RESUMEN
Brucellosis is a contagious disease caused by bacteria of the genus Brucella. Platelets (PLTs) have been widely involved in the modulation of the immune response. We have previously reported the modulation of Brucella abortus-mediated infection of monocytes. As a result, PLTs cooperate with monocytes and increase their inflammatory capacity, promoting the resolution of the infection. Extending these results, in this study we demonstrate that patients with brucellosis present slightly elevated levels of complexes between PLTs and both monocytes and neutrophils. We then assessed whether PLTs were capable of modulating functional aspects of neutrophils. The presence of PLTs throughout neutrophil infection increased the production of interleukin-8, CD11b surface expression and reactive oxygen species formation, whereas it decreased the expression of CD62L, indicating an activated status of these cells. We next analyzed whether this modulation was mediated by released factors. To discriminate between these options, neutrophils were treated with supernatants collected from B. abortus-infected PLTs. Our results show that CD11b expression was induced by soluble factors of PLTs but direct contact between cell populations was needed to enhance the respiratory burst. Additionally, B. abortus-infected PLTs recruit polymorphonuclear (PMN) cells to the site of infection. Finally, the presence of PLTs did not modify the initial invasion of PMN cells by B. abortus but improved the control of the infection at extended times. Altogether, our results demonstrate that PLTs interact with neutrophils and promote a proinflammatory phenotype which could also contribute to the resolution of the infection.
Asunto(s)
Plaquetas/microbiología , Brucella abortus , Brucelosis , Monocitos/inmunología , Neutrófilos/inmunología , HumanosRESUMEN
Vaccine developmental strategies are utilizing antigens encapsulated in biodegradable polymeric nanoparticles. Here, we developed a Chlamydia nanovaccine (PLGA-rMOMP) by encapsulating its recombinant major outer membrane protein (rMOMP) in the extended-releasing and self-adjuvanting PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. PLGA-rMOMP was small (nanometer size), round and smooth, thermally stable, and exhibited a sustained release of rMOMP. Stimulation of mouse primary dendritic cells (DCs) with PLGA-rMOMP augmented endosome processing, induced Th1 cytokines (IL-6 and IL-12p40), and expression of MHC-II and co-stimulatory (CD40, CD80, and CD86) molecules. BALB/c mice immunized with PLGA-rMOMP produced enhanced CD4+ T-cells-derived memory (CD44high CD62Lhigh), and effector (CD44high CD62Llow) phenotypes and functional antigen-specific serum IgG antibodies. In vivo biodistribution of PLGA-rMOMP revealed its localization within lymph nodes, suggesting migration from the injection site via DCs. Our data provide evidence that the PLGA (85:15) nanovaccine activates DCs and augments Chlamydia-specific rMOMP adaptive immune responses that are worthy of efficacy testing.
Asunto(s)
Inmunidad Adaptativa/genética , Proteínas de la Membrana Bacteriana Externa/genética , Nanopartículas/química , Vacunas/inmunología , Inmunidad Adaptativa/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Antígenos CD4/química , Antígenos CD4/inmunología , Chlamydia/genética , Chlamydia/inmunología , Chlamydia/patogenicidad , Células Dendríticas/inmunología , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Receptores de Hialuranos/química , Receptores de Hialuranos/inmunología , Subunidad p40 de la Interleucina-12/genética , Subunidad p40 de la Interleucina-12/inmunología , Interleucina-6/genética , Interleucina-6/inmunología , Selectina L/química , Selectina L/inmunología , Ratones , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/inmunología , Linfocitos T/inmunología , Vacunas/genéticaRESUMEN
Despite eliciting a potent CD8+ T cell response, Brucella abortus is able to persist and establish a chronic infection inside its host. We have previously reported that the infection of human monocytes/macrophages with B. abortus inhibits the IFN-γ-induced MHC-I cell surface expression down-modulating cytotoxic CD8+ T cell responses. MHC-I down-modulation depends on bacterial viability and results from the capacity of B. abortus to retain the MHC-I molecules within the Golgi apparatus. Furthermore, we recently demonstrated that epidermal growth factor receptor (EGFR) pathway is involved in this phenomenon and that this is an early event during infection. However, the components and mechanisms whereby B. abortus is able to down-modulate MHC-I remained to be elucidated. In this study we demonstrated that the down-modulation of MHC-I expression is not mediated by well-known Brucella virulence factors but instead by B. abortus RNA, a PAMP associated to viability (vita-PAMP). Surprisingly, completely degraded RNA was also able to inhibit MHC-I expression to the same extent as intact RNA. Accordingly, B. abortus RNA and its degradation products were able to mimic the MHC-I intracellular retention within the Golgi apparatus observed upon infection. We further demonstrated that TLR8, a single-stranded RNA and RNA degradation products sensor, was involved in MHC-I inhibition. On the other hand, neutralization of the EGFR reversed the MHC-I inhibition, suggesting a connection between the TLR8 and EGFR pathways. Finally, B. abortus RNA-treated macrophages display diminished capacity of antigen presentation to CD8+ T cells. Overall, our results indicate that the vita-PAMP RNA as well as its degradation products constitute novel virulence factors whereby B. abortus, by a TLR8-dependent mechanism and through the EGFR pathway, inhibits the IFN-γ-induced MHC-I surface expression on human monocytes/macrophages. Thus, bacteria can hide within infected cells and avoid the immunological surveillance of cytotoxic CD8+ T cells.
Asunto(s)
Brucelosis/inmunología , Receptores ErbB/inmunología , Evasión Inmune/inmunología , Monocitos/inmunología , ARN Bacteriano/inmunología , Receptor Toll-Like 8/inmunología , Animales , Brucella abortus/inmunología , Reactividad Cruzada/inmunología , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Antígenos de Histocompatibilidad Clase I/biosíntesis , Humanos , Ratones , Ratones Endogámicos C57BL , Microscopía Confocal , Monocitos/microbiología , Transducción de Señal/inmunologíaRESUMEN
Blood-brain barrier activation and/or dysfunction are a common feature of human neurobrucellosis, but the underlying pathogenic mechanisms are largely unknown. In this article, we describe an immune mechanism for inflammatory activation of human brain microvascular endothelial cells (HBMEC) in response to infection with Brucella abortus Infection of HBMEC with B. abortus induced the secretion of IL-6, IL-8, and MCP-1, and the upregulation of CD54 (ICAM-1), consistent with a state of activation. Culture supernatants (CS) from glial cells (astrocytes and microglia) infected with B. abortus also induced activation of HBMEC, but to a greater extent. Although B. abortus-infected glial cells secreted IL-1ß and TNF-α, activation of HBMEC was dependent on IL-1ß because CS from B. abortus-infected astrocytes and microglia deficient in caspase-1 and apoptosis-associated speck-like protein containing a CARD failed to induce HBMEC activation. Consistently, treatment of CS with neutralizing anti-IL-1ß inhibited HBMEC activation. Both absent in melanoma 2 and Nod-like receptor containing a pyrin domain 3 are partially required for caspase-1 activation and IL-1ß secretion, suggesting that multiple apoptosis-associated speck-like protein containing CARD-dependent inflammasomes contribute to IL-1ß-induced activation of the brain microvasculature. Inflammasome-mediated IL-1ß secretion in glial cells depends on TLR2 and MyD88 adapter-like/TIRAP. Finally, neutrophil and monocyte migration across HBMEC monolayers was increased by CS from Brucella-infected glial cells in an IL-1ß-dependent fashion, and the infiltration of neutrophils into the brain parenchyma upon intracranial injection of B. abortus was diminished in the absence of Nod-like receptor containing a pyrin domain 3 and absent in melanoma 2. Our results indicate that innate immunity of the CNS set in motion by B. abortus contributes to the activation of the blood-brain barrier in neurobrucellosis and IL-1ß mediates this phenomenon.
Asunto(s)
Encéfalo/inmunología , Brucella abortus/inmunología , Brucelosis/inmunología , Neuroglía/inmunología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Barrera Hematoencefálica/patología , Encéfalo/microbiología , Proteínas Adaptadoras de Señalización CARD , Movimiento Celular , Células Cultivadas , Femenino , Humanos , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microvasos/patología , Neuroglía/microbiologíaRESUMEN
In this study, we demonstrate that the unlipidated (U) outer membrane protein (Omp) 19 from Brucella spp. is a competitive inhibitor of human cathepsin L. U-Omp19 inhibits lysosome cathepsins and APC-derived microsome activity in vitro and partially inhibits lysosomal cathepsin L activity within live APCs. Codelivery of U-Omp19 with the Ag can reduce intracellular Ag digestion and increases Ag half-life in dendritic cells (DCs). U-Omp19 retains the Ag in Lamp-2(+) compartments after its internalization and promotes a sustained expression of MHC class I/peptide complexes in the cell surface of DCs. Consequently, U-Omp19 enhances Ag cross-presentation by DCs to CD8(+) T cells. U-Omp19 s.c. delivery induces the recruitment of CD11c(+)CD8α(+) DCs and monocytes to lymph nodes whereas it partially limits in vivo Ag proteolysis inside DCs. Accordingly, this protein is able to induce CD8(+) T cell responses in vivo against codelivered Ag. Antitumor responses were elicited after U-Omp19 coadministration, increasing survival of mice in a murine melanoma challenge model. Collectively, these results indicate that a cysteine protease inhibitor from bacterial origin could be a suitable component of vaccine formulations against tumors.
Asunto(s)
Antígenos Bacterianos/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Brucella/inmunología , Brucelosis/inmunología , Linfocitos T CD8-positivos/fisiología , Vacunas contra el Cáncer/inmunología , Catepsinas/metabolismo , Células Dendríticas/inmunología , Inmunoterapia/métodos , Lipoproteínas/metabolismo , Lisosomas/metabolismo , Melanoma/terapia , Animales , Antígenos de Neoplasias/inmunología , Reactividad Cruzada , Femenino , Activación de Linfocitos , Proteína 2 de la Membrana Asociada a los Lisosomas/metabolismo , Melanoma/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Inflammation has long been implicated as a contributor to pathogenesis in neurobrucellosis. Many of the associated neurocognitive symptoms of neurobrucellosis may be the result of neuronal dysfunction resulting from the inflammatory response induced by Brucella abortus infection in the central nervous system. In this manuscript, we describe an immune mechanism for inflammatory activation of microglia that leads to neuronal death upon B. abortus infection. B. abortus was unable to infect or harm primary cultures of mouse neurons. However, when neurons were co-cultured with microglia and infected with B. abortus significant neuronal loss occurred. This phenomenon was dependent on TLR2 activation by Brucella lipoproteins. Neuronal death was not due to apoptosis, but it was dependent on the microglial release of nitric oxide (NO). B. abortus infection stimulated microglial proliferation, phagocytic activity and engulfment of neurons. NO secreted by B. abortus-activated microglia induced neuronal exposure of the "eat-me" signal phosphatidylserine (PS). Blocking of PS-binding to protein milk fat globule epidermal growth factor-8 (MFG-E8) or microglial vitronectin receptor-MFG-E8 interaction was sufficient to prevent neuronal loss by inhibiting microglial phagocytosis without affecting their activation. Taken together, our results indicate that B. abortus is not directly toxic to neurons; rather, these cells become distressed and are killed by phagocytosis in the inflammatory surroundings generated by infected microglia. Neuronal loss induced by B. abortus-activated microglia may explain, in part, the neurological deficits observed during neurobrucellosis.
Asunto(s)
Brucella abortus/patogenicidad , Muerte Celular/fisiología , Inflamación/metabolismo , Microglía/microbiología , Microglía/fisiología , Neuronas/patología , Fagocitosis/fisiología , Animales , Antígenos Bacterianos/toxicidad , Proteínas de la Membrana Bacteriana Externa/toxicidad , Muerte Celular/genética , Células Cultivadas , Embrión de Mamíferos , Regulación Bacteriana de la Expresión Génica/fisiología , Inflamación/inducido químicamente , Inflamación/patología , Lipopolisacáridos/farmacología , Lipoproteínas/metabolismo , Lipoproteínas/toxicidad , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Óxido Nítrico/metabolismo , Prosencéfalo/citología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genéticaRESUMEN
Brucella abortus is able to persist inside the host despite the development of potent CD8+ T-cell responses. We have recently reported the ability of B. abortus to inhibit the interferon-γ-induced major histocompatibility complex (MHC)-I cell surface expression on human monocytes. This phenomenon was due to the B. abortus-mediated retention of MHC-I molecules within the Golgi apparatus and was dependent on bacterial viability. However, the implications of bacterial virulence or replicative capacity and the signaling pathways remained unknown. Here we demonstrated that the B. abortus mutant strains RB51 and virB10- are able to inhibit MHC-I expression in the same manner as wild-type B. abortus, even though they are unable to persist inside human monocytes for a long period of time. Consistent with this, the phenomenon was triggered early in time and could be observed at 8 h postinfection. At 24 and 48 h, it was even stronger. Regarding the signaling pathway, targeting epidermal growth factor (EGF) receptor (EGFR), ErbB2 (HER2) or inhibition of tumor necrosis factor-α-converting enzyme, one of the enzymes which generates soluble EGF-like ligands, resulted in partial recovery of MHC-I surface expression. Moreover, recombinant EGF and transforming growth factor-α as well as the combination of both were also able to reproduce the B. abortus-induced MHC-I downmodulation. Finally, when infection was performed in the presence of an extracellular signal-regulated kinase 1/2 (Erk1/2) inhibitor, MHC-I surface expression was significantly recovered. Overall, these results describe how B. abortus evades CD8+ T-cell responses early during infection and exploits the EGFR-ERK signaling pathway to escape from the immune system and favor chronicity.
Asunto(s)
Brucella abortus/inmunología , Brucelosis/inmunología , Linfocitos T CD8-positivos/inmunología , Receptores ErbB/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Monocitos/inmunología , Animales , Brucella abortus/patogenicidad , Brucelosis/microbiología , Linfocitos T CD8-positivos/microbiología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Evasión Inmune , Ratones , Ratones Endogámicos C57BL , Microbiología , Transducción de Señal , Células THP-1 , Regulación hacia ArribaRESUMEN
Alveolar macrophages (AM) seem to constitute the main cellular target of inhaled brucellae. Here, we show that Brucella abortus invades and replicates in murine AM without inducing cytotoxicity. B. abortus infection induced a statistically significant increase of tumor necrosis factor alpha (TNF-α), CXCL1 or keratinocyte chemoattractant (KC), interleukin-1ß (IL-1ß), IL-6, and IL-12 in AM from C57BL/6 mice and BALB/c mice, but these responses were generally weaker and/or delayed compared to those elicited in peritoneal macrophages. Studies using knockout mice for TLR2, TLR4, and TLR9 revealed that TNF-α and KC responses were mediated by TLR2 recognition. Brucella infection reduced in a multiplicity of infection-dependent manner the expression of major histocompatibility complex class II (MHC-II) molecules induced by gamma interferon (IFN-γ) in AM. The same phenomenon was induced by incubation with heat-killed B. abortus (HKBA) or the lipidated form of the 19-kDa outer membrane protein of Brucella (L-Omp19), and it was shown to be mediated by TLR2 recognition. In contrast, no significant downregulation of MHC-II was induced by either unlipidated Omp19 or Brucella LPS. In a functional assay, treatment of AM with either L-Omp19 or HKBA reduced the MHC-II-restricted presentation of OVA peptides to specific T cells. One week after intratracheal infection, viable B. abortus was detected in AM from both wild-type and TLR2 KO mice, but CFU counts were higher in the latter. These results suggest that B. abortus survives in AM after inhalatory infection in spite of a certain degree of immune control exerted by the TLR2-mediated inflammatory response. Both the modest nature of the latter and the modulation of MHC-II expression by the bacterium may contribute to such survival.
Asunto(s)
Brucella abortus/inmunología , Antígenos de Histocompatibilidad Clase II/biosíntesis , Antígenos de Histocompatibilidad Clase II/inmunología , Macrófagos Alveolares/inmunología , Macrófagos Alveolares/microbiología , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Animales , Citocinas/metabolismo , Regulación hacia Abajo , Femenino , Evasión Inmune , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Viabilidad Microbiana , Receptor Toll-Like 2/genéticaRESUMEN
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
Brucella abortus elicits a vigorous Th1 immune response which activates cytotoxic T lymphocytes. However, B. abortus persists in its hosts in the presence of CD8(+) T cells, establishing a chronic infection. Here, we report that B. abortus infection of human monocytes/macrophages inhibited the IFN-γ-induced MHC-I cell surface expression. This phenomenon was dependent on metabolically active viable bacteria. MHC-I down-modulation correlated with the development of diminished CD8(+) cytotoxic T cell response as evidenced by the reduced expression of the activation marker CD107a on CD8(+) T lymphocytes and a diminished percentage of IFN-γ-producing CD8(+) T cells. Inhibition of MHC-I expression was not due to changes in protein synthesis. Rather, we observed that upon B. abortus infection MHC-I molecules were retained within the Golgi apparatus. Overall, these results describe a novel mechanism based on the intracellular sequestration of MHC-I molecules whereby B. abortus would avoid CD8(+) cytotoxic T cell responses, evading their immunological surveillance.
