RESUMEN
The widely administered tuberculosis (TB) vaccine, Bacillus Calmette-Guerin (BCG), is the only licensed vaccine, but has highly variable efficiency against childhood and pulmonary TB. Therefore, the BCG prime-boost strategy is a rational solution for the development of new TB vaccines. Studies have shown that Mycobacterium tuberculosis (Mtb) culture filtrates contain proteins that have promising vaccine potential. In this study, Rv1876 bacterioferritin was identified from the culture filtrate fraction with strong immunoreactivity. Its immunobiological potential has not been reported previously. We found that recombinant Rv1876 protein induced dendritic cells' (DCs) maturation by MAPK and NF-κB signaling activation, induced a T helper type 1 cell-immune response, and expanded the population of the effector/memory T cell. Boosting BCG with Rv1876 protein enhanced the BCG-primed Th1 immune response and reduced the bacterial load in the lung compared to those of BCG alone. Thus, Rv1876 is a good target for the prime-boost strategy.
Asunto(s)
Proteínas Bacterianas/inmunología , Células Dendríticas/inmunología , Inmunidad , Mycobacterium bovis/inmunología , Células TH1/inmunología , Animales , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/genética , Proliferación Celular , Citocinas/metabolismo , Femenino , Memoria Inmunológica , Activación de Linfocitos/inmunología , Ratones Endogámicos C57BL , Mutación/genética , Mycobacterium bovis/crecimiento & desarrollo , VacunaciónRESUMEN
Prostaglandin E2 (PGE2) is an important biological mediator involved in the defense against Mycobacterium tuberculosis (Mtb) infection. Currently, there are no reports on the mycobacterial components that regulate PGE2 production. Previously, we have reported that RpfE-treated dendritic cells (DCs) effectively expanded the Th1 and Th17 cell responses simultaneously; however, the mechanism underlying Th1 and Th17 cell differentiation is unclear. Here, we show that PGE2 produced by RpfE-activated DCs via the MAPK and cyclooxygenase 2 signaling pathways induces Th1 and Th17 cell responses mainly via the EP4 receptor. Furthermore, mice administered intranasally with PGE2 displayed RpfE-induced antigen-specific Th1 and Th17 responses with a significant reduction in bacterial load in the lungs. Furthermore, the addition of optimal PGE2 amount to IL-2-IL-6-IL-23p19-IL-1ß was essential for promoting differentiation into Th1/Th17 cells with strong bactericidal activity. These results suggest that RpfE-matured DCs produce PGE2 that induces Th1 and Th17 cell differentiation with potent anti-mycobacterial activity.
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Proteínas Bacterianas/metabolismo , Diferenciación Celular , Células Dendríticas/metabolismo , Dinoprostona/metabolismo , Mycobacterium tuberculosis/fisiología , Células TH1/citología , Células Th17/citología , Animales , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Femenino , Ratones , Ratones Endogámicos C57BL , Transducción de Señal , Células TH1/inmunología , Células Th17/inmunología , Tuberculosis/inmunología , Tuberculosis/metabolismo , Tuberculosis/microbiologíaRESUMEN
Although Mycobacterium tuberculosis (Mtb) is an intracellular pathogen in phagocytic cells, the factors and mechanisms by which they invade and persist in host cells are still not well understood. Characterization of the bacterial proteins modulating macrophage function is essential for understanding tuberculosis pathogenesis and bacterial virulence. Here we investigated the pathogenic role of the Rv2145c protein in stimulating IL-10 production. We first found that recombinant Rv2145c stimulated bone marrow-derived macrophages (BMDMs) to secrete IL-10, IL-6 and TNF-α but not IL-12p70 and to increase the expression of surface molecules through the MAPK, NF-κB, and TLR4 pathways and enhanced STAT3 activation and the expression of IL-10 receptor in Mtb-infected BMDMs. Rv2145c significantly enhanced intracellular Mtb growth in BMDMs compared with that in untreated cells, which was abrogated by STAT3 inhibition and IL-10 receptor (IL-10R) blockade. Expression of Rv2145c in Mycobacterium smegmatis (M. smegmatis) led to STAT3-dependent IL-10 production and enhancement of intracellular growth in BMDMs. Furthermore, the clearance of Rv2145c-expressing M. smegmatis in the lungs and spleens of mice was delayed, and these effects were abrogated by administration of anti-IL-10R antibodies. Finally, all mice infected with Rv2145c-expressing M. smegmatis died, but those infected with the vector control strain did not. Our data suggest that Rv2145c plays a role in creating a favorable environment for bacterial survival by modulating host signals.
