Modulatory Impact of the sRNA Mcr11 in Two Clinical Isolates of Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is a successful pathogen causing tuberculosis (TB) disease in humans. It has been shown, that some circulating strains of Mtb in TB endemic populations, are more virulent and more transmissible than others, which may be related to their evolved adaptations to modulate the host immune responses. Underlying these adaptations to the stressful conditions, different genetic regulatory networks involved sRNAs that are mostly unknown for Mtb. We have previously shown that Mcr11 is one of the main sRNAs that determine transcriptomic differences among the Colombian clinical isolates UT127 and UT205 compared to the laboratory strain H37Rv. We found that the knock-down of mcr11 using CRISPRi has a major impact on phenotypic traits, especially in the clinical isolate UT205. Through the analysis of RNA-seq during the knock-down of mcr11 in UT205, we found a downregulation of genes mainly involved in lipid synthesis, lipid metabolism, ribosomal proteins, transport systems, respiratory and energy systems, membrane and cell wall components, intermediary metabolism, lipoproteins and virulence genes. One of the most interesting genes showing transcriptomic changes is OprA (encoded by the gene rv0516c), which has been involved in the K+ regulation. Overall, our data may suggest that one of the prominent roles of the sRNA Mcr11 is to regulate genes that control Mtb growth and osmoregulation.

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.

A systematic evaluation of Mycobacterium tuberculosis Genome-Scale Metabolic Networks.

Metabolism underpins the pathogenic strategy of the causative agent of TB, Mycobacterium tuberculosis (Mtb), and therefore metabolic pathways have recently re-emerged as attractive drug targets. A powerful approach to study Mtb metabolism as a whole, rather than just individual enzymatic components, is to use a systems biology framework, such as a Genome-Scale Metabolic Network (GSMN) that allows the dynamic interactions of all the components of metabolism to be interrogated together. Several GSMNs networks have been constructed for Mtb and used to study the complex relationship between the Mtb genotype and its phenotype. However, the utility of this approach is hampered by the existence of multiple models, each with varying properties and performances. Here we systematically evaluate eight recently published metabolic models of Mtb-H37Rv to facilitate model choice. The best performing models, sMtb2018 and iEK1011, were refined and improved for use in future studies by the TB research community.

Potential of DosR and Rpf antigens from Mycobacterium tuberculosis to discriminate between latent and active tuberculosis in a tuberculosis endemic population of Medellin Colombia.

BACKGROUND: Tuberculosis (TB) remains one of the most deadly infectious diseases. One-third to one-fourth of the human population is estimated to be infected with Mycobacterium tuberculosis (Mtb) without showing clinical symptoms, a condition called latent TB infection (LTBI). Diagnosis of Mtb infection is based on the immune response to a mixture of mycobacterial antigens (PPD) or to Mtb specific ESAT-6/CFP10 antigens (IGRA), highly expressed during the initial phase of infection. However, the immune response to PPD and IGRA antigens has a low power to discriminate between LTBI and PTB. The T-cell response to a group of so-called latency (DosR-regulon-encoded) and Resuscitation Promoting (Rpf) antigens of Mtb has been proved to be significantly higher in LTBI compared to active TB across many populations, suggesting their potential use as biomarkers to differentiate latent from active TB. METHODS: PBMCs from a group LTBI (n = 20) and pulmonary TB patients (PTB, n = 21) from an endemic community for TB of the city of Medellín, Colombia, were in vitro stimulated for 7 days with DosR- (Rv1737c, Rv2029c, and Rv2628), Rpf- (Rv0867c and Rv2389c), the recombinant fusion protein ESAT-6-CFP10 (E6-C10)-, or PPD-antigen. The induced IFNγ levels detectable in the supernatants of the antigen-stimulated cells were then used to calculate specificity and sensitivity in discriminating LTBI from PTB, using different statistical approaches. RESULTS: IFNγ production in response to DosR and Rpf antigens was significantly higher in LTBI compared to PTB. ROC curve analyses of IFNγ production allowed differentiation of LTBI from PTB with areas under the curve higher than 0.70. Furthermore, Multiple Correspondence Analysis (MCA) revealed that LTBI is associated with higher levels of IFNγ in response to the different antigens compared to PTB. Analysis based on decision trees showed that the IFNγ levels produced in response to Rv2029c was the leading variable that best-classified disease status. Finally, logistic regression analysis predicted that IFNγ produced by PBMCs in response to E6-C10, Rv2029c, Rv0867c (RpfA) and Rv2389c (RpfA) antigens correlates best with the probability of being latently infected. CONCLUSIONS: The Mtb antigens E6-C10, Rv2029c (PfkB), Rv0867c (RpfA) and Rv2389c (RpfA), may be potential candidates to discriminate LTBI from PTB.

