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
BACKGROUND: Coccidiosis is a poultry disease that occurs worldwide and is caused by Eimeria species. The infection is associated with reduced feed efficiency, body weight gain, and egg production. This study aimed to investigate the current status of coccidiosis and anticoccidial resistance to anticoccidial drugs used as part of control strategies for this disease in Korean chicken farms. RESULTS: An overall prevalence of 75% (291/388) was found. Positive farms contained several Eimeria species (mean = 4.2). Of the positive samples, E. acervulina (98.6%), E. maxima (84.8%), and E. tenella (82.8%) were the most prevalent species. Compared with cage-fed chickens, broilers and native chickens reared in free-range management were more at risk of acquiring an Eimeria infection. Sensitivities to six anticoccidial drugs (clopidol, diclazuril, maduramycin, monensin, salinomycin, and toltrazuril) were tested using nine field samples. Compared with untreated healthy control chickens, the body weight gains of infected chickens and treated/infected chickens were significantly reduced in all groups. Fecal oocyst shedding was significantly reduced in four clopidol-treated/infected groups, three diclazuril-treated/infected groups, two toltrazuril-treated/infected groups, one monensin-treated/infected group, and one salinomycin-treated/infected group, compared with the respective untreated/infected control groups. Intestinal lesion scores were also reduced in three clopidol-treated/infected groups, one monensin-treated/infected group, and one toltrazuril-treated/infected group. However, an overall assessment using the anticoccidial index, percent optimum anticoccidial activity, relative oocyst production, and reduced lesion score index found that all field samples had strong resistance to all tested anticoccidial drugs. CONCLUSION: The results of this large-scale epidemiological investigation and anticoccidial sensitivity testing showed a high prevalence of coccidiosis and the presence of severe drug resistant Eimeria species in the field. These findings will be useful for optimizing the control of coccidiosis in the poultry industry.
Assuntos
Coccidiose , Coccidiostáticos , Eimeria , Doenças das Aves Domésticas , Animais , Galinhas , Clopidol , Coccidiose/tratamento farmacológico , Coccidiose/epidemiologia , Coccidiose/veterinária , Coccidiostáticos/farmacologia , Coccidiostáticos/uso terapêutico , Resistência a Medicamentos , Fazendas , Monensin , Oocistos , Doenças das Aves Domésticas/tratamento farmacológico , Doenças das Aves Domésticas/epidemiologia , República da Coreia/epidemiologia , Aumento de PesoRESUMO
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
To date, the antimicrobial activity of arachidonic acid (AA) with regard to pathogenesis of Brucella in macrophages is unknown. We found that AA is highly toxic to B. abortus in a time- and dose-dependent manner. Transcription profiling of different groups of phospholipases A2 (PLA2) was examined, ten PLA2 were detected including cPLA2-IV-A, cPLA2-IV-B, iPLA2-VI, sPLA2-I-B, sPLA2-II-C, sPLA2-II-D, sPLA2-II-E, sPLA2-V, sPLA2-X, sPLA2-XII-A. Phagocytic signaling investigation indicated that AA treatment attenuated p38α activity in infected culture macrophages possibly leading to inhibition of Brucella internalization. Post-treatment with the fatty acid did not influence bacterial intracellular multiplication or alter production of antimicrobial effectors like ROS and NO in RAW 264.7 cells. On the other hand, AA administration significantly reduced bacterial load and modestly inhibited pro-inflammatory cytokine secretion including TNF, IFN-γ and IL-6 in mice plasma. To our knowledge, we are the first to suggest that B. abortus invasion to RAW 264.7 macrophages is impaired by AA.