Asunto(s)
Proteínas Bacterianas/inmunología , Mycobacterium tuberculosis/patogenicidad , Receptores de Interleucina-10/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Proteínas Bacterianas/genética , Interleucina-10/metabolismo , Activación de Macrófagos , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Viabilidad Microbiana/genética , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/inmunología , Mycobacterium smegmatis/patogenicidad , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/inmunología , Receptores de Interleucina-10/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Factor de Transcripción STAT3/antagonistas & inhibidores , Transducción de Señal , Receptor Toll-Like 4/metabolismo , VirulenciaRESUMEN
Mycobacterium tuberculosis contains diverse immunologically active components. This study investigated the biological function of a newly identified component, Rv1654, with the potential to induce apoptosis in macrophages. Recombinant Rv1654 induced macrophage apoptosis in a caspase-9/3-dependent manner through the production of reactive oxygen species (ROS) and interaction with Toll-like receptor 4. In addition, Rv1654 induced the production of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein-1 through the mitogen-activated protein kinase pathway. Furthermore, Rv1654-induced c-Jun N-terminal kinase (JNK) activation was inhibited by the ROS scavenger and Rv1654-induced apoptosis was inhibited by the JNK inhibitor. Moreover, it was found that treatment of macrophages with Rv1654 led to the loss of mitochondrial membrane potential, release of cytochrome c into the cytosol, and translocation of Bax into the mitochondria. Finally, Rv1654-mediated apoptosis was inhibited in macrophages transfected with Bax siRNA. These results suggest that Rv1654 induces macrophage apoptosis through a mitochondrial-dependent pathway and ROS-mediated JNK activation.
Asunto(s)
Apoptosis , Proteínas Bacterianas/inmunología , Macrófagos/microbiología , Mitocondrias , Mycobacterium tuberculosis , Caspasas , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteínas Recombinantes/inmunología , Receptores Toll-LikeRESUMEN
In Mycobacterium tuberculosis infection, naïve T cells that encounter mycobacterial antigens through dendritic cells (DCs) induce various CD4+ T-cell responses; therefore, appropriate DC activation is the key for protective immunity against tuberculosis. We previously found that Rv2299c-matured DCs induce Th1 differentiation with bactericidal activity. In this study, to prove that Rv2299c could enhance the protective immunity of other vaccine candidates comprising T-cell-stimulating antigens, Ag85B-ESAT6, a well-known vaccine candidate, was selected as a fusion partner of Rv2299c. Recombinant Rv2299c-Ag85B-ESAT6 protein induced DC maturation and activation. Furthermore, fusion of Rv2299c enhanced the protective efficacy of the Ag85B-ESAT6 vaccine in a mouse model and significantly higher production of TNF-α and IL-2 was detected in the lungs, spleen, and lymph nodes of the group immunized with the Rv2299c-fused protein than with Ag85B-ESAT6. In addition, fusion of Rv2299c enhanced the Ag85B-ESAT6-mediated expansion of multifunctional CD4+ T cells. These data suggested that the DC-activating protein Rv2299c may potentiate the protective immunity of the vaccine candidate comprising T cell antigens.
