Establishment of a porcine bronchial epithelial cell line and its application to study innate immunity in the respiratory epithelium

Abstract

In vitro culture models that precisely mirror the porcine respiratory epithelium are needed to gain insight into how pathogens and host interact. In this study, a new porcine bronchial epithelial cell line, designated as PBE cells, was established from the respiratory tract of a neonatal pig. PBE cells assumed a cobblestone-epithelial like morphology with close contacts between the cells when they reached confluence. The PBE cell line was characterized in terms of its expression of pattern recognition receptors (PRRs) and its ability to respond to the activation of the Toll-like receptor 3 (TLR3) and TLR4 signaling pathways, which are key PRRs involved in the defense of the respiratory epithelium against pathogens. PBE cells stimulated with poly(I:C) were able to up-regulate the expression of IFN-β, IFN-λ1 (IL-29), IFN-λ3 (IL-28B), the antiviral factors Mx1, OAS1, and PKR, as well as the viral PRRs RIG-1 and MDA5. The expression kinetics studies of immune factors in PBE cells allow us to speculate that this cell line can be a useful in vitro tool to investigate treatments that help to potentiate antiviral immunity in the respiratory epithelium of the porcine host. In addition, poly(I:C) and LPS treatments increased the expression of the inflammatory cytokines TNF-α, IL-6, IL-8, and MCP-1/CCL2 and differentially modulated the expression of negative regulators of the TLR signaling pathways. Then, PBE cells may also allow the evaluation of treatments that can regulate TLR3- and TLR4-mediated inflammatory injury in the porcine airway, thereby protecting the host against harmful overresponses.

Keywords: TLR3; TLR4; epithelial cells; porcine respiratory epithelial cell line; respiratory epithelium; viral immunity.

Figure 1

Growth kinetics of the originally established porcine bronchial epithelial (PBE) cell line. PBE cells were seeded at an initial concentration of 0.5 x 10⁴ or 1.0 x 10⁴ cells/cm² and evaluated during six days to determine cell density. Microscope photographs show the growth of cells seeded at an initial concentration of 0.5 x 10⁴ cells/cm² from day 1 to day 6, in which the cells assumed a cobblestone-epithelial like morphology with close contacts between the cells. Results represent data from three independent experiments. Asterisks indicate significant differences in cell densities between the curves with distinct initial concentrations. * (P < 0.05).

Figure 2

Scanning electron microscopy (SEM) characterization of the originally established porcine bronchial epithelial (PBE) cell line. PBE cells were seeded at an initial concentration of 1.0 x 10⁴ cells/cm² and evaluated at days 2, 10 and 20 by SEM analysis. SEM photographs show the surface of PBE cells in which the formation of cilia that increase in size over time can be distinguished. Results represent data from two independent experiments.

Figure 3

Immunohistochemical characterization of the originally established porcine bronchial epithelial (PBE) cell line. PBE cells were seeded at an initial concentration of 1.0 x 10⁴ cells/cm² and evaluated at day 6. Photographs show the PBE cells stained with fluorescent antibodies directed to cytokeratin, tubulin, muc5B protein, the peripheral membrane phosphoprotein zona occludens 1 (ZO-1), occludin or E-cadherin. Cell nuclei were stained with DAPI. Antibody controls were performed by incubating cells with secondary antibodies without the addition of primary antibodies. The photographs show antibody control for ZO-1. Results represent data from two independent experiments.

Figure 4

Expression of Pattern Recognition Receptors (PRRs) in the originally established porcine bronchial epithelial (PBE) cell line. PBE cells were seeded at an initial concentration of 1.0 x 10⁴ cells/cm² and evaluated at day 6. The expressions of PRRs from the Toll-like receptor (TLR) and the nucleotide-binding oligomerization domain-containing protein (NOD) families were evaluated by qPCR. Results are expressed as copy numbers of the PRRS genes per 25 ng of cDNA. Photographs show the PBE cells stained with fluorescent antibodies directed to TLR3, TLR4, or TLR7. Cell nuclei were stained with DAPI. Results represent data from three independent experiments.