Assuntos
Brucella abortus , Transcriptoma , Animais , Ácido Araquidônico , Brucella abortus/genética , Camundongos , Fosfolipases A2/genética , Transdução de SinaisRESUMO
Avian coccidiosis has a major economic impact on the poultry industry, it is caused by 7 species of Eimeria, and has been primarily controlled using chemotherapeutic agents. Due to the emergence of drug-resistant strains, alternative control strategies are needed. We assessed anticoccidial effects of berberine-based diets in broiler chickens following oral infection with 5 Eimeria species (E. acervulina, E. maxima, E. tenella, E. mitis, and E. praecox). When 0.2% berberine, a concentration that does not affect weight gain, was added to the diet, the 4 groups infected with E. acervulina, E. tenella, E. mitis, or E. praecox showed significant reductions in fecal oocyst shedding (P<0.05) compared to their respective infected and untreated controls. In chickens treated 0.5% berberine instead of 0.2% and infected with E. maxima, fecal oocyst production was significantly reduced, but body weight deceased, indicating that berberine treatment was not useful for E. maxima infection. Taken together, these results illustrate the applicability of berberine for prophylactic use to control most Eimeria infections except E. maxima. Further studies on the mechanisms underlying the differences in anticoccidial susceptibility to berberine, particularly E. maxima, are remained.
Assuntos
Berberina , Coccidiose , Eimeria , Doenças das Aves Domésticas , Animais , Galinhas , Coccidiose/tratamento farmacológico , Coccidiose/veterinária , Doenças das Aves Domésticas/tratamento farmacológicoRESUMO
Coccidiosis in chickens is an intestinal parasitic disease caused by protozoan parasites named Eimeria spp. In some Eimeria infections, intestinal lymphocytes are known to highly express chicken NK-lysin (cNK-lysin), an antimicrobial peptide with anticoccidial activity. Therefore, this study aims to investigate the expression of cNK-lysin in E. necatrix-infected chickens and its role in E. necatrix infection. The expression of cNK-lysin transcript was significantly increased in E. necatrix sporozoites-treated lymphocytes. In E. necatrix infection, cNK-lysin transcript was induced in intestinal lymphocytes but not in the spleen. The recombinant cNK-lysin exhibited anticoccidial activity against E. necatrix sporozoites as well as immunomodulatory activity on macrophages by inducing proinflammatory cytokines. These results indicated that E. necatrix infection induces high local expression of cNK-lysin and the secreted cNK-lysin helps protect coccidiosis.
Assuntos
Coccidiose , Eimeria , Doenças das Aves Domésticas , Animais , Galinhas , Coccidiose/veterinária , ProteolipídeosRESUMO
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
Brucella abortus is a Gram-negative zoonotic pathogen for which there is no 100% effective vaccine. Phagosomes in B. abortus-infected cells fail to mature, allowing the pathogen to survive and proliferate. Interleukin 10 (IL10) promotes B. abortus persistence in macrophages by mechanisms that are not fully understood. In this study, we investigated the regulatory role of IL10 in the immune response to B. abortus infection. B. abortus-infected macrophages were treated with either IL10 siRNA or recombinant IL10 (rIL10), and the expression of phagolysosome- or inflammation-related genes was evaluated by qRT-PCR and Western blotting. Phagolysosome fusion was monitored by fluorescence microscopy. We found that the synthesis of several membrane-trafficking regulators and lysosomal enzymes was suppressed by IL10 during infection, resulting in a significant increase in the recruitment of hydrolytic enzymes by Brucella-containing phagosomes (BCPs) when IL10 signaling was blocked. Moreover, blocking IL10 signaling also enhanced proinflammatory cytokine production. Finally, concomitant treatment with STAT3 siRNA significantly reduced the suppression of proinflammatory brucellacidal activity but not phagolysosome fusion by rIL10. Thus, our data provide the first evidence that clearly indicates the suppressive role of IL10 on phagolysosome fusion and inflammation in response to B. abortus infection through two distinct mechanisms, STAT3-independent and -dependent pathways, respectively, in murine macrophages.
