Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Feb 23;19(2):635.
doi: 10.3390/ijms19020635.

The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process

Affiliations
Free PMC article

The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process

Federico Manai et al. Int J Mol Sci. .
Free PMC article

Abstract

Gliadin, 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.

Keywords: Caco-2 cells; autophagosome; celiac disease; gluten.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Electrophoretic characterization of enzymatically digested (PT)-gliadin fragments after peptic-tryptic digestion. (A) Protein gel electrophoresis after Coomassie staining of whole gliadin and PT-gliadin (both 100 µg) (B) Immunoblotting analysis of PT-gliadin (40 µg) using a mouse monoclonal anti-gliadin antibody; and, (C) Protein gel electrophoretic analysis of fluorescently labelled PT-gliadin (GLIA-488 and GLIA-555) and ovalbumin (OVA-488) proteins (40 µg). MW, in kDa are reported.
Figure 2
Figure 2
PT-gliadin fluorescent and ultrastructural visualization. (A,B) Labelled PT-gliadin (GLIA-488, 10 µg) was added in 1 mL DMEM medium and visualized by fluorescent inverted microscope Eclipse Nikon TS100 (scale bars = 10 µm). (C,D) TEM evaluation of PT-gliadin aggregates, indicated by arrows.
Figure 3
Figure 3
Morphological evaluation of PT-gliadin administration in Caco-2 cells by optical microscopy analysis. Caco-2 cells were incubated with PT-gliadin (right, 1 µg/µL) or not- (NT) (left) and visualized using inverted optical microscope Eclipse Nikon TS100 at 24–48 h p.t. Continuous arrows indicate intra-cellular vesicles, dashed arrows point for extra-cellular PT-gliadin aggregates in proximity to the plasma membranes (asterisks) (scale bars = 10 µm). (A,B) are magnifications of a and b panels, respectively.
Figure 4
Figure 4
Internalization and extra-cellular release of fluorescent PT-gliadin in Caco-2 cells. Analysis of GLIA-488 traffic in Caco-2 cells through fluorescent inverted microscope Eclipse Nikon TS100 at T0–48 h p.t. Visualizations were performed through an inverted microscope Eclipse Nikon TS100, 40× objective. Asterisk indicates a large intra-cellular vesicle containing large GLIA-488 aggregates (scale bars = 30 µm).
Figure 5
Figure 5
Ultrastructural and molecular characterization of intracellular large vesicles after PT-gliadin administration in Caco-2 cells. (A,B) TEM analysis of Caco-2 cells at 24 h after PT-gliadin administration (1 µg/µL) visualized using a Zeiss EM900 electron microscope. Scale bars = 2 µm. (C,D) Caco-2 cells incubated with PT-gliadin as above were transduced with the Premo Autophagy Sensor LC3B-GFP system, according to the guidelines and visualized using an inverted microscope Eclipse Nikon TS100, 100× oil immersion Plan Fluor objective. Scale bars = 2 µm (computer magnification); (E,F) For intra-vesicular pH content of Caco-2 vesicles, after PT-gliadin administration (1 µg/µL for 24 h), cells were incubated with acridine orange (1 µg/mL) for 10 min. Cells were then visualized under the optical microscope and using an inverted microscope Eclipse Nikon TS100, 40×, respectively. (scale bars = 10 µm).
Figure 6
Figure 6
Viability assays after PT-gliadin administration in Caco-2 cells. (A) Cytofluorimetric plots of viability profiles (L: cells live; D: cells death) summarized in Panel (B) (asterisk indicates p < 0.05, Anova One-way, compared to T0 untreated sample).
Figure 6
Figure 6
Viability assays after PT-gliadin administration in Caco-2 cells. (A) Cytofluorimetric plots of viability profiles (L: cells live; D: cells death) summarized in Panel (B) (asterisk indicates p < 0.05, Anova One-way, compared to T0 untreated sample).
Figure 7
Figure 7
Apoptotic analysis of Caco-2 cells after PT-gliadin administration. (A) Cytofluorimetric plots of apoptotic profiles of Annexin V expression, summarized in Panel (B).
Figure 8
Figure 8
LC3-II expression in Caco-2 cells after PT-gliadin administration. (A) Cytofluorimetric plots representing LC3-II expression in Caco-2 cells incubated with 1 or 4 µg/µL of PT-gliadin at different time intervals (h); (B) Graphical summary of LC3-II expression trends. Asterisks indicate p < 0.05, Anova One-way, as compared to untreated NT samples at T0.
Figure 9
Figure 9
Immunoblotting analysis of LC3-II and p62 expression in Caco-2 cells after PT-gliadin or ovalbumine administration. (A) PT-gliadin (left) or ovalbumin (right, both at 1 µg/µL) were administered to Caco-2 cells. (B) LC3-II, p62 and BACT protein expression was analysed using immunoblotting and densitometric analyses. LC3-II and p62 were normalized to BACT levels as recommended [22]. Molecular weights (in kDa) are indicated. Asterisks indicate p < 0.05, Anova One-way, compared to T0 untreated sample.
Figure 10
Figure 10
LC3-II expression following modulation of autophagy in Caco-2 cells incubated with PT-gliadin. LC3-II expression in Caco-2 cells treated with PT-gliadin (1 µg/µL), after starvation (A), rapamycyn (5 µM) (B) or 3-methyladenine (5 mM) (C) administration, during different time intervals (hours). Asterisks indicate statistical significance p < 0.05, Anova One-way, as compared to untreated NT samples at T0.
Figure 11
Figure 11
Intra-cellular fluorescent PT-gliadin (GLIA-555) content following autophagy modulation in Caco-2 cells. PT-gliadin was administered as fluorescent GLIA-555 (1 µg/µL) moieties and assayed at different p.t. intervals by cytofluorimetric evaluations, following different autophagy activation protocols (starvation using HBSS medium; rapamycin 5 µM; 3-MA 5 mM). Asterisks indicates statistical significance p < 0.05, Anova One-way, when compared to the respective time interval; of Caco-2 cells incubated with PT-gliadin.
Figure 12
Figure 12
Extra- and intra-cellular evaluation of fluorescent PT-gliadin in Caco-2 cells. (A) Different time intervals of GLIA-488 administration (1 µg/µL) were reported (left, 30 min. p.t.: right, 24 h p.t.). Note a large autophagosome-like vesicle in the right Panel storing large fluorescent aggregates (ea: extra-cellular aggregates; pv: perimembrane exocytic vesicles; iv: intra-cellular vesicles). Scale bars = 10 µm; (B) GLIA-488 fluorescent labelled digested gliadin (1 µg/µL) was administered to growing Caco-2 cells in presence of starvation conditions, rapamycyn (5 µM) and 3-MA (5 mM) treatments. Media were collected and analysed by fluorimeter (ext. 492 nm–emis. 517 nm). Asterisks 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.
Figure 13
Figure 13
Apoptotic analysis of Caco-2 cells after PT-gliadin administration and autophagy modulation protocols. (A) Cytofluorimetric plots of apoptotic profiles of Annexin V expression, for single and combined treatments (starvation, rapamycin and 3-methyladenine) summarized, as total apoptotic events (Early + Late) in Panel (B).

