RESUMEN
Macrophages (MФ) are an essential immune cell for defense and repair that travel to different tissues and adapt based on local stimuli. A critical factor that may govern their polarization is the crosstalk between metabolism and epigenetics. However, simultaneous measurements of metabolites, epigenetics, and proteins (phenotype) have been a major technical challenge. To address this, we have developed a novel triomics approach using mass spectrometry to comprehensively analyze metabolites, proteins, and histone modifications in a single sample. To demonstrate this technique, we investigated the metabolic-epigenetic-phenotype axis following polarization of human blood-derived monocytes into either 'proinflammatory M1-' or 'anti-inflammatory M2-' MФs. We report here a complex relationship between arginine, tryptophan, glucose, and the citric acid cycle metabolism, protein and histone post-translational modifications, and human macrophage polarization that was previously not described. Surprisingly, M1-MФs had globally reduced histone acetylation levels but high levels of acetylated amino acids. This suggests acetyl-CoA was diverted, in part, toward acetylated amino acids. Consistent with this, stable isotope tracing of glucose revealed reduced usage of acetyl-CoA for histone acetylation in M1-MФs. Furthermore, isotope tracing also revealed MФs uncoupled glycolysis from the tricarboxylic acid cycle, as evidenced by poor isotope enrichment of succinate. M2-MФs had high levels of kynurenine and serotonin, which are reported to have immune-suppressive effects. Kynurenine is upstream of de novo NAD+ metabolism that is a necessary cofactor for Sirtuin-type histone deacetylases. Taken together, we demonstrate a complex interplay between metabolism and epigenetics that may ultimately influence cell phenotype.
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Polaridad Celular , Quinurenina , Macrófagos , Humanos , Acetilcoenzima A/metabolismo , Epigénesis Genética , Glucosa/metabolismo , Histonas/genética , Histonas/metabolismo , Quinurenina/metabolismo , Macrófagos/metabolismo , Polaridad Celular/genéticaRESUMEN
Autophagy and phagocytosis are conserved cellular functions involved in innate immunity. However, the nature of their interactions remains unclear. We evaluated the role of autophagy in regulating phagocytosis in macrophages from myeloid-specific autophagy-related gene 7-deficient (Atg7â»/â») mice. Atg7â»/â» macrophages exhibited higher bacterial uptake when infected with Mycobacterium tuberculosis (Mtb) or with M. tuberculosis var. bovis BCG (BCG). In addition, BCG-infected Atg7â»/â» mice showed increased bacterial loads and exacerbated lung inflammatory responses. Atg7â»/â» macrophages had increased expression of two class A scavenger receptors: macrophage receptor with collagenous structure (MARCO) and macrophage scavenger receptor 1 (MSR1). The increase in scavenger receptors was caused by increased activity of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) transcription factor resulting from accumulated sequestosome 1 (SQSTM1 or p62) in Atg7â»/â» macrophages. These insights increase our understanding of the host-pathogen relationship and suggest that therapeutic strategies should be designed to include modulation of both phagocytosis and autophagy.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagia/inmunología , Proteínas de Choque Térmico/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Fagocitosis/inmunología , Receptores Inmunológicos/biosíntesis , Receptores Depuradores de Clase A/biosíntesis , Animales , Proteína 7 Relacionada con la Autofagia , Carga Bacteriana/inmunología , Células Cultivadas , Macrófagos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/genética , Infecciones por Mycobacterium/inmunología , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium bovis/inmunología , Mycobacterium tuberculosis/crecimiento & desarrollo , Mycobacterium tuberculosis/inmunología , Neumonía/inmunología , Neumonía/microbiología , Receptores Depuradores/biosíntesis , Receptores Depuradores/inmunología , Proteína Sequestosoma-1 , Regulación hacia ArribaRESUMEN
Tuberculosis is a major cause of death in mankind and BCG vaccine protects against childhood but not adult tuberculosis. BCG avoids lysosomal fusion in macrophages decreasing peptides required for activating CD4 T cells and Th1 immunity while suppressing the expression of MHC-II by antigen presenting cells (APCs). An in vitro model of antigen presentation showed that ligands for TLR-9, 7, 4 and 1/2 increased the ability of APCs to present antigen-85B of BCG to CD4 T cells, which correlated with an increase in MHC-II expression. TLR-activation led to a down-regulation of MARCH1 ubiquitin ligase which prevents the degradation of MHC-II and decreased IL-10 also contributed to an increase in MHC-II. TLR-activation induced up-regulation of MHC-II was inhibited by the blockade of IRAK, NF-kB, and MAPKs. TLR-7 and TLR-9 ligands had the most effective adjuvant like effect on MHC-II of APCs which allowed BCG vaccine mediated activation of CD4 T cells.
