Defining the effects of transplant immunosuppressants on innate immunity to fungi

Organ transplantation is an increasingly common treatment for a range of diseases, but requires that individuals take life-long immunosuppressive medication. These drugs stop the immune system rejecting the transplant, but increase susceptibility to life-threatening invasive fungal infections. These infections have a mortality in excess of 40% overall, and require a long duration of highly toxic, expensive anti-fungal therapy. Understanding the immunological mechanisms that predispose some patients to these infections is critical to improving outcome.

The research is relevant to invasive fungal infection and specifically invasive aspergillosis. Invasive aspergillosis has emerged as the commonest fungal infection causing death in developed countries, with a mortality of 40% to 90%. There are estimated to be 3000 to 4000 deaths from this disease per annum in the UK. Invasive aspergillosis almost exclusively occurs in immunocompromised patients, and solid organ transplant patients have emerged as a rapidly growing at risk population.

The research is trying to define the mechanisms that lead to increased risk of fungal infection in transplant patients. The main immunosuppressive drugs these patients receive are called calcineurin inhibitors which are thought to primarily suppress T cell adaptive immune responses. However recent studies have demonstrated that calcineurin inhibitors could have major effects on immune cells called phagocytes. This is because the calcineurin pathway is required for signalling through a group of phagocyte immune sensing molecules called C-type lectins. This is very important because a range of fungal pathogens are sensed through C-type lectin receptors such as Dectin-1. The research will investigate how these drugs interfere with phagocyte sensing and killing of fungal pathogens and will further define the calcineurin-dependent mechanisms that mediate killing once a fungal pathogen has been recognised.

This research is important because invasive fungal infections are a major cause of mortality in an expanding group of transplant patients and within other patient groups on maintenance therapy with calcineurin inhibitor immunosuppressive drugs. Understanding the mechanisms that lead to increased susceptibility to these infections will enable us to develop new strategies to identify when patients become at risk, and how best to restore fungal immunity when it becomes critically impaired. Furthermore defining the effects of these drugs on phagocyte function may lead to further insights into transplant rejection and is likely to have wider implications for transplant immunity as well as fungal immunity.

The research will be carried out by Dr Anand Shah through a Clinical Research Fellowship. Dr Shah is a junior doctor specialising in respiratory infection and transplantation at Harefield Hospital. His fellowship will be supported by Prof. Sunil Shaunak, Dr Darius Armstrong-James and Dr Elaine Bignell at Imperial College, Professor Gordon Brown at the University of Aberdeen, and Dr. Anna Reed at Harefield Hospital. Professor Shaunak is a leading Infectious Diseases Clinical Academic focussed on translational medicine development. Dr Armstrong-James is an MRC Clinician Scientist in fungal immunology who has developed a relevant transplant model of fungal infection. Professor Brown is a renowned fungal immunologist who discovered the C-type lectin fungal sensor Dectin-1. Dr. Reed is a lung transplant physician at Harefield Hospital.

The research will be primarily focussed on a new calcineurin inhibitor based mouse model of invasive aspergillosis. This model will be used to define the effects of calcineurin inhibitor drugs on phagocyte killing of fungi, and how this is affected in aspergillosis. In addition, we have ethical approval to confirm the model-based findings through targeted observational studies of lung transplant patients with invasive aspergillosis at Harefield Hospital.

Aim: To define the effects of the calcineurin inhibitor FK506 on innate immunity to aspergillosis

Objectives:

1. Define the effects of FK506 on innate immunity to A. fumigatus in vivo.
Methodology: Outcome from pulmonary aspergillosis will be characterised in wild-type C57BL/6, Rag2 -/-, Dectin-1 -/- and Myd88 -/- mice immunosuppressed with FK506/control. This will enable the effects of FK506 on the innate host response to aspergillosis to be determined and whether these effects are mediated via TLRs or Dectin-1 fungal pattern recognition receptors.

2: Characterise the effects of FK506 on A. fumigatus phagocytosis
Methodology: Alveolar macrophages and neutrophils will be isolated from C57BL/6, Dectin-1 -/- and Myd88 -/- mice, treated with FK506/control, and challenged with A. fumigatus conidia. Phagocytosis efficiency and killing will be determined by flourescence microscopy and culture. Killing defects will be further characterised by time-lapse microscopy, ROS and NO assays.

3: Identify the pathways required for calcineurin-dependent phagocytosis of A. fumigatus
Alveolar macrophages/neutrophils will be isolated from C57BL/6 mice and challenged with A. fumigatus after treatment with FK506/control. Calcineurin-dependent signalling pathways will be identified by transcript profiling. The role of identified pathways in fungal killing will be defined by siRNA.

Aim 4: Validate key observations in lung transplant patients with aspergillosis
Key observations identified in components 1 to 3 will be validated in alveolar macrophages and neutrophils from lung transplant patients with pulmonary aspergillosis and controls.

Scientific and medical opportunities:
These studies will advance our understanding of how the main transplant immunosuppressive FK506 impairs innate fungal immunity. These findings will have broader implications for the role of the calcineurin pathway in innate immunity to other infectious diseases and transplant rejection.

Beneficiaries:

-The Royal Brompton and Harefield NHS Trust
For this study we have created a novel, unique collaboration between eminent clinical and laboratory mycologists, transplant and fungal immunologists, infectious disease specialists and transplant physicians enabling a long-term academic partnership to flourish for future clinical and basic science research. This will have a significant impact on the ability to create and deliver high-quality translational research projects in the future in the field of clinical mycology, infection and immunology over the next 20 years.

-The NHS
The research described will elucidate the mechanisms by which calcineurin inhibitors impair innate fungal immunity. This will lead to a better understanding of when patients are likely to develop invasive fungal infections, and how immunity can be augmented to improve outcomes. This will directly benefit the NHS, by reducing morbidity and mortality from these life-threatening infections and the very high costs of antifungal therapy. In addition improved outcomes from immunosuppression in transplantation will remove some of the existing barriers to transplantation and make this approach to treatment of organ dysfunction more acceptable.

- General public
By better defining the abnormalities in host immunity that predispose to invasive fungal infections (IFIs) within solid-organ transplantation, we will provide targets for future research investigating potential immunodiagnostic or immunotherapeutic strategies. If successful this would have significant impact on the morbidity and mortality from IFIs amongst the transplant population and enable a more confident diagnosis with reduced empirical antifungal treatment. Additionally by better defining the host immune risk for developing IFIs amongst the transplant population this will lead to more targeted preventative strategies and antifungal prophylaxis. This will not only reduce mortality and morbidity but will have significant economic cost benefit compared to utilising a blanket antifungal strategy that is often applied. Morbidity from antifungal therapy which have significant toxicity will also be greatly reduced. This study is the first step towards a translational mycology diagnostic and therapeutic goal which should be achievable within ten years.

- The private sector
The research is translational and aimed at identifying key immune deficits in transplant patients. The research output is likely to be useful for the development of novel immuno-diagnostics and immuno-therapeutics to improve outcomes in transplant infectious diseases.