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

Barone MV, Zanzi D, Maglio M, Nanayakkara M, Santagata S, Lania G, Miele E, Ribecco MT, Maurano F, Auricchio R, Gianfrani C, Ferrini S, Troncone R, Auricchio S - PLoS ONE (2011)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, European Laboratory for the Investigation of Food-Induced Diseases, University of Naples Federico II, Naples, Italy. mv.barone@unina.it

ABSTRACT

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.

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Related in: MedlinePlus

Overnight treatment with gliadin peptide P31-43, but not P57-68, increased levels of IL-15 mRNA in CaCo-2 cells.Quantitative PCR analysis shows an increase of IL-15 mRNA after O/N treatment of CaCo-2 cells with P31-43 but not after 30 min, 3 h and 6 h. This increase can be prevented by IL-15 blocking antibodies. RQ = relative quantity of IL-15 mRNA. Columns represent means, and bars are standard deviations of a representative experiment done in triplicate. Four separate experiments show similar results. UN = untreated. For methods, see supplementary material.
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pone-0017039-g003: Overnight treatment with gliadin peptide P31-43, but not P57-68, increased levels of IL-15 mRNA in CaCo-2 cells.Quantitative PCR analysis shows an increase of IL-15 mRNA after O/N treatment of CaCo-2 cells with P31-43 but not after 30 min, 3 h and 6 h. This increase can be prevented by IL-15 blocking antibodies. RQ = relative quantity of IL-15 mRNA. Columns represent means, and bars are standard deviations of a representative experiment done in triplicate. Four separate experiments show similar results. UN = untreated. For methods, see supplementary material.

Mentions: We treated CaCo-2 cells with P31-43 for 30 min, 3 h, 6 h or O/N to determine whether the peptide affected IL-15 mRNA levels. Quantitative PCR analysis showed an increase in IL-15 mRNA only after O/N treatment with P31-43, the control peptide P57-68 was not able to increase IL15 mRNA at the same levels (Fig. 3). Intriguingly, this increase in IL-15 mRNA is IL-15-dependent as it can be prevented by IL-15 blocking antibodies. This finding suggested that P31-43 acts on pre-existing IL-15 protein to further increase IL-15 mRNA accumulation in CaCo-2 cells. Indeed, exogenous IL-15 induced an even greater increase of IL-15 mRNA than did P31-43. (Methods are described in Text S2)


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

Barone MV, Zanzi D, Maglio M, Nanayakkara M, Santagata S, Lania G, Miele E, Ribecco MT, Maurano F, Auricchio R, Gianfrani C, Ferrini S, Troncone R, Auricchio S - PLoS ONE (2011)

Overnight treatment with gliadin peptide P31-43, but not P57-68, increased levels of IL-15 mRNA in CaCo-2 cells.Quantitative PCR analysis shows an increase of IL-15 mRNA after O/N treatment of CaCo-2 cells with P31-43 but not after 30 min, 3 h and 6 h. This increase can be prevented by IL-15 blocking antibodies. RQ = relative quantity of IL-15 mRNA. Columns represent means, and bars are standard deviations of a representative experiment done in triplicate. Four separate experiments show similar results. UN = untreated. For methods, see supplementary material.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3045409&req=5

pone-0017039-g003: Overnight treatment with gliadin peptide P31-43, but not P57-68, increased levels of IL-15 mRNA in CaCo-2 cells.Quantitative PCR analysis shows an increase of IL-15 mRNA after O/N treatment of CaCo-2 cells with P31-43 but not after 30 min, 3 h and 6 h. This increase can be prevented by IL-15 blocking antibodies. RQ = relative quantity of IL-15 mRNA. Columns represent means, and bars are standard deviations of a representative experiment done in triplicate. Four separate experiments show similar results. UN = untreated. For methods, see supplementary material.
Mentions: We treated CaCo-2 cells with P31-43 for 30 min, 3 h, 6 h or O/N to determine whether the peptide affected IL-15 mRNA levels. Quantitative PCR analysis showed an increase in IL-15 mRNA only after O/N treatment with P31-43, the control peptide P57-68 was not able to increase IL15 mRNA at the same levels (Fig. 3). Intriguingly, this increase in IL-15 mRNA is IL-15-dependent as it can be prevented by IL-15 blocking antibodies. This finding suggested that P31-43 acts on pre-existing IL-15 protein to further increase IL-15 mRNA accumulation in CaCo-2 cells. Indeed, exogenous IL-15 induced an even greater increase of IL-15 mRNA than did P31-43. (Methods are described in Text S2)

Bottom Line: 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.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, European Laboratory for the Investigation of Food-Induced Diseases, University of Naples Federico II, Naples, Italy. mv.barone@unina.it

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus