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Neospora caninum is a parasite relevant to the veterinary field. Innate and adaptive responses against N. caninum induce effector mechanisms that limit parasite replication, but little is known about their role in humoral response. Our work aimed to verify whether key molecules in the TLR2/MyD88-mediated response would impact the production of specific IgM and IgG antibodies in mice during immunization with soluble antigens of N. caninum. We observed that lack of IFN-gamma did not negatively affect the production of specific antibodies. However, mice genetically deficient in Toll-like receptor 2, Myeloid differentiation factor 88, Interleukin 12 and inducible nitric oxide synthase presented significant decrease in antibody levels against N. caninum antigens, which also reflected in the diversity of the antigen recognized by their serum. In that sense, we show here that molecules within this innate recognition pathway may present a direct impact in the induction of an antibody response against N. caninum.

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.

Neospora caninum poses as a considerable threat to animal health and generates significant economic impact in livestock production worldwide. Here, we have investigated the mechanism that underlies the participation of the inflammasome complex and Reactive Oxygen Species (ROS) in the regulation of immune responses during N. caninum infection. For that purpose, we used in vitro (bone marrow derived macrophages) and in vivo mouse models of infection. Our results show that NLRP3 and NLRC4 receptors, alongside with ASC and Caspase-1, are required for proper activation of the inflammasome during N. caninum infection. As expected, the engagement of these pathways is crucial for IL-1α, IL-1β, and IL-18 production, as well as the induction of pyroptosis. Our results also show that N. caninum induces ROS production dependent of the inflammasome assembly, which in its turn also depends on MyD88/NF-κB-induced ROS to maintain its activation and, ultimately, lead to restriction of parasite replication.