Assuntos
Brucella abortus/fisiologia , Interleucina-10/metabolismo , Lisossomos/metabolismo , Macrófagos/citologia , Macrófagos/microbiologia , Animais , Camundongos , Fagossomos/metabolismo , Células RAW 264.7 , Fator de Transcrição STAT3/metabolismo , Regulação para CimaRESUMO
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
Lipocalin 2 (Lcn2) is an important innate immunity component against bacterial pathogens. In this study, we report that Lcn2 is induced by Brucella (B.) abortus infection and significantly contributes to the restriction of intracellular survival of Brucella in macrophages. We found that Lcn2 prevented iron uptake by B. abortus through two distinct mechanisms. First, Lcn2 is secreted to capture bacterial siderophore(s) and abrogate iron import by Brucella. Second, Lcn2 decreases the intracellular iron levels during Brucella infection, which probably deprives the invading Brucella of the iron source needed for growth. Suppression of Lcn2 signalling resulted in a marked induction of anti-inflammatory cytokine, interleukin 10, which was shown to play a major role in Lcn2-induced antibrucella immunity. Similarly, interleukin 6 was also found to be increased when Lcn2 signalling is abrogated; however, this induction was thought to be an alternative pathway that rescues the cell from infection when the effective Lnc2 pathway is repressed. Furthermore, Lcn2 deficiency also caused a marked decrease in brucellacidal effectors, such as reactive oxygen species and nitric oxide but not the phagolysosome fusion. Taken together, our results indicate that Lcn2 is required for the efficient restriction of intracellular B. abortus growth that is through limiting iron acquisition and shifting cells to pro-inflammatory brucellacidal activity in murine macrophages.
Assuntos
Brucella abortus/metabolismo , Ferro/metabolismo , Lipocalina-2/metabolismo , Animais , Brucella abortus/imunologia , Brucella abortus/patogenicidade , Proteínas de Transporte de Cátions/metabolismo , Imunidade Inata/fisiologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Células RAW 264.7RESUMO
In this study, we investigated the effects of gallic acid (GA) in intracellular signaling within murine macrophages and its contribution to host immunity during Brucella infection. In vitro analysis revealed that GA treatment decreased F-actin content and suppressed p38α phosphorylation level. In vivo analysis showed that GA treatment reduced inflammation and proliferation of Brucella in spleens of mice in comparison to PBS treatment yielding a significant protection unit. For the analysis of immune response, the uninfected GA-treated mice showed increased production of IFN-γ and MCP-1, and the Brucella-infected GA-treated mice showed elevated levels of IL-12p70, TNF, IFN-γ, MCP-1, IL-10 and IL-6 in comparison to negative and positive control groups, respectively. These findings demonstrate the therapeutic effects of GA against Brucella infection through interference on intracellular signaling pathway, induction of cytokine production and protection from bacterial proliferation in spleens of mice.
Assuntos
Brucelose/imunologia , Ácido Gálico/farmacologia , Interleucina-12/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Transdução de Sinais/efeitos dos fármacos , Actinas/metabolismo , Animais , Brucella abortus/imunologia , Brucelose/microbiologia , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2 , Citocinas/metabolismo , Feminino , Inflamação , Interferon gama , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Camundongos , Fosforilação , Células RAW 264.7/efeitos dos fármacos , Baço/microbiologiaRESUMO
A novel avian paramyxovirus (APMV), Cheonsu1510, was isolated from wild bird feces in South Korea and serologically and genetically characterized. In hemagglutination inhibition tests, antiserum against Cheonsu1510 showed low reactivity with other APMVs and vice versa. The complete genome of Cheonsu1510 comprised 15,408 nucleotides, contained six open reading frames (3'-N-P-M-F-HN-L-5'), and showed low sequence identity to other APMVs (< 63%) and a unique genomic composition. Phylogenetic analysis revealed that Cheonsu1510 was related to but distinct from APMV-1, -9, and -15. These results suggest that Cheonsu1510 represents a new APMV serotype, APMV-17.
