Clinical School (Royal Melbourne Hospital) - Theses
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ItemUnderstanding innate immunity to Plasmodium falciparum malaria( 2017)The malaria parasite Plasmodium falciparum still causes an incredibly high number of deaths in young children under the age of five and pregnant women annually. The pathogenesis of human malaria is due to a combination of several parasite and host factors that simultaneously influence the severity and outcome of disease. Thus, a better understanding of the roles played by innate immune cells in both pregnancy and non-pregnancy related malaria infection is crucial. In Aim 1, an in vitro model of human peripheral blood mononuclear cells (PBMCs) derived from non-immune individuals were exposed to infected erythrocytes (IEs) expressing different P. falciparum erythrocyte membrane protein 1 (PfEMP1) binding phenotypes. This included IT parasites lines (CS2, a placental parasite line expressing VAR2CSA which binds to chondroitin sulfate A (CSA) and E8B (CD36 binding)) and NF54 lines (3C (non-CD36 binding) and 3D7 (CD36 binding)). The objectives were to identify whether PfEMP1 binding phenotypes influence PBMCs cytokine response, and also to determine whether CD36 binding is parasite characteristic that influences the innate immune response. Higher cytokines secretions were observed when PBMCs were stimulated with non-CD36 binding IEs in comparison with CD36 binding IEs. Furthermore, γδ T cells represented the predominant source of IFNγ in PBMCs from naïve donors stimulated with P. falciparum IEs. The focus of Aim 2 was to investigate the ability of purified in vitro expanded γδ T cells to produce cytokines when exposed to P. falciparum IEs with different variant surface antigens. It was found that these cells can be directly activated by P. falciparum IEs. Furthermore, cytokine secretion was not substantially different in response to parasite lines expressing different PfEMP1 types. Moreover, the contribution of Vδ2+ γδ T cells to the production of IFNγ was significantly higher than that of Vδ2- γδ T cells. Nonetheless, the activation of Vδ2- γδ T cells represented an unanticipated and intriguing result in the present study. Examining the culture of Vδ2 depleted γδ T cells, a significant ability of them to produce cytokines (IFNγ, GMCSF and TNFα) was observed following exposure to P. falciparum IEs. Furthermore, activation of these cells by P. falciparum IEs for IFNγ production was highly dependent on the direct contact between the cells and the whole IEs. This activation was shown to be sensitive to trypsin digestion but not lack of expression of PfEMP1. Considering both complete and Vδ2 depleted γδ T cell cultures, it was found that the activation modes of γδ T cells subsets appeared to be different in presence or absence of Vδ2. Two different mechanisms of activation of Vδ1 and Vδ1-Vδ2- γδ T cell subsets in presence or absence of Vδ2 γδ T cells were also proposed in this study. The goal of Aim 3 was to assess the effect of various factors such as active malaria infection, life time malaria exposure and gravidity on the maternal innate immune responses. PBMCs derived from Papua New Guinea (PNG) (highly endemic area) pregnant women with and without current malaria infection and Melbourne pregnant women (non-endemic area) were exposed to P. falciparum CS2-IEs for 24 hours. The present study found that among PBMCs derived from PNG pregnant women, presence of microscopic malaria infection did not alter either secretion of IFNγ or proportion of innate immune cells positive for induction of IFNγ. With regard to malaria exposure, a significant increase in IFNγ production was observed in response to both P. falciparum CS2-IEs and Phytohaemagglutinin (PHA) in PNG samples compared to Melbourne samples. This observation was shown to be associated with significantly more active natural killer cells (NK cells) in pregnant women living in PNG. Considering the effect of gravidity, a significant elevation of IFNγ secretion in multigravidae group was observed compared to primigravidae group in PNG pregnant women (a similar, non-significant trend in Melbourne pregnant women) in response to P. falciparum CS2-IEs. This observation could be explained by the observed percentages of IFNγ producing γδ T cells and specially their Vδ2 subset being substantially higher in multigravidae women compared to primigravid women in PNG group. Together, these results suggested that the combination of exposure, not entirely specific to malaria, and gravidity significantly impacts innate immune response in pregnant women. The outcomes of these studies led to new insights that might contribute to development of new strategies in modulating the innate immune system and particularly to increasing proportions of IFNγ producing γδ T cells to safely prevent or diminish P. falciparum-induced immunopathology.