Gliadin-Mediated Proliferation and Innate Immune Activation in Celiac Disease Are Due to Alterations in Vesicular Trafficking

Background and Objectives: Damage to intestinal mucosa in celiac disease (CD) is mediated both by inflammation due to adaptive and innate immune responses, with IL-15 as a major mediator of the innate immune response, and by proliferation of crypt enterocytes as an early alteration of CD mucosa causing crypts hyperplasia. We have previously shown that gliadin peptide P31-43 induces proliferation of cell lines and celiac enterocytes by delaying degradation of the active epidermal growth factor receptor (EGFR) due to delayed maturation of endocytic vesicles. IL-15 is increased in the intestine of patients affected by CD and has pleiotropic activity that ultimately results in immunoregulatory cross-talk between cells belonging to the innate and adaptive branches of the immune response. Aims of this study were to investigate the role of P31-43 in the induction of cellular proliferation and innate immune activation. Methods/Principal Findings: Cell proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation both in CaCo-2 cells and in biopsies from active CD cases and controls. We used real-time PCR to evaluate IL-15 mRNA levels and FACS as well as ELISA and Western Blot (WB) analysis to measure protein levels and distribution in CaCo-2 cells. Gliadin and P31-43 induce a proliferation of both CaCo-2 cells and CD crypt enterocytes that is dependent on both EGFR and IL-15 activity. In CaCo-2 cells, P31-43 increased IL-15 levels on the cell surface by altering intracellular trafficking. The increased IL-15 protein was bound to IL15 receptor (IL-15R) alpha, did not require new protein synthesis and functioned as a growth factor. Conclusion: In this study, we have shown that P31-43 induces both increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by altering the trafficking of the vesicular compartments as well as proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR

Gliadin Peptide P31-43 Localises to Endocytic Vesicles and Interferes with Their Maturation

BACKGROUND: Celiac Disease (CD) is both a frequent disease (1:100) and an interesting model of a disease induced by food. It consists in an immunogenic reaction to wheat gluten and glutenins that has been found to arise in a specific genetic background; however, this reaction is still only partially understood. Activation of innate immunity by gliadin peptides is an important component of the early events of the disease. In particular the so-called "toxic" A-gliadin peptide P31-43 induces several pleiotropic effects including Epidermal Growth Factor Receptor (EGFR)-dependent actin remodelling and proliferation in cultured cell lines and in enterocytes from CD patients. These effects are mediated by delayed EGFR degradation and prolonged EGFR activation in endocytic vesicles. In the present study we investigated the effects of gliadin peptides on the trafficking and maturation of endocytic vesicles. METHODS/PRINCIPAL FINDINGS: Both P31-43 and the control P57-68 peptide labelled with fluorochromes were found to enter CaCo-2 cells and interact with the endocytic compartment in pulse and chase, time-lapse, experiments. P31-43 was localised to vesicles carrying early endocytic markers at time points when P57-68-carrying vesicles mature into late endosomes. In time-lapse experiments the trafficking of P31-43-labelled vesicles was delayed, regardless of the cargo they were carrying. Furthermore in celiac enterocytes, from cultured duodenal biopsies, P31-43 trafficking is delayed in early endocytic vesicles. A sequence similarity search revealed that P31-43 is strikingly similar to Hrs, a key molecule regulating endocytic maturation. A-gliadin peptide P31-43 interfered with Hrs correct localisation to early endosomes as revealed by western blot and immunofluorescence microscopy. CONCLUSIONS: P31-43 and P57-68 enter cells by endocytosis. Only P31-43 localises at the endocytic membranes and delays vesicle trafficking by interfering with Hrs-mediated maturation to late endosomes in cells and intestinal biopsies. Consequently, in P31-43-treated cells, Receptor Tyrosine Kinase (RTK) activation is extended. This finding may explain the role played by gliadin peptides in inducing proliferation and other effects in enterocytes from CD biopsies

M.V. Barone, M. Nanayakkara, D. Zanzi, S. Santagata, G. Lania, V. Discepolo, M. Ten-Eikelder, S. Auricchio

