Peritoneal murine macrophages stimulated with recombinant TSA-1-C4 plus Tc24-C4 antigen combination display a Th1-immune response
We were also interested in evaluating the Th1 and Th2 immune profiles of peritoneal murine macrophages activated by TSA-1-C4 plus Tc24-C4 recombinant antigen combination, hence, we measured levels of the principal pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and the anti-inflammatory cytokine IL-10 by ELISA kit-test as we described previously.
When we measured TNF-α, IL-1β and IL-6 production by TSA-1-C4 plus Tc24-C4 combination, we observed significant cytokine-production for all concentrations evaluated, except for 6.25 µg/mL compared to non-stimulated macrophages (Table 1 ), which values are represented by the purple dotted line in the Fig. 2,A-C . Moreover, peritoneal murine macrophages stimulated with LPS induced the highest production of TNF-α, IL-1β, and IL-6 cytokines as we expected (Table 1 ). According to the Fig. 2,A-C , we observed a dose-dependent-effect, while higher is the stimulation, higher is the production of each cytokine, besides, TSA-1-C4 plus Tc24-C4 stimulated macrophages induced a significant increase in the pro-inflammatory cytokine-production compared to individuals TSA-1-C4 or Tc24-C4 stimulated cells for all concentrations evaluated, except for 6.25 µg/mL. This finding suggests that, when we stimulate macrophages with 12.5 µg/mL onwards of TSA-1-C4 plus Tc24-C4 recombinant antigen combination there is a synergistic effect in order to induce a Th1 immune-response compared to macrophages stimulated with TSA-1-C4 or Tc24-C4 individual antigens.
In order to have a perspective for the Th2 immune response, we measured levels of IL-10 cytokine-production by TSA-1-C4 plus Tc24-C4 stimulated macrophages. According to the Table 1 , we observed that, peritoneal macrophages stimulated with either 100, 50, 25, 12.5 and 6.25 µg/mL of TSA-1-C4 plus Tc24-C4 recombinant antigen combination showed significant IL-10 production compared to non-stimulated macrophages. As we expected MTX stimulation induced the highest levels of the cytokine. By the other hand, we observed an inverse dose-dependent-effect, while higher is the concentration, lower is the IL-10 production (Fig. 2,D ). No significant differences were found to compare TSA-1-C4 plus Tc24-C4 stimulated macrophages with individuals TSA-1-C4 or Tc24-C4 stimulated cells for all concentrations evaluated. In consequence, the recombinant TSA-1-C4 plus Tc24-C4 antigen combination induced an anti-inflammatory response mediated by IL-10 production byin-vitro assays, however, there was not a benefit to stimulate with the bivalent recombinant protein strategy.