The Diacetyl-Exposed Human Airway Epithelial Secretome: New Insights into Flavoring-Induced Airways Disease

Abstract

Bronchiolitis obliterans (BO) is an increasingly important lung disease characterized by fibroproliferative airway lesions and decrements in lung function. Occupational exposure to the artificial food flavoring ingredient diacetyl, commonly used to impart a buttery flavor to microwave popcorn, has been associated with BO development. In the occupational setting, diacetyl vapor is first encountered by the airway epithelium. To better understand the effects of diacetyl vapor on the airway epithelium, we used an unbiased proteomic approach to characterize both the apical and basolateral secretomes of air-liquid interface cultures of primary human airway epithelial cells from four unique donors after exposure to an occupationally relevant concentration (∼1,100 ppm) of diacetyl vapor or phosphate-buffered saline as a control on alternating days. Basolateral and apical supernatants collected 48 h after the third exposure were analyzed using one-dimensional liquid chromatography tandem mass spectrometry. Paired t tests adjusted for multiple comparisons were used to assess differential expression between diacetyl and phosphate-buffered saline exposure. Of the significantly differentially expressed proteins identified, 61 were unique to the apical secretome, 81 were unique to the basolateral secretome, and 11 were present in both. Pathway enrichment analysis using publicly available databases revealed that proteins associated with matrix remodeling, including degradation, assembly, and new matrix organization, were overrepresented in the data sets. Similarly, protein modifiers of epidermal growth factor receptor signaling were significantly altered. The ordered changes in protein expression suggest that the airway epithelial response to diacetyl may contribute to BO pathogenesis.

Keywords: 2,3-butanedione; bronchiolitis obliterans; diacetyl; occupational lung disease; proteomics.

Figure 1.

Two-dimensional (2D) agglomerative clustering of apical proteins. Protein expression values were converted to Z-scores, followed by 2D agglomerative clustering using the Ward method. DA, diacetyl.

Figure 2.

2D agglomerative clustering of basolateral proteins. Protein expression values were converted to Z-scores, followed by 2D agglomerative clustering using the Ward method.

Figure 3.

The apical and basolateral secretomes of DA-exposed primary human airway epithelial cells are highly polarized. The Venn diagram shows the overlap of differentially expressed protein sets (significant at the level of false discovery rate [FDR] < 0.1).

Figure 4.

Known or hypothesized modifiers of EGFR signaling show mRNA expression consistent with protein changes identified by proteomic analysis. FBLN3 (A) and DDB1 (B) mRNA expression is significantly attenuated by DA exposure, whereas ECM1 (C) and GDF15 (D) mRNA expression is significantly increased by DA exposure. *P < 0.05 versus PBS. DDB1, DNA damage-binding protein 1; ECM1, extracellular matrix protein 1; EGFR, epidermal growth factor receptor; FBLN3, EGF-containing fibulin-like extracellular matrix protein 1; GDF15: growth/differentiation factor 15.