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-β 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.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico FAPEMIG - Fundação de Amparo a Pesquisa do Estado de Minas Gerais Neospora caninum é um parasito intracelular obrigatório com distribuição mundial, gerando abortamentos em bovinos e perdas bilionárias anuais na área da produção animal. Pesquisas sobre a resposta imune contra esta infecção adotam geralmente modelos murinos através da infecção parenteral. Entretanto, compreender os mecanismos imunológicos após a infecção oral é essencial, uma vez que mimetiza a infecção natural com maior precisão. A resposta imune contra protozoários intracelulares é baseada em respostas imunes de padrão Th1, reguladas através da produção de IFN-γ. Assim, neste trabalho, objetivou-se avaliar a resposta imune desencadeada pela infecção oral por N. caninum em camundongos, com especial atenção na produção de IFN-γ através de vias inatas TLR-MyD88 e Inflamassoma. Para estes experimentos, foram utilizados camundongos C57BL/6 infectados com taquizoítos pela via oral, após o bloqueio do pH estomacal. Os camundongos foram analisados quanto à sobrevivência, lesões teciduais, produção de citocinas e produção de anticorpos específicos. Observamos que camundongos WT infectados via oral apresentaram níveis crescentes de IgG sérica específica, composta principalmente por anticorpos IgG2. DNA de parasitos foi detectado principalmente no jejuno distal e no íleo dos camundongos, embora nenhuma alteração histológica tenha sido detectada em todo o intestino. No entanto, lesões inflamatórias agudas foram observadas nos fígados e pulmões de camundongos infectados por via oral, associados à presença, in situ, de DNA de parasitos e produção de IFN-γ. A fim de analisar o papel dessa citocina chave, nós infectamos camundongos geneticamente deficientes em IFN-γ com taquizoítos de N. caninum utilizando a mesma via. Observamos que camundongos IFN-γ-/- apresentaram pouco ou nenhum sinal de morbidade durante os primeiros dias de infecção. No entanto, a partir do sexto dia, camundongos IFN-γ-/- sucumbiram rapidamente à infecção, sem sinais de lesão inflamatória nos tecidos analisados. Com tal confirmação, nós investigamos a via responsável pela produção da citocina IFN-γ durante a infecção oral, utilizando camundongos deficientes em MyD88 e Caspase-1/11 infectados com N. caninum. Foi observado neste experimento uma redução significativa da produção de IFN-γ em camundongos Caspase-1/11-/-, enquanto camundongos deficientes em MyD88 não apresentaram alterações nos níveis desta citocina. Tal resultado foi corroborado pela análise histológica dos tecidos, com redução da lesão de tecidos pulmonares e hepáticos em camundongos Caspase-1/11-/-, de modo semelhante ao anteriormente observado na ausência de IFN-γ. Em conclusão os resultados aqui apresentados demonstram a rápida passagem do protozoário N. caninum pelo intestino delgado de camundongos, lesionando órgãos abdominais e torácicos por meio de processos replicativos e inflamatórios mediados por IFN-γ, induzido pela ativação do complexo Inflamassoma. Por meio dos conhecimentos gerados, abre-se a possibilidade de melhor controle da patogenia induzida pela infecção por N. caninum. Neospora caninum is an obligate intracellular parasite with worldwide distribution that causes abortions in cattle and billionaire losses in livestock. The investigation of immune response against this infection usually adopts parenteral infection in murine models. However, it is essential to understand the immune mechanisms after oral infection wich accurately mimics natural condition. Th1 adaptative immune response is crucial to protect against this intracellular protozoan, wich is mainly regulated by IFN-γ production. Thus, in this work, we aimed to evaluate the immune response triggered by N. caninum oral infection in mice, focusing in the IFN- γ production induced by innate pathway TLR-MyD88 and inflamassome. For this purpose, we used C57BL/6 mice orally infected with tachyzoites after neutralization of the gastric pH. We analyzed the survival, tissue lesions, cytokine production, and specific antibody production in infected mice. We observed that WT mice infected by oral route presented higher levels of serum specific IgG, composed mainly by IgG2 antibodies. DNA of the parasite was detected mainly in the distal jejunum and ileum, however, no histological alterations were detected through the gut. Meanwhile, acute inflammatory lesions were observed in livers and lungs of orally infected mice, related with the presence, in situ, of parasite DNA and IFN-γ production. In order to analyze the role of these key cytokine, we infected IFN-γ genetically deficient mice with N. caninum tachyzoites by gavage. We observed that IFN γ-/- mice presented reduced signs of morbidity during the beginning of infection. However, after six days of infection, IFN γ-/- mice succumbed quickly the infection, with no signs of inflammatory lesions in analyzed tissues. After this results, we investigated the pathway responsible to IFN-γ production during oral infection using MyD88-/- and Caspase-1/11-/- mice infected with N. caninum. A significant reduction in the IFN-γ production in Caspase-1/11-/- mice was observed in this experiment, whereas MyD88 deficient mice did not present alterations in the levels of this cytokine. These findings are supported by histological analyses that demonstrated a reduction of lung and liver tissue lesions in Caspase-1/11-/- mice, similar to the result previously observed in the absence of IFN-γ. In conclusion, this work demonstrated the quick passage of the N. caninum protozoa into the small intestine of mice, and then injuring abdominal and thoracic organs through replicative and inflammatory processes mediated by IFN-γ. Also, this cytokine is induced by the activation of the inflamassome complex. Overall, we can get the possibility to enhance the control of pathogenicity induced by N. caninum infection. Tese (Doutorado)

