UNIVERSITY OF CINCINNATI

Date: 26-Apr-2010

I, Gang Chen , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Developmental Biology It is entitled: Critical roles of Foxa2 and Spdef in regulating innate immunity and goblet cell

differentiation in the lung

Student Signature: Gang Chen

This work and its defense approved by: Committee Chair: Jeffrey Whitsett, MD Jeffrey Whitsett, MD

6/10/2010 576 Critical Roles of Foxa2 and Spdef in Regulating Innate Immunity and

Goblet Cell Differentiation in the Lung

A dissertation submitted to the

Division of Research and Advanced Studies of

the University of Cincinnati

In partial fulfillment of the requirements for the degree of

DOCTORATE OF PHILOSOPHY (Ph.D.)

In the Program of Molecular and Developmental Biology

of the College of Medicine

2010

by

Gang Chen

Committee Chair: Jeffrey A. Whitsett, M.D.

John M. Shannon, PhD

George D. Leikauf, Ph.D.

James M. Wells, Ph.D.

Gurjit K. Hershey, M.D., Ph.D. Abstract:

The respiratory epithelial cells lining the conducting airways play critical roles in mediating

innate and acquired immune responses by participating mucociliary clearance, secreting anti-bacterial

peptides, and release cytokines and chemokines to interact with immune cells. The forkhead box

transcription factor, Foxa2, is normally expressed in respiratory epithelial cells lining conducting

airways and in alveolar type II cells. Foxa2 is required for normal lung maturation, surfactant protein

and lipid synthesis during lung development. However, the role of Foxa2 in regulating crosstalk between

epithelium and immune system during lung development was unknown. In the present thesis, selective

deletion of Foxa2 allele in the respiratory epithelium mediated by human surfactant protein C (SFTPC) promoter driven Cre recombinase during embryonic stage (E6.5-E12.5) was found to cause asthma-like phenotype including eosinophilic inflammation and goblet cell metaplasia in the neonatal mice. Loss of

Foxa2 induced the recruitment and activation of myeloid dendritic cells (mDCs) and T helper 2 (Th2) cells in the lung, resulting in increased production of Th2 cytokines and chemokines, including interleukin 4 (IL-4), IL-13, IL-5 and thymus and activation-regulated chemokine (Tarc), and induced expression of goblet cell transcription factor Spdef. Expression of Foxa2 in the non-ciliated secretory epithelial cells (Clara cells) inhibited Spdef expression and goblet cell differentiation after allergen exposure, suggesting that Foxa2 and Spdef interacted within a genetic network that was associated with goblet cell differentiation. Spdef, SAM pointed domain Ets-like factor, normally expressed at low level in tracheal and bronchial epithelium, is significantly induced in goblet cells after IL-13 or allergen exposure at a Stat6 dependent manner. Expression of Spdef in Clara cells caused rapid and reversible goblet cell differentiation in the absence of cell proliferation in vivo. Spdef enhanced the expression of genes associated with goblet cell differentiation and pathogenesis of asthma, including Muc16, Agr2,

Clca1, Ptger3, Muc5ac, and a group of genes mediating mucin glycosylation, including Gcnt3. Deletion

1 of the murine Spdef gene resulted in the absence of goblet cells in tracheal-laryngeal submucosal glands and in the surface epithelium after pulmonary allergen exposure in vivo, demonstrating its requirement for normal goblet cell differentiation in the lung. Spdef inhibited Foxa2 and TTF-1, and induced Foxa3 demonstrating its pivotal role in transcriptional control of airway epithelial cell differentiation. Spdef and Foxa3 were increased after sensitization with pulmonary allergen and were co-localized in goblet cells in normal human bronchial glands and in goblet cells lining airways of patients with chronic lung diseases. Our current findings provided new evidence that the innate immune system is strongly determined by respiratory epithelial cells during early neonatal period via the transcription factor Foxa2.

Loss of Foxa2 induced expression of Spdef that was found to play a critical role in the regulation of a transcriptional network mediating goblet cell differentiation and mucus hyperproduction, a process that is highly relevant to the pathogenesis of asthma, cystic fibrosis, and other inflammatory lung diseases.

2

Acknowledgments:

I would like to express my gratitude to the following people for their support and assistance

during the last five and half years.

I would like to give special thank to my mentor, Dr. Whitsett who gave me an extraordinary

training in last 5 years. I feel very privileged and lucky that he accepted me as a graduate student 5 years

ago, so that I was able to learn from him and work on the exciting projects. He guided, encouraged and

inspired me with his never-ending wisdom, enthusiasm and great patience. With his knowledge,

experience and attitude towards sciences, he has had a fundamental impact on my scientific thinking and development.

I would like to express my sincere gratitude to my thesis committee members for their continuous guidance and helpful discussions through my thesis work. I also thank the Molecular and

Developmental Biology Graduate Program for offering me the exceptional graduate training.

I appreciate the help from the co-workers in the Division of Neonatology and Pulmonary

Biology, especially the colleagues in Whitsett lab, for their openness and honesty with which they shared their life, feelings and experience with me.

I am deeply indebted to my loving wife, Zhenglei Pei, for her unwavering support. Lastly, I am grateful for the encouragement, love and care my parents have shown to me.

3

Table of Contents:

Abstract...... 1

Acknowledgements...... 3

Table of Contents...... 4

List of Symbols and Abbreviations...... 6

Chapter I General Introduction...... 8

Reference...... 29

Chapter II Foxa2 programs Th2-cell mediated innate immunity in the developing lung………36

Abstract...... 37

Introduction...... 38

Materials and Methods……………………………………………………………………………………...40

Results...... 45

Discussion...... 50

Acknowledgements...... 53

References...... 54.

Figures and Legends………………………………………………………………………………………...58

Supplementary Figures and Legends………………………………………………………………………..66

4

Chapter III Spdef is required for pulmonary goblet cell differentiation and regulates a network of genes associated with mucus production……………………………………………………………….……….……80

Abstract...... 81

Introduction...... 82

Results…………………………………………………………………………………….………………….…..84

Discussion…………………………………………………………………………….………………………….87.

Materials and Methods………………………………………………………….………………………………..92

Acknowledgements………………………………………………………………………………………………98

References………………………………………………………………………………………………………..99

Figures and Legends……………………………………………………………………………………………..103

Supplementary Figures and Legends…………………………………………………………………………….118

Chapter IV General Discussion...... 122

Reference...... 136

5

List of Symbols and Abbreviations

Abca3 ATP-binding cassette, sub-family A (ABC1), member 3 Agr2 anterior gradient 2 (Xenopus laevis) AHR airway hyperresponsiveness Alox5 arachidonate 5-lipoxygenase Aqp5 aquaporin 5 Arg1 arginase 1 BALF bronchiolar-alveolar lavage fluid Ccl11 chemokine (C-C motif) ligand 11 Ccl17 chemokine (C-C motif) ligand 17 Ccl20 chemokine (C-C motif) ligand 20 Ccl24 chemokine (C-C motif) ligand 24 CD4 cluster of differentiation 4 Chi3l1 chitinase 3-like 1 Chia chitinase Clca1 chloride channel calcium activated 1 CMV cytomegalovirus DCs dendritic cells DEPC diethylpyrocarbonate Dox doxycycline EGFP Enhanced Green Fluorescent Proteins EGFR epidermal growth factor receptor Epx eosinophil peroxidase Foxa2 forkhead box A2 Foxa3 forkhead box A3 Gcnt3 glucosaminyl (N-acetyl) transferase 3, mucin type GFI1 growth factor independent 1 IFN-γ interferon gamma IL-13 interleukin 13 Il1rn interleukin 1 receptor antagonist IL-25 interleukin 25 IL-33 interleukin 33 IL-4 interleukin 4 IL-4Ra IL-4 receptor α subunit IL-5 interleukin 5 IL-6 interleukin 6 IRES internal ribosome entry site Itgb2 integrin beta 2 Kng1 kininogen 1 Lgals3 lectin, galactose binding, soluble 3 Ltc4s leukotriene C4 synthase

6 MAPK mitogen activated protein kinase MATH1 also termed ATOH1, atonal homolog 1 (Drosophila) mDCs Myeloid dendritic cells MDI median fluorescent intensity MLE15 mouse lung epithelial cell line 15 Muc16 mucin 16 Nkx2-1 NK2 homeobox 1 Ova ovalbumin OX40 also termed as Tnfrsf4 (tumor necrosis factor receptor superfamily, member 4) pDCs plasmacytoid dendritic cells PGK phosphoglycerate kinase (promoter) PMA Phorbol myristate acetate PN postnatal day Por P450 (cytochrome) oxidoreductase Prg2 proteoglycan 2, bone marrow (eosinophil major basic protein) Ptger3/4 prostaglandin E receptor 3 (subtype EP3)/ prostaglandin E receptor 4 (subtype EP4) Rag1 recombination activating gene 1 Scgb1a1 secretoglobin, family 1A, member 1 (uteroglobin) Scnn1b sodium channel, nonvoltage-gated 1 beta Scnn1g sodium channel, nonvoltage-gated 1 gamma Serpinb11 serine (or cysteine) peptidase inhibitor, clade B (ovalbumin), member 11 Sftpa1 surfactant associated protein A1 Sftpb surfactant associated protein B Sftpd surfactant associated protein D STAT6 signal transducer and activator of transcription 6 Tarc thymus and activation-regulated chemokine Th1 T helper 1 Th17 T helper 17 Th2 T helper 2 Titf1 (gene symbol) thyroid transcription factor 1, also termed as Nkx2-1 (NK2 homeobox 1) TLR toll-like receptor TNF tumor necrosis factor TTF-1 (protein symbol) thyroid transcription factor 1, also termed as Nkx2-1 (NK2 homeobox 1) UDP uridine diphosphate UEA-I Ulex europaeus agglutinin I UTR untranslated region

7 Chapter I General Introduction

The current thesis was conducted to test two major hypotheses: 1) that the respiratory epithelium regulates Th2-cell mediated innate immunity in the developing lung via transcription factor Foxa2, and 2) that Spdef and Foxa2 regulate a transcription network that is associated with goblet cell differentiation in the lung.

After birth, newborns face the transition from a sterile intrauterine environment to the extrauterine environment that provides a myriad of challenges from microbes, pathogens as antigens.

The respiratory epithelium is constantly exposed to inhaled microorganisms, toxicants, particles and allergens. Development of innate host defense system in the lung is critical for the neonates to adapt to environment exposures, and in turn influences the maturation of acquired immune system. The respiratory epithelium is positioned at the interface of environment and host, and plays critical roles in participating and regulating innate and adaptive immunity in response to insults in the mature lung.

However, the mechanism of controlling interactions between respiratory epithelium and innate immune system during postnatal lung maturation remained largely unknown. Understanding the regulation of neonatal innate immunity related to infection provides the framework for reducing infant morbidity and mortality, as well as the prevention and treatment of allergic diseases that maybe caused by exposures to pathogens and antigens during early life (1). Foxa2 is the transcription factor expressed in the respiratory epithelium lining the conducting airways and peripheral type II cells, and is required for lung morphogenesis and surfactant protein synthesis during development. Deletion of Foxa2 in respiratory epithelium during development (E6.5-E12.5) caused goblet cell metaplasia in the early postnatal stage.

The current thesis discovered an essential role of respiratory epithelium in suppression of Th2 cell

8 mediated inflammation in the developing lung via transcription factor Foxa2. Loss of Foxa2 induced goblet cell transcription factor Spdef, and conditional expression of Foxa2 inhibited Spdef and goblet cell differentiation following pulmonary allergen sensitization, indicating that Foxa2 and Spdef are interacting in a genetic network that regulates goblet cell differentiation and mucus production in vivo.

Goblet cell metaplasia and mucus hypersecretion are the common features of a number of major chronic respiratory diseases, including asthma, cystic fibrosis (CF) and chronic pulmonary obstructive disease (COPD). Scientific advancements achieved by modern technologies during recent decades have helped to elucidate the key genes mediating mucus hypersecretion associated chronic pulmonary diseases. These genes include those encoding Th2 cytokines/chemokines and their receptors, epidermal, neuronal, vascular and fibroblast growth factors, ion channels, epithelial cell membrane receptors, and transcription factors. Treatment of mucus hypersecretion in chronic lung diseases has inconsistent success varied in asthma, COPD and CF (2), partially due to lack of the knowledge of molecular mechanism underlying goblet cell differentiation in these diseases. Identification of new targets that are common to chronic lung diseases will be informative to develop therapeutic intervention. The current thesis demonstrated that goblet cell differentiation and mucus production were controlled by a transcriptional network mediated by the transcription factors Spdef and Foxa2. Spdef inhibits Foxa2 expression, and transdifferentiates Clara cells into goblet cells in a reversible manner. Spdef was detected in goblet cells of human lung tissue with chronic pulmonary diseases, suggesting that Spdef is a potential therapeutic target in treatment of mucus hypersection associated diseases like asthma, CF and

COPD.

9 Respiratory epithelial cells regulate innate immunity in the lung

The respiratory epithelium lining the conducting airways provides a physical barrier that is

strategically positioned to interact with external environment. Evolutionarily, this unique position makes

the epithelium a prime candidate for participating in host defense machinery. The host defense system of

the lung includes components of innate and adaptive immunity. The respiratory epithelium actively

participates in innate immune response by removing pathogens and particles from airway via

mucociliary clearance, sensing exposure of microorganisms or allergens, secreting antimicrobial peptides into airway lumen, and releasing cytokines and chemokines which initiate inflammatory responses. This inflammatory reaction includes recruitment of phagocytes to remove microorganisms that are not cleared by epithelium itself, dendritic cells and lymphocytes that interact to mount acquired immune responses (3). Failure to mount proper immune responses to inhaled pathogens, particles and antigens by epithelium may plays a role in pathogenesis of chronic pulmonary diseases including asthma,

COPD, CF, fibrosis and lung cancer.

Mucociliary clearance by the airway epithelium The conducting airway is lined primarily by a pseudostratified epithelium composed of ciliated, goblet, basal, serous, non-ciliated secretory (Clara), neuroendocrine and brush cells. In humans, the major cell types lining in tracheal and bronchial regions are ciliated cells (expressing FOXJ1and β-tubulin IV), goblet cells (expressing MUC5AC and MUC5B) and basal cells (expressing P63). Most of the mouse conducting airways is lined by a single layer of columnar epithelium comprised of ciliated and Clara cell (expressing Scgb1a1). (see Table 1 (4) for comparison of different types of respiratory epithelial cell populations in the conducting airways in humans and mice). Due to the anatomical differences, epithelial cell populations and upper airway structures are somewhat distinct in human and mouse lung. Disease models developed in mouse resemble but do not share all features characteristic of human airway diseases.

10 Population Densities (%) of the Population Densities (%) of the Population Densities (%) of the Terminal Tracheal Surface Epithelium Bronchial Surface Epithelium Bronchiolar Surface Epithelium Basal Ciliated Goblet Clara Basal Ciliated Goblet Clara Basal Ciliated Goblet Clara Human 33 49 9 0 6±1 56±10 26±10 0 + + 1.2* 8.3* Mouse 10 39 <1 49 4 47 1 46 0 <50 0 >50 Table 1 Comparison of epithelial cell composition in the conducting airways of normal adult human and mouse.

+, present. *(5)

The airway epithelial cells contribute to the first-line of host defense by responding to inhalation of airborne irritants, particles and pathogens with production of the mucus layer overlies and protects the airway epithelial cells from damage. Mucus is the collective term for mucin, ions, water and other substances normally present on the surface of the airways. By its virtue of highly glycosylated secreted mucins, lipids and soluble proteins, mucus binds and entraps a broad array of inhaled pathogens and particles, and removes them from the airways (6). This process, termed mucociliary clearance, is accomplished by the coordination of both ciliated and goblet cells that transport the mucus on the tips of beating cilia towards the throat. This elaborate system of host defense maintains the sterility of the lung

(7). Impairment of mucociliary clearance leads to airflow restriction, bacterial infection, and is associated with increased morbidity and mortality in chronic pulmonary diseases including asthma (8),

COPD (9), and CF (10). The effective mucociliary clearance depends on the normal function of ciliated cells and goblet cells. The high molecular weight glycoconjugates in the mucus are mucins that are produced and secreted mainly by goblet cells in the surface epithelium and by the submucosal glands in the submucosa. Mucus is also synthesized by serous, and possibly ciliated cells (11). Increased secretion of mucus glycoconjugates is associated with increased goblet cell numbers and submucosal gland size that are the common pathological features of chronic pulmonary diseases. Chronic mucus hypersecretion was found to be a significant predictor of COPD-related death from pulmonary infection (12).

Numerous pharmaceutical interventions and treatments have been developed to improve mucus clearance or to reverse the hypersecretory phenotype associated with chronic obstructive pulmonary

11 diseases (13). Recently, studies from our laboratory discovered that Spdef was the transcription factor

that promoted goblet cell differentiation and mucus production, and was induced by Th2 cytokines

including IL-13 and IL-4. These same Th2 cytokines inhibited Foxa2 and induced goblet cell

differentiation. Loss of Foxa2 in respiratory epithelium was sufficient to induce goblet cell

differentiation in the developing lung. The previous studies indicated that epithelial transcription factors

Spdef and Foxa2 were Th2 cytokine responsive genes, and were involved a circuit of regulating goblet

cell differentiation which was associated with mucociliary clearance, the innate host defense machinery

of the lung.

Airway epithelial cells express innate host defense molecules in response to pathogen

exposure. Besides participating in mucociliary clearance to remove pathogen and particles from the airway, respiratory epithelium actively engages in killing microorganisms by secretion of antimicrobial host defense molecules into airway lumen. The airway epithelium produces various innate host defense proteins, including collectins, such as the surfactant protein (SP-A and SP-D) bind and clear microbes

(14), defensins and cathelicidins that kill microorganism to regulate immune responses, inflammation and wound repair (15, 16). Expression of many of these innate host defense molecules, including SP-A,

SP-D, CCSP and intelectin are regulated by epithelial transcription factor Foxa2 and TTF-1, which are required for lung morphogenesis and epithelial cell differentiation during development (17, 18). Since both Foxa2 and Spdef are involved in regulating goblet cell differentiation and influencing host defense, it was unclear whether Spdef regulated expression of these proteins.

Recognition and response to invasive microorganisms are dependent on expression of pathogen recognition receptors in respiratory epithelium (19), which in turn produces proinflammatory cytokines and chemokines to crosstalk with dendritic cells and T cells at a NF-κB dependent manner (20). The discovery of Toll-like receptors (TLRs), one type of the pathogen recognition receptors, was a

12 breakthrough for understanding the ability of the innate system to rapidly recognize the pathogen related components, known as pathogen-associated molecular pattern. To date, 11 human TLRs have been identified. TLR4 has been most intensively studied because of its central role in response to bacterial lipopolysaccharide (LPS). The binding of LPS to TLR4 is mediated by the TLR4 complex that recognizes LPS, and is followed by activation of the signaling complex associated with intracellular domain of TLR4 and the adaptor protein MyD88. Expression of TLR4 in airway epithelium, but not in alveolar macrophages, is required for mediating allergic response via recruitment and activation of mDC after exposure to house dust mite (21), normally a harmless airborne allergen that is tolerogenic in the normal human lung, but causes airway inflammation, mucus hyperproduction and airway hyperresponsiveness in asthmatic subjects. Discovery of the critical role of TLR4 in epithelial cells opened up avenues of research for studying genetic susceptibly to asthma. Studies of polymorphisms in the TLR4 locus hint at an association with asthma susceptibility, particularly in children (22). In current thesis, the crosstalk between epithelial cells and mDC was found to be regulated by transcription factor

Foxa2, loss of which induced Th2 dominant inflammation in the neonatal lung. Discovery of the critical role of Foxa2 in suppression of Th2 inflammation provides a new mechanism that influences the pathogenesis of asthma, particularly in young children. Spdef influenced expression of intelectin1, SP-A,

SP-D, CCSP, Scnn1g, innate host defense genes that were also regulated by Foxa2 and TTF-1, suggesting that Spdef and Foxa2 interacted in the genetic network mediating innate host defense in the respiratory epithelium.

