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P005. Autophagy induces immune tolerance by regulating interactions between dendritic cells–epithelial cell in the gut

C. Strisciuglio1,2, M. Duijvestein1, A.C.W. Vos1, A.P. Verhaar1, E. Miele2, R. Troncone2, G.R. van den Brink1, D.W. Hommes1

1Leiden University Medical Center, Leiden, The Netherlands; 2University Federico II, Naples, Italy

Aim: Over the last few years, various single nucleotide polymorphisms (SNPs) in the autophagy related genes ATG16L1 and IRGM have been associated with the development of Crohn's disease. These SNPs lead to decreased autophagic activity, suggesting a regulatory role for this mechanism in immunity. In the intestine, DC are capable of sampling luminal antigen by protruding dendrites through the epithelial cell layer, while maintaining barrier integrity. It has been shown that the proper sampling of antigen, as well as the interactions between DC and the epithelium is crucial for maintenance of intestinal tolerance. We have previously shown that autophagy contributes to regulation of cell–cell interactions between dendritic cells (DC) and T cells and therefore, the aim of this study was to elucidate the role of autophagy in the regulation of cell-cell interactions between DC and intestinal epithelium.

Materials and Methods: An in vitro model system for luminal sampling by DC was set up, in which an epithelial colon cancer cell line was cultured on one side of a transwell insert, and human dendritic cells (DC) on the other side of the insert, at the basolateral side of the epithelium. Modulation of autophagy was achieved using siRNA. In this assay, DC phenotype and luminal antigen sampling were measured by flow cytometry. The capacity of DC to form transepithelial protrusions was determined by confocal microscopy.

Results: In the gut DC–epithelial interactions are characterized by the presence of tight junctions and adhesion molecules. Interestingly, the adhesion molecule E-Cadherin partly localized to autophagosomes and decreased autophagy resulted in increased levels of this protein, suggesting autophagy is involved in proper DC–epithelial interactions. Indeed, reduced autophagy in either DC, epithelial cells or both resulted in the decreased formation of transepithelial protusions by DC as well as a reduction in antigen sampling.

Moreover, when autophagy was inhibited in either DC or epithelial cells in the co-culture model, DC expressed increased levels of HLA-DR and costimulatory molecules. Furthermore, decreased levels of autophagy resulted in an altered cytokine profile of DC and these cells induced significantly more T-cell proliferation in an allogenic mixed lymphocyte reaction. This data suggest that decreased autophagy is also involved in increased maturation and increased immunoreactivity.

Conclusions: Decreased autophagy is involved in the formation of proper interactions between DC and intestinal epithelium. Autophagy deficiency leads to aberrant interactions resulting in decreased antigen sampling, increased DC maturation and a more pro-inflammatory type of DC. Therefore, autophagy-related SNPs may contribute to CD pathogenesis through dysregulation of the interactions between DC and epithelial cells resulting in a lack of immune tolerance.