Background and AimsWe previously observed that A-gliadin peptide P31-43 induces effects similar to Epidermal growthfactor (EGF) both in cultured cell lines and in enterocytes from celiac disease (CD) patients. Wealso showed that the effect is mediated by delayed EGF degradation and prolonged EGF receptor(EGFR) activation in endocytic vesicles. Here we address the molecular mechanisms underlyinggliadin peptide effects on trafficking and maturation of vesicles responsible for EGFR endocytosis.MethodsA sequence similarity search revealed that P31-43 is strikingly similar to a region of Hrs, a keymolecule involved in endocytic maturation. Western blot and immunofluorescence microscopy wereused to determine Hrs localization to endocytic vesicles and cytosol. Pulse and chase labelling intime-lapse experiments allowed to follow uptake and sub-cellular localization of gliadin peptides inCaCo 2 cells and enterocytes from CD patients and controls.ResultsA-gliadin peptide P31-43 interferes with Hrs localization to early endosomes. Both P31-43 and thecontrol P56-68 peptide enter CaCo 2 cells and interact with the endocytic compartment, but P31-43 is localized to vesicles carrying early endocytic markers at time points when P56-68-carryingvesicles mature into late endosomes. Dynamic analysis shows that P31-43 labelled vesicles areslowed down in time-lapse experiments. The effect is independent of the cargo they carry: dextrancontaining vesicles, behave similarly to EGFR containing ones. Markers of recycling pathwayTrasferrin receptor and Lamp, are increased on the surface of P31-43 treated cells together withEGFR. Gliadin peptide P31-43 enters epithelial cells and colocalize with EEA1 both in CD biopsiesand controls coltured for 24 hours. Pulse and chase experiments show a delay of trafficking of P31-43 peptide in EEA1 positive vesicles only in CD biopsies, but not in controlsConclusionsGliadin peptide P31-43 delays vesicle trafficking by interfering with Hrs mediated maturation to lateendosomes and also promotes the recycling pathway. As a consequence, in P31-43 treated cells,EGFR activation is extended, more transferrin and EGFR receptors find their way to the cellsurface. In CD biopsies trafficking of this peptide is delayed. This may help explaining the roleplayed by gliadin peptides in CD pathogenesis

Gliadin peptide P31-43 interferes Hrs (hepatocyte growth factor-regulated tyrosine kinasesubstrate) localization to endocytic vesicles: implications for Celiac Disease (CD)

Maria Vittoria Barone1, Merlin Nanayakkara2, Delia Zanzi2, Sara Santagata2, ValentinaDiscepolo2, Maria ten Eikelder2, Giuliana Lania2, Salvatore Auricchio21Pediatric Department (ELFID), Università degli Studi di Napoli, Federico II, Napoli2Pediatric Department (ELFID), Università degli Studi di Napoli,Background and AimsWe previously observed that A-gliadin peptide P31-43 induces effects similar to Epidermal growthfactor (EGF) both in cultured cell lines and in enterocytes from celiac disease (CD) patients. Wealso showed that the effect is mediated by delayed EGF degradation and prolonged EGF receptor(EGFR) activation in endocytic vesicles. Here we address the molecular mechanisms underlyinggliadin peptide effects on trafficking and maturation of vesicles responsible for EGFR endocytosis.MethodsA sequence similarity search revealed that P31-43 is strikingly similar to a region of Hrs, a keymolecule involved in endocytic maturation. Western blot and immunofluorescence microscopy wereused to determine Hrs localization to endocytic vesicles and cytosol. Pulse and chase labelling intime-lapse experiments allowed to follow uptake and sub-cellular localization of gliadin peptides inCaCo 2 cells and enterocytes from CD patients and controls.ResultsA-gliadin peptide P31-43 interferes with Hrs localization to early endosomes. Both P31-43 and thecontrol P56-68 peptide enter CaCo 2 cells and interact with the endocytic compartment, but P31-43 is localized to vesicles carrying early endocytic markers at time points when P56-68-carryingvesicles mature into late endosomes. Dynamic analysis shows that P31-43 labelled vesicles areslowed down in time-lapse experiments. The effect is independent of the cargo they carry: dextrancontaining vesicles, behave similarly to EGFR containing ones. Markers of recycling pathwayTrasferrin receptor and Lamp, are increased on the surface of P31-43 treated cells together withEGFR. Gliadin peptide P31-43 enters epithelial cells and colocalize with EEA1 both in CD biopsiesand controls coltured for 24 hours. Pulse and chase experiments show a delay of trafficking of P31-43 peptide in EEA1 positive vesicles only in CD biopsies, but not in controlsConclusionsGliadin peptide P31-43 delays vesicle trafficking by interfering with Hrs mediated maturation to lateendosomes and also promotes the recycling pathway. As a consequence, in P31-43 treated cells,EGFR activation is extended, more transferrin and EGFR receptors find their way to the cellsurface. In CD biopsies trafficking of this peptide is delayed. This may help explaining the roleplayed by gliadin peptides in CD pathogenesis.NOTE: T