Airway epithelial cells interact with immune cells to regulate innate immunity in the lung.

Interactions between airway epithelium and immune cells, including dendritic cell, T cells and B cells, are required to initiate and maintain immune response following pathogen or allergen exposure. Many

13 cytokines/chemokines are produced by epithelial cells that attract and activate DCs or Th2 cells after allergen exposure, including thymic stromal lymphopoietin (TSLP), granulocyte macrophage colony- stimulating factor (GM-CSF), IL-1β, IL-33, osteopontin, IL-25 (20), CCL17 and CCL20 (Figure 1).

Dendritic cells mediate Th2 immune response. DCs are the professional antigen presenting cells that are generated from Flt3-expressing myeloid and lymphoid progenitors produced in bone marrow from hematopoietic stem cells (23). DCs are present in the respiratory tract, and form an intra- epithelial network by interacting with epithelial cells via tight junction proteins (ZO1, claudin and E- cadherin) (Figure 1). Airway DCs are capable of capturing and processing antigens. After DCs uptake antigen in the presence of a danger signal from neighboring cells (24), they undergo maturation characterized by expressing co-stimulatory molecules and chemokines to attract and stimulate naïve T cells in draining lymph nodes of the lung. Upon encountering specific T cells, DCs interact with T cells, leading to cell activation, proliferation and differentiation. DCs influence T cell polarization dependent on their lineage, their maturation status and the consequent expression pattern of co-stimulatory molecules (25). Airway DCs mainly consist of two cell types, myeloid dendritic cells (mDCs) which express CD11c+, MHCII+, CD11b+, and plasmacytoid dendritic cells (pDCs) which express CD11clow,

MHCIIlow, CD11b-, 128G8+, Gr-1(Ly6G/C)+, B220+. mDCs are primarily responsible for induction of

Th2 effector cells and allergic response (26, 27), while pDCs inhibit Th2 effector cell differentiation induced by mDCs (28) and play an anti-inflammatory role in allergic inflammation (29). Expression of major histocompatibility complex II (MHCII) molecule in DCs is responsible for naïve T cell prolieration(30), and expression of co-stimulatory molecules in DCs including the B7 super-family

(CD80, CD86, B7-H1 and B7-DC) that are critical for activating or inhibiting effector T cells differentiation (31-34). Recently, recruitment and activation of immature mucosal DCs was found to be dependent on the signaling coming from the neighboring airway epithelium (20). Following allergen

14 sensitization, activation of TLRs initiates a NF-κB dependent secretion of cytokines and chemokines in

the epithelial cells that attract and activate DCs, and Th2 cells. Epithelial derived cytokines and

chemokines are required for initiation and maintenance of DC mediated Th2 responses, including TSLP

that directly activates DCs to prime naïve T cells to differentiate into Th2 cells. CCL20, a

chemoattractant, recruits immature DCs to the airways, and GM-CFS that promotes maturation of DCs

(20) (Figure1).

The respiratory epithelium produces cytokines and chemokines that attract and activate

DCs and Th2 cells during allergic responses. TSLP is a 140 amino acid IL-7-like 4-helix bundle cytokine mainly produced by epithelial cells, and strongly activates myeloid DCs to induce a Th2 response. TSLP-activated DCs Expression of MHC class II, CD80, CD86, CCL17 (TARC) that induce

Th2 polarization, but not IL-12 family members IL-12, IL-23 and IL-27, nor type I interferons (IFNs)-all cytokines that induced in Th1 differentiation [reviewed in (35, 36)]. Conditional expression of TSLP by surfactant protein C (SFTPC) promoter in lung resulted spontaneous and progressive asthma-like disease with DC-driven Th2-cell response in the airways (37). In contrast, TSLP receptor (Tslpr) deficient mice

(Tslpr -/-) failed to develop Th2 response in airways unless they are supplemented with wild type CD4+

T cells (37, 38). GM-CSF is a cytokine produced by airway epithelial cells after allergen exposure (such

as house dust mite (39)), and is required for eosinophil recruitment, goblet cell metaplasia, and Th1 and

Th2 cytokine production following pathogen infection (40). Expression of GM-CSF in mouse lung

markedly increased airway inflammation characterized by eosinophilia and goblet cell hyperplasia after

allergen exposure (41). DC maturation is dependent on epithelial-derived GM-CSF in response to

allergen stimulation like diesel exhaust particles (42). IL-1β and IL-33 belong to the IL-1 family.

Following allergen (HDM) or diesel exhaust particles exposure, normal airway epithelial cells produce

IL-1β (43), which in turn rapidly promotes expression of TSLP in epithelial cells (44). Induced

15 expression of IL-1β in respiratory epithelium under the control of Clara cell secretory protein (Scgb1a1,

also termed CCSP) promoter caused lung inflammation, enlargement of distal airspaces, mucus

metaplasia, and airway fibrosis in the adult mouse (45). IL-33 is expressed in airway epithelial cells, and

promotes initiation of Th2 response. Interestingly, unlike other inducible cytokines, IL-33 is

constitutively expressed in normal human airway epithelial cells with a nuclear localization pattern (46).

IL-33 exerts its cytokine activity through a signaling receptor ST2, which is expressed predominantly,

but not exclusively on immune cells associated with allergic inflammation, including polarized Th2 cells

(47). IL-33 is a selective Th2 cell chemoattractant (48). Administration of IL-33 in vivo induced

expression of Th2 associated cytokines, IL-5 and IL-13 (49), inhibited Th1 cytokine IFN-γ (50).

Expression of IL-25 in airway epithelial cells is induced by allergen exposure (51). Conditional expression of IL-25 driven by CCSP promoter induced infiltration of macrophages and eosinophils, and mucus hyperproduction in vivo. With its receptor highly expressed in Th2 cells, IL-25 promotes Th2 cell differentiation in an IL-4 and STAT6 dependent manner (51). Neutralization of IL-25 by soluble IL-25 receptor decreased antigen-induced eosinophil and CD4+ T-cell recruitment into the airways (52).

Epithelial cell produced chemokines include CCL17 and CCL20. CCL17, chemokine (C-C motif) ligand 17, also termed thymus and activation regulated chemokine (TARC) is expressed by airway epithelium of both normal and asthmatic subjects. Its expression was induced by TNF-α treatment at a NF-kappa B dependent manner (53, 54). CCL17 is responsible for chemoattraction of

CCR4+ Th2 cells to mucosal sites (55). CCL20, a chemokine produced by epithelial cells in response to cytokine or HDM stimulation (56, 57), induces DCs migration to epithelium via the interaction with

CCR6 expressed on immature DCs (58).

In summary, the respiratory epithelial cells actively participate innate immune response via different ways, including mucociliary clearance, secreting anti-microbial peptides to remove and/or kill

16 Figure 1 Respiratory epithelial cells attract and activate DCs and Th2 cells via expression of cytokines and chemokines. Activation of TLRs in airway epithelium after allergen exposure triggers a NF-κB dependent cascade to secrete cytokines and chemokines that are responsible to attraction and activation of immature DCs and Th2 cell, including TSLP, IL- 25, IL33, CCL17 and CCL20. The crosstalk between epithelial cells and DCs, Th2 cells are required for initiation and maintain the allergic microorganisms, and releasing cytokines and chemokines to interact with DCs and Th2 cells, which leads to initiate adaptive immune response. Toll like receptors, especially TLR4, expressed in epithelial cells are essential for initiation of innate response by epithelial cells after LPS or HDM exposure (21).

Nuclear factor-κB (NF-κB), a transcription factor that is widely expressed in almost all cell types, is found to play a central role in mediating inflammation response following TLR activation. NF-κB is required for expression of cytokines and chemokines to recruit and activate DC and Th2 cells in respiratory epithelium (Figure 1), including TSLP (59), GM-CSF (60), TARC (61), CCL20 (62).

However, the molecular events that control expression of these innate host defense molecules following activation of TLRs were still largely unknown, especially those regulating epithelial specific cytokines, like TSLP. The current thesis was conducted to investigate a novel mechanism underlying regulation of

Th2 cell mediated innate immune response via the respiratory epithelial cell transcription factor Foxa2 in the developing lung. Foxa2 was found to be critical in suppression of Th2 inflammation and goblet cell differentiation during neonatal development, via regulation of expression of CCL17 and CCL20.

Foxa2 expression in epithelium is required for inhibition of DC and Th2 cell infiltration and activation in an IL-4Rα dependent manner. In the mature lung, Foxa2 mediates a transcription network that is associated with goblet cell differentiation and mucus production together with Spdef. Goblet cell

17 metaplasia is induced by inflammation caused by Th2 cells or neutrophils that are recruited into the lung in human chronic pulmonary diseases, and regulation of this process is still unclear in terms of cellular origin and molecular mechanism of goblet cell differentiation. The second part of this thesis demonstrated Th2 cytokines induced goblet cell differentiation via the transcription factor Spdef, which inhibited Foxa2 and transdifferentiated normal Clara cells into goblet cells.

Regulation of airway goblet cell differentiation in chronic pulmonary diseases

Goblet (mucus) cells are the major source of mucus that protects the respiratory epithelium from damage. The numbers of goblet cells lining conducting airways varies among species, interage, as well as environmental conditions. In the human lung goblet cells are found in tracheal, bronchial and bronchiolar regions. In contrast, goblet cells are normally absent in the conducting airways of adult mice

(see Table1 for comparison). Increased goblet cell numbers and excessive mucus secretion to the airway lumens contribute to the airflow restriction and bacterial infection, the common pathological features in chronic pulmonary diseases, including asthma, COPD and CF. Cellular and transcriptional mechanisms underlying goblet cell “hyperplasia” and mucin gene expression in the healthy and disease conditions have been intensively studied; however, the critical signaling pathways and transcriptional programs that regulate this process have yet to be identified.

The origin of goblet cells in the lung In normal adult lung, the airway epithelium turnovers slowly (63). In response to inhalation of allergens and pathogens, epithelium rapidly changes the composition of its cell types, with increased population of mucus producing goblet cells, often described as “goblet cell hyperplasia”. The cellular mechanism underlying this process in the airway epithelium is poorly understood. It was unclear whether the increased number of goblet cells was the result of differentiation of progenitor cells, or by proliferation of existing goblet cells. To distinguish airway

18 epithelial cell proliferation verse differentiation, mitosis was monitored by [3H] thymidine incorporation

in epithelium after exposure to pathogen in rat (64). Mucin gene expression and goblet cell numbers

were not associated with mitosis. Consistent with this finding, using colchicine to block metaphase, did

not inhibit production of goblet cell in rat after endotoxin treatment [reviewed in (65)]. These evidence

suggested that goblet cells appear to arise from pre-existing progenitor cells, perhaps including basal,

Clara and ciliated cells, rather than from division of goblet cells (66). The plasticity of airway

epithelium has been studied in vivo and in vitro for its potential of differentiation into goblet cells. Basal

cells expressing Trp63 (p63), cytokeratin 5 and 14 (Krt5/14) (67) comprise about 33% and 10% of total

epithelial population in tracheal regions of human and mouse, respectively. Despite the slow turnover

rate of the respiratory epithelium under normal adult, the multi-potentiality and self-renewing ability of

basal cells was observed after injury (68, 69). Evidence supporting basal-to-goblet cell

transdifferentiation was observed using rat tracheal as a model system, supporting the concept that basal

cells served as stem or progenitor cell in the lung (70). Non-ciliated secretory (Clara) cells, express

secretoglobin 1a1 (Scgb1a1, also termed CCSP, CC10) line upper and lower conducting airway of

mouse, but are absent in human airways except in terminal bronchioles. In the postnatal period, Clara

cells self-renew and give rise to ciliated cells in bronchiole, as revealed by the lineage tracing studies in mouse (71). The transition of Clara-to-goblet cell during allergen exposure has been studied by

immunohistochemistry, electromicroscopy (72-74). Selective deletion of the gene encoding IL-4

receptor α subunit (IL-4Rα) in Clara cells inhibited goblet cell differentiation after ovalbumin

sensitization, suggesting that activated IL-4Rα signaling in Clara cells was required for them to

differentiate into goblet cells (75). Differentiation from ciliated to goblet cell was reported in mice

infected with Sendai virus in the respiratory tract (76). Tyner and colleagues found that virus infection

19 caused activation of epidermal growth factor receptor (EGFR), one of the critical signaling pathways that induced goblet cell differentiation.

Present evidence supports the concept “goblet cell metaplasia”, rather than a proliferative process. So, the term of “goblet cell hyperplasia”, which has been widely used in the field, may be a misleading. Precise clarification of the cellular origins of goblet cells in the lung using cellular lineage tracing system is required to identify the mechanism underlying goblet cell metaplasia or hyperplasia. In the current thesis, a Clara cell lineage tracing transgenic mouse line was used to identify the lineage relationships between Clara cells and goblet cells. Clara-to-goblet cell differentiation occurred in the absence of proliferation.

The critical signaling pathways that regulate goblet cell differentiation in the lung

Differentiation of goblet cells in the conducting airways is regulated by a variety of stimuli, including neuronal control (77, 78), chemical irritants (79), reactive oxygen species (80), as well as numerous cytokines. Th2 cells infiltrating into the lung produce Th2 cytokines (IL-4 and IL-13) that play the dominant roles in inducing goblet cell metaplasia and airway hyperresponsiveness in allergic asthma. In

COPD, goblet cell metaplasia is induced through a different mechanism, which neutrophils are recruited into the lung to activate EGFR dependent pathway that induces goblet cell metaplasia (81).

Role of Th2 cytokines in regulating goblet cell differentiation The major Th2 cytokines including IL-4, IL-13 and IL-9 are well known for their ability of inducing goblet cell differentiation as well as mucin gene expression both in vivo and in vitro. Among these, IL-4 and IL-13 were the most intensively studied and well understood because of their roles in asthma pathogenesis. IL-4 binds to two distinct receptor complexes (type I and type II receptor), whereas IL-13 only binds to one receptor complex (type II receptor). The type I receptor is composed of two subunits, IL-4Rα chain and the γc chain. The type II receptor is a heterodimer of IL-4Rα and IL-13Rα1 (82-84). Due to the selective

20 expression patterns of these subunits, differential roles of IL-4 and IL-13 contribute pathogenesis of asthma were identified using transgenic mice. IL-4 is primarily involved in promoting the differentiation and proliferation of Th2 cells and the synthesis of immunoglobulin E (IgE), whereas IL-13 has a critical role in mediating airway hyperresponsiveness, goblet cell metaplasia and mucus hypersecretion, the elements that are most closely linked to clinical manifestations of asthma (82, 85, 86). Selective loss of

IL-4Rα expression in Clara cells inhibited goblet cell differentiation after allergen exposure, suggesting that the pivotal role of IL-4 receptor mediated signaling in regulating differentiation of goblet cells in the lung (75). Activation of IL-4 receptor by IL-4 or IL-13 initiates the transcriptional programs that control the differentiation of T cell, B cell and epithelial cells. This differentiation process is dependent on phosphorylation of signal transducer and activator of transcription factor 6 (STAT6). Mice lacking Stat6 in resident lung cells, but not in T cells, were protected from eosinophilia, goblet cell metaplasia and airway hyperresposiveness (AHR) after allergen exposure (87). Reconstitution of Stat6 expression only in respiratory epithelial cells in Stat6 -/- mice was sufficient to induce AHR and goblet cell metaplasia in the absence of inflammation after IL-13 treatment (88). This direct effect of activation of Stat6 in epithelial cells by IL-13 leads to differentiation of goblet cells in the conducting airways. Activation of p38 MAPK (mitogen-activated protein kinases) is Stat6 dependent after IL-13 stimulation, and is required for Muc5ac expression and goblet cell differentiation in primary mouse tracheal epithelial cells

(89).

Goblet cell differentiation and mucin gene expression (MUC2 and MUC5AC) are induced by IL-

9 in vivo and in vitro (90), through a mechanism independent of IL-13 (91). The strong correlation between expression of IL-9 and calcium-activated chloride channel 1 (hCLCA1), the ortholog of which is calcium-activated chloride channel 3 in mouse (mClca3, also termed gob5), has been reported

(92-94), suggesting that IL-9 may regulate expression of this particular the membrane ion channel that

21 required for goblet cell differentiation (95, 96). Indirectly, IL-6 regulates goblet cell differentiation via

its effect on IL-13 production in vivo. Loss of IL-6 reduced IL-13 secretion in BALF and decreased

goblet cell numbers in airway after Aspergillus fumigates exposure (97).

Role of activation of EGFR in inducing goblet cell differentiation Activation of EGFR by its

ligand EGF or transforming growth factor alpha (TGF-α) was sufficient to induce mucin gene

(MUC5AC) expression and goblet cell metaplasia both in vivo and in vitro (98). Treatment with a

selective EGFR tyrosine kinase inhibitor prevented IL-13-induced goblet cell metaplasia in a dose

dependent manner in rat (99). The mechanisms underlying EGFR signaling pathway promoting goblet

cell differentiation was proposed by Tyner and colleagues (76). They suggested that anti-apoptosis

pathways were activated by EGFR-PI3K-Akt cascade. Anti-apoptosis proteins are required for

regulation of goblet cell differentiation in various model systems. Bcl-2, the anti-apoptotic protein, was

induced in metaplastic mucous cells (100-102). Reduced expression of Bax, the pro-apoptotic protein,

was observed in mucus cells of asthmatic patients (103). Activation of EGFR pathway in respiratory

epithelium is associated with pathogenesis of chronic cigarette smoking induced COPD (cigarette

smoke-derived reactive oxygen species activate EGFR via tyrosine phosphorylation) (104). To form the

active form of EGFR ligands, the pro-EGFR ligands need being cleavaged by matrix metalloproteinases

(MMPs) and a disintegrin and metalloproteinase domain proteins (ADAMs) that are necessary for

inducing mucus production mediated via EGFR dependent pathway. MMP9, MMP14 and ADAM17

were shown to be critical in activating EGFR mediated mucin gene expression and goblet cell

metaplasia after exposure to acrolein, the component of cigarette smoke, in vitro and in vivo (105, 106).

