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 hypersecre- tion 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 air- way. 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 clear- ance, 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