RESUMO
Catalase, an antioxidant enzyme widely produced in mammalian cells and bacteria, is crucial to mitigating oxidative stress in hostile environments. This function enhances the intracellular survivability of various intracellular growth pathogens, including Brucella (B.) abortus. In this study, to determine whether the suppression of catalase can inhibit the intracellular growth of B. abortus, we employed 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor, in both RAW 264.7 macrophage cells and an ICR mouse model during Brucella infection. The intracellular growth assay indicated that 3-AT exerts growth-inhibitory effects on B. abortus within macrophages. Moreover, it contributes to the accumulation of reactive oxygen species and the formation of nitric oxide. Notably, 3-AT diminishes the activation of the nucleus transcription factor (NF-κB) and modulates the cytokine secretion within infected cells. In our mouse model, the administration of 3-AT reduced the B. abortus proliferation within the spleens and livers of infected mice. This reduction was accompanied by a diminished immune response to infection, as indicated by the lowered levels of TNF-α, IL-6, and IL-10 and altered CD4+/CD8+ T-cell ratio. These results suggest the protective and immunomodulatory effects of 3-AT treatment against Brucella infection.
Assuntos
Brucella abortus , Brucelose , Animais , Camundongos , Amitrol (Herbicida)/farmacologia , Catalase , Camundongos Endogâmicos ICR , Brucelose/tratamento farmacológico , Brucelose/microbiologia , Imunidade , MamíferosRESUMO
Bacteria of the genus Brucella cause brucellosis, one of the world's most common zoonotic diseases. A major contributor to Brucella's virulence is the ability to circumvent host immune defense mechanisms. Here, we find that the DNA-binding protein Dps from Brucella is secreted within the macrophage cytosol, modulating host iron homeostasis and mediating intracellular growth of Brucella. In addition to dampening iron-dependent production of reactive oxygen species (ROS), a key immune effector required for immediate bacterial clearance, cytosolic Dps mediates ferritinophagy activation to elevate intracellular free-iron levels, thereby promoting Brucella growth and inducing host cell necrosis. Inactivation of the ferritinophagy pathway by Ncoa4 gene knockout significantly inhibits intracellular growth of Brucella and host cell death. Our study uncovers an unconventional role of bacterial Dps, identifying a crucial virulence mechanism used by Brucella to adapt to the harsh environment inside macrophages.
Assuntos
Brucella , Brucelose , Humanos , Brucelose/metabolismo , Brucelose/microbiologia , Macrófagos/metabolismo , Morte Celular , Ferro/metabolismoRESUMO
In this study, we investigated the effects of sodium propionate (SP) treatment on intracellular mechanism of murine macrophages and its contribution to host immunity during Brucella abortus 544 infection. The intracellular growth assay revealed that SP inhibited Brucella replication inside the macrophages. To determine intracellular signaling involved during SP treatment after Brucella infection, we analyzed the change of five different cytokines production relevant to SP such as TNF-α, IL-10, IFN-γ, IL-1ß, and IL-6, and the results indicated that the boost with IL-10 was apparent throughout the culture period for 48 h as well as IL-1ß which was apparent at 24 h post-infection and IFN-γ which was apparent at 24 h and 48 h in comparison to SP untreated groups. On the other way, SP-treated cells displayed suppressed production of TNF-α and IL-6 at all time points tested and 48 h post-infection, respectively. Furthermore, we conducted western blot to establish a cellular mechanism, and the result suggested that SP treatment attenuated p50 phosphorylation, part of the NF-κB pathway. These findings indicated that the inhibitory effect of SP against Brucella infection could be attributed through induction of cytokine production and interference on intracellular pathway, suggesting SP as a potential candidate for treating brucellosis.
