SAM-Pointed Domain ETS Factor Mediates Epithelial Cell–Intrinsic Innate Immune Signaling During Airway Mucous Metaplasia

SAM-pointed domain ETS factor mediates epithelial cell–intrinsic innate immune signaling during airway mucous metaplasia

Thomas R. Korfhagena, Joseph Kitzmillera, Gang Chena, Anusha Sridharana, Hans-Michael Haitchid, Rashmi S. Hegdeb, Senad Divanovicc, Christopher L. Karpc,1, and Jeffrey A. Whitsetta,2

aDivisions of Neonatology, Perinatal, and Pulmonary Biology, bDivision of Developmental Biology, and cDivision of Molecular Immunology, Cincinnati Children’s Hospital Medical Center and the University of Cincinnati College of Medicine, Cincinnati, OH 45229; and dFaculty of Medicine, University of Southampton, Southampton, S017 1BJ, United Kingdom

Edited by Michael J. Welsh, Howard Hughes Medical Institute, Iowa City, IA, and approved August 29, 2012 (received for review May 14, 2012)

Airway mucus plays a critical role in clearing inhaled toxins, specification, and resolution after infection (8). Dysregulation of particles, and pathogens. Diverse toxic, inflammatory, and in- any of these parameters can impair gas exchange, either acutely fectious insults induce airway mucus secretion and goblet cell or through the destructive, chronic remodeling processes that metaplasia to preserve airway sterility and homeostasis. However, often occur when pulmonary infection and inflammation fail goblet cell metaplasia, mucus hypersecretion, and airway obstruc- to resolve. tion are integral features of inflammatory lung diseases, including The barrier, clearance, and hydration functions of airway asthma, chronic obstructive lung disease, and cystic fibrosis, which mucus, produced by secretory epithelial cells lining the airways cause an immense burden of morbidity and mortality. These chronic (as well as by submucosal glands in large airways), play critical fl lung diseases are united by susceptibility to microbial coloni- roles in protecting the lung from microbial, toxic, and in am- zation and recurrent airway infections. Whether these twinned matory injury (1). Exposure to such insults leads to induction of phenomena (mucous metaplasia, compromised host defenses) are airway mucus production along with recruitment of mucus se- causally related has been unclear. Here, we demonstrate that SAM cretion capacity, the latter through submucosal gland hyperplasia pointed domain ETS factor (SPDEF) was induced by rhinoviral as well as through mucous metaplasia, which is the rapid, re- versible differentiation of basal cells and other secretory cell infection of primary human airway cells and that cytoplasmic ac- progenitors into airway goblet cells. A transcriptional program tivities of SPDEF, a transcriptional regulator of airway goblet cell driving airway mucous metaplasia in the airway epithelium has metaplasia, inhibited Toll-like receptor (TLR) activation of epithe- recently been defined. SAM pointed domain ETS factor (SPDEF) lial cells. SPDEF bound to and inhibited activities of TLR signaling is critical to this process, functioning in a cell-autonomous manner adapters, MyD88 and TRIF, inhibiting MyD88-induced cytokine β in the respiratory epithelium. Increased SPDEF expression is production and TRIF-induced interferon production. Conditional closely associated with mucus cell metaplasia and mucus hyper- expression of SPDEF in airway epithelial cells in vivo inhibited LPS- production in lungs of asthmatics, patients with cystic fibrosis, fi induced neutrophilic in ltration and bacterial clearance. SPDEF- and those with chronic obstructive lung disease and in mouse mediated inhibition of both TLR and type I interferon signaling models of