Augmentation and regulation of immune responses to Tuberculosis by vitamin D

Tuberculosis remains a major threat to global health, with one third of the world's population estimated to have been exposed, causing 9 million cases of active disease and 2 million deaths every year. The human immune system is our major defence against TB. In some cases our immune system can clear the infection completely, but in most it is thought to control the infection so that we remain healthy despite the persistence of some TB bacteria, resulting in latent infection. The risk of latent TB is that it can re-activate at a later date, particularly if the immune system weakens. Some aspects of the immune response to TB may also be harmful by causing the damage that leads to disease in active TB. Consequently, we hope to find ways to increase the ability of the immune system to fight TB without causing excessive inflammation that exacerbates the disease. There is evidence to suggest that treatment with vitamin D can achieve these goals and I aim to extend these studies in order to inform new strategies to prevent or treat TB. Vitamin D deficiency is widespread and is associated with greater risk of active TB. We have shown previously that oral vitamin D supplements can benefit people with active TB, and may increase the ability of the immune system to kill the TB bacteria. In laboratory experiments, addition of vitamin D to immune cells called macrophages infected with TB helps the cells kill TB bacteria. However, in the body as a whole, the immune response to TB requires co-operation between macrophages and other immune cells called T cells. The addition of vitamin D to T cells can increase or decrease their activity, but how vitamin D affects the interaction between macrophages and T cells against TB is not known, and is the overarching aim of my fellowship.
I will perform laboratory experiments using purified human T cells and macrophages infected with TB, to test the effect of vitamin D on bacterial killing and identify the mechanism by which vitamin D works. I will also study the effect of vitamin D on immune responses within the body: firstly, by taking skin biopsies after injecting extracts of dead TB bacteria in patients with active TB, latent TB or no TB infection, before or after receiving vitamin D supplements; secondly by testing the effects of vitamin D in blood samples from patients with active TB in a recent clinical trial of the effect of combining vitamin D with antibiotic treatment for TB. The blood and skin samples from these studies will be used to measure gene expression patterns throughout the human DNA code, giving a comprehensive and unbiased view of the effect of vitamin D on immune responses to TB. These experiments will test the hypothesis that vitamin D supplements can both augment immune defence against TB and function to reduce excessive inflammation, depending on whether someone has active or latent TB infection. My results will inform our understanding of the mechanisms by which vitamin D affects the immune system and hence lead to more rational use of vitamin D supplements in clinical practice, with a major impact on the management of TB in the UK and across the world.
The use of human samples in this research is essential in order to represent the complex human response to this pathogen. Ethical approval is in place for much of this work and will be sought for the remainder of this work prior to the onset of the research. I will carry out this research myself during a PhD, hosted by University College London (UCL). A new collaboration between my supervisors, Dr Noursadeghi and Dr Martineau, at this internationally renowned institution, creates a perfect setting for this work. Dr Noursadeghi (of UCL) has extensive experience with experimental models of immune responses as well as the complex analysis required for this study, whilst, as an international expert in vitamin D research, Dr Martineau (of Queen Mary University of London) will provide unrivalled expertise to support my fellowship.

Augmentation of host defences to protect against tuberculosis (TB), by enhancing bacillary clearance coupled to regulatory control of inflammation to minimize immunopathogenesis of active diseases is a global research priority. Vitamin D can enhance intracellular killing of Mycobacterium tuberculosis (Mtb) and promote T cell regulation. Therefore, I aim to test the hypothesis that vitamin D affords the benefits of enhanced Mtb clearance and homeostatic regulation of immune responses in active disease. Immunological control of Mtb is dependent on the interaction between macrophages and T cells, but the influence of vitamin D in this context is not known. I will test the effect of vitamin D on T cell mediated killing of Mtb within macrophages in vitro and identify the changes to host cell response that mediate these effects. I will then test the effect of vitamin D on in vivo immune responses to TB, by genome-wide transcriptional profiling of skin biopsies at the site of tuberculin tests before or after vitamin D supplementation in people with active TB, latent TB or no evidence of infection. This approach will provide comprehensive and unbiased assessment of in vivo immune responses to TB at systems level and with molecular resolution, with which I will test the hypothesis that vitamin D supplementation attenuates adaptive responses in patients with active TB, but not in uninfected patients or those with latent TB, and to define the molecular detail of the effects of vitamin D on cutaneous responses to tuberculin in each group. Finally, I will take advantage of peripheral blood RNA samples collected during the ADJUVIT clinical trial of vitamin D in the treatment of active TB, to test the hypothesis that supplementation of antimicrobial therapy with vitamin D induces qualitatively different gene expression changes in peripheral blood compared to antimicrobial therapy alone, which reflect the immunoregulatory effects of vitamin D.

The primary beneficiaries of the proposed work will be patients with active or latent TB and the physicians managing these patients. By investigating the role of vitamin D in the immune response to both latent and active TB, we more clearly define the potential role for the therapeutic use of vitamin D in both settings. For patients with active TB, there remains the potential for supplementation of vitamin D to enhance the efficacy of antituberculous therapy (ATT), with greater benefits to be found in those with difficult to treat TB such as MDR TB and HIV co-infected patients. This has clear benefits in terms of reduction in the significant associated morbidity and mortality. In those contacts with latent TB, the role of vitamin D may be to prevent reactivation to active TB. Not only does this reduce morbidity in these patients, it reduces risks of transmission to previously uninfected individuals with consequent public health benefits and an improvement in the health and wellbeing of the wider population. Furthermore, if the hypothesis that vitamin D has differential effects in latent to active TB is proven, the interpretation of the tuberculin skin test (TST) will require consideration of the patients' vitamin D status to interpret it appropriately; i.e. we may be underestimating the incidence of latent TB in contacts of TB when relying on the TST in vitamin D deficient patients.

The application of the use of microarray data in the longitudinal analysis of patients on TB treatment, with or without vitamin D, will also provide a basis from which to identify new target molecules for drug therapy or immunomodulation, as well as provide insight into potential new uses of vitamin D, such as, as an adjuvant to vaccination. Again, the wider impact of this will be vast given the huge worldwide burden of TB.

Study of the effect of vitamin D in tuberculosis will also provide insight into its roles in the immune response in general, thereby informing research in other immune disorders, including multiple sclerosis and diabetes. This will, therefore, benefit a wider patient population.
Although often used in practice, there remains a need for further scientific evidence for the use of vitamin D in patients with TB infection. My project will add to this body of evidence, informing policy (from bodies such as SIGN and NICE) and national management guidelines. At present, there is no comprehensive national screening programme for new entrants on arrival to the UK. If shown to augment the immune response in patients with latent TB, we highlight the role for screening new entrants from high endemicity countries, and supplementation of vitamin D in order to prevent active TB. As a safe and cheap medication, the use would not need to be limited to resource-rich countries alone. In the longer term, this will result in health economic savings and an increase in productivity of the predominantly young population affected by TB in the UK. This work will provide data to inform later use of clinical trials required to take such an intervention forward.

In a wider sense, the publication and communication of our work in peer-reviewed journals, conference presentations and press statements, will increase international recognition of TB research in the UK. Close collaboration with clinicians running the TB services will also raise the profile of research and its vital role in the practice of clinical medicine and improving patient care.
On a personal level, I will gain many useful research skills, including critical appraisal and review of literature, leadership and management skills, which are directly transferable to clinical work and will enhance my practice of evidence-based medicine and that of my junior team. This in itself will have indirect effects by encouraging juniors interested in academic medicine to themselves undertake research.