Efficacy of macozinone in mice with genetically diverse susceptibility to Mycobacterium tuberculosis infection.

Host heterogeneity in pulmonary tuberculosis leads to varied responses to infection and drug treatment. The present portfolio of anti-TB drugs needs to be boosted with new drugs and drug regimens. Macozinone, a clinical-stage molecule targeting the essential enzyme, DprE1, represents an attractive option. Mice (I/St, B6, (AKRxI/St)F1, B6.I-100 and B6.I-139) genetically diverse susceptibility to Mycobacterium tuberculosis (Mtb) H37Rv infection were subjected to aerosol- or intravenous infection to determine the efficacy of macozinone (MCZ). They were treated with macozinone or reference drugs (isoniazid, rifampicin). Lung and spleen bacterial burdens were measured at four and eight weeks post-infection. Lung histology was evaluated at four weeks of treatment. Treatment with macozinone resulted in a statistically significant reduction in the bacterial load in the lungs and spleen as early as four weeks after treatment initiation in mice susceptible or resistant to Mtb infection. In the TB hypoxic granuloma model, macozinone was more potent than rifampicin in reducing the CFU counts. However, histopathological analysis revealed significant lung changes in I/St mice after eight weeks of treatment initiation. Macozinone demonstrated efficacy to varying degrees across all mouse models of Mtb infection used. These results should facilitate its further development and potential introduction into clinical practice.

The H2-A Class II molecule α/ß-chain cis-mismatch severely affects cell surface expression, selection of conventional CD4+ T cells and protection against TB infection.

Introduction: To dissect the role of the part of the H2 complex comprised of the MHC-II genes in the control of tuberculosis (TB) infection, we previously established a panel of recombinant congenic mouse strains bearing different segments of the H2 j haplotype on the B6 (H2 b) genetic background. Fine genetic mapping, gene sequencing and assessment of TB phenotypes resulted in identification of the H2-Ab gene as a major factor of TB control. Methods: We further narrowed the MHC-II H2 j interval by spotting a new recombination event, sequencing newly established DNA configuration and establishing a mouse strain B6.I-103 in which j/b recombination occurred within the coding sequence of the H2-Ab gene. Results: Unexpectedly, a novel H2-Aα b/AßjE0 haplotype provided exclusively high susceptibility to TB challenge. Immunologic analysis revealed an altered CD4+ T-cell selection and maintenance in B6.I-103 mice, as well as seriously impaired expression of the H2-Aαb/Aßj molecule on the surface of antigen presenting cells. Unlike previously reported cases of Class II malfunctioning, the defective phenotype arose not from strong structural mutations, but from regular recombination events within the MHC-II recombination hot spot region. Discussion: Our findings provide evidence that Class II α/ß-chain cis-allelic mismatches created by regular genetic recombination may severely affect immune system functioning. This issue is discussed in the context of the MHC evolution.

Prolonged infection triggered by dormant Mycobacterium tuberculosis: Immune and inflammatory responses in lungs of genetically susceptible and resistant mice.

We developed an approach for substantial attenuation of Mycobacterium tuberculosis by prolonged culturing under gradually acidifying conditions. Bacteria subjected to acidification lost the capacity to form colonies on solid media, but readily resuscitated their growth in the murine host, providing a useful model to study in vivo development of infection mimicking latent and reactivation tuberculosis (TB) in humans. Here we characterize biomarkers of lung pathology and immune responses triggered by such attenuated bacteria in genetically TB-susceptible and resistant mice. In susceptible I/St mice, CFU counts in lungs and spleens were ~1.5-log higher than in resistant B6 mice, accompanied by diffuse pneumonia and excessive lung infiltration with highly activated CD44+CD62L- T-lymphocytes resulting in death between months 7-9 post challenge. B6 mice were characterized by development of local inflammatory foci, higher production of pro-inflammatory IL-6 and IL-11 cytokines and a more balanced T-cell activation in their lungs. CFU counts remained stable in B6 mice during the whole 18-mo observation period, and all mice survived. Thus, we established a mouse model of fatal reactivation TB vs. indefinite mycobacterial possession after identical challenge and characterized the features of immune responses in the lung tissue underlining these polar phenotypes.

A new model for chronic and reactivation tuberculosis: Infection with genetically attenuated Mycobacterium tuberculosis in mice with polar susceptibility.