RESUMEN
Immunotherapy represents a promising approach for improving current antibiotic treatments through the engagement of the host's immune system. Latency-associated antigens have been included as components of multistage subunit tuberculosis vaccines. We first identified Rv2005c, a DosR regulon-encoded protein, as a seroreactive protein. In this study, we found that Rv2005c induced dendritic cell (DC) maturation and Th1 responses, and its expression by Mycobacterium tuberculosis (Mtb) within macrophages was enhanced by treatment with CoCl2, a hypoxia-mimetic agent. T cells activated by Rv2005c-matured DCs induced antimycobacterial activity in macrophages under hypoxic conditions but not under normoxic conditions. However, Rv2005c alone did not exhibit any significant vaccine efficacy in our mouse model. The fusion of Rv2005c to the macrophage-activating protein Rv2882c resulted in significant activation of DCs and antimycobacterial activity in macrophages, which were enhanced under hypoxic conditions. Furthermore, the Rv2882c-Rv2005c fusion protein showed significant adjunctive immunotherapeutic effects and led to the generation of long-lasting, antigen-specific, multifunctional CD4+ T cells that coproduced TNF-α, IFN-γ and IL-2 in the lungs of our established mouse model. Overall, these results provide a novel fusion protein with immunotherapeutic potential as adjunctive chemotherapy for tuberculosis.
RESUMEN
The antigen-specific Th17 responses in the lungs for improved immunity against Mycobacterium tuberculosis (Mtb) infection are incompletely understood. Tuberculosis (TB) vaccine candidate HSP90-ESAT-6 (E6), given as a Bacillus Calmette-Guérin (BCG)-prime boost regimen, confers superior long-term protection against the hypervirulent Mtb HN878 infection, compared to BCG or BCG-E6. Taking advantage of protective efficacy lead-out, we found that ESAT-6-specific multifunctional CD4+IFN-γ+IL-17+ T-cells optimally correlated with protection level against Mtb infection both pre-and post-challenge. Macrophages treated with the supernatant of re-stimulated lung cells from HSP90-E6-immunised mice significantly restricted Mtb growth, and this phenomenon was abrogated by neutralising anti-IFN-γ and/or anti-IL-17 antibodies. We identified a previously unrecognised role for IFN-γ/IL-17 synergism in linking anti-mycobacterial phagosomal activity to enhance host control against Mtb infection. The implications of our findings highlight the fundamental rationale for why and how Th17 responses are essential in the control of Mtb, and for the development of novel anti-TB subunit vaccines.
RESUMEN
Mycobacterium tuberculosis (Mtb) is an intracellular pathogen known to persist in host cells. The apoptotic response of macrophages serves as a defense mechanism to inhibit the growth of intracellular bacteria, the failure of which can favor the spread of the pathogen to new cells. However, the mycobacterial components that regulate cell death and the related underlying mechanisms remain poorly understood. In this study, we investigated protein Rv3261, isolated from an Mtb culture filtrate, for its apoptotic potential using multidimensional fractionation. Rv3261 was found to induce macrophage apoptosis through the caspase-3/-9-dependent pathway. Furthermore, the ROS-dependent JNK activation pathway was found to be critical in Rv3261-mediated apoptosis. Rv3261 inhibited the growth of intracellular Mtb, which was significantly abrogated by pre-treatment with the ROS scavenger N-acetylcysteine (NAC), suggesting that Rv3261-mediated apoptosis may act as a host defense response. These findings suggest that Rv3261 is involved in the apoptotic modulation of Mtb-infected macrophages.