22 Figure 2 Hypothesized model of goblet cell differentiation: activation of EGFR and IL-13 are required for transdifferentiation of ciliated cell to goblet cell. In response to viral infection, EGFR is activated in ciliated cells, leading to inhibition of apoptosis via PI3K/Akt signaling pathway. The survival ciliated cells response to the Th2 cytokine IL-13, which is required for expression of goblet cell specific genes including MUC5AC at a STAT6 dependent manner to produces mucus in the airway lumen.
In summary, two major signaling pathways have been proposed in regulating goblet cell differentiation are IL-13 and activation of EGFR. Production of IL-13 by Th2 cells and activation of

EGFR by various ligands are thought to be the major causes of induction of goblet cell metaplasia in asthma patients and chronic smokers, respectively. IL-13 and EGFR pathways target to different subset of genes, IL-13 regulates genes associated with allergic asthma, including FOXA3, CCL26 and

SERPINB10, while EGFR pathways regulate genes responsible for cell metabolism, survival and differentiation (107). A model of airway goblet cell differentiation was proposed by Tyner (76), Cohn

(108) and their colleagues, in which they suggested that both IL-13 and EGFR signaling were required for this coordinated, 2-step process (Figure 2). Specifically, EGFR activation inhibits epithelial cell apoptosis, allowing IL-13 to stimulate the differentiation of these cells into goblet cells, which secrete mucus. In contrast, in the present thesis, a lineage tracing transgenic mouse line was used, in which

Clara cells were permanently label with β-galactosidase and identified as the cellular progenitors of goblet cells following pulmonary allergen exposure. Th2 cytokine IL-13 induced Spdef and inhibited

Foxa2 in an IL-4Rα/Stat6 axis dependent manner to promote goblet cell differentiation from present

Clara cell. Blocking EGFR activation using pharmacological inhibitors was not sufficient to inhibit

Spdef expression or goblet cell differentiation in allergic asthma model, suggesting that neither Spdef

23 expression nor goblet cell differentiation was EGFR dependent following pulmonary allergen (house

dust mite) exposure.

Transcriptional control of goblet cell differentiation in the lung Compared to signaling

pathways, the transcriptional programs that control goblet cell differentiation in the airways are less

known. The early knowledge of transcription factors in goblet cells was learned from those mediating in

IL-4 and IL-13 signaling pathways, such as STAT6. Activation of STAT6 is dependent on

phosphorylation of the cytoplasmic domain of IL-4R after ligation of IL-13 or IL-4 with the receptor

complex. After recruited to the IL-4Rα through its SH2 domain, STAT6 becomes tyrosyl

phosphorylated at its C-terminus through the action of Janus family of protein kinases (Jaks), leading to

its dimerization and translocation to the nucleus, where it activates the transcription of IL-4- and IL-13-

responsive genes (109). Expression of Stat6 in epithelium is required for IL-13 or allergen induced

goblet cell differentiation (87, 88, 110). Stat6 is also required for induction of Spdef and inhibition of

Foxa2 in the goblet cells following Th2 cytokine sensitization (111, 112).

Role of Foxa2 in regulating goblet cell differentiation The roles of a number of transcription

factors that mediate goblet cell differentiation were recently discovered by our laboratory (Foxa2, Spdef

and Sox2) and others. Foxa2 is the forkhead box transcription factor normally expressed in respiratory

epithelial cells lining conducting airway and peripheral type II cells, and is required for normal lung

maturation, surfactant protein and lipid synthesis during lung development (17). Postnatally, mice

lacking Foxa2 in respiratory epithelium (SFTPC/Cre/Foxa2Δ/Δ, Foxa2 allele was deleted during E6.5-

E12.5) developed goblet cell metaplasia and inflammation, suggesting its critical role in regulating homeostasis and immune response in the neonatal lung. Foxa2 expression was lost in the airway goblet cells after IL-13 or allergen exposure in a Stat6 dependent manner in mouse models. The reverse correlation between Foxa2 and mucin gene expression was revealed by inhibition of MUC5AC promoter

24 activity following transfection of Foxa2 expression plasmid in H292 cells (111). Loss of FOXA2 was

also observed in the goblet cells of lung tissues from human patients of CF, chronic pulmonary infection

and bronchiectasis (111). Recent studies showed that FOXA2 was a common target that responded to

IL-13 stimulation and EGFR activation in normal human bronchial epithelial cells in vitro (107). Foxa2

expression is lost or reduced in the goblet cells of lung tissues from asthma (113), CF (111) and chronic

cigarette smoking patients (114, 115), and Foxa2 expression is negatively correlated to mucin gene

expression (111, 113). It is rational to design pharmacological interventions to maintain normal levels of

Foxa2 expression in respiratory epithelial cell as a therapeutic tool to treat mucus hypersecretion

associated pulmonary diseases. However, transcriptional mechanism controlling Foxa2 expression in

respiratory epithelium following IL-13 exposure and activation of EGFR was unknown. Mechanism

underlying loss of Foxa2 expression in goblet cell was not clear. In the current thesis, expression of

Foxa2 mRNA and protein was found to be inhibited by Spdef, the transcription factor that was sufficient

to promote goblet cell differentiation independent of allergen or inflammation. Forced expression of

Foxa2 in Clara cells, the progenitors of goblet cells in the airways, inhibited goblet cell differentiation

and Spdef expression following allergen exposure, suggesting loss of Foxa2 was required for goblet cell

differentiation in asthma model. The current study of the role of Foxa2 in respiratory epithelium

provides the molecular mechanism underlying mucus hypersecretion, associated with the pathogenesis

of human asthma, CF and COPD.

Role of SPDEF in promoting goblet cell metaplasia in the lung SPDEF, also termed PDEF

(prostate-derived Ets family transcription factor) was initially identified in prostate epithelium (116).

Later on, its expression was also found in human breast (117) and non-small cell lung cancer (NSCLC) primary tumor (118). Dramatic induction of Spdef mRNA (15-20 folds, http://research.cchmc.org/pbge) in the lung was found in the mouse had mutation of phosphorylation sites of Nkx2-1 (also termed as

25 TTF-1) protein (112), the master transcription factor regulated lung development (119). It is still unclear

the mechanism of induction of Spdef mRNA in response to mutagenesis of TTF-1 in the lung. By

performing in situ hybridization, we identified that Spdef was selectively expressed in the epithelium-

enriched tissues, including colon, prostate, seminal vesicle, oviduct and lung. Spdef mRNA was

normally expressed at submucosal glands of tracheal-laryngeal regions and at low levels in a subset of

surface epithelium lining tracheal-bronchial region in normal adult mice (Figure 3). Conditional

expression of Spdef in Clara cells promotes goblet cell differentiation in adult mice, in the absence of

inflammation or Th2 cytokines (IL-4, IL-13) expression changes. In response to allergen or IL-13

intrapulmonary exposure, endogenous Spdef expression (both mRNA and protein) is dramatically

induced in the goblet cell that extended to bronchiolar epithelium in a Stat6 dependent manner (112).

The previous studies strongly suggested that Spdef was the transcription factor that regulated goblet cell

differentiation in Th2 inflammation (112). However, the molecular mechanism underlying Spdef-driven

Clara-to-goblet cell differentiation and the requirement of Spdef in goblet cell differentiation were

unclear. In the current thesis, Spdef expression was found in goblet cells of lung tissues of the patients

with asthma, CF and history of chronic smoking. Spdef regulated a transcription network that promoted

goblet cell differentiation by inducing expression of mucin genes and genes responsible for mucin

biosynthesis, while inhibiting expression of TTF-1 and Foxa2, that were absent in the goblet cells.

Deletion of Spdef (germline deletion of Spdef) caused absence of goblet cell differentiation in submucosal glands and surface epithelium after allergen exposure in Spdef -/- mice, indicating it was

required for goblet cell differentiation in vivo. SPDEF was expressed in goblet cells of in airways of

various chronic pulmonary diseases in human, and was associated with MUC5AC expression, the major

gel-form mucin excessively produced in the airway diseases, and induced expression of FOXA3, AGR2,

ClCA1 that regulate goblet cell metaplasia. The current study highlights that Spdef is both sufficient and

26 required for goblet cell differentiation in the respiratory epithelium, and opens a new opportunity for

treatment of mucus hypersecretion associated diseases by pharmaceutical interventions.

Role of epithelial ion channels in regulating goblet cell differentiation In addition to cytokine signaling pathways and transcriptional factors, the cell membrane receptors and ion channels are also critical in promoting mucus production. The epithelial sodium channel (ENaC, also termed as sodium channel non-neuronal 1 (SCNN1)) is a membrane-bound ion-channel that is permeable for protons, and especially the Na+-ions. It is a heteromultimeric protein composed of three subunits (α, β

and γ) that are encoded by the Scnn1a, Scnn1b and Scnn1c genes, respectively. Transgenic mice

overexpressed the epithelial Na+ channel β subunit (βENaC protein, Scnn1b gene) driven by the CCSP

promoter resulted in epithelial Na+ hyperabsorption, airway surface liquid (ASL) dehydration, impaired mucus clearance, airway inflammation, and early postnatal mortality. And in surviving mice, mucus plugging and mucous secretory cell metaplasia progressively extend into the intrapulmonary bronchi, the features of which recapitulate cystic fibrosis and other human airway diseases associated with relative

dehydration of airway surfaces including chronic bronchitis and COPD (120, 121). Th2 cytokines (IL-4 and IL-13) and activation of EGFR (TGF-α and EGF) inhibited ENaC expression and its mediated Na+

absorption (122, 123), suggesting potential role of ENac in contributing to goblet cell differentiation and

mucociliary clearance in the lung. In current study, ENac protein subunits (Scnn1b and Scnn1g) were

found to be inhibited by Spdef in current study, indicating the Spdef transcriptional regulated expression

of ion channel that contributed in goblet cell differentiation.

Summary

27 Respiratory epithelial cells are positioned at the interface between host and environment, and

serve as the front-line host defense machinery to remove inhaled microorganisms, particles and toxicants.

In response to pathogen or allergen exposure, they actively participate innate immune response by

producing anti-microbial peptides. They secrete cytokines and chemokines in a TLR4-NFκB dependent

manner to recruit and activate DCs and Th2 cells leading to mount adaptive immune response. The

molecular mechanism underlying regulation of innate immune system development in neonatal lung,

however, was still largely unknown. The current thesis was conducted to test the hypothesis that

respiratory epithelial cells play a critical role in programming the Th2 cell-mediated innate immunity in

the developing lung via transcription factor Foxa2.

Previous study showed that loss of Foxa2 induced goblet cell differentiation and Spdef

expression in the neonatal lung. Spdef is the transcription factor that is sufficient to promote goblet cell

differentiation in vivo. Goblet cell metaplasia and mucus hyperproduction are the common features of chronic pulmonary diseases including asthma, CF and COPD. In allergic asthma, Th2 cytokines including IL-13 and IL-4 are known for their ability to induce goblet cell differentiation. In COPD, infiltrated neutrophils activate EGFR dependent signaling to induce mucus production. Spdef expression is induced in goblet cells in an IL-13/IL-4R/STAT6 dependent manner in allergic asthma models, and this same signaling cascade inhibits Foxa2 expression in goblet cells, indicating that Spdef and Foxa2 are involved in a transcriptional network regulating goblet cell differentiation. The present thesis tested the hypothesis that Spdef and Foxa2 mediated a transcriptional network that was associated with goblet cell differentiation and mucus production in the lung.

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35 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§ &

Jeffrey A. Whitsett*

*Perinatal Institute, Division of Neonatology, Perinatal and Pulmonary Biology, §Division of Immunobiology,

Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati,

OH, USA. †(Present address) West China Developmental & Stem Cell Institute, Sichuan University Second

University Hospital, Chengdu, P.R. China, ‡West China Hospital of Sichuan University, Chengdu, P.R. China

There is no conflict of interest.

Correspondence:

Jeffrey A. Whitsett, M.D., Cincinnati Children’s Hospital Medical Center, Division of Pulmonary Biology,

MLC7029, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, Phone: 513-803-2790, FAX: 513-636-7868,

Email: [email protected]

Supporting Grants:

This work was supported by ALA (H.W.), the National Institutes of Health HL095580, HL090156 (J.A.W. and

Y.X.) and NHLBI grant AR47363 that supported the flow cytometry core at Cincinnati Children’s Hospital

Medical Center.

‡ manuscript accepted for publication in Journal of Immunology 2010

36 Abstract

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. Herein, we

show that Foxa2, expressed selectively in the respiratory epithelium, plays a critical role in regulating genetic

programs influencing T helper 2 (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 (mDCs) 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 interleukin-13 (IL-13), IL-4, eotaxins,

thymus and activation-regulated chemokine (Tarc), Il33, Ccl20, and SAM pointed domain containing Ets

transcription factor (Spdef). Pulmonary inflammation and goblet cell differentiation were abrogated by treatment

of neonatal Foxa2Δ/Δ mice with monoclonal antibody against IL-4 receptor α subunit (IL-4Rα). 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.

37 Introduction

After 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-4 receptor/STAT6 (1), Toll- like receptor (TLR)/NF-κB (2), EGFR/MAP kinase (3, 4) pathways. Allergen induced 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, antibody production, goblet cell metaplasia, airway remodeling, and hyper-reactivity (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-4Rα chain, the functional receptor subunit of both the type I receptor, which is a heterodimer of

IL-4Rα and the γc chain, and the type II receptor, which is a heterodimer of IL-4Rα and IL-13Rα1. IL-13 does not bind to IL-4Rα directly but binds to IL-13Rα1 and can only activate the type II receptor. As the common subunit of receptor complexes for both IL-4 and IL-13, IL-4Rα is required for mediating signal transduction of these two cytokines (1, 6, 7). Targeting IL-4Rα function by antagonist 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-4Rα 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 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

38 and caused spontaneous inflammation and mucus 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 Spdef and Foxa3 indicating that these transcription factors interact in a network influencing airway epithelial 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.

Herein, we demonstrate that respiratory epithelial cell specific 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 monoclonal antibody to block IL-4 receptor signaling in neonatal mice. Deletion of Foxa2 induced expression of a network of genes influencing or associated with Th2-cell mediated inflammation, and dendritic cell 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-4Rα signaling.

39 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. Mice were housed in a pathogen-free barrier

facility in humidity and temperature controlled rooms on a 12:12 hour light: dark cycle and were allowed food

LoxP/LoxP and water ad libitum. SFTPC-rtTA/tetO7CMV-Cre/Foxa2 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 E6.5 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 5´UTR and 3´UTR was isolated from pRc/CMV-rFoxa2 (14) at EcoR I sites and cloning into Bam HI site of pOtet7-ERES-EGFP vector (21) (that 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: 5´-AGC AAA GAC CCC AAC GAG AAG C-3´ and 5´-CAA ACA ACA

GAT GGC TGG CAA C-3´.

Histology and Immunohistochemistry

Immunohistochemistry was performed on 5 µm 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 Inc., Santa Cruz, CA), mouse monoclonal antibody against Muc5ac (1:500, ab3649, Abcam) (18).

Sections were processed with antigen retrieval using heating in citrate buffer. Anti-mouse IL-4Rα monoclonal

antibody (Lot: 9382-25B, 11/12/03, AS/CH) was kindly provided by Amgen Inc (Thousand Oaks, CA).

Anti-IL-4 Receptor Treatment

40 Neonatal SFTPC/Foxa2Δ/Δ pups and control littermates were injected intraperitoneally on PN2 and PN9

with either anti-IL-4Rα antibody (50 µg/g of body weight) or equal volume of sterile saline. On PN15, mice were anesthetized and the lungs lavaged five times with 0.3 ml of saline, and the cells were counted from BALF.

Cytospins of BALF cells (5x104) were stained with Diff-Quik kit (Cat. 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 Ovalbumin Sensitization

Mice were sensitized to ovalbumin by systemic and pulmonary administration using protocols previously

described (18). Expression of Foxa2 in the Scgb1a1/Foxa2-IE mice was induced at 6 weeks of age by provision

of doxycycline 24 hours before mice were intranasally sensitized with ovalbumin (Supplementary Figure 8). The

control mice were the single transgenic littermates (Scgb1a1-rtTA) that received the same doxycycline and ovalbumin treatments. Doxycycline was continued until the time of sacrifice. mRNA Microarray Analysis

Lung cRNA was hybridized to the murine genome MOE430 chips (consisting of ≈ 45000 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 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 from R/Bioconductor package. Differentially expressed genes were selected

with the threshold of p-value < 0.01, fold change ≥ 1.5 and a minimum of 2 “Present Calls” in 3 samples with

relatively higher expression. Gene Ontology 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 after deletion of FOXA2 and all genes in MOE430

41 mouse genome. Gene sets associated with known pathways and disease states were identified from KEGG

(http://www.genome.ad.jp/kegg/), GenMAPP (http://www.genmapp.org/) and GEArrays

(http://www.sobiosciences.com/microarrays.php/). A pathway was considered to be over-represented when a

probability p value ≤ 0.01 and gene hits ≥ 5. Potential protein/protein or protein/DNA interactions were identified using Ingenuity Pathway Analysis (IPA, Ingenuity). IPA 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 QRT-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, Cat.VS0413, Sartorius, Bohemia, NY) and subjected to

ELISA to determine the concentrations cytokines using the mouse IL4, IL5, IL13 and IFN-γ ELISA kits from

eBioscience, San Diego, CA (Cat. # 88-7044, 88-7054, 88-7137, 88-7104 and 88-7384, respectively). The Ccl17

ELISA kit was purchased from R&D System (MCC170, Minneapolis, MN). The ELISA was performed with n of

6 to 8 mice of each genotype (Foxa2Δ/Δ mice and their littermate controls) at PN15. Whole lung total RNA was

purified by RNeasy Mini Kit (Cat. 74104, Qiagen, Valencia, CA), and reverse transcribed into cDNA by Verso

cDNA kit (Cat. AB-1453/A, Thermo Fisher Scientific, Waltham, MA). Quantitative RT-PCR was performed on

7300 realtime-PCR system (Applied Biosystem, Forster 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-γ (Mm00801778_m1) and normalized to endogenous 18s

42 ribosomal RNA for control (probe part number: 4352930E). The quantitative RT-PCR was performed with n of 3

mice for each genotype at postnatal day 11 and 15 (PN11 and PN15).

Flow Cytometry

Lung cell suspensions were incubated with anti-CD16/32 (clone 2.4G2) for 30 minutes and then staining

reactions were performed at 4°C. Myeloid DCs (CD11c+, CD11b+, Gr1neg, CD317neg) and plasmacytoid DCs

(CD11clow, CD11bneg, Gr1+, 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 co-stimulatory 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 µg/ml). Cytokine secretion was

blocked with a combination of Brefeldin A and Monensin (eBioscience) for 4 hours. Cells were stained with PE-

Cy7 conjugated anti-CD4 (RM4-5) and PE-conjugated anti IL-13 (eBio13A). All mAbs were purchased from

eBioscience. Data were acquired with an LSRII flow cytometer (BD Biosciences) 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 (Treestar Inc., Ashland, OR).

