Duodenal intraepithelial lymphocytes of children with cow milk allergy preferentially bind the glycan-binding protein galectin-3

Abstract

A breakdown in intestinal homeostasis results in inflammatory bowel diseases including coeliac disease and allergy. Galectins, evolutionarily conserved beta-galactoside-binding proteins, can modulate immune-epithelial cell interactions by influencing immune cell fate and cytokine secretion. In this study we investigated the glycosylation signature, as well as the regulated expression of galectin-1 and -3 in human duodenal samples of allergic and non-allergic children. Whereas galectin-1 was predominantly localized in the epithelial compartment (epithelial cells and intraepithelial lymphocytes) and the underlying lamina propria (T cells, macrophages and plasma cells), galectin-3 was mainly expressed by crypt epithelial cells and macrophages in the lamina propria. Remarkably, expression of these galectins was not significantly altered in allergic versus non-allergic patients. Investigation of the glycophenotype of the duodenal inflammatory microenvironment revealed substantial alpha2-6-linked sialic acid bound to galactose in lamina propria plasma cells, macrophages and intraepithelial lymphocytes and significant levels of asialo core 1 O-glycans in CD68+ macrophages and enterocytes. Galectin-1 preferentially bound to neutrophils, plasma cells and enterocytes, while galectin-3 binding sites were mainly distributed on macrophages and intraepithelial lymphocytes. Notably, galectin-3, but not galectin-1 binding, was substantially increased in intraepithelial gut lymphocytes of allergic patients compared to non-allergic subjects, suggesting a potential role of galectin-3-glycan interactions in shaping epithelial-immune cell connections during allergic inflammatory processes.

Fig. 1. Histopathologic analysis of duodenal samples of allergic and non-allergic patients

Mast cells, eosinophils, IgE⁺ cells, CD45RO⁺ cells, CD3⁺ intraepithelial (IELs) and CD3⁺ lamina propria (LPLs) cells were counted in 6 villi of duodenal samples of allergic (N: 8) and non-allergic patients (N: 8). Cells were detected by Giemsa or hematoxylin/eosin staining (mast cells and eosinophils, respectively) or by immunohistochemistry using anti-IgE, anti-CD45RO or anti-CD3 monoclonal antibodies. Statistical analysis revealed no significant differences between different groups of patients. (Original magnification: 1000x except for CD45RO).

Fig. 2. Expression of Gal-1 and Gal-3 in duodenal specimens of allergic and non-allergic patients

Gal-1 and Gal-3 expression was assessed by immuno-histochemistry using specific polyclonal antibodies against Gal-1 and Gal-3 (purified rabbit IgGs) in duodenal samples of allergic and non-allergic patients. Representative photographs of mucosa from allergic and non allergic patients were selected to show Gal-1 (A) and Gal-3 (B) positive cells. Control with serum from non-immunized rabbits is depicted. All samples were analyzed at least three times in independent experiments (Original magnification: 100x and 400x).

Fig. 3. SNA binding pattern on duodenal samples: Pattern of α2,6 sialylation in mucosal tissue of allergic versus non-allergic patients

A) Lectin-histochemistry using biotinylated-SNA and hematoxylin staining was done to identify SAα2–6-galactose residues. A photograph with stained IEL from an allergic patient is shown. B) Co-localization studies using a Cy3-conjugated anti-CD3 antibody (red), biotinylated SNA/ FITC-conjugated streptavidin (green) and DAPI (blue) was performed by confocal microscopy. C) Lectin binding inhibition assessed by confocal microscopy. Cells were incubated with biotinylated SNA or biotinylated SNA in the presence of 100 mM lactose. D) SNA⁺ cells were quantified by lectin-histochemistry in biopsy specimens of allergic (N=8) and non-allergic (N=8) patients and no significant difference was found (p= 0.11). E) ST6Gal1 expression was detected using a polyclonal specific antibody by immunohistochemistry. A control of immunoreactivity is shown using sera from non-immunized rabbits. All samples were analyzed at least three times in independent experiments and representative images are shown (Original magnifications: 200x and 1000x).

Fig. 4. PNA binding pattern in duodenal samples: Detection of asialo core-1-O-glycans in intestinal glycoproteins of allergic and non-allergic patients

A) Lectin-histochemistry with biotinylated PNA in gut biopsies. B) Co-localization studies by confocal microscopy using Cy3-conjugated anti-CD3 antibody (red), biotinylated PNA/ FITC-conjugated streptavidin (green) and DAPI (blue nuclear staining); (C) Co-localization studies by confocal microscopy using biotinylated PNA/ FITC-conjugated streptavidin (green) and PE-conjugated anti-CD68 antibody (red). D) Lectin binding inhibition studies assessed by confocal microscopy using biotinylated PNA/ FITC-labeled streptavidin or biotinylated PNA/ FITC-labeled streptavidin in the presence of 200 mM galactose. E) PNA⁺ cells were quantified by lectin-histochemistry in biopsy specimens of allergic and non-allergic patients; no significant differences were found (P:0.14). All samples were analyzed at least three times in independent experiments and representative images are shown (Original magnification: 200x).

Fig. 5. Gal-1-specific ligands on duodenal samples of allergic and non-allergic patients

A) Lectin-histochemistry with biotinylated Gal-1/ HRP-conjugated streptavidin in gut biopsies. B) Co-localization studies by confocal microscopy using biotinylated Gal-1/ FITC-conjugated streptavidin (green), Cy3-labeled anti-CD3 or PE-labeled anti-CD138 (red) antibodies and DAPI (blue nuclear staining). C) Lectin binding inhibition studies assessed by confocal microscopy with biotinylated Gal-1/ FITC-labeled streptavidin or biotinylated Gal-1/ FITC-conjugated streptavidin in the presence of 100 mM lactose. D) Gal-1⁺ cells were quantified by lectin-histochemistry in biopsy specimens of allergic and non-allergic patients; no significant differences were found (P= 0.8). All samples were analyzed at least three times in independent experiments and representative images are shown (Original magnifications: 200x and 400x).

Fig. 6. Gal-3-specific ligands in duodenal samples of allergic versus non-allergic patients

A) Lectin-histochemistry with biotinylated Gal-3/ HRP-conjugated streptavidin in gut biopsies from allergic and non-allergic patients. A control omitting biotinylated Gal-3 is shown. B) Co-localization studies by confocal microscopy using biotinylated Gal-3/ FITC-labeled streptavidin (green), Cy3-conjugated anti-CD3 (red) and DAPI (blue nuclear staining). C) Lectin binding inhibition studies by confocal microscopy with biotinylated Gal-3/ FITC-labeled streptavidin or biotinylated Gal-3 / FITC-labeled streptavidin in the presence of 100 mM lactose. D) CD3⁺ IELs (upper panel; P= 0.9) and Gal-3⁺ IELs (middle panel; P<0.0001) were quantified in samples of allergic and non-allergic patients. In the lower panel the ratio of Gal-3⁺ IELs/total CD3⁺IELs is depicted (P<0.001). All samples were analyzed at least three times in independent experiments and representative images are shown (Original magnifications: 200x, 400x and 1000x)