RESUMEN
Trophoblasts are targets of infection by Brucella spp. but their role in the pathophysiology of pregnancy complications of brucellosis is unknown. Here we show that Brucella abortus invades and replicates in the human trophoblastic cell line Swan-71 and that the intracellular survival of the bacterium depends on a functional virB operon. The infection elicited significant increments of interleukin 8 (IL8), monocyte chemotactic protein 1 (MCP-1), and IL6 secretion, but levels of IL1beta and tumor necrosis factor-alpha (TNF-alpha) did not vary significantly. Such proinflammatory response was not modified by the absence of the Brucella TIR domain-containing proteins BtpA and BtpB. The stimulation of Swan-71 cells with conditioned medium (CM) from B. abortus-infected human monocytes (THP-1 cells) or macrophages induced a significant increase of IL8, MCP-1 and IL6 as compared to stimulation with CM from non-infected cells. Similar results were obtained when stimulation was performed with CM from infected neutrophils. Neutralization studies showed that IL1beta and/or TNF-alpha mediated the stimulating effects of CM from infected phagocytes. Reciprocally, stimulation of monocytes and neutrophils with CM from Brucella-infected trophoblasts increased IL8 and/or IL6 secretion. These results suggest that human trophoblasts may provide a local inflammatory environment during B. abortus infections either through a direct response to the pathogen or through interactions with monocytes/macrophages or neutrophils, potentially contributing to the pregnancy complications of brucellosis.
Asunto(s)
Brucella abortus , Brucelosis/patología , Inflamación/microbiología , Fagocitos/microbiología , Trofoblastos/microbiología , Brucelosis/metabolismo , Línea Celular , Quimiocina CCL2/metabolismo , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Macrófagos/patología , Monocitos/metabolismo , Monocitos/microbiología , Monocitos/patología , Fagocitos/metabolismo , Fagocitos/patología , Trofoblastos/metabolismo , Trofoblastos/patología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In patients with active brucellosis, the liver is frequently affected by histopathologic lesions, such as granulomas, inflammatory infiltrations, and parenchymal necrosis. Herein, we examine some potential mechanisms of liver damage in brucellosis. We demonstrate that Brucella abortus infection inhibits matrix metalloproteinase-9 (MMP-9) secretion and induces collagen deposition and tissue inhibitor of matrix metalloproteinase-1 secretion induced by hepatic stellate cells (LX-2). These phenomena depend on transforming growth factor-ß1 induction. In contrast, supernatants from B. abortus-infected hepatocytes and monocytes induce MMP-9 secretion and inhibit collagen deposition in hepatic stellate cells. Yet, if LX-2 cells are infected with B. abortus, the capacity of supernatants from B. abortus-infected hepatocytes and monocytes to induce MMP-9 secretion and inhibit collagen deposition is abrogated. These results indicate that depending on the balance between interacting cells and cytokines of the surrounding milieu, the response of LX-2 cells could be turned into an inflammatory or fibrogenic phenotype. Livers from mice infected with B. abortus displayed a fibrogenic phenotype with patches of collagen deposition and transforming growth factor-ß1 induction. This study provides potential mechanisms of liver immune response induced by B. abortus-infected hepatic stellate cells. In addition, these results demonstrate that the cross talk of these cells with hepatocytes and macrophages implements a series of interactions that may contribute to explaining some of mechanisms of liver damage observed in human brucellosis.
Asunto(s)
Brucella abortus , Brucelosis , Colágeno/metabolismo , Regulación Enzimológica de la Expresión Génica , Células Estrelladas Hepáticas , Cirrosis Hepática , Metaloproteinasa 9 de la Matriz/biosíntesis , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Brucelosis/metabolismo , Brucelosis/patología , Línea Celular , Regulación hacia Abajo , Femenino , Células Estrelladas Hepáticas/metabolismo , Células Estrelladas Hepáticas/patología , Humanos , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones , Ratones Endogámicos BALB CRESUMEN
The pathogenic mechanisms of bone loss caused by Brucella species have not been completely deciphered. Although T lymphocytes (LTs) are considered important to control infection, the mechanism of Brucella-induced T-cell responses to immunopathological features is not known. We present in vitro and in vivo evidence showing that Brucella abortus-induced inflammatory response leads to the activation of LTs, which further promote osteoclastogenesis. Pre-activated murine LTs treated with culture supernatant from macrophages infected with B. abortus induced bone marrow-derived monocytes (BMMs) to undergo osteoclastogenesis. Furthermore, osteoclastogenesis was mediated by CD4(+) T cells. Although B. abortus-activated T cells actively secreted the pro-osteoclastogenic cytokines RANKL and IL-17, osteoclastogenesis depended on IL-17, because osteoclast generation induced by Brucella-activated T cells was completely abrogated when these cells were cultured with BMMs from IL-17 receptor knockout mice. Neutralization experiments indicated that IL-6, generated by Brucella infection, induced the production of pro-osteoclastogenic IL-17 from LTs. By using BMMs from tumor necrosis factor receptor p55 knockout mice, we also demonstrated that IL-17 indirectly induced osteoclastogenesis through the induction of tumor necrosis factor-α from osteoclast precursors. Finally, extensive and widespread osteoclastogenesis was observed in the knee joints of mice injected with Brucella-activated T cells. Our results indicate that activated T cells, elicited by B. abortus-infected macrophages and influenced by the inflammatory milieu, promote the generation of osteoclasts, leading to bone loss.
