Macrophage migration inhibitory factor favors Neospora caninum infection in mice.

Neospora caninum is a protozoan parasite with worldwide incidence, acting as a major cause of reproductive failures in ruminants and neuromuscular symptoms in dogs. Macrophage Migration Inhibitory Factor (MIF) is produced by several cell types and exhibits a central role in immune responses against intracellular pathogens. The present study aimed to comprehend the role of MIF in the relationship between N. caninum and its host. We used in vivo, in vitro and ex vivo experiments in a model of infection based on genetically deficient mice to analyze the infection kinetics and inflammatory markers. MIF production was measured in response to N. caninum during the acute and chronic phases of the infection. While Mif-/- mice survived lethal doses of NcLiv tachyzoites, sublethal infections in these mice showed that parasite burden was controlled in target tissues, alongside with reduced inflammatory infiltrates detected in lung and brain sections. TNF was increased at the initial site of the infection in genetically deficient mice and the MIF-dependent reduction was confirmed in vitro with macrophages and ex vivo with primed spleen cells. In sum, MIF negatively regulated host immunity against N. caninum, favoring disease progression.

TNF-TNFR1 Signaling Enhances the Protection Against Neospora caninum Infection.

Neospora caninum is a protozoan associated with abortions in ruminants and neuromuscular disease in dogs. Classically, the immune response against apicomplexan parasites is characterized by the production of proinflammatory cytokines, such as IL-12, IFN-γ and TNF. TNF is mainly produced during the acute phases of the infections and binds to TNF receptor 1 (CD120a, p55, TNFR1) activating a variety of cells, hence playing an important role in the induction of the inflammatory process against diverse pathogens. Thus, in this study, we aimed to evaluate the role of TNF in cellular and humoral immune responses during N. caninum infection. For this purpose, we used a mouse model of infection based on wildtype (WT) and genetically deficient C57BL/6 mice in TNFR1 (Tnfr1-/-). We observed that Tnfr1-/- mice presented higher mortality associated with inflammatory lesions and increased parasite burden in the brain after the infection with N. caninum tachyzoites. Moreover, Tnfr1-/- mice showed a reduction in nitric oxide (NO) levels in vivo. We also observed that Tnfr1-/- mice showed enhanced serum concentration of antigen-specific IgG2 subclass, while IgG1 production was significantly reduced compared to WT mice, suggesting that TNFR1 is required for regular IgG subclass production and antigen recognition. Based on our results, we conclude that the TNF-TNFR1 complex is crucial for mediating host resistance during the infection by N. caninum.

Inducible Nitric Oxide Synthase is required for parasite restriction and inflammatory modulation during Neospora caninum infection.

Neospora caninum infection is an important cause of neuromuscular disease in dogs and abortion in cattle, leading to significant economic losses in beef and dairy industries. The protective immunity against apicomplexan parasites, specifically Toxoplasma gondii and N. caninum, is typically achieved by inducing an IL-12-driven Th1 immune response. IL-12 stimulates IFN-γ production, which activates Inducible Nitric Oxide Synthase (iNOS) and promotes consequent Nitric Oxide (NO) synthesis, classically described as one of the main effector mechanisms for parasite elimination. Here, we aimed to evaluate the role played by iNOS during N. caninum infection. Our results show that N. caninum infection in C57BL/6 wild type (WT) mice induce NO production in vivo and in vitro. In agreement, iNOS deficient mice, as well as WT mice treated with iNOS inhibitor aminoguanidine, succumbed during acute infection with a dose lethal to 50 % of the WT mice, and presented significant increase in parasite load when submitted to sub-lethal infection protocols. Interestingly, the lack of control of parasite proliferation observed in iNOS-/- mice was associated with notable CNS inflammation and increased production of the main systemic proinflammatory cytokines (IL-12, IFN-γ, IL-6, TNF and IL-17A). Taken together, our findings show that iNOS plays an important role in restricting N. caninum replication, while also modulates the inflammatory process induced by the infection.

Toll-Like Receptor 3-TRIF Pathway Activation by Neospora caninum RNA Enhances Infection Control in Mice.

Neospora caninum is a protozoan parasite closely related to Toxoplasma gondii and has been studied for causing neuromuscular disease in dogs and abortions in cattle. It is recognized as one of the main transmissible causes of reproductive failure in cattle and consequent economic losses to the sector. In that sense, this study aimed to evaluate the role of Toll-like receptor 3 (TLR3)-TRIF-dependent resistance against N. caninum infection in mice. We observed that TLR3-/- and TRIF-/- mice presented higher parasite burdens, increased inflammatory lesions, and reduced production of interleukin 12p40 (IL-12p40), tumor necrosis factor (TNF), gamma interferon (IFN-γ), and nitric oxide (NO). Unlike those of T. gondii, N. caninum tachyzoites and RNA recruited TLR3 to the parasitophorous vacuole (PV) and translocated interferon response factor 3 (IRF3) to the nucleus. We also observed that N. caninum upregulated the expression of TRIF in murine macrophages, which in turn upregulated IFN-α and IFN-ß in the presence of the parasite. Furthermore, TRIF-/- infected macrophages produced lower levels of IL-12p40, while exogenous IFN-α replacement was able to completely restore the production of this key cytokine. Our results show that the TLR3-TRIF signaling pathway enhances resistance against N. caninum infection in mice, since it improves Th1 immune responses that result in controlled parasitism and reduced tissue inflammation, which are hallmarks of the disease.

Biological and Enzymatic Characterization of Proteases from Crude Venom of the Ant Odontomachus bauri.

Hymenoptera venoms constitute an interesting source of natural toxins that may lead to the development of novel therapeutic agents. The present study investigated the enzymatic and biological characteristics of the crude venom of the ant Odontomachus bauri. Its crude venom presents several protein bands, with higher staining for six proteins with gelatinolytic activity (17, 20, 26, 29, 43 and 48 kDa). The crude venom showed high proteolytic activity on azocasein at optimal pH 8.0 and 37 °C. In the presence of protease inhibitors as aprotinin, leupeptin and EDTA, the azocaseinolytic activity was reduced by 45%, 29% and 9%, respectively, suggesting that the enzymes present in the crude venom belong to the three classes of proteases, with the serine proteases in greater intensity. The crude venom degraded the fibrinogen α-chain faster than the ß-chain, while the fibrinogen γ-chain remained unchanged. In biological assays, O. bauri venom showed hemolytic and coagulant activity in vitro, and defibrinating activity in vivo. In addition, the venom showed antimicrobial activity against Staphylococcus aureus and Escherichia coli as well as antiparasitic activity on Toxoplasma gondii infection in vitro. In that sense, this study sheds perspectives for pharmacological applications of O. bauri venom enzymes.