Drug-Tolerant Mycobacterium tuberculosis Adopt Different Survival Strategies in Alveolar Macrophages of Patients with Pulmonary Tuberculosis.

The rapid spread of drug-resistant M. tuberculosis (Mtb) strains and the phenomenon of phenotypic tolerance to drugs present challenges toward achieving the goal of tuberculosis (TB) elimination worldwide. By using the ex vivo cultures of alveolar macrophages obtained from lung tissues of TB patients after intensive antimicrobial chemotherapy before surgery, different subpopulations of multidrug-tolerant Mtb with a spectrum of phenotypic and growth features were identified in the same TB lesions. Our results are indicative of not only passive mechanisms generating nonheritable resistance of Mtb to antibiotics, which are associated mainly with a lack of Mtb growth, but also some active mechanisms of Mtb persistence, such as cell wall and metabolic pathway remodeling. In one of the subpopulations, non-acid-fast Mtb have undergone significant reprogramming with the restoration of acid-fastness, lipoarabinomannan expression and replication in host cells of some patients after withdrawal of anti-TB drugs. Our data indicate the universal stress protein Rv2623 as a clinically relevant biomarker of Mtb that has lost acid-fastness in human lungs. The studies of Mtb survival, persistence, dormancy, and resumption and the identification of biomarkers characterizing these phenomena are very important concerning the development of vaccines and drug regimens with individualized management of patients for overcoming the resistance/tolerance crisis in anti-TB therapy.

Mycobacterium tuberculosis Load in Host Cells and the Antibacterial Activity of Alveolar Macrophages Are Linked and Differentially Regulated in Various Lung Lesions of Patients with Pulmonary Tuberculosis.

Tuberculosis (TB) is a disease caused by Mycobacterium tuberculosis (Mtb) infection with the formation of a broad range of abnormal lung lesions within a single patient. Although host-pathogen interactions determine disease outcome, they are poorly understood within individual lesions at different stages of maturation. We compared Mtb load in a tuberculoma wall and the lung tissue distant from tuberculomas in TB patients. These data were combined with an analysis of activation and bactericidal statuses of alveolar macrophages and other cell subtypes examined both in ex vivo culture and on the histological sections obtained from the same lung lesions. The expression of pattern recognition receptors CD14, CD11b, and TLR-2, transcription factors HIF-1α, HIF-2α, and NF-κB p50 and p65, enzymes iNOS and COX-2, reactive oxygen species (ROS) biosynthesis, and lipid production were detected for various lung lesions, with individual Mtb loads in them. The walls of tuberculomas with insufficient inflammation and excessive fibrosis were identified as being the main niche for Mtb survival (single or as colonies) in non-foamy alveolar macrophages among various lung lesions examined. The identification of factors engaged in the control of Mtb infection and tissue pathology in local lung microenvironments, where host-pathogen relationships take place, is critical for the development of new therapeutic strategies.

Analysis of Mycobacterium tuberculosis Uptake by Alveolar Macrophages after Ex vivo Expansion Indicates Processing Host Cells with Pathogen Actually from Lung Tissue of Patients with Pulmonary Tuberculosis.

Background: Previously, the ex vivo cultures of alveolar macrophages were developed from the surgical samples of the lungs in patients with pulmonary tuberculosis (TB) to establish the unique features of Mycobacterium tuberculosis (Mtb) lifestyle in host cells, but the question has remained whether Mtb-infected cells are isolated from the human lungs or they may be the result of Mtb phagocytosis in ex vivo culture. The study was aimed to investigate Mtb uptake by TB patients' cells after ex vivo expansion. Methods: Alveolar macrophages were infected with the Mtb clinical isolates in ex vivo culture, and the acid-fast Mtb loads in the cells were analyzed. Immunofluorescent staining and the examination of cytological and histological preparations by confocal microscopy were applied to detect Mtb ligands and macrophage surface markers. Results: The studies shown the lack of Mtb uptake by patients' alveolar macrophages during experimental infection with highly virulent Mtb clinical isolates containing pathogen-associated molecular patterns lipoarabinomannan and Ag38 at all used multiplicity of infection including a very high dose of infection. This fact was probably determined by the absence of pattern recognition receptors CD14, TLR2, and CD11b on the plasma membrane of human cells, likely, as a result of cellular processing from the resected lung tissues of patients. Conclusion: The findings indicate that alveolar macrophages with single Mtb or Mtb in colonies, including those with cord-morphology, found in the ex vivo cell cultures of all TB patients examined, were isolated from the lungs, and they characterize the Mtb infection in patients at the time of surgery.

