P672. T300A variant of autophagy ATG16L1 gene is associated with decreased antigen sampling and processing by dendritic cells in pediatric IBD
C. Strisciuglio1, E. Miele1, M.E. Wildenberg2, F.P. Giugliano1, M. Andreozzi1, A. Vitale1, F. Capasso1, M.V. Barone1, A. Staiano1, R. Troncone1, C. Gianfrani1, 1Federico II, Pediatrics, Naples, Italy, 2Intestinal and Liver Research, Academic Medical Center, Gastroenterology and Hepatology and Tytgat, Amsterdam, Netherlands
Intestinal dendritic cells (DC) sample luminal antigens by protruding dendrites through the epithelial cell layer. The single nucleotide polymorphism T300A (rs2241880) of ATG16L1, which has been associated with Crohn's disease (CD), is responsible for a decreased autophagic activity. The aim of this study was to elucidate the role of autophagy in DC uptake and processing of antigens and in the interaction between DC and intestinal epithelium in pediatric IBD patients.
We recruited CD paediatric patients that homozygously carry either the protective (wt, n = 7) or risk allele (var, n = 13) of ATG16L1, as well as heterozygous patients (het, n = 13). DC obtained from peripheral blood monocytes were studied after 2-hours of incubation with bacteria particles, 4-hour of incubation with DQ-Ovalbumin (DQ-OVA), or 24-hours of co-culture with Caco2 epithelial cells and bacteria particles in transwell culture. DC phenotype, antigen sampling and processing were measured by flow cytometry. The capability of DC to form transepithelial protrusions was determined by confocal microscopy.
DC generated from wt patients showed significant higher bacterial sampling and initial antigen processing compared to var patients (p = 0.01 and p = 0.03, respectively). Additionally, after exposure to either bacterial particles or the model antigen DQ-OVA, wt DC showed a significant increase in the expression of the activation markers HLA-DR and CD86 when compared to var DC. Interestingly, also het patients showed an impairment in bacterial uptake and expression of activation marker compared to wt, except for DQ-OVA processing that was reduced but did not reach a statististical significance, suggesting a dose dependent effect of the T300A allele on DC functionality. To model antigen sampling in the intestine, DC were co-cultured with colonic Caco-2 cells. In this set-up, formation of transepithelial protrusions by DC was less efficient in var DC compared to wt. In accordance, antigen uptake and processing by DC derived from var patients was decreased significantly.
DCs of pediatric CD patients showed a marked impairment of bacterial uptake and antigen processing, as well as a decreased maturation. Moreover autophagy is involved in the proper interactions between DC and intestinal epithelium. Our results indicate that an autophagy defect is associated with an impairment of intestinal innate immunity in pediatric CD.