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Vacuna BCG , Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Macrófagos/inmunología , Mycobacterium tuberculosis/inmunología , Receptor Toll-Like 7/metabolismo , Receptor Toll-Like 9/metabolismo , Tuberculosis/inmunología , Aciltransferasas/inmunología , Animales , Presentación de Antígeno/genética , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/farmacología , Linfocitos T CD4-Positivos/microbiología , Células Cultivadas , Células Dendríticas/microbiología , Femenino , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/metabolismo , Humanos , Interleucina-10/genética , Interleucina-10/metabolismo , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas , Macrófagos/microbiología , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Arginine and tryptophan are pivotal in orchestrating cytokine-driven macrophage polarization and immune activation. Specifically, interferon-gamma (IFN-γ) stimulates inducible nitric oxide synthase (iNOS) expression), leading to the conversion of arginine into citrulline and nitric oxide (NO), while Interleukin-4 (IL4) promotes arginase activation, shifting arginine metabolism toward ornithine. Concomitantly, IFN-γ triggers indoleamine 2,3-dioxygenase 1 (IDO1) and Interleukin-4 induced 1 (IL4i1), resulting in the conversion of tryptophan into kynurenine and indole-3-pyruvic acid. These metabolic pathways are tightly regulated by NAD+-dependent sirtuin proteins, with Sirt2 and Sirt5 playing integral roles. In this review, we present novel insights that augment our understanding of the metabolic pathways of arginine and tryptophan following Mycobacterium tuberculosis infection, particularly their relevance in macrophage responses. Additionally, we discuss arginine methylation and demethylation and the role of Sirt2 and Sirt5 in regulating tryptophan metabolism and arginine metabolism, potentially driving macrophage polarization.
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Arginina , Tuberculosis , Humanos , Arginina/metabolismo , Triptófano/metabolismo , Interleucina-4 , Sirtuina 2 , Activación de Macrófagos , Interferón gamma/farmacologíaRESUMEN
Tuberculosis (TB), a chronic infectious disease affecting humans, causes over 1.3 million deaths per year throughout the world. The current preventive vaccine BCG provides protection against childhood TB, but it fails to protect against pulmonary TB. Multiple candidates have been evaluated to either replace or boost the efficacy of the BCG vaccine, including subunit protein, DNA, virus vector-based vaccines, etc., most of which provide only short-term immunity. Several live attenuated vaccines derived from Mycobacterium tuberculosis (Mtb) and BCG have also been developed to induce long-term immunity. Since Mtb mediates its virulence through multiple secreted proteins, these proteins have been targeted to produce attenuated but immunogenic vaccines. In this review, we discuss the characteristics and prospects of live attenuated vaccines generated by targeting the disruption of the genes encoding secretory mycobacterial proteins.
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The nucleoprotein (NP) is a vital target for the heterosubtypic immunity of CD8+ cytotoxic T lymphocytes (CTLs) due to its conservation among influenza virus subtypes. To further enhance the T cell immunity of NP, autophagy-inducing peptide C5 (AIP-C5) from the CFP10 protein of Mycobacterium tuberculosis was used. Mice were immunized intranasally (i.n.) with human adenoviral vectors, HAd-C5-NP(H7N9) or HAd-NP(H7N9), expressing NP of an H7N9 influenza virus with or without the AIP-C5, respectively. Both vaccines developed similar levels of NP-specific systemic and mucosal antibody titers; however, there was a significantly higher number of NP-specific CD8 T cells secreting interferon-gamma (IFN-γ) in the HAd-C5-NP(H7N9) group than in the HAd-NP(H7N9) group. The HAd-C5-NP(H7N9) vaccine provided better protection following the challenge with A/Puerto Rico/8/1934(H1N1), A/Hong Kong/1/68(H3N2), A/chukkar/MN/14951-7/1998(H5N2), A/goose/Nebraska/17097/2011(H7N9), or A/Hong Kong/1073/1999(H9N2) influenza viruses compared to the HAd-NP(H7N9) group. The autophagy transcriptomic gene analysis of the HAd-C5-NP(H7N9) group revealed the upregulation of some genes involved in the positive regulation of the autophagy process. The results support further exploring the use of NP and AIP-C5 for developing a universal influenza vaccine for pandemic preparedness.
