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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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).