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.

Intracellular delivery of antimicrobial agents by nanoparticles, such as mesoporous silica particles (MSPs), offers an interesting strategy to treat intracellular infections. In tuberculosis (TB), Mycobacterium tuberculosis avoids components of the immune system by residing primarily inside alveolar macrophages, which are the desired target for TB therapy. We have previously identified a peptide, called NZX, capable of inhibiting both clinical and multi-drug resistant strains of M. tuberculosis at therapeutic concentrations. In this study we analysed the potential of MSPs containing NZX for the treatment of tuberculosis. The MSPs released functional NZX gradually into simulated lung fluid and the peptide filled MSPs were easily taken up by primary macrophages. In an intracellular infection model, the peptide containing particles showed increased mycobacterial killing compared to free peptide. The therapeutic potential of peptide containing MSPs was investigated in a murine infection model, showing that MSPs preserved the effect to eliminate M. tuberculosis in vivo. In this study we found that loading the antimicrobial peptide NZX into MSPs increased the inhibition of intracellular mycobacteria in primary macrophages and preserved the ability to eliminate M. tuberculosis in vivo in a murine model. Our studies provide evidence for the feasibility of using MSPs for treatment of tuberculosis.

The widely used animal models for tuberculosis (TB) display fundamental differences from human TB. Therefore, a validated model that recapitulates human lung TB is attractive for TB research. Here, we describe a unique method for establishment of TB infection in an experimental human lung tissue model. The model is based on cell lines derived from human lungs and primary macrophages from peripheral blood, and displays characteristics of human lung tissue, including evenly integrated macrophages throughout the epithelium, production of extracellular matrix, stratified epithelia and mucus secretion. Establishment of experimental infection in the model tissue with Mycobacterium tuberculosis, the bacterium that causes TB, resulted in clustering of macrophages at the site of infection, reminiscent of early TB granuloma formation. We quantitated the extent of granuloma formation induced by different strains of mycobacteria and validated our model against findings in other TB models. We found that early granuloma formation is dependent on ESAT-6, which is secreted via the type VII secretion machinery of virulent mycobacteria. Our model, which can facilitate the discovery of the interactions between mycobacteria and host cells in a physiological environment, is the first lung tissue model described for TB.

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 protection against tuberculosis induced by the Bacille Calmette Guérin (BCG) vaccine is unpredictable. In our previous study, altered DNA methylation pattern in peripheral blood mononuclear cells (PBMCs) in response to BCG was observed in a subgroup of individuals, whose macrophages killed mycobacteria effectively (‘responders’). These macrophages also showed production of Interleukin-1β (IL-1β) in response to mycobacterial stimuli before vaccination. Here, we hypothesized that the propensity to respond to the BCG vaccine is reflected in the DNA methylome. We mapped the differentially methylated genes (DMGs) in PBMCs isolated from responders/non-responders at the time point before vaccination aiming to identify possible predictors of BCG responsiveness. We identified 43 DMGs and subsequent bioinformatic analyses showed that these were enriched for actin-modulating pathways, predicting differences in phagocytosis. This could be validated by experiments showing that phagocytosis of mycobacteria, which is an event preceding mycobacteria-induced IL-1β production, was strongly correlated with the DMG pattern.

Granulomas are hallmarks of pulmonary tuberculosis (TB) and traditionally viewed as host-protective structures. However, recent evidence suggest that Mycobacterium tuberculosis (Mtb) uses its virulence factors to stimulate the formation of granuloma. In the present study, we investigated the contribution of matrix metalloproteinases (MMPs), host enzymes that cause degradation of the extracellular matrix, to granuloma formation and bacterial load in Mtb-infected tissue. To this end, we used our lung tissue model for TB, which is based on human lung-derived cells and primary human monocyte-derived macrophages. Global inhibition of MMPs in the Mtb-infected tissue model reduced both granuloma formation and bacterial load. The infection caused upregulation of a set of MMPs (MMP1, 3, 9, and 12), and this finding could be validated in lung biopsies from patients with non-cavitary TB. Data from this study indicate that MMP activation contributes to early TB granuloma formation, suggesting that host-directed, MMP-targeted intervention could be considered as adjunct therapy to TB treatment.