Asunto(s)
Proteínas Bacterianas/metabolismo , Macrófagos/microbiología , Mitocondrias/metabolismo , Mycobacterium tuberculosis/fisiología , Acetilcisteína/farmacología , Animales , Apoptosis , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Procesos de Crecimiento Celular , Evasión Inmune , Inmunidad Innata , Espacio Intracelular , MAP Quinasa Quinasa 4/metabolismo , Macrófagos/inmunología , Ratones , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Transducción de SeñalRESUMEN
:Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of chronic granulomatous bowel disease in animals and is associated with various autoimmune diseases in humans including Crohn's disease. A good understanding of the host-protective immune response and antibacterial immunity controlled by MAP and its components may contribute to the development of effective control strategies. MAP1889c was identified as a seroreactive antigen in Crohn's disease patients. In this study, we investigated the immunological function of MAP1889c in dendritic cells (DCs). MAP1889c stimulated DCs to increase expression of co-stimulatory molecules (CD80 and CD86) and major histocompatibility complex (MHC) class molecules and to secret higher interleukin (IL)-10 and moderate IL-6, tumor necrosis factor (TNF)-α, and IL-12p70 levels through the Toll-like receptor (TLR) 4 pathway. MAP1889c-induced DC activation was mediated by mitogen-activated protein kinases (MAPKs), cAMPp-response element binding protein (CREB), and nuclear factor kappa B (NF-κB). In particular, the CREB signal was essential for MAP1889c-mediated IL-10 production but not TNF-α and IL-12p70. In addition, MAP1889c-matured DCs induced T cell proliferation and drove the Th2 response. Production of lipopolysaccharide (LPS)-mediated pro-inflammatory cytokines and anti-inflammatory cytokines was suppressed and enhanced respectively by MAP1889c pretreatment in DCs and T cells. Furthermore, treatment of MAP1889c in M. avium-infected macrophages promoted intracellular bacterial growth and IL-10 production. These findings suggest that MAP1889c modulates the host antimycobacterial response and may be a potential virulence factor during MAP infection.
Asunto(s)
Células Dendríticas/metabolismo , Interleucina-10/inmunología , Mycobacterium avium subsp. paratuberculosis/patogenicidad , Células Th2/inmunología , Receptor Toll-Like 4/inmunología , Animales , Células Dendríticas/citología , Femenino , Humanos , Ratones , Transducción de Señal , TransfecciónRESUMEN
Macrophages are responsible for innate and adaptive immune response activation necessary for eliminating infections. Optimal activation of macrophages to phagocytize Mycobacterium tuberculosis is critical in anti-mycobacterial defense. Here, we identified a novel Rv3463 hypothetical protein that induces macrophage activation in Mtb culture filtrate. Recombinant Rv3463 activated mouse bone marrow-derived macrophages to induce the expression of surface molecules and secrete pro-inflammatory cytokines via the TLR2 and TLR4 pathways. Mitogen activated protein kinase, phospatidylinositol-4,5-bisphosphate 3-kinases, and the NF-κB signaling pathways are involved in Rv3463-mediated macrophage activation. Furthermore, Rv3463 induced bactericidal effects in Mtb-infected macrophages through phagosome maturation and phagolysosomal fusion enhanced by phospatidylinositol-4,5-bisphosphate 3-kinases and Ca2+ signaling pathways and exhibited therapeutic effects in a short-term Mtb-infection mouse model. Overexpression of Rv3463 in M. smegmatis caused rapid clearance of bacteria in macrophages and mice. Our study suggests that Rv3463 is a promising target for the development of post-exposure tuberculosis vaccines or adjunct immune-therapy.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/uso terapéutico , Tuberculosis/prevención & control , Animales , Antígenos Bacterianos/metabolismo , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Lisosomas/inmunología , Lisosomas/microbiología , Activación de Macrófagos , Macrófagos/microbiología , Ratones , Fagocitosis/inmunología , Profilaxis Posexposición/métodos , Transducción de Señal/inmunología , Células THP-1 , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismo , Tuberculosis/inmunología , Tuberculosis/microbiología , Vacunas contra la Tuberculosis/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/uso terapéuticoRESUMEN
A safe and effective adjuvant is necessary to induce reliable protective efficacy of the protein-based vaccines against tuberculosis (TB). Mycobacterial components, such as synthetic cord factor and arabinogalactan, have been used as one of the adjuvant components. Mycobacterium bovis bacillus Calmette- Guérin cell-wall skeleton (BCG-CWS) has been used as an effective immune-stimulator. However, it is not proven whether BCG-CWS can be an effective adjuvant for the subunit protein vaccine of TB. In this study, we demonstrated that the BCG-CWS effectively coupled with Ag85B and enhanced the conjugated Ag85B activity on the maturation of dendritic cells (DCs). Ag85B-BCG-CWS-matured DCs induced significant Th1 and Th17 responses when compared to BCG-CWS or Ag85B alone. In addition, significant Ag85B-specific Th1 and Th17 responses were induced in Ag85B-BCG-CWS-immunized mice before infection with M. tuberculosis and maintained after infection. Moreover, Ag85B-BCG-CWS showed significant protective effect comparable to live BCG at 6 weeks after infection and maintained its protective efficacy at 32 weeks post-challenge, whereas live BCG did not. These results suggest that the BCG-CWS may be an effective adjuvant candidate for a protein-based vaccine against TB.
