Imaging analysis of the gliadin direct effect on tight junctions in an in vitro three-dimensional Lovo cell line culture system

Tight junctions play a pivotal role in maintaining the integrity of the intestinal barrier. Their alteration is involved in the pathogenesis of celiac disease. Our aim was to investigate the gliadin effect on the tight junction proteins in an in vitro three-dimensional cell culture model through imaging analyses. Lovo multicellular spheroids were treated with enzymatically digested (PT) gliadin 500 μg/mL and its effect on actin, occludin and zonula occludens-1, was evaluated by means of confocal laser microscopy, transmission electron microscopy and image capture analysis. Compared to untreated spheroids, PT-gliadin-treated ones showed enlargement of the paracellular spaces (9.0 ± 6.9 vs. 6.2 ± 1.7 nm, p<0.05) at transmission electron microscopy and tight junction protein alterations at confocal microscopy and image analyses. In untreated cell cultures thickness of the fluorescence contour of actin, zonula occludens-1 and occludin appeared significantly larger and more intense than in the treated ones. In occludin planimetric analysis the lengths of the integral uninterrupted cellular contour appeared longer in untreated than in PT-gliadin treated spheroids (71.8 ± 42.8 vs. 23.4 ± 25.9 μm, p<0.01). Our data demonstrated that tight junction proteins are directly damaged by gliadin as shown by means of quantitative imaging analysis.