Mara Rosa Bono, Dr. (E) MFI quantification of NIH3T3-Dectin-2 + FcR SCH28080 uptake shown in (C). Data are presented as the mean SEM of two impartial experiments. Two-way ANOVA followed by the Bonferroni posttest was performed to compare control vs. either Dectin-1 or Dectin-2: ns = not significant. Image_1.TIFF (548K) GUID:?183C296A-DC51-4DF4-9130-1EB50F8BF5A4 Abstract Mollusk hemocyanins have biomedical uses as carriers/adjuvants and nonspecific immunostimulants with beneficial clinical outcomes by triggering the production of proinflammatory cytokines in antigen-presenting cells (APCs) and driving immune responses toward type 1 T helper (Th1) polarization. Significant structural features of hemocyanins as a model antigen are their glycosylation patterns. Indeed, hemocyanins have a multivalent nature as highly mannosylated antigens. We have previously shown that hemocyanins are internalized by APCs through receptor-mediated endocytosis with proteins that contain C-type lectin domains, such as mannose receptor (MR). However, the contribution of other innate immune receptors to the proinflammatory signaling pathway brought on by hemocyanins is usually unknown. Thus, we studied the roles of Dectin-1, Dectin-2, and Toll-like receptor 4 (TLR4) in the hemocyanin activation of murine APCs, both in dendritic cells (DCs) and macrophages, using hemocyanins from (CCH) and (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors [CCH (9)] and [FLH (10)] from the Chilean coasts and from from the Black Sea (RvH), previously referred to as [RtH (11)], among others (12, 13). Hemocyanins are extensively used in clinical and biotechnological applications because they do not produce unwanted side effects; however, the mechanisms involved in their immunomodulatory effects are poorly comprehended (3). Understanding those mechanisms will be crucial to improve the adjuvant and immunostimulant properties of hemocyanins, which would allow the development of better vaccines and immunotherapies (14, 15). Different characteristics have been suggested to explain the immunological properties of hemocyanins, such as their xenogenicity, large size, complex quaternary structure, presence of a quasi-symmetrical arrangement of repetitive epitopes, and glycan moiety content (16C18). All of these glycoproteins fold as large structures, with molecular weights ranging from 4 to 8 MDa. The basic structure comprises ten subunits that self-assemble in pairs to form a hollow cylinder known as decamer, which can interact to form didecamers (17, 19, 20). However, isolated subunits of CCH (21) and RtH (11) also have immunological effects, suggesting that large size and complex quaternary structure are not the only factors SCH28080 involved in the immunogenicity and antitumor effect of these hemocyanins. In this context, the glycosylation patterns in hemocyanins have an important role in the stability of their quaternary structure, supporting the conversation between the hemocyanin subunits, but these patterns have also been related to their immunomodulatory effects (18, 22, 23). Mollusk hemocyanins are highly glycosylated proteins, reaching up to 9% (w/w) (24). KLH, CCH, and FLH have different N- and O-glycosylation patterns, with mannose-rich and fucose-rich N-glycans being the most abundant types (10, 22). KLH immunization induces cross-reactive antibodies against T antigen due to the presence of oligosaccharides with the terminal moiety Gal(1-3)-GalNAc in its structure (25). Moreover, sugars on KLH share the structural motif Gal(1-6)-Man with side chains on liposaccharide (LPS) from pathogens such as and based on fucose-shared glycans (26C28). Furthermore, sera from mice immunized with KLH present cross-reactivity with lipoarabinomannan (LAM) from mycobacteria, the polysaccharide capsule of and LPS from (29). The MALDI-TOF-MS oligosaccharide profile of CCH and FLH revealed abundant diversity in their mannose-rich branched structures (10). Altogether, these data indicate that mollusk hemocyanins possess characteristic and heterogeneous glycosylation patterns and, therefore, could generate different immune responses. Indeed, the effects of mollusk hemocyanins on antigen-presenting cells (APCs) are variable. Hemocyanins stimulate innate immunity by SCH28080 inducing different temporal patterns of proinflammatory cytokine expression in murine peritoneal macrophages, leading to a classically activated macrophage (M1) profile (30). Remarkably, native FLJ42958 FLH induces the activation of murine bone marrow-derived dendritic cells (BMDCs) with Th1-inducing cytokine expression and enhances major histocompatibility complex class SCH28080 II (MHC-II) levels (10). Our current data show that hemocyanins are internalized by APCs through macropinocytosis and receptor-mediated endocytosis in a calcium-dependent manner, which normally leads to the secretion of proinflammatory cytokines. However, this cytokine secretion significantly decreased using deglycosylated FLH, suggesting the participation of specific glycan receptors in this process (10, 30). APCs have been described to recognize pathogen-derived glycans through C-type lectin receptors (CLR) and Toll-like receptors (TLR), among others, which are able to.