allergic pulmonary inflammation, wherein SPDEF is likely protects the lung against inflammatory damage when incit- induced by TH2-cytokine signaling via Stat6 (9). SPDEF is both ing stimuli are not eradicated. Present findings provide, at least in necessary and sufficient to regulate a transcriptional network part, a molecular explanation for increased susceptibility to infec- that drives the expression of genes regulating goblet cell differ- tion in lung diseases associated with mucous metaplasia and a mech- entiation, including those mediating mucin production, glycosyl- anism by which patients with florid mucous metaplasia may tolerate ation, packaging, and secretion and those inhibiting the expression microbial burdens that are usually associated with fulminant in- of genes characteristically expressed by normal airway epithelial flammatory disease in normal hosts. cells (9, 10). Although likely beneficial in microbial clearance in the short airway epithelium | innate immunity term, when chronic, airway mucous metaplasia and hypersecretion are thought to contribute to the pathogenesis of diverse fl or efficient gas exchange to occur, the lung maintains an in ammatory diseases, including chronic obstructive pulmonary ∼ 2 disease, bronchiectasis, asthma, and cystic fibrosis. Paradoxically, Fenormous ( 100 m in humans), thin (two cells thick, from fl bloodstream to airstream) membrane in direct contact with the despite ongoing in ammation, patients with both acute and chronic outside environment. This interface is susceptible to compromise disease associated with mucous metaplasia exhibit heightened by the destructive power of microbes introduced with ∼10 L/min susceptibility to microbial colonization and infection of the airway. Likewise, secondary or mixed viral and bacterial infections of inspired air. The lung is just as susceptible to compromise by – the destructive power of inflammatory responses generated in are common complications in chronic lung diseases (11 13). The response to microbial insult. It is thus unsurprising that the air- present study demonstrates that the Ets family transcription ways and alveoli of the lung are equipped with a wide array of factor SPDEF, a transcriptional regulator required for airway innate immune defenses that facilitate microbial clearance and mucous metaplasia, plays an unexpected role in suppressing killing without inducing inflammation, including mucociliary clearance, abundant noninflammatory macrophages, and the constitu- tive (and inducible) presence of diverse opsonic and antimicrobial Author contributions: T.R.K., J.K., G.C., H.-M.H., R.S.H., S.D., C.L.K., and J.A.W. designed proteins in airway fluids (1–3). In turn, immune activation is tightly research; J.K., G.C., and A.S. performed research; T.R.K., J.K., R.S.H., C.L.K., and J.A.W. regulated in the lung by a variety of mechanisms (4–7). When analyzed data; and T.R.K., J.K., R.S.H., C.L.K., and J.A.W. wrote the paper. pulmonary inflammatory responses are necessary, their class, The authors declare no conflict of interest. amplitude, and time course are kept under tight control in the This article is a PNAS Direct Submission. lung. In all of this, airway epithelial cells play a central role, effecting 1Present address: The Bill & Melinda Gates Foundation, Seattle, WA 98102. mucociliary clearance, acting as critical sentinels and effectors of the 2To whom correspondence should be addressed. E-mail: [email protected]. pulmonary innate immune system, and interacting in complex ways This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. with hematopoietic cells to regulate immune activation, class 1073/pnas.1208092109/-/DCSupplemental.