Asunto(s)
Antígenos Bacterianos/inmunología , Pared Celular/inmunología , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , Células TH1/inmunología , Células Th2/inmunología , Vacunas contra la Tuberculosis/inmunología , Tuberculosis/inmunología , Adyuvantes Inmunológicos/farmacología , Animales , Antígenos Bacterianos/farmacología , Femenino , Ratones , Células Th2/patología , Tuberculosis/patología , Tuberculosis/prevención & control , Vacunas contra la Tuberculosis/farmacologíaRESUMEN
Mycobacterial acyl carrier protein (AcpM; Rv2244) is a meromycolate extension acyl carrier protein of Mycobacterium tuberculosis (Mtb), which participates in multistep mycolic acid biosynthesis. However, the function of AcpM in host-mycobacterium interactions during infection remains largely uncharacterized. Here we show that AcpM inhibits host cell apoptosis during mycobacterial infection. To examine the function of AcpM during infection, we generated a recombinant Mycobacterium smegmatis (M. smegmatis) strain overexpressing AcpM (Ms_AcpM) and a strain transformed with an empty vector (Ms_Vec). Ms_AcpM promoted intracellular survival of M. smegmatis and led to a significant decrease in the death rate of primary bone marrow-derived macrophages (BMDMs). Importantly, Ms_AcpM showed significantly decreased reactive oxygen species (ROS) generation and activation of c-Jun N-terminal kinase (JNK) signaling compared with Ms_Vec. In addition, treatment of BMDMs with recombinant AcpM significantly inhibited the apoptosis and ROS/JNK signaling induced by M. smegmatis. Moreover, recombinant AcpM enhanced intracellular survival of Mtb H37Rv. Taken together, these results indicate that AcpM plays a role as a virulence factor by modulating host cell apoptosis during mycobacterial infection.
Asunto(s)
Apoptosis/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/patología , Mycobacterium tuberculosis/química , Especies Reactivas de Oxígeno/metabolismo , Animales , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Bacterianas/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/farmacología , Células Cultivadas , Femenino , Expresión Génica , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Macrófagos/metabolismo , Macrófagos/microbiología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Viabilidad Microbiana/efectos de los fármacos , Infecciones por Mycobacterium/inmunología , Infecciones por Mycobacterium/metabolismo , Infecciones por Mycobacterium/microbiología , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/fisiología , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/fisiología , Células RAW 264.7 , Transducción de Señal/efectos de los fármacosRESUMEN
The attenuated vaccine Mycobacterium bovis BCG (Bacille Calmette Guerin) has limited protective efficacy against TB. The development of more effective TB vaccines has focused on the mycobacterial antigens that cause strong T helper 1 (Th1) responses. Mtb protein Rv3841 (bacterioferritin B; BfrB) is known to play a crucial role in the growth of Mtb. Nonetheless, it is unclear whether Rv3841 can induce protective immunity against Mtb. Here, we studied the action of Rv3841 in maturation of dendritic cells (DCs) and its engagement in the development of T-cell immunity. We found that Rv3841 functionally activated DCs by upregulating costimulatory molecules and increased secretion of proinflammatory cytokines. Activation of DCs by Rv3841 was mediated by Toll-like receptor 4 (TLR4), followed by triggering of mitogen-activated protein kinase and nuclear factor-κB signaling pathways. In addition, Rv3841-matured DCs effectively proliferated and polarized Th1 immune response of naïve CD4+ and CD8+ T-cells. Moreover, Rv3841 specifically caused the expansion of CD4+CD44highCD62Llow T-cells from Mtb-infected mice; besides, the T-cells activated by Rv3841-matured DCs inhibited intracellular mycobacterial growth. Our data suggest that Rv3841 induces DC maturation and protective immune responses, a finding that may provide candidate of effective TB vaccines.