Figure 5

Expression of interferons (IFNs) and antiviral factors in the originally established porcine bronchial epithelial (PBE) cell line in response to the activation of the Toll-like receptor 3 (TLR3) signaling pathway. PBE cells were seeded at an initial concentration of 1.0 x 10⁴ cells/cm². At day 6, PBE cells were stimulated with the TLR3 synthetic agonist poly(I:C) (100 ng/ml) and the expressions of IFN-β, IFN-λ1 (IL-29), IFN-λ3 (IL-28B) and the antiviral factors Mx1, OAS1, and PKR were evaluated by qPCR at the indicated time points. Results represent data from three independent experiments. Asterisks indicate significant differences between the control and the poly(I:C)-treated PBE cells. * (P < 0.05), ** (P < 0.01).

Figure 6

Expression of antiviral Pattern Recognition Receptors (PRRs) in the originally established porcine bronchial epithelial (PBE) cell line in response to the activation of the Toll-like receptor 3 (TLR3) signaling pathway. PBE cells were seeded at an initial concentration of 1.0 x 10⁴ cells/cm². At day 6, PBE cells were stimulated with the TLR3 synthetic agonist poly(I:C) (100 ng/ml) and the expressions of the PRRs RIG-1, and MDA5 were evaluated by qPCR at the indicated time points. Results represent data from three independent experiments. Asterisks indicate significant differences between the control and the poly(I:C)-treated PBE cells. * (P < 0.05), ** (P < 0.01).

Figure 7

Expression of inflammatory cytokines and chemokines in the originally established porcine bronchial epithelial (PBE) cell line in response to the activation of the Toll-like receptor 3 (TLR3) signaling pathway. PBE cells were seeded at an initial concentration of 1.0 x 10⁴ cells/cm². At day 6, PBE cells were stimulated with the TLR3 synthetic agonist poly(I:C) (100 ng/ml) and the expressions of the inflammatory factors TNF-α, IL-6, IL-8, and MCP-1 were evaluated by qPCR at the indicated time points. Results represent data from three independent experiments. Asterisks indicate significant differences between the control and the poly(I:C)-treated PBE cells. * (P < 0.05), ** (P < 0.01).

Figure 8

Expression of negative regulators of the Toll-like receptor (TLR) signaling pathway in the originally established porcine bronchial epithelial (PBE) cell line in response to the activation of TLR3. PBE cells were seeded at an initial concentration of 1.0 x 10⁴ cells/cm². At day 6, PBE cells were stimulated with the TLR3 synthetic agonist poly(I:C) (100 ng/ml) and the expressions of the TLR negative regulators A20 and Bcl-3 were evaluated by qPCR at the indicated time points. Results represent data from three independent experiments. Asterisks indicate significant differences between the control and the poly(I:C)-treated PBE cells. * (P < 0.05), ** (P < 0.01).

Figure 9

Expression of inflammatory cytokines and chemokines in the originally established porcine bronchial epithelial (PBE) cell line in response to the activation of the Toll-like receptor 4 (TLR4) signaling pathway. PBE cells were seeded at an initial concentration of 1.0 x 10⁴ cells/cm². At day 6, PBE cells were stimulated with the TLR4 agonist LPS (1000 ng/ml) and the expressions of the inflammatory factors TNF-α, IL-6, IL-8, and MCP-1 were evaluated by qPCR at the indicated time points. Results represent data from three independent experiments. Asterisks indicate significant differences between the control and the poly(I:C)-treated PBE cells. * (P < 0.05), ** (P < 0.01).

Figure 10

Expression of negative regulators of the Toll-like receptor (TLR) signaling pathway in the originally established porcine bronchial epithelial (PBE) cell line in response to the activation of TLR4. PBE cells were seeded at an initial concentration of 1.0 x 10⁴ cells/cm². At day 6, PBE cells were stimulated with the TLR4 agonist LPS (1000 ng/ml) and the expressions of the TLR negative regulators A20, IRAK-M and Bcl-3 were evaluated by qPCR at the indicated time points. Results represent data from three independent experiments. Asterisks indicate significant differences between the control and the poly(I:C)-treated PBE cells. * (P < 0.05), ** (P < 0.01).