Asunto(s)
Brucella abortus/inmunología , Interleucina-17/metabolismo , Macrófagos/microbiología , Osteoclastos/inmunología , Osteogénesis/inmunología , Linfocitos T/inmunología , Animales , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Brucelosis/inmunología , Brucelosis/microbiología , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Interleucina-17/biosíntesis , Interleucina-6/metabolismo , Activación de Linfocitos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoclastos/metabolismo , Ligando RANK/metabolismo , Receptores de Interleucina-17/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Fracciones Subcelulares/metabolismo , Tibia/inmunología , Tibia/patología , Receptores Señuelo del Factor de Necrosis Tumoral/deficiencia , Receptores Señuelo del Factor de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Fabry disease is an X-linked lysosomal disorder (LD) due to deficiency of the enzyme α-galactosidase A (αGal), which leads to the accumulation of neutral glycosphingolipids, mainly globotriaosylceramide (Gb3). Several mechanisms contribute to the diverse physiopathological alterations observed in this disease, and it has been suggested that an underlying proinflammatory state could play a significant role. The aim of this study is to investigate the presence of a proinflammatory state in the different subsets of peripheral blood mononuclear cells (PBMC) and to understand the mechanisms that contribute to its onset and perpetuation. We have shown that cultured PBMC from Fabry patients present a higher proinflammatory cytokine expression and production. Moreover, we determined that among PBMC, dendritic cells and monocytes present a basal proinflammatory cytokine production profile, which is further exacerbated with an inflammatory stimulus. Finally we established that normal, monocyte-derived dendritic cells and macrophages display the same proinflammatory profile when cultured in the presence of Gb3 and an inhibitor of αGal. Furthermore, this effect can be abolished using a TLR4 blocking antibody, indicating that TLR4 is necessary in the process. In summary, our results demonstrate the presence of a proinflammatory state involving two key subsets of innate immunity, and provide direct evidence of Gb3 having a proinflammatory role, likely mediated by TLR4, a finding that could help in the understanding of the underlying causes of the inflammatory pathogenesis of Fabry disease.
Asunto(s)
Citocinas/metabolismo , Enfermedad de Fabry/sangre , Trihexosilceramidas/sangre , alfa-Galactosidasa/sangre , Adolescente , Adulto , Anciano , Niño , Preescolar , Células Dendríticas/metabolismo , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/inmunología , Enfermedad de Fabry/patología , Femenino , Humanos , Inflamación/metabolismo , Inflamación/patología , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Masculino , Persona de Mediana Edad , Receptor Toll-Like 4/metabolismo , Trihexosilceramidas/inmunologíaRESUMEN
Osteoarticular brucellosis is the most common presentation of the active disease in humans. Loss of bone is a serious complication of localized bacterial infection of bones or the adjacent tissue, and brucellosis proved not to be the exception. The skeleton is a dynamic organ system which is constantly remodeled. Osteoblasts are responsible for the deposition of bone matrix and are thought to facilitate the calcification and mineralization of the bone matrix, and their function could be altered under infectious conditions. In this article, we describe immune mechanisms whereby Brucella abortus may invade and replicate within osteoblasts, inducing apoptosis, inhibiting mineral and organic matrix deposition, and inducing upregulation of RANKL expression. Additionally, all of these mechanisms contributed in different ways to bone loss. These processes implicate the activation of signaling pathways (mitogen-activated protein kinases [MAPK] and caspases) involved in cytokine secretion, expression of activating molecules, and cell death of osteoblasts. In addition, considering the relevance of macrophages in intracellular Brucella survival and proinflammatory cytokine secretion in response to infection, we also investigated the role of these cells as modulators of osteoblast survival, differentiation, and function. We demonstrated that supernatants from B. abortus-infected macrophages may also mediate osteoblast apoptosis and inhibit osteoblast function in a process that is dependent on the presence of tumor necrosis factor alpha (TNF-α). These results indicate that B. abortus may directly and indirectly harm osteoblast function, contributing to the bone and joint destruction observed in patients with osteoarticular complications of brucellosis.
