RESUMO
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.
Assuntos
Anticorpos Antibacterianos , Vacinas Bacterianas , Infecções por Chlamydia , Chlamydia muridarum , Citocinas , Camundongos Endogâmicos BALB C , Animais , Feminino , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Chlamydia muridarum/imunologia , Citocinas/metabolismo , Infecções por Chlamydia/prevenção & controle , Infecções por Chlamydia/imunologia , Camundongos , Anticorpos Antibacterianos/sangue , Injeções Intramusculares , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Sintéticas/imunologia , Vacinas Sintéticas/administração & dosagem , Imunização Secundária , Modelos Animais de Doenças , Imunogenicidade da Vacina , Injeções Subcutâneas , Nanopartículas/administração & dosagem , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/administração & dosagem , Eficácia de Vacinas , Células Th1/imunologia , NanovacinasRESUMO
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.
Assuntos
Brucella abortus , Microglia , Humanos , Microglia/fisiologia , Astrócitos/metabolismo , Interleucina-6/metabolismo , Inflamação/metabolismoRESUMO
Recently we reported the immune-potentiating capacity of a Chlamydia nanovaccine (PLGA-rMOMP) comprising rMOMP (recombinant major outer membrane protein) encapsulated in extended-releasing PLGA [poly (D, L-lactide-co-glycolide) (85:15)] nanoparticles. Here we hypothesized that PLGA-rMOMP would bolster immune-effector mechanisms to confer protective efficacy in mice against a Chlamydia muridarum genital challenge and re-challenge. Female BALB/c mice received three immunizations, either subcutaneously (SC) or intranasally (IN), before receiving an intravaginal challenge with C. muridarum on day 49 and a re-challenge on day 170. Both the SC and IN immunization routes protected mice against genital challenge with enhanced protection after a re-challenge, especially in the SC mice. The nanovaccine induced robust antigen-specific Th1 (IFN-γ, IL-2) and IL-17 cytokines plus CD4+ proliferating T-cells and memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) phenotypes in immunized mice. Parallel induction of antigen-specific systemic and mucosal Th1 (IgG2a, IgG2b), Th2 (IgG1), and IgA antibodies were also noted. Importantly, immunized mice produced highly functional Th1 avidity and serum antibodies that neutralized C. muridarum infectivity of McCoy fibroblasts in-vitro that correlated with their respective protection levels. The SC, rather than the IN immunization route, triggered higher cellular and humoral immune effectors that improved mice protection against genital C. muridarum. We report for the first time that the extended-releasing PLGA 85:15 encapsulated rMOMP nanovaccine confers protective immunity in mice against genital Chlamydia and advances the potential towards acquiring a nano-based Chlamydia vaccine.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Infecções por Chlamydia/prevenção & controle , Chlamydia muridarum/efeitos dos fármacos , Preparações de Ação Retardada/administração & dosagem , Genitália/efeitos dos fármacos , Nanopartículas/química , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/sangue , Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Peptídeos Catiônicos Antimicrobianos/química , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/genética , Vacinas Bacterianas/administração & dosagem , Citocinas/imunologia , Feminino , Genitália/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , VacinaçãoRESUMO
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.
Assuntos
Plaquetas/microbiologia , Brucella abortus , Brucelose , Monócitos/imunologia , Neutrófilos/imunologia , HumanosRESUMO
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.
Assuntos
Imunidade Adaptativa/genética , Proteínas da Membrana Bacteriana Externa/genética , Nanopartículas/química , Vacinas/imunologia , Imunidade Adaptativa/imunologia , Animais , Proteínas da Membrana Bacteriana Externa/imunologia , Antígenos CD4/química , Antígenos CD4/imunologia , Chlamydia/genética , Chlamydia/imunologia , Chlamydia/patogenicidade , Células Dendríticas/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Receptores de Hialuronatos/química , Receptores de Hialuronatos/imunologia , Subunidade p40 da Interleucina-12/genética , Subunidade p40 da Interleucina-12/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Selectina L/química , Selectina L/imunologia , Camundongos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/imunologia , Linfócitos T/imunologia , Vacinas/genéticaRESUMO
Brucella abortus, the causative agent of brucellosis, displays many resources to evade T cell responses conducive to persist inside the host. Our laboratory has previously showed that infection of human monocytes with B. abortus down-modulates the IFN-γ-induced MHC-II expression. Brucella outer membrane lipoproteins are structural components involved in this phenomenon. Moreover, IL-6 is the soluble factor that mediated MHC-II down-regulation. Yet, the MHC-II down-regulation exerted by lipoproteins was less marked than the one observed as consequence of infection. This led us to postulate that there should be other components associated with viable bacteria that may act together with lipoproteins in order to diminish MHC-II. Our group has recently demonstrated that B. abortus RNA (PAMP related to pathogens' viability or vita-PAMP) is involved in MHC-I down-regulation. Therefore, in this study we investigated if B. abortus RNA could be contributing to the down-regulation of MHC-II. This PAMP significantly down-modulated the IFN-γ-induced MHC-II surface expression on THP-1 cells as well as in primary human monocytes and murine bone marrow macrophages. The expression of other molecules up-regulated by IFN-γ (such as co-stimulatory molecules) was stimulated on monocytes treated with B. abortus RNA. This result shows that this PAMP does not alter all IFN-γ-induced molecules globally. We also showed that other bacterial and parasitic RNAs caused MHC-II surface expression down-modulation indicating that this phenomenon is not restricted to B. abortus. Moreover, completely degraded RNA was also able to reproduce the phenomenon. MHC-II down-regulation on monocytes treated with RNA and L-Omp19 (a prototypical lipoprotein of B. abortus) was more pronounced than in monocytes stimulated with both components separately. We also demonstrated that B. abortus RNA along with its lipoproteins decrease MHC-II surface expression predominantly by a mechanism of inhibition of MHC-II expression. Regarding the signaling pathway, we demonstrated that IL-6 is a soluble factor implicated in B. abortus RNA and lipoproteins-triggered MHC-II surface down-regulation. Finally, CD4+ T cells functionality was affected as macrophages treated with these components showed lower antigen presentation capacity. Therefore, B. abortus RNA and lipoproteins are two PAMPs that contribute to MHC-II down-regulation on monocytes/macrophages diminishing CD4+ T cell responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Macrófagos/imunologia , Monócitos/imunologia , RNA Bacteriano/imunologia , Animais , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Brucella abortus/genética , Brucella abortus/imunologia , Brucella abortus/fisiologia , Brucelose/imunologia , Brucelose/microbiologia , Linfócitos T CD4-Positivos/metabolismo , Células Cultivadas , Regulação para Baixo/imunologia , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Monócitos/metabolismo , Moléculas com Motivos Associados a Patógenos/imunologia , Moléculas com Motivos Associados a Patógenos/metabolismo , RNA Bacteriano/genética , Células THP-1RESUMO
Neurobrucellosis, which is the most morbid form of brucellosis disease, presents with inflammatory signs and symptoms. Recent experimental evidence clearly indicates that deregulation of astrocytes and microglia caused by Brucella infection creates a microenvironment in the central nervous system (CNS) in which secretion of pro-inflammatory mediators lead to destabilization of the glial structure, the damage of the blood brain barrier (BBB) and neuronal demise. This review of Brucella interactions with cells of the CNS and the BBB is intended to present recent immunological findings that can explain, at least in part, the basis for the inflammatory pathogenesis of the nervous system that takes place upon Brucella infection.
Assuntos
Barreira Hematoencefálica/imunologia , Brucelose/imunologia , Sistema Nervoso Central/imunologia , Imunidade Inata/fisiologia , Fatores Imunológicos/imunologia , Animais , Barreira Hematoencefálica/metabolismo , Brucelose/metabolismo , Sistema Nervoso Central/metabolismo , Humanos , Fatores Imunológicos/metabolismoRESUMO
Brucella infection activates the immune system and favors the differentiation of CD4+ and CD8+ T cells. To persist during a long time inside macrophages evading immune surveillance of these T cells the pathogen must exploit different evasion strategies. We review the mechanisms whereby Brucella, through TLR signaling, inhibits MHC class I and II antigen presentation, allowing infected macrophages to become effective niches for Brucella survival.
