The haematological consequences of experimental visceral leishmaniasis

Visceral leishmaniasis (VL) is a neglected tropical disease with high morbidity and mortality, presents with hepatosplenomegaly, immune dysregulation, haematological complications and high risk of co-/secondary infections. Thrombocytopenia is one of the predominant haematological features in infected humans and experimental models but the underlying mechanisms remain poorly understood. In this thesis, key aspects of platelet production and removal were examined in an experimental murine model of VL First, ultrastructural morphology of bone marrow megakaryocytes (MKs) in L. donovani-infected mice was examined using transmission electron microscopy and quantitative image analysis. The cytoplasmic demarcation membranous system and associated platelet territories, features of normal thrombopoiesis, were reduced in MKs. Despite this, MKs retained the functional capacity to produce platelets when placed under stress following anti-CD41 mediated platelet removal. Thrombopoietin, a key regulator of thrombopoiesis, was differentially expressed in hepatocytes around the hepatic granulomas in chronically infected mice. To address platelet removal, depletion of tissue macrophages was performed. In uninfected mice, no change in platelet counts was suggestive of other clearance mechanisms under homeostatic conditions. Full depletion of macrophages could not be achieved in infected mice, leaving open the question of their role in platelet clearance during infection. However, infected mice had significant numbers of IgG bound platelets, suggestive of macrophage dependent clearance. Excessive platelet desialylation in infected mice was also observed, providing an alternate mechanism involving enhanced platelet clearance by hepatocytes. To address the reversibility of these changes, a model of drug-induced parasite clearance was used. Blood parameters and tissue microarchitecture were restored after AmBisome® treatment. Finally, this model was extended to determine how previous VL affected the course of experimental malaria, as these two infections are commonly found in endemic countries. Malaria induced pronounced thrombocytopenia in mice with previous VL, but parasitaemia was delayed compared to control mice, suggestive of a residual cross protective immune response. Together, these data suggest a reversible multifactorial pathogenesis of thrombocytopenia with defective production and enhanced clearance. Delayed malaria kinetics post-treatment could be useful to study in human patients to avoid delayed and wrong diagnosis due to overlap of clinical presentations.