Plasmid and Luciferase Reporter Assay

The control plasmid used in luciferase reporter assay expressing EGFP was made by isolation of IRES-

EGFP fragment from pIRES2-EGFP (Cat.6029-1, Clontech, Mountain View, CA) by BamHI and Not I sites and

subcloned into BamHI site of p3XFLAG-Myc-CMV plasmid (Cat. 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: 5’-ATG AAT TCA ATG CTG GGA GCC GTG AAG-3’ and 5’- ATG AAT TCG CGG ACG

AGT TCA TAA TAGG-3’ and inserting into the EcoR I site of the control vector. AccuPrime Taq DNA

Polymerase (Cat.12339-016, Invitrogen) was used to amplification of DNA fragments. The mouse Ccl17-pGL3

plasmid was cloned by amplification of 2 kb promoter region from C57/B6 mouse genomic DNA using primers:

43 5’-GGA CCT GAA ATA GTC AGC ATCC-3’ and 5’-CTG AGG TGA AGG TCT TCA TGGG-3’ and cloned into pGL3-basic plasmid (Cat. E1751, Promega). The luciferase assay was performed by transfection of MLE15 cells with mCcl17-pGL3 plasmid along with either control plasmid expressing EGFP or Foxa2-EGFP at 1:1 ratio using the transfection reagent lipofectamine 2000 (Cat. 11668-019). CMV-Gal plasmid was cotransfected to serve as an internal control and the luciferase activity was normalized to -galactosidase activity after 24 hours of transfection.

Statistics

Statistical differences in cell counts and concentrations of proteins assessed by ELISA were determined using Student’s t test (2-tailed and unpaired). The statistic method applied to analyze quantitative RT-PCR was the Mood’s Median test. Difference between two groups was considered significant when the p value was less than 0.05 for all tests.

44 Results

Conditional deletion of Foxa2 caused Th2-cell mediated pulmonary inflammation. To delete Foxa2

LoxP/LoxP expression in respiratory epithelium, dams of SFTPC-rtTA, tetO7-CMV-Cre, Foxa2 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 two weeks of life (Figure 1A). On

postnatal day 15 (PN15), pulmonary inflammation was seen histologically and supported by quantitation of

inflammatory cells in bronchiolar-alveolar lavage fluid (BALF). Numbers of both eosinophils lymphocytes and

macrophages were increased in BALF from Foxa2Δ/Δ mice (Figure 1B). Deletion of Foxa2 was associated with

increased expression of Th2 cytokines and chemokines, including IL-13, IL-4, IL-5 and Ccl17 (Tarc) as measured

in BALF (Figure 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 (MFI) of IL-13, IL-17a and abundance of Th2

and Th17 cell populations in the lungs of Foxa2Δ/Δ mice. Expression of IL-6, a cytokine required for Th17 cell differentiation (23, 24), was significantly induced. In contrast, Ifn-γ mRNA was not altered (Supplementary

Figure 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.

Inhibition of IL-4R mediated signaling inhibited eosinophilic inflammation and goblet cell differentiation

induced by Foxa2 deletion. A key pathway that regulates allergic inflammation and tissue remodeling in the

airways involves the Th2 cytokines IL-4 and IL-13 and the activation of the IL-4 receptor α subunit (IL-4Rα) (25).

IL-4 is primarily involved in promoting the differentiation and proliferation of Th2 cells and the synthesis of

immunoglobulin E (IgE), whereas IL-13 has a critical role in mediating airway hyperresponsiveness (AHR),

goblet cell metaplasia and mucus hypersecretion, the elements of asthma most closely linked to the manifestations

of the disease. Blocking IL-4 receptor signaling pathway with neutralizing antibody against IL-4Rα or

mutagenesis of IL-4Rα was sufficient to inhibit responses to both IL-4 and IL-13 cytokines in vivo and in vitro

(26-28). Specifically, loss of IL-4Rα in Clara cells, inhibited mucus production after ovalbumin intrapulmonary

45 exposure in adult mice (29), and loss of IL-4Rα 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-4Rα monoclonal antibody at PN2 and PN9. Eosinophilic inflammation was significantly inhibited by anti-IL-4Rα antibody, as shown by the reduction of inflammatory cells and eosinophils in BALF (Figure 2A). Consistent with inhibition of Th2 inflammation, goblet cell metaplasia and mucus hyperproduction in Foxa2Δ/Δ mice were inhibited by the IL-4Rα antibody. Mucus cell

markers, including Alcian blue, Spdef, Foxa3, and Muc5ac staining were also inhibited by neutralizing antibody

against IL-4Rα (Figure 2B), demonstrating that the goblet cell metaplasia was dependent upon activated IL-4Rα

signaling caused by deletion of Foxa2.

Loss of Foxa2 in respiratory epithelium enhances myeloid dendritic cell recruitment, and activation. Th2-

cell mediated inflammation is dependent on activation and migration of pulmonary dendritic cells (DCs) to the

respiratory epithelium (31). As professional antigen presenting cells, dendritic cells 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. Myeloid DCs (mDCs, defined as CD11c+, CD11b+, Gr1neg, CD317neg cells) promote robust T cell activation and efficiently promote allergen-induced airway hyperresponsiveness (AHR)

(32). In contrast, plasmacytoid DCs (pDCs, defined as CD11clow, CD11bneg, Gr1+, 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 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

(Figure 3A). While 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

46 associated co-stimulatory molecule expression on mDCs from control and Foxa2Δ/Δ mice demonstrated significantly elevated frequencies of mDCs expressing B7-DC (Figure 3B), B7-H1 (Figure 3C), CD86 (Figure

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 (Supplementary Figure 2).

Interestingly, while pDCs were also generally more activated in Foxa2Δ/Δ mice than in control mice, by PN15, a

time when there is robust activation of T cell cytokine production, the levels of pDC activation had decreased and

were indistinguishable from controls (Figure 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 (Supplementary Figure 3 and Figure 4B). Mechanisms underlying the recruitment and activation of dendritic cells in the lung are not well established at present. The IL-7-like cytokine TSLP,

produced by epithelial cells, is known to influence DCs expressing OX40 ligand (OX40L), causing differentiation

of naïve 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 (Supplementary Figure 4), suggesting a potential mechanism by which dendritic cells are recruited and activated in the lungs of Foxa2Δ/Δ mice. While Tslp mRNA was not increased in whole lung at

PN11 or PN15 (data not shown), it remains possible that time dependent or focal changes in its expression would

not be detected in the present 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 target s 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

gene ontology were analyzed. Deletion of Foxa2 significantly influenced the expression of 665 mRNAs. Among

these, 516 of 665 mRNAs were induced and 148 were reduced in response to deletion of Foxa2 in the respiratory

47 epithelium (Figure 4A). The complete microarray dataset has been submitted to Gene Expression Omnibus;

Accession no. GSE19204 (http://www.ncbi.nlm.nih.gov/geo/query/). Gene Ontology (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 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 (Supplementary 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 &

Antigen 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 over-represented canonical pathway in mRNAs decreased after

deletion of Foxa2 (Supplementary Table II). Expression of mRNAs of major Th2 cytokines and chemokines in

the whole lung was confirmed by quantitative RT-PCR. Consistent with the severe eosinophilic inflammation

and Th2 lymphocytes infiltration seen in Foxa2Δ/Δ mice, Il4, Il5, Il13 mRNAs in the whole lung were significantly

increased; while expression of Th1 cytokine Ifn-γ was not changed (Figure 4B and Supplementary Figure 1).

Epithelial cells lining respiratory and gastrointestinal tract play pivotal role of initiation, regulation and resolution of innate and adaptive immune response by expressing a wide range of immune response genes including co- stimulatory molecules, chemokines, cytokines and prostaglandins (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 (Figure 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 Mouse Lung Epithelial (MLE-15) cells in vitro. Foxa2 inhibited the Tarc promoter by approximately 40% (Supplementary Figure 5).

48 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 (Supplementary Figure 6). Double transgenic mice Scgb1a1- rtTA/tetO7-Foxa2-IE (Scgb1a1/Foxa2-IE) were treated with doxycycline and the induction of Foxa2 and EGFP expression was confirmed by immunohistochemistry and fluorescence microscopy (Figure 5A and Supplementary

Figure 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 (Supplementary Figure 7). Conditional expression of

Foxa2 in the respiratory epithelium in adult mice inhibited goblet cell differentiation (Supplementary Figure 8 and

Figure 5B). However, expression of Foxa2 in respiratory epithelial cells of the mature lung prior to ovalbumin 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, Ifn-γ concentrations were similar in BALF from Foxa2 expressing and control mice after ovalbumin exposure (Supplementary Figure 9A, B). 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.

49 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 lung, and

associated with increased Th2 cytokines (IL-4, IL-5 and IL-13) and Ccl17 (Tarc) production, 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. 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 after deletion 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-4 receptor signaling pathway, e.g. IL-4, IL-13, and IL-4Rα 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 hyper-reactivity (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-4Rα signaling substantially

inhibited pulmonary eosinophilic inflammation and goblet cell metaplasia after deletion of Foxa2, supports the

important role of Foxa2 in inhibiting development of Th2-mediated 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, 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 (46). mRNAs associated with eosinophilic inflammation were induced in lungs of

50 Foxa2Δ/Δ mice, including Ccl11, Ccl24, Epx, Itgb2, Kng1, Lgals3, Ltc4s, Alox5, Arg1, Il1rn and Rag1. Among

these, eosinophil specific chemokines Ccl11 and Ccl24 were increased 7 and 12 fold, respectively. Epx mRNA

(eosinophil peroxidase), a heme-containing glycoprotein that may contribute to the pathogenesis of epithelial

damage and bronchial hyperreactivity in human asthma (47), was markedly increased in Foxa2Δ/Δ mice.

Eosinophil major basic protein (Prg2, 11 fold) and eosinophil associated ribonuclease (Ear11, 90 fold) mRNAs

were also dramatically increased.

Th2 associated inflammatory mediators, including acidic chitinase (Chia), chitinase 3-like 1 (Chi3l1),

chitinase 3-like 3 (Chi3l3) and chitinase 3-like 4 (Chi3l4) mRNAs were increased in Foxa2Δ/Δ mice. Chitinases

and chitinase-like proteins are believed to play a key role in the innate immunity to parasites and other infectious

agents, may play an important role in the pathogenesis of allergy and/or asthma and regulation of eosinophilia and

eotaxin induction (48-50). CHIA is induced via a Th2 specific, IL13-mediated pathway in lung epithelial cells

and macrophages and is increased in lungs of asthmatics (48). CHIA stimulates chemokine production by

pulmonary epithelial cells (49).

Deletion of Foxa2 from lung epithelium disrupted Th1-Th2 balance inducing a large number of Th2

cytokines and associated signaling pathways. Il6 mRNA, a cytokine regulating Th1, Th2, and Th17 cell

differentiation (51, 52) was increased 4.6 fold in Foxa2Δ/Δ mice. Recent studies demonstrated that Foxa2 bound to

the Il6 promoter and suppressed Il6 expression in liver (53). Since multiple Th1-associated genes are altered in the Foxa2Δ/Δ mice, it is presently unclear which play the dominant roles in the observed phenotype. Of genes expressed in the respiratory epithelium, both Il33 and the chemokine Ccl17 were significantly induced after deletion of Foxa2. IL-33 and CCL17 both promote Th2 cytokine responses (38, 39) and are expressed in respiratory epithelial cells. CCL17 attract Th2 lymphocytes to mucosal sites (54) and is increased in the bronchial epithelium of asthmatic patients (55). The present finding, that Tarc promoter activity in MLE-15 cells was

inhibited by Foxa2 in vitro, provides a potential mechanism by which Foxa2 influences Th2 cell recruitment and

activation in the lung.

51 Role of Foxa2 in the regulation of pulmonary dendritic cells. A network of dendritic cells is closely associated

with the respiratory epithelium (56). Both mDC and pDC are present in the lung, but mDCs subsets generally

dominate the airway mucosa (57). In the present 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 while the level of mDC activation increased from PN8 to PN15 in control mice, the activation status of pulmonary pDCs peaked at PN day 11, and decreased thereafter, suggesting that between PN11 and PN15, there is a shift towards 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. Figure 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 present 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 ovalbumin sensitization in the mature mouse.

52 Acknowledgements

We acknowledge Ann Maher for preparation of the manuscript, AMGen Inc. for providing the IL-4Rα monoclonal antibody, and Dr. Fred Finkelman for scientific input.

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57 Figures and Lengends:

Figure 1 Deletion of Foxa2 in respiratory epithelium caused pulmonary eosinophilic inflammation and goblet cell differentiation. The Foxa2Δ/Δ mice spontaneously developed airway inflammation, eosinophilic infiltration and goblet cell metaplasia at postnatal day 15. Deletion of Foxa2 was confirmed by immunohistochemical staining. Increased eosinophils in BALF were revealed by Diff-Quik staining (A). 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 Foxa2Δ/Δ mice (B). Expression of Th2 cytokines (IL-4, IL-5 and IL-13)

and chemokine Ccl17 (Tarc) was increased in BALF of Foxa2Δ/Δ mice as determined by ELISA (C). Inserts in (A) are the higher magnification of the regions indicated by arrows. The graphs represent means ± S.E. *, <0.05 versus controls using Student’s-t test (2-tailed, unpaired), n = 6 for each genotype. Eos: eosinophil, Mac:

macrophage, Lym: lymphocytes, Neu: Neutrophils. Scale bar: 50 µm.

58

Figure 2 Inhibition of IL-4R suppressed goblet cell differentiation and pulmonary inflammation in Foxa2Δ/Δ mice.

IL-4Rα monoclonal antibody was administered i.p. at PN2 and PN9. Eosinophilic inflammation was inhibited,

shown by reduced total cell and eosinophil numbers. Grey bar: saline treatment, black bar: IL-4Rα monoclonal

antibody treatment (A). Goblet cell differentiation was inhibited by antibody treatment, indicated by lack of

Spdef, Muc5ac, and Foxa3 staining in Foxa2Δ/Δ mice (B). Inserts show the higher magnification (4×) of eosinophils in perivascular regions pointed by arrows (left bottom of left panels), Scale bar: 50 µm. Graphs represent means ± S.D. *, <0.01 versus controls using Student’s-t test (2-tailed, unpaired). n=6 for each genotype.

i.p., intraperitoneal.

59

Figure 3 Loss of Foxa2 in respiratory epithelium enhanced recruitment and activation of mDC. The percentages

of both mDCs and pDCs were increased in the lung of Foxa2Δ/Δ mice at PN11 to PN15 (A). While 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 co-stimulatory 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 to control mice. n=8 at PN8, n=5 at PN11 and n=4 at PN15 of each genotype. Graphs represent means ± S.E. *, <0.05; **, <0.01 and ***,

<0.001 versus controls as determined by Student’s t-test (2 tailed, unpaired).

60

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 revealed increased expression of Th2 cytokines, chemokines including Il4, Il13, Ccl17, Ccl22,

61 Ccl11, and Ccl24, as well as goblet cell associated genes, Clca3 and Agr2 after deletion of Foxa2. (B) Expression of mRNAs of Th2 cytokines (Il4, Il5, Il13 and Il33), chemokines (Ccl11, Ccl24 and Ccl17), were increased in

Foxa2Δ/Δ mice at PN15 as detected by quantitative RT-PCR. Graphs represent mean ± SEM at arbitrary units

(a.u.). *, <0.05 versus control determined by Mood’s Median test, n=3 for each genotype for quantitative RT-PCR assay.

62

63 Figure 5 Foxa2 inhibited allergen induced goblet cell differentiation. Scgb1a1-rtTA, Foxa2-IE transgenic mice

were treated with doxycycline for 3 days, increasing Foxa2 expression as detected by immunohistochemistry

using a high dilution of Foxa2 antibody at 4 weeks of age (panel A). Foxa2 was not prominent at this antibody

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 one day before intranasal ovalbumin exposure. Foxa2 markedly inhibited goblet cell differentiation as shown by decreased Alcian blue,

Muc5ac, Spdef, and Foxa3 staining (B). Inserts are the higher magnification (4×) of the regions indicated by arrows. Scale bar: 50 µm. Figures are representative of n=3 for each genotype.

64

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, naïve 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 monoclonal antibody against IL-4Rα 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.

65

Supplementary Figure 1 Increased Th2 cytokine expression and Th2 cell infiltration in lungs of Foxa2Δ/Δ mice.

After mice were sacrificed at each postnatal time (PN), lung cells were stimulated overnight with

66 phorbolmyristate-acetate (PMA) and in ionomycin to stimulate T cell cytokine production. The following

morning, monensin and brefeldin A were added to stop cytokine secretion, allowing the cytokine signal to

accumulate inside the cell. Cells were then fixed, permeabilized, and stained with mAbs to CD4, and IFNγ, IL-13

and IL-17a to indentify the frequency of Th1, Th2 and Th17 cells in the lung. There was significant increase of

median fluorescent intensity (MFI) of both IL-13 and IL-17a cytokines, as well as increased population

abundance of IL-13+ and IL-17+ cells among all CD4+ cells in Foxa2Δ/Δ mice. There was no change of Ifn-γ MFI,

although there was a slightly increase of abundance of Ifn-γ+ cells of total CD4+ cell in Foxa2Δ/Δ mice. The ratio of recruitment of IL-13+ CD4+ cells versus Ifn-γ+ CD4+ cells was approximately 8:1, indicating a influx of majority of Th2 cells in the Foxa2Δ/Δ mice lung (A). Quantitative RT-PCR confirmed that Il6 mRNA was

significantly induced, but Ifn-γ (p=0.46) mRNA was not changed in Foxa2Δ/Δ mice (B). Graphs represent means ±

S.E. *, <0.05 versus controls assessed by Student’s-t test (2-tailed, unpaired), n=8 at PN8, n=5 at PN11 and n=4 at

PN15 of each genotype were assayed. a.u., arbitrary unit.

67

Supplementary Figure 2 Expression of CD80 and MHC II on DC was not significantly affected by loss of Foxa2 in respiratory epithelium. Expression of co-stimulatory molecules was analyzed by FACS at postnatal day 8, 11

and 15. There was no significant difference of CD80 and MHC II expression on mDC or pDC populations in

Foxa2Δ/Δ mice, n=8 at PN8, n=5 at PN11 and n=4 at PN15 of each genotype. Differences were assessed using

Student’s-t test (2-tailed, unpaired).

68

Supplementary Figure 3 Increased expression of Th2 cytokine in lungs of Foxa2Δ/Δ mice at PN11. Consistent

with recruitment and activation of DC at PN11, Th2 cytokines (Il4 and Il13) mRNAs were increased in Foxa2Δ/Δ at PN11. n of 3 for each genotype. Graphs represent mean ± S.E. at arbitrary units (a.u.). * <0.05 versus controls, using Mood’s Median test.

69

Supplementary Figure 4 Increased expression of Ccl20, but not Tslp mRNA in Foxa2Δ/Δ mice. mRNAs were

collected from whole lungs of Foxa2Δ/Δ mice and control littermates, and detected by quantitative RT-PCR.

Ccl20 but not Tslp mRNA were increased in Foxa2Δ/Δ mice at PN15 (n of 3 for each genotype). Graphs represent mean ± SEM at arbitrary units (a.u.). * <0.05 versus controls, using Mood’s Median test.

70

Supplementary Figure 5 Foxa2 inhibited Tarc promoter activity in vitro. MLE-15 cells were cotransfected luciferase reporter plasmid mCcl17-pGL3 contained the 2 kb of mouse Ccl17 gene promoter region with internal control plasmid CMV-
Gal and a plasmid expressing either EGFP or Foxa2-EGFP. The luciferase activity was measured 24 hours after transfection and normalized to -galactosidase activity. Expression of Foxa2 in MLE cells inhibited mouse Ccl17 gene promoter activity by 40%. Graph represents 3 independent experiments at means ± S.E. * <0.05 versus controls, using Student’s-t test (2-tailed, unpaired).