TB infection in mice develops relatively rapidly which interferes with experimental dissection of immune responses and lung pathology features that differ between genetically susceptible and resistant hosts. Earlier we have shown that the M. tuberculosis strain lacking four of five Rpf genes (ΔACDE) is seriously attenuated for growth in vivo. Using this strain, we assessed key parameters of lung pathology, immune and inflammatory responses in chronic and reactivation TB infections in highly susceptible I/St and more resistant B6 mice. ΔACDE mycobacteria progressively multiplied only in I/St lungs, whilst in B6 lung CFU counts decreased with time. Condensed TB foci apeared in B6 lungs at week 4 of infection, whilst in I/St their formation was delayed. At the late phase of infection, in I/St lungs TB foci fused resulting in extensive pneumonia, whereas in B6 lungs pathology was limited to condensed foci. Macrophage and neutrophil populations characteristically differed between I/St and B6 mice at early and late stages of infection: more neutrophils accumulated in I/St and more macrophages in B6 lungs. The expression level of chemokine genes involved in neutrophil influx was higher in I/St compared to B6 lungs. B6 lung cells produced more IFN-γ, IL-6 and IL-11 at the early and late phases of infection. Overall, using a new mouse model of slow TB progression, we demonstrate two important features of ineffective infection control underlined by shifts in lung inflammation: delay in early granuloma formation and fusion of granulomas resulting in consolidated pneumonia late in the infectious course.

Capacity of lung stroma to educate dendritic cells inhibiting mycobacteria-specific T-cell response depends upon genetic susceptibility to tuberculosis.

The balance between activation and inhibition of local immune responses in affected tissues during prolonged chronic infections is important for host protection. There is ample evidence that regulatory, tolerogenic dendritic cells (DC) are developed and present in tissues and inhibit overwhelming inflammatory reactions. Also, it was firmly established that stromal microenvironment of many organs is able to induce development of immature regulatory DC (DCreg), an essential element of a general immune regulatory network. However, direct experimental data demonstrating inhibition of immune responses by stroma-instructed immature DCreg in infectious models are scarce, and virtually nothing is known about functioning of this axis of immunity during tuberculosis (TB) infection. In this study, we demonstrate that lung stromal cells are capable of supporting the development in culture of immature CD11b(+)CD11c(low)CD103(-) DCreg from lineage-negative (lin(-)) bone marrow precursors. DCreg developed on lung stroma isolated from mice of genetically TB-hyper-susceptible I/St and relatively resistant B6 inbred strains inhibited proliferative response of mycobacteria-specific CD4(+) T-cell lines a dose-dependent manner. Importantly, the inhibitory activity of B6 DCreg was substantially higher than that of I/St Dcreg. Moreover, when the donors of stromal cells were chronically infected with virulent mycobacteria, the capacity to instruct inhibitory DCreg was retained in B6, but further diminished in I/St stromal cells. DCreg-provided suppression was mediated by a few soluble mediators, including PGE2, NO and IL-10. The content of CD4(+)Foxp3(+) Treg cells in the mediastinal, lung-draining lymph nodes at the advanced stages of chronic infection did not change in I/St, but increased 2-fold in B6 mice, and lung pathology was much more pronounced in the former mice. Taken together, these data provide genetic evidence that the capacity to maintain populations of regulatory cells during M. tuberculosis infection is a part of the host protective strategy.

Interleukin-11 drives early lung inflammation during Mycobacterium tuberculosis infection in genetically susceptible mice.

IL-11 is multifunctional cytokine whose physiological role in the lungs during pulmonary tuberculosis (TB) is poorly understood. Here, using in vivo administration of specific antibodies against IL-11, we demonstrate for the first time that blocking IL-11 diminishes histopathology and neutrophilic infiltration of the lung tissue in TB-infected genetically susceptible mice. Antibody treatment decreased the pulmonary levels of IL-11 and other key inflammatory cytokines not belonging to the Th1 axis, and down-regulated IL-11 mRNA expression. This suggests the existence of a positive feedback loop at the transcriptional level, which is further supported by up-regulation of IL-11 mRNA expression in the presence of rIL-11 in in vitro cultures of lung cells. These findings imply a pathogenic role for IL-11 during the early phase of Mycobacterium tuberculosis-triggered disease in a genetically susceptible host.

In mice, tuberculosis progression is associated with intensive inflammatory response and the accumulation of Gr-1 cells in the lungs.