16630–16635 | PNAS | October 9, 2012 | vol. 109 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1208092109 Downloaded by guest on September 30, 2021 innate immune signaling, suggesting a molecular mechanism that immune function could occur either indirectly, such as through could inhibit inflammation while contributing to susceptibility to rheologic alteration to influence the access of exogenous stimuli infection associated with chronic airway disease. to responding cells related to changes on the mucus barrier or directly, such as through alteration of the responsiveness of Results airway epithelial cells to LPS (e.g., by regulating Muc1 expres- SPDEF-Driven Mucous Metaplasia Blunts Lipopolysaccharide-Driven sion, a known regulator of TLR signaling) (15). However, Inflammation in the Airway. The susceptibility to pulmonary in- SPDEF did not induce Muc1 mRNA in airway epithelial cells in fection associated with chronic airway diseases, together with the the mouse (10). To test whether SPDEF directly influenced TLR centrality of airway epithelial cells to host defense of the lung, signaling, we examined the effect of SPDEF on TLR signaling in suggested that mucous metaplasia might directly modulate the HEK293T cells, which are tractable cells with fully functional immune responsiveness of airway epithelial cells. To address this, TLR signaling machinery that have been extensively used to we used transgenic mice with conditional expression of SPDEF identify components of the complex TLR signaling pathways under control of the Clara cell secretory protein promoter driven (16). Expression of SPDEF in HEK293T cells caused dose-de- reverse tetracycline transactivator (Scgb1a1[CCSP]-rtTA/Tre2- pendent inhibition of a nuclear factor (NF)-κB reporter con- Spdef mice). Treatment of these mice with doxycycline selectively struct, pELAM luc, in response to TLR2, TLR3, TLR4, and drives robust goblet cell metaplasia in the conducting airways (9) TLR5 stimulation (Fig. 3 A–D). Because SPDEF inhibited sig- without the confounding effects associated with airway in- naling via multiple TLRs, we tested whether it influenced the flammation caused by noxious external stimuli that are required activity of the TLR/IL-1/resistance (TIR)-containing adaptor to induce mucus production in the murine airway. In the normal proteins, MyD88 and TRIF (17). Of note, SPDEF expression mouse, Toll-like receptor (TLR) challenge of the airway leads to significantly suppressed NF-κB transactivation in response to a brisk, neutrophilic inflammatory response (Fig. 1). In mice MyD88 in both HEK293T (Fig. 3E) and primary human bron- expressing SPDEF in the respiratory epithelium, mucous meta- chial epithelial cells (Fig. 3F) and significantly suppressed both plasia was associated with significant inhibition of production of NF-κB– and IFN response factor–dependent promoter trans- the neutrophil chemokine, KC, and neutrophil infiltration into activation in response to TRIF expression in HEK293T cells the airways in response to both acute and subacute repeated (Fig. 3 G and H). Consistent with these inhibitory effects, challenge of the airway with LPS (Fig. 1 A–C and Fig. S1). The SPDEF significantly inhibited IL-8 mRNA in response to LPS reverse experiment, lipopolysaccharide (LPS) challenge of air- stimulation or MyD88 expression in HEK293T cells and inhibi- − − ways of Spdef / mice (10) is impractical, because wild-type ted IL-6, IL-8, and IL-1β proinflammatory cytokine mRNA ex- controls kept under specific pathogen-free conditions do not pression in A549 cells, a human airway epithelial cell line (Fig. have airway mucous metaplasia and lack SPDEF expression. 4). SPDEF also inhibited IFN-β mRNA expression in response MEDICAL SCIENCES Furthermore, known strategies to induce goblet cell metaplasia, to expression of TRIF in HEK293T cells (Fig. 4F). Thus, SPDEF e.g., expression of Th2-cytokines or pulmonary allergic sensiti- caused cell-autonomous inhibition of TLR signaling in HEK293T zation, require chronic inflammatory stimuli, and the associated cells, in primary, nontransformed bronchial epithelial cells, and inflammation influences innate defenses. SPDEF inhibited LPS- in A549 adenocarcinoma cells, supporting its counter-regulatory induced inflammation, demonstrating that SPDEF-induced mu- role in innate immune activation in respiratory epithelial cells. cous metaplasia plays a counter-regulatory role in restraining innate immune responses to TLR challenge of the airway. SPDEF Interacts Directly with MyD88 and TRIF. The ability of SPDEF to restrain signaling driven by expression of MyD88 and TRIF SPDEF Inhibits TLR Signaling in Airway Epithelial Cells. SPDEF ex- suggested that the locus of SPDEF-mediated inhibition of TLR pression and mucous metaplasia are induced both by interleukin signaling was at the level of, or downstream of, these critical (IL)-13 in Th2-mediated inflammatory processes, after loss of adaptor proteins. In addition to the four signaling TIR-con- FOXA2, and in clinical settings associated with chronic lung taining adaptors (MyD88, TRIF, TRAM, and Mal), a fifth TIR- inflammation (9, 10, 14). We found that mucous metaplasia, containing adaptor protein, SARM, negatively regulates TLR SPDEF, and genes associated with mucus hyperproduction were signaling via effects on both TRIF and MyD88 (18, 19). In the markedly induced by IL-13 in primary human airway epithelial case of TRIF, direct interactions with SARM have been dem- cells in vitro (Fig. 2 A–E). Likewise, infection by rhinovirus in- onstrated by coimmunoprecipitation techniques (18). Intrigu- duced SPDEF and MUC5AC mRNAs (Fig. 2F). This association ingly, the SAM domain of SPDEF exhibits considerable se- between SPDEF and mucous metaplasia is consistent with pre- quence homology with the SAM domain of SARM, suggesting vious studies in murine models of experimental asthma and the possibility of direct interactions between SPDEF and these immunohistochemistry in lung tissue from patients with asthma, adaptor proteins. Direct association of SPDEF with MyD88 was cystic fibrosis, and smoking habits (9, 10). The effects of SPDEF demonstrated by bidirectional coimmunoprecipitation in lysates and associated mucous metaplasia on airway epithelial innate of HEK293T cells coexpressing epitope-tagged SPDEF along