Assuntos
Brucelose , Citocinas , Animais , Camundongos , Citocinas/metabolismo , Brucella abortus , Células RAW 264.7 , Interleucina-10 , Fator de Necrose Tumoral alfa , Interleucina-6/metabolismo , Brucelose/tratamento farmacológicoRESUMO
Brucellosis is a contagious zoonotic disease that infects millions of people annually with hundreds of millions more being exposed. It is caused by Brucella, a highly infectious bacterial species capable of infecting humans with an estimated dose of 10-100 organisms. Sirtuin 1 (SIRT1) has been reported to contribute to prevention of viral diseases as well as a chronic infection caused by Mycobacterium bovis. Here, we investigated the role of SIRT1 in the establishment of Brucella abortus infection in both in vitro and in vivo systems using the reported SIRT1 activators resveratrol (RES), piceatannol (PIC), and ginsenoside Rg3 (Rg3). In RAW264.7 cells, SIRT1 activators did not alter the adherence of Brucella or Salmonella Typhimurium. However, reduced uptake of Brucella was observed in cells treated with PIC and Rg3, and survival of Brucella within the cells was only observed to decrease in cells that were treated with Rg3, while PIC treatment reduced the intracellular survival of Salmonella. SIRT1 treatment in mice via oral route resulted in augmented Brucella resistance for PIC and Rg3, but not RES. PIC treatment favors Th2 immune response despite reduced serum proinflammatory cytokine production, while Rg3-treated mice displayed high IL-12 and IFN-γ serum production. Overall, our findings encourage further investigation into the complete mechanisms of action of the different SIRT1 activators used as well as their potential benefit as an effective alternative approach against intracellular and extracellular pathogens.
Assuntos
Brucella abortus , Brucelose , Humanos , Animais , Camundongos , Brucella abortus/fisiologia , Sirtuína 1/metabolismo , Resveratrol/farmacologia , Resveratrol/metabolismo , Brucelose/tratamento farmacológico , Brucelose/prevenção & controle , Macrófagos/metabolismo , Linhagem CelularRESUMO
This study investigated the contribution of lipoxygenase (LOX) inhibitors, nordihydroguaiaretic acid (NDGA), tetra-O-methyl nordihydroguaiaretic acid (M4N) and zileuton (ZIL), and thromboxane A2 (TXA2) inhibitor 4,5-diphenylimidazole (DPI) in the proliferation of Brucella abortus infection. None of the compounds affected the uptake of Brucella into the macrophages. We determined the effect of neutralizing leukotriene B4 (LTB4) receptor and showed that the uptake of the bacteria was inhibited at 30 min post-infection. M4N treatment attenuated intracellular survival of Brucella at 2 h post-incubation but it was not observed in the succeeding time points. DPI treatment showed reduced survival of Brucella at 24 h post-incubation while blocking LTB4 receptor was observed to have a lower intracellular growth at 48 h post-incubation suggesting different action of the inhibitors in the course of the survival of Brucella within the cells. Reduced proliferation of the bacteria in the spleens of mice was observed in animals treated with ZIL or DPI. Increased serum cytokine level of TNF-α and MCP-1 was observed in mice treated with M4N or ZIL while a lower IFN-γ level in ZIL-treated mice and a higher IL-12 serum level in DPI-treated mice were observed at 7 d post-infection. At 14 d post-infection, ZIL-treated mice displayed reduced serum level of IL-12 and IL-10. Overall, inhibition of 5-LOX or TXA2 or a combination therapy promises a potential alternative therapy against B. abortus infection. Furthermore, strong ligands for LTB4 receptor could also be a good candidate for the control of Brucella infection.
