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1. Autophagy contributes to immune regulation through destabilization of the immunological synapse

M.E. Wildenberg, A.C.W. Vos, M. Duijvestein, A.P. Verhaar, C. Strisciuglio, G.R. van den Brink, D.W. Hommes

Leiden University Medical Center, Leiden, The Netherlands

Various polymorphisms in the autophagy related genes ATG16L1 and IRGM have been associated with the development of Crohn's disease (CD). Although the link between decreased autophagy and an inflammatory disorder like IBD suggests a role for this process in the regulation of immune responses, no data has been available on this topic. Therefore, this study focused on the effects of decreased autophagy on the immunogenicity of dendritic cells (DC).

Methods: Gene knockdown was achieved in monocyte derived (human) or bone marrow (mouse) DC using siRNA technology. DC-T cell interactions were induced in mixed lymphocytes reactions (MLR), and cytoskeletal changes and interaction times were studied by immunofluorescence and time-lapse microscopy. T cell reactivity was determined by 3H incorporation and cytokine production assays.

Results: ATG16L1low and IRGMlow DC induced significantly more T-cell proliferation in both an allogeneic MLR and an antigen specific proliferation assay. This finding was consistent in human and mouse cells, suggesting a conserved role for autophagy in the regulation of immune responses. No alterations in cytokine production or surface marker expression of autophagylow DC were seen, indicating the immunostimulatory phenotype is not due to increased maturation. However, aberrancies occured at the site of contact between DC and T cells, the so-called immunological synapse (IS). Autophagylow DC displayed increased cytoskeletal polarization towards interacting T cells, and interaction times between DC and lymphocytes were prolonged, pointing to IS hyperstability. Furthermore, autophagosomes close to the IS contained the IS components MHC II and ICAM-1, suggesting a role for autophagy in IS degradation.

Finally, autophagylow DC more effectively induced Th17 polarization without affecting Th1 cells, thus increasing the Th17/Th1 balance. This effect was likely due to the IS hyperstability observed, as destabilizing the synapse pharmacologically had the opposite effect and tilted the balance towards Th1.

Conclusion: Decreased levels of autophagy results in an increased pro-inflammatory capacity in both human and mouse DC. This effect is regulated through hyperstabilization of the IS, leading to increased T cell activation and tilting of immune responses towards a more Th17 phenotype. This mechanism is likely to contribute to the increased immune activation as seen in CD patients carrying mutations in the various autophagy related genes.