Tuberculosis Severity Predictive Model Using Mtb Variants and Serum Biomarkers in a Colombian Cohort of APTB Patients.

Currently, tuberculosis (TB) is a bacterial infection caused by Mycobacterium tuberculosis (Mtb) that primarily affects the lungs. The severity of active pulmonary TB (APTB) is an important determinant of transmission, morbidity, mortality, disease experience, and treatment outcomes. Several publications have shown a high prevalence of disabling complications in individuals who have had severe APTB. Furthermore, certain strains of Mtb were associated with more severe disease outcomes. The use of biomarkers to predict severe APTB patients who are candidates for host-directed therapies, due to the high risk of developing post-tuberculous lung disease (PTLD), has not yet been implemented in the management of TB patients. We followed 108 individuals with APTB for 6 months using clinical tools, flow cytometry, and whole-genome sequencing (WGS). The median age of the study population was 26.5 years, and the frequency of women was 53.7%. In this study, we aimed to identify biomarkers that could help us to recognize individuals with APTB and improve our understanding of the immunopathology in these individuals. In this study, we conducted a follow-up on the treatment progress of 121 cases of APTB. The follow-up process commenced at the time of diagnosis (T0), continued with a control visit at 2 months (T2), and culminated in an exit appointment at 6 months following the completion of medical treatment (T6). People classified with severe APTB showed significantly higher levels of IL-6 (14.7 pg/mL; p < 0.05) compared to those with mild APTB (7.7 pg/mL) at T0. The AUCs for the ROC curves and the Matthews correlation coefficient values (MCC) demonstrate correlations ranging from moderate to very strong. We conducted WGS on 88 clinical isolates of Mtb, and our analysis revealed a total of 325 genes with insertions and deletions (Indels) within their coding regions when compared to the Mtb H37Rv reference genome. The pattern of association was found between serum levels of CHIT1 and the presence of Indels in Mtb isolates from patients with severe APTB. A key finding in our study was the high levels of CHIT1 in severe APTB patients. We identified a biomarker profile (IL-6, IFN-γ, IL-33, and CHIT1) that allows us to identify individuals with severe APTB, as well as the identification of a panel of polymorphisms (125) in clinical isolates of Mtb from individuals with severe APTB. Integrating these findings into a predictive model of severity would show promise for the management of APTB patients in the future, to guide host-directed therapy and reduce the prevalence of PTLD.

Large genomic deletions delineate Mycobacterium tuberculosis L4 sublineages in South American countries.

Mycobacterium tuberculosis (Mtb) is still one of the primary pathogens of humans causing tuberculosis (TB) disease. Mtb embraces nine well-defined phylogenetic lineages with biological and geographical disparities. The lineage L4 is the most globally widespread of all lineages and was introduced to America with European colonization. Taking advantage of many genome projects available in public repositories, we undertake an evolutionary and comparative genomic analysis of 522 L4 Latin American Mtb genomes. Initially, we performed careful quality control of public read datasets and applied several thresholds to filter out low-quality data. Using a genome de novo assembly strategy and phylogenomic methods, we spotted novel south American clades that have not been revealed yet. Additionally, we describe genomic deletion profiles of these strains from an evolutionary perspective and report Mycobacterium tuberculosis L4 sublineages signature-like gene deletions, some of the novel. One is a specific deletion of 6.5 kbp that is only present in sublineage 4.1.2.1. This deletion affects a complex group of 10 genes with putative products annotated, among others, as a lipoprotein, transmembrane protein, and toxin/antitoxin system proteins. The second novel deletion spans for 4.9 kbp and specific of a particular clade of the 4.8 sublineage and affects 7 genes. The last novel deletion affects 4 genes, extends for 4.8 kbp., and is specific to some strains within the 4.1.2.1 sublineage that are present in Colombia, Peru and Brasil.

