Dual RNA Sequencing of Mycobacterium tuberculosis-Infected Human Splenic Macrophages Reveals a Strain-Dependent Host-Pathogen Response to Infection.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), leading to pulmonary and extrapulmonary TB, whereby Mtb is disseminated to many other organs and tissues. Dissemination occurs early during the disease, and bacteria can be found first in the lymph nodes adjacent to the lungs and then later in the extrapulmonary organs, including the spleen. The early global gene expression response of human tissue macrophages and intracellular clinical isolates of Mtb has been poorly studied. Using dual RNA-seq, we have explored the mRNA profiles of two closely related clinical strains of the Latin American and Mediterranean (LAM) family of Mtb in infected human splenic macrophages (hSMs). This work shows that these pathogens mediate a distinct host response despite their genetic similarity. Using a genome-scale host-pathogen metabolic reconstruction to analyze the data further, we highlight that the infecting Mtb strain also determines the metabolic response of both the host and pathogen. Thus, macrophage ontogeny and the genetic-derived program of Mtb direct the host-pathogen interaction.

Metabolic adaptation of two in silico mutants of Mycobacterium tuberculosis during infection.

BACKGROUND: Up to date, Mycobacterium tuberculosis (Mtb) remains as the worst intracellular killer pathogen. To establish infection, inside the granuloma, Mtb reprograms its metabolism to support both growth and survival, keeping a balance between catabolism, anabolism and energy supply. Mtb knockouts with the faculty of being essential on a wide range of nutritional conditions are deemed as target candidates for tuberculosis (TB) treatment. Constraint-based genome-scale modeling is considered as a promising tool for evaluating genetic and nutritional perturbations on Mtb metabolic reprogramming. Nonetheless, few in silico assessments of the effect of nutritional conditions on Mtb's vulnerability and metabolic adaptation have been carried out. RESULTS: A genome-scale model (GEM) of Mtb, modified from the H37Rv iOSDD890, was used to explore the metabolic reprogramming of two Mtb knockout mutants (pfkA- and icl-mutants), lacking key enzymes of central carbon metabolism, while exposed to changing nutritional conditions (oxygen, and carbon and nitrogen sources). A combination of shadow pricing, sensitivity analysis, and flux distributions patterns allowed us to identify metabolic behaviors that are in agreement with phenotypes reported in the literature. During hypoxia, at high glucose consumption, the Mtb pfkA-mutant showed a detrimental growth effect derived from the accumulation of toxic sugar phosphate intermediates (glucose-6-phosphate and fructose-6-phosphate) along with an increment of carbon fluxes towards the reductive direction of the tricarboxylic acid cycle (TCA). Furthermore, metabolic reprogramming of the icl-mutant (icl1&icl2) showed the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA, during growth at high fatty acid consumption rates and aerobic conditions. At elevated levels of fatty acid uptake and hypoxia, we found a drop in TCA cycle intermediate accumulation that might create redox imbalance. Finally, findings regarding Mtb-mutant metabolic adaptation associated with asparagine consumption and acetate, succinate and alanine production, were in agreement with literature reports. CONCLUSIONS: This study demonstrates the potential application of genome-scale modeling, flux balance analysis (FBA), phenotypic phase plane (PhPP) analysis and shadow pricing to generate valuable insights about Mtb metabolic reprogramming in the context of human granulomas.

Innate immune cells for immunotherapy of autoimmune and cancer disorders.

Modulation of the immune system has been widely targeted for the treatment of several immune-related diseases, such as autoimmune disorders and cancer, due to its crucial role in these pathologies. Current available therapies focus mainly on symptomatic treatment and are often associated with undesirable secondary effects. For several years, remission of disease and subsequently recovery of immune homeostasis has been a major goal for immunotherapy. Most current immunotherapeutic strategies are aimed to inhibit or potentiate directly the adaptive immune response by modulating antibody production and B cell memory, as well as the effector potential and memory of T cells. Although these immunomodulatory approaches have shown some success in the clinic with promising therapeutic potential, they have some limitations related to their effectiveness in disease models and clinical trials, as well as elevated costs. In the recent years, a renewed interest has emerged on targeting innate immune cells for immunotherapy, due to their high plasticity and ability to exert a potent and extremely rapid response, which can influence the outcome of the adaptive immune response. In this review, we discuss the immunomodulatory potential of several innate immune cells, as well as they use for immunotherapy, especially in autoimmunity and cancer.