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Tuberculosis (TB) remains a significant global health challenge, with approximately 1.5 million deaths per year. The Bacillus Calmette-Guérin (BCG) vaccine against TB is used in infants but shows variable protection. Here, we introduce a novel approach using a double gene knockout mutant (DKO) from wild-type Mycobacterium tuberculosis (Mtb) targeting fbpA and sapM genes. DKO exhibited enhanced anti-TB gene expression in mouse antigen-presenting cells, activating autophagy and inflammasomes. This heightened immune response improved ex vivo antigen presentation to T cells. Subcutaneous vaccination with DKO led to increased protection against TB in wild-type C57Bl/6 mice, surpassing the protection observed in caspase 1/11-deficient C57Bl/6 mice and highlighting the critical role of inflammasomes in TB protection. The DKO vaccine also generated stronger and longer-lasting protection than the BCG vaccine in C57Bl/6 mice, expanding both CD62L-CCR7-CD44+/-CD127+ effector T cells and CD62L+CCR7+/-CD44+CD127+ central memory T cells. These immune responses correlated with a substantial ≥ 1.7-log10 reduction in Mtb lung burden. The DKO vaccine represents a promising new approach for TB immunization that mediates protection through autophagy and inflammasome pathways.
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Macrófagos , Ratones Endogámicos C57BL , Mycobacterium tuberculosis , Vacunas contra la Tuberculosis , Tuberculosis , Animales , Mycobacterium tuberculosis/inmunología , Ratones , Macrófagos/inmunología , Tuberculosis/inmunología , Tuberculosis/prevención & control , Vacunas contra la Tuberculosis/inmunología , Antígenos Bacterianos/inmunología , Antígenos Bacterianos/genética , Inflamasomas/inmunología , Femenino , Vacuna BCG/inmunología , Autofagia/inmunología , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/genética , Modelos Animales de EnfermedadRESUMEN
Background: Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. HIV infection decreases CD4+ T cell levels markedly increasing Mtb co-infections. An appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans during co-infection would facilitate basic and translational research in HIV/Mtb infections. Herein, we describe a novel humanized mouse model. Methods: The irradiated NSG-SGM3 mice were transplanted with human CD34+ hematopoietic stem cells, and the humanization was monitored by staining various immune cell markers for flow cytometry. They were challenged with HIV and/or Mtb, and the CD4+ T cell depletion and HIV viral load were monitored over time. Before necropsy, the live mice were subjected to pulmonary function test and CT scan, and after sacrifice, the lung and spleen homogenates were used to determine Mtb load (CFU) and cytokine/chemokine levels by multiplex assay, and lung sections were analyzed for histopathology. The mouse sera were subjected to metabolomics analysis. Results: Our humanized NSG-SGM3 mice were able to engraft human CD34+ stem cells, which then differentiated into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced granulomatous lesions in the lungs. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. Conclusion: The humanized NSG-SGM3 mice are able to recapitulate the pathogenic effects of HIV and Mtb infections and co-infection at the pathological, immunological and metabolism levels and are therefore a reproducible small animal model for studying HIV/Mtb co-infection.
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Coinfección , Modelos Animales de Enfermedad , Infecciones por VIH , Mycobacterium tuberculosis , Tuberculosis , Animales , Coinfección/inmunología , Coinfección/microbiología , Infecciones por VIH/inmunología , Infecciones por VIH/complicaciones , Humanos , Ratones , Tuberculosis/inmunología , Mycobacterium tuberculosis/inmunología , Linfocitos T CD4-Positivos/inmunología , Trasplante de Células Madre Hematopoyéticas , Carga Viral , VIH-1/inmunología , Pulmón/inmunología , Pulmón/patología , Pulmón/virología , Células Madre Hematopoyéticas/inmunología , Ratones SCIDRESUMEN
Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), continues to be a major public health problem worldwide. The human immunodeficiency virus (HIV) is another equally important life-threatening pathogen. Further, co-infections with HIV and Mtb have severe effects in the host, with people infected with HIV being fifteen to twenty-one times more likely to develop active TB. The use of an appropriate animal model for HIV/Mtb co-infection that can recapitulate the diversity of the immune response in humans would be a useful tool for conducting basic and translational research in HIV/Mtb infections. The present study was focused on developing a humanized mouse model for investigations on HIV-Mtb co-infection. Using NSG-SGM3 mice that can engraft human stem cells, our studies showed that they were able to engraft human CD34+ stem cells which then differentiate into a full-lineage of human immune cell subsets. After co-infection with HIV and Mtb, these mice showed decrease in CD4+ T cell counts overtime and elevated HIV load in the sera, similar to the infection pattern of humans. Additionally, Mtb caused infections in both lungs and spleen, and induced the development of granulomatous lesions in the lungs, detected by CT scan and histopathology. Distinct metabolomic profiles were also observed in the tissues from different mouse groups after co-infections. Our results suggest that the humanized NSG-SGM3 mice are able to recapitulate the effects of HIV and Mtb infections and co-infection in the human host at pathological, immunological and metabolism levels, providing a dependable small animal model for studying HIV/Mtb co-infection.
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Mycobacterium tuberculosis is one of the strongest reducers of nitrate among all mycobacteria. Reduction of nitrate to nitrite, mediated by nitrate reductase (NarGHJI) of M. tuberculosis, is induced during the dormant stage, and the enzyme has a respiratory function in the absence of oxygen. Nitrite reductase (NirBD) is also functional during aerobic growth when nitrite is the sole nitrogen source. However, the role of NirBD-mediated nitrite reduction during the dormancy is not yet characterized. Here, we analyzed nitrite reduction during aerobic growth as well as in a hypoxic dormancy model of M. tuberculosis in vitro. When nitrite was used as the sole nitrogen source in the medium, the organism grew and the reduction of nitrite was evident in both hypoxic and aerobic cultures of M. tuberculosis. Remarkably, the hypoxic culture of M. tuberculosis, compared to the aerobic culture, showed 32- and 4-fold-increased expression of nitrite reductase (NirBD) at the transcription and protein levels, respectively. More importantly, a nirBD mutant of M. tuberculosis was unable to reduce nitrite and compared to the wild-type (WT) strain had a >2-log reduction in viability after 240 h in the Wayne model of hypoxic dormancy. Dependence of M. tuberculosis on nitrite reductase (NirBD) was also seen in a human macrophage-based dormancy model where the nirBD mutant was impaired for survival compared to the WT strain. Overall, the increased expression and essentiality of nitrite reductase in the in vitro dormancy models suggested that NirBD-mediated nitrite reduction could be critical during the persistent stage of M. tuberculosis.
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Monocitos/enzimología , Mycobacterium tuberculosis/enzimología , Nitrito Reductasas/metabolismo , Línea Celular , Regulación Bacteriana de la Expresión Génica , Humanos , Mutación , Nitrito Reductasas/genética , Nitritos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins and contributes to the pathogenesis of many human diseases. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits unfolded protein response (UPR) that serves to attenuate protein translation, and increase protein refolding or degradation. In contrast to UPR in the ER, the regulatory molecules operative in cytosolic responses and their potential relation to ER stress are not well elucidated. Aggresome-like induced structures (ALIS) have been described as transient aggregation of ubiquitinated proteins in the cytosol. In this study, we show that cells respond to inflammation, infection or ER stress by cytosolic formation of ALIS, indicating that ALIS formation represents an early event in cellular adjustment to altered proteostasis that occurs under these conditions. This response was aided by rapid transcriptional up-regulation of polyubiqutin-binding protein p62. NF-κB and mTOR activation were also required for ALIS formation. Importantly, we show a cross talk between UPR in the ER and cytosolic ALIS. Down-regulation of ER UPR in XBP1 deficient cells increases cyotosolic ALIS formation. Furthermore, lysosomal activity but not macroautophagy is responsible for ALIS clearance. This study reveals the underlying regulatory mechanisms of ALIS formation and clearance, and provides a previously unrecognized common adaptive mechanism for cellular responses against inflammation and ER stress.
