Investigating local determinants of outcome in human tuberculosis

Tuberculosis is an infection which kills approximately one and a half million people each year, and is becoming increasingly resistant to the antibiotics used to treat it. A central challenge in understanding tuberculosis is that the body's immune response is needed to control infection, but also causes the inflammation that leads to lung damage and spread of infection. In addition, in the same individual, some areas of infection can progress whilst other regress, indicating that it is local factors that determine outcome. Tuberculosis is exclusively a disease of humans, and therefore to fully understand this complex interaction requires a human laboratory system that can be studied to determine the underlying processes.

We have developed an entirely new way of investigating infection in the laboratory by developing a method where we infect human cells and then encapsulate them into mini-spheres for long-term analysis. This has the benefit that we can monitor the infection for much longer than the usual laboratory systems, and we have shown that key features like the behaviour of human cells and the response to antibiotics is more similar to that observed in patients than traditional systems. In this programme of work, we will use two different but complementary approaches to determine what regulates the outcome of tuberculosis infection in individual lesions. First, we will modify the mini-sphere environment and composition to determine which factors lead to optimal control of infection. We will add inflammatory mediators around the spheres and measure the progress of infection. Next, we will modulate the number and activation of immune cells that we encapsulate in the mini-spheres. Finally, we will change the fibres within the spheres. Together, these experiments will tell us which conditions help the immune system control infection most effectively.

In parallel, we will design a highly sensitive camera system, which allows us to measure infection within each mini-sphere, as the bacteria have been genetically modified to spontaneously produce light. We will measure the progress of infection over time, and then study the cells within mini-spheres where infection is being controlled versus those where the infection is progressing. We will extract the cells and measure their activation using the latest single cell profiling techniques, to understand which characteristics are needed to control infection. Finally, we will study tissue from patients with tuberculosis, again comparing progressive and controlling lesions, to confirm our observations in mini-spheres at the site of disease in patients.

Taken together, our findings will tell us why some tuberculosis lesions progress and some regress, which is essential knowledge to inform new strategies such as vaccination and lung-protective treatments to control the global epidemic.

Tuberculosis (TB) remains a global pandemic, killing 1.4 million people a year. An enduring challenge limiting development of transformative interventions is that the host immune response is necessary to control Mycobacterium tuberculosis (Mtb) infection, but also causes immunopathology that leads to tissue destruction and spread. Understanding the relationship between protective and pathological host immune responses is a critical open question in the field. Mtb is exclusively a pathogen of man, and therefore analysis of a human system is necessary to fully dissect the underlying mechanisms.

We have developed an advanced cell culture model of human granulomas, validated expression against clinical samples and demonstrated its ability to investigate the host-pathogen interaction in TB. We have shown that antibiotic sensitivity mirrors that in patients, excessive inflammation is harmful and have performed detailed mechanistic translational studies. Here, we will utilise this highly tractable system to systematically analyse factors that regulate control or progression of infection. First, we will modulate cytokine concentrations, T cell numbers and specificity, and extracellular matrix composition, to determine which factors skew the host-pathogen relationship towards control or progression. In parallel, we will design a microsphere imaging system, and quantitate Mtb growth in individual spheres longitudinally. We will characterise controlling versus progressing microspheres by single cell RNAseq to define determinants of outcome by an unbiased methodology. Finally, we will validate key regulatory points by RNA analysis and immunohistochemistry using human TB granulomas biopsied from patients before the initiation of antibiotics. Taken together, these findings will determine the protective versus pathological components of the host immune response to TB, which is essential to inform new vaccination or host-directed therapies.