Asunto(s)
Apoptosis , Brucella abortus/patogenicidad , Osteoblastos/metabolismo , Osteoblastos/microbiología , Osteogénesis , Animales , Células Cultivadas , Macrófagos/inmunología , Macrófagos/microbiología , RatonesRESUMEN
Osteoarticular complications are common in human brucellosis, but the pathogenic mechanisms involved are largely unknown. Since matrix metalloproteinases (MMPs) are involved in joint and bone damage in inflammatory and infectious diseases, we investigated the production of MMPs by human osteoblasts and monocytes, either upon Brucella abortus infection or upon reciprocal stimulation with factors produced by each infected cell type. B. abortus infection of the normal human osteoblastic cell line hFOB 1.19 triggered a significant release of MMP-2, which was mediated in part by granulocyte-macrophage colony-stimulating factor (GM-CSF) acting on these same cells. Supernatants from infected osteoblasts exhibited increased levels of monocyte chemoattractant protein 1 and induced the migration of human monocytes (THP-1 cell line). Infection with B. abortus induced a high MMP-9 secretion in monocytes, which was also induced by heat-killed B. abortus and by the Omp19 lipoprotein from B. abortus. These effects were mediated by Toll-like receptor 2 and by the action of tumor necrosis factor alpha (TNF-α) produced by these same cells. Supernatants from B. abortus-infected monocytes induced MMP-2 secretion in uninfected osteoblasts, and this effect was mediated by TNF-α. Similarly, supernatants from infected osteoblasts induced MMP-9 secretion in uninfected monocytes. This effect was mediated by GM-CSF, which induced TNF-α production by monocytes, which in turn induced MMP-9 in these cells. These results suggest that MMPs could be potentially involved in the tissue damage observed in osteoarticular brucellosis.
Asunto(s)
Brucella abortus/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Metaloproteinasas de la Matriz/metabolismo , Monocitos/microbiología , Osteoblastos/microbiología , Factor de Necrosis Tumoral alfa/metabolismo , Línea Celular , Regulación de la Expresión Génica/fisiología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Monocitos/metabolismo , Osteoblastos/metabolismoRESUMEN
Arthritis is one of the most common complications of human brucellosis, but its pathogenic mechanisms have not been elucidated. Fibroblast-like synoviocytes (FLS) are known to be central mediators of joint damage in inflammatory arthritides through the production of matrix metalloproteinases (MMPs) that degrade collagen and of cytokines and chemokines that mediate the recruitment and activation of leukocytes. In this study we show that Brucella abortus infects and replicates in human FLS (SW982 cell line) in vitro and that infection results in the production of MMP-2 and proinflammatory mediators (interleukin-6 [IL-6], IL-8, monocyte chemotactic protein 1 [MCP-1], and granulocyte-macrophage colony-stimulating factor [GM-CSF]). Culture supernatants from Brucella-infected FLS induced the migration of monocytes and neutrophils in vitro and also induced these cells to secrete MMP-9 in a GM-CSF- and IL-6-dependent fashion, respectively. Reciprocally, culture supernatants from Brucella-infected monocytes and neutrophils induced FLS to produce MMP-2 in a tumor necrosis factor alpha (TNF-α)-dependent fashion. The secretion of proinflammatory mediators and MMP-2 by FLS did not depend on bacterial viability, since it was also induced by heat-killed B. abortus (HKBA) and by a model Brucella lipoprotein (L-Omp19). These responses were mediated by the recognition of B. abortus antigens through Toll-like receptor 2. The intra-articular injection of HKBA or L-Omp19 into the knee joint of mice resulted in the local induction of the proinflammatory mediators MMP-2 and MMP-9 and in the generation of a mixed inflammatory infiltrate. These results suggest that FLS, and phagocytes recruited by them to the infection focus, may be involved in joint damage during brucellar arthritis through the production of MMPs and proinflammatory mediators.
Asunto(s)
Artritis Infecciosa/inmunología , Brucella abortus/inmunología , Brucelosis/inmunología , Articulaciones/microbiología , Articulaciones/patología , Metaloproteinasas de la Matriz/biosíntesis , Membrana Sinovial/inmunología , Animales , Antígenos Bacterianos/inmunología , Artritis Infecciosa/enzimología , Artritis Infecciosa/microbiología , Artritis Infecciosa/patología , Proteínas de la Membrana Bacteriana Externa/inmunología , Brucella abortus/crecimiento & desarrollo , Brucella abortus/patogenicidad , Brucelosis/enzimología , Brucelosis/microbiología , Brucelosis/patología , Línea Celular , Movimiento Celular/efectos de los fármacos , Quimiocinas/biosíntesis , Medios de Cultivo Condicionados , Citocinas/biosíntesis , Citocinas/metabolismo , Inducción Enzimática , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Articulación de la Rodilla/microbiología , Lipoproteínas/inmunología , Activación de Linfocitos , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Endogámicos BALB C , Monocitos/fisiología , Neutrófilos/fisiología , Membrana Sinovial/citología , Membrana Sinovial/microbiología , Receptor Toll-Like 2/metabolismoRESUMEN
Fabry disease is an X-linked lysosomal storage disorder (LSD) due to deficiency of the enzyme α-galactosidase A, resulting in intracellular deposition of globotriaosylceramide (Gb3). Accumulation of Gb3 is probably related to tissue and organ dysfunctions. Diverse pathological mechanisms are elicited in LSDs, giving together the phenotypic expression of each disease. The purpose of the present study is to investigate if apoptosis could play a role in Fabry disease pathogenesis and to understand the mechanisms involved in the proapoptotic state. We have demonstrated that Fabry disease peripheral blood mononuclear cells display a higher apoptotic state, which is reduced by enzyme replacement therapy (ERT), and is mediated, at least in part, by activation of the intrinsic pathway of caspases. We could rule out the implication of "unfolded protein response-ER stress" in this apoptotic process. To further confirm the suggestion that Gb3 is associated to apoptotic cell death, we treated normal cells with Gb3 at concentrations found in Fabry patients. Addition of Gb3 resulted in a dose-dependent induction of apoptosis involving the intrinsic pathway. In summary, PBMC from Fabry patients display a higher apoptotic state, which could be mainly related to elevated Gb3.
Asunto(s)
Apoptosis/fisiología , Enfermedad de Fabry/fisiopatología , Leucocitos Mononucleares/metabolismo , Trihexosilceramidas/metabolismo , Adolescente , Adulto , Análisis de Varianza , Anexina A5 , Apoptosis/efectos de los fármacos , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Terapia de Reemplazo Enzimático , Enfermedad de Fabry/tratamiento farmacológico , Femenino , Citometría de Flujo , Humanos , Immunoblotting , Etiquetado Corte-Fin in Situ , Isoenzimas , Leucocitos Mononucleares/fisiología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Trihexosilceramidas/farmacología , alfa-GalactosidasaRESUMEN
BACKGROUND & AIMS: Hepatic involvement is frequent in human brucellosis. While different histopathological lesions have been reported in these patients, the underlying cellular and molecular mechanisms have not been addressed. METHODS: This study assessed whether Brucella abortus can infect a human hepatoma cell line and induce a proinflammatory response in these cells. RESULTS: The bacterium not only infected the human hepatoma cell line HepG2 but also exhibited intracellular replication. The infection induced hepatoma cells to secrete IL-8, and supernatants from Brucella-infected hepatoma cells were shown to induce the migration of human neutrophils. The infection also induced the expression of the intercellular adhesion molecule ICAM-1 on hepatoma cells, and the adhesion of neutrophils to these cells was significantly higher than to uninfected hepatoma cells. ICAM-1 expression was also induced by stimulation of hepatoma cells with supernatants from Brucella-infected neutrophils. While Brucella infection did not induce the expression of matrix metalloproteinases (MMPs) in hepatoma cells, it significantly induced MMP-9 in neutrophils. Hepatoma cell apoptosis was significantly induced by B. abortus infection and also by stimulation with supernatants from Brucella-infected neutrophils. CONCLUSIONS: The present study provides clues regarding potential mechanisms of tissue damage during liver brucellosis.