Tuberculin skin test reactivity is dependent on host genetic background in Colombian tuberculosis household contacts.

The tuberculin skin test (TST) measures the intensity of antimycobacterial acquired immunity and is used to diagnose latent infection with Mycobacterium tuberculosis. We report evidence for a codominant gene explaining ∼65% of the TST variability. Disregarding the host genetic background may lead to misclassifications of TST-based diagnosis of latent M. tuberculosis infection.

Mycobacterium tuberculosis alters the differentiation of monocytes into macrophages in vitro.

This paper shows that in vitro infection of human monocytes by Mycobacterium tuberculosis affected monocyte to macrophage differentiation. Despite the low bacterial load used, M. tuberculosis-infected monocytes had fewer granules, displayed a reduced number of cytoplasmic projections and decreased HLA class II, CD68, CD86 and CD36 expression compared to cells differentiated in the absence of mycobacteria. Infected cells produced less IL-12p70, TNF-α, IL-10, IL-6 and high IL-1ß in response to lipopolysaccharide and purified protein M. tuberculosis-derived. Reduced T-cell proliferative response and IFN-γ secretion in response to phytohemagglutinin and culture filtrate proteins from M. tuberculosis was also observed in infected cells when compared to non-infected ones. The ability of monocytes differentiated in the presence of M. tuberculosis to control mycobacterial growth in response to IFN-γ stimulation was attenuated, as determined by bacterial plate count; however, they had a similar ability to uptake fluorescent M. tuberculosis and latex beads compared to non-infected cells. Recombinant IL-1ß partially altered monocyte differentiation into macrophages; however, treating M. tuberculosis-infected monocytes with IL-1RA did not reverse the effects of infection during differentiation. The results indicated that M. tuberculosis infection altered monocyte differentiation into macrophages and affected their ability to respond to innate stimuli and activate T-cells.

Role of TLR2- and TLR4-mediated signaling in Mycobacterium tuberculosis-induced macrophage death.

Infection of macrophages with Mycobacterium tuberculosis (Mtb) induces cell death by apoptosis or necrosis. TLRs 2 and 4 recognition of mycobacterial ligands has been independently associated to apoptosis induction. To try to understand the particular contribution of these receptors to apoptotic or necrotic signaling upon infection with live Mtb H37Rv, we used macrophage lines derived from wild-type or TLR2-, TLR4-, and MyD88-deficient mouse strains. Mtb-infection triggered apoptosis depending on a TLR2/TLR4/MyD88/p38/ERK/PI-3K/NF-kB pathway; however, necrosis was favored in absence of TLR4 signaling independently of p38, ERK1/2, PI-3K or NF-kappaB activity. In conclusion, our results indicate that cooperation between TLR2- and TLR4-dependent mediated signals play a critical role in macrophage apoptosis induced by Mtb and the TLR4-mediated signaling has important role in the maintenance of the balance between apoptotic vs. necrotic cell death induced by macrophage infection with Mtb.

Infection of Monocytes From Tuberculosis Patients With Two Virulent Clinical Isolates of Mycobacterium tuberculosis Induces Alterations in Myeloid Effector Functions.

Monocytes play a critical role during infection with Mycobacterium tuberculosis (Mtb). They are recruited to the lung, where they participate in the control of infection during active tuberculosis (TB). Alternatively, inflammatory monocytes may participate in inflammation or serve as niches for Mtb infection. Monocytes response to infection may vary depending on the particularities of the clinical isolate of Mtb from which they are infected. In this pilot study, we have examined the baseline mRNA profiles of circulating human monocytes from patients with active TB (MoTB) compared with monocytes from healthy individuals (MoCT). Circulating MoTB displayed a pro-inflammatory transcriptome characterized by increased gene expression of genes associated with cytokines, monocytopoiesis, and down-regulation of MHC class II gene expression. In response to in vitro infection with two clinical isolates of the LAM family of Mtb (UT127 and UT205), MoTB displayed an attenuated inflammatory mRNA profile associated with down-regulation the TREM1 signaling pathway. Furthermore, the gene expression signature induced by Mtb UT205 clinical strain was characterized by the enrichment of genes in pathways and biological processes mainly associated with a signature of IFN-inducible genes and the inhibition of cell death mechanisms compared to MoTB-127, which could favor the establishment and survival of Mtb within the monocytes. These results suggest that circulating MoTB have an altered transcriptome that upon infection with Mtb may help to maintain chronic inflammation and infection. Moreover, this functional abnormality of monocytes may also depend on potential differences in virulence of circulating clinical strains of Mtb.

Human Alveolar and Splenic Macrophage Populations Display a Distinct Transcriptomic Response to Infection With Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) infects alveolar macrophages (AMs), causing pulmonary tuberculosis (PTB), the most common form of the disease. Less frequently, Mtb is disseminated to many other organs and tissues, resulting in different extrapulmonary forms of TB. Nevertheless, very few studies have addressed the global mRNA response of human AMs, particularly from humans with the active form of the disease. Strikingly, almost no studies have addressed the response of human extrapulmonary macrophages to Mtb infection. In this pilot study, using microarray technology, we examined the transcriptomic ex vivo response of AMs from PTB patients (AMTBs) and AMs from control subjects (AMCTs) infected with two clinical isolates of Mtb. Furthermore, we also studied the infection response of human splenic macrophages (SMs) to Mtb isolates, as a model for extrapulmonary infection, and compared the transcriptomic response between AMs and SMs. Our results showed a striking difference in global mRNA profiles in response to infection between AMs and SMs, implicating a tissue-specific macrophage response to Mtb.

New Conjugated Compound T5 Epidioxy-Sterol-ANB Inhibits the Growth of Mycobacterium tuberculosis Affecting the Cholesterol and Folate Pathways.

The upsurge and persistence of drug resistant strains of Mycobacterium tuberculosis (Mtb) is an important limitant to the battery of drugs available for the elimination of tuberculosis (TB). To avoid future scarcity of antibiotics against Mtb, it is important to discover new effective anti-mycobacterial agents. In this study, we present data from a series of experiments to determine in vitro and in vivo anti-mycobacterial activity of a library of epidioxy-sterol analogs. We test 15 compounds for their ability to reduce the viability of Mtb. We found that one compound called T5 epidioxy-sterol-ANB display significant potency against Mtb in vitro specifically inside macrophages but without effectivity in axenic cultures. A viability assay confirms that this T5 compound is less toxic for macrophages in vitro as compared to the current Mtb drug Rifampicin at higher concentrations. We use a transcriptomic analysis of Mtb inside macrophages after T5 epidioxy-sterol-ANB treatment, and we found a significant down-regulation of enzymes involved in the cholesterol and folic acid pathways. In vivo, significant differences were found in the lungs and spleen CFUs of Mtb infected mice treated with the T5 epidioxy-sterol-ANB as compared with the untreated control group, which provides additional evidence of the effectivity of the T5 compound. Altogether these results confirm the potential of this T5 epidioxy-sterol-ANB compound against Mtb.

Differential determinants of virulence in two Mycobacterium tuberculosis Colombian clinical isolates of the LAM09 family.

The heterogeneity of the clinical outcome of Mycobacterium tuberculosis (Mtb) infection may be due in part to different strategies used by circulating strains to cause disease. This heterogeneity is one of the main limitations to eradicate tuberculosis disease. In this study, we have compared the transcriptional response of two closely related Colombian clinical isolates (UT127 and UT205) of the LAM family under two axenic media conditions. These clinical isolates are phenotypically different at the level of cell death, cytokine production, growth kinetics upon in vitro infection of human tissue macrophages, and membrane vesicle secretion upon culture in synthetic medium. Using RNA-seq, we have identified different pathways that account for two different strategies to cope with the stressful condition of a carbon-poor media such as Sauton's. We showed that the clinical isolate UT205 focus mainly in the activation of virulence systems such as the ESX-1, synthesis of diacyl-trehalose, polyacyl-trehalose, and sulfolipids, while UT127 concentrates its efforts mainly in the survival mode by the activation of the DNA replication, cell division, and lipid biosynthesis. This is an example of two Mtb isolates that belong to the same family and lineage, and even though they have a very similar genome, its transcriptional regulation showed important differences. This results in summary highlight the necessity to reach a better understanding of the heterogeneity in the behavior of these circulating Mtb strains which may help us to design better treatments and vaccines and to identify new targets for drugs.

