dependent upon IL-4R signaling. inflammatory responses in the mature lung. Consistent with the observed Th2-mediated inflammation in Foxa2Δ/Δ mice, a number of factors involved in T cell and eo- Discussion sinophil recruitment, including colony-stimulating factor receptors Deletion of Foxa2 in respiratory epithelial cells caused sponta- Csf2ra and Csf2rb (2- and 4-fold), Il1b (2-fold), tumor necrosis neous pulmonary inflammation associated with goblet cell meta- signaling factor-associated genes Tnfrsf1b,-4,-9,-13,-14, and plasia and enhanced expression of Th2 cell-associated cytokines, -18, were induced (2–10-fold). Likewise, mRNAs encoding lym- chemokines, and their receptors. Recruitment and activation of phocyte chemoattractants, Ccl13 (3.4-fold), Ccl22 (4.6-fold), Ccr9 mDCs and Th2 lymphocytes were increased in Foxa2Δ/Δ mice (4-fold), Ccr5 (4-fold), and Ccr2 (2.6-fold), were induced, the lungs and associated with increased Th2 cytokines (IL-4, IL-5, latter being required for pulmonary DC accumulation in asthma 6140 Foxa2 AND Th2-MEDIATED LUNG INFLAMMATION

(46). mRNAs associated with eosinophilic inflammation were absence of Foxa2. Fig. 6 summarizes the present findings re- induced in lungs of Foxa2Δ/Δ mice, including Ccl11, Ccl24, Epx, garding mechanisms by which Foxa2 influences lung inflam- Itgb2, Kng1, Lgals3, Ltc4s, Alox5, Arg1, Il1rn, and Rag1. Among mation in the developing mouse. these, eosinophil-specific chemokines Ccl11 and Ccl24 were in- Goblet cell differentiation caused by deletion of Foxa2 in the creased 7- and 12-fold, respectively. Epx mRNA (eosinophil developing lung seen in the current study was associated with Th2 peroxidase), a heme-containing glycoprotein that may contribute cell activation and the induction of Spdef and Foxa3 that appear to to the pathogenesis of epithelial damage and bronchial hyperre- function in a transcriptional network in the respiratory epithelium activity in human asthma (47), was markedly increased in Foxa2D/D (18). Consistent with recently published studies (59), we demon- mice. Eosinophil major basic protein (Prg2, 11-fold) and eosinophil- strated that forced expression of Foxa2 in the adult mouse in- associated RNase (Ear11, 90-fold) mRNAs were also dramatically hibited goblet cell differentiation, blocking Spdef and Foxa3, but increased. did not influence allergen-induced inflammation or eosinophilic Th2-associated inflammatory mediators, including acidic chi- infiltration. These findings support the role of Foxa2 in the in- tinase (Chia), chitinase 3-like 1 (Chi3l1), chitinase 3-like 3 struction of innate immunity during development rather than (Chi3l3), and chitinase 3-like 4 (Chi3l4) mRNAs, were increased in a direct role for Foxa2 in suppressing inflammation during OVA Foxa2Δ/Δ mice. Chitinases and chitinase-like proteins are believed sensitization in the mature mouse. to play a key role in the innate immunity to parasites and other infectious agents and may play an important role in the pathogenesis of allergy and/or asthma and regulation of eosinophilia Acknowledgments and eotaxin induction (48–50). Chia is induced via a Th2-specific, We thank Ann Maher for preparation of the manuscript, Amgen for provid- Downloaded from ing the IL-4Ra mAb, and Dr. Fred Finkelman for scientific input. IL-13–mediated pathway in lung epithelial cells and macrophages and is increased in lungs of asthmatics (48). Chia stimulates chemokine production by pulmonary epithelial cells (50). Disclosures Deletion of Foxa2 from lung epithelium disrupted Th1-Th2 The authors have no financial conflicts of interest. balance, inducing a large number of Th2 cytokines and associated signaling pathways. 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