Mycobacterium tuberculosis with different virulence reside within intact phagosomes and inhibit phagolysosomal biogenesis in alveolar macrophages of patients with pulmonary tuberculosis.

Tuberculosis (TB) is a dangerous airborne disease caused by Mycobacterium tuberculosis (Mtb) and characterized by a tight interplay between pathogen and host cells, mainly alveolar macrophages. Studies of the mechanisms of Mtb survival within human cells during TB disease are extremely important for the development of new strategies and drugs for TB treatment. We have used the ex vivo cultures of alveolar macrophages and histological sections obtained from the resected lungs of patients with pulmonary TB to establish the unique features of Mtb lifestyle in host cells. Our data indicate that Mtb with different virulence, as single and in colonies, with or without cording morphology, are exclusively intravacuolar pathogens with intact phagosomal membranes in viable host cells of TB patients and Mtb-infected guinea pig. Mycobacteria were detected in the cytoplasm and/or damaged vacuoles only in alveolar macrophages with morphological signs of cell death after prolonged ex vivo culture, however Mtb were found inside phagosomes in viable alveolar macrophages or cells with apoptotic/necrotic morphology in the same ex vivo cell culture. The Mtb phagosomes interacted with human different endocytic pathways, but inhibited phagolysosomal biogenesis, while intracellular vesicles containing Mtb products were fused with lysosomes in the same host cells.

Mycobacterium tuberculosis cording in alveolar macrophages of patients with pulmonary tuberculosis is likely associated with increased mycobacterial virulence.

Mycobacterium tuberculosis (Mtb) is an infectious agent that causes tuberculosis (TB) in humans. A study of the volume of Mtb population and the detection of Mtb virulence in the lungs of patients with pulmonary TB are of great importance for understanding the infectious process and the outcome of the disease. We analyzed the functional state of Mtb and their number in alveolar macrophages obtained from the resected lungs of patients with TB in ex vivo culture and determined that the number of Mtb, referred mainly to the Beijing genotype family (A0 and B0/W148 clusters), were significantly different in cells between different patients. Only single Mtb were found in alveolar macrophages of some patients, while Mtb were actively replicated in colonies in alveolar macrophages of other patients, including cord morphology of Mtb growth (the indicator of Mtb virulence). Our data demonstrated association between the formation of Mtb cording in alveolar macrophages of patients and increased virulence of Mtb from the lungs of these patients in guinea pig TB model. The find of cording formation by replicating Mtb in human alveolar macrophages may be used for preliminary quick estimation of increased Mtb virulence in individual patients with pulmonary TB.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute tuberculosis. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of animals with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized animals do not develop the symptoms of acute tuberculosis and their body weight gain was five times more as compared with the non-immunized-infected animals. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

Ex vivo expansion of alveolar macrophages with Mycobacterium tuberculosis from the resected lungs of patients with pulmonary tuberculosis.

Tuberculosis (TB), with the Mycobacterium tuberculosis (Mtb) as the causative agent, remains to be a serious world health problem. Traditional methods used for the study of Mtb in the lungs of TB patients do not provide information about the number and functional status of Mtb, especially if Mtb are located in alveolar macrophages. We have developed a technique to produce ex vivo cultures of cells from different parts of lung tissues surgically removed from patients with pulmonary TB and compared data on the number of cells with Mtb inferred by the proposed technique to the results of bacteriological and histological analyses used for examination of the resected lungs. The ex vivo cultures of cells obtained from the resected lungs of all patients were largely composed of CD14-positive alveolar macrophages, foamy or not, with or without Mtb. Lymphocytes, fibroblasts, neutrophils, and multinucleate Langhans giant cells were also observed. We found alveolar macrophages with Mtb in the ex vivo cultures of cells from the resected lungs of even those TB patients, whose sputum smears and lung tissues did not contain acid-fast Mtb or reveal growing Mtb colonies on dense medium. The detection of alveolar macrophages with Mtb in ex vivo culture as soon as 16-18 h after isolation of cells from the resected lungs of all TB patients suggests that the technique proposed for assessing the level of infection in alveolar macrophages of TB patients has higher sensitivity than do prolonged bacteriological or pathomorphological methods. The proposed technique allowed us to rapidly (in two days after surgery) determine the level of infection with Mtb in the cells of the resected lungs of TB patients and, by the presence or absence of Mtb colonies, including those with cording morphology, the functional status of the TB agent at the time of surgery.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

Differences between Mycobacterium-Host Cell Relationships in Latent Tuberculous Infection of Mice Ex Vivo and Mycobacterial Infection of Mouse Cells In Vitro.