Assuntos
Brucella abortus , Brucelose , Animais , Brucelose/tratamento farmacológico , Brucelose/microbiologia , Citocinas/metabolismo , Interleucina-10 , Interleucina-12 , Leucotrieno B4/farmacologia , Inibidores de Lipoxigenase/farmacologia , Lipoxigenases , Masoprocol/análogos & derivados , Masoprocol/farmacologia , Camundongos , Receptores do Leucotrieno B4 , Tromboxano A2/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Simvastatin is an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase and has been found to have protective effects against several bacterial infections. In this study, we investigate the effects of simvastatin treatment on RAW 264.7 macrophage cells and ICR mice against Brucella (B.) abortus infections. The invasion assay revealed that simvastatin inhibited the Brucella invasion into macrophage cells by blocking the mevalonic pathway. The treatment of simvastatin enhanced the trafficking of Toll-like receptor 4 in membrane lipid raft microdomains, accompanied by the increased phosphorylation of its downstream signaling pathways, including JAK2 and MAPKs, upon =Brucella infection. Notably, the suppressive effect of simvastatin treatment on Brucella invasion was not dependent on the reduction of cholesterol synthesis but probably on the decline of farnesyl pyrophosphate and geranylgeranyl pyrophosphate synthesis. In addition to a direct brucellacidal ability, simvastatin administration showed increased cytokine TNF-α and differentiation of CD8+ T cells, accompanied by reduced bacterial survival in spleens of ICR mice. These data suggested the involvement of the mevalonate pathway in the phagocytosis of B. abortus into RAW 264.7 macrophage cells and the regulation of simvastatin on the host immune system against Brucella infections. Therefore, simvastatin is a potential candidate for studying alternative therapy against animal brucellosis.
Assuntos
Brucella abortus , Brucelose , Animais , Brucella abortus/metabolismo , Brucelose/tratamento farmacológico , Linfócitos T CD8-Positivos/metabolismo , Modelos Animais de Doenças , Ácido Mevalônico/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Células RAW 264.7 , Sinvastatina/farmacologia , Sinvastatina/uso terapêuticoRESUMO
Brucella abortus, one of the most important members of the genus Brucella responsible for human disease, is an intracellular pathogen capable of avoiding or interfering components of the host immune responses that are critical for its virulence. GPR84, on the other hand, is a seven-transmembrane GPCR involved in the inflammatory response and its induced expression was associated with B. abortus infection of RAW264.7 cells. Here we examined the effects of the reported GPR84 surrogate and endogenous agonists, namely 6-n-octylaminouracil (6-OAU) and lauric acid (LU), respectively in the progression of B. abortus infection in a cell and mouse models. The in vitro studies revealed the LU had bactericidal effect against Brucella starting at 24 h post-incubation. Adhesion of Brucella to RAW264.7 cells was attenuated in both 6-OAU and LU treatments. Brucella uptake was observed to be inhibited in a dose and time-dependent manner in 6-OAU but only at the highest non-cytotoxic concentration in LU-treated cells. However, survival of Brucella within the cells was reduced only in LU-treated cells. We also investigated the possible inhibitory effects of the agonist in other Gram-negative bacterium, Salmonella Typhimurium and we found that both adhesion and uptake were inhibited in 6-OAU treatment and only the intracellular survival for LU treatment. Furthermore, 6-OAU treatment reduced ERK phosphorylation and MCP-1 secretion during Brucella infection as well as reduced MALT1 protein expression and ROS production in cells without infection. LU treatment attenuated ERK and JNK phosphorylation, MCP-1 secretion and NO accumulation but increased ROS production during infection, and similar pattern with MALT1 protein expression. The in vivo studies showed that both treatments via oral route augmented resistance to Brucella infection but more pronounced with 6-AOU as observed with reduced bacterial proliferation in spleens and livers. At 7 d post-treatment and 14 d post-infection, 6-OAU-treated mice displayed reduced IFN-γ serum level. At 7 d post-infection, high serum level of MCP-1 was observed in both treatments with the addition of TNF-α in LU group. IL-6 was increased in both treatments at 14 d post-infection with higher TNF-α, MCP-1 and IL-10 in LU group. Taken together, 6-OAU and LU are potential candidates representing pharmaceutical strategy against brucellosis and possibly other intracellular pathogens or inflammatory diseases.
