Long-term evolution of the epithelial cell secretome in preclinical 3D models of the human bronchial epithelium

Abstract

The human bronchial epithelium is the first line of defense against atmospheric particles, pollutants, and respiratory pathogens such as the novel SARS-CoV-2. The epithelial cells form a tight barrier and secrete proteins that are major components of the mucosal immune response. Functional in vitro models of the human lung are essential for screening the epithelial response and assessing the toxicity and barrier crossing of drugs, inhaled particles, and pollutants. However, there is a lack of models to investigate the effect of chronic exposure without resorting to animal testing. Here, we developed a 3D model of the human bronchial epithelium using Calu-3 cell line and demonstrated its viability and functionality for 21 days without subculturing. We investigated the effect of reduced Fetal Bovine Serum supplementation in the basal medium and defined the minimal supplementation needed to maintain a functional epithelium, so that the amount of exogenous serum proteins could be reduced during drug testing. The long-term evolution of the epithelial cell secretome was fully characterized by quantitative mass spectrometry in two preclinical models using Calu-3 or primary NHBE cells. 408 common secreted proteins were identified while significant differences in protein abundance were observed with time, suggesting that 7-10 days are necessary to establish a mature secretome in the Calu-3 model. The associated Reactome pathways highlight the role of the secreted proteins in the immune response of the bronchial epithelium. We suggest this preclinical 3D model can be used to evaluate the long-term toxicity of drugs or particles on the human bronchial epithelium, and subsequently to investigate their effect on the epithelial cell secretions.

Figure 1

Development of a 3D model of the human bronchial epithelial using Calu-3 cells for long-term toxicity studies. (a) Schematic of the experimental protocol to establish long-term cultures of Calu-3 cells. Cells were grown on Transwell inserts with a 3-µm pore diameter. Cell differentiation was induced by changing culture conditions from submerged to ALI. The barrier integrity of the epithelium was assessed by the measurement of the trans-epithelial electrical resistance (TEER) and the paracellular permeability (Lucifer Yellow (LY) permeability assay). (b) Evolution of the TEER for 21 days in a representative experiment (n = 3). A TEER > 300 Ω cm² (horizontal line) is associated with a tight epithelium (n = 3). (c) Measurement of the paracellular permeability with the LY assay at 11, 17, and 21 days. An empty Transwell insert was used as a negative control. A LY permeability < 2% (dotted line) is associated with a tight epithelium (n = 3). Significant differences between conditions (FBS %) *P < 0.01. **P < 0.05, ***P < 0.001.

Figure 2

Effect of lower FBS supplementation on the apical secretion by Calu-3 cells in long-term cultures. (a) Total protein and (b) glycoprotein concentration in the apical secretome of Calu-3 cells supplemented with 0, 2, 4, 8% FBS at day 3, 10 and 17 after ALI. The glycoprotein concentration was normalized to the total protein concentration. (c–f) Immunolabelling of ZO-1 (green) and MUC5AC (red) in DAPI-stained Calu-3 cells at day 17 after ALI. The cell cultures were supplemented with 0 (c), 2 (d), 4 (e), and 8% FBS. Each image is the z-projection of 10 slides. (n = 3) *P < 0.01 ^a,bstatistically different between days.

Figure 3

Proteomic analysis of the apical secretome of the human bronchial epithelium in Calu-3 and NHBE models. (a) Qualitative analysis of the extracellular proteins of Calu-3 and NHBE cells at day 4, day 11 (Calu-3) or 12 (NHBE), and day 18 after ALI. The total number of proteins and the percentage of common proteins are shown in a Venn diagram. (b) Comparison of the abundance of extracellular proteins by Principal Component Analysis (PCA). The analysis was performed at day 4, day 11 (Calu-3) or 12 (NHBE), and day 18. The different donors (NHBE cells) and biological replicates (Calu-3 cells) are represented for each condition and circled on the graph. The percentage associated with each principal component is indicated in the axis legend. (c) Heat map of extracellular proteins identified in the apical secretome of Calu-3 cells and NHBE cells at day 4, 11–12, and 18 after ALI. A protein set showing a large difference at day 4 in the secretome of Calu-3 cells is highlighted by a black rectangle.

Figure 4

Time evolution of the secretome of Calu-3 and NHBE cells. Schematic representing the proteins secreted by Calu-3 (a) and NHBE (b) cells as a function of their abundance at day 4, 11–12, and 18 after ALI. A larger sphere denotes a higher protein abundance. Proteins are designated by their corresponding gene. Human serum albumin (alb), neutrophil gelatinase-associated lipocalin (lcn2), polymeric immunoglobulin receptor (pigr), alpha-1-antitrypsin (serpina1), BPI fold containing family B member 1 (bpfib1). The full protein list is detailed in Table S1.

Figure 5

Analysis of the biological pathways associated with the secreted proteins in the Calu-3 and NHBE models. (a) Heat map of the 20 most abundant proteins secreted by Calu-3 and NHBE cells at day 4, 11–12, and 18 after ALI. Proteins are designated by their corresponding gene. (b) Reactome pathways associated with the extracellular proteins identified in the apical secretome of Calu-3 cells (in green) and NHBE cells (in blue) at day 11–12 and 18 after ALI. (P < 0.05 for each fold enrichment).