Assuntos
Animais Selvagens , Infecções por Avulavirus/virologia , Avulavirus/genética , Doenças das Aves/virologia , Genoma Viral , Animais , Infecções por Avulavirus/epidemiologia , Sequência de Bases , Doenças das Aves/epidemiologia , Fezes/virologia , Filogenia , República da CoreiaRESUMO
In this study, we investigated the protective effects of tannin-derived components, gallic acid (GA) and tannic acid (TA), in vitro and in vivo against Salmonella infection in mice. Both GA and TA showed antibacterial effects against Salmonella (S.) Typhimurium as well as inhibitory effects on the adherence, invasion, and intracellular growth of the pathogens in macrophages. Following a lethal dose of Salmonella infection in mice, reduced virulence in both GA- and TA-treated groups was observed based on reduced mortality rates. In the non-infected groups, the average weights of the spleens and livers of GA- or TA-treated mice were not significantly different with the control group. In addition, the average weights of these organs in all of the Salmonella-infected groups were not significantly different but the numbers of bacteria in the spleens and livers in both GA- and TA-treated mice were significantly reduced. The levels of cytokine production in non-infected mice revealed that GA-treated and TA-treated mice elicited an increased level of IFN-γ, and both IFN-γ and MCP-1, respectively, as compared with the PBS-treated group. These findings highlight the potential of GA and TA as alternatives for the treatment of salmonellosis and as supplements to conventional antimicrobial food additives.
Assuntos
Antibacterianos/farmacologia , Ácido Gálico/farmacologia , Infecções por Salmonella/tratamento farmacológico , Salmonella typhimurium/efeitos dos fármacos , Taninos/farmacologia , Adesinas Bacterianas/efeitos dos fármacos , Animais , Carga Bacteriana , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2 , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Interferon gama/metabolismo , Fígado/microbiologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Mortalidade , Fagocitose/efeitos dos fármacos , Células RAW 264.7 , Infecções por Salmonella/imunologia , Infecções por Salmonella/mortalidade , Salmonella typhimurium/crescimento & desenvolvimento , Baço/microbiologia , Virulência/efeitos dos fármacosRESUMO
Brucella is a zoonotic pathogen that survives within macrophages; however the replicative mechanisms involved are not fully understood. We describe the isolation of sufficient Brucella abortus RNA from primary host cell environment using modified reported methods for RNA-seq analysis, and simultaneously characterize the transcriptional profiles of intracellular B. abortus and bone marrow-derived macrophages (BMM) from BALB/c mice at 24 h (replicative phase) post-infection. Our results revealed that 25.12% (801/3190) and 16.16% (515/3190) of the total B. abortus genes were up-regulated and down-regulated at >2-fold, respectively as compared to the free-living B. abortus. Among >5-fold differentially expressed genes, the up-regulated genes are mostly involved in DNA, RNA manipulations as well as protein biosynthesis and secretion while the down-regulated genes are mainly involved in energy production and metabolism. On the other hand, the host responses during B. abortus infection revealed that 14.01% (6071/43,346) of BMM genes were reproducibly transcribed at >5-fold during infection. Transcription of cytokines, chemokines and transcriptional factors, such as tumor necrosis factor (Tnf), interleukin-1α (Il1α), interleukin-1ß (Il1ß), interleukin-6 (Il6), interleukin-12 (Il12), chemokine C-X-C motif (CXCL) family, nuclear factor kappa B (Nf-κb), signal transducer and activator of transcription 1 (Stat1), that may contribute to host defense were markedly induced while transcription of various genes involved in cell proliferation and metabolism were suppressed upon B. abortus infection. In conclusion, these data suggest that Brucella modulates gene expression in hostile intracellular environment while simultaneously alters the host pathways that may lead to the pathogen's intracellular survival and infection.
