Chemokine expression in the early response to injury in human airway epithelial cells

<div><p>Basal airway epithelial cells (AEC) constitute stem/progenitor cells within the central airways and respond to mucosal injury in an ordered sequence of spreading, migration, proliferation, and differentiation to needed cell types. However, dynamic gene transcription in the early events after mucosal injury has not been studied in AEC. We examined gene expression using microarrays following mechanical injury (MI) in primary human AEC grown in submersion culture to generate basal cells and in the air-liquid interface to generate differentiated AEC (dAEC) that include goblet and ciliated cells. A select group of ~150 genes was in differential expression (DE) within 2–24 hr after MI, and enrichment analysis of these genes showed over-representation of functional categories related to inflammatory cytokines and chemokines. Network-based gene prioritization and network reconstruction using the PINTA heat kernel diffusion algorithm demonstrated highly connected networks that were richer in differentiated AEC compared to basal cells. Similar experiments done in basal AEC collected from asthmatic donor lungs demonstrated substantial changes in DE genes and functional categories related to inflammation compared to basal AEC from normal donors. In dAEC, similar but more modest differences were observed. We demonstrate that the AEC transcription signature after MI identifies genes and pathways that are important to the initiation and perpetuation of airway mucosal inflammation. Gene expression occurs quickly after injury and is more profound in differentiated AEC, and is altered in AEC from asthmatic airways. Our data suggest that the early response to injury is substantially different in asthmatic airways, particularly in basal airway epithelial cells.</p></div

Enrichment analysis of the DE genes in normal basal AEC and dAEC 8 hours after mechanical injury.

<p>Enrichment analysis of the DE genes in normal basal AEC and dAEC 8 hours after mechanical injury.</p

Reconstruction of molecular networks for normal basal and differentiated AEC after mechanical injury.

<p>String networks generated from WGCNA modules using the genes in significant DE at 2–24 hr after injury for each cell type to demonstrate functional relationships in molecular networks. Highly connected network activation was richer and more substantial in differentiated (dAEC) versus basal AEC, particularly at 2 and 8 hr after MI, but less so at 24 hr. Probe labels are as shown.</p

Expression of genes clustered for other physiologic processes important to the epithelial reparative response in the first 24 hr after MI such as cell cycle and proliferation, migration, and differentiation.

<p>Heat maps of these time points and conditions demonstrated few genes in DE.</p

Increased differential expression of genes in select inflammatory functional pathways after mechanical injury.

<p>The number of expressed genes and calculated Bayes factors from enrichment analysis for each functional category in differentiated (dAEC) and basal AEC from normal and asthmatic airways. There are more expressed genes in normal versus asthmatic cells, and differentiated versus basal cells, in most categories.</p

Comparison of differential gene expression for selected inflammatory and signaling genes in basal and differentiated AEC collected from normal and asthmatic donors.

<p>In each graph, expression is arranged in increasing expression in asthma AEC. Gene expression patterns differ between the two disease states for cytokines and chemokines. N = 4 in normal donors and 6 in asthmatic donors.</p

Selected functional genetic ontology (GO) pathways identified in asthmatic airway epithelial differentiated cells after mechanical injury in 8 hours.

<p>Selected functional genetic ontology (GO) pathways identified in asthmatic airway epithelial differentiated cells after mechanical injury in 8 hours.</p

Normal basal and differentiated airway epithelial cells differ in gene expression patterns after mechanical injury.

<p>A. Volcano plots in AEC from normal donor lungs to demonstrate differential expression in probes in the resting (without mechanical injury) state (N = 4 in each group) between differentiated (dAEC) and basal AEC, using all expressed gene probe sets (n = 35,530) as an input dataset. For each, the number of up- and down-regulated probes (≥1.5 fold change, vertical dashed lines, adjusted P < 0.05, horizontal dashed line) is provided. B. Differential expression for selected gene probes involved in inflammation and inflammation signaling at 2–24 hr after injury versus non-injured cells is provided as a heat map.</p

Normal basal and differentiated airway epithelial cells differ in gene expression patterns after mechanical injury.

<p>A. Volcano plots in AEC from asthmatic donor lungs to demonstrate differential expression in probes in the resting (without mechanical injury) state (N = 6 in each group) between differentiated (dAEC) and basal AEC, using all expressed gene probe sets (n = 41,733) as an input dataset. For each, the number of up- and down-regulated probes (≥1.5 fold change, vertical dashed lines, adjusted P < 0.05, horizontal dashed line) is provided. B. Differential expression for selected gene probes involved in inflammation and inflammation signaling at 2–24 hr after injury versus non-injured cells is provided as a heat map.</p

Reconstruction of molecular networks for asthmatic basal and differentiated AEC after mechanical injury.

<p>String networks generated from WGCNA modules using the genes in significant DE at 2–24 hr after injury for each cell type to demonstrate functional relationships in molecular networks. Highly connected network activation was similar at 2 hr, richer and more substantial in differentiated (dAEC) versus basal AEC at 8 hr, and less significant at 24 hr after MI in dAEC compared to basal AEC. Probe labels are as shown.</p