Clinical School (Royal Melbourne Hospital) - Theses
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ItemInnate cellular responses to malaria in children and pregnant women( 2018)Malaria is one of the leading causes of morbidity and mortality in young African children and pregnant women. Clinical presentations and outcomes of P. falciparum infections are influenced by a combination of host, parasite and environmental factors. In the absence of an effective vaccine, it is important to understand how parasite and environmental factors interact with host innate immunity to influence disease severity in paediatric and pregnancy-related malaria. Aim 1, investigated the association between P. falciparum erythrocyte membrane protein 1 (PfEMP1) and inflammatory responses in clinical malaria syndromes. Malaria-infected erythrocytes (IE) were isolated from Malawian children with cerebral or uncomplicated malaria to compare their PfEMP1 profiles and cytokine elicitation capacity on malaria naïve PBMC. Also, Monocyte-derived macrophages (MDM) from Melbourne donors were exposed to isogenic parasite lines with different PfEMP1 phenotypes including (CS2: CSA binder; P6A1: CD36 binder; P6G2: CD36; E8B: CD36 and ICAM-1 binders) to determine whether PfEMP1 binding characteristics influence cytokine production. According to the var typing data of the clinical parasites from Malawi, the transcription of group A PfEMP1 was significantly higher in cerebral malaria isolates compared to the uncomplicated malaria isolates. Following in vitro stimulation of PBMC, IE from cerebral malaria isolates induced higher pro-inflammatory cytokines compared to the uncomplicated malaria isolates, suggesting a relationship between PfEMP1 expression and the elicited cytokine responses. Furthermore, MDM stimulations with CD36 binding parasites generated significantly lower inflammatory cytokines compared to the non-CD36 binding line. Together, findings of this study suggest that PfEMP1 binding phenotypes influence inflammatory responses and consequently disease severity. In Aim 2, innate cellular responses in acute and convalescence were compared between children with cerebral and uncomplicated malaria from Malawi. The objective was to evaluate the influence of inflammatory responses and monocyte subpopulations in malaria severity. Plasma levels of inflammatory cytokines and the proportion of monocyte subsets were analysed at presentation (day 0) and follow-up (day 28). Although inflammatory cytokine levels were significantly elevated during acute infection, responses were similar between cerebral and uncomplicated malaria. Consistently, monocyte profiles were not different between the two malaria groups. Aim 3, was to investigate the effects of factors such as gravidity, lifetime malaria exposure, active malaria infection and different P. falciparum strains on maternal innate immunity. PBMC from primigravidae and multigravidae pregnant women from Papua New Guinea (PNG) (with or without current infection) and Australia were stimulated with CS2-IE (CSA binder), P6A1-IE (CD36 binder) or controls (PHA & uRBC). In this study, PBMC responses were not influenced by gravidity in both study cohort or current malaria infection among PNG women. Compared to samples for Australia, PBMC from PNG produced significantly higher cytokine levels to both CS2-IE and PHA, suggesting a geography-related influence not entirely specific to malaria exposure. Between the two parasite lines, PBMC stimulations with CS2-IE elicited higher responses for some cytokines compared to P6A1-IE. These observations suggest that maternal innate immunity to malaria is influenced by geographic origin and different P. falciparum strains. The findings from these studies have added to our current understanding of P. falciparum-induced immunopathology and identified mechanisms that could be exploited for the development of new intervention strategies targeted towards innate immune modulation.
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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.