Assuntos
Apresentação de Antígeno/imunologia , Brucella/imunologia , Brucelose/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Evasão da Resposta Imune , Animais , Brucelose/microbiologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Humanos , Macrófagos/imunologia , Macrófagos/microbiologia , Transdução de Sinais , Receptores Toll-Like/metabolismoRESUMO
Recently, we reported that our PPM chlamydial nanovaccine [a biodegradable co-polymeric PLA-PEG (poly(lactic acid)-poly(ethylene glycol))-encapsulated M278 peptide (derived from the major outer membrane protein (MOMP) of Chlamydia)] exploits the caveolin-mediated endocytosis pathway for endosomal processing and MHC class II presentation to immune-potentiate Chlamydia-specific CD4+ T-cell immune effector responses. In the present study, we employed the Chlamydia muridarum mouse infection model to evaluate the protective efficacy of PPM against a genital tract challenge. Our results show that mice immunized with PPM were significantly protected against a homologous genital tract challenge evidently by reduced vaginal bacterial loads. Protection of mice correlated with enhanced Chlamydia-specific adaptive immune responses predominated by IFN-γ along with CD4+ T-cells proliferation and their differentiation to CD4+ memory (CD44high CD62Lhigh) and effector (CD44high CD62Llow) T-cell phenotypes. We observed the elevation of M278- and MOMP-specific serum antibodies with high avidity in the ascending order IgG1 > IgG2b > IgG2a. A key finding was the elevated mucosal IgG1 and IgA antibody titers followed by an increase in MOMP-specific IgA after the challenge. The Th1/Th2 antibody titer ratios (IgG2a/IgG1 and IgG2b/IgG1) revealed that PPM evoked a Th2-directed response, which skewed to a Th1-dominated antibody response after the bacterial challenge of mice. In addition, PPM immune sera neutralized the infectivity of C. muridarum in McCoy cells, suggesting the triggering of functional neutralizing antibodies. Herein, we reveal for the first time that subcutaneous immunization with the self-adjuvanting biodegradable co-polymeric PPM nanovaccine immune-potentiated robust CD4+ T cell-mediated immune effector responses; a mixed Th1 and Th2 antibody response and local mucosal IgA to protect mice against a chlamydial genital tract challenge.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Vacinas Bacterianas/imunologia , Infecções por Chlamydia/imunologia , Infecções por Chlamydia/microbiologia , Chlamydia muridarum/imunologia , Imunidade nas Mucosas , Adjuvantes Imunológicos , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos/imunologia , Vacinas Bacterianas/administração & dosagem , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Imunização , Imunoglobulina G/imunologia , Memória Imunológica , Lactatos , Camundongos , Testes de Neutralização , Polietilenoglicóis , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vagina/imunologia , Vagina/microbiologiaRESUMO
Neurobrucellosis is an inflammatory disease caused by the invasion of Brucella spp. to the central nervous system (CNS). The pathogenesis of the disease is not well characterized; however, for Brucella to gain access to the brain parenchyma, traversing of the blood-brain barrier (BBB) must take place. To understand the CNS determinants of the pathogenesis of B. abortus, we have used the in vitro BBB model of human brain microvascular endothelial cells (HBMEC) to study the interactions between B. abortus and brain endothelial cells. In this study, we showed that B. abortus is able to adhere and invade HBMEC which was dependent on microtubules, microfilaments, endosome acidification and de novo protein synthesis. After infection, B. abortus rapidly escapes the endosomal compartment of HBMEC and forms a replicative Brucella-containing vacuole that involves interactions with the endoplasmic reticulum. Despite the ability of B. abortus to invade and replicate in HBMEC, the bacterium was unable by itself to traverse HBMEC, but could traverse polarized HBMEC monolayers within infected monocytes. Importantly, infected monocytes that traversed the HBMEC monolayer were a bacterial source for de novo infection of glial cells. This is the first demonstration of the mechanism whereby B. abortus is able to traverse the BBB and infect cells of the CNS. These results may have important implications in our understanding of the pathogenesis of neurobrucellosis.
