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

Cyclooxygenase (COX)-2 Inhibitors Reduce Toxoplasma gondii Infection and Upregulate the Pro-inflammatory Immune Response in Calomys callosus Rodents and Human Monocyte Cell Line

Toxoplasma gondii is able to infect a wide range of vertebrates, including humans. Studies show that cyclooxygenase-2 (COX-2) is a modulator of immune response in multiple types of infection, such as Trypanosoma cruzi. However, the role of COX-2 during T. gondii infection is still unclear. The aim of this study was to investigate the role of COX-2 during infection by moderately or highly virulent strains of T. gondii in Calomys callosus rodents and human THP-1 cells. C. callosus were infected with 50 cysts of T. gondii (ME49), treated with COX-2 inhibitors (meloxicam or celecoxib) and evaluated to check body weight and morbidity. After 40 days, brain and serum were collected for detection of T. gondii by real-time PCR and immunohistochemistry or cytokines by CBA. Furthermore, peritoneal macrophages or THP-1 cells, infected with RH strain or uninfected, were treated with meloxicam or celecoxib to evaluate the parasite proliferation by colorimetric assay and cytokine production by ELISA. Finally, in order to verify the role of prostaglandin E2 in COX-2 mechanism, THP-1 cells were infected, treated with meloxicam or celecoxib plus PGE2, and analyzed to parasite proliferation and cytokine production. The data showed that body weight and morbidity of the animals changed after infection by T. gondii, under both treatments. Immunohistochemistry and real-time PCR showed a reduction of T. gondii in brains of animals treated with both COX-2 inhibitors. Additionally, it was observed that both COX-2 inhibitors controlled the T. gondii proliferation in peritoneal macrophages and THP-1 cells, and the treatment with PGE2 restored the parasite growth in THP-1 cells blocked to COX-2. In the serum of Calomys, upregulation of pro-inflammatory cytokines was detected, while the supernatants of peritoneal macrophages and THP-1 cells demonstrated significant production of TNF and nitrite, or TNF, nitrite and MIF, respectively, under both COX-2 inhibitors. Finally, PGE2 treatment in THP-1 cells triggered downmodulation of pro-inflammatory mediators and upregulation of IL-8 and IL-10. Thus, COX-2 is an immune mediator involved in the susceptibility to T. gondii regardless of strain or cell types, since inhibition of this enzyme induced control of infection by upregulating important pro-inflammatory mediators against Toxoplasma

NcROP4 como um marcador de fase crônica no sorodiagnóstico da infecção por Neospora caninum