Suppression of autophagy and antigen presentation by Mycobacterium tuberculosis PE_PGRS47.

Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection.

Mecanismos de activación de las células T asesinas naturales invariantes (iNKT) / Mecanismos de ativação das células T assassinas naturais invariantes (iNKT) / Activation mechanisms of invariant natural killer T cells (iNKTs)

Aunque se ha logrado un conocimiento amplio acerca de las células T asesinas naturales (iNKT), aún no existe consenso sobre sus mecanismos de activación. Dichas células reconocen diferentes antígenos glicolipídicos presentados por medio de la molécula CD1d, los cuales pueden ser endógenos, exógenos derivados de organismos como bacterias y sintéticos desarrollados para aplicaciones clínicas. Existe mucho interés en entender cómo estas distintas variantes glicolipídicas inducen diferentes tipos de polarización, pero ha sido muy difícil llegar a un consenso, debido a que la respuesta depende de varios factores como la naturaleza, la internalización y el procesamiento de los glicolípidos. Además, la activación de las células iNKT la determinan el tipo y estado de activación de la célula presentadora de antígeno, las moléculas coestimuladoras, los mecanismos de transactivación y la localización de los complejos CD1d-glicolípido en distintas microrregiones de la membrana plasmática, como las balsas lipídicas. Esta revisión explora la evidencia sobre los factores que afectan la activación de las células iNKT con el fin de entender su potencial inmunomodulador.

A great amount of knowledge on natural killer T cells (iNKTs) is now available, but a consensus about their activation mechanisms has not been reached. These cells recognize different glycolipid antigens through the CD1d molecule. Such antigens may be endogenous, derived from bacteria (foreign) and synthetic, the latter have been developed for clinical applications. There exists much interest in understanding how these different glycolipid compounds induce different types of polarization, but it has been difficult to reach a consensus due to the fact that responses depend on different factors such as: the nature of the molecule, the internalization process and the presentation of the glycolipids. Moreover, activation of iNKT cells is determined by the type and state of the antigen presenting cell, the co-stimulatory molecules, the transactivation mechanisms and the location of the glycolipid-CD1d complexes on the plasma membrane, such as the lipid rafts. This review explores the evidence about the factors that affect activation of iNKT cells in order to understand their immune-modulatory potential.

Ainda que se conseguiu um conhecimento amplo a respeito das células T assassinas naturais (iNKT), ainda não existe consenso sobre seus mecanismos de ativação. Ditas células reconhecem diferentes antígenos glicolipídicos apresentados por meio da molécula CD1d, os quais pode ser: endógenos, exógenos derivados de organismos como bactérias e sintéticos desenvolvidos para aplicações clínicas. Existe muito interesse em entender como estas diferentes variantes glicolipídicas induzem diferentes tipos de polarização, mas foi muito difícil chegar a um consenso, devido a que a resposta depende de vários fatores como a natureza, a internalização e o processamento dos glicolípidos. Ademais, a ativação das células iNKT a determinam o tipo e estado de ativação da célula apresentadora de antígeno, as moléculas co-estimuladoras, os mecanismos de transativação e a localização dos complexos CD1d-glicolípido em diferentes microrregiões da membrana plasmática, como as balsas lipídicas. Esta revisão explora a evidência sobre os fatores que afetam a ativação das células iNKT com o fim de entender seu potencial imunomodulador.

Encephalitozoon intestinalis Inhibits Dendritic Cell Differentiation through an IL-6-Dependent Mechanism.