Assuntos
Barreira Hematoencefálica/microbiologia , Brucella abortus/crescimento & desenvolvimento , Células Endoteliais/microbiologia , Leucócitos Mononucleares/microbiologia , Microvasos/microbiologia , Animais , Barreira Hematoencefálica/citologia , Brucella abortus/fisiologia , Brucelose/microbiologia , Retículo Endoplasmático/microbiologia , Endossomos/microbiologia , Células Endoteliais/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microvasos/citologia , Cultura Primária de Células , Transcitose/fisiologia , Vacúolos/microbiologiaRESUMO
Brucellosis is an infectious disease elicited by bacteria of the genus Brucella. Platelets have been extensively described as mediators of hemostasis and responsible for maintaining vascular integrity. Nevertheless, they have been recently involved in the modulation of innate and adaptive immune responses. Although many interactions have been described between Brucella abortus and monocytes/macrophages, the role of platelets during monocyte/macrophage infection by these bacteria remained unknown. The aim of this study was to investigate the role of platelets in the immune response against B. abortus. We first focused on the possible interactions between B. abortus and platelets. Bacteria were able to directly interact with platelets. Moreover, this interaction triggered platelet activation, measured as fibrinogen binding and P-selectin expression. We further investigated whether platelets were involved in Brucella-mediated monocyte/macrophage early infection. The presence of platelets promoted the invasion of monocytes/macrophages by B. abortus. Moreover, platelets established complexes with infected monocytes/macrophages as a result of a carrier function elicited by platelets. We also evaluated the ability of platelets to modulate functional aspects of monocytes in the context of the infection. The presence of platelets during monocyte infection enhanced IL-1ß, TNF-α, IL-8, and MCP-1 secretion while it inhibited the secretion of IL-10. At the same time, platelets increased the expression of CD54 (ICAM-1) and CD40. Furthermore, we showed that soluble factors released by B. abortus-activated platelets, such as soluble CD40L, platelet factor 4, platelet-activating factor, and thromboxane A2, were involved in CD54 induction. Overall, our results indicate that platelets can directly sense and react to B. abortus presence and modulate B. abortus-mediated infection of monocytes/macrophages increasing their pro-inflammatory capacity, which could promote the resolution of the infection.
Assuntos
Plaquetas/citologia , Brucella abortus/fisiologia , Comunicação Celular/imunologia , Monócitos/imunologia , Brucella abortus/imunologia , Antígeno CD56/imunologia , Linhagem Celular , Células Cultivadas , Quimiocina CCL2/imunologia , Humanos , Interleucina-10/imunologia , Interleucina-8/imunologia , Monócitos/microbiologia , Células THP-1 , Fator de Necrose Tumoral alfa/imunologiaRESUMO
We previously developed a Chlamydia trachomatis nanovaccine (PPM) by encapsulating a chlamydial M278 peptide within poly(lactic acid)-poly(ethylene glycol) biodegradable nanoparticles that immunopotentiated Chlamydia-specific immune effector responses in mice. Herein, we investigated the mechanistic interactions of PPM with mouse bone marrow-derived dendritic cells (DCs) for its uptake, trafficking, and T cell activation. Our results reveal that PPM triggered enhanced expression of effector cytokines and chemokines, surface activation markers (Cd1d2, Fcgr1), pathogen-sensing receptors (TLR2, Nod1), co-stimulatory (CD40, CD80, CD86) and MHC class I and II molecules. Co-culturing of PPM-primed DCs with T cells from C. muridarum vaccinated mice yielded an increase in Chlamydia-specific immune effector responses including CD3+ lymphoproliferation, CD3+CD4+ IFN-γ-secreting cells along with CD3+CD4+ memory (CD44high and CD62Lhigh) and effector (CD44high and CD62Llow) phenotypes. Intracellular trafficking analyses revealed an intense expression and colocalization of PPM predominantly in endosomes. PPM also upregulated the transcriptional and protein expression of the endocytic mediator, caveolin-1 in DCs. More importantly, the specific inhibition of caveolin-1 led to decreased expression of PPM-induced cytokines and co-stimulatory molecules. Our investigation shows that PPM provided enhancement of uptake, probably by exploiting the caveolin-mediated endocytosis pathway, endosomal processing, and MHC II presentation to immunopotentiate Chlamydia-specific immune effector responses mediated by CD4+ T cells.