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Deter R.L., De Duve C. Influence of glucagon, an inducer of cellular autophagy, on some physical properties of rat liver lysosomes. J. Cell Biol. 1967;33:437–449. doi: 10.1083/jcb.33.2.437. - DOI - PMC - PubMed
    1. Pfeifer U., Warmuth-Metz M. Inhibition by insulin of cellular autophagy in proximal tubular cells of rat kidney. Am. J. Physiol. 1983;244:E109–E114. doi: 10.1152/ajpendo.1983.244.2.E109. - DOI - PubMed
    1. Ravikumar B., Sarkar S., Davies J.E., Futter M., Garcia-Arencibia M., Green-Thompson Z.W., Jimenez-Sanchez M., Korolchuk V.I., Lichtenberg M., Luo S., et al. Regulation of mammalian autophagy in physiology and pathophysiology. Physiol. Rev. 2010;90:1383–1435. doi: 10.1152/physrev.00030.2009. - DOI - PubMed
    1. Bah A., Vergne I. Macrophage Autophagy and Bacterial Infections. Front. Immunol. 2017;8:1483. doi: 10.3389/fimmu.2017.01483. - DOI - PMC - PubMed
    1. Palumbo S., Comincini S. Autophagy and ionizing radiation in tumors: The “survive or not survive” dilemma. J. Cell. Physiol. 2013;228:1–8. doi: 10.1002/jcp.24118. - DOI - PubMed
Feedback