Microsporidia are a group of intracellular pathogens causing self-limited and severe diseases in immunocompetent and immunocompromised individuals, respectively. A cellular type 1 adaptive response, mediated by IL-12, IFNγ, CD4+, and CD8+ T cells has been shown to be essential for host resistance, and dendritic cells (DC) play a key role at eliciting anti-microsporidial immunity. We investigated the in vitro response of DC and DC precursors/progenitors to infection with Encephalitozoon intestinalis (Ei), a common agent of human microsporidosis. Ei-exposed DC cultures up-regulated the surface expression of MHC class II and the costimulatory molecules CD86 and CD40, only when high loads of spores were used. A vigorous secretion of IL-6 but not of IL-1ß or IL-12p70 was also observed in these cultures. Ei-exposed DC cultures consisted of immature infected and mature bystander DC, as assessed by MHC class II and costimulatory molecules expression, suggesting that intracellular Ei spores deliver inhibitory signals in DC. Moreover, Ei selectively inhibited the secretion of IL-12p70 in LPS-stimulated DC. Whereas Ei-exposed DC promoted allogeneic naïve T cell proliferation and IL-2 and IFNγ secretion in DC-CD4+ T cell co-cultures, separated co-cultures with bystander or infected DCs showed stimulation or inhibition of IFNγ secretion, respectively. When DC precursors/progenitors were exposed to Ei spores, a significant inhibition of DC differentiation was observed without shifting the development toward cells phenotypically or functionally compatible with myeloid-derived suppressor cells. Neutralization experiments demonstrated that this inhibitory effect is IL-6-dependent. Altogether this investigation reveals a novel potential mechanism of immune escape of microsporidian parasites through the modulation of DC differentiation and maturation.

Murine invariant natural killer T cells recognize glycolipids derived from extracts of the lichen Stereocaulon ramulosum / Células T asesinas naturales invariantes murinas reconocen glicolipidos derivados de extractos del liquen Stereocaulon ramulosum

Background: Invariant natural killer T cells (iNKT) can be activated by certain types of glycolipids that have the potential to generate adjuvant effects which could be used to develop effective and safe immunotherapies. Many of these glycolipids have been isolated from natural organisms, but there is a great amount of these organisms completely unexplored as a source of these types of compounds. Some of these organisms are lichens which are complex symbiotic organisms that have been showed to contain glycolipids. Objectives: We decide to test if glycolipids isolated from lichens would be able to activate iNKT cells in vitro and in vivo. Methods: We have used extracted glycolipids from 43 different species of lichens from Colombia. We have used iNKT hybridoma cells, C57BL/6 mice, IL-2 ELISA and the B16 melanoma to test for the adjuvant capabilities of glycolipids isolated from lichens. Results: In this study we have found two glycolipids with the capacity to activate iNKT cells in vivo. One of the glycolipids was able to activate iNKT cells in vivo, and was competent to induce protection against the B16 melanoma in the mouse model. Conclusions: We propose a possible chemical structure for a novel glycolipid called ß-GalCer-lich (1) derived from the lichen Stereocaulon ramulosum.

Antecedentes: Las células asesinas naturales T (iNKT) pueden ser activadas por ciertos tipos de glicolípidos que tienen el potencial para generar efectos adyuvantes los cuales pueden ser usados para desarrollar inmunoterapias efectivas. Muchos de estos glicolípidos han sido aislados de organismos naturales, pero hay una gran cantidad de organismos completamente inexplorados como fuente de este tipo de compuestos. Algunos de estos organismos son los líquenes, los cuales son organismos simbiontes complejos para los que se ha mostrado que contienen glicolípidos. Objetivos: Nosotros decidimos probar si los glicolípidos aislados de líquenes podrían ser capaces de activar alas celulas iNKT in vitro e in vivo. Metodos: Nosotros hemos extraído glicolípidos de 43 especies de líquenes de Colombia. Nosotros hemos usado células de un hibridoma de iNKTs, ratones C57BL/6, ELISA para IL-2 y el melanoma B16 para probar la capacidad adyuvante de los glicolipidos aislados de los líquenes. Resultados: En este estudio nosotros hemos encontrado dos glicolípidos con la capacidad de activar iNKTs in vitro. Uno de los glicolípidos fue capaz de activar células iNKT in vivo, y fue competente para inducir protección contra el melanoma B16 en el modelo de ratón. Conclusiones: Nosotros proponemos una posible estructura química para el nuevo glicolípido llamado ß-GalCer-lich (1) derivado del liquen Stereocaulon ramulosum.

A single subset of dendritic cells controls the cytokine bias of natural killer T cell responses to diverse glycolipid antigens.

Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α(+) DEC-205(+) dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α(+) dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses.