71

Supplementary Figure 6 Conditional expression of Foxa2 and EGFP in respiratory epithelial cells. Foxa2 was expressed under control of Scgb1a1 promoter in Scgb1a1/Foxa2-IE transgenic mice exposed to doxycycline (A).

Transgene expression was induced by provision of doxycycline from PN1 to PN7. EGFP derived from the transgene was observed by fluorescence-microscopy at PN7 (B).

72

Supplementary Figure 7 Foxa2 inhibited normal goblet cell differentiation during postnatal development.

Goblet cells were not present in mice expressing Foxa2 for 2 weeks from PN4 to PN18. In contrast, goblet cells were readily detected in single transgenic control mice at this developmental stage as shown by hemetoxylin- eosin and Foxa2 staining of adjacent sections. Foxa2 antibody was adjusted to barely detect endogenous Foxa2 expression. Inserts are the higher magnification (4×) of the regions indicated by arrows. Scale bar: 50 µm.

Figures are representative of n=3 for each genotype.

73

Supplementary Figure 8 Ovalbumin sensitization protocol. Scgb1a1/Foxa2-IE transgenic mice and Scgb1a1- rtTA single transgenic littermate (6 weeks of age) were sensitized with intraperitoneal (i.p.) injection of ovalbumin with adjuvant (100 μg ovalbumin and 1 mg Imject Alum/mouse) at day 1 and day 14. Doxycycline

(Dox) food was provided to mice 1 day before the mice received the first intranasal (i.n.) exposure to ovalbumin

(50 μg in 50 μl sterile saline/mice) until sacrifice at day 29.

74

Supplementary Figure 9. Expression of Foxa2 did not alter inflammatory cell numbers or Th2 cytokine production in BALF following intrapulmonary ovalbumin sensitization. At 6 weeks of age, Scgb1a1-rtTA or

Scgb1a1/Foxa2-IE double transgenic mice, were treated with doxycycline for 24 hours before they were treated with intrapulmonary ovalbumin. BALF was collected 48 hours after second intrapulmonary ovalbumin sensitization. The total and differential cell number in Scgb1a1-rtTA and Scgb1a1/Foxa2-IE mice were counted and cytokine expression was determined by ELISA. There were no significant changes in inflammatory cell numbers (A) nor Th1, Th2 cytokine expression in BALF (B) after expression of Foxa2 followed by pulmonary ovalbumin exposure. n=6 for each genotype. Graphs represent means ± S.E., compared by Student’s-t test (2- tailed, unpaired). Grey bars: control mice, black bars: Foxa2 Δ/Δ mice.

75

76

77 Supplementary Table I Loss of Foxa2 influenced the expression of genes associated with allergic inflammation

and asthma. mRNAs changed by deletion of Foxa2 were compared with those associated with asthma related genes (genes causing, predisposing or protecting from asthma) that were compiled from the literature, mRNA microarray analyses, and other data in the public domain including OMIM and HGMD. The overlap between the known “asthma” related genes and those responding to the deletion of Foxa2 was highly statistically significant.

The p-value for the same or a stronger association = 0.000000 by random chance, supporting the concept Foxa2 plays a critical role in the regulation of Th2-cytokine mediated inflammation.

78

Supplementary Table II Overrepresented Pathway analysis: mRNAs influenced by deletion of Foxa2 in respiratory epithelial cells were categorized and compared with canconical pathways including all genes in

MOE430 mouse genome associated with known pathways and disease states from KEGG, GenMAPP, and

GEArrays. Pathways regulating dendritic cell activation, antigen presentation, T-cell activation and inflammatory cytokines/chemokines and their receptors were significantly induced in lungs from Foxa2Δ/Δ mice.

79 SPDEF is required for mouse pulmonary goblet cell differentiation and

regulates a network of genes associated with mucus production*

Gang Chen1, Thomas R. Korfhagen1, Yan Xu1, Joseph Kitzmiller1, Susan E. Wert1,

Yutaka Maeda1, Alexander Gregorieff 2, Hans Clevers 2 and Jeffrey A. Whitsett1, 3

1The Perinatal Institute, Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center and the

University of Cincinnati School of Medicine, Cincinnati, OH 45229; 2Netherlands Institute of Developmental

Biology, Utrecht, The Netherlands

Conflict of Interest: The authors have declared that no conflict of interest exists.

3Correspondence:

Jeffrey A. Whitsett, M.D.

Cincinnati Children’s Hospital Medical Center

Division of Pulmonary Biology, MLC 7029

3333 Burnet Avenue

Cincinnati, OH 45229-3039

Phone: (513) 803-2790 FAX: (513) 636-7868

Email: [email protected]

* Manuscript is published in Journal of Clinical Investigation 2009;119 (10):2914–2924.

80 Abstract Goblet cell hyperplasia and excess mucus production are central to the pathogenesis of chronic pulmonary

diseases, including asthma, cystic fibrosis (CF), and chronic obstructive pulmonary disease (COPD). Here we

show that SPDEF (SAM Pointed Domain Ets-like Factor) controls a transcriptional program critical for

pulmonary goblet cell differentiation. Expression of SPDEF in non-ciliated secretory epithelial cells (Clara cells)

caused rapid and reversible goblet cell differentiation in the absence of cell proliferation in vivo. Cell lineage

tracing was used to identify Clara cells as the progenitors of goblet cells induced by pulmonary allergen exposure

in vivo. Deletion of the murine Spdef gene resulted in the absence of goblet cells in tracheal/laryngeal submucosal glands and in the conducting airway epithelium after pulmonary allergen exposure in vivo, demonstrating its requirement for normal goblet cell differentiation in the lung. SPDEF enhanced the expression of genes associated with goblet cell differentiation and allergen sensitization, including Muc16, Agr2, Clca1,

Gcnt3, Ptger3, Muc5ac, and a group of genes mediating protein glycosylation. SPDEF inhibited FOXA2 and

TTF-1 and induced FOXA3 demonstrating its pivotal role in transcriptional control of airway epithelial cell

differentiation. SPDEF and FOXA3 were increased after sensitization with pulmonary allergen and were co-

localized in goblet cells in normal human bronchial glands and in goblet cells lining airways of patients with

chronic lung diseases. SPDEF plays a critical role in the regulation of a transcriptional network mediating goblet

cell differentiation and mucus hyperproduction associated with chronic pulmonary disorders.

81 Introduction

Goblet cell “hyperplasia” and mucus hypersecretion contribute to the pathogenesis of common pulmonary disorders, including asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF). Allergens, cigarette smoke, inhaled toxicants, and chronic infections induce mucus hyperproduction in conducting airways, causing airway obstruction and tissue remodeling. While initially derived from shared endodermal progenitors during lung morphogenesis (1), the conducting regions of the respiratory tract are lined by a diversity of epithelial cell types, including Clara, serous, basal, goblet (mucous), neuroepithelial cells, and ciliated cells, that together mediate innate host defense and mucociliary clearance to maintain sterility of the lung. While goblet cells are generally not abundant in the normal lung, goblet cell hyperplasia is induced by acute and chronic inflammation, influencing mucociliary clearance and innate host defense in the lung (2, 3). The differentiation of various pulmonary epithelial cell types is determined by both genetic and environmental factors that, in turn, regulate transcriptional programs controlling epithelial cell differentiation and behavior. During development, alveolar type II and Clara cell differentiation is dependent upon interactions of a number of transcription factors that regulate a group of genes mediating host defense and other aspects of lung function (4). In contrast, there is a paucity of knowledge regarding transcriptional programs regulating goblet cell differentiation. Goblet cells are found in many epithelial-enriched tissues where they synthesize, store and secrete large mucopolysaccharide-rich mucins that influence mucociliary clearance and innate defense of the lung (5, 6). In the gastrointestinal tract, goblet cells are relatively abundant, and their differentiation is regulated by the Notch signaling pathway (7, 8).

In the lung, goblet cells (mucous cells) are present in submucosal glands but are not abundant in conducting airways in the absence of inflammation. Numbers and activity of goblet cells are induced by a variety of acute and chronic inflammatory stimuli. Goblet cells are observed following pulmonary allergen sensitization, mediated primarily by the TH2-associated cytokines, IL-4 and IL-13, that activate the IL-4 receptor, STAT6 phosphorylation, and subsequent gene expression (9-12). At the transcriptional level, pulmonary goblet cells are increased by allergens, dust mite or IL-13 exposure and are associated with the decrease of FOXA2 mRNA in bronchial and bronchiolar epithelial cells (13). In the mouse lung, deletion of the Foxa2 gene in respiratory

82 epithelial cells is sufficient to induce goblet cell differentiation in vivo (14). The potential role of SPDEF, a member of the Ets family of transcription factors (15), in goblet cell differentiation is supported by the finding that SPDEF is induced following pulmonary allergen and IL-13 exposure (16). Chronic expression of SPDEF in epithelial cells of the mouse lung is associated with extensive goblet cell differentiation in the airways of transgenic mice (16).

The present study was undertaken to identify the role and mechanisms by which SPDEF regulates goblet cell differentiation in conducting airways of the lung. SPDEF was required for normal goblet cell differentiation in laryngeal-tracheal submucosal glands and in the bronchial-bronchiolar epithelium during experimental pulmonary allergen sensitization. SPDEF regulates a transcriptional network inhibiting Clara cell differentiation and inducing goblet cell differentiation and mucus production.

83 Results

Cellular origins of goblet cells induced by allergen exposure. To define the cellular precursors of goblet cells in the respiratory epithelium, lineage tracing was performed by conditionally labeling Clara cells using Cre- recombinase to recombine the ROSA26 loxP/stop locus thereby permanently expressing ß-galactosidase in Clara cells or their derivatives, Figure 1A. Administration of doxycycline to Scgb1a1-rtTA/Otet-Cre/R26R transgenic mice caused extensive recombination in Clara cells within 5 days, at which time ß-galactosidase staining was confined to Clara cells and not detected in ciliated cells. Goblet cell differentiation was then induced by pulmonary sensitization with ovalbumin, Figure 1B. The goblet cells induced by the allergen expressed ß- galactosidase, indicating their derivation from Clara cell progenitors, Figure 1C. Goblet cell differentiation, including increased MUC5AC staining (Figure 1C) occurred without evidence of cellular proliferation as assessed by BrdU and phosphohistone H3 labeling, Supplementary Figure 1. Consistent with these findings, conditional expression of SPDEF using the Clara cell-specific promoter (Scgb1a1-rtTA/TRE2-Spdef) induced marked goblet cell differentiation without causing proliferation in the conducting airways within 3 days. Neither BrdU labeling, phosphohistone H3 nor the number of cells per unit length of the bronchioles (latter data not shown) was increased during allergen sensitization or after expression of SPDEF in vivo. This process was rapidly reversible and associated with the restoration of Clara cell morphology and CCSP (Clara cell secretory protein) expression,

Figure 1D. Thus, goblet cell differentiation in this model occurs without proliferation, a finding that does not support the term “hyperplasia.”

SPDEF regulates gene expression in the respiratory epithelium. To identify mRNAs regulated by SPDEF during goblet cell differentiation, laser capture microdissection was used to obtain proximal bronchiolar epithelial cells before and 3 days after treatment with doxycycline to induce SPDEF expression in Clara cells (Supplementary

Figure 2). SPDEF influenced the expression of 306 unique mRNAs (p=<0.01 level, using differences of 2-fold),

Figure 2. SPDEF induced expression of a number of genes involved in the regulation of many aspects of mucus production, including protein glycosylation and mucin secretion, as well as genes that are highly represented in experiments in which mice were exposed to pulmonary allergens or IL-13. For example, Muc16 (17), Agr2 (18,

84 19), Clca1 (20), Ptger3/4 (21, 22), Gcnt3 (23), Foxa3 (13), Serpinb11 (24), lumican and versican (25) were markedly induced by SPDEF. Although Muc5ac mRNA was increased two-fold by SPDEF, it was pre-filtered out because of the low hybridization signals (close to background signal). In contrast, SPDEF inhibited the expression of groups of genes associated with Clara and type II alveolar cell differentiation, including Foxa2,

Titf1, and a number of genes known to be directly regulated by FOXA2 and TTF-1, including Abca3, Sftpa, Sftpb,

Sftpd, Par, Aqp5, and Scgb1a1 (26, 27), Figure 2A. SPDEF inhibited Scnn1b, and Scnnlg, consistent with a role for SPDEF in the regulation of fluid and electrolyte transport that are important for mucociliary clearance in the lung. Quantitative RT-PCR was used to confirm the SPDEF related changes in a number of these mRNAs, Figure

2B-F. Of note, mouse Clca1 mRNA was significantly induced by SPDEF in vivo. Human CLCA1, a calcium activated chloride channel associated with mucus production (28), is the closest ortholog of mouse CLCA1 (29)

(Accession Number Q9D7Z6, Swiss-Prot). The finding that SPDEF inhibited Titf-1 and Foxa2 mRNAs was confirmed by immunohistochemistry. Staining for FOXA2 and TTF-1 were markedly inhibited in goblet cells induced by SPDEF or after pulmonary ovalbumin sensitization, while FOXA3 and AGR2 were induced, Figure

3A-D. Consistent with the role of SPDEF in promoting mucin biosynthesis, protein glycosylation in the goblet cells induced by SPDEF or pulmonary ovalbumin sensitization was detected by staining of the lectin, UEA-I, which recognized the L-fucose moiety of glycoproteins (30), Figure 3E. The induction of AGR2 was of particular interest, since Agr2 encodes a potential chaperone required for mucin packaging in goblet cells in vivo (31).

SPDEF was colocalized with FOXA3 and AGR2 in goblet cells in vivo, indicating that SPDEF and FOXA3 may cooperate in the regulation of gene expression, Figure 4A, B. SPDEF and FOXA3 synergistically activated the

Agr2-luciferase promoter and endogenous Agr2 mRNA expression in vitro, Figure 4C, D. Taken together,

SPDEF induces goblet cell differentiation, increasing the expression of genes associated with mucin biosynthesis, glycosylation, and packaging, while inhibiting genes characteristic of Clara cells in the normal bronchiolar epithelium, including genes regulating fluid and electrolyte transport, as well as innate host defense. Since TTF-1 and FOXA2 are important transcriptional regulators of genes expressed selectively in Clara cells, their inhibition by SPDEF likely accounts for changes of gene expression associated with Clara cells.

85 Co-expression of SPDEF, FOXA3, and AGR2 in pulmonary diseases. Immunohistochemical staining for SPDEF,

FOXA3, and AGR2 was assessed in bronchial tissues from patients with pulmonary diseases caused by cystic

fibrosis or cigarette smoking, disorders in which goblet cell hyperplasia and mucus hypersecretion are prominent.

Alcian blue, SPDEF, FOXA3, and AGR2 staining was markedly increased at sites of goblet cell hyperplasia and was not readily detected in normal airway epithelium, Figure 5. In contrast, SPDEF, FOXA3 and AGR2 were expressed in goblet cells in the normal submucosal glands in both human and in the mouse, Figure 5F and data

not shown.

SPDEF is required for allergen induced goblet cell differentiation. Spdef -/- mice breed and survive normally in the vivarium. Spdef -/-, Spdef +/-, and Spdef +/+ mice were sensitized by repeated systemic injection followed by

intrapulmonary administration of ovalbumin. Lung histology was unaltered in Spdef -/- mice as assessed by light microscopy; however, goblet cells were absent in the tracheal and laryngeal submucosal glands of Spdef -/- mice

prior to allergen exposure, Figure 6. As in wild type mice, ovalbumin sensitization induced goblet cell

differentiation in Spdef +/- mice as indicated by Alcian blue staining of acidic mucopolysaccharides and increased

staining of SPDEF, FOXA3, MUC5AC and UEA-I lectin, Figure 7. In contrast, neither goblet cell morphology

nor the goblet cell associated transcription factor, FOXA3, were detected in the bronchial-bronchiolar epithelial

cells in Spdef -/- mice before or after allergen sensitization. MUC5AC and staining for mucus associated fucosyl

glycoconjugates with UEA-I lectin were markedly inhibited, but not absent in the Spdef -/- mice after allergen

exposure, Figure 7. Pulmonary inflammation, eosinophilic and lymphocytic infiltration associated with allergen

exposure were similar in Spdef +/- and Spdef -/- mice, Supplementary Figure 3. To assess whether expression of

SPDEF in airway epithelial cells was sufficient to induce MUC5AC expression in vitro, NCI-H292 cells and

HBEC 3KT (Human Bronchial Epithelial Cells) (32) were infected with lentivirus expressing the Spdef cDNA.

SPDEF induced endogenous MUC5AC expression in both cell lines, Figure 8. SPDEF was induced by IL-13 in primary mouse tracheal epithelial cells grown at air-liquid interface (Supplementary Figure 4), consistent with the role for TH2 cytokine signaling in the regulation of SPDEF.

86 Discussion

The respiratory tract is continuously exposed to inhaled irritants, particles, and pathogens necessitating the evolution of robust innate and acquired host defense systems to maintain pulmonary sterility and homeostasis.

Mucociliary clearance, the production of innate defense proteins, and the activity of the immune system all serve to maintain lung function after birth. The respiratory epithelium adapts to inflammatory stimuli, often dramatically increasing the number and activity of goblet cells. Goblet cell differentiation and excessive production of mucus are common features of many chronic pulmonary diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease. The present study demonstrates that goblet cells induced by allergens or by the expression of SPDEF are derived by differentiation of existing Clara cells and not by proliferation.

Thus, the findings support the concept that goblet cells formed in the present model occur by metaplasia rather than by “hyperplasia.” SPDEF is required for goblet cell differentiation in normal tracheal-laryngeal submucosal glands and is required for goblet cell differentiation following allergen exposure. SPDEF regulates a group of genes associated with goblet cell differentiation, mucin biosynthesis and secretion. SPDEF plays a central role in the regulation of goblet cell differentiation in epithelial cells in the respiratory tract, interacting with a transcriptional network that includes suppression of TTF-1 and FOXA2 and induction of FOXA3.

SPDEF was initially identified in prostate epithelium where it interacts with NKX-3.1 in the regulation of prostate specific antigen (PSA) (33). Spdef mRNA and protein are expressed in a number of organs that are enriched in epithelial cells of mucous and other secretory lineages, including breast, pancreas, and both reproductive and gastrointestinal tracts, supporting its potential role in goblet cell differentiation. In the intestine, secretory cell (goblet and Paneth cells) differentiation is regulated by the Notch signaling pathway (7, 8) and transcription factors including MATH1/ATOH1 (34), GFI1 (35); while in the colon, KLF4 regulates goblet cell differentiation (36). Loss of MATH1/ATOH1 caused loss of GFI1, a zinc-finger transcription factor required for intestinal secretory lineage specification, which in turn decreased Spdef mRNA and reduced goblet cell number

(35). Thus, expression of Spdef in post-mitotic intestinal epithelial cells is regulated by the Notch signaling pathway and its downstream transcription factors MATH1/ATOH1 and GFI1 which are required for secretory

87 lineage specification. However, it is still unclear that expression of SPDEF is regulated by the Notch in the

mature respiratory epithelium. Notch signaling pathway has been recently reported to promote airway goblet cell

differentiation in a STAT6 independent manner in fetal tracheal explants in vitro (37). In the adult respiratory

epithelium, SPDEF expression and goblet cell differentiation were induced by Th2 cytokines in a STAT6-

dependent manner in vivo (10, 16). In the lung, GFI1 expression is confined to pulmonary neuroendocrine cells

(38), and Klf4 mRNA was not induced in association with goblet cell differentiation in the pulmonary allergy and

IL-13 microarray databases presently analyzed, suggesting that at least some aspects of the transcriptional regulation of goblet cell differentiation may be tissue specific. Nevertheless, SPDEF is expressed in many tissues containing goblet cells including the respiratory and gastrointestinal epithelium (16, 39).