Asunto(s)
Brucella abortus/inmunología , Brucella abortus/fisiología , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Evasión Inmune , Macrófagos/inmunología , Macrófagos/microbiología , Células Cultivadas , Aparato de Golgi/química , Humanos , Interferón gamma/metabolismo , Transporte de ProteínasRESUMEN
To begin to optimize the immunization routes for our reported PLGA-rMOMP nanovaccine [PLGA-encapsulated Chlamydia muridarum (Cm) recombinant major outer membrane protein (rMOMP)], we compared two prime-boost immunization strategies [subcutaneous (SC) and intramuscular (IM-p) prime routes followed by two SC-boosts)] to evaluate the nanovaccine-induced protective efficacy and immunogenicity in female BALB/c mice. Our results showed that mice immunized via the SC and IM-p routes were protected against a Cm genital challenge by a reduction in bacterial burden and with fewer bacteria in the SC mice. Protection of mice correlated with rMOMP-specific Th1 (IL-2 and IFN-γ) and not Th2 (IL-4, IL-9, and IL-13) cytokines, and CD4+ memory (CD44highCD62Lhigh) T-cells, especially in the SC mice. We also observed higher levels of IL-1α, IL-6, IL-17, CCL-2, and G-CSF in SC-immunized mice. Notably, an increase of cytokines/chemokines was seen after the challenge in the SC, IM-p, and control mice (rMOMP and PBS), suggesting a Cm stimulation. In parallel, rMOMP-specific Th1 (IgG2a and IgG2b) and Th2 (IgG1) serum, mucosal, serum avidity, and neutralizing antibodies were more elevated in SC than in IM-p mice. Overall, the homologous SC prime-boost immunization of mice induced enhanced cellular and antibody responses with better protection against a genital challenge compared to the heterologous IM-p.
Asunto(s)
Anticuerpos Antibacterianos , Vacunas Bacterianas , Infecciones por Chlamydia , Chlamydia muridarum , Citocinas , Ratones Endogámicos BALB C , Animales , Femenino , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/administración & dosificación , Chlamydia muridarum/inmunología , Citocinas/metabolismo , Infecciones por Chlamydia/prevención & control , Infecciones por Chlamydia/inmunología , Ratones , Anticuerpos Antibacterianos/sangre , Inyecciones Intramusculares , Copolímero de Ácido Poliláctico-Ácido Poliglicólico/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/administración & dosificación , Inmunización Secundaria , Modelos Animales de Enfermedad , Inmunogenicidad Vacunal , Inyecciones Subcutáneas , Nanopartículas/administración & dosificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/administración & dosificación , Eficacia de las Vacunas , Células TH1/inmunología , NanovacunasRESUMEN
Brucella abortus is recognized by several Toll-like receptor (TLR)-associated pathways triggering proinflammatory responses that affect both the nature and intensity of the immune response. Previously, we demonstrated that B. abortus-mediated dendritic cell (DC) maturation and control of infection are dependent on the adaptor molecule MyD88. However, the involvement of all TLRs in response to B. abortus infection is not completely understood. Therefore, we decided to evaluate the requirement for TLR6 in host resistance to B. abortus. Here, we demonstrated that TLR6 is an important component for triggering an innate immune response against B. abortus. An in vitro luciferase assay indicated that TLR6 cooperates with TLR2 to sense Brucella and further activates NF-κB signaling. However, in vivo analysis showed that TLR6, not TLR2, is required for the efficient control of B. abortus infection. Additionally, B. abortus-infected dendritic cells require TLR6 to induce tumor necrosis factor alpha (TNF-α) and interleukin-12 (IL-12). Furthermore, our findings demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway is impaired in TLR2, TLR6, and TLR2/6 knockout (KO) DCs when infected with B. abortus, which may account for the lower proinflammatory cytokine production observed in TLR6 KO mouse dendritic cells. In summary, the results presented here indicate that TLR6 is required to trigger innate immune responses against B. abortus in vivo and is required for the full activation of DCs to induce robust proinflammatory cytokine production.
Asunto(s)
Brucella abortus/inmunología , Brucelosis/inmunología , Inmunidad Innata/fisiología , Receptor Toll-Like 6/fisiología , Análisis de Varianza , Animales , Citocinas/metabolismo , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/fisiología , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Bazo/microbiología , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 6/deficienciaRESUMEN
BACKGROUND: Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. We have recently demonstrated that B. abortus infects microglia and astrocytes, eliciting the production of a variety of pro-inflammatory cytokines which contribute to CNS damage. Matrix metalloproteinases (MMP) have been implicated in inflammatory tissue destruction in a range of pathological situations in the CNS. Increased MMP secretion is induced by pro-inflammatory cytokines in a variety of CNS diseases characterized by tissue-destructive pathology. METHODS: In this study, the molecular mechanisms that regulate MMP secretion from Brucella-infected astrocytes in vitro were investigated. MMP-9 was evaluated in culture supernatants by ELISA, zymography and gelatinolytic activity. Involvement of mitogen-activated protein kinases (MAPK) signaling pathways was evaluated by Western blot and using specific inhibitors. The role of TNF-α was evaluated by ELISA and by assays with neutralizing antibodies. RESULTS: B. abortus infection induced the secretion of MMP-9 from murine astrocytes in a dose-dependent fashion. The phenomenon was independent of bacterial viability and was recapitulated by L-Omp19, a B. abortus lipoprotein model, but not its LPS. B. abortus and L-Omp19 readily activated p38 and Erk1/2 MAPK, thus enlisting these pathways among the kinase pathways that the bacteria may address as they invade astrocytes. Inhibition of p38 or Erk1/2 significantly diminished MMP-9 secretion, and totally abrogated production of this MMP when both MAPK pathways were inhibited simultaneously. A concomitant abrogation of B. abortus- and L-Omp19-induced TNF-α production was observed when p38 and Erk1/2 pathways were inhibited, indicating that TNF-α could be implicated in MMP-9 secretion. MMP-9 secretion induced by B. abortus or L-Omp19 was completely abrogated when experiments were conducted in the presence of a TNF-α neutralizing antibody. MMP-9 activity was detected in cerebrospinal fluid (CSF) samples from patients suffering from neurobrucellosis. CONCLUSIONS: Our results indicate that the inflammatory response elicited by B. abortus in astrocytes would lead to the production of MMP-9 and that MAPK may play a role in this phenomenon. MAPK inhibition may thus be considered as a strategy to control inflammation and CNS damage in neurobrucellosis.
Asunto(s)
Brucella abortus , Brucelosis/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Proteínas Quinasas Activadas por Mitógenos/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/fisiología , Animales , Anticuerpos Bloqueadores/farmacología , Antígenos Bacterianos/fisiología , Astrocitos/metabolismo , Astrocitos/microbiología , Astrocitos/fisiología , Proteínas de la Membrana Bacteriana Externa/fisiología , Citocinas/metabolismo , Gelatinasas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , Lipopolisacáridos/farmacología , Lipoproteínas/farmacología , Lipoproteínas/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Endogámicos BALB C , Cultivo Primario de Células , Transducción de Señal/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/fisiologíaRESUMEN
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
Inflammation plays a key role in the pathogenesis of neurobrucellosis where glial cell interactions are at the root of this pathological condition. In this study, we present evidence indicating that soluble factors secreted by Brucella abortus-infected astrocytes activate microglia to induce neuronal death. Culture supernatants (SN) from B. abortus-infected astrocytes induce the release of pro-inflammatory mediators and the increase of the microglial phagocytic capacity, which are two key features in the execution of live neurons by primary phagocytosis, a recently described mechanism whereby B. abortus-activated microglia kills neurons by phagocytosing them. IL-6 neutralization completely abrogates neuronal loss. IL-6 is solely involved in increasing the phagocytic capacity of activated microglia as induced by SN from B. abortus-infected astrocytes and does not participate in their inflammatory activation. Both autocrine microglia-derived and paracrine astrocyte-secreted IL-6 endow microglial cells with up-regulated phagocytic capacity that allows them to phagocytose neurons. Blocking of IL-6 signaling by soluble gp130 abrogates microglial phagocytosis and concomitant neuronal death, indicating that IL-6 activates microglia via trans-signaling. Altogether, these results demonstrate that soluble factors secreted by B. abortus-infected astrocytes activate microglia to induce, via IL-6 trans-signaling, the death of neurons. IL-6 signaling inhibition may thus be considered a strategy to control inflammation and CNS damage in neurobrucellosis.
