The recombinant proteins TSA-1-C4 and Tc24-C4 have been proposed as potential antigens for the development of vaccines candidates, with previous studies demonstrating their therapeutic effect. However additional work is needed to understand their immunologic mechanisms and evaluate their immunomodulatory effects through specific assays.
In this study, we found that peritoneal murine macrophages stimulated with either TSA-1-C4, Tc24-C4 or the combination of recombinant antigens induces releasing of host NO, H2O2 molecules, as well as, TNF-α, IL-1β, IL-6 and IL-10 cytokines, which are indicator of activation and functionality. This is the first report showing releasing levels of NO and H2O2 molecules using in-vitro cultures of macrophages exposes to recombinant TSA-1-C4 and Tc24-C4 proteins. Particularly, we observed that Tc24-C4 alone is a good inducer of NO and H2O2 molecules, even compared with the combination of TSA-1-C4 plus Tc24-C4 proteins, which induction was slightly lower. We assume that TSA-1-C4 antigen could have a downregulating effect on cytotoxic molecules releasing by macrophages. Previous studies suggest that, ROS and NO molecules are essential for parasite proliferation and growth, due to provide ideal conditions (e.g., iron availability in macrophages) for parasite-replication inducing a toxic effect in the host (Goes et al. 2016; Paiva et al. 2018). In our study, the amounts of NO and H2O2 molecules reported are similar to those reported in previous studies (Alonso-Castro et al. 20192020; Arana-Argáez et al. 2021), which peritoneal murine macrophages were stimulated with extracts of plants or microorganisms with antiparasitic activity, and according to their data, not significant cytotoxic activity was observed.
We also evaluated the inflammatory cytokine profile from supernatants of TSA-1-C4 plus Tc24-C4 stimulated macrophages. Here, we observed that when we increase the concentration of either, TSA-1-C4, Tc24-C4 or their combination, the production of TNF-α, IL-1β and IL-6 increase, while the regulatory cytokine IL-10 tends to reduce, therefore, we suggest that macrophages stimulated with the recombinant antigens can induce an inflammatory profile which is it a dose-dependent effect. Moreover, we demonstrated that the combination TSA-1-C4 plus Tc24-C4 increase significatively the production of TNF-α, IL-1β and IL-6 compared to individual TSA-1-C4 or Tc24-C4 stimulated-macrophages from naïve mice evidencing the capacity to induce a strong inflammatory response. It has been reported during early stages of T. cruzi infection, macrophages release cytokines, such as TNF-α, IL-1β, and IL-6, which induce the expression of adhesion molecules, causing chemotaxis and migration of T cells, hence are critical for control of parasite-replication and protective immunity (Kumar and Tarleton 2001; Barton 2008; Arango-Duque and Descoteaux 2014). Other studies have reported similar data to our findings. After stimulation with Tc24-C4 recombinant protein, production of cytokines as TNF-α or IL-6 was reported in supernatants of spleen cells from T. cruzi-infected Balb/c mice immunized with a vaccine formulated with the recombinant Tc24-C4 antigen in addition with TLR-4 agonist adjuvants (Jones et al. 2018; Cruz-Chan et al. 2021; Jones et al. 2023; Poveda et al. 2023). On the other hand, limited information is currently published related to TSA-1-C4, however, pro-inflammatory profile was observed in supernatants of spleen mononuclear cells from T. cruzi-infected and TSA-1-C4 + E6020-SE vaccinated mice after TSA-1-C4 stimulation (data in publication process).
Protective immunity against intracellular parasites as T. cruzi is mediated by CD8+ T cells, which release cytotoxic molecules such as, perforin and granzymes from cytotoxic granules (Kumar and Tarleton 1998; de Alencar et al. 2009). Moreover, CD8+ T cells modulate the immune response through secretion of cytokines, which are required for activation of APCs, macrophages and T cells or downregulate the extensive inflammatory response by production of regulatory cytokines (Martin and Tarleton 2004; Padilla et al. 2009). In this study, we measured the major cytokines representing for pro-inflammatory (IFN-γ and TNF-α) and anti-inflammatory (IL-10 and IL-4) immune responses in supernatants from co-cultures of TSA-1-C4 and Tc24-C4 stimulated macrophages and CD8+ T cells from naïve mice. All stimulus conditions (regardless of if the recombinant proteins were used in combination or not) induced pro-inflammatory and anti-inflammatory cytokines responses, through a dose-dependent effect. As we hypothesized, we also observed that there is a benefit to use the bivalent recombinant protein combination, our data reveals that the macrophages stimulated with recombinant TSA-1-C4 and Tc24-C4 combination can activate CD8+ T cells which are characterized by inducing a significant Th1-type response mediated by a strong production of IFN-γ and TNF-α compared with individual TSA-1-C4 or Tc24-C4 antigen stimulation. In accordance with our data, some studies have reported immunological profiles mediated by Tc24 antigen-specific CD8+ T cells in in-vitro spleen cells culture from T. cruzi-infected Balb/c mice and immunized with Tc24-C4 recombinant vaccines (Jones et al. 2018; Cruz-Chan et al. 2021), while in a recent study it was demonstrated that re-stimulation with recombinant Tc24-C4 protein in spleen cells from Tc24-C4 + GLA-SE vaccinated mice induces an antigen-specific CD8+IFN-γ+ immune profile (Poveda et al., 2023). Similarly, TSA-1 antigen-specific CD8+IFN-γ+ T cells was observed in spleen cells cultures from Balb/c mice immunized with TSA-1 in conjunction with Monophosphoryl-Lipid A (MPLA) adjuvant (de la Cruz et al. 2019).
This study supports the immunogenic effect of the bivalent recombinant protein strategy; and it is accordance with a previous study which evaluated the therapeutic efficacy of a vaccine-linked chemotherapy formulated by the recombinants TSA-1-C4 and Tc24-C4 proteins given during T. cruzi-chronic infection using a pre-clinical model (Dzul-Huchim et al. 2022).
Some limitations were observed in this study. Despite we recovered peritoneal macrophages from cavity peritoneal of mice, is possible that there were also other cells lines in our environment, such as dendritic cells, which could be responsible for the activation of CD8+ T cells in our in-vitro assay. Measurement of cytokines by other techniques highly sensitives as cytometric-bead-array (CBA) assay also could support our findings. Furthermore, it has been demonstrated that IL-17 family of cytokines plays a critical role in host survival by regulating exuberant inflammation during T. cruzi infection, suggesting that IL-17 cytokine have protective roles during adaptive immunity (Tosello-Boari et al. 2012, 2018; Cruz-Chan et al. 2021). For these reasons, we believe that further studies, would be relevant to evaluate the Th17 profile induced by the recombinant TSA-1-C4 plus Tc24-C4 antigen combination.