Asunto(s)
Brucella abortus/patogenicidad , Brucelosis/inmunología , Brucelosis/microbiología , Hepatocitos/inmunología , Hepatocitos/microbiología , Hepatopatías/inmunología , Hepatopatías/microbiología , Apoptosis , Brucella abortus/inmunología , Brucelosis/patología , Adhesión Celular , Línea Celular Tumoral , Movimiento Celular , Medios de Cultivo Condicionados , Hepatocitos/patología , Humanos , Molécula 1 de Adhesión Intercelular/biosíntesis , Interleucina-8/biosíntesis , Hepatopatías/patología , Metaloproteinasa 9 de la Matriz/biosíntesis , Neutrófilos/enzimología , Neutrófilos/inmunología , Neutrófilos/patologíaRESUMEN
The ability of Brucella spp. to infect human osteoblasts and the cytokine response of these cells to infection were investigated in vitro. Brucella abortus, B. suis, B. melitensis, and B. canis were able to infect the SaOS-2 and MG-63 osteoblastic cell lines, and the first three species exhibited intracellular replication. B. abortus internalization was not significantly affected by pretreatment of cells with cytochalasin D but was inhibited up to 92% by colchicine. A virB10 mutant of B. abortus could infect but not replicate within osteoblasts, suggesting a role for the type IV secretion system in intracellular survival. Infected osteoblasts produced low levels of chemokines (interleukin-8 [IL-8] and macrophage chemoattractant protein 1 [MCP-1]) and did not produce proinflammatory cytokines (IL-1beta, IL-6, and tumor necrosis factor alpha [TNF-alpha]). However, osteoblasts stimulated with culture supernatants from Brucella-infected human monocytes (THP-1 cell line) produced chemokines at levels 12-fold (MCP-1) to 17-fold (IL-8) higher than those of infected osteoblasts and also produced IL-6. In the inverse experiment, culture supernatants from Brucella-infected osteoblasts induced the production of IL-8, IL-1beta, IL-6, and TNF-alpha by THP-1 cells. The induction of TNF-alpha and IL-1beta was largely due to granulocyte-macrophage colony-stimulating factor produced by infected osteoblasts, as demonstrated by inhibition with a specific neutralizing antibody. This study shows that Brucella can invade and replicate within human osteoblastic cell lines, which can directly and indirectly mount a proinflammatory response. Both phenomena may have a role in the chronic inflammation and bone and joint destruction observed in osteoarticular brucellosis.
Asunto(s)
Brucelosis/inmunología , Monocitos/inmunología , Monocitos/microbiología , Osteoblastos/inmunología , Osteoblastos/microbiología , Brucella/inmunología , Línea Celular , Citocinas/biosíntesis , Citometría de Flujo , Humanos , Microscopía ConfocalRESUMEN
Available vaccines against Brucella spp. are live attenuated Brucella strains. In order to engineer a better vaccine to be used in animals and humans, our laboratory aims to develop an innocuous subunit vaccine. Particularly, we are interested in the outer membrane proteins (OMPs) of B. abortus: Omp16 and Omp19. In this study, we assessed the use of these proteins as vaccines against Brucella in BALB/c mice. Immunization with lipidated Omp16 (L-Omp16) or L-Omp19 in incomplete Freund's adjuvant (IFA) conferred significant protection against B. abortus infection. Vaccination with unlipidated Omp16 (U-Omp16) or U-Omp19 in IFA induced a higher degree of protection than the respective lipidated versions. Moreover, the level of protection induced after U-Omp16 or U-Omp19 immunization in IFA was similar to that elicited by live B. abortus S19 immunization. Flow cytometric analysis showed that immunization with U-Omp16 or U-Omp19 induced antigen-specific CD4(+) as well as CD8(+) T cells producing gamma interferon. In vivo depletion of CD4(+) or CD8(+) T cells in mice immunized with U-Omp16 or U-Omp19 plus IFA resulted in a loss of the elicited protection, indicating that both cell types are mediating immune protection. U-Omp16 or U-Omp19 vaccination induced a T helper 1 response, systemic protection in aluminum hydroxide formulation, and oral protection with cholera toxin adjuvant against B. abortus infection. Both immunization routes exhibited a similar degree of protection to attenuated Brucella vaccines (S19 and RB51, respectively). Overall these results indicate that U-Omp16 or U-Omp19 would be a useful candidate for a subunit vaccine against human and animal brucellosis.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacuna contra la Brucelosis/inmunología , Brucella abortus/inmunología , Brucelosis/prevención & control , Adyuvante de Freund/administración & dosificación , Adyuvantes Inmunológicos/farmacología , Hidróxido de Aluminio/administración & dosificación , Hidróxido de Aluminio/farmacología , Animales , Anticuerpos Antibacterianos/sangre , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Toxina del Cólera/administración & dosificación , Toxina del Cólera/farmacología , Recuento de Colonia Microbiana , Femenino , Citometría de Flujo , Adyuvante de Freund/farmacología , Inmunoglobulina G/sangre , Interferón gamma/biosíntesis , Procedimientos de Reducción del Leucocitos , Ratones , Ratones Endogámicos BALB C , Bazo/microbiología , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunologíaRESUMEN
The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.