Assuntos
Brucelose , Ácidos Láuricos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Uracila/análogos & derivados , Animais , Brucella abortus , Bovinos , Humanos , Camundongos , Células RAW 264.7 , Uracila/farmacologiaRESUMO
BACKGROUND: We previously elucidated the protective mechanism of Korean red ginseng oil (RGO) against Brucella abortus infection, and our phytochemical analysis revealed that palmitic acid (PA) was an abundant component of RGO. Consequently, we investigated the contribution of PA against B. abortus. OBJECTIVES: We aimed to investigate the efficacy of PA against B. abortus infection using a murine cell line and a murine model. METHODS: Cell viability, bactericidal, internalization, and intracellular replication, western blot, nitric oxide (NO), and superoxide (O2â») analyses and flow cytometry were performed to determine the effects of PA on the progression of B. abortus infection in macrophages. Flow cytometry for cytokine analysis of serum samples and bacterial counts from the spleens were performed to determine the effect of PA in a mouse model. RESULTS: PA did not affect the growth of B. abortus. PA treatment in macrophages did not change B. abortus uptake but it did attenuate the intracellular survivability of B. abortus. Incubation of cells with PA resulted in a modest increase in sirtuin 1 (SIRT1) expression. Compared to control cells, reduced nitrite accumulation, augmented O2â», and enhanced pro-inflammatory cytokine production were observed in PA-treated B. abortus-infected cells. Mice orally treated with PA displayed a decreased serum interleukin-10 level and enhanced bacterial resistance. CONCLUSIONS: Our results suggest that PA participates in the control of B. abortus within murine macrophages, and the in vivo study results confirm its efficacy against the infection. However, further investigations are encouraged to completely characterize the mechanisms involved in the inhibition of B. abortus infection by fatty acids.
Assuntos
Antibacterianos/farmacologia , Brucella abortus/efeitos dos fármacos , Brucelose/prevenção & controle , Ácido Palmítico/farmacologia , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Substâncias Protetoras/farmacologia , Células RAW 264.7 , Organismos Livres de Patógenos EspecíficosRESUMO
Here, we explore the potential role of formyl peptide receptor 2 (FPR2) during Brucella abortus infection. FPR2 manipulation affected B. abortus internalization but not its growth within macrophages. During the activation of FPR2 induced by its agonist AGP-8694, a high level of Brucella uptake was accompanied by an increase in ERK phosphorylation, while intracellular survival at 24 h postincubation was observed to be associated with slightly reduced nitrite accumulation but augmented superoxide anion production. Attenuated secretion of IL-6 and IL-10 were observed 48 h postincubation in the bone marrow-derived macrophages (BMDMs) treated with the FPR2 antagonist WRW4. An opposite pattern of bacterial uptake was observed upon treatment with the FPR2 antagonist, but no significant changes in the activation of MAPKs or the production of nitrite or superoxide anion were observed. Interestingly, AGP-8694 treatment of mice did not lead to differences in spleen or liver weight but slightly enhanced bacterial proliferation was observed in the spleen. Although the weights of the spleen or liver did not differ, WRW4 treatment led to reduced bacterial proliferation in the spleen. Furthermore, FPR2 antagonist treatment was associated with high serum levels of the proinflammatory cytokines IL-12, TNF-α, IFN-γ and MCP-1, while the production of TNF-α was inhibited in AGP-8694-treated mice. IL-6 and IL-10 levels were slightly increased in AGP-8694-treated mice at 24 h postinfection. Our findings demonstrated the contribution of FPR2 via manipulating this receptor using its reported agonist AGP-8694 and antagonist WRW4 in both in vitro and in vivo systems. Although activation of the receptor did not consistently induced Brucella infection, FPR2 inhibition may be a promising strategy to treat brucellosis in animals which encourages further investigation.