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Estrés del Retículo Endoplásmico/fisiología , Proteínas Ubiquitinadas/metabolismo , Respuesta de Proteína Desplegada/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Línea Celular , Citosol/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/fisiología , Ratones , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Factores de Transcripción del Factor Regulador X , Proteína Sequestosoma-1 , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , Proteínas Ubiquitinadas/genética , Regulación hacia Arriba/fisiología , Proteína 1 de Unión a la X-BoxRESUMEN
Bacillus Calmette-Guérin (BCG), the antituberculosis vaccine, localizes within immature phagosomes of macrophages and dendritic cells (APCs), and avoids lysosomal degradation. BCG-derived antigenic peptides are thus inefficiently processed by APCs, and we investigated alternate mechanisms of Ag processing. Proteomics identified that BCG phagosomes are enriched for nicastrin, APH, and presenilin components of γ-secretase, a multimeric protease. Using an in vitro Ag presentation assay and BCG-infected APCs, we found γ-secretase components to cleave BCG-derived Ag85B to produce a peptide epitope, which, in turn, primed IL-2 release from Ag85B-specific T cell hybridoma. siRNA knockdown or chemical inhibition of γ-secretase components using L685458 decreased the ability of BCG or Mycobacterium tuberculosis-infected APCs to present Ag85B. In addition, L685485 inhibition of γ-secretase led to a decreased ability of BCG-dendritic cells to immunize mice and induce Ag85B-specific CD4 T cells in vivo. Because BCG and M. tuberculosis sequester within APCs preventing immune recognition, γ-secretase components appear to fortuitously process the immunodominant Ag85B, facilitating immune recognition.
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Secretasas de la Proteína Precursora del Amiloide/inmunología , Presentación de Antígeno/inmunología , Antígenos Bacterianos/inmunología , Vacuna BCG/inmunología , Epítopos de Linfocito T/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Células Presentadoras de Antígenos/inmunología , Células Presentadoras de Antígenos/metabolismo , Células Presentadoras de Antígenos/microbiología , Antígenos Bacterianos/metabolismo , Endopeptidasas/inmunología , Endopeptidasas/metabolismo , Técnica del Anticuerpo Fluorescente , Antígenos de Histocompatibilidad Clase II/inmunología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Mycobacterium bovis/inmunología , Mycobacterium bovis/metabolismo , Fagosomas/inmunología , Fagosomas/metabolismo , Presenilinas/inmunología , Presenilinas/metabolismo , Proteómica , Linfocitos T/metabolismoRESUMEN
Macrophages are the preeminent phagocytic cells which control multiple infections. Tuberculosis a leading cause of death in mankind and the causative organism Mycobacterium tuberculosis (MTB) infects and persists in macrophages. Macrophages use reactive oxygen and nitrogen species (ROS/RNS) and autophagy to kill and degrade microbes including MTB. Glucose metabolism regulates the macrophage-mediated antimicrobial mechanisms. Whereas glucose is essential for the growth of cells in immune cells, glucose metabolism and its downsteam metabolic pathways generate key mediators which are essential co-substrates for post-translational modifications of histone proteins, which in turn, epigenetically regulate gene expression. Herein, we describe the role of sirtuins which are NAD+-dependent histone histone/protein deacetylases during the epigenetic regulation of autophagy, the production of ROS/RNS, acetyl-CoA, NAD+, and S-adenosine methionine (SAM), and illustrate the cross-talk between immunometabolism and epigenetics on macrophage activation. We highlight sirtuins as emerging therapeutic targets for modifying immunometabolism to alter macrophage phenotype and antimicrobial function.