Assuntos
Endocitose/fisiologia , Lactatos/química , Nanopartículas/química , Polietilenoglicóis/química , Animais , Linfócitos T CD4-Positivos , Caveolinas/metabolismo , Proliferação de Células/fisiologia , Chlamydia muridarum/imunologia , Células Dendríticas/imunologia , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Brucelose/imunologia , Receptores ErbB/imunologia , Evasão da Resposta Imune/imunologia , Monócitos/imunologia , RNA Bacteriano/imunologia , Receptor 8 Toll-Like/imunologia , Animais , Brucella abortus/imunologia , Apresentação Cruzada/imunologia , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe I/biossíntese , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Monócitos/microbiologia , Transdução de Sinais/imunologiaRESUMO
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.
Assuntos
Brucella abortus/patogenicidade , Morte Celular/fisiologia , Inflamação/metabolismo , Microglia/microbiologia , Microglia/fisiologia , Neurônios/patologia , Fagocitose/fisiologia , Animais , Antígenos de Bactérias/toxicidade , Proteínas da Membrana Bacteriana Externa/toxicidade , Morte Celular/genética , Células Cultivadas , Embrião de Mamíferos , Regulação Bacteriana da Expressão Gênica/fisiologia , Inflamação/induzido quimicamente , Inflamação/patologia , Lipopolissacarídeos/farmacologia , Lipoproteínas/metabolismo , Lipoproteínas/toxicidade , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Óxido Nítrico/metabolismo , Prosencéfalo/citologia , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/deficiência , Receptor 4 Toll-Like/genéticaRESUMO
Osteoarticular brucellosis is the most common presentation of human active disease although its prevalence varies widely. The three most common forms of osteoarticular involvement are sacroiliitis, spondylitis, and peripheral arthritis. The molecular mechanisms implicated in bone damage have been recently elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and the receptor activator of nuclear factor kappa-B ligand (RANKL)-the natural modulator of bone homeostasis are involved. These processes are driven by inflammatory cells, like monocytes/macrophages, neutrophils, Th17 CD4+ T, and B cells. In addition, Brucella abortus has a direct effect on osteoarticular cells and tilts homeostatic bone remodeling. These bacteria inhibit bone matrix deposition by osteoblasts (the only bone cells involved in bone deposition), and modify the phenotype of these cells to produce matrix metalloproteinases (MMPs) and cytokine secretion, contributing to bone matrix degradation. B. abortus also affects osteoclasts (cells naturally involved in bone resorption) by inducing an increase in osteoclastogenesis and osteoclast activation; thus, increasing mineral and organic bone matrix resorption, contributing to bone damage. Given that the pathology induced by Brucella species involved joint tissue, experiments conducted on synoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits synoviocyte apoptosis. Brucella is an intracellular bacterium that replicates preferentially in the endoplasmic reticulum of macrophages. The analysis of B. abortus-infected synoviocytes indicated that bacteria also replicate in their reticulum suggesting that they could use this cell type for intracellular replication during the osteoarticular localization of the disease. Finally, the molecular mechanisms of osteoarticular brucellosis discovered recently shed light on how the interaction between B. abortus and immune and osteoarticular cells may play an important role in producing damage in joint and bone.
RESUMO
Brucella abortus is an intracellular pathogen capable of surviving inside of macrophages. The success of B. abortus as a chronic pathogen relies on its ability to orchestrate different strategies to evade the adaptive CD4+ T cell responses that it elicits. Previously, we demonstrated that B. abortus inhibits the IFN-γ-induced surface expression of MHC class II (MHC-II) molecules on human monocytes, and this phenomenon correlated with a reduction in antigen presentation. However, the molecular mechanisms, whereby B. abortus is able to down-regulate the expression of MHC-II, remained to be elucidated. In this study, we demonstrated that B. abortus infection inhibits the IFN-γ-induced transcription of MHC-II, transactivator (CIITA) and MHC-II genes. Accordingly, we observed that the synthesis of MHC-II proteins was also diminished. B. abortus was not only able to reduce the expression of mature MHC-II, but it also inhibited the expression of invariant chain (Ii)-associated immature MHC-II molecules. Outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, diminished the expression of MHC-II and CIITA transcripts to the same extent as B. abortus infection. IL-6 contributes to these down-regulatory phenomena. In addition, B. abortus and its lipoproteins, through IL-6 secretion, induced the transcription of the negative regulators of IFN-γ signaling, suppressor of cytokine signaling (SOCS)-1 and -3, without interfering with STAT1 activation. Yet, B. abortus lipoproteins via IL-6 inhibit the expression of IFN regulatory factor 1 (IRF-1), a critical regulatory transcription factor for CIITA induction. Overall, these results indicate that B. abortus inhibits the expression of MHC-II molecules at very early points in their synthesis and in this way, may prevent recognition by T cells establishing a chronic infection.
