Airway epithelial cells provide barrier function, mucociliary clearance, and secrete diverse antimicrobials, mucins, chemokines, and cytokines that recruit and instruct inflammatory cells that serve to limit infection. Chronic epithelial cell injury and inflammation cause tissue remodeling and increased susceptibility to infection that together ultimately impair lung function in chronic airway diseases, including asthma, cystic fibrosis, and chronic obstructive pulmonary disease. Although transcriptional networks integrating mucous cell metaplasia and chemokine production by airway epithelial cells are presently poorly understood, the importance of the respiratory epithelium in the instruction of various aspects of innate immune system of the lung is highly relevant to the pathogenesis of common chronic lung diseases. Here, we observed that a transcription factor NKX2-1 in airway epithelial cells was suppressed in patients with asthma. In mouse models and in human epithelial cells, NKX2-1 inhibited genes associated with mucous metaplasia and Th2 inflammation.
To further understand the role of NKX2-1 in airway epithelial cells, we used Ingenuity Pathway Analysis and identified biological relationships of NKX2-1 among gene nodes within a network of corelated genes, the “network” being defined as a group of biologically related genes, proteins, or other molecules. Biological relationships were derived from the combined analyses of the present mRNA microarray data and previously published mRNA arrays obtained from transgenic mice in which Spdef
, or NKX2-1
expression was selectively induced (9
), deleted (23
), or inhibited (12
); from published and present experimental data that included mRNA, protein analysis, and immunohistochemistry; and from peer-reviewed publications in the Ingenuity knowledge base (). The network analyses revealed important interrelated cellular processes influenced by NKX2-1: (1
) suppression of mucous cell metaplasia via inhibition of SPDEF-regulated mucous genes (e.g., Muc5ac
, and Tff2
) inhibition of mRNAs associated with Th2-mediated inflammation (e.g., Ccl17
, and Icos
); and (3
) a complementary role of NKX2-1 with FOXA2 in the inhibition of mucous cell metaplasia and Th2-mediated inflammation.
Figure 7. NK2 homeobox 1 (NKX2-1)–Sam Pointed Domain Ets-like Factor (SPDEF)–associated gene network analyzed using Ingenuity Pathway Analysis. (A) Genes/proteins are represented as nodes, and the biological relationship between two nodes is represented (more ...)
mRNAs decreased by NKX2-1 during allergen exposure partially overlap with those induced by expression of SPDEF in airway epithelial cells (9
) as shown schematically in . The finding that NKX2-1 inhibited allergen-induced expression of SPDEF provides a mechanism by which a number of genes associated with mucin production were coordinately regulated ( and ). The concept that NKX2-1 and SPDEF counter-regulate each other and associated target genes is consistent with previous studies in embryonic lungs from transgenic mice bearing phosphorylation sites mutant NKX2-1
), in which Spdef
mRNA was markedly induced in lung before birth (Figure E5). In the present study, SPDEF induced the expression of mucins, including Muc5ac
, and a number of genes mediating various aspects of mucin biosynthesis and packaging, (e.g., glycotransferases, Agr2
, and Clca3/1
), which were inhibited by NKX2-1. Thus, reciprocal interactions between SPDEF and NKX2-1 likely serve as a switch to control the differentiation of airway epithelial cells to mucous cells.
Th2 chemokines and cytokines induced by allergen sensitization were also inhibited by NKX2-1 in vivo and in vitro, indicating that NKX2-1 suppresses some aspects of Th2-mediated responses in airway epithelial cells in a cell-autonomous manner. Although a number of mRNAs associated with Th2-mediated inflammation were inhibited by NKX2-1, expression of a number of mRNAs associated with innate immune mediated inflammation, including Il6, Saa3, Saa4, Orm1, and Orm2, were induced ( and ). Thus, the inhibitory effects of NKX2-1 on mucous cell metaplasia are likely mediated, at least in part, by inhibition of Th2-associated chemokines/cytokines and increased expression of a subset of mRNAs associated with acute inflammation.
FOXA2 is a member of the forkhead family of transcription factors that is normally expressed in both conducting airway and alveolar respiratory epithelial cells. Conditional deletion of Foxa2
in the developing respiratory epithelium caused severe eosinophilic lung inflammation, mucous metaplasia, and increased expression of mRNAs associated with mucous cell metaplasia and Th2-mediated inflammation, including Spdef
, and Ccl22
). Expression of FOXA2 in airway epithelial cells blocked allergen-induced mucous cell metaplasia and expression of SPDEF in adult mice (23
). In the present study, expression of NKX2-1 prevented the loss of FOXA2 and inhibited various mediators of Th2-mediated inflammation and mucous cell metaplasia after aeroallergen sensitization (), supporting the concept that NKX2-1 and FOXA2 play complementary roles in the inhibition of mucous metaplasia and lung inflammation ().
Glucocorticoids are often used to treat patients with asthma; thus, the decreased expression of NKX2-1 in patients with asthma might be mediated, in part, by their treatment. Our patient cohort, however, included three patients not treated with glucocorticoids, and the decreased expression of NKX2-1 was also evident in these patients (data not shown). The numbers are small, and to conclusively address this larger numbers of patients would be needed to assess the potential role of glucocorticoid treatment in the expression of NKX2-1. Nevertheless, in the mouse experiments, NKX2-1
mRNA was decreased by allergen challenge in the absence of exogenous treatments (Figure E1). Furthermore, NKX2-1 was not reduced by treatment of dexamethasone, a corticosteroid, in H441 human lung epithelial cells (25
). These results indicate that the expression of NKX2-1 is influenced by aeroallergen sensitization.
NKX2-1 inhibited aeroallergen-induced mucous cell metaplasia, in part, by inhibiting SPDEF and by maintaining expression of FOXA2. mRNA microarray analysis indicated that NKX2-1 and SPDEF act in an opposing manner within a gene network influencing both respiratory epithelial differentiation and the Th2 immunoregulatory axis. NKX2-1 inhibited allergen-induced Ccl17, Il13, CCL26, and other genes that play important roles in the pathogenesis of asthma and mucous metaplasia (). The present study demonstrates a novel function of NKX2-1 as an inhibitor of aeroallergen-induced airway mucous cell metaplasia in the adult lung and provides further support for the important role of the respiratory epithelium in the regulation of pulmonary inflammation.