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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.

Copper and copper oxide nanoparticles (CuNP) have been utilized as anti-microbial agent, metal catalyst, diet supplement, high thermal conductive material, lubricant additive, among others. Although the uses of CuNP are relatively new, there is an increasing interest to know their immunotoxicology. CuNP have shown to induce one of the most potent acute inflammatory reactions among several types of nanoparticles. Recently, in a pulmonary infection model, it was demonstrated that mice, which had been previously exposed to CuNP, presented an impaired bacterial clearance. These effects were due to a reduction in macrophage function; however, the precise molecular mechanism underlying these effects is still unknown. Here, we described the effects of CuNP in the response of peritoneal macrophages against microbial products. Transmission electron microscopy (TEM) data showed that CuNP were rapidly internalized by macrophages. The internalization of the particles induced a significant reduction of cell viability in comparison with other nanoparticles. The challenge with CuNP inhibited LPS-mediated nitric oxide (NO) production in a dose dependent manner. These results showed that CuNP modulate the expression of a key inflammatory mediator that is crucial to eliminate bacterial infection.

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.