Assuntos
Brucella abortus/patogenicidade , Regulação da Expressão Gênica/genética , Interações Hospedeiro-Patógeno/genética , Macrófagos/metabolismo , Macrófagos/microbiologia , Animais , Sequência de Bases , Brucelose/patologia , Células Cultivadas , Quimiocinas/biossíntese , Feminino , Perfilação da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , RNA/genética , Análise de Sequência de RNA , Fatores de Transcrição/biossínteseRESUMO
Brucellosis is one of the most important and widespread zoonosis worldwide responsible for serious economic losses and considerable public health burden. In this study, we investigated the modulatory effect of a microtubule-inhibitor, nocodazole, on B. abortus infection in murine macrophages and in a mouse model. Nocodazole activated macrophages and directly inhibited the growth of Brucella in a dose-dependent manner. Nocodazole increased adhesion but reduced invasion and intracellular growth of Brucella in macrophages although it did not affect co-localization of Brucella with LAMP-1. In addition, nocodazole negatively affected actin polymerization, and weakly activated ERK and p38α but significantly activated JNK in non-infected cells. After subsequent infection, nocodazole weakly inhibited activation of ERK and p38α. For the in vivo tests, nocodazole -treated mice displayed elevated levels of IFN-γ, MCP-1 and IL-10 while Brucella-infected nocodazole -treated mice showed high levels of TNF, IFN-γ, MCP-1, IL-10 and IL-6 as compared to controls. Furthermore, nocodazole treatment reduced inflammation and Brucella proliferation in the spleens of mice. These findings highlight the potential use of nocodazole for the control of brucellosis although further investigations are encouraged to validate its therapeutic use in animal hosts.
Assuntos
Antibacterianos/farmacologia , Brucella abortus/efeitos dos fármacos , Brucelose/microbiologia , Nocodazol/farmacologia , Baço/microbiologia , Actinas/metabolismo , Animais , Aderência Bacteriana/efeitos dos fármacos , Carga Bacteriana , Brucella abortus/patogenicidade , Brucelose/tratamento farmacológico , Brucelose/imunologia , Brucelose/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Células RAW 264.7 , Baço/imunologia , Baço/metabolismo , Baço/patologiaRESUMO
BACKGROUND: Brucella abortus is an intracellular pathogen which can infect and persist in host cells through multiple interactions. Above all, its interaction to host cell receptor is important to understand the pathogenic mechanisms of B. abortus. Accordingly, we demonstrated that platelet-activating factor receptor (PAFR) affects host cell response against B. abortus infection. RESULTS: First of all, B. abortus infection to macrophage induces secretion of platelet-activating factor (PAF), which is a PAFR agonist. The stimulation of PAFR by PAF remarkably increases B. abortus uptake into macrophages. It induces Janus kinase 2 (JAK2) and p38α phosphorylation, indicating that PAFR-mediated activation of JAK2 signaling leads to enhanced uptake of B. abortus. Moreover, the dynamics of F-actin polymerization revealed that PAFR-mediated B. abortus uptake is related with the reorganization of F-actin and JAK2. Upon B. abortus phagocytosis, reduced PAFR in the membrane and subsequently increased levels of PAFR colocalization with endosomes were observed which indicate that B. abortus uptake into macrophages allowed PAFR trafficking to endosomes. CONCLUSIONS: This study demonstrated that PAFR has a compelling involvement in B. abortus uptake as a promoter of phagocytosis, which is associated with JAK2 activation. Thus, our findings establish a novel insight into a receptor-related phagocytic mechanism of B. abortus.