Neospora caninum is an obligate intracellular parasite whick is able to infect a wide range of hosts. Since its first description, N. caninum has emerged as an important disease of cattle and dogs worldwide. The neosporosis diagnosis is classically performed by serological tests as indirect immunofluorescence (RIFI) and ELISA, being the first gold standard for serodiagnosis that aim to detect the presence of antibodies against the parasite. However, there are no commercial tests designed to assess the stage of infection, once the main clinical signs are reported during the acute phase or during infection reactivation. Several protein targets of this pathogen has been tested in order to check the phase of host infection with satisfactory results. However, new targets should be evaluated in order to develop an effective diagnostic for the stages of infection. Thus, the present study aimed to studied the potential of NcROP4 protein as a stage marker of N. caninum infection. For this purpose, we evaluated the expression and localization of NcROP4 during infection in HeLa cells to investigate the possible exposure of this protein to antibodies. Subsequently, bioinformatic analysis and phage display were developed aiming to predict and determine B cell epitopes of the NcROP4. Mice and cattle were experimentally infected to investigate the producing of IgG through indirect and reverse immunoenzyme assay against NcROP4 during infection. It was observed that the NcROP4 protein is secreted into the cytoplasm of HeLa cells during the invasion process, which allows its extravasation to the extracellular environment after cell lysis. Bioinformatics analysis showed that NcROP4 displays 23 regions that are potential B-cell epitopes and analysis of phage display have showed that mAb 20D2 binds to the region 360-400 amino acids of this protein. Also made sure that mice produce high avidity antibodies against recombinant NcROP4 (rNcROP4) after 30 days of infection (chronic phase) and that there is no difference between the IgG subclass (IgG1 and IgG2) in this recognition process. Moreover, experimentally infected cattle recognize rNcROP4 after 44 days of infection, featuring the recognizing of chronic phase. It was also observed that NcROP4 is recognized only during reinfection in reverse ELISA, in which there is the block an epitope of the protein. Thus, it is concluded that NcROP4 can be used as chronic phase marker during N. caninum infection, being an additional strategy in the serodiagnosis of neosporosis.Mestre em Imunologia e Parasitologia AplicadasNeospora caninum é um parasito intracelular obrigatório, capaz de causar doença em uma variedade de hospedeiros. Desde sua primeira descrição, a infecção por N. caninum tem emergido como uma importante doença em cães e bovinos em todo o mundo. Testes sorológicos como imunofluorescência indireta (RIFI) e ELISA são classicamente utilizados no diagnóstico da neosporose, sendo o primeiro considerado padrão ouro no sorodiagnóstico. Entretanto, ainda não há testes comerciais que visam avaliar o estágio da infecção, uma vez que os principais sinais clínicos são relatados durante a fase aguda ou reativação do parasito. Diversos alvos proteicos do patógeno vem sendo testados a fim de verificar a fase de infecção do hospedeiro, apresentando resultados satisfatórios, todavia, novos alvos devem ser avaliados para desenvolver um diagnóstico efetivo para os estágios da infecção. Desse modo, o presente trabalho objetivou avaliar o potencial da proteína NcROP4 de N. caninum como marcador de fase da infecção pelo protozoário. Para tanto, avaliou-se a expressão e localização de NcROP4 durante o processo de infecção em células HeLa para investigar a possível exposição da proteína a anticorpos. Posteriormente, análises de bioinformática e phage display foram desenvolvidas visando predizer e determinar os epítopos de células B da proteína. Camundongos e bovinos foram infectados experimentalmente a fim de investigar através de ensaios imunoenzimáticos indireto e reverso a produção de anticorpos IgG anti-NcROP4 durante a infecção. Observou-se que a proteína NcROP4 é secretada no citoplasma de células HeLa durante o processo de invasão, o que permite seu extravasamento para o ambiente extracelular após lise das células. Análises de bioinformática demonstrou que NcROP4 exibe 23 regiões que são potenciais epítopos de célula B e as análises de phage display constataram que o mAb 20D2 se liga a região de 360 400 aminoácidos da proteína. Certificou-se também que camundongos produzem anticorpos de alta avidez contra NcROP4 recombinante (rNcROP4) após 30 dias de infecção (fase crônica) e que não há diferença entre as subclasses de IgG (IgG1 e IgG2a) neste processo de reconhecimento. Além disso, anticorpos de bovinos infectados experimentalmente reconheceram rNcROP4 após 44 dias de infecção, caracterizando um reconhecimento de fase crônica. Observou-se ainda que NcROP4 é reconhecida somente durante reinfecção em ELISA reverso, no qual há o bloqueio de um epítopo da proteína. Desse modo, conclui-se que NcROP4 pode ser utilizada como marcador de fase crônica na infecção por N. caninum, sendo uma estratégia adicional no sorodiagnóstico da neosporose

Stimulation of innate pathways in uterine cells and immunoprophylaxis assays in mice model to the bovine neosporosis control