BACKGROUND: Infection with Mycobacterium tuberculosis (Mtb) results in different clinical outcomes ranging from asymptomatic containment to rapidly progressing tuberculosis (TB). The mechanisms controlling TB progression in immunologically-competent hosts remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: To address these mechanisms, we analyzed TB progression in a panel of genetically heterogeneous (A/SnxI/St) F2 mice, originating from TB-highly-susceptible I/St and more resistant A/Sn mice. In F2 mice the rates of TB progression differed. In mice that did not reach terminal stage of infection, TB progression did not correlate with lung Mtb loads. Nor was TB progression correlated with lung expression of factors involved in antibacterial immunity, such as iNOS, IFN-gamma, or IL-12p40. The major characteristics of progressing TB was high lung expression of the inflammation-related factors IL-1beta, IL-6, IL-11 (p<0.0003); CCL3, CCL4, CXCL2 (p<0.002); MMP-8 (p<0.0001). The major predictors of TB progression were high expressions of IL-1beta and IL-11. TNF-alpha had both protective and harmful effects. Factors associated with TB progression were expressed mainly by macrophages (F4-80(+) cells) and granulocytes (Gr-1(hi)/Ly-6G(hi) cells). Macrophages and granulocytes from I/St and A/Sn parental strains exhibited intrinsic differences in the expression of inflammatory factors, suggesting that genetically determined peculiarities of phagocytes transcriptional response could account for the peculiarities of gene expression in the infected lungs. Another characteristic feature of progressing TB was the accumulation in the infected lungs of Gr-1(dim) cells that could contribute to TB progression. CONCLUSIONS/SIGNIFICANCE: In a population of immunocompetent hosts, the outcome of TB depends on quantitatively- and genetically-controlled differences in the intensity of inflammatory responses, rather than being a direct consequence of mycobacterial colonization. Local accumulation of Gr-1(dim) cells is a newly identified feature of progressing TB. High expression of IL-1beta and IL-11 are potential risk factors for TB progression and possible targets for TB immunomodulation.

CD27low CD4 T lymphocytes that accumulate in the mouse lungs during mycobacterial infection differentiate from CD27high precursors in situ, produce IFN-gamma, and protect the host against tuberculosis infection.

The generation of effector, IFN-gamma producing T lymphocytes and their accumulation at sites of infection are critical for host protection against various infectious diseases. The activation and differentiation of naive T lymphocytes into effector memory cells starts in lymphoid tissues, but it is not clear whether the Ag-experienced cells that leave lymph nodes (LN) are mature or if they undergo further changes in the periphery. We have previously shown that CD44(high)CD62L(low) effector CD4 T lymphocytes generated during the course of mycobacterial infection can be segregated into two subsets on the basis of CD27 receptor expression. Only the CD27(low) subset exhibited a high capacity for IFN-gamma secretion, indicating that low CD27 expression is characteristic of fully differentiated effector CD4 T lymphocytes. We demonstrate now that CD27(low) IFN-gamma-producing CD4 T lymphocytes accumulate in the lungs but are rare in LNs. Several factors contribute to their preferential accumulation. First, CD27(low) CD4 T lymphocytes present in the LN are highly susceptible to apoptosis. Second, circulating CD27(low) CD4 T cells do not enter the LN but efficiently migrate to the lungs. Third, CD27(high) effector CD4 T cells that enter the lungs down-regulate CD27 expression in situ. In genetically heterogeneous mice that exhibit varying susceptibility to tuberculosis, the accumulation of mature CD27(low) CD4 T cells in the lungs correlates with the degree of protection against infection. Thus, we propose that terminal maturation of effector CD4 T lymphocytes in the periphery provides the host with efficient local defense and avoids potentially harmful actions of inflammatory cytokines in lymphoid organs.

Mycobacterial dissemination and cellular responses after 1-lobe restricted tuberculosis infection of genetically susceptible and resistant mice.

BACKGROUND AND METHODS: To study mycobacterial dissemination and immune-cell trafficking in tuberculosis, we developed a mouse model in which we introduced 1 microL of Mycobacterium tuberculosis directly into the middle lobe of the right lung. We investigated the kinetics of both mycobacterial spread to different anatomical sites and recruitment of phagocytes and activated lymphocytes. RESULTS: Mycobacterial dissemination was independent of susceptibility to infection and was identical in H-2-congenic mouse strains with high and low resistance to tuberculosis. In resistant mice, recruitment of phagocytic cells to the uninfected lung occurred before the appearance of mycobacteria and decreased shortly thereafter. In susceptible mice, this recruitment was delayed in both lungs but increased during a 10-week period. Recruitment of CD4+ and CD8+ lymphocytes to the contralateral lung was observed before mycobacterial dissemination in both strains, so mycobacterial seeding of secondary tissues occurred in the presence of immune lymphocytes. In resistant mice, more T cells expressed the CD44hi CD62lo activation phenotype, and higher levels of interferon- gamma were produced. CONCLUSIONS: Mycobacterial spread to lymphoid organs preceded spread to the initially uninfected contralateral lung. Genetic differences in susceptibility to tuberculosis are associated with differences in dynamics of the immune response, rather than differences in mycobacterial trafficking.