The present data support potential roles of FOXA2, TTF-1 and FOXA3 in a transcriptional network associated with goblet cell differentiation or function, Figure 9. SPDEF inhibited FOXA2 and TTF-1 and induced

FOXA3, the latter a transcription factor that is not abundantly expressed in the normal lung (40). We observed

reduced expression of FOXA2 and increased expression of SPDEF and FOXA3 during goblet cell differentiation

caused by ovalbumin sensitization, and in lung tissue from patients with CF and chronic smoking. FOXA2 and

FOXA3 staining was mutually exclusive following expression of SPDEF or its induction by allergen. Their

colocalization in goblet cells and the finding that SPDEF and FOXA3 synergistically induced Agr2, indicates

their potential roles in goblet cell differentiation.

SPDEF regulates expression of genes regulating mucin biosynthesis. The mRNA microarray analysis of SPDEF

induced genes in the conducting airways, indicated that the expression of genes associated with mucin

biosynthesis were induced most dramatically. Mucins are large molecular weight glycoproteins with 10 to 20%

protein and 80 to 90% carbohydrate components. Biosynthesis of mature glycosylated mucin requires 1)

transcription of a MUC gene to encode a MUC mRNA in nucleus, 2) translation into a MUC protein backbone on

a ribosome that is inserted into the endoplasmic reticulum (ER), a cellular compartment for protein folding, and 3)

posttranslational modification of mucin core proteins by one or several glycosyltransferases in the Golgi. In the

present study, mRNA microarray analysis supports the concept that SPDEF influences mucus production at least

88 in part by regulation of AGR2, a mucin chaperone protein in the ER and peptidyl N-acetylgalactosaminyl transferases in the Golgi. AGR2 was identified as an endoplasmic reticulum (ER) protein that belongs to the protein disulfide isomerase family (PDI) of chaperones that facilitate the folding of proteins targeted for the secretory pathway (41). AGR2 binds to unfolded parts of mucin protein in the ER, to enhance protein folding and the posttranslational modification of serine and threonine residues, the sites of O-glycosylation in Golgi (31).

Mucin biosynthesis begins with the attachment of N-acetylgalactosamine (GalNAc) to a serine or threonine residue, a process catalyzed by various UDP-GalNAc:polypeptide GalNAc-transferases (pGalNAc-T subfamily) (42). The synthesis of more complex, functional mucin carbohydrate requires the formation of core 2 beta1, 6 branched structure in the mucin glycan chain by beta-1,6-N-acetylglucosaminyltransferase family of enzymes (also known as the C2GnT family) (43). mRNAs of all three members in the C2GnT family that mediate production of functional mucins (Gcnt1, 2, and 3) were significantly induced by SPDEF (increased 3.4,

1.8, and 9.3 fold respectively), strongly supporting its regulatory role in mucin biosynthesis and secretion.

Among these enzymes, GCNT3 was induced the most by the expression of SPDEF in the airway epithelium

(Figure 3E). GCNT3 mediates both core 2 and core 4 O-glycan branching, two important steps in mucin-type biosynthesis (44). Gcnt3 mRNA is expressed primarily in mucus secreting tissues, including the gastrointestinal tract and trachea and is induced by retinoic acid, TNF-α and IL-13 (45, 46). Previous studies demonstrated that

GCNT1 and 3 enzyme activities and mRNA levels were induced in airway epithelial cells following exposure to

Th2 cytokines (IL-4 and IL-13) in a process mediated by the JAK-STAT signaling pathway (23, 45). Consistent with the important role of SPDEF in the regulation of mucin glycosylation, staining for UEA-I lectin was induced by SPDEF.

In the present study, goblet cell differentiation was associated with the increased expression of SPDEF and FOXA3 in bronchiolar epithelial cells, consistent with the microarray results showing the induction of many asthma related and IL-13 responsive genes. Muc5ac, encoding a major mucin type secreted from airway goblet cells, was induced by SPDEF expression in vitro and in vivo. Expression of SPDEF was sufficient to induce

MUC5AC in H292 and HBEC cells in vitro. Muc16 mRNA was markedly induced by SPDEF in vivo. MUC16

89 expression in tracheal epithelium, submucosal glands and cultured bronchial epithelial cells was recently reported

(17). Taken together, SPDEF regulates a number of genes involved in mucin synthesis, its glycosylation, and packaging. It remains unclear whether the effects of SPDEF represent direct cell autonomous effects on target gene expression or more complex effects on other regulators of goblet cell differentiation and function.

Expression of SPDEF in Clara cells caused their differentiation into goblet cells and inhibited a number of genes regulating host defense, as well as fluid and electrolyte transport which are normally expressed in Clara cells and are known transcriptional targets of FOXA2 and TTF-1. This inhibitory effect is mediated in part by loss of FOXA2 and TTF-1 expression caused by induction of SPDEF in the airway epithelium. It is presently unclear if this inhibition is mediated by direct or indirect effects of SPDEF on these transcriptional targets, Figure

9.

SPDEF, FOXA3 and AGR2 were increased in both the mouse models and in human lung tissue wherein their expression was associated with the presence of goblet cells, supporting the conservation of the proposed regulatory network. Since the cellular composition of the mouse airway differs from that of the human, the cellular origin of goblet cells in the human airway in which basal rather than Clara cells are more prominent, remains unclear.

Pulmonary allergen exposure and chronic inflammatory diseases of the lung are associated with infiltration by many cell types and the expression of numerous cytokines, chemokines, and other inflammatory mediators. The present findings indicate that these complex signals influence goblet cell differentiation in the respiratory epithelium via the transcription factor SPDEF and its influence on an extended transcriptional network,

Figure 9. Changes in gene expression, cell differentiation, and morphology caused by SPDEF occur rapidly and reversibly, without the activation of cell proliferation. Goblet cell differentiation and mucin secretion also occurs following acute inflammation. Thus, SPDEF plays a central role in the regulation of a gene network that responds to pathogens or toxicants, in turn changing epithelial cell differentiation and mucociliary clearance that together play a role in innate host defense of the lung. Such changes in cell differentiation and function may represent an

90 adaptive change in the epithelium that occurs without cell death, minimizing the need to activate cell proliferation.

Since mucus hyperproduction contributes to the pathogenesis of acute and chronic pulmonary disorders, knowledge regarding the regulation and function of SPDEF in the respiratory tract provides a framework for the development of new strategies for diagnosis and therapy for chronic lung diseases.

91 Materials and Methods

Mouse models, ovalbumin sensitization and BrdU administration: Mouse strains included in this study were

Spdef -/- mice produced in the laboratory of Dr. Hans Clevers, Netherlands Institute of Developmental Biology.

Young adults of approximately 6-8 weeks of age were utilized in most studies. Scgb1a1-rtTA (line 2) (47)/TRE2-

Spdef (16) mice in FVB/N strain were treated with doxycycline (625 mg/kg of food). RosaA26 reporter mice

(R26R), kindly provided by Dr. Soriano, Fred Hutchinson Cancer Research Center (48), were bred to Scgb1a1- rtTA (line 2), (Otet)7CMV-Cre mice for lineage tracing (Figure 1A). The ovalbumin sensitization protocol was

described previously (14). Wild type FVB/N mice were obtained from The Jackson Laboratory, Bar Harbor, ME.

To induce pulmonary goblet cell hyperplasia, mice received intraperitoneal injection with 100 μg ovalbumin

(grade V, Sigma-Aldrich, St. Louis, MO) and 1 mg Imject Alum (Thermo Scientific, Waltham, MA) as adjuvant,

followed by two intranasal instillations of 50 µg of ovalbumin or saline 3 days apart, starting at least 10 days after the second sensitization. BrdU (B5002, Sigma, St. Louis, MO) was administered daily by intraperitoneal injection (50 µg/g of body weight at a concentration of 10 mg/ml in sterile saline) during treatment with doxycycline (over 3 days) and nasal sensitization with ovalbumin (day 24 through day 29, Figure 1B). Animal protocols were approved by the Institutional Animal Care and Use Committee in accordance with NIH guidelines.

Laser capture, RNA purification and amplification: Laser capture microdissection (LCM) was performed as

described (49). To induce Spdef expression, 8 weeks old adult male Scgb1a1-rtTA/TRE2-Spdef transgenic mice

were treated with doxycycline for 3 days (n=3). The control mice were the same age, sex, and genotype but were

not treated with doxycycline (n=3). Mice were anesthetized, exsanguinated, and the lungs were inflated with

OCT (Fisher Scientific, Pittsburgh, PA)/DEPC-PBS with 10% sucrose (50% v/v) (sucrose, S0389, Sigma, St.).

After inflation, lungs were dissected, lobes were separated, embedded in OCT, and snap frozen in isopentane, and

stored at -80°C. Tissue was sectioned at -20ºC in the cryostat. Thin sections (10 µm) were collected on 1:20

poly-L-lysine (P8920, Sigma) coated slides and stored at -80ºC. Prior to laser capture microscopy, slides were

fixed in iced DEPC 70% ethanol, washed in DEPC-H20, and dehydrated in 95% and 100% ethanol, xylene and air dried. Bronchiolar cells were captured by Veritas Microdissection Instrument (Model 704) (Molecular Devices,

92 Sunnyvale, CA) with a laser set at 15 µm. Total RNAs were purified by Arcturus PicoPure RNA Isolation Kit

(Molecular Devices). RNAs were then subjected to two rounds of amplification using TargetAmp 2-Round

Aminoallyl-aRNA Amplification Kit 1.0 (Epicentre biotechnologies, Madison, WI).

RNA microarray analysis: The RNAs were then hybridized to the murine genome MOE430 chips (consists of ≈

45000 gene entries, Affymetrix, Santa Clara, CA) according to the manufacturer's protocol. The RNA quality and

quantity assessment, probe preparation, labeling, hybridization and image scan were carried out in the CCHMC

Affymetrix Core using standard procedures. Affymetrix Microarray Suite 5.0 was used to scan and quantitate the

gene chips under default scan settings. Hybridization data were subjected sequentially to normalization,

transformation, filtration, and functional classification and pathway analysis as previously described (50, 51).

Data analysis was performed with BRB Array Tools software package

(http://linus.nci.nih.gov/BRBArrayTools.html). Differentially expressed genes between the with or without

doxycycline treatment groups were identified using a random-variance t-test, which is an improvement over the

standard t-test as it permits sharing information among genes within-class variation without assuming that all

genes have the same variance (52). Genes were considered statistically significant if their p values were less than

0.01 and fold changes were greater than 1.5. We also performed a permutation test to provide 90% confidence

that the false discovery rate was less than 10%. The false discovery rate is the proportion of the list of genes

claimed to be differentially expressed that are false positives. In addition, Affymetrix "Present Call" in at least

two of three replicates and coefficient of variation among replicates of ≤50% were set as a requirement for gene selection.

Functional classification and pathway analysis: Gene Ontology Analysis was performed using the publicly available web-based tool DAVID (database for annotation, visualization, and integrated discovery). Gene sets associated with known pathways and disease states from KEGG (http://www.genome.ad.jp/kegg/), GenMAPP

(http://www.genmapp.org/) and GEArrays (http://www.superarray.com/) were identified by comparing the overlap of pathway genes to the differentially expressed genes and all genes in the MOE430 mouse genome. A gene ontology term is considered to be overly-represented when a Fisher’s exact test p value of ≤0.01 and gene

93 hits of ≥10. Potential protein/protein or protein/DNA interactions were identified using Ingenuity Pathway

Analisis (IPA, Ingenuity). Genetic networks preferentially enriched for input genes 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, which takes into account the number of network-eligible genes and the size of the network.

Human Specimens: Anonymous, deidentified, human adult and pediatric lung samples were obtained through

the Department of Pathology, University of Cincinnati College of Medicine, and the Division of Pathology,

Cincinnati Children’s Hospital Medical Center, in accordance with institutional guidelines for use of human tissue

for research purposes (courtesy of Drs. Gail Deutsch, Kathryn Wikenheiser-Brokamp, and Robert Baughman).

Immunohistochemistry, Immunofluorescence, X-gal and Alcian blue staining: Adult mouse lung was inflation

fixed, embedded, sectioned, and immunostained. Alcian blue and immunohistochemical staining for SPDEF,

CCSP, and phosphohistone H3 followed previously described methods (16). TTF-1 was detected by a mouse

monoclonal TTF-1 antibody (8G7G3/1) as previously described (53). AGR2, MUC5AC (ab47044 and ab3649,

respectively, Abcam, Cambridge, MA), FOXA3 (SC-5361, Santa Cruz Biotechnology, Santa Cruz, CA), BrdU

(Zymed BrdU staining Kit, Invitrogen, Carlsbad, CA), UEA-I (Sigma, L9006) staining followed standard

procedures, as recommended by the manufacturers. Rat anti-mouse CD68 antibody (AbD Serotec) was used to

detect monocytes and macrophages on frozen sections. Human SPDEF antibody (54) used on all human

specimens was kindly provided by Dr. Dennis Watson (Medical University of South Carolina), and was

performed at dilution of 1:500 after antigen retrieval with citrate buffer and heat. Major Basic Protein (MBP)

antibody was kindly provided by Drs. Jamie and Nancy Lee (Mayo Clinic, Scottsdale, AZ). To detect β-

galactosidase expression, lungs were inflation fixed with 2% paraformaldehyde (PFA) fixative for 10 hours at 4°C

and then processed for preparation of frozen sections. X-gal enzymatic reaction was performed by incubating the

lung sections with 5 mM K4Fe(CN)6, 5 mM K3(CN)6, 1 mg/ml Xgal in PBS (pH 7.2) at 30°C for 4-8 hours.

After Xgal staining, the same slides were subjected to immunofluorescence staining for CCSP and FOXJ1

following previously described methods (16). To identify goblet cells, Alcian blue staining was performed, after

94 immunohistochemical staining, using a 3 minutes incubation in 3% acetic acid and a 10 minutes incubation in 1%

Alcian blue (Poly Scientific, Bay Shore, NY). Then slides were rinsed with running water for 5 minutes followed by 2 minutes in nuclear fast red, dehydration and coverslipping with mounting media.

Plasmids and cell lines: Promoter regions were selected based on the sequence similarity and the conservation of the predicted transcription factor binding sites present in human, mouse and rat genome. All the PCR reactions were performed using GC-Rich PCR system (Roche Applied Science, Indianapolis, IN), and then cloned into TA cloning vector, pSC-A, (StrataClone PCR cloning kit, Stratagene, La Jolla, CA) for sequencing. The primer sets for cloning human FOXA3 3kb promoter were: 5’-GCT CGA GCC TGC AGG AGC TAG ATT TTA TGC-3’,

5’-ATC TCG AGT CTG GAT CTC TCA GCG GGC ACGG-3’. The PCR product was cloned into pSC-A vector, isolated by cutting with Hind III and SpeI, and cloned into Hind III and NheI sites of the pGL3 basic vector

(Promega, Madison, WI). The primer sets for cloning the mouse Agr2 1.6 kb promoter were: 5’-TTC TCG AGA

ATG GGT GGG ATT TCG GGTC-3’, 5’-ATC TCG AGT GCT TGT CAA TTG CCT TACC-3’, mouse Muc16

2.6 kb promoter were: 5’- TTC TCG AGT ACT CCA CTT ATA AAT GAG-3’, 5’-TTC TCG AGG AAA ACT

CAT ATC ATA AGC-3’. The PCR fragments were then cloned into Xho I site of pGL3-basic vector. The

FOXA3 expression vector was made by amplifying the mouse 1 kb Foxa3 cDNA using the primer sets: 5’-TTG

GAT CCA TGC TGG GCT CAG TGA AGA TG-3’, 5’-TGG ATC CCT AGG ATG CAT TAA GCA GAG

AGCG-3’, and subcloned into Bam HI site of pcDNA5/TO (Invitrogen). SPDEF expression vector was made by cloning 1kb Spdef cDNA from TRE-Spdef (16) plasmid using the primer sets: 5’-AAT TCT AGA GAT GGG

CAG TGC CAG CCC AGG-3’, 5’- ATT CTA GAT CAG ACT GGA TGC ACA AATT-3’, and subcloned into

Xba I site of pcDNA5/TO. To make SPDEF expressing lentivirus, Spdef cDNA was cloned into p3×FLAG-myc-

CMV-26 expression vector (E6401, Sigma), and cut out from Sac I and Bam HI to make FLAG-Spdef-myc fusion- protein fragment. This fragment was inserted into Bam HI site of PGK-IRES-EGFP backbone modified from a previously described lentiviral vector (55) via blunt-end ligation to make the plasmid that was packaged to generate the SPDEF expressing lentivirus. The control virus was made by cutting out the FLAG-myc fragment from p3×FLAG-myc-CMV-26 expression vector and cloned into the PGK-IRES-EGFP backbone with the same

95 cloning strategy to make the SPDEF virus. Air-liquid interface culture of mouse tracheal epithelial cells was performed as previous described (56). The mouse IL-13 used for in vitro culture was purchased from R&D

System (Minneapolis, MN).

Three dimensional air-liquid interface culture of the HEBCs and culture of NCI-H292 cells: HBECs were

stably infected with either control virus or SPDEF expressing virus. The three dimensional cultures were

established as previously described (57). The HBECs were seeded in growth media on top of the gels. When

cultures became confluent in submerged conditions, they were switched to differentiation media (58) with few

alterations (2 μM hydrocortisone, 2 μg/μl transferrin, 0.55 μM epinephrine, 25 mg bovine pituitary extract) for 2

days. The cultures were then raised to an air-liquid interface and grown for 4 weeks. The cells were then fixed in

4% paraformaldehyde (20 mM Tris-HCl, pH7.6, 137 mM NaCl), processed, embedded in paraffin and prepared

for immunohistochemistry. Human bronchial epithelial NCl-H292 cells obtained from ATCC were grown in

RPMI supplemented with 10% fetal calf serum and antibiotics penicillin/streptomycin. Cells at 30% confluence

in six-well plates were infected with either control virus or SPDEF expressing virus. Five days after infection,

cells were harvested for mRNA (MUC5AC) and protein (SPDEF and actin) analysis.

Promoter reporter assays: Promoter reporter constructs were co-transfected into primary sheep tracheal

epithelial cells (aSTEpC) with CMV-β-Gal plasmid (Clontech, Palo Alto, CA) and/or transcription factor

expression plasmid using Lipofectamine 2000 (Invitrogen) as previously described (16). Growth media was

changed into a differentiation media (MTEC/Nu, mouse tracheal epithelial cells culture media, 2% Nu serum) (56)

after transfection. Cell lysates were collected for luciferase activity assay 24 hours after transfection. All

transfection assays were performed with primary sheep adult tracheal epithelial cells at passage 3 or 4. Relative

promoter activities were normalized to internal control, the β-galactosidase activity, and shown as mean±S.D.

