P668. ATG16L1 genotype is associated with response to anti-TNF in vitro
M. Wildenberg1, A. Levin2, C. Vos2, J. Brandse1, J. de Bruyn1, G. D'Haens1, G. van den Brink1, 1Academic Medical Center, Gastroenterology, Amsterdam, Netherlands, 2Academic Medical Center, Tytgat Institute for Intestinal and Liver Research, Netherlands
We have previously shown that infliximab (IFX) treatment of mixed lymphocytes reactions results in the induction of macrophages with immunosuppressive and wound healing properties. Strikingly, the number of these macrophages increases significantly in the intestine of patients who respond to IFX treatment compared to non-responders. Genetic studies have shown an association between various autophagy related genes and the development of Crohn's disease. The aim of this study was to determine whether autophagy is involved in the induction of regulatory macrophages by IFX and whether autophagy related polymorphisms influence the response to IFX in vitro.
Peripheral blood was isolated from 29 healthy volunteers and rs_2241880 genotype was determined by pcr. After isolation of PBMC mixed lymphocyte reactions containing cells from 2 donors were established for 150 separate donor combinations. Cultures were treated with IFX or control IgG and incubated for 6–7 days. Cells were analyzed by gene array, immunofluorescence, flow cytometry and thymidine incorporation.
Anti-TNF induced regulatory macrophages displayed increased numbers of autophagosomes as well as an increased expression of autophagy related transcripts including atg5, atg7, atg9 and atg16l2, suggesting induction of autophagy by IFX treatment.
Of all donors, 7 were homozygously carrying the CD associated risk allele, 14 were heterozygous and 7 were homozygous for the WT allele.
The number of CD14+ regulatory macrophages correlated significantly with the number of WT alleles present in each individual culture, with the largest number of macrophages found in cultures containing 3 or 4 WT alleles (2 WT donors or 1 WT and 1 heterozygous). Similarly, expression of CD206, which is associated with the immunosuppressive function of macrophages, positively correlated with the number of WT alleles present. To confirm the functional consequences of these findings, IFX mediated suppression of T cell proliferation was determined. Again, the level of suppression correlated significantly with the number of WT alleles present in the respective donor combinations.
Induction of regulatory macrophages by IFX is associated with induction of autophagy. In vitro, the number and function of IFX induced macrophages correlated with the number of WT alleles for the autophagy related gene ATG16L1. This suggests that an intact autophagy pathway is important for effectiveness of anti-TNF therapy.