It has been described that the metalloprotease BmooMP-alpha-I purified from Bothrops moojeni snake venom is able to hydrolyze the TNF molecule. However, this observation has been based mainly on in vitro investigation, in addition to molecular modeling and docking approaches. Considering that there is no in vivo study to demonstrate the biological effects of this enzyme, the major aim to the present work was to investigate whether the BmooMP-alpha-I has any anti-inflammatory efficacy by setting up a murine experimental design of colitis induced by dextran sulfate sodium (DSS). For this purpose, C57BL/6 mice were divided into six groups, as follows: (i) animals without intestinal inflammation, (ii) animals without intestinal inflammation treated with BmooMP-alpha-I (50 μg/animal/day), and (iii) animals with intestinal inflammation induced by 3% of DSS, (iv) mice with intestinal inflammation induced by DSS and treated with BmooMP-alpha-I enzyme at the 50, 25, or 12.5 μg/animal/day dosages by intraperitoneal route. Clinical signs of colitis were observed daily for calculating the morbidity scores, cytokine measurements, and histological features. We observed that the animals treated with different doses of the enzyme presented a remarkable improvement of colitis signs, as confirmed by a significant increase of the intestine length in comparison to the DSS group. Also, no difference was observed between the groups treated with the enzyme or vehicle, as the colon length of these animals was slightly lower than that of the group of healthy animals, without induction of intestinal inflammation. The cytokine quantification in supernatants of intestinal tissue homogenates showed a significant reduction of 38% in IFN-gamma levels, when the animals were treated with 50 μg of the BmooMP-alpha-I compared to the animals receiving DSS only. A significant reduction of 39% in TNF levels was also observed in all doses of treatment with BmooMP-alpha-I, in addition to a significant reduction of 35% in the amount of IL-12p40. Histological examinations revealed that the BmooMP-alpha-I 50 μg treated group preserved colon architecture and goblet cells and reduced the ulcer area, when compared with DSS mice, which showed typical inflammatory changes in tissue architecture, such as ulceration, crypt dilation, loss of tissue architecture, and goblet cell depletion, accompanied by a significant cell infiltration. In conclusion, our results suggest that the improvement of clinical scores and histological findings related to BmooMP-alpha-I treatment in this experimental model could be attributed to the metalloprotease ability to modulate cytokine production locally at the inflamed intestine. These findings highlight the potential anti-inflammatory role and effectiveness of this enzyme as a therapeutic alternative in this type of immunopathological condition.

It has been described that the metalloprotease BmooMP-alpha-I purified from Bothrops moojeni snake venom is able to hydrolyze the TNF molecule. However, this observation has been based mainly on in vitro investigation, in addition to molecular modeling and docking approaches. Considering that there is no in vivo study to demonstrate the biological effects of this enzyme, the major aim to the present work was to investigate whether the BmooMP-alpha-I has any anti-inflammatory efficacy by setting up a murine experimental design of colitis induced by dextran sulfate sodium (DSS). For this purpose, C57BL/6 mice were divided into six groups, as follows: (i) animals without intestinal inflammation, (ii) animals without intestinal inflammation treated with BmooMP-alpha-I (50 μg/animal/day), and (iii) animals with intestinal inflammation induced by 3% of DSS, (iv) mice with intestinal inflammation induced by DSS and treated with BmooMP-alpha-I enzyme at the 50, 25, or 12.5 μg/animal/day dosages by intraperitoneal route. Clinical signs of colitis were observed daily for calculating the morbidity scores, cytokine measurements, and histological features. We observed that the animals treated with different doses of the enzyme presented a remarkable improvement of colitis signs, as confirmed by a significant increase of the intestine length in comparison to the DSS group. Also, no difference was observed between the groups treated with the enzyme or vehicle, as the colon length of these animals was slightly lower than that of the group of healthy animals, without induction of intestinal inflammation. The cytokine quantification in supernatants of intestinal tissue homogenates showed a significant reduction of 38% in IFN-gamma levels, when the animals were treated with 50 μg of the BmooMP-alpha-I compared to the animals receiving DSS only. A significant reduction of 39% in TNF levels was also observed in all doses of treatment with BmooMP-alpha-I, in addition to a significant reduction of 35% in the amount of IL-12p40. Histological examinations revealed that the BmooMP-alpha-I 50 μg treated group preserved colon architecture and goblet cells and reduced the ulcer area, when compared with DSS mice, which showed typical inflammatory changes in tissue architecture, such as ulceration, crypt dilation, loss of tissue architecture, and goblet cell depletion, accompanied by a significant cell infiltration. In conclusion, our results suggest that the improvement of clinical scores and histological findings related to BmooMP-alpha-I treatment in this experimental model could be attributed to the metalloprotease ability to modulate cytokine production locally at the inflamed intestine. These findings highlight the potential anti-inflammatory role and effectiveness of this enzyme as a therapeutic alternative in this type of immunopathological condition.