Correlation between the response to Mycobacterium tuberculosis antigens and the tuberculin skin test in patients with rheumatoid arthritis in Colombia / Correlación de la respuesta a antígenos de Mycobacterium tuberculosis y la prueba cutánea de tuberculina en pacientes con artritis reumatoide

Introduction. Rheumatoid arthritis patients under treatment with anti-TNF-α are at a high risk of developing active tuberculosis, and therefore, screening for latent tuberculosis infection is recommended before anti-TNF-α therapy. Objective. To compare the tuberculin test and IFNγ production induced by culture filtrate proteins(CFPs) and Mycobacterium tuberculosis-specific CFP-10 antigens in rheumatoid arthritis patients. Materials and methods. An analytic transversal study was conducted in rheumatoid arthritis patients treated at Hospital Universitario San Vicente Fundación between January and December 2007. IFNγ production in response to CFPs and CFP-10 was measured in the supernatants of whole blood cultures and evaluated for correlations with tuberculin reactivity. The degree of concordance between both tests was also established. Results. Forty-five patients were included, of which 14 (31.1%) had a tuberculin reaction of ≥10 mm of induration, 9 (20%) produced IFNγ in response to CFP-10, and 7 were positive for both tests. The correlation between tests was r=0.53 (IC 95%:0.28-0.72), and the global concordance between tests was80%, with a Kappa coefficient of 0.48 (IC95%:0.20-0.76). Conclusions. Only two tuberculin (-)/CFP-10+ "anergic" patients were observed. By contrast, six tuberculin +/CFP-10(-) "tuberculin false-positive" patients were observed. These data suggest that the tuberculin test is not an appropriate tool for determining the need for tuberculosis prophylaxis.

Introducción. Los pacientes con artritis reumatoide bajo tratamiento con anti-TNFα están en alto riesgo de desarrollar tuberculosis activa, por lo cual se recomienda hacer la tamización para infección latente de tuberculosis, antes de iniciar el tratamiento. Objetivo. Comparar la prueba de tuberculina y la producción de IFNγ inducida por antígenos CFP (Culture Filtrate Protein) y antígenos específicos de Mycobacterium tuberculosis (CFP-10) para el diagnóstico de infección latente de tuberculosis en pacientes con artritis reumatoide. Materiales y métodos. Se llevó a cabo un estudio transversal analítico en pacientes con artritis reumatoide atendidos en el Hospital Universitario San Vicente Fundación, entre enero y diciembre de 2007, a los cuales se les determinó la producción de IFNγ en respuesta a CFP y CFP-10 en sobrenadantes de cultivos de sangre total, y se correlacionó con la reacción en la prueba de tuberculina. Además, se estableció el grado de concordancia entre ambas pruebas. Resultados. Se incluyeron 45 pacientes, de los cuales, 14 (31,1 %) tuvieron un diámetro de induración ≥10 mm (tuberculina positiva), nueve (20 %) produjeron IFNγ en respuesta a CFP-10, y siete fueron positivos para ambas pruebas. La correlación entre las pruebas fue de r=0,53 (IC95%: 0,28-0,72) y la concordancia global entre pruebas fue de 80 %, con un coeficiente kappa de 0,48 (IC95%: 0,20-0,76). Conclusiones. Solo se observaron dos pacientes con tuberculina positiva y CFP-10 positivo "anérgicos" y se encontraron seis pacientes con tuberculina positiva y CFP-10 negativa "falsos positivos para tuberculina", lo cual sugiere que la prueba de la tuberculina no es la más adecuada para indicar profilaxis para tuberculosis.

Correlation between the response to Mycobacterium tuberculosis antigens and the tuberculin skin test in patients with rheumatoid arthritis in Colombia.

INTRODUCTION: Rheumatoid arthritis patients under treatment with anti-TNF-α are at a high risk of developing active tuberculosis, and therefore, screening for latent tuberculosis infection is recommended before anti-TNF-α therapy. OBJECTIVE: To compare the tuberculin test and IFNγ production induced by culture filtrate proteins(CFPs) and Mycobacterium tuberculosis-specific CFP-10 antigens in rheumatoid arthritis patients. MATERIALS AND METHODS: An analytic transversal study was conducted in rheumatoid arthritis patients treated at Hospital Universitario San Vicente Fundación between January and December 2007. IFNγ production in response to CFPs and CFP-10 was measured in the supernatants of whole blood cultures and evaluated for correlations with tuberculin reactivity. The degree of concordance between both tests was also established. RESULTS: Forty-five patients were included, of which 14 (31.1%) had a tuberculin reaction of ≥10 mm of induration, 9 (20%) produced IFNγ in response to CFP-10, and 7 were positive for both tests. The correlation between tests was r=0.53 (IC 95%:0.28-0.72), and the global concordance between tests was80%, with a Kappa coefficient of 0.48 (IC95%:0.20-0.76). CONCLUSIONS: Only two tuberculin (-)/CFP-10+ "anergic" patients were observed. By contrast, six tuberculin +/CFP-10(-) "tuberculin false-positive" patients were observed. These data suggest that the tuberculin test is not an appropriate tool for determining the need for tuberculosis prophylaxis.