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Antiinfecciosos , Sirtuinas , Tuberculosis , Humanos , Histonas/metabolismo , Sirtuinas/genética , Sirtuinas/metabolismo , Epigénesis Genética , Especies Reactivas de Oxígeno/metabolismo , NAD/metabolismo , Macrófagos , Antiinfecciosos/metabolismoRESUMEN
An improved vaccine is urgently needed to replace the now more than 100-year-old Bacillus Calmette-Guérin (BCG) vaccine against tuberculosis (TB) disease, which represents a significant burden on global public health. Mycolic acid, or cord factor trehalose 6,6' dimycolate (TDM), a lipid component abundant in the cell wall of the pathogen Mycobacterium tuberculosis (MTB), has been shown to have strong immunostimulatory activity but remains underexplored due to its high toxicity and poor solubility. Herein, we employed a novel strategy to encapsulate TDM within a cubosome lipid nanocarrier as a potential subunit nanovaccine candidate against TB. This strategy not only increased the solubility and reduced the toxicity of TDM but also elicited a protective immune response to control MTB growth in macrophages. Both pre-treatment and concurrent treatment of the TDM encapsulated in lipid monoolein (MO) cubosomes (MO-TDM) (1 mol %) induced a strong proinflammatory cytokine response in MTB-infected macrophages, due to epigenetic changes at the promoters of tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) in comparison to the untreated control. Furthermore, treatment with MO-TDM (1 mol %) cubosomes significantly improved antigen processing and presentation capabilities of MTB-infected macrophages to CD4 T cells. The ability of MO-TDM (1 mol %) cubosomes to induce a robust innate and adaptive response in vitro was further supported by a mathematical modeling study predicting the vaccine efficacy in vivo. Overall, these results indicate a strong immunostimulatory effect of TDM when delivered through the lipid nanocarrier, suggesting its potential as a novel TB vaccine.
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Mycobacterium tuberculosis , Tuberculosis , Humanos , Anciano de 80 o más Años , Factores Cordón/farmacología , Estudios Prospectivos , Tuberculosis/tratamiento farmacológico , Tuberculosis/prevención & control , CitocinasRESUMEN
Because of continual generation of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is critical to design the next generation of vaccines to combat the threat posed by SARS-CoV-2 variants. We developed human adenovirus (HAd) vector-based vaccines (HAd-Spike/C5 and HAd-Spike) that express the whole Spike (S) protein of SARS-CoV-2 with or without autophagy-inducing peptide C5 (AIP-C5), respectively. Mice or golden Syrian hamsters immunized intranasally (i.n.) with HAd-Spike/C5 induced similar levels of S-specific humoral immune responses and significantly higher levels of S-specific cell-mediated immune (CMI) responses compared with HAd-Spike vaccinated groups. These results indicated that inclusion of AIP-C5 induced enhanced S-specific CMI responses and similar levels of virus-neutralizing titers against SARS-CoV-2 variants. To investigate the protection efficacy, golden Syrian hamsters immunized i.n. either with HAd-Spike/C5 or HAd-Spike were challenged with SARS-CoV-2. The lungs and nasal turbinates were collected 3, 5, 7, and 14 days post challenge. Significant reductions in morbidity, virus titers, and lung histopathological scores were observed in immunized groups compared with the mock- or empty vector-inoculated groups. Overall, slightly better protection was seen in the HAd-Spike/C5 group compared with the HAd-Spike group.
