Beyond epithelial-to-mesenchymal transition: Common suppression of differentiation programs underlies epithelial barrier dysfunction in mild, moderate, and severe asthma.

BACKGROUND: Epithelial barrier dysfunction is a central feature in the pathogenesis of allergic disease. Epithelial-to-mesenchymal transition (EMT) has been proposed as one mechanism afflicting barrier in asthma. However, genes and pathways involved in aberrant epithelial-mesenchymal signaling, and their relationship to asthma severity, are poorly understood. METHODS: We used unbiased gene network analysis to evaluate functional convergence in epithelial gene expression signatures across multiple public access transcriptomics datasets of human asthma, followed by text mining to evaluate functional marker relevance of discovered genes. We objectively confirmed these findings in epithelial brushings and primary asthmatic epithelial cells cultured in different biological contexts. RESULTS: We found a striking suppression of epithelial differentiation in asthma, overrepresented by insufficiency in insulin and Notch signaling, but with the absence of conventional EMT markers. We identified EFNB2, FGFR1, FGFR2, INSR, IRS2, NOTCH2, TLE1, and NTRK2 as novel markers central to dysregulation of epithelial-mesenchymal signaling, but surprisingly overlooked in asthma research. We found that this "core" signature of asthma is shared by mild, moderate, and severe forms of disease, progressing with severity. Loss of epithelial differentiation induced by insulin deprivation in normal human bronchial epithelial cells cultured in organotypic conditions closely approximated gene expression in asthmatic epithelial brushings. CONCLUSIONS: The comparative analysis of publically available transcriptomes demonstrated that epithelial barrier dysfunction in asthma is characterized by persistent underlying de-differentiation program with complex etiology. The lasting alteration of the asthmatic epithelial cell transcriptome implicates regulation involving metabolism and epigenetics, beyond EMT driven by injury and repair in chronic inflammation.

Phenotypic plasticity and targeting of Siglec-F(high) CD11c(low) eosinophils to the airway in a murine model of asthma.

Eosinophil recruitment in asthma is a multistep process, involving both trans-endothelial migration to the lung interstitium and trans-epithelial migration into the airways. While the trans-endothelial step is well studied, trans-epithelial recruitment is less understood. To contrast eosinophil recruitment between these two compartments, we employed a murine kinetics model of asthma. Eosinophils were phenotyped by multicolor flow cytometry in digested lung tissue and bronchoalveolar lavage (BAL) simultaneously, 6 h after each ovalbumin (OVA) challenge. There was an early expansion of tissue eosinophils after OVA challenge followed by eosinophil buildup in both compartments and a shift in phenotype over the course of the asthma model. Gradual transition from a Siglec-F(med) CD11c(-) to a Siglec-F(high) CD11c(low) phenotype in lung tissue was associated with eosinophil recruitment to the airways, as all BAL eosinophils were of the latter phenotype. Secondary microarray analysis of tissue-activated eosinophils demonstrated upregulation of specific integrin and chemokine receptor signature suggesting interaction with the mucosa. Using adhesion assays, we demonstrated that integrin CD11c mediated adhesion of eosinophils to fibrinogen, a significant component of epithelial barrier repair and remodeling. To the best of our knowledge, this is the only report to date dissecting compartmentalization of eosinophil recruitment as it unfolds during allergic inflammation. By capturing the kinetics of eosinophil phenotypic change in both tissue and BAL using flow cytometry and sorting, we were able to demonstrate a previously undocumented association between phenotypic shift of tissue-recruited eosinophils and their trans-epithelial movement, which implicates the existence of a specific mechanism targeting these cells to mucosal airways.