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1.
Cell Rep ; 28(1): 218-230.e7, 2019 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-31269442

RESUMEN

Classical activation of macrophages (M(LPS+IFNγ)) elicits the expression of inducible nitric oxide synthase (iNOS), generating large amounts of NO and inhibiting mitochondrial respiration. Upregulation of glycolysis and a disrupted tricarboxylic acid (TCA) cycle underpin this switch to a pro-inflammatory phenotype. We show that the NOS cofactor tetrahydrobiopterin (BH4) modulates IL-1ß production and key aspects of metabolic remodeling in activated murine macrophages via NO production. Using two complementary genetic models, we reveal that NO modulates levels of the essential TCA cycle metabolites citrate and succinate, as well as the inflammatory mediator itaconate. Furthermore, NO regulates macrophage respiratory function via changes in the abundance of critical N-module subunits in Complex I. However, NO-deficient cells can still upregulate glycolysis despite changes in the abundance of glycolytic intermediates and proteins involved in glucose metabolism. Our findings reveal a fundamental role for iNOS-derived NO in regulating metabolic remodeling and cytokine production in the pro-inflammatory macrophage.


Asunto(s)
Ciclo del Ácido Cítrico , Inflamación/metabolismo , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Succinatos/metabolismo , Animales , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Ciclo del Ácido Cítrico/efectos de los fármacos , Transporte de Electrón/efectos de los fármacos , Endotoxemia/inducido químicamente , Endotoxemia/metabolismo , GTP Ciclohidrolasa/genética , GTP Ciclohidrolasa/metabolismo , Glucólisis/efectos de los fármacos , Interferón gamma/farmacología , Interleucina-1beta/metabolismo , Isocitrato Deshidrogenasa/metabolismo , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Mitocondrias/metabolismo , Infecciones por Mycobacterium/metabolismo , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Fragmentos de Péptidos/metabolismo , Proteoma/genética , Proteoma/metabolismo , Ácido Succínico/metabolismo , Espectrometría de Masas en Tándem
2.
BMC Infect Dis ; 16: 412, 2016 08 12.
Artículo en Inglés | MEDLINE | ID: mdl-27519524

RESUMEN

BACKGROUND: In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed. METHODS: We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen. RESULTS: Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay. CONCLUSIONS: We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms.


Asunto(s)
Vacuna BCG/inmunología , Recuento de Colonia Microbiana/métodos , Vacunas contra la Tuberculosis/farmacología , Tuberculosis/prevención & control , Animales , Vacuna BCG/farmacología , Técnicas de Cocultivo , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Femenino , Interferón gamma/genética , Interferón gamma/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mycobacterium bovis/inmunología , Mycobacterium smegmatis/crecimiento & desarrollo , Mycobacterium smegmatis/inmunología , Mycobacterium tuberculosis/inmunología , Mycobacterium tuberculosis/patogenicidad , Bazo/citología , Bazo/inmunología , Bazo/microbiología , Tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Vacunación
3.
Plant Biotechnol J ; 12(7): 840-50, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-24629003

RESUMEN

Progress with protein-based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency-associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self-polymerization of the complexes. TB-RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen-presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB-RICs developed antigen-specific serum IgG antibody responses with mean end-point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN-γ, IL-2 and TNF-α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG-immunized mice with TB-RICs led to a reduced M. tuberculosis infection in their lungs from log10 mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant-expressed TB-RICs represent a novel molecular platform for developing self-adjuvanting mucosal vaccines.


Asunto(s)
Adyuvantes Inmunológicos/biosíntesis , Complejo Antígeno-Anticuerpo/metabolismo , Mycobacterium tuberculosis/inmunología , Nicotiana/genética , Vacunas contra la Tuberculosis/inmunología , Adyuvantes Inmunológicos/metabolismo , Administración Intranasal , Animales , Formación de Anticuerpos , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Clonación Molecular , Humanos , Interleucina-2/metabolismo , Ratones , Plantas Modificadas Genéticamente/metabolismo , Nicotiana/metabolismo , Vacunas contra la Tuberculosis/administración & dosificación , Factor de Necrosis Tumoral alfa/metabolismo
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