Gliadin Peptide P31-43 Research Articles

Inflammation Is Present, Persistent and More Sensitive to Proinflammatory Triggers in Celiac Disease Enterocytes.

Celiac disease (CD) is a chronic inflammatory disease caused by a genetic predisposition to an abnormal T cell-mediated immune response to the gluten in the diet. Different environmental proinflammatory factors can influence and amplify the T cell-mediated response to gluten. The aim of this manuscript was to study the role of enterocytes in CD intestinal inflammation and their response to different proinflammatory factors, such as gliadin and viruses. Intestinal biopsies from CD patients on a gluten-containing (GCD-CD) or a gluten-free diet (GFD-CD) as well as biopsies from potential CD patients (Pot-CD) before the onset of intestinal lesions and controls (CTR) were used to investigate IL-1β and IL-6 mRNA levels in situ. Organoids from CD patients were used to test the levels of NF-κB, ERK, IL-6, and IL-1β by Western blot (WB), ELISA, and quantitative PCR. The Toll-like receptor ligand loxoribine (Lox) and gliadin peptide P31-43 were used as proinflammatory stimuli. In CD biopsies inflammation markers IL-1β and IL-6 were increased in the enterocytes, and also in Pot-CD before the onset of the intestinal lesion and in GFD-CD. The inflammatory markers pNF-κB, pERK, IL-1β, and IL-6 were increased and persistent in CD organoids; these organoids were more sensitive to P31-43 and Lox stimuli compared with CTR organoids. Taken together, these observations point to constitutive inflammation in CD enterocytes, which are more sensitive to inflammatory stimuli such as food components and viruses.

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.

Effect of pH control during rice fermentation in preventing a gliadin P31-43 entrance in epithelial cells

Coeliac disease is an increasingly recognised pathology, induced by the ingestion of gluten in genetically predisposed patients. Undigested gliadin peptide can induce adaptive and innate immune response that unleash the typical intestinal mucosal alterations. A growing attention is paid to alternative therapeutic approaches to the gluten-free diet: one of these approaches is the use of probiotics and/or postbiotics. We performed lactic fermentation of rice flour with and without pH control, using Lactobacillus paracasei CBA L74 as fermenting strain. We evaluated bacterial growth, lactic acid production during fermentation and gliadin peptide P31-43 entrance in CaCo-2 cells with and without pH control. When pH control was applied no differences were observed in terms of bacterial growth; on the contrary, lactic acid production was greater, as expected. Both samples could inhibit the P31-43 entrance in CaCo-2 cells but the effect was significantly greater for samples obtained when the pH control was applied.

A Peptide from Kiwifruit Exerts Anti-Inflammatory Effects in Celiac Disease Mucosa

Objective: Celiac disease is an immune-mediated disease of the intestine triggered by gluten. Gluten elicits, in genetically susceptible individuals, cytokine responses that are then transmitted to the immunocompetent cells. Vegetables and fruit have anti-inflammatory and antioxidant properties with a protective effect on intestinal epithelium. Kiwifruit is known to have beneficial effects on the intestinal tissues, and it is the only plant food containing the peptide kissper, with anti-inflammatory properties. The aim of this study was the evaluation of the kissper effect on the gluten-induced inflammation in celiac disease.Methods: We used an in vitro model of intestinal culture explant from celiac disease patients and non-celiac disease patients, cultured for 24 hours with the toxic gliadin peptide P31-43 and kissper preincubation.Results: Our data showed HLA-DR and TG2 reduction in the celiac disease mucosa pretreated with kissper, as well as a reduction of COX-2 in two patients. No differences we observed for the TGF-b1 and IL-15 levels in supernatants upon kissper pretreatment.Conclusions: The preliminary results suggest that kissper has a potential anti-inflammatory role in celiac disease.

Mechanisms of innate immune activation by gluten peptide p31-43 in mice.

Celiac disease (CD) is an immune-mediated enteropathy triggered by gluten in genetically susceptible individuals. Innate immunity contributes to the pathogenesis of CD, but the mechanisms remain poorly understood. Although previous in vitro work suggests that gliadin peptide p31-43 acts as an innate immune trigger, the underlying pathways are unclear and have not been explored in vivo. Here we show that intraluminal delivery of p31-43 induces morphological changes in the small intestinal mucosa of normal mice consistent with those seen in CD, including increased cell death and expression of inflammatory mediators. The effects of p31-43 were dependent on MyD88 and type I IFNs, but not Toll-like receptor 4 (TLR4), and were enhanced by coadministration of the TLR3 agonist polyinosinic:polycytidylic acid. Together, these results indicate that gliadin peptide p31-43 activates the innate immune pathways in vivo, such as IFN-dependent inflammation, relevant to CD. Our findings also suggest a common mechanism for the potential interaction between dietary gluten and viral infections in the pathogenesis of CD.

