Estimulação de vias inatas em células uterinas e ensaios de imunoprofilaxia em modelo murino visando o controle da neosporose bovina

Abstract

Neospora caninum has caused great interest in the research due to its association with repeated abortion in the cattle herd, entailing billions of dollars of loss in livestock annually. Classified as a mandatory intracellular protozoan, vertical transmission is common in bovine hosts and the consequences generated will depend on the host immune response profile. In addition, N. caninum presents several secretory molecules important in the invasion process. Among them, NcROP4 is a rhoptry protein that has immunogenic character important for the process of invasion and evasion in the host cell. Here we aimed at two manuscripts to understand the innate mechanisms involved in the response against N. caninum in bovine uterine cells and to evaluate the immunomodulatory capacity of recombinant NcROP4 (rNcROP4) in the development of vaccine protocols and from there to test the immunotherapeutic protocols of an acute inflammatory pathology. The results set forth in Chapter II of the thesis demonstrated that N. caninum is able to induce IFNγ, IL-10, IL-6 and IL-8 production in bovine endometrial cells. In addition, the IFNγ production was TLR3 and MyD88-dependent, because when these molecules were silenced there was a decrease in the production of this mediator. This silencing also generated an increase in the IL-10, IL-6 and IL-8 production after infection. In contrast, inhibition of MAPK p38 and ERK induced a reduction of IFNγ, and an increase in IL-10, IL-6 and IL-8 production, exhibiting an opposite effect to TLR3 and MyD88. In addition, we have demonstrated that IFNγ production is STAT1-dependent whereas IL-10 production is STAT3-dependent on bovine uterine cells studied. There was also an increase in IL-10 production when STAT1 was silenced and the same was observed with IFNγ when STAT3 was silenced, showing a balance between these two cytokines during pathogen infection in bovine endometrial cells. In chapter III we initially identified that NcROP4 binding to mAb20D2. This binding region was chemically synthesized and named (PepNcROP4). rNcROP4 was produced in Escherichia coli. Both the peptide and the protein were placed in mice macrophage and culture peritoneum and induced a high IL-10 production and low IFNγ production compared to the parasite extract (NLA). We also used rNcROP4, PepNcROP4 and NLA in immunization protocols and found that all groups induced specific antibody production. After challenge with the parasite, rNcROP4 induced increase in body weight while the other antigens used induced reduction of body weight. In addition, immunization with this protein resulted in a reduction of parasite burden and a considerable increase in survival compared to the other groups. Finally, the regulatory effect of rNcROP4 was tested in the treatment of Toxoplasma gondii-induced ileitis in murine model. The results showed that the treatment with this protein reduced the inflammatory process in the ileum, evidencing that rNcROP4 presents regulatory effect in other inflammatory diseases in mice. From the foregoing, we can conclude that innate signaling receptors and pathways participate in the response to N. caninum in bovine endometrial cells and the rNcROP4 protein shows regulatory potential able to increase the protective capacity in neosporosis and reducing T. gondii-induced ileitis in murine model.