Persistent activation of interlinked type 2 airway epithelial gene networks in sputum-derived cells from aeroallergen-sensitized symptomatic asthmatics

Abstract

Atopic asthma is a persistent disease characterized by intermittent wheeze and progressive loss of lung function. The disease is thought to be driven primarily by chronic aeroallergen-induced type 2-associated inflammation. However, the vast majority of atopics do not develop asthma despite ongoing aeroallergen exposure, suggesting additional mechanisms operate in conjunction with type 2 immunity to drive asthma pathogenesis. We employed RNA-Seq profiling of sputum-derived cells to identify gene networks operative at baseline in house dust mite-sensitized (HDMS) subjects with/without wheezing history that are characteristic of the ongoing asthmatic state. The expression of type 2 effectors (IL-5, IL-13) was equivalent in both cohorts of subjects. However, in HDMS-wheezers they were associated with upregulation of two coexpression modules comprising multiple type 2- and epithelial-associated genes. The first module was interlinked by the hubs EGFR, ERBB2, CDH1 and IL-13. The second module was associated with CDHR3 and mucociliary clearance genes. Our findings provide new insight into the molecular mechanisms operative at baseline in the airway mucosa in atopic asthmatics undergoing natural aeroallergen exposure, and suggest that susceptibility to asthma amongst these subjects involves complex interactions between type 2- and epithelial-associated gene networks, which are not operative in equivalently sensitized/exposed atopic non-asthmatics.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1

Differential gene network comparing HDMS nonwheezers versus nonatopic controls. Differentially expressed genes were identified with an edgeR analysis. Gene expression patterns in sputum were compared between HDMS nonwheezers and nonatopic controls. The wiring diagram of the underlying gene network was reconstructed employing prior knowledge from the literature (Ingenuity Knowledge Base). Genes highlighted in red denote upregulation, whilst green indicates downregulation in HDMS nonwheezers.

Figure 2

Differential gene network comparing HDMS wheezers versus nonatopic controls. Differentially expressed genes were identified with an edgeR analysis. Gene expression patterns in sputum were compared between HDMS wheezers and nonatopic controls. The wiring diagram of the underlying gene network was reconstructed using prior knowledge from the literature (Ingenuity Knowledge Base). Genes highlighted in red denote upregulation, whilst molecules in green indicate downregulation in HDMS wheezers.

Figure 3

Differential gene network comparing HDMS wheezers versus HDMS nonwheezers. Differentially expressed genes were identified with an edgeR analysis. Gene expression patterns in sputum were compared between HDMS wheezers versus HDMS nonwheezers. The wiring diagram of the underlying gene network was reconstructed employing prior knowledge from the literature (Ingenuity Knowledge Base). Genes highlighted in red denote upregulation, whilst molecules in green indicate downregulation in HDMS wheezers.

Figure 4

Common and unique genes networked with the 4 hub genes: EGFR, ERBB2, CDH1 and IL-13. Venn diagram illustrating common and unique genes that are networked with each hub gene in merged modules “P” and “Q”.

Figure 5

Differential gene network comparing HDMS wheezers versus nonatopic controls/HDMS nonwheezers. Gene co-expression network analysis (WGCNA) was employed to construct the gene networks and unbiased correlation patterns were utilized to reconstruct the underlying wiring diagram of the mucociliary clearance module “A”. CDHR3 is identified as a hub and dominant hubs are highlighted in red.

Figure 6

Increased expression of CDHR3 and EGFR in bronchial epithelial cells from HDM sensitized asthmatics versus nonatopic controls. The expression of the hub genes CDHR3, EGFR and ERBB2 was validated at the protein-level in an independent cohort. Bronchial epithelial cells were obtained from HDM sentitized atopics with asthma and nonatopic controls. (a) Bronchial epithelial cells were immunofluorescently stained for CDHR3 expression, EGFR expression, ERBB2 expression and nuclei with DAPI (blue). Staining images were then overlaid over bright field images taken of the same field of view. Note: mag 200×; inset 400×. (b) Quantification of the images demonstrated that the expression of CDHR3 and EGFR was more intense in the atopics with asthma. The expression of ERBB2 was not different between the groups. Mann-Whitney U Test was utilized to test for statistical significance. ***p-value < 0.001, **p-value < 0.01.