The search for factors that account for the reproduction and survival of mycobacteria, including vaccine strains, in host cells is the priority for studies on tuberculosis. A comparison of BCG-mycobacterial loads in granuloma cells obtained from bone marrow and spleens of mice with latent tuberculous infection and cells from mouse bone marrow and peritoneal macrophage cultures infected with the BCG vaccine in vitro has demonstrated that granuloma macrophages each normally contained a single BCG-Mycobacterium, while those acutely infected in vitro had increased mycobacterial loads and death rates. Mouse granuloma cells were observed to produce the IFNγ, IL-1α, GM-CSF, CD1d, CD25, CD31, СD35, and S100 proteins. None of these activation markers were found in mouse cell cultures infected in vitro or in intact macrophages. Lack of colocalization of lipoarabinomannan-labeled BCG-mycobacteria with the lysosomotropic LysoTracker dye in activated granuloma macrophages suggests that these macrophages were unable to destroy BCG-mycobacteria. However, activated mouse granuloma macrophages could control mycobacterial reproduction in cells both in vivo and in ex vivo culture. By contrast, a considerable increase in the number of BCG-mycobacteria was observed in mouse bone marrow and peritoneal macrophages after BCG infection in vitro, when no expression of the activation-related molecules was detected in these cells.

Mycobacterium-Host Cell Relationships in Granulomatous Lesions in a Mouse Model of Latent Tuberculous Infection.

Tuberculosis (TB) is a dangerous infectious disease characterized by a tight interplay between mycobacteria and host cells in granulomatous lesions (granulomas) during the latent, asymptomatic stage of infection. Mycobacterium-host cell relationships were analyzed in granulomas obtained from various organs of BALB/c mice with chronic TB infection caused by in vivo exposure to the Bacillus Calmette-Guérin (BCG) vaccine. Acid-fast BCG-mycobacteria were found to be morphologically and functionally heterogeneous (in size, shape, and replication rates in colonies) in granuloma macrophages, dendritic cells, and multinucleate Langhans giant cells. Cord formation by BCG-mycobacteria in granuloma cells has been observed. Granuloma macrophages retained their ability to ingest damaged lymphocytes and thrombocytes in the phagosomes; however, their ability to destroy BCG-mycobacteria contained in these cells was compromised. No colocalization of BCG-mycobacteria and the LysoTracker dye was observed in the mouse cells. Various relationships between granuloma cells and BCG-mycobacteria were observed in different mice belonging to the same line. Several mice totally eliminated mycobacterial infection. Granulomas in the other mice had mycobacteria actively replicating in cells of different types and forming cords, which is an indicator of mycobacterial virulence and, probably, a marker of the activation of tuberculous infection in animals.

Investigation of functional activity of cells in granulomatous inflammatory lesions from mice with latent tuberculous infection in the new ex vivo model.

The new ex vivo model system measuring functional input of individual granuloma cells to formation of granulomatous inflammatory lesions in mice with latent tuberculous infection has been developed and described in the current study. Monolayer cultures of cells that migrated from individual granulomas were established in the proposed culture settings for mouse spleen and lung granulomas induced by in vivo exposure to BCG vaccine. The cellular composition of individual granulomas was analyzed. The expression of the leukocyte surface markers such as phagocytic receptors CD11b, CD11c, CD14, and CD16/CD32 and the expression of the costimulatory molecules CD80, CD83, and CD86 were tested as well as the production of proinflammatory cytokines (IFN γ and IL-1 α) and growth factors (GM-CSF and FGFb) for cells of individual granulomas. The colocalization of the phagocytic receptors and costimulatory molecules in the surface microdomains of granuloma cells (with and without acid-fast BCG-mycobacteria) has also been detected. It was found that some part of cytokine macrophage producers have carried acid-fast mycobacteria. Detected modulation in dynamics of production of pro-inflammatory cytokines, growth factors, and leukocyte surface markers by granuloma cells has indicated continued processes of activation and deactivation of granuloma inflammation cells during the latent tuberculous infection progress in mice.