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We describe a role of CD44-mediated signaling during host-defense against tuberculosis (TB) using a mouse model of TB and studies in M. tuberculosis (Mtb) infected human macrophage (MФ). Liposomes targeting CD44 using thioaptamers (CD44TA-LIP) were designed and tested as new vaccines to boost host immunity in TB. CD44TA-LIP enhanced killing of Mtb in human MФ, which correlated with an increased production of pro-inflammatory cytokines IL-1ß, TNF-α and IL-12. CD44TA-LIP activated MФ showed an enhanced MHC-II dependent antigen presentation to CD4 T-cells. Inhibition of cellular proliferation and cytoskeleton rearrangement pathways downstream of CD44 signaling abrogated CD44TA-LIP-induced antimicrobial effects. Blockade of inflammatory pathways also reduced antigen presentation by MФ and activation of CD4 T cells. Mtb infected MФ treated with CD44TA-LIP exhibited increased nitric oxide and HßD2 defensin peptide production. Among Mtb infected mice with increased lung and spleen loads of organisms, intranasal administration of CD44TA-LIP led to a ten-fold reduction of colony forming units of Mtb and elevated IFN-γ + CD4, effector, central and resident memory T cells. Biodistribution studies demonstrated that CD44TA-LIP preferentially accumulated in the lungs and were associated with CD11b + cells. CD44TA-LIP treated mice showed no weight loss or increased liver LDH levels. This study highlights the importance of CD44-mediated signaling in host-defense during TB and the therapeutic potential of CD44TA-LIP.
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Antiinfecciosos , Receptores de Hialuranos/metabolismo , Mycobacterium tuberculosis , Nanopartículas , Tuberculosis , Defensinas , Humanos , Interleucina-12 , Liposomas , Antígeno de Macrófago-1 , Óxido Nítrico , Distribución Tisular , Tuberculosis/tratamiento farmacológico , Factor de Necrosis Tumoral alfaRESUMEN
GM-CSF is an important cytokine that regulates the proliferation of monocytes/macrophages and its various functions during health and disease. Although growing evidences support the notion that GM-CSF could play a major role in immunity against tuberculosis (TB) infection, the mechanism of GM-CSF mediated protective effect against TB remains largely unknown. Here in this study we examined the secreted levels of GM-CSF by human macrophages from different donors along with the GM-CSF dependent cellular processes that are critical for control of M. tuberculosis infection. While macrophage of different donors varied in their ability to produce GM-CSF, a significant correlation was observed between secreted levels of GM-CSF, survial of macrophages and intra-macrophage control of Mycobacterium tuberculosis bacilli. GM-CSF levels secreted by macrophages negatively correlated with the intra-macrophage M. tuberculosis burden, survival of infected host macrophages positively correlated with their GM-CSF levels. GM-CSF-dependent prolonged survival of human macrophages also correlated with significantly decreased bacterial burden and increased expression of self-renewal/cell-survival associated genes such as BCL-2 and HSP27. Antibody-mediated depletion of GM-CSF in macrophages resulted in induction of significantly elevated levels of apoptotic/necrotic cell death and a simultaneous decrease in autophagic flux. Additionally, protective macrophages against M. tuberculosis that produced more GM-CSF, induced a stronger granulomatous response and produced significantly increased levels of IL-1ß, IL-12 and IL-10 and decreased levels of TNF-α and IL-6. In parallel, macrophages isolated from the peripheral blood of active TB patients exhibited reduced capacity to control the intracellular growth of M. tuberculosis and produced significantly lower levels of GM-CSF. Remarkably, as compared to healthy controls, macrophages of active TB patients exhibited significantly altered metabolic state correlating with their GM-CSF secretion levels. Altogether, these results suggest that relative levels of GM-CSF produced by human macrophages plays a critical role in preventing cell death and maintaining a protective differentiation and metabolic state of the host cell against M. tuberculosis infection.
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Factor Estimulante de Colonias de Granulocitos y Macrófagos , Macrófagos , Mycobacterium tuberculosis , Tuberculosis , Diferenciación Celular , Citocinas/metabolismo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Macrófagos/citología , Macrófagos/microbiología , Tuberculosis/inmunologíaRESUMEN
Tuberculosis is a leading cause of death in mankind due to infectious agents, and Mycobacterium tuberculosis (Mtb) infects and survives in macrophages (MФs). Although MФs are a major niche, myeloid-derived suppressor cells (MDSCs) are an alternative site for pathogen persistence. Both MФs and MDSCs express varying levels of leukocyte immunoglobulin-like receptor B (LILRB), which regulate the myeloid cell suppressive function. Herein, we demonstrate that antagonism of LILRB2 by a monoclonal antibody (mab) induced a switch of human MDSCs towards an M1-macrophage phenotype, increasing the killing of intracellular Mtb. Mab-mediated antagonism of LILRB2 alone and its combination with a pharmacological blockade of SHP1/2 phosphatase increased proinflammatory cytokine responses and phosphorylation of ERK1/2, p38 MAPK, and NF-kB in Mtb-infected MDSCs. LILRB2 antagonism also upregulated anti-mycobacterial iNOS gene expression and an increase in both nitric oxide and reactive oxygen species synthesis. Because genes associated with the anti-mycobacterial function of M1-MФs were enhanced in MDSCs following mab treatment, we propose that LILRB2 antagonism reprograms MDSCs from an immunosuppressive state towards a pro-inflammatory phenotype that kills Mtb. LILRB2 is therefore a novel therapeutic target for eradicating Mtb in MDSCs.
