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Étude de l'interaction entre les immunoglobulines A sécrétoires et la cellule épithéliale intestinale humaine

Abstract : Among the five isotypes of antibodies found in Humans, immunoglobulins A (IgA) are the most abundant in the mucosae. In the lamina propria, IgA are produced mainly as polymeric IgA (pIgA) and then secreted in the lumen. In fact, pIgA are translocated across the epithelium via the polymeric immunoglobulin receptor (pIgR), which is expressed at the basolateral side of the epithelium. Once secreted in the lumen, pIgA retain the extracellular domain of the pIgR and are called secretory IgA (SIgA). One of the main functions of intestinal SIgA is to restrain microorganisms and dietary antigens in the lumen, therefore, preventing their uptake through the epithelial barrier. However, retrotranscytosis of SIgA conjugated to bacteria has been described in Peyer’s patches where it participates to immune response. Moreover, previous works from the laboratory have suggested that transferrin receptor (CD71), which is overexpressed at the apical side of enterocytes from coeliac patients, interacts with SIgA bound to gliadin peptides and allows their retrotranscytosis across the intestinal epithelium. This thesis work aimed to further characterize the interaction between SIgA and CD71 in human enterocytes. We, first, generated a human epithelial intestinal cell line (Caco-2 TC7) devoid of CD71 expression (CD71KO) using the CRISPR/Cas9 genome editing method. Unexpectedly, flow cytometry experiments did not reveal a significant reduction of SIgA binding at the cell surface of CD71KO cells. Overall, our results indicated that CD71-SIgA interaction is indirect and may occur via additional protein partners through the assembly of a multifactorial protein complex. Therefore, we screened IgA receptors already known in the literature and showed that all are dispensable for SIgA binding at the surface of Caco-2 TC7 cells. In the effort to identify partner(s) within SIgA-CD71 complex, we set out mass spectrometry-based immunoprecipitation proteomics experiments and identified secretory carrier membrane protein 3 (SCAMP3), B-cell receptor-associated protein 31 (BCAP31) and histocompatibility minor 13 (HM13) as members of SIgA-CD71 complex. By generating knockout cell lines with the CRISPR/Cas9 system, we showed that none of these partners directly interacts with SIgA. However, our results suggest that SCAMP3 is required for the oligomerization of SIgA complexes at cell surface. Finally, we did not find any role in SIgA internalization for the different members of the complex, suggesting that they may play a role later on during SIgA retrotranscytosis. In conclusion, our work shows that SIgA interact with the intestinal epithelium via a proteic complex composed of at least five members: CD71, SCAMP3, BCAP31, HM13 and one or more unknown SIgA receptor(s). These results complement the previous works on the pathophysiologic role of SIgA in coeliac disease and underline the highly complex interaction between IgA and enterocytes. An important point to address will be to identify SIgA receptor(s) and to determine the role of the four other identified partners in SIgA retrotranscytosis across the intestinal epithelium.
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Benoît Clément. Étude de l'interaction entre les immunoglobulines A sécrétoires et la cellule épithéliale intestinale humaine. Immunologie. Université Sorbonne Paris Cité, 2017. Français. ⟨NNT : 2017USPCB048⟩. ⟨tel-02426225⟩

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