Relationship between proliferative effects and activation of innate immunity induced by gliadin

Santagata, Sara (2010) Relationship between proliferative effects and activation of innate immunity induced by gliadin. [Tesi di dottorato] (Inedito)

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Abstract

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. We observed that 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. 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 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. Damage to the intestinal mucosa in celiac disease is mediated both by inflammation due to the 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 investigated the role of P31-43 in the induction of cellular proliferation and innate immune activation. In this work it has been shown that gliadin and P31-43 induce a proliferation of both CaCo2 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 interfering with its intracellular trafficking. The increased IL-15 protein was linked to IL15 receptor (IL-15R) alpha, which did not require new protein synthesis and therefore functioned as a growth factor. In this study, we have shown that P31-43 induces both an increase of the trans-presented IL-15/IL5R alpha complex on cell surfaces by interfering with trafficking of vesicular compartments as well as a proliferation of crypt enterocytes with consequent remodelling of CD mucosa due to a cooperation of IL-15 and EGFR. Celiac disease is condition where the regulation of the mucosal immune response to dietary gliadin might be altered. The transcription factor Foxp3 has been identified as a marker of a subset of regulatory T cells (Treg). In this study we have investigated the presence and the suppressive function of Treg cells in the celiac small intestinal mucosa, their correlation with the disease state and the inducibility by gliadin in an organ culture system; moreover, we tried to define whether interleukin 15, overexpressed in CD, could influence the regulatory activity of such cells. We observed a higher density of CD4+CD25+Foxp3+ T cells were seen in duodenal biopsies from active CD patients in comparison to treated CD and non-CD controls. In coculture CD4+CD25+ T cells were functionally suppressive, but their activity was impaired by IL-15. Cells from CD subjects showed increased sensitivity to the IL15 action likely due to enhanced expression of IL15 receptor. Finally, we demonstrated an expansion of Foxp3 in treated CD mucosa following in vitro challenge with gliadin. These data suggest that CD4+CD25+Foxp3+ T cells are induced in situ by gliadin. However, their suppressor capacity might be impaired in vivo by IL-15, this phenomenon contributing to maintain and expand the local inflammatory response in CD.

Tipologia di documento:Tesi di dottorato
Parole chiave:Celiac disease
Settori scientifico-disciplinari MIUR:Area 06 Scienze mediche > MED/12 GASTROENTEROLOGIA
Area 06 Scienze mediche > MED/38 PEDIATRIA GENERALE E SPECIALISTICA
Coordinatori della Scuola di dottorato:
Coordinatore del Corso di dottoratoe-mail (se nota)
Pignata, Claudiopignata@unina.it
Tutor della Scuola di dottorato:
Tutor del Corso di dottoratoe-mail (se nota)
Barone, Maria Vittoria
Stato del full text:Inedito
Data:29 Novembre 2010
Numero di pagine:142
Istituzione:Università degli studi di Napoli Federico II
Dipartimento o Struttura:Pediatria
Tipo di tesi:Dottorato
Stato dell'Eprint:Inedito
Scuola di dottorato:Medicina clinica e sperimentale
Denominazione del dottorato:Riproduzione, sviluppo e accrescimento dell'uomo
Ciclo di dottorato:23
Numero di sistema:8071
Depositato il:03 Dicembre 2010 10:37
Ultima modifica:18 Maggio 2011 12:22

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