dStrains of the Beijing genotype family of Mycobacterium tuberculosis are a cause of particular concern because of their increasing dissemination in the world and their association with drug resistance. Phylogenetically, this family includes distinct ancient and modern sublineages. The modern strains, contrary to the ancestral counterparts, demonstrated increasing prevalence in many world regions that suggest an enhanced bacterial pathogenicity. We therefore evaluated virulence of modern versus ancient Beijing strains with similar epidemiological and genotype characteristics. For this, we selected six strains that had very similar 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing profiles and belonged to the region of difference 181 (RD181) subgroup but differed using markers (mutT2 and mutT4 genes and NTF locus) that discriminate between modern and ancient Beijing sublineages. The strains were isolated from native patients in Brazil and Mozambique, countries with a low prevalence of Beijing strains. The virulence levels of these strains were determined in models of pulmonary infection in mice and in vitro macrophage infection and compared with that of a strain from Russia, part of the epidemic and hypervirulent Beijing clone B0/W148, and of the laboratory strain H37Rv. The results showed that two of the three modern Beijing strains were highly pathogenic, exhibiting levels of virulence comparable with that of the epidemic Russian strain. In contrast, all isolates of the ancient sublineage displayed intermediate or low virulence. The data obtained demonstrate that the strains of the modern Beijing sublineage are more likely to exhibit highly virulent phenotypes than ancient strains and suggest that genetic alterations characteristic of the modern Beijing sublineage favor selection of highly virulent bacteria. Despite extensive surveillance, tuberculosis (TB) remains a serious public health problem. In different parts of the world, there is concern about TB caused by the East Asian/Beijing lineage of Mycobacterium tuberculosis, demonstrating increasing prevalence in the global M. tuberculosis population (1). Clinical and epidemiological studies demonstrated that emergence of the Beijing strains could be associated with high levels of bacterial resistance to multiple drugs (2, 3) and enhanced pathogenicity of these strains, leading to increased transmissibility (4) and rapid progression from infection to active disease (5). However, the data on evaluation of the virulence of Beijing isolates were inconclusive, demonstrating a wide range of inflammatory and virulence phenotypes, as determined in animal models (6,7,8) and in vitro models of macrophage infection (9, 10).Such differences in the virulence of Beijing strains could be associated with genetic heterogeneity of the Beijing M. tuberculosis lineage. Indeed, bacterial genotyping and sequencing demonstrated that the Beijing lineage, having in common a characteristic spoligotype signature and lack of the region ...

The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R−/− mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R−/− mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis.

Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb) that in most cases induces irreversible necrosis of lung tissue as a result of excessive inflammatory reactions. The murine model of TB in resistant C57BL/6 mice infected with reference Mtb strains is widely used in TB studies; however, these mice do not show a necrotic pathology, which restricts their use in studies of irreversible tissue damage. Recently, we demonstrated that necrotic lung lesions could be induced in the C57BL/6 mice by highly virulent Mtb strains belonging to the modern Beijing sublineage. However, the pathogenic mechanisms leading to necrosis in this model were not elucidated. In this study, we investigated the dynamics of lung lesions in mice infected with highly virulent Beijing Mtb strain M299, compared with those infected with laboratory Mtb strain H37Rv. The data demonstrate that necrotic lung lesions in mice infected by the strain M299 were associated with enhanced recruitment of myeloid cells, especially neutrophils, and increased levels of proinflammatory cytokines, consistent with exacerbated inflammation. High levels of IFN-γ production contributed to the control of bacterial growth. Further progression to chronic disease was associated with a reduction in the levels of inflammatory mediators in the lungs, the accumulation of foamy macrophages and partial healing of the necrotic tissue by fibrosis. At a late stage of disease, degradation of foamy cells resulted in the liberation of accumulated lipids and persisting bacilli and further activation of inflammation, which promoted lung consolidation. Overall, our studies show that C57BL/6 mice infected with highly virulent Mtb strain may serve as a TB model reproducing an exacerbated inflammatory response in a resistant host to hypervirulent mycobacteria, leading to irreversible necrotic lung lesions.

BackgroundTuberculosis, caused by Mycobacterium tuberculosis or Mycobacterium bovis, remains one of the leading infectious diseases worldwide. The ability of mycobacteria to rapidly grow in host macrophages is a factor contributing to enhanced virulence of the bacteria and disease progression. Bactericidal functions of phagocytes are strictly dependent on activation status of these cells, regulated by the infecting agent and cytokines. Pathogenic mycobacteria can survive the hostile environment of the phagosome through interference with activation of bactericidal responses. To study the mechanisms employed by highly virulent mycobacteria to promote their intracellular survival, we investigated modulating effects of two pathogenic M. bovis isolates and a reference M. tuberculosis H37Rv strain, differing in their ability to multiply in macrophages, on activation phenotypes of the cells primed with major cytokines regulating proinflammatory macrophage activity.ResultsBone marrow- derived macrophages obtained from C57BL/6 mice were infected by mycobacteria after a period of cell incubation with or without treatment with IFN-γ, inducing proinflammatory type-1 macrophages (M1), or IL-10, inducing anti-inflammatory type-2 cells (M2). Phenotypic profiling of M1 and M2 was then evaluated. The M. bovis strain MP287/03 was able to grow more efficiently in the untreated macrophages, compared with the strains B2 or H37Rv. This strain induced weaker secretion of proinflammatory cytokines, coinciding with higher expression of M2 cell markers, mannose receptor (MR) and arginase-1 (Arg-1). Treatment of macrophages with IFN-γ and infection by the strains B2 and H37Rv synergistically induced M1 polarization, leading to high levels of inducible nitric oxide synthase (iNOS) expression, and reduced expression of the Arg-1. In contrast, the cells infected with the strain MP287/03 expressed high levels of Arg-1 which competed with iNOS for the common substrate arginine, leading to lower levels of NO production.ConclusionsThe data obtained demonstrated that the strain, characterized by increased growth in macrophages, down- modulated classical macrophage activation, through induction of an atypical mixed M1/M2 phenotype.

Background The role of myeloid-derived suppressor cells (MDSCs) in severe tuberculosis patients who suffer from uncontrolled pulmonary inflammation caused by hypervirulent mycobacterial infection remains unclear. Methods This issue was addressed using C57BL/6 mice infected with highly virulent Mycobacterium bovis strain MP287/03. Results CD11b +GR1 int population increased in the bone marrow, blood and lungs during advanced disease. Pulmonary CD11b +GR1 int (Ly6G intLy6C int) cells showed granularity similar to neutrophils and expressed immature myeloid cell markers. These immature neutrophils harbored intracellular bacilli and were preferentially located in the alveoli. T cell suppression occurred concomitantly with CD11b +GR1 int cell accumulation in the lungs. Furthermore, lung and bone-marrow GR1 + cells suppressed both T cell proliferation and IFN-γ production in vitro. Anti-GR1 therapy given when MDSCs infiltrated the lungs prevented expansion and fusion of primary pulmonary lesions and the development of intragranulomatous caseous necrosis, along with increased mouse survival and partial recovery of T cell function. Lung bacterial load was reduced by anti-GR1 treatment, but mycobacteria released from the depleted cells proliferated extracellularly in the alveoli, forming cords and clumps. Conclusions Granulocytic MDSCs massively infiltrate the lungs during infection with hypervirulent mycobacteria, promoting bacterial growth and the development of inflammatory and necrotic lesions, and are promising targets for host-directed therapies.