Risk of infection and disease progression in children exposed to tuberculosis at home, Colombia.

AIM: To assess the risk of tuberculosis (infection and disease) in children less than 15 years' old who are household contacts of pulmonary tuberculosis patients in three Colombian cities (Medellín, Cali, and Popayán). METHODS: A cohort of 1,040 children household contacts of 380 adults with smear-positive pulmonary tuberculosis was followed up for 24 months. Study period 2005-2009. RESULTS: Tuberculin skin test was positive (≥10 mm) in 43.7% (95% CI: 39.2-48.2). Tuberculin skin test positivity was associated with age 10-14 years (Prevalence Ratio -PR= 1.43, 95% CI: 1.1-1.9), having a BCG vaccine scar (PR= 1.52, 95% CI: 1.1-2.1), underweight, closer proximity to the index case and exposure time >3 months. The annual risk of infection (tuberculin skin test induration increase of 6 mm or more per year) was 17% (95% CI: 11.8-22.2) and was associated with a bacillary load of the adult index case (Relative Risk -RR= 2.12, 95% CI: 1.0-4.3). The incidence rate of active tuberculosis was 12.4 cases per 1,000 persons-year. Children <5 years without BCG vaccine scar had a greater risk of developing active disease (Hazard Ratio -HR= 6.00, 95% CI: 1.3-28.3) than those with scar (HR= 1.33, 95% CI: 0.5-3.4). The risk of developing active tuberculosis augmented along with the increase from initial tuberculin skin test (tuberculin skin test 5-9 mm HR= 8.55, 95% CI: 2.5-29.2; tuberculin skin test ≥10 mm HR= 8.16, 95% CI: 2.0-32.9). CONCLUSIONS: There is a need for prompt interruption of adult-to-children tuberculosis transmission within households. Conducting proper contact investigation and offering chemoprophylaxis to infected children could reduce tuberculosis transmission.

OBJETIVO: Evaluar el riesgo de tuberculosis (infección y enfermedad) en niños menores de 15 años de edad convivientes de pacientes con tuberculosis pulmonar en tres ciudades colombianas (Medellín, Cali y Popayán). MÉTODOS: Se siguió durante 24 meses una cohorte de 1,040 niños convivientes de 380 adultos con tuberculosis pulmonar bacilífera. Periodo de estudio 2005-2009. Resultados: La prueba de tuberculina fue positiva (≥10 mm) en el 43.7% (IC 95%: 39.2-48.2), y estuvo asociada con la edad de 10-14 años (Razón de Prevalencia-RP= 1.43, IC 95%: 1.1-1.9), tener cicatriz de la vacuna BCG (RP= 1.52, IC 95%: 1.1-2.1). El riesgo anual de infección (aumento de la induración en la prueba de tuberculina de 6 mm o más al año) fue 17% (IC 95%: 11.8-22.2), y estuvo asociado con mayor carga bacilar en el adulto con tuberculosis pulmonar (Riesgo Relativo-RR= 2.12, IC 95%: 1.0-4.3). La tasa de incidencia de tuberculosis activa fue de 12.4 casos por 1,000 años-persona de seguimiento. Los niños menores de 5 años sin cicatriz de vacuna BCG tuvieron un mayor riesgo de desarrollar tuberculosis activa (Razón de Peligro -HR= 6.00, IC 95%: 1.3-28.3), que quienes tenían cicatriz (HR= 1.33, IC 95%: 0.5-3.4). El riesgo de desarrollar tuberculosis activa aumentó conforme el aumento de la prueba de tuberculina inicial (prueba de tuberculina 5-9 mm HR= 8.55, IC 95%: 2.5-29.2; prueba de tuberculina ≥10 mm HR= 8.16, IC 95%: 2.0-32.9). CONCLUSIÓN: Es necesario interrumpir rápidamente la transmisión de tuberculosis de adultos a niños en los hogares. Realizar investigaciones de contacto apropiadas y ofrecer quimioprofilaxis a los niños infectados podría reducir la transmisión de la tuberculosis.

Human splenic macrophages as a model for in vitro infection with Mycobacterium tuberculosis.

Macrophages play an important role during Mycobacterium tuberculosis (MTB) infection. In humans most of the studies on MTB-macrophage interactions have been performed using circulating monocytes and monocyte-derived macrophages. However, little research has been performed on this interaction using tissue macrophages. Herein, we used human splenic macrophages to characterize particular responses to MTB infection. Based on morphological, biochemical, and immunological markers, splenic adherent cells exhibit characteristics of tissue macrophages. They were able to efficiently phagocytose both live and heat-killed (h-k) MTB H37Rv. Upon infection with live, but not h-k MTB, an increase in secreted TNF-alpha was elicited. Splenic macrophages produced high basal levels of IL-10; however, infection with live or h-k MTB resulted in decrease IL-10 secretion. Both IL-12p40 and IL-12p70 basal levels were also decreased upon infection with live or h-k MTB; however, while the reduction for IL-12p40 levels was observed at earlier time points (4h) for both live and h-k MTB, infection with live MTB, but not h-k MTB, resulted in a time-dependent secretion of IL-12p40 at 24 and 48h after infection. IL-12p70 levels were completely reduced upon infection by either live or h-k MTB. These results support that human splenic macrophages may represent a potential useful model to study MTB-macrophage interactions in vitro.

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.

Alveolar macrophages from tuberculosis patients display an altered inflammatory gene expression profile.

Alveolar macrophages (AMs) are major targets of Mycobacterium tuberculosis (Mtb) infection, critical during the progression of active tuberculosis (TB). The complex immunopathology of TB generates diverse microenvironments in the lung, which shape immune responses by AMs. In the current study, we perform whole genome microarray transcriptional profiling on RNA isolated from AMs from TB patients (AMsTB) compared to AMs from control subjects (AMsCT) using bronchoalveolar lavage (BAL). Our hypothesis was that systemic effects on the local lung microenvironment during TB affect the transcriptional response of AMsTB. We found a unique gene expression profile of 51 genes, including up-regulated CHIT1, CHI3L1, CCL5, CCL22, CCL8, CXCL9, MMP9, MMP7 and MMP12, associated with a robust pro-inflammatory response, cell recruitment and tissue damage, and genes of the cyclin family (CCND1, CCND2, and CCNA1) associated with cell proliferation. These expression profiles may account for the inflammatory condition in the lungs of TB patients. CXCL5, IL1B, CAMP, and TGFB1 were down-regulated, suggesting an altered control of Mtb infection. Also, MARCO and COLEC12, affecting phagocytosis, and CES1, associated with an increase in free cholesterol, were down-regulated. The observed changes in mRNA expression profiles may partially account for the inability of AMsTB to effectively control Mtb infection, suggesting that a balanced control of pro- and anti-inflammatory immune responses is crucial for infection control.

Differences in IgG responses against infection phase related Mycobacterium tuberculosis (Mtb) specific antigens in individuals exposed or not to Mtb correlate with control of TB infection and progression.

