Mechanisms underlying the mollusk hemocyanin processing and presentation through MHC-dependent pathways in antigen presenting cells of mammals

Hemocyanins are oligomeric glycoproteins widely used as immunomodulators because they bias immunity towards a Th1 profile when inoculated in mammals. We have demonstrated that hemocyanins are internalized through receptor-mediated endocytosis, and TLR4 and C-type lectin receptors (MR, DC-SIGN, MGL) participate in the hemocyanin-mediated proinflammatory response in mouse and human dendritic cells (DCs). However, despite the massive use of hemocyanins, their intracellular processing route for MHC presentation to T lymphocytes has been scarcely studied. Therefore, we hypothesized that hemocyanins follow the MHC-II pathway as a classical T-cell-dependent antigen. Interestingly, our results analyzing the processing pathway of hemocyanins in mouse DCs showed that hemocyanins from Fissurella latimarginata (FLH) and Megathura crenulata (KLH) co-localized with Rab5+, Rab7+, and Lamp-1+ compartments. This observation strongly suggests that hemocyanins could be cross-presented by MHC-I molecules. Furthermore, DCs incubated with FLH showed an increase in the percentage of MHC-I+ cells versus the control cells. FLH-induced cytokine secretion decreased in J774.2 macrophages treated with pharmacological inhibitors of both MHC-II and MHC-I pathways, supporting our previous results on hemocyanin cross-presentation but also the MHC-II pathway. Furthermore, immunoblot confirmed different FLH proteolysis patterns in macrophages treated with MHC-I and MHC-II pathway inhibitors. Hence, we postulate that hemocyanins undergo both MHC-I and MHC-II dependent antigen presentation pathways in antigen-presenting cells. These findings offer molecular clues to underlying hemocyanin processing and presentation mechanisms. Supported by grants from FONDECYT N° 1151337 and N° 1201600 (MIB), ANID/Beca Doctorado Nacional N° 21210946 (MLS) and N° 21200880 (DDD).