C‐type lectin receptors MR and DC‐SIGN are involved in recognition of hemocyanins, shaping their immunostimulatory effects on human dendritic cells

Abstract

Hemocyanins are used as immunomodulators in clinical applications because they induce a strong Th1‐biased cell‐mediated immunity, which has beneficial effects. They are multiligand glycosylated molecules with abundant and complex mannose‐rich structures. It remains unclear whether these structures influence hemocyanin‐induced immunostimulatory processes in human APCs. We have previously shown that hemocyanin glycans from Concholepas concholepas (CCH), Fissurella latimarginata (FLH), and Megathura crenulata (KLH), participate in their immune recognition and immunogenicity in mice, interacting with murine C‐type lectin receptors (CLRs). Here, we studied the interactions of these hemocyanins with two major mannose‐binding CLRs on monocyte‐derived human DCs: MR (mannose receptor) and DC‐SIGN (DC‐specific ICAM‐3–grabbing nonintegrin). Diverse analyses showed that hemocyanins are internalized by a mannose‐sensitive mechanism. This process was calcium dependent. Moreover, hemocyanins colocalized with MR and DC‐SIGN, and were partly internalized through clathrin‐mediated endocytosis. The hemocyanin‐mediated proinflammatory cytokine response was impaired when using deglycosylated FLH and KLH compared to CCH. We further showed that hemocyanins bind to human MR and DC‐SIGN in a carbohydrate‐dependent manner with affinity constants in the physiological concentration range. Overall, we showed that these three clinically valuable hemocyanins interact with human mannose‐sensitive CLRs, initiating an immune response and promoting a Th1 cell‐driving potential.

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

“…A series of papers describing and characterising these cells were published in the Journal of Experimental Medicine from 1973-1975. These cells were shown to be unique potent activators of naïve T cells in comparison to other Ag presenting cell [1395,1396] and were regarded as 'accessory' cells that linked innate and adaptive immunity [1397,1398]. Human DCs traditionally include cDCs, namely CD1c + cDC2 and CLEC9A + XCR1 + CADM1 + CD141 + cDC1 and CD123 + pDCs [1399].…”

Surface immunogenic protein from Streptococcus agalactiae and Fissurella latimarginata hemocyanin are TLR4 ligands and activate MyD88- and TRIF dependent signaling pathways

“…F. latimarginata hemocyanin (FLH) was provided by Biosonda SA (Santiago, Chile). This protein was isolated and purified under sterile, pyrogen-free conditions in phosphate-buffered saline ([PBS] containing sodium phosphate 0.1 M NaCl), pH 7.2 ( 24 ), and Tris buffer for FLH containing 50 mM Tris, pH 7.4, 5 mM CaCl 2.5 mM MgCl 2 , and 0.15 mM NaCl ( 25 ). All chemicals were analytical reagent grade, and solutions were prepared with human irrigation water (Baxter Healthcare, Charlotte, NC, USA) and filtered through a 0.2 µm membrane filter (Millipore).…”

“…Studies performed in our laboratory showed that FLH has antitumor effects in murine melanoma and oral cancer models ( 20 ). In addition, FLH binds to TLR4 and induces the expression and secretion of Th1-type proinflammatory cytokines ( 17 , 23 , 25 ). A remarkable characteristic of hemocyanins is their carbohydrate content, which is fundamental to their structure and immunological efficacy ( 23 , 26 ).…”

“…A remarkable characteristic of hemocyanins is their carbohydrate content, which is fundamental to their structure and immunological efficacy ( 23 , 26 ). Recent work showed that the enzymatic N-deglycosylation of FLH influences its immunogenic effects on macrophages ( 23 ), leading to a decrease in its binding to C-type lectin receptors, such as mannose receptor (MR), macrophage galactose lectin receptor (MGL), DC-specific intracellular adhesion molecule (ICAM)-grabbing nonintegrin (DC-SIGN), and TLR4 ( 17 , 23 , 25 ).…”

Assessment of the In Vitro and In Vivo Antitumor Activity of Hemocyanins from Helix aspersa, Helix lucorum, and Rapana venosa in a Graffi Myeloid Tumor Model

“…The monosaccharide composition of mollusc glycans includes mannose, D-galactose, fucose, N-acetyl-D-galactosamine, and N-acetyl-glucosamine residues, of which mannose is the most abundant carbohydrate [ 13 ]. Research data have demonstrated that hemocyanin glycans act as multivalent ligands that bind with high affinities to innate immune receptors on the surface of the antigen-presenting cells (APCs), such as mannose receptors and Toll-like receptors, and triggers proinflammatory responses [ 11 , 14 , 15 ]. Hemocyanins are incorporated by APCs by both macropinocytosis and receptor-mediated endocytosis, then slowly processed, which facilitates a prolonged antigen presentation to T- or B-lymphocytes, and promotes potent cell-mediated and humoral immune responses [ 13 ].…”