Dendritic cells are the most powerful antigen-presenting cells of the immune system. They present exogenous antigens associated with Major Histocompatibility Complex (MHC) Class II molecules through the classical pathway to stimulate CD4+ T cells, or with MHC-I to activate CD8+ T lymphocytes through the cross-presentation pathway. DCs represent one of the main cellular targets during infection by Toxoplasma gondii. This intracellular parasite incorporates essential nutrients, such as cholesterol, to grow and proliferate inside a highly specialized organelle, the parasitophorous vacuole (PV). While doing so, T. gondii modulates the host immune response through multiple interactions with proteins and lipids. Cholesterol is an important cellular component that regulates cellular physiology at the structural and functional levels. Although different studies describe the relevance of cholesterol transport for exogenous antigen presentation, the molecular mechanism underlying this process is not defined. Here, we focus our study on the inhibitor U18666A, a drug widely used to arrest multivesicular bodies biogenesis that interrupts cholesterol trafficking and changes the lipid composition of intracellular membranes. Upon bone marrow-derived DC (BMDC) treatment with U18666A, we evidenced a drastic disruption in the ability to present exogenous soluble and particulate antigens to CD4+ and CD8+ T cells. Strikingly, the presentation of T. gondii-associated antigens and parasite proliferation were hampered in treated cells. However, neither antigen uptake nor BMDC viability was significantly affected by the U18666A treatment. By contrast, this drug altered the transport of MHC-I and MHC-II molecules to the plasma membrane. Since U18666A impairs the formation of MVBs, we analyzed in T. gondii infected BMDCs the ESCRT machinery responsible for the generation of intraluminal vesicles. We observed that different MVBs markers, including ESCRT proteins, were recruited to the PV. Surprisingly, the main ESCRT-III component CHMP4b was massively recruited to the PV, and its expression level was upregulated upon BMDC infection by T. gondii. Finally, we demonstrated that BMDC treatment with U18666A interrupted cholesterol delivery and CHMP4b recruitment to the PV, which interfered with an efficient parasite replication. Altogether, our results highlight the importance of cholesterol trafficking and MVBs formation in DCs for optimal antigen presentation and T. gondii proliferation.

Cross-presentation is the process whereby antigenic peptides derived from exogenous antigens are associated to MHC class I molecules triggering the activation of CD8+ T lymphocytes. The endocytic route of dendritic cells (DCs) is strongly specialized to achieve antigen cross-presentation efficiently, which is crucial to initiate cytotoxic immune responses against many pathogens (i.e. Toxoplasma gondii) and tumors. Nevertheless, the endosomal molecular effectors involved in this process are not completely understood. In particular, the role of sorting nexin (SNX) proteins in cross-presentation has never been addressed. In this work, we identify the endosomal protein SNX17 as a key regulator of antigen internalization and cross-presentation by DCs. Our results demonstrate that SNX17 expression in DCs is essential to guarantee a normal cross-presentation of soluble, particulate and T. gondii-associated antigens. The silencing of SNX17 expression in DCs significantly affected the uptake of exogenous antigens by fluid-phase endocytosis and phagocytosis, but not by receptor-mediated endocytosis. Moreover, the knock-down of SNX17 impaired T. gondii invasion, CD11b integrin recycling and hampered the organization of the actin cytoskeleton. Finally, we show that SNX17 controls the proper maturation of DC phagosomes. Our findings provide compelling evidence that SNX17 plays a central role in the modulation of DC endocytic network, which is crucial for competent antigen internalization and cross-presentation.

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