Asunto(s)
Proteínas Bacterianas/inmunología , Grupo Citocromo b/inmunología , Células Dendríticas/inmunología , Ferritinas/inmunología , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/inmunología , Células TH1/inmunología , Tuberculosis/inmunología , Animales , Diferenciación Celular , Células Cultivadas , Femenino , Humanos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/metabolismo , Transducción de Señal , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismoRESUMEN
Autophagy is known to be a vital homeostatic defense process that controls mycobacterial infection. However, the relationship between autophagy response and the virulence of Mycobacterium abscessus strain UC22 has not been reported. Here, we demonstrate that M. abscessus induces autophagy and inhibits autophagy flux in murine macrophages. Further, the rough variant of M. abscessus, UC22 that is a highly virulent clinical isolate, significantly inhibited autophagic flux than the smooth variant of M. abscessus ATCC 19977. In addition, it was noticed that the intracellular survival of UC22 is significantly enhanced by blocking the autophagosome-lysosome fusion in macrophages compared to the smooth variant. However, Mycobacterium smegmatis did not block autophagy flux in murine macrophages. Besides, we confirmed that the lipid components of M. abscessus UC22 play a role in autophagosome formation. These data suggest that the virulent M. abscessus might be able to survive and grow within autophagosomes by preventing the autophagosome-lysosome fusion and their clearance from the cells.
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Autofagia , Macrófagos/microbiología , Mycobacterium abscessus/fisiología , Animales , Femenino , Lípidos , Macrófagos/fisiología , Ratones , Ratones Endogámicos C57BL , Células RAW 264.7RESUMEN
Mycobacterium abscessus (MAB) is a species of nontuberculous mycobacteria (NTM) and a major causative pathogen of pulmonary diseases especially in patients with cystic fibrosis. MAB infection is notoriously difficult to treat because of its intrinsic or inducible resistance to most antibiotics. The rough (R) morphotype of MAB, lacking cell surface glycopeptidolipids (GPLs), is associated with more severe and persistent infection than the smooth (S) type; however, the mechanisms underlying the R type's virulence and the relation with GPLs remain unclear. In this study, we found that R-type MAB is much more proapoptotic than the S type, as a result of GPL-mediated inhibition of macrophage apoptosis. Polar GPLs inhibited an apoptotic response (induced by proapoptotic stimuli) by suppressing ROS production and the cytochrome c release and by preserving mitochondrial transmembrane potential. Furthermore, GPLs were found to be targeted to mitochondria and interacted with cyclophilin D; their acetylation was essential for this interaction. Finally, GPLs inhibited the intracellular growth and bacterial spreading of R-type MAB among macrophages via apoptosis inhibition. These findings suggest that GPLs limit MAB virulence by inhibiting apoptosis and the spread of bacteria and therefore provide a novel insight into the mechanism underlying virulence of MAB.
