Volume 143, Issue 3, Pages 698-707.e4 (September 2012) Interactions Among Secretory Immunoglobulin A, CD71, and Transglutaminase-2 Affect Permeability of Intestinal Epithelial Cells to Gliadin Peptides  Corinne Lebreton, Sandrine Ménard, Juliette Abed, Ivan Cruz Moura, Rosanna Coppo, Christophe Dugave, Renato C. Monteiro, Aurélie Fricot, Meriem Garfa Traore, Martin Griffin, Christophe Cellier, Georgia Malamut, Nadine Cerf–Bensussan, Martine Heyman  Gastroenterology  Volume 143, Issue 3, Pages 698-707.e4 (September 2012) DOI: 10.1053/j.gastro.2012.05.051 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Analysis of molecular interactions of IgA with CD71 in duodenal biopsy specimens by Duolink PLA and FRET. (A) Duolink PLA labeling of duodenal biopsy specimens reveals IgA/CD71 interactions as red dots at the surface epithelium and to some extent in the lamina propria of patients with ACD (n = 4) but not of controls (n = 4). Duolink PLA is described in the Materials and Methods section and is illustrated in the right panel. Signals are analyzed by confocal microscopy. Cell nuclei are stained in blue by 4′,6-diamidino-2-phenylindole. (B and C) FRET experiments on duodenal biopsy specimens from ACD are shown before and after bleaching CD71 (red) and IgA (green) (B) or alkaline phosphatase (AP) (red) and IgA (green) (C). Physical interactions of IgA with CD71 at the epithelial surface is attested by 20% FRET in ACD (n = 4) compared with 5% in controls (n = 4) (*P < .001). Specificity is shown by the lack of FRET between IgA and AP despite apparent co-localization (n = 2). FRET is described in the Materials and Methods section and is depicted in panel C. Gastroenterology 2012 143, 698-707.e4DOI: (10.1053/j.gastro.2012.05.051) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Analysis of molecular interactions of CD71 with IgA and transglutaminase 2 by Duolink PLA and FRET. (A and B) FRET analysis of duodenal biopsy specimens from ACD patients (n = 4) before and after bleaching of (A) CD71 (green) and Tgase2 (red), or (B) CD71 (green) and alkaline phosphatase (AP) (red). Interaction between CD71 and Tgase2 is shown by 18% FRET in ACD compared with 0% in controls (n = 4) (*P < .001). Specificity of interaction between CD71 and Tgase2 is shown by the absence of FRET between CD71 and alkaline phosphatase despite their apparent colocalization. (C) Labeling of cryosections of duodenal biopsy specimens (n = 3) with anti-CD71 and anti-Tgase2 (ACD) by Duolink PLA confirms the formation of CD71/Tgase2 complexes revealed as red dots in the surface epithelium and lamina propria in ACD but not in controls (n = 3). Apical and basal sides of epithelium are highlighted with arrows. Cell nuclei are stained in blue by 4′,6-diamidino-2-phenylindole. Gastroenterology 2012 143, 698-707.e4DOI: (10.1053/j.gastro.2012.05.051) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Interactions between IgA, CD71, and Tgase2 in Caco-2 epithelial cell line. (A) Flow cytometry analysis of Caco-2 cells labeled with anti-CD71 antibody (left panel) or SIgA (as described in the Materials and Methods section) after transfection with CD71-siRNA (red dotted line) or with control siRNA (blue line) (n = 4). Control staining with mouse isotype and monomeric human IgA1 are shown in black in cells transfected with CD71-siRNA. (B) Confocal analysis showing apical localization of CD71 in polarized Caco-2 cells (upper panel) and Duolink PLA labeling of polarized Caco-2 cells after pre-incubation with SIgA at 4°C for 1 hour. Physical interactions revealed by red dots are seen at the apical surface between IgA and CD71, CD71 and Tgase2, but not between IgA and Tgase2. (C) Duolink PLA in nonpolarized Caco-2 cells transfected or not with CD71 or control siRNA and pre-incubated with SIgA at 4°C. Red dots revealing CD71/SIgA interactions are less numerous in Caco-2 cells transfected with CD71 siRNA than in not-transfected cells or in cells transfected by control siRNA. No staining was seen with control isotypes. (B and C) Results are representative of 3 experiments in triplicate. Nuclei are stained in blue with 4′,6-diamidino-2-phenylindole. Gastroenterology 2012 143, 698-707.e4DOI: (10.1053/j.gastro.2012.05.051) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 Immunoprecipitation of CD71-SIgA-transglutaminase 2 complexes in Caco-2 cells. (A) Immunoprecipitation of Caco-2 cell lysates pre-incubated or not with SIgA (250 μg/mL) for 1 hour at 4°C and CNBr-activated sepharose beads bound to rabbit anti-human IgA (2 h, 4°C) followed by Western blot with indicated antibodies. Controls include lysates from Caco-2 cells pre-incubated or not with SIgA, and anti-IgA beads alone (n = 3). (B) Immunoprecipitation of Caco-2 cell lysate by indicated preformed complexes bound to protein A/G–coated beads. Western blot with anti-CD71 (n = 2). Gastroenterology 2012 143, 698-707.e4DOI: (10.1053/j.gastro.2012.05.051) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 Transcytosis of SIgA/CD71 complexes in Caco-2 monolayers via early and recycling endosomes. (A) Localization by confocal microscopy of SIgA/CD71 or CD71/Tgase 2 complexes stained with Duolink PLA in polarized