Fig. 1. Mucus metaplasia induced by SAM pointed domain ETS factor (SPDEF) restrains airway neutrophilic inflammation of the airway. Mice were treated with doxycycline in utero and thereafter until sacrifice at 8–10 wk of age. Lipopolysaccharide (LPS) (20 μg) or an equivalent volume of phosphate-buffered saline (PBS) was administered intratracheally to mice with quantification of the following in bronchoalveolar lavage fluid 16 h later: (A) Total cells. (B) Total neu- trophils. (C) Keratinocyte chemoattractantdetermined by enzyme-linked immunosorbent assay. Data represent means ± SEM, two experiments, n = 8 (LPS) or 3 (PBS) per condition. White bars represent Scgb1a1-rtTA mice; black bars represent Scgb1a1-rtTA/Tre2-Spdef mice.*P < 0.00001, **P = 0.025. Student’s two-sample t test.

Korfhagen et al. PNAS | October 9, 2012 | vol. 109 | no. 41 | 16631 Downloaded by guest on September 30, 2021 Fig. 2. Interleukin (IL)-13 and rhinovirus induce SAM pointed domain ETS factor–and mucus-associated genes in primary human airway epithelial cells (HAECs). HAECs cultured at ALI were treated with IL-13 for 5 d and stained with Alcian Blue (A and B) and for MUC5AC (C and D). mRNA in HAECs cultured at ALI at 5 d of IL-13 treatment was assessed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) (E and F). White bars represent cells treated without IL-13, black bars those treated with 10 ng/mL of IL-13. Experiments were performed in at least triplicate using independent donor samples (n ≥ 3), mean fold change ± SEM. HAEC ALI cultures from normal donor samples were exposed to rhinovirus RV16 and mRNA assessed by qRT-PCR 48 h after infection. White bars represent un- infected cells, black bars cells infected with rhinovirus at a multiplicity of infection of 5. Data are mean ± SEM from two independent donor samples, with 2–3 technical replicates per condition. *P = 0.001, **P = 0.04, ***P = 0.05. Student’s t test.