Assuntos
Brucella abortus/patogenicidade , Macrófagos/imunologia , Macrófagos/microbiologia , Fagocitose , Glicoproteínas da Membrana de Plaquetas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Brucella abortus/metabolismo , Brucelose/metabolismo , Brucelose/microbiologia , Endossomos/metabolismo , Interações Hospedeiro-Patógeno , Janus Quinase 2/metabolismo , Macrófagos/enzimologia , Camundongos , Células RAW 264.7 , Transdução de SinaisRESUMO
Brucellosis is one of the major zoonoses worldwide that inflicts important health problems in animal and human. Here, we demonstrated that dextran sulfate sodium (DSS) significantly increased adhesion of Brucella (B.) abortus in murine macrophages compared to untreated cells. Even without infection, Brucella uptake into macrophages increased and F-actin reorganization was induced compared with untreated cells. Furthermore, DSS increased the phosphorylation of MAPKs (ERK1/2 and p38α) in Brucella-infected, DSS-treated cells compared with the control cells. Lastly, DSS markedly increased the intracellular survival of Brucella abortus in macrophages by up to 48 h. These results suggest that DSS enhanced the adhesion and phagocytosis of B. abortus into murine macrophages by stimulating the MAPK signaling proteins phospho-ERK1/2 and p38α and that DSS increased the intracellular survival of B. abortus by inhibiting colocalization of Brucella-containing vacuoles (BCVs) with the late endosome marker LAMP-1. This study emphasizes the enhancement of the phagocytic and intracellular modulatory effects of DSS, which may suppress the innate immune system and contribute to prolonged Brucella survival and chronic infection.
Assuntos
Aderência Bacteriana/efeitos dos fármacos , Brucella abortus/efeitos dos fármacos , Brucella abortus/crescimento & desenvolvimento , Brucelose/microbiologia , Sulfato de Dextrana/farmacologia , Macrófagos/microbiologia , Animais , Brucella abortus/fisiologia , Brucelose/genética , Brucelose/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Viabilidade Microbiana/efeitos dos fármacos , Células RAW 264.7 , Regulação para Cima/efeitos dos fármacosRESUMO
Currently, there are several serodiagnostic tools available for brucellosis, however, it is difficult to differentiate an active infection from vaccination. Hence, there is a great need to develop alternative means that can distinguish between these two conditions without utilizing lipopolysaccharide (LPS). This study was an attempt to determine the efficacy of combined recombinant Brucella (B.) abortus outer membrane proteins (rOmps) and individual rOmps in the serodiagnosis of brucellosis by enzyme linked immunosorbent assay (ELISA), utilizing both that standard tube agglutination test (TAT)-positive and -negative serum samples from Korean native cattle. The results are very interesting and promising because the combined rOmp antigens used in the study were highly reactive with the TAT-positive serum samples. The combined rOmps sensitivity, specificity and accuracy were 215/232 (92.67%), 294/298 (98.66%) and 509/530 (96.04%), respectively. While these results are preliminary, the tests performed have very high potential in the serodiagnosis of brucellosis and likewise, the combined rOmps can be used for future vaccine production.
Assuntos
Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Brucelose Bovina/diagnóstico , Proteínas Recombinantes/imunologia , Testes Sorológicos/métodos , Medicina Veterinária/métodos , Testes de Aglutinação , Animais , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/genética , Bovinos , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas Recombinantes/genética , República da Coreia , Sensibilidade e EspecificidadeRESUMO
Brucellosis is a vital zoonotic disease caused by Brucella, which infects a wide range of animals and humans. Accurate diagnosis and reliable vaccination can control brucellosis in domestic animals. This study examined novel immunogenic proteins that can be used to detect Brucella abortus infection or as an effective subcellular vaccine. In an immunoproteomic assay, 55 immunodominant proteins from B. abortus 544 were observed using two dimensional electrophoresis (2DE) and immunoblot profiles with antisera from B. abortus-infected cattle at the early (week 3), middle (week 7), and late (week 10) periods, after excluding protein spots reacting with antisera from Yersinia enterocolitica O:9-infected and non-infected cattle. Twenty-three selected immunodominant proteins whose spots were observed at all three infection periods were identified using MALDI-MS/MS. Most of these proteins identified by immunoblot and mass spectrometry were determined by their subcellular localization and predicted function. We suggest that the detection of prominent immunogenic proteins during the infection period can support the development of advanced diagnostic methods with high specificity and accuracy; subsidiarily, these proteins can provide supporting data to aid in developing novel vaccine candidates.