Asunto(s)
Proteínas Bacterianas/metabolismo , Brucella abortus/genética , Proteómica , Factores de Virulencia/metabolismo , Amidohidrolasas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Brucella abortus/metabolismo , Brucella abortus/patogenicidad , Línea Celular , Medios de Cultivo , Electroforesis en Gel Bidimensional , Genes Bacterianos , Proteínas HSP70 de Choque Térmico/metabolismo , Ratones , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil/metabolismo , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Virulencia/genéticaRESUMEN
The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-gamma)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-gamma production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection.
Asunto(s)
Presentación de Antígeno , Brucella abortus/fisiología , Regulación de la Expresión Génica , Genes MHC Clase II/genética , Interleucina-6/metabolismo , Receptor Toll-Like 2/metabolismo , Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Brucella abortus/inmunología , Células Cultivadas , Regulación hacia Abajo , Humanos , Interferón gamma , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/microbiología , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Monocitos/metabolismo , Monocitos/microbiologíaRESUMEN
Infection with Brucella abortus induces a pro-inflammatory response that drives T cell responses toward a Th1 profile. The mechanism by which this bacterium triggers this response is unknown. Dendritic cells (DC) are crucial mediators at the host-pathogen interface and are potent Th1-inducing antigen-presenting cells. Thus, we examined the mechanism whereby B. abortus stimulate human DC maturation. B. abortus-infected DC increased the expression of CD86, CD80, CCR7, CD83, MHCII, MHCI and CD40 and induced the production of TNF-alpha, IL-6, IL-10 and IL-12. Both phenomena were not dependent on bacterial viability since they were also induced by heat-killed B. abortus (HKBA). B. abortus LPS was unable to induce markers up-regulation or cytokine production. We next investigated the capacity of the outer membrane protein 19 (Omp19) as a B. abortus lipoprotein model to induce DC maturation. Lipidated Omp19 (L-Omp19), but not its unlipidated form, increased the expression of cell surface markers and the secretion of cytokines. L-Omp19-matured DC also have decreased endocytic activity and displayed enhanced T cell stimulatory activity in a MLR. Pre-incubation of DC with anti-TLR2 mAb blocked L-Omp19-mediated cytokine production. These results demonstrate that B. abortus lipoproteins can stimulate DC maturation providing a mechanism by which these bacteria generate a Th1-type immune response.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Brucella abortus/patogenicidad , Diferenciación Celular , Células Dendríticas , Lipoproteínas/inmunología , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Citometría de Flujo , Humanos , Receptores de Superficie Celular/metabolismoRESUMEN
Brucella canis infection is an important cause of late-term abortion in pregnant bitches. The pathophysiological mechanisms leading to B. canis-induced abortion are unknown, but heavily infected trophoblasts are consistently observed. As trophoblasts responses to other pathogens contribute to placental inflammation leading to abortion, the aim of the present study was to characterize the cytokine response of canine trophoblasts to B. canis infection. To achieve this, trophoblasts isolated from term placenta of healthy female dogs were infected with B. canis, culture supernatants were harvested for cytokine determinations, and the load of intracellular viable B. canis was determined at different times post-infection. Additionally, cytokine responses were assessed in non-infected trophoblasts stimulated with conditioned media (CM) from B. canis-infected canine monocytes and neutrophils. Finally, cytokine response and bacteria replication were assessed in canine placental explants infected ex vivo. B. canis successfully infected and replicated in primary canine trophoblasts, eliciting an increase in IL-8 and RANTES (CCL5) secretion. Moreover, the stimulation of trophoblasts with CM from B. canis-infected monocytes and neutrophils induced a significant increase in IL-8, IL-6 and RANTES secretion. B. canis replication was confirmed in infected placental explants and the infection elicited an increased secretion of TNF-α, IL-8, IL-6 and RANTES. This study shows that canine trophoblasts produce proinflammatory cytokines in response to B. canis infection and/or to stimulation with factors produced by infected monocytes and neutrophils. These cytokines may contribute to placental inflammation leading to abortion in B. canis-infected pregnant bitches.