Assuntos
Brucella abortus/fisiologia , Regulação para Baixo , Antígenos de Histocompatibilidade Classe II/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Interleucina-6/metabolismo , Proteínas Nucleares/antagonistas & inibidores , Transativadores/antagonistas & inibidores , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Brucelose/imunologia , Brucelose/microbiologia , Brucelose/patologia , Catepsinas/metabolismo , Linhagem Celular , Antígenos HLA-DR/imunologia , Humanos , Interferon gama/metabolismo , Espaço Intracelular/metabolismo , Lipoproteínas/imunologia , Lipoproteínas/metabolismo , Modelos Biológicos , Monócitos/microbiologia , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , Fator de Transcrição STAT1/metabolismo , Proteína 1 Supressora da Sinalização de Citocina/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transcrição GênicaRESUMO
The innate immune system is essential for the detection and elimination of bacterial pathogens. Upon inflammasome activation, caspase-1 cleaves pro-IL-1ß and pro-IL-18 to their mature forms IL-1ß and IL-18, respectively, and the cell undergoes inflammatory death termed pyroptosis. Here, we reviewed recent findings demonstrating that Brucella abortus ligands activate NLRP3 and AIM2 inflammasomes which lead to control of infection. This protective effect is due to the inflammatory response caused by IL-1ß and IL-18 rather than cell death. Brucella DNA is sensed by AIM2 and bacteria-induced mitochondrial reactive oxygen species is detected by NLRP3. However, deregulation of pro-inflammatory cytokine production can lead to immunopathology. Nervous system invasion by bacteria of the genus Brucella results in an inflammatory disorder termed neurobrucellosis. Herein, we discuss the mechanism of caspase-1 activation and IL-1ß secretion in glial cells infected with B. abortus. Our results demonstrate that the ASC inflammasome is indispensable for inducing the activation of caspase-1 and secretion of IL-1ß upon infection of astrocytes and microglia with Brucella. Moreover, our results demonstrate that secretion of IL-1ß by Brucella-infected glial cells depends on NLRP3 and AIM2 and leads to neurobrucellosis. Further, the inhibition of the host cell inflammasome as an immune evasion strategy has been described for bacterial pathogens. We discuss here that the bacterial type IV secretion system VirB is required for inflammasome activation in host cells during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes mainly NLRP3 and AIM2 that collectively orchestrate a robust caspase-1 activation and pro-inflammatory response.
Assuntos
Brucella abortus/imunologia , Brucelose/imunologia , Brucelose/metabolismo , Proteínas de Ligação a DNA/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Brucelose/microbiologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , DNA Bacteriano/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Imunidade InataRESUMO
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.
Assuntos
Brucella abortus/imunologia , Brucelose/imunologia , Linfócitos T CD8-Positivos/imunologia , Receptores ErbB/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Monócitos/imunologia , Animais , Brucella abortus/patogenicidade , Brucelose/microbiologia , Linfócitos T CD8-Positivos/microbiologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Evasão da Resposta Imune , Camundongos , Camundongos Endogâmicos C57BL , Microbiologia , Transdução de Sinais , Células THP-1 , Regulação para CimaRESUMO
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.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Brucella/imunologia , Brucelose/imunologia , Linfócitos T CD8-Positivos/fisiologia , Vacinas Anticâncer/imunologia , Catepsinas/metabolismo , Células Dendríticas/imunologia , Imunoterapia/métodos , Lipoproteínas/metabolismo , Lisossomos/metabolismo , Melanoma/terapia , Animais , Antígenos de Neoplasias/imunologia , Apresentação Cruzada , Feminino , Ativação Linfocitária , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
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.