Gliadin fragments promote migration of dendritic cells

Abstract

In genetically predisposed individuals, ingestion of wheat gliadin provokes a T-cell-mediated enteropathy, celiac disease. Gliadin fragments were previously reported to induce phenotypic maturation and Th1 cytokine production by human dendritic cells (DCs) and to boost their capacity to stimulate allogeneic T cells. Here, we monitor the effects of gliadin on migratory capacities of DCs. Using transwell assays, we show that gliadin peptic digest stimulates migration of human DCs and their chemotactic responsiveness to the lymph node-homing chemokines CCL19 and CCL21. The gliadin-induced migration is accompanied by extensive alterations of the cytoskeletal organization, with dissolution of adhesion structures, podosomes, as well as up-regulation of the CC chemokine receptor (CCR) 7 on cell surface and induction of cyclooxygenase (COX)-2 enzyme that mediates prostaglandin E2 (PGE₂) production. Blocking experiments confirmed that gliadin-induced migration is independent of the TLR4 signalling. Moreover, we showed that the α-gliadin-derived 31-43 peptide is an active migration-inducing component of the digest. The migration promoted by gliadin fragments or the 31-43 peptide required activation of p38 mitogen-activated protein kinase (MAPK). As revealed using p38 MAPK inhibitor SB203580, this was responsible for DC cytoskeletal transition, CCR7 up-regulation and PGE₂ production in particular. Taken together, this study provides a new insight into pathogenic features of gliadin fragments by demonstrating their ability to promote DC migration, which is a prerequisite for efficient priming of naive T cells, contributing to celiac disease pathology.

Fig 1

Gliadin fragments promote spontaneous as well as chemotactic DC migration. (A) Spontaneous DC migration (migration of DCs towards the chamber without chemokines) and chemotactic DC migration (migration towards the chamber supplemented with 200 ng/ml of CCL19 or CCL21) were measured after 24 or 48 hrs of cell exposure to gliadin fragments (Gl100, 100 μg/ml; Gl200, 200 μg/ml), or LPS (1 μg/ml). Data are expressed as a migration index and represent the means ± S.D. of at least six independent experiments. *P < 0.05 versus iDC (unstimulated DCs). (B) To confirm that migration was not due to any potential LPS contamination of the gliadin digest, DCs were pretreated with blocking TLR4 mAb at 20 μg/ml for 1 hr and then stimulated for 24 hrs with gliadin fragments (Gl200 μg/ml) or LPS (1 μg/ml). The DC migration was assessed as above. Data represent the means ± S.D. for at least two independent experiments. With versus without anti-TLR4 mAb; *P < 0.05.

Fig 2

Gliadin fragments induce cytoskeletal remodelling in DCs. (A) DCs plated on fibronectin-coated cover slips were stimulated with gliadin fragments (Gl200, 200 μg/ml) or LPS (1 μg/ml) for 24 hrs. Following fixation with 4% paraformaldehyde, the cells were stained with TRITC-conjugated phalloidin to detect F-actin (red) and anti-vinculin Ab (green). The micrographs are representative of at least six independent experiments. (B) The percentage of cells with podosomes was determined after cell treatment with gliadin fragments (Gl100, 100 μg/ml; Gl200, 200 μg/ml), or LPS (1 μg/ml) for 24 hrs. Results represent the means ± S.D. of at least three independent experiments, in which at least 100 cells per condition per experiment were counted (the percentage of cells containing podosomes of untreated control iDC was arbitrarily set to 100%). *P < 0.05 versus iDC.

Fig 3

Gliadin fragments up-regulate CCR7 in DCs. (A) Total RNA was isolated from unstimulated control iDC or DCs stimulated with gliadin fragments (Gl200, 200 μg/ml) for 24 hrs. The levels of CCR7 mRNA and total GAPDH mRNA (internal control) were determined by RT-PCR. The shown data are representative of four independent experiments that yield similar results. (B) After cell stimulation for 24 or 48 hrs with gliadin fragments (Gl100, 100 μg/ml; Gl200, 200 μg/ml), or LPS (1 μg/ml), the surface expression of CCR7 was determined by flow cytometry. Data represent the means ± S.D. of at least eight independent experiments, in which the MFI of untreated control iDC was arbitrarily set to 100%. *P < 0.05 versus iDC.