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.CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorTese (Doutorado)Neospora caninum tem causado grande interesse na pesquisa devido a sua associação com repetidos abortamentos no rebanho de bovinos, gerando bilhões de dólares de prejuízo na pecuária anualmente. Classificado como um protozoário intracelular obrigatório, a transmissão vertical é comum em hospedeiros bovinos e as consequências geradas dependerá do perfil de resposta imune do hospedeiro. Além disso, N. caninum apresenta diversas moléculas secretórias importante no processo de invasão. Dentre elas, a NcROP4 é uma proteína de roptria que possui caráter imunogênico importante para o processo de invasão e evasão na célula hospedeira. Aqui nós objetivamos em dois manuscritos compreender os mecanismos inatos envolvidos na resposta contra N. caninum em células uterinas de bovinos, além de avaliar a capacidade imunomodulatória de NcROP4 recombinante (rNcROP4) no desenvolvimento de protocolos vacinais e a partir disso, testar protocolos imunoterapêuticos de doenças inflamatórias agudas. Os resultados expostos no capítulo II da tese demonstraram que N. caninum é capaz de induzir produção de IFNγ, IL-10, IL-6 e IL-8 em células epiteliais e estromais de endométrio bovino. Além disso, a produção de IFNγ foi dependente das moléculas TLR3 e MyD88, pois quando essas moléculas foram silenciadas houve a diminuição da produção desse mediador. Esse silenciamento gerou também um aumento na produção de IL-10, IL-6 e IL-8 após infecção. Em contrapartida, a inibição da MAPK p38 e ERK induziram uma redução de IFNγ, e aumento de IL-10, IL-6 e IL-8, exibindo efeito antagônico à TLR3 e MyD88. Adicionalmente, nós demonstramos que a produção de IFNγ é dependente de STAT1 enquanto que a produção de IL-10 é dependente de STAT3 nas células uterinas bovinas estudadas. Houve ainda um aumento na produção de IL-10 quando STAT1 foi silenciada e o mesmo se observou com IFNγ quando STAT3 foi silenciada, evidenciando um balanço entre essas duas citocinas durante a infecção pelo patógeno em células de endométrio bovinos. No capítulo III nós identificamos inicialmente que NcROP4 ligante ao mAb20D2. Essa região de ligação foi quimicamente sintetizada e denominada (PepNcROP4). A rNcROP4 foi produzida em Escherichia coli. Tanto o peptídeo quanto a proteína foram colocados em cultura de macrófagos e no peritônio murino e induziram uma alta na produção de IL-10 e pouca produção de IFNγ em comparação com o extrato do parasito (NLA). Nós usamos rNcROP4, PepNcROP4 e NLA em protocolos de imunização e verificamos que todos os grupos induziram produção de anticorpos específicos. Após o desafio com o parasito, rNcROP4 induziu aumento no peso corpóreo enquanto os demais antígenos utilizados. Adicionalmente, a imunização com esta proteína resultou na redução da carga parasitária e considerável aumento na sobrevida em comparação aos demais grupos. Por fim, o efeito regulatório de rNcROP4 foi testado no tratamento de ileíte induzida por Toxoplasma gondii em modelo murino. Os resultados evidenciaram que o tratamento com esta proteína reduziu o processo inflamatório no íleo, evidenciando que rNcROP4 apresenta efeito regulatório em outras patologias inflamatórias em camundongos. A partir do exposto acima podemos concluir que receptores e vias de sinalização inatas participam da resposta à N. caninum em células endometriais bovinas e a proteína rNcROP4 exibe potencial regulatório capaz de aumentar a capacidade protetiva na neosporose e reduzir a ileíte induzida por T. gondii em camundongos

Role of TLR2/MyD88 in the production of specific IgM and IgG antibodies during the immunization of mice against Neospora caninum

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

Lectins from Synadenium carinatum (ScLL) and Artocarpus heterophyllus (ArtinM) are able to induce beneficial immunomodulatory effects in a murine model for treatment of Toxoplasma gondii infection

Infection by Toxoplasma gondii affects around one-third of world population and the treatment for patients presenting toxoplasmosis clinically manifested disease is mainly based by a combination of sulfadiazine, pyrimethamine and folinic acid. However, this therapeutic protocol is significantly toxic, causing relevant dose-related bone marrow damage. Thus, it is necessary to improve new approaches to investigate the usefulness of more effective and non-toxic agents for treatment of patients with toxoplasmosis. It has been described that lectins from plants can control parasite infections, when used as immunological adjuvants in vaccination procedures. This type of lectins, such as ArtinM and ScLL is able to induce immunostimulatory activities, including efficient immune response against parasites. The present study aimed to evaluate the potential immunostimulatory effect of ScLL and ArtinM for treatment of T. gondii infection during acute phase, considering that there is no study in the literature accomplishing this issue. For this purpose, bone marrow-derived macrophages (BMDMs) were treated with different concentrations from each lectin to determine the maximum concentration without or with lowest cytotoxic effect. After, it was also measured the cytokine levels produced by these cells when stimulated by the selected concentrations of lectins. We found that ScLL showed high capacity to induce of pro-inflammatory cytokine production, while ArtinM was able to induce especially an anti-inflammatory cytokines production. Furthermore, both lectins were able to increase NO levels. Next, we evaluated the treatment effect of ScLL and ArtinM in C57BL/6 mice infected by ME49 strain from T. gondii. The animals were infected and treated with ScLL, ArtinM, ArtinM plus ScLL or sulfadiazine, and the following parameters analyzed: cytokines production, brain parasite burden and survival rates. Our results demonstrated that the ScLL or ScLL plus ArtinM treatment induced production of pro-inflammatory and anti-inflammatory cytokines, showing differential but complementary profiles. Moreover, when compared with non treated mice, the parasite burden was significantly lower and survival rates higher in mice treated with ScLL or ScLL plus ArtinM, similarly with sulfadiazine treatment. In conclusion, the results demonstrated the suitable potential immunotherapeutic effect of ScLL and ArtinM lectins to control acute toxoplasmosis in this experimental murine model