with MyD88 (Fig. 5A). Direct association of SPDEF with TRIF was shown by similar means (Fig. 5C). Confocal microscopy revealed cytoplasmic colocalization of SPDEF with MyD88 (Fig. 5B) and TRIF (Fig. 5D) in HEK293T cells. Coimmunoprecipi- tation and confocal microscopy revealed that coexpression with MyD88 and TRIF led to increased SPDEF concentrations in these cells. Thus, SPDEF can interact directly with MyD88 and TRIF in the nonnuclear compartment, a finding that supports the concept that SPDEF may restrain inflammation via Fig. 3. SAM pointed domain ETS factor (SPDEF) inhibits Toll-like receptor interactions with these adaptor proteins, thereby limiting TLR (TLR) signaling. HEK293T cells (A, E, G,andH) were cotransfected with signaling. We also tested whether MyD88 might influence tran- reporters (pELAM-luc or pISRE-luc and pRL-TK) and plasmids described for each condition. Effects of SPDEF expression plasmids were assessed by scriptional activity of SPDEF on the Agr2 promoter in trans- luciferase assay. Thirty hours after transfection, cells were stimulated with fection experiments in BEAS2B cells. Although SPDEF and TLR ligand or with phosphate-buffered saline for 18 h. (A)Cellswere SPDEF in the presence of FOXA3 induced the Agr2 promoter in cotransfected with plasmids expressing TLR4, CD-14, and MD-2 with or vitro, MyD88 did not alter these responses (Fig. S2). As might be without SPDEF and treated with 10 ng/mL Escherichia coli lipopolysac- expected, initial mutational analysis of SPDEF indicated a clear charide, four experiments, n = 10–12 per condition. (B) HEK293/TLR2/CD- role for the SAM domain of SPDEF in suppression of MyD88- 14 cells were transfected with or without SPDEF and exposed to 100 ng/mL dependent signaling (Fig. S3). In structural studies, six MyD88 Pam3Cys, two experiments, n = 6 per condition. (C) HEK293/TLR5 cells molecules oligomerize and form a helical tower with IRAK4 and were transfected with or without SPDEF and treated with 100 ng/mL fla- IRAK2 (20). SAM domains are also known to oligomerize and gellin, four experiments, n = 12 per condition. (D) HEK293/TLR3 cells were transfected with or without pCMV-Flag-SPDEF and treated with 10 μg/mL form helical assemblies (21, 22). One possibility suggested by = these analogies and our experimental observations is that a he- Poly (I:C), two experiments, n 4 per condition. HEK293T cells were cotransfected with pEF-BOS-MyD88 (E) with or without SPDEF, six lical arrangement of the SPDEF SAM domains may assemble on experiments, n = 18 per condition. (F) Primary human airway epithelial cells a MyD88 helix to form a helical tower analogous to the MyD88- were cotransfected with pEF-BOS-MyD88 with or without SPDEF, two IRAK4-IRAK2 complex. experiments, n = 6 per condition. (G) Cells were cotransfected with pEF-BOS- TRIF with or without SPDEF, two experiments, n = 6 per condition. (H) Cells Mucous Metaplasia Compromises Pulmonary Antibacterial Responses. were cotransfected with pISRE-Luc and pEF-BOS-TRIF with or without SPDEF, Although the mechanistic focus in these studies was on regula- three experiments, n = 9 per condition. Data are mean ± SEM. *P ≤ 0.05, tion of TLR signaling, the finding that rhinovirus induced SPDEF **P ≤ 0.001. Student’s t test.

16632 | www.pnas.org/cgi/doi/10.1073/pnas.1208092109 Korfhagen et al. Downloaded by guest on September 30, 2021 blunting of pulmonary antimicrobial responses required for bacterial clearance (Fig. S5). Discussion The ﬁnding that SPDEF plays an epithelial cell autonomous role in the regulation of innate immune responses in the respiratory epithelium provides an unanticipated causal link between mucous metaplasia, suppression of innate immune signaling, in- ﬂammation, and susceptibility to pulmonary infection. Patients with chronic pulmonary goblet cell metaplasia are highly susceptible MEDICAL SCIENCES

Fig. 4. SAM pointed domain ETS factor (SPDEF) inhibits Toll-like receptor (TLR) and interferon (IFN) target gene expression. Effects of SPDEF expression vector on mRNAs were assessed by quantitative reverse transcription polymerase chain reaction. (A) HEK293T cells with (black bars) or without (white bars) SPDEF expression plasmid were cotransfected with TLR4, CD14, and MD-2 and treated with lipopolysaccharide (LPS) or phosphate-buffered saline (PBS) (100 ng/mL). (B and F) HEK293T cells were cotransfected with control pEF-BOS or pEF-BOS-MyD88 or pEF-BOS-TRIF. (C–E)A549cellswere stably transfected with lentivirus-eGFP (white bars) or lentivirus-SPDEF- IRES-eGFP (black bars) and stimulated for 2 h with 1 μg/mL LPS or PBS. (F) SPDEF inhibited INF-β mRNA induced by pEF-BOS-TRIF. Data are mean ± SEM, n = 3 per condition, each in triplicate. *P ≤ 0.04, **P ≤ 0.001. Student’s t test.

