The outcome of the interaction between Mycobacterium tuberculosis (Mtb) and a macrophage depends on the interplay between host defense and bacterial immune subversion mechanisms. MicroRNAs critically regulate several host defense mechanisms, but their role in the Mtb-macrophage interplay remains unclear. MicroRNA profiling of Mtb-infected macrophages revealed the downregulation of miR-let-7f in a manner dependent on the Mtb secreted effector ESAT-6. We establish that let-7f targets A20, a feedback inhibitor of the NF-κB pathway. Expression of let-7f decreases and A20 increases with progression of Mtb infection in mice. Mtb survival is attenuated in A20-deficient macrophages, and the production of TNF, IL-1β, and nitrite, which are mediators of immunity to Mtb, is correspondingly increased. Further, let-7f overexpression diminishes Mtb survival and augments the production of cytokines including TNF and IL-1β. These results uncover a role for let-7f and its target A20 in regulating immune responses to Mtb and controlling bacterial burden.

The reason for the largely variable protective effect against TB of the vaccine Bacille Calmette-Guerin (BCG) is not understood. In this study, we investigated whether epigenetic mechanisms are involved in the response of immune cells to the BCG vaccine. We isolated peripheral blood mononuclear cells (PBMCs) from BCG-vaccinated subjects and performed global DNA methylation analysis in combination with functional assays representative of innate immunity against Mycobacterium tuberculosis infection. Enhanced containment of replication was observed in monocyte-derived macrophages from a sub-group of BCG-vaccinated individuals (identified as ‘responders’). A stable and robust differential DNA methylation pattern in response to BCG could be observed in PBMCs isolated from the responders but not from the non-responders. Gene ontology analysis revealed that promoters with altered DNA methylation pattern were strongly enriched among genes belonging to immune pathways in responders, however no enrichments could be observed in the non-responders. Our findings suggest that BCG-induced epigenetic reprogramming of immune cell function can enhance anti-mycobacterial immunity in macrophages. Understanding why BCG induces this response in responders but not in non-responders could provide clues to improvement of TB vaccine efficacy.

The best characterized survival mechanism of Mycobacterium tuberculosis inside the macrophage is the inhibition of phagosomal maturation. Phagosomal maturation involves several steps including fusion with lysosomes and acidification. However, it has not been elucidated which components of phagosomal maturation correlate with growth restriction of virulent mycobacteria in human macrophages, and we aimed to study this. We infected human monocyte-derived macrophages with M. tuberculosis and assessed bacterial replication, translocation of CD63 to the phagosome, and phagosomal acidification. We found that unstimulated human macrophages were able to control infection with M. tuberculosis upon inoculation at a low multiplicity of infection (MOI) of 1, but not at a high MOI of 10. The low MOI resulted in a macrophage-controlled balance between host cells and bacteria. Both H37Rv and H37Ra infection, at high and low MOI, led to equally ineffective translocation of CD63 to the phagosome. On the other hand, acidification of mycobacterial phagosomes was more efficient at MOI 1 than 10 with both mycobacterial strains, consistent with a direct or indirect role for phagosomal acidification in restricting M. tuberculosis growth. Furthermore, inhibition of the vacuolar H⁺-ATPase as well as of cathepsin D led to enhanced mycobacterial replication inside the macrophage. This again shows the importance of phagosomal acidification for control of mycobacterial growth, through the activation of lysosomal hydrolases. We conclude that acidification and related functional aspects of the mature phagosome are important factors for restriction of M. tuberculosis replication in human macrophages.