Asunto(s)
Brucella abortus , Microglía , Humanos , Microglía/fisiología , Astrocitos/metabolismo , Interleucina-6/metabolismo , Inflamación/metabolismoRESUMEN
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
Knowing the inherent stimulatory properties of the lipid moiety of bacterial lipoproteins, we first hypothesized that Brucella abortus outer membrane protein (Omp)16 lipoprotein would be able to elicit a protective immune response without the need of external adjuvants. In this study, we demonstrate that Omp16 administered by the i.p. route confers significant protection against B. abortus infection and that the protective response evoked is independent of the protein lipidation. To date, Omp16 is the first Brucella protein that without the requirement of external adjuvants is able to induce similar protection levels to the control live vaccine S19. Moreover, the protein portion of Omp16 (unlipidated Omp16 [U-Omp16]) elicits a protective response when administered by the oral route. Either systemic or oral immunization with U-Omp16 elicits a Th1-specific response. These abilities of U-Omp16 indicate that it is endowed with self-adjuvanting properties. The adjuvanticity of U-Omp16 could be explained, at least in part, by its capacity to activate dendritic cells in vivo. U-Omp16 is also able to stimulate dendritic cells and macrophages in vitro. The latter property and its ability to induce a protective Th1 immune response against B. abortus infection have been found to be TLR4 dependent. The facts that U-Omp16 is an oral protective Ag and possesses a mucosal self-adjuvanting property led us to develop a plant-made vaccine expressing U-Omp16. Our results indicate that plant-expressed recombinant U-Omp16 is able to confer protective immunity, when given orally, indicating that a plant-based oral vaccine expressing U-Omp16 could be a valuable approach to controlling this disease.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacuna contra la Brucelosis/inmunología , Brucelosis/prevención & control , Células Dendríticas/inmunología , Interacciones Huésped-Patógeno/inmunología , Células TH1/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/genética , Administración Oral , Animales , Antígenos Bacterianos/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , Vacuna contra la Brucelosis/administración & dosificación , Brucelosis/inmunología , Diferenciación Celular/inmunología , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Adyuvante de Freund/administración & dosificación , Interacciones Huésped-Patógeno/genética , Inmunidad Celular , Inyecciones Intraperitoneales , Lípidos/administración & dosificación , Lipoproteínas/administración & dosificación , Lipoproteínas/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Células TH1/microbiología , Nicotiana/genética , Nicotiana/inmunologíaRESUMEN
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
Central nervous system (CNS) invasion by bacteria of the genus Brucella results in an inflammatory disorder called neurobrucellosis. In this study we present in vivo and in vitro evidence that B. abortus and its lipoproteins activate the innate immunity of the CNS, eliciting an inflammatory response that leads to astrogliosis, a characteristic feature of neurobrucellosis. Intracranial injection of heat-killed B. abortus (HKBA) or outer membrane protein 19 (Omp19), a B. abortus lipoprotein model, induced astrogliosis in mouse striatum. Moreover, infection of astrocytes and microglia with B. abortus induced the secretion of interleukin (IL)-6, IL-1beta, tumor necrosis factor (TNF)-alpha, macrophage chemoattractant protein-1, and KC (CXCL1). HKBA also induced these inflammatory mediators, suggesting the involvement of a structural component of the bacterium. Accordingly, Omp19 induced the same cytokine and chemokine secretion pattern. B. abortus infection induced astrocyte, but not microglia, apoptosis. Indeed, HKBA and Omp19 elicited not only astrocyte apoptosis but also proliferation, two features observed during astrogliosis. Apoptosis induced by HKBA and L-Omp19 was completely suppressed in cells of TNF receptor p55-/- mice or when the general caspase inhibitor Z-VAD-FMK was added to cultures. Hence, TNF-alpha signaling via TNF receptor (TNFR) 1 through the coupling of caspases determines apoptosis. Our results provide proof of the principle that Brucella lipoproteins could be key virulence factors in neurobrucellosis and that astrogliosis might contribute to neurobrucellosis pathogenesis.