Hemocyanins induce a potent Th1-dominant immune response with beneficial clinical outcomes when used as a carrier/adjuvant in vaccines and nonspecific immunostimulant in cancer. However, the mechanisms by which hemocyanins trigger innate immune responses, leading to beneficial adaptive immune responses, are unknown. This response is triggered by a proinflammatory signal from various components, of which macrophages are an essential part. To understand how these proteins influence macrophage response, we investigated the effects of mollusks hemocyanins with varying structural and immunological properties, including hemocyanins from Concholepas concholepas, Fissurella latimarginata, and Megathura crenulata (keyhole limpet hemocyanin), on cultures of peritoneal macrophages. Hemocyanins were phagocytosed and slowly processed. Analysis of this process showed differential gene expression along with protein levels of proinflammatory markers, including IL-1β, IL-6, IL-12p40, and TNF-α. An extended expression analysis of 84 cytokines during a 24-h period showed a robust proinflammatory response for F. latimarginata hemocyanin in comparison with keyhole limpet hemocyanin and C. concholepas hemocyanin, which was characterized by an increase in the transcript levels of M1 cytokines involved in leukocyte recruitment. These cytokine genes included chemokines (Cxcl1, Cxcl3, Cxcl5, Ccl2, and Ccl3), ILs (Il1b and Ifng), growth factors (Csf2 and Csf3), and TNF family members (Cd40lg). The protein levels of certain cytokines were increased. However, every hemocyanin maintains downregulated key M2 cytokine genes, including Il4 and Il5. Collectively, our data demonstrate that hemocyanins are able to trigger the release of proinflammatory factors with different patterns of cytokine expression, suggesting differential signaling pathways and transcriptional network mechanisms that lead to the activation of M1-polarized macrophages.

Hemocyanins inoculated in mammals induce a Th1-dominant response with beneficial clinical outcomes, being used as carrier/adjuvant in vaccines and as immunostimulant for the treatment of bladder cancer. However, its effects during the early phases of the immune response are unknown. We hypothesized that when hemocyanins are incorporated by antigen presenting cells, they induce an inflammatory milieu, producing a bystander effect, key event in its non-specific immunostimulatory effects. Here, in vitro cultures of murine peritoneal macrophages were incubated with different hemocyanins from M. crenulata (KLH) or C. concholepas (CCH) or F. latimarginata (FLH), to study the correlation between mRNA and protein levels from a panel of proinflammatory cytokines and chemokines, using qPCR and ELISA. The results showed that hemocyanins were endocyted and slowly processed by macrophages, stimulating the expression of IL-6, IL-12p40 and TNF-α at different times and intensities. FLH and KLH but not CCH at 24 h, induced an increased in the expression of chemokines related to leukocyte extravasations, such as CCL-3, CXCL-1 and CXCL-5. Moreover, hemocyanins kept down-regulated key Th2 cytokines such as IL-4 and TGF-β2. We concluded that each hemocyanin can stimulate different kinetic patterns of proinflammatory cytokines and chemokines expression in macrophages, suggesting different signaling pathways, which can be explained in part, by its structural differences.

The versatile properties of mollusk hemocyanins in biomedical applications, including non-specific immunostimulant during the therapy of bladder cancer and carrier/adjuvant in cutting-edge therapeutic cancer vaccines, has increased the interest in finding new hemocyanins with better immunomodulatory properties. Here, we evaluate the physicochemical and immunological properties of the hemocyanin from Fissurella latimarginata (FLH). This protein shares with keyhole limpet hemocyanin (KLH) and Concholepas hemocyanin (CCH) the typical hollow cylinder structure and it is also made by two subunits (~350 KDa). ConA lectin staining of FLH, demonstrated the presence of mannosylated carbohydrate structures as well as in KLH and CCH. The humoral responses in mice demonstrated that antibody titers induced by FLH itself were significantly higher than KLH and CCH. However, it performance as a carrier protein to induce antibodies to the hapten DNFB, was similar to the other carriers using Freund’s adjuvant as a repository. More interestingly, FLH exhibits an outstanding antitumor activity prolonging mice survival, when was evaluated and compared with KLH and CCH in the B16F10 mouse melanoma model. In addition, flow cytometry analysis showed that FLH induced a significant number of tumor-infiltrating CD4+ cells and also, IFN-δ secretion. Together, these findings introduce a novel hemocyanin, with intrinsically more immunogenic and immunomodulatory capacities for antitumor therapy.