Assuntos
Antibacterianos/farmacologia , Brucella abortus/efeitos dos fármacos , Brucelose/microbiologia , Brucelose/prevenção & controle , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Terapia de Alvo Molecular , Receptores de Formil Peptídeo/antagonistas & inibidores , Animais , Biomarcadores , Brucelose/metabolismo , Citocinas/biossíntese , Gerenciamento Clínico , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismoRESUMO
To better understanding Brucella abortus infection, serum metabolites of B. abortus-infected and -uninfected mice were analyzed and twenty-one metabolites were tentatively identified at 3 and 14 days post-infection (d.p.i.). Level of most lysophosphatidylcholines (LPCs) was found to increase in infected mice at 3 d.p.i., while it was decreased at 14 d.p.i. as compared to uninfected mice. In contrast, acylcarnitines were initially reduced at 3 d.p.i then elevated after two-weeks of infection, while hydroxysanthine was increased at 14 d.p.i. in infected mice. Our findings suggest that the significant changes in LPCs and other identified metabolites may serve as potential biomarkers in acute phase of B. abortus infection.
Assuntos
Brucelose Bovina , Brucelose , Doenças dos Bovinos , Doenças dos Roedores , Animais , Brucella abortus , Brucelose/veterinária , Bovinos , Metabolômica , CamundongosRESUMO
In this study, we investigated the effects of linoleic acid (LA) treatment on Brucella abortus infection in professional phagocyte RAW264.7 cells, particularly during the pathogens invasion and intracellular growth in these cells, as well as in murine model BALB/c mice focusing on bacterial splenic proliferation and immunoregulatory activities. LA inhibited the growth of Brucella in a doseand time-dependent manner. The ability of the pathogen to enter the phagocytes was inhibited as was its survival within these cells. This was accompanied by increased nitrite accumulation in these cells at 24 h post-infection. The concentration of LA used in the present study did not affect the total body weight or liver function of the mice. During Brucella infection, the total splenic weight of these animals was not changed; rather, resistance to bacterial proliferation was enhanced in the spleen. Furthermore, mice treated with LA displayed elevated levels of IL-12 and IFN-γ but reduced levels of IL-10 during infection. The findings in this study showed the regulatory role of LA against B. abortus infection suggesting its potential use in designing intervention strategy for brucellosis.
Assuntos
Antibacterianos/farmacologia , Brucella abortus/efeitos dos fármacos , Brucelose/microbiologia , Ácido Linoleico/farmacologia , Animais , Antibacterianos/química , Citocinas/metabolismo , Modelos Animais de Doenças , Ácido Linoleico/química , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Baço/efeitos dos fármacosRESUMO
Brucella as a stealthy intracellular pathogen avoids activation of innate immune response. Here we investigated the contribution of an adenosine receptor, Adora2b, during Brucella infection in professional phagocyte RAW 264.7 cells and in a murine model. Adora2b-deficient cells showed attenuated Brucella internalization and intracellular survival with enhanced release of IL-6, TNF-α, IL-12 and MCP-1. In addition, blockade of Adora2b using MRS 1754 treatment in mice resulted in increased total weight of the spleens but suppressed bacterial burden in these organs accompanied by elevated levels of IL-6, IFN-γ, TNF-α, IL-12 and MCP-1, while reduced IL-10. Overall, we proposed that the Adora2b participates in the successful phagocytic pathway and intracellular survival of Brucella in RAW 264.7 cells, and could be a potential therapeutic target for the treatment of acute brucellosis in animals.