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Ciclofilinas/metabolismo , Macrófagos/metabolismo , Mitocondrias/metabolismo , Mycobacterium abscessus/patogenicidad , Micobacterias no Tuberculosas/patogenicidad , Apoptosis , Peptidil-Prolil Isomerasa F , HumanosRESUMEN
Understanding functional interactions between DCs and antigens is necessary for achieving an optimal and desired immune response during vaccine development. Here, we identified and characterized protein Rv2299c (heat-shock protein 90 family), which effectively induced DC maturation. The Rv2299c-maturated DCs showed increased expression of surface molecules and production of proinflammatory cytokines. Rv2299c induced these effects by binding to TLR4 and stimulating the downstream MyD88-, MAPK- and NF-κB-dependent signaling pathways. The Rv2299c-maturated DCs also showed an induced Th1 cell response with bactericidal activity and expansion of effector/memory T cells. The Rv2299c-ESAT-6 fused protein had greater immunoreactivity than ESAT-6. Furthermore, boosting BCG with the fused protein significantly reduced hypervirulent Mycobacterium tuberculosis HN878 burdens post-challenge. The pathological study of the lung from the challenged mice assured the efficacy of the fused protein. The fused protein boosting also induced Rv2299c-ESAT-6-specific multifunctional CD4+ T-cell response in the lungs of the challenged mice. Our findings suggest that Rv2299c is an excellent candidate for the rational design of an effective multiantigenic TB vaccine.
Asunto(s)
Antígenos Bacterianos/inmunología , Vacuna BCG/inmunología , Proteínas Bacterianas/inmunología , Células Dendríticas/inmunología , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/prevención & control , Vacunas de Subunidad/uso terapéutico , Animales , Vacuna BCG/administración & dosificación , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Células Cultivadas , Citocinas/metabolismo , Femenino , Memoria Inmunológica/inmunología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/patogenicidad , Proteínas Recombinantes de Fusión/inmunología , Células TH1/inmunología , Tuberculosis Pulmonar/inmunología , Tuberculosis Pulmonar/microbiologíaRESUMEN
Mycobacterium avium complex induces macrophage apoptosis. However, the M. avium components that inhibit or trigger apoptosis and their regulating mechanisms remain unclear. We recently identified the immunodominant MAV2054 protein by fractionating M. avium culture filtrate protein by multistep chromatography; this protein showed strong immuno-reactivity in M. avium complex pulmonary disease and in patients with tuberculosis. Here, we investigated the biological effects of MAV2054 on murine macrophages. Recombinant MAV2054 induced caspase-dependent macrophage apoptosis. Enhanced reactive oxygen species production and JNK activation were essential for MAV2054-mediated apoptosis and MAV2054-induced interleukin-6, tumour necrosis factor, and monocyte chemoattractant protein-1 production. MAV2054 was targeted to the mitochondrial compartment of macrophages treated with MAV2054 and infected with M. avium. Dissipation of the mitochondrial transmembrane potential (ΔΨm) and depletion of cytochrome c also occurred in MAV2054-treated macrophages. Apoptotic response, reactive oxygen species production, and ΔΨm collapse were significantly increased in bone marrow-derived macrophages infected with Mycobacterium smegmatis expressing MAV2054, compared to that in M. smegmatis control. Furthermore, MAV2054 expression suppressed intracellular growth of M. smegmatis and increased the survival rate of M. smegmatis-infected mice. Thus, MAV2054 induces apoptosis via a mitochondrial pathway in macrophages, which may be an innate cellular response to limit intracellular M. avium multiplication.
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Apoptosis/fisiología , Proteínas Bacterianas/metabolismo , Macrófagos/metabolismo , Mitocondrias/metabolismo , Mycobacterium avium/metabolismo , Mycobacterium avium/fisiología , Animales , Citocromos c/metabolismo , Femenino , Interleucina-6/metabolismo , Macrófagos/fisiología , Potencial de la Membrana Mitocondrial/fisiología , Ratones , Ratones Endogámicos C57BL , Mycobacterium smegmatis/metabolismo , Mycobacterium smegmatis/fisiología , Células RAW 264.7 , Especies Reactivas de Oxígeno/metabolismo , Tasa de Supervivencia , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Macrophages constitute the first line of defense against Mycobacterium tuberculosis and are critical in linking innate and adaptive immunity. Therefore, the identification and characterization of mycobacterial proteins that modulate macrophage function are essential for understanding tuberculosis pathogenesis. In this study, we identified the novel macrophage-activating protein, Rv2882c, from M. tuberculosis culture filtrate proteins. Recombinant Rv2882c protein activated macrophages to secrete pro-inflammatory cytokines and express co-stimulatory and major histocompatibility complex molecules via Toll-like receptor 4, myeloid differentiation primary response protein 88, and Toll/IL-1 receptor-domain-containing adaptor inducing IFN-beta. Mitogen-activated protein kinases and NF-κB signaling pathways were involved in Rv2882c-induced macrophage activation. Further, Rv2882c-treated macrophages induced expansion of the effector/memory T cell population and Th1 immune responses. In addition, boosting Bacillus Calmette-Guerin vaccination with Rv2882c improved protective efficacy against M. tuberculosis in our model system. These results suggest that Rv2882c is an antigen that could be used for tuberculosis vaccine development.