Gliadin peptide P31-43 interferes with Hrs localization to endocytic vesicles

Gliadin peptide P31-43 induces Epidermal Growth Factor Receptor (EGFR) dependent proliferation and actinmodifications in cultured cell lines and in enterocytes from celiac disease (CD) patients. These effects aremediated by delayed degradation of the EGFR in endocytic vesicles. We investigated the molecular mechanismsunderlying the effects of gliadin peptides on trafficking of endocytic vesicles responsible for EGFR degradation.P31-43 peptide entered CaCo-2 cells and localized in early endosomes, maturation to late endosomes of P31-43 containing vesicles was delayed. A sequence similarity search revealed that P31-43 is similar to a region ofHrs (HGF-regulated tyrosine kinase substrate) needed for its correct localization to the endocytic vesicles. P31-43 interfering with Hrs localization to early endosomes induced delay of endocytic maturation and increase ofrecycling pathway markers, transferrin receptor and LAMP, on cells surface. In the celiac enterocytes P31-43 isdelayed in early endocytic vesicles. In conclusion P31-43 delays vesicle trafficking by interfering with Hrsmediatedmaturation to late endosomes, and promotes the recycling pathway in cells and intestinal biopsies.Consequently, in P31-43-treated cells and biopsies, EGFR activation is extended and more transferrin receptorreaches the surface. This may explain the role played by gliadin peptides in CD

Celiac disease patients dendritic cells have a different shape when interacting with the substrate compared to controls.

Dendritic cells from CD patients have a different shape compared to controls when interacting with fibronectin without any treatment.CD patients DC independently from the phase of the disease have a different shape when interacting with the substrate. Suggesting that genetic environment more than inflammatory state seems influence DC shape and actin cytoskeleton. Gliadin peptide P31-43 interferes with DC shape in healthy controls

CELL SHAPE DISCRIMINATES BETWEEN NORMAL AND CELIAC DISEASE (CD) DENDRITIC CELLS (DC).

AIM: The interaction with fibronectin of dendritic cells (DC) from celiac disease (CD) patients and controls was investigated. In particular, the ability of these cells to interact with the substrate by analyzing their morphology and actin rearrangement when in contact with fibronectin, which is a physiological substrate for DC. Furthermore, we investigated the role of anti-tTg and anti-β1 integrin antibodies on the interaction of DC with fibronectin. DC from controls and from CD patients both in the active and remission phase of the disease were compared. MATERIALS AND METHODS: Immature DC were generated by extracting monocytes from peripheral blood and stimulating them with IL-4 and GM-CSF for 6 days. Cell shape and adhesion was determined by crystal violet staining. Confocal microscopy of phalloidin staining was used to highlight the actin cytoskeleton.RESULTS: We showed that cells of CD patients have a different shape when interacting with the substrate compared to controls cells, even without any treatment. This alteration is observed in patients in the active as well in the remission fase of the disease. Gliadin peptide P31-43 interferes with cells shape mainly in controls, inducing DC to make more dendrites. Gliadin peptide P56-68 has less effect on cell shape.Moreover we found that tTg antibodies can interfere with the shape of the cells, by making them round and small with almost no dendrite formation, both in CD patients and controls, without interfering with the possibility to adhere. This indicates a new biological role for these antibodies in the disease.Also anti-β1integrin antibodies interfere with the shape of the cells in the same way tTg antibodies does, suggesting a common mechanism for both antibodies. CONCLUSIONS: These results suggest that genetic background in CD patients seems to influence DC shape and actin cytoskeleton more than inflammatory state. Moreover the treatment of controls cells with gliadin peptides, mainly P31-43, are able to alter the shape of the cells by inducing them to generate more dendrites. Anti-tTg and Anti-β1 integrin antibodies can interfere with DC morphology both in CD patients and controls. The biological activity of these antibodies on DC may have a role in the CD pathogenesis