Fig 4

Gliadin fragments up-regulate COX-2 expression and PGE2 production. (A) After DC stimulation with gliadin fragments (Gl200, 200 μg/ml) or LPS (1 μg/ml), COX-2 mRNA levels were determined by RT-PCR. GAPDH mRNA was used as an internal control. The shown data are representative of four independent experiments that gave similar results. (B) COX-2 protein levels and total actin (loading control) in whole cell lysates were detected by Western blot. Results are representative of at least six independent experiments. (C) Levels of PGE2 in culture supernatants were measured by EIA. The shown data are representative experiment of two independent experiments. *P < 0.05 versus iDC.

Fig 5

The α-gliadin-derived 31–43 peptide is an active migration-inducing component of the digest. DC migration was measured in transwell assays at 24 hrs of cell treatment with the synthetic 31–43 or 33mer peptides (100 μg/ml) or a mixture of both peptides (100:100 μg/ml, ratio 1:1) for 24 hrs. Data are expressed as the means ± S.D. of at least five independent experiments. *P < 0.05 versus iDC.

Fig 6

p38 MAPK activation is crucial for gliadin-induced DC migration. Following 30 min. of pre-incubation with 20 μM SB203580 (+SB20) or without treatment with SB203580 (–SB20), DCs were exposed to gliadin fragments (Gl200, 200 μg/ml) or LPS (1 μg/ml) for 24 or 48 hrs, or to the synthetic 31–43 peptide (100 μg/ml) for 24 hrs, washed, and evaluated for spontaneous (A) and chemotactic migration (B, C) in transwell assays, respectively. Data represent the means ± S.D. for at least three independent experiments. With versus without 20 μM SB203580 (SB20); *P < 0.05.

Fig 7

Gliadin fragments signalling through the p38 MAPK pathway promotes cytoskeletal remodelling, CCR7 up-regulation and PGE2 production. (A, B) DCs plated on fibronectin-coated cover slips were pretreated with 10 μM SB203580 (+SB10) for 30 min. or left without SB203580 treatment (–SB10), exposed to gliadin fragments (Gl200, 200 μg/ml) or LPS (1 μg/ml) for 24 hrs, and stained for F-actin (red) and vinculin (green). The percentage of cells with podosomes was counted from at least 100 cells per condition per experiment (the percentage of cells containing podosomes of untreated control iDC was arbitrarily set to 100%) and the shown data represent the means ± S.D. for three independent experiments. With versus without 10 μM SB203580 (SB10); *P < 0.05; (C) Following 30 min. of pretreatment with 20 μM SB203580 (+SB20) or without pretreatment (–SB20), cells were exposed to gliadin fragments (Gl200, 200 μg/ml) or LPS (1 μg/ml) for 24 or 48 hrs, and the surface expression of CCR7 was determined by flow cytometry. Data represent the means ± S.D. of at least four independent experiments, in which the MFI of untreated control iDC was arbitrarily set to 100%. With versus without 20 μM SB203580 (SB20); *P < 0.05; (D, E) Cells were treated with 20 μM SB203580 (+SB20) or not (–SB20) for 30 min. prior to exposure to gliadin fragments (Gl200, 200 μg/ml) or LPS (1 μg/ml). After 12 hrs, COX-2 protein levels and total actin (loading control) in whole cell lysates were detected by Western blot (D), and the level of PGE2 in the culture supernatants was measured by EIA (E). The Western blot is representative of four independent experiments that gave similar results. The PGE2 levels are from representative experiment of two independent experiments, performed in duplicate. With versus without 20 μM SB203580 (SB20); *P < 0.05.

Fig 8

Model of gliadin-induced DC migratory behaviour and chemotactic responsiveness. The migration of DCs promoted by gliadin fragments is under control of the p38 MAPK pathway, and would result from cytoskeletal remodelling, involving dissolution of adhesion structures podosomes, as well as surface CCR7 up-regulation, and induction of COX-2 enzyme that mediates PGE production, respectively.