Quantitative RT-PCR and statistics: Total RNAs obtained from LCM of bronchiolar tissue were reverse

transcribed to cDNA by Verso cDNA Kit (Thermo Scientific, Waltham, MA). Quantitative RT-PCR was

performed using Taqman probes and primer sets (Applied BioSystem, Forster City, CA) specific for Spdef (Assay

96 ID: Mm00600221_m1), Muc16 (Mm01177119_g1), Ptger3 (Mm01316856_m1), Clca1 (Mm00777368_m1),

Agr2 (Mm00507853_m1), Gcnt3 (Mm00511233_m1) and MUC5AC (Hs01365616_m1). A probe and primer set for ribosomal 18S (part number: 4352930E) was used as normalization control. PCR reactions were performed by using 25 ng cDNA per reaction in a 7300 Realtime PCR System (Applied Biosystem, Forster City, CA). The quantification RT-PCR data to confirm microarray results were analyzed by Hsu’s MCB (best) test (59). Other quantification data were analyzed by 2 tailed, type 1 Student’s t-test in this study; p values of <0.05 were considered significant.

97 Acknowledgements

The authors acknowledge support from Ann Maher for preparation of the manuscript, the Morphology

Core, Division of Pulmonary Biology, Michael Mucenski, Michael Bruno, and Angela Keiser, for technical support, and Dr. Johannes C.M. van der Loo, Dr. Punam Malik, and Ms. Anusha Sridharan for assistance with lentiviral vectors. Grant support was provided by the National Institutes of Health HL090156 and HL095580

(J.A.W. and T.R.K.).

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102 Figures and Legends

103 Figure 1 SPDEF caused differentiation of goblet cells from Clara cells. (A) Adult Scgb1a1-rtTA/Otet-Cre mice were mated to R26R mice. (B) To permanently label Clara cells, doxycycline was administered from days 12 to

17 during i.p. sensitization with ovalbumin. Mice were sacrificed either before receiving the first i.n. sensitization

(day 24) to assess Clara cell labeling with β-galactosidase or after the second i.n. sensitization (day 29) with ovalbumin to induce goblet cell hyperplasia. (C) Before pulmonary ovalbumin sensitization, β-galactosidase was expressed in non-ciliated epithelial cells, as shown by its exclusion from FOXJ1 expressing cells and co- localization with CCSP. After sensitization, β-galactosidase was detected (white pseudocolor) in most goblet cells, identified by MUC5AC, indicating their derivation from Clara cells. MUC5AC, β-galactosidase and

FOXJ1 were not co-localized. (D) Scgb1a1-rtTA/TRE2-Spdef mice were treated 3 days with or without doxycycline. SPDEF induced goblet cell differentiation as detected by Alcian blue (AB) and by changes in cell morphology. SPDEF staining decreased 4 and 8 days after withdrawal of doxycycline, at which time goblet cell differentiation was substantially resolved. CCSP was decreased in the conducting airway epithelium 3 days after induction of SPDEF. Four to eight days after withdrawal of doxycycline, CCSP staining was restored. Inserts show higher magnifications of the regions indicated at the arrows. i.p.: intraperitoneal. i.n.: intranasal. All scale bars: 25 µm.

104

Figure 2 mRNA microarray analysis of bronchiolar epithelial cells: heatmap and partial list of SPDEF regulated genes. Bronchiolar cells were isolated by laser capture microscopy (LCM) and mRNAs isolated and subjected to mRNA microarray analysis after treating Scgb1a1-rtTA/TRE2-Spdef mice for 3 days with or without doxycycline.

A heatmap of the mRNAs is shown in (A). Red indicates mRNAs increased by SPDEF; green indicates those decreased. A number of mRNAs that were previously associated with pulmonary allergen exposure, including

Foxa3, Gcnt3, Clca1, Agr2, Ptger3, and Muc16 were induced by SPDEF. SPDEF inhibited genes selectively expressed in normal airway epithelial cells including Abca3, Sftpa1, Sftpb, Sftpd and Foxa2 (A). Quantitative RT-

PCR was performed in triplicate using cDNAs obtained from bronchiolar cells by LCM. Spdef mRNA was

105 induced by doxycycline treatment (B). SPDEF induced Muc16 (C), Gcnt3 (D), Clca1 (E) and Ptger3 (F) mRNAs.

Results were expressed as the means ± S.D. of 3 independent mice of each treatment. *, p< 0.05 versus off doxycycline control littermates (Hsu’s MCB test). a.u.: arbitrary unit.

106

107

Figure 3 SPDEF or allergen sensitization induced FOXA3, AGR2 and UEA-I lectin staining, and inhibited

FOXA2 and TTF-1 staining in goblet cells in vivo. Expression of SPDEF in Scgb1a1-rtTA/TRE2-Spdef mice or intrapulmonary ovalbumin sensitization induced FOXA3 (A), AGR2 (B) and UEA-I lectin (E) staining, and decreased staining of FOXA2 (C) and TTF-1 (D) in goblet cells. Arrows indicate regions selected in the inserts.

Scale bar: 25 μm.

108

Figure 4 SPDEF and FOXA3 are co-expressed and induce Agr2 expression. Scgb1a1-rtTA/TRE2-Spdef

transgenic mice were treated with doxycycline for 3 days to induce SPDEF in bronchiolar epithelial cells (A-B).

SPDEF was colocalized with FOXA3 in nuclei (A) and AGR2 in the cytoplasm (B) of goblet cells as assayed by

immunofluorescence microscopy (yellow indicates colocalization of nuclear SPDEF and FOXA3). Luciferase

reporter constructs containing the promoter region from mouse Agr2 (1.6 kb) gene was transfected into primary

sheep tracheal epithelial cells. Synergistic activation of Agr2 promoter was observed when SPDEF and FOXA3

were co-transfected (C). Endogenous Agr2 mRNA expression was induced by transfection with both SPDEF and

109 FOXA3 expression plasmids in the mouse lung epithelial cell line, MLE15 (D). Results were expressed as the means ± S.D. of 3 independent experiments. *, p< 0.02 and **, p<0.01 versus control constructs. Scale bar 25 μm.

110

Figure 5 SPDEF, FOXA3 and AGR2 in human lung tissue. SPDEF, FOXA3 and AGR2 were detected by

immunostaining of human lung tissue. Tissue from a pediatric patient with bronchiolitis obliterans lacked goblet

cells; SPDEF, FOXA3 and AGR2 staining were absent (A). In normal lung, SPDEF staining was not detected

and FOXA3 staining was observed in relatively few bronchial epithelial cells in regions lacking goblet cells,

where AGR2 was weakly expressed (B, n=3). Intense SPDEF staining was seen in bronchial tissue from patients

with cystic fibrosis (CF, n=5), particularly in regions of goblet cell hyperplasia (top panel of C) as indicated on

adjacent section stained with Alcian blue (top panel of D), and in tissue from patients with history of chronic

smoking (top panel of E). FOXA3 staining was also increased in the nuclei of goblet cells from lung tissues of

CF patients (n=4) and patients with the history of chronic smoking (middle panel of C, D and E, respectively).

AGR2 was increased in bronchial epithelial cells of CF patients’ and smokers’ lungs (lower panel of C, D and E, respectively). SPDEF, FOXA3 and AGR2 were detected in mucous cells of bronchial submucosal glands of normal human lung (F, n=3), as well as in smokers’ lung (data not shown). Arrows indicate regions selected in the inserts. White and black scale bars: 25 μm.

111

Figure 6 Absence of mucous (goblet) cells in tracheal and laryngeal submucosal glands in Spdef -/- mice.

Tracheal/laryngeal glands in wild type mouse are shown after hematoxylin-eosin (H&E, and Alcian blue staining.

Distinct mucous cells (black arrow heads in A) and serous cells (red arrows in A and B) are shown from wild type mice. Mucous cells were not detected in the submucosal glands of Spdef -/- mice, although serous glands were present (B). Alcian blue staining was readily detected in submucosal glands of wild type mice (arrow) (C), but rarely observed in submucosal glands of Spdef -/- mice (arrow) (D). Arrows indicate regions of higher magnification shown in inserts (C, D). Figures are representative of n=3 individual mice of each genotype. Scale bar: 50 µm.

112

113 Figure 7 SPDEF is required for goblet cell differentiation following intrapulmonary allergen sensitization. After

intrapulmonary sensitization with ovalbumin (as described in Figure 1), SPDEF was readily detected in the

bronchiolar epithelial cells in Spdef +/- but not in Spdef -/- mice. The increased Alcian blue, MUC5AC, FOXA3 and UEA-I lectin staining in goblet cells after ovalbumin sensitization seen in Spdef +/- mice was markedly

inhibited in the Spdef -/- littermates. Arrows indicate regions selected in the inserts. All scale bars: 50 μm.

Figures are representative of n=4 mice of each genotype.

114

Figure 8 SPDEF induced MUC5AC mRNA and protein expression. (A) SPDEF induced MUC5AC mRNA expression in NCI-H292 cells in vitro. Lentivirus expressing mouse SPDEF or GFP (control) protein was used to infect H292 cells. After 5 days, MUC5AC mRNA was increased approximately 12 fold in the cells expressing

SPDEF compared to uninfected cells or those expressing GFP. Expression of SPDEF is shown by Western blot the lower panel. Graphs were expressed as the means ± S.D. of 3 independent experiments. *, p< 0.01 versus control virus. (B) SPDEF induced MUC5AC expression in HBECs. Cells were infected with lentivirus expressing SPDEF or control virus (same as in panel a) followed by culture at air-liquid interface for 4 weeks, and assayed by immunohistochemistry. MUC5AC was induced by expression of SPDEF. Figures are the representatives of 3 independent experiments. Scale bar: 25 µm.

115

Figure 9 Schematic representation of genomic responses induced by conditional expression of SPDEF in the airway epithelium. SPDEF promotes goblet cell differentiation and mucus production, while suppressing expression of genes associated with Clara cells. SPDEF interacts in a regulatory network mediated, in part, by the inhibition of FOXA2 and TTF-1 and the induction of FOXA3. SPDEF is induced, while FOXA2 is inhibited by pulmonary allergen or IL-13 in a STAT6 dependent manner (A). SPDEF induced the expression of a number of genes regulating mucin biosynthesis, particularly mucin glycosylation (B) and goblet cell differentiation (C),

116 while suppressing those regulating fluid and electroylte transport, and innate host defense in part by its inhibitory effects on TTF-1 and FOXA2 transcription factors that control differentiation and function of the normal bronchiolar epithelium (D). The network indicates a regulatory relationship but does not imply direct transcriptional control of each gene by SPDEF or other transcription factors.

117

Supplementary Figure 1 Absence of proliferation during goblet cell hyperplasia induced by SPDEF or ovalbumin sensitization. Scgb1a1-rtTA/TRE2-Spdef mice were treated with doxycycline for 3 days, as described in Figure 1D. Wild type mice were sensitized with ovalbumin. Cell proliferation was assayed by detection of

BrdU uptake. BrdU was administered daily by i.p. injection during treatment with doxycycline and nasal sensitization with ovalbumin (day 24 through day 29, Figure 1B). Goblet cell differentiation indicated by Alcian blue staining was induced in both models. Neither SPDEF nor ovalbumin sensitization increased phosphohistone

118 H3 (pHH3) or BrdU staining in goblet cells. Intestinal tissue collected from the same animal receiving BrdU

substrate served as a positive control for proliferation (lower panels). All scale bars: 25 μm.

Supplementary Figure 2 Isolation of bronchiolar cells using laser capture microdissection (LCM).

Immunofluorescence staining of SPDEF (red in A) in bronchiolar epithelial cells is shown after the Scgb1a1-

rtTA/TRE2-Spdef transgenic mice were treated with doxycycline for 3 days (A). Tissue was counterstained with

DAPI to detect nuclei (blue in A). Adjacent lung sections were used for LCM as shown in (B-D). After

dehydration of the 10 μm frozen sections (B), bronchiolar cells were isolated on the laser caps (C). Tissue remaining after removal of airway cells by LCM is shown in (D). Scale bar 50 μm.

119

Supplementary Figure 3 Pulmonary ovalbumin sensitization caused pulmonary inflammation in the presence or

absence of SPDEF. (A) Eosinophil infiltration was observed in both Spdef +/- and Spdef -/- mice as revealed by

Major Basic Protein (MBP) staining, a eosinophil specific marker. Goblet cell differentiation was observed in

Spdef +/- but not in Spdef -/- mice. (B) Monocytes and macrophages were recruited to the lungs of both Spdef +/- and Spdef -/- mice, as indicated by CD68 staining. Scale bar: 25 µm.

120

Supplementary Figure 4 IL-13 induces SPDEF in primary mouse tracheal epithelial cells in vitro.

Immunohistochemical staining of SPDEF in primary mouse tracheal epithelial cells cultured under air-liquid interface (ALI) condition in the presence or absence of IL-13 (10 ng/ml). Expression of SPDEF was detected after 3 days (upper panel) and 7 days (lower panel) after ALI culture. Scale bar: 25 µm.

121 Chapter IV General Conclusion and Discussion

Foxa2 is the transcription factor normally expressed in the epithelium lining the conducting airways and peripheral type II cells in the lung. Deletion of Foxa2 allele in respiratory epithelium by

SFTPC promoter driven Cre recombinase during embryonic stage (E6.5-E12.5) caused goblet cell metaplasia in the neonatal mice. In the current thesis, a novel role of Foxa2 in regulating Th2 cell- mediated innate immunity was discovered. Expression of Foxa2 in respiratory epithelium is required for suppression of Th2 cell-mediated inflammation and goblet cell metaplasia in the neonatal lung. Loss of

Foxa2 induced an IL-4Rα dependent Th2 inflammation via recruitment and activation of mDCs, Th2 cells, eosinophils, production of Th2 cytokines/chemokines, including IL-13 and IL-4 that induce expression of Spdef and goblet cell differentiation. In the mature lung, Foxa2 inhibits Spdef expression and goblet cell differentiation induced by pulmonary allergen exposure, demonstrating that Foxa2 and

Spdef interact in a genetic network that regulates goblet cell differentiation.

Previous studies showed that Spdef was the transcription factor that promoted goblet cell differentiation in the conducting airways. The present thesis identified a transcriptional network that was regulated by Spdef. Spdef is sufficient to induce normal Clara cell differentiate into goblet cell in the absence of allergen exposure or inflammation in a reversible process once Spdef expression is inhibited.

To induced transdifferentiation from Clara cells to goblet cells, Spdef inhibits transcription factors

Foxa2 and TTF-1 and their target genes that are expressed in Clara cells, including Sftpa1, Sftpd, Por.

On the other hand, Spdef induces expression of mucin genes (Muc16, MUC5AC) and others that are required for mucin packaging (Agr2), and glycosylation (Gcnt3) to promote goblet cell differentiation.

Lack of Spdef causes absence of mucus cell in submucosal glands, and inhibits goblet cell metaplasia

122 and mucus production after allergen exposure in the respiratory tract of Spdef -/- mice, demonstrating

that Spdef is both sufficient and required for goblet cell differentiation in vivo.

Foxa2 regulates Th2-cell mediated inflammation in the neonatal lung.

Loss of Foxa2 in respiratory epithelium induced production of Th2 cytokines in the

neonatal lung. Loss of Foxa2 in the respiratory epithelium induced expressing a number of genes that encoded Th2 cytokines and their receptors, including IL-13, IL-4, IL-5, IL-13Rα1, IL-13Rα2 and IL-4Rα.

These genes are also involved in allergic airway inflammation which is characterized by recruitment and

activation of Th2 lymphocytes and eosinophils, goblet cell metaplasia, and mucus hyperproduction.

Among all the Th2 cytokines, IL-13 is the one that necessary and sufficient to induce major features of

allergic asthma (1). IL-13 administration in mouse respiratory tract is sufficient to induce marked goblet

cell metaplasia and mucus plugging of the airways, the thickened airway smooth muscle layer, and the

infiltration of inflammatory cells around the airway wall (2). IL-13 deficient mice failed to develop

allergen induced goblet cell metaplasia and airway hyperresponsiveness, despite the presence of

vigorous Th2-biased, eosinophilic pulmonary inflammation (3). Therefore, deletion of Foxa2 caused

Th2 inflammation and goblet cell metaplasia is mediated by, at least partially, by the effect of IL-13 in

the lung.

Deletion of Foxa2 caused eosinophilic inflammation in the neonatal lung. Eosinophila is one

of the major features of allergic asthma, and eosinophils contribute to the pathogenesis of asthma by

releasing highly basic and cytotoxic proteins such as major basic protein. Eosinophils are recruited to

mucosa via the interaction between chemokine receptor CCR3 and chemoattractant eotaxin1 (CCL11)

and eotaxin 2 (CCL24) (4). Respiratory epithelial cells produce both eotaxin 1 and 2 in response to Th2

123 cytokine (IL-4 and IL-13) stimulation (5). Loss of Foxa2 in respiratory epithelium caused severe

eosinophilic inflammation in the neonatal lung. In consistent with this phenotype, genes responsible for

chemoattraction for eosinophils and eosinophil-associated genes were induced. Ccl11, Ccl24 and Ccr3

were increased 7, 12 and 4 folds, respectively. Epx mRNA (eosinophil peroxidase), a heme-containing

glycoprotein that may contribute to the pathogenesis of epithelial damage and bronchial hyperreactivity

in human asthma (6), was markedly increased in Foxa2Δ/Δ mice. Eosinophil major basic protein (Prg2,

11 fold) and eosinophil associated ribonuclease (Ear11, 90 fold) mRNAs were also dramatically

increased.

Increased production of epithelial cell derived cytokines and chemokines in lungs of Foxa2

Δ/Δ mice Of genes expressed in the respiratory epithelium, the cytokine gene Il33, the chemokine genes

Ccl17 and Ccl20 were significantly induced after deletion of Foxa2. IL-33 and CCL17 both promote

Th2 cytokine responses (7, 8) and are expressed in respiratory epithelial cells. CCL17 attract Th2 lymphocytes to mucosal sites (9) and is increased in the bronchial epithelium of asthmatic patients (10).

The present finding, that Tarc promoter activity in MLE-15 cells was inhibited by Foxa2 in vitro, provides a potential mechanism by which Foxa2 influences Th2 cell recruitment and activation in the lung. CCL20 is a chemokine produced in epithelial cells and capable of inducing DC migration via interaction with CCR6 expressed on the immature DCs (11, 12). Ccl20 mRNA expression was induced in the lungs of Foxa2Δ/Δ mice at PN15, suggesting a potential mechanism by which dendritic cells were

recruited and activated in the lungs of Foxa2Δ/Δ mice.

Foxa2 and hygiene hypothesis Discovery of the suppressive role of Foxa2 in Th2 inflammation provided a potential alternative mechanism underlying pathogenesis of asthma in young children, and supported the “hygiene hypothesis”. The hygiene hypothesis states that a lack of early childhood exposure to infectious agents, symbiotic microorganisms (e.g., gut flora or probiotics), and parasites

124 increases susceptibility to allergic diseases by modulating immune system development (13, 14). Future study using transgenic mice conditionally deleting only one Foxa2 allele (Foxa2 +/Δ) will be useful in

testing their susceptibility to spontaneous induction of Th2 inflammation and will be useful in

understanding how epithelial cells influence development of innate immunity in the neonatal lung.

Foxa2 and Spdef regulate innate host defense in the lung.

