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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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P31-43 alters trafficking of IL-15-containing recycling vesicles and increases recycling markers expressed on CaCo-2 cell surfaces.(A) IL-15-EGFP and Transferrin-Tex Red accumulate and co-localise after P31-43 treatment in a recycling vesicular compartment. IL-15-EGFP was transfected into CaCo-2 cells and observed by microscope after treatment with Transferrin-Tex Red and P31-43. White lines show the area of a single cell. (63x objective and 2x zoom). IL-15-EGFP (green) co-localises with Transferrin-Tex-Red (red) positive vesicles. Merge of the red and green panels is shown with yellow/orange colour indicating co-localisation. The co-localisation coefficient was calculated as reported under “Methods”. The results are representative of three independent experiments. For methods, see supplementary material. (B) Statistical analysis of fluorescence intensity/cell. For treated and untreated samples, three independent experiments were done, measuring fluorescence intensity of 10 cells in random fields in each experiment. ** = p<0.01, *** = p<0.001 (Student t-test). For methods, see supplementary material.
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pone-0017039-g009: P31-43 alters trafficking of IL-15-containing recycling vesicles and increases recycling markers expressed on CaCo-2 cell surfaces.(A) IL-15-EGFP and Transferrin-Tex Red accumulate and co-localise after P31-43 treatment in a recycling vesicular compartment. IL-15-EGFP was transfected into CaCo-2 cells and observed by microscope after treatment with Transferrin-Tex Red and P31-43. White lines show the area of a single cell. (63x objective and 2x zoom). IL-15-EGFP (green) co-localises with Transferrin-Tex-Red (red) positive vesicles. Merge of the red and green panels is shown with yellow/orange colour indicating co-localisation. The co-localisation coefficient was calculated as reported under “Methods”. The results are representative of three independent experiments. For methods, see supplementary material. (B) Statistical analysis of fluorescence intensity/cell. For treated and untreated samples, three independent experiments were done, measuring fluorescence intensity of 10 cells in random fields in each experiment. ** = p<0.01, *** = p<0.001 (Student t-test). For methods, see supplementary material.

Mentions: IL-15 has been found in the Golgi complex and in transferrin-carrying endocytic vesicles [25], [26]. We previously demonstrated that P31-43 alters the vesicular trafficking [9]. Therefore, we evaluated whether P31-43 affects the recycling pathway by carrying more IL-15 to the cell surface. IL15-EGFP localises to a recycling vesicular compartment when it is transfected in CaCo-2 cells [25]. After treatment with P31-43, IL-15EGFP-containing vesicles accumulated in the cytosol as shown in Fig. 9 A,B. The fluorescence intensity of the P31-43-treated cells exhibited a statistically significant increase from 54±2.9 to 79.3±4.7 after P31-43 treatment. To identify the IL-15EGFP-containing vesicular compartment, we treated CaCo-2 cells transfected with IL-15-EGFP with the recycling marker transferrin-Tex-Red for 90 min. [36]. As shown in Fig. 9A and B, treatment with P31-43 increased the number of transferring-containing vesicles, indicating that P31-43 can alter the trafficking of the recycling vesicles (fluorescence intensity/cell increased from 52.25±6.8 to 73.3±5.6 after P31-43 treatment). Treatment with P57-68 had no effect on the number of transferring-carrying vesicles. Furthermore, IL-15-EGFP co-localised with transferrin-Tex red in the same vesicular compartment before and after P31-43 treatment. To confirm P31-43 induced alterations of the recycling vesicular compartment, we investigated the levels of recycling marker transferrin receptor on the cell surface by FACS analysis before and after overnight treatment with P31-43 or P57-68. As shown in Fig. 10, the percentage of cells displaying the transferrin receptor on their surfaces significantly increased (from 18%±7% to 34.4%±13%) after P31-43 treatment while P57-68 treatment had no effect on the cell surface levels of transferrin receptor (from 18%±7% to 13%±4.7%). Therefore, P31-43 increased the expression of recycling vesicle markers on the cell surface, suggesting that the increase of IL-15 on the cell surface may relate to re-distribution of IL-15 from an intracellular vesicular compartment to the cell membrane. (Methods are described in Text S4.)