Gliadin-dependent cytokine production in a bidimensional cellular model of celiac intestinal mucosa.

The downstream cascade of the inflammatory response to gliadin in celiac intestinal mucosa encompasses the early activation of the innate immunity that triggers the adaptive response. Therefore, the in vitro study of the pathogenic mechanism of celiac disease (CD) on enterocytes alone or mucosal T lymphocytes alone does not fully consider all the aspects of gliadin-dependent inflammation. Although the in vitro culture of specimens of intestinal mucosa obtained from celiac patients is the gold standard for the study of CD, this technique presents several technical challenges and the bioptic specimens are not easily available. So, in this paper, we described the gliadin-dependent cytokine production in a bidimensional cellular system, which is able to mimic both the innate and the adaptive steps of the mucosal immune response of CD. In the upper compartment, the intestinal epithelial cells are grown on a filter, and in the lower compartment, the mononuclear cells isolated from peripheral blood of celiac patients are cultured. Cells were apically exposed to the toxic gliadin peptide p31-43 for 3 h and then with the immunodominant gliadin fragment pα-9 for 21 h. The incubation with gliadin peptides resulted in increased levels of IL-15, INF-γ, IL-6, tumor necrosis factor (TNF)-α, IL-1β, and CCL 2, 3 and 4 in the basal supernatants, with respect to cells exposed to medium alone. The p31-43-driven epithelial priming of mucosal response consists of transglutaminase (TG2)-mediated deamidation of the immunostimulatory gliadin peptides, as demonstrated by the inhibition of pα-9 activity, when the system is exposed to blocking anti-TG2 antibody.

A Celiac Cellular Phenotype, with Altered LPP Sub-Cellular Distribution, Is Inducible in Controls by the Toxic Gliadin Peptide P31-43

Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP) gene was identified as strongly associated with CD using genome-wide association studies (GWAS). The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD) and controls, without and with treatment with A-gliadin peptide P31-43. We observed a “CD cellular phenotype” in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis.

Enterocyte proliferation and signaling are constitutively altered in celiac disease.

Celiac disease (CD) occurs frequently, and is caused by ingestion of prolamins from cereals in subjects with a genetic predisposition. The small intestinal damage depends on an intestinal stress/innate immune response to certain gliadin peptides (e.g., A-gliadin P31-43) in association with an adaptive immune response to other gliadin peptides (e.g., A-gliadin P57-68). Gliadin and peptide P31-43 affect epithelial growth factor receptor (EGFR) signaling and CD enterocyte proliferation. The reason why the stress/innate immune and proliferative responses to certain gliadin peptides are present in CD and not in control intestine is so far unknown. The aim of this work is to investigate if, in CD, a constitutive alteration of enterocyte proliferation and signaling exists that may represent a predisposing condition to the damaging effects of gliadin. Immunofluorescence and immunohistochemistry were used to study signaling in CD fibroblasts and intestinal biopsies. Western blot (WB) analysis, immunoprecipitation, and quantitative PCR were also used. We found in CD enterocytes enhancement of both proliferation and Epidermal Growth Factor Receptor (EGFR)/ligand system. In CD enterocytes and fibroblasts we found increase of the phosphorylated downstream signaling molecule Extracellular Signal Regulated Kinase (ERK); block of the ERK activation normalizes enterocytes proliferation in CD mucosa. In conclusion the same pathway, which gliadin and gliadin peptide P31-43 can interfere with, is constitutively altered in CD cells. This observation potentially explains the specificity of the damaging effects of certain gliadin peptides on CD intestine.

An undigested gliadin peptide activates innate immunity and proliferative signaling in enterocytes: the role in celiac disease

On ingestion of gliadin, the major protein component of wheat and other cereals, the celiac intestine is characterized by the proliferation of crypt enterocytes with an inversion of the differentiation/proliferation program. Gliadins and A-gliadin peptide P31-43, in particular, act as growth factors for crypt enterocytes in patients with celiac disease (CD). The effects of gliadin on crypt enterocyte proliferation and activation of innate immunity are mediated by epidermal growth factors (EGFs) and innate immunity mediators [interleukin 15 (IL15)]. The aim of this study was to determine the molecular basis of proliferation and innate immune response to gliadin peptides in enterocytes. The CaCo-2 cell line was used to study EGF-, IL15-, and P31-43-induced proliferation. Silencing messenger RNAs and blocking EGF receptor and IL15 antibodies have been used to study proliferation in CaCo-2 cells and intestinal biopsy samples from patients with CD and control subjects. In the CaCo-2 cell model, IL15 and EGF cooperated to induce proliferation in intestinal epithelial cells at both the transcriptional and posttranscriptional levels, and the respective receptors interacted to activate each other's signaling. In addition, the effects of the P31-43 peptide on CaCo-2 cell proliferation and downstream signaling were mediated by cooperation between EGF and IL15. The increased crypt enterocyte proliferation in intestinal biopsy samples from patients with CD was reduced by EGF receptor and IL15 blocking antibodies only when used in combination. EGF receptor/IL15R-α cooperation regulates intestinal epithelial cell proliferation induced by EGF, IL15, and the gliadin peptide P31-43. Increased proliferation of crypt enterocytes in the intestine of CD patients is mediated by EGF/IL15 cooperation.