The neonatal immune system is immature as characterized by lack of Th1 cell function, but a

Th2 biased immune response (15). This feature of neonatal immune system makes newborns susceptible

to infection and allergic reactions (16). The limited Th1 response is due, in part, to delayed maturation

of the subset of DCs that produce IL-12 and lead to apoptosis of Th1 cells (15, 16).

Foxa2 influences expression of host defense molecules that contribute to Th2 immune

response. Loss of Foxa2 in the respiratory epithelium reduced expression of host defense protein

including surfactant protein A (SP-A), CCSP and lysozyme in vivo. Activating of Scgb1a1 (Scgb1a1

encodes CCSP) and lysozyme (both M and P subtypes) promoters was induced by Foxa2 in vitro (17, 18),

suggesting the critical role of Foxa2 in influencing host defense molecule expression. SP-A belongs to

the collectin family of the host defense proteins of the lung that remove pathogens. This process is

mediated through carbohydrate-dependent interaction, aggregating, opsonizing, and enhancing clearance

of the organisms by alveolar macrophages in the lung (19). The role of SP-A was recently reported to

inhibit Th2 response in the allergic asthma model (20). SP-A inhibited CD4+ lymophcyte proliferation

in a dose dependent manner in vitro and suppressed Aspergillus fumigates induced Th2 cytokine (IL-4

and IL-5) production in vivo (20). CCSP is a host defense molecule expressed by Clara cells. Expression

of CCSP was found to inhibit Th2 cell differentiation, decrease GATA3 expression (Th2 cell

125 transcription factor) and inhibit eosinophilic inflammation in a mouse allergic asthma model (21). The

polymorphism (A38G) in the CCSP protein was associated with asthma patients (22). In addition, CCSP

inhibits Th2 cell differentiation from naïve neonatal human T cells (23), suggesting the pivotal role of

CCSP in regulating neonatal innate immunity. So, loss of Foxa2 caused decreased expression of SP-A

and CCSP in turn enhancing Th2 cell differentiation and Th2 cytokine production that may influence

Th2 responses in the neonatal lung. The current evidence supports the concept that the neonatal innate

immunity is Th2 biased (16).

Spdef inhibits Foxa2 and TTF-1 expression, and influences innate host defense

Differentiation of respiratory epithelium is control by signaling pathways during lung development and

the transcription factors that regulate differentiation of epithelial cells from progenitors. Thyroid

transcription factor-1 (TTF-1) is a 43-kDa, a phosphorylated member of the Nkx2 family of

homeodomain-containing proteins, also termed as Nkx2-1. TTF-1 is detected in respiratory epithelial

cells lining the conducting airways and peripheral type II cells, and plays a critical role in the regulation of lung morphogenesis, epithelial cell differentiation, and the expression of genes upon which perinatal

respiratory adaptation depends (24). Mice containing mutant Titf-1 (the gene encoding TTF-1) allele, in

which seven serine phosphorylation sites were mutated, died immediately following birth (25). By

performing microarray analysis, a number of genes that maintain Clara cell characteristics and influence

innate host defense were found to be regulated by TTF-1, including Sftpa, Sftpb, Scgb1a1 and lysozyme

(25). Loss of Foxa2 in respiratory epithelium also caused reduced expressed of Sftpa, Sftpb and Scgb1a1

(17). Spdef mRNA was significantly induced (15~20 fold induction of mRNA) in TTF-1

phosphorylation mutant mice lung (http://research.cchmc.org/pbge/), and Spdef was also induced in goblet cells in the Foxa2 Δ/Δ mice. Spdef inhibits Foxa2 and TTF-1 mRNA and protein expression in the

goblet cells, suggesting a negative correlation between TTF-1, Foxa2 and Spdef in the lung. Spdef

126 inhibits Sftpa, CCSP, the host defense protein that play the suppressive roles in Th2 inflammation (20,

23), partially through the inhibition of TTF-1 and Foxa2.

In addition, Spdef inhibits Scnn1b and Scnn1g (TTF-1 regulated gene (25)), which encode the β

and γ subunits of the E-Nac protein, a Na+ channel in epithelial cells. Over-expression of Scnn1b driven by CCSP promoter caused increased airway Na+ absorption, which in turn led to airway surface liquid

volume depletion (dehydration), increased mucus concentration, delayed mucus transport and mucus

adhesion to airway surface (26). Half of the Scnn1b transgenic mice died within 3 week after birth, and

the surviving mice developed goblet cell metaplasia, mucus plugging, neutrophilic inflammation and

slow bacterial clearance, the phenotype of which similar to CF lung in human (26). Spdef inhibits both β

and γ subunits, suggesting that Spdef may influence function of E-Nac, including transepithelial Na+

transport in the airway epithelium, the airway surface liquid volume and regulation of mucociliary

clearance.

In summary, Spdef promotes goblet cell differentiation by inducing mucin biosynthesis and

inhibiting normal Clara cell differentiation, at least partially, by suppression of TTF-1 and Foxa2. TTF-1

and Foxa2 are the transcription factors that regulate expression of surfactant proteins and host defense

molecules in Clara cells, including Sftpa and CCSP. In addition, Spdef represses expression of β and γ

subunits of Na+ channel protein E-Nac that is expressed in the respiratory epithelial cells that in turn

regulates mucociliary clearance in the conducting airways.

The counter-regulatory roles of Spdef and Foxa2 mediate the transcriptional

network that is associated with goblet cell differentiation

127 Deletion of Foxa2 caused goblet cell metaplasia, and conversely, the expression of Foxa2 in

Clara cells inhibited goblet cell differentiation in response to pulmonary ovalbumin sensitization. The loss of Foxa2 enhanced expression of Spdef and Foxa3, transcription factors associated with both allergen and IL-13 induced goblet cell differentiation. Recent studies demonstrated that Spdef itself was sufficient to induce goblet cell differentiation (27, 28). Thus, Spdef and Foxa2 function in opposition in a transcriptional network regulating goblet cell differentiation from Clara cells. Expression of Spdef inhibited Foxa2 and induced Foxa3 in the respiratory epithelium, Spdef and Foxa3 transcriptionally regulated Agr2, the gene that was required for mucus production. In the present study, loss of Foxa2 also induced expression of mucin genes and proteins/genes associated with mucin biosynthesis and packaging, e.g., Spdef, Foxa3, Muc5ac, Agr2, and Clac3 (29, 30). This group of genes was induced during Th2-mediated inflammation in mouse asthma models using house dust mite or pulmonary ovalbumin exposure (28, 31, 32). Many of the genes associated with mucus production were induced by expression of Spdef in airway epithelial cells of transgenic mice; however, expression of Spdef did not cause lung inflammation (27, 28). Goblet cell differentiation caused by deletion of Foxa2 is likely driven in part by the enhanced activity of Spdef; however, Foxa2 plays an additional role in the suppression of lung Th2-like inflammation during postnatal lung development. Expression of Foxa2 in

Clara cells in transgenic mice inhibited Spdef, Foxa3, Muc5ac and goblet cell differentiation in response to allergens, consistent with a recent study that showed inhibition of mucus production by Foxa2 in vivo

(33). This same transcriptional circuit is operative after exposure of the lung to IL-13 or allergens, wherein Spdef is induced and Foxa2 is lost in a Stat6-dependent manner (18, 27). The increase in Th2 cytokine production (e.g. IL-4, IL-5 and IL-13) associated with deletion of Foxa2 likely plays an important role in the activation of goblet cell metaplasia seen in the present model.

128 Spdef regulates mucin gene expression, mucin biosynthesis and secretion to promote

goblet cell differentiation.

Spdef regulates mucin gene expression. Recent studies from our laboratory and others demonstrated that Spdef is a common transcription factor that is sufficient and required for goblet cell differentiation in respiratory and gastrointestinal tracts (27, 28, 34, 35). Mucus secreted by goblet cells is one of the components that participates mucociliary clearance, the host defense machinery to protect respiratory epithelium from injury and keep lung sterile. However, excessive mucus production by airway epithelium which causes airflow restriction and bacterial infection is associated with chronic pulmonary diseases like asthma, CDOP and CF. Mucus is a collective term of highly glycosylated mucin, lipids, soluble proteins, ions and water. Spdef was found to regulate mucin gene expression, mucin packaging and glycosylation, the process that was required for synthesis and secretion of the functional mucin. Spdef significantly induces expression of MUC5AC in vivo and in vitro (27, 28).

MUC5AC is the major gel-forming mucin that is dramatically induced and contributes to the pathogenesis of the chronic pulmonary diseases, like CF (36, 37), asthma (38), and COPD (39). Loss of

Spdef causes reduced Muc5ac expression in the airway epithelium after allergen exposure in vivo,

suggesting the Spdef is required for Muc5ac expression. However, Spdef does not activate the

MUC5AC promoter in vitro, and it takes 5 days to detect MUC5AC mRNA induction after infection of

Spdef expressing virus in H292 cells. Expression of MUC5AC protein was detected 4 weeks after

infection of Spdef expressing virus in HBECs. The current evidence indicates that Spdef may not

directly regulate MUC5AC gene expression through activation of its promoter. In addition, Spdef

induces Muc16 expression, the membrane tethered mucin that is normally expressed in human tracheal

bronchial epithelial cells and submucosal glands (40), as well as the primary tracheobronchial air-liquid

interface cell culture (41), suggesting the potential role of Spdef in regulating mucin expression in

129 normal epithelium. MUC2 is the most abundant mucin expressed in the gastrointestinal goblet cells (42).

Regulation of Muc2 expression in the gastrointestinal tract and colon epithelial cell line was recently

reported to be dependent on Spdef, in vivo and in vitro. Significantly loss of mature goblet cell and

associated Muc2 staining was observed in the small intestine of Spdef -/- mice (34). SPDEF shRNA

inhibited γ-secretase induced goblet cell differentiation in human colorectal cell line LS174T, and

repressed Muc2 mRNA induction (35). The genomic locus of Muc2 allele is located next to Muc5ac

(Muc2 and Muc5ac are both on the chromosome 7 F5 in mouse, and chromosome 11p15.5 in human)

and the distance between 2 genes is less than 0.1 Mb. Therefore, it is possible that expression of Spdef

causes this region of the genome accessible to tissue specific transcription factors to bind DNA and

initiate transcription (e.g. activate Muc5ac promoter in respiratory epithelium, but Muc2 promoter in

gastrointestinal epithelium).

Spdef and Foxa3 transactivate mucin chaperon protein Agr2 expression. In addition to its roles in regulating mucin gene expression, Spdef also influences expression of mucin chaperon protein

Agr2, a protein mediating correct folding of mucin protein backbones. Spdef induces Agr2 expression in the goblet cells in vivo, and Agr2 promoter is synergistically activated by Spdef and Foxa3 in vitro (28).

The present evidence suggests that the transcriptional activation of Agr2 gene is controlled by Spdef and its regulated gene Foxa3 in respiratory epithelium. Agr2 was identified as an endoplasmic reticulum (ER) protein that belongs to the protein disulfide isomerase family (PDI) of chaperones that facilitate the folding of proteins targeted for the secretory pathway (43). Agr2 binds to unfolded parts of mucin protein in the ER, to enhance protein folding and the posttranslational modification of serine and threonine residues, the sites of O-glycosylation in Golgi (30). Loss of Agr2 causes impaired mucin production (Muc2) and goblet cell maturation in the small intestine and colon (29), suggesting its requirement in goblet cell differentiation.

130 Spdef induces mucin glycosyltransferases expression. Glycosylation is the major post- translation modification of mucin, and this process is mediated by specific glycosyltransferase to transfer O-glycans to serine and theroine residues of mucin backbones. The diversity of glycosylation of airway mucins may be important in facilitating adherence of microorganisms to mucus prior to mucociliary clearance (44). Spdef significantly induced all three members in the protein glycosyltransferase C2GnT family that mediate production of functional mucins (Gcnt1, 2, and 3), strongly supporting its regulatory role in mucin biosynthesis and secretion. Among these enzymes,

Gcnt3 was induced the most by the expression of Spdef in the airway epithelium (9.3-fold). GCNT3, also named as C2GnT2, mediates both core 2 and core 4 O-glycan branching, two important steps in mucin-type biosynthesis (45). Gcnt3 mRNA is expressed primarily in mucus secreting tissues, including the gastrointestinal tract and trachea and is induced by retinoic acid, TNF-α and IL-13 (46, 47).

A recent study showed that loss of Gcnt3 in mouse caused increased mucosal permeability in the gastrointestinal epithelium, and impaired epithelial barrier function in the Gcnt3 -/- mice (48). Consistent

with the important role of Spdef in the regulation of mucin glycosylation, staining for UEA-I lectin

which binds to fucosylated protein, was induced by Spdef.

Taken together, Spdef is a transcription factor that regulates mucin biosynthesis in the airway

epithelium. Specifically, Spdef induces expression of mucin genes including Muc16, a gene expressed in

airway epithelium, and MUC5AC which is significantly induced by Th2 cytokines and activation of

EGFR in chronic pulmonary diseases. Spdef promotes mucin packaging and folding by inducing Agr2, a

protein essential for mucus production and goblet cell differentiation. In addition, Spdef regulates mucin

post-translation modification of mucins by influencing expression of 3 major glycosyltransferases

(Gcnt1,2,3) in airway epithelium.

131 Interaction of Spdef and IL-13, EGFR signaling pathways

Spdef is induced at an IL-13/IL-4Rα/Stat6 dependent manner in goblet cells. IL-13 and

EGFR signaling are two major pathways that mediate goblet cell differentiation in asthma and COPD,

respectively. Although function through the same receptor subunit IL-4Rα, IL-13 binds to the type II

receptor (heterodimer of IL-13Rα1 and IL-4Rα), and promotes goblet cell metaplasia and development

of airway hyperresponsiveness, while IL-4 binds to both type I receptor (heterodimer of IL-4Rα and γC)

and type II receptor to regulate IgE production and Th2 cell proliferation and differentiation (49).

Induction of Spdef is mediated by Stat6 following IL-13 or allergen exposure in vivo (27), and

administration of IL-13 in the culture media is sufficient to induce Spdef expression (both mRNA and

protein) in the primary mouse tracheal epithelial cells in vitro (28). Loss of IL-13 inhibited both Spdef

mRNA and protein expression and goblet cell differentiation after ovalbumin or house dust mite

exposure in IL-13 -/- mice, suggesting that IL-13, but not IL-4, was required for Spdef expression (27) and (NCBI GEOprofiles, GDS958). Blockade of IL-4 receptor alpha that mediates signal from both IL-

13 and IL-4 by treatment of IL-4Rα monoclonal antibody is sufficient to inhibit goblet cell differentiation and associated Spdef induction caused by Th2 inflammation. The current evidence supports that Spdef is induced by IL-13/IL-4Rα/Stat6 signaling axis in the airway epithelial cells.

Spdef expression and goblet cell differentiation are EGFR activation independent following

allergen sensitization. Activation of EGFR via various ligands is major cause of goblet cell differentiation and mucus production in COPD and CF, which is induced by neutrophilic, but not Th2 cell-mediated eosinophilic inflammation in the lung (50, 51). Previous studies using human lung epithelial cell line H292 demonstrated that activation of EGFR by EGF or TGF-α was sufficient to induce MUC5AC expression (52). Instillation of TGF-α was able to induce goblet cell differentiation.

This process was blocked by EGFR inhibitor (EGFR tyrosine kinase inhibitor BIBX1522) in rat

132 respiratory tract in vivo (53). Spdef is sufficient to induce MUC5AC mRNA in H292 cells, and SPDEF is expressed in the goblet cells of CF patients and chronic smokers (28), suggesting that SPDEF contributes to the goblet cell differentiation in these diseases. However, endogenous SPDEF expression was not significantly induced by EGFR activation. Treatment of TGF-α, the EGFR ligand, caused marginal increase of SPDEF mRNA (2-fold) in H292 cells, and there was no synergistic effect on

MUC5AC mRNA induction by infection of SPDEF lentivirus and treating with TGF-α together in H292 cells (data not shown). The EGFR inhibitor Tarceva, the medicine used in treatment of non-small cell lung cancer and pancreatic cancer, is effective to block TGF-α induced lung fibrosis in mice (54).

However, Tarceva failed to inhibit goblet cell differentiation or Spdef induction following house dust mite exposure in mice in vivo (data not shown), indicating that goblet cell differentiation and Spdef expression is independent of activation of EGFR in Th2 inflammation. It is currently unclear the mechanism and genetic pathway that induce Spdef expression in goblet cells induced by activation of

EGFR.

In summary, Spdef is induced by Th2 cytokine IL-13 at an IL-4Rα and Stat6 dependent manner.

Spdef induction is independent of activation of EGFR, and SPDEF is sufficient to drive MUC5AC expression in the absence of IL-13 or inflammation in vivo (27)and in vitro (28).

133 Conclusion

In conclusion, Foxa2 is required for

suppression of Th2 inflammation, goblet cell

metaplasia and expression of Spdef and

Foxa3 in the neonatal lung. Selective

deletion of Foxa2 in respiratory epithelium

induces recruitment and activation of mDCs

and Th2 cells (Figure 1A), causing increased

Figure 1 Epithelial cell transcription factors Foxa2 production of Th2 cytokines and chemokines and Spdef mediate a network that is associated at an IL-4Rα dependent manner. The current with regulation of innate immunity and goblet cell differentiation in the lung. Foxa2 is required for findings provided new evidence that the inhibition of mDC and Th2 cell mediated pulmonary inflammation, and goblet cell metaplasia in the innate immune system is strongly determined developing lung (A). Foxa2 is required for maintaining Clara cell gene expression (E), and by respiratory epithelial cells during early inhibits Spdef (B) and its mediated goblet cell neonatal period via the transcription factor differentiation (C). Spdef suppresses Foxa2 (D) and transdifferentiates Clara cell to goblet cell by Foxa2, a process that is relevant to the inducing expression of mucin biosynthesis associated genes (C) pathogenesis of asthma and other inflammatory lung diseases. Studies of polymorphisms in the Foxa2 locus and its association with asthma susceptibility, particularly in newborns, would be informative in understanding the genetic processes involved in the pathogenesis of allergic asthma. Expression of Foxa2 is required for Clara cell differentiation, inducing expression of Clara cell genes (CCSP, Sftpa) during development (Figure 1E).

Foxa2 inhibits Spdef (Figure 1B), Foxa3 and goblet cell differentiation following Th2 cytokine sensitization (Figure 1C), suggesting that Foxa2 and Spdef play opposing roles in regulating goblet cell differentiation.

134 Spdef regulates a transcriptional network that promotes goblet cell differentiation by inducing mucin genes and others required for mucin biosynthesis, meanwhile inhibits genes maintain normal

Clara cell differentiation, partially via suppression of transcription factors Nkx2-1 and Foxa2 (Figure

1D). Spdef is required for mucous cell differentiation in submucosal glands, and goblet cell metaplasia and mucus production after allergen exposure in mouse model. Thus, Spdef and Foxa2 play central roles in the regulation of a genetic network that responds to pathogens or toxicants, in turn changing epithelial cell differentiation and mucociliary clearance that together play a role in innate host defense of the lung.

Since mucus hyperproduction contributes to the pathogenesis of acute and chronic pulmonary disorders, knowledge regarding the regulation and function of Spdef and Foxa2 in the respiratory tract provides a framework for the development of new strategies for diagnosis and therapy for chronic lung diseases in children and adults worldwide.

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