The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process
AbstractGliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for celiac disease (CD), and its toxicity is not reduced by digestion with gastro-pancreatic enzymes. Importantly, it is proved that an innate immunity to gliadin plays a key role in the development of CD. The immune response induces epithelial stress and reprograms intraepithelial lymphocytes into natural killer (NK)-like cells, leading to enterocyte apoptosis and an increase in epithelium permeability. In this contribution, we have reported that in Caco-2 cells the administration of enzymatically digested gliadin (PT-gliadin) reduced significantly the expression of the autophagy-related marker LC3-II. Furthermore, electron and fluorescent microscope analysis suggested a compromised functionality of the autophagosome apparatus. The rescue of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was obtained with a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce a significant improvement in the clearance of extra- and intra-cellular fluorescent PT-gliadin amount. Altogether, our results highlighted the possible contribution of the autophagy process in the degradation and in the reduction of extra-cellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD. View Full-Text
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Description: Supplementary Figure 1 LC3 and Lamp1 expression in Caco-2 cells after PT-gliadin administration. (A,B) Immunofluorescence analysis of LC3 or Lamp1 (green) and gliadin (red) expression, visualized using an inverted microscope Eclipse Nikon TS100, 100X oil immersion Plan Flor objective. Scale bars=10 m. (C) Immunoblotting expression and densitometric analysis of Lamp1 normalized with BACT housekeeping values. Asterisks indicate p<0.05, Anova One-way, compared to T0 untreated sample. Supplementary Figure 2. Micronuclei formation after PT-gliadin administration in Caco-2 cells. Fluorescent DAPI staining and analysis of the number of micronuclei in Caco-2 cells treated with PT-gliadin (1 g/l). For each condition, 1000 nuclei were considered. Scale bars=10 m. Asterisks indicates p<0.05, Anova One-way, compared to NT untreated samples. Supplementary Figure 3. Effect of BECN1 silencing on Caco-2 cells after PT-gliadin administration. PT-gliadin (1 g/l) was administered to Caco-2 cells, transfected with a pool of validated siBECN1 molecules. (A) Immunoblotting and densitometric analysis of BECN1 protein expression. (B) Collected media were analysed by fluorimeter (ext. 492 nm – emis. 517 nm). Asterisk indicates statistical significance p<0.05, Anova One-way, compared to untreated sample (nt). Fluorescence was reported as arbitrary units. SD bars (n=3) are reported.
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Manai, F.; Azzalin, A.; Gabriele, F.; Martinelli, C.; Morandi, M.; Biggiogera, M.; Bozzola, M.; Comincini, S. The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process. Int. J. Mol. Sci. 2018, 19, 635.
Manai F, Azzalin A, Gabriele F, Martinelli C, Morandi M, Biggiogera M, Bozzola M, Comincini S. The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process. International Journal of Molecular Sciences. 2018; 19(2):635.Chicago/Turabian Style
Manai, Federico; Azzalin, Alberto; Gabriele, Fabio; Martinelli, Carolina; Morandi, Martina; Biggiogera, Marco; Bozzola, Mauro; Comincini, Sergio. 2018. "The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process." Int. J. Mol. Sci. 19, no. 2: 635.
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