Gliadin-mediated proliferation and innate immune activation in celiac disease are due to alterations in vesicular trafficking.

Background and ObjectivesDamage 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 FindingsCell 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.ConclusionIn 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.

BackgroundCeliac 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 FindingsBoth 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.ConclusionsP31-43 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 Tyrosin 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.

Gliadin peptide P31-43 presents IL15 in trans to the neighbouring cells

Background and aim. We have previously observed that gliadin peptide P31-43 can induce proliferation of NIH3T3 cells and of enterocytes of untreated celiac mucosa due to an interference of this peptide with the degradation of the ligand loaded EGF receptor [ [1] Barone et al. Growth factor-like activity of gliadin, an alimentary protein: implications for coeliac disease. Gut. 2007; 56 (Epub 2006 Aug 4): 480-488 Crossref PubMed Scopus (90) Google Scholar ]. We have also shown that in presence of P31-43 the EGF receptor stays longer activated as a consequence of a delay of the endocytic maturation. Aim of our study was to investigate whether proliferative effects induced by gliadin peptide P31-43 could be mediated also by activation of innate immunity. In particular we tested P31-43 effects on IL15 induction at level of transcription, translation, intracellular trafficking and role in P31-43 induced proliferation of both Caco2 cells and celiac enterocytes.

Rotavirus and Celiac Disease: Clues to the Pathogenesis and Perspectives on Prevention

Rotavirus and Celiac Disease: Clues to the Pathogenesis and Perspectives on Prevention

EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) DEPENDENT PROLIFERATION IN CULTURED BIOPSIES FROM CELIAC DISEASE (CD) PATIENTS CHALLENGED WITH GLIADIN

Aim: Caracterization of the role of EGFR signalling on the early effects of gliadin peptides on jejunal colture biopsies from CD untreated patients. Method: Biopsies from untreated CD patients were cultured over-night in an organ culture system in the presence of peptic-triptic digest of gliadin (PTG) or of ovalbumin (PTO). In other experiments gliadin peptides P31-43 or P56-68 were used. To test the EGFR involvement in PTG and P31-43 effects on cell morphology and proliferation two inhibitors were used. ZD … and the bloking antibody anti EGFR 528. Bromodeoxiuridine was added to the medium to detect cells in to the S-fase of the cell cycle. Actin cytoskeleton rearrangements (by phalloidin), number of cells in S-fase (by bromodeoxiuridine) and in apoptosis (Tunel staining) were examined; EGFR was stained by specific antibody (SC 1005). Results: Incubation of untreated celiac biopsies in presence of PTG or P31-43 induces an alteration of the epitelial morphology and the actin cytoskeleton and a diffused, more intense EGFR staining. An increase of both apoptotic and proliferating epitelial cells is detectable with BrdU incorporation rising to 38% (−/ + 8%) versus 15% (−/ + 5%) of the control, and Tunel positive cells rising to 28% (−/ + 10%) versus 8% (−/ + 4%) of the control. The EGFR inhibitors were able to prevent the morphology alteration, the actin cytoskeleton modifications and the increase of the BrdU incorporation obtained in presence of PTG and P31-43. In contrast PTG and P31-43 induced apoptosis was not greatly altered in presence of the EGFR inhibitors. Summary: Peptic-tryptic digests of gliadin (PTG) and P31-43 gliadin peptide mimic EGFR (Epidermal Growth Factor Receptor) signalling and induce actin rearrangements and proliferation in several cell lines (nota bibliografica). As EGFR signalling is known to act on cell proliferation and morphology, PTG ability to exert EGF effects on biopsies from florid CD patients in colture was tested. Gliadin induced alteration of epitelial cells morphology, actin cytoskeleton and proliferation are prevented by EGFR inhibitors; strongly suggesting that this pathway is ingaged by gliadin peptides at least for these read outs. Probably, other receptors may be involved in gliadin induced apoptosis since this effect is not prevented by EGFR inhibitors. Conclusion: Gliadin and gliadin peptide P31-43 induce EGFR dependent proliferation but not apoptosis in cultured biopsies from CD patients.