expression suggested a possible role for SPDEF in modulating IFN-β signaling as well. Indeed, SPDEF inhibited IFN- β–induced expression of pISRE-luciferase in HEK293T cells, indicating that SPDEF may play a wider role in restraining innate immune activation that includes inhibition of IFN-β responses (Fig. S4). Such broad counter-regulation of innate immune responses by SPDEF suggests a possible mechanism underlying the ability of patients with diseases marked by florid, chronic mucous metaplasia, such as chronic obstructive pulmonary disease, bronchiectasis, and cystic fibrosis, to tolerate lung microbial burdens that would be associated with fulminant in- fl ammatory disease in normal hosts or be susceptible to sec- Fig. 5. SAM pointed domain ETS factor interacts directly with MyD88 and ondary infection following acute viral illness that caused mucus TRIF. (A) Bidirectional coimmunoprecipitation of MyD88 and Flag-SPDEF in metaplasia. We therefore directly tested whether SPDEF might lysates of HEK293T cells transfected with the indicated constructs or empty provide a molecular explanation for the increased susceptibility pCMV-Flag vector controls. (B) Confocal microscopy of pEF-BOS-MyD88 to colonization and infection that are associated with mucous (red), GFP-SPDEF (green), and TOPRO-3 (blue) in HEK293T cells transfected metaplasia. As previously described, treatment of Scgb1a1-rtTA/ with the indicated constructs, colocalization (yellow). (C) Bidirectional coimmunoprecipitation of HA-Trif and Flag-SPDEF in lysates of HEK293T cells transfected Tre2-Spdef transgenic mice induced diffuse mucous metaplasia with the indicated constructs or empty vector controls. (D) Confocal mi- in conducting airways (9, 10). Control- and SPDEF-expressing croscopy of pEF-BOS-TRIF (red), GFP-SPDEF (green), and TOPRO-3 (blue) in transgenic mice were treated intratracheally with Klebsiella HEK293T cells transfected with the indicated constructs. All data repre- pneumoniae. Expression of SPDEF was associated with significant sent two to three experiments.