Assuntos
Antagonistas do Receptor A2 de Adenosina/farmacologia , Brucelose/tratamento farmacológico , Imunidade Inata , Macrófagos/microbiologia , Receptor A2B de Adenosina/imunologia , Acetamidas/farmacologia , Agonistas do Receptor A2 de Adenosina/farmacologia , Aminopiridinas/farmacologia , Animais , Brucella abortus/efeitos dos fármacos , Brucella abortus/fisiologia , Brucelose/microbiologia , Citocinas/imunologia , Feminino , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Fagocitose , Purinas/farmacologia , Células RAW 264.7 , Receptor A2B de Adenosina/genética , Transdução de SinaisRESUMO
In this study, two recombinant proteins encoded by Brucella abortus genes Adk and SecB were evaluated as single subunit vaccine (SSV) as well as combined subunit vaccine (CSV) against B. abortus infection in BALB/c mice. These genes were cloned into pcold-TF expression system and recombinant proteins were expressed in Escherichia coli DH5α. The immunoreactivity of purified rAdk and rSecB was analyzed by immunoblotting showing that purified rAdk and rSecB as well as pcold-TF vector strongly reacted with Brucella-positive serum. Mice were immunized intraperitoneally with SSVs, CSV, pcold-TF, RB51 and PBS. The analysis of cytokine revealed that SSVs and CSV can strongly induce production of proinflammatory cytokines TNF and IL-6, suggesting that these subunit vaccines elicited innate immune response, particularly, activated antimicrobial mechanism of macrophages to limit the initial infection. On the other hand, immunization with SSVs and CSV elicited strong IFN-γ production and decreased IL-10 production compared to PBS group. The secretion profiles of IFN-γ and IL-10 together with an enhancement of blood CD4+ population and significantly induced specific IgG1 and IgG2a antibodies indicated that SSVs and CSV induced not only humoral immunity but also T helper 1 T cell immunity. Finally, spleen proliferation and bacterial burden in the spleen of mice vaccinated with these subunit vaccines were significantly lower than those of PBS group, which conferred significant protection against B. abortus infection. Altogether, the potential of these antigens of B. abortus could be prospective candidates to develop subunit vaccines against brucellosis.
Assuntos
Vacinas Bacterianas/uso terapêutico , Brucella abortus/imunologia , Brucelose/prevenção & controle , Animais , Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Western Blotting , Brucelose/imunologia , Citocinas/sangue , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Proteínas Recombinantes , Vacinas SintéticasRESUMO
We previously identified ß-sitosterol (BS) as one of the most abundant compounds found in Korean red ginseng oil. BS is a widely prevalent vegetable-derived phytosterol with many known health benefits. Here, we investigated the efficacy of BS against Brucella (B.) abortus infection. BS showed no effect on bacterial growth but attenuated internalization, intracellular survival and MAPKs-linked intracellular signaling in RAW264.7 cells. BS treatment in cells is also associated with increased nitrite concentration during infection at 24 h. Slightly enhanced resistance to B. abortus infection was observed in mice orally given BS, which could be mediated by induced production of proinflammatory cytokines. Taken together, our study demonstrates the contribution of BS treatment against B. abortus infection although further investigation is encouraged to maximize its beneficial effects against intracellular infection.
Assuntos
Brucella abortus/efeitos dos fármacos , Brucelose/prevenção & controle , Citocinas/sangue , Sitosteroides/administração & dosagem , Animais , Brucella abortus/fisiologia , Brucelose/imunologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/efeitos dos fármacos , Nitratos/metabolismo , Células RAW 264.7 , Sitosteroides/farmacologiaRESUMO
Protective efficiency of a combination of four recombinant Brucella abortus (B. abortus) proteins, namely outer membrane protein (Omp) 16, Omp19, Omp28, and 50S ribosomal protein L7/L12 was evaluated as a combined subunit vaccine (CSV) against B. abortus infection in RAW 264.7 cell line and murine model. The immunoreactivity of these four recombinant proteins as well as pCold-TF vector reacted with Brucella-positive serum individually, but not with Brucella-negative serum by immunoblotting assay. CSV-treated RAW 264.7 cells significantly induced production of IFN-γ and IL-12 while decreased IL-10 production at the late stage of infection compared to PBS-treated control cells. In addition, the enhancement of nitric oxide production together with cytokines secretion profile in CSV-treated cells proved that CSV notably activated bactericidal mechanisms in macrophages. Consistently, mice immunized with CSV strongly elicited production of pro-inflammatory cytokines TNF-α, IL-6 and MCP-1 compared to PBS control group. Moreover, the concentration of IFN-γ was >IL-10 and titers of IgG2a were also heightened compared to IgG1 in CSV-immunized mice which suggest that CSV induced predominantly T helper 1 T cell. These results suggest that the CSV used in the present study is a potential candidate as a preventive therapy against brucellosis.