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Glicoproteínas de Membrana , Mycobacterium tuberculosis , Células Supresoras de Origen Mieloide , Receptores Inmunológicos , Tuberculosis Ganglionar , Citocinas/inmunología , Humanos , Macrófagos/inmunología , Glicoproteínas de Membrana/inmunología , Mycobacterium tuberculosis/inmunología , Células Supresoras de Origen Mieloide/inmunología , Receptores Inmunológicos/inmunologíaRESUMEN
Mycobacterium tuberculosis (Mtb) is responsible for approximately 1.5 million deaths each year. Though 10% of patients develop tuberculosis (TB) after infection, 90% of these infections are latent. Further, mice are nearly uniformly susceptible to Mtb but their M1-polarized macrophages (M1-MΦs) can inhibit Mtb in vitro, suggesting that M1-MΦs may be able to regulate anti-TB immunity. We sought to determine whether human MΦ heterogeneity contributes to TB immunity. Here we show that IFN-γ-programmed M1-MΦs degrade Mtb through increased expression of innate immunity regulatory genes (Inregs). In contrast, IL-4-programmed M2-polarized MΦs (M2-MΦs) are permissive for Mtb proliferation and exhibit reduced Inregs expression. M1-MΦs and M2-MΦs express pro- and anti-inflammatory cytokine-chemokines, respectively, and M1-MΦs show nitric oxide and autophagy-dependent degradation of Mtb, leading to increased antigen presentation to T cells through an ATG-RAB7-cathepsin pathway. Despite Mtb infection, M1-MΦs show increased histone acetylation at the ATG5 promoter and pro-autophagy phenotypes, while increased histone deacetylases lead to decreased autophagy in M2-MΦs. Finally, Mtb-infected neonatal macaques express human Inregs in their lymph nodes and macrophages, suggesting that M1 and M2 phenotypes can mediate immunity to TB in both humans and macaques. We conclude that human MФ subsets show unique patterns of gene expression that enable differential control of TB after infection. These genes could serve as targets for diagnosis and immunotherapy of TB.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Animales , Citocinas/genética , Citocinas/metabolismo , Humanos , Inmunidad Innata/genética , Macrófagos/metabolismo , Ratones , Tuberculosis/metabolismoRESUMEN
Proteomics has been applied to study intracellular bacteria and phagocytic vacuoles in different host cell lines, especially macrophages (Mφs). For mycobacterial phagosomes, few studies have identified over several hundred proteins for systems assessment of the phagosome maturation and antigen presentation pathways. More importantly, there has been a scarcity in publication on proteomic characterization of mycobacterial phagosomes in dendritic cells (DCs). In this work, we report a global proteomic analysis of Mφ and DC phagosomes infected with a virulent, an attenuated, and a vaccine strain of mycobacteria. We used label-free quantitative proteomics and bioinformatics tools to decipher the regulation of phagosome maturation and antigen presentation pathways in Mφs and DCs. We found that the phagosomal antigen presentation pathways are repressed more in DCs than in Mφs. The results suggest that virulent mycobacteria might co-opt the host immune system to stimulate granuloma formation for persistence while minimizing the antimicrobial immune response to enhance mycobacterial survival. The studies on phagosomal proteomes have also shown promise in discovering new antigen presentation mechanisms that a professional antigen presentation cell might use to overcome the mycobacterial blockade of conventional antigen presentation pathways.