TLR4, but Neither Dectin-1 nor Dectin-2, Participates in the Mollusk Hemocyanin-Induced Proinflammatory Effects in Antigen-Presenting Cells From Mammals

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 triggered by hemocyanins is 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 Megathura crenulata ( KLH), Concholepas concholepas (CCH) and Fissurella latimarginata (FLH). The results showed that these hemocyanins bound to chimeric Dectin-1 and Dectin-2 receptors in vitro ; which significantly decreased when the glycoproteins were deglycosylated. However, hemocyanin-induced proinflammatory effects in APCs from Dectin-1 knock-out (KO) and Dectin-2 KO mice were independent of both receptors. Moreover, when wild-type APCs were cultured in the presence of hemocyanins, phosphorylation of Syk kinase was not detected. We further showed that KLH and FLH induced ERK1/2 phosphorylation, a key event involved in the TLR signaling pathway. We confirmed a glycan-dependent binding of hemocyanins to chimeric TLR4 in vitro . Moreover, DCs from mice deficient for MyD88-adapter-like (Mal), a downstream adapter molecule of TLR4, were partially activated by FLH, suggesting a role of the TLR pathway in hemocyanin recognition to activate APCs. The participation of TLR4 was confirmed through a decrease in IL-12p40 and IL-6 secretion induced by FLH when a TLR4 blocking antibody was used; a reduction was also observed in DCs from C3H/HeJ mice, a mouse strain with a nonfunctional mutation for this receptor. Moreover, IL-6 secretion induced by FLH was abolished in macrophages deficient for TLR4. Our data showed the involvement of TLR4 in the hemocyanin-mediated proinflammatory response in APCs, which could cooperate with MR in innate immune recognition of these glycoproteins.

A heat-shocked melanoma cell lysate vaccine enhances tumor infiltration by prototypic effector T cells inhibiting tumor growth

“…Unlikely, KLH is formed by homododecamers, whose stability depends on Ca +2 and Mg +2 for maintenance. 25 Our results showed that TRIMELVax immunizations activate effective cell-mediated and humoral immune responses against B16F10 tumors, inhibiting tumor growth and prolonging mice survival, even in the absence of combined therapy with anti-PD-1 antibodies.…”

“…9 10 In contrast, TRIMEL seems to generate the appropriate combination of DAMPs capable of inducing an effective DC maturation. 8 9 CCH has potent immunostimulatory effects, whose adjuvant properties could be partially explained by its carbohydrate-based high complexity and stability, 24 which activate APCs via the interaction with the C-type lectin and Toll-like receptors (TLRs) such as mannose receptor and TLR4, respectively, 25 strengthening innate and adaptive immune responses in mammals, making them useful in cancer immunotherapy. 26 30 Notably, treatment of B16F10 tumor-bearing mice with TRIMEL/CCH or combined cell lysates in the absence of CCH did not prevent tumor growth ( figure 2B), indicating that every vaccine component is crucial for induction of protective responses in our model.…”

“…It has been described that the highly complex CCH molecule shows a structural stability that contribute to their potent immunostimulatory effects, 24 strengthening innate and adaptive immunity in mammals and making it useful in cancer immunotherapy. 25 26 Although both hemocyanins show comparable immunogenic characteristics, 27 CCH is made up of intermixed subunits organized as heterododecamers that do not require divalent ions, given greater comparative stability. Unlikely, KLH is formed by homododecamers, whose stability depends on Ca +2 and Mg +2 for maintenance.…”

“…Unlikely, KLH is formed by homododecamers, whose stability depends on Ca +2 and Mg +2 for maintenance. 25 …”

N-Glycosylation of mollusk hemocyanins contributes to their structural stability and immunomodulatory properties in mammals

“…Moreover, KLH binding to human MR triggers proinflammatory responses in APCs (29). Similarly, TLR4 has been shown to participate in the immunostimulatory effects of CCH, FLH, and KLH in murine APCs (30). All these receptors recognize glycosylated structures from pathogens and promote endocytosis, proinflammatory responses, and antigen presentation to T lymphocytes (31–33).…”