The triterpene glycoside glycyrrhizin is the main active compound in liquorice. It is used as a herbal medicine owing to its anticancer, antiviral and anti-inflammatory properties. Its mode of action, however, remains widely unknown. In the present study, we aimed to elucidate the molecular mechanism of glycyrrhizin in attenuating inflammatory responses in macrophages. Using microarray analysis, we found that glycyrrhizin caused a broad block in the induction of pro-inflammatory mediators induced by the TLR (Toll-like receptor) 9 agonist CpG-DNA in RAW 264.7 cells. Furthermore, we found that glycyrrhizin also strongly attenuated inflammatory responses induced by TLR3 and TLR4 ligands. The inhibition was accompanied by decreased activation not only of the NF-kappaB (nuclear factor kappaB) pathway but also of the parallel MAPK (mitogen-activated protein kinase) signalling cascade upon stimulation with TLR9 and TLR4 agonists. Further analysis of upstream events revealed that glycyrrhizin treatment decreased cellular attachment and/or uptake of CpG-DNA and strongly impaired TLR4 internalization. Moreover, we found that the anti-inflammatory effects were specific for membrane-dependent receptor-mediated stimuli, as glycyrrhizin was ineffective in blocking Tnfa (tumour necrosis factor alpha gene) induction upon stimulation with PMA, a receptor- and membrane-independent stimulus. These observations suggest that the broad anti-inflammatory activity of glycyrrhizin is mediated by the interaction with the lipid bilayer, thereby attenuating receptor-mediated signalling.

To survive and replicate in macrophages Mycobacterium tuberculosis (Mtb) has developed strategies to subvert host defence mechanisms, including autophagy. Autophagy induction has the potential to clear Mtb, but little is known about its effect during controlled tuberculosis and HIV co-infection. Mammalian target of rapamycin complex1 (mTORC1) inhibitors were used to induce autophagy in human macrophages pre-infected with HIV-1BaL and infected with a low dose of Mtb (co-infected), or single Mtb infected (single infected). The controlled Mtb infection was disrupted upon mTOR inhibition resulting in increased Mtb replication in a dose-dependent manner which was more pronounced during co-infection. The increased Mtb replication could be explained by the marked reduction in phagosome acidification upon mTOR inhibition. Autophagy stimulation targeting mTORC1 clearly induced a basal autophagy with flux that was unlinked to the subcellular environment of the Mtb vacuoles, which showed a concurrent suppression in acidification and maturation/flux. Overall our findings indicate that mTOR inhibition during Mtb or HIV/Mtb co-infection interferes with phagosomal maturation, thereby supporting mycobacterial growth during low-dose and controlled infection. Therefore pharmacological induction of autophagy through targeting of the canonical mTORC1-pathway should be handled with caution during controlled tuberculosis, since this could have serious consequences for patients with HIV/Mtb co-infection.

The causative agent of tuberculosis, Mycobacterium tuberculosis, shares several characteristics with organisms that produce biofilms during infections. One of these is the ability to form tight bundles also known as cords. However, little is known of the physiological relevance of the cording phenotype. In this study, we investigated whether cord-forming M. tuberculosis induce the formation of macrophage extracellular traps (METs) in human monocyte-derived macrophages. Macrophages have previously been shown to produce extracellular traps in response to various stimuli. We optimized bacterial culturing conditions that favored the formation of the cord-forming phenotype as verified by scanning electron microscopy. Microscopy analysis of METs formation during experimental infection of macrophages with M. tuberculosis revealed that cord-forming M. tuberculosis induced significantly more METs compared to the non-cording phenotype. Deletion of early secreted antigenic target-6 which is an important virulence factor of M. tuberculosis, abrogated the ability of the bacteria to induce METs. The release of extracellular DNA from host cells during infection may represent a defense mechanism against pathogens that are difficult to internalize, including cord-forming M. tuberculosis.

The standard treatment of tuberculosis (TB) takes six to nine months to complete and this lengthy therapy contributes to the emergence of drug-resistant TB. TB is caused by Mycobacterium tuberculosis (Mtb) and the ability of this bacterium to switch to a dormant phenotype has been suggested to be responsible for the slow clearance during treatment. A recent study showed that the replication rate of a non-virulent mycobacterium, Mycobacterium smegmatis, did not correlate with antibiotic susceptibility. However, the question whether this observation also holds true for Mtb remains unanswered. Here, in order to mimic physiological conditions of TB infection, we established a protocol based on long-term infection of primary human macrophages, featuring Mtb replicating at different rates inside the cells. During conditions that restricted Mtb replication, the bacterial phenotype was associated with reduced acid-fastness. However, these phenotypically altered bacteria were as sensitive to isoniazid, pyrazinamide and ethambutol as intracellularly replicating Mtb. In support of the recent findings with M. smegmatis, we conclude that replication rates of Mtb do not correlate with antibiotic tolerance.

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