Asunto(s)
Proteínas Bacterianas/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/metabolismo , Receptor Toll-Like 4/metabolismo , Vacunas contra la Tuberculosis/inmunología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Citocinas/análisis , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Lipopolisacáridos/toxicidad , Pulmón/metabolismo , Activación de Macrófagos , Macrófagos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/inmunología , Factor 88 de Diferenciación Mieloide/metabolismo , FN-kappa B/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Transducción de Señal , Bazo/metabolismo , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/genéticaRESUMEN
Mycobacterium avium and its sonic extracts induce apoptosis in macrophages. However, little is known about the M. avium components regulating macrophage apoptosis. In this study, using multidimensional fractionation, we identified MAV2052 protein, which induced macrophage apoptosis in M. avium culture filtrates. The recombinant MAV2052 induced macrophage apoptosis in a caspase-dependent manner. The loss of mitochondrial transmembrane potential (ΔΨm), mitochondrial translocation of Bax, and release of cytochrome c from mitochondria were observed in macrophages treated with MAV2052. Further, reactive oxygen species (ROS) production was required for the apoptosis induced by MAV2052. In addition, ROS and mitogen-activated protein kinases were involved in MAV2052-mediated TNF-α and IL-6 production. ROS-mediated activation of apoptosis signal-regulating kinase 1 (ASK1)-JNK pathway was a major signaling pathway for MAV2052-induced apoptosis. Moreover, MAV2052 bound to Toll-like receptor (TLR) 4 molecule and MAV2052-induced ROS production, ΔΨm loss, and apoptosis were all significantly reduced in TLR4(-/-) macrophages. Altogether, our results suggest that MAV2052 induces apoptotic cell death through TLR4 dependent ROS production and JNK pathway in murine macrophages.
Asunto(s)
Apoptosis/fisiología , Proteínas Bacterianas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Macrófagos/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Receptor Toll-Like 4/metabolismo , Animales , Línea Celular , Citocromos c/metabolismo , Femenino , Interleucina-6/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Potencial de la Membrana Mitocondrial , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mycobacterium avium/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Reciprocal induction of the Th1 and Th17 immune responses is essential for optimal protection against Mycobacterium tuberculosis (Mtb); however, only a few Mtb antigens are known to fulfill this task. A functional role for resuscitation-promoting factor (Rpf) E, a latency-associated member of the Rpf family, in promoting naïve CD4(+) T-cell differentiation toward both Th1 and Th17 cell fates through interaction with dendritic cells (DCs) was identified in this study. RpfE induces DC maturation by increasing expression of surface molecules and the production of IL-6, IL-1ß, IL-23p19, IL-12p70, and TNF-α but not IL-10. This induction is mediated through TLR4 binding and subsequent activation of ERK, p38 MAPKs, and NF-κB signaling. RpfE-treated DCs effectively caused naïve CD4(+) T cells to secrete IFN-γ, IL-2, and IL-17A, which resulted in reciprocal expansions of the Th1 and Th17 cell response along with activation of T-bet and RORγt but not GATA-3. Furthermore, lung and spleen cells from Mtb-infected WT mice but not from TLR4(-/-) mice exhibited Th1 and Th17 polarization upon RpfE stimulation. Taken together, our data suggest that RpfE has the potential to be an effective Mtb vaccine because of its ability to activate DCs that simultaneously induce both Th1- and Th17-polarized T-cell expansion.