monolayers incubated with SIgA at 4°C for 1 hour and then at 37°C for 5, 10, and 20 minutes. (B) Colocalization of PLA-labeled SIgA/CD71 (i and ii) or CD71/Tgase2 (iii and iv) complexes with early endosome marker EEA1 (i and iii) and with Rab11, a marker of recycling endosomes (iii) but not with lysosomal marker LAMP2 (ii and iv). Extensive co-localization of individually labeled SIgA with LAMP2, indicating that free SIgA are addressed to the degradative lysosomal compartment (v). (B) All results are shown after 10 minutes at 37°C. (D) Co-localization analysis performed on original images using cross-correlation coefficient (CCF) of Van Steensel et al17 between reds dots corresponding to CD71/IgA or CD71/Tgase2 complexes and FITC-EEA1, FITC-Rab11, or FITC-Lamp2 (upper panel) or with JACop (ImageJ) (lower panel). Perfect bell-shaped curves and Pearson coefficients ranging from ∼0.58 to 0.8 are observed for CD71/SIgA or CD71/Tgase2 complexes and EEA1 or rab11, respectively (left panel), but not with Lamp2 (right panel). Gastroenterology 2012 143, 698-707.e4DOI: (10.1053/j.gastro.2012.05.051) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Transport of 3H-p31-49 across filter-grown Caco-2 monolayers. Polarized filter-grown Caco-2 monolayers were incubated apically with 3H-p31-49 either or not after a 30-minute pre-incubation in the presence of IgA with or without inhibitors. Transport and processing of 3H-p31-49 was assessed by RP-HPLC with online detection of radioactivity in the serosal compartment. (A) Permeability to intact (black columns), degraded (white columns), and total (black + white) 3H-p31-49 gliadin peptide in the presence or not of IgA purified from control or ACD serum IgA1 at indicated concentrations (n = 6). Statistical significance compared with p31-49 alone is shown for intact p31-49 permeability (above black column) and total permeability (top of column). #P < .05, *P < .01, §P < .001. (B) Same as in panel A in the presence of various concentrations of colostrum SIgA, total or either depleted or enriched in reactivity toward p31-49. #P < .05, §P < .001 when compared with p31-49 alone (n = 6). (C) Permeability to intact and total 3H-p31-49 in the presence of 250 μg/mL ACD IgA1 and Tgase inhibitors R281 or R294 (specific for Tgase2) or indicated concentrations of soluble CD71 (n = 8). §P <. 001; **P < .01; §§P < .05 when compared with p31-49 and ACD IgA1. (D) Radio-HPLC elution profiles of 3H-p31-49 in the basal compartment of filter-grown Caco-2 monolayers treated in indicated conditions. Gastroenterology 2012 143, 698-707.e4DOI: (10.1053/j.gastro.2012.05.051) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 1 Analysis of permeability to horseradish peroxidase (HRP) and mannitol in polarized Caco-2 monolayers. Permeability to HRP and 14C-mannitol, taken as markers of transcellular and paracellular permeabilities, respectively, was measured in the presence of ACD IgA1. IgA1 did not modify permeability to intact HRP, indicating that IgA-dependent permeability of p31-49 is antigen-specific. In addition, no paracellular leakage was induced by ACD-IgA1 because mannitol permeability did not increase (and even decreased for unknown reasons). Gastroenterology 2012 143, 698-707.e4DOI: (10.1053/j.gastro.2012.05.051) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 2 Characteristics of serum IgA in celiac disease. (A and B) Total serum IgA, antigliadin, anti–p31-49, and anti-33mer IgA were compared by enzyme-linked immunosorbent assay in ACD patients (n = 21), treated CD patients (n = 15), and controls (n = 49). (A) Patients with ACD have, as expected, higher titers of antigliadin IgA than controls and treated patients. (B) No significant increase in the specificity of IgA against p31-49 or against the 33 mer was observed between ACD patients and controls although IgA titers against p31-49 were significantly less in treated CD. (C) Serum IgA were purified on Jacalin gel and their galactosylation was analyzed by enzyme-linked immunosorbent assay using Helix aspersa lectin (which binds hypogalactosylated glycan chains) in adults and children with CD, Crohn's disease, or in age-matched controls. IgA are significantly less glycosylated in adults with active CD (P < .0001) or treated CD compared with IgA in adult controls. *P < .002, significantly different from adult controls. †P < .0001, significantly different from adults with active CD. Gastroenterology 2012 143, 698-707.e4DOI: (10.1053/j.gastro.2012.05.051) Copyright © 2012 AGA Institute Terms and Conditions

Supplementary Figure 3 Transport of 3H-p31-49 across duodenal biopsy specimens from control or celiac patients. Biopsy specimens from active CD patients were mounted in Ussing chambers and incubated apically with 3H-p31-49 for 3 hours. Typical chromatographic (RP-HPLC) elution profiles of 3H-p31-49 in the serosal compartment are shown. Amounts of intact (black columns) and total peptide (white + black columns) are shown as histograms. Data are shown for 4 controls, 4 adults with ACD, and 1 child with ACD. Gastroenterology 2012 143, 698-707.e4DOI: (10.1053/j.gastro.2012.05.051) Copyright © 2012 AGA Institute Terms and Conditions