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)

P31-43 alters trafficking of IL-15-containing recycling vesicles and increases recycling markers expressed on CaCo-2 cell surfaces.(A) IL-15-EGFP and Transferrin-Tex Red accumulate and co-localise after P31-43 treatment in a recycling vesicular compartment. IL-15-EGFP was transfected into CaCo-2 cells and observed by microscope after treatment with Transferrin-Tex Red and P31-43. White lines show the area of a single cell. (63x objective and 2x zoom). IL-15-EGFP (green) co-localises with Transferrin-Tex-Red (red) positive vesicles. Merge of the red and green panels is shown with yellow/orange colour indicating co-localisation. The co-localisation coefficient was calculated as reported under “Methods”. The results are representative of three independent experiments. For methods, see supplementary material. (B) Statistical analysis of fluorescence intensity/cell. For treated and untreated samples, three independent experiments were done, measuring fluorescence intensity of 10 cells in random fields in each experiment. ** = p<0.01, *** = p<0.001 (Student t-test). For methods, see supplementary material.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045409&req=5

pone-0017039-g009: P31-43 alters trafficking of IL-15-containing recycling vesicles and increases recycling markers expressed on CaCo-2 cell surfaces.(A) IL-15-EGFP and Transferrin-Tex Red accumulate and co-localise after P31-43 treatment in a recycling vesicular compartment. IL-15-EGFP was transfected into CaCo-2 cells and observed by microscope after treatment with Transferrin-Tex Red and P31-43. White lines show the area of a single cell. (63x objective and 2x zoom). IL-15-EGFP (green) co-localises with Transferrin-Tex-Red (red) positive vesicles. Merge of the red and green panels is shown with yellow/orange colour indicating co-localisation. The co-localisation coefficient was calculated as reported under “Methods”. The results are representative of three independent experiments. For methods, see supplementary material. (B) Statistical analysis of fluorescence intensity/cell. For treated and untreated samples, three independent experiments were done, measuring fluorescence intensity of 10 cells in random fields in each experiment. ** = p<0.01, *** = p<0.001 (Student t-test). For methods, see supplementary material.
Mentions: IL-15 has been found in the Golgi complex and in transferrin-carrying endocytic vesicles [25], [26]. We previously demonstrated that P31-43 alters the vesicular trafficking [9]. Therefore, we evaluated whether P31-43 affects the recycling pathway by carrying more IL-15 to the cell surface. IL15-EGFP localises to a recycling vesicular compartment when it is transfected in CaCo-2 cells [25]. After treatment with P31-43, IL-15EGFP-containing vesicles accumulated in the cytosol as shown in Fig. 9 A,B. The fluorescence intensity of the P31-43-treated cells exhibited a statistically significant increase from 54±2.9 to 79.3±4.7 after P31-43 treatment. To identify the IL-15EGFP-containing vesicular compartment, we treated CaCo-2 cells transfected with IL-15-EGFP with the recycling marker transferrin-Tex-Red for 90 min. [36]. As shown in Fig. 9A and B, treatment with P31-43 increased the number of transferring-containing vesicles, indicating that P31-43 can alter the trafficking of the recycling vesicles (fluorescence intensity/cell increased from 52.25±6.8 to 73.3±5.6 after P31-43 treatment). Treatment with P57-68 had no effect on the number of transferring-carrying vesicles. Furthermore, IL-15-EGFP co-localised with transferrin-Tex red in the same vesicular compartment before and after P31-43 treatment. To confirm P31-43 induced alterations of the recycling vesicular compartment, we investigated the levels of recycling marker transferrin receptor on the cell surface by FACS analysis before and after overnight treatment with P31-43 or P57-68. As shown in Fig. 10, the percentage of cells displaying the transferrin receptor on their surfaces significantly increased (from 18%±7% to 34.4%±13%) after P31-43 treatment while P57-68 treatment had no effect on the cell surface levels of transferrin receptor (from 18%±7% to 13%±4.7%). Therefore, P31-43 increased the expression of recycling vesicle markers on the cell surface, suggesting that the increase of IL-15 on the cell surface may relate to re-distribution of IL-15 from an intracellular vesicular compartment to the cell membrane. (Methods are described in Text S4.)

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