Korfhagen et al. PNAS | October 9, 2012 | vol. 109 | no. 41 | 16633 Downloaded by guest on September 30, 2021 to viral and bacterial infections and develop disease exacer- Neither mucous metaplasia nor expression of SPDEF is typical bations that lead to further deterioration in lung function. of the normal respiratory tract, but both are rapidly induced by Likewise, secondary bacterial infections commonly complicate Th2-inflammatory process, infection, and chronic injury. Thus, recovery from antecedent viral infections, infections themselves expression of SPDEF is closely linked to mucous metaplasia in associated with mucus hyperproduction. The present study shows chronic airway diseases, including asthma, cystic fibrosis, and that SPDEF restrains intracellular signaling from pathogen-as- chronic obstructive pulmonary disease, and is likely to be in- sociated molecular patterns from Gram-positive and -negative volved in the pathogenesis of mucus hyperproduction following bacteria and viruses that signal through TLRs. SPDEF inhibited acute respiratory viral infections. The present findings demon- TLR signaling pathways and IFN signaling pathways that origi- strate that SPDEF is rapidly induced following rhinovirus innate from TLRs by binding and inhibiting the activity of critical fection of human airway epithelial cells. The expression of adaptor proteins that regulate NF-κB and IFN transcriptional SPDEF causes mucous metaplasia and enhances the expression responses. The present finding that SPDEF plays an epithelial of groups of genes associated with the synthesis, glycosylation, cell autonomous role in the regulation of innate immunity in the and packaging of mucus and also influences the expression of lung provides a potential direct link between the process of a diverse array of airway peptides that play roles in innate im- mucous metaplasia and susceptibility to pulmonary infection and munity (10). Thus, although many effects of SPDEF are likely inflammation. SPDEF regulates expression of genes critical for mediated by its role in gene transcription in the nuclear com- the production of mucus, including Muc5AC, Muc16, Claca1, partment, the present study identifies a distinct, nonnuclear and Agr2, which influence mucocillary clearance of infectious role for SPDEF. Consistent with the proposed role of SPDEF pathogens from the airways. The present findings demonstrate in the cytoplasm, we colocalized SPDEF with both TRIF and that SPDEF also plays an anti-inflammatory role in respiratory MyD88 in cytoplasmic vesicles in epithelial cells. The presence of epithelial cells by binding to and inhibiting adaptor proteins SPDEF in the cytoplasm is consistent with the immunohisto- MyD88 and TRIF. These anti-inflammatory effects of SPDEF chemistry of airway epithelial cells in mouse models associated may play an important role in suppressing the potential delete- with mucous metaplasia and in patients with chronic lung dis- rious effects of uncontrolled inflammation during the resolution ease, in which both nuclear and nonnuclear staining of SPDEF of airway infections or injuries, a phase typically associated with was observed (9, 10). The nuclear and nonnuclear localization of persistent mucus production following acute respiratory infections. SPDEF varies among diverse organs and cell types, presumably On the other hand, in acute and chronic pulmonary disorders as- reflecting tissue-specific regulatory functions. The proposed im- sociated with mucous metaplasia, SPDEF-mediated suppression of mune counter-regulatory role of SPDEF in the airway under- inflammation may inhibit epithelial innate immune responses scores that tight linkage between the developmental transcrip- needed for clearance of microbial pathogens. Thus, these dual tional pathways and those involved in responses to environmental functions of SPDEF in the airway epithelium provide a potential stressors. In this regard, the role of SPDEF in the differentiation molecular explanation for both susceptibility to infection and fail- of intestinal goblet cells (30, 31) supports the concept that ure to completely clear pulmonary pathogens that is commonly SPDEF may play an important role in restraining immune acti- associated with chronic airway diseases. Although the present study vation in response to the normal intestinal microbiota. supports a cell autonomous role for SPDEF in the respiratory epithelium, it is also possible that chronic expression of SPDEF Materials and Methods alters inflammatory processes indirectly by affecting other lung cell Mouse Models. Scgb1a1-rtTA and Scgb1a1-rtTA/TRE2-Spdef, described pre- types. Because SPDEF induces the expression of mucins and viously (9, 10), were treated with doxycycline as described (10). Escherichia mucus-related genes, changes in bacterial clearance are likely coli 055:B5 LPS (20 μgin100μL; purified by ion-exchange chromatography; influenced by rheologic consequences of mucous metaplasia and Sigma) was administered intratracheally for 16 h as previously described μ μ associated airway obstruction that may contribute to the persistent (32). For chronic challenge, LPS (30 gin30 L) was administered intranasally infections