Asunto(s)
Brucella canis/fisiología , Trofoblastos/microbiología , Animales , Antígenos Bacterianos/inmunología , Brucella canis/inmunología , Quimiocinas/metabolismo , Perros , Femenino , Inflamación/microbiología , Fagocitos/citología , Placenta/microbiología , Embarazo , Receptores Toll-Like/agonistas , Trofoblastos/citología , Trofoblastos/metabolismoRESUMEN
The expression of haemin-binding proteins (HBPs) in the outer membrane is one of the strategies used by Gram-negative bacteria to obtain iron from the host. No HBP has been described in Brucella spp. We investigated whether Omp31, an outer membrane protein from Brucella with homology to HBPs from Bartonella quintana, is an HBP. Soluble recombinant Omp31 bound specifically to haemin-agarose, while an unrelated Brucella protein (SurA) did not. A similar experiment showed that native Omp31 found in the Brucella suis membrane fraction also binds to haemin-agarose. Recombinant Omp31 was electrophoresed by SDS-PAGE, transferred to nitrocellulose, and incubated with a haemin solution. Haemin bound to Omp31 and to albumin (positive control) but not to SurA. IPTG-induced recombinant Escherichia coli cells expressing Omp31 on their membrane bound significantly more haemin than uninduced cells or controls carrying a similar plasmid without the omp31 gene, showing that Omp31 also binds haemin in a bacterial membrane environment. Viable Brucella ovis cells bound haemin in solution, and this binding was markedly inhibited by preincubation of cells with antibodies to Omp31 and to an exposed prominent loop of the protein, thus showing that Omp31 functions as an HBP in brucellae. To test whether the expression of Omp31 is iron-regulated, B. suis was grown in trypticase-soy broth (TSB) and in iron-depleted TSB. The expression of Omp31, as assessed by Western blot, was significantly higher in bacteria grown under iron limitation. Overall, these results show that Omp31 from B. suis, B. melitensis and B. ovis is an HBP, whose expression seems to be induced by iron limitation.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Brucella/metabolismo , Proteínas Portadoras/metabolismo , Hemoproteínas/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Brucella/clasificación , Brucella/genética , Brucella/crecimiento & desarrollo , Medios de Cultivo , Regulación Bacteriana de la Expresión Génica , Proteínas de Unión al Hemo , Hemina/metabolismo , Hierro/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs.
Asunto(s)
Proteínas Bacterianas/inmunología , Sistemas de Secreción Bacterianos , Vacuna contra la Brucelosis/inmunología , Brucella/crecimiento & desarrollo , Brucella/inmunología , Bazo/microbiología , Células TH1/inmunología , Adyuvantes Inmunológicos , Animales , Anticuerpos Antibacterianos/inmunología , Carga Bacteriana , Proteínas Bacterianas/administración & dosificación , Bacteriólisis , Brucella/patogenicidad , Vacuna contra la Brucelosis/administración & dosificación , Brucella abortus/inmunología , Brucella canis/inmunología , Perros , Hipersensibilidad Tardía , Inyecciones Subcutáneas , Interferón gamma/inmunología , Interleucina-4/inmunología , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Bazo/citología , Bazo/inmunología , VacunaciónRESUMEN
Both CCL20 and human ß-defensin 2 (hBD2) interact with the same membrane receptor and display chemotactic and antimicrobial activities. They are produced by airway epithelia in response to infectious agents and proinflammatory cytokines. Whereas Brucella spp. can infect humans through inhalation, their ability to induce CCL20 and hBD2 in lung cells is unknown. Here we show that B. abortus induces CCL20 expression in human alveolar (A549) or bronchial (Calu-6) epithelial cell lines, primary alveolar epithelial cells, primary human monocytes, monocyte-derived macrophages and the monocytic cell line THP-1. CCL20 expression was mainly mediated by JNK1/2 and NF-kB in both Calu-6 and THP-1 cells. CCL20 secretion was markedly induced in A549, Calu-6 and THP-1 cells by heat-killed B. abortus or a model Brucella lipoprotein (L-Omp19) but not by the B. abortus lipopolysaccharide (LPS). Accordingly, CCL20 production by B. abortus-infected cells was strongly TLR2-dependent. Whereas hBD2 expression was not induced by B. abortus infection, it was significantly induced in A549 cells by conditioned media from B. abortus-infected THP-1 monocytes (CMB). A similar inducing effect was observed on CCL20 secretion. Experiments using blocking agents revealed that IL-1ß, but not TNF-α, was involved in the induction of hBD2 and CCL20 secretion by CMB. In the in vitro antimicrobial assay, the lethal dose (LD) 50 of CCL20 for B. abortus (>50 µg/ml) was markedly higher than that against E. coli (1.5 µg/ml) or a B. abortus mutant lacking the O polysaccharide in its LPS (8.7 ug/ml). hBD2 did not kill any of the B. abortus strains at the tested concentrations. These results show that human lung epithelial cells secrete CCL20 and hBD2 in response to B. abortus and/or to cytokines produced by infected monocytes. Whereas these molecules do not seem to exert antimicrobial activity against this pathogen, they could recruit immune cells to the infection site.
