Lactobacillus GG restoration of the gliadin induced epithelial barrier disruption: the role of cellular polyamines

Abstract

Background: Celiac disease is characterized by enhanced intestinal paracellular permeability due to alterations of function and expression of tight junction (TJ) proteins including ZO-1, Claudin-1 and Occludin. Polyamines are pivotal in the control of intestinal barrier function and are also involved in the regulation of intercellular junction proteins. Different probiotic strains may inhibit gliadin-induced toxic effects and the Lactobacillus rhamnosus GG (L.GG) is effective in the prevention and treatment of gastrointestinal diseases. Aims of the study were to establish in epithelial Caco-2 cells whether i) gliadin affects paracellular permeability and polyamine profile; ii) co-administration of viable L.GG, heat-killed L.GG (L.GG-HK) or its conditioned medium (L.GG-CM) preserves the intestinal epithelial barrier integrity. Additionally, the effects of L.GG on TJ protein expression were tested in presence or absence of polyamines.

Results: Administration of gliadin (1 mg/ml) to Caco-2 cells for 6 h caused a significant alteration of paracellular permeability as demonstrated by the rapid decrease in transepithelial resistance with a concomitant zonulin release. These events were followed by a significant increase in lactulose paracellular transport and a slight lowering in ZO-1 and Occludin expression without affecting Claudin-1. Besides, the single and total polyamine content increased significantly. The co-administration of viable L.GG (10(8) CFU/ml), L.GG-HK and L.GG-CM with gliadin significantly restored barrier function as demonstrated by transepithelial resistance, lactulose flux and zonulin release. Viable L.GG and L.GG-HK, but not L.GG-CM, led to a significant reduction in the single and total polyamine levels. Additionally, only the co-administration of viable L.GG with gliadin significantly increased ZO-1, Claudin-1 and Occludin gene expression compared to control cells. When Caco-2 cells treated with viable L.GG and gliadin were deprived in the polyamine content by α-Difluoromethylornithine, the expression of TJ protein mRNAs was not significantly different from that in controls or cells treated with gliadin alone.

Conclusions: Gliadin modifies the intestinal paracellular permeability and significantly increases the polyamine content in Caco-2 cells. Concomitant administration of L.GG is able to counteract these effects. Interestingly, the presence of cellular polyamines is necessary for this probiotic to exert its capability in restoring paracellular permeability by affecting the expression of different TJ proteins.

Figure 1

Effects of supplementation of viable L.GG (10⁸ CFU/ml), L.GG-HK and L.GG-CM on gliadin-induced (1 mg/ml) TER decrease. All data represent the results of three different experiments (mean ± SEM). For each time of treatment, data were analyzed by Kruskal-Wallis analysis of variance and Dunn’s Multiple Comparison Test. (*) P < 0.05 compared to gliadin treated cells.

Figure 2

Zonulin release in Caco-2 monolayers exposed to gliadin (1 mg/ml) alone or in combination with viable L.GG (10⁸ CFU/ml), heat killed L.GG (L.GG-HK) and L.GG conditioned medium (L.GG-CM). All data represent the results of three different experiments (mean ± SEM). For each time of treatment, data were analyzed by Kruskal-Wallis analysis of variance and Dunn’s Multiple Comparison Test. (*) P < 0.05 gliadin vs. gliadin + Viable L.GG.

Figure 3

ZO-1, Claudin-1 and Occludin mRNA levels in Caco-2 monolayers after 6 h of exposure to different probiotic and gliadin treatments. Panels A, B, and C report ZO-1, Claudin-1 and Occludin mRNA levels in Caco-2 monolayers after 6 h of exposure to viable L.GG (10⁸ CFU/ml), heat killed L.GG (L.GG-HK) and L.GG conditioned medium (L.GG-CM). Data were analyzed by Kruskal-Wallis analysis of variance and Dunn’s Multiple Comparison Test. (*) P < 0.05 compared to control cells. Panels D, E and F report ZO-1, Claudin-1 and Occludin mRNA levels in Caco-2 monolayers after 6 h of exposure to gliadin (1 mg/ml) alone or in combination with viable L.GG, L.GG-HK and L.GG-CM. Data were analyzed by Kruskal-Wallis analysis of variance and Dunn’s Multiple Comparison Test. (*) P < 0.05 compared to gliadin treated cells. All data represent the results of three different experiments (mean ± SEM).

Figure 4

ZO-1, Claudin-1 and Occludin mRNA levels in Caco-2 monolayers after 6 h of exposure to gliadin (1 mg/ml) alone or in combination with viable L.GG (10⁸ CFU/ml), in presence or absence of polyamines following administration of α-Difluoromethylornithine (DFMO). All data represent the results of three different experiments (mean ± SEM). A. ZO-1 mRNA levels; B. Claudin-1 mRNA levels; C. Occludin mRNA levels. Data were analyzed by Kruskal-Wallis analysis of variance and Dunn’s Multiple Comparison Test. (*) P < 0.05 compared to gliadin treated cells.

Figure 5

Western Blot analysis of ZO-1, Claudin-1 and Occludin (using their specific antibodies as specified in Methods) in Caco-2 monolayers after 6 h of exposure to gliadin (1 mg/ml) alone or in combination with viable L.GG (10⁸ CFU/ml), heat killed L.GG (L.GG-HK) and L.GG conditioned medium (L.GG-CM). Immunoreactive bands were quantified using Quantity One programme. The diagrams show quantification of the intensity of bands, calibrated to the intensity of the β-actin bands. All data represent the results of three different experiments (mean ± SEM). Data were analyzed by Kruskal-Wallis analysis of variance and Dunn’s Multiple Comparison Test. (*) P < 0.05 compared to gliadin treated cells.