associated with cystic fibrosis and other chronic lung on days 0, 2, 4, 7, and 9. Infection of the lung with Klebsiella pneumoniae disorders. Whether SPDEF and associated mucous metaplasia (KPA1 serotype, cps K2) was via intratracheal delivery (33, 34). Lung bacterial fl fl burden was quantified by plating serial dilutions of lung homogenates (34, in uence processes in ciliated or basal cells to alter in ammation 35) onto blood agar plates. BALF cell counts and differential were per- and mucociliary clearance is presently unknown. formed as described (35). KC was quantified by enzyme-linked immunosor- The counter-regulatory mechanism supported by the present bent assay (R&D Systems). findings provides support for a tissue-specific transcriptional fl pathway to limit in ammatory processes. This is in contrast to In Vitro Studies. Luciferase assays in cell lines were performed essentially, as the general pathways by which immune activation is resolved previously described using Effectene (Qiagen) instead of Fugene (35). All that also render the host susceptible to secondary infections. For transfections included pELAM-luc or pISRE-luc reporter plasmids. Fireflylucif- example, endotoxin tolerance, the inhibition of subsets of TLR- erase from pELAM-luc or pISRE-luc was quantified relative to renilla luciferase driven responses associated with ongoing infection or bacterial from pRL-TK as previously described (32, 35). In experiments in Fig. 3 A–D,and product exposure, provides protection from the pathologic Fig. S3C, 30 h after transfection, cells were stimulated with bacterial pathogen- hyperactivation of innate immunity at the cost of risk of super- associated molecular patterns [LPS, Pam3Cys, Flagellin, and Poly (I:C)] or infection (23). Likewise, in the adaptive immune system, T-cell phosphate-buffered saline as control. Luciferase activity was determined after exhaustion protects against immunopathology at the risk of 18 h by comparison with cells transfected identically but not stimulated with sustained viral replication and viral persistence (24). Addition- bacterial pathogen-associated molecular patterns. Data are reported as relative luciferase activity by comparing cells without SPDEF to cells with SPDEF. In ally, SPDEF is induced in a Th2 environment representing a shift from the TH1/17 antibacterial environment. SPDEF expression Fig. 3 E, G, and H and Fig. S3B, cells were transfected with pEF-BOS-MyD88 or may contribute to the bacterial susceptibility described in an pEF-BOS-TRIF or pEF-BOS (without MyD88 or TRIF) for 24 h and luciferase allergic environment in the lung (25, 26). activity determined by comparing cells transfected with MyD88 or TRIF with cells with the empty vector pEF-BOS. Data are reported as relative luciferase SPDEF is a member of a large family of transcription factors activity determined by comparing cells without SPDEF with cells with SPDEF. that share the ETS DNA binding domain. SPDEF is distin- Primary human airway epithelial cells were electroporated using the NEON guished from many ETS transcription factors in containing dis- Transfection System (Invitrogen). Firefly luciferase from pELAM-luc was tinct SAM pointed domain and noncanonical transcriptional quantified relative to renilla luciferase from pRL-TK as previously described binding motifs that place it as a distant member of this family of (32, 35). Significance was determined using Microsoft Excel software. transcription factors (27, 28). SPDEF is also expressed in epithelial cells in the prostate, mammary gland, and gastrointestinal IL-13 Treatment and Rhinovirus Infection of Human Airway Epithelial Cells. tract (29–31), all sites that must maintain careful immune sur- Well-differentiated primary human airway epithelial cells were cultured at veillance to maintain sterility or normal homeostasis in the face air–liquid interface performed as previously described (36) and treated with of pathogens or normal bacterial colonization, respectively. IL-13 (10 ng/mL) or phosphate-buffered saline in culture medium for 5 d. Alcian

16634 | www.pnas.org/cgi/doi/10.1073/pnas.1208092109 Korfhagen et al. Downloaded by guest on September 30, 2021 blue and immunoﬂuorescence for MUC5AC was performed (9). For rhinovirus ACKNOWLEDGMENTS. We thank E. Kurt-Jones, Kate Fitzgerald, Albert experiments, ALI cultures were exposed apically to ∼5 MOI of rhinovirus RV16 Senft, and M. Fenton for reagents; Y. Maeda, C. L. Na, and S. Wert for for 2 h and total RNA was isolated 48 h after infection. Rhinovirus RV16 was technical assistance. Human bronchiolar epithelial cells were provided by S. Randell with support by the Cystic Fibrosis Foundation Research De- kindly provided by Dr. Albert Senft, Lovelace Respiratory Research Institute, velopment program at Cincinnati Children’s Hospital Medical Center. This Albuquerque, NM. work was funded by National Institute Heart Lung and Blood Grants Descriptions of reagents and methods are provided in SI Materials HL110964, HL108907 and HL095580 (to J.A.W. and T.R.K.) and National In- and Methods. stitute of Allergy and Infectious Diseases Grant AI088372 (to C.L.K.).

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