Constitutive alterations in vesicular trafficking increase the sensitivity of cells from celiac disease patients to gliadin

Celiac Disease (CD) is an autoimmune disease characterized by inflammation of the intestinal mucosa due to an immune response to wheat gliadins. Some gliadin peptides (e.g., A-gliadin P57-68) induce an adaptive Th1 pro-inflammatory response. Other gliadin peptides (e.g., A-gliadin P31-43) induce a stress/innate immune response involving interleukin 15 (IL15) and interferon α (IFN-α). In the present study, we describe a stressed/inflamed celiac cellular phenotype in enterocytes and fibroblasts probably due to an alteration in the early-recycling endosomal system. Celiac cells are more sensitive to the gliadin peptide P31-43 and IL15 than controls. This phenotype is reproduced in control cells by inducing a delay in early vesicular trafficking. This constitutive lesion might mediate the stress/innate immune response to gliadin, which can be one of the triggers of the gliadin-specific T-cell response.


EGF induced EGFR endocytic trafficking in late vesicular compartments in skin fibroblasts from patients with CD and controls cultured in absence or presence of P31-43.
A. Less EGFR co-localized with LAMP2-positive vesicles in CD fibroblasts than in control fibroblasts at all time points after EGF treatment. The yellow colour in the merge panels indicates co-localization of EGFR (red) with late endocytic marker LAMP2 (green) in fibroblasts from patients and controls treated with EGF at the indicated times. Representative fields are shown. Scale bar = 20µm. B. In both control and CD fibroblasts, co-localization of EGFR with the late endocytic marker LAMP2 increased at 30 min and 1 h compared to the not treated sample (NT) and decreased at later time points. EGFR/LAMP co-localization was decreased at all time points in CD fibroblasts compared to CTR fibroblasts. These results suggested a delay in EGFR trafficking in the early endocytic compartment in CD fibroblasts. C. EGF induced EGFR trafficking in the late endocytic compartment in fibroblasts from patients with CD and controls after treatment with P31-43 for 10' (T0) and then with both EGF and P31-43 for 30 min, 1 h, 3 h, 6 h or 24 h. An increase in EGFR co-localization with LAMP2 was not observed in control fibroblasts after 30 min or 1 h of P31-43 treatment compared to the untreated control fibroblasts (B), as expected from a delay in EGFR trafficking in the early endocytic compartment. No variation in EGFR-LAMP2 co-localization was observed in CD fibroblasts treated with P31-43.

Co-localization analysis
Samples were examined with a Zeiss LSM 510 laser scanning confocal microscope. We used Argon/2 (458, 477, 488, 514 nanometers) and HeNe1 (543 nanometers) excitation lasers, which were switched on separately to reduce cross-talk of the two fluorochromes. The green and the red emissions were separated by a dichroic splitter (FT 560) and filtered (515-to 540-nm band-pass filter for green and 610-nm long pass filter for red emission). A threshold was applied to the images to exclude about 99% of the signal found in control images. The weighted co-localisation coefficient represents the sum of intensity of co-localising pixels in channels 1 and 2 as compared to the overall sum of pixel intensities above threshold. This value could be 0 (no colocalisation) or 1 (all pixels co-localise). Bright pixels contribute more than faint pixels. The co-localisation coefficient represents the weighted co localisation coefficients of Ch1 (red) with respect to Ch2 (green) for each experiment (1,2). In the GCD biopsies, when the villi were too flat, the surface epithelium, between the crypts, was evaluated.

Primary fibroblast culture.
Skin explants or duodenal biopsies were immediately placed in Dulbecco's Modified Eagle's Seven-ten days later, the fibroblasts began to emerge from the fragments. When the fibroblasts reached confluence, the cells were harvested with trypsin and immediately frozen. In all experiments, the fibroblasts were used between the 2nd and 4th passage.

Immunofluorescence staining of fibroblasts
Fibroblasts were seeded on glass coverslips and then starved in starvation medium (DMEM, 0.5% FBS, 100 units/ml penicillin-streptomycin and 1 mM glutamine) after 2 days to construct EGFR degradation curves. After approximately 18 h, the control coverslips were collected and fixed.
Afterwards, EGF (100 ng/ml) was added, to the coverslips and they were incubated for different

Electro Microscopy
Fibroblasts were grown in 10-cm petri dishes and fixed with 1% glutaraldehyde in 0.15 M Hepes for 2 h at room temperature. Samples were then stained with OsO4/potassium ferrocyanide, dehydrated and embedded in epon, as described previously (3). Ultrathin sections (60-70 nm) were obtained, examined using a Tecnai-12 (FEI, Eindhoven) electron microscope, and images were acquired using a Veletta CCD digital camera. Random sampling of cellular profiles for the endocytic organelles was performed and the structures were classified based on the following morphological criteria, 1) endocytic structures with less than 8 intraluminal vesicles were classified as early endosomes (EE) and 2) the remaining endocytic structures (with more than 8 intraluminal vesicles or other membranes) were classified as lysosome-like structures (LLS) (3).