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

麻风病是一种由麻风分枝杆菌引起的慢性传染病，在包括巴西在内的发展中国家仍然是一种公共卫生威胁，每年报告的新病例超过3万例。麻风病患者的一个主要健康问题是，这种疾病主要影响皮肤和周围神经系统，导致感觉障碍和永久性残疾。事实上，免疫系统对麻风杆菌的急性炎症反应性麻风反应是神经损伤的主要原因。这些事件可能发生在个体患者身上，在他们被认为治愈并终止抗麻风药物治疗很久之后。然而，我们对麻风病反应的生理病理学理解仍然有限，如果我们要了解这种被忽视疾病的生物学基础，迫切需要进一步的研究。本研究的总体目的是研究麻风病患者反应性发作的分子基础。我们的假设是麻风分枝杆菌成分可以在宿主组织中停留很长一段时间，并负责这些患者在反应期间发生的免疫激活。这项研究将汇集来自FIOCRUZ， Oswaldo Cruz基金会（巴西）和伯明翰大学（英国）的研究人员的各种和互补的专业知识，以调查分枝杆菌脂质，细胞壁碎片和DNA作为反应发作的触发因素的参与。巴西的调查人员是麻风病专家，特别是与感染有关的免疫反应。英国的研究人员是分枝杆菌细胞壁分子遗传学和生物化学方面的专家。鉴于麻风病影响许多发展中国家，本研究中产生的知识可有助于制定更有效的麻风病反应治疗和管理战略，不仅在巴西，而且在全球对生活质量产生更大影响。该研究的主要目标是：目的一：英国团队（伯明翰大学，UoB）将定义标准麻风分枝杆菌、替代血友病分枝杆菌和麻风活检材料的诊断性脂质和细胞包膜成分概况，并评估这些化合物在麻风患者的活检中持续存在，这些麻风患者在抗麻风治疗结束后至少两年出现反应。目的二：巴西队（FIOCRUZ）将通过分析麻风病反应性病变活检中炎性体激活的标记物，研究麻风分枝杆菌DNA水平和炎性体通路的参与。目标III：在UoB使用完善的提取和纯化方案，为FIOCRUZ（巴西团队）的合作者在目标IV中进一步研究提供特定的细胞包膜成分。这将包括麻风分枝杆菌成分（如PGL、PDIM、霉菌酸、多肽），包括英国团队和BEI资源提供的先前纯化的材料（如全细胞、LAM）。由于麻风杆菌是不可培养的，血友病分枝杆菌，麻风分枝杆菌的一种系统发育相关的替代物，也将被用作结构相关的麻风分枝杆菌型脂质和LAM的来源，用于大量纯化。血友病分枝杆菌PGL和LAM的截短版本也将准备。目的四：巴西小组（FIOCRUZ）将调查目的一和目的三中鉴定的分枝杆菌成分在反应性患者全血样本中诱导炎症介质的生物活性。最有希望的免疫活性成分将用于研究它们在体外分化的巨噬细胞和树突状细胞中激活炎性体途径的能力。活性分子的截断版本也将被测试以绘制其功能部分。

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.