Mycobacteria release active membrane vesicles that modulate immune responses in a TLR2-dependent manner in mice.

Bacteria naturally release membrane vesicles (MVs) under a variety of growth environments. Their production is associated with virulence due to their capacity to concentrate toxins and immunomodulatory molecules. In this report, we show that the 2 medically important species of mycobacteria, Mycobacterium tuberculosis and Mycobacterium bovis bacille Calmette-Guérin, release MVs when growing in both liquid culture and within murine phagocytic cells in vitro and in vivo. We documented MV production in a variety of virulent and nonvirulent mycobacterial species, indicating that release of MVs is a property conserved among mycobacterial species. Extensive proteomic analysis revealed that only MVs from the virulent strains contained TLR2 lipoprotein agonists. The interaction of MVs with macrophages isolated from mice stimulated the release of cytokines and chemokines in a TLR2-dependent fashion, and infusion of MVs into mouse lungs elicited a florid inflammatory response in WT but not TLR2-deficient mice. When MVs were administered to mice before M. tuberculosis pulmonary infection, an accelerated local inflammatory response with increased bacterial replication was seen in the lungs and spleens. Our results provide strong evidence that actively released mycobacterial vesicles are a delivery mechanism for immunologically active molecules that contribute to mycobacterial virulence. These findings may open up new horizons for understanding the pathogenesis of tuberculosis and developing vaccines.

Primate TNF promoters reveal markers of phylogeny and evolution of innate immunity.

BACKGROUND: Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry. METHODOLOGY/PRINCIPAL FINDINGS: Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages. CONCLUSIONS/SIGNIFICANCE: Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF.

The -1030/-862-linked TNF promoter single-nucleotide polymorphisms are associated with the inability to control HIV-1 viremia.

Control of HIV-1 viremia and progression to AIDS has been associated with specific HLA genes. The tumor necrosis factor ( TNF) and the non-classical major histocompatibility (MHC) class I chain-related A ( MICA) genes are located in the genomic segment between the HLA class I and II genes and variants of both genes have been identified. We thus analyzed TNF promoter and MICA variants in a well-characterized group of HIV-1 infected individuals with different abilities to control HIV-1 viremia. In our cohort, the -1030/-862-linked TNF promoter single-nucleotide polymorphisms (SNPs), but not MICA variants, are significantly associated with lack of control of HIV-1 viremia ( P=0.03). This association is independent of those HLA-B35 alleles associated with HIV-1 disease progression with which the -862 TNF SNP has previously been independently associated. Thus, non-randomly associated genes near the TNF locus are likely involved in control of HIV-1 viremia.

Ethnic-specific genetic associations with pulmonary tuberculosis.

Several susceptibility-associated genetic polymorphisms have been proposed to explain differential susceptibility to tuberculosis (TB) disease progression in different populations. Here, polymorphisms in the natural resistance-associated macrophage protein 1 (NRAMP1), vitamin D receptor, tumor necrosis factor-alpha, interleukin (IL)-1, and IL-10 genes were evaluated in 358 Cambodian patients with pulmonary TB and 106 tuberculin-positive control subjects. Heterozygosity for the -1082 polymorphism of the IL-10 promoter and heterozygosity for 2 linked polymorphic NRAMP1 variants, D543N and 3' untranslated region, were associated with TB susceptibility and resistance, respectively. Other polymorphisms associated with differential susceptibility to TB were not associated with susceptibility or resistance to TB in Cambodians. The novel pattern of genetic associations with susceptibility and resistance to TB detected in Cambodia is consistent with the conclusion that unique environmental and natural selective factors have resulted in the development of ethnic-specific host genetic factors associated with TB susceptibility and resistance worldwide.