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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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Gliadin peptide P31-43 increased IL-15/IL-15R alpha complex on the cell surface in CaCo-2 cells.(A) P31-43 increased IL-15 on the cell surface in CaCo-2 cells. FACS analysis of IL-15 on the cell surface after overnight (O/N), or 3 h or 6 h of treatment (B) with P31-43. Columns represent means and bars are the standard deviations of ten independent experiments for panel A and three independent experiments for panel B; * = p<0.05 (Student's t-test), ** = p<0.01 (Student's t-test) (C) Histogram of one representative experiment of CaCo-2 cells treated O/N with P31-43. Black dotted curve corresponds to negative control (isotype-matched Ab), the green open curve depicts specific IL-15 staining after medium treatment and pink open curve is specific IL-15 staining after O/N culture with P31-43.
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pone-0017039-g004: Gliadin peptide P31-43 increased IL-15/IL-15R alpha complex on the cell surface in CaCo-2 cells.(A) P31-43 increased IL-15 on the cell surface in CaCo-2 cells. FACS analysis of IL-15 on the cell surface after overnight (O/N), or 3 h or 6 h of treatment (B) with P31-43. Columns represent means and bars are the standard deviations of ten independent experiments for panel A and three independent experiments for panel B; * = p<0.05 (Student's t-test), ** = p<0.01 (Student's t-test) (C) Histogram of one representative experiment of CaCo-2 cells treated O/N with P31-43. Black dotted curve corresponds to negative control (isotype-matched Ab), the green open curve depicts specific IL-15 staining after medium treatment and pink open curve is specific IL-15 staining after O/N culture with P31-43.

Mentions: To investigate whether P31-43 affects the expression of IL-15 protein, we evaluated (by FACS analysis) the intracellular and surface pools of IL-15 in CaCo-2 cells before and after exposure to P31-43. Overnight treatment with P31-43 did not affect the intracellular pool of IL-15 (Figure S2) and neither did shorter treatment times (not shown). We next evaluated whether P31-43 affects the extra-cellular release of IL-15 by CaCo-2 cells. After overnight incubation with P31-43, there was no statistically significant increase in IL-15 in the supernatant as measured by ELISA assay (Figure S3). However, the percentage of IL-15-positive cells on the surface increased from 22.92%±22.24% to 53.20%±18.26% after overnight treatment (Fig. 4A). This increase is specific for P31-43 because the control peptide P57-68 did not affect the percentage of cells expressing IL-15 on the surface (from 22.92%±22.24% to 17.09%±11.98%). IL-15 on the cell surface appeared to increase in expression after only 3 h of incubation with P31-43. This increase became statistically significant after 6 h of incubation, when it was comparable to that observed after overnight treatment (Fig. 4B). These findings indicate that either P31-43 increases the production of the IL-15 protein or mobilizes, from a pre-existing protein pool, IL-15 on the surface of the cells. We next analysed whether protein synthesis blockade induced by cycloheximide treatment was able to interfere with P31-43-induced increase of IL-15 on cell surfaces. Cycloheximide treatment failed to prevent the P31-43-mediated expression of IL-15 on the cell surface (57.42%+/−10.52% vs. 52.40+/−8.35, in the absence of cycloheximide) (Fig. 4B), suggesting that protein synthesis is not required for the P31-43 effect and that IL-15 is mobilized from an existing intracellular pool to the cell surface.


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)

Gliadin peptide P31-43 increased IL-15/IL-15R alpha complex on the cell surface in CaCo-2 cells.(A) P31-43 increased IL-15 on the cell surface in CaCo-2 cells. FACS analysis of IL-15 on the cell surface after overnight (O/N), or 3 h or 6 h of treatment (B) with P31-43. Columns represent means and bars are the standard deviations of ten independent experiments for panel A and three independent experiments for panel B; * = p<0.05 (Student's t-test), ** = p<0.01 (Student's t-test) (C) Histogram of one representative experiment of CaCo-2 cells treated O/N with P31-43. Black dotted curve corresponds to negative control (isotype-matched Ab), the green open curve depicts specific IL-15 staining after medium treatment and pink open curve is specific IL-15 staining after O/N culture with P31-43.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045409&req=5

pone-0017039-g004: Gliadin peptide P31-43 increased IL-15/IL-15R alpha complex on the cell surface in CaCo-2 cells.(A) P31-43 increased IL-15 on the cell surface in CaCo-2 cells. FACS analysis of IL-15 on the cell surface after overnight (O/N), or 3 h or 6 h of treatment (B) with P31-43. Columns represent means and bars are the standard deviations of ten independent experiments for panel A and three independent experiments for panel B; * = p<0.05 (Student's t-test), ** = p<0.01 (Student's t-test) (C) Histogram of one representative experiment of CaCo-2 cells treated O/N with P31-43. Black dotted curve corresponds to negative control (isotype-matched Ab), the green open curve depicts specific IL-15 staining after medium treatment and pink open curve is specific IL-15 staining after O/N culture with P31-43.
Mentions: To investigate whether P31-43 affects the expression of IL-15 protein, we evaluated (by FACS analysis) the intracellular and surface pools of IL-15 in CaCo-2 cells before and after exposure to P31-43. Overnight treatment with P31-43 did not affect the intracellular pool of IL-15 (Figure S2) and neither did shorter treatment times (not shown). We next evaluated whether P31-43 affects the extra-cellular release of IL-15 by CaCo-2 cells. After overnight incubation with P31-43, there was no statistically significant increase in IL-15 in the supernatant as measured by ELISA assay (Figure S3). However, the percentage of IL-15-positive cells on the surface increased from 22.92%±22.24% to 53.20%±18.26% after overnight treatment (Fig. 4A). This increase is specific for P31-43 because the control peptide P57-68 did not affect the percentage of cells expressing IL-15 on the surface (from 22.92%±22.24% to 17.09%±11.98%). IL-15 on the cell surface appeared to increase in expression after only 3 h of incubation with P31-43. This increase became statistically significant after 6 h of incubation, when it was comparable to that observed after overnight treatment (Fig. 4B). These findings indicate that either P31-43 increases the production of the IL-15 protein or mobilizes, from a pre-existing protein pool, IL-15 on the surface of the cells. We next analysed whether protein synthesis blockade induced by cycloheximide treatment was able to interfere with P31-43-induced increase of IL-15 on cell surfaces. Cycloheximide treatment failed to prevent the P31-43-mediated expression of IL-15 on the cell surface (57.42%+/−10.52% vs. 52.40+/−8.35, in the absence of cycloheximide) (Fig. 4B), suggesting that protein synthesis is not required for the P31-43 effect and that IL-15 is mobilized from an existing intracellular pool to the cell surface.

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