Tuberculosis (TB) occurs in only 3-10% of Mycobacterium tuberculosis (Mtb) infected individuals, suggesting that natural immunity can contain Mtb infection, although this remains poorly understood. Next to T-cells, a potentially protective role for B-cells and antibodies has emerged recently. However, the Mtb antigens involved remain ill-defined. Here, we investigated in a TB-endemic setting IgG levels against 15 Mtb antigens, representing various phases of Mtb infection and known to be potent human T-cell antigens. IgG levels against ESAT6/CFP10, Rv0440, Rv0867c, Rv1737c, Rv2029c, Rv2215, Rv2389c, Rv3616c and Mtb purified protein derivative (PPD) were higher in TB patients than in endemic and non-endemic controls. The only exception was Rv1733c that was preferentially recognized by antibodies from endemic controls compared to TB patients and non-endemic controls, suggesting a potential correlation with control of TB infection and progression. In patients, IgG levels against Ag85B and Rv2029c correlated with Mtb loads, while immunoglobulins against Rv0440 differed between genders. Our results support the potential role of certain Mtb antigen-(Rv1733c) specific antibodies in the control of TB infection and progression, while other Mtb antigen-specific antibodies correlate with TB disease activity and bacillary loads. The findings for Rv1733c agree with previous T-cell results and have implications for including antibody-mediated immunity in designing new strategies to control TB.

Functional and phenotypic changes in monocytes from patients with tuberculosis are reversed with treatment.

Alterations of monocyte/macrophages have been reported in patients with tuberculosis (TB), but their significance is poorly understood. Blood mononuclear cells from patients with different clinical forms of TB, at various times of anti-TB treatment, and healthy tuberculin positive individuals, were double-stained for CD14 plus CD206, TLR-2, IFN-gammaR1, CD40, HLA-DR, CD36 and CD163, and analyzed by flow cytometry. Monocytes were infected with Mycobacterium tuberculosis H37Rv and 24h later the phenotype, induction of necrosis and apoptosis and production of tumor necrosis factor TNFalpha, interleukin (IL)-10 and IL-12p40 were determined. TB patients presented higher percentage of CD14+ cells but lower percentage of CD14+DR+ and CD14+CD36+ cells. Expression of CD14, HLA-DR and CD36 was decreased in TB patients. Normal percentages and expression were restored during anti-TB treatment. Monocytes from TB patients underwent necrosis and apoptosis after M. tuberculosis infection, whereas monocytes from healthy controls exhibited only apoptosis. Anti-TB treatment reverted necrosis. There were no differences between the various clinical forms of TB. In vitro M. tuberculosis infection decreased expression of the membrane molecules studied. HLA-DR and CD36 inhibition correlated with induction of apoptosis. Restoration of monocyte alterations during anti-TB treatment suggests that such alterations may be caused by the high M. tuberculosis load present during active disease.

Multifunctional T Cell Response to DosR and Rpf Antigens Is Associated with Protection in Long-Term Mycobacterium tuberculosis-Infected Individuals in Colombia.

Multifunctional T cells have been shown to be protective in chronic viral infections. In mycobacterial infections, however, evidence for a protective role of multifunctional T cells remains inconclusive. Short-term cultures of peripheral blood mononuclear cells stimulated with the Mycobacterium tuberculosis RD1 antigens 6-kDa early secretory antigenic target (ESAT6) and 10-kDa culture filtrate antigen (CFP10), which are induced in the early infection phase, have been mainly used to assess T cell multifunctionality, although long-term culture assays have been proposed to be more sensitive than short-term assays for assessment of memory T cells, which are essential for long-term immunity. Here we used a long-term culture assay system to study the T cell immune responses to the M. tuberculosis latency-associated DosR antigens and reactivation-associated Rpf antigens, compared to ESAT6 and CFP10, in patients with pulmonary tuberculosis (PTB) and household contacts of PTB patients with long-term latent tuberculosis infection (ltLTBI), in a community in which M. tuberculosis is endemic. Our results showed that the DosR antigens Rv1737c (narK2) and Rv2029c (pfkB) and the Rv2389c (rpfD) antigen of M. tuberculosis induced higher frequencies of CD4+ or CD8+ mono- or bifunctional (but not multifunctional) T cells producing interferon gamma (IFN-γ) and/or tumor necrosis alpha (TNF-α) in ltLTBI, compared to PTB. Moreover, the frequencies of CD4+ and/or CD8+ T cells with a CD45RO+ CD27+ phenotype were higher in ltLTBI than in PTB. Thus, the immune responses to selected DosR and Rpf antigens may be associated with long-term latency, correlating with protection from M. tuberculosis reactivation in ltLTBI. Further study of the functional and memory phenotypes may contribute to further discrimination between the different states of M. tuberculosis infections.