RESUMO
We investigated the involvement of chemokine receptor type 4 (CXCR4) signaling on the outcome of Brucella (B.) abortus 544 infection in murine macrophages and in a mouse model. CXCR4 manipulation were first evaluated for Brucella invasion and intracellular survival efficiency, mitogen-activated protein kinases (ERK1/2, JNK, p38α) activation and generation of nitric oxide (NO), and then in the splenic bacterial proliferation and cytokine production in BALB/c mice. CXCR4 blockade is involved in the successful control of Brucella invasion, reduction of ERK1/2 phosphorylation and inhibition of nitric oxide release from macrophages. Furthermore, using a reported CXCR4-specific antagonist AMD3100 resulted in splenomegaly but attenuated Brucella proliferation in these organs with elevated serum levels of MCP-1, TNF and IL-12. These findings provide insights on the contribution of CXCR4 signaling in the phagocytic pathway and immune modulation during B. abortus infection.
Assuntos
Brucella abortus , Brucelose/imunologia , Compostos Heterocíclicos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Animais , Benzilaminas , Sobrevivência Celular , Ciclamos , Suscetibilidade a Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Modelos Biológicos , Células RAW 264.7 , Organismos Livres de Patógenos EspecíficosRESUMO
To date, the implications of interleukin 6 (IL-6) for immune responses in the context of Brucella infection are still unknown. In the present study, we found that Brucella abortus infection induced marked production of IL-6 in mice that was important for sufficient differentiation of CD8+ T cells, a key factor in Brucella clearance. Blocking IL-6 signaling also significantly induced serum IL-4 and IL-10, together with a decreased gamma interferon (IFN-γ) level, suggesting that IL-6 is essential for priming the T-helper (Th) 1 cell immune response during Brucella infection. The IL-6 pathway also activated the bactericidal activity of primary and cultured macrophages. Bacterial killing was markedly abrogated when IL-6 signaling was suppressed, and this phenomenon was mainly associated with decreased activity of lysosome-mediated killing. Interestingly, suppressor of cytokine signaling 3 (SOCS3) was important for regulating the IL-6-dependent anti-Brucella activity through the JAK/STAT pathway. During early infection, in the absence of SOCS3, IL-6 exhibited anti-inflammatory effects and lysosome-mediated killing inhibition; however, the increase in SOCS3 successfully shifted functional IL-6 toward proinflammatory brucellacidal activity in the late stage. Our data clearly indicate that IL-6 contributes to host resistance against B. abortus infection by controlling brucellacidal activity in macrophages and priming cellular immune responses.
Assuntos
Brucella abortus/fisiologia , Citocinas/metabolismo , Interleucina-6/metabolismo , Macrófagos/microbiologia , Animais , Anticorpos , Células Apresentadoras de Antígenos , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Citocinas/genética , Interleucina-6/genética , Camundongos , Células RAW 264.7 , Interferência de RNA , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Células Th1/metabolismoRESUMO
The interleukin-1 (IL-1) family of cytokines, particularly IL-1α and IL-1ß, are potent regulators of innate immunity that play key roles in host defense against infection, hence we evaluated the role of these cytokines in the control of brucellosis within RAW 264.7 cells. Marked expression and secretion of IL-1α and IL-1ß were observed during Brucella infection in macrophages. Blocking of IL-1α and IL-1ß reduced induction of IL-10, IL-1ß and TNF, and IL-6 and TNF, respectively. However, interference of IL-1α and not IL-1ß signaling notably augmented susceptibility of macrophages to Brucella infection which indicates that IL-1α is required for a downstream signaling cascade of innate immunity for efficient clearance of Brucella. This protection requires binding to interleukin-1 receptor (IL-1R) mediated by myeloid differentiation factor 88 (MyD88) signaling and associated with increased lysosomal-mediated killing and nitric oxide (NO) production. Expression of pro-inflammatory cytokines was observed to be mediated via NF-κB-p50, HIF-1α and CEBPA, but negatively controlled by CEBPB while transcription of some important phagolysosomal genes was regulated via CEBPA and c-Jun which indicates the important role of these transcription factors in the control of Brucella infection in macrophages via IL-1α signaling pathway.
