Dissecting the role of mycobacterial cell envelope components and DNA in leprosy reactions

Leprosy, a chronic infectious disease caused by the bacterium Mycobacterium leprae, remains a public health threat in developing countries, including Brazil, where more than 30,000 new cases are reported annually. A major health concern for individuals suffering from leprosy is that the disease principally affects the skin and the peripheral nervous system, leading to sensorial impairment and permanent disabilities. Indeed, acute inflammatory reactive leprosy responses of the immune system to the leprosy bacillus are the leading cause of nerve damage. These episodes can occur in individual patients, long after they are considered cured and termination of anti-leprosy drug treatment. However, our understanding of the physiopathology of leprosy reactions remains limited, and further research is urgently needed if we are to understand the biology underlying this neglected disease. The overall aim of this study is to investigate the molecular basis of reactional episodes in leprosy patients. Our hypothesis is that M. leprae constituents can remain in host tissues for long periods of time, and are responsible for the immune activation that occurs in these patients during reaction. This study will bring together the varied and complementary expertise of investigators from The FIOCRUZ, Oswaldo Cruz Foundation (Brazil) and The University of Birmingham (UK) to investigate the involvement of mycobacterial lipids, cell wall fragments and DNA as triggers of the reactional episodes. The investigators based in Brazil are experts in leprosy, specifically the immune responses involved in the infection. Investigators in the UK are experts in the molecular genetics and biochemistry of the mycobacterial cell wall. The knowledge generated in this study can contribute to more effective treatments and management strategies for leprosy reactions, with a greater impact on the quality of life, not just in Brazil but globally, given that leprosy affects many developing countries.

The main objectives of the study are:

Objective I: The UK Team (University of Birmingham, UoB) will define diagnostic lipid and cell envelope component profiles of standard M. leprae, surrogate Mycobacterium haemophilum and leprosy biopsy material and assess the persistence of these compounds in biopsies from leprosy patients who have experienced a reaction at least two years after the conclusion of anti-leprosy treatment.

Objective II: The Brazil Team (FIOCRUZ) will investigate the levels of M. leprae DNA and the involvement of the inflammasome pathways by analysing markers of inflammasome activation in leprosy biopsies of reactional lesions.

Objective III: To use well-established extraction and purification protocols at UoB to provide specific cell envelope components for further studies in Objective IV by collaborators at FIOCRUZ (Brazil Team). This will include M. leprae components (e.g. PGL, PDIM, mycolic acids, muropeptides), including available previously purified material by the UK team and through BEI Resources (e.g. whole cells, LAM). Since, leprosy bacilli are uncultivable, M. haemophilum, a phylogenetically related surrogate of M. leprae, will also be used as a source of structurally-related M. leprae-type lipids and LAM for bulk purification. Truncated versions of M. haemophilum PGL and LAM will also be prepared.

Objective IV: The Brazil Team (FIOCRUZ) will investigate which mycobacterial components identified in Objectives I and III are bioactive in inducing inflammatory mediators in whole blood samples of reactional patients. The most promising immune active components will be used to investigate their capacity to activate inflammasome pathways in in vitro differentiated macrophages and dendritic cells. Truncated versions of the active molecules will also be tested to map their functional moieties.

The aim of this UK-Brazil joint research grant is to delineate the molecular basis of reactional episodes in leprosy patients. We will specifically examine the potential role of the inflammasome pathways and the involvement of M. leprae cell envelope components and DNA in triggering the reactional episodes. The main objectives of the study are:

Objective I: The UK Team (UoB) will define diagnostic lipid and cell envelope component profiles of standard M. leprae, surrogate M. haemophilum and leprosy biopsy material and assess the persistence of these in leprosy biopsies.

Objective II: The Brazil Team (FIOCRUZ) will investigate in biopsies of reactional skin lesions levels of M. leprae DNA, the phenotype of immune cells composing the inflammatory infiltrate and markers of inflammasome activation.

Objective III: To use well-established extraction and purification protocols at UoB to provide specific cell envelope components for further studies in Objective IV by collaborators at FIOCRUZ. This will include M. leprae components (e.g. PGL, PDIM, mycolic acids, muropeptides), including available previously purified material by the UK team and through BEI Resources (e.g. whole cells, LAM). Since, leprosy bacilli are uncultivable, M. haemophilum, a related surrogate of M. leprae, will also be used as a source of structurally-related M. leprae-type lipids and LAM for bulk purification.

Objective IV: To investigate by multiplex analysis the profile of inflammatory mediators secreted by whole blood samples of reactional patients in response to mycobacterial components identified in Objective I and prepared in Objective III. Use functional in vitro studies with purified mycobacterial components in order to investigate their capacity to activate inflammasome pathways in in vitro differentiated macrophages and dendritic cells. Truncated versions of the active molecules will also be tested to map their functional moieties.

Who will benefit from this research: The outputs from the proposed research will increase our fundamental understanding of the biology underlying leprosy and the leprosy bacillus. While research on this neglected disease continues, it lags behind that of other mycobacterial diseases, such as, tuberculosis. The proposed work in this research grant will provide the much-needed boost to advance leprosy research. The immediate beneficiaries will thus be researchers studying leprosy and its etiological agent. In particular, the dedicated researchers who will be working on this project should the proposal be funded. However, leprosy researchers would by no means be the only academic beneficiaries, as the work has the potential to add to our understanding of how bacteria use different strategies to cause infection and reactivation. Academic beneficiaries will also include immunologists studying mycobacterial diseases. Long-term beneficiaries will include individuals affected by leprosy as the proposed work has the potential to inform therapeutics and better diagnostics. Given that leprosy affects individuals not just in Brazil, but also several other developing countries in nearly all continents, this research would impact both on local, as well as global health.

How will they benefit from this research: Career development of the researchers. Regular meetings, in addition to laboratory meetings, will be held with the researchers to set goals and for trouble-shooting, and if necessary revising the experimental approach. This will ensure that outputs are maximised and the research is focussed. Additionally, for professional development, University of Birmingham has a mentoring scheme termed 'Personal Best' that ensures that research training needs and career opportunities are identified. Career training presentations are also regularly held in the University to bolster the professional development of early career stage researchers. PDRAs, both from Brazil and the UK, would also benefit from training in specialist methodology at each partner institution during exchange visits.

Other academic beneficiaries: Our work will potentially further our understanding of how reactional episodes occur in leprosy and thus leprosy researchers will be the initial beneficiaries of the findings from this research grant. As the study aims to look at the immune response in reactional episodes of leprosy, immunologists studying mycobacterial diseases in particular, and infection in general, will benefit from our potential findings as this is a poorly understood area of research in leprosy. The proposal also aims to develop lipids as biomarkers that is especially relevant to the efforts of researchers to develop improvised diagnostics for leprosy and other mycobacterial diseases.

The wider public: Research in leprosy remains, as mentioned above, neglected and as a result diagnostics and therapeutics in their present state are not optimal. Our research aims to have a long-term impact on these two aspects by furthering our understanding of how mycobacterial cell envelope components potentially drive reactional leprosy episodes, highlighting also lipids as potential biomarkers for eventual use in diagnostics. In the longer-term, this information will thus inform anti-leprosy therapeutics and diagnosis, impacting global health. We expect immediate impact activities will be measured in the number of publications, invited talks, sustaining the proposed collaborations via further grant applications and career progression of the trained researchers. These are expected to take place during the time of the proposed research grant. Long term impacts like commercial exploitation of findings are likely to occur beyond the timeline of the award and will be key for future impact on national (Brazil) and global health.