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 the major cause of economic losses in bovine products. In that sense, this study aimed to evaluate the role of TLR3-TRIF dependent resistance against N. caninum infection. We observed that TLR3−/− and TRIF−/− mice presented higher parasite burden, increased inflammatory lesions and reduced production of IL-12p40, TNF, IFN-γ, and NO. Differently from T. gondii, N. caninum tachyzoites and its RNA recruited TLR3 and IRF3 to the parasitophorous vacuole (PV). We observed that N. caninum upregulated the expression of TRIF in macrophages, which by its turn upregulated IFN-α and IFN-β in the presence of the parasite. Furthermore, TRIF−/− infected macrophages produced lower levels of IL-12p40 and IFN-α replacement was able to completely restore the production of this key cytokine. Our results have shown that TLR3-TRIF signaling pathway enhances resistance against N. caninum infection, since it improves Th1 immune responses that control parasitism and tissue inflammation, which are hallmarks of the disease.

Neospora caninum is an intracellular protozoan parasite that has drawn increasing interest due to its association with worldwide repetitive bovine abortions, which cause billionaire losses to the meat and dairy industries annually. Innate immunity plays an important role in infection control, and N. caninum activates the production of inflammatory mediators through Toll-like Receptors (TLR), Nod-Like Receptors (NLR) and Mitogen Activated Protein Kinase (MAPK) signaling pathways. Advances in the knowledge of initial host-parasite interactions are desirable for the design of control measures against the infection, obliterating its pathogenesis. In that sense, we here aimed to describe the role of the innate C-type lectin receptor (CLR) Dectin-1 during the infection by N. caninum. With that intent, we observed that the absence of Dectin-1, observed in genetically depleted (Dectin-1-/-) mice or competitively inhibited by an inert agonist (Laminarin, LAM), rescued 50% of the mice infected with lethal doses of N. caninum. Dectin-1-/- and LAM-treated mice also presented a reduction in the parasite load during acute and chronic phases, associated to decreased inflammatory scores in the central nervous system. Amongst all the cell phenotypes that migrated to the initial site of infection, dendritic cells and macrophages gained subpopulations with high Dectin-1 surface expression. The impairment of the receptor in these cells led to a decreased parasite burden, as well as augmented production of IL-12p40. We also found that Dectin-1+ cells produced less reactive oxygen species (ROS) at the initial site of the infection, while mice deficient in NAPDH oxidase isoform 2 (NOX2-/-) were not able to control parasite replication and produce IL-12p40, even upon LAM treatment. Interestingly, the absence of functional Dectin-1 did not alter the susceptibility of mice against closely related T. gondii. In conclusion, the gathered data suggests that Dectin-1 is involved in the parasite-induced downmodulation of ROS and other key molecules triggered for the control of N. caninum infection, and is a promising target for future development of protocols intended for intervention against neosporosis.

Neospora caninum is an intracellular protozoan parasite that has drawn increasing interest due to its association with worldwide repetitive bovine abortions, which cause billionaire losses to the meat and dairy industries annually. Innate immunity plays an important role in infection control, and N. caninum activates the production of inflammatory mediators through toll-like receptors, NOD-like receptors, and mitogen-activated protein kinase signaling pathways. Advances in the knowledge of initial host–parasite interactions are desirable for the design of control measures against the infection, obliterating its pathogenesis. In that sense, we here aimed to describe the role of the innate C-type lectin receptor Dectin-1 during the infection by N. caninum. With that intent, we observed that the absence of Dectin-1, observed in genetically depleted (Dectin-1−/−) mice or competitively inhibited by an inert agonist [laminarin (LAM)], rescued 50% of the mice infected with lethal doses of N. caninum. Dectin-1−/− and LAM-treated mice also presented a reduction in the parasite load during acute and chronic phases, associated with decreased inflammatory scores in the central nervous system. Among all the cell phenotypes that migrated to the initial site of infection, dendritic cells and macrophages gained subpopulations with high Dectin-1 surface expression. The impairment of the receptor in these cells led to a decreased parasite burden, as well as augmented production of IL-12p40. We also found that Dectin-1+ cells produced less reactive oxygen species (ROS) at the initial site of the infection, while mice deficient in NADPH oxidase isoform 2 (NOX2−/−) were not able to control parasite replication and produce IL-12p40, even upon LAM treatment. Interestingly, the absence of functional Dectin-1 did not alter the susceptibility of mice against closely related Toxoplasma gondii. In conclusion, the gathered data suggest that Dectin-1 is involved in the parasite-induced downmodulation of ROS, and other key molecules triggered for the control of N. caninum infection and are a promising target for future development of protocols intended for intervention against neosporosis.