Assuntos
Brucella abortus/patogenicidade , Interleucina-1alfa/imunologia , Macrófagos/imunologia , Óxido Nítrico/imunologia , Animais , Imunidade Inata , Interleucina-1alfa/genética , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Células RAW 264.7 , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/imunologiaRESUMO
Chronic infection with intracellular Brucella abortus (B. abortus) in livestock remains as a major problem worldwide. Thus, the search for an ideal vaccine is still ongoing. In this study, we evaluated the protective efficacy of a combination of B. abortus recombinant proteins; superoxide dismutase (rSodC), riboflavin synthase subunit beta (rRibH), nucleoside diphosphate kinase (rNdk), 50S ribosomal protein (rL7/L12) and malate dehydrogenase (rMDH), cloned and expressed into a pMal vector system and DH5α, respectively, and further purified and applied intraperitoneally into BALB/c mice. After first immunization and two boosters, mice were infected intraperitoneally (IP) with 5 × 104 CFU of virulent B. abortus 544. Spleens were harvested and bacterial loads were evaluated at two weeks post-infection. Results revealed that this combination showed significant reduction in bacterial colonization in the spleen with a log protection unit of 1.31, which is comparable to the average protection conferred by the widely used live attenuated vaccine RB51. Cytokine analysis exhibited enhancement of cell-mediated immune response as IFN-γ is significantly elevated while IL-10, which is considered beneficial to the pathogen's survival, was reduced compared to control group. Furthermore, both titers of IgG1 and IgG2a were significantly elevated at three and four-week time points from first immunization. In summary, our in vivo data revealed that vaccination with a combination of five different proteins conferred a heightened host response to Brucella infection through cell-mediated immunity which is desirable in the control of intracellular pathogens. Thus, this combination might be considered for further improvement as a potential candidate vaccine against Brucella infection.
Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Vacina contra Brucelose/imunologia , Brucella abortus/imunologia , Brucelose/prevenção & controle , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/genética , Carga Bacteriana , Proteínas de Bactérias/genética , Feminino , Imunidade Celular , Imunização , Camundongos Endogâmicos BALB C , Proteínas Recombinantes/imunologia , Baço/microbiologiaRESUMO
The aim of this work is to investigate the protective efficacy of emodin, an active, naturally-occurring anthraquinone derivative of several traditional Chinese herbs, against Brucella abortus infection in macrophages. Brucella were incubated with different concentrations of emodin and showed that bacterial survival rates were markedly reduced in a dose-dependent manner at increasing incubation time points. Through bacterial infection assay, the highest non-cytotoxic concentration of emodin demonstrated attenuated invasion of Brucella into macrophages, however it did not inhibit the growth of these pathogens within the host cells. On the other hand, emodin effectively decreased the number of bacteria that adhered to host cells, which indicated its potential as an anti-adhesin agent. Furthermore, using immunoblotting and FACS assay for detecting MAPK signaling proteins and F-actin polymerization, respectively, the results showed that the emodin-incubated cells displayed modest reduction in the phosphorylation levels of ERK1/2 and inhibition of F-actin polymerization as compared to control cells. These findings indicate the potential use of emodin as a naturally-occurring alternative method for the prevention of animal brucellosis although this requires confirmation of safe clinical doses.