Asunto(s)
Células Epiteliales Alveolares/metabolismo , Brucella abortus/inmunología , Brucelosis/metabolismo , Quimiocina CCL20/biosíntesis , beta-Defensinas/biosíntesis , Células Epiteliales Alveolares/microbiología , Antibacterianos/farmacología , Brucelosis/inmunología , Brucelosis/microbiología , Línea Celular , Línea Celular Tumoral , Quimiocina CCL20/metabolismo , Quimiocina CCL20/farmacología , Humanos , Inmunidad Innata , Lipopolisacáridos/farmacología , Pulmón/inmunología , Pulmón/microbiología , Pulmón/patología , Sistema de Señalización de MAP Quinasas , Pruebas de Sensibilidad Microbiana , Monocitos/inmunología , Monocitos/metabolismo , Monocitos/microbiología , Receptor Toll-Like 2/metabolismo , beta-Defensinas/metabolismo , beta-Defensinas/farmacologíaRESUMEN
It has been proposed that anti-myocardial antibodies (Ab) against neurotransmitter (NT) receptors are involved in the immunopathology of chronic Chagas' heart disease. We demonstrated that an anti-Trypanosoma cruzi monoclonal Ab (mAb), CAK20.12, binds to murine cardiac beta-adrenergic and muscarinic acetyl choline (mACh) receptors eliciting abnormal physiological responses on normal heart. No cross-linking requirement for mAb actions was demonstrated using Fab fragment derived from CAK20.12. mAb binding to synthetic peptides from the second extracellular loop of both beta1-adrenergic and mACh receptors, demonstrated by ELISA, identified the region of NT receptors involved. Cross-reactivity between these peptides and T. cruzi antigen was confirmed by binding inhibition assays. These results support the existence of cross-reactivity due to molecular mimicry between a parasite antigen and the major antigenic epitopes present on both beta1-adrenergic and M2-ACh receptors. Its possible relationship with cardiac dysfunction during chronic stage of Chagas' disease is also discussed.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Antiprotozoarios/farmacología , Contracción Miocárdica/efectos de los fármacos , Pindolol/análogos & derivados , Receptor Muscarínico M2/inmunología , Receptores Adrenérgicos beta 1/inmunología , Trypanosoma cruzi/inmunología , Antagonistas Adrenérgicos beta , Análisis de Varianza , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ensayo de Inmunoadsorción Enzimática/métodos , Epítopos/metabolismo , Epítopos/farmacología , Fragmentos Fab de Inmunoglobulinas/metabolismo , Técnicas In Vitro , Isótopos de Yodo/farmacocinética , Ratones , Ratones Endogámicos BALB C , Antagonistas Muscarínicos/farmacocinética , Contracción Miocárdica/fisiología , Pindolol/farmacocinética , Quinuclidinil Bencilato/farmacocinética , Radioinmunoensayo/métodos , Ensayo de Unión Radioligante/métodos , Receptor Muscarínico M2/química , Receptores Adrenérgicos beta 1/química , Volumetría/métodos , Trypanosoma cruzi/químicaRESUMEN
Previous studies have shown that the detection of antibodies to an 18-kDa cytoplasmic protein of Brucella spp. is useful for the diagnosis of human and animal brucellosis. This protein has now been expressed in recombinant form in Escherichia coli. The recombinant protein is soluble only under reducing conditions, but alkylation with iodoacetamide renders it soluble in non-reducing media. As shown by gel exclusion chromatography, this soluble form arranges in pentamers of 90 kDa. The reactivity of human and animal sera against the recombinant protein was similar to that found with the native protein present in brucella cytoplasmic fraction, suggesting that the recombinant protein is correctly folded. The protein has low but significant homology (30%) with lumazine synthases involved in bacterial riboflavin biosynthesis, which also arrange as pentamers. Biological tests on the crude extract of the recombinant bacteria and on the purified recombinant protein showed that the biological activity of the Brucella spp. 18-kDa protein is that of lumazine synthase. Preliminary crystallographic analysis showed that the Brucella spp. lumazine synthase arranges in icosahedric capsids similar to those formed by the lumazine synthases of other bacteria. The high immunogenicity of this protein, potentially useful for the design of acellular vaccines, could be explained by this polymeric arrangement.