Recombinant TSA-1-C4 plus Tc24-C4 protein combination activates peritoneal murine macrophages
In this study, we evaluated the immunomodulatory effect of the recombinant TSA-1-C4 plus Tc24-C4 antigen combination at different concentrations (100, 50, 25, 12.5 and 6.25 µg/mL) by activation of peritoneal macrophages and CD8+ T cells from naïve mice by in-vitro assays. In order to evaluate the synergistic effect of the bivalent recombinant protein strategy, we also determine the effect induced by the stimulation with TSA-1-C4 and Tc24-C4 antigens.
Nitric oxide and hydrogen peroxide are considered as indicators of macrophage activation; hence, we measured levels of NO indirectly by nitrites concentration and H2O2production of supernatants from cultures of naïve peritoneal macrophages stimulated with TSA-1-C4 and Tc24-C4 combination.
According to our data, peritoneal murine macrophages stimulated with 100, 50, 25, and 12.5 µg/mL of TSA-1-C4 plus Tc24-C4 combination or either individuals’ antigens, exhibited significant NO and H2O2 production compared to non-stimulated macrophages (Fig. 1A, 1B ). A no significant increment was observed in peritoneal macrophages stimulated with 6.25 µg/mL of recombinant proteins. As we expected, LPS stimulation induced the highest production of NO and H2O2molecules. Moreover, we observed a dose-dependent effect, while higher is the concentration of antigen-stimulation, higher is the production of NO or H2O2 molecules by peritoneal macrophages. Nevertheless, NO and H2O2production by Tc24-C4 stimulated macrophages (100, 50, 25, and 12.5 µg/mL) was significantly higher compared to TSA-1-C4 plus Tc24-C4 or individual TSA-1-C4 stimulated cells (Fig. 1A, 1B ). Hence, all this data indicates that, TSA-1-C4 plus Tc24-C4 recombinant antigen combination active peritoneal macrophages from naïve mice, however, Tc24-C4 stimulation induced the higher production of NO and H2O2 for all concentrations evaluated. Our results suggest that Tc24-C4 alone is a good producer of NO and H2O2 molecules, even compared with the combination of TSA-1-C4 plus Tc24-C4 proteins, which production was slightly lower.