OP006 Autophagy regulates dendritic cell migration through Rac1: implications for thiopurine therapy
M. Wildenberg*1, P. Koelink2, K. Diederen2, A. te Velde2, V. Nuij3, M. Peppelenbosch3, M. Nobis4, O. Sansom4, C.J. van der Woude3, G. D’Haens1, G. van den Brink1
1Academic Medical Centre, Gastroenterology and Hepatology, Amsterdam, Netherlands, 2Academic Medical Centre, Tytgat Institute, Amsterdam, Netherlands, 3Erasmus Medical Centre, Department of Gastroenterology and Hepatology, Rotterdam, Netherlands, 4Beatson Institute for Cancer Research, Glasgow, United Kingdom
The T300A variant of the ATG16L1 gene that reduces autophagy is one of the few highly prevalent risk factors associated specifically with Crohn’s disease but not with ulcerative colitis or other immune-mediated diseases. We have previously shown a regulatory role for autophagy during dendritic cell (DC)–T-cell interactions, where the T300A allele results in enhanced contact time, T-cell hyper activation, and Th17 skewing (Wildenberg et al, Gastroenterology 2012). Because a key element of the DC-T-cell interaction is regulation of the cytoskeleton, we have further investigated the role of autophagy in DC cytoskeletal organisation and function, and correlated our findings to thiopurine therapy in inflammatory bowel disease (IBD) patients.
Autophagy deficient dendritic cells (DC) were generated using small interfering RNA, pharmacological inhibition or CD11cCre-Atg5fl/fl bone marrow or generated from T300A allele carrier monocytes. DC phenotype and migratory capacity were analysed using migration assays. Correlation between ATG16L1 genotype and response to thiopurines in patients with IBD was determined in 2 retrospective cohort studies.
Reduced autophagy in DC resulted in loss of filo podia and increased membrane ruffling. This cytoskeletal phenotype was mediated by increases in Rac1 activity. As a result, autophagy deficient DC showed increased adhesion to various substrates. Random, as well as directional, migration were impaired in human DC where autophagy was inhibited, independent of the method of inhibition (ATG16L1 silencing, 3MA, or carrying the T300A single nucleotide polymorphism [SNP]). This was confirmed in vivo, where murine bone-marrow derived DC lacking autophagy showed defects in migration between peripheral tissue and draining lymph nodes. Strikingly, migration of autophagy deficient DC could be restored by addition of the Rac1 inhibitor 6-thioguanin (6-TG), whereas 6-TG did not influence migration in autophagy sufficient DC. Similar data were obtained using monocytes carrying the ATG16L1 T300A risk allele. Finally, we correlated ATG16L1 genotype and response to thiopurines in 2 inflammatory bowel disease (IBD) cohorts and found that the ATG16L1 risk variant associates with response to thiopurine treatment specifically in patients with Crohn’s disease but not with ulcerative colitis.
Our results suggest that a defect in the autophagosomal regulation of active Rac1 underlies the association between ATG16L1 and Crohn’s disease through decreased myeloid cell migration. Because thiopurine can inhibit Rac1 activity, ATG16L1 genotyping may be used to identify patients that would benefit from thiopurine treatment.