Effect of lipopolysaccharide on diesel exhaust particle-induced junctional dysfunction in primary human nasal epithelial cells.

OBJECTIVES

Tight junctions (TJs) in the epithelium play a critical role in the formation of a paracellular epithelial barrier against the extracellular environment. Diesel exhaust particles (DEPs) disrupt the epithelial barrier. The aim of this study was to investigate how DEPs disrupt the epithelial barrier and whether Toll-like receptor 4 (TLR4) is involved in DEP-induced epithelial barrier dysfunction in primary human nasal epithelial (PHNE) cells.

METHODS

PHNE cells were cultured at an air-liquid interface (ALI) to create a fully differentiated in vivo-like model of the epithelium and then exposed to DEPs (particulate matter <4 μm) or lipopolysaccharide (LPS) alone (mono-exposure) and DEPs plus LPS (co-exposure) at the apical side of the PHNE. TJ formation and integrity were monitored by measuring transepithelial electric resistance (TEER) and fluorescently labeled dextran permeability. The expression of TJ proteins was assessed by confocal microscopy and a biochemical assay.

RESULTS

PHNE cell viability was reduced in a time- and dose-dependent manner following DEP exposure. TEER was significantly decreased at ALI day 20 but not at day 12 following DEP exposure. The dextran permeability of the PHNE was significantly increased at both ALI day 12 and day 20 following DEP exposure. The increased dextran permeability recovered to that of the control following co-exposure to DEPs plus LPS. In the presence of DEPs, the membrane expression of myosin light chain kinase (MLCK) was dramatically increased, and the expression of occludin, ZO1, claudin-1, and E-cadherin was significantly decreased. Co-exposure to DEPs plus LPS significantly reduced membrane MLCK, claudin-1, and E-cadherin but increased occludin and ZO1 expression at ALI day 12.

CONCLUSION

The activation of TLR4 by LPS inhibits MLCK trafficking to the plasma membrane, and this increased during DEP exposure, resulting in increased occludin expression at the plasma membrane that partially recovered TJ barrier dysfunction following DEP exposure.