How do functional and metabolic characteristics of trained monocytes affect their anti-bacterial activity?

Led by Asst Prof Steven Smith (LSHTM), with Dr Javier Sanchez-Garcia (Instituto Politécnico Nacional), Prof Jo Prior (dstl), and Prof Gregory Bancroft (LSHTM)

Project Aims

The human immune response has two components, the innate and the adaptive responses. This project will investigate the potential of the innate response to contribute to protection against tuberculosis (TB) and melioidosis. TB remains a major global problem being one of the world’s leading causes of death from infectious disease. In addition to better drugs and better means of diagnosing TB, we also need a better vaccine. The current vaccine, BCG, is only partially effective and an improved version is needed. However, many novel TB vaccine candidates focus on the adaptive immune response involving T-lymphocytes as this is where immune memory is found which is essential for vaccine-induced protection. We think that an optimal innate response is also essential. It has recently been discovered that the innate response can be “trained” by some vaccines to respond better to later infections. In this project, we aim to investigate this training effect on the innate response to a) determine how best to train innate cells to prevent the growth of the causative organisms of TB and melioidosis; b) characterise these trained innate cells to find which immune molecules are important for their protective effect and how the metabolism (or biochemical energy production pathways) of these cells affects the trained response and c) to investigate why innate cells from different people do not respond in the same way to training – to do this we will look at differences in the DNA of immune cells (termed epigenetic marks) which cause these cells to produce different immune molecules when exposed to the same microorganism. The data produced by this project should help us understand better how different components of the immune response contribute to protection against TB and meliodosis and should aid the design of better vaccination regimes for bacterial diseases.

Project Outcomes

Tuberculosis remains the deadliest infectious disease worldwide and thus, any effort to understand the biological basis of disease progression as well as to provide the means to boost anti-Mycobacterium tuberculosis immune responses should be considered, as it might have a positive impact on overcoming this human health burden.

Innate immune training offers an interesting theoretical framework for the understanding of tuberculosis, since monocyte/macrophages are the main target cells for infection by M. tuberculosis. Innate immune training allow these cells to respond faster and better to a second antigenic challenge, even if the first and second challenges are qualitatively different. An in vitro model of trained immunity in human monocytes was established in the laboratory based on a previous publication. This involved the isolation of CD14+ monocytes from peripheral blood and co-culture with training stimuli including live BCG, heat-killed BCG or beta-glucan followed by a rest period of 6 days. Training of monocytes was demonstrated by their enhanced ability to produce cytokines (TNF and IL-6) in response to heterologous ligands. Trained monocytes were also cocultured with BCG to determine their capacity to inhibit the growth of mycobacteria which was observed for some, but not all donor monocytes. Monocyte aliquots were stored for analysis of DNA methylation patterns.

Recent developments in the field show that, in some instances, innate immune training correlates with intracellular fumarate accumulation. Of note, fumarate-stimulation of monocyte/macrophages induces the functional and epigenetic signatures of innate immune training.

As part of this project we aimed to characterize the ‘’mitochondrial signature’’ of this training, as well as to explore some other monocyte/macrophage functions relevant for the control of mycobacterial infection.

Human peripheral blood monocytes were first stimulated with monomethyl fumarate and then with LPS or BCG, and mitochondrial dynamics; mitochondrial membrane potential was followed for 3 h post-stimulation and mitochondrial calcium uptake followed for 15min after the addition of BCG. These three functional activities of mitochondria were increased in fumarate-treated monocytes as compared to un-treated cells. Fumarate-stimulated monocyte/macrophages (plus the appropriate controls) were subjected to transmission electron microscopy in order to analyse the mitochondrial cristae.

In addition, RAW 264.7 cells (a murine macrophage cell line) were stimulated with monomethyl fumarate in order to study mitochondria and then mitochondrial proteins. Anti-phosphoserine Western blot analyses showed a different pattern of serine-phosphorylated mitochondrial proteins in cells stimulated with a fumarate as compared to un-stimulated cells. All these mitochondrial functional parameters contribute to define the ‘’mitochondrial signature’’ of innate training.

As for the function of trained versus non-trained monocytes, the ability of these cells to process antigen was explored. Results showed that fumarate-treated cells have an increased capacity for antigen processing.

Taken together, these results provide evidence of a distinctive mitochondrial function (‘’mitochondrial signature’’) in trained monocytes and also show that trained monocytes process exogenous antigens more efficiently that un-trained monocytes. Such a ‘’mitochondrial signature’’ may identify one way to boost anti-mycobacterial immune responses, maintain innate immune training, and understand the basis of mycobacterial dormancy and reactivation.

Project Outputs

Butkeviciute E, Jones CE, Smith SG. Heterologous effects of infant BCG vaccination: potential mechanisms of immunity. Future Microbiol. 2018 Aug; 13:1193-1208.

C Angélica Pérez-Hernández et al 2020. Mitochondrial signature in human monocytes and